phylum xvi. tenericutes murray 1984a, 356 (effective...

Phylum XVI. Tenericutes Murray 1984a, 356VP (Effective publication: Murray 1984b, 33.)

Daniel R. BRown

Ten.er¢i.cutes. L. adj. tener tender; L. fem. n. cutis skin; N.L. fem. n. Tenericutes prokaryotes of a soft pliable nature indicative of a lack of a rigid cell wall.

Members of the Tenericutes are wall-less bacteria that do not syn-thesize precursors of peptidoglycan.

Further descriptive information

The nomenclatural type by monotypy (Murray, 1984a) is the class Mollicutes, which consists of very small prokaryotes that are devoid of cell walls. Electron microscopic evidence for the absence of a cell wall was mandatory for describing novel species of mollicutes until very recently. Genes encoding the pathways for peptidoglycan biosynthesis are absent from the genomes of more than 15 species that have been annotated to date. Some species do possess an extracellular glycocalyx. The absence of a cell wall confers such mechanical plasticity that most molli-cutes are readily filterable through 450 nm pores and many spe-cies have some cells in their populations that are able to pass through 220 nm or even 100 nm filters. However, they may vary in shape from coccoid to flask-shaped cells or helical filaments that reflect flexible cytoskeletal elements.

Taxonomic comments

To provide greater definition and formal nomenclature for vernacular names used in the 8th edition of Bergey’s Manual of Determinative Bacteriology (Bergey VIII; Buchanan and Gibbons, 1974), Gibbons and Murray (1978) proposed that the higher taxa of prokaryotes be subdivided primarily according to the presence and character, or absence, of a rigid or semirigid cell wall as reflected in the determinative Gram reaction. Similar to the non-hierarchical groupings of Bergey VIII, which were based on a few readily determined criteria, the “wall-deficient” organisms grouped together in the first edition of The Prokary-otes included the mollicutes (Starr et al., 1981). While acknowl-edging the emerging 16S rRNA-based evidence that indicated a phylogenetic relationship between mollicutes and certain Gram-stain-positive bacteria in the division Firmicutes, Murray (1984b) proposed the separate division Tenericutes for the stable and distinctive group of wall-less species that are not simply an obvious subset of the Firmicutes.

The approved divisional rank of Tenericutes and the assignment of class Mollicutes as its nomenclatural type (Murray, 1984a) were adopted by the International Committee on Systematic Bacte-riology’s Subcommittee on the Taxonomy of Mollicutes (Tully, 1988) and subsequent valid taxonomic descriptions assigned novel species of mollicutes to the Tenericutes. However, the second (1992) and third (2007) editions of The Prokaryotes described the mollicutes instead as Firmicutes with low G+C DNA. The Subcom-mittee considered this to be an unfortunate grouping: “While

the organisms are evolutionarily related to certain clostridia, the absence of a cell wall cannot be equated with Gram reac-tion positivity or with other members of the Firmicutes. It is unfortunate that workers involved in determinative bacteriology have a reference in which wall-free prokaryotes are described as Gram-positive bacteria” (Tully, 1993a). Despite numerous valid assignments of novel species of mollicutes to the Tenericutes dur-ing the intervening years, the class Mollicutes was still included in the phylum Firmicutes in the most recent revision of the Taxo-nomic Outline of Bacteria and Archaea (TOBA), which is based solely on the phylogeny of 16S rRNA genes (Garrity et al., 2007). The taxon Tenericutes is not recognized in the TOBA, although paradoxically it is the phylum consisting of the Mollicutes in the most current release of the Ribosomal Database Project (Cole et al., 2009). Mollicutes are specifically excluded from the most recently emended description of the Firmicutes in Bergey’s Manual of Systematic Bacteriology (2nd edition, volume 3; De Vos et al., 2009) on the grounds of their lack of rigid cell walls plus analyses of strongly supported alternative universal phylogenetic markers, including RNA polymerase subunit B, the chaperonin GroEL, several different aminoacyl tRNA synthetases, and subunits of F0F1-ATPase (Ludwig et al., 2009; Ludwig and Schleifer, 2005). The taxonomic dignity of Tenericutes bestowed by its original formal validation, and upheld by a quarter of a century of valid descriptions of novel species of mollicutes, has therefore been respected in this volume of Bergey’s Manual.

Type order: Mycoplasmatales Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993b, 382.

References

Buchanan, R.E. and N.E. Gibbons (editors). 1974. Bergey’s Manual of Determinative Bacteriology, 8th edn. Williams & Wilkins, Baltimore.

Cole, J.R., Q. Wang, E. Cardenas, J. Fish, B. Chai, R.J. Farris, A.S. Kulam-Syed-Mohideen, D.M. McGarrell, T. Marsh, G.M. Garrity and J.M. Tiedje. 2009. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 37: (Database issue): D141–D145.

De Vos, P., G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer and W.B. Whitman. 2009. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer, New York.

Freundt, E.A. 1955. The classification of the pleuropneumoniae group of organisms (Borrelomycetales). Int. Bull. Bacteriol. Nomencl. Taxon. 5: 67–78.

Garrity, G.M., T.G. Lilburn, J.R. Cole, S.H. Harrison, J. Euzéby and B.J. Tindall. 2007. The Taxonomic Outline of the Bacteria and Archaea, Release 7.7, Part 11 – The Bacteria: Phyla Planctomycetes, Chlamyd-iae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, Fusobacteria,

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PhyLum XVI. TeNeRIcuTes

Verrucomicrobia, Dictyoglomi, Gemmatomonadetes, and Lentisphaerae. pp. 540–595. (http://www.taxonomicoutline.org/).

Gibbons, N.E. and R.G.E. Murray. 1978. Proposals concerning the higher taxa of bacteria. Int. J. Syst. Bacteriol. 28: 1–6.

Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacte-ria based on comparative sequence analysis of conserved genes. In Microbial Phylogeny and Evolution, Concepts and Controversies, (edited by Sapp). Oxford University Press, New York, pp. 70–98.

Ludwig, W., K.H. Schleifer and W.B. Whitman. 2009. Revised road map to the phylum Firmicutes. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3, The Firmicutes (edited by De Vos, Garrity, Jones, Krieg, Lud-wig, Rainey, Schleifer and Whitman). Springer, New York, pp. 1–13.

Murray, R.G.E. 1984a. In Validation of the publication of new names and new combinations previously effectively published outside the IJSB. List no. 15. Int. J. Syst. Bacteriol. 34: 355–357.

Murray, R.G.E. 1984b. The higher taxa, or, a place for everything…? In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 31–34.

Starr, M.P., H. Stolp, H.G. Trüper, A. Balows and H.G. Schlegel (editors). 1981. The Prokaryotes. Springer, Berlin.

Tully, J.G. 1988. International Committee on Systematic Bacteriol-ogy, Subcommittee on the Taxonomy of Mollicutes, Minutes of the Interim Meeting, 25 and 28 August 1986, Birmingham, Alabama. Int. J. Syst. Bacteriol. 38: 226–230.

Tully, J.G. 1993a. International Committee on Systematic Bacteriol-ogy, Subcommittee on the Taxonomy of Mollicutes, Minutes of the Interim Meetings, 1 and 2 August, 1992, Ames, Iowa. Int. J. Syst. Bacteriol. 43: 394–397.

Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993b. Revised taxonomy of the class Mollicutes – proposed elevation of a monophyl-etic cluster of arthropod-associated mollicutes to ordinal rank (Ento-moplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385.

class I. Mollicutes edward and Freundt 1967, 267AL

Daniel R. BRown, Meghan May, Janet M. BRaDBuRy anD KaRl-eRiK Johansson

mol¢li.cutes or mol.li.cu¢tes. L. adj. mollis soft, pliable; L. fem. n. cutis skin; N.L. fem. pl. n. Mollicutes class with pliable cell boundary.

Very small prokaryotes totally devoid of cell walls. Bounded by a plasma membrane only. Incapable of synthesis of pepti-doglycan or its precursors. Consequently resistant to penicillin and its derivatives and sensitive to lysis by osmotic shock, deter-gents, alcohols, and specific antibody plus complement. Gram-stain-negative due to lack of cell wall, but constitute a distinct phylogenetic lineage within the Gram-stain-positive bacteria (Woese et al., 1980). Pleomorphic, varying from spherical or flask-shaped structures to branched or helical filaments. The coccoid and flask-shaped cells usually range from 200–500 nm in diameter, although cells as large as 2000 nm have been seen. Replicate by binary fission, but genome replication may pre-cede cytoplasmic division, leading to the formation of multi-nucleated filaments. Colonies on solid media are very small, usually much less than 1 mm in diameter. The organisms tend to penetrate and grow inside the solid medium. Under suitable conditions, almost all species form colonies that have a char-acteristic fried-egg appearance. Usually nonmotile, but some species show gliding motility. Species that occur as helical fila-ments show rotary, flexional, and translational motility. No rest-ing stages are known.

The species recognized so far can be grown on artificial cell-free media of varying complexity, although certain strains may be more readily isolated by cell-culture procedures. Many “Can-didatus” species have been proposed and characterized at the molecular level, but not yet cultivated axenically. Most cultiva-ble species require sterols and fatty acids for growth. However, members of some genera can grow well in either serum-free media or serum-free media supplemented with polyoxyethyl-ene sorbitan. Most species are facultative anaerobes, but some are obligate anaerobes that are killed by exposure to minute quantities of oxygen. No tricarboxylic acid cycle enzymes, qui-nones, or cytochromes have been found.

All mollicutes are commensals or parasites, occurring in a wide range of vertebrate, insect, and plant hosts. Many are significant

pathogens of humans, animals, insects, or plants. Genome sizes range from 580 to 2200 kbp, among the smallest recorded in prokaryotes. The genomes of more than 20 species have been completely sequenced and annotated to date (Table 135). The G+C content of the DNA is usually low, ~23–34 mol%, but in some species is as high as ~40 mol% (Bd, Tm). Can be distinguished from other bacteria in having only one or two rRNA operons (one spe-cies of Mesoplasma has three) and an RNA polymerase that is resis-tant to rifampin. The 5S rRNA contains fewer nucleotides than that of other bacteria and there are fewer tRNA genes. In some genera, instead of a stop, the UGA codon encodes tryptophan. Plasmids and viruses (phage) occur in some species.

Type order: Mycoplasmatales Freundt 1955, 71AL emend. Tully, Bové, Laigret and Whitcomb 1993, 382.


Table 136 summarizes the present classification of the Mollicutes into families and genera and provides the major distinguishing characteristics of these taxa. The trivial term mycoplasma has been used to denote any species included in the class Mollicutes, but the term mollicute(s) is now considered most appropriate as the trivial name for all members of the class, so that the triv-ial name mycoplasma can be retained only for members of the genus Mycoplasma. Hemotropic mycoplasmas are referred to by the trivial name hemoplasmas. The trivial names ureaplasma, entomoplasma, mesoplasma, spiroplasma, acholeplasma, anaeroplasma, and asteroleplasma are commonly used when reference is made to members of the corresponding genus.

Their 16S rRNA gene sequences usefully place the mol-licutes into phylogenetic groups ( Johansson, 2002; Weisburg et al., 1989) and an analysis of 16S rRNA gene sequences is now mandatory for characterization of novel species (Brown et al., 2007). 16S rRNA gene sequences have also shown that certain hemotropic bacteria, previously considered to be members of the Rickettsia, belong to the order Mycoplasmatales.

568

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Tab

lE

135

. C

har

acte

rist

ics

of s

eque

nce

d m

ollic

ute

gen

omes

a

Spec

ies

Mycoplasma agalactiae

Mycoplasma arthritidis

Mycoplasma capricolum subsp. capricolum

Mycoplasma conjunctivae

Mycoplasma gallisepticum

Mycoplasma genitalium

Mycoplasma hominis

Mycoplasma hyopneumoniaeb

Mycoplasma mobile

Mycoplasma mycoides subsp. mycoides SC

Mycoplasma penetrans

Mycoplasma pneumoniae

Mycoplasma pulmonis

Mycoplasma synoviae

Mesoplasma florum

Ureaplasma parvumc

Ureaplasma urealyticumc

Acholeplasma laidlawii

“Candidatus Phytoplasma asteris”d

Stra

inPG

2T15

8L3-

1A

TC

C

2734

3T

HR

C/5

81T

RG

-37T

PG21

TJT

163K

TPG

1TH

F-2

M12

9U

AB

CT

IP53

L1T

AT

CC

70

0970

AT

CC

33

699

PG8T

AYW

B

Size

(m

b)0.

880.

821.

00.

850.

990.

580.

670.

900.

781.

211.

360.

820.

960.

800.

790.

750.

871.

50.

71D

NA

G+C

co

nte

nt

(mol

%)

2930

2328

3131

2728

2524

2640

2628

2725

2531

26

Ope

n r

eadi

ng

fram

es74

263

181

272

772

647

553

765

763

310

1610

3768

978

265

968

765

369

214

3367

1

Hyp

oth

etic

al

gen

es (

%)

50n

dn

d45

3721

36n

d27

4141

3838

33n

d33

nd

nd

nd

Cod

ing

de

nsi

ty (

%)

8780

8890

9190

9087

9080

8887

9091

9291

8990

73

Ref

eren

ces

Sira

nd-

Pu

gnet

et

al.

(2

007)

Dyb

vig

et

al.

(200

8)

J. G

lass

an

d

oth

ers,

un

pub-

lis

hed

.

Cal

dero

n-

Cop

ete

et a

l. (2

009)

Papa

zisi

et

al.

(200

3)

Fras

er

et a

l. (1

995)

Pere

yre

et a

l. (2

009)

Vasc

once

los

et a

l. (2

005)

Jaff

e

et a

l. (2

004)

Wes

tber

g et

al.

(200

4)

Sasa

ki

et a

l. (2

002)

Him

mel

reic

h

et a

l. (1

996)

Ch

am ba

ud

et a

l. (2

001)

Vasc

once

los

et a

l. (2

005)

Kn

igh

t et

al.

(200

4)

Gla

ss

et a

l. (2

000)

na

na

Bai

et

al.

(200

6)

a nd,

Not

det

erm

ined

; na,

not

ava

ilabl

e.b M

ycop

lasm

a hy

opne

umon

iae

stra

ins

232

and

7448

wer

e se

quen

ced

by M

inio

n e

t al.

(200

4) a

nd

Vasc

once

los

et a

l. (2

005)

.c D

ata

for

Ure

apla

sma

parv

um a

nd

Ure

apla

sma

urea

lytic

um r

efer

to s

erov

ars

3 an

d 10

, res

pect

ivel

y; s

erov

ars

1–14

wer

e se

quen

ced

and

depo

site

d di

rect

ly in

to G

enB

ank.

d AYW

B, A

ster

yel

low

s w

itch

es’ b

room

; th

e on

ion

yel

low

s st

rain

was

seq

uen

ced

by O

shim

a et

al.

(200

4).


Tab

lE

136

. D

escr

ipti

on o

f th

e cl

ass

Mol

licut

esa

Ord

erFa

mily

Gen

usSp

ecie

sbG

enom

e si

ze r

ange

(kb

p)C

hol

este

rol

requ

irem

ent

Hab

itat

cD

efin

ing

feat

ures

I. M

ycop

lasm

atal

esM

ycop

lasm

atac

eae

Myc

opla

sma

116,

9, 1

, 458

0–1,

350

+H

, AI.

Myc

opla

smat

ales

Myc

opla

smat

acea

eU

reap

lasm

a7,

0, 0

, 076

0–1,

140

+H

, AU

rea

hyd

roly

sis

I. M

ycop

lasm

atal

esd

Ince

rtae

sed

isEp

eryt

hroz

oon

4, 0

, 0, 0

Nd

nd

AH

emot

ropi

cI.

Myc

opla

smat

ales

dIn

cert

ae s

edis

Hae

mob

arto

nella

1, 0

, 0, 0

Nd

nd

AH

emot

ropi

cII

. Ent

omop

lasm

atal

esEn

tom

opla

smat

acea

eEn

tom

opla

sma

6, 0

, 0, 0

870–

900

+N

, PII

. Ent

omop

lasm

atal

esEn

tom

opla

smat

acea

eM

esop

lasm

a11

, 0, 0

, 082

5–93

0−

N, P

Gro

wth

wit

h P

ES

II. E

ntom

opla

smat

ales

Spir

opla

smat

acea

eSp

irop

lasm

a37

, 0, 0

, 078

0–2,

220

+N

, PH

elic

al m

orph

olog

yII

I. A

chol

epla

smat

ales

Ach

olep

lasm

atac

eae

Ach

olep

lasm

a18

, 0, 0

, 01,

500–

1,65

0−

A, N

, PII

I. A

chol

epla

smat

ales

Ince

rtae

sed

is“C

andi

datu

s Ph

ytop

lasm

a”0,

27,

0, 0

530–

1,35

0n

dN

, PN

ot y

et c

ultu

red

in v

itro

IV. A

naer

opla

smat

ales

Ana

erop

lasm

atac

eae

Ana

erop

lasm

a4,

0, 0

, 01,

500–

1,60

0+

ASt

rict

ly a

nae

robi

cIV

. Ana

erop

lasm

atal

esA

naer

opla

smat

acea

eA

ster

olep

lasm

a1,

0, 0

, 01,

500

−A

Stri

ctly

an

aero

bic

a nd,

Not

det

erm

ined

; PE

S, p

olyo

xyet

hyl

ene

sorb

itan

.b N

umbe

rs o

f spe

cies

: val

id, C

andi

datu

s, in

cert

ae s

edis

, in

valid

.c H

, hum

an; A

, ver

tebr

ate

anim

al; N

, in

vert

ebra

te a

nim

al; P

, pla

nt.

d Aff

iliat

ion

of t

he

con

stit

uen

t gen

era

wit

hin

the

Myc

opla

smat

ales

has

not

bee

n fo

rmal

ized

.

570


The phytoplasmas, a large group of uncultivated mollicutes occurring as agents that can cycle between plant and inver-tebrate hosts, have been given a provisional “Candidatus Phy-toplasma” genus designation. The 16S rRNA gene sequences from at least ten unique phylotypes, recently discovered among the human microbial flora through global 16S rRNA gene PCR (Eckburg et al., 2005), cluster distinctly enough to suggest the existence of a yet-uncircumscribed order within the class (May et al., 2009).

Non-helical mollicutes isolated from insects and plants have been placed in the order Entomoplasmatales, the two genera of which are distinguished by their requirement for choles-terol (Tully et al., 1993). Members of the genus Entomoplasma require cholesterol; those of the genus Mesoplasma do not. However, sterol requirements do not correlate well with phy-logenetic analyses in other groups. At least four species of spiroplasmas do not require sterol for growth, but they do not form a phylogenetic group. Within the order including the obligately anaerobic mollicutes Anaeroplasmatales, members of the genus Anaeroplasma require sterols for growth, whereas members of the genus Asteroleplasma do not (Robinson et al., 1975; Robinson and Freundt, 1987). Thus, sterol requirement is a useful phylogenetic marker only in the Acholeplasmatales and Anaeroplasmatales.

In the past, there was some risk of confusing mollicutes with wall-less “L (Lister)-phase” variants of certain other bacteria, but simple PCR-based analyses of 16S rRNA or other gene sequences now obviate that concern. Wall-less members of the genus Ther-moplasma, previously assigned to the Mollicutes, are Archaea and differ from all other members of this class in their 16S rRNA nucleotide sequences plus a number of important features relat-ing to their mode of life and metabolism. Thus, they are quite unrelated to this class (Fox et al., 1980; Razin and Freundt, 1984; Woese et al., 1980). Members of the Erysipelothrix line of descent, also formerly assigned to the Mollicutes, are now assigned to the class Erysipelotrichi in the phylum Firmicutes (Stackebrandt, 2009; Verbarg et al., 2004).

Taxonomic comments

The origin of mollicutes and their relationships to other prokaryotes was controversial for many years, especially since their small genomes and comparative phenotypic simplicity suggested that they might have descended from a primitive organism. The first comparative phylogenetic analysis of the origin of mollicutes was carried out by oligonucleotide map-ping of 16S rRNA gene sequences by Woese et al. (1980). The organisms then assigned to the genera Mycoplasma, Spiroplasma, and Acholeplasma seemed to have arisen by reductive evolution as a deep branch of the clostridial lineage leading to the genera Bacillus and Lactobacillus. This relationship had been proposed earlier (Neimark, 1979) because the low G+C mollicutes, strep-tococci, and lactic acid bacteria share characteristic enzymes. In particular, acholeplasma and streptococcus aldolases show high amino acid sequence similarity.

These findings were generally confirmed by studies of 5S rRNA gene sequences (Rogers et al., 1985), which included a number of acholeplasmas, anaeroplasmas, mycoplasmas, ure-aplasmas, and Clostridium innocuum. Dendrograms constructed from evolutionary distance matrices indicated that the mol-licutes form a coherent phylogenetic group that developed as

a branch of the Firmicutes. The initial event in this evolution was proposed to be the formation of the Acholeplasma branch, although the position of the Anaeroplasma species (Anaero-plasma bactoclasticum and Anaeroplasma abactoclasticum) was not definitely established within these dendrograms. Formation of the acholeplasmas may have coincided with a reduction in genome size to about 1500–1700 kb and loss of the cell wall. With a genome size similar to the acholeplasmas, the spiroplas-mas may have formed from the acholeplasmas. Later indepen-dent genome reductions to 500–1000 kb may have led to the origins of the sterol-requiring mycoplasma and ureaplasma lin-eages. The more extensive phylogenetic analysis of Weisburg et al. (1989) examined the 16S rRNA gene sequences of about 50 species of mollicutes and confirmed a number of these observations and provided additional insights into mollicute evolution. These results also indicated that the acholeplasmas formed upon the initial divergence of mollicutes from clostrid-ial ancestors. Further divergence of this stem led to the sterol-requiring, anaerobic Anaeroplasma and the non-sterol-requiring Asteroleplasma branches. The Spiroplasma branch also appeared to originate from within the acholeplasmas, with further evolu-tion leading to a series of repeated and independent genome reductions from nearly 2000 kb to 600–1200 kb to yield the Mycoplasma and Ureaplasma lineages.

Based on the phylogeny of 16S rRNA genes, the class Mollicutes was included in the phylum Firmicutes in the most recent revision of the Taxonomic Outline of Bacteria and Archaea (Garrity et al., 2007). However, the Mollicutes are excluded from the most recently emended description of the Firmicutes (De Vos et al., 2009) based on alternative phylogenetic markers, including RNA polymerase subunit B, the chaperonin GroEL, several different aminoacyl tRNA synthetases, and subunits of F

0F1-ATPase (Ludwig and Schleifer, 2005).The Weisburg et al. (1989) study also proposed five addi-

tional phylogenetic groupings within the mollicutes, including the anaeroplasma, asteroleplasma, spiroplasma, pneumoniae, and hominis groups (Figure 105). Phytoplasmas are similar to acholeplasmas in their 16S rRNA gene sequences and UGA codon usage (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). They probably diverged from acholeplasmas at about the same time as the split of spiroplasmas into helical and non-helical lineages (Maniloff, 2002). The modern species concept for mollicutes is justified principally by DNA–DNA hybridization, serology, and 16S rRNA gene sequence similarity (Brown et al., 2007). A large number of individual species have been assigned to phylogenetic groups, clusters, and subclusters that also share other characteristics, although the cluster boundaries are sometimes subjective (Harasawa and Cassell, 1996; Johansson, 2002; Pettersson et al., 2000, 2001).

Lastly, the type order Mycoplasmatales is assigned to the class as this clearly appeared to be the intention of Edward and Fre-undt (1967) in their paper entitled “Proposal for Mollicutes as name of the class established for the order Mycoplasmatales”.

Acknowledgements

The lifetime achievements in mycoplasmology and major con-tributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged. Daniel R. Brown and Meghan May were supported by NIH grant 5R01GM076584.

571


Further reading

Barile, M.F., S. Razin, J.G. Tully and R.F. Whitcomb (Editors). 1979, 1985, 1989. The Mycoplasmas (five volumes). Academic Press, New York.

Maniloff, J., R.N. McElhaney, L.R. Finch and J.B. Baseman (edi-tors). 1992. Mycoplasmas: Molecular Biology and Pathogen-esis. American Society for Microbiology, Washington, D.C.

Murray, R.G.E. 1984. The higher taxa, or, a place for every-thing…?. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 31–34.

Razin, S. and J.G.E. Tully. 1995. Molecular and Diagnostic Proce-dures in Mycoplasmology, vol. 1. Academic Press, San Diego.

Taylor-Robinson, D. and J. Bradbury. 1998. Mycoplasma diseases. In Topley and Wilson’s Principles and Practice of Microbiol-ogy, vol. 3 (edited by Hausler and Sussman). Edward Arnold, London, pp. 1013–1037.

Taylor-Robinson, D. and J.G. Tully. 1998. Mycoplasmas, ure-aplasmas, spiroplasmas, and related organisms. In Topley and Wilson, Principles and Practice of Microbiology, 9th edn, vol. 2 (edited by Balows and Duerden). Arnold Publishers, Lon-don, pp. 799–827.

Tully, J.G. and S. Razin (editors). 1996. Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2. Academic Press, San Diego, CA.

Mycoplasma hominisUreaplasma urealyticum

Mycoplasma pneumoniaeMycoplasma coccoides

Spiroplasma apisMycoplasma mycoides subsp. mycoidesEntomoplasma ellychniae

Mesoplasma florumSpiroplasma citri

Spiroplasma ixodetisAcholeplasma laidlawii

‘Candidatus Phytoplasma’ strain OY-MAnaeroplasma abactoclasticum

Asteroleplasma anaerobiumClostridium innocuum

Scale:0.1 substitutions/site

*

FIgurE 105. Phylogenetic grouping of the class Mollicutes. The phylogram was based on a Jukes–Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type genera, plus representatives of other major clusters within the Mycoplas-matales and Entomoplasmatales and a phytoplasma. Clostridium innocuum was the outgroup. All bootstrap values (100 replicates) are >50% except where indicated (asterisk).

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De Vos, P., G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K. H. Schleifer and W.B. Whitman. 2009. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3. Springer, New York.

Dybvig, K., C. Zuhua, P. Lao, D.S. Jordan, C.T. French, A.H. Tu and A.E. Loraine. 2008. Genome of Mycoplasma arthritidis. Infect. Immun. 76: 4000–4008.

Eckburg, P., E. Bik, C. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S. Gill, K. Nelson and D. Relman. 2005. Diversity of the human intes-tinal microbial flora. Science 308: 1635–1638.

Edward, D.G.ff. and E.A. Freundt. 1967. Proposal for Mollicutes as name of the class established for the order Mycoplasmatales. Int. J. Syst. Bacteriol. 17: 267–268.

Fox, G.E., E. Stackebrandt, R.B. Hespell, J. Gibson, J. Maniloff, T.A. Dyer, R.S. Wolfe, W.E. Balch, R.S. Tanner, L.J. Magrum, L.B. Zablen, R. Blakemore, R. Gupta, L. Bonen, B.J. Lewis, D.A. Stahl, K.R. Luehrsen, K.N. Chen and C.R. Woese. 1980. The phylogeny of prokaryotes. Science 209 : 457–463.

Fraser, C.M., J.D. Gocayne, O. White, M.D. Adams, R.A. Clayton, R.D. Fleischmann, C.J. Bult, A.R. Kerlavage, G. Sutton, J.M. Kelley and

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Garrity, G.M., T.G. Lilburn, J.R. Cole, S.H. Harrison, J. Euzéby and B.J. Tindall. 2007. The Taxonomic Outline of the Bacteria and Archaea, Release 7.7, Part 11 – The Bacteria: phyla Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, Fusobacteria, Verrucomicrobia, Dictyoglomi, Gemmatomonadetes, and Lentisphaerae. pp. 540–595. (http://www.taxonomicoutline.org/).

Glass, J.I., E.J. Lefkowitz, J.S. Glass, C.R. Heiner, E.Y. Chen and G.H. Cassell. 2000. The complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature 407: 757–762.

Harasawa, R. and G.H. Cassell. 1996. Phylogenetic analysis of genes coding for 16S rRNA in mammalian ureaplasmas. Int. J. Syst. Bacte-riol. 46: 827–829.

Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B.C. Li and R. Herrmann. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24: 4420–4449.

IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma Taxon-omy Group. 2004. Description of the genus ‘Candidatus Phytoplasma’, a taxon for the wall-less non-helical prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243–1255.

Jaffe, J.D., N. Stange-Thomann, C. Smith, D. DeCaprio, S. Fisher, J. Butler, S. Calvo, T. Elkins, M.G. Fitzgerald, N. Hafez, C.D. Kodira, J. Major, S. Wang, J. Wilkinson, R. Nicol, C. Nusbaum, B. Birren, H.C. Berg and G.M. Church. 2004. The complete genome and proteome of Mycoplasma mobile. Genome Res. 14: 1447–1461.

Johansson, K.-E. 2002. Taxonomy of Mollicutes. In Molecular Biology and Pathogenicity of Mycoplasmas (edited by Razin and Herrmann). Kluwer Academic/Plenum Publishers, New York, pp. 1–29.

Knight, T.F., Jr. 2004. Reclassification of Mesoplasma pleciae as Achole-plasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data. Int. J. Syst. Evol. Microbiol. 54: 1951–1952.

Ludwig, W. and K.H. Schleifer. 2005. Molecular phylogeny of bacte-ria based on comparative sequence analysis of conserved genes. In Microbial Phylogeny and Evolution, Concepts and Controversies (edited by Sapp). Oxford University Press, New York, pp. 70–98.

Maniloff, J. 2002. Phylogeny and Evolution. In Molecular Biology and Pathogenicity of Mycoplasmas. Kluwer Academic/Plenum Publish-ers, pp. 31–43.

May, M., R.F. Whitcomb and D.R. Brown. 2009. Mycoplasma and related organisms. In CRC Practical Handbook of Microbiology, 2nd edn. (edited by Goldman and Green). Taylor & Francis, pp. 456–479.

Minion, F.C., E.J. Lefkowitz, M.L. Madsen, B.J. Cleary, S.M. Swartzell and G.G. Mahairas. 2004. The genome sequence of Mycoplasma hyo-pneumoniae strain 232, the agent of swine mycoplasmosis. J. Bacte-riol. 186: 7123–7133.

Neimark, H.C. 1979. Phylogenetic relationships between mycoplasmas and other prokaryotes. In The Mycoplasmas, vol. 1 (edited by Barile and Razin). Academic Press, New York, pp. 43–61.

Oshima, K., S. Kakizawa, H. Nishigawa, H.Y. Jung, W. Wei, S. Suzuki, R. Arashida, D. Nakata, S. Miyata, M. Ugaki and S. Namba. 2004. Reduc-tive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nat. Genet. 36: 27–29.

Papazisi, L., T. Gorton, G. Kutish, P. Markham, G. Browning, D. Nguyen, S. Swartzell, A. Madan, G. Mahairas and S. Geary. 2003. The com-plete genome sequence of the avian pathogen Mycoplasma gallisepti-cum strain R(low). Microbiology 149 : 2307–2316.

Pereyre, S., P. Sirand-Pugnet, L. Beven, A. Charron, H. Renaudin, A. Barre, P. Avenaud, D. Jacob, A. Couloux, V. Barbe, A. de Daruvar, A. Blanchard and C. Bebear. 2009. Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas. PLoS. Genet. 5: e1000677.

Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2000. Updated phylogenetic description of the Mycoplasma hominis cluster (Weisburg et al. 1989) based on 16S rDNA sequences. Int. J. Syst. Evol. Micro-biol. 50 : 291–301.

Pettersson, B., J.G. Tully, G. Bolske and K.E. Johansson. 2001. Re-evaluation of the classical Mycoplasma lipophilum cluster (Weisburg et al. 1989) and description of two new clusters in the hominis group based on 16S rDNA sequences. Int. J. Syst. Evol. Microbiol. 51: 633–643.

Razin, S. and E.A. Freundt. 1984. The Mollicutes, Mycoplasmatales, and Mycoplasmataceae. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 740–742.

Robinson, I.M., M.J. Allison and P.A. Hartman. 1975. Anaeroplasma abac-toclasticum gen. nov., sp. nov., obligately anaerobic mycoplasma from rumen. Int. J. Syst. Bacteriol. 25: 173–181.

Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended clas-sification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81.

Rogers, M.J., J. Simmons, R.T. Walker, W.G. Weisburg, C.R. Woese, R.S. Tanner, I.M. Robinson, D.A. Stahl, G. Olsen, R.H. Leach and J. Maniloff. 1985. Construction of the mycoplasma evolutionary tree from 5S rRNA sequence data. Proc. Natl. Acad. Sci. U.S.A. 82: 1160–1164.

Sasaki, Y., J. Ishikawa, A. Yamashita, K. Oshima, T. Kenri, K. Furuya, C. Yoshino, A. Horino, T. Shiba, T. Sasaki and M. Hattori. 2002. The complete genomic sequence of Mycoplasma penetrans, an intra-cellular bacterial pathogen in humans. Nucleic Acids Res. 30 : 5293–5300.

Sirand-Pugnet, P., C. Lartigue, M. Marenda, D. Jacob, A. Barre, V. Barbe, C. Schenowitz, S. Mangenot, A. Couloux, B. Segurens, A. de Daru-var, A. Blanchard and C. Citti. 2007. Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome. PLoS. Genet. 3: e75.

Stackebrandt, E. 2009. Class III. Erysipelotrichia. In Bergey’s Manual of Systematic Bacteriology, vol. 3 (edited by de Vos, Garrity, Jones, Krieg, Ludwig, Rainey, Schleifer and Whitman). Springer, New York, p. 1298.

Tully, J.G., J.M. Bove, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes - proposed elevation of a mono-phyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385.

Vasconcelos, A.T. and a. coauthors. 2005. Swine and poultry patho-gens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J. Bacteriol. 187: 5568–5577.

Verbarg, S., H. Rheims, S. Emus, A. Fruhling, R.M. Kroppenstedt, E. Stackebrandt and P. Schumann. 2004. Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of vegetable peptone broth, and description of Erysipelotrichaceae fam. nov. Int. J. Syst. Evol. Microbiol. 54: 221–225.

Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Man-delco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467.

Westberg, J., A. Persson, A. Holmberg, A. Goesmann, J. Lundeberg, K.E. Johansson, B. Pettersson and M. Uhlen. 2004. The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res. 14: 221–227.

Woese, C.R., J. Maniloff and L.B. Zablen. 1980. Phylogenetic analysis of the mycoplasmas. Proc. Natl. Acad. Sci. U. S. A. 77: 494–498.

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http://www.taxonomicoutline.org/

Phylum XVI. TenerIcuTes

Order I. Mycoplasmatales Freundt 1955, 71Al emend. Tully, Bové, laigret and Whitcomb 1993, 382

Daniel R. BRown, Meghan May, Janet M. BRaDBuRy, KaRl-eRiK Johansson anD haRolD neiMaRK

my.co.plas.ma.ta¢les. n.l. neut. n. Mycoplasma, -atos type genus of the order; -ales ending to denote an order; n.l. fem. pl. n. Mycoplasmatales the Mycoplasma order.

The first order in the class Mollicutes is assigned to a group of sterol-requiring, wall-less prokaryotes that occur as commensals or pathogens in a wide range of vertebrate hosts. The descrip-tion of the order is essentially the same as for the class. A single family Mycoplasmataceae with two genera, Mycoplasma and Ureaplasma, recognizes the prominent and distinct characteristics of the assigned organisms, based on their sterol requirements for growth, the capacity of some to hydrolyze urea, and conserved 16S rRNA gene sequences.

Type genus: Mycoplasma Nowak 1929, 1349 nom. cons. Jud. Comm. Opin. 22, 1958, 166.


The entire class Mollicutes was encompassed initially by a single order. The elevation of acholeplasmas to ordinal rank (Acholeplasmatales Freundt, Whitcomb, Barile, Razin and Tully 1984) recognized their major distinctions in nutritional, biochemical, physiological, and genetic characteristics from other members of the class Mollicutes. Subsequently, additional orders were proposed to recognize the anaerobic mollicutes and the wall-less prokaryotes from plants and insects which were phyloge-netically related to the remaining Mycoplasmatales. Thus, the Anaeroplasmatales (Robinson and Freundt, 1987) recognized the strictly anaerobic, wall-less prokaryotes first isolated from the bovine and ovine rumen, and Entomoplasmatales (Tully et al., 1993) provided a classification for a number of the mol-licutes regularly associated with plant and insect hosts. On the basis of 16S rRNA gene sequence similarities (Johansson and

Pettersson, 2002), the Mycoplasmatales and Entomoplasmatales represent a clade deeply split from the Acholeplasmatales and Anaeroplasmatales.

A growth requirement for cholesterol or serum is shared by the organisms assigned to the order Mycoplasmatales, as well as most other organisms within the class Mollicutes. Therefore, tests for cholesterol requirements are essential to classification. Earlier assessments of the growth requirements for cholesterol were based upon the capacity of organisms to grow in a number of serum-free broth preparations to which various concentra-tions of cholesterol were added (Edward, 1971; Razin and Tully, 1970). In this test, species that do not require exogenous ste-rol usually show no significant growth response to increasing cholesterol concentrations. Polyoxyethylene sorbitan (Tween 80) and palmitic acid should be included in the base medium because acholeplasmas such as Acholeplasma axanthum and Acholeplasma morum require additional fatty acids for adequate growth. A modified method utilizing serial passage in selective medium has been applied successfully to a large number of mollicutes (Rose et al., 1993; Tully, 1995). The Acholeplasmatales grow through end-point dilutions in serum-containing medium and in serum-free preparations, or occasionally in serum-free medium supplemented with Tween 80. Mesoplasmas from the order Entomoplasmatales grow in serum-containing medium and in serum-free medium supplemented only with Tween 80. Most spiroplasmas, also from the Entomoplasmatales, and all members of the order Mycoplasmatales grow only in serum-containing medium.

references

Edward, D.G. 1971. Determination of sterol requirement for Mycoplasmatales. J. Gen. Microbiol. 69 : 205–210.


Freundt, E.A., R.F. Whitcomb, M.F. Barile, S. Razin and J.G. Tully. 1984. Proposal for elevation of the family Acholeplasmataceae to ordinal rank: Acholeplasmatales. Int. J. Syst. Bacteriol. 34: 346–349.

Johansson, K.E., Pettersson B. 2002. Taxonomy of Mollicutes. In Molecu-lar biology and pathogenicity of mycoplasmas (edited by Razin and Herrmann). Kluwer Academic, New York, pp. 1–30.

Judicial Commission. 1958. Opinion 22. Status of the generic name Asterococcus and conservation of the generic name Mycoplasma. Int. Bull. Bacteriol. Nomencl. Taxon. 8: 166–168.

Nowak, J. 1929. Morphologie, nature et cycle évolutif du microbe de la péripneumonie des bovidés. Ann. Inst. Pasteur (Paris) 43: 1330–1352.

Razin, S. and J.G. Tully. 1970. Cholesterol requirement of mycoplasmas. J. Bacteriol. 102: 306–310.

Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended clas-sification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81.

Rose, D.L., J.G. Tully, J.M. Bove and R.F. Whitcomb. 1993. A test for measuring growth responses of Mollicutes to serum and polyoxyethyl-ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532.

Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised tax-onomy of the class Mollicutes - proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bac-teriol. 43: 378–385.

Tully, J.G. 1995. Determination of cholesterol and polyoxyethylene sor-bitan growth requirements of mollicutes. In Molecular and Diagnos-tic Procedures in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, San Diego, pp. 381–389.

574

Genus I. MycoplasMa

Family I. Mycoplasmataceae Freundt 1955, 71al emend. Tully, Bové, laigret and Whitcomb 1993, 382

Daniel R. BRown, Meghan May, Janet M. BRaDBuRy, KaRl-eRiK Johansson anD haRolD neiMaRK

My.co.plas.ma.ta.ce′ae. n.l. neut. n. Mycoplasma, -atos type genus of the family; -aceae ending to denote a family; n.l. fem. pl. n. Mycoplasmataceae the Mycoplasma family.

Pleomorphic usually coccoid cells, 300–800 nm in diameter, to slender branched filaments of uniform diameter. Some cells have a terminal bleb or tip structure that mediates adhesion to certain surfaces. Cells lack a cell wall and are bounded only by a plasma membrane. Gram-stain-negative due to the absence of a cell wall. Usually nonmotile. Facultatively anaerobic in most instances, possessing a truncated flavin-terminated electron transport chain devoid of quinones and cytochromes. Colonies of Mycoplasma are usually less than l mm in diameter and colo-nies of Ureaplasma are much smaller than that. The typical col-ony has a fried-egg or “cauliflower head” appearance. Usually catalase-negative. Chemo-organotrophic, usually using either sugars or arginine, but sometimes both, or having an obligate requirement for urea as the major energy source. Require cho-lesterol or related sterols for growth. Commensals or pathogens of a wide range of vertebrate hosts. The genome size ranges from about 580 to 1350 kbp, as measured by pulsed field gel electrophoresis (PFGE) or complete DNA sequencing.

DNA G+C content (mol%): about 23–40 (Bd, Tm).

Type genus: Mycoplasma Nowak 1929, 1349 nom. cons. Jud. Comm. Opin. 22, 1958, 166.


This family and its type genus Mycoplasma are polyphyletic. Two genera, Mycoplasma and Ureaplasma, are currently accepted within the family. The genus Mycoplasma is further divisible into phylogenetic groups on the basis of 16S rRNA gene sequence similarities (Johansson and Pettersson, 2002), including an eco-logically, phenotypically, and genetically cohesive group called the mycoides cluster, which includes the type species Mycoplasma mycoides and other major pathogens of ruminant animals. The taxonomic position of the mycoides cluster is an important anomaly because molecular markers based upon rRNA and other gene sequences indicate that it is closely related to other genera usually associated with plant and insect hosts and cur-rently classified within the order Entomoplasmatales. Members of the genus Ureaplasma are distinguished by their tiny colony size and capacity to hydrolyze urea.

Genus I. Mycoplasma nowak 1929, 1349 nom. cons. Jud. comm. opin. 22, 1958, 166al

Daniel R. BRown, Meghan May, Janet M. BRaDBuRy, Mitchell F. Balish, Michael J. calcutt, John i. glass, séveRine tasKeR, Joanne B. MessicK, KaRl-eRiK Johansson anD haRolD neiMaRK

My.co.plas¢ma. Gr. masc. n. myces a fungus; Gr. neut. n. plasma something formed or molded, a form; n.l. neut. n. Mycoplasma fungus form.

Pleomorphic cells, 300–800 nm in diameter, varying in shape from spherical, ovoid or flask-shaped, or twisted rods, to slender branched filaments ranging in length from 50 to 500 nm. Cells lack a cell wall and are bounded by a single plasma membrane. Gram-stain-negative due to the absence of a cell wall. Some have a complex internal cytoskeleton. Some have a specific tip struc-ture that mediates attachment to host cells or other surfaces. Usually nonmotile, but gliding motility has been demonstrated in some species. Aerobic or facultatively anaerobic. Optimum growth at 37°C is common, but permissive growth temperatures range from 20 to 45°C. Chemo-organotrophic, usually using either sugars or arginine as the major energy source. Require cholesterol or related sterols for growth. Colonies are usually less than l mm in diameter. The typical colony has a fried-egg appearance. The genome size of species examined ranges from 580 kbp to about 1350 kbp. The codon UGA encodes trypto-phan in all species examined. Commensals or pathogens in a wide range of vertebrate hosts.

DNA G+C content (mol%): 23–40.Type species: Mycoplasma mycoides (Borrel, Dujardin-

Beaumetz, Jeantet and Jouan 1910) Freundt 1955, 73 (Astero-coccus mycoides Borrel, Dujardin-Beaumetz, Jeantet and Jouan 1910, 179).


The shape of these organisms (trivial name, mycoplasmas) can depend on the osmotic pressure, nutritional quality of the

culture medium, and the growth phase. Some mycoplasmas are filamentous in their early and exponential growth phases or when attached to surfaces or other cells. This form can be transitory, and the filaments may branch or fragment into chains of cocci or individual vegetative cells. Many species are typically coccoid and never develop a filamentous phase. Some species develop specialized attachment tip structures involved in colonization and virulence (Figure 106). In Romanowsky-type stained blood smears, hemotropic species (trivial name, hemoplasmas) appear as round to oval cells on the surface of erythrocytes (Figure 107). They may be found individually or, during periods of high parasitemia, in pairs or chains giving the appearance of pleomorphism. Their small size and the absence of cell wall components provide considerable plasticity to the organisms, so that cells of most species are readily filterable through 450 nm pores, and many species have some cells in the population that are able to pass through 220 nm or even 100 nm filters (Tully, 1983). Descriptions of the morphology, ultra-structure, and motility of mycoplasmas should be based on cor-relation of the appearance of young exponential-phase broth cultures under phase-contrast or dark-field microscopy with their appearance using negative-staining or electron micros-copy (Biberfeld and Biberfeld, 1970; Boatman, 1979; Carson et al., 1992; Cole, 1983). Special attention to the osmolarity of the fixatives and buffers is required since these may alter the size and shape of the organisms. The classical isolated colony is umbonate with a fried-egg appearance, but others may have

575

FaMIly I. MycoplasMaTaceae

either cauliflower-like or smooth colony surfaces (Figure 108), with smooth, irregular or scalloped margins, depending on the species, agar concentration, and other growth conditions.

A significant minority of species exhibit cell polarization. This depends on Triton X-100-insoluble cytoskeletal structures

involved in morphogenesis, motility, cytadherence, and cell divi-sion (Balish and Krause, 2006). In the distantly related species Mycoplasma pneumoniae and Mycoplasma mobile, the cytoskeleton underlies a terminal organelle. This prominent extension of the cytoplasm and cell membrane is the principal focus of adherence

Figure 106. Diverse cellular morphology in the genus Mycoplasma. Scanning electron micrographs of cells of (a) Mycoplasma penetrans, (b) Mycoplasma pneumoniae, (c) “Mycoplasma insons”, and (d) Mycoplasma genitalium. Bar = 1 mm. Images provided by Dominika Jurkovic, Jennifer Hatchel, Ryan Relich and Mitchell Balish.

Figure 107. Hemotropic mycoplasmas. (a) Scanning electron micrograph of Mycoplasma ovis cells colonizing the surface of an erythrocyte (Neimark et al., 2004); bar = 500 nm. (b) Transmission electron micrograph showing fibrils bridging the space between a “Candidatus Mycoplasma kahaneii” cell and a depression in the surface of a colonized erythrocyte (Neimark et al., 2002a); bar = 250 nm. Images used with permission.

576

Genus I. MycoplasMa

and is the leading end of cells engaged in gliding motility. In Mycoplasma pneumoniae, adhesin proteins are located either all over the surface of the organelle or at its distal tip; an unrelated adhesin is concentrated at the base of the terminal organelle in Mycoplasma mobile (Balish, 2006). Both the formation of this attachment organelle of Mycoplasma pneumoniae and the local-ization of the adhesins depend upon cytoskeletal proteins that form an electron-dense core within its cytoplasm, which is sur-rounded by an electron-lucent space (Krause and Balish, 2004). The overall appearance of this core is that of two parallel, flat rods of differing thickness, with a bend near the cell-proximal end (Henderson and Jensen, 2006; Seybert et al., 2006). A bi-lobed button constitutes its distal end and its proximal base ter-minates in a bowl-like structure. Overall, both the core and the attachment organelle are 270–300 nm in length (Hatchel and Balish, 2008). Around the onset of DNA replication, a second attachment organelle is constructed (Seto et al., 2001). The motile force provided by the first organelle reorganizes the cell such that the new organelle is moved to the opposite cell pole before cell division (Hasselbring et al., 2006). These observa-tions suggest that complex coordination exists between attach-ment organelle biogenesis, motor activity, the DNA replication machinery, and the cytokinetic machinery. Similar structures are present in other species of the Mycoplasma pneumoniae cluster, but in most cases the attachment organelle is shorter, resulting in much of the core protruding into the cell body (Hatchel and Bal-ish, 2008). In Mycoplasma mobile, the terminal organelle is com-pletely dissimilar, consisting of a cell-distal sphere with numerous tentacle-like strands extending into the cytoplasm (Nakane and Miyata, 2007). It is comprised of proteins unrelated to those

found in Mycoplasma pneumoniae, suggesting that it has evolved independently. Further distinct cytoskeletal structures appear in Mycoplasma penetrans (Jurkovic and Balish, unpublished), “Myco-plasma insons” (Relich et al., 2009), and several species of the mycoides cluster (Peterson et al., 1973).

Attachment to eukaryotic host cells is important for the natu-ral survival and transmission of mycoplasmas. The prominent attachment organelle of species in the Mycoplasma pneumoniae cluster is the most extensively characterized determinant of cytad-herence. In other species, multiple adhesin proteins are involved in cytadherence. When one adhesin is blocked, cytadherence is reduced, but not completely lost. For this reason, the adhesins appear to be functionally redundant rather than synergistic in action. Numerous species possess multigene families of antigeni-cally variable proteins, some of which have been implicated in host cell attachment or hemagglutination. While this attach-ment may serve as a supplemental binding mechanism in species such as Mycoplasma gallisepticum and Mycoplasma hominis, variable surface proteins are currently the only known mechanism for cytadherence and hemagglutination of Mycoplasma synoviae and Mycoplasma pulmonis. The avidity of adherence may differ among variants in Mycoplasma pulmonis and Mycoplasma hominis. Though one or more attachment mechanisms have been described for numerous species, there remains a greater number of species with no documented system for cytadherence. Strains that lose the capacity to cytadhere are almost invariably unable to survive in their hosts, but highly invasive species such as Mycoplasma alligatoris may not require host cell attachment for infection.

The mycoplasmas possess a typical prokaryotic plasma membrane composed of amphipathic lipids and proteins

Figure 108. Diverse colonial morphology in the genus Mycoplasma. (a) Mycoplasma mycoides PG1T (diameter 0.50–0.75 mm), (b) Mycoplasma hyopneumoniae NCTC 10110T (diameter 0.15–0.20 mm), (c) Mycoplasma pneumoniae NCTC 10119T (diameter 0.05–0.10 mm), and (d) Mycoplasma hyorhinis ATCC 29052 (diameter 0.25–0.30 mm) after 3, 7, 5, and 6 d growth, respectively, on Mycoplasma Experience Solid Medium at 36°C in 95% nitrogen/5% carbon dioxide. Original magnification 25×. Images provided by Helena Windsor and David Windsor.

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(McElhaney, 1992a, b, c; Smith, 1992; Wieslander et al., 1992). At one time, demonstration of a single unit membrane was mandatory for defining all novel species of mollicutes (Tully, 1995a). Now, when the 16S rRNA gene sequence of a novel spe-cies is determined and the candidate is placed in one of the phy-logenetic clusters of mollicutes, in the majority of cases it can be safely inferred that the organism lacks a cell wall, because the majority of others in that cluster will have been shown to be solely membrane-bound (Brown et al., 2007). The lack of a cell wall explains the resistance of the organisms to lysis by lysozyme and their susceptibility to lysis by osmotic shock and various agents causing the lysis of bacterial protoplasts (Razin, 1979, 1983). In certain species, the extracellular surface is tex-tured with capsular material or a nap, which can be stained with ruthenium red in some cases (Rosenbusch and Minion, 1992).

These organisms represent some of the most nutritionally fastidious prokaryotes, as expected from their greatly reduced or minimalist genomes, close association with vertebrate hosts as commensals and pathogens, and total dependence upon the host to meet all nutritional requirements. They have very limited capacity for intermediary metabolism, which restricts the utility of conventional biochemical tests for identification. Detailed infor-mation on carbohydrate (Pollack, 1992, 1997, 2002; Pollack et al., 1996), lipid (McElhaney, 1992a), and amino acid (Fischer et al., 1992) metabolism is available. All species examined have trun-cated respiratory systems, lack a complete tricarboxylic acid cycle, and lack quinones or cytochromes, which precludes their capacity to carry out oxidative phosphorylation. Instead only low levels of ATP may be generated through glycolysis or the arginine dihy-drolase pathway (Miles, 1992a, b). Fermentative species catabo-lize glucose or other carbohydrates to produce ATP and acid and, consequently, lower the pH of the medium. Non-fermentative spe-cies hydrolyze arginine to yield ammonia, some ATP, and carbon dioxide, and consequently raise the pH of the medium. Species such as Mycoplasma fermentans have both pathways. Species such as Mycoplasma bovis evidently lack both pathways, but are capable oxi-dizing pyruvate or lactate to yield ATP (Miles, 1992a; Taylor et al., 1994). Some species cause a pronounced “film and spots” reac-tion on media incorporating heat-inactivated horse serum or egg yolk: a wrinkled film composed of cholesterol and phospholipids forms on the surface of the medium and dark spots containing salts of fatty acids appear around the colonies.

Most mycoplasmas are aerobes or facultative anaerobes, but some species such as Mycoplasma muris prefer an anaerobic environment. The optimum growth of species isolated from homeothermic hosts is commonly at 37°C and the permissive temperature range of species from poikilothermic fish and reptiles is always above 20–25°C. Thus, growth of the myco-plasmas is restricted to mesophilic temperatures. Growth in liquid cultures usually produces at most light turbidity and few sedimented cells, except for the heavy turbidity and sediments usually observed with members of the Mycoplasma mycoides clus-ter. Tully (1995b) described in detail the most commonly used culture media formulations. Although colonies are occasionally first detected on blood agar, complex undefined media such as American Type Culture Collection (ATCC) medium 988 (SP-4) are usually required for primary isolation and maintenance. Cell-wall-targeting antibiotics are included to discourage growth of other bacteria. Phenol red facilitates detection of species that excrete acidic or alkaline metabolites. Growth of

arginine-hydrolyzing species can be enhanced by supplementing media with arginine. Commonly used alternatives such as Frey’s, Hayflick’s and Friis’ media differ from SP-4 mainly in the propor-tions of inorganic salts, amino acids, serum sources, and types of antibiotics. For species that utilize both sugars and arginine as carbon sources, the pH of the medium may initially decrease before rising later during the course of growth (Razin et al., 1998). Defined mycoplasma culture media have been described in detail (Rodwell, 1983), but provision of lipids and amino acids in the appropriate ratios is difficult technically (Miles, 1992b).

Many mobile genetic elements occur in the genus. Four plas-mids have been identified in members of the mycoides clus-ter (Bergemann and Finch, 1988; Djordjevic et al., 2001; King and Dybvig, 1994). Each plasmid is apparently cryptic, with no discernible determinants for virulence or antibiotic resistance. DNA viruses have been isolated from Mycoplasma bovirhinis (Howard et al., 1980), Mycoplasma hyorhinis (Gourlay et al., 1983) Mycoplasma pulmonis (Tu et al., 2001), and Mycoplasma arthritidis (Voelker and Dybvig, 1999). The Mycoplasma pulmonis P1 virus and the lysogenic bacteriophage MAV1 of Mycoplasma arthritidis do not share sequence similarity (Tu et al., 2001; Voelker and Dybvig, 1999), whereas the Mycoplasma fermentans MFV1 prophage is strikingly similar in genetic organization to MAV1 (Röske et al., 2004). No role in pathobiology has been demonstrated for any virus or prophage.

The most abundant mobile DNAs in Mycoplasma are inser-tion sequence (IS) elements. The first identified units (IS1138 of Mycoplasma pulmonis, IS1221 of Mycoplasma hyorhinis, IS1296 of Mycoplasma mycoides subsp. mycoides and ISMi1 of Mycoplasma fermentans) are members of the IS3 family (Bhugra and Dyb-vig, 1993; Ferrell et al., 1989; Frey et al., 1995; Hu et al., 1990). More recently, multiple IS elements of divergent subgroups have been identified. Members of the IS4 family include IS1634 and ISMmy1 of Mycoplasma mycoides subsp. mycoides (Vilei et al., 1999; Westberg et al., 2002), ISMhp1 of Mycoplasma hyopneumoniae, ISMhp1-like unit of Mycoplasma synoviae, and four distinct ele-ments of Mycoplasma bovis (Lysnyansky et al., 2009). Among the IS30 family members identified are IS1630 of Mycoplasma fermen-tans, ISMhom1 from Mycoplasma hominis, ISMag1 of Mycoplasma agalactiae (Pilo et al., 2003), and two IS units of Mycoplasma bovis. IS-like elements have also been identified in Mycoplasma leachii, Mycoplasma penetrans (belonging to four different families), Myco-plasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma orale. Transposases that reside within IS units are also discernable in the genome of Mycoplasma gallisepticum (Papazisi et al., 2003). In select instances, almost identical IS units have been found in species from different phylogenetic clades, which strongly suggests lateral gene transfer between species. Despite their widespread distribution, IS elements are not ubiquitous in the genus. Although the type strains of Mycoplasma bovis (54 IS units of seven different types) and Mycoplasma mycoides subsp. mycoides (97 elements of three different types) possess large numbers of elements, the sequenced genomes of Mycoplasma arthritidis, Myco-plasma genitalium, Mycoplasma pneumoniae, and Mycoplasma mobile lack detectable IS units.

Although IS units only encode genes related to transposition, large integrating elements have also been identified in diverse Mycoplasma species. The Integrative Conjugal Elements (ICE) of Mycoplasma fermentans strain PG18T comprise >8% of the genome and related units have been identified in Mycoplasma

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agalactiae, Mycoplasma bovis, Mycoplasma capricolum, Mycoplasma hyopneumoniae, and Mycoplasma mycoides subsp. mycoides. In gen-eral, such units encode 18–30 genes, can be detected in extra-chromosomal forms, and are strain-variable in distribution and chromosomal insertion site. Two additional large mobile DNAs, designated Tra Islands, were identified in Mycoplasma capricolum California kidT. The presence of putative conjugation genes and the variability in genomic location of Tra Islands and ICE suggest that these are agents of lateral gene transfer.

The best-studied mycoplasmas are primary pathogens of humans or domesticated animals (Baseman and Tully, 1997). About half of the listed species occur in the absence of disease, but are occasional opportunistic or secondary pathogens. The principal human pathogens are Mycoplasma pneumoniae, Myco-plasma hominis, and Mycoplasma genitalium, with Mycoplasma pen-etrans added to this list due to its association with HIV infections (Blanchard, 1997; Blanchard et al., 1997; Tully, 1993; Waites and Talkington, 2005). Mycoplasma pneumoniae is one of the main agents of community-acquired pneumonia, bronchitis, and other respiratory complications (Atkinson et al., 2008). Myco-plasma pneumoniae infections can also involve extra-pulmonary complications including central nervous system, cardiovascular, and dermatological manifestations. Outbreaks cause consider-able morbidity and require rapid and effective therapeutic inter-vention (Hyde et al., 2001; Meyer and Clough, 1993). Mycoplasma hominis occurs more frequently in the urogenital tract of women than men and is often found in the genital tract of women with vaginitis, bacterial vaginosis, or localized intrauterine infections (Keane et al., 2000). The organism can gain access to a fetus from uterine sites and it is associated with perinatal morbidity and mortality (Gonçalves et al., 2002; Waites et al., 1988). It is also clearly associated with septicemias and respiratory infections and with transplant or joint infections in immunosuppressed persons (Brunner et al., 2000; Busch et al., 2000; Fernandez Guerrero et al., 1999; Garcia-Porrua et al., 1997; Gass et al., 1996; Hopkins et al., 2002; Mattila et al., 1999; Tully, 1993; Zheng et al., 1997). Mycoplasma genitalium has been associated with non-gonococcal urethritis in men (Gambini et al., 2000; Jensen, 2004; Jensen et al., 2004; Taylor-Robinson et al., 2004; Taylor-Robinson and Horner, 2001; Totten et al., 2001) and urogenital disease in women (Baseman et al., 2004; Blaylock et al., 2004). Mycoplasma genitalium occurs more frequently in the vagina than in the cer-vix or urethra, but it may be involved in cervicitis (Casin et al., 2002; Manhart et al., 2001). Mycoplasmas are common agents of chronic joint inflammation in a wide variety of hosts (Cole et al., 1985). Species associated with arthritis in humans include Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma salivarium, and possibly Mycoplasma pneumoniae in juvenile arthritis (Waites and Talkington, 2005). Humans are also susceptible to opportunistic zoonotic mycoplasmosis; immuno-suppressed persons are highly susceptible. The recent molecular confirmation of a Mycoplasma haemofelis-like infection in an HIV-positive patient (dos Santos et al., 2008) highlights the zoonotic potential of the hemoplasmas.

Mycoplasmas colonize fish, reptiles, birds, and terrestrial and aquatic mammals. Some cause significant diseases of cat-tle and other ruminants, swine, poultry, or wildlife, and others are opportunistic or secondary veterinary pathogens (Simecka et al., 1992; Tully and Whitcomb, 1979). The principal bovine pathogens include the serovars historically called “Small Colony”

types of Mycoplasma mycoides subsp. mycoides, and Mycoplasma bovis. Mycoplasma mycoides subsp. mycoides has caused major losses of livestock globally in the twentieth century due to contagious bovine pleuropneumonia and currently remains a problem in Asia and Africa (Lesnoff et al., 2004). Mycoplasma bovis is a wide-spread agent of otitis media, pneumonia, mastitis, polyarthri-tis, and urogenital disease in cattle and buffaloes. Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum, and Mycoplasma agalactiae are important causes of arthritis, mastitis, and agalactia in goats and sheep. Mycoplasma mycoides subsp. capri (type strain PG3T) properly includes all of the serovars histori-cally called “Large Colony” types of subspecies mycoides (Manso-Silván et al., 2009; Shahram et al., 2010). Mycoplasma capricolum subsp. capripneumoniae (type strain F38T) causes severe conta-gious pleuropneumonia in goats (Leach et al., 1993; McMartin et al., 1980). Contagious bovine and caprine pleuropneumonia, and mycoplasmal agalactia of sheep or goats are subject to con-trol through listing in the Terrestrial Animal Health Code of the Office International des Epizooties (http://oie.int) as well as strict notification and export regulations by individual countries. Mycoplasma hyopneumoniae, one of the most difficult species to cultivate, causes primary enzootic pneumonia in pigs and exac-erbates other porcine respiratory diseases leading to substantial economic burdens. Mycoplasma hyosynoviae is carried in the upper respiratory tract, but causes nonsuppurative polyarthritis, usually without other serositis, especially in growing pigs.

The most important poultry pathogens are Mycoplasma gal-lisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis, but more than 20 other species have been isolated from birds as diverse as ostriches, raptors, and penguins (Bradbury and Morrow, 2008). Mycoplasma gallisepticum can be transmitted ver-tically, venereally, by other direct contact, or by aerosol to cause respiratory disease and its sequelae in chickens, turkeys, and other birds. It also causes decreased egg production and egg quality in chickens. Mycoplasma synoviae can cause a syndrome of synovitis, tendonitis, and bursitis in addition to respiratory disease in chickens and turkeys, whereas the developmental abnormalities and airsacculitis associated with congenital or acquired Mycoplasma meleagridis infection seem restricted to tur-keys. Mycoplasma gallisepticum and Mycoplasma synoviae are also listed in the OIE’s Terrestrial Animal Health Code.

Pathogenicity of specific mycoplasmas has also been reported for companion animals (Chalker, 2005; Lemcke, 1979; Messick, 2003) and a number of wild animal hosts (Brown et al., 2005). The respiratory, reproductive, and joint diseases caused in rodents by Mycoplasma pulmonis and Mycoplasma arthritidis (Schoeb, 2000) are important models of infection and immu-nity in humans and other animals.

Hemoplasmas infect a variety of wild and domesticated animals and are relatively host-specific, although cross- infection of related hosts has been reported. Transmission can be achieved by ingestion of infected blood or by percutaneous inoculation. Arthropod vector transmission of some species is also supported by experimentation and by the clustered geographic distribu-tion of hemoplasmosis in some studies (Sykes et al., 2007; Willi et al., 2006a). The pathogenicity of different hemoplasma spe-cies is variable and strain virulence also likely plays a key role in the development of disease. For example, Mycoplasma haemofelis can induce acute clinical disease in non-splenectomized, immu-nocompetent cats, whereas Mycoplasma haemocanis appears able

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to induce disease only in immunosuppressed or splenectomized dogs. Clinical syndromes range from acute fatal hemolytic ane-mia to chronic insidious anemia and ill-thrift. Signs may include anemia, pyrexia, anorexia, dehydration, weight loss, and infer-tility. The presence of erythrocyte-bound antibodies (including cold agglutinins), indicated by positive Coombs’ testing, has been demonstrated in some hemoplasma-infected animals and may contribute to anemia. Animals can remain chronic asymp-tomatic carriers of hemoplasmas after acute infection. PCR is the diagnostic test of choice for hemoplasma infection.

Contamination of eukaryotic cell cultures with mollicutes is still a common and important yet often unrecognized problem (Tully and Razin, 1996). More than 20 species have been isolated from contaminated cell lines, but more than 90% of the contamination is thought to be caused by just five species of mycoplasma: Myco-plasma arginini, Mycoplasma fermentans, Mycoplasma hominis, Myco-plasma hyorhinis, and Mycoplasma orale, plus Acholeplasma laidlawii. Mycoplasma pirum and Mycoplasma salivarium account for most of the remainder (Drexler and Uphoff, 2002). Culture medium com-ponents of animal origin, passage of contaminated cultures, and laboratory personnel are likely to be the most significant sources of cell culture contaminants. PCR-based approaches to detection achieve sensitivity and specificity far superior to fluorescent stain-ing methods (Masover and Becker, 1996). Another method of detection is based on mycoplasma-specific ATP synthesis activity present in contaminated culture medium (Robertson and Stemke, 1995; MycoAlert, Lonza Group). Eradication through treatment of contaminated cultures with antibiotics (Del Giudice and Gardella, 1996) is rarely successful. Strategies for prevention and control of mycoplasmal contamination of cell cultures have been described in detail (Smith and Mowles, 1996).

Several categories of potential virulence determinants are encoded in the metagenome of pathogenic mycoplasmas. Some species possess multiple types of virulence factors. Determinants such as adhesins and accessory proteins, extracellular polysaccha-ride structures, and pro-inflammatory or pro- apoptotic membrane lipoproteins are produced by multiple species. Several species excrete potentially toxic by-products of intermediary metabolism, including hydrogen peroxide, superoxide radicals, or ammonia. Other determinants such as extracellular endopeptidases, nucle-ases, and glycosidases seem irregularly distributed in the genus, whereas the ADP-ribosylating and vacuolating cytotoxin (pertus-sis exotoxin S1 subunit analog) of Mycoplasma pneumoniae and the T-lymphocyte mitogen (superantigen) of Mycoplasma arthritidis are evidently unique to those species. Reports of a putative exotoxin elaborated by Mycoplasma neurolyticum have not been substantiated by later work (Tryon and Baseman, 1992).

Candidate virulence mechanisms, such as motility, biofilm formation, or facultative intracellular invasion, are expressed by a range of pathogenic species. Several species possess systems of variable surface antigens that are thought to be important in evasion of the hosts’ adaptive immune responses. In addition, a large number of species can suppress or inappro-priately stimulate host immune cells and their receptors and cytokines through diverse, poorly characterized mycoplasmal components. Although candidate virulence factor discovery has accelerated significantly in recent years through whole genome annotation, the molecular basis for pathogenicity and causal relationships with disease still remain to be definitively estab-lished for most of these factors (Razin and Herrmann, 2002; Razin et al., 1998).

Because they lack lipopolysaccharide and a cell wall, and do not synthesize their own nucleotides, mycoplasmas are intrin-sically resistant to polymixins, b-lactams, vancomycin, fosfomy-cin, sulfonamides, and trimethoprim. They are also resistant to rifampin because their RNA polymerase is not affected by that antibiotic (Bébéar and Kempf, 2005). Individual species exhibit an even broader spectrum of antibiotic resistance, such as the resistance to erythromycin and azithromycin exhibited by sev-eral species, which is apparently mediated by mutation in the 23S rRNA (Pereyre et al., 2002). Treatment of mycoplasmosis often involves the use of antibiotics that inhibit protein syn-thesis or DNA replication. Certain macrolides or ketolides are used when tetracyclines or fluoroquinolones are inappropri-ate. Fluoroquinolones, aminoglycosides, pleuromutilins, and phenicols are not widely used to treat human mycoplasmosis at present, with the exception of chloramphenicol for neonates with mycoplasmosis of the central nervous system unresponsive to other antibiotics (Waites et al., 1992), but their use in veteri-nary medicine is more common. The long-term antimicrobial therapy often required may be due to mycoplasmal sequestra-tion in privileged sites, potentially including inside host cells. Mycoplasmosis in immunodeficient patients is very difficult to control with antibiotic drugs (Baseman and Tully, 1997).

enrichment and isolation procedures

Techniques for isolation of mycoplasmas from humans, various species of animals, and from cell cultures have been described (Neimark et al., 2001; Tully and Razin, 1983). Typical steps in the isolation of mycoplasmas were outlined in the recently revised minimal standards for descriptions of new species (Brown et al., 2007). Initial isolates may contain a mixture of species, so cloning by repeated filtration through membrane filters with a pore size of 450 or 220 nm is essential. The initial filtrate and dilutions of it are cultured on solid medium and an isolated colony is subse-quently picked from a plate on which only a few colonies have developed. This colony is used to found a new cultural line, which is then expanded, filtered, plated, and picked two additional times. Hemoplasmas have not yet been successfully grown in continuous culture in vitro, although recent work (Li et al., 2008) suggests that in vitro maintenance of Mycoplasma suis may be possible.

Maintenance procedures

Cultures of mycoplasma can be preserved by lyophilization or cryo-genic storage (Leach, 1983). The serum in the culture medium provides effective cryoprotection, but addition of sucrose may enhance survival following lyophilization. Hemoplasmas can be frozen in heparin- or EDTA-anticoagulated blood cryopreserved with dimethylsulfoxide. Most species can be recovered with little loss of viability even after storage for many years.

Taxonomic comments

This polyphyletic genus is divisible on the basis of 16S rRNA and other gene sequence similarities into a large paraphyletic clade of over 100 species in two groups called hominis and pneumo-niae (Johansson and Pettersson, 2002; Figure 109), plus the ecologically, phenotypically, and genetically cohesive “mycoides cluster” of five species including the type species Mycoplasma mycoides (Cottew et al., 1987; Manso-Silván et al., 2009; Shahram et al., 2010). The priority of Mycoplasma mycoides as the type spe-cies of the genus Mycoplasma and, hence, the family Mycoplasmata-ceae and the order Mycoplasmatales is, in retrospect, unfortunate.

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The phylogenetic position of the mycoides cluster is eccentrically situated to the remaining species of the order Mycoplasmatales, amidst genera that are properly classified in the order Entomoplas-matales. When the order Entomoplasmatales was established, a cen-tury after the discovery of Mycoplasma mycoides, it was explicitly accepted that the taxonomic anomaly created by the phyloge-netic position of the mycoides cluster will remain impractical to resolve (Tully et al., 1993). The few species in the mycoides cluster cannot simply be renamed, because confusion and peril would result, especially regarding “Small Colony” PG1T-like strains of Mycoplasma mycoides subsp. mycoides and F38T-like strains of Myco-plasma capricolum subsp. capripneumoniae, which are highly virulent pathogens and subject to strict international regulations.

Another controversy involves the nomenclature of uncultivated hemotropic bacteria originally assigned to the genera Eperythro-zoon or Haemobartonella. It is now undisputed that, on the bases of their lack of a cell wall, small cell size, low G+C content, use of the codon UGA to encode tryptophan, regular association with vertebrate hosts, and 16S rRNA gene sequences that are most simi-lar (80–84%) to species in the pneumoniae group of Mycoplasma, these organisms are properly affiliated with the Mycoplasmatales. However, the proposed transfers of Eperythrozoon and Haemobarto-nella species to the genus Mycoplasma (Neimark et al., 2001, 2005) were opposed on the grounds that the degree of 16S rRNA gene sequence similarity is insufficient (Uilenberg et al., 2004, 2006). The principal objection to establishing the hemoplasmas in a third genus in the Mycoplasmataceae (Uilenberg et al., 2006) is that this would compound the polyphyly within the pneumoniae group solely on the basis of a capacity to adhere to erythrocytes in vivo. In addition, the transfer of the type species Eperythrozoon coccoides to the genus Mycoplasma is complicated by priority because Eperythro-zoon predates Mycoplasma. The alternative, to transfer all mycoplas-mas to the genus Eperythrozoon, would be completely impractical and perilous in part because the epithet Eperythrozoon does not indicate an affiliation with Mycoplasmatales. The Judicial Commis-sion of the International Committee on Systematics of Prokaryotes (ICSP) declined to rule on a request for an opinion in this matter (Neimark et al., 2005) during their 2008 meeting, but a provisional placement in the genus Mycoplasma has otherwise been embraced by specialists in the molecular biology and clinical pathogenicity of these and similar hemotropic organisms. At present, the designa-tion “Candidatus” must still be used for new types.

Mycoplasma feliminutum was first described during a time when the only named genus of mollicutes was Mycoplasma. Its publication coincided with the first proposal of the genus Acholeplasma (Edward and Freundt, 1969, 1970), with which Mycoplasma feliminutum is properly affiliated through established phenotypic (Heyward et al., 1969) and 16S rRNA gene sequence (Brown et al., 1995) similari-ties. This explains the apparent inconsistencies with its assignment to the genus Mycoplasma. The name Mycoplasma feliminutum should therefore be revised to Acholeplasma feliminutum comb. nov. The type strain is BenT (=ATCC 25749T; Heyward et al., 1969).

Recent work at the J. Craig Venter Institute, including com-plete chemical synthesis and cloning of an intact Mycoplasma genitalium chromosome (Gibson et al., 2008) and other work with Mycoplasma mycoides “Large Colony” (Lartigue et al., 2009), suggests that de novo synthesis of two species of mollicute is immi-nent. Transplantation of isolated deproteinized tetR-selectable chromosomes from donor Mycoplasma mycoides “Large Colony” into recipient Mycoplasma capricolum cells displaced the recipient genome and conferred the genotype and phenotype of the donor

(Lartigue et al., 2007). Cloning in yeast and subsequent resur-rection of Mycoplasma mycoides “Large Colony” genomes as living bacteria demonstrate that it is possible to enliven a prokaryotic genome constructed in a eukaryotic cell (Lartigue et al., 2009). The ICSP subcommittee on the taxonomy of Mollicutes may be the first to accommodate a system of nomenclature and classifi-cation for species of novel prokaryotes that originate by entirely artificial speciation events (Brown and Bradbury., 2008).

Differentiation of the genus Mycoplasma from other genera

Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. Aerobic or facultatively anaerobic growth in artificial medium and a growth requirement for sterols exclude assign-ment to the genera Anaeroplasma, Asteroleplasma, Acholeplasma, or “Candidatus Phytoplasma”. Non-spiral cellular morphology and regular association with a vertebrate host or fluids of ver-tebrate origin support exclusion from the genera Spiroplasma, Entomoplasma, or Mesoplasma. The inability to hydrolyze urea excludes assignment to the genus Ureaplasma.

acknowledgements

The lifetime achievements in mycoplasmology and substantial contributions to the preparation of this material by Joseph G. Tully are gratefully acknowledged. Daniel R. Brown and Meghan May were supported by NIH grant 5R01GM076584. Séverine Tasker was supported by Wellcome Trust grant WT077718.

Further reading

Blanchard, A. and G. Browning (editors). 2005. Mycoplasmas: Molecular Biology, Pathogenicity, and Strategies for Control. Horizon Press, Norwich, UK.

Maniloff, J., R.N. McElhaney, L.R. Finch and J.B. Baseman (edi-tors). 1992. Mycoplasmas: Molecular Biology and Pathogen-esis. American Society for Microbiology, Washington, D.C.

Razin, S. and J.G. Tully (editors). 1995. Molecular and Diagnos-tic Procedures in Mycoplasmology, vol. 1, Molecular Charac-terization. Academic Press, San Diego.

Tully, J.G. and S. Razin (editors). 1996. Molecular and Diagnos-tic Procedures in Mycoplasmology, vol. 2, Diagnostic Proce-dures. Academic Press, San Diego.

Differentiation of the species of the genus Mycoplasma

Glucose fermentation and arginine hydrolysis are discriminat-ing phenotypic markers (Table 137), but the pleomorphism and metabolic simplicity of mycoplasmas has led to a current reliance principally on the combination of 16S rRNA gene sequencing and reciprocal serology for species differentiation. Failure to cross-react with antisera against previously recognized species provides substantial evidence for species novelty. For this reason, deposi-tion of antiserum against a novel type strain into a recognized collection is still mandatory for novel species descriptions (Brown et al., 2007). Preliminary differentiation can be by PCR and DNA sequencing using primers specific for bacterial 16S rRNA genes or the 16S–23S intergenic region. A similarity matrix relating the candidate strain to its closest neighbors, usually species with >94% 16S rRNA gene sequence similarity, will suggest related species that should be examined for serological cross-reactivities.

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Mycoplasma equigenitaliumMycoplasma elephantis

Mycoplasma bovisMycoplasma agalactiae

Mycoplasma primatumMycoplasma opalescens

Mycoplasma spermatophilumMycoplasma fermentans

Mycoplasma caviaeMycoplasma adleri

Mycoplasma felifauciumMycoplasma leopharyngis

Mycoplasma maculosumMycoplasma lipofaciensMycoplasma bovigenitaliumMycoplasma californicum

Mycoplasma simbaeMycoplasma phocirhinis

Mycoplasma meleagridisMycoplasma gallinarum

Mycoplasma inersMycoplasma columbinasale

Mycoplasma columbinumMycoplasma lipophilum

Mycoplasma hyopharyngisMycoplasma sphenisci

Mycoplasma synoviaeMycoplasma verecundum

Mycoplasma gallinaceumMycoplasma corogypsi

Mycoplasma glycophilumMycoplasma gallopavonis

Mycoplasma buteonisMycoplasma felis

Mycoplasma mustelaeMycoplasma leonicaptivi

Mycoplasma bovirhinisMycoplasma cynos

Mycoplasma edwardiiMycoplasma canis

Mycoplasma columboraleMycoplasma oxoniensisMycoplasma citelli

Mycoplasma sturniMycoplasma pullorum

Mycoplasma anatisMycoplasma crocodyli

Mycoplasma alligatorisMycoplasma hominisMycoplasma equirhinis

Mycoplasma phocidaeMycoplasma falconis

Mycoplasma spumansMycoplasma arthritidis

Mycoplasma phocicerebraleMycoplasma aurisMycoplasma alkalescens

Mycoplasma canadenseMycoplasma gateae

Mycoplasma argininiMycoplasma cloacale

Mycoplasma anserisMycoplasma buccale

Mycoplasma hyosynoviaeMycoplasma orale

Mycoplasma indienseMycoplasma faucium

Mycoplasma subdolumMycoplasma gypis

Mycoplasma pulmonis strain UAB CTIPMycoplasma agassizii

Mycoplasma testudineumMycoplasma sualvi

Mycoplasma moatsiiMycoplasma mobile

Mycoplasma neurolyticumMycoplasma cricetuliMycoplasma collis

Mycoplasma molareMycoplasma lagogenitalium

Mycoplasma iguanaeMycoplasma hyopneumoniae

Mycoplasma flocculareMycoplasma ovipneumoniae

Mycoplasma disparMycoplasma bovoculi

Mycoplasma conjunctivaeMycoplasma hyorhinis

Mycoplasma vulturis

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equigenitalium cluster

bovis cluster

lipophilum cluster

synoviae cluster

hominis cluster

pulmonis cluster

sualvi cluster

neurolyticum cluster

hom

inis

gro

up


Figure 109. (Continued)

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Genus I. MycoplasMa

1. Mycoplasma mycoides (Borrel, Dujardin-Beaumetz, Jeantet and Jouan 1910) Freundt 1955, 73AL (Asterococcus mycoides Borrel, Dujardin-Beaumetz, Jeantet and Jouan 1910, 179)

my.co.i¢des. Gr. n. mukês -êtos mushroom or other fungus; L. suff. -oides (from Gr. suff. -eides, from Gr. n. eidos that which is seen, form, shape, figure) resembling, similar; N.L. neut. adj. mycoides fungus-like.

This is the type species of the genus. Cells are pleomor-phic and capable of forming long filaments. Nonmotile. An extracellular capsule can be visualized by electron micros-copy following staining with ruthenium red. Colonies on solid agar have a characteristic fried-egg appearance. Grows in modified Hayflick medium supplemented with glucose at 37°C.

The species has subsequently been divided as follows.

1a. Mycoplasma mycoides subsp. capri Manso-Silván, Vilei, Sachse, Djordjevic, Thiaucourt and Frey 2009, 1357VP (Asterococcus mycoides var. capri Edward 1953, 874; Mycoplasma mycoides subsp. mycoides var. large colony Cottew and Yeats 1978, 294)

ca¢pri. L. n. caper, -pri goat; L. gen. n. capri of the goat.

Cells are pleomorphic and capable of forming long fila-ments and long, helical rods known as rho forms. Nonmo-tile. An extracellular capsule can be visualized by electron microscopy following staining with ruthenium red. Colonies on solid agar have a characteristic fried-egg appearance and are notably larger than those of Mycoplasma mycoides subsp. mycoides. Grows in modified Hayflick medium supplemented with glucose at 37°C. Formation of biofilms has been dem-onstrated (McAuliffe et al., 2006).

Pathogenic; causes polyarthritis, mastitis, conjunctivi-tis (a syndrome collectively termed contagious agalactia), pneumonia, peritonitis, and septicemia in goats; and bal-anitis and vulvitis in sheep. Transmission occurs via direct contact between animals or with fomites, or can be vector-borne by the common ear mite (Psoroptes cuniculi).

Tetracyclines are effective therapeutic agents. Eradica-tion from herds is difficult due to the tendency of healthy animals to harbor the organism in the ear canal without seroconverting. Antigenic cross-reactivity with Mycoplasma

Figure 109. Phylogenetic relationships in the Mycoplasma hominis and Mycoplasma pneumoniae groups of the order Mycoplasmatales. The phylogram was based on a Jukes–Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type strains, except where noted. Acholeplasma (formerly Mycoplasma) feliminutum was the outgroup. The major groups and clusters are defined in terms of posi-tions in 16S rRNA showing characteristic base composition and signature positions, plus higher-order structural synapomorphies (Johansson and Pettersson, 2002; Weisburg et al., 1989). Bootstrap values (100 replicates) <50% are indicated (*); the branching order is considered to be equivocal.


Mycoplasma coccoidesMycoplasma haemofelis

Mycoplasma haemocanis‘Candidatus Mycoplasma haemobos’

Mycoplasma haemomurisMycoplasma suis

Mycoplasma wenyoniiMycoplasma ovis

*

hemotropic cluster

Mycoplasma insonsMycoplasma cavipharyngis

Mycoplasma fastidiosum

Mycoplasma pneumoniaeMycoplasma genitalium

Mycoplasma amphoriformeMycoplasma testudinis

Mycoplasma alviMycoplasma pirumMycoplasma gallisepticum strain RMycoplasma imitans

Ureaplasma urealyticumUreaplasma parvum serovar 3

Ureaplasma galloraleUreaplasma diversum

Ureaplasma felinumUreaplasma canigenitalium

Mycoplasma penetrans strain HF-2Mycoplasma iowae

Mycoplasma murisMycoplasma microti

*

*

fastidiosum cluster

pneumoniae cluster

muris cluster

Ureaplasma cluster

hemotropic cluster

pneu

mon

iae

grou

p

list of species of the genus Mycoplasma

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Table 137. Descriptive characteristics of species of Mycoplasma a

Species Morphology

DNA G+C content (mol%)

Energy source

Medium pH shift Serum source

Representative host Relation to host

M. mycoides subsp. mycoides Pleomorphic 24 G A FB Cattle PathogenM. mycoides subsp. capri Pleomorphic 24 G A FB Goats PathogenM. adleri Coccoidal 29.6 R K E Goats PathogenM. agalactiae Coccoidal 29.7 G A FB, E Goats PathogenM. agassizii Pleomorphic nr G A FB Tortoises PathogenM. alkalescens Coccobacillary 25.9 R K FB Cattle PathogenM. alligatoris Coccoidal nr G A FB Alligators PathogenM. alvi Flask-shaped 26.4 G, R V FB Cattle CommensalM. amphoriforme Flask-shaped 34 G A FB Humans OpportunisticM. anatis Coccoidal 26.6 G A FB Ducks OpportunisticM. anseris Spherical 26 R K E Goose OpportunisticM. arginini Coccoidal 27.6 R K E Mammals PathogenM. arthritidis Filamentous 30.7 R K FB Rats PathogenM. auris Pleomorphic 26.9 R K E Goats CommensalM. bovigenitalium Pleomorphic 30.4 OH, OA N FB Cattle PathogenM. bovirhinis nr 27.3 G A FB Cattle OpportunisticM. bovis Coccobacillary 32.9 OH, OA N FB, E Cattle PathogenM. bovoculi Coccobacillary 29 G A E Cattle PathogenM. buccale Coccobacillary 26.4 R K E Humans CommensalM. buteonis Coccoidal 27 G A P Raptors CommensalM. californicum Pleomorphic 31.9 OH, OA N E Cattle PathogenM. canadense Coccobacillary 29 R K FB Cattle PathogenM. canis Pleomorphic 28.4 G A FB Dogs OpportunisticM. capricolum subsp. capricolum Coccobacillary 23 G A FB, E Goats PathogenM. capricolum subsp.

capripneumoniaeCoccobacillary 24.4 G A FB, E Goats Pathogen

M. caviae nr nr G A FB Guinea pigs CommensalM. cavipharyngis Twisted rod 30 G A E Guinea pigs CommensalM. citelli Pleomorphic 27.4 G A FB Squirrels CommensalM. cloacale Spherical 26 R K E Galliforms CommensalM. coccoidesb Coccoidal nr U na na Mice PathogenM. collis Coccoidal 28 G A E Rodents CommensalM. columbinasale Coccobacillary 32 R K FB Pigeons CommensalM. columbinum Pleomorphic 27.3 R K P Pigeons CommensalM. columborale Coccoidal 29.2 G A P Pigeons CommensalM. conjunctivae Coccobacillary nr G A FB Goats PathogenM. corogypsi Pleomorphic 28 G A P Vultures PathogenM. cottewii Coccoid 27 G A E Goats CommensalM. cricetuli Pleomorphic nr G A E Hamsters CommensalM. crocodyli Coccoidal 27.6 G A FB Crocodiles PathogenM. cynos Coccobacillary 25.8 G A FB Dogs PathogenM. dispar Pleomorphic 29.3 G A FB, P Cattle PathogenM. edwardii Coccobacillary 29.2 G A FB Dogs OpportunisticM. elephantis Coccoidal 24 G A E Elephants CommensalM. equigenitalium Pleomorphic 31.5 G A E Horses OpportunisticM. equirhinis Coccobacillary nr R K E Horses OpportunisticM. falconis Coccoidal 27.5 R K P Falcons OpportunisticM. fastidiosum Twisted rod 32.3 G A P Horses CommensalM. faucium Coccoidal nr R K FB Humans CommensalM. felifaucium Coccoidal 31 R K FB, E Pumas CommensalM. feliminutum nr 29.1 G A FB Cats CommensalM. felis Filamentous 25.2 G A FB Cats PathogenM. fermentans Filamentous 28.7 R, G V FB, E Humans UnclearM. flocculare Coccobacillary 33 U N P Pigs OpportunisticM. gallinaceum Coccobacillary 28 G A P Galliforms PathogenM. gallinarum Coccobacillary 28 R K P Galliforms CommensalM. gallisepticum Flask-shaped 31 G A FB, E Galliforms PathogenM. gallopavonis Coccobacillary 27 G A P Turkeys OpportunisticM. gateae nr 28.5 U N FB Cats Opportunistic

(Continued)

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Species Morphology


Energy source



M. genitalium Flask-shaped 31 G A FB Humans PathogenM. glycophilum Elliptical 27.5 G A E Galliforms CommensalM. gypis Coccoidal 27.1 R K P Vultures OpportunisticM. haemocanisb Coccoidal nr U na na Dogs PathogenM. haemofelisb Coccoidal 38.8 U na na Cats PathogenM. haemomurisb Coccoidal nr U na na Mice OpportunisticM. hominis Coccobacillary 33.7 R K FB Humans PathogenM. hyopharyngis Pleomorphic 24 R K P, E Pigs CommensalM. hyopneumoniae Coccobacillary 28 G A P, FB Pigs PathogenM. hyorhinis Coccobacillary 27.8 G A FB Pigs PathogenM. hyosynoviae Pleomorphic 28 G A FB Pigs PathogenM. iguanae Coccoidal nr G A FB Iguanas PathogenM. imitans Flask-shaped 31.9 G A FB Ducks, geese PathogenM. indiense Pleomorphic 32 R K FB Monkeys CommensalM. iners nr 29.6 R K P Galliforms CommensalM. insonsc Twisted rod nr G A FB Iguanas CommensalM. iowae Pleomorphic 25 G, R V FB Turkeys PathogenM. lagogenitalium Coccoidal 23 G A FB Pikas CommensalM. leachii Pleomorphic nr G A E Cattle PathogenM. leonicaptivi Pleomorphic 27 G A FB Lions CommensalM. leopharyngis Pleomorphic 28 G A FB Lions CommensalM. lipofaciens Elliptical 24.5 G, R V P Galliforms CommensalM. lipophilum Pleomorphic 29.7 R K FB, E Humans UnclearM. maculosum Coccobacillary 29.6 R K FB, E Dogs OpportunisticM. meleagridis Coccobacillary 28.6 R K P Turkeys PathogenM. microti Coccoidal nr G A FB Voles CommensalM. moatsii Spheroidal 25.7 G, R V FB, E Monkeys CommensalM. mobile Flask-shaped 25 G, R V E Tench PathogenM. molare Coccoidal 26 G A FB Dogs OpportunisticM. mucosicanisc Coccoidal nr U nr E Dogs CommensalM. muris Coccoidal 24.9 R K FB Mice CommensalM. mustelae Pleomorphic 28 G A E Minks CommensalM. neurolyticum Filamentous 26.2 G A E Mice UnclearM. opalescens nr 29.2 R K FB Dogs CommensalM. orale Pleomorphic 28.2 R K FB, E Humans CommensalM. ovipneumoniae nr 25.7 G A FB, P Sheep PathogenM. ovisb Coccoidal nr U na na Sheep PathogenM. oxoniensis Coccoidal 29 G A FB Hamsters CommensalM. penetrans Flask-shaped 25.7 G, R V FB Humans OpportunisticM. phocicerebrale Dumbbell 25.9 R K FB Seals PathogenM. phocidae Coccoidal 27.8 G, R V FB, E Seals OpportunisticM. phocirhinis Coccoidal 26.5 R K E, P Seals PathogenM. pirum Flask-shaped 25.5 G A FB Humans CommensalM. pneumoniae Flask-shaped 40 G A FB Humans PathogenM. primatum Coccobacillary 28.6 R K FB, E Monkeys OpportunisticM. pullorum Coccobacillary 29 G A P, E Galliforms PathogenM. pulmonis Flask-shaped 26.6 G A FB, E Mice PathogenM. putrefaciens Coccobacillary 28.9 G A FB, E Goats PathogenM. salivarium Coccoidal 27.3 R K E Humans OpportunisticM. simbae Pleomorphic 37 R K FB Lions CommensalM. spermatophilum Coccoidal 32 R K FB Humans PathogenM. spheniscic Pleomorphic 28 G A P Penguins PathogenM. spumans Pleomorphic 28.4 R K FB, E Dogs OpportunisticM. sturni Pleomorphic 31 G A FB Songbirds PathogenM. sualvi Coccobacillary 23.7 R, G V FB Pigs CommensalM. subdolum Coccoidal 28.8 R K FB, E, P Horses OpportunisticM. suisb Coccoidal 31.1 U na na Pigs PathogenM. synoviae Coccoidal 34.2 G A P Galliforms PathogenM. testudineum Coccoidal nr G A FB Tortoises Pathogen

Table 137. (Continued)

(Continued)

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Species Morphology


Energy source



M. testudinis Flask-shaped 35 G A FB Tortoises CommensalM. verecundum Pleomorphic 27 OA N FB, E Cattle Commensal“M. vulturis”b, c Coccoidal nr U na na Vultures UnclearM. wenyoniib Coccoidal nr U na na Cattle PathogenM. yeatsii Coccoidal 26.6 G na FB Goats OpportunisticM. zalophic nr nr G na FB Sea lions Pathogen“Candidatus M.

haematoparvum”b

Coccoidal nr U na na Dogs nr

“Candidatus M. haemobos”b Coccoidal nr U na na Cattle nr“Candidatus M.

haemodidelphidis”b

Coccoidal nr U na na Opossum nr

“Candidatus M. haemolamae”b Coccoidal nr U na na Llamas nr“Candidatus M.

haemominutum”b

Coccoidal nr U na na Cats nr

“Candidatus M. kahaneii”b Coccoidal nr U na na Monkeys nr“Candidatus M. ravipulmonis”b Coccoidal nr U na na Mice Pathogen“Candidatus M.

haemotarandirangiferis”b

Coccoidal nr U na na Reindeer nr

“Candidatus M. turicensis”b Coccoidal nr U na na Cats nr

anr, Not reported; na, not applicable; G, glucose; R, arginine; OH, alcohols; OA, organic acids; U, undefined; A, acidic pH shift; K, alkaline pH shift; N, pH remains neutral; V, pH shift can be acidic or alkaline depending on the energy source provided; FB, fetal bovine serum; E, equine serum; P, porcine serum.bNot yet cultivated in cell-free artificial medium. The putative organism “Candidatus M. haemotarandirangiferis” remains to be definitively established and the name has no standing in nomenclature.cHas been grown only in co-culture with eukaryotic cells.

Table 137. (Continued)

mycoides subsp. mycoides precludes the exclusive reliance on serological-based diagnostics. Experimental vaccines using formalin-inactivated Mycoplasma mycoides subsp. capri appear to protect goats from subsequent challenge (Bar-Moshe et al., 1984; de la Fe et al., 2007). This organism is under certain quarantine regulations in most non-endemic countries and is a List B pathogen in the World Organiza-tion for Animal Health (OIE) disease classification (http://oie.int).

Source: isolated from the synovial fluid, synovial mem-branes, udders, expelled milk, conjunctivae, lungs, blood, and ear canals of goats; and the urogenital tract of sheep (Bergonier et al., 1997; Cottew, 1979; Kidanemariam et al., 2005; Thiaucourt et al., 1996).

DNA G+C content (mol%): 24 (Tm).Type strain: PG3, NCTC 10137, CIP 71.25.Sequence accession nos (16S rRNA gene): U26037 (strain

PG3T), U26044 (strain Y-goat).Further comment: Mycoplasma mycoides subsp. capri now

refers to strains once known as Mycoplasma mycoides subsp. mycoides var. large colony as well as strains known as Myco-plasma mycoides subsp. capri (Manso-Silván et al., 2009; Shahram et al., 2010).

1b. Mycoplasma mycoides subsp. mycoides Manso-Silván, Vilei, Sachse, Djordjevic, Thiaucourt and Frey 2009, 1356VP (Myco-plasma mycoides subsp. mycoides var. small colony Cottew and Yeats 1978, 294)

my.co.i¢des. Gr. n. mukês -êtos mushroom or other fungus; L. suff. -oides (from Gr. suff. -eides from Gr. n. eidos that which is seen, form, shape, figure) resembling, similar; N.L. neut. adj. mycoides fungus-like.

Cells are pleomorphic and capable of forming long filaments, but do not produce rho forms. Nonmotile. An extracellular capsule can be visualized by electron micros-copy following staining with ruthenium red. Colonies on solid agar have a characteristic fried-egg appearance and are notably smaller than those of Mycoplasma mycoides subsp. capri. Grows in modified Hayflick medium supplemented with glucose at 37°C. Formation of biofilms has been dem-onstrated (McAuliffe et al., 2008).

Pathogenic; causes a characteristic, highly lethal fibrin-ous interstitial pneumonia and pleurisy known as conta-gious bovine pleuropneumonia (CBPP) in adult cattle and severe polyarthritis in calves. Transmission occurs primarily via direct contact, but can occur by droplet aerosol as well.

Tetracyclines, chloramphenicol, and fluoroquinolones are effective chemotherapeutic agents; however, treat-ment of endemic herds is often counterproductive as resis-tance can develop in carrier animals. Organisms are often sequestered in areas of coagulative necrosis in subclinically infected animals and can serve as a reservoir for reintroduc-tion of resistant clones of Mycoplasma mycoides subsp. mycoides into a herd. Culling of infecting herds and restricting the movement of infected animals are more effective strategies for controlling spread of the disease (Windsor and Masiga, 1977). Killed and live vaccines are available for the preven-tion of infection, but suffer from low antigenicity, poor efficacy, and residual pathogenesis (Brown et al., 2005). Antigenic cross-reactivity with Mycoplasma mycoides subsp. capri and Mycoplasma leachii preclude the exclusive reliance on serological-based diagnostics. Multiple molecular diag-nostics have been described (Gorton et al., 2005; Loren-zon et al., 2008; Persson et al., 1999) and many additional

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Genus I. MycoplasMa

molecular tools such as insertion sequence typing have led to a greater understanding of the epidemiology of outbreaks (Cheng et al., 1995; Frey et al., 1995; Vilei et al., 1999). This organism is under certain quarantine regulations in most non-endemic countries and is listed in the Terrestrial Ani-mal Health Code of the Office International des Epizooties (http://oie.int).

Source: isolated from the lungs, pleural fluid, lymph nodes, sinuses, kidneys, urine, synovial fluid, and syn-ovial membranes of cattle and water buffalo (Gourlay and Howard, 1979; Scanziani et al., 1997; Scudamore, 1976); the respiratory tract of bison; the respiratory tract of yak; and the lungs, nasopharynx, and pleural fluid of sheep and goats (Brandao, 1995; Kusiluka et al., 2000).

DNA G+C content (mol%): 26.1 (Tm), 24.0 (strain PG1T complete genome sequence).

Type strain: PG1, NCTC 10114, CCUG 32753.Sequence accession nos: U26039 (16S rRNA gene),

BX293980 (strain PG1T complete genome sequence).Further comment: Mycoplasma mycoides subsp. mycoides now

refers exclusively to the agent of CBPP (Manso-Silván et al., 2009).

2. Mycoplasma adleri Del Giudice, Rose and Tully 1995, 31VP

ad¢le.ri. N.L. masc. gen. n. adleri of Adler, referring to Henry Adler, a Californian veterinarian whose studies contributed much new information concerning the pathogenic role of caprine and avian mycoplasmas.

Cells are primarily coccoid. Nonmotile. Colonies on solid media have a typical fried-egg appearance. Grows well in Hayflick medium supplemented with arginine at 35–37°C.

Pathogenic; associated with suppurative arthritis and joint abscesses. Mode of transmission is unknown.

Source: isolated from an abscessed joint of a goat with sup-purative arthritis (Del Giudice et al., 1995).

DNA G+C content (mol%): 29.6 (Bd).Type strain: G145, ATCC 27948, CIP 105676.Sequence accession no. (16S rRNA gene): U67943.

3. Mycoplasma agalactiae (Wróblewski 1931) Freundt 1955, 73AL (Anulomyces agalaxiae Wróblewski 1931, 111)

a.ga.lac.ti¢ae. Gr. n. agalactia want of milk, agalactia; N.L. gen. n. agalactiae of agalactia.

Cells are primarily coccoid, but are occasionally branched and filamentous. Nonmotile. Colonies on solid media have a typical fried-egg appearance. Grows well in SP-4 or Hay-flick medium supplemented with glucose at 37°C. Forma-tion of biofilms has been demonstrated (McAuliffe et al., 2006).

Pathogenic; causes polyarthritis, mastitis, conjunctivi-tis (a syndrome collectively termed contagious agalactia; Bergonier et al., 1997), nonsuppurative arthritis, pneumo-nia, abortion, and granular vulvovaginitis (Cottew, 1983; DaMassa, 1996) in goats and sheep. Transmission occurs via direct contact, most commonly during feeding (kids and lambs) or milking (dams and ewes).

Macrolides and fluoroquinolones are effective chemo-therapeutic agents; however, antimicrobial therapy is not often utilized in widespread outbreaks due to the potential for infected animals to develop carrier states with resistant

strains and the tendency of antimicrobials to be excreted in milk. Control measures such as disinfection of fomites (endemic areas) and culling of infected animals (acute out-breaks) are more common practices. Mycoplasma agalactiae reportedly shares surface antigens with Mycoplasma bovis and Mycoplasma capricolum subsp. capricolum (Alberti et al., 2008; Boothby et al., 1981), potentially complicating serol-ogy-based diagnosis of infection. Molecular diagnostics that can distinguish Mycoplasma agalactiae from Mycoplasma bovis have been described (Chávez Gonzalez et al., 1995). Com-mercially available vaccines are widely used, but exhibit poor efficacy. Numerous experimental vaccines have been described. This organism is under certain quarantine regu-lations in some countries and is listed in the Terrestrial Ani-mal Health Code of the Office International des Epizooties (http://oie.int).

Source: isolated from the joints, udders, milk, conjuncti-vae, lungs, vagina, liver, spleen, kidneys, and small intestine of sheep and goats.

DNA G+C content (mol%): 30.5 (Tm), 29.7 (strain PG2T complete genome sequence).

Type strain: PG2, NCTC 10123, CIP 59.7.Sequence accession nos: M24290 (16S rRNA gene),

NC_009497 (strain PG2T complete genome sequence).

4. Mycoplasma agassizii Brown, Brown, Klein, McLaughlin, Schumacher, Jacobson, Adams and Tully 2001c, 417VP

a.gas.si¢zi.i. N.L. masc. gen. n. agassizii of Agassiz, referring to Louis Agassiz, a naturalist whose name was assigned to a species of desert tortoise (Gopherus agassizii) from which the organism was isolated.

Cells are coccoid to pleomorphic, with some strains appearing to possess a rudimentary terminal structure. Cells exhibit gliding motility. Colony forms on solid medium vary from those with a fried-egg appearance to some with mul-berry characteristics. Grows well in SP-4 medium supple-mented with glucose at 30°C.

Pathogenic; causes chronic upper respiratory tract dis-ease characterized by severe rhinitis in desert tortoises, gopher tortoises, Russian tortoises, and leopard tortoises. Mode of transmission appears to be intranasal inhalation (Brown et al., 1994).

Source: isolated from the nares and choanae of desert tortoises, gopher tortoises, Russian tortoises, and leopard tortoises (Brown et al., 2001c).

DNA G+C content (mol%): not determined.Type strain: PS6, ATCC 700616.Sequence accession no. (16S rRNA gene): U09786.

5. Mycoplasma alkalescens Leach 1973, 149AL

al.ka.les¢cens. N.L. v. alkalesco to make alkaline, referring to the reaction produced in arginine-containing media; N.L. part. adj. alkalescens alkaline-making.

Coccoid to coccobacillary cells. Motility and colony mor-phology have not been described for this species. Grows well in SP-4 medium supplemented with arginine at 37°C.

Pathogenic; causes febrile arthritis and sometimes masti-tis, pneumonia, and otitis in cattle (Kokotovic et al., 2007; Lamm et al., 2004; Leach, 1973). Mode of transmission has not been established.

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Tetracyclines and pleuromutilins are effective chemother-apeutic agents (Hirose et al., 2003). Mycoplasma alkalescens reportedly shares surface antigens with many arginine- fermenting Mycoplasma species; however, this is only likely to interfere with accurate diagnosis of infection in the case of Mycoplasma arginini.

Source: isolated from the synovial fluid, expelled milk, lungs, ears, prepuce, and semen of cattle.

DNA G+C content (mol%): 25.9 (Tm).Type strain: D12, PG51, NCTC 10135, ATCC 29103.Sequence accession no. (16S rRNA gene): U44764.Further comment: Bovine serogroup 8 of Leach (1967).

6. Mycoplasma alligatoris Brown, Farley, Zacher, Carlton, Clip-pinger, Tully and Brown 2001a, 423VP

al.li.ga.to¢ris. N.L. n. alligator, -oris an alligator; N.L. gen. n. alligatoris of/from an alligator.

Cells are primarily coccoid. Nonmotile. Colonies on solid medium exhibit typical fried-egg morphology. Growth is very rapid in SP-4 medium supplemented with glucose at 30°C.

Pathogenic; causes a multisystemic inflammatory illness with a lethality unprecedented among mycoplasmas. Patho-logic lesions in infected animals include meningitis, intersti-tial pneumonia, fibrinous pleuritis, polyserositis, fibrinous pericarditis, myocarditis, endocarditis, synovitis, and splenic and hepatic necrosis. A high level of mortality of naturally and experimentally infected American alligators (Alligator missisippiensis) and broad-nosed caimans (Caiman latiro-stris) occurs. In contrast, Mycoplasma alligatoris colonizes the tonsils of experimentally infected Siamese crocodiles (Crocodylus siamensis) without causing overt pathology. Such findings led to the hypothesis that Mycoplasma alligatoris is a natural commensal of a species more closely related to crocodiles than alligators and that the extreme disease state observed in alligators results from an enzoonotic infection (Brown et al., 1996; Pye et al., 2001). The natural mode of transmission has not been established definitively; however, animals can be experimentally infected via inoculation of the glottis.

Source: isolated from the blood, synovial fluid, cerebrospi-nal fluid, lungs, brain, heart, liver, and spleen of naturally and experimentally infected American alligators, experi-mentally infected broad-nosed caimans, and from the ton-sils of experimentally infected Siamese crocodiles.

DNA G+C content (mol%): not determined.Type strain: A21JP2, ATCC 700619.Sequence accession no. (16S rRNA gene): U56733.Further comment: the name Mycoplasma alligatoris was

assigned for this organism in consideration of the initial isolation from an alligator (Order: Crocodylia). The Eng-lish word “alligator” is from the Spanish el lagarto (Latin ille lacertu the lizard). However, the specific epithet lacerti, originally proposed for this taxon, was ultimately rejected because of the modern phylogenetic distinction between lizards (Order: Lacertilia) and crocodilians.

7. Mycoplasma alvi Gourlay, Wyld and Leach 1977, 95AL

al¢vi. L. n. alvus bowel, womb, stomach; L. gen. n. alvi of the bowel.

Cells are primarily coccoid; however, subsets of the population display elongated, flask-shaped cells with well-defined terminal structures. Unlike most species that exhibit polar structures, Mycoplasma alvi appears to be nonmotile (Bredt, 1979; Hatchel and Balish, 2008). Colonies on solid medium exhibit typical fried-egg morphology. Grows well in SP-4 medium supplemented with either glucose or arginine at 37°C.

No evidence of pathogenicity. Mode of transmission has not been established definitively.

Source: isolated from the lower alimentary tract, feces, bladder, and vagina of cows, and the intestinal tract of voles (Gourlay and Howard, 1979).

DNA G+C content (mol%): 26.4 (Bd).Type strain: Ilsley, NCTC 10157, ATCC 29626.Sequence accession no. (16S rRNA gene): U44765.

8. Mycoplasma amphoriforme Pitcher, Windsor, Windsor, Bradbury, Yavari, Jensen, Ling and Webster 2005, 2592VP

am.pho.ri.for¢me. L. n. amphora amphora; L. adj. suff. -formis -e like, of the shape of; N.L. neut. adj. amphoriforme amphora-shaped, having the form of an amphora.

Cells are flask-shaped with a distinct terminal structure reminiscent of Mycoplasma gallisepticum. Cells exhibit low-speed gliding motility and move in the direction of the ter-minal structure (Hatchel et al., 2006). Colony morphology variable, from a typical fried-egg morphology to a “ground glass” appearance. Grows well in SP-4 medium supple-mented with glucose at 37°C.

Pathogenicity and mode of transmission have not been established definitively.

Despite showing sensitivity to fluoroquinolones, tetra-cyclines, and macrolides in vitro, Mycoplasma amphoriforme appears to be successfully evasive during treatment of patients with these antibiotics. The veterinary antibiotic valnemulin is successful at controlling infection (Webster et al., 2003).

Source: isolated from the sputum of immunocompro-mised humans with bronchitis and related lower respira-tory tract disease (Pitcher et al., 2005; Webster et al., 2003). The prevalence of Mycoplasma amphoriforme in the general human population is unsubstantiated.

DNA G+C content (mol%): 34.0 (fluorescent intensity).Type strain: A39, NCTC 11740, ATCC BAA-992.Sequence accession no. (16S rRNA gene): FJ226575.

9. Mycoplasma anatis Roberts 1964, 471AL

a.na¢tis. L. n. anas, -atis a duck; L. gen. n. anatis of a duck.

Cells have been described as coccoid with ring forms, but cellular and colony morphology is generally poorly described. Motility for this species has not been assessed. Grows well in SP-4 medium supplemented with glucose 37°C.

Isolated from pathologic lesions, but attempts to repro-duce disease following experimental infection have been equivocal (Amin and Jordan, 1978; Roberts, 1964). The mode of transmission has not been established definitively.

Source: isolated from pathologic lesions, the respiratory tract, hock joint, pericardium, cloaca, and meninges of ducks (Goldberg et al., 1995; Ivanics et al., 1988; Tiong, 1990).

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DNA G+C content (mol%): 26.6 (Bd).Type strain: 1340, ATCC 25524, NCTC 10156.Sequence accession no. (16S rRNA gene): AF412970.

10. Mycoplasma anseris Bradbury, Jordan, Shimizu, Stipkovits and Varga 1988, 76VP

an¢se.ris. L. gen. n. anseris of the goose.

Cells are primarily spherical. Nonmotile. Colonies on solid medium have a typical fried-egg appearance. Grows well in Hayflick medium supplemented with arginine at 37°C.

Opportunistic pathogen; associated with balanitis of geese, but can be isolated from clinically normal animals. Mode of transmission has not been established definitively.

Source: isolated from the phallus and cloaca of geese (Hinz et al., 1994; Stipkovits et al., 1984a).

DNA G+C content (mol%): 24.7–26.0 (Bd/Tm).Type strain: 1219, ATCC 49234.Sequence accession no. (16S rRNA gene): AF125584.

11. Mycoplasma arginini Barile, Del Giudice, Carski, Gibbs and Morris 1968, 490AL

ar.gi.ni¢ni. N.L. n. argininum arginine, an amino acid; N.L. gen. n. arginini of arginine, referring to its hydrolysis.

Cells are primarily coccoid. Motility for this species has not been assessed. Colonies have either the typical fried-egg morphology or a granular, “berry-like” appearance. Grows well in modified Hayflick medium supplemented with argi-nine at 37°C.

Pathogenic; associated with pneumonia, vesiculitis, kera-toconjunctivitis, and mastitis in cattle; pneumonia and ker-atoconjunctivitis in sheep; possibly with arthritis in goats; and septicemia in immunocompromised humans (Tully and Whitcomb, 1979; Yechouron et al., 1992). Modes of transmission have not been definitively assessed and likely vary by anatomical site.

Mycoplasma arginini is commonly associated with contam-ination of eukaryotic cell culture and is frequently removed by treatment of cells with antibiotics and/or maintenance of cell lines in antibiotic-containing medium. The most effec-tive classes of antibiotics for cell culture eradication are tet-racyclines, macrolides, and fluoroquinolones. Additionally, passage of eukaryotic cells in hyperimmune serum raised against Mycoplasma arginini has been shown to be an effec-tive method of eradication (Jeansson and Brorson, 1985).

Source: isolated from a wide array of mammalian hosts including cattle, sheep, goats, pigs, horses, domestic dogs, domestic cats, lions, lynxes, cheetahs, chamois, camels, ibexes, humans, and mice.

DNA G+C content (mol%): 27.6 (Tm).Type strain: G230, ATCC 23838, NCTC 10129, CIP 71.23,

NBRC 14476.Sequence accession no. (16S rRNA gene): U15794.

12. Mycoplasma arthritidis (Sabin 1941) Freundt 1955, 73AL (Murimyces arthritidis Sabin 1941, 57)

ar.thri¢ti.dis. Gr. n. arthritis -idos gout, arthritis; N.L. gen. n. arthritidis of arthritis.

Cells are filamentous and vary in length. Motility for this species has not been assessed. Colonies on solid medium

have a typical fried-egg appearance. Grows well in SP-4 medium supplemented with arginine.

Pathogenic; causes purulent polyarthritis, rhinitis, otitis media, ocular lesions, and abscesses in rats. Mycoplasma arthritidis is also known to superinfect lung lesions initiated by Mycoplasma pulmonis. Experimental inoculations via vari-ous routes can result in septicemia, acute flaccid paralysis, and pyelonephritis in rats, and chronic arthritis in mice and rabbits. Mycoplasma arthritidis is unique among mycoplasmas in harboring the lysogenized bacteriophage MAV1, whose contribution to virulence is equivocal, and producing the potent mitogen MAM, which appears to confer increased toxicity and lethality but to be irrelevant to arthritogenicity (Clapper et al., 2004; Luo et al., 2008; Voelker et al., 1995). The mechanism of transmission is largely dependent on the tissue infected.

Source: isolated from the synovial membranes, synovial fluid, middle ear, eye, abscessed bone, abscessed ovary, and oropharynx of wild and captive rats. Isolations have also been reported from non-human primates including rhesus monkeys and bush babies (Somerson and Cole, 1979); and from joint fluid of wild boars (Binder et al., 1990). The true origins of putative isolates from the human urethra, pros-tate, and cervix have been questioned (Cassell and Hill, 1979; Washburn et al., 1995).

DNA G+C content (mol%): 30.0 (Tm; strain PG6T), 30.7 (strain 158L3-1 complete genome sequence).

Type strain: PG6, ATCC 19611, NCTC 10162, CIP 104678, NBRC 14860.

Sequence accession nos: M24580 (16S rRNA gene), CP001047 (strain 158L3-1 complete genome sequence).

13. Mycoplasma auris DaMassa, Tully, Rose, Pitcher, Leach and Cottew 1994, 483VP

au¢ris. L. gen. n. auris of the ear, referring to the provenance of the organism, the ears of goats.

Cells are coccoid to pleomorphic. Nonmotile. Colo-nies on solid medium have a typical fried-egg appearance. Grows well in Hayflick medium supplemented with argin-ine at 37°C.

No evidence of pathogenicity. Mechanism of transmis-sion has not been established.

Source: isolated from the external ear canals of goats (Damassa et al., 1994).

DNA G+C content (mol%): 26.9 (Tm).Type strain: UIA, ATCC 51348, NCTC 11731, CIP

105677.Sequence accession no. (16S rRNA gene): U67944.

14. Mycoplasma bovigenitalium Freundt 1955, 73AL

bo.vi.ge.ni.ta¢li.um. L. n. bos, bovis the ox, bull, cow; L. pl. n. genitalia the genitals; N.L. pl. gen. n. bovigenitalium of bovine genitalia.

Cells range from coccoid to filamentous. Motility for this species has not been assessed. Colonies on solid medium have a typical fried-egg appearance. Grows well in SP-4 broth supplemented with glucose and/or arginine at 37°C and produces a “film and spots” reaction.

Pathogenic; causes vulvovaginitis, vesiculitis, epididymi-tis, abortion, infertility, mastitis, pneumonia, conjunctivitis,

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and arthritis in cattle; and pneumonia and conjunctivitis in domestic dogs. Mode of transmission is via sexual contact and/or droplet aerosol.

Control measures during outbreaks of Mycoplasma bovi-genitalium infection include suspension of natural breeding in favor of artificial insemination with disposable instru-ments and, in severe cases, culling of infected animals.

Source: isolated from the udders, seminal vesicles, pre-puce, semen, vagina, cervix, lungs, conjunctivae, and joint capsule of cattle; and from the lungs, prepuce, prostate, vagina, cervix, and conjunctivae of domestic dogs (Chalker, 2005; Gourlay and Howard, 1979).

DNA G+C content (mol%): 30.4 (Tm).Type strain: PG11, ATCC 19852, NCTC 10122, NBRC

14862.Sequence accession nos (16S rRNA gene): M24291,

AY121098.Further comment: the collection of strains formerly referred

to as “Mycoplasma ovine/caprine serogroup 11” have been reclassified as Mycoplasma bovigenitalium (Nicholas et al., 2008).

15. Mycoplasma bovirhinis Leach 1967, 313AL

bo.vi.rhi¢nis. L. n. bos, bovis the ox; Gr. n. rhis, rhinos nose; N.L. gen. n. bovirhinis of the nose of the ox.

Cell and colony morphology and motility for this spe-cies are poorly defined. Grows well in SP-4 medium supple-mented with glucose at 37°C.

Pathogenic; causes pneumonia, otitis, conjunctivitis, and mastitis in cattle. Mycoplasma bovirhinis is often found in co-infections with other pathogens, leading to speculation that it often acts as a superinfecting agent. Mode of transmission has not been established definitively.

Source: isolated from the lungs, nasopharynx, trachea, udders, expelled milk, ears, conjunctivae, and rarely from the urogenital tract of cattle (Gourlay and Howard, 1979).

DNA G+C content (mol%): 27.3 (Tm).Type strain: PG43, ATCC 27748, NCTC 10118, CIP 71.24,

NBRC 14857.Sequence accession no. (16S rRNA gene): U44766.

16. Mycoplasma bovis (Hale, Hemboldt, Plastridge and Stula 1962) Askaa and Ernø 1976, 325AL (Mycoplasma agalactiae var. bovis Hale, Hemboldt, Plastridge and Stula 1962, 591; Mycoplasma bovimastitidis Jain, Jasper and Dellinger 1967, 409)

bo¢vis. L. n. bos the ox; L. gen. n. bovis of the ox.

Cells range from coccoidal to short filaments. Nonmotile. Formation of biofilms has been demonstrated ( McAuliffe et al., 2006). Colonies on solid medium have the typical fried-egg appearance, with notably large centers. Grows well in SP-4 medium supplemented with glucose at 37°C and produces a “film and spots” reaction.

Pathogenic; causes mastitis, polyarthritis, keratoconjunc-tivitis (Gourlay and Howard, 1979), pneumonia, and otitis media (Caswell and Archambault, 2007; Maeda et al., 2003), and is rarely associated with infertility, abortion, endometri-tis, salpingitis, and vesiculitis (Doig, 1981; Gourlay and How-ard, 1979) in cattle; pneumonia and polyarthritis in bison (Dyer et al., 2008); and is rarely associated with pneumonia,

mastitis, and arthritis in goats (Egwu et al., 2001; Gourlay and Howard, 1979). Mode of transmission is via direct con-tact with infected animals or fomites, most commonly dur-ing feeding (suckling or trough), milking (cows), aerosol, or sexual contact.

Macrolides and fluoroquinolones are effective chemo-therapeutic agents in vitro; however, antimicrobial therapy is not often utilized in animals with advanced disease due to poor efficacy and the tendency of antimicrobials to be excreted in milk. Control measures such as disinfection of fomites, isolation of infected animals, and euthanasia of animals showing clinical signs are more common prac-tices. Mycoplasma bovis reportedly shares surface antigens with Mycoplasma agalactiae (Boothby et al., 1981), poten-tially complicating serology-based diagnosis of infection. Molecular diagnostics that can distinguish Mycoplasma aga-lactiae from Mycoplasma bovis have been described (Chávez Gonzalez et al., 1995). Several vaccines are commercially available, but exhibit poor efficacy in that they tend to allow for the establishment of infection while only preventing overt clinical signs.

Source: isolated from the udders, expelled milk, synovial fluid, synovial membranes, conjunctivae, lungs, ear canals, tympanic membranes, aborted calves, uterus, cervix, vagina, and semen of cattle; from the lungs and synovial fluid of bison; and from the lungs and udders of goats.

DNA G+C content (mol%): 32.9 (Tm).Type strain: Donetta, PG45, ATCC 25523, NCTC 10131.Sequence accession no. (16S rRNA gene): AJ419905.Further comment: Bovine serotype 5 of Leach (1967).

17. Mycoplasma bovoculi Langford and Leach 1973, 1443AL

bo.vo¢cu.li. L. n. bos, bovis ox, bull, cow; L. n. oculus the eye; N.L. gen. n. bovoculi of the bovine eye.

Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid medium have a typical fried-egg appearance. Grows well in Hayflick medium supplemented with glucose at 37°C.

Pathogenic; causes conjunctivitis and keratoconjunctivi-tis in cattle. Face flies are the suggested mechanism of trans-mission, though this has yet to be established definitively. Topical application of oxytetracycline is an effective treat-ment for infection.

Source: isolated from the conjunctivae and semen of cat-tle, and from aborted calves (Langford and Leach, 1973; Singh et al., 2004).

DNA G+C content (mol%): 29.0 (Tm).Type strain: M165/69, NCTC 10141, ATCC 29104.Sequence accession no. (16S rRNA gene): U44768.Further comment: Mycoplasma bovoculi was originally

described as Mycoplasma oculi by Leach in 1973, wherein the defining publication referring to the species as Myco-plasma bovoculi (Langford and Leach, 1973) was cited as “in press”.

18. Mycoplasma buccale Freundt, Taylor-Robinson, Purcell, Chanock and Black 1974, 252AL

buc.ca¢le. L. n. bucca the mouth; L. neut. suff. -ale suffix denoting pertaining to; N.L. neut. adj. buccale buccal, per-taining to the mouth.

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Genus I. MycoplasMa

Cells range from coccoid to filamentous. Motility for this species has not been assessed. Colonies on solid medium have a typical fried-egg appearance. Grows well in Hayflick medium supplemented with arginine and herring sperm DNA at 37°C.

No evidence of pathogenicity. Mode of transmission has not been assessed definitively.

Source: isolated from the oropharynx of humans, rhesus macaques, chimpanzees, orangutans, baboons, African green monkeys, crab-eating macaques, and patas monkeys (Somerson and Cole, 1979).

DNA G+C content (mol%): 26.4 (Tm).Type strain: CH20247, ATCC 23636, NCTC 10136, CIP

105530, NBRC 14851.Sequence accession no. (16S rRNA gene): AF125586.

19. Mycoplasma buteonis Poveda, Giebel, Flossdorf, Meier and Kirchhoff 1994, 97VP

bu.te.o¢nis. L. masc. n. buteo, -onis buzzard; L. masc. gen. n. buteonis of the buzzard.

Cells are coccoid. Motility for this species has not been assessed. Colonies on solid medium have typical fried-egg appearance. Grows well in modified Frey’s medium supple-mented with glucose at 37°C.

Mycoplasma buteonis may be pathogenic for saker falcons, as it was found in the respiratory tract, nervous system, and bone of a nestling with pneumonia, hepatitis, ataxia, and dyschondroplasia. No evidence of pathogenicity for buz-zards. Mode of transmission has not been assessed.

Source: isolated from the trachea of buzzards; from the eggs of the lesser kestrel; and the trachea, lungs, brain, and bone marrow of the saker falcon. Has been detected in the common kestrel and the Western marsh harrier (Erdélyi et al., 1999; Lierz et al., 2008a, 2008c).

DNA G+C content (mol%): 27.0 (Bd).Type strain: Bb/T2g, ATCC 51371.Sequence accession no. (16S rRNA gene): AF412971.

20. Mycoplasma californicum Jasper, Ernø, Dellinger and Christiansen 1981, 344VP

ca.li.for¢ni.cum. N.L. neut. adj. californicum pertaining to California.

Cells are coccoid to filamentous. Motility for this spe-cies has not been assessed. Colonies are conical in shape with distinct small centers. Grows well in modified Hayflick broth at 37°C.

Pathogenic; causes purulent mastitis in cows and rarely in sheep. Mode of transmission has not been established definitively.

Source: isolated from the udders and expelled milk of cows and ewes.

DNA G+C content (mol%): 31.9 (Bd).Type strain: ST-6, ATCC 33461, AMRC-C 1077, NCTC

10189.Sequence accession no. (16S rRNA gene): M24582.

21. Mycoplasma canadense Langford, Ruhnke and Onoviran 1976, 218AL

ca.na.den¢se. N.L. neut. adj. canadense pertaining to Canada.

Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid agar have a characteristic fried-egg appearance. Grows well in SP-4 medium supplemented with arginine at 37°C.

Pathogenic; causes mastitis and arthritis, and may be asso-ciated with infertility, abortion, and pneumonia of cattle. Mode of transmission has not been established definitively.

Source: isolated from the udders, expelled milk, synovial membranes, aborted calves, vagina, semen, and lungs (Boughton et al., 1983; Friis and Blom, 1983; Gourlay and Howard, 1979; Jackson et al., 1981).

DNA G+C content (mol%): 29.0 (Tm).Type strain: 275C, NCTC 10152, ATCC 29418.Sequence accession no. (16S rRNA gene): U44769.

22. Mycoplasma canis Edward 1955, 90AL

ca¢nis. L. n. canis, -is a dog; L. gen. n. canis of a dog.

Cells are pleomorphic, exhibiting branched and filamen-tous forms. Motility for this species has not been assessed. Colonies on solid medium exhibit two stable forms: “smooth” colonies with a nongranular appearance and round edges; and “rough” colonies with a granular appearance and irreg-ular or crenated edges. Each form maintains its characteris-tic appearance during repeated subculturing. Grows well in SP-4 medium supplemented with glucose at 37°C.

Opportunistic pathogen; associated with infertility and adverse pregnancy outcomes, endometritis, epididymi-tis, urethritis, cystitis, and pneumonia of domestic dogs (Chalker, 2005); pneumonia of cattle (ter Laak et al., 1992b); and pneumonia in immunocompromised humans (Armstrong et al., 1971). Mode of transmission is via sex-ual contact or aerosol. Mycoplasma canis appears to have a greater tendency toward upper respiratory tract commen-salism and urogenital tract pathogenicity in dogs, while exhibiting pathogenicity for the respiratory tract of cattle.

Source: isolated from the cervix, vagina, prepuce, epididymis, prostate, semen, urine, bladder, oropharynx, nares, lungs, trachea, conjunctivae, kidneys, spleen, peri-cardium, liver, and lymph nodes of domestic dogs; from the lungs and oropharynx of cattle; from the lungs and phar-ynx of humans; and from the throat and rectum of baboons and African green monkeys.


14846.Sequence accession no. (16S rRNA gene): AF412972.

23. Mycoplasma capricolum Tully, Barile, Edward, Theodore and Ernø 1974, 116AL

ca.pri.co¢lum. L. n. caper, -pri the male goat; N.L. -suff. colus, -a, -um (from L. v. incolere to dwell) dwelling; N.L. neut. adj. capricolum dwelling in a male goat.

Cells are coccobacillary. Nonmotile. Colonies on solid agar have a characteristic fried-egg appearance. Grows in SP-4 or modified Hayflick medium supplemented with glu-cose at 37°C.

DNA G+C content (mol%): 24.1 (Tm).Type strain: California kid, ATCC 27343, NCTC 10154,

CIP 104620.The species has subsequently been divided as follows.

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23a. Mycoplasma capricolum subsp. capricolum (Tully, Barile, Edward, Theodore and Ernø 1974) Leach, Ernø and MacOwan 1993, 604VP (Mycoplasma capricolum Tully, Barile, Edward, Theodore and Ernø 1974, 116)

ca.pri.co¢lum. L. n. caper, -pri the male goat; N.L. -suff. colus, -a, -um (from L. v. incolere to dwell) dwelling; N.L. neut. adj. capricolum dwelling in a male goat.

Cells are coccobacillary and can produce long, helical rods known as rho forms. Nonmotile. Colonies on solid agar have a characteristic fried-egg appearance. Grows in SP-4 or modified Hayflick medium supplemented with glu-cose at 37°C.

Pathogenic; causes fibrinopurulent polyarthritis, mastitis, conjunctivitis (a syndrome collectively termed contagious agalactia) and septicemia in goats. Transmission occurs via direct contact between animals or with fomites.

Tetracyclines, macrolides, and tylosin are effective che-motherapeutic agents. Treatment of acutely infected animals often leads to the eradication of the organism, whereas treatment of chronically infected animals does not. Early intervention with antibiotics and improved sanitation are effective control measures (Thiaucourt et al., 1996). Antigenic cross-reactivity with Mycoplasma capricolum subsp. capripneumoniae and Mycoplasma leachii preclude the exclu-sive reliance on serological-based diagnostics. Multiple molecular diagnostics have been described (Fitzmaurice et al., 2008; Greco et al., 2001). This organism is under cer-tain quarantine regulations in some countries and is listed in the Terrestrial Animal Health Code of the Office Interna-tional des Epizooties (http://oie.int).

Source: isolated from the synovial fluid, synovial mem-branes, udders, expelled milk, conjunctivae, spleen, nasopharynx, oral cavity, and ear canal of goats; and the nasopharynx of sheep (Cottew, 1979).

DNA G+C content (mol%): 24.1 (Tm), 23 (strain California kidT complete genome).

Type strain: California kid, ATCC 27343, NCTC 10154, CIP 104620.

Sequence accession nos: U26046 (16S rRNA gene), NC_007633 (strain California kidT complete genome).

23b. Mycoplasma capricolum subsp. capripneumoniae Leach, Ernø and MacOwan 1993, 604VP

ca.pri.pneu.mo.ni¢ae. L. n. capra, -ae a goat; Gr. n. pneumonia disease of the lungs, pneumonia; N.L. gen. n. capripneumo-niae of a pneumonia of a goat.

Cells are coccobacillary. Nonmotile. Colonies on solid agar have a characteristic fried-egg appearance. Grows in SP-4 or modified Hayflick medium supplemented with glu-cose at 37°C.

Pathogenic; causes characteristic, highly lethal fibrin-ous pleuropneumonia known as contagious caprine pleu-ropneumonia (CCPP) in goats (McMartin et al., 1980). A respiratory tract disease of similar pathology found in association with Mycoplasma capricolum subsp. capripneumo-niae has been reported in sheep, mouflon, and ibex (Arif et al., 2007; Shiferaw et al., 2006). Transmission occurs via droplet aerosol.

Tetracyclines and tylosin are effective chemotherapeutic agents; however, treatment of endemic herds is not often

undertaken. Culling of infected herds and restricting the movement of infected animals are more common strate-gies for controlling spread of the disease (Thiaucourt et al., 1996). Live and killed vaccines have been described; how-ever, each appear to afford delayed or partial protection from morbidity, and few have been completely successful at preventing infection (Browning et al., 2005). Antigenic cross-reactivity with Mycoplasma capricolum subsp. capri-colum and Mycoplasma leachii preclude the exclusive reli-ance on serological-based diagnostics. Multiple molecular diagnostics have been described (Lorenzon et al., 2008; March et al., 2000; Woubit et al., 2004). This organism is under certain quarantine regulations in some countries and is listed in the Terrestrial Animal Health Code of the Office International des Epizooties (http://www.oie.int).

Source: isolated from the lower respiratory tract of goats, sheep, mouflon, and ibex.

DNA G+C content (mol%): 24.4 (Bd).Type strain: F38, NCTC 10192.Sequence accession no. (16S rRNA gene): U26042.Further comment: previously known as the F38-type caprine

mycoplasmas.

24. Mycoplasma caviae Hill 1971, 112AL

ca.vi¢ae. N.L. n. cavia guinea pig (Cavia cobaya); N.L. gen. n. caviae of a guinea pig.

Cell morphology for this species has not been described and motility has not been assessed. Colonies on solid agar have a characteristic fried-egg appearance. Grows in SP-4 medium supplemented with glucose at 37°C.


Source: isolated from the nasopharynx and urogenital tract of guinea pigs (Hill, 1971).

DNA G+C content (mol%): not determined.Type strain: G122, ATCC 27108, NCTC 10126.Sequence accession no. (16S rRNA gene): AF221111.

25. Mycoplasma cavipharyngis Hill 1989, 371VP (Effective publi-cation: Hill 1984, 3187)

ca.vi.pha.ryn¢gis. N.L. n. cavia the guinea pig (Cavia cobaya); N.L. n. pharynx -yngis (from Gr. n. pharugx pharuggos throat) throat; N.L. gen. n. cavipharyngis of the throat of a guinea pig.

Cells are highly filamentous and filaments are twisted at intervals along their length. Regular helical forms like those of Spiroplasma species are not produced. Nonmotile. Growth on solid medium shows small, granular colonies with poorly defined centers. Grows in Hayflick medium supplemented with glucose at 35°C.


Source: isolated from the nasopharynx of guinea pigs (Hill, 1984).

DNA G+C content (mol%): 30 (Tm).Type strain: 117C, NCTC 11700, ATCC 43016.Sequence accession no. (16S rRNA gene): AF125879.

26. Mycoplasma citelli Rose, Tully and Langford 1978, 571AL

ci.tel¢li. N.L. n. Citellus a genus of ground squirrel; N.L. gen. n. citelli of Citellus.

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http://oie.int

http://www.oie.int

Genus I. MycoplasMa

Cells are highly pleomorphic. Motility for this species has not been assessed. Colonies on solid agar have a char-acteristic fried-egg appearance. Grows well in SP-4 medium supplemented with glucose at 37°C.


Source: isolated from the trachea, lung, spleen, and liver of ground squirrels (Rose et al., 1978).

DNA G+C content (mol%): 27.4 (Bd).Type strain: RG-2C, ATCC 29760, NCTC 10181.Sequence accession no. (16S rRNA gene): AF412973.

27. Mycoplasma cloacale Bradbury and Forrest 1984, 392VP

clo.a.ca¢le. L. neut. adj. cloacale pertaining to a cloaca.

Cells are primarily spherical. Nonmotile. Colonies on solid medium exhibit typical fried-egg appearance. Grows well in Hayflick medium supplemented with arginine at 37°C.

No evidence of pathogenicity. Mode of transmission has not been established definitively

Source: isolated from the cloaca of a turkey; from the lungs, trachea, ovaries, and eggs of ducks; and from chickens, pheasants, and geese (Bencina et al., 1987, 1988; Bradbury et al., 1987; Goldberg et al., 1995; Hinz et al., 1994).

DNA G+C content (mol%): 26 (Bd).Type strain: 383, ATCC 35276, NCTC 10199.Sequence accession no. (16S rRNA gene): AF125592.

28. Mycoplasma collis Hill 1983b, 849VP

col¢lis. L. gen. n. collis of a hill, alluding to the author who described the species.

Cells are primarily coccoidal and are nonmotile. Growth on a solid medium shows colonies with a typical fried-egg appearance. Grows in Hayflick medium supplemented with glucose at 35–37°C.

No evidence of pathogenicity. Mode of transmission has not been assessed.

Source: isolated from the conjunctivae of captive rats and mice (Hill, 1983b). References to isolation of Mycoplasma collis from domestic dogs appear to have been in error (Chalker and Brownlie, 2004).

DNA G+C content (mol%): 28 (Tm).Type strain: 58B, NCTC 10197, ATCC 35278.Sequence accession no. (16S rRNA gene): AF538681.

29. Mycoplasma columbinasale Jordan, Ernø, Cottew, Hinz and Stipkovits 1982, 114VP

co.lum.bi.na.sa¢le. L. n. columbus a pigeon; L. n. nasus nose; L. neut. suff. -ale suffix used with the sense of pertaining to; N.L. neut. adj. nasale pertaining to the nose; N.L. neut. adj. columbinasale pertaining to the nose of a pigeon.

Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid medium exhibit typical fried-egg appearance. Grows well in SP-4 medium supplemented with arginine at 35–37°C. Produces a “film and spots” reaction.


Source: isolated from the turbinates of rock pigeons, racing pigeons, and fantail pigeons (Bencina et al., 1987; Keymer et al., 1984; Nagatomo et al., 1997; Yoder and Hofstad, 1964).

DNA G+C content (mol%): 32 (Bd).Type strain: 694, ATCC 33549, NCTC 10184.Sequence accession no. (16S rRNA gene): AF221112.Further comment: previously known as avian serovar (sero-

type) L (Yoder and Hofstad, 1964).

30. Mycoplasma columbinum Shimizu, Ernø and Nagatomo 1978, 545AL

co.lum.bi¢num. L. neut. adj. columbinum pertaining to a pigeon.

Cells are pleomorphic and vary from coccoid to ring forms. Motility for this species has not been assessed. Colo-nies on solid medium have typical fried-egg morphology. Grows in Frey’s medium supplemented with arginine at 37°C. Produces a “film and spots” reaction.


Source: isolated from the trachea and oropharynx of feral pigeons and from the brain and lungs of racing pigeons (Bencina et al., 1987; Jordan et al., 1981; Keymer et al., 1984; Reece et al., 1986).

DNA G+C content (mol%): 27.3 (Bd).Type strain: MMP1, ATCC 29257, NCTC 10178.Sequence accession no. (16S rRNA gene): AF221113.

31. Mycoplasma columborale Shimizu, Ernø and Nagatomo 1978, 545AL

co.lum.bo.ra¢le. L. n. columba pigeon; L. n. os, oris the mouth; L. neut. suff. -ale suffix used with the sense of pertaining to; N.L. neut. adj. orale of or pertaining to the mouth; N.L. neut. adj. columborale of the pigeon mouth.

Cells are pleomorphic but predominantly coccoid or exhibiting ring forms. Motility for this species has not been assessed. Growth on solid medium yields medium to large colonies with very small central zones. Grows in Frey’s medium supplemented with glucose at 37°C.

Pathogenicity for pigeons is unconfirmed, but one report described airsacculitis in experimentally inoculated chick-ens. Mode of transmission has not been assessed defini-tively.

Source: isolated from the trachea and oropharynx of feral pigeons and fantail pigeons; from the oropharynx and sinuses of racing pigeons; and from corvids and house flies (Bencina et al., 1987; Bradbury et al., 2000; Jordan et al., 1981; Kempf et al., 2000; Keymer et al., 1984; MacOwan et al., 1981; Nagatomo et al., 1997; Reece et al., 1986).

DNA G+C content (mol%): 29.2 (Bd).Type strain: MMP4, ATCC 29258, NCTC 10179.Sequence accession no. (16S rRNA gene): AF412975.

32. Mycoplasma conjunctivae Barile, Del Giudice and Tully 1972, 74AL

con.junc.ti¢va.e. N.L. n. conjunctiva the membrane joining the eyeball to the lids; N.L. gen. n. conjunctivae of conjunctiva.

Cells are coccoid to coccobacillary. Motility for this spe-cies has not been assessed. Colonies grown on solid medium may have elevated centers and a greenish, brownish, or olive color. Grows well in SP-4 medium supplemented with glucose at 37°C.

Pathogenic; causes infectious keratoconjunctivitis that can either resolve into a carrier state or result in complete

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or near-complete blindness in goats, sheep, chamois, and ibex (Cottew, 1979; Mayer et al., 1997). Mode of transmis-sion is via direct contact.

Though tetracyclines are an effective antimicrobial ther-apy in vivo, treatment to eradicate Mycoplasma conjunctivae from herds is not often attempted as the economic burden of infection is low. Topical treatment of secondary infec-tions is often necessary (Slatter, 2001). Several commercial diagnostic assays have been described.

Source: isolated from the conjunctivae of goats, sheep, chamois, and Alpine ibex.

DNA G+C content (mol%): not determined.Type strain: HRC581, ATCC 25834, NCTC 10147.Sequence accession no. (16S rRNA gene): AY816349.

33. Mycoplasma corogypsi Panangala, Stringfellow, Dybvig, Woodard, Sun, Rose and Gresham 1993, 589VP

co.ro.gyp¢si. Gr. n. korax -acos a raven (black); Gr. n. gyps gypos a vulture; N.L. gen. n. corogypsi (sic) of a raven vulture.

Cells are highly pleomorphic and show small circular budding processes abutting elongated cells. Motility for this species has not been assessed. Colonies on solid medium have a fried-egg appearance. Grows in Frey’s medium sup-plemented with glucose at 37°C.

Pathogenicity has not been established, although asso-ciated with abscess formation in a black vulture. Myco-plasma corogypsi has been isolated from clinically normal captive falcons, and may represent a commensal of this species. Mode of transmission has not been assessed definitively.

Source: isolated from the abscessed footpad of a black vul-ture, and from captive falcons (Lierz et al., 2002; Panangala et al., 1993).

DNA G+C content (mol%): 28 (Bd).Type strain: BV1, ATCC 51148.Sequence accession no. (16S rRNA gene): L08054.

34. Mycoplasma cottewii DaMassa, Tully, Rose, Pitcher, Leach and Cottew 1994, 483VP

cot.te¢wi.i. N.L. masc. gen. n. cottewii of Cottew, named for Geoffrey S. Cottew, an Australian veterinarian who was a co-isolator of the organism.

Cells are primarily coccoid. Nonmotile. Formation of biofilms has been demonstrated (McAuliffe et al., 2006). Growth on solid medium shows colonies with a typical fried-egg appearance. Grows well in Hayflick medium supple-mented with glucose at 37°C.

No evidence of pathogenicity. Mode of transmission has not been established.

Source: isolated from the external ear canals and rarely the sinuses of goats (Damassa et al., 1994).

DNA G+C content (mol%): 27 (Tm).Type strain: VIS, ATCC 51347, NCTC 11732, CIP 105678.Sequence accession no. (16S rRNA gene): U67945.

35. Mycoplasma cricetuli Hill 1983a, 117VP

cri.ce.tu¢li. N.L. n. Cricetulus generic name of the Chinese hamster, Cricetulus griseus; N.L. gen. n. cricetuli of Cricetulus.

Cells are coccoid to pleomorphic. Nonmotile. Colony growth on solid medium has a fried-egg appearance with

markedly small centers. Grows well in Hayflick broth sup-plemented with glucose at 37°C.


Source: isolated from the conjunctivae and nasopharynx of Chinese hamsters (Hill, 1983a).

DNA G+C content (mol%): not determined.Type strain: CH, NCTC 10190, ATCC 35279.Sequence accession no. (16S rRNA gene): AF412976.

36. Mycoplasma crocodyli Kirchhoff, Mohan, Schmidt, Runge, Brown, Brown, Foggin, Muvavarirwa, Lehmann and Floss-dorf 1997, 746VP

cro.co.dy¢li. N.L. n. Crocodylus (from L. n. crocodilus croco-dile) generic name of the crocodile; N.L. gen. n. crocodyli of Crocodylus.

Cells are coccoid. Nonmotile. Colonies on solid medium show a typical fried-egg appearance. Grows very rapidly in SP-4 medium supplemented with glucose at 30°C.

Pathogenic; causes exudative polyarthritis and rarely pneumonia in crocodiles. The natural mode of transmis-sion has not been assessed definitively; however, experimen-tal infection resulting in the reproduction of disease was achieved by intracoelomic and/or intrapulmonary inocula-tion.

Tetracyclines are effectively used to alleviate clinical signs in farmed crocodiles. A bacterin vaccine effective at control-ling infection and preventing disease has been described (Mohan et al., 2001).

Source: isolated from the joints and lungs of Nile croco-diles (Mohan et al., 1997).

DNA G+C content (mol%): 27.6 (Bd).Type strain: MP145, ATCC 51981.Sequence accession no. (16S rRNA gene): AF412977.

37. Mycoplasma cynos Røsendal 1973, 53AL

cy¢nos. Gr. n. cyon, cynos a dog; N.L. gen. n. cynos of a dog.

Cells are coccoid to coccobacillary. Motility for this spe-cies has not been assessed. Colonies on solid medium have defined centers and scalloped perimeters. Grows well in SP-4 medium supplemented with arginine at 37°C.

Pathogenic; causes pneumonia, bronchitis, and rarely cys-titis in domestic dogs. The mode of transmission is via drop-let aerosol, as demonstrated by studies housing infected and sentinel dogs (Røsendal and Vinther, 1977).

Source: isolated from the lungs, trachea, nasopharynx, urine, prepuce, prostate, cervix, vagina, and conjunctivae of domestic dogs (Chalker, 2005).

DNA G+C content (mol%): 25.8 (Bd).Type strain: H 831, ATCC 27544, NCTC 10142.Sequence accession no. (16S rRNA gene): AF538682.

38. Mycoplasma dispar Gourlay and Leach 1970, 121AL

dis¢par. L. neut. adj. dispar dissimilar, different.

Cells range from coccoid to short and filamentous. Motil-ity for this species has not been assessed. An extracellular capsule can be visualized by electron microscopy following staining with ruthenium red. Colonies on solid medium have a granular, lacy, or reticulated appearance with no or a poorly defined central area. Grows in SP-4 medium or

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Genus I. MycoplasMa

modified Friis medium supplemented with glucose and calf thymus DNA at 37°C.

Pathogenic; causes pneumonia and rarely mastitis in cat-tle. Mode of transmission is by droplet aerosol.

Source: isolated from the lower respiratory tract and udders of cattle (Gourlay and Howard, 1979; Hodges et al., 1983).

DNA G+C content (mol%): 28.5–29.3 (Tm).Type strain: 462/2, ATCC 27140, NCTC 10125.Sequence accession no. (16S rRNA gene): AF412979.

39. Mycoplasma edwardii Tully, Barile, Del Giudice, Carski, Armstrong and Razin 1970, 349AL

ed.war¢di.i. N.L. masc. gen. n. edwardii of Edward, named after Derrick Graham ff. Edward (1910–1978), who first iso-lated this organism.

Cells are coccobacillary to short and filamentous. Motil-ity for this species has not been assessed. Colonies on solid medium show a typical fried-egg appearance. Grows well in SP-4 medium supplemented with glucose at 37°C.

Opportunistic pathogen; commonly found as a commen-sal of the oral and/or nasal cavities and urogenital tract of domestic dogs. Mycoplasma edwardii is rarely associated with pneumonia, arthritis, and septicemia of domestic dogs, often as a secondary pathogen compounding an existing lesion. Mode of transmission has not been established definitively.

Source: isolated from the oropharynx, nasopharynx, trachea, lungs, prepuce, vagina, cervix, blood, and syn-ovial fluid of domestic dogs (Chalker, 2005; Stenske et al., 2005).

DNA G+C content (mol%): 29.2 (Tm).Type strain: PG-24, ATCC 23462, NCTC 10132.Sequence accession no. (16S rRNA gene): U73903.

40. Mycoplasma elephantis Kirchhoff, Schmidt, Lehmann, Clark and Hill 1996, 440VP

e.le.phan¢tis. L. n. elephas, -antis elephant; L. gen. n. elephan-tis of the elephant.

Cells are coccoidal. Nonmotile. Colonies on solid medium show a typical fried-egg appearance. Grows well in Hayflick medium supplemented with glucose at 37°C.

Probable commensal. No pathology was observed at the site of isolation (i.e., the vagina and urethra); however, isolation was achieved almost exclusively from arthritic animals with evidence of rheumatoid factor. The possibil-ity thus exists that the clinical status of the animals was due to sexually acquired reactive arthritis, which has been observed with other Mycoplasma species known to parasitize the urogenital tract (Blanchard and Bébéar, 2002). Mode of transmission has not been established definitively.

Source: isolated from the vagina and urethra of captive elephants (Clark et al., 1980, 1978).

DNA G+C content (mol%): 24 (Bd).Type strain: E42, ATCC 51980.Sequence accession no. (16S rRNA gene): AF221121.

41. Mycoplasma equigenitalium Kirchhoff 1978, 500AL

e.qui.ge.ni.ta¢li.um. L. n. equus, equi the horse; L. pl. n. geni-talia the genitals; N.L. pl. gen. n. equigenitalium of equine genitalia.

Cells are pleomorphic. Motility for this species has not been assessed. Colonies on solid medium show a typical fried-egg appearance. Grows in Hayflick medium supple-mented with glucose at 37°C.

Opportunistic pathogen. Associated with endometri-tis, vulvitis, balanoposthitis, impaired fecundity, and abor-tion in horses; however, Mycoplasma equigenitalium is highly prevalent in clinically normal horses (Spergser et al., 2002). Mode of transmission is via sexual contact.

Source: isolated from the cervix, semen, and aborted foals, and rarely from the trachea, of horses (Lemcke, 1979).

DNA G+C content (mol%): 31.5 (Bd).Type strain: T37, ATCC 29869, NCTC 10176.Sequence accession no. (16S rRNA gene): AF221120.

42. Mycoplasma equirhinis Allam and Lemcke 1975, 405AL

e.qui.rhi¢nis. L. n. equus, equi a horse; Gr. n. rhis, rhinos nose; N.L. gen. n. equirhinis of the nose of a horse.

Cells are coccoid to coccobacillary. Motility for this spe-cies has not been assessed. Colonies on solid medium show a typical fried-egg appearance. Grows in SP-4 or Hayflick medium supplemented with arginine at 37°C.

Opportunistic pathogen; associated with rhinitis and pneumonitis in horses, but can also be found in clinically normal animals. Mode of transmission is via droplet aero-sol.

Source: isolated from the nasopharynx, nasal turbinates, trachea, tonsils, and semen of horses, and from the nasopharynx of cattle (Lemcke, 1979; Spergser et al., 2002; ter Laak et al., 1992a).

DNA G+C content (mol%): not determined.Type strain: M432/72, ATCC 29420, NCTC 10148.Sequence accession no. (16S rRNA gene): AF125585.

43. Mycoplasma falconis Poveda, Giebel, Flossdorf, Meier and Kirchhoff 1994, 97VP

fal.co¢nis. L. gen. n. falconis of the falcon, the host from which the organism was first isolated.

Cells are coccoid. Motility for this species has not been assessed. Colonies on solid medium have a fried-egg appear-ance. Grows well in modified Frey’s medium supplemented with arginine at 37°C.

Pathogenicity has not been established. Associated with respiratory tract infections of saker falcons, although can also be isolated from clinically normal birds. Mode of trans-mission has not been established definitively.

Source: isolated from the trachea of falcons (Lierz et al., 2002, 2008a, b).

DNA G+C content (mol%): 27.5 (Bd).Type strain: H/T1, ATCC 51372.Sequence accession no. (16S rRNA gene): AF125591.

44. Mycoplasma fastidiosum Lemcke and Poland 1980, 161VP

fas.ti.di.o¢sum. L. neut. adj. fastidiosum fastidious, referring to the nutritionally fastidious nature of the organism on pri-mary isolation.

Cells are highly filamentous and filaments are twisted at regular intervals along their length. Helical forms like those of Spiroplasma species are not produced. Nonmotile. Colo-nies on solid medium show a typical fried-egg appearance.

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Grows in SP-4 or Frey’s medium supplemented with glucose at 37°C.


Source: isolated from the nasopharynx of horses (Lemcke and Poland, 1980).

DNA G+C content (mol%): 32.3 (Bd).Type strain: 4822, NCTC 10180, ATCC 33229.Sequence accession no. (16S rRNA gene): AF125878.

45. Mycoplasma faucium Freundt, Taylor-Robinson, Purcell, Chanock and Black 1974, 253AL

fau¢ci.um. L. pl. n. fauces, -ium the throat; L. gen. pl. n. fau-cium of the throat.

Cells are coccoidal. Motility for this species has not been assessed. Colonies on solid medium show a typical fried-egg appearance, but are more loosely attached to the agar surface than are the colonies of most other mycoplasmas. Grows well in SP-4 medium supplemented with arginine at 37°C. Produces a “film and spots” reaction.

Probable commensal. Most commonly found as a com-mensal of the human oropharynx; however, recent isola-tions of Mycoplasma faucium have been made from brain abscesses (Al Masalma et al., 2009). Mode of transmission has not been established definitively.

Source: isolated from the oropharynx and brain of humans, and from the oral cavity of numerous species of nonhuman primates (Freundt et al., 1974; Somerson and Cole, 1979).

DNA G+C content (mol%): not determined.Type strain: DC-333, ATCC 25293, NCTC 10174.Sequence accession no. (16S rRNA gene): AF125590.

46. Mycoplasma felifaucium Hill 1988, 449VP (Effective publica-tion: Hill 1986, 1927)

fe.li.fau¢ci.um. L. n. felis cat; L. pl. n. fauces, -ium throat; N.L. gen. pl. n. felifaucium of the feline throat.

Cells are primarily coccoidal. Nonmotile. Colonies on solid medium show a typical fried-egg appearance. Grows well in SP-4 or Hayflick medium supplemented with argin-ine at 37°C. Produces a “film and spots” reaction.


Source: isolated from the oropharynx of captive pumas (Felis concolor; Hill, 1986).

DNA G+C content (mol%): 31 (Tm).Type strain: PU, NCTC 11703, ATCC 43428.Sequence accession no. (16S rRNA gene): U15795.

47. Mycoplasma feliminutum Heyward, Sabry and Dowdle 1969, 621AL

fe.li.mi.nu¢tum. L. n. felis a cat; L. neut. part. adj. minutum small; N.L. neut. adj. feliminutum a small colony organism isolated from cats.

Morphology is poorly defined. Motility for this species has not been assessed. Colonies are relatively small and irregu-lar in shape. Grows well in SP-4 medium supplemented with glucose at 37°C.


Source: isolated from the oropharynx of domestic cats; from the nasopharynx, lungs, and urogenital tract of domes-tic dogs; and from the respiratory tract of horses (Chalker, 2005; Heyward et al., 1969; Lemcke, 1979).

DNA G+C content (mol%): 29.1 (Bd).Type strain: Ben, ATCC 25749, NCTC 10159.Sequence accession no. (16S rRNA gene): U16758.Further comment: this organism was first described during

a time when the only named genus of mollicutes was Myco-plasma. Its publication coincided with the first proposal of the genus Acholeplasma (Edward and Freundt, 1969, 1970), with which Mycoplasma feliminutum is properly affiliated through established phenotypic (Heyward et al., 1969) and 16S rRNA gene sequence (Brown et al., 1995) simi-larities. This explains the apparent inconsistencies with its assignment to the genus Mycoplasma. The name Mycoplasma feliminutum should therefore be revised to Acholeplasma feliminutum comb. nov.

48. Mycoplasma felis Cole, Golightly and Ward 1967, 1456AL

fe¢lis. L. n. felis a cat, L. gen. n. felis of a cat.

Cells are coccobacillary to filamentous. Motility for this species has not been assessed. Colonies on solid media dis-play the typical fried-egg morphology. Grows well in SP-4 medium supplemented with glucose at 37°C.

Pathogenic; associated with conjunctivitis, rhinitis, ulcer-ative keratitis, and polyarthritis in domestic cats, and upper and lower respiratory tract infection in horses. Mycoplasma felis can also be isolated from clinically normal domestic cats, domestic dogs, and horses. The mode of transmission has not been established definitively.

Source: isolated from the conjunctivae, nasopharynx, lungs, and urogenital tract of domestic cats; from the lungs, tonsils, trachea nasopharynx of horses; from the orophar-ynx and trachea of domestic dogs; and from the synovial fluid of an immunocompromised human (Lemcke, 1979) Røsendal, 1979; (Bonilla et al., 1997; Gray et al., 2005; Hooper et al., 1985).

DNA G+C content (mol%): 25.2 (Tm).Type strain: CO, ATCC 23391, NCTC 10160.Sequence accession no. (16S rRNA gene): U09787.Further comment: the proposed species “Mycoplasma

equipharyngis” (Kirchoff, 1974) has been reported in horses. Further characterization has demonstrated unequivocally that these isolates are Mycoplasma felis and all mention of “Mycoplasma equipharyngis” should be considered equivalent to Mycoplasma felis (Lemcke, 1979).

49. Mycoplasma fermentans Edward 1955, 90AL

fer.men¢tans. L. part. adj. fermentans fermenting.

Cells are filamentous. Motility has not been established for this species. Colonies on solid media display typical fried-egg morphology. Grows well in SP-4 or Hayflick medium supplemented with either arginine or glucose at 37°C.

Pathogenicity unclear; associated with balanitis, vulvovag-initis, salpingitis, respiratory distress syndrome, pneumo-nia, and development of rheumatoid arthritis. Mycoplasma fermentans has also been tenuously linked with the progres-sion of AIDS, chronic fatigue syndrome, Gulf War syn-drome, Adamantiades-Behçet’s disease, and fibromyalgia.

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The connection of the preceding clinical syndromes with Mycoplasma fermentans is highly equivocal, as different stud-ies have reached markedly different conclusions. Mode of transmission has not been established definitively.

Source: isolated from the urine, urethra, rectum, penis, cervix, vagina, fallopian tube, amniotic fluid, blood, synovial fluid, and throat of humans; from the cervix of an African green monkey (Chlorocebus sp.); and from the vagina of a sheep (Blanchard et al., 1993; Nicholas et al., 1998; Taylor-Robinson and Furr, 1997; Waites and Talkington, 2005).

DNA G+C content (mol%): 28.7 (Tm)Type strain: PG18, ATCC 19989, NCTC 10117, NBRC


50. Mycoplasma flocculare Meyling and Friis 1972, 289AL

floc.cu.la¢re. L. dim. n. flocculus a small flock or tuft of wool; L. neut. suff. -are suffix denoting pertaining to; N.L. neut. adj. flocculare resembling a small floc of wool, referring to the tendency of the organism to form clumps of flocculent material in broth culture.

Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid media are slightly convex with a coarsely granular surface and lack a defined center. Aggregates of cells may be produced during growth in broth, appearing as small floccular elements upon gentle shaking of the culture. Grows slowly in Friis medium at 37°C.

Opportunistic pathogen; normally regarded as a com-mensal of the nasopharynx that can cause pneumonia in association with other pathogens, most notably Mycoplasma hyopneumoniae. Mode of transmission is via droplet aerosol. Mycoplasma flocculare reportedly shares surface antigens with Mycoplasma hyopneumoniae, potentially complicating serology-based diagnosis of infection (Whittlestone, 1979).

Source: isolated from the nasopharynx, lungs, pericar-dium, and conjunctivae of pigs.

DNA G+C content (mol%): 33 (Bd).Type strain: Ms42, ATCC 27399, NCTC 10143.Sequence accession no. (16S rRNA gene): L22210.

51. Mycoplasma gallinaceum Jordan, Ernø, Cottew, Hinz and Stipkovits 1982, 114VP

gal.li.na¢ce.um. L. neut. adj. gallinaceum pertaining to a domestic fowl.

Cells are coccoid to coccobacillary. Motility for this spe-cies has not been assessed. Colonies on solid medium have typical fried-egg morphology although some are devoid of a central core. Grows well in Frey’s medium supplemented with glucose at 37°C.

Opportunistic pathogen associated with tracheitis, air-sacculitis, or conjunctivitis in chickens, turkeys, ducks, and pheasants. Mycoplasma gallinaceum has been reported to complicate cases of infectious synovitis due to Mycoplasma synoviae in chickens. The mode of transmission has not been assessed definitively.

Source: isolated from upper and lower respiratory tract of chickens, turkeys, pheasants, partridges, and ducks; from the conjunctivae of pheasants; and from the synovial fluid of chickens (Bradbury et al., 2001; Tiong, 1990; Welchman et al., 2002; Yagihashi et al., 1983).

DNA G+C content (mol%): 28 (Bd).Type strain: DD, ATCC 33550, NCTC 10183.Sequence accession no. (16S rRNA gene): L24104.Further comment: previously known as avian serotype D

(Kleckner, 1960).

52. Mycoplasma gallinarum Freundt 1955, 73AL

gal.li.na¢rum. L. n. gallina a hen; L. gen. pl. n. gallinarum of hens.

Cells are coccoid to coccobacillary. Nonmotile. Colo-nies on solid medium have a typical fried-egg appearance. Grows well in Frey’s medium supplemented with arginine at 37°C. The organism shares some antigens in immunodif-fusion tests with Mycoplasma iners, Mycoplasma columbinasale, and Mycoplasma meleagridis.

Commensal of gallinaceous birds; little evidence exists for the pathogenicity of isolates in such hosts. Mycoplasma gallinarum may have a role in airsacculitis of geese and par-ticipate in complex infection of chickens. Mode of transmis-sion has not been assessed definitively.

Source: isolated from the respiratory tract of chickens, tur-keys, ducks, geese, red jungle fowl, bamboo partridge, spar-row, swan, and demoisella crane; and from sheep (Kisary et al., 1976; Kleven et al., 1978; Shimizu et al., 1979; Singh and Uppal, 1987).

DNA G+C content (mol%): 26.5–28.0 (Tm, Bd).Type strain: PG16, ATCC 19708, NCTC 10120.Sequence accession no. (16S rRNA gene): L24105.Further comment: previously known as avian serotype B

(Kleckner, 1960).

53. Mycoplasma gallisepticum Edward and Kanarek 1960, 699AL

gal.li.sep¢ti.cum. L. n. gallus rooster, chicken; L. adj. septi-cus -a -um producing a putrefaction, putrefying, septic; N.L. neut. adj. gallisepticum hen-putrefying (infecting).

Cells are coccoid, ovoid, and elongated pear-shaped with a highly structured polar body, called the bleb. Cells are motile and glide in the direction of the terminal bleb. Glid-ing speed varies among strains. Colonies on solid medium may be small and not necessarily of typical fried-egg appear-ance. Grows well in SP-4 or Hayflick medium supplemented with glucose at 37°C (Balish and Krause, 2006; Hatchel et al., 2006; Nakane and Miyata, 2009).

Pathogenic. Causes a characteristic combination of pneumonia, tracheitis, and airsacculitis (collectively termed chronic respiratory disease); salpingitis and atro-phy of the ovaries, isthmus, and cloaca resulting in poor egg quality and reduced hatchability; arthritis or synovitis; and keratoconjunctivitis in chickens; infectious sinusitis, coryza, airsacculitis, arthritis or synovitis, encephalitis, meningitis, ataxia, and torticollis in turkeys; conjunctivi-tis and coryza featuring a high mortality rate in finches and grosbeaks; and respiratory disease in additional game birds including quail, partridges, pheasants, and pea-fowl. Lesions established by Mycoplasma gallisepticum are often complicated by additional avian pathogens includ-ing Mycoplasma synoviae, avian strains of Escherichia coli, Newcastle disease virus, and infectious bronchitis virus. Established mechanisms of transmission include droplet

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aerosols, direct contact with infected animals or fomites, and vertical transmission.

Tetracyclines, macrolides, aminoglycosides, fluoroqui-nolones, and pleuromutilins are effective chemotherapeu-tic agents; however, treatment is typically only sought for individual birds, as medicating a commercial flock is not considered an effective control strategy. Vaccination and management strategies (i.e., single age “all in/all out” sys-tems and culling of endemic flocks) are more commonly utilized. Multiple live and killed vaccines are commercially available, but suffer from residual pathogenicity, the need to develop a carrier state to provide protective immunity, adverse reactions, or low efficacy. Experimental vaccines have also been described. Mycoplasma gallisepticum shares surface antigens with Mycoplasma imitans and Mycoplasma synoviae, potentially complicating serology-based diagnosis of infection. Numerous molecular diagnostics have been described. This organism is listed in the Terrestrial Animal Health Code of the Office International des Epizooties (http://oie.int; Yogev et al., 1989; Kempf, 1998; Markham et al., 1999; Gautier-Bouchardon et al., 2002; Ferguson et al., 2004; Browning et al., 2005; Crespo and McMillan, 2008; Gates et al., 2008; Gerchman et al., 2008; Kleven, 2008).

Source: isolated from the trachea, lungs, air sacs, ovaries, oviducts, brain, arterial walls, synovial membranes, synovial fluid, conjunctivae, and eggs of chickens; from the infraor-bital sinuses, air sacs, brain, meninges, conjunctivae, syn-ovial membranes, and synovial fluid of turkeys; from the conjunctivae, infraorbital sinuses, and trachea of finches; and from the respiratory tract of quail, partridges, pheas-ants, peafowl, ducks, grosbeak, crows, robins, and blue jays (Bencina et al., 2003, 1988; Bradbury and Morrow, 2008; Bradbury et al., 2001; Dhondt et al., 2005, 2007; Levisohn and Kleven, 2000; Ley et al., 1996; Mikaelian et al., 2001; Murakami et al., 2002; Nolan et al., 2004; Nunoya et al., 1995; Welchman et al., 2002; Wellehan et al., 2001).

DNA G+C content (mol%): 31.8 (Tm), 31 (strain R com-plete genome sequence).

Type strain: PG31, X95, ATCC 19610, NCTC 10115.Sequence accession nos: M22441 (16S rRNA gene of

strain A5969), NC_004829 (strain R complete genome sequence).

Further comment: previously known as avian serotype A (Kleckner, 1960).

54. Mycoplasma gallopavonis Jordan, Ernø, Cottew, Hinz and Stipkovits 1982, 114VP

gal.lo.pa.vo¢nis. N.L. n. gallopavo, -onis a turkey (Meleagris gallopavo); N.L. gen. n. gallopavonis of a turkey.

Cells are coccoid to coccobacillary. Motility has not been assessed for this species. Colonies on solid medium have typical fried-egg morphology. Grows well in Frey’s medium supplemented with glucose at 37°C.

Opportunistic pathogen; occasionally associated with airsacculitis in turkeys, but is also isolated from clinically normal turkeys. Mode of transmission has not been assessed definitively.

Source: isolated from the choanae, trachea, and air sacs of domestic and wild turkeys (Bencina et al., 1987; Cobb et al., 1992; Hoffman et al., 1997; Luttrell et al., 1992).

DNA G+C content (mol%): 27 (Bd).Type strain: WR1, ATCC 33551, NCTC 10186.Sequence accession no. (16S rRNA gene): AF412980.Further comment: previously known as avian serotype F

(Kleckner, 1960).

55. Mycoplasma gateae Cole, Golightly and Ward 1967, 1456AL

ga.te¢ae. N.L. gen. n. gateae (probably from Spanish gato, a cat) of a cat.

Morphology is poorly defined. Motility for this species has not been assessed. Colonies on solid medium are vacu-olated and lack a well-defined central spot. Grows well in SP-4 medium at 37°C.

Opportunistic pathogen; can cause polyarthritis in domestic cats (Moise et al., 1983), but appears to be pri-marily a commensal species of the oral cavity. Mycoplasma gateae also appears to be a commensal species of domestic dogs and cattle. Mode of transmission has not been assessed definitively.

Source: isolated from the synovial membrane, orophar-ynx, saliva, and urogenital tract of domestic cats; from the lungs, oropharynx, trachea, and urogenital tract of domes-tic dogs; and from the urogenital tract of cattle (Chalker, 2005; Gourlay and Howard, 1979; Røsendal, 1979).

DNA G+C content (mol%): 28.5 (Tm).Type strain: CS, ATCC 23392, NCTC 10161.Sequence accession no. (16S rRNA gene): U15796.Further comment: the original specific epithet “gateae”,

which has been perpetuated in lists of bacterial names approved by the International Committee on Systematics of Prokaryotes, the American Type Culture Collection’s Cata-log of Bacteria and Bacteriophages, and in GenBank, is illegiti-mate because the genitive of the medieval Latin word gata (female cat) would have been gatae and there is no word for which gateae would have been a legitimate genitive (Brown et al., 1995).

56. Mycoplasma genitalium Tully, Taylor-Robinson, Rose, Cole and Bové 1983, 395VP

ge.ni.ta¢li.um. L. pl. n. genitalia, -ium the genitals; L. gen. pl. n. genitalium of the genitals.

Cells are predominantly flask-shaped with a terminal organelle protruding from the cell pole that is narrower than that of Mycoplasma gallisepticum and shorter than that of Mycoplasma pneumoniae. The leading end of the terminal structure is often curved. Cells exhibit gliding motility in circular patterns and glide in the direction of the terminal organelle’s curvature (Hatchel and Balish, 2008). Colonies on solid media are round and possess a defined center that is somewhat less distinct than most mycoplasma species. Grows well in SP-4 medium supplemented with glucose at 37°C.

Pathogenic; causes urethritis, cervicitis, endometritis, and pelvic inflammatory disease. Mycoplasma genitalium is associated with infertility in humans. Mode of transmis-sion is via sexual contact, congenitally, and possibly, in rare instances, via droplet aerosol.

Macrolides and fluoroquinolones are effective chemo-therapeutic agents; however, reports indicate that treat-ment should be extensive, as clinical signs and detection

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http://oie.int

Genus I. MycoplasMa

of Mycoplasma genitalium tend to recur following cessation. This is potentially due to sequestration within host cells. Mycoplasma genitalium shares numerous surface antigens with Mycoplasma pneumoniae, complicating serology-based diagnosis of infection. Numerous molecular diagnostics have been described, but few have been developed com-mercially (Jensen, 2004; Waites and Talkington, 2005).

Source: isolated or detected in the urogenital tract, urine, rectum, synovial fluid, conjunctiva, and nasopharynx of humans (Baseman et al., 1988; de Barbeyrac et al., 1993; Jensen, 2004; Waites and Talkington, 2005).

DNA G+C content (mol%): 32.4 (Bd), 31 (strain G-37T com-plete genome sequence).

Type strain: G-37, ATCC 33530, CIP 103767, NCTC 10195.

Sequence accession nos: X77334 (16S rRNA gene), NC_000908 (strain G-37T complete genome sequence), CP000925 (strain JCVI-1.0 complete genome sequence).

57. Mycoplasma glycophilum Forrest and Bradbury 1984, 355VP (Effective publication: Forrest and Bradbury 1984, 602)

gly.co.phi¢lum. Gr. adj. glykys sweet (this adjective was used to coin the noun glucose); N.L. neut. adj. philum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. glycophilum sweet-loving, intended to mean glucose-loving.

Cells are spherical or elliptical with an extracellular layer. Nonmotile. Growth on solid medium shows colonies with typical fried-egg appearance. Grows well in Hayflick medium supplemented with glucose at 37°C.

Pathogenicity has not been established, although there may be an association with a slight decrease in hatchability. Mode of transmission has not been assessed definitively.

Source: isolated from the respiratory tract and cloaca of chickens; and from the respiratory tract of turkeys, pheas-ants, partridges, ducks, and geese (Forrest and Bradbury, 1984).

DNA G+C content (mol%): 27.5 (Bd).Type strain: 486, ATCC 35277, NCTC 10194.Sequence accession no. (16S rRNA gene): AF412981.

58. Mycoplasma gypis Poveda, Giebel, Flossdorf, Meier and Kirchhoff 1994, 98VP

gy¢pis. Gr. n. gyps, gypos vulture; N.L. gen. n. gypis of the vul-ture, the host from which the organism was first isolated.

Cells are coccoid or round. Motility for this species has not been assessed. Colonies on solid medium have a fried-egg appearance. Grows in Frey’s medium supplemented with arginine at 37°C. Produces a “film and spots” reac-tion.

Pathogenicity has not been established. Associated with respiratory tract disease of griffon vultures, but has also been isolated from healthy birds of prey. Mode of transmis-sion has not been assessed definitively.

Source: isolated from the trachea of griffon vultures (Grif-fon fulvus), and from the trachea and air sacs of Eurasian buzzards, red kites, and Western marsh harriers (Lierz et al., 2008a, 2000; Poveda et al., 1994).

DNA G+C content (mol%): 27.1 (Bd).Type strain: B1/T1, ATCC 51370.Sequence accession no. (16S rRNA gene): AF125589.

59. Mycoplasma haemocanis (Kikuth 1928) Messick, Walker, Raphael, Berent and Shi 2002, 697VP [Bartonella canis Kikuth 1928, 1730; Haemobartonella (Bartonella) canis (Kikuth 1928) Tyzzer and Weinman 1939, 151; Kreier and Ristic 1984, 726]

ha.e.mo.ca¢nis Gr. neut. n. haema blood; L. fem. gen. n. canis of the dog; N.L. gen. n. haemocanis of dog blood.

Cells are coccoid to pleomorphic. Motility for this species has not been assessed. The morphology of infected erythro-cytes is altered, demonstrating a marked depression at the site of Mycoplasma haemocanis attachment. This species has not been grown on any artificial medium; therefore, nota-ble biochemical parameters are not known.

Pathogenic; causes hemolytic anemia in domestic dogs. Transmission is vector-borne and mediated by the brown dog tick (Rhipicephalus sanguineus).

Source: observed in association with erythrocytes of domestic dogs (Hoskins, 1991).

DNA G+C content (mol%): not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): AF197337.

60. Mycoplasma haemofelis (Clark 1942) Neimark, Johansson, Rikihisa and Tully 2002b, 683VP [Eperythrozoon felis Clark 1942, 16; Haemobartonella felis (Clark 1942) Flint and McKelvie 1956, 240 and Kreier and Ristic 1984, 725]

ha.e.mo.fe¢lis. Gr. neut. n. haema blood; L. fem. gen. n. felis of the cat; N.L. gen. n. haemofelis of cat blood.

Cells are coccoid. Motility for this species has not been assessed. This species has not been grown on artificial medium; therefore, notable biochemical parameters are not known.

Pathogenic; causes hemolytic anemia in cats. The mode of transmission is percutaneous or oral; an insect vector has not been identified, although fleas have been implicated (Woods et al., 2005).

Tetracyclines and fluoroquinolones are effective thera-peutic agents (Dowers et al., 2002; Tasker et al., 2006).

Source: observed in association with erythrocytes of domestic cats.

DNA G+C content (mol%): 38.5–38.8 (genome sequence survey of strain OH; Berent and Messick, 2003; J.B. Messick et al., unpublished).

Type strain: not establishedSequence accession no. (16S rRNA gene): U88563.

61. Mycoplasma haemomuris (Mayer 1921) Neimark, Johans-son, Rikihisa and Tully 2002b, 683VP (Bartonella muris Mayer 1921, 151; Bartonella muris ratti Regendanz and Kikuth 1928, 1578; Haemobartonella muris Tyzzer and Weinman 1939, 143)

ha.e.mo.mu¢ris. Gr. neut. n. haema blood; L. masc. gen. n. muris of the mouse; N.L. gen. n. haemomuris of mouse blood.

Cells are coccoid and some display dense inclusion par-ticles. Motility for this species has not been assessed. The morphology of infected erythrocytes is altered, demonstrat-ing a marked depression at the site of Mycoplasma haemo-muris attachment. This species has not been grown on any artificial medium; therefore, notable biochemical para-meters are not known.

599


Opportunistic pathogen; causes anemia in splenectomized or otherwise immunosuppressed mice. Transmission is vector-borne and mediated by the rat louse (Polypax spinulosa).

Source: observed in association with erythrocytes of wild and captive mice, and hamsters.

DNA G+C content (mol%): not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): U82963.

62. Mycoplasma hominis (Freundt 1953) Edward 1955, 90AL (Micromyces hominis Freundt 1953, 471)

ho¢mi.nis. L. n. homo, -inis man; L. gen. n. hominis of man.

Cells are coccoid to filamentous. Motility for this spe-cies has not been assessed. Colonies on solid media have a typical fried-egg appearance. Grows well in SP-4 medium supplemented with arginine at 37°C.

Pathogenic; causes pyelonephritis, pelvic inflamma-tory disease, chorioamnionitis, and postpartum fevers in women; congenital pneumonia, meningitis, and abscesses in newborns; and rarely extragenital pathologies including bacteremia, arthritis, osteomyelitis, abscesses and wound infections, mediastinitis, pneumonia, peritonitis, pros-thetic- and catheter-associated infections, and infection of hematomas. Extragenital manifestations of Mycoplasma hominis infection are more commonly seen in immunosup-pressed individuals, but can be seen in immunocompetent patients as well. Synergism between Mycoplasma hominis and Trichomonas vaginalis infections has been reported and a recent report documents the intraprotozooal location and transmission of Mycoplasma hominis with Trichomonas vagi-nalis (Dessi et al., 2006; Germain et al., 1994; Vancini and Benchimol, 2008). Mode of transmission is via sexual con-tact, congenitally, or by artificial introduction on foreign objects (e.g., catheters) or transplanted tissues.

Macrolides and fluoroquinolones are effective chemo-therapeutic agents. Combination therapy with metronida-zole is required for complex infections involving Trichomonas vaginalis. Many commercial diagnostics are available for routine clinical use.

Source: isolated from the urogenital tract, amniotic fluid, placenta, umbilical cord blood, urine, semen, bloodstream, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage fluid, peritoneal aspirates, conjunctivae, bone abscesses, and hematoma aspirates of humans; and from several species of nonhuman primates (Somerson and Cole, 1979; Taylor-Robinson and McCormack, 1979; Waites and Talkington, 2005).

DNA G+C content (mol%): 33.7 (Tm)Type strain: PG21, ATCC 23114, NCTC 10111, CIP 103715,

NBRC 14850.Sequence accession no. (16S rRNA gene): M24473.

63. Mycoplasma hyopharyngis Erickson, Ross, Rose, Tully and Bové 1986, 58VP

hy.o.pha.ryn¢gis. Gr. n. hys, hyos a swine; N.L. n. pharynx -yngis (from Gr. n. pharugx, pharuggos throat) throat; N.L. gen. n. hyopharyngis of a hog’s throat.

Cells are pleomorphic. Motility for this species has not been assessed. Colonies on solid medium have a typical fried-egg appearance. Grows in medium D-TS or Hayflick

medium supplemented with arginine at 37°C and produces a “film and spots” reaction.


Source: isolated from the nasopharynx of pigs (Erickson et al., 1986).

DNA G+C content (mol%): 24 (Bd, Tm).Type strain: H3-6B F, ATCC 51909, NCTC 11705.Sequence accession no. (16S rRNA gene): U58997.

64. Mycoplasma hyopneumoniae (Goodwin, Pomeroy and Whittlestone 1965) Maré and Switzer 1965, 841AL (Myco-plasma suipneumoniae Goodwin, Pomeroy and Whittlestone 1965, 1249)

hy.o.pneu.mo¢ni.ae. Gr. n. hys, hyos a swine; Gr. n. pneumonia pneumonia; N.L. gen. n. hyopneumoniae of swine pneumonia.

Cells are coccoid to coccobacillary. Nonmotile. Colo-nies on solid medium are very small, lack a defined cen-tral region, and are usually convex with a granular surface. Grows very slowly in modified Friis medium, medium A26, and modified SP-4 medium supplemented with glucose at 37°C. Produces a “film and spots” reaction.

Pathogenic; causes a very characteristic chronic pneu-monitis associated with ciliostasis and marked sloughing of the epithelial lining in pigs. This collection of lesions in conjunction with Mycoplasma hyopneumoniae is referred to as enzootic pneumonia of pigs (EPP), and is associated with high morbidity and poor feed conversion (with propor-tional economic loss). The mechanism of transmission is via droplet aerosol.

Tetracyclines and tylosin do not typically eradicate Mycoplasma hyopneumoniae from infected animals, but are effective in limiting sequelae. Maintenance of pigs on anti-biotics in conjunction with management practices involv-ing adequate nutrition, air quality, and stress reduction are commonly employed to control the effects of disease in endemic herds. Many molecular diagnostics have been described (Dubosson et al., 2004). Serological diagnostic techniques utilizing monoclonal antibodies have proven successful at distinguishing Mycoplasma hyopneumoniae from Mycoplasma flocculare, though the two share surface antigens (Armstrong et al., 1987). Numerous experimental vaccines have been described and several commercial vaccines are available. The latter appear to reduce or eliminate clinical signs rather than prevent infection (Browning et al., 2005).

Source: isolated from the lungs, nasopharynx, tonsils, tra-chea, and bronchiolar lavage fluid of pigs (Marois et al., 2007; Whittlestone, 1979).

DNA G+C content (mol%): 27.5 (Bd), 28 (strain JT and strain 7448 complete genome sequence), 28.6 (strain 232 complete genome sequence).

Type strain: J, ATCC 25934, NCTC 10110.Sequence accession nos: AY737012 (16S rRNA gene),

AE017243 (strain JT complete genome sequence), AE017244 (strain 7448 complete genome sequence), AE017332 (strain 232 complete genome sequence).

65. Mycoplasma hyorhinis Switzer 1955, 544AL

hy.o.rhi¢nis. Gr. n. hys, hyos a swine; Gr. n. rhis, rhinos nose; N.L. gen. n. hyorhinis of a hog’s nose.

600

Genus I. MycoplasMa

Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid media display typi-cal fried-egg morphology. Grows well on S-4 supplemented with glucose at 37°C.

Mycoplasma hyorhinis is associated with contamination of eukaryotic cell culture and can be removed by treat-ment of cells with antibiotics and/or maintenance of cell lines in antibiotic-containing medium. The noted effects of Mycoplasma hyorhinis on cell-cycle regulation make the detection and elimination of this organism particularly per-tinent (Goodison et al., 2007; Schmidhauser et al., 1990). The most effective classes of antibiotics for cell culture eradication are tetracyclines and fluoroquinolones (Borup- Christensen et al., 1988; Schmitt et al., 1988).

Pathogenic; associated with arthritis, polyserositis, and otitis media in pigs. Mycoplasma hyorhinis is also regarded as a commensal of the nasopharynx that can occasionally cause pneumonia, often in association with other patho-gens (most notably Mycoplasma hyopneumoniae and Bordetella bronchiseptica). Mode of transmission is via droplet aerosol.

Source: isolated from the nasopharynx, lungs, ear canal, synovial fluid, serous cavity, and pericardium of pigs (Friis and Szancer, 1994; Ross, 1992; Whittlestone, 1979).

DNA G+C content (mol%): 27.8 (Tm).Type strain: BTS-7, ATCC 17981, NCTC 10130, CIP

104968, NBRC 14858.Sequence accession no. (16S rRNA gene): M24658.

66. Mycoplasma hyosynoviae Ross and Karmon 1970, 710AL

hy.o.sy.no.vi¢ae. Gr. n. hys, hyos a swine; N.L. n. synovia fluid in the joints; N.L. gen. n. hyosynoviae of joint fluid of swine.

Cells are coccoid to filamentous. Motility of this species has not been assessed. Colonies display a typical fried-egg appearance at 37°C. Grows well in SP-4 medium supple-mented with glucose at 37°C. A granular deposit and a waxy surface pellicle are produced during growth in broth.

Pathogenic; causes infectious synovitis, arthritis, and rarely pericarditis in pigs. Transmission occurs from sows to piglets or between adults via aerosol.

Lincosamides, fluoroquinolones, and macrolides have been used effectively for treatment in conjunction with improved disinfection and quarantine during husbandry.

Source: isolated from the synovial fluid, nasopharynx, ton-sils, lymph nodes, and pericardium of pigs (Whittlestone, 1979).

DNA G+C content (mol%): 28.0 (Bd).Type strain: S16, ATCC 25591, NCTC 10167.Sequence accession no. (16S rRNA gene): U26730.

67. Mycoplasma iguanae Brown, Demcovitz, Plourdé, Potter, Hunt, Jones and Rotstein 2006, 763VP

i.gua¢nae. N.L. gen. n. iguanae of the iguana lizard.

Cells are predominantly coccoid. Nonmotile. Colonies on solid medium exhibit variable (convex to umbonate) forms; mature colonies display sectored centers. Grows well in SP-4 medium supplemented with glucose between 25 and 42°C.

Associated with pathologic lesions, but unable to repro-duce disease following experimental inoculation (Brown et al., 2007). Mechanism of transmission has not been estab-lished.

Source: isolated from vertebral abscesses of green iguanas.

DNA G+C content (mol%): not determined.Type strain: 2327, ATCC BAA-1050, NCTC 11745.Sequence accession no. (partial 16S rRNA gene sequence):

AY714305.

68. Mycoplasma imitans Bradbury, Abdul-Wahab, Yavari, Dupiellet and Bové 1993, 726VP

i¢mi.tans. L. part. adj. imitans imitating, mimicking, refer-ring to the organism’s phenotypic resemblance to Myco-plasma gallisepticum.

Cells are oval and flask-shaped with a short, wide attach-ment organelle. Cells are motile and exhibit gliding motil-ity in the direction of the attachment organelle (Hatchel and Balish, 2008). Colonies have typical fried-egg morphol-ogy on solid medium. Grows well in SP-4 medium supple-mented with glucose at 37°C.

Pathogenic; causes sinusitis in ducks, geese, and par-tridges. Disease has been reproduced experimentally. Mode of transmission has not been assessed definitively.

Diagnosis of infection is potentially complicated by numerous factors. Serological cross-reactions occur with Mycoplasma gallisepticum due to known epitopes including PvpA, the VlhA hemagglutinins, pyruvate dehydrogenase, lactate dehydrogenase, and elongation factor Tu (Jan et al., 2001; Lavric et al., 2005; Markham et al., 1999; Rosengarten et al., 1995). In addition, the 16S rRNA genes share 99.9% identity, despite whole genome hybridization showing a relatedness of only 40–46%, potentially complicating molec-ular diagnostics based on the 16S rRNA gene. Two molecu-lar methods for distinguishing these two species have been described (Harasawa et al., 2004; Marois et al., 2001).

Source: isolated from the nasal turbinates and sinuses of ducks, geese, and partridges (Dupiellet, 1984; Ganapathy and Bradbury, 1998; Kleven, 2003).

DNA G+C content (mol%): 31.9 (Bd).Type strain: 4229, NCTC 11733, ATCC 51306.Sequence accession no. (16S rRNA gene): L24103.

69. Mycoplasma indiense Hill 1993, 39VP

in.di.en¢se. N.L. neut. adj. indiense pertaining to India (source of the infected primates).

Cells are pleomorphic. Nonmotile. Colonies on agar have a characteristic fried-egg appearance. Grows well in SP-4 medium supplemented with arginine at 37°C.


Source: isolated from the throats of a rhesus monkey and a baboon (Hill, 1993).

DNA G+C content (mol%): 32 (Bd).Type strain: 3T, NCTC 11728, ATCC 51125.Sequence accession no. (16S rRNA gene): AF125593.

70. Mycoplasma iners Edward and Kanarek 1960, 699AL

i¢ners. L. neut. adj. iners inactive, inert.

The cell morphology is poorly defined. Motility for this species has not been assessed. Colonies on solid medium are relatively small and of a typical fried-egg appearance. Grows in Frey’s medium supplemented with arginine at 37°C.

601



Source: isolated from the respiratory tract of chickens, tur-keys, geese pigeons, pheasants, and partridges; and from tissues of swine (Bencina et al., 1987; Bradbury et al., 2001; Taylor-Robinson and Dinter, 1968).

DNA G+C content (mol%): 29.1 (Tm), 29.6 (Bd).Type strain: PG30, ATCC 19705, NCTC 10165.Sequence accession no. (16S rRNA gene): AF221114.Further comment: previously known as avian serotype E

(Kleckner, 1960).

71. Mycoplasma iowae Jordan, Ernø, Cottew, Hinz and Stipko-vits 1982, 114VP

i.o¢wa.e. N.L. gen. n. iowae of Iowa.

Cells are pleomorphic and some display a terminal protrusion with possible attachment properties (Gallagher and Rhoades, 1983; Mirsalimi et al., 1989). Motile. Colonies on agar show typical fried-egg appearance. Grows well in SP-4 medium supplemented with either glucose or arginine at 41–43°C (Grau et al., 1991; Yoder and Hofstad, 1964).

Pathogenic; causes airsacculitis and embryo lethality resulting in reduced hatchability in turkeys. Transmission occurs vertically and by direct contact.

Tetracyclines, macrolides, and fluoroquinolones are effective chemotherapeutic agents; however, medicating a commercial flock is not considered an effective control strategy. Management tactics are more commonly uti-lized. Molecular diagnostic methods have been described (Ramírez et al., 2008; Raviv and Kleven, 2009). Mycoplasma iowae strains show considerable intra-species antigenic heterogeneity and a cross-reactive epitope with both Myco-plasma gallisepticum and Mycoplasma imitans potentially com-plicating serology-based diagnosis of infection (Al-Ankari and Bradbury, 1996; Dierks et al., 1967; Rosengarten et al., 1995).

Source: isolated from the air sacs, intestinal tract, and eggs of turkeys, and from the seed of an apple tree with apple proliferation disease (Bradbury and Kleven, 2008; Grau et al., 1991; Mirsalimi et al., 1989).

DNA G+C content (mol%): 25 (Bd).Type strain: 695, ATCC 33552, NCTC 10185.Sequence accession no. (16S rRNA gene): M24293.Further comment: previously known as avian serotype I

(Yoder and Hofstad, 1964).

72. Mycoplasma lagogenitalium Kobayashi, Runge, Schmidt, Kubo, Yamamoto and Kirchhoff 1997, 1211VP

la.go.ge.ni.ta¢li.um. Gr. masc. n. lagos hare; L. neut. pl. gen. n. genitalium of genitals; N.L. gen. pl. n. lagogenitalium of hare’s genitals.

Cells are primarily coccoid. Nonmotile. Colonies on agar have a characteristic fried-egg appearance. Grows well in SP-4 medium supplemented with glucose at 37°C.


Source: isolated from the preputial smegma of Afghan pikas (Ochotona rufescens; Kobayashi et al., 1997).

DNA G+C content (mol%): 23 (Tm).

Type strain: 12MS, ATCC 700289, CIP 105489, DSM 22062.

Sequence accession no. (16S rRNA gene): AF412983.

73. Mycoplasma leachii Manso-Silván, Vilei, Sachse, Djordjevic, Thiaucourt and Frey 2009, 1356VP

le.a.chi¢i. N.L. masc. gen. n. leachii of Leach, named in honor of Dr R.H. Leach, who first characterized this taxon.

Cells are pleomorphic. Nonmotile. Colonies on solid agar have a characteristic fried-egg appearance. Grows in modi-fied Hayflick medium supplemented with glucose at 37°C.

Pathogenic; causes polyarthritis, mastitis, abortion, and pneumonia in cattle. Mode of transmission has not been established definitively.

Tetracyclines appear to control infection during acute outbreaks (Hum et al., 2000). Mycoplasma leachii shares surface antigens with Mycoplasma mycoides subsp. mycoides, Mycoplasma capricolum subsp. capripneumoniae, and Myco-plasma capricolum subsp. capricolum, potentially compound-ing serology-based diagnosis of infection.

Source: isolated from the synovial fluid, udders, expelled milk, lungs, lymph nodes, pericardium, cervix, vagina, pre-puce, semen, and aborted calves of cattle (Alexander et al., 1985; Gourlay and Howard, 1979; Hum et al., 2000).

DNA G+C content (mol%): not determined.Type strain: PG50, NCTC 10133, DSM 21131.Sequence accession no. (16S rRNA gene): AF261730.Further comment: the assignment of strains formerly called

“Mycoplasma species bovine group 7 of Leach” to the species Mycoplasma leachii came in response to a request from the Sub-committee on the Taxonomy of Mollicutes of the International Committee on Systematics of Prokaryotes for a proposal for an emended taxonomy for the members of the Mycoplasma mycoides phylogenetic cluster (Manso-Silván et al., 2009).

74. Mycoplasma leonicaptivi corrig. Hill 1992, 521VP

le.o.ni.cap¢ti.vi. L. n. leo, -onis the lion; L. adj. captivus cap-tive; N.L. gen. n. leonicaptivi of the captive lion.

Cells are pleomorphic (primarily coccoid). Nonmotile. Colonies on solid medium have a typical fried-egg appear-ance. Growth in SP-4 broth supplemented with glucose occurs between 35 and 37°C.


Source: isolated from the throat and respiratory tract of captive lions and leopards (Hill, 1992).

DNA G+C content (mol%): 27 (Bd).Type strain: 3L2, NCTC 11726, ATCC 49890.Sequence accession no. (16S rRNA gene): U16759.Further comment: the original spelling of the specific epi-

thet, leocaptivus (sic), has been corrected by Trüper and De’Clari (1998).

75. Mycoplasma leopharyngis Hill 1992, 521VP

le.o.pha.ryn¢gis. L. masc. n. leo, -onis lion; N.L. n. pharynx, -yngis (from Gr. n. pharugx, pharuggos throat) throat; N.L. gen. n. leopharyngis (sic) of the throat of a lion.

Cells are coccoid to pleomorphic. Nonmotile. Colonies on solid medium have a typical fried-egg appearance under

602

Genus I. MycoplasMa

anaerobic conditions. Grows in SP-4 broth supplemented with glucose at optimum temperatures of 35–37°C. Grows well under both aerobic and anaerobic conditions.


Source: isolated from the throat of lions (Hill, 1992).DNA G+C content (mol%): 28 (Bd).Type strain: LL2, NCTC 11725, ATCC 49889.Sequence accession no. (16S rRNA gene): U16760.

77. Mycoplasma lipofaciens Bradbury, Forrest and Williams 1983, 334VP

li.po.fa¢ci.ens. Gr. n. lipos animal fat, lard, tallow; L. v. facio to make; N.L. part. adj. lipofaciens fat-making, intended to refer to the production of a lipid film on solid media.

Cells are mainly spherical and elliptical. Nonmotile. Colo-nies on solid medium have typical fried-egg appearance. Grows in Hayflick medium supplemented with glucose or arginine at 37°C. Produces a strong “film and spots” reaction.

Commensal of birds; little evidence exists for naturally occurring pathogenicity of the isolates, although experi-mental inoculation of chicken or turkey eggs can result in embryo mortality. Inadvertent transmission to an investi-gator during experimental inoculation studies resulted in clinical signs including rhinitis and pharyngitis. Aerosol transmission has been documented in turkeys.

Source: isolated from the infraorbital sinuses of chickens; from tissues or eggs of turkeys and ducks; and from eggs of Northern goshawks (Bencina et al., 1987; Bradbury et al., 1983; Lierz et al., 2007a, b, c, 2008b).

DNA G+C content (mol%): 24.5 (Bd).Type strain: R171, ATCC 35015, NCTC 10191.Sequence accession no. (16S rRNA gene): AF221115.

78. Mycoplasma lipophilum Del Giudice, Purcell, Carski and Chanock 1974, 152AL

li.po.phi¢lum. Gr. n. lipos animal fat; N.L. neut. adj. philum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. lipophilum fat-loving.

Cells are pleomorphic and granular. Motility for this spe-cies has not been assessed. Colonies display typical fried-egg morphology. Growth on solid medium is associated with heavy production of film that spreads over the surface of the agar, with the development of numerous internal par-ticles in the colonies. A film similar to that produced on agar medium develops on the surface of broth-grown cul-tures (Del Giudice et al., 1974). Grows in SP-4 or Hayflick medium supplemented with arginine at 37°C.

Pathogenicity for this species is unclear. This species was first isolated from a human patient with primary atypical pneumonia; however, subsequent isolations from similarly symptomatic patients have not been achieved. Mode of transmission has not been formally assessed.

Source: isolated from the upper and lower respiratory tract of a human with primary atypical pneumonia and the lower respiratory tract of rhesus monkeys (Hill, 1977).

DNA G+C content (mol%): 29.7 (Bd).Type strain: MaBy, ATCC 27104, NCTC 10173, NBRC


79. Mycoplasma maculosum Edward 1955, 90AL

ma.cu.lo¢sum. L. neut. adj. maculosum spotted, alluding to a crinkled film covering the colonies and spreading between them, and spots appearing in the medium beneath and around the colonies.

Cells are short and filamentous, with occasional branch-ing. Motility for this species has not been assessed. Colo-nies on solid medium have a typical fried-egg appearance. Grows well in Hayflick or SP-4 medium supplemented with arginine at 37°C.

Opportunistic pathogen. Cause of pneumonia in domes-tic dogs and rarely of meningitis in immunocompromised humans. The route of transmission is via droplet aerosol.

Source: isolated from the nasopharynx, lungs, conjuncti-vae, and urogenital tract of domestic dogs and the cerebro-spinal fluid of an immunocompromised human (Chalker, 2005).

DNA G+C content (mol%): 26.7 (Tm), 29.6 (Bd).Type strain: PG15, ATCC 19327, NCTC 10168, NBRC


80. Mycoplasma meleagridis Yamamoto, Bigland and Ortmayer 1965, 47AL

me.le.a¢gri.dis. L. n. meleagris, -idis a turkey; L. gen. n. melea-gridis of a turkey.

Cells are coccoid to coccobacillary. Motility for this spe-cies has not been assessed. An extracellular capsule can be visualized by electron microscopy following staining with ruthenium red (Green III and Hanson, 1973). Colonies on solid medium are not necessarily of typical fried-egg appear-ance. Grows in Frey’s medium supplemented with arginine at 37–38°C (Yamamoto et al., 1965).

Pathogenic; causes airsacculitis, pneumonia, sinusitis, perosis, chondrodystrophy, bursitis, synovitis, and reduced hatchability due to embryo lethality in turkeys. Transmis-sion is primarily vertical, but can also occur through droplet aerosol or sexual contact.

Tetracyclines, macrolides, and fluoroquinolones are effective chemotherapeutic agents; however, medicating a commercial flock is not considered an effective control strategy. The temporary use of in ovo antimicrobial therapy can be used to eradicate Mycoplasma meleagridis from a flock. Management tactics (e.g., single age “all in/all out” systems and culling of endemic flocks) are more commonly utilized (Kleven, 2008). Serological and molecular diagnostic meth-ods have been described (Ben Abdelmoumen Mardassi et al., 2007; Ramírez et al., 2008; Raviv and Kleven, 2009).

Source: isolated from the air sacs, trachea, infraorbital sinuses, oviduct, cloaca, phallus, and eggs of turkeys, and the air sacs of buzzards, kites, and kestrels (Chin et al., 2008; Jordan, 1979; Lam et al., 2004; Lierz et al., 2000).

DNA G+C content (mol%): 27.0 (Tm), 28.6 (Bd).Type strain: 17529, ATCC 25294, NCTC 10153.Sequence accession no. (16S rRNA gene): L24106.Further comment: previously known as avian serotype H

(Kleckner, 1960).

81. Mycoplasma microti (Dillehay, Sander, Talkington, Thacker and Brown 1995) Brown, Talkington, Thacker, Brown,

603


Dillehay and Tully 2001b, 412VP (Mycoplasma volis Dillehay, Sander, Talkington, Thacker and Brown 1995, 633)

mi.cro¢ti. N.L. n. Microtus a genus of field vole; N.L. gen. n. microti of Microtus.

Cells are predominantly coccoid in shape. Nonmo-tile. Colonies on solid medium exhibit a typical fried-egg appearance. Grows well in SP-4 supplemented with glucose in temperatures ranging from 35 to 37°C.

Opportunistic pathogen. No evidence exists for patho-genicity in the natural host; however, pneumonitis was experimentally induced in mice and rats (Evans-Davis et al., 1998).

Source: isolated from the nasopharynx and lung of prairie voles (Dillehay et al., 1995).

DNA G+C content (mol%): not determined.Type strain: IL371, ATCC 700935.Sequence accession no. (16S rRNA gene): AF212859.

82. Mycoplasma moatsii Madden, Moats, London, Matthew and Sever 1974, 464AL

mo.at¢si.i. N.L. gen. masc. n. moatsii of Moats, named after Kenneth E. Moats, whose primary interest has been in the mycoplasmas of nonhuman primates.

Cells are spheroidal and some exhibit protrusions from the membrane. Motility for this species has not been assessed. Colonies exhibit typical fried-egg morphology. Grows readily in SP-4 or Hayflick broth supplemented with either arginine or glucose at an optimum temperature of 37°C.

No evidence of pathogenicity.Source: isolated from the respiratory and reproductive

tracts of grivet monkeys and from the cecum, jejunum, and colon of wild Norway rats (Giebel et al., 1990).

DNA G+C content (mol%): 25.7 (Bd).Type strain: MK 405, ATCC 27625, NCTC 10158.Sequence accession no. (16S rRNA gene): AF412984.

83. Mycoplasma mobile Kirchhoff, Beyene, Fischer, Flossdorf, Heitmann, Khattab, Lopatta, Rosengarten, Seidel and Yousef 1987, 197VP

mo¢bi.le. L. neut. adj. mobile motile.

Cells are conical or flask-shaped and have a distinct ter-minal protrusion referred to as the “head-like structure”. Cells demonstrate rapid gliding motility when adhering to charged surfaces and move in the directional of the head-like structure (Miyata et al., 2002, 2000). Colonies on solid medium have a typically fried-egg appearance. Grows well in Aluotto’s medium supplemented with glucose or argi-nine. The temperature range for growth is 17–30°C, with optimum growth at 30°C.

Pathogenic; causes necrotic erythrodermatitis in tench. The mode of transmission has not been established.

Source: isolated from the gills of a freshwater fish (Tinca tinca) with “red disease” (Kirchhoff et al., 1987).

DNA G+C content (mol%): 23.5 (Bd), 24.9.Type strain: 163K, ATCC 43663, NCTC 11711.Sequence accession nos: M24480 (16S rRNA gene),

NC_006908 (complete genome sequence of strain 163K).

84. Mycoplasma molare Røsendal 1974, 130AL

mo.la¢re. L. neut. adj. molare of or belonging to a mill, here millstone-like, referring to the heavy film reaction, which resembles the pattern on the surface of a millstone.

Cells are coccoid to pleomorphic. Nonmotile. Colonies have a typical fried-egg appearance. Grows well in SP-4 medium supplemented with glucose at 37°C. A lipid film of characteristic appearance develops on the surface and along the circumference of colonies grown on egg-yolk agar.

Opportunistic pathogen. Associated with pharyngitis and mild inflammatory lesions of the lower respiratory tract and may be associated with infertility, vaginitis, and posthitis of domestic dogs. No clear evidence for primary pathogenicity of the species. Mode of transmission has not been estab-lished definitively.

Source: isolated from the oral cavity, pharynx, cervix, vagina, and prepuce of domestic dogs (Røsendal, 1979; (Chalker, 2005).

DNA G+C content (mol%): 26.0 (Bd).Type strain: H 542, ATCC 27746, NCTC 10144.Sequence accession no. (16S rRNA gene): AF412985.

85. “Mycoplasma mucosicanis” Spergser, Langer, Muck, Macher, Szostak, Rosengarten and Busse 2010

mu.co.si.ca¢nis. N.L. n. mucosa mucous membrane; L. n. canis a dog; N.L. gen. n. mucosicanis of mucous membranes of a dog.

Cells are pleomorphic, but primarily coccoid. Nonmo-tile. Colonies on solid media have a typical fried-egg mor-phology. Grows wells in modified Hayflick medium at 37°C and produces a “film and spots” reaction.


Source: isolated from the prepuce, semen, vagina, cervix, and oral cavity of domestic dogs (Spergser et al., 2010).

DNA G+C content (mol%): not determined.Type strain: 1642, ATCC BAA-1895, DSM 22457.Sequence accession no. (16S rRNA gene): AM774638.

86. Mycoplasma muris McGarrity, Rose, Kwiatkowski, Dion, Phillips and Tully 1983, 355VP

mu¢ris. L. n. mus, muris mouse; L. gen. n. muris of a mouse.

Cells are primarily coccoid or coccobacillary, but exhibit a few other pleomorphic forms. Motility for this species has not been assessed. Colonies usually have a granular appear-ance and few colonies demonstrate the typical fried-egg appearance. Grow well in SP-4 broth supplemented with arginine at 37°C and produces a “film and spots” reaction.

No evidence of pathogenicity.Source: isolated from the vagina of a pregnant mouse (lab-

oratory strain RIII; McGarrity et al., 1983).DNA G+C content (mol%): 24.9 (TLC).Type strain: RIII-4, ATCC 33757, NCTC 10196.Sequence accession no. (16S rRNA gene): M23939.

87. Mycoplasma mustelae Salih, Friis, Arseculeratne, Freundt and Christiansen 1983, 478VP

mu.ste¢lae. N.L. n. Mustela (from L. n. mustela a weasel) the generic name of the mink Mustela vison; N.L. gen. n. muste-lae of Mustela.

604

Genus I. MycoplasMa

Cells are highly pleomorphic; most common morpholo-gies include pleomorphic rings, short filamentous forms, and coccoid elements. Nonmotile. Colonies on solid medium show a typical fried-egg appearance. Growth in Hayflick medium supplemented with glucose occurs at 37°C.

No evidence of pathogenicity.Source: isolated from the trachea and lungs of juvenile

minks (Mustela vison; Salih et al., 1983).DNA G+C content (mol%): 28.2 (Bd).Type strain: MX9, ATCC 35214, NCTC 10193, AMRC-C


88. Mycoplasma neurolyticum (Sabin 1941) Freundt 1955, 73AL (Musculomyces neurolyticus Sabin 1941, 57)

neu.ro.ly¢ti.cum. Gr. n. neuron nerve; N.L. adj. lyticus -a -um (from Gr. adj. lutikos -ê -on) able to loosen, able to dissolve; N.L. neut. adj. neurolyticum nerve-destroying.

Cells are filamentous and highly variable length. Nonmo-tile (Nelson and Lyons, 1965). Colonies show a typical fried-egg appearance after incubation at 37°C. Grows in Hayflick medium supplemented with glucose at 37°C (Naot et al., 1977).

Pathogenicity is currently uncertain. Potentially associ-ated with spongiform encephalopathy and ischemic necro-sis of the brain resulting in a clinical state referred to as “rolling disease” in mice and rats. Pathology may be exac-erbated in the presence of additional neurotropic organ-isms (i.e., Toxoplasma gondii, Chlamydia spp., Plasmodium spp., and yellow fever virus) or during leukemic syndromes. Transmission to suckling rodents occurs shortly after birth. Treatment of Mycoplasma neurolyticum infections is uncom-mon, as pathology is typically not resolvable after the onset of clinical signs.

Source: isolated from the brain, conjunctivae, nasophar-ynx, and middle ear of captive mice and rats.

DNA G+C content (mol%): 22.8 (Bd), 26.2 (Tm).Type strain: Type A, ATCC 19988, NCTC 10166, CIP

103926, NBRC 14799.Sequence accession no. (16S rRNA gene): M23944.Further comment: a putative exotoxin with neurological

effects on rodents was formerly thought to be produced by most freshly isolated strains, although a few non-toxic strains were described (Tully and Ruchman, 1964). The findings were not substantiated by later work (Tryon and Baseman, 1992).

89. Mycoplasma opalescens Røsendal 1975, 469AL

o.pa.les¢cens. L. n. opalus precious stone; N.L. neut. adj. opalescens opalescent, referring to the opalescent film pro-duced on solid medium.

Morphology by light microscopy or ultrastructural exam-ination is not defined. Motility for this species has not been assessed. Colonies on solid medium have a typical fried-egg appearance and possess an iridescent quality. Grows well in SP-4 medium supplemented with arginine at 37°C.

No evidence of pathogenicity.Source: isolated from the oral cavity, prepuce, and pros-

tate gland of domestic dogs (Røsendal, 1975).

DNA G+C content (mol%): 29.2 (Bd).Type strain: MH5408, ATCC 27921, NCTC 10149.Sequence accession no. (16S rRNA gene): AF538961.

90. Mycoplasma orale Taylor-Robinson, Canchola, Fox and Chanock 1964, 141AL

o.ra¢le. L. n. os, oris the mouth; L. neut. suff. -ale suffix denoting pertaining to; N.L. neut. adj. orale pertaining to the mouth.

Cells can be either coccoid or filamentous. Motility for this species has not been assessed. Colonies on solid medium have a typical fried-egg appearance. Grows well in Hayflick or SP-4 medium supplemented with arginine at 37°C.

Mycoplasma orale is most commonly associated with con-tamination of eukaryotic cell culture and is frequently removed by treatment of cells with antibiotics and/or main-tenance of cell lines in antibiotic-containing medium. The most effective classes of antibiotics for cell culture eradica-tion are tetracyclines, macrolides, and fluoroquinolones. Additionally, passage of eukaryotic cells in hyperimmune serum raised against Mycoplasma orale has been shown to be an effective method of eradication (Vogelzang and Com-peer-Dekker, 1969).

Commensal/opportunistic pathogen. Commonly found as a commensal of the human oral cavity; can cause respira-tory tract infections, osteomyelitis, infectious synovitis, and abscesses in immunocompromised individuals (Paessler et al., 2002; Roifman et al., 1986).

Source: isolated from the oral cavity of subclinical humans, the sputum of an immunocompromised human with acute respiratory illness, and from synovial fluid, bone, and splenic abscesses of another immunocompromised individual.

DNA G+C content (mol%): 24.0–28.2 (Tm, Bd).Type strain: CH19299, ATCC 23714, NCTC 10112, CIP

104969, NBRC 14477.Sequence accession no. (16S rRNA gene): M24659.

91. Mycoplasma ovipneumoniae Carmichael, St George, Sullivan and Horsfall 1972, 677AL

o.vi.pneu.mo.ni′ae. L. fem. n. ovis a sheep; Gr. n. pneumonia pneumonia; N.L. gen. n. ovipneumoniae of sheep pneumo-nia.

Morphology and motility are poorly described. The organism produces a polysaccharide capsule with variable thickness that is dependent upon culture conditions and strain (Niang et al., 1998). Colonies grown on standard agar are convex and have a lacy or vacuolated appearance. Grows well in Friis medium or SP-4 broth supplemented with glucose at 37°C.

Pathogenic; causes chronic proliferative interstitial pneu-monia, pulmonary adenomatosis, conjunctivitis (Jones et al., 1976), and mastitis under experimental conditions (Jones, 1985) of sheep and goats. Transmission occurs via droplet aerosol and can occur via intravenous inoculation in experimental infection studies.

Source: isolated from the lungs, trachea, nose, and con-junctivae of sheep and goats.

DNA G+C content (mol%): 25.7 (Bd).Type strain: Y98, NCTC 10151, ATCC 29419.Sequence accession no. (16S rRNA gene): U44771.

605


92. Mycoplasma ovis (Neitz, Alexander and du Toit 1934) Neimark, Hoff and Ganter 2004, 369VP (Eperythrozoon ovis Neitz, Alexander and du Toit 1934, 267)

o¢vis. L. fem. n. ovis, -is a sheep; L. gen. n. ovis of a sheep.

Cells are coccoid and motility for this species has not been assessed. The morphology of infected erythrocytes is altered demonstrating a marked depression at the site of Mycoplasma ovis attachment. This species has not been grown on artificial medium; therefore, notable biochemical parameters are not known.

Neoarsphenamine is an effective therapeutic agent. Myco-plasma ovis is reported to share antigens with Mycoplasma wenyonii (Kreier and Ristic, 1963), potentially complicating serology-based diagnosis of infection.

Pathogenic; causes mild to severe anemia in sheep and goats that often results in poor feed conversion. Transmis-sion occurs via blood-feeding arthropods, e.g., Haemophysalis plumbeum, Rhipicephalus bursa, Aedes camptorhynchus, and Culex annulirostris (Daddow, 1980; Howard, 1975; Nikol’skii and Slipchenko, 1969), and likely via fomites such as reused needles, shearing tools, and ear-tagging equipment (Brun-Hansen et al., 1997; Mason and Statham, 1991).

Source: observed in association with erythrocytes or unat-tached in suspension in the blood of sheep, goats, and rarely in eland and splenectomized deer.


93. Mycoplasma oxoniensis Hill 1991b, 24VP

oxo.ni.en¢sis. N.L. adj. oxoniensis (sic) pertaining to Oxon, an abbreviation of Oxfordshire, where the mycoplasma was first isolated.

Cells are primarily coccoid. Nonmotile. Colonies on agar have a typical fried-egg appearance. Growth in SP-4 broth supplemented with glucose occurs at 35–37°C.

No evidence of pathogenicity. Mode of transmission is unknown.

Source: isolated from the conjunctivae of the Chinese hamster (Cricetulus griseus; Hill, 1991b).

DNA G+C content (mol%): 29 (Bd).Type strain: 128, NCTC 11712, ATCC 49694.Sequence accession no. (16S rRNA gene): AF412987.

94. Mycoplasma penetrans Lo, Hayes, Tully, Wang, Kotani, Pierce, Rose and Shih 1992, 363VP

pe.ne¢trans. L. part. adj. penetrans penetrating, referring to the ability of the organism to penetrate into mammalian cells.

Cells are flask-shaped, with a distinct terminal struc-ture reminiscent of the Mycoplasma pneumoniae attachment organelle. Cells demonstrate gliding motility when adher-ing to charged surfaces and move in the direction of the terminal structure. Colonies on agar plates display a typi-cal fried-egg appearance. Grows well in SP-4 broth supple-mented with either glucose or arginine at 37°C.

Opportunistic pathogen; found in the urogenital tract of immunocompromised humans, most notably HIV-positive individuals (serological detection in HIV-negative individu-als is rare). Speculation regarding the ability of Mycoplasma

penetrans to act as a cofactor in the progression of AIDS by modulation of the immune system remains intriguing, but in need of further substantiation (Blanchard, 1997). Trans-mission is presumed to be via sexual contact.

Source: isolated from the urine of HIV-positive humans, and from the blood, respiratory secretions, and trachea of an HIV-negative patient with multiple autoimmune syn-dromes (Yanez et al., 1999).

DNA G+C content (mol%): 30.5 (Tm), 25.7 (HF-2 genome sequence; Sasaki et al., 2002).

Type strain: GTU-54-6A1, ATCC 55252.Sequence accession nos: L10839 (16S rRNA gene),

NC_004432 (HF-2 complete genome sequence).

95. Mycoplasma phocicerebrale corrig. Giebel, Meier, Binder, Flossdorf, Poveda, Schmidt and Kirchhoff 1991, 43VP

pho.ci.ce.re.bra¢le. L. n. phoca seal; N.L. neut. adj. cerebrale of or pertaining to the brain; N.L. neut. adj. phocicerebrale pertaining to the brain of a seal.

Cells are coccoid or exhibit a dumbbell shape. Motility for this species has not been assessed. Colonies on solid medium typically show a fried-egg appearance. Grows well in SP-4 medium supplemented with arginine at 37°C.

Pathogenic; associated with respiratory disease and con-junctivitis in harbor seals (Kirchhoff et al., 1989) and a dis-tinctive ulcerative keratitis subsequent to seal bites (known as “seal finger”) and secondary arthritis in humans (Baker et al., 1998; Ståby, 2004). Mode of transmission between harbor seals has not been established definitively; transmis-sion to humans appears to be zoonotic following seal bites.

Source: isolated from the brains, noses, throats, lungs, and hearts of seals (Phoca vitulina) during an outbreak of respi-ratory disease (Kirchhoff et al., 1989), and from cutaneous lesions of humans with seal finger (Baker et al., 1998).

DNA G+C content (mol%): 25.9 (Bd).Type strain: 1049, ATCC 49640, NCTC 11721.Sequence accession no. (16S rRNA gene): AF304323.Further comment: the original spelling of the specific epi-

thet, phocacerebrale (sic), has been corrected by Königsson et al. (2001).

96. Mycoplasma phocidae Ruhnke and Madoff 1992, 213VP

pho.ci¢da.e. L. n. phoca seal; N.L. gen. n. phocidae (sic) of a seal.

Cells are primarily coccoid. Motility for this species has not been assessed. Colonies on solid medium have a typi-cal fried-egg appearance. Grows well in SP-4 or Hayflick medium supplemented with arginine at 37°C. Produces “film and spots” reaction.

Opportunistic pathogen; associated with secondary pneumonia of harbor seals subsequent to influenza infec-tion. Attempts to produce disease in gray or harp seals with Mycoplasma phocidae in pure culture were not successful (Geraci et al., 1982). Mode of transmission has not been established definitively.

Source: isolated from the lungs, tracheae, and heart of harbor seals.

DNA G+C content (mol%): 27.8 (Bd).Type strain: 105, ATCC 33657.Sequence accession no. (16S rRNA gene): AF304325.

606

Genus I. MycoplasMa

Further comment: the species designation “Mycoplasma phocae” was suggested by Königsson et al. (2001), but the original epithet “phocidae” should be retained. The sug-gested change is forbidden by Rule 61 (Note) of the Bacte-riological Code because it would change the first syllable of the original epithet without correcting any orthographic or typographical error.

97. Mycoplasma phocirhinis corrig. Giebel, Meier, Binder, Flossdorf, Poveda, Schmidt and Kirchhoff 1991, 43VP

pho.ci.rhi¢nis. L. n. phoca seal; Gr. n. rhis, rhinos nose; N.L. gen. n. phocirhinis of the nose of a seal.

Cells are coccoid; motility for this species has not been assessed. Colonies on solid medium usually have a fried-egg appearance. Grows well in Friis or Hayflick medium at 37°C. Produces “film and spots” reaction.

Pathogenic; associated with respiratory disease and con-junctivitis of harbor seals (Kirchhoff et al., 1989). Mode of transmission has not been established definitively.

Source: isolated from the nose, pharynx, trachea, lungs, and heart of seals (Phoca vitulina; Kirchhoff et al., 1989).

DNA G+C content (mol%): 26.5 (Bd).Type strain: 852, ATCC 49639, NCTC 11722.Sequence accession no. (16S rRNA gene): AF304324.Further comment: the original spelling of the specific

epithet, phocarhinis (sic), has been corrected by Königsson et al. (2001)

98. Mycoplasma pirum Del Giudice, Tully, Rose and Cole 1985, 290VP

pi¢rum. L. neut. n. pirum (nominative in apposition) pear, referring to the pear-shaped morphology of the cells.

Cells are predominantly flask or pear-shaped and possess an organized terminal structure, with an outer, finely par-ticulate nap covering the entire surface of the cell. Cells exhibit low-speed gliding motility and move in the direc-tion of the terminal organelle (Hatchel and Balish, 2008). Colonies display a typical fried-egg appearance. Grows well in SP-4 medium supplemented with glucose at 37°C.

No evidence of pathogenicity.Source: isolated from the rectum of immunocompetent

humans and whole blood and circulating lymphocytes of HIV-positive humans (Montagnier et al., 1990). Originally isolated from cultured eukaryotic cells that were of human origin (Del Giudice et al., 1985).

DNA G+C content (mol%): 25.5 (Bd).Type strain: HRC 70-159, ATCC 25960, NCTC 11702.Sequence accession no. (16S rRNA gene): M23940.

99. Mycoplasma pneumoniae Somerson, Taylor-Robinson and Chanock 1963, 122AL

pneu.mo.ni¢ae. Gr. n. pneumonia pneumonia; N.L. gen. n. pneumoniae of pneumonia.

Cells are highly pleomorphic; the predominant shape includes a long, thin terminal structure at one cell pole, with or without a trailing filament at the opposite pole. Cells are motile and glide in the direction of the terminal organelle when attached to cell surfaces, plastic, or glass. Colonies on solid medium usually lack the light peripheral zone, appearing rather as circular dome-shaped, granular

structures. Growth is best achieved in SP-4 medium supple-mented with glucose at 37°C.

Pathogenic; causes interstitial pneumonitis, tracheobron-chitis, desquamative bronchitis, and pharyngitis [collectively referred to as primary atypical pneumonia (PAP); Krause and Taylor-Robinson (1992)]. Less commonly, Mycoplasma pneumoniae causes meningoencephalitis, otitis media, bullous myringitis, infectious synovitis, glomerulonephritis, pancrea-titis, hepatitis, myocarditis, pericarditis, hemolytic anemia, and rhabdomyolysis (Waites and Talkington, 2005). The preceding can be primary lesions, but are often secondary to respiratory disease. Dysfunction of the immune system by inappropriate cytokine responses or possibly molecular mim-icry following infection are associated with long-term seque-lae including the development or exacerbation of asthma and chronic obstructive pulmonary disease; Stevens-Johnson syn-drome and other exanthemas; and Guillain-Barre syndrome, Bell’s palsy, and demyelinating neuropathies (Atkinson et al., 2008). Mode of transmission is via droplet aerosols (PAP) or sexual contact (urogential colonization).

Clinical manifestations are successfully treated with tet-racyclines, fluoroquinolones, macrolides, and lincosamides (Waites and Talkington, 2005). Signs can be treated with inhaled or injected steroids. Experimental vaccinations aimed at preventing infection have been unsuccessful due to failure to elicit immune responses, retention of viru-lence, or invocation of immune responses that exacerbated clinical signs (Barile, 1984; Jacobs et al., 1988). Mycoplasma pneumoniae is reported to share antigens with Mycoplasma genitalium (Taylor-Robinson, 1983a), potentially complicat-ing serology-based diagnosis of infection.

Source: isolated from the upper and lower respiratory tract, cerebrospinal fluid, synovial fluid, and urogential tract of humans.

DNA G+C content (mol%): 38.6 (Tm), 40.0 (strain M129 genome sequence).

Type strain: FH, ATCC 15531, NCTC 10119, CIP 103766, NBRC 14401.

Sequence accession nos: M29061 (16S rRNA gene), U00089 (strain M129 genome sequence).

100. Mycoplasma primatum Del Giudice, Carski, Barile, Lemcke and Tully 1971, 442AL

pri.ma¢tum. L. n. primas, primatis chief, from which pri-mates, the highest order of mammals originates; L. pl. gen. n. primatum of chiefs, of primates.

Cells are both spherical and coccobacillary. Motility for this species has not been assessed. Colony morphology has a fried-egg appearance. Grows well in SP-4 or Hayflick medium supplemented with arginine at 37°C.

Opportunistic pathogen; rarely associated with keratitis in humans (Ruiter and Wentholt, 1955). Mode of trans-mission has not been established.

Source: isolated from the oral cavity and/or urogenital tract of baboons, African green monkeys, rhesus macaques, squirrel monkeys, and humans (Hill, 1977; Somerson and Cole, 1979; Thomsen, 1974).

DNA G+C content (mol%): 28.6 (Tm).Type strain: HRC292, ATCC 25948, NCTC 10163.Sequence accession no. (16S rRNA gene): AF221118.

607


101. Mycoplasma pullorum Jordan, Ernø, Cottew, Hinz and Stipkovits 1982, 114VP

pul.lo¢rum. L. n. pullus a young animal, especially chicken; L. gen. pl. n. pullorum of young chickens.

Cells are coccoid to coccobacillary. Motility for this species has not been assessed. Colonies on solid medium display typical fried-egg appearance. Grows in Frey’s or Hayflick medium supplemented with glucose at 37°C.

Pathogenicity not fully established, but has been asso-ciated with tracheitis and airsacculitis in chickens, and embryo lethality resulting in reduced hatchability in chickens and turkeys. Mode of transmission has not been assessed definitively.

Source: isolated from the trachea, air sacs, and eggs of chickens; from the eggs of turkeys; and from tissues of pheasants, partridges, pigeons, and quail (Bencina et al., 1987; Bradbury et al., 2001; Kempf et al., 1991; Kleven, 2003; Lobo et al., 2004; Moalic et al., 1997; Poveda et al., 1990).

DNA G+C content (mol%): 29 (Bd).Type strain: CKK, ATCC 33553, NCTC 10187.Sequence accession no. (16S rRNA gene): U58504.Further comment: previously known as avian serotype C

(Adler et al., 1958).

102. Mycoplasma pulmonis (Sabin 1941) Freundt 1955, 73AL (Murimyces pulmonis Sabin 1941, 57)

pul.mo¢nis. L. n. pulmo, -onis the lung; L. gen. n. pulmonis of the lung.

Cells are predominantly coccoid with a well-organized terminal structure. Cells are motile and glide in the direc-tion of the terminal structure. An extracellular capsular matrix can be demonstrated by staining with ruthenium red and formation of biofilms has been demonstrated. Colonies on solid medium have a coarsely granulated and vacuolated appearance, with a lesser tendency to grow into the agar, and the central spot is consistently less well defined than in most other Mycoplasma species. Grows in SP-4 or modified Hayflick broth supplemented with glu-cose at an optimum temperature of 37°C.

Pathogenic; causes rhinitis, laryngotracheitis, broncho-pneumonia (collectively described as murine respiratory mycoplasmosis in mice), otitis media, conjunctivitis, acute and chronic arthritis, oophoritis, salpingitis, epididymitis, and urethritis of rodents (chiefly mice, rats, guinea pigs, and hamsters). Transmission occurs via aerosol, fomites, sexual contact, or vertically during gestation.

Macrolides, fluoroquinolones, and tetracyclines are effective against Mycoplasma pulmonis in vitro; however, con-trol measures such as decontamination of fomites, culling of infected colonies, and treatment of clinical signs with steroids are more commonly employed in clinical settings. Several candidate vaccines have been described.

Source: isolated from the respiratory and urogenital tracts, eyes, synovial fluid, and synovial membranes of (principally captive) rodents, and rarely from the nasopharynx of rab-bits and horses (Allam and Lemcke, 1975; Cassell and Hill, 1979; Deeb and Kenny, 1967; Simecka et al., 1992).

DNA G+C content (mol%): 27.5–29.2 (Bd), 26.6 (strain UAB CTIP genome sequence).

Type strain: Ash, PG34, ATCC 19612, NCTC 10139, CIP 75.26, NBRC 14896.

Sequence accession nos: M23941 (16S rRNA gene sequence), NC_002771 (strain UAB CTIP genome sequence).

103. Mycoplasma putrefaciens Tully, Barile, Edward, Theodore and Ernø 1974, 116AL

pu.tre.fa¢ci.ens. L. v. putrefacio to make rotten; L. part. adj. putrefaciens making rotten or putrefying, connot-ing the production of a putrid odor in broth and agar cultures.

Cells are predominantly coccobacillary to pleomorphic. Nonmotile. Formation of biofilms has been demonstrated (McAuliffe et al., 2006). Colony morphology has a typical fried-egg appearance. Grows well in SP-4 medium supple-mented with glucose at 37°C.

Pathogenic; causes polyarthritis, mastitis, conjunctivi-tis (a syndrome collectively termed contagious agalactia) (Bergonier et al., 1997), abortion, salpingitis, metritis, and testicular atrophy (Gil et al., 2003) in goats.

Macrolides, fluoroquinolones, lincosamides, and tetra-cyclines are effective against Mycoplasma putrefaciens; how-ever, control measures such as decontamination of fomites and culling of infected herds are typically recommended to discourage the development of antimicrobial-resistant strains in carrier animals (Antunes et al., 2007; Bergonier et al., 1997).

Source: isolated from the synovial fluid, udders, expelled milk, conjunctivae, ear canal, uterus, and testes of goats.

DNA G+C content (mol%): 28.9 (Tm).Type strain: KS1, ATCC 15718, NCTC 10155.Sequence accession no. (16S rRNA gene): M23938.

104. Mycoplasma salivarium Edward 1955, 90AL

sa.li.va¢ri.um. L. neut. adj. salivarium slimy, saliva-like, intended to denote of saliva.

Cells are coccoid to coccobacillary. Nonmotile. Colonies are large with a typical fried-egg appearance. Grows well in Hayflick medium supplemented with arginine at 37°C and produces a “film and spots” reaction.

Mycoplasma salivarium is most frequently associated with contamination of eukaryotic cell culture and is fre-quently removed by treatment of cells with antibiotics and/or maintenance of cell lines in antibiotic-containing medium. The most effective classes of antibiotics for cell culture eradication are tetracyclines, macrolides, and flu-oroquinolones.

Opportunistic pathogen; primarily found as a commen-sal of the human oral cavity, and rarely associated with arthritis, submasseteric abscesses, gingivitis, and periodon-titis in immunocompromised patients (Grisold et al., 2008; Lamster et al., 1997; So et al., 1983). Mode of transmission is via direct contact with human saliva.

Source: isolated from the oral cavity, synovial fluid, den-tal plaque, and abscessed mandibles of humans, and the nasopharynx of pigs (Erickson et al., 1988).

DNA G+C content (mol%): 27.3 (Bd).Type strain: PG20, H110, ATCC 23064, NCTC 10113,

NBRC 14478.Sequence accession no. (16S rRNA gene): M24661.

608

Genus I. MycoplasMa

105. Mycoplasma simbae Hill 1992, 520VP

sim¢bae. Swahili n. simba lion; N.L. gen. n. simbae of a lion.

Cells are pleomorphic and nonmotile. Colonies on solid medium have a typical fried-egg appearance. Film is pro-duced by cultivation on egg yolk agar. Grows well in SP-4 medium at 37°C.


Source: isolated from the throats of lions (Hill, 1992).DNA G+C content (mol%): 37 (Bd).Type strain: LX, NCTC 11724, ATCC 49888.Sequence accession no. (16S rRNA gene): U16323.

106. Mycoplasma spermatophilum Hill 1991a, 232VP

sper.ma.to.phi¢lum. Gr. n. sperma, -atos sperm or seed; N.L. neut. adj. philum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. spermatophilum sperm-loving.

Cells are primarily coccoid. Nonmotile. Colonies are convex to fried-egg shaped and are of below-average size. Grows well in SP-4 medium supplemented with added argi-nine under anaerobic conditions at 37°C.

Pathogenic; potentially associated with infertility, as infected spermatozoa do not fertilize ova and infected fertilized ova were unable to implant following in vitro fertilization (Hill et al., 1987; Hill, 1991a). Mode of trans-mission is via sexual contact.

Source: isolated from the semen and cervix of humans with impaired fertility.

DNA G+C content (mol%): 32 (Bd).Type strain: AH159, NCTC 11720, ATCC 49695, CIP


107. Mycoplasma spumans Edward 1955, 90AL

spu¢mans. L. part. adj. spumans foaming, presumably allud-ing to thick dark markings that suggest the presence of globules inside the coarsely reticulated colonies.

Cells are coccoid to filamentous. Motility for this species has not been assessed. Colonies in early subcultures have a coarsely reticulated and vacuolated appearance. A typical fried-egg appearance of the colonies develops on repeated subculturing. Grows well in SP-4 or modified Hayflick medium supplemented with arginine.

Opportunistic pathogen; primarily found as a commen-sal of the nasopharynx, but has also been associated with pneumonia and arthritis of domestic dogs. Mode of trans-mission has not been established definitively.

Source: isolated from the lungs, nasopharynx, synovial fluid, cerebrospinal fluid, trachea, prepuce, prostate, bladder, cer-vix, vagina, and urine of domestic dogs (Chalker, 2005).



108. Mycoplasma sturni Forsyth, Tully, Gorton, Hinckley, Frasca, van Kruiningen and Geary 1996, 719VP

stur¢ni. N.L. n. Sturnus (from L. n. sturnus a starling or stare) a genus of birds, N.L. gen. n. sturni of the genus Sturnus, the genus of the bird from which the organism was isolated.

Cells are primarily coccoid with some irregular flask-shaped and filamentous forms seen. Motility for this species has not been assessed. Colonies on agar usually have a fried-egg appearance when grown at 37°C. Grows well in SP-4 medium supplemented with glucose at 34–37°C.

Pathogenicity has not been fully established, but it is associated with conjunctivitis in the European starling, mockingbirds, blue jays, and American crows. The organ-ism is also found in clinically normal birds. Mode of trans-mission has not been established definitively.

Source: isolated from the conjunctivae of European star-lings, mockingbirds, blue jays, American crows, American robins, blackbirds, rooks, carrion crows, and magpies (Frasca et al., 1997; Ley et al., 1998; Pennycott et al., 2005; Wellehan et al., 2001).

DNA G+C content (mol%): 31 (Bd).Type strain: UCMF, ATCC 51945.Sequence accession no. (16S rRNA gene): U22013.

109. Mycoplasma sualvi Gourlay, Wyld and Leach 1978, 292AL

su.al¢vi. L. n. sus, suis swine; L. n. alvus bowel, womb, stom-ach; N.L. gen. n. sualvi of the bowel of swine.

Cells are coccobacillary and many possess organized terminal structures. Nonmotile. Colonies have a typical fried-egg appearance. Grows well in SP-4 medium supple-mented with either arginine or glucose at 37°C.

No evidence of pathogenicity.Source: isolated from the rectum, colon, small intestines,

and vagina of pigs (Gourlay et al., 1978).DNA G+C content (mol%): 23.7 (Bd).Type strain: Mayfield B, NCTC 10170, ATCC 33004.Sequence accession no. (16S rRNA gene): AF412988.

110. Mycoplasma subdolum Lemcke and Kirchhoff 1979, 49AL

sub.do¢lum. L. neut. adj. subdolum somewhat deceptive, alludes to the deceptive color change that led to the original erroneous description of the strains as urea-hydrolyzing.

Cells are coccoid to coccobacilliary. Motility for this species has not been assessed. Colony growth on solid medium exhibits the typical fried-egg appearance. Grows well in SP-4, Frey’s, or Hayflick medium supplemented with arginine at 37°C.

Opportunistic pathogen; equivocal evidence for viru-lence may represent variation among strains. Associated with impaired fecundity and abortion in horses; however, is highly prevalent in clinically normal horses (Spergser et al., 2002). Mode of transmission is via sexual contact.

Source: isolated from the cervix, semen, and aborted foals of horses (Lemcke and Kirchhoff, 1979).

DNA G+C content (mol%): 28.8 (Bd).Type strain: TB, ATCC 29870, NCTC 10175.Sequence accession no. (16S rRNA gene): AF125588.

111. Mycoplasma suis corrig. (Splitter 1950) Neimark, Johansson, Rikihisa and Tully 2002b, 683VP (Eperythrozoon suis Splitter 1950, 513)

su¢is. L. gen. n. suis of the pig.

Cells are coccoid. Motility for this species has not been assessed. This species has not been grown on any artificial

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medium; therefore, notable biochemical parameters are not known.

Neoarsphenamine and tetracyclines are effective thera-peutic agents. An enzyme-linked immunosorbant assay (ELISA) and PCR-based detection assays to enable diagno-sis of infection have been described (Groebel et al., 2009; Gwaltney and Oberst, 1994; Hoelzle, 2008; Hsu et al., 1992).

Pathogenic; causes febrile icteroanemia in pigs. Trans-mission occurs via insect vectors including Stomoxys calci-trans and Aedes aegypti (Prullage et al., 1993).

Source: observed in association with the erythrocytes of pigs.

DNA G+C content (mol%): 31.1 (complete genome sequence of strain Illinois; J.B. Messick et al., unpublished).

Type strain: not established.Sequence accession no. (16S rRNA gene): AF029394.Further comment: the original spelling of the specific epi-

thet, haemosuis (sic), has been corrected by the List Editor.

112. Mycoplasma synoviae Olson, Kerr and Campbell 1964, 209AL

sy.novi¢ae. N.L. n. synovia the joint fluid; N.L. gen. n. syn-oviae of joint fluid.

Cells are coccoid and pleomorphic. Nonmotile. An amorphous extracellular layer is described. Colony appear-ance on solid medium is variable with some showing typi-cal fried-egg type colonies. Grows well in Frey’s medium supplemented with glucose, l-cysteine, and nicotinamide adenine dinucleotide at 37°C. Produces a “film and spots” reaction (Ajufo and Whithear, 1980; Frey et al., 1968).

Pathogenic; causes infectious synovitis, osteoarthritis, and upper respiratory disease which is often subclinical in chickens and turkeys. Also associated with a reduction in egg quality in chickens. Mycoplasma synoviae is often found in association with additional avian pathogens including Mycoplasma gallisepticum, avian strains of Escherichia coli, Newcastle disease virus, and infectious bronchitis virus. Direct contact with fomites and droplet aerosols are the primary mechanisms of transmission.

Tetracyclines and fluoroquinolones are effective che-motherapeutic agents; however, treatment is typically only sought for individual birds, as medicating a commercial flock is not considered an effective control strategy. Vac-cination and management strategies (i.e., single age “all in/all out” systems and culling of endemic flocks) are more commonly utilized. A live vaccine is commercially available. Mycoplasma synoviae shares surface antigens with Mycoplasma gallisepticum, potentially complicating serology-based diagnosis of infection. Numerous molecular diag-nostics have been described. This organism is listed in the Terrestrial Animal Health Code of the Office International des Epizooties (http://oie.int; Yogev et al., 1989; Browning et al., 2005; Kleven, 2008; Hammond et al., 2009; Raviv and Kleven, 2009)

Source: isolated from the synovial fluid, synovial mem-branes, and respiratory tract tissues of chickens and tur-keys, and from ducks, geese, pigeons, Japanese quail, pheasants, red-legged partridges, wild turkeys, and house sparrows (Bradbury and Morrow, 2008; Feberwee et al., 2009; Jordan, 1979; Kleven, 1998).

DNA G+C content (mol%): 34.2 (Bd).Type strain: WVU 1853, ATCC 25204, NCTC 10124.Sequence accession nos: X52083 (16S rRNA gene),

NC_007294 (strain 53 complete genome sequence).

113. Mycoplasma testudineum Brown, Merritt, Jacobson, Klein, Tully and Brown 2004, 1529VP

tes.tu.di¢ne.um. L. neut. adj. testudineum of or pertaining to a tortoise.

Cells are predominantly coccoid in shape, though some exhibit a terminal protrusion. Cells exhibit gliding motility. Colonies on solid medium exhibit typical fried-egg forms. Grows well in SP-4 medium supplemented with glucose at 22–30°C.

Pathogenic; causes rhinitis and conjunctivitis in desert and gopher tortoises. Mode of transmission appears to be intranasal inhalation (Brown et al., 2004).

Source: isolated from the nares of desert tortoises (Gopherus agassizii) and gopher tortoises (Gopherus polyphemus).

DNA G+C content (mol%): not determined.Type strain: BH29, ATCC 700618, MCCM 03231.Sequence accession no. (16S rRNA gene): AY366210.

114. Mycoplasma testudinis Hill 1985, 491VP

tes.tu¢di.nis. L. n. testudo, -inis tortoise; L. gen. n. testudinis of a tortoise.

Cells are pleomorphic, with many possessing a terminal organelle similar to those of Mycoplasma gallisepticum and Mycoplasma amphoriforme that periodically exhibits curva-ture (Hatchel et al., 2006). A subset of cells are motile and glide at high speed in the direction of the terminal struc-ture. Colonies on solid medium have a typical fried-egg appearance. Grows well in SP-4 medium supplemented with glucose at 25–37°C, with optimum growth at 30°C.


Source: isolated from the cloaca of a Greek tortoise (Hill, 1985).

DNA G+C content (mol%): 35 (Tm).Type strain: 01008, NCTC 11701, ATCC 43263.Sequence accession no. (16S rRNA gene): U09788.

115. Mycoplasma verecundum Gourlay, Leach and Howard 1974, 483AL

ve.re¢cun.dum. L. neut. adj. verecundum shy, unobtru-sive, free from extravagance, alluding to the lack of obvi-ous biochemical characteristics of the species.

Cells are highly pleomorphic, exhibiting coccoid bod-ies, ring forms, and branched filaments. Motility for this species has not been assessed. Growth on solid medium produces colonies with the typical fried-egg appearance. Grows well in SP-4 or Hayflick medium at 37°C.

Probable commensal; attempts to produce disease experi-mentally have been unsuccessful (Gourlay and Howard, 1979). Mode of transmission has not been established.

Source: isolated from the eyes of calves with conjunc-tivitis, the prepuce of clinically normal bulls, and from in-market kale (Gourlay and Howard, 1979; Gourlay et al., 1974; Somerson et al., 1982).

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http://oie.int

Genus I. MycoplasMa

DNA G+C content (mol%): 27 (Tm).Type strain: 107, ATCC 27862, NCTC 10145.Sequence accession no. (16S rRNA gene): AF412989.

116. Mycoplasma wenyonii (Adler and Ellenbogen 1934) Neimark, Johansson, Rikihisa and Tully 2002b, 683VP (Eperythrozoon wenyonii Adler and Ellenbogen 1934, 220)

we.ny.o¢ni.i. N.L. masc. gen. n. wenyonii of Wenyon, named after Charles Morley Wenyon (1878–1948), an investigator of these organisms.

Cells are coccoid. Motility for this species has not been assessed. This species has not been grown on any artificial medium; therefore, notable biochemical parameters are not known.

Pathogenic; causes anemia and subsequent lameness and/or infertility in cattle. Transmission is primarily vector-mediated by Dermacentor andersoni and reportedly can also occur vertically during gestation. Oxytetracycline is an effective therapeutic agent (Montes et al., 1994). Mycoplasma wenyonii is reported to share antigens with Mycoplasma ovis (Kreier and Ristic, 1963), potentially complicating serology-based diagnosis of infection.

Source: observed in association with the erythrocytes of cattle; Kreier and Ristic (1968) reported in addition to erythrocytes an association with platelets.


117. Mycoplasma yeatsii DaMassa, Tully, Rose, Pitcher, Leach and Cottew 1994, 483VP

ye.at¢si.i. N.L. masc. gen. n. yeatsii of Yeats, named after F.R. Yeats, an Australian veterinarian who was a co-isolator of the organism.

Cells are coccoid and nonmotile. Colonies on agar have a fried-egg appearance. Grows well in SP-4 medium sup-plemented with glucose at 37°C. Formation of biofilms has been demonstrated (McAuliffe et al., 2006).

Opportunistic pathogen; commensal of the ear canal of goats that has rarely been found in association with masti-tis and arthritis (DaMassa et al., 1991). The mode of trans-mission has not been established.

Source: isolated from the external ear canals, retropha-ryngeal lymph node, nasal cavity, udders, and milk of goats.

DNA G+C content (mol%): 26.6 (Tm).Type strain: GIH, ATCC 51346, NCTC 11730, CIP

105675.Sequence accession no. (16S rRNA gene): U67946.

species incertae sedis

1. Mycoplasma coccoides (Schilling 1928) Neimark, Peters, Robinson and Stewart 2005, 1389VP (Eperythrozoon coccoides Schilling 1928, 1854)

coc.co¢ides. N.L. masc. n. coccus (from Gr. masc. n. kokkos grain, seed) coccus; L. suff. -oides (from Gr. suff. eides, from Gr. n. eidos that which is seen, form, shape, figure), resem-bling, similar; N.L. neut. adj. coccoides coccus-shaped.


Pathogenic; causes anemia in wild and captive mice, and captive rats, hamsters, and rabbits. Transmission is believed

to be vector-borne and mediated by the rat louse Polyplex spinulosa and the mouse louse Polyplex serrata.

Neoarsphenamine and oxophenarsine were thought to be effective chemotherapeutic agents for treatment of Myco-plasma coccoides infection in captive rodents, whereas tetracy-clines are effective only at keeping infection at subclinical levels (Thurston, 1953).

Source: observed in association with the erythrocytes of wild and captive rodents.

DNA G+C content (mol%): not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): AY171918.

species Candidatus

1. “Candidatus Mycoplasma haematoparvum” Sykes, Ball, Bailiff and Fry 2005, 29

ha.e.ma.to.par¢vum. Gr. neut. n. haema, -atos blood; L. neut. adj. parvum small; N.L. neut. adj. haemoatoparvum small (mycoplasma) from blood.

Source: blood of infected canines (Sykes et al., 2005).Host habitat: circulation of infected canines.Phylogeny: assignment to the hemoplasma cluster of

the pneumoniae group of Mollicutes (Foley and Pedersen, 2001).

Cell morphology: wall-less; coccoid in shape.Optimum growth temperature: not applicable.Cultivation status: non-culturable.Sequence accession no. (16S rRNA gene): AY854037.

2. “Candidatus Mycoplasma haemobos” Tagawa, Matsumoto and Inokuma 2008, 179

ha.e.mo¢bos. Gr. neut. n. haema blood; L. n. bos an ox, a bull, a cow; N.L. n. haemobos (sic) intended to mean of cattle blood.

Source: blood of infected cattle.Host habitat: blood of cattle.Phylogeny: assignment to the hemoplasma cluster of the

pneumoniae group of the genus Mycoplasma.Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): EF460765.Further comment: this organism is synonymous with “Candidatus

Mycoplasma haemobovis” (sequence accession no. EF616468).

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3. “Candidatus Mycoplasma haemodidelphidis” Messick, Walker, Raphael, Berent and Shi 2002, 697

ha.e.mo.di.del¢phi.dis. Gr. neut n. haema blood; N.L. fem. gen. n. didelphidis of the opossum; N.L. gen. n. haemodidel-phidis of opossum blood.

Source: blood of an infected opossum.Host habitat: circulation of an infected opossum.Phylogeny: assignment to the hemoplasma cluster of the

pneumoniae group of mollicutes (Messick et al., 2002).Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): AF178676.

4. “Candidatus Mycoplasma haemolamae” Messick, Walker, Raphael, Berent and Shi 2002, 697

ha.e.mo.la¢ma.e. Gr. neut n. haema blood; N.L. gen. n. lamae of the alpaca; N.L. fem. gen. n. haemolamae of alpaca blood.

Source: blood of infected llamas.Host habitat: circulation of infected llamas (McLaughlin

et al., 1991).Phylogeny: assignment to the hemoplasma cluster of the

pneumoniae group of mollicutes (Messick et al., 2002).Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): AF306346.

5. “Candidatus Mycoplasma haemominutum” Foley and Pedersen 2001, 817

ha.e.mo.mi¢nu.tum. Gr. neut n. haema blood; L. neut. part. adj. minutum small in size; N.L. neut. adj. haemominutum small (mycoplasma) from blood.

Source: blood of infected felines (George et al., 2002; Tasker et al., 2003).

Host habitat: circulation of infected felines.Phylogeny: assignment to the hemoplasma cluster of the

pneumoniae group of mollicutes (Foley and Pedersen, 2001).

Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape; 300–600 nm in

diameter.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): U88564.

6. “Candidatus Mycoplasma kahaneii” Neimark, Barnaud, Gounon, Michel and Contamin 2002a, 697

ka.ha.ne¢i.i. N.L. masc. gen. n. kahaneii of Kahane, named for I. Kahane.

Source: blood of infected monkeys (Saimiri sciureus) (Michel et al., 2000).

Host habitat: circulation of infected monkeys.Phylogeny: assignment to the hemoplasma cluster of

the pneumoniae group of mycoplasmas (Neimark et al., 2002a).

Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): AF338269.

7. “Candidatus Mycoplasma ravipulmonis” Neimark, Mitchel-more and Leach 1998, 393

ra.vi.pul.mo¢nis. L. adj. ravus grayish; L. n. pulmo, -onis the lung; N.L. gen. n. ravipulmonis of a gray lung.

Source: lung tissue of mouse with respiratory infection (gray lung disease).

Host habitat: respiratory tissue of mice with pneumonia.Phylogeny: forms a single species line in the hominis group

of mollicutes (Neimark et al., 1998; Pettersson et al., 2000).Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape; 650 nm in

diameter.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): AF001173.

8. “Candidatus Mycoplasma turicensis” Willi, Boretti, Baum-gartner, Tasker, Wenger, Cattori, Meli, Reusch, Lutz and Hofmann-Lehmann 2006, 4430

tu.ri.cen¢sis. L. masc. (sic) adj. turicensis pertaining to Turi-cum, the Latin name of Zurich, the site of the organism’s initial detection.

Source: blood of infected domestic cats.Host habitat: blood of domestic cats.Phylogeny: assignment to the hemoplasma cluster of the

pneumoniae group of the genus Mycoplasma.Cultivation status: non-culturable.Cell morphology: wall-less; coccoid in shape.Optimum growth temperature: not applicable.Sequence accession no. (16S rRNA gene): DQ157150.

The following proposed species has been incidentally cited, but the putative organism remains to be established definitively and the name has no standing in nomenclature.

1. “Candidatus Mycoplasma haemotarandirangiferis” Stoff-regen, Alt, Palmer, Olsen, Waters and Stasko 2006, 254

ha.e.mo.ta.ran.di.ran.gi¢fe.ris. Gr. neut n. haema blood; Rangifer tarandus scientific name of the reindeer; N.L. gen. n. haemotarandirangiferis epithet intended to indicate occur-rence in blood of reindeer.

Source: blood of reindeer.Host habitat: blood of reindeer.Phylogeny: partial 16S rRNA gene sequences suggest possi-

ble relationships to Mycoplasma ovis and Mycoplasma wenyonii (suis cluster), and/or to Mycoplasma haemofelis (haemofelis cluster).

Cultivation status: non-culturable.Cell morphology: single punctate, chaining punctate, clus-

tering punctate, single bacillary, chaining bacillary, single rings, chaining rings, and clustering rings.

Optimum growth temperature: not applicable.Sequence accession nos (16S rRNA gene): DQ524812–

DQ524818.Further comment : sequence accession no. DQ524819, rep-

resenting clone 107LSIA (Stoffregen et al., 2006), does not support assignment of that clone to the genus Mycoplasma because it is most similar to 16S rRNA genes of Fusobacte-rium spp. Several different partial 16S rRNA gene sequences obtained from other clones are too variable to establish their coherence as a species.

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Genus II. ureaplasMa

Coccoid cells about 500 nm in diameter; may appear as coc-cobacillary forms in exponential growth phase; filaments are rare. Nonmotile. Facultative anaerobes. Form exceptionally small colonies on solid media that are described either as tiny (T) “fried-egg” colonies or as “cauliflower head” colonies hav-ing a lobed periphery. Unusual pH required for growth (about 6.0–6.5). Optimal incubation temperature for examined spe-cies is 35–37°C. Chemo-organotrophic. Like Mycoplasma, species of Ureaplasma lack oxygen-dependent, NADH oxidase activity. Unlike Mycoplasma, species of Ureaplasma lack hexokinase or arginine deiminase activities but have a unique and obligate requirement for urea and produce potent ureases that hydro-lyze urea to CO2 and NH3 for energy generation and growth. Genome sizes range from 760 to 1170 kbp (PFGE). Commensals or opportunistic pathogens in vertebrate hosts, primarily birds and mammals (mainly primates, ungulates, and carnivores).

DNA G+C content (mol%): 25–32 (Bd, Tm).Type species: Ureaplasma urealyticum Shepard, Lunceford,

Ford, Purcell, Taylor-Robinson, Razin and Black 1974, 167 emend. Robertson, Stemke, Davis, Harasawa, Thirkell, Kong, Shepard and Ford 2002, 593.


Although cellular diameters as small as 100 nm and as large as 1000 nm, and minimal reproductive units of about 330 nm in diameter have been reported (Taylor-Robinson and Gourlay, 1984), published thin sections of these organisms (trivial name, ureaplasmas) show diameters only as large as 450–500 nm. The exceptions are feline ureaplasmas with thin section diameters of up to 800 nm (Harasawa et al., 1990a). Morphometric analy-sis of cells of the type strain of Ureaplasma urealyticum fixed in the exponential phase of growth showed coccoid cells with

other organisms

1. “Mycoplasma insons” May, Ortiz, Wendland, Rotstein, Relich, Balish and Brown 2007, 298

in¢sons. L. neut. adj. insons guiltless, innocent.

Source: trachea and choanae of a healthy green iguana (Iguana iguana).

Host habitat: respiratory tract and blood of green iguanas (Iguana iguana).

Phylogeny: assignment to the Mycoplasma fastidiosum cluster of the pneumoniae group of the genus Mycoplasma.

Cultivation status: cells are culturable in SP-4 medium sup-plemented with glucose.

Cell morphology: pleomorphic, but many have a highly atypi-cal shape for a mycoplasma, often resembling a twisted rod.

Optimum growth temperature: 30°C.Sequence accession no. (16S rRNA gene): DQ522159.

2. “Mycoplasma sphenisci” Frasca, Weber, Urquhart, Liao, Gladd, Cecchini, Hudson, May, Gast, Gorton and Geary 2005, 2979

sphe.nis¢ci. N.L. gen. n. sphenisci of Spheniscus, the genus of penguin that includes the jackass penguin (Spheniscus dem-ersus) from which this mycoplasma was isolated.

Source: choanae of a jackass penguin (Spheniscus demersus) with choanal discharge and halitosis.

Host habitat: upper respiratory tract of the jackass pen-guin.

Phylogeny: assignment to the Mycoplasma lipophilum cluster of the hominis group of the genus Mycoplasma.

Cultivation status: cells are culturable in Frey’s medium supplemented with glucose.

Cell morphology: pleomorphic; some cells exhibit terminal structures.

Optimum growth temperature: 37°C.Sequence accession no. (16S rRNA gene): AY756171.

3. “Mycoplasma vulturis” corrig. Oaks, Donahoe, Rurangirwa, Rideout, Gilbert and Virani 2004, 5911

vul.tu¢ri.i. L. gen. n. vulturis of a vultures, named for the host animal (Oriental white-backed vulture).

Source: lung and spleen tissue of an Oriental white-backed vulture.

Host habitat: upper and lower respiratory tract of Oriental white-backed vulture, where it replicates intracellularly.

Phylogeny: assignment to the Mycoplasma neurolyticum clus-ter of the hominis group of the genus Mycoplasma.

Cultivation status: cells can be grown in co-culture with chicken embryo fibroblasts, but have not been grown in pure in vitro culture.

Cell morphology: coccoid; cells display intracellular vacuoles and intracellular granules of electron-dense material.

Optimum growth temperature: 37°C.Sequence accession no. (16S rRNA gene): AY191226.

4. “Mycoplasma zalophi” Haulena, Gulland, Lawrence, Fauquier, Jang, Aldridge, Spraker, Thomas, Brown, Wendland and Davidson 2006, 43

za.lo¢phi. N.L. gen. n. zalophi of Zalophus, the genus of sea lion that includes the California sea lion (Zalophus califor-nianus) from which this mycoplasma was isolated.

Source: subdermal abscesses of captive sea lions.Host habitat: subdermal and intramuscular abscesses,

joints, lungs, and lymph nodes of captive sea lions.Phylogeny: assignment to the Mycoplasma hominis cluster of

the hominis group of the genus Mycoplasma.Cultivation status: cells are culturable in SP-4 medium sup-

plemented with glucose.Cell morphology: not yet described.Optimum growth temperature: 37°C.Sequence accession no. (16S rRNA gene): AF493543.

Genus II. ureaplasma shepard, lunceford, Ford, purcell, Taylor-robinson, razin and Black 1974, 167al

Janet a. RoBeRtson anD DaviD tayloR-RoBinson

u.re.a.plas¢ma. n.l. fem. n. urea urea; Gr. neut. n. plasma anything formed or moulded, image, figure; n.l. neut. n. Ureaplasma urea form.

613


diameters of about 500 nm (Robertson et al., 1983). Similar cellular diameters have been seen in hemadsorption studies in which cells were pre-fixed during incubation before usual fixa-tion for electron microscopy. Reports of budding and filamen-tous forms probably reflect the effect of cultural and handling conditions on these highly plastic cells. Although the sequence of the Ureaplasma parvum genome lacks any recognizable FtsZ genes (Glass et al., 2000), ureaplasmas appear to reproduce by binary fission. Because of their minute size, ureaplasma cells are rarely seen by light microscopy. Although they are of the Gram-stain-positive lineage, the lack of cell wall results in the organisms appearing Gram-stain-negative. They are more easily detected if stained with crystal violet alone. Electron micro-graphs have indicated hair-like structures, possibly pili, 5–8 nm long, radiating from the membrane (Whitescarver and Furness, 1975). An extramembranous capsule was expected from light microscopic studies. In cytochemical studies, a carbohydrate-containing, capsular structure has been demonstrated in a strain of Ureaplasma urealyticum (Robertson and Smook, 1976; Figure 110). The structure is a lipoglycan that has also been demonstrated on the type strain T960T of Ureaplasma urealyticum and on a serovar 3 strain of Ureaplasma parvum; the lipoglycan composition was strain-variable (Smith, 1986). No viruses have been seen nor has viral or plasmid nucleic acid been reported in any ureaplasma.

Ureaplasma colonies are significantly smaller in diameter (£10–175 nm) than those of Mycoplasma species (300–800 nm; Figure 110). For this reason, they were first described as “tiny (T) form PPLO (pleuropneumonia-like organisms) colonies” (Shepard, 1954) and later called T-mycoplasmas (Meloni et al., 1980; Shepard et al., 1974). Cultures on solid media may grow in air, but more numerous and larger colonies result in 5–15% CO2 in N2 or H2 (Robertson, 1982). Colonies of many strains are detectable after overnight incubation and reach maximum dimensions within 2 d. Isolates from ungulates may require

longer incubation. Temperatures of 20–40°C are permissive for growth of examined strains, but their optimal incubation tem-peratures are 35–37°C (Black, 1973). Ureaplasma cultures in liquid media are incubated aerobically with growth occurring in the bottom of the tube, as revealed by changes in the pH indicator. Mean generation times of 10 isolates from humans ranged from 50 to 105 min (Furness, 1975). Maximum titers of £108 organisms per ml of culture produce insufficient cell mass for detectable turbidity, precluding growth measurement by turbidometric or spectrophotometric methods. Growth is best measured by broth dilution methods (Ford, 1972; Rodwell and Whitcomb, 1983; Stemke and Robertson, 1982). When immediate estimation of populations is required, ATP lumi-nometry (Stemler et al., 1987) may be useful. An indicator sys-tem enhances colony detection and ureaplasma identification. Ammonia from urea degradation causes a rise in pH and cer-tain cations to form a golden to deep brown precipitate on the colorless colonies, making them visible when viewed by directly transmitted light. Initially, the urease spot test used a solution of urea and 1 mM Mn2+ (as MnCl2 or MnSO4) dropped onto agar (Shepard and Howard, 1970); later, Mn2+ was incorpo-rated into the agar itself to create a differential solid medium (e.g., Shepard, 1983). However, Mn2+ is toxic for ureaplasmas (Robertson and Chen, 1984). Equimolar CaCl2 (Shepard and Robertson, 1986) gave a similar response, but allowed the recov-ery of live cells. Manganese susceptibility has taxonomic value (Table 138); animal isolates show differing responses (Stemke et al., 1984; Stemler et al., 1987).

Ureaplasma urealyticum has some elements of the glyco-lytic cycle and pentose shunt (Cocks et al., 1985), but can-not degrade glucose and lacks arginine deiminase (Woodson et al., 1965). Instead, ureaplasmas have a unique and absolute requirement for urea (0.4–1.0 mM) and a slightly acidic envi-ronment (pH 6.0–7.0); pH values outside this range can be associated with growth inhibition. The essential cytoplasmic

Figure 110. Ureaplasma colonial size and cellular morphology. (a) Many isolated Ureaplasma urealyticum colonies, accentuated by a urease spot test, surround a single large Mycoplasma hominis colony on a solid genital mycoplasma (GM) agar surface. Colonies of Ureaplasma urealyticum commonly have diameters of 15–125 mm; the diameter of the Mycoplasma hominis colony shown is approximately 0.9 mm. (Reproduced with permission from Robertson et al., 1983. Sexually Transmitted Diseases 10 (October-December Suppl.): 232–239 © Lippincott Williams & Wilkins.) (b) Transmission electron micrograph of Ureaplasma urealyticum, serovar 4, strain 381/74 cells, showing coccoid morphology, extramembranous capsule stained with ruthenium red, lack of a cell wall, the single limiting mem-brane, and apparently simple cytoplasmic contents in which only ribosomes are clearly evident. Cell diameters were 485–585 nm. (Reproduced with permission from Robertson and Smook, 1976. Journal of Bacteriology 128: 658–660.)

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urease, comprising three subunits (Blanchard, 1990), pro-duces a transmembrane potential, which leads to ATP syn-thesis (Romano et al., 1980; Smith et al., 1993). Ureaplasma dependence upon catalysis of urea for energy is the basis of growth inhibition by the urease inhibitor hydroxamic acid and its derivatives (Ford, 1972) and by fluorofamide (Kenny, 1983). The proton pump inhibitor lansoprazole and its metabolites interfere with ATP synthesis at micromolar concentrations (Nagata et al., 1995). Their small genomes make ureaplasmas dependent upon the host for amino acids, amino acid precursors, lipids, and other growth components. Ureaplasmas from humans exhibit minimal levels of acetate kinase activity (Muhlrad et al., 1981). Like other Mycoplasmata-ceae, ureaplasmas make superoxide dismutase (O’Brien et al., 1983), but, unlike the rest of this family, oxygen-dependent NADH oxidase has not been detected (Masover et al., 1977). The Ureaplasma parvum genome sequence includes genes for six hemin and/or Fe3+ transporters which are believed to be related to respiration (Glass et al., 2000). For information

on additional physiological traits, see: Black (1973); Shepard et al., (1974); Shepard and Masover (1979); Taylor-Robinson and Gourlay (1984); and Pollack (1986).

Except for the obligatory requirements for supplementary urea and lower pH, ureaplasma growth requirements are simi-lar to those of members of the genus Mycoplasma. The sterol requirement is met by horse, bovine, or fetal bovine serum. Because heat-sensitive pantothenic acid is a growth factor sup-plied by serum (Shepard and Lunceford, cited by Shepard and Masover, 1979), the serum supplement should not be “inacti-vated” by heating to reduce complement activity. The effect of yeast extract is variable, perhaps depending upon the particular strain requirements or the batch of extract. Other defined addi-tives have been reported to enhance growth but, in the absence of a defined medium, evaluation is difficult. Dependence on sterols for the integrity of the cell membrane renders ureaplas-mas susceptible to digitonin and certain antifungal agents, but most strains tolerate the polyene nystatin (50 U/ml) that pre-vents overgrowth by yeasts.

Table 138. Phenotypic characteristics that partition the human serovar-standard strains of ureaplasmas to Ureaplasma speciesa

Characteristic U. urealyticum serovar U. parvum serovar Reference

Isoelectric focusing and SDS-PAGE at pH 5.3 polypeptide patterns:

Sayed and Kenny (1980)

Absent 1, 3T, 6Present 2, 4, 5, 7, 8T

1-D SDS-PAGE T960T biovar band:b Howard et al. (1981)Absent 1, 3T, 6Present 2, 4, 5, 7, 8T

2-D SDS-PAGE:b

Biovar 1 pattern 1, 3T, 6, 14 Mouches et al. (1981)Biovar 2 pattern 2, 4, 5, 7, 8T, 9, 11, 12 Swensen et al. (1983)

Growth inhibition by 1 mM Mn2+: Robertson and Chen (1984)Temporary 1, 3T, 6, 14Permanent 2, 4, 5, 7, 8T, 9–12c

Polypeptide recognized by immunoblots:51 and 58 kDa 1, 3T, 6, 14 Horowitz et al. (1986)47 kDa 2, 4, 5, 7, 8T, 9–13Biovar 1 pattern 1, 3T Lee and Kenny (1987)Biovar 2 pattern 2, 4, 8T

mAb UU8/39 recognition of membrane proteins: Thirkell et al. (1989)17 kDa only 1, 3T, 6, 1416/17 kDa 2, 4, 5, 7, 8T, 9–13

mAb UU8/17 recognition of 72 kDa urease subunit:d Thirkell et al. (1990)No 1, 3T, 6, 14Yes 8T

mAb VB10 recognition of 72 kDa urease subunit: MacKenzie et al. (1996)Yes 1, 3T, 6, 14No 2, 4, 5, 7, 8T, 9–13

Urease dimorphism: Davis et al. (1987)Lower MW 1, 3T, 6, 14Higher MW 2, 4, 5, 7, 8T, 9–13

Pyrophosphatase dimorphism: Davis and Villanueva (1990)Lower MW 1, 3T, 6, 14Higher MW 2, 4, 5, 7, 8T, 9–13

Diaphorase bands dimorphism: Davis and Villanueva (1990)Inapparent 1, 3T, 6, 14Apparent 2, 4, 5, 7, 8T, 9–13

aT, Type strain of species.b1-D, One-dimensional; 2-D, two-dimensional.cSerovar 13 gave an intermediate response and was excluded from the initial partition scheme.dHyperimmune rabbit sera and acute and convalescent sera from women with postpartum fever.

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Information about ureaplasma genetics is now abundant. The genomes of 26 strains of Ureaplasma, including six of the seven named species and six unnamed strains, range in size from 760 to 1170 kbp (Kakulphimp et al., 1991; Robertson et al., 1990). The largest genome belongs to Ureaplasma felinum. The G+C content of ureaplasmal DNA is in the range 25.5–31.6 mol%, which is lower than that for other Mollicutes and for all other prokaryotes, greatly limiting the degeneracy in the genetic code of ureaplasmas. For the type strain of Ureaplasma urealyticum, UGA is a codon for tryptophan (Blanchard, 1990). The entire sequences of the genomes of two strains of Ureaplasma parvum serovar 3 (formerly known as Ureaplasma urealyticum biovar 1; Glass et al., 2000) have been determined. At 752 kbp, the organ-isms share with other obligate symbions, such as Mycoplasma genitalium (580 kbp) and Buchnera aphidicola (641 kbp), greatly reduced genomes. Of 641–653 total genes, 32–39 code for struc-tural RNAs and about 610 code for proteins, about 47% of which have been classified as hypothetical genes of unknown function. Some (19%) resemble genes present in other genomes, but many (28%) appear unique. Unexpectedly absent in the Ureaplasma parvum genome are recognizable genes for FtsZ, for the GroEl and GroES chaperones, and for ribonucleoside-diphosphate reductase. Attempts have been made to reconcile the anomalies of gene functions assigned to this serovar of Ureaplasma parvum with the activities and pathways found in various ureaplasmas of humans (Glass et al., 2000; Pollack, 2001). One limitation to such analysis is that most of the physiological data that has been accumulated pertain not to Ureaplasma parvum but instead to the type species, Ureaplasma urealyticum strain T960T, possess-ing a 7% larger genome. The entire sequences of the genomes of Ureaplasma urealyticum serovars 8 and 10 (Glass et al., 2000, 2008) have also been determined and the genomes of the type strains of all remaining Ureaplasma urealyticum and Ureaplasma parvum serovars are nearly completely annotated (Glass et al., 2008). Preliminary comparisons found that in addition to “core” and “dispensable” genomes for each species, Ureaplasma urealyti-cum had partly duplicated multiple-banded antigen (mba) genes (Kong et al., 1999a, 2000), and up to twice the number of genes that Ureaplasma parvum has for lipoproteins. Such comparisons are expected to substantially improve understanding of ure-aplasmal pathogenicity and differential strain virulence.

Clinical studies based entirely on qualitative assessments of ureaplasmas are often difficult to interpret and only a few inves-tigators have presented quantitative data (Bowie et al., 1977; De Francesco et al., 2009; Heggie et al., 2001; Taylor-Robinson et al., 1977). Factors such as hormonal levels, specific genetic attributes, and even socio-economic conditions may encour-age urogenital colonization and proliferation. Nevertheless, it is clear that ureaplasmas are commensals that, on occasion, contribute to disease in susceptible human hosts. Infections attributed to ureaplasmas are often associated with an immu-nological component (Bowie et al., 1977), including the subset of the human population with common variable hypogam-maglobulinemia (Cordtz and Jensen, 2006; Furr et al., 1994; Lehmer et al., 1991; Webster et al., 1978), in which ureaplasma-induced septic arthritis and occasionally persistent ureaplasmal urethritis is seen (Taylor-Robinson, 1985). Sexually acquired, reactive arthritis is usually linked to Chlamydia trachomatis, but immunological evidence of ureaplasmal involvement exists (Horowitz et al., 1994). Through urea metabolism, ureaplasmas can induce crystallization of struvite and calcium phosphates

in urine in vitro and produce urinary calculi in animal models (Reyes et al., 2009). They are found in patients with infection stones more often than in those with metabolic stones (Grenabo et al., 1988). A statistical association with infection stones has been made (Kaya et al., 2003).

Evidence for the association of ureaplasmas with acute nongonococcal urethritis in men has been controversial, but a significant association of Ureaplasma urealyticum (but not Ure-aplasma parvum) with this disease in two of three recent studies suggests a way forward to resolving this issue. Evidence for a role for ureaplasmas in acute epididymitis is, at the most, mea-ger and it is very unlikely that they have a role in chronic prosta-titis. Ureaplasmas have been associated with bacterial vaginosis and pelvic inflammatory disease, but are unlikely to be causal in either condition. It follows, therefore, that there is no convinc-ing evidence to implicate ureaplasmas as an important cause of infertility in men or in couples. The most convincing data relating ureaplasmas to poor pregnancy outcomes have been seen when the organisms have been detected in amniotic fluid prior to membrane rupture, but there are conflicting opinions about the role of Ureaplasma urealyticum vs Ureaplasma parvum. Data since the 1980s have supported the association of neonatal ureaplasma infection with chronic lung disease and sometimes death in very low birth-weight infants. The ability of Ureaplasma parvum to induce chorioamnionitis and to contribute to pre-term labor and fetal lung injury is supported by experimental studies in rhesus monkeys (Novy et al., 2009).

Information on the range of animal species infected with ureaplasmas and their geographic distribution is patchy, possi-bly because ureaplasmas are largely avirulent or, at least, not an economic threat. Isolations have been reported from squirrel, talapoin, patas, macaque and green monkeys; as well as mar-mosets and chimpanzees (Taylor-Robison and Gourlay, 1984). Also, there are reports of isolation from domestic dogs, raccoon dogs, cats and mink; cattle, sheep, goats, and camels; chickens and other fowl; and swine (the latter needing confirmation). Baboons, rats and mice are susceptible to experimental infec-tion with ureaplasmas. While Ureaplasma diversum can inhabit all mucosal membranes of cattle, the two natural diseases it causes are subclinical respiratory infections in young calves, which occasionally develop into bronchopneumonia, and the eco-nomically important urogenital infections transmitted by bulls or their semen. The latter present as vulvovaginitis or ascend to cause infertility or abortion (Ruhnke et al., 1984; ter Laak et al., 1993). A species-specific PCR assay (Vasconcellos Cardosa et al., 2000) can circumvent culture insensitivity. As in humans, ureaplasmal diseases in animals may be influenced by the par-ticular ureaplasma strain or many other factors. Although ure-aplasmas may be present initially in certain organ and primary cell cultures, the lack of urea and higher pH in most eukaryotic cell culture systems would discourage persistence.

Although the spectrum of diseases of primary ureaplasmal etiology remains controversial, many potential virulence factors have been identified. Structural elements include a capsule, pilus-like fibrils, and the antigens of the outer membrane that constitute the serovar determinants. Erythrocytes from several animal species adhere firmly to colonies of certain strains of Ureaplasma parvum (Shepard and Masover, 1979), but most human isolates exhibit transient or no binding (Robertson and Sherburne, 1991). Strains of both Ureaplasma urealyticum and Ureaplasma parvum adhere to HeLa cells (Manchee and

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Taylor-Robinson, 1969) and to spermatozoa (Knox et al., 2003). Demonstration of beta-hemolysis of erythrocytes by ure-aplasmal products depends upon several variables; hemolysis of guinea pig erythrocytes has been most consistently observed (Black, 1973; Manchee and Taylor-Robinson, 1970; Shepard, 1967). Manchee and Taylor-Robinson (1970) described hemo-lysis of homologous erythrocytes by a canine ureaplasma as per-oxide-associated and blocked lysis by adding catalase, except in the presence of a catalase inhibitor. Genome sequencing has revealed genes resembling those for the hemolysins HlyA and HlyC of enterohemorrhagic Escherichia coli (Glass et al., 2000). Ammonia and ammonium ions generated by urea hydrolysis and the alkaline environment that they create are inhibitory to the ureaplasmas (Ford and MacDonald, 1967; Shepard and Lunceford, 1967). They have well-established deleterious effects on eukaryotic cell and mammalian tissue cultures and may be the “toxin” described but never substantiated (Furness, 1973). An IgA1 protease activity has been demonstrated by ureaplas-mas from both humans and canines. Human IgA1-specific pro-tease activity, apparently similar to the type 2 serine protease of certain bacterial pathogens of humans (Spooner et al., 1992), is produced by both Ureaplasma parvum and Ureaplasma urealyticum (Kilian et al., 1984; Robertson et al., 1984), but a gene responsi-ble for the activity has not yet been identified. Putative phopho-lipase A1, A2, and C activities (De Silva and Quinn, 1986) could not be confirmed, nor could such gene sequences be identified (J. Glass, unpublished). Biofilm production has been described recently (García-Castillo et al., 2008). Ureaplasmas have been seen intracellularly during studies in cell cultures (Mazzali and Taylor-Robinson, 1971), but may be there transiently after phagocytosis. Cells in culture (Li et al., 2000), in either experi-mental infection models (Moss et al., 2008) or epidemiological study subjects (Buss et al., 2003; Dammann et al., 2003; Jacobs-son et al., 2003; Shobokshi and Shaarawy, 2002), have exhibited proinflammatory responses. While assessment of clinical studies is sometimes difficult because of inherent reporting bias (Klas-sen et al., 2002; Schelonka et al., 2005), it is anticipated that the application of bioarray technologies will lead to improved understanding of ureaplasmal mechanisms of pathogenesis.

Reviews of in vitro methodologies used for antimicrobial susceptibility testing for both human (Bébéar and Robertson, 1996; Waites et al., 2001) and animal (Hannan, 2000) isolates are available. Antimicrobial susceptibility patterns of Ureaplasma urealyticum and Ureaplasma parvum appear to be similar (Matlow et al., 1998). The choice of therapeutic agents active against them is limited. Tetracyclines or the macrolides (excluding lin-comycin and clindamycin) are the bacteriostatic agents usually employed. Ureaplasmas from human, simian, bovine, caprine, feline, and avian sources (Koshimizu et al., 1983) withstand relatively high levels of lincomycin (10 mg/ml), to which most mycoplasmas are susceptible. Ureaplasmas also show in vitro resistance to rifampin and sulfonamides. Clinical resistance of ureaplasmas to tetracyclines has been long known (Ford and Smith, 1974). This high-level resistance is determined by the presence of the tetM determinant (Roberts and Kenny, 1986), which is now readily identified by PCR (Blanchard et al., 1997). Tetracycline-resistant strains exposed to a variety of antibiot-ics demonstrate a broad range of responses (Robertson et al., 1988). Clinical resistance to macrolides has also been reported (e.g., Taylor-Robinson and Furr, 1986). Certain aminoglyco-

sides, chloramphenicol, and newer fluoroquinolones inhibit ureaplasmas, but are inappropriate for broad clinical use. Fluo-roquinolone resistance is increasing (Xie and Zhang, 2006) and has been found in a previously susceptible strain (Duffy et al., 2006). Ureaplasma strains exhibiting resistance to multiple anti-biotics have been found in immunocompromised hosts. In one case, the isolates were of the same serovar, but exhibited differ-ent susceptibility patterns at different anatomical sites (Lehmer et al., 1991). Because of ongoing changes in antimicrobial sus-ceptibility patterns, the recent literature should be consulted (e.g., Beeton et al., 2009). To treat natural Ureaplasma diversum infections, tiamulin hydrogen fumarate, a diterpene agent in common use in veterinary medicine, may be at least as effective as the macrolide tylosin (Stipkovits et al., 1984). Others have examined the efficacy of single or combinations of antibiotics in eradicating ureaplasmas from various sites in cattle as well as from semen used for artificial insemination (ter Laak et al., 1993). The urease inhibitor, fluorofamide, has been used with varying success in eliminating ureaplasmas from animals.

When establishing antibiograms for ureaplasmas, the require-ments established for common bacterial pathogens do not suf-fice. First, medium components may affect antibiotic activity. For instance, serum-binding reduces tetracycline activity, while the initial acidic culture reduces macrolide activity against ure-aplasmas (Robertson et al., 1981). Conventional bacteria of established, low-level susceptibility can be used to measure the effect of the ureaplasmal medium on a particular antimicro-bial agent. Second, the relatively slow growth of ureaplasmas as compared with many pathogenic bacteria requires that the half-life of the antibiotic be considered when test inoculum and incubation period are established. Lastly, the end points of the sensitivity tests on agar are about four-fold lower than for tests in broth (Waites et al., 1991). On consideration of the in vivo environment in which these organisms naturally occur, inter-pretation of susceptibility test end points continues to present a challenge. An international subcommittee under the aegis of the National Committee for Clinical Laboratory Standards Institute (USA) is currently finalizing a “Final Report for Devel-opment of Quality Control Reference Standards and Methods for Antimicrobial Susceptibility Testing” for Ureaplasma and Mycoplasma species infecting humans that have demonstrated variability in response to antimicrobial agents.


Media formulations in current use for the cultivation of ureaplasmas from human sources include, in order of decreasing supplementation: the 10B broth of Shepard and Lunceford (Shepard, 1983); Taylor-Robinson’s broth (Taylor-Robinson, 1983a), made without thallium acetate; and bromothymol blue broth (Robertson, 1978). Ureaplasmas are exquisitely sensitive to thallium acetate and it should not be used to inhibit other bacte-ria. The U4 formulation for ungulate isolates was developed by Howard et al. (1978). Consult Waites et al. (1991) for additional media formulations for isolation from humans, and Shepard (1983), Livingston and Gauer (1974), or Hannan (2000) for iso-lation from animals. The quality of medium components may be as important as the medium formulation. Serum supplements should be tested for their ability to support growth of the ure-aplasmas of interest. Liquid medium may be stored at −20°C until required. Transport medium (e.g., 2SP) should be free of antibi-

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otics inhibitory to ureaplasmas. Broth cultures should be diluted to ³1:100 to reduce effects of any growth inhibitors present in the specimen. In general, Ureaplasma parvum is less demanding nutri-tionally than Ureaplasma urealyticum and isolates from human and other animal sources together are less demanding than those from ungulate hosts. Agar surfaces are examined at ³40× magni-fication. The less fastidious strains (e.g., Ureaplasma parvum type strain) are more likely to produce recognizable “fried-egg” colo-nies than are the more fastidious strains (e.g., Ureaplasma urealyti-cum type strain), which are more likely to produce “cauliflower head” or core colonies. Cultures may be thrice cloned and the resultant culture used to initiate stocks.

Maintenance procedures

Broth cultures of ureaplasmas commonly become sterile within 12–24 h incubation at 35–37°C. “Red is dead” was the mantra of Shepard in regard to his phenol red-containing broth medium. One way to lessen this problem is to change to an indicator that changes color at a lower pH (Robertson, 1978). To Shepard’s mantra we might add, “the higher the urea concentration, the steeper the death phase”, an effect not countered by buffer. Incu-bation of diluted cultures at 30–34°C slows growth and main-tains viability for up to 1 week, reducing the frequency of culture transfer and helping in transport. Short exposure of broth cul-tures to refrigeration reduces viability. Cells within colonies on solid medium may be recovered for approximately 1 week if the cultures are removed from incubation when growth is detect-able and stored under cool, humidified conditions. For long-term storage, ultra-low temperatures (−80°C or liquid nitrogen) with a cryoprotectant [e.g., 10–20% (v/v) sterile glycerol] can maintain viability for well over a decade. For lyophilization, the pellets from broth cultures centrifuged at high speed are resus-pended in a minimal volume of liquid medium or the serum used for its supplementation before processing; on reconstitu-tion, urease activity occasionally is not immediately evident.

Differentiation of the genus Ureaplasma from other genera

Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. Aerobic or facultatively anaerobic growth in artificial media and the necessity for sterols for growth exclude assignment to the genera Anaeroplasma, Asteroleplasma, Achole-plasma, and “Candidatus Phytoplasma”. Non-spiral cellular mor-phology and regular association with a vertebrate host or fluids of vertebrate origin support exclusion from the genera Spiro-plasma, Entomoplasma, and Mesoplasma. The ability to hydrolyze urea, with the inability to metabolize either glucose or arginine, excludes assignment to the genus Mycoplasma. For routine pur-poses, the colonial morphology characteristic of ureaplasmas and demonstration of the isolate’s ability to catabolize urea, using the urease spot test or indicator agar, suffice for prelimi-nary differentiation from other mollicutes. Other methods for urease detection have been largely replaced by PCRs that detect urease genes.

Taxonomic comments

The hypothetical evolutionary relationships of the Mollicutes have been based primarily upon 16S rRNA gene sequences (Weisburg et al., 1989). The nucleotide sequence for the 16S rRNA genes (Kong et al., 1999b; Robertson et al., 1993;

Robertson et al., 1994) and the 16S–23S rRNA intergenic spacer regions of all named species plus the 14 serovars associated with humans (Ureaplasma urealyticum and Ureaplasma parvum) have been determined (Harasawa and Kanamoto, 1999; Kong et al., 1999b). The genus Ureaplasma comprises two subclusters within the highly diverse pneumoniae group of the family Mycoplasma-taceae (Johansson, 2002). One subcluster contains the human, avian, and mink isolates and the other contains feline, canine, and bovine strains (see genus Mycoplasma Figure 109, pneumo-nia group) Although the three serovars A, B, and C of Ureaplasma diversum are antigenically heterogeneous, the strains examined meet the 70% DNA–DNA hybridization benchmark used as an arbitrary species criterion. However, the available DNA–DNA hybridization values suggest that Ureaplasma gallorale and Ure-aplasma canigenitalium might each represent more than a single species. The 16S rRNA gene sequences of ureaplasmas isolated from nonhuman primates and some less-studied vertebrates are unknown. The range of G+C contents of ureaplasmal DNA is too narrow to have much taxonomic utility. Nevertheless, the values for isolates from cattle, sheep, and goats are between 28.7 and 31.6 mol% (Howard et al., 1978), at the higher end of the range for the genus.

It is generally assumed that all ureaplasmas from avian sources belong to the species Ureaplasma gallorale. The seven avian isolates examined were similar to each other serologically and in SDS-PAGE, immunoblot, and RFLP profiles, and they were distinct from Ureaplasma urealyticum and Ureaplasma diver-sum, although resemblances between Ureaplasma gallorale and Ureaplasma urealyticum based upon one- and two-dimensional PAGE analyses have been reported (Mouches et al., 1981). How-ever, the DNA–DNA hybridization values among Ureaplasma gal-lorale strains fall into two clusters. Homology was 70–100% for the five strains within cluster A and 96–100% for the two strains within cluster B that were studied. Between strains of cluster A and B, homology was only 51–59% and, vice versa, 52–69% (Harasawa et al., 1985). The taxonomic status of avian ureaplas-mas, therefore, might benefit from examination of additional strains and reconsideration. The clusters appear to be more closely related than Ureaplasma urealyticum and Ureaplasma par-vum (Table 139).

The taxonomy of ureaplasmas of canines is also problematic. The first report indicated G+C contents of 27.2–27.8 mol% (Bd; Howard et al., 1978). Later, the representatives of four serogroups (SI to SIV for strains DIM-C, D29M, D11N-A, and D6P-CT, respectively) were reported to have G+C contents of 28.3–29.4 mol% (HPLC). However, the published data regard-ing their DNA reassociation values are confusing. Initially, Barile (1986) and Harasawa et al. (1990b) reported that the serogroup SI representative, DIM-C, had 73% homology with the serotype 2 SII representative, D29M, indicating that these two strains likely belong to the same species. However, perhaps because of the borderline value, further work was undertaken, also using [3H]DNA–DNA hybridization procedures (Harasawa et al., 1993). The values obtained ranged from 41 to 63% homol-ogy among the four serogroups, i.e., each serogroup seemed to represent a distinct species. At present, the only named canine ureaplasma species is Ureaplasma canigenitalium; the SIV rep-resentative D6P-CT is the type strain (Harasawa et al., 1993). Its degree of distinctiveness from the other serogroups is not emphasized in the literature.

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Table 139. Genotypic characteristics that partition the serovar-standard strains of ureaplasmas isolated from humans to the level of speciesa

Characteristic U. urealyticum serovars U. parvum serovars Reference(s)

DNA–DNA relatedness with strain 27T DNA: Christiansen et al. (1981)91–102% 1, 3T, 638–60% 2, 4, 5, 7, 8T

DNA–DNA relatedness with strain T960T DNA: Christiansen et al. (1981)49–52% 1, 3T, 669–100% 2, 4, 5, 7, 8T

DNA–DNA relatedness with strain 27T DNA: Harasawa et al. (1991)75–100% 1, 3T, 6, 1438–57% 2, 4, 5, 7, 8T, 9–13

DNA–DNA relatedness with strain T960T DNA: Harasawa et al. (1991)48–59% 1, 3T, 6, 1476–101% 2, 4, 5, 7, 8T, 9–13

Cleavage of DNA by Fnu4HI: Cocks and Finch (1987)Yes 1, 3T, 6No 2, 4, 5, 7, 8T, 9

BamHI, HindIII, and PstI RFLP probed with RNA genes: Razin (1983)Biovar l pattern 1, 3T, 6Biovar 2 pattern 2, 4, 5, 7, 8T, 9

EcoRI and HindIII RFLP probed with RNA genes: Harasawa et al. (1991)Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13b

Genome sizes determined by PFGE: Robertson et al. (1990)~760 kbp 1, 3T, 6, 14840–1140 kbp 2, 4, 5, 7, 8T, 9–13

Heterogeneity of alpha polypeptide-associated urease genes: Blanchard (1990)Yes 1, 3T, 6, 10c, 12c, 14No 2, 4, 5, 7, 8T, 9, 13

Heterogeneity of HindIII site in subunit ureC of urease gene: Neyrolles et al. (1996)Absent 1, 3T, 6Present 2, 8T

Heterogeneity of HindIII fragments probed with serovar 8IC61 urease probe:

Neyrolles et al. (1996)

Biovar 1 pattern 1, 3T, 6Biovar 2 pattern 2, 8T

Heterogeneity of urease subunit-associated genes:d Kong et al. (1999b)Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

Heterogeneity of biovar-specific 16S rRNA genes determined by PCR:d

Robertson et al. (1993)

Strain 27T 1, 3T, 6, 14Strain T960T 2, 4, 5, 7, 8T, 9–13

Biovar-specific 16S rRNA gene sequences:d Robertson et al. (1994)Strain 27T sequence 1, 3T, 6, 14Strain T960T sequence 2, 5, 8T

16S rRNA, spacer regions, and urease subunit sequences:d Kong et al. (1999b)Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

RFLP of 5¢ region of mba genes: Teng et al. (1994)Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

5¢ end sequences of mba genes:d,e Kong et al. (1999b)Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

16S–23S intergenic spacer region:d Harasawa and Kanamoto (1999), Kong et al. (1999b)

Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

Arbitrarily primed PCR:d Grattard et al. (1995)Biovar 1 pattern 1, 3T, 6, 14Biovar 2 pattern 2, 4, 5, 7, 8T, 9–13

aT, Type strain of species.bSerovar 13 response to EcoRI was anomalous.cThis anomalous pattern resulted from cultures being misidentified as serovars 10 and 12; the error was corrected by Teng et al. (1994). Kong et al. (1999b) have con-firmed the efficacy of the Blanchard (1990) primers.dPrimers used for biovar-defining PCR(s).eThe multiple band (MB) antigens seen by PAGE are putative virulence markers. See text for details.


While DNA–DNA hybridization has been key to species level identification for the genus Ureaplasma, serology led to our knowl-edge of intra-species relationships (Robertson et al., 2002) and still figures importantly. Much interest has been focused specifi-cally on the multiple-banded antigens (MBA) of the ureaplas-mal cell surface. Watson et al. (1990), studying the type strain (serovar 3) of Ureaplasma parvum, identified the predominant ureaplasmal antigens recognized by the host during human infection. The MBA were first seen as unusual, laddered bands in immunoblots. These lipoproteins exhibited epitopes for both serovar specificity and cross-reactivity, and showed in vitro size variation (Teng et al., 1994; Zheng et al., 1995). Monecke et al. (2003) added evidence that the MBA are involved in a phase-switching process similar to that identified in several Mycoplasma species. The mba gene sequences have since been used to further define ureaplasma phylogeny (Knox et al., 1998; Kong et al., 1999b), as well as to characterize isolates (Knox et al., 1998; Knox and Timms, 1998; Kong et al., 1999a; Pitcher et al., 2001). The sequences of the 5¢ ends of the mba genes of all 14 serovars have been defined (Kong et al., 1999a, 2000). Kong et al. (2000) found that for Ureaplasma parvum, a specific site on the gene determines serovar identity, whereas for Ureaplasma urealyticum at least the sequence of the 3¢ end is required and, for certain other serovars, the entire sequence is involved.

The genes for the three urease subunits, ureA, ureB, and ureC, and adjoining regions from many isolates have been sequenced (Blanchard, 1990; Kong et al., 1999b; Neyrolles et al., 1996; Ruifu et al., 1997). Rocha and Blanchard (2002) applied bioin-formatics to predict how certain gene products (e.g., the MBA, restriction and modification systems, transcription anti-termi-nation elements, and GTP-binding proteins) might exhibit species specificity. PCRs based upon the 16S rRNA genes are the most highly conserved, followed by the 16S–23S intergenic region, the urease-associated genes, the mba genes, and the region upstream of them.

acknowledgements

We thank J. Glass and collaborators at the J. Craig Venter Insti-tute and the University of Alabama - Birmingham for the gift of the genome sequences of Ureaplasma parvum and Ureaplasma urealyticum, and J.G. Tully, K.E. Johansson, and C. Williams for their suggestions regarding the initial chapter.

Differentiation of the species of the genus Ureaplasma

The first paper on ureaplasma taxonomy stated that “An advan-tage of forming a new genus is that it confers freedom to classify new species within the genus without adhering to the principles formulated for the other genera. A numbered serovar of a Ure-aplasma from humans is broadly equivalent to a named species within the genera Mycoplasma or Acholeplasma” (Shepard et al., 1974). The serological diversity within the type strain, Ureaplasma urealyticum, was regarded as no more than antigenic heterogene-ity, possibly reflecting minor differences within a single epitope. To ensure that taxonomy would develop rationally, official rec-ognition of Ureaplasma subspecies was avoided. Thirty-five years later, this taxonomic restraint can be appreciated.

When the first genus and species, Ureaplasma urealyticum, was named, it had eight known antigenic specificities (Shepard et al., 1974); 6 years later, the number had reached 14 (Robert-son and Stemke, 1982) where, surprisingly, it has remained. It is surprising because the serotypes were isolated over a 20-year

period representing antigens in Vancouver, BC, Canada (Ford, 1967), Camp Lejeune, NC (Shepard, 1954), and Boston, MA, USA (Lin et al., 1972), i.e., on the west and east coasts of North America. While putative untypable strains are occasionally encountered, after cloning, these have usually turned out to be serovar 3, probably the most likely to dominate in a mixed cul-ture. However, additional serovars/genovars can be expected to emerge, especially from other parts of the world.

The named species were identified primarily by DNA–DNA hybridization and by serological tests. For taxonomic studies, the metabolism inhibition test (Purcell et al., 1966; Robertson and Stemke, 1979; Taylor-Robinson, 1983b) and either a direct or indirect immunofluorescence test (Black and Krogsgaard-Jensen, 1974; Piot, 1977; Stemke and Robertson, 1981) have been most useful. However, serological tests sometimes gave confusing cross-reactivity patterns and were not ideal for differ-entiating strains within a single species (Stemke and Robertson, 1985). In an attempt to circumvent problems of cross-reactions obtained with polyclonal antisera, monoclonal antibodies (mAbs) to all 14 serovars of human isolates were developed (e.g., Echahidi et al., 2001). The use of mAbs coincided with and has been largely overshadowed by the genomic revolution.

In the 1980s, the same pattern of partitioning of the serovars of human isolates was demonstrated by other phenotypic traits, traits primarily related to protein structures and functions (Table 138). When phenotypy failed to deliver a clear and convenient means of discrimination, genotypy did. The initial DNA–DNA relatedness studies of ureaplasmas from humans (Christiansen et al., 1981) confirmed the two, distinct clusters; however, the large cell biomass, special equipment, and (then) rare expertise required resulted in few strains being tested. New techniques, especially PCR, became more easily performed and less expensive so that more strains were examined and non-ambiguous results were obtained (Table 139). Supported by these strong data, the taxonomy of Ureaplasma urealyticum was emended and extended (Robertson et al., 2002). The ten anti-genic specificities of the larger cluster (known as group 2 or as the T960 biovar) retained the Ureaplasma urealyticum designa-tion with strain T960T as the type strain. The remaining four antigenic specificities (known as group 1 or the parvo biovar) were renamed Ureaplasma parvum in recognition of that clus-ter’s considerably smaller genome size; strain 27T, the serovar 3 standard, was designated the type strain. Many PCR primers to identify species and strains of ureaplasmas have been pub-lished; commercial PCR-based kits are currently available for all named Ureaplasma species.

In summary, current ureaplasma taxonomy is based upon pragmatic, polyphasic criteria, i.e., a synthesis of phylogeny, phenotypy, and genotypy. For the specific requirements for taxonomic studies of Mollicutes, consult the most recent mini-mal standards document (Brown et al., 2007). Some serological testing is mandatory. Rabbit antisera to the 14 serovars of ure-aplasmas of humans and certain animal species are currently available from Jerry K. Davis, Curator of the Mollicutes Collec-tion, School of Veterinary Medicine, Purdue University, West Lafayette, IN, USA. Expertise in determining phenotypic traits specific to ureaplasmas may be accessible through collaboration with the appropriate working team of the International Research Programme for Comparative Mycoplasmology (IRPCM) of the International Organization for Mycoplasmology (IOM) at www.the-iom.org.

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http://www.the-iom.org

http://www.the-iom.org


list of species of the genus Ureaplasma

1. Ureaplasma urealyticum Shepard, Lunceford, Ford, Purcell, Taylor-Robinson, Razin and Black 1974, 167AL emend. Rob-ertson, Stemke, Davis, Harasawa, Thirkell, Kong, Shepard and Ford 2002, 593

u.re.a.ly¢ti.cum. N.L. fem. n. urea urea; N.L. adj. lyticus -a -um (from Gr. adj. lutikos -ê -on) able to loosen, able to dissolve; N.L. neut. adj. urealyticum urea- dissolving or urea-digesting.

Cells are coccoid and approximately 500 nm in diameter. Coccobacillary forms are seen in exponential phase cultures. One strain has been shown to have a carbohydrate- containing capsule; it and others contain lipoglycans. Grows at tempera-tures between 20 and 40°C, grows better at 30–35°C and best at 36–37°C. Colonies are £20–50 mm in diameter with com-plete or partial fried-egg morphology.

Serologically distinct from all other named species in the genus, but serologically heterogeneous. Ten specific anti-genic determinants are known: 2, 4, 5, and 7–13. Multiple-banded antigens are serovar-related and recognized by the host. Like Ureaplasma parvum, it has human IgA-specific pro-tease activity that specifically cleaves human IgA1, but not human IgA2. Has distinctive PAGE and RFLP patterns. DNA is not restricted by endonuclease Uur9601. Genome size of the type strain is 890 kbp, whereas the sizes of the 10 known serovar standard strains range from 840 to 1140 kbp (PFGE). DNA reassociation values: within the species (serovars 2, 4, 5, and 7), 69–100%; with Ureaplasma parvum (serovars 1, 3, and 6), 49–52%. Opportunistic pathogen of humans; causes some cases of nongonococcal urethritis, infectious kidney stones, systemic infection in immunologically compromised hosts. Associated with a broad variety of urogenital infections for which causality remains to be established.

Source: primarily found in the genitourinary tract of female and male humans; occasionally in the oral cavity and rectum.

DNA G+C content (mol%): 25.5–27.8 (Tm; type strain) and 27.7–28.5 (Bd; serovars 2, 4, 5, and 7).

Type strain: T960, (CX8), ATCC 27618, NCTC 10177.Sequence accession nos: M23935 and AF073450 (type strain 16S

rRNA gene), AB028088 and AF059330 (type strain 16S–23S rRNA intergenic region). Complete and near-complete (>99%) genomes: serovar 2 strain ATCC 27814, NZ_ABFL00000000; serovar 4 strain ATCC 27816, NZ_AAYO00000000; serovar 5 strain ATCC 27817, NZ_AAZR00000000; serovar 7 strain ATCC 27819, NZ_AAYP00000000; serovar 8 strain ATCC 27618, NZ_AAYN00000000; serovar 9 strain ATCC 33175, NZ_AAYQ00000000; serovar 10 strain ATCC 33699, NC_011374; serovar 11 strain ATCC 33695, NZ_AAZS00000000; serovar 12 strain ATCC 33696, NZ_AAZT00000000; serovar 13 strain ATCC 33698, NZ_ABEV00000000.

2. Ureaplasma canigenitalium Harasawa, Imada, Kotani, Koshi-mizu and Barile 1993, 644VP

ca.ni.ge.ni.ta¢li.um. L. n. canis dog; L. pl. n. genitalia the geni-tals; N.L. pl. gen. n. canigenitalium of canine genitals.

Cells are coccoid and about 500 nm in diameter; cocco-bacillary forms are seen. Colonies are £20–140 mm diameter with fried-egg morphology. Serogroup I strains represented by D6P-CT are serologically distinct from all other established species in the genus and from the other three serogroups of

ureaplasmas isolated from dogs (represented by the strains DIM-C, D29M, and D11N-A). The species designation refers only to serogroup I strains, although strain D11N-A shows a one-way, serological cross-reaction with D6P-CT. It produces an IgA protease which specifically cleaves canine myeloma IgA, but not human or murine IgA. Genome size is 860 kbp (PFGE). DNA reassociation values: between D6P-CT and the other three canine strains (DIM-C, D29M, and D11N-A) are 41–63% versus 33% with Ureaplasma urealyticum (strain T960T).

Source: habitat is the prepuce, vagina, and oral and nasal cavities of canines.

DNA G+C content (mol%): 29.4 (HPLC).Type strain: D6P-C, ATCC 51252, CIP 106087.Sequence accession no. (16S rRNA gene): D78648 (type strain).

3. Ureaplasma cati Harasawa, Imada, Ito, Koshimizu, Cassell and Barile 1990a, 50VP

ca¢ti. L. gen. n. cati of a cat.

Cells are coccoid and ³675 nm diameter, exceeding the 450–550 nm range of most named Ureaplasma species. Coc-cobacillary forms are seen and occasionally filaments. Col-onies are £15–140 mm in diameter with diffuse, granular appearance; some fried-egg colonies may appear after pas-saging. Distinct from other established species in the genus, including Ureaplasma felinum, antigenically and in PAGE (Harasawa et al., 1990a) and RFLP patterns (Harasawa et al., 1984). Genome size has not been determined. DNA reasso-ciation values: 83–100% within feline serogroup SII strains (Ureaplasma cati) versus <10% with serogroup SI strains (Ure-aplasma felinum).

Source: found in the oral cavity of healthy domestic cats (Felis domestica).

DNA G+C content (mol%): 27.9 (Bd), 28.1 (HPLC) for strain F2T.

Type strain: F2, ATCC 49228, NCTC 11710, CIP 106088.Sequence accession nos: D78649 (type strain 16S rRNA gene),

D63685 (type strain 16S–23S rRNA intergenic spacer region).

4. Ureaplasma diversum Howard and Gourlay 1982, 450VP

di.ver¢sum. L. neut. part. adj. diversum different, distinct, het-erogeneous, referring to the difference in polypeptides and G+C content as compared to Ureaplasma urealyticum and to the heterogeneous antigenic structure of the species.

Cells are coccoid or coccobacillary and appear to be within the size range of other named Ureaplasma species although no measurements have been published. Colonies are £100–175 mm in diameter based on photomicrographs.

Serologically distinct from other named species but anti-genically heterogeneous, comprising serogroups A, B, and C, and represented by strains A417T, D48, and T44. These show three distinctive PAGE patterns (Howard and Gourlay, 1982), but only one RFLP pattern (Harasawa et al., 1984), and, based upon the latter criterion, were considered homogeneous.

Genome size range is 1100–1160 kbp for strains 95 TX, 1763, and 2065-B202 (PFGE). No DNA reassociation values are available.

Source: the type strain originated from a pneumonic calf lung.

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DNA G+C content (mol%): 29.0 and 28.7–30.2 (Bd) for the type strain and 10 bovine isolates, respectively; thus, higher than and not overlapping the values for Ureaplasma urealyti-cum and Ureaplasma parvum.

Type strain: A417, ATCC 43321, NCTC 10182, CIP 106089.Sequence accession nos: D78650 (type strain 16S rRNA

gene), D63686 (type strain 16S–23S rRNA intergenic spacer region).

5. Ureaplasma felinum Harasawa, Imada, Ito, Koshimizu, Cassell and Barile 1990a, 50VP

fe.li¢num. L. neut. adj. felinum of or belonging to a cat.

Coccoid cells of ³800 nm diameter exceed the 450–500 nm range of most Ureaplasma species. Coccobacillary forms and occasional filaments are seen. Colonies are £15–140 mm diam-eter with diffuse, granular appearance; some fried-egg colo-nies may appear after passaging. Distinct antigenically and by PAGE and RFLP patterns (Harasawa et al., 1984) from other established species in the genus, including Ureaplasma cati. The genome size is 1170 kbp (PFGE). It is the largest of any ure-aplasma strain examined. DNA reassociation values: 89–100% within feline serogroup SI strains (Ureaplasma felinum) versus <10% with serogroup II strains (Ureaplasma cati).

Source: found in the oral cavity of healthy domestic cats (Felis domestica).

DNA G+C content (mol%): 27.9 (HPLC).Type strain: FT2-B, ATCC 49229, NCTC 11709, CIP

106090.Sequence accession nos: D78651 (type strain 16S rRNA gene),

D63687 (type strain 16S–23S rRNA intergenic spacer region).

6. Ureaplasma gallorale Koshimizu, Harasawa, Pan, Kotani, Ogata, Stephens and Barile 1987, 337VP

gal.lo.ra¢le. L. n. gallus a barnyard fowl; L. n. os, oris the mouth; L. neut. suff. -ale suffix used with the sense of per-taining to; N.L. neut. adj. gallorale relating to the mouth of barnyard fowl.

Cells are coccoid and about 500 nm in diameter; coccoba-cillary forms are seen. Colonies are £15–60 mm in diameter with fried-egg morphology. Serologically distinct from all other established species in the genus. Isolates have similar SDS-PAGE, immunoblot, and RFLP patterns, but demon-strate some species heterogeneity based on reassociation val-ues (cluster A strains D6-1T and T9-1; cluster B strain Y8-1). Genome size is 760 kbp (PFGE). DNA reassociation values fall into two clusters: within cluster A (strains D6-1T, D23, F2, F5, and T9-1), values are 70–100%; within cluster B (strains Y8-1 and Y4-2), values are 96–100%; between these clusters, values are 51–69%. Although these values are below expectations for a single species status, they exceed the 19–27% reassociation values with Ureaplasma urealyticum and Ureaplasma diversum.

Source: found only in oropharynx of healthy red jungle fowl (Gallus gallus) and chickens (Gallus gallus var. domesticus) kept as laboratory or zoo animals in Japan and in chickens and turkeys with pneumonia or airsacculitis in Hungary.

DNA G+C content (mol%): 27.6 (HPLC).Type strain: D6-1, ATCC 43346, NCTC 11707.Sequence accession nos: U62937 (type strain 16S rRNA

gene), D63688 (type strain 16S–23S rRNA intergenic spacer region).

7. Ureaplasma parvum Robertson, Stemke, Davis, Harasawa, Thirkell, Kong, Shepard and Ford 2002, 593VP

par¢vum. L. neut. adj. parvum small, referring to its significantly smaller genome sizes compared to Ureaplasma urealyticum, the other species from humans.

Cells are coccoid and about 500 nm in diameter. Cocco-bacillary forms are present in exponential phase cultures. Lipoglycans have been identified in a serovar 3 strain. Colo-nies are £20–140 mm in diameter with complete or partial fried-egg morphology.

Serologically distinct from all other named species in the genus, but serological heterogeneity is exhibited within the species. Four specific antigenic determinants are known: 1, 3, 6, and 14. Serovar specificities are related to the multiple-banded antigens recognized by the host. Like Ureaplasma urealyticum, Ureaplasma parvum has an IgA protease activity that specifically cleaves human IgA1, but not human IgA2. Distinctive PAGE and RFLP patterns. DNA restricted by endonuclease Uur9601. Genome size: 751,719 kbp for the type strain. Complete genomic sequence of the organism has been reported (Glass et al., 2000). DNA reassociation values: within the species (serovars 1, 3, and 6), 91–102%; with Urea-plasma urealyticum (serovars 2, 4, 5, 7–13), 38–60%.

Source: primary habitat is the genitourinary tract of female and male humans; occasionally found in the oral cavity and rectum. As yet, unclear whether an opportunistic pathogen in non-gonococcal urethritis, but likely to be so in systemic infection in immunologically compromised hosts. Associ-ated with a broad variety of urogenital infections for which causality remains to be established.

DNA G+C content (mol%): 25.5 (from genome sequence) and 27.8–28.2 (Tm) for serovars 1 and 6.

Type strain: 27, ATCC 27815, NCTC 11736.Sequence accession nos: L08642 and AF073456 (type strain

16S rRNA gene), AB028083 and AF059323 (type strain 16S–23S rRNA intergenic spacer region). Complete and near-complete (>99%) genomes: serovar 1 strain ATCC 27813, NZ_ABES00000000; serovar 3 strain ATCC 27815, NC_010503; serovar 3 strain ATCC 700970, NC_002162; serovar 6 strain ATCC 27818, NZ_AAZQ00000000; serovar 14 strain ATCC 33697, NZ_ABER00000000.

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638

FaMIly II. IncerTae seDIs

Family II. incertae sedis

Daniel R. BRown, séveRine tasKeR, Joanne B. MessicK anD haRolD neiMaRK

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Yechouron, A., J. Lefebvre, H.G. Robson, D.L. Rose and J.G. Tully. 1992. Fatal septicemia due to Mycoplasma arginini: a new human zoonosis. Clin. Infect. Dis. 15: 434–438.

Yoder, H.W. and M.S. Hofstad. 1964. Characterization of avian myco-plasmas. Avian Dis. 8: 481–512.

Yogev, D., S. Levisohn and S. Razin. 1989. Genetic and antigenic relat-edness between Mycoplasma gallisepticum and Mycoplasma synoviae. Vet. Microbiol. 19: 75–84.

Zheng, X., L. Teng, H. Watson, J. Glass, A. Blanchard and G. Cassell. 1995. Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with anti-gen size variation. Infect. Immun. 63: 891–898.

Zheng, X., D.A. Olson, J.G. Tully, H.L. Watson, G.H. Cassell, D.R. Gustafson, K.A. Svien and T.F. Smith. 1997. Isolation of Myco-plasma hominis from a brain abscess. J. Clin. Microbiol. 35: 992–994.

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Family ii. incertae sedis

Genus i. Eperythrozoon schilling 1928, 293al

Daniel R. BRown, SéveRine TaSkeR, Joanne B. MeSSick anD HaRolD neiMaRk

e.pe.ry.thro.zo¢on. Gr. pref. epi on; Gr. adj. erythros red; Gr. neut. n. zoon living being, animal; n.l. neut. n. Eperythrozoon (presumably intended to mean) animals on red (blood cells).

Cells adherent to host erythrocyte surfaces are coccoid and about 350 nm in diameter, but may arrange to appear as chains or deform to appear rod- or ring-shaped in stained blood smears.

Type species: Eperythrozoon coccoides Schilling 1928, 1854.


Hemotropic mollicutes such as the species formerly called Eperythrozoon coccoides (trivial name, hemoplasmas; Neimark et al., 2005) infect a variety of mammals occasionally includ-ing humans. Transmission can be through ingestion of infected blood, percutaneous inoculation, or by arthropod vectors (Sykes et al., 2007; Willi et al., 2006). The pathogenicity of dif-ferent hemoplasma species is variable, and strain virulence and host immunocompetence likely play roles in the development of disease. Clinical syndromes range from acute fatal hemolytic anemia to chronic insidious anemia. Signs may include ane-mia, pyrexia, anorexia, dehydration, weight loss, and infertil-ity. The presence of erythrocyte-bound antibodies has been demonstrated in some hemoplasma-infected animals and may contribute to anemia. Animals can remain chronic asymptom-atic carriers of hemoplasmas after acute infection. PCR is the diagnostic test of choice for hemoplasmosis. Tetracycline treat-ment reduces the number of organisms in peripheral blood, but probably does not eradicate the organisms from infected animals.


Hemoplasmas have not yet been successfully grown in continu-ous culture in vitro, although recent work (Li et al., 2008) sug-gests that in vitro maintenance of the species Mycoplasma suis may be possible.

maintenance procedures

Hemoplasmas can be frozen in heparin- or EDTA-anticoagu-lated blood cryopreserved with dimethylsulfoxide.

differentiation of the genus Eperythrozoon from other genera

A distinctive characteristic of these organisms is that they are found only in the blood of vertebrate hosts or transiently in arthropod vectors of transmission. The tenuous distinction between species of Eperythrozoon and those of Haemobartonella was based on the relatively more common visualization of eperythrozoa as ring forms (now known to be artifactual) in stained blood smears and the perception that eperythrozoa were observed with about equal frequency on erythrocytes and free in plasma, while haemobartonellae were thought to occur less often free in plasma. Properties that partially fulfill criteria for assignment of this genus to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. Presumptive use of the codon UGA to encode tryptophan (Berent and Messick, 2003) supports exclusion from the genera Anaeroplasma, Asteroleplasma, Acholeplasma, and “Candidatus Phytoplasma”. Non- spiral cellular

morphology and regular association with vertebrate hosts support exclusion from the genera Spiroplasma, Entomoplasma, and Mesoplasma, but sterol requirement, the degree of aerobio-sis, and the capacity to hydrolyze arginine, characteristics that would help to confirm their provisional 16S rRNA-based place-ment in the genus Mycoplasma, remain unknown.

taxonomic comments

The taxonomy and nomenclature of the uncultivated hemotro-pic bacteria originally assigned to the genus Eperythrozoon remain matters of current controversy. It is now undisputed that, on the basis of their lack of a cell wall, small cell size, low G+C content, use of the codon UGA to encode tryptophan, regular association with vertebrate hosts, and 16S rRNA gene sequences that are most similar (80–84%) to species in the pneumoniae group of genus Mycoplasma, these organisms are properly affili-ated with the Mycoplasmatales. However, the proposed trans-fers of Eperythrozoon and Haemobartonella species to the genus Mycoplasma (Neimark et al., 2001, 2005) were opposed on the grounds that the degree of 16S rRNA gene sequence similarity is insufficient (Uilenberg et al., 2004, 2006). The alternative of situating the hemoplasmas in a new genus in the Mycoplasmata-ceae (Uilenberg et al., 2006) would regrettably compound the 16S rRNA gene-based polyphyly within Mycoplasma on no other basis than a capacity to adhere to the surface of erythrocytes in vivo.

The proposed transfer of the type species Eperythrozoon coccoides to the genus Mycoplasma (Neimark et al., 2005) is complicated by priority because Eperythrozoon predates Myco-plasma. However, the alternative of uniting the genera by trans-ferring all mycoplasmas to the genus Eperythrozoon is completely unjustifiable considering the biological characteristics of the non-hemotropic majority of Mycoplasma species. The Judicial Commission of the International Committee on Systematics of Prokaryotes declined to rule on a request for an opinion in this matter during their 2008 meeting, but a provisional placement of the former Eperythrozoon species in the genus Mycoplasma has otherwise been embraced by specialists in the molecular biology and clinical pathogenicity of these and similar hemotropic organisms. At present, the designation “Candidatus Mycoplasma” must still be used for new types.

Further reading

Kreier, J.P. and M. Ristic. 1974. Genus IV. Haemobartonella Tyzzer and Weinman 1939, 143AL; Genus V. Eperythrozoon Schilling 1928, 1854AL. In Bergey’s Manual of Determinative Bacteriol-ogy, 8th edn (edited by Buchanan and Gibbons). Williams & Wilkins, Baltimore, pp. 910–914.

differentiation of the species of the genus Eperythrozoon

Species differentiation relies principally on 16S rRNA gene sequencing. Some species exhibit a degree of host specificity, although cross-infection of related hosts has been reported.

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Genus i. eperythrozoon

list of species of the genus Eperythrozoon

1. Mycoplasma coccoides (Schilling 1928) Neimark, Peters, Robinson and Stewart 2005, 1389VP (Eperythrozoon coccoides Schilling 1928, 1854)

coc.co′ides. N.L. masc. n. coccus (from Gr. masc. n. kokkos grain, seed) coccus; L. suff. -oides (from Gr. suff. eides from Gr. n. eidos that which is seen, form, shape, figure), resem-bling, similar; N.L. neut. adj. coccoides coccus-shaped.

Pathogenic; causes anemia in wild and captive mice, and captive rats, hamsters, and rabbits. Transmission is believed to be vector-borne and mediated by the rat louse Polyplex spinu-losa and the mouse louse Polyplex serrata. Neoarsphenamine and oxophenarsine were thought to be effective chemother-apeutic agents for treatment of Mycoplasma coccoides infection in captive rodents, whereas tetracyclines are effective only at keeping infection at subclinical levels (Thurston, 1953).

Source: observed in association with the erythrocytes of wild and captive rodents.

DNA G+C content (mol%): not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): AY171918.

2. Eperythrozoon parvum Splitter 1950, 513AL

par¢vum. L. neut. adj. parvum small.

A nonpathogenic epierythrocytic parasite of pigs. Organic arsenicals are effective; tetracyclines suppress infection. Transmissible by parenteral inoculation and sometimes by massive oral inoculation. This is the only remaining species of Eperythrozoon whose name has standing in nomenclature that has not yet been examined by molecular genetic meth-ods. It seems likely that, if a specimen of this organism can be found, it will prove to be a mycoplasma.

3. Mycoplasma ovis (Neitz, Alexander and de Toit 1934) Nei-mark, Hoff and Ganter 2004, 369VP (Eperythrozoon ovis Neitz, Alexander and de Toit 1934, 267)

o¢vis. L. fem. gen. n. ovis of a sheep.

Cells are coccoid and motility for this species has not been assessed. The morphology of infected erythrocytes is altered demonstrating a marked depression at the site of Myplasma ovis attachment. This species has not been grown on artificial medium; therefore, notable biochemi-cal parameters are not known.

Neoarsphenamine is an effective therapeutic agent. Myco-plasma ovis is reported to share antigens with Mycoplasma wenyonii (Kreier and Ristic, 1963), potentially complicating serology-based diagnosis of infection.

Pathogenic; causes mild to severe anemia in sheep and goats that often results in poor feed conversion. Transmis-sion occurs via blood-feeding arthropods, e.g., Haemophysa-lis plumbeum, Rhipicephalus bursa, Aedes camptorhynchus, and Culex annulirostris (Daddow, 1980; Howard, 1975; Nikol’skii and Slipchenko, 1969), and likely via fomites such as reused needles, shearing tools, and ear-tagging equipment (Brun-Hansen et al., 1997; Mason and Statham, 1991).

Source: observed in association with erythrocytes or unat-tached in suspension in the blood of sheep, goats, and rarely in eland and splenectomized deer.


4. Mycoplasma suis corrig. (Splitter 1950) Neimark, Johansson, Rikihisa and Tully 2002, 683VP (Eperythrozoon suis Splitter 1950, 513)

su¢is. L. gen. n. suis of the pig.


Neoarsphenamine and tetracyclines are effective thera-peutic agents. An enzyme-linked immunosorbant assay (ELISA) and PCR-based detection assays to enable diagnosis of infection have been described (Groebel et al., 2009; Gwalt-ney and Oberst, 1994; Hoelzle, 2008; Hsu et al., 1992).

Pathogenic; causes febrile icteroanemia in pigs. Trans-mission occurs via insect vectors including Stomoxys calci-trans and Aedes aegypti (Prullage et al., 1993).

Source: observed in association with the erythrocytes of pigs.DNA G+C content (mol%): not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): AF029394.Further comment: the original spelling of the specific

epithet, haemosuis (sic), has been corrected by the List Editor.

5. Mycoplasma wenyonii (Adler and Ellenbogen 1934) Nei-mark, Johansson, Rikihisa and Tully 2002, 683VP (Eperythro-zoon wenyonii Adler and Ellenbogen 1934, 220)

we.ny.o¢ni.i. N.L. masc. gen. n. wenyonii of Wenyon, named after Charles Morley Wenyon (1878–1948), an investigator of these organisms.


Pathogenic; causes anemia and subsequent lameness and/or infertility in cattle. Transmission is primarily vector-mediated by Dermacentor andersoni and reportedly can also occur vertically during gestation. Oxytetracycline is an effec-tive therapeutic agent (Montes et al., 1994). Mycoplasma wenyonii is reported to share antigens with Mycoplasma ovis (Kreier and Ristic, 1963), potentially complicating serology-based diagnosis of infection.

Source: observed in association with the erythrocytes and platelets of cattle (Kreier and Ristic, 1968).


species of unknown phylogenetic affiliation

The phylogenetic affiliations of the following proposed organ-isms are unknown and their names do not have standing in

nomenclature. They are listed here merely because they have been incidentally cited as species of Eperythrozoon.

641


1. “Eperythrozoon mariboi” Ewers 1971

The name given to uncultivated polymorphic structures observed on or in erythrocytes from flying foxes (Pteropus macrotis) following splenectomy (Ewers, 1971). The structures, described as fine lines, lines with rings, and rows of rings that span the diameter of the erythrocytes, differ from those of hemotropic mycoplasmas.

2. “Eperythrozoon teganodes” Hoyte 1962

The name given to uncultivated serially transmissible bod-ies observed in Giemsa-stained blood smears from cattle. The bodies only occur free in the blood plasma and do not

attach to erythrocytes (Hoyte, 1962). The bodies differ from Mycoplasma wenyonii in morphology and include “frying-pan” shaped structures.

3. “Eperythrozoon tuomii” Tuomi and Von Bonsdorff 1967

Uncultivated transmissible cell wall-less bodies observed in Giemsa-stained blood smears and electron micrographs of blood from splenectomized calves. The bodies appeared in blood smears predominantly as delicate rings that did not attach to erythrocytes but were associated exclusively with thrombocytes (Tuomi and Von Bonsdorff, 1967; Uilenberg, 1967; Zwart et al., 1969).

Genus ii. Haemobartonella tyzzer and Weinman 1939, 305al

Daniel R. BRown, SéveRine TaSkeR, Joanne B. MeSSick anD HaRolD neiMaRk

ha.e.mo.bar.to.nel′la. Gr. n. haima (l. transliteration haema) blood; n.l. fem. n. Bartonella a bacterial genus; n.l. fem. n. Haemobartonella the blood (-inhabiting) Bartonella.

Cells adherent to host erythrocyte surfaces are coccoid and about 350 nm in diameter, but may occur as chains or deform to appear rod- or ring-shaped in stained blood smears.

Type species: Haemobartonella muris (Mayer 1921) Tyzzer and Weinman 1939AL (Bartonella muris Mayer 1921, 151; Bartonella muris ratti Regendanz and Kikuth 1928, 1578; Haemobartonella muris Tyzzer and Weinman 1939, 143).


Those organisms originally assigned to the genus Haemobartonella are properly affiliated with the Mycoplasmatales, but their transfer to the order has not yet been formalized. Any distinction between Haemobartonella and Eperythrozoon is tenuous and possibly arbitrary (Kreier and Ristic, 1974; Uilenberg et al., 2004). Enrichment, iso-lation and maintenance procedures, and methods of differentia-tion are essentially the same as those for genus Eperythrozoon.

list of species of the genus Haemobartonella

1. Mycoplasma haemomuris (Mayer 1921) Neimark, Johansson, Rikihisa and Tully 2002, 683VP (Bartonella muris Mayer 1921, 151; Bartonella muris ratti Regendanz and Kikuth 1928, 1578; Haemobartonella muris Tyzzer and Weinman 1939, 143)

ha.e.mo.mu¢ris. Gr. neut. n. haema blood; L. masc. gen. n. muris of the mouse; N.L. gen. n. haemomuris of mouse blood.

Cells are coccoid and some display dense inclusion particles. Motility for this species has not been assessed. The morphology of infected erythrocytes is altered, demonstrating a marked depression at the site of Mycoplasma haemomuris attachment. This species has not been grown on any artificial medium; therefore, notable biochemical parameters are not known.

Opportunistic pathogen; causes anemia in splenecto-mized or otherwise immunosuppressed mice. Transmis-sion is vector-borne and mediated by the rat louse (Polypax spinulosa).

Source: observed in association with erythrocytes of wild and captive mice, and hamsters.

DNA G+C content (mol%): not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): U82963.

2. Mycoplasma haemocanis (Kikuth 1928) Messick, Walker, Raphael, Berent and Shi 2002, 697VP [Bartonella canis Kikuth 1928, 1730; Haemobartonella (Bartonella) canis (Kikuth 1928) Tyzzer and Weinman 1939, 151; Kreier and Ristic 1984, 726]

ha.e.mo.ca¢nis Gr. neut. n. haema blood; L. fem. gen. n. canis of the dog; N.L. gen. n. haemocanis of dog blood.

Cells are coccoid to pleomorphic. Motility for this species has not been assessed. The morphology of infected erythro-cytes is altered, demonstrating a marked depression at the site of Mycoplasma haemocanis attachment. This species has not been grown on any artificial medium; therefore, nota-ble biochemical parameters are not known.

Pathogenic; causes hemolytic anemia in domestic dogs. Transmission is vector-borne and mediated by the brown dog tick (Rhipicephalus sanguineus).

Source: observed in association with erythrocytes of domestic dogs (Hoskins, 1991).

DNA G+C content (mol%): Not determined.Type strain: not established.Sequence accession no. (16S rRNA gene): AF197337.

3. Mycoplasma haemofelis (Clark 1942) Neimark, Johansson, Rikihisa and Tully 2002, 683VP [Eperythrozoon felis Clark 1942, 16; Haemobartonella felis (Clark 1942) Flint and McKelvie 1956, 240 and Kreier and Ristic 1984, 725]

ha.e.mo.fe¢lis. Gr. neut. n. haema blood; L. fem. gen. n. felis of the cat; N.L. gen. n. haemofelis of cat blood.


Pathogenic; causes hemolytic anemia in cats. The mode of transmission is percutaneous or oral; an insect vector has not been identified although fleas have been implicated (Woods et al., 2005).

Tetracyclines and fluoroquinolones are effective thera-peutic agents (Dowers et al., 2002; Tasker et al., 2006).

642

Genus ii. haemobartonella

references

Adler, S. and V. Ellenbogen. 1934. A note on two new blood parasites of cattle: Eperythrozoon and Bartonella. J. Comp. Pathol. 47: 220–221.

Berent, L.M. and J.B. Messick. 2003. Physical map and genome sequenc-ing survey of Mycoplasma haemofelis (Haemobartonella felis). Infect. Immun. 71: 3657–3662.

Brown, D., R. Whitcomb and J. Bradbury. 2007. Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes). Int. J. Syst. Evol. Microbiol. 57: 2703–2719.

Brun-Hansen, H., H. Gronstol, H. Waldeland and B. Hoff. 1997. Eperyth-rozoon ovis infection in a commercial flock of sheep. Zentralbl. Veteri-narmed. B 44: 295–299.

Clark, R. 1942. Eperythrozoon felis (sp. nov.) in a cat. J. Afr. Vet. Med. Assoc. 13: 15–16.

Daddow, K.N. 1980. Culex annulirostris as a vector of Eperythrozoon ovis infection in sheep. Vet. Parasitol. 7: 313–317.

Dowers, K.L., C. Olver, S.V. Radecki and M.R. Lappin. 2002. Use of enrofloxacin for treatment of large-form Haemobartonella felis in experimentally infected cats. J. Am. Vet. Med. Assoc. 221 : 250–253.

Ewers, W.H. 1971. Eperythrozoon mariboi sp. nov. (Protophyta: order Richettsiales) a parasite of red blood cells of the flying fox Pteropus macrotis epularius in New Guinea. Parasitology 63 : 261–269.

Flint, J.C. and McKelvie D.H.. 1956. Feline infectious anemia-diagnosis and treatment. Proc. 92nd Ann. Meet. Am. Vet. Med. Assoc. 1955 240–242.

Frerichs, W.M. and A.A. Holbrook. 1971. Haemobartonella procyoni sp. n. in the raccoon, Procyon lotor. J. Parasitol. 57 : 1309–1310.

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Howard, G.W. 1975. The experimental transmission of Eperythrozoon ovis by mosquitoes. Parasitology 71: xxxiii.

Hoyte, H.M.D. 1962. Eperythrozoon teganodes sp. nov. (Rickettsiales), para-sitic in cattle. Parasitology 52: 527–532.

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Kikuth, W. 1928. Über Einen neuen Anämeerreger; Bartonella canis nov. spec. Klin. Wochenschr. 7: 1729–1730.

Kreier, J.P. and M. Ristic. 1963. Morphologic, antigenic, and pathogenic characteristics of Eperythrozoon ovis and Eperythrozoon wenyoni. Am. J. Vet. Res. 24: 488–500.

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Man and Animals (edited by Weinman and Ristic). Academic Press, New York, pp. 387–472.

Kreier, J.P. and M. Ristic. 1974. Genus IV. Haemobartonella Tyzzer and Weinman 1939, 143AL; Genus V. Eperythrozoon Schilling 1928, 1854AL. In Bergey’s Manual of Determinative Bacteriology, 8th edn (edited by Buchanan and Gibbons). Williams & Wilkins, Baltimore, pp. 910–914.

Kreier, J.P. and M. Ristic. 1984. Genus III. Haemobartonella; Genus IV. Eperythrozoon. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 724–729.

Li, X., X. Jia, D. Shi, Y. Xiao, S. Hu, M. Liu, Z. Yuan and D. Bi. 2008. Continuous in vitro Cultivation of Mycoplasma suis. Acta Vet. Zootech. Sinica 38: 1142–1146.

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Source: observed in association with erythrocytes of domestic cats.

DNA G+C content (mol%): 38.5 (genome sequence survey of strain OH; Berent and Messick, 2003).

Type strain: not established.Sequence accession no. (16S rRNA gene): U88563.

The phylogenetic affiliations of the following proposed organism are unknown and its name does not have standing

in nomenclature. It is listed here merely because it has been incidentally cited as a species of Haemobartonella.

1. “Haemobartonella procyoni” Frerichs and Holbrook 1971

Electron microscopy shows this epierythrocytic organism from a raccoon (Procyon lotor) is wall-less and its description indicates it probably will prove to be a hemotropic myco-plasma (Frerichs and Holbrook, 1971).

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Uilenberg, G., F. Thiaucourt and F. Jongejan. 2006. Mycoplasma and Eperythrozoon (Mycoplasmataceae). Comments on a recent paper. Int. J. Syst. Evol. Microbiol. 56: 13–14.

Willi, B., F.S. Boretti, C. Baumgartner, S. Tasker, B. Wenger, V. Cattori, M.L. Meli, C.E. Reusch, H. Lutz and R. Hofmann-Lehmann. 2006. Prevalence, risk factor analysis, and follow-up of infections caused by three feline hemoplasma species in cats in Switzerland. J. Clin. Microbiol. 44 : 961–969.

Woods, J.E., M.M. Brewer, J.R. Hawley, N. Wisnewski and M.R. Lappin. 2005. Evaluation of experimental transmission of Candidatus Myco-plasma haemominutum and Mycoplasma haemofelis by Ctenocephalides felis to cats. Am. J. Vet. Res. 66: 1008–1012.

Zwart, D., P. Leeflang and C.J. van Vorstenbosch. 1969. Studies on an Eperythrozoon associated with bovine thrombocytes. Zentralbl. Bakteriol. [Orig.] 210 : 82–105.

order ii. Entomoplasmatales tully, bové, laigret and Whitcomb 1993, 381Vp

Daniel R. BRown, JaneT M. BRaDBuRy anD RoBeRT F. wHiTcoMB*

en.to.mo.plas.ma.ta¢les. n.l. neut. n. Entomoplasma type genus of the order; -ales ending to denote an order: n.l. fem. pl. n. Entomoplasmatales the Entomoplasma order.

This order in the class Mollicutes has been assigned to a group of nonhelical and helical mollicutes that are regularly associated with arthropod or plant hosts. The description of organisms in the order is essentially the same as for the class. Two families are designated, Entomoplasmataceae for nonhelical mollicutes and Spiroplasmataceae for helical ones. The order consists of four major phylogenetic clades: the paraphyletic entomoplasmataceae clade, which consists of the genera Entomoplasma and Mesoplasma; and the Apis, Citri–Chrysopicola–Mirum, and Ixodetis clades of the genus Spiroplasma. All cells are chemo-organotrophic, usually fer-menting glucose through the phosphoenolpyruvate-dependent sugar transferase system. Arginine may be hydrolyzed, but urea is not. Cells may require sterol for growth. Nonhelical strains that grow in serum-free media supplemented with polyoxyethylene sorbitan (PES) are currently assigned to the genus Mesoplasma. Temperature optimum for growth is usually 30–32°C, with a few species able to grow at 37°C. Genome sizes range from 780 to 2220 kbp by pulsed-field gel electrophoresis (PFGE), with DNA G+C contents ranging from 25 to 34 mol%. Like members of the Mycoplasmatales, all organisms in this order are thought to utilize the UGA codon to encode tryptophan.

Type genus: Entomoplasma Tully, Bové, Laigret and Whitcomb 1993, 379VP.


The basis for the proposal for the order Entomoplasmatales (Tully et al., 1993) was the distinctive phylogenetic and phenotypic characteristics of culturable mollicutes regularly associated with arthropods or plants. Members of the family Entomoplas-mataceae are nonhelical mollicutes that differ in their choles-terol or serum requirements for growth. Nonhelical organisms with a strict requirement for cholesterol were placed in the genus Entomoplasma (trivial name, entomoplasmas), whereas nonhelical strains able to grow in a sterol-free medium supple-mented with PES were assigned to the genus Mesoplasma (trivial name, mesoplasmas). The proposal also included the transfer of the family Spiroplasmataceae from the family Mycoplasmatales to the family Entomoplasmatales. The helical organisms assigned to the genus Spiroplasma were within the Spiroplasmataceae, and genus and family descriptions of these organisms remained as proposed previously (Skripal, 1983; Whitcomb and Tully, 1984). The order Entomoplasmatales is a phylogenetic sister to the order Mycoplasmatales. These two orders together form a lineage with several unique properties, including the use of UGA as a tryptophan codon rather than a stop codon.

taxonomic comments

The genera Entomoplasma and Mesoplasma constitute a poly-phyletic sister lineage of the mycoides cluster of mycoplasmas that are eccentrically situated in the paraphyletic family Ento-moplasmataceae (Gasparich et al., 2004). There is no current *Deceased 21 December 2007.

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Family i. entomoplasmataceae

phylogenetic support for separation of Entomoplasma and Mesoplasma species based on neighbor-joining or maximum-parsimony methods of 16S rRNA gene sequence similarity analysis because they do not form coherent clusters, but are instead intermixed in one paraphyletic group (Johansson and Pettersson, 2002; Tully et al., 1998). No DNA–DNA reas-sociation experiments have been performed nor is there any other polyphasic taxonomic basis to support the separation. In particular, the growth requirement for sterols is not as pro-found a character as was initially believed and fails to justify these two species (Gasparich et al., 2004; Rose et al., 1993). For these reasons, and because Entomoplasma has priority

(Tully et al., 1993), the species currently assigned to the genus Mesoplasma should most likely be transferred to the genus Ento-moplasma. Because the transfer would include its type species, the genus Mesoplasma would then become illegitimate. More-over, Knight (2004) showed that the species formerly called Mesoplasma pleciae (Tully et al., 1994) is properly affiliated with the genus Acholeplasma on undisputed grounds of 16S rRNA gene sequence similarity and preferred use of UGG rather than UGA as the codon for tryptophan. Therefore, transfer of the currently remaining members of genus Mesoplasma cannot be endorsed until similar analyses have been completed for all of those organisms (D.V. Volokhov, unpublished).

references

Gasparich, G.E., R.F. Whitcomb, D. Dodge, F.E. French, J. Glass and D.L. Williamson. 2004. The genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation with phenotype and roots of the Mycoplasma mycoides clade. Int. J. Syst. Evol. Micro-biol. 54: 893–918.

Johansson, K.E. and B. Pettersson. 2002. Taxonomy of Mollicutes. In Molec-ular Biology and Pathogenicity of Mycoplasmas (edited by Razin and Hermann). Kluwer Academic/Plenum Publishers, London, pp. 1–31.



Skripal, I.G. 1983. Revival of the name Spiroplasmataceae fam. nov., nom. rev., omitted from the 1980 Approved Lists of Bacterial Names. Int. J. Syst. Bacteriol. 33: 408.

Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes–proposed elevation of a mono-

phyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385.

Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Rose, R.B. Henegar, J.M. Bove, P. Carle, D.L. Williamson and T.B. Clark. 1994. Taxonomic descriptions of eight new non-sterol-requiring Mollicutes assigned to the genus Mesoplasma. Int. J. Syst. Bacteriol. 44: 685–693.

Tully, J.G., R.F. Whitcomb, K.J. Hackett, D.L. Williamson, F. Laigret, P. Carle, J.M. Bove, R.B. Henegar, N.M. Ellis, D.E. Dodge and J. Adams. 1998. Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae). Int. J. Syst. Bacteriol. 48: 1197–1204.

Whitcomb, R.F. and J.G. Tully. 1984. Family III. Spiroplasmataceae Skripal 1983, 408VP. Genus I. Spiroplasma Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt. In Bergey’s Manual of Systematic Bacteriology, vol. 1 (edited by Krieg and Holt). Williams & Wilkins, Baltimore, pp. 781–787.

Family i. Entomoplasmataceae tully, bové, laigret and Whitcomb 1993, 380Vp

Daniel R. BRown, JaneT M. BRaDBuRy anD RoBeRT F. wHiTcoMB*

en.to.mo.plas.ma.ta.ce¢ae. n.l. neut. n. Entomoplasma, -atos type genus of the family; -aceae ending to denote a family; n.l. fem. pl. n. Entomoplasmataceae the Entomoplasma family.

Cells are usually coccoid or occur as short, branched or unbranched, pleomorphic, nonhelical filaments. Filterable through membranes with a mean pore diameter of 220–450 nm. Cells lack a cell wall and are bounded only by a plasma membrane. Nonmotile. Facultatively anaerobic. The tempera-ture range for growth varies from 10 to 37°C, with the optimum usually at 30°C. The typical colony has a “fried-egg” appear-ance. Chemo-organotrophic; acid is produced from glucose, with evidence of a phosphoenolpyruvate-dependent sugar transport system(s) in some members. Arginine and urea are not hydrolyzed. The organisms may require serum or choles-terol for growth or may grow in serum-free media plus 0.04% PES. The genome sizes range from 790 to 1140 kbp.

DNA G+C content (mol%): 26–34.

Type genus: Entomoplasma Tully, Bové, Laigret and Whitcomb 1993, 379VP.


All members of this paraphyletic family are nonhelical and are regularly associated with arthropod or plant hosts. They may require cholesterol or serum for growth, and most have an optimal growth temperature near 30°C. Separation of members of the genera Entomoplasma and Mesoplasma within the Entomoplasmataceae is based on the capacity of the Meso-plasma species to grow in a serum-free or cholesterol-free medium supplemented with PES (Rose et al., 1993; Tully et al., 1995), whereas Entomoplasma species have a growth requirement for cholesterol. The family is derived from the Spiroplasma lineage and is most closely related to the Apis cluster of that group. The mycoides cluster of species in the genus Mycoplasma is related to this family and seems to have evolved from it.*Deceased 21 December 2007.

645

Family i. EntomoplasmatacEaE

Cells are nonhelical and nonmotile, frequently pleomorphic and range in size from 200 to 1200 nm in diameter. Some cells exhibit short filamentous forms. Most species ferment glucose. Species possess the phosphoenolpyruvate-dependent sugar-phosphotransferase system. Organisms require serum or cho-lesterol for growth. The temperature range for growth ranges from 10 to 32°C, with the optimum usually at 30–32°C. The genome sizes range from 870 to 900 kbp (PFGE). All currently assigned species were isolated from insects or from plant sur-faces where they were presumably deposited by insects.

DNA G+C content (mol%): 27–34.Type species: Entomoplasma ellychniae Tully, Rose, Hackett,

Whitcomb, Carle, Bové, Colflesh and Williamson (Tully et al., 1989) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Myco-plasma ellychniae Tully, Rose, Hackett, Whitcomb, Carle, Bové, Colflesh and Williamson 1989, 288).


Cells of these organisms vary from coccoid to pleomorphic forms exhibiting short, branching, nonhelical filaments. Round cells are usually in the size range of 200–300 nm, but may be larger. Most strains were initially isolated in either M1D or SP-4 medium and all entomoplasmas grow well in SP-4 broth containing a sup-plement of 17% fetal bovine serum. Some strains are able to grow on media with reduced serum content. Most established species have an optimal growth temperature of 30°C, but some species grow better in broth medium maintained at 23–25°C or at 32°C. Colony growth on solid medium is best obtained on SP-4 medium incubated under anaerobic conditions at about 30°C. Under these conditions, most species produce colonies with a classic fried-egg appearance, although Entomoplasma freundtii is notable for its granular colony morphology.

All species show strong fermentation of glucose with produc-tion of acid and a reduction in medium pH (Table 140). Actively growing cultures in broth medium containing glucose may rap-idly acidify the medium, causing partial or complete loss of

viability after 7–10 d. Arginine hydrolysis and “film and spot” lipase reactions are rare among species described to date. Ento-moplasmas were shown to lack some key metabolic activities found in other mollicutes, especially PPi-dependent phospho-fructokinase and dUTPase, and to possess uracil DNA glycosy-lase activity. Although the latter pyrimidine enzymic activity distinguished Entomoplasma from Mesoplasma species, only two Entomoplasma species and three Mesoplasma species have been tested so far for these activities (Pollack et al., 1996).

Antisera to whole cell antigens of entomoplasmas have been used extensively to provide specific identification to the species level with a variety of serologic techniques, including growth inhibition, metabolism inhibition, and agar plate immunofluo-rescence (Tully et al., 1989, 1990, 1998). There is no evidence for the pathogenicity of entomoplasmas to either plant or insect hosts. Like other mollicutes, the entomoplasmas are resistant to 500 U/ml penicillin G.

Enrichment and isolation procedures

Flowers and other plant material should be cut in the field and placed in plastic bags without touching by hand. In the labora-tory, plant materials are rinsed briefly in either SP-4 or M1D media (May et al., 2008). In both of these media, fetal bovine serum is a critical component for successful growth of these organisms (Hackett and Whitcomb, 1995; Tully, 1995). The rinse medium is immediately decanted and passed through a sterile membrane filter, usually of 450 nm porosity. The filtrate is then passed through at least several tenfold dilutions in the selected culture medium. The retentate may be frozen at −70°C for later use or for retesting. The cultures are incubated at 27–30°C and monitored by dark-field microscopy and/or by observing acidifi-cation of the medium. It is important to note that several non-sterol-requiring Acholeplasma species have also been isolated from plant and insect material (Tully et al., 1994b).

Insect material, primarily from gut contents or hemolymph obtained by dissection or by fine-pointed glass pipettes, should be added to small volumes of SP-4 or M1D medium and filtered through a 450 nm membrane filter. Serial tenfold dilutions of the filtrate should be incubated at 27–30°C and observed for a decrease in pH of the medium. After two to three serial pas-sages, the organisms should be purified by conventional filter-cloning techniques (Tully, 1983) and stocks of various clones and early passage isolates frozen for further identification pro-cedures (Whitcomb and Hackett, 1996).


Stock cultures of entomoplasmas can be maintained well in SP-4 and/or M1D broth medium containing about 17% fetal bovine serum. Most strains in the group can be adapted to grow in a broth medium containing bovine serum. Stock cul-tures in broth medium can be stored at −70°C for indefinite periods. For optimum preservation, the organisms should be lyophilized as broth cultures in the early exponential phase of growth and the dried cultures should be sealed under vacuum and stored at 4°C.

Table 140. Differential characteristics of species of the genus Entomoplasma a

Characteristic E. e

llych

niae

E. fr

eund

tii

E. lu

civo

rax

E. lu

min

osum

E. m

elal

euca

e

E. s

omni

lux

Glucose fermentation + + + + + +Arginine hydrolysis − + − − − −“Film and spots” − nd + + − −Hemadsorption of guinea

pig red blood cells− nd − + − −

DNA G+C content (mol%) 27.7 34 27.4 28.8 27 27

aSymbols: +, >85% positive; −, 0–15% positive; nd, not determined.

Genus i. entomoplasma tully, Bové, laigret and Whitcomb 1993, 379Vp

Daniel R. BRown, Janet M. BRaDBuRy anD RoBeRt F. whitcoMB*

En.to.mo.plas¢ma. Gr. n. entomon insect; Gr. neut. n. plasma something formed or molded, a form; n.l. neut. n. Entomoplasma name intended to show association with insects.

*Deceased 21 December 2007.

646

GEnus i. Entomoplasma

Differentiation of the genus Entomoplasma from other genera

Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. Aerobic or facultative anaerobic growth in artificial media and the necessity for sterols for growth exclude assign-ment to the genera Anaeroplasma, Asteroleplasma, Acholeplasma, Mesoplasma, or “Candidatus Phytoplasma”. Non-helical cellular morphology and regular association with arthropod or plant hosts support exclusion from the genera Spiroplasma or Myco-plasma. The inability to hydrolyze urea excludes assignment to the genus Ureaplasma. However, the difficulty in assigning novel species to this genus is well demonstrated by the ear-lier difficulties in establishing accurately the taxonomic status of these organisms (Tully et al., 1993). The availability of 16S rRNA gene sequence analyses was critical to the differentiation of these organisms from other mollicutes. Although isolates from vertebrates are very unlikely to be entomoplasmas, two bona fide Mycoplasma species, Mycoplasma iowae and Mycoplasma equigenitalium, have been isolated from plants [Grau et al., 1991; J.C. Vignault, J.M. Bové and J.G. Tully, unpublished (see ATCC 49192)].

taxonomic comments

The landmark studies of Weisburg et al. (1989), using 16S rRNA gene sequences of about 50 species of mollicutes, were critical in the resolution of certain taxonomic conflicts regarding the species that became Entomoplasma. The first entomoplasmas to be recognized were serologically related isolates from the flow-ers of Melaleuca and Grevillea trees (McCoy et al., 1979). Others, found in a wide range of insect species (Tully et al., 1987), included strain ELCN-1T from the hemolymph of the firefly beetle Ellychnia corrusca (Tully et al., 1989) and three serologi-cally distinct strains isolated from gut contents of Pyractomena and Photinus beetles (Williamson et al., 1990). Although these nonhelical, sterol-requiring mollicutes were initially placed in the genus Mycoplasma, 16S rRNA gene sequence analysis clearly indicated that strain M1T, previously designated Mycoplasma melaleucae, and strain ELCN-1T, previously designated Myco-plasma ellychniae, were most closely affiliated with the Spiroplasma lineage of helical organisms isolated primarily from arthro-pods. These findings prompted a proposal to reclassify the non-helical mollicutes from arthropods and plants in a new order, Entomoplasmatales, and new family, Entomoplasmataceae, with the genus Entomoplasma reserved for sterol-requiring species (Tully et al., 1993). Strains M1T and ELCN-1T were renamed as Entomoplasma melaleucae and Entomoplasma ellychniae, respec-tively. Subsequent phylogenetic analysis of Mycoplasma freundtii, later renamed Entomoplasma freundtii, confirmed the placement (Tully et al., 1998).

The paraphyletic relationship between the genera Ento-moplasma and Mesoplasma is currently an unresolved problem in the systematics of this genus. It is possible that these genera, sepa-rated by the single criterion of sterol requirement, should be combined into the single genus Entomoplasma. However, Knight (2004) showed that Mesoplasma pleciae (Tully et al., 1994b) should belong to the genus Acholeplasma based on 16S rRNA gene sequence similarity and the preferred use of UGG rather than UGA as the codon for tryptophan. Therefore, transfer of the cur-rently remaining members of genus Mesoplasma to other genera cannot be endorsed until similar analyses have been completed for all of those species (D.V. Volokhov, unpublished).

acknowledgements

We thank Karl-Erik Johansson for helpful comments and sug-gestions and Gail E. Gasparich for her landmark contributions regarding the phylogenetics of the Entomoplasmatales. The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading

Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, San Diego, pp. 1–31.

Tully, J.G. 1996. Mollicute–host interrelationships: current con-cepts and diagnostic implications. In Molecular and Diagnos-tic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 1–21.

Differentiation of the species of the genus Entomoplasma

The primary technique for differentiation of Entomoplasma species is 16S rRNA gene sequence comparisons, confirmed by serology (Brown et al., 2007). Nonhelical mollicutes that belong to a known species isolated from arthropods or plants can be readily identified serologically provided that a battery of potent antisera for classified species is available. Growth inhibition tests, performed by placing paper discs saturated with type-specific antisera on agar plates inoculated with the organism, are perhaps the most convenient and rapid serological technique to differentiate species (Clyde, 1983). The agar plate immunofluorescence test is also a convenient and rapid means of mollicute species identification. In the absence of specific conjugated antiserum, an indirect immu-nofluorescence test can be performed with type-specific anti-serum and a fluorescein-conjugated secondary antibody. The metabolism inhibition test (Taylor-Robinson, 1983) has also been applied to differentiation of Entomoplasma species (Tully et al., 1998).

list of species of the genus Entomoplasma

1. Entomoplasma ellychniae (Tully, Rose, Hackett, Whitcomb, Carle, Bové, Colflesh and Williamson 1989) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma ellychniae Tully, Rose, Hackett, Whitcomb, Carle, Bové, Colflesh and Williamson 1989, 288)

el.lych.ni¢ae. N.L. n. Ellychnia a genus of firefly beetles; N.L. gen. n. ellychniae of Ellychnia, from which the organism was first isolated.

This is the type species of the genus Entomoplasma. Cells are nonhelical, pleomorphic filaments, with some branching;

647


small coccoid forms, ranging in diameter from 200 to 300 nm, also occur. Passage of broth cultures through 450 and 300 nm porosity membrane filters does not reduce viable cell numbers, whereas passage through 220 nm porosity reduces cell popula-tions by about 10%. Grows well in SP-4 medium with fetal bovine serum supplements. Does not grow well in horse serum-supplemented broth or agar media. Optimum temperature for broth growth is 30°C; can grow at 18–32°C. Colonies incu-bated at 30°C under anaerobic conditions have a fried-egg appearance. Does not hemadsorb guinea pig erythrocytes.

No evidence for pathogenicity for insects.Source: isolated from the hemolymph of the firefly beetle

Ellychniae corrusca.DNA G+C content (mol%): 27.5 (Bd).Type strain: ELCN-1, ATCC 43707, NCTC 11714.Sequence accession no. (16S rRNA gene): M24292.

2. Entomoplasma freundtii Tully, Whitcomb, Hackett, Williamson, Laigret, Carle, Bové, Henegar, Ellis, Dodge and Adams 1998, 1203VP

freund¢ti.i. N.L. masc. gen. n. freundtii of Freundt, named after Eyvind Freundt, a Danish pioneer in the taxonomy and classification of mollicutes.

Cells are predominantly coccoid in shape, ranging from 300 to 1200 nm in diameter. Organisms are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm; more than 90% of viable cells in broth culture are able to pass 220 nm porosity membranes. The tempera-ture range for growth is 10–32°C, with an optimum at 30°C. Colonies under anaerobic conditions are granular and fre-quently exhibit multiple satellite forms although the organ-ism is considered nonmotile. The organism grows well in SP-4 broth medium or other media containing horse serum supplements.

No evidence for pathogenicity for insects.Source: isolated from the gut contents of a green tiger bee-

tle (Coleoptera: Cicindelidae).DNA G+C content (mol%): 34.1 (Bd).Type strain: BARC 318, ATCC 51999.Sequence accession no. (16S rRNA gene): AF036954.

3. Entomoplasma lucivorax (Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma lucivo-rax Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990, 164)

lu.ci.vo¢rax. L. fem. n. lux lucis light; L. neut. adj. vorax glut-tonous, devouring; N.L. neut. adj. lucivorax light devouring, referring to the predacious habit of the host insect, which preys on other luminescent firefly species.

Cells are either pleomorphic coccoidal or subcoccoidal, with a diameter of 200–300 nm, or are short, branched or unbranched filaments. Cells are readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm, but do not pass 100 nm porosity membranes. Opti-mum temperature for growth is 30°C; can grow at 10–32°C. Nonmotile. Colonies under anaerobic conditions usually have a fried-egg appearance. Grows well in SP-4 broth medium or other media containing horse serum supplements. Colonies do not hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects or plants.Source: first isolated from the gut of a firefly beetle (Photi-

nus pyralis); also isolated from a flower (Spirea ulmaria; C. Chastel, unpublished).

DNA G+C content (mol%): 27.4 (Bd).Type strain: PIPN-2, ATCC 49196, NCTC 11716.Sequence accession no. (16S rRNA gene): AF547212.

4. Entomoplasma luminosum (Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma luminosum Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990, 163)

lu.mi.no¢sum. L. neut. adj. luminosum luminous, emitting light, referring to the luminescence of the adult host from which the organism was isolated.

Cells are pleomorphic and coccoidal or subcoccoidal with a diameter of 200–300 nm. Cells also occur as short, branched or unbranched filaments. The organisms are read-ily filterable through membranes with mean pore diameters of 450, 300, and 220 nm, but do not pass 100 nm porosity membranes. The temperature range for growth is 10–32°C, with an optimum at 32°C. Nonmotile. Colonies under anaer-obic conditions have a fried-egg appearance. The organism grows well in SP-4 broth medium or other media containing horse serum supplements. Colonies hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects.Source: isolated from the gut of the firefly beetle (Photinus

marginata).DNA G+C content (mol%): 28.8 (Bd).Type strain: PIMN-1, ATCC 49195, NCTC 11717.Sequence accession no. (16S rRNA gene): AY155670.

5. Entomoplasma melaleucae (Tully, Rose, McCoy, Carle, Bové, Whitcomb and Weisburg 1990) Tully, Bové, Laigret and Whit-comb 1993, 380VP (Mycoplasma melaleucae Tully, Rose, McCoy, Carle, Bové, Whitcomb and Weisburg 1990, 146)

me la.leu¢cae. N.L. n. Melaleuca a genus of tropical trees hav-ing white flowers with sweet fragrance; N.L. gen. n. melaleucae of Melaleuca, the plant from which the type strain was iso-lated.

Cells are pleomorphic and coccoidal or subcoccoidal, with few filamentous forms. Coccoidal forms have mean diameters of 250–300 nm. Cells are readily filterable through 450 and 300 nm porosity membrane filters, with few cells passing 220 nm porosity membranes. The temperature range for growth is 10–30°C, with an optimum at about 23°C. Nonmotile. Col-onies under anaerobic conditions at 23–30°C display a fried-egg appearance. Grows well in SP-4 broth or in modified Edward medium containing fetal bovine serum. The organ-ism does not grow well in horse serum-based broth medium. Agar colonies do not adsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects or plants.Source: isolated from flower surfaces of a subtropical plant,

Melaleuca quinquenervia, in south Florida. Related strains have been isolated from flowers of other subtropical trees in Florida, Melaleuca decora and Grevillea robusta (silk oak), and from an anthophorine bee (Xylocopa micans) in the same geographic area.

648

GEnus ii. mEsoplasma

DNA G+C content (mol%): 27.0 (Bd).Type strain: M1, ATCC 49191, NCTC 11715.Sequence accession nos (16S rRNA gene): M24478, AY345990.Further comment: the 16S rRNA gene sequence is more sim-

ilar to that of members of genus Mesoplasma than to others in the genus Entomoplasma.

6. Entomoplasma somnilux (Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma somnilux Williamson, Tully, Rose, Hackett, Henegar, Carle, Bové, Colflesh and Whitcomb 1990, 163)

som.ni¢lux. L. masc. n. somnus sleep; L. fem. n. lux light; N.L. n. somnilux intended to mean sleeping light, referring to the quiescent pupal stage of the host from which the organism was isolated, which precedes the luminescent adult stage.

Cells are pleomorphic and coccoidal or subcoccoidal, with a diameter of 200–300 nm; also occur as short, branched or unbranched filaments. Readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm. The tem-perature range for growth is 10–32°C, with optimum growth at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. The organ-ism grows well in SP-4 broth medium or other media con-taining horse serum supplements. Colonies do not adsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects.Source: isolated from a pupal gut of the firefly beetle (Pyra-

ctomena angulata).DNA G+C content (mol%): 27.4 (Bd).Type strain: PYAN-1, ATCC 49194, NCTC 11719.Sequence accession no. (16S rRNA gene): AY157871.

Genus ii. Mesoplasma tully, Bové, laigret and Whitcomb 1993, 380Vp

Daniel R. BRown, Janet M. BRaDBuRy anD RoBeRt F. whitcoMB*

me.so.plas¢ma. Gr. adj. mesos middle; Gr. neut. n. plasma something formed or molded, a form; n.l. neut. n. Mesoplasma middle form, name intended to denote a middle position with respect to sterol or cholesterol requirement.

Cells are nonhelical and nonmotile, generally coccoid or short filamentous forms. Coccoid cells are usually 220–300 nm in dia-meter, but some cells in some species can be as large as 400–500 nm. Most strains ferment glucose and most, but not all, lack the ability to hydrolyze arginine. Species possess the phosphoenolpyruvate-dependent sugar-phosphotransferase system. Neither serum nor cholesterol is required for growth, but strains show sustained growth in a serum-free or cholesterol-free medium when the medium is supplemented with 0.04% PES. The optimum temper-ature for growth is usually near 28–32°C, with some strains able to grow well at temperatures as low as 23°C or as high as 37°C. Genome sizes range from 825 to 930 kbp (PFGE).

DNA G+C content (mol%): 26–32.Type species: Mesoplasma florum (McCoy, Basham, Tully, Rose,

Carle and Bové 1984) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and Bové 1984, 14).


Cells are predominantly coccoid in the exponential phase of growth when examined by dark-field microscopy. Cells from broth cultures examined by transmission electron microscopy are also coccoid, with individual cells usually 220–500 nm in diameter and clearly defined by a single cytoplasmic mem-brane. Colony growth is best obtained on SP-4 agar medium. Plates incubated under anaerobic conditions at about 30°C usually display characteristic fried-egg type colonies after 5–7 d incubation.

Several mesoplasmas lack certain key metabolic activities found in other mollicutes, especially PPi-dependent phospho-fructokinase, dUTPase, and uracil DNA glycosylase activity (Pollack et al., 1996). Most mesoplasmas were isolated in M1D medium containing 15% fetal bovine serum (Whitcomb, 1983), but adapt well to growth in SP-4 broth containing 15–17% fetal

bovine serum, or in broth medium containing a 1% bovine serum fraction supplement (Tully, 1984; Tully et al., 1994a). All species show strong fermentation of glucose with acid produc-tion (Table 141), with a rapid decline in pH of the medium and loss of viability. Arginine hydrolysis has been observed only with the type strain (PUPA-2T) of Mesoplasma photuris.

Antisera directed against whole-cell antigens of filter-cloned mesoplasmas have been used extensively to establish species and to provide species identifications. There is no evidence of pathogenicity of any currently established species in the genus for either an insect or plant host. Mesoplasmas are resistant to 500 U/ml penicillin.

Enrichment, isolation, and maintenance procedures

The culture media and procedures for isolation and mainte-nance of entomoplasmas from plant and insect sources can also be effectively applied for mesoplasmas.

Differentiation of the genus Mesoplasma from other genera

Properties that fulfill criteria for assignment to this genus are the same as those for the genus Entomoplasma, with the excep-tion that the genus Mesoplasma is currently reserved for species that are able to grow in serum-free medium supplemented with PES (Tully et al., 1993).

taxonomic comments

The existence of a flora of nonhelical, wall-less prokaryotes associated with arthropod or plant hosts was first documented by T.B. Clark, S. Eden-Green, and R.E. McCoy and colleagues. Some of the plant isolates were clearly related to previously described Acholeplasma species, such as Acholeplasma oculi (Eden-Green and Tully, 1979), whereas others were established as novel Acholeplasma species, able to grow well in broth media without any cholesterol, serum, or fatty acid supplements. However, a significant group of other similarly derived strains were able to *Deceased 21 December 2007.

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grow in serum-free or cholesterol-free media only when small amounts of PES were added to the medium. Because these strains grew in the absence of cholesterol or serum, several of them were initially described as Acholeplasma species, including Acholeplasma florum (McCoy et al., 1984), Acholeplasma entomophi-lum (Tully et al., 1988), and Acholeplasma seiffertii (Bonnet et al., 1991). Although the growth response to PES in serum-free or cholesterol-free media suggested that there were fundamental differences between such mollicutes and classic acholeplasmas, conclusive taxonomic evidence was lacking. The subsequent analysis of 16S rRNA gene sequences by Weisburg et al. (1989) showed that the PES-requiring organisms were closely related to the spiroplasma group of mollicutes and were phylogeneti-cally distant from acholeplasmas. On the basis of these findings and additional phylogenetic data, a proposal was made that the plant- and insect-derived mollicutes with growth responses to PES in serum-free or cholesterol-free media would be assigned to a new family, Entomoplasmataceae, and a new genus, Mesoplasma (Tully et al., 1993). Three of the plant-derived strains previously described as Acholeplasma species (Acholeplasma f lorum, Achole-plasma entomophilum, and Acholeplasma seiffertii) were transferred to the genus Mesoplasma, with retention of their species epithets. A single plant-derived strain that had previously been described as Mycoplasma lactucae, and later found to grow in serum-free or cholesterol-free media supplemented with PES, was renamed Mesoplasma lactucae. Later, eight novel Mesoplasma species were described (Tully et al., 1994a).

The paraphyletic relationship between the genera Ento-moplasma and Mesoplasma is a currently unresolved problem in the systematics of this genus. It is possible that these genera,

separated by the single criterion of sterol requirement, should be combined into the single genus Entomoplasma. However, Knight (2004) showed that Mesoplasma pleciae (Tully et al., 1994a) should belong to the genus Acholeplasma based on 16S rRNA gene sequence similarity and the preferred use of UGG rather than UGA as the codon for tryptophan. Therefore, trans-fer of the currently remaining members of the genus Meso-plasma to other genera cannot be endorsed until similar analyses have been completed for all of those species (D.V. Volokhov, unpublished).

acknowledgements

We thank Karl-Erik Johansson for helpful comments and sug-gestions and Gail E. Gasparich for her landmark contributions regarding the phylogenetics of the Entomoplasmatales. The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading

Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas, vol. 5 (edited by Whitcomb and Tully). Academic Press, San Diego, pp. 1–31.

Tully, J.G. 1996. Mollicute-host interrelationships: current con-cepts and diagnostic implications. In Molecular and Diagnos-tic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 1–21.

Differentiation of the species of the genus Mesoplasma

The techniques for differentiation of Mesoplasma species are the same as those for genus Entomoplasma.

Table 141. Differential characteristics of species of the genus Mesoplasma a

Characteristic M. f

loru

m

M. c

haul

ioco

la

M. c

oleo

pter

ae

M. c

orru

scae

M. e

ntom

ophi

lum

M. g

ram

mop

tera

e

M. l

actu

cae

M. p

hotu

ris

M. s

eiffe

rtii

M. s

yrph

idae

M. t

aban

idae

Glucose fermentation + + + + + + + + + + +Arginine hydrolysis − − − − − − − + − − −Hemadsorption of guinea pig red blood cells − + − + + − + − + + −DNA G+C content (mol%) 27.3 28.3 27.7 26.4 30 29.1 30 28.8 30 27.6 28.3

aSymbols: +, >85% positive; −, 0–15% positive.

list of species of the genus Mesoplasma

1. Mesoplasma florum (McCoy, Basham, Tully, Rose, Carle and Bové 1984) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and Bové 1984, 14)flo¢rum. L. gen. pl. n. florum of flowers, indicating the recov-ery site of the organism.

This is the type species of the genus. Cells are oval or coc-coid. The organism is readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm, but does not pass a membrane with 100 nm porosity. Temperature range for growth is 18–37°C, with an optimum at 28–30°C. Colonies on agar medium containing horse serum supple-ments have a typical fried-egg appearance after anaerobic

incubation at 37°C. Colonies on agar do not hemadsorb guinea pig erythrocytes.

The 16S rRNA gene sequence is identical to that of Meso-plasma entomophilum (GenBank accession no. AF305693), but antiserum against Mesoplasma florum did not inhibit growth of Mesoplasma entomophilum or label the surfaces of Mesoplasma entomophilum colonies on agar (Tully et al., 1988). There are additional phenotypic distinctions between the two species.

No evidence of pathogenicity for plants or insects.Source: first isolated from surface of flowers on a

lemon tree (Citrus limon) in Florida, with subsequent isolations from floral surfaces of grapefruit (Citrus

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paradisi) and powderpuff trees (Albizia julibrissin) in Florida (McCoy et al., 1979). Also isolated from a variety of plants and from the gut tissues of numerous species of insects (Clark et al., 1986; Tully et al., 1990; Whitcomb et al., 1982).

DNA G+C content (mol%): 27.3 (Bd, whole genome sequence).

Type strain: L1, ATCC 33453, NCTC 11704.Sequence accession nos: AF300327 (16S rRNA gene),

NC_006055 (strain L1T genome sequence).

2. Mesoplasma chauliocola Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP

chau.li.o¢co.la. N.L. n. chaulio first part of the genus name of goldenrod beetle (Chauliognathus); L. suff. -cola (from L. masc. or fem. n. incola) inhabitant; N.L. masc. n. chauliocola inhabitant of the goldenrod beetle.

Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm, with a small number of cells able to pass through 100 nm porosity filters. Temperature range for growth is 10–37°C, with an optimum of 32–37°C. Nonmotile. Colo-nies incubated anaerobically at 32–37°C show fried-egg morphology. Colonies hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for plants or insects.Source: originally isolated from gut fluid of an adult gold-

enrod soldier beetle (Chauliognathus pennsylvanicus).DNA G+C content (mol%): 28.3 (Bd, Tm, HPLC).Type strain: CHPA-2, ATCC 49578.Sequence accession no. (16S rRNA gene): AY166704.

3. Mesoplasma coleopterae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 692VP

co.le.op.te¢rae. N.L. fem. gen. n. coleopterae of Coleoptera, referring to the order of insects (Coleoptera) from which the organism was first isolated.

Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Organisms are readily filterable through membranes with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–37°C, with an optimum of 30–37°C. Nonmotile. Colonies incubated anaerobically at 30°C usually have a fried-egg appearance. Agar colonies do not hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for plants or insects.Source: original isolation was from the gut of an adult

soldier beetle (Chauliognathus sp.).DNA G+C content (mol%): 27.7 (Bd, Tm, HPLC).Type strain: BARC 779, ATCC 49583.Sequence accession no. (16S rRNA gene): DQ514605 (partial

sequence).

4. Mesoplasma corruscae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP

cor.rus¢cae. N.L. fem. gen. n. corruscae of corrusca, refer-ring to the species of firefly beetle (Ellychnia corrusca) from which the organism was first isolated.

Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Cells are readily filterable through mem-branes with mean pore diameters of 450, 300, and 220 nm.

Temperature range for growth is 10–32°C, with an optimum of 30°C. Nonmotile. Colonies incubated anaerobically at 30°C usually have a fried-egg appearance. Colonies hemad-sorb guinea pig erythrocytes.

No evidence of pathogenicity for plants or insects.Source: original isolation was from the gut of an adult

firefly (Ellychnia corrusca).DNA G+C content (mol%): 26.4 (Bd, Tm, HPLC).Type strain: ELCA-2, ATCC 49579.Sequence accession no. (16S rRNA gene): AY168929.

5. Mesoplasma entomophilum (Tully, Rose, Carle, Bové, Hackett and Whitcomb 1988) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma entomophilum Tully, Rose, Carle, Bové, Hackett and Whitcomb 1988, 166)

en.to.mo.phi¢lum. Gr. n. entomon insect; N.L. neut. adj. philum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. entomophilum insect-loving.

Cells are pleomorphic, but primarily coccoid, ranging from 300 to 500 nm in diameter. Cells are readily filterable through 220 nm porosity membrane filters. The tempera-ture range for growth is 23–32°C, with an optimum at 30°C. Nonmotile. Colonies incubated under anaerobic condi-tions at 30°C usually have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes.

The 16S rRNA gene sequence is identical to that of Mesoplasma florum (GenBank accession no. AF300327), but antiserum against Mesoplasma florum did not inhibit growth of Mesoplasma entomophilum or label the surfaces of Meso-plasma entomophilum colonies on agar (Tully et al., 1988). There are additional phenotypic distinctions between the two species.

No evidence of pathogenicity for plants or insects.Source: original isolation was from the gut contents of a

tabanid fly (Tabanus catenatus). Also isolated from a variety of other species of insects.

DNA G+C content (mol%): 30 (Bd).Type strain: TAC, ATCC 43706, NCTC 11713.Sequence accession no. (16S rRNA gene): AF305693.

6. Mesoplasma grammopterae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP

gram.mop.te¢rae. N.L. fem. gen. n. grammopterae of Gram-moptera, referring to the genus of beetle (Grammoptera) from which the organism was first isolated.

Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Cells are readily filterable through mem-brane filters with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–37°C, with an optimum at 30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for plants or insects.Source: original isolation was from the gut contents of an

adult long-horned beetle (Grammoptera sp.). Other isola-tions were made from adult soldier beetle (Cantharidae sp.) and from an adult mining bee (Andrena sp.).

DNA G+C content (mol%): 29.1 (Bd, Tm, HPLC).Type strain: GRUA-1, ATCC 49580.Sequence accession no. (16S rRNA gene): AY174170.

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7. Mesoplasma lactucae (Rose, Kocka, Somerson, Tully, Whitcomb, Carle, Bové, Colflesh and Williamson 1990) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Mycoplasma lactucae Rose, Kocka, Somerson, Tully, Whitcomb, Carle, Bové, Colflesh and Williamson 1990, 141)

lac.tu¢cae. L. fem. n. lactuca lettuce; L. gen. n. lactucae of lettuce, referring to the plant from which the organism was first isolated.

Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter, with only occasional short, nonhelical, pleomorphic filaments. Cells are readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm, and a few cells are able to pass 100 nm poros-ity membranes. Temperature range for growth is 18–37°C, with optimal growth at 30°C. Nonmotile. Colonies incu-bated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for plants or insects.Source: original isolation was from lettuce (Lactuca

sativa).DNA G+C content (mol%): 30 (Bd).Type strain: 831-C4, ATCC 49193, NCTC 11718.Sequence accession no. (16S rRNA gene): AF303132. Has been

reported to possess three rRNA operons (Grau, 1991).

8. Mesoplasma photuris Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP

pho.tu¢ris. N.L. gen. n. photuris of Photuris, referring to the genus of firefly beetle (Photuris sp.) from which the organ-ism was first isolated.

Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Readily filterable through membrane filters with mean pore diameters of 450, 300, and 220 nm. Tem-perature range for growth is 10–32°C, with optimum at 30°C. Nonmotile. Colonies incubated under anaerobic con-ditions at 30°C have a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for plants or insects.Source: original isolation was from gut fluids of larval and

adult fireflies (Photuris lucicrescens and other Photuris spp.). One isolate (BARC 1976) was obtained by F.E. French from the gut of a horse fly (Tabanus americanus).

DNA G+C content (mol%): 28.8 (Bd, Tm, HPLC).Type strain: PUPA-2, ATCC 49581.Sequence accession no. (16S rRNA gene): AY177627.

9. Mesoplasma seiffertii (Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully and Whitcomb 1991) Tully, Bové, Laigret and Whitcomb 1993, 380VP (Acholeplasma seiffertii Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully and Whitcomb 1991, 48)

seif.fer¢ti.i. N.L. masc. gen. n. seiffertii of Seiffert, in honor of Gustav Seiffert, a German microbiologist who performed pioneering studies on mollicutes that occur in soil and com-post and do not require sterols for growth.

Cells are primarily coccoid, ranging in diameter from 300 to 500 nm. Cells are readily filterable through membranes with mean pore diameters of 450, 300, and

220 nm. Temperature range for growth is 20–35°C, with optimum at about 28–30°C. Nonmotile. Colonies incubated under anaerobic conditions at 30°C have a fried-egg appearance. Colonies hemadsorb guinea pig erythrocytes.

Three insect isolates of Mesoplasma seiffertii, two from mosquitoes and one from a horse fly, were compared to strain F7T of plant origin. High relatedness values of 78–98% DNA–DNA reassociation under high stringency conditions were obtained (Gros et al., 1996).

No evidence of pathogenicity for plants or insects.Source: first isolated from floral surfaces of a sweet orange

tree (Citrus sinensis) and from wild angelica (Angelica sylves-tris). Also isolated from insects.

DNA G+C content (mol%): 30 (Bd).Type strain: F7, ATCC 49495.Sequence accession no. (16S rRNA gene): L12056.

10. Mesoplasma syrphidae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 691VP

syr.phi¢dae. N.L. fem. gen. n. syrphidae of a syrphid, refer-ring to the syrphid fly family (Syrphidae), from which the organism was first isolated.

Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells readily pass membrane filters with mean pore diameters of 450, 300, and 220 nm. Tem-perature range for growth is 10–32°C, with optimum at 23–25°C. Nonmotile. Colonies incubated under anaerobic conditions at 23–25°C have a fried-egg appearance. Colo-nies hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects.Source: original isolation was from the gut of an adult

syrphid fly (Diptera: Syrphidae). Similar strains have been isolated from a bumblebee (Bombus sp.) and a skipper (Lepidoptera: Hesperiidae).

DNA G+C content (mol%): 27.6 (Bd, Tm, HPLC).Type strain: YJS, ATCC 51578.Sequence accession no. (16S rRNA gene): AY231458.

11. Mesoplasma tabanidae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 692VP

ta.ba.ni.dae. N.L. fem. gen. n. tabanidae of a tabanid, refer-ring to the horse fly family (Tabanidae), the host from which the organism was first isolated.

Cells are primarily coccoid, ranging in size from 300 to 500 nm in diameter. Cells readily pass membrane fil-ters with mean pore diameters of 450, 300, and 220 nm. Temperature range for growth is 10–37°C, with optimum at 37°C. Nonmotile. Colonies incubated under anaerobic conditions at 37°C display a fried-egg appearance. Colonies do not hemadsorb guinea pig erythrocytes.

No evidence of pathogenicity for insects.Source: original isolation was from the gut of an adult

horse fly (Tabanus abactor).DNA G+C content (mol%): 28.3 (Bd, Tm, HPLC).Type strain: BARC 857, ATCC 49584.Sequence accession no. (16S rRNA gene): AY187288.

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Whitcomb, R.F. 1983. Culture media for spiroplasmas. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 147–158.

Whitcomb, R.F. and K.J. Hackett. 1996. Identification of mollicutes from insects. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 2 (edited by Tully and Razin). Academic Press, San Diego, pp. 313–322.

Whitcomb, R.F., J.G. Tully, D.L. Rose, E.B. Stephens, A. Smith, R.E. McCoy and M.F. Barile. 1982. Wall-less prokaryotes from fall flow-ers in central United States and Maryland. Curr. Microbiol. 7: 285–290.

Williamson, D.L., J.G. Tully, D.L. Rose, K.J. Hackett, R. Henegar, P. Carle, J.M. Bové, D.E. Colflesh and R.F. Whitcomb. 1990. Mycoplasma somnilux sp. nov., Mycoplasma luminosum sp. nov., and Mycoplasma lucivorax sp. nov., new sterol-requiring mollicutes from firefly beetles (Coleoptera, Lampyridae). Int. J. Syst. Bacteriol. 40 : 160–164.

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Family ii. SpiroplaSmataceae

Family ii. Spiroplasmataceae Skripal 1983, 408Vp

DaviD L. WiLLiamson, GaiL E. Gasparich, Laura B. rEGassa, coLLEttE saiLLarD, JoëL rEnauDin, JosEph m. Bové anD roBErt F. WhitcomB*

Spi.ro.plas.ma.ta.ce′ae. N.l. neut. n. Spiroplasma, -atos type genus of the family; -aceae ending to denote a family; N.l. fem. pl. n. Spiroplasmataceae the Spiroplasma family.

Cells are helical during exponential growth, with rotatory, flexional, and translational motility. Genome size is variable: 780–2220 kbp. Variable sterol requirement for growth. Pro-cedures for determining sterol requirement are as described for Family I (Entomoplasmataceae). Possess a phosphoenolpyru-vate phosphotransferase system for glucose uptake. Reduced

nicotinamide adenine dinucleotide (NADH) oxidase activity is located only in the cytoplasm. Unable to synthesize fatty acids from acetate. Other characteristics are as described for the class and order.

Type genus: Spiroplasma Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt 1973, 201AL.

Genus i. Spiroplasma Saglio, l’Hospital, laflèche, Dupont, Bové, tully and Freundt 1973, 201al

DaviD L. WiLLiamson, GaiL E. Gasparich, Laura B. rEGassa, coLLEttE saiLLarD, JoëL rEnauDin, JosEph m. Bové anD roBErt F. WhitcomB*

Spi.ro.plas¢ma. Gr. n. speira (l. transliteration spira) a coil, spiral; Gr. neut. n. plasma something formed or molded, a form; N.l. neut. n. Spiroplasma spiral form.

Cells are pleomorphic, varying in size and shape from helical and branched nonhelical filaments to spherical or ovoid. The helical forms, usually 100–200 nm in diameter and 3–5 mm in length, generally occur during the exponential phase of growth and in some species persist during stationary phase. The cells of some species are short (1–2 mm). In certain cases, helical cells may be very tightly coiled, or the coils may show continu-ous variation in amplitude. Spherical cells ~300 nm in diameter and nonhelical filaments are frequently seen in the stationary phase, where they may not be viable, and in all growth phases in suboptimal growth media, where they may or may not be viable. In some species during certain phases, spherical forms may be the replicating form. Helical filaments are motile, with flexional and twitching movements, and often show an appar-ent rotatory motility. Fibrils are associated with the membrane, but flagellae, periplasmic fibrils, or other organelles of locomo-tion are absent. Fimbriae and pili observed on the cell surface of insect- and plant-pathogenic spiroplasmas are believed to be involved in host-cell attachment and conjugation (Ammar et al., 2004; Özbek et al., 2003), but not in locomotion. Cells divide by binary fission, with doubling times of 0.7–37 h. Fac-ultatively anaerobic. The temperature growth range varies among species, from 5 to 41°C. Colonies on solid media are frequently diffuse, with irregular shapes and borders, a condi-tion that reflects the motility of the cells during active growth (Figure 111). Colony type is strongly dependent on the agar concentration. Colony sizes vary from 0.1 to 4.0 mm in diam-eter. Colonies formed by nonmotile variants or mutants, or by cultures growing on inadequate media are typically umbonate with diameters of 200 mm or less. Some species, such as Spiro-plasma platyhelix, have barely visible helicity along most of their length and display little rotatory or flexing motility. Colonies of motile, fast-growing spiroplasmas are diffuse, often with sat-ellite colonies developing from foci adjacent to the initial site of colony development. Light turbidity may be produced in liquid cultures. Chemo-organotrophic. Acid is produced from glucose. Hydrolysis of arginine is variable. Urea, arbutin, and

esculin are not hydrolyzed. Sterol requirements are variable. An optimum osmolality, usually in the range of 300–800 mOsm, has been demonstrated for some spiroplasmas. Media contain-ing mycoplasma broth base, serum, and other supplements are required for primary growth, but after adaptation, growth often occurs in less complex media. Defined or semi-defined media are available for some species. Resistant to 10,000 U/ml peni-cillin. Insensitive to rifampicin, sensitive to erythromycin and tetracycline. Isolated from the surfaces of flowers and other plant parts, from the guts and hemolymph of various insects and crustaceans, and from tick triturates. Also isolated from vascular plant fluids (phloem sap) and insects that feed on the fluids. Specific host associations are common. The type spe-cies, Spiroplasma citri, is pathogenic for citrus (e.g., orange and

FIGURE 111. Colonial morphology of Spiroplasma lampyridicola strain PUP-lT grown on SP-4 agar under anaerobic conditions for 4 d at 30°C. The diffuse appearance and indistinct margins reflect the motility of spiroplasmas during active growth. Bar = 50 mm. (Reprinted with permis-sion from Stevens et al., 1997. Int. J. Syst. Bacteriol. 47: 709–712.)

*Deceased 21 December 2007.

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GeNuS i. SpiroplaSma

grapefruit), producing “stubborn” disease. Experimental or natural infections also occur in horseradish, periwinkle, radish, broad bean, carrot, and other plant species. Spiroplasma kunkelii is a maize pathogen. Some species are pathogenic for insects. Certain species are pathogenic, under experimental condi-tions, for a variety of suckling rodents (rats, mice, hamsters and rabbits) and/or chicken embryos. Genome sizes vary from 780 to 2220 kbp (PFGE).

DNA G+C content (mol%): 24–31 (Tm, Bd).Type species: Spiroplasma citri Saglio, L’Hospital, Laflèche,

Dupont, Bové, Tully and Freundt 1973, 202AL.


Morphology. The morphology of spiroplasmas is most eas-ily observed in suspensions with the light microscope under dark-field illumination (Williamson and Poulson, 1979). In the exponential phase in liquid media, most spiroplasma cells are helical filaments 90–250 nm in diameter and of variable length (Figure 112). Fixed and negatively stained cells usually show a blunt and a tapered end (Williamson, 1969; Williamson and Whitcomb, 1974). The tapered ends of the cells are a conse-quence of the constriction process preceding division (Garnier et al., 1981, 1984). However, they are adapted as attachment sites in some species (Ammar et al., 2004).

Motility. Helical spiroplasma cells exhibit flexing, twitching, and apparent rotation about the longitudinal axis (Cole et al., 1973; Davis and Worley, 1973). Spiroplasmas exhibit tempera-ture-dependent chemotactic movement toward higher concen-trations of nutrients, such as carbohydrates and amino acids

(Daniels and Longland, 1984, 1980); but motility is random in the absence of attractants (Daniels and Longland, 1984). Both natural (Townsend et al., 1980b, 1977) and engineered (Cohen et al., 1989; Duret et al., 1999; Jacob et al., 1997) motil-ity mutants have been described. These mutants form perfectly umbonate colonies on solid medium. Mutational analysis has highlighted the involvement of the smc1 gene in motility. Jacob et al. (1997) demonstrated that a Tn4001 insertion mutant with reduced flexional motility and no rotational motility could be complemented with the wild-type scm1 gene. The scm1 gene encodes a 409 amino acid polypeptide having ten transmem-brane domains but no significant homology with known pro-teins. In another study, the scm1 gene was inactivated through homologous recombination, abolishing motility (Duret et al., 1999). The disrupted scm1− mutant was injected into the leaf-hopper vector (Circulifer haematoceps); it multiplied actively in the insect vector and was then transmitted to periwinkle plants. The mutant induced symptoms that were indistinguishable from those caused by the motile wild-type strain showing that spiroplasma motility is not essential for phytopathogenicity and transmission to the plant host (Duret et al., 1999).

Fibrils and motility. Microfibrils 3.6 nm in width have been envisioned in the membranes of some spiroplasmas. These structures have repeat intervals of 9 nm along their lengths (Williamson, 1974) and form a ribbon that extends the entire length of the helix (Charbonneau and Ghiorse, 1984; William-son et al., 1984). The sequence of the fibril protein gene has been determined (Williamson et al., 1991) and the calculated mass of the fibril protein is 59 kDa. The flat, monolayered, membrane-bound ribbon composed of several well-ordered fibrils represents the internal spiroplasmal cytoskeleton. The spiroplasmal cytoskeletal ribbon follows the shortest helical line on the cellular coil. Recent studies have focused on the detailed cellular and molecular organization of the cytoskel-eton in Spiroplasma melliferum and Spiroplasma citri (Gilad et al., 2003; Trachtenberg, 2004; Trachtenberg et al., 2003a, b; Tra-chtenberg and Gilad, 2001). Each cytoskeletal ribbon contains seven fibril pairs (or 14 fibrils) and the functional unit is a pair of aligned fibrils (Trachtenberg et al., 2003a). Paired fibrils can be viewed as chains of tetramers composed of 59 kDa mono-mers. Cryo-electron tomography has been used to elucidate the native state, cytoskeletal structure of Spiroplasma melliferum and suggested the presence of three parallel ribbons under the membrane: two appear to be composed of the fibril pro-tein and the third is composed of the actin-like MreB protein (Kürner et al., 2005). Subsequent studies suggest the presence of a single ribbon structure (Trachtenberg et al., 2008). The subunits in the fibrils undergo conformational changes from circular to elliptical, which results in shortening of the fibrils and helix contraction, or from elliptical to circular, leading to a length increase of the fibrils and cell helix. The cytoskeleton, which is bound to the spiroplasmal membrane over its entire length, acts as a scaffold and controls the helical shape of the cell. The cell shape is therefore dynamic. Movement appears to be driven by the propagation of a pair of kinks that travel down the length of the cell along the fibril ribbons (Shaevitz et al., 2005; Wada and Netz, 2007; Wolgemuth and Charon, 2005). The contractile cytoskeleton can thus be seen as a “linear motor” in contrast to the common “rotary motor” that is part of the flagellar apparatus in bacteria (Trachtenberg, 2006).

FIGURE 112. Electron micrograph of Drosophila willistoni strain B3SR sex-ratio spiroplasmas. Hemolymph suspension in phosphate buffered saline, glutaraldehyde vapor-fixed, and negatively stained with 1% phos-photungstic acid, pH 7.2. (Reprinted with permission from Whitcomb et al., 2007. Biodiversity and Conservation 16: 3877–3894.)

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There are several adherent proteins that copurify with the cytoskeleton, ranging in size from 26 to 170 kDa (Townsend et al., 1980a; Trachtenberg, 2006; Trachtenberg and Gilad, 2001). These proteins are apparently membrane-associated and may function as anchor proteins (Trachtenberg and Gilad, 2001). The structural organization of the cytoskeleton-associ-ated proteins of Spiroplasma melliferum is beginning to be elu-cidated (Trachtenberg et al., 2008). The 59 kDa polypeptide is the cytoskeletal fibril protein. The 26 kDa polypeptide is probably spiralin, the major spiroplasmal membrane protein. However, the involvement of spiralin in helicity and motility is unlikely (see “Spiralin” section below), especially since spira-lin is anchored on the outside surface of the cell (Bévén et al., 1996; Bové, 1993; Brenner et al., 1995; Foissac et al., 1996) and spiralin-deficient mutants maintain helicity and motility (Duret et al., 2003). The 45 kDa protein may correspond to the prod-uct of the scm1 gene, shown to be essential for motility (Jacob et al., 1997), and the 34 kDa protein may be the product of the mreB1 gene (W. Maccheroni and J. Renaudin, unpublished).

MreB is the bacterial homolog to eukaryotic actin (Jones et al., 2001; Van den Ent et al., 2001). Early work provided evidence for the presence of actin-like proteins in spiroplasmas. Antisera prepared against SDS-denatured invertebrate actin coupled to horseradish peroxidase specifically stained cells of Spiroplasma citri (Williamson et al., 1979a). Also, a protein with a molecular mass similar to that of actin (protein P25) was isolated from Spiroplasma citri and reacted with IgG directed against rabbit actin (Mouches et al., 1982b, c, 1983b). Monospecific antibodies raised against the P25 protein recognized not only P25 of Spiroplasma citri, but also a homologous protein from Mycoplasma mycoides PG50 and Ureaplasma urealyticum serotype V (Mouches et al., 1983b). More recent work has focused on the molecular organization of the genes. mreB genes are present in rod-shaped, filamentous, and helical bacteria, but not in coccoid, spherical bacteria, regard-less of whether or not they are Gram-stain-positive or Gram-stain-negative. mreB genes are also absent from the pleomorphic mycoplasmas. However, Spiroplasma citri contains five homologs of Bacillus subtilis mreB genes (Maccheroni et al., 2002). Four of these (mreB2, 3, 4, and 5) form a cluster on the genome and are transcribed in two separate operons. Gene mreB1 is transcribed as a monocistronic operon and at a much higher level.

Growth characteristics. Spiroplasma cells increase in length and divide by constriction. Pulse labeling of the membrane with tritiated amino acids revealed a polar growth of the helix. Polar-ity was also observed by tellurium-labeling of oxido-reduction sites (Garnier et al., 1984). In the stationary or death phase, the cells are usually distorted, often forming either subovoid bod-ies or nonhelical filaments. Within cultured insect cells, all the spiroplasma cells were subovoid, but presumably viable (Waya-dande and Fletcher, 1998). Thus, the ability of cells to grow and divide is not linked inextricably to helicity.

Growth rate. Enumerated microscopically (Rodwell and Whitcomb, 1983), spiroplasmas reach titers of 108–1011 cells/ml in medium containing horse or fetal bovine serum. Growth rates of related strains tend to be similar. Konai et al. (1996a) calculated doubling times from the time required for medium acidification. In general, spiroplasmas adapted to complex cycles or single hosts had slower growth rates than spiroplasmas known or suspected to be transmitted on plant surfaces.

Temperature. Konai et al. (1996a) determined temperature ranges and optima for a large number of spiroplasma strains. The ranges of some strains (e.g., Spiroplasma apis) were very wide (5–41°C), but some group I strains from leafhoppers and plants grew only at 25° and 30°C. Although some spiroplasmas grew well at 41°C, none grew at 43°C.

Biochemical reactions. All tested spiroplasmas ferment glucose with concomitant acid production, although the utili-zation rates may vary. Some strains of group I (e.g., members of subgroups I-4 and I-6) and all strains of Spiroplasma mirum ferment glucose slowly. With Spiroplasma citri, all strains tested grew actively on fructose and strain GII3 grew on fructose, glu-cose, or trehalose. The ability of spiroplasmas to utilize argin-ine varies (Hackett et al., 1996a). Arginine hydrolysis by some spiroplasmas can be observed only if glucose is also present in the medium. In other cases, aggressive glucose metabolism interferes with detection of arginine hydrolysis (Hackett et al., 1996a).

Regulation of the fructose and trehalose operons of Spiroplasma citri. The fructose operon of Spiroplasma citri (Gaurivaud et al., 2000a) became of special interest when fructose utiliza-tion was implicated in Spiroplasma citri phytopathogenicity (see “Mechanism of Spiroplasma citri phytopathogenicity” below). In particular, the role of the first gene of the operon, fruR, was investigated. In vivo transcription of the operon is greatly enhanced by the presence of fructose in the growth medium, whereas glucose has no effect. When fruR is not expressed (fruR− mutants), transcription of the operon is not stimulated by fructose and the rate of fructose fermentation is decreased, indicating that FruR is an activator of the fructose operon (Gau-rivaud et al., 2001). Trehalose is the major sugar in leafhoppers and other insects. The trehalose operon of Spiroplasma citri has a gene organization very similar to that of the fructose operon and the first gene of the trehalose operon, treR, also encodes a transcriptional activator of the operon (André et al., 2003).

Sterol utilization. It was originally thought that all spiroplas-mas require sterol for growth. Subsequent screening by Rose et al. (1993) showed that a minority of the spiroplasmas tested were able to sustain growth in mycoplasma broth base medium without sterols. The discovery that the sterol requirement in Mollicutes is polyphyletic greatly diminished the significance of sterol requirements in mollicute taxonomy (Tully et al., 1993).

Metabolic pathways and enzymes. The intermediary meta-bolism of Mollicutes has been reviewed (Miles, 1992; Pollack, 2002a, b; Pollack et al., 1997). Like all mollicutes, Spiroplasma species apparently lack both cytochromes and, except for malate dehydrogenase, the enzymes of the tricarboxylic acid cycle. They do not have an electron-transport system and their res-piration is characterized as being flavin-terminated. McElwain et al. (1988) studied Spiroplasma citri and Pollack et al. (1989) screened ten spiroplasma species for 67 enzyme activities. All spiroplasmas were fermentative; their 6-phosphofructokinases (6-PFKs) required ATP for substrate phosphorylation during glycolysis. This enzymic requirement is common to all molli-cutes except Acholeplasma and Anaeroplasma spp. The 6-PFKs of the species in these genera require pyrophosphate and cannot use ATP. Additionally, except for Spiroplasma floricola, all Spiro-plasma species have dUTPase activity. Pollack et al. (1989) also

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reported that all spiroplasmas except Spiroplasma floricola have deoxyguanosine kinase activity. They found that deoxyguanos-ine, but no other nucleoside, could be phosphorylated to GMP with ATP.

Spiroplasmal proteins with multiple functions have been described. The CpG-specific methylase from Spiroplasma mono-biae appears to also have topoisomerase activity (Matsuo et al., 1994). Protein P46 of Spiroplasma citri is a bifunctional protein in which the N-terminal domain represents ribosomal pro-tein L29, whereas the C-terminal domain is capable of bind-ing a specific inverted repeat sequence. It could be involved in regulation (Le Dantec et al., 1998). Such protein multifunc-tionality may reflect genomic economy in the small mollicute genome (Pollack, 2002b). However, functional redundancy has also been reported; Spiroplasma citri apparently has two distinct membrane ATPases (Simoneau and Labarère, 1991).

Genome size, genomic maps, and chromosomal rearrange-ments. PFGE revealed that the genome size range for spiro-plasmas varied continuously (Pyle and Finch, 1988) from 780 kbp for Spiroplasma platyhelix to 2220 kbp for Spiroplasma ixodetis (Carle et al., 1995, 1990). There is a general trend for genomic simplification in Spiroplasma lineages. This trend culminated in loss of helicity and motility in the Entomoplasmataceae and even-tually to the host transfer events forming the mycoides group of mycoplasmas (Gasparich et al., 2004).

The genome size of Spiroplasma citri varies among strains from 1650 to 1910 kbp (Ye et al., 1995). It was found that the relative positions of mapped loci were conserved in most of the strains, but that differences in the sizes of certain fragments permitted genome size variation. Genome size can fluctuate rapidly in spiroplasma cultures after a relatively short number of in vitro passages (Melcher and Fletcher, 1999; Ye et al., 1996). The genome of Spiroplasma melliferum is 360 kbp shorter than that of Spiroplasma citri strain R8-A2T, but DNA hybridization has shown that the two spiroplasmas share extensive DNA hybridiza-tion (65%). Comparison of their genomic maps revealed that the genome region, which is shorter in Spiroplasma melliferum, corresponds to a variable region in the genomes of Spiroplasma citri strains and that a large region of the Spiroplasma melliferum genome is inverted in comparison with Spiroplasma citri. There-fore, chromosomal rearrangements and deletions were proba-bly major events during evolution of the genomes of Spiroplasma citri and Spiroplasma melliferum. In addition, a large amount of noncoding DNA is present as repeat sequences (McIntosh et al., 1992; Nur et al., 1986, 1987) and integrated viral DNA (Bébéar et al., 1996) may also account for differences in genome sizes of closely related species.

Base composition. The DNA G+C content for most spiro-plasma groups and subgroups has been determined (Carle et al., 1995, 1990; Williamson et al., 1998). Most group I spiro-plasmas and Spiroplasma poulsonii have a G+C content of 25–27 mol%. However, the G+C content of subgroup I-6 Spiroplasma insolitum is significantly higher, indicating that the base compo-sition of spiroplasmal DNA may shift over relatively short evo-lutionary periods. The range of G+C content of 25–27 mol% is modal for Spiroplasma and is also common in the Apis clade. However, Spiroplasma mirum (group V), strains of Spiroplasma apis (group IV), and group VIII strains have a G+C content of about 29–31 mol%. Restriction sites containing only G and C

nucleotides are not uniformly distributed over the genome (Ye et al., 1992).

Methylated bases. Methylated bases have been detected in spiroplasmal DNA (Nur et al., 1985). The gene encoding the CpG methylase in Spiroplasma monobiae has been cloned (Ren-baum et al., 1990) and its mode of action studied (Renbaum and Razin, 1992).

DNA restriction patterns. Restriction patterns of spiroplas-mal DNA, as determined by polyacrylamide gel electrophoresis, may be highly similar among strains of a given species (Bové et al., 1989). Variations in restriction fragment length patterns among strains of Spiroplasma corruscae correlated imperfectly with serological variation, so their significance was uncertain (Gasparich et al., 1998).

RNA genes. Some spiroplasmas, such as Spiroplasma citri, have only one rRNA operon, whereas others, such as Spiro-plasma apis, have two (Amikam et al., 1984, 1982; Bové, 1993; Grau et al., 1988; Razin, 1985). The three rRNA genes are linked in the classical order found in bacteria: 5¢-16S–23S-5S-3¢. The sequence of the 16S rRNA gene (rDNA) of most spiro-plasma species has been determined for phylogenetic studies (Gasparich et al., 2004; Weisburg et al., 1989). A gene cluster of ten tRNAs (Cys, Arg, Pro, Ala, Met, Ile, Ser, fMet, Asp, Phe) was identified in Spiroplasma melliferum (Rogers et al., 1987). Simi-lar tRNA gene clusters have been cloned and sequenced from Spiroplasma citri (Citti et al., 1992).

Codon usage. In spiroplasmas, UGA is not a stop codon but encodes tryptophan. The universal tryptophan codon, UGG, is also used (Citti et al., 1992; Renaudin et al., 1986). Codon usage also reflects the A+T richness of spiroplasmal DNA (usually about 74 mol% A+T). For example, in Spiroplasma citri, UGA is used to code for tryptophan eight times more frequently than the universal tryptophan codon UGG (Bové, 1993; Citti et al., 1992; Navas-Castillo et al., 1992). Also, synonymous codons with U or A at the 5¢ or 3¢ ends are preferentially used over those with a C or G in that position.

RNA polymerase and spiroplasmal insensitivity to rifam-picin. Spiroplasmas are insensitive to rifampicin. DNA-dependent RNA polymerases from Spiroplasma melliferum and Spiroplasma apis were at least 1000 times less sensitive to rifam-picin than the corresponding Escherichia coli enzyme (Gadeau et al., 1986). Rifampicin insensitivity of Spiroplasma citri and all other mollicutes tested was found to be associated with the presence of an asparagine residue at position 526 in RpoB. The importance of the asparagine residue was confirmed by site-directed mutagenesis of the histidine codon (CAC) to an asparagine codon (AAC) at position 526 of Escherichia coli RpoB, resulting in a rifampicin-resistant mutant (Gaurivaud et al., 1996). The genetic organization surrounding the rpoB gene in spiroplasmas is also atypical. In many bacteria, rpoB is part of the b operon in which the four genes rplK, rplA, rplJ, and rplL, encoding ribosomal proteins L11, L1, L10, and L12, respectively, are located immediately upstream of rpoB; rpoC is immediately downstream of rpoB. In Spiroplasma citri, the gene organization is different in that the hsdS gene, encoding a com-ponent of a type I restriction-modification system, is upstream of rpoB. Sequences showing similarities with insertion elements are found between hsdS and rpoB (Laigret et al., 1996).

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DNA polymerases and other proteins involved in DNA rep-lication and repair. From genomic studies, it appears that Mycoplasma species carry the essential, multimeric enzyme for genomic DNA replication, DNA polymerase III. The subunit responsible for actual DNA biosynthesis is subunit a, encoded by polC (dnaE). The polC gene has been identified in all sequenced mollicute genomes, including Spiroplasma citri. The genes encoding the other subunits, dnaN (subunit b) and dnaX (subunits t and g), are also shared by the Spiroplasma and Myco-plasma species studied to date. So, it seems that spiroplasmas, like other mollicutes, possess DNA polymerase III and that it is probably the major DNA replication enzyme. However, there is also evidence for two additional DNA polymerases. A second gene for a DNA polymerase (enzyme B) was found in the Spiro-plasma citri genome and there is evidence that the Spiroplasma kunkelii polA gene may encode a full-length DNA polymerase I protein (Bai and Hogenhout, 2002). DNA polymerase I is a single polypeptide that has, in addition to DNA synthesis activ-ity, two exonuclease activities: exo-3¢ to 5¢ as well as exo-5¢ to 3¢. At this stage, it is not possible to determine the equivalence between the three spiroplasmal DNA polymerases identified by sequencing (Pol III, enzyme B, and Pol I) and those originally detected biochemically (ScA, ScB, ScC) (Charron et al., 1979, 1982). As the Spiroplasma citri genome sequencing project has progressed, the following Spiroplasma citri genes involved in DNA replication have been detected: dnaA, dnaB, polA, dnaE, polC, dnaN, dnaX, holB, dinB (truncated), dnaJ, dnaK, gyrA, gyrB, parC, parE, topA, rnhB, rnhC, rnpA, rnR, rnc, yrrc, xseA, xseB, and ssb (Carle et al., 2010; accession numbers AM285301–AM285339). Genes encoding DNA replication proteins have also been iden-tified in Spiroplasma kunkelii (Bai and Hogenhout, 2002). Spiro-plasma citri is highly sensitive to UV irradiation (Labarère and Barroso, 1989) and the organism has no functional recA gene, since a significant portion of the C-terminal part of the gene is lacking (Marais et al., 1996).

Origin of DNA replication. Even before the Spiroplasma citri genome project was initiated, some fragments with mul-tiple open reading frames had been completely sequenced. For example, Ye et al. (1994b) sequenced a 5.6 kbp fragment containing genes for the replication initiation protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA and gyrB). Several dnaA-box con-sensus sequences were found upstream and downstream of the dnaA gene. From these data, it was established that the dnaA region was the origin of replication in Spiroplasma citri (Ye et al., 1994b). Zhao et al. (2004a) cloned a cell division gene cluster from Spiroplasma kunkelii and functionally characterized the key division gene, ftsZsk, and showed that it encodes a cell division protein similar to FtsZ proteins from other bacteria.

Spiralin. Spiralin, encoded by the spi gene, is the major membrane protein of Spiroplasma citri (Wróblewski et al., 1977, 1989). The deduced amino acid sequence of the protein (Bové et al., 1993; Chevalier et al., 1990; Saillard et al., 1990) corre-sponds well with the experimentally determined amino acid composition (Wróblewski et al., 1984). In particular, spiralin lacks tryptophan and, thus, has no UGG and/or UGA codons, which facilitates gene expression in Escherichia coli. Detailed analyses showed that all Spiroplasma citri spiralins were 241–242 amino acids long (Foissac et al., 1996). A conserved central

region and an amino acid sequence repetition, including a VTKXE consensus sequence, are present in all spiralins analyzed (Foissac et al., 1997a). Spiralin confers a significant amount of the antigenic activity in group I spiroplasmas (Whitcomb et al., 1983) and has a high degree of species specificity, although minor cross-reactions have been detected (Zaaria et al., 1990). The spiralin genes of Spiroplasma citri and Spiroplasma melliferum species, which have about 65% overall DNA–DNA hybridization, shared 89% nucleotide sequence identity and 75% deduced amino acid sequence similarity (Bové et al., 1993).

Spiralin mutants were constructed through homologous recombination in Spiroplasma citri to examine the role of spi-ralin in vivo (Duret et al., 2003). Phenotypic characterization of mutant 9a2 showed that, in spite of a total lack of spiralin, it maintained helicity and motility similar to the wild-type strain GII3 (Duret et al., 2003). When injected into the leafhopper vector, Circulifer haematoceps, the mutant multiplied to a high titer, but transmission efficiency to periwinkle plants was very low compared to the wild-type strain. In the infected plants, however, the spiralin-deficient mutant multiplied well and pro-duced the typical symptoms of the disease. In addition, prelimi-nary results indicated that the mutant could not be acquired by insects feeding on 9a2-infected plants, suggesting that spira-lin may mediate spiroplasma invasion of insect tissues (Duret et al., 2003). In order to test this possibility, Circulifer haemato-ceps leafhopper proteins were screened as putative Spiroplasma citri-binding molecules using Far-Western analysis (Killiny et al., 2005).These experiments showed that spiralin is a lectin capa-ble of binding to insect 50 and 60 kDa mannose glycoproteins. Hence, spiralin could play a key role in insect transmission of Spiroplasma citri by mediating spiroplasma adherence to epithe-lial cells of the insect vector gut or salivary gland (Killiny et al., 2005). This would also explain why the spiralin-negative mutant 9a2 is poorly transmitted by the vector and is not acquired by insects feeding on 9a2-infected plants.

Viruses. Four different virus types have been found in Spiro-plasma, SpV1-SpV4. Use of SpV1 viruses for recombinant DNA studies in Spiroplasma citri is described later in the section on “Tools for molecular genetics of Spiroplasma citri ”.

Cells of many spiroplasma species contain filamentous/rod-shaped viruses (SVC1 = SpV1) that are associated with nonlytic infections (Bové et al., 1989; Ranhand et al., 1980; Renaudin and Bové, 1994). They belong to the Plectrovirus group within the Inoviridae. SpV1 viruses have circular, single-stranded DNA genomes (7.5 to 8.5 kbp), some of which have been sequenced (Renaudin and Bové, 1994). SpV1 sequences also occur as prophages in the genome of the majority of Spiroplasma citri strains studied (Renaudin and Bové, 1994). These insertions take place at numerous sites in the chromosomes of Spiroplasma citri (Ye et al., 1992) and Spiroplasma melliferum (Ye et al., 1994a). The SpV1-ORF3 and the repeat sequences could be part of an IS-like element of chromosomal origin. Resistance of spiroplas-mas to virus infection may be associated with integration of viral DNA sequences in the chromosome or extrachromosomal elements (Sha et al., 1995). The evolutionary history of these viruses is unclear, but there is some evidence for virus and plas-mid co-evolution in the group I Spiroplasma species (Gasparich et al., 1993a) and indications of potentially widespread horizon-tal transmission (Vaughn and de Vos, 1995). Virus infection of spiroplasma cells can pose problems in cultures. For example,

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lyophilized early passages of Spiroplasma citri R8-A2T proved difficult to grow and electron microscopy revealed that these cells carried large numbers of virions of virus SpV1-R8A2 (Cole et al., 1974). Likewise, SpV1 viruses have been found in Spiro-plasma poulsonii (Cohen et al., 1987) and Spiroplasma melliferum (Liss and Cole., 1981); the Spiroplasma melliferum SpV1-KC3 virus forms plaques on various strains of Spiroplasma melliferum, including the type strain BC-3T.

A second virus, reminiscent of a type B tailed bacterial virus, occurs in a small number of Spiroplasma citri strains (Cole et al., 1973). This SCV2 (= SpV2) virus is a polyhedron with a long, noncontractile tail. It may be associated with lytic infection. Infections in which large numbers of virions of SpV2 viruses are produced tend to be irregular and difficult to maintain under experimental conditions, so this is the least studied of the spiro-plasma viruses.

A third virus (SpV3) forms polyhedral virions with short tails and has been found in many strains of Spiroplasma citri (Cole, 1979, 1977, 1974). The SpV3 genome is a linear dou-ble-stranded DNA molecule of 16 kbp, which can circularize to form a covalently closed molecule with single-stranded gaps, indicating that the linear molecule has cohesive ends. There is significant diversity among SpV3 viruses, extending even to major differences in genome sizes. Virus SpV3-AV9/3 was iso-lated from Spiroplasma citri strain ASP-9 (Stephens, 1980). Dick-inson and Townsend (1984) isolated the SpV3 virus from plants infected with Spiroplasma citri. This virus, when plated on cells of Spiroplasma citri, had a plaque morphology typical of temperate phages. In spiroplasma cells that have been lysogenized, com-plete virus genomes may be integrated into the spiroplasma chromosome. These cells are then immune to superinfection by the lysogenizing virus, but susceptible to other SpV3 viruses. It is possible that lysogenization of Spiroplasma citri by SpV3-ai affects spiroplasma pathogenicity, particularly with respect to attenuation. Drosophila spiroplasmas, male-lethal or nonlethal, usually carry SpV3 viruses. Each strain of Drosophila spiroplasma carries an associated virus that is lytic to certain other strains (Oishi et al., 1984).

A fourth virus (SpV4), with a naked, icosahedral nucleo-capsid 25 nm in diameter, was discovered (Ricard et al., 1982) in the B63 strain of Spiroplasma melliferum. SpV4 has a circu-lar, single-stranded DNA genome (Renaudin and Bové, 1994; Renaudin et al., 1984a, b) and is a lytic Spiromicrovirus within the Microviridae (Chipman et al., 1998). Infection with this virus results in very clear plaques, indicating a lytic process. Host range studies (Renaudin et al., 1984a, b) have shown that only Spiroplasma melliferum is susceptible to SpV4. Two strains of Spiro-plasma melliferum, including the type strain BC-3T and B63, are not susceptible, as no plaques were formed on lawns of these spiroplasmas. These strains could be infected by transfection suggesting that resistance to the whole virus occurred at the level of adsorption or penetration of the virus (Renaudin and Bové, 1994; Renaudin et al., 1984b).

Genome sequencing. Genomic DNA sequencing efforts for two Spiroplasma species are in progress. For Spiroplasma citri GII3 (Carle et al., 2010; Saillard et al., 2008), assembly of 20,000 sequencing reads obtained from shotgun and chromosome specific libraries yielded: (1) 39 chromosomal contigs totalling 1525 kbp of the 1820 kbp Spiroplasma citri GII3 chromosome as well as (2) 8 circular contigs, which proved to represent seven

plasmids: pSciA (7.8 kbp), pSci1 to pSci6 (12.9 to 35.3 kbp), and one viral RF DNA (SVTS2). The chromosomal contigs con-tained 1905 putative genes or coding sequences (CDS). Of the CDS-encoded proteins, 29% are involved in cellular processes, cell metabolism, or cell structure. CDS for viral proteins and mobile elements represented 24% of the total, whereas 47% of the CDS were for hypothetical proteins with no known function; 21% of the total CDS appeared truncated as compared to their bacterial orthologs. Families of paralogs were mainly clustered in a large region of the chromosome opposite the origin of rep-lication. Eighty-four CDS were assigned to transport functions, including phosphoenolpyruvate phosphotransferase systems (PTS), ATP binding cassette (ABC) transporters, and ferritin. In addition to the general enzymes EI and HPr, glucose- fructose- and trehalose-specific PTS permeases, and glycolytic and ATP synthesis pathways, Spiroplasma citri possesses a Sec-dependent protein export system and a nearly complete pathway for ter-penoid biosynthesis. The sequencing of the Spiroplasma kunkelii CR2-3x genome (1.55 Mb) is also nearing completion (http://www.genome.ou.edu/spiro.html); the physical and genetic maps have been published (Dally et al., 2006). Several studies have begun to focus on gene content and genomic organiza-tion (Zhao et al., 2003, 2004a, b). Results show that, in addition to virus SpV1 DNA insertions, the Spiroplasma kunkelii genome harbors more purine and amino acid biosynthesis, transcrip-tional regulation, cell envelope, and DNA transport/binding genes than Mycoplasmataceae (e.g., Mycoplasma genitalium and Mycoplasma pneumoniae) genomes (Bai and Hogenhout, 2002).

Plasmids. Several plasmids have been discovered in spiro-plasmas (Archer et al., 1981; Gasparich and Hackett, 1994; Gasparich et al., 1993a; Mouches et al., 1984a; Ranhand et al., 1980). They are especially common in spiroplasmas of group I. Eight extrachromosomal elements, including seven plas-mids, were discovered during the Spiroplasma citri GII3 genome sequencing project. The six largest plasmids, pSci1 to pSci6, range from 12.9 to 35.3 kb (Saillard et al., 2008). In silico analyses of plasmid sequences revealed that they share exten-sive regions of homology and display a mosaic gene organiza-tion. Genes encoding proteins of the TraD-TraG, TrsE-TraE, and Soj-ParA protein families, were predicted in most of the pSci sequences. The presence of such genes, usually involved in chromosome integration, cell to cell DNA transfer, or DNA element partitioning; suggests that these molecules could be inherited vertically as well as horizontally. The largest plasmid, pSci6, encodes P32 (Killiny et al., 2006), a membrane-associ-ated protein interestingly absent in all insect non-transmissible strains tested so far. The five remaining plasmids (pSci1 to pSci5) encode eight different Spiroplasma citri adhesion-related proteins. The complete sequences of plasmids pSKU146 from Spiroplasma kunkelii CR2-3x and pBJS-O from Spiroplasma citri BR3 have been reported (Davis et al., 2005; Joshi et al., 2005). These large plasmids, like the above Spiroplasma citri plasmids, encode an adhesin and components of a type IV translocation-related conjugation system. Characterizing the replication and stability regions of Spiroplasma citri plasmids resulted in the iden-tification of a novel replication protein, suggesting that Spiro-plasma citri plasmids belong to a new plasmid family and that the soj gene is involved in segregational stability of these plas-mids (Breton et al., 2008a). Similar replicons were detected in various spiroplasmas of group I, such as Spiroplasma melliferum,

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http://www.genome.ou.edu/spiro.html

http://www.genome.ou.edu/spiro.html


Spiroplasma kunkelii, Spiroplasma sp. 277F, and Spiroplasma phoeni-ceum, showing that they are not restricted to plant pathogenic spiroplasmas.

Tools for molecular genetics of Spiroplasma citri. Recent recombinant DNA tools are described in this section.

Several reports have been published concerning the use of SpV1 viruses as tools to introduce recombinant DNA into spiroplasmas, including optimization of transfection conditions (Gasparich et al., 1993b). The replicative form of SpV1 was used to clone and express the Escherichia coli-derived chloram-phenicol acetyltransferase (cat) gene in Spiroplasma citri. Both the replicative form (RF) and the virion DNA produced by the transfected cells contained the cat gene sequences (Stambur-ski et al., 1991). The G fragment of the Mycoplasma pneumoniae cytadhesin P1 gene could also be expressed in Spiroplasma citri (Marais et al., 1993) using a similar method. However, the recombinant RF proved unstable, resulting in the loss of the DNA insert (Marais et al., 1996).

Recombinant plasmids have also been developed to intro-duce genes into Spiroplasma citri cells. The introduced genes include antibiotic resistance markers and wild-type genes to complement auxotrophic mutants. Most recombinant plas-mids contain the origin of DNA replication (oriC) of the Spiro-plasma citri chromosome (Ye et al., 1994b). One such plasmid is pBOT1 (Renaudin, 2002; Renaudin et al., 1995). This plas-mid contains a 2 kbp oriC region, a tetracycline resistance gene (tetM) from Tn916, and the linearized Escherichia coli plasmid pBS with a colE1 origin of replication. Because of its two ori-gins of replication, oriC and colE1, pBOT1 is able to shuttle between Spiroplasma citri and Escherichia coli. When introduced into Spiroplasma citri, pBOT1 replicates first as a free extrachro-mosomal element, but later integrates into the chromosome via homologous recombination involving a single crossover event in the oriC region. Once integrated into the host chromosome, the whole plasmid is stably maintained. Recent studies suggest that the broad host range Spiroplasma citri GII3 plasmids and their shuttle derivatives may have significant advantages over oriC plasmids for gene transfer and expression in spiroplasmas (Breton et al., 2008a). They transform Spiroplasma citri (as well as Spiroplasma kunkelii and Spiroplasma phoeniceum) strains at relatively high efficiencies, the growth of the transformants is not significantly affected, they do not integrate into the chro-mosome, and their stability/loss can be modulated depending upon the presence/absence of the soj gene.

Spiroplasma citri mutants have been produced by random and targeted approaches. The transposon Tn4001 has been used successfully for random mutagenesis of Spiroplasma citri (Foissac et al., 1997c). For targeted gene inactivation, plasmids derived from pBOT1 have been used to disrupt genes (e.g., fructose operon, motility gene scm1) through homologous recombi-nation involving a single crossover event (Duret et al., 1999; Gaurivaud et al., 2000c). More recently, Lartigue et al. (2002) developed vector pC2, in which the oriC fragment was reduced to the minimal sequence needed to promote plasmid replica-tion; this vector increases recombination frequency at the tar-get gene. To avoid the extensive passaging that was required for recombination prior to transformant screening, vector pC55 was designed using a selective tetracycline resistance marker that is only expressed after the plasmid has integrated into the chromosome at the target gene. This approach was used to inactivate the spiralin gene (spi) and the gene encoding the

IICB component of the glucose phosphotransferase system permease (ptsG) (André et al., 2005; Duret et al., 2003; Lar-tigue et al., 2002). Another series of recombinant plasmids, the pGOT vectors, allow for selection of rare recombination events by using two distinct selective markers. First, transformants are screened for their resistance to gentamicin and next, site-spe-cific recombinants are selected for based on their resistance to tetracycline, which can only be expressed through recombina-tion at the target gene. In this way, inactivation of the crr gene, encoding the glucose phosphotransferase permease IIA compo-nent, was obtained (Duret et al., 2005). The use of the transpo-son gd TnpR/res recombination system to produce unmarked mutations (i.e., without insertion of antibiotic markers) in Spiro-plasma citri was demonstrated by the production of a disrupted arcA mutant (Duret et al., 2005); arcA encodes arginine deimi-nase. In this system, the target gene is disrupted by integration of a plasmid containing target gene sequences along with the tetM gene flanked by binding-specific recombination (res) sites. After integration of the plasmid, a second plasmid is introduced that encodes the resolvase TnpR. TnpR mediates the resolution of the cointegrate at the res sites, thereby removing tetM but leaving behind a mutated version of the target gene. The TnpR-encoding plasmid is lost spontaneously when selective pressure is removed.

Antigenic structure. Growth inhibition tests (Whitcomb et al., 1982) were used in the early years to identify spiroplasma species or groups, but metabolism inhibition (Williamson et al., 1979b; Williamson and Whitcomb, 1983) and deformation tests (Williamson et al., 1978) are now used almost exclusively (see below).

Antigenic variability, which has been described for some Mycoplasma species (Rosengarten and Wise, 1990; Yogev et al., 1991), has not been demonstrated in spiroplasmas (R. Rosen-garten, personal communication).

Group classification. The classification of spiroplasmas was first proposed by Junca et al. (1980) and has been revised peri-odically (Tully et al., 1987; Williamson et al., 1998). These clas-sifications are based on serological reactions of the organisms in growth inhibition, deformation and metabolism inhibition tests and/or characteristics of their genomes. Development of a classification scheme has resulted in the delineation of spiroplasma groups and subgroups (Table 142). In the scheme, “groups” have been defined as clusters of similar organisms, all of which possess negligible DNA–DNA hybridization with representatives of other groups, but moderate to high levels of hybridization (20–100%) with each other. Groups are, there-fore, putative species. This level of genomic differentiation cor-relates well with substantial differences in serology. Thirty-four groups were presented in a revised classification of spiroplas-mas in 1998 (Williamson et al., 1998). Four additional groups (XXXV–XXXVIII) were proposed recently as the result of a global spiroplasma environmental survey (Whitcomb et al., 2007) and more are anticipated (Jandhyam et al., 2008). Sub-groups have been defined by the International Committee on Systematics of Bacteria (ICSB) Subcommittee on the Taxonomy of Mollicutes (ICSB, 1984) as clusters of spiroplasma strains showing intermediate levels of intragroup DNA–DNA hybrid-ization (10–70%) and possessing corollary serological relation-ships. Three spiroplasma groups [group I (Junca et al., 1980; Saillard et al., 1987), group VIII (Gasparich et al., 1993c), and

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TablE 142. Biological properties of spiroplasmasa

Group Spiroplasma Strain ATCC no. Morphologyb Genomec G+Cd Arge Dtf OptTg Host

I-1 S. citri R8-A2T 27556T Long helix 1820 26 + 4.1 32 Phloem/leafhopperI-2 S. melliferum BC-3T 33219T Long helix 1460 26 + 1.5 37 Honey beeI-3 S. kunkelii E275T 29320T Long helix 1610 26 + 27.3 30 Phloem/leafhopperI-4 Spiroplasma sp. 277F 29761 Long helix 1620 26 + 2.3 32 Rabbit tickI-5 Spiroplasma sp. LB-12 33649 Long helix 1020 26 − 26.3 30 Plant bugI-6 S. insolitum M55T 33502T Long helix 1810 28 − 7.2 30 Flower surfaceI-7 Spiroplasma sp. N525 33287 Long helix 1780 26 + 4.7 32 Green June beetleI-8 S. phoeniceum P40T 43115T Long helix 1860 26 + 16.8 30 Phloem/vectorI-9 S. penaei SHRIMPT BAA-1082T

(CAIM 1252T)Helix nd 29 + nd 28 Pacific white shrimp

II S. poulsonii DW-1T 43153T Long helix 1040 26 nd 15.8 30 Drosophila hemolymph

III S. floricola OMBG 29989T Helix 1270 26 − 0.9 37 Plant surfaceIV S. apis B31T 33834T Helix 1300 30 + 1.1 34.5 Honey beeV S. mirum SMCAT 29335T Helix 1300 30 + 7.8 37 Rabbit tickVI S. ixodetis Y32T 33835T Tight coil 2220 25 − 9.2 30 Ixodid tickVII S. monobiae MQ-1T 33825T Helix 940 28 − 1.9 32 Monobia waspVIII-1 S. syrphidicola EA-1T 33826T Minute helix 1230 30 + 1.0 32 Syrphid flyVIII-3 Spiroplasma sp. TAAS-1 51123 Minute helix 1170 31 + 1.4 37 Horse flyVIII-2 S. chrysopicola DF-1T 43209T Minute helix 1270 29 + 6.4 30 Deer flyIX S. clarkii CN-5T 33827T Helix 1720 29 + 4.3 30 Green June beetleX S. culicicola AES-1T 35112T Short helix 1350 26 − 1.0 37 MosquitoXI S. velocicrescens MQ-4T 35262T Short helix 1480 26 − 0.6 37 Monobia waspXII S. diabroticae DU-1T 43210T Helix 1350 25 + 0.9 32 BeetleXIII S. sabaudiense Ar-1343T 43303T Helix 1175 29 + 4.1 30 MosquitoXIV S. corruscae EC-1T 43212T Helix nd 26 − 1.5 32 Horse fly/beetleXV Spiroplasma sp. I-25 43262 Wave-coil 1380 26 − 3.4 30 LeafhopperXVI-1 S. cantharicola CC-1T 43207T Helix nd 26 − 2.6 32 Cantharid beetleXVI-2 Spiroplasma sp. CB-1 43208 Helix 1320 26 − 2.6 32 Cantharid beetleXVI-3 Spiroplasma sp. Ar-1357 51126 Helix nd 26 − 3.4 30 MosquitoXVII S. turonicum Tab4cT 700271T Helix 1305 25 − nd 30 Horse flyXVIII S. litorale TN-1T 43211T Helix 1370 25 − 1.7 32 Horse flyXIX S. lampyridicola PUP-1T 43206T Unstable helix 1375 25 − 9.8 30 FireflyXX S. leptinotarsae LD-1T 43213T Motile funnel 1085 25 + 7.2 30 Colorado potato

beetleXXI Spiroplasma sp. W115 43260 Helix 980 24 − 4.0 30 Flower surfaceXXII S. taiwanense CT-1T 43302T Helix 1195 26 − 4.8 30 MosquitoXXIII S. gladiatoris TG-1T 43525T Helix nd 26 − 4.1 31 Horse flyXXIV S. chinense CCHT 43960T Helix 1530 29 − 0.8 37 Flower surfaceXXV S. diminutum CUAS-1T 49235T Short helix 1080 26 − 1.0 32 MosquitoXXVI S. alleghenense PLHS-1T 51752T Helix 1465 31 + 6.4 30 Scorpion flyXXVII S. lineolae TALS-2T 51749T Helix 1390 25 − 5.6 30 Horse flyXXVIII S. platyhelix PALS-1T 51748T Wave-coil 780 29 + 6.4 30 DragonflyXXIX Spiroplasma sp. TIUS-1 51751 Rare helices 840 28 − 3.6 30 Tiphiid waspXXX Spiroplasma sp. BIUS-1 51750 Late helices nd 28 − 0.9 37 Flower surfaceXXXI S. montanense HYOS-1T 51745T Helix 1225 28 + 0.7 32 Horse flyXXXII S. helicoides TABS-2T 51746T Helix nd 27 − 3.0 32 Horse flyXXXIII S. tabanidicola TAUS-1T 51747T Helix 1375 26 − 3.7 30 Horse flyXXXIV Spiroplasma sp. B1901 700283 Helix 1295 25 − nd nd Horse flyXXXV Spiroplasma sp. BARC 4886 BAA-1183 Helix nd nd − 0.6 32 Horse flyXXXVI Spiroplasma sp. BARC 4900 BAA-1184 Helix nd nd − 1.0 30 Horse flyXXXVII Spiroplasma sp. BARC 4908 BAA-1187 Helix nd nd − 1.2 32 Horse flyXXXVIII Spiroplasma sp. GSU5450 BAA-1188 Helix nd nd − 1.5 32 Horse flyNd S. atrichopogonis GNAT3597T BAA-520T (NBRC

100390T)Helix nd 28 + nd 30 Biting midge

Nd S. leucomae SMAT BAA-521T (NBRC 100392T)

Helix nd 24 + nd 30 Satin moth

and, Not determined.bFor descriptions of morphotypes, see text.cGenome size (kbp).dDNA G+C content (mol%).e+, Catabolizes arginine.fDoubling time (h) (Konai et al., 1996a).gOptimum growth temperature (°C).

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group XVI (Abalain-Colloc et al., 1993)] have been divided into a total of 15 subgroups. “Serovars” have been defined as geno-typic clusters varying substantially in metabolism inhibition and deformation serology, but that are insufficiently differentiated from members of existing groups or subgroups to warrant sepa-ration. However, with the discovery of a large number of strains for some groups (e.g., group VIII), the serovar/subgroup pic-ture has become very confused (Regassa et al., 2004; see Phylog-eny, below).

Procedures for species descriptions and minimal standards. Species descriptions of spiroplasmas have been in accord with recommendations of minimum standards proposed by the ICSP (International Committee on Systematics of Prokaryotes) Sub-committee on the Taxonomy of Mollicutes (Brown et al., 2007).

Cloning. Production of spiroplasma lineages produced from a single cell or clonings are performed largely by serial dilution of filtered cultures using 96-well microtiter plates (Whitcomb et al., 1986; Whitcomb and Hackett, 1987). At a certain dilution, which varies from plating to plating, the mean number of cells per well decreases so that fewer than about 8 of the 96-wells support growth of a spiroplasma clone. Very prob-ably, such clones arise from a single spiroplasmal cell.

Cellular morphology. Using dark-field microscopy, cultures should appear helical and motile during at least one growth phase (see “Morphology” and “Motility” above). However, mor-phological exceptions do occur (see “Differentiating charac-ters” below and reviewed by Gasparich et al., 2004).

16S rRNA gene sequence analysis. Preliminary identifica-tion is performed by PCR amplification using universal 16S rRNA (Gasparich et al., 2004) or other described primers (e.g., Fukatsu and Nikoh, 1998; Jandhyam, 2008). DNA sequence analysis using a blast search provides preliminary placement within the genus Spiroplasma. Those strains showing close phylo-genetic relationships based on 16S rRNA gene sequence analy-ses should then be screened using serological tests.

Serological tests. The deformation test (Williamson et al., 1978) is used routinely for serological analyses. Reciprocal titers of ³320 are generally required for definitive group placement. Deformation is defined as entire or partial loss of helicity. At the end point, cells are often seen in which an unaffected part of the helical filament exhibits flexing motility despite the presence of a bleb on another part of the cell. The deformation titer is the reciprocal of the final antiserum dilution that exhibits defor-mation of ³50% of the cells. Antiserum should be produced for any strain thought to represent a novel serogroup and any positive test against characterized groups requires a recriprocal test using the newly prepared antiserum.

The high levels of specificity and sensitivity of the metabo-lism inhibition test make it especially useful for defining groups and subgroups (Williamson et al., 1979b; Williamson and Whit-comb, 1983). Other serological tests have also been employed for characterization of spiroplasmas. Growth inhibition tests were used for delineation of spiroplasma groups I through XI (Whitcomb et al., 1982), but were not used thereafter. Growth inhibition tests are problematic for spiroplasmas because they require development of procedures for obtaining colonies. The spiroplasma motility inhibition test (Hackett et al., 1997) has proved useful for determination of intraspecific variation in

Spiroplasma leptinotarsae. ELISA has been used for detection of Spiroplasma kunkelii (Gordon et al., 1985) and Spiroplasma citri (Saillard and Bové, 1983).

Optimum growth temperature. Optimal growth tempera-tures between 10 and 41°C have been determined (Konai et al., 1996a).

Substrate metabolism. The ability to ferment glucose and produce acid must be examined (Aluotto et al., 1970). The ability to hydrolyze arginine and produce ammonia should be assessed (Barile, 1983). See the section on “Biochemical reac-tions” above for more details.

Ecology. The species description must include ecological information such as isolation site within the host and cultiva-tion conditions, common and binomial host name, geographi-cal location of host (with GPS), any known interaction between the spiroplasma and its host, and, in the case of a pathogen, disease symptoms observed.

Antibiotic sensitivities. In early studies (Bowyer and Cala-van, 1974; Liao and Chen, 1981b), spiroplasmas proved to be especially sensitive in vitro to tetracycline, erythromycin, tylosin, tobramycin, and lincomycin. Strains have been isolated that are permanently resistant to kanamycin, neomycin, gentamicin, erythromycin, and several tetracycline antibiotics (Liao and Chen, 1981b). Insensitivity to rifampicin has been studied in relation to its inhibition of transcription (see “RNA polymerase and spiroplasmal insensitivity to rifampicin” above) and penicil-lin insensitivity is seen for all spiroplasmas due to the lack of a cell wall. Natural amphipathic peptides such as Gramicidin S alter the membrane potential of spiroplasma cells and induce the loss of cell motility and helicity (Bévén and Wróblewski, 1997). The toxicity of the lipopeptide antibiotic globomycin was found to be correlated with an inhibition of spiralin processing (Bévén et al., 1996). As with Gramicidin S, the antibiotic was effective against spiroplasmas, but not Mycoplasma mycoides. Nat-ural 18-residue peptaibols (trichorzins PA) are bacteriocidal to spiroplasmas (Bévén et al., 1998). The mode of action appears to be permeabilization of the host cell membrane.

Hosts, ecology, and pathogenicity

Hosts. Almost all spiroplasmas have been found to be asso-ciated with arthropods or an arthropod connection is strongly suspected. Hackett et al. (1990) searched for mollicutes in a wide variety of insect orders. Isolates were obtained from six orders and 14 insect families. Only one of these orders, Odo-nata (dragonflies), was primitive (heterometabolous) and it was speculated that the spiroplasma from a dragonfly host might have been acquired via predation. Hackett et al. (1990) sug-gested that the Spiroplasma/Entomoplasma clade may have arisen in a paraneopteran-holometabolan ancestor, coevolved with these orders, and never adapted to more primitive insect orders. Some insect families have an especially rich spiroplasma, ento-moplasma, and mesoplasma flora.

Insect gut. The majority of spiroplasmas appear to be main-tained in an insect gut/plant surface cycle. Clark (1984) hypoth-esized several types of gut infection in which persistence in the gut and the ability to invade hemolymph varied among spiro-plasma species. It has been hypothesized (Hackett and Clark, 1989) that the gut cycle was primitive and that other cycles were derived from it. Spiroplasmas have been isolated from guts of

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tabanids (Diptera: Tabanidae) worldwide (French et al., 1997, 1990, 1996; Jandhyam et al., 2008; Le Goff et al., 1991, 1993; Regassa and Gasparich, 2006; Vazeille-Falcoz et al., 1997; Whit-comb et al., 1997a). Examination of diversity trends among the tabanid isolates suggests that spiroplasma diversity increases with temperature, resulting in more diversity in southern climes in the Northern Hemisphere (Whitcomb et al., 2007). Although evidence points strongly to multiple cycles of hori-zontal transmission, the sites where such transmission occurs remain unknown. However, some tabanids utilize honeydew (excreta of sucking insects) deposited on leaf surfaces, suggest-ing a possible transmission mechanism. Mosquitoes (Chastel and Humphery-Smith, 1991) are also common spiroplasma hosts (Lindh et al., 2005). Additionally, spiroplasmas inhabit the gut of ground beetles (Harpalus pensylvanicus and Anisodactylus sanctaecrucis) as evidenced by 16S rRNA gene sequence analysis of the digestive tract bacterial flora (Lundgren et al., 2007).

Plant surfaces. Flowers and other plant surfaces represent a major site where spiroplasmas and other microbes are transmit-ted from insect to insect (Clark, 1978; Davis, 1978; McCoy et al., 1979). Members of several spiroplasma groups have been iso-lated only from flowers and strains of several other spiroplasmas have been isolated from both insects and flowers. Biological evidence suggests that mosquito spiroplasmas are transmitted from insect to insect on flowers (Chastel et al., 1990; Le Goff et al., 1990). It is not known whether any of the so-called “flower spiroplasmas” can exist as true epiphytes. Isolations of spiroplas-mas from a variety of insects (Clark, 1982; Hackett et al., 1990) suggest that it is likely that many or most of these flower isolates are deposited passively by visiting arthropods.

Plant phloem and sucking insects. Several spiroplasmas pos-sess a life cycle that involves infection of plant phloem and homopterous insects (Bové, 1997; Fletcher et al., 1998; Garnier et al., 2001; Saglio and Whitcomb, 1979). In the course of pas-sage through the insect, spiroplasmas pass through, accumu-late, or multiply in gut epithelial cells and salivary cells. They also accumulate in the insect neurolemma. Large accumula-tions of spiroplasma cells occur frequently in the hemolymph, where they undoubtedly multiply (Whitcomb and Williamson, 1979). Spiroplasmas may multiply in a number of sucking insect species that have been exposed to diseased plants, but often only a single vector or several vector species transmit spiroplas-mal pathogens from plant to plant (summarized in Calavan and Bové, 1989; Whitcomb, 1989; Kersting and Sengonca, 1992).

Sex ratio organisms. Once thought to be a genetic factor, the sex ratio trait in Drosophila was shown by Poulson and Sakaguchi (1961) to be induced by a micro-organism, Spiroplasma poulsonii (Williamson et al., 1999). A number of other spiroplasmas in a variety of insect hosts have been identified that also cause sex ratio distortions, including isolates from the chrysomelid beetle Adalia bipunctata (Hurst and Jiggins, 2000; Hurst et al., 1999) and the butterfly Danaus chrysippus (Jiggins et al., 2000). In addition, 16S rRNA gene sequence analysis identified spiroplas-mas as the causative agent for male-killing: in a population of Harmonia axyridis (ladybird beetle) in Japan (Nakamura et al., 2005); in populations of Drosophila neocardini, Drosophila ornati-frons and Drosophila paraguayensis from Brazil (Montenegro et al., 2006, 2005); in populations of Anisosticta novemdecimpunc-tata (ladybird beetle) in Britain (Tinsley and Majerus, 2006); in

a population of Adalia bipunctata (Sokolova et al., 2002); in sev-eral strains from the Tucson Drosophila stock culture collection (Mateos et al., 2006); and in Drosophila melanogaster populations from Uganda and Brazil (Pool et al., 2006). Other organisms closely associated with their insect hosts were discovered infer-entially by PCR studies (Fukatsu and Nikoh, 2000, 2001) and also appear to be related to Spiroplasma mirum. They also cause preferential male killing in an infected Drosophila population (Anbutsu and Fukatsu, 2003). Natural infection rates of male-killing spiroplasmas in Drosophila melanogaster are about 2.3%, as determined for a Brazilian population (Montenegro et al., 2005), and vary between 0.1 and 3% for Japanese populations of Drosophila hydei (Kageyama et al., 2006). The male-killing spiroplasma strain isolated from Adalia bipunctata was used to artificially infect eight different coccinellid beetle species. The data suggest that host range could serve to limit horizontal transfer to closely related host species (Tinsley and Majerus, 2007). Supporting this hypothesis was the study that showed the interspecific lateral transmission of spiroplasmas from Drosophila nebulosa to Drosophila willistoni via ectoparasitic mites (Jaenike et al., 2007). A recent multilocus analysis by Haselkorn et al. (2009) showed that Drosophila species are infected with at least four distinct spiroplasma haplotypes.

Studies on Drosophila infections by the sex-ratio organism showed that it did not induce the innate immunity of the insect (Hurst et al., 2003). The sex-ratio spiroplasmas have been shown to be vertically transmitted through female hosts, with spiroplas-mas present during oogenesis (Anbutsu and Fukatsu, 2003). Although the exact mechanism of male-killing has not been determined, studies have shown that male killing occurs shortly after formation of the host dosage compensation complex (Bent-ley et al., 2007) and that male Drosophila melanogaster mutants lacking any of the five genes involved in the dosage compensa-tion complex are not killed (Veneti et al., 2005). In the Kenyan butterfly Danaus chrysippus, a correlation between male killing and a recessive allele for a gene controlling infection suscepti-bility has been reported. Moreover, infections seemed to have a negative effect on body size (Herren et al., 2007).

Ticks. Three Spiroplasma species have been isolated from ticks. Two of these, Spiroplasma mirum and Spiroplasma sp. 277F, are from the rabbit tick Haemaphysalis leporispalustris (Tully et al., 1982; Williamson et al., 1989). The third species was isolated from Ixodes pacificus ticks and named Spiroplasma ixodetis (Tully et al., 1995). 16S rRNA gene sequence analysis of spiroplasmas originally isolated from Ixodes ticks and growing in a Buffalo Green Monkey mammalian cell culture line showed a high degree of identity with the Spiroplasma ixodetis 16S rRNA gene (Henning et al., 2006). Analysis of the 16S rRNA gene sequence from DNA extracted from unfed Ixodes ovatus from Japan indi-cated the presence of spiroplasmas that were also closely related to Spiroplasma ixodetis (Taroura et al., 2005). The ability of tick spiroplasmas, including Spiroplasma ixodetis, to multiply at 37°C reflects the role of vertebrates as tick hosts. The ability of Spiro-plasma ixodetis to grow at 32°C as well as 37°C (Tully et al., 1982) may reflect the ecology of some of the cold-blooded vertebrate hosts of these ticks. There is no evidence that any of these spiro-plasmas are transmitted to vertebrate hosts of the ticks.

Crustaceans. Spiroplasma sp. have recently been isolated in both freshwater and salt-water crustaceans.

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Spiroplasma penaei (strain SHRIMPT) was isolated from the hemolymph of Pacific white shrimp (Penaeus vannamei) after high mortalities were observed in an aquaculture pond in Columbia, South America (Nunan et al., 2004). The patho-genic agent was the spiroplasma (Nunan et al., 2005). Although not cultivated, 16S rRNA gene sequence analysis also revealed the presence of spiroplasmas in the gut of the hydrothermal shrimp Rimicaris exoculata (Zbinden and Cambon-Bonavita, 2003). In another outbreak, Chinese mitten crab (Eriocheir sin-ensis) reared in aquaculture ponds in China became infected with tremor disease. The causative agent was determined to be a spiroplasma with 99% 16S rRNA gene sequence identity to Spiroplasma mirum (Wang et al., 2004a, b). However, recent stud-ies suggest that the infective agent may be a species similar to, but distinct from, Spiroplasma mirum (Bi et al., 2008). The same organism also infects red swamp crayfish (Procambarus clarkii) that are co-reared with the Chinese mitten crab (Bi et al., 2008; Wang et al., 2005) as well as the shrimp Penaeus vannamei (Bi et al., 2008).

Other hosts. Spiroplasmas have been identified in a variety of other hosts, although not necessarily linked to the gut habitat. The first spiroplasma isolated from a lepidopteran came from the hemolymph of white satin moth larvae (Leucoma salicis L.) from Poland (designated strain SMAT) and was serologically dis-tinct from any previously described spiroplasma group (Oduori et al., 2005). Another novel spiroplasma (designated strain GNAT3597T) was isolated from biting midges from the genus Atrichopogon (Koerber et al., 2005). Spiroplasmas that are closely related to the male-killing spiroplasmas in ladybird beetles (Majerus et al., 1999; Tinsley and Majerus, 2006) have also been identified in the predatory mite Neoseiulus californicus using 16S rRNA gene sequence analysis (Enigl and Schausberger, 2007). A broad survey of 16 spider families for the presence of endo-symbionts using 16S rRNA gene sequence analysis revealed that six families contained spiroplasmas, including Agelenidae, Araneidae, Gnaphosidae, Linyphiidae, Lycosidae, and Tetragnathidae (Goodacre et al., 2006).

Biogeography. Spiroplasmas have been identified from hosts in Africa, Asia, Australia, Europe, South America, and North America. While they are worldwide in distribution, studies sug-gest that biodiversity may be greatest in warm climates (Whit-comb et al., 2007). Because spiroplasmas are host-associated, it seems reasonable that Spiroplasma species distribution would be limited by host biogeography. Early studies indicated that some spiroplasmas have discrete geographic distributions (Whitcomb et al., 1990). As the diversity of sampling sites increases, the view of spiroplasma biogeography is likely to shift (Regassa and Gasparich, 2006). Distinct distributions may exist, but probably on a larger geographic scale. While it is not clear what factors account for spiroplasma ranges, the level of host specificity and host overwintering ranges may contribute to the biogeography of Spiroplasma species (Whitcomb et al., 2007).

Pathogenicity. Symptoms of infection and confirmation of Koch’s postulates have been reported for the etiologic roles of: Spiroplasma citri in “stubborn” disease of citrus (Calavan and Bové, 1989; Markham et al., 1974); corn stunt spiroplasma (Chen and Liao, 1975; Nault and Bradfute, 1979; William-son and Whitcomb, 1975); Spiroplasma phoeniceum in aster, an experimental host (Saillard et al., 1987); Spiroplasma poulsonii

in Drosophila pseudoobscura (Williamson et al., 1989); Spiroplasma penaei in Penaeus vannamei (Nunan et al., 2005); and Spiroplasma eriocheiris (Wang et al., 2010) in the Chinese mitten crab, Eriocheir sinensis (Wang et al., 2004b). Recent studies have focused on spiroplasma infection and replication in the midgut and Mal-pighian tubules of leafhoppers (Özbek et al., 2003). The use of immunofluorescence confocal laser scanning microscopy has revealed the presence of Spiroplasma kunkelii in the midgut, filter chamber, Malpighian tubules, hindgut, fat tissues, hemo-cytes, muscle, trachea, and salivary glands of leafhopper hosts, but not in the nerve cells of the brain or nerve ganglia (Ammar and Hogenhout., 2005). Plant spiroplasmas may also be patho-genic for unusual vectors (Whitcomb and Williamson, 1979), but are much less so for their usual host (Madden and Nault, 1983; Nault et al., 1984). In fact, some spiroplasmas are benefi-cial to their leafhopper hosts (Ebbert and Nault, 1994) and it has been hypothesized that infection plays an important role in the host’s overwintering strategies (Moya-Raygoza et al., 2007a, b; Summers et al., 2004).

Spiroplasma mirum is experimentally pathogenic for a variety of suckling animals, causing cataract and other ocular symp-toms, neural pathology (Clark and Rorke, 1979), and malig-nant transformation in cultured cells (Kotani et al., 1990). Spiroplasma melliferum also persists and causes pathology in suck-ling mice (Chastel et al., 1990, 1991). Spiroplasma eriocheiris is neurotropic to brain tissue in experimentally injected chicken embryos (Wang et al., 2003). There are two recent reports of spiroplasmas in aquatic invertebrates. Nunan et al. (2005) char-acterized a spiroplasma in commercially raised shrimp that led to a lethal disease. Spiroplasma melliferum and Spiroplasma apis cause disease in honey bees (Clark, 1977; Mouches et al., 1982a, 1983a). Intrathoracic inoculation of Spiroplasma taiwanense reduced the survival and impaired the flight capacity of inocu-lated mosquitoes (Humphery-Smith et al., 1991a), and inocula-tion of Spiroplasma taiwanense per os decreased the survival of mosquito larvae in laboratory trials (Humphery-Smith et al., 1991b). Spiroplasma poulsonii causes sex ratio abnormalities (male-killing) in Drosophila (Williamson and Poulson, 1979). Male-killing spiroplasma strains related to Spiroplasma poulsonii cause necrosis in neuroblastic and fibroblastic cells (Kuroda et al., 1992). The significance of some biological properties of spiroplasmas is incompletely understood. For example, mem-branes of Spiroplasma monobiae are potent inducers of tumor necrosis factor alpha secretion and of blast transformation (Sher et al., 1990a, b) in insect cell culture.

Spiroplasmas are implicated by circumstantial evidence, in the view of some workers, to be associated with human disease. Bastian first claimed in 1979 that spiroplasmas were associ-ated with Creutzfeldt–Jakob Disease (CJD), an extremely rare scrapie-like disease of humans (Bastian, 1979). Bastian and Foster (2001) reported finding spiroplasma 16S rRNA genes in CJD- and scrapie-infected brains that were not observed in con-trols. More recent studies (Bastian et al., 2004) presented evi-dence to show that spiroplasma 16S rRNA genes were found in brain tissue samples from scrapie-infected sheep, chronic wast-ing disease-infected cervids, and CJD-infected humans. All the brain tissues from non-infected controls were negative for spiro-plasmal DNA. These authors further showed that the sequence of the PCR products from the infected brains was 96% identi-cal to the Spiroplasma mirum 16S rRNA gene. However, these

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results could not be replicated in an independent blind study of uninfected and Scrapie-infected hamster brains using the same primers (Alexeeva et al., 2006). A recent study to fulfill Koch’s postulate reported the transfer of spiroplasma from transmis-sible spongiform encephalopathy (TSE) brains and Spiroplasma mirum to induce spongiform encephalopathy in ruminants (Bastian et al., 2007). The current status of the involvement of spiroplasmas in TSE is the subject of recent reviews (Bastian, 2005; Bastian and Fermin, 2005). Other proposed connections between mollicutes and human disease have been evaluated by Baseman and Tully (1997).

Mechanism of Spiroplasma citri phytopathogenicity. Transpo-son (Tn4001) mutants have been examined extensively to elu-cidate the molecular mechanisms associated with Spiroplasma citri phytopathogenicity. One of these mutants, GMT553, high-lighted the involvement of selective carbohydrate utilization in Spiroplasma citri pathogenicity (see review by Bové et al., 2003). When introduced into periwinkle plants via injected leafhop-pers (Circulifer haematoceps), GMT553 multiplied in the plants as actively as wild-type Spiroplasma citri strain GII3, but did not induce symptoms (Foissac et al., 1997b, c; Gaurivaud et al., 2000b). In this mutant, the transposon was found to be inserted in fruR, a transcriptional activator of the fructose operon (fru-RAK; Gaurivaud et al., 2000a). The second gene of the operon, fruA, encodes fructose permease, which enables uptake of fructose; and the third gene, fruK, encodes 1-phosphofructoki-nase. In mutant GMT553, transcription of the fructose operon is abolished and, hence, the mutant cannot utilize fructose as a carbon or energy source (Gaurivaud et al., 2000a). Mutant GMT553 was functionally complemented for fructose utiliza-tion and phytopathogenicity in trans by a recombinant fruR–fruA–fruK operon, fruA–fruK partial operon, or fruA alone, but not fruR or fruR–fruA (Gaurivaud et al., 2000a, b). It should be pointed out that both fructose+ and fructose− spiroplasmas are able to utilize glucose.

Further insight into Spiroplasma citri phytopathogenicity in rela-tion to sugar metabolism comes from the production of a spiro-plasma mutant unable to use glucose (André et al., 2005). The import of glucose into Spiroplasma citri cells involves a phospho-transferase (PTS) system composed of two distinct polypeptides encoded by (1) crr (glucose PTS permease IIAGlc component) and (2) ptsG (glucose PTS permease IICBGlc component). A ptsG mutant (GII3-glc1) proved unable to import glucose. When introduced into periwinkle (Catharanthus roseus) plants through leafhopper transmission, the mutant induced severe symptoms similar to those obtained with wild-type GII3, in strong contrast to the fructose operon mutant, GMT553, which was virtually non-pathogenic. These results indicated that fructose and glu-cose utilization were not equally involved in pathogenicity and are consistent with biochemical data showing that, in the pres-ence of both sugars, Spiroplasma citri preferentially used fructose. NMR analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by wild-type Spiroplasma citri GII3 or GII3-glc1, but not in those infected by GMT553. In the infected plant, Spiroplasma citri cells are restricted to the sieve tubes. In the companion cell, sucrose is cleaved by invertase to fructose and glucose. In the sieve tube, wild-type Spiroplasma citri cells will use fructose prefer-entially over glucose leading to a decreased fructose concentra-tion and, consequently, to an increase of invertase activity, which

in turn results in glucose accumulation. Glucose accumulation is known to induce stunting and repression of photosynthesis genes in Arabidopsis thaliana. Such symptoms are precisely those observed in periwinkle plants infected by wild-type Spiroplasma citri (André et al., 2005).

Genes that are up- or down-regulated in plants following infection with Spiroplasma citri have been studied by differential display analysis of mRNAs in healthy and symptomatic periwin-kle plants (Jagoueix-Eveillard et al., 2001). Expression of the transketolase gene was inhibited in plants infected by the wild-type spiroplasma, but not by the non-phytopathogenic mutant GMT553, further indicating that sugar metabolism and trans-port are important factors in pathogenicity. Sugar PTS system permeases have been shown to be important in rapid adapta-tion to sugar differences between plant host and insect vector (André et al., 2003).

Leafhopper transmission of Spiroplasma citri. Spiroplasmas are acquired by leafhopper vectors that imbibe sap from the sieve tubes of infected plants. However, in order to be trans-mitted to a plant, the mollicutes need first to multiply in the insect vector after crossing the gut barrier (Wayadande and Fletcher, 1995). They multiply to high titers (106–107/ml) in the insect hemolymph, but only when they have reached the salivary glands can they be inoculated into a plant. One gene required for efficient transmission, sc76, was inactivated in a transposon mutant (G76) with reduced transmissibility (Bou-tareaud et al., 2004); sc76 encodes a putative lipoprotein. Plants infected with the G76 mutant showed symptoms 4–5 weeks later than those infected with wild-type GII3, but when they appeared, the symptoms induced were severe. Mutant G76 mul-tiplied in plants and leafhoppers as efficiently as the wild-type strain. However, leafhoppers injected with the wild-type spiro-plasma transmitted the spiroplasma to 100% of exposed plants. In contrast, those injected with mutant G76 infected only 50% of the plants. This inefficiency was shown to be associated with a numerical decrease in spiroplasma cells in the salivary glands that correlated with reduced output from the stylets of trans-mitting leafhoppers; the number of mutant cells transmitted through Parafilm membranes was less than 5% of numbers of wild-type cells transmitted based on colony-forming units. Func-tional complementation of the G76 mutant with the sc76 gene restored the wild-type phenotype. Because both wild-type and mutant cells multiplied to equally high titers in the hemolymph, the results suggest that the mutant is inefficiently passed from the hemolymph into the salivary glands or that it may multiply to a lower titer in the glands.

Transmission of Spiroplasma citri by leafhopper vectors must involve adherence to and invasion of insect host cells. Elec-tron microscopic studies of leafhopper midgut by Ammar et al. (2004) have demonstrated the attachment of Spiroplasma kunkelii cells by a tip structure to the cell membrane between microvilli of epithelial cells. Spiroplasma citri surface protein P89 was shown to mediate adhesion of the spiroplasma to cells of the vector Circulifer tenellus and was designated SARP1 (Berg et al., 2001; Yu et al., 2000). The gene encoding SARP1, arp1, was cloned and characterized from Spiroplasma citri BR3-T. The putative gene product SARP1 contains a novel domain at the N terminus, called “sarpin” (Berg et al., 2001). The arp1 gene is located on plasmid pBJS-O in Spiroplasma citri (Joshi et al., 2005). The Spiroplasma kunkelii plasmid pSKU146 encodes an

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adhesin that is a homolog of SARP1 (Davis et al., 2005). Other spiroplasma plasmids encode additional adhesin-related pro-teins. As indicated above (see Plasmids), Spiroplasma citri GII3 contains six large plasmids, pSci1 to pSci6 (Saillard et al., 2008). Although plasmids pSci1 to pSci5 encode eight differ-ent Spiroplasma citri adhesin-related proteins (ScARPs), they are not required for insect transmission (Berho et al., 2006b). One of the ScARPs, protein P80, shared 63% similarity and 45% identity with SARP1. Protein P80 is carried by plasmid pSci4 and has been named ScARP4a. The ScARP-encoding genes could not be detected in DNA from non-transmissible strains (Berho et al., 2006b). Sequence alignments of ScARP proteins revealed that they share common features including a conserved signal peptide followed by six to eight repeats of 39–42 amino acids each, a central conserved region of 330 amino acids, and a transmembrane domain at the C terminus (Saillard et al., 2008).

Plasmid pSci6 carries the gene for protein P32, which is pres-ent in all Spiroplasma citri strains capable of being transmitted by the leafhopper vector Circulifer haematoceps, but absent from all non-transmissible strains (Killiny et al., 2006). Complementa-tion studies with P32 alone did not restore transmissibility (Kill-iny et al., 2006). However, if the pSci6 plasmid was transferred to an insect-non-transmissible Spiroplasma citri strain, then the phenotype could be converted to insect-transmissible, indicat-ing the likely presence of additional transmissibility factors on pSci6 (Berho et al., 2006a). Indeed, recent data indicates that factors essential for transmissibility are encoded by a 10 kbp fragment of pSci6 (Breton et al., 2010). The finding that the insect-transmissible strain Spiroplasma citri Alc254 contains only a single plasmid, pSci6 (S. Richard and J. Renaudin, unpub-lished) also reinforces the hypothesis that pSci6-encoded deter-minants play a key role in insect transmission of Spiroplasma citri by its leafhopper vector.


Isolation. Success in the isolation of fastidious spiroplasmas is influenced strongly by the titer of the inoculum. Spiroplasmas have been isolated from salivary glands, gut, and nerve tissues of their insect hosts. Many spiroplasmas envisioned by dark-field microscopy have proved to be noncultivable (Hackett and Clark, 1989). Initial insect extracts in growth media are passed through a 0.45 mm filter. The filtrate is then observed daily for pH indicator change. An alternative to filtration involves the use of antibiotics or other inhibitors (Grulet et al., 1993; Markham et al., 1983; Whitcomb et al., 1973). Spiroplasma isolations from infected plants are best obtained from sap expressed from vas-cular bundles of hosts showing early disease symptoms. Plant sap often contains spiroplasmal substances (Liao et al., 1979) whose presence in primary cultures may necessitate blind pas-sage or serial dilution.

Isolation media. M1D medium (Whitcomb, 1983) has been used for primary isolations of the large proportion of spiro-plasma species. SP-4 medium, a rich formulation derived from experiments with M1D, is necessary for isolation of Spiroplasma mirum from fluids of the embryonated egg (Tully et al., 1982). SP-4 medium is also required for isolation of Spiroplasma ixode-tis (Tully et al., 1981). Some very fastidious spiroplasmas such as Spiroplasma poulsonii (Hackett et al., 1986) and Spiroplasma

leptinotarsae (Hackett and Lynn, 1985) were isolated by co-cultivation with insect cells. However, the requirement for co-cultivation of Spiroplasma leptinotarsae can be circumvented by placing the primary cultures in BBL anaerobic GasPak jar sys-tems with low redox potential and enhanced CO2 atmosphere (Konai et al., 1996b). By lowering the pH of the growth medium from 7.4 to 6.2 and using bromocresol purple as a pH indicator (pH 5.2 yellow to pH 6.8 purple), it was possible to perform metabolism inhibition tests involving Spiroplasma leptinotarsae as the antigen. The same low-pH medium containing 2.0% Noble agar permitted the growth of colonies (Williamson, unpub-lished data). Cohen and Williamson (1988) reported that a fortuitous contamination of H-2 medium by a slow-growing, pink-colored yeast (Rhodotorula rubra) permitted primary iso-lation of the non-male-lethal variant of the Dorsophila willistoni spiroplasma. After 10–12 passages with yeast, the spiroplasmas were able to grow in yeast-free H-2 medium.

Maintenance procedures. Adaptation. Most spiroplas-mas can be adapted to a wide variety of media formulations. Spiroplasmas commonly grow more slowly upon transfer to new media. Initial reduction in growth rate is probably related to a combination of differences in nutrients, pH, osmolality, etc. Isolates may grow at only slightly reduced rates during the first 1–5 passages in a new medium. However, if the new medium is markedly deficient, the growth rate may decrease precipi-tously after 5–10 passages. Continuous careful passaging may result in growth rate recovery to levels similar to that in the initial medium. For such adaptations, best results are achieved by starting with a 1:1 ratio of old and new media and gradually withdrawing the old formulation. Spiroplasma clarkii, after con-tinuous passage for hundreds of generations, finally adapted to extremely simple media (Hackett et al., 1994). Adaptation may involve mutation and/or activation of adaptive enzymes, or, possibly, other mechanisms. Growth rates in such simple media were much slower than those in rich media.

Maintenance media. Spiroplasma citri can be cultivated in a relatively simple medium that utilizes sorbitol to maintain osmolality (Saglio et al., 1971). A modification of this medium (BSR) has been used extensively for Spiroplasma citri (Bové and Saillard, 1979), in which the horse serum content was lowered to 10% and the fresh yeast extract was omitted. Other simple media, such as C-3G (Liao and Chen, 1977), are suitable for maintenance or large-batch cultivation of fast-growing spiro-plasmas. This medium is also adequate for primary isolation of Spiroplasma kunkelii (Alivizatos, 1988). However, cultivation of more fastidious spiroplasmas is best achieved in M1D medium (Hackett and Whitcomb, 1995; Whitcomb, 1983; Williamson and Whitcomb, 1975) if they derive from plant or insect habi-tats. SP-4 medium (Tully et al., 1977) is very suitable if spiroplas-mas derive from tick habitats. SM-1 medium (Clark, 1982) has also been successfully employed for many insect spiroplasmas.

Defined media. Spiroplasma floricola and some strains of Spiroplasma apis have been cultivated in chemically defined media (Chang, 1989, 1982).

Preservation. Spiroplasmas are routinely preserved by lyophilization (FAO/WHO, 1974). Most spiroplasmas can be maintained at −70°C indefinitely. Preservation success at −20°C is irregular and uncertain.

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Differentiation of the genus Spiroplasma from other closely related taxa

Spiroplasmas can be clearly differentiated from all other micro-organisms by their unique properties of helicity and motility, combined with the complete absence of periplasmic fibrils, cell walls, or cell wall precursors. However, spiroplasmas may be nonhelical under some environmental conditions or when cultures are in the stationary phase of growth. Morphological study of the organisms in the exponential phase of growth usu-ally reveals characteristic helical forms. However, the existence of spiroplasmas that appear entirely or largely as nonhelical forms (e.g., Spiroplasma ixodetis and group XXIII strain TIUS-1) raises the theoretical possibility that an organism situated at an apomorphic (advanced) position on the spiroplasma phylogenetic tree could totally lack helicity or motility. In fact, the clade containing Mycoplasma mycoides and the Entomoplas-mataceae has apparently done exactly that. Spiroplasma floricola produces nonhelical, but viable, cells early in stationary phase, which can begin within 24 h of medium inoculation. For rea-sons such as this, it is necessary to examine cultures throughout the growth cycle to ensure that an adequate search for helical cells has been made.

taxonomic comments

Early history. The term “spiroplasma” was first coined as a trivial term to describe helical organisms shown to be associ-ated with corn stunt disease (Davis et al., 1972a, b) that could not, at that time, be cultivated (Davis and Worley, 1973). Shortly thereafter, when similar organisms associated with citrus stub-born disease were characterized (Saglio et al., 1973), the triv-ial term was adopted as the generic name and the stubborn organism was named Spiroplasma citri. This species was the first cultured spiroplasma and the first cultured mollicute of plant origin. Shortly after the stubborn agent was named, the genus Spiroplasma was elevated to the status of a family (Skri-pal, 1974) and added to the Approved Lists of Bacterial Names (Skripal, 1983). The organism that was eventually named Spiro-plasma mirum (Tully et al., 1982) was isolated by Clark (1964) in embryonated chicken eggs soon after the discovery of the organism later named Spiroplasma poulsonii. Because Spiroplasma mirum readily passed through filters, it was first mistaken for a virus. The subgroup I-4 277F spiroplasma was cultivated in 1968, but was mistaken for a spirochete (Pickens et al., 1968). The first organism to be initially recognized as a spiroplasma was Spiroplasma kunkelii, which was envisioned by dark-field and electron microscopy in 1971–1972 and cultivated in 1975 (Liao and Chen, 1977; Williamson and Whitcomb, 1975). More than a decade passed before Clark (1982) showed that spiroplasmas, many of them fast-growing, occurred principally in insects.

Species concept. The species concept in spiroplasmas, as in all bacteria, was based on DNA–DNA reassociation (ICSB Sub-committee on the Taxonomy of Mollicutes, 1995; Johnson, 1994; Rosselló-Mora and Amann, 2001; Stackebrandt et al., 2002; Wayne et al., 1987). In practice, DNA–DNA reassociation results with spiroplasmas have proven difficult to standardize. Estimates of reassociation between Spiroplasma citri (subgroup I-1) and Spiroplasma kunkelii (subgroup I-3) varied between 30 and 70%, depending on the method employed and the degree of strin-gency (Bové and Saillard, 1979; Christiansen et al., 1979; Lee

and Davis, 1980; Liao and Chen, 1981a; Rahimian and Gumpf, 1980). Given these challenges, an alternative method was identi-fied in serology. Surface serology of spiroplasmas has proven to be a robust surrogate for DNA–DNA hybridization assays.

Phylogeny. Phylogenetic studies of Spiroplasma became possible when Carl Woese and colleagues, searching for a molecular chronometer by which microbial evolution could be reconstructed, found that rRNA met most or all of the desired criteria (reviewed by Woese, 1987). Today, sequencing of rRNA genes has become a universal tool for phylogenetic reconstruc-tion. Early phylogenetic analyses involved distance estimates (DeSoete, 1983). Later, neighbor-joining (Saitou and Nei, 1987) was introduced into mollicute phylogeny (Maniloff, 1992) and several mollicute workers have used maximum-likelihood (Felsenstein, 1993). The extensive and classical studies of K.-E. Johansson’s group (Johansson et al., 1998; Pettersson et al., 2000) were completed using neighbor-joining, but selectively confirmed by maximum-likelihood and maximum-parsimony (Swofford, 1998). Gasparich et al. (2004) studied the phylogeny of Spiroplasma and its nonhelical descendants using parsimony, maximum-likelihood, distance, and neighbor-joining analyses, which generated 24 phylogenetic inferences that were com-mon to all, or almost all, of the trees. More recently, Bayesian analysis [MrBayes (http://mrbayes.csit.fsu.edu/index.php)] was used to examine an expanded Spiroplasma Apis clade based on 16S rRNA and 16S–23S ITS sequences; the analyses showed congruency between Bayesian and maximum-parsimony trees (Jandhyam et al., 2008).

Woese et al. (1980) presented a 16S rRNA gene-based phylo-genetic tree for Mollicutes, including Spiroplasma, indicating that these wall-less bacteria were related to members of the phylum Firmicutes such as Lactobacillus spp. and Clostridium innocuum. The tree suggested that Mollicutes might be monophyletic. However, a later study by Weisburg et al. (1989) with 40 addi-tional species of Mollicutes including ten spiroplasmas, failed to confirm the monophyly of Mollicutes at the deepest branch-ing orders. The Woese et al. (1980) model also suggested that the genus Mycoplasma might not be monophyletic, in that the type species, Mycoplasma mycoides, and two related species, Myco-plasma capricolum and Mycoplasma putrefaciens, appeared to be more closely related to the Apis clade of Spiroplasma than to the other Mycoplasma species. This conclusion was supported by analyses of the 5S rRNA genes (Rogers et al., 1985). All trees so far obtained indicate that the acholeplasma-anaeroplasma (Acholeplasmatales–Anaeroplasmatales) and spiroplasma-myco-plasma (Mycoplasmatales–Entomoplasmatales) lineages are mono-phyletic, but are separated by an ancient divergence.

In-depth analysis of characterized spiroplasmas and their non-helical descendants indicates the existence of four major clades within the monophyletic spiroplasma-mycoplasma lineage (Gas-parich et al., 2004; Figure 113). One of the four clades consists of the nonhelical species of the mycoides group (as defined by Johansson, 2002) as well as the six species of Entomoplasma and twelve species of Mesoplasma (the Entomoplasmataceae); this assemblage was designated the Mycoides-Entomoplasmataceae clade. The analyses indicated that the remaining three clades represented Spiroplasma species. One of these clades, the Apis clade, was found to be a sister to the Mycoides-Entomoplasma-taceae clade. The Apis clade contains a large number of spe-cies from diverse insect hosts, many of which possess life cycles

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http://mrbayes.csit.fsu.edu/index.php


Spiroplasma chrysopicolaSpiroplasma syrphidicolaSpiroplasma sp. TAAS-1

Spiroplasma mirumSpiroplasma sp. LB-12Spiroplasma sp. 277FSpiroplasma sp. N525

Spiroplasma poulsoniiSpiroplasma penaei

Spiroplasma insolitumSpiroplasma phoeniceum P40

Spiroplasma kunkelii CR2-3xSpiroplasma citri

Spiroplasma melliferumEntomoplasma freundtii

Mycoplasma mycoidesMesoplasma seiffertii

Spiroplasma monobiaeSpiroplasma diabroticaeSpiroplasma floricolaSpiroplasma sp. BIUS-1Spiroplasma sp. W115

Spiroplasma cantharicola CC-1Spiroplasma sp. CB-1

Spiroplasma sp. Ar-1357Spiroplasma diminutumSpiroplasma taiwanenseSpiroplasma gladiatoris

Spiroplasma lineolae TALS-2 Spiroplasma sp. BARC 1901

Spiroplasma helicoidesSpiroplasma clarkii

Spiroplasma apisSpiroplasma montanense

Spiroplasma litoraleSpiroplasma turonicumSpiroplasma corruscae

Spiroplasma culicicolaSpiroplasma velocicrescensSpiroplasma chinense

Spiroplasma leptinotarsaeSpiroplasma lampyridicola

Spiroplasma sabaudienseSpiroplasma alleghenense

Spiroplasma ixodetisMycoplasma pneumoniaeUreaplasma urealyticum

Acholeplasma laidlawii’Candidatus Phytoplasma’ sp. vigna Il

Anaeroplasma bactoclasticumClostridium innocuum

Bacillus subtilis TB11 Asteroleplasma anaerobiumEscherichia coli

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FIGURE 113. Phylogenetic relationships of members of the class Mollicutes and selected members of the phylum Firmicutes. The phylogram was based on a Jukes-Cantor corrected distance matrix and weighted neighbor-joining analysis of the 16S rRNA gene sequences of the type strains, except where noted. Escherichia coli was the outgroup. Bootstrap values (100 replicates) <50% are indicated (*). The GenBank accession numbers for 16S rRNA gene sequences used are: Mycoplasma mycoides (U26039); Mycoplasma pneumoniae (M29061); Entomoplasma freundtii (AF036954); Mesoplasma seiffertii (AY351331); Spiroplasma apis (M23937); Spiroplasma clarkii (M 24474); Spiroplasma gladiatoris (M24475); Spiroplasma taiwan-ense (M24476); Spiroplasma monobiae (M24481); Spiroplasma diabroticae (M24482); Spiroplasma melliferum (AY325304); Spiroplasma citri (M23942); Spiroplasma mirum (M24662); Spiroplasma ixodetis (M24477); Spiroplasma sp. strain N525 (DQ186642); Spiroplasma poulsonii (M24483); Spiroplasma penaei (AY771927); Spiroplasma phoeniceum (AY772395); Spiroplasma kunkelii (DQ319068); Spiroplasma cantharicola (DQ861914); Spiroplasma lineolae (DQ860100); Spiroplasma sp. strain 277F (AY189312); Spiroplasma sp. strain LB-12 (AY189313); Spiroplasma insolitum (AY189133); Spiroplasma flori-cola (AY189131); Spiroplasma syrphidicola (AY189309); Spiroplasma chrysopicola (AY189127); Spiroplasma sp. strain TAAS-1 (AY189314); Spiroplasma culicicola (AY189129); Spiroplasma velocicrescens (AY189311); Spiroplasma sabaudiense (AY189308); Spiroplasma corruscae (AY189128); Spiroplasma sp. strain CB-1 (AY189315); Spiroplasma sp. strain Ar-1357 (AY189316); Spiroplasma turonicum (AY189310); Spiroplasma litorale (AY189306); Spiroplasma lampyridicola (AY189134); Spiroplasma leptinotarsae (AY189305); Spiroplasma sp. strain W115 (AY189317); Spiroplasma chinense (AY189126); Spiro-plasma diminutum (AY189130); Spiroplasma alleghenense (AY189125); Spiroplasma sp. strain BIUS-1 (AY189319); Spiroplasma montanense (AY189307); Spiroplasma helicoides (AY189132); Spiroplasma sp. strain BARC 1901 (AY189320); Ureaplasma urealyticum (M23935); “Candidatus Phytoplasma” sp. Vigna II (AJ289195); Acholeplasma laidlawii (M23932); Anaeroplasma bactoclasticum (M25049); Clostridium innocuum (M23732); Asteroleplasma anaero-bium (M22351); Bacillus subtilis (AF058766); Escherichia coli (J01859).


involving transmission between the guts of insects and plant surfaces. One of these species, Spiroplasma sp. TIUS-1 (group XXVIII) has very poor helicity and a genome size of 840 kbp, smaller than that of most other spiroplasmas. This species diverged from the spiroplasma lineage close to the node of entomoplasmal divergence and can be envisioned as a “missing link” in the evolutionary development of the Mycoides-Ento-moplasmataceae clade. The other two Spiroplasma clades are the monospecific Ixodetis clade (group VI) and the Citri-Chrysopi-cola-Mirum clade (with representatives from groups I, II, V, and VIII). The Citri-Chrysopicola-Mirum clade contains Spiro-plasma mirum, Spiroplasma poulsonii, the three subgroups of the Chrysopicola (group VIII) clade, and the nine subgroups of the Citri (group I) clade. Members of group I and group VIII show close intragroup relationships, as indicated by the similarities of their 16S rRNA gene sequences (Gasparich et al., 2004). DNA–DNA reassociation studies for group I (Bové et al., 1983, 1982; Junca et al., 1980) spiroplasmas supported the subgroup clus-ter. The Chrysopicola clade (group VIII) subgroups have met a different fate. Although their DNA–DNA similarities in reasso-ciation procedures were slightly less than 70%, their 16S rRNA gene sequence similarities were >99% (Gasparich et al., 1993c). The strains of this group, including not only the subgroups, but a plethora of isolates from the same ecological context, appear to form a matrix of interrelated strains. Boundaries that seemed clear when the subgroups were initially described, eventually eroded beyond recognition. The 16S rRNA gene sequence sim-ilarities are too high to permit cladistic analysis and even 16S–23S rRNA spacer region sequence analysis failed to resolve the

existing subgroups (Regassa et al., 2004). Over time, the con-cept of the microbial species has undergone a subtle change. It is now recognized (Rosselló-Mora and Amann, 2001; Stack-ebrandt et al., 2002) that microbial species must at times consist of strain clusters that may contain species with <70% similarity as determined by DNA–DNA reassociation. Group VIII spiro-plasmas may comprise such a cluster and efforts to subdivide this cluster may have been inadvisable.

Character mapping of non-genetic features has been com-pleted in conjunction with phylogenetic analyses (Gasparich et al., 2004). Serological classifications of spiroplasmas are gen-erally supported by the trees, but the resolution of genetic anal-yses appears to be much greater than that of serology. Genome size and G+C content were moderately conserved among closely related strains. Apparent conservation of slower growth rates in some clades was most likely attributable to host affiliation; spiroplasmas of all groups that were well adapted to a specific host had slower growth rates. Sterol requirements were poly-phyletic, as was the ability to grow in the presence of PES, but not serum.

acknowledgements

We gratefully acknowledge J. Dennis Pollack for assistance on sections concerning intermediary metabolism.

Further reading

Whitcomb, R.F. and J.G. Tully (editors). 1989. The Mycoplas-mas, vol. 5, Spiroplasmas, acholeplasmas, and mycoplasmas of plants and arthropods. Academic Press, New York.

list of species of the genus Spiroplasma

1. Spiroplasma citri Saglio, L’Hospital, Laflèche, Dupont, Bové, Tully and Freundt 1973, 202AL

cit¢ri. L. masc. n. citrus the citrus; N.L. masc. n. Citrus generic name; N.L. gen. n. citri of Citrus, to denote the plant host.

Cells are helices that divide in mid-exponential phase when they have four turns. Helical filaments are usually 100–200 nm in diameter and 2–4 mm in length. Cells are longer in late exponential phase and early stationary phase. Nonviable cells in late exponential phase are non-helical.

Colonies on solid media containing 20% horse serum and 0.8% Noble agar (Difco) are umbonate, 60–150 mm in diameter. Moderate turbidity is produced in liquid cultures. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some cross-reactivity with members of other group I subgroups. Has close phy-logenetic affinities with other group I members, and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences.

Pathogenic for citrus plants and a variety of plant hosts (aster, periwinkle, broad bean) following transmission by infected insects (leafhoppers).

DNA–DNA renaturation experiments confirm serologi-cal data that indicate that the differences between the type strain (subgroup I-1) and other subgroups of group I are great enough to warrant its designation as a distinct species. The genome size is 1820 kbp (PFGE).

Source: isolated from leaves, seed coats, and fruits of citrus plants (orange and grapefruit) infected with stub-born disease, and from other naturally infected plants (e.g., periwinkle, horseradish or brassicaceous weeds) or insects. Known from Mediterranean and other warm climates of Europe, North Africa, Near and Middle East, and the West-ern United States (California and Arizona).

DNA G+C content (mol%): 25–27 (Tm, Bd).Type strain: ATCC 27556, Morocco strain, R8-A2.Sequence accession no. (16S rRNA gene): M23942.

2. Spiroplasma alleghenense Adams, Whitcomb, Tully, Clark, Rose, Carle, Konai, Bové, Henegar and Williamson 1997, 762VP

al.le.ghen.en¢se. N.L. neut. adj. alleghenense of the Allegheny Mountains, referring to the geographic origin of the type strain, the range of the Appalachian Mountains from which it was derived.

Cells are motile helical filaments, 100–300 nm in diameter. Under many growth conditions, cells in medium are deformed. Colonies on solid medium containing 3.0% Noble agar are small and granular and never have a fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. When tested as an antigen, cross-reacts broadly with many nonspecific sera (one-way reac-tion). Has close phylogenetic relationship to Spiroplasma

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sabaudiense (group XIII) and strain TIUS-1 (group XXVIII) in trees constructed using 16S rRNA gene sequences. The genome size is 1,465 kbp (PFGE).

Source: isolated from the hemolymph of a common scor-pion fly, Panorpa helena in West Virginia, USA.

DNA G+C content (mol%): 31 ± 1 (Tm, Bd).Type strain: ATCC 51752, PLHS-1.Sequence accession no. (16S rRNA gene): AY189125.

3. Spiroplasma apis Mouches, Bové, Tully, Rose, McCoy, Carle-Junca, Garnier and Saillard 1984b, 91VP (Effective publica-tion: Mouches, Bové, Tully, Rose, McCoy, Carle-Junca, Gar-nier and Saillard 1983a, 383.)

a¢pis. L. fem. n. apis, -is a bee, and also the genus name of the honey bee, Apis mellifera; L. gen. n. apis of a bee, of Apis mellifera, the insect host for this species.

The morphology is as described for the genus. Helical filaments are usually 100–150 nm in diameter and 3–10 mm in length. Colonies on solid medium containing 20% fetal bovine serum and 0.8% Noble agar (Difco) are usually dif-fuse, rarely exhibiting central zones of growth into the agar. Colonies on solid medium with 2.25% Noble agar and 1–5% bovine serum fraction are smaller, but exhibit central zones of growth into the agar and some peripheral growth on the agar surface around the central zones. Marked turbidity is produced during growth in most spiroplasma media (BSR, M1A, SP-4). Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Many strains show partial cross-reactions when tested against sera to strain B31T (Tully et al., 1980). These strains show more than 80% DNA–DNA reassociation with strain B31T, but their exact taxonomic status is unclear. Some strains show a very low level recipro-cal cross-reaction with Spiroplasma montanense in deforma-tion serology. In accordance with serology, Spiroplasma apis and Spiroplasma montanense are sister species in phylogenetic trees constructed using 16S rRNA gene sequences. The genome size is 1300 kbp (PFGE).

Etiologic agent of May disease of honey bees in south-western France. Various strains of the organism exhibit experimental pathogenicity for young honey bees in feed-ing experiments.

Source: isolated from honey bees (Apis mellifera) and from flower surfaces in widely separated geographic regions (France, Corsica, Morocco, USA).

DNA G+C content (mol%): 29–31 (Tm, Bd).Type strain: ATCC 33834, B31.Sequence accession no. (16S rRNA gene): AY736030.

4. Spiroplasma atrichopogonis Koerber, Gasparich, Frana and Grogan 2005, 291VP

a.tri.cho.po.go¢nis. N.L. gen. n. atrichopogonis of Atrichopogon, systematic genus name of a biting midge (Diptera: Cer-atopogonidae).

The morphology is as described for the genus. Cells are helical and motile. Biological properties are listed in Table 142.

Serologically distinct from previously established Spiro-plasma species, groups, and subgroups. The genome size has not been determined.

Source: isolated from a pooled sample of two nearly iden-tical species of biting midges (Atrichopogon geminus and Atrichopogon levis).

DNA G+C content (mol%): 28.8 ± 1 (Tm).Type strain: ATTC BAA-520, NBRC 100390, GNAT3597.Sequence accession no. (16S rRNA gene): not available.

5. Spiroplasma cantharicola Whitcomb, Chastel, Abalain-Colloc, Stevens, Tully, Rose, Carle, Bové, Henegar, Hackett, Clark, Konai and Williamson 1993a, 423VP

can.thar.i¢co.la. Gr. kantharos scarab beetle; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. cantharicola an inhabitant of a family of beetles.

The morphology is as described for the genus. Cells are helical and motile. Colonies on solid medium containing 0.8% Noble agar are diffuse, without fried-egg morphology. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some reciprocal cross-reactivity with members of other group XVI subgroups.

Not yet classified phylogenetically, but no doubt closely related to subgroups XVI-2 and XVI-3, which are sisters form-ing a clade related to Spiroplasma diminutum in phylogenetic trees constructed using 16S rRNA gene sequences. More-over, DNA–DNA renaturation experiments confirm that the differences between the type strain and other subgroups of group XVI are great enough to warrant its designation as a distinct species. The genome size is 1320 kbp (PFGE).

Source: isolated from the gut of an adult cantharid beetle (Cantharis carolinus) in Maryland, USA. Based on its resi-dence in the gut of a flower-visiting insect, this species is thought to be transmitted on flowers.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 43207, CC-1.Sequence accession no. (16S rRNA gene): DQ861914.

6. Spiroplasma chinense Guo, Chen, Whitcomb, Rose, Tully, Williamson, Ye and Chen 1990, 424VP

chi.nen¢se. N.L. neut. adj. chinense of China, the location where the organism was first isolated.

The morphology is as described for the genus. Cells are motile helical filaments ~160 nm in diameter. Colonies on solid medium containing 0.8–1.0% Noble agar are diffuse with many small satellite colonies; growth on 2.25% agar produces smaller rough or granular colonies and fewer sat-ellite forms. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is closely related to Spiroplasma velocicrescens in phylogenetic trees constructed using 16S rRNA gene sequences. The genome size is 1530 kbp (PFGE).

Source: isolated from flower surfaces of bindweed (Calyste-gia hederacea) in Jiangsu, People’s Republic of China.

DNA G+C content (mol%): 29 ± 1 (Tm).Type strain: ATCC 43960, CCH.Sequence accession no. (16S rRNA gene): AY189126.

7. Spiroplasma chrysopicola Whitcomb, French, Tully, Gas-parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP

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chry.so.pi¢co.la. N.L. n. Chrysops a genus of deer flies in the Tabanidae; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. chrysopicola inhabiting Chrysops spp.

Helical motile filaments are short and thin, passing a 220 nm filter quantitatively. Grows to titers as high as 1011/ml. Colonies on solid medium containing 2.25% Noble agar have dense centers and smooth edges (a fried-egg appear-ance) and do not have satellites. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but exhibits some reciprocal or one-way cross-reactivity with members of other group VIII subgroups. Some strains of group VIII spiroplasmas may be difficult to identify to subgroup. Shares less than 70% DNA–DNA reassociation with Spiroplasma syrphidicola and strain TAAS-1 (subgroup VIII-3). Phylogenetically, this spe-cies is closely related to other group VIII strains in trees constructed using 16S rRNA gene sequences. The 16S rRNA gene similarity coefficients of group VIII spiroplas-mas are >0.99, so this gene is insufficient for distinguishing species in group VIII. The genome size is 1270 kbp (PFGE). Pathogenicity for insects has not been determined.

Source: isolated from the gut of a deer fly (Chrysops sp.) in Maryland, USA. Other strains from deer flies have been collected from as far west as Wyoming, from New England, and very rarely, as far south as Georgia, USA.

DNA G+C content (mol%): 30 ± 1 (Bd).Type strain: ATCC 43209, DF-1.Sequence accession no. (16S rRNA gene): AY189127.

8. Spiroplasma clarkii Whitcomb, Vignault, Tully, Rose, Car-le, Bové, Hackett, Henegar, Konai and Williamson 1993c, 264VP

clar¢ki.i. N.L. masc. gen. n. clarkii of Clark, in honor of Tru-man B. Clark, a pioneer spiroplasma ecologist.

The morphology is as described for the genus. The heli-cal motile filaments remain stable throughout exponential growth. Colonies on solid medium containing 0.8% Noble agar are diffuse, without fried-egg morphology. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is placed in the classical Apis cluster of spiroplasmas, but it does not have an especially close neighbor in trees constructed using 16S rRNA gene sequences. The genome size is 1720 kbp (PFGE). Patho-genicity for insects has not been determined.

Source: isolated from the gut of a larval scarabaeid beetle (Cotinus nitida) in Maryland, USA.

DNA G+C content (mol%): 29 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 33827, CN-5.Sequence accession no. (16S rRNA gene): M24474.

9. Spiroplasma corruscae Hackett, Whitcomb, French, Tully, Gasparich, Rose, Carle, Bové, Henegar, Clark, Konai, Clark and Williamson 1996c, 949VP

cor.rus¢cae. N.L. gen. n. corruscae of corrusca, referring to the species of firefly beetle (Ellychnia corrusca) from which the organism was first isolated.

The morphology is as described for the genus. Cells are helical and motile. Colonies on solid medium containing

2.25% Noble agar are slightly diffuse to discrete and gen-erally without the characteristic fried-egg morphology. Bio-logical properties are listed in Table 142.

Serologically distinct from previously established Spiro-plasma species, groups, and subgroups. Phylogenetically, closely related to Spiroplasma turonicum and Spiroplasma lito-rale in trees constructed using 16S rRNA gene sequences. The genome size has not been determined.

Source: isolated from the gut of an adult lampyrid beetle (Ellychnia corrusca) in Maryland in early spring, but found much more frequently in horse flies in summer months. Other strains have been collected from Canada and Geor-gia, Connecticut, South Dakota, and Texas, USA.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd).Type strain: ATCC 43212, EC-1.Sequence accession no. (16S rRNA gene): AY189128.

10. Spiroplasma culicicola Hung, Chen, Whitcomb, Tully and Chen 1987, 368VP

cu.li.ci′co.la. L. n. culex, -icis a gnat, midge, and also a genus of mosquitoes (Culex, family Culicidae); L. suffix -cola (from L. n. incola) inhabitant, dweller; N.L. n. culicicola intended to mean an inhabitant of the Culicidae.

Cells are pleomorphic, but are commonly very short motile helices, 1–2 mm in length. Colonies on solid medium containing 1% Noble agar have a fried-egg appearance with satellites. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is placed in the classical Apis cluster of spiroplasmas, but does not have an especially close neighbor in trees constructed using 16S rRNA gene sequences. The genome size is 1350 kbp (PFGE).

Source: isolated from a triturate of a salt marsh mosquito (Aedes sollicitans) collected in New Jersey, USA.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd).Type strain: ATCC 35112, AES-1.Sequence accession no. (16S rRNA gene): AY189129.

11. Spiroplasma diabroticae Carle, Whitcomb, Hackett, Tully, Rose, Bové, Henegar, Konai and Williamson 1997, 80VP

di.a.bro.ti′cae. N.L. gen. n. diabroticae of Diabrotica, referring to Diabrotica undecimpunctata, the chrysomelid beetle from which the organism was isolated.

The morphology is as described for the genus. Cells are helical, motile filaments, 200–300 nm in diameter. Colonies on solid medium containing 0.8% Noble agar are diffuse, without fried-egg morphology. Biological properties are listed in Table 142.

Serologically distinct from other established Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to Spiroplasma floricola in trees constructed using 16S rRNA gene sequences. The genome size is 1350 kbp (PFGE).

Source: isolated from the hemolymph of an adult chry-somelid beetle, Diabroticae undecimpunctata howardi.

DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 43210, DU-1.Sequence accession no. (16S rRNA gene): M24482.

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12. Spiroplasma diminutum Williamson, Tully, Rosen, Rose, Whitcomb, Abalain-Colloc, Carle, Bové and Smyth 1996, 232VP

di.min.u¢tum. L. v. deminuere to break into small pieces, make smaller; L. neut. part. adj. diminutum made smaller, reflecting a smaller size.

The morphology is as described for the genus. Cells are short (1–2 mm), helical filaments, 100–200 nm in diameter that appear to be rapidly moving, irregularly spherical bod-ies when exponential phase broth cultures are examined under dark-field illumination. Colonies on solid medium containing 1.6% Noble agar have dense centers, granular perimeters, and nondistinct edges with satellite colonies. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to group XVI spiroplasmas in trees constructed using 16S rRNA gene sequences. The genome size is 1080 kbp (PFGE).

Source: isolated from a frozen triturate of adult female Culex annulus mosquitoes collected in Taishan, Taiwan.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 49235, CUAS-1.Sequence accession no. (16S rRNA gene): AY189130.

13. Spiroplasma floricola Davis, Lee and Worley 1981, 462VP

flor.i¢co.la. L. n. flos, -oris a flower; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. floricola flower-dweller.

The morphology is as described for the genus. Helical cells are 150–200 nm in diameter and 2–5 mm in length. Colonies on solid media have granular central regions surrounded by satellite colonies that probably form after migration of cells from the central focus. Biological proper-ties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to Spiroplasma diabroticae and various flower spiroplasmas in trees constructed using 16S rRNA gene sequences. The genome size of strain OBMG is 1270 kbp. Experimentally pathogenic for insects and embryonated chicken eggs.

Source: isolated from flowers of tulip tree and magnolia trees in Maryland, USA. Other strains have been collected from coleopterous insects.

DNA G+C content (mol%): 25 (Tm).Type strain: ATCC 29989, 23-6.Sequence accession no. (16S rRNA gene): AY189131.

14. Spiroplasma gladiatoris Whitcomb, French, Tully, Gas-parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP

gla.di.a¢to.ris. L. gen. n. gladiatoris of a gladiator, reflect-ing the initial isolation of the organism from the horse fly Tabanus gladiator.

Morphology is as described for the genus. Cells are motile helical filaments. Colonies on solid medium contain-ing 3% Noble agar are granular with dense centers and dif-fuse edges, do not have satellites, and never have a fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. This species has a specific antigen,

common to several spiroplasmal inhabitants of horse flies, that confers a high level of one way cross-reactivity when it is used as an antigen. Phylogenetically, closely related to two other tabanid spiroplasmas, Spiroplasma helicoides and group XXXIV strain B1901, in phylogenetic trees constructed using 16S rRNA gene sequences. The genome size has not been determined.

Source: isolated from the gut of a horse fly (Tabanus gladi-ator) in Maryland, USA. Other strains have been collected at various locations in the southeastern United States.

DNA G+C content (mol%): 26 ± 1 (Bd).Type strain: ATCC 43525, TG-1.Sequence accession no. (16S rRNA gene): M24475.

15. Spiroplasma helicoides Whitcomb, French, Tully, Gas-parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP

he.li.co.i¢des. Gr. n. helix spiral; Gr. suff. -oides like, resem-bling, similar; N.L. neut. adj. helicoides spiral-like.

The morphology is as described for the genus. Cells are motile helical filaments that lack a cell wall. Colonies on solid medium containing 2.25% Noble agar have dense centers and smooth edges, do not have satellites, and have a perfect fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. This species has a specific antigen, common to several spiroplasmal inhabitants of horse flies, that confers a high level one-way cross-reaction when it is used as antigen. Phylogenetically, closely related to two other tabanid spiroplasmas, Spiroplasma gladiatoris and Spiro-plasma sp. BARC 1901, in trees constructed using 16S rRNA gene sequences. Genome size has not been determined.

Source: isolated from the gut of a horse fly Tabanus abactor collected in Oklahoma, USA. Other strains have been col-lected in Georgia, USA.

DNA G+C content (mol%): 26 ± 1 (Bd).Type strain: ATCC 51746, TABS-2.Sequence accession no. (16S rRNA gene): AY189132.

16. Spiroplasma insolitum Hackett, Whitcomb, Tully, Rose, Carle, Bové, Henegar, Clark, Clark, Konai, Adams and Wil-liamson 1993, 276VP

in.so′li.tum. L. neut. adj. insolitum unusual or uncommon, to denote unusual base composition.

Cells in exponential phase are long, motile, helical cells that lack true cell walls and periplasmic fibrils. Colonies on solid SP-4 medium containing 0.8 or 2.25% Noble agar are diffuse, with small central zones of growth surrounded by small satellite colonies. Colonies on solid SP-4 medium con-taining horse serum and 0.8% Noble agar show fried-egg morphology. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species and groups, but cross-reacts reciprocally in complex patterns of relatedness with group I subgroups and Spiroplasma poulsonii. DNA–DNA renaturation experiments confirm that the differences between the type strain and other sub-groups of group I are great enough to warrant its designa-tion as a distinct species. Has close phylogenetic affinities with other group I members and with Spiroplasma poulsonii

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in trees constructed using 16S rRNA gene sequences. The genome size is 1850 kbp (PFGE). Pathogenicity for insects has not been determined.

Source: the type strain was isolated from a fall flower (Aster-aceae: Bidens sp.) collected in Maryland, USA. Similar iso-lates have been found in the hemocoel of click beetles. Also isolated from other composite and onagracead flowers and from the guts of many insects visiting these flowers, includ-ing cantharid and meloid beetles; syrphid flies; andrenid and megachilid bees; and four families of butterflies.

DNA G+C content (mol%): 28 ± 1 (Tm, Bd).Type strain: ATCC 33502, M55.Sequence accession no. (16S rRNA gene): AY189133.

17. Spiroplasma ixodetis Tully, Rose, Yunker, Carle, Bové, Wil-liamson and Whitcomb 1995, 27VP

ix.o.de¢tis. N.L. gen. n. ixodetis of Ixodes, the genus name of Ixodes pacificus ticks, from which the organism was first isolated.

Cells are coccoid forms, 300–500 nm in diameter, straight and branched filaments, or tightly coiled helical organisms. Motility is flexional, but not translational. Colonies on solid medium containing 2.25% Noble agar usually have the appearance of fried eggs. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically unique; occurs at base of spiroplasma lineage in trees constructed using 16S rRNA gene sequences. The genome size is 2220 kbp (PFGE).

Source: isolated from macerated tissue suspensions pre-pared from pooled adult Ixodes pacificus ticks (Ixodidae) collected in Oregon, USA.

DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 33835, Y32.Sequence accession no. (16S rRNA gene): M24477.

18. Spiroplasma kunkelii Whitcomb, Chen, Williamson, Liao, Tully, Bové, Mouches, Rose, Coan and Clark 1986, 175VP

kun.kel¢i.i. N.L. masc. gen. n. kunkelii of Kunkel, named after Louis Otto Kunkel (1884–1960), to honor his major and fundamental contributions to the study of plant mol-licutes.

Cells in exponential phase are helical, motile filaments, 100–150 nm in diameter and 3–10 mm long to nonhelical filaments or spherical cells, 300–800 nm in diameter. Colo-nies on solid medium containing 0.8% Noble agar are usu-ally diffuse, rarely exhibiting central zones of growth into agar. Colonies on solid C-3G medium containing 5% horse serum or on media containing 2.25% Noble agar frequently have a fried-egg morphology. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares complex patterns of reciprocal cross-reactivity with members of other group I subgroups and Spiroplasma poulsonii. DNA–DNA renatur-ation experiments confirm that the serological differences between the type strain and other subgroups of group I are great enough to warrant its designation as a distinct spe-cies. Has close phylogenetic affinities with other group I

members and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. The genome size is 1610 kbp (PFGE). Pathogenicity for plants and insects has been experimentally verified.

Source: isolated from maize displaying symptoms of corn stunt disease and from leafhoppers associated with diseased maize, largely in the neotropics.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd).Type strain: ATCC 29320, E275.Sequence accession no. (16S rRNA gene): DQ319068 (strain

CR2-3x).

19. Spiroplasma lampyridicola Stevens, Tang, Jenkins, Goins, Tully, Rose, Konai, Williamson, Carle, Bové, Hackett, French, Wedincamp, Henegar and Whitcomb 1997, 711VP

lam.py.ri.di¢co.la. N.L. n. Lampyridae the firefly beetle family; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. lampyridicola an inhabitant of members of the Lampyridae.

The morphology is as described for the genus. Cells are motile helical filaments. Colonies on solid medium contain-ing 3.0% Noble agar are small and granular with dense cen-ters, but do not have a true fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. When tested as antigen, cross-reacts (one-way) with many specific spiroplasma antisera. Phylo-genetically, a sister to Spiroplasma leptinotarsae in trees con-structed using 16S rRNA gene sequences. The genome size is 1375 kbp (PFGE).

Source: isolated from the gut fluids of a firefly beetle (Photuris pennsylvanicus) collected in Maryland, USA. Also known from Georgia and New Jersey, USA.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd).Type strain: ATCC 43206, PUP-1.Sequence accession no. (16S rRNA gene): AY189134.

20. Spiroplasma leptinotarsae Hackett, Whitcomb, Clark, Hen-egar, Lynn, Wagner, Tully, Gasparich, Rose, Carle, Bové, Konai, Clark, Adams and Williamson 1996b, 910VP

lep.ti.no.tar¢sae. N.L. gen. n. leptinotarsae of Leptinotarsa, referring to Leptinotarsa decemlineata, the Colorado potato beetle.

Cells in vivo are usually seen in the resting stage, in which they consist of coin-like compressed coils. When placed in fresh medium, these bodies turn immediately into “spring”- or “funnel”-shaped spirals, which are capable of very rapid translational motility. After a relatively small number of passes in vitro, this spectacular morphology is lost and the cells return to the modal morphology as described for the genus. Colonies on solid medium containing 2.0% Noble agar are slightly diffuse to discrete and produce numerous satellites. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. When tested as antigen, cross-reacts with many spiroplasma antisera (one-way). Phylogeneti-cally, a sister to Spiroplasma lampyridicola in trees constructed using 16S rRNA gene sequences. The genome size is 1,085 kbp (PFGE).

Source: isolated from the gut of Colorado potato beetle (Leptinotarsa decemlineata) larvae in Maryland, USA. Also

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isolated from beetles collected in Maryland, Michigan, New Mexico, North Carolina, Texas, Canada, and Poland.

DNA G+C content (mol%): 25 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 43213, LD-1.Sequence accession no. (16S rRNA gene): AY189305.

21. Spiroplasma leucomae Oduori, Lipa and Gasparich 2005, 2449VP

leu.co¢mae. N.L. gen. n. leucomae of Leucoma, systematic genus name of the white satin moth (Lepidoptera: Lyman-triidae), the source of the type strain.

Morphology is as described for the genus. Cells are fila-mentous, helical, motile, and approximately 150 nm in diameter. They freely pass through filters with pores of 450 and 220 nm, but do not pass through filters with 100 nm pores. Biological properties are listed in Table 142.

Serologically distinct from previously established Spiro-plasma species, groups, and subgroups. The genome size has not been determined. Pathogenicity for the moth lar-vae is not known.

Source: isolated from fifth instar satin moth larvae (Leu-coma salicis).

DNA G+C content (mol%): 24 ± 1 (Tm).Type strain: ATCC BAA-521, NBRC 100392, SMA.Sequence accession no. (16S rRNA gene): DQ101278.

22. Spiroplasma lineolae French, Whitcomb, Tully, Carle, Bové, Henegar, Adams, Gasparich and Williamson 1997, 1080VP

lin.e.o¢lae. N.L. n. lineola a species of tabanid fly; N.L. gen. n. lineolae of Tabanus lineola, from which the organism was isolated.

The morphology is as described for the genus. Cells are motile, helical filaments, 200–300 nm in diameter. Colo-nies on solid medium containing 3% Noble agar are small, granular, and never have a fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetic position has not been determined, but its other taxonomic properties suggest that it may be related to other tabanid spiroplasmas of the Apis cluster. The genome size is 1390 kbp (PFGE).

Source: type strain isolated from the viscera of the tabanid fly Tabanus lineola collected in coastal Georgia. A strain from Tabanus lineola has been collected in Costa Rica (Whitcomb et al., 2007).

DNA G+C content (mol%): 25 ± 1 (Tm, Bd).Type strain: ATCC 51749, TALS-2.Sequence accession no. (16S rRNA gene): DQ860100.

23. Spiroplasma litorale Konai, Whitcomb, French, Tully, Rose, Carle, Bové, Hackett, Henegar, Clark and Williamson 1997, 361VP

li.to.ra¢le. L. neut. adj. litorale of the shore or coastal area.

The morphology is as described for the genus. Cells are motile, helical filaments. Colonies on solid medium contain-ing 2.25% Noble agar are granular with dense centers, uneven margins, and multiple satellites, and never have fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, closely related to

two other tabanid spiroplasmas, Spiroplasma turonicum and Spiroplasma litorale, in trees constructed using 16S rRNA gene sequences. The genome size is 1370 kbp (PFGE).

Source: isolated from the gut of a female green-eyed horse fly (Tabanus nigrovittatus) from the Outer Banks of North Carolina. Also collected from coastal Georgia and both Atlantic and Pacific coasts of Costa Rica.

DNA G+C content (mol%): 25 ± 1 (Bd).Type strain: ATCC 34211, TN-1.Sequence accession no. (16S rRNA gene): AY189306.

24. Spiroplasma melliferum Clark, Whitcomb, Tully, Mouches, Saillard, Bové, Wróblewski, Carle, Rose, Henegar and Wil-liamson 1985, 305VP

mel.li′fe.rum. L. adj. mellifer, -fera, -ferum honey-bearing, honey-producing; L. neut. adj. melliferum intended to mean isolated from the honey bee (Apis mellifera).

Morphology is as described for the genus. Cells are pleo-morphic, varying from helical filaments that are 100–150 nm in diameter and 3–10 mm in length to nonhelical fila-ments or spherical cells that are 300–800 nm in diameter. The motile cells lack true cell wells and periplasmic fibrils. Colonies on solid medium supplemented with 0.8% Noble agar are usually diffuse, rarely exhibiting central zones of growth into agar. Colonies on solid medium containing 2.25% Noble agar are smaller, but frequently have a fried-egg morphology. Physiological and genomic properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares complex patterns of reciprocal cross-reactivity with members of other group I subgroups and Spiroplasma poulsonii. Has close phyloge-netic affinities with other group I members and with Spiro-plasma poulsonii in trees constructed using 16S rRNA gene sequences. DNA–DNA renaturation experiments confirm that the serological differences between the type strain and other subgroups of group I are great enough to warrant its designation as a distinct species. The genome size is 1460 kbp (PFGE). Pathogenic for honey bees in natural and experimental oral infections.

Source: isolated from hemolymph and gut of honey bees (Apis mellifera) in widely separated geographic regions. Also recovered from hemolymph of bumble bees, leafcutter bees, and a robber fly, and the intestinal contents of sweat bees, digger bees, bumble bees, and a butterfly. Also recov-ered from a variety of plant surfaces (flowers) in widely separated geographic regions.

DNA G+C content (mol%): 26–28 (Tm, Bd).Type strain: ATCC 33219, BC-3.Sequence accession no. (16S rRNA gene): AY325304.

25. Spiroplasma mirum Tully, Whitcomb, Rose and Bové 1982, 99VP

mi′rum. L. neut. adj. mirum extraordinary.

The morphology is as described for the genus. Helical filaments measure 100–200 nm in diameter and 3–8 mm in length. Colonies on solid media containing fetal bovine serum and 0.8–2.25% Noble agar (Difco) are diffuse and without central zones of growth into the agar. Solid media prepared with 1.25% agar and in which fetal bovine serum

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has been replaced with bovine serum fraction yield colonies with central zones of growth into the agar and no peripheral growth on the surface of the medium. Moderate turbidity is produced during growth in liquid media. Biological proper-ties are listed in Table 142. This species has been cultivated in a defined medium.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, in trees con-structed using 16S rRNA gene sequences, this species is basal to group I and group VIII spiroplasmas on the one hand, and to the Apis cluster and Entomoplasmataceae on the other. It is the most primitive (plesiomorphic) spiroplasma with modal helicity. The genome size is 1300 kbp (PFGE). Produces experimental ocular and nervous system disease and death in intracerebrally inoculated suckling animals (rats, mice, hamsters, and rabbits). Pathogenic for chicken embryos via yolk sac inoculation. Experimentally patho-genic for the wax moth (Galleria mellonella).

Source: the type strain was isolated from rabbit ticks (Hae-maphysalis leporispalustris) collected in Georgia, USA. Other strains have been collected in Georgia, Maryland, and New York, USA.

DNA G+C content (mol%): 30–31 (Tm).Type strain: ATCC 29335, SMCA.Sequence accession no. (16S rRNA gene): M24662.

26. Spiroplasma monobiae Whitcomb, Tully, Rose, Carle, Bové, Henegar, Hackett, Clark, Konai, Adams and Williamson 1993b, 259VP

mo.no.bi′ae. N.L. n. Monobia a genus of vespid wasps; N.L. gen. n. monobiae of the genus Monobia, from which the organism was isolated.

The morphology is as described for the genus, with motile helical filaments. Colonies on solid medium contain-ing 2.25% Noble agar are diffuse and never have a fried-egg appearance. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, a member of the Apis clade, but with no especially close neighbors in trees constructed using 16S rRNA gene sequences. The genome size is 940 kbp (PFGE).

Source: isolated from the hemolymph of an adult vespid wasp (Monobia quadridens) collected in Maryland, USA. Based on its residence in the gut of a flower-visiting insect, this species is thought to be transmitted on flowers.

DNA G+C content (mol%): 28 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 33825, MQ-1.Sequence accession no. (16S rRNA gene): M24481.

27. Spiroplasma montanense Whitcomb, French, Tully, Rose, Carle, Bové, Clark, Henegar, Konai, Hackett, Adams and Williamson 1997c, 722VP

mon.ta.nen¢se. N.L. neut. adj. montanense pertaining to Montana, where the species was first isolated.

The morphology is as described for the genus. Cells are motile, helical filaments that lack a cell wall. Colo-nies on solid medium containing 2.25% Noble agar are granular and have dense centers, irregular margins, and numerous small satellites. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Reacts reciprocally in deformation serology at very low levels in deformation tests with Spiro-plasma apis. “Bridge strains” have been isolated in Georgia with substantial cross-reactivity with both Spiroplasma mon-tanense and Spiroplasma apis. Sister to Spiroplasma apis in trees constructed using 16S rRNA gene sequences. The genome size is 1225 kbp (PFGE).

Source: isolated from the gut of the tabanid fly Hybomitra opaca, in southwestern Montana. Other isolates have been obtained from New England, Connecticut, and southeast-ern Canada.

DNA G+C content (mol%): 28 ± 1 (Bd).Type strain: ATCC 51745, HYOS-1.Sequence accession no. (16S rRNA gene): AY189307.

28. Spiroplasma penaei Nunan, Lightner, Oduori and Gas-parich 2005, 2320VP

pe.na′e.i. N.L. n. Penaeus a species of shrimp; N.L. gen. penaei of Penaeus, referring to Penaeus vannamei, from which the organism was isolated.

The morphology is as described for the genus. Cells are heli-cal and motile. Biological properties are listed in Table 142.

Serologically distinct from previously characterized Spiro-plasma species, groups, and subgroups, but shares some cross-reactivity with members of other group I subgroups. Has close phylogenetic affinities with other group I mem-bers and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. The genome size has not been determined. Pathogenicity has been indicated by injection into Penaeus vannamei.

Source: isolated from the hemolymph of the Pacific white shrimp, Penaeus vannamei.

DNA G+C content (mol%): 29 ± 1 (Tm).Type strain: CAIM 1252, SHRIMP, ATCC BAA-1082.Sequence accession no. (16S rRNA gene): AY771927.

29. Spiroplasma phoeniceum Saillard, Vignault, Bové, Raie, Tully, Williamson, Fos, Garnier, Gadeau, Carle and Whit-comb 1987, 113VP

phoe.ni¢ce.um. N.L. neut. adj. phoeniceum (from L. neut. adj. phonicium) of Phoenice, an ancient country that was located on today’s Syrian coast, referring to the geographi-cal origin of the isolates.

Morphology is as described for the genus. Colonies on solid medium containing 0.8% Noble agar show fried-egg morphology. Physiological and genomic properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some cross-reactivity with members of other group I subgroups and Spiroplasma poul-sonii. Has close phylogenetic affinities with other group I members and with Spiroplasma poulsonii in trees constructed using 16S rRNA gene sequences. Has been shown to be transmissible to leafhoppers by injection and experimen-tally pathogenic to aster inoculated by the injected leafhop-pers. DNA–DNA renaturation experiments confirm that the differences between the type strain and other subgroups of group I are great enough to warrant its designation as a dis-tinct species. The genome size is 1860 kbp (PFGE).

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Source: isolated from periwinkles that were naturally infected in various locations along the Syrian coastal area.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd).Type strain: ATCC 43115, P40.Sequence accession no. (16S rRNA gene): AY772395.

30. Spiroplasma platyhelix Williamson, Adams, Whitcomb, Tul-ly, Carle, Konai, Bové and Henegar 1997, 766VP

pla.ty.he¢lix. Gr. adj. platys flat; Gr. fem. n. helix a coil or spi-ral; N.L. fem. n. platyhelix flat coil, referring to the flattened nature of the helical filament.

Cells are flattened, helical filaments, 200–300 nm in diameter. They show no rotatory or translational motility, but exhibit contractile movements in which tightness of coil-ing moves along the axis of the filament. Colonies on solid medium containing 2.25% Noble agar form perfect fried-egg colonies with dense centers, smooth edges, and without satellites. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma spe-cies, groups, and subgroups. The genome size is 780 kbp (PFGE).

Source: isolated from the gut of a dragonfly, Pachydiplax longipennis, collected in Maryland, USA.

DNA G+C content (mol%): 29 ± 1 (Bd).Type strain: ATCC 51748, PALS-1.Sequence accession no. (16S rRNA gene): AY800347.

31. Spiroplasma poulsonii Williamson, Sakaguchi, Hackett, Whitcomb, Tully, Carle, Bové, Adams, Konai and Henegar 1999, 616VP

poul.so′ni.i. N.L. masc. gen. n. poulsonii of Poulson, named in memory of Donald F. Poulson, in whose laboratory at Yale University this spiroplasma was discovered and studied intensively.

Morphology is as described for the genus. Long, motile, helical filaments, 200–250 nm in diameter occur in vivo in Drosophila hemolymph and in vitro. Colonies on solid medium containing 1.8% Noble agar are small (60–70 mm in diameter), have dense centers and uneven edges, and are without satellites. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some reciprocal cross-reactivity with members of group I subgroups. Phylogeneti-cally related to group I spiroplasmas in trees constructed using 16S rRNA gene sequences. The genome size is 2040 kbp (PFGE). Spiroplasmas causing sex-ratio abnormalities occur naturally in Drosophila spp. collected in Brazil, Colom-bia, Dominican Republic, Haiti, Jamaica, and the West Indies. Non-male-lethal spiroplasmas also occur in natu-ral populations of Drosophila hydei in Japan. Pathogenicity (lethality to male progeny) has been confirmed by injection into Drosophila pseudoobscura female flies. Vertical transmis-sibility is lost after cultivation and cloning.

Source: isolated from the hemolymph of Drosophila pseu-doobscura females infected by hemolymph transfer of the Barbados-3 strain of Drosophila willistoni SR organism.

DNA G+C content (mol%): 26 ± 1 (Tm, Bd).Type strain: ATCC 43153, DW-1.Sequence accession no. (16S rRNA gene): M24483.

32. Spiroplasma sabaudiense Abalain-Colloc, Chastel, Tully, Bové, Whitcomb, Gilot and Williamson 1987, 264VP

sa.bau.di.en¢se. L. neut. adj. sabaudiense of Sabaudia, an ancient country of Gaul, corresponding to present day Savoy, referring to the geographic origin of the isolate.

The morphology is as described for the genus. Cells are helical filaments, 100–160 nm in diameter and 3.1–3.8 mm long. Motile. Colonies on solid medium containing 1.6% Noble agar are diffuse, rarely exhibiting fried-egg morphol-ogy, with numerous satellite colonies. Physiological and genomic properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, related to Spiro-plasma alleghenense and Spiroplasma sp. TIUS-1 in trees con-structed using 16S rRNA gene sequences. The genome size is 1175 kbp (PFGE).

Source: isolated from a triturate of female Aedes spp. mos-quitoes in Savoy, France.

DNA G+C content (mol%): 30 ± 1 (Tm, Bd).Type strain: ATCC 43303, Ar-1343.Sequence accession no. (16S rRNA gene): AY189308.

33. Spiroplasma syrphidicola Whitcomb, Gasparich, French, Tully, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1996, 799VP

syr.phi.di¢co.la. N.L. pl. n. Syrphidae a family of flies; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant, dweller; N.L. masc. n. syrphidicola inhabitant of syrphid flies, the insects from which the organism was isolated.

Helical motile filaments are short and thin, passing a 220 nm filter quantitatively. Grows to titers as high as 1011/ml. These short, thin, abundant cells are provisionally diag-nostic for group VIII. Colonies on solid medium contain-ing 2.25% Noble agar are irregular with satellites, diffuse, and never have a fried-egg appearance. Growth on solid medium containing 1.6% Noble agar is diffuse. Biological properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups, but shares some reciprocal cross-reactivity with members of other group VIII subgroups. Placement of group VIII strains into subgroups has become increasingly difficult as more strains have accumulated. Phylogenetically, this species is closely related to other group VIII strains in trees constructed using 16S rRNA gene sequences. The 16S rRNA gene sequence similarity coefficients of group VIII spiroplasmas are >0.99, so this gene is insufficient for species separations in group VIII. DNA–DNA renaturation experiments confirm that the dif-ferences between the type strain and other subgroups of group VIII are great enough to warrant its designation as a distinct species. Genome size is 1230 kbp (PFGE).

Source: isolated from the hemolymph of the syrphid fly Eristalis arbustorum in Maryland, USA. Strains that are pro-visionally identified as Spiroplasma syrphidicola have been obtained from horse flies collected from several locations in the southeastern United States.

DNA G+C content (mol%): 30 ± 1 (Bd).Type strain: ATCC 33826, EA-1.Sequence accession no. (16S rRNA gene): AY189309.

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34. Spiroplasma tabanidicola Whitcomb, French, Tully, Gas-parich, Rose, Carle, Bové, Henegar, Konai, Hackett, Adams, Clark and Williamson 1997b, 718VP

ta.ba.ni.di¢co.la. N.L. n. Tabanidae family name for horse flies; L. suff. -cola (from L. n. incola) inhabitant, dweller; N.L. n. tabanidicola an inhabitant of horse flies.

The morphology is as described for the genus. Cells are motile, helical filaments that lack a cell wall. Colonies on solid medium containing 3% Noble agar are uneven and granular with dense centers and irregular edges, do not have satellites, and never have a fried-egg appearance. Phys-iological and genomic properties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. However, some strains may show a very low level reciprocal serological cross-reaction in deformation serology with Spiroplasma gladiatoris. This spe-cies has a specific antigen, common to several spiroplasmal inhabitants of horse flies, that confers a high level one-way cross-reaction when it is used as antigen. The genome size is 1375 kbp (PFGE).

Source: isolated from the gut of a horse fly belonging to the Tabanus abdominalis-limbatinevris complex.

DNA G+C content (mol%): 26 ± 1 (Bd).Type strain: ATCC 51747, TAUS-1.Sequence accession no. (16S rRNA gene): DQ004931.

35. Spiroplasma taiwanense Abalain-Colloc, Rosen, Tully, Bové, Chastel and Williamson 1988, 105VP

tai.wan.en¢se. N.L. neut. adj. taiwanense of or belonging to Taiwan, referring to the geographic origin of the isolate.

The morphology is as described for the genus. Cells are motile, helical filaments, 100–160 nm in diameter and 3.1–3.8 mm long. Colonies on solid medium containing 1.6% Noble agar have fried-egg morphology. Biological proper-ties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is in the classical Apis cluster of spiroplasmas, but does not have an especially close neighbor in trees constructed using 16S rRNA gene sequences. The genome size is 1195 kbp (PFGE).

Source: isolated from a triturate of female mosquitoes (Culex tritaeniorhynchus) at Taishan, Taiwan, Republic of China.

DNA G+C content (mol%): 25 ± 1 (Tm, Bd).Type strain: ATCC 43302, CT-1.Sequence accession no. (16S rRNA gene): M24476.

36. Spiroplasma turonicum Hélias, Vazeille-Falcoz, Le Goff, Ab-alain-Colloc, Rodhain, Carle, Whitcomb, Williamson, Tully, Bové and Chastel 1998, 460VP

tu.ro¢ni.cum. L. neut. adj. turonicum of Touraine, the province in France from which the organism was first iso-lated.

The morphology is as described for the genus. Cells are motile, helical filaments. Colonies on solid medium con-taining 3% Noble agar exhibit a “cauliflower-like” appear-ance and do not have a fried-egg morphology. Biological properties are listed in Table 142.

Serologically distinct from previously established Spiro-plasma species. Phylogenetically, related to two other tabanid spiroplasmas, Spiroplasma corruscae and Spiroplasma litorale, in trees constructed using 16S rRNA gene sequences. The genome size is 1305 kbp (PFGE).

Source: isolated from a triturate of a single horse fly (Hae-matopota pluvialis) collected in France.

DNA G+C content (mol%): 25 ± 1 (Bd).Type strain: ATCC 700271, Tab4c.Sequence accession no. (16S rRNA gene): AY189310.

37. Spiroplasma velocicrescens Konai, Whitcomb, Tully, Rose, Carle, Bové, Henegar, Hackett, Clark and Williamson 1995, 205VP

ve.lo.ci.cres¢cens. L. adj. velox, -ocis fast, quick; L. part. adj. crescens growing; N.L. n. part. adj. velocicrescens fast-growing.

The morphology is as described for the genus. Cells are helical, motile filaments, 200–300 nm in diameter. Colonies on solid medium containing 0.8% Noble agar are diffuse and never have a fried-egg appearance. Biological proper-ties are listed in Table 142.

Serologically distinct from other Spiroplasma species, groups, and subgroups. Phylogenetically, this species is sister to Spiroplasma chinense in trees constructed using 16S rRNA gene sequences. The genome size is 1480 kbp (PFGE).

Source: isolated from the gut of a vespid wasp, Monobia quadridens, collected in Maryland, USA. Based on its resi-dence in the gut of a flower-visiting insect, this species is thought to be transmitted on flowers.

DNA G+C content (mol%): 27 ± 1 (Tm, Bd, HPLC).Type strain: ATCC 35262, MQ-4.Sequence accession no. (16S rRNA gene): AY189311.

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Whitcomb, R.F., C. Chastel, M. Abalain-Colloc, C. Stevens, J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, R.B. Henegar, K.J. Hackett, T.B. Clark, M. Konai and D.L. Williamson. 1993a. Spiroplasma cantharicola sp. nov., from cantharid beetles (Coleoptera: Cantharidae). Int. J. Syst. Bacteriol. 43: 421–424.

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Whitcomb, R.F., J.C. Vignault, J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, K.J. Hackett, R.B. Henegar, M. Konai and D.L. Williamson. 1993c. Spiroplasma clarkii sp. nov. from the green June beetle (Coleoptera, Scarabaeidae). Int. J. Syst. Bacteriol. 43: 261–265.

Whitcomb, R.F., G.E. Gasparich, F.E. French, J.G. Tully, D.L. Rose, P. Carle, J.M. Bové, R.B. Henegar, M. Konai, K.J. Hackett, J.R. Adams, T.B. Clark and D.L. Williamson. 1996. Spiroplasma syrphidicola sp.

685


nov., from a syrphid fly (Diptera: Syrphidae). Int. J. Syst. Bacteriol. 46: 797–801.

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686

Family i. acholeplasmataceae

order iii. Acholeplasmatales Freundt, Whitcomb, Barile, Razin and tully 1984, 348Vp

Daniel R. BRown, Janet M. BRaDBuRy anD KaRl-eRiK Johansson

a.cho.le.plas.ma.ta¢les. N.l. neut. n. Acholeplasma type genus of the order; -ales ending to denote an order; N.l. fem. pl. n. Acholeplasmatales the Acholeplasma order.

This order in the class Mollicutes is assigned to a group of wall-less prokaryotes that do not require sterol for growth and occur in a wide variety of habitats, including many vertebrate hosts, insects, and plants. A single family, Acholeplasmataceae, and a sin-gle genus, Acholeplasma, recognize the prominent and distinct characteristics of the assigned organisms.

Type genus: Acholeplasma Edward and Freundt 1970, 1AL.


The trivial name acholeplasma(s) is commonly used when reference is made to species of this order. The initial pro-posal for elevation of the acholeplasmas to ordinal rank (Freundt et al., 1984) was based primarily on the universal lack of a sterol requirement for growth of Acholeplasma species, in addition to other major genetic, nutritional, bio-chemical, and physiological characteristics that distinguish them from other members of the class Mollicutes. A subse-quent proposal for an additional order, Entomoplasmatales (Tully et al., 1993), within the class to distinguish a group of mollicutes that are phylogenetically more closely related to the Mycoplasmatales than to acholeplasmas necessitated further revisions within the class.

Although most mollicutes require exogenous cholesterol or serum for growth, all species within the genus Acholeplasma and some assigned to the genera Asteroleplasma, Spiroplasma, and Meso-plasma do not have that requirement. The species that do not have a sterol requirement can easily be excluded from the sterol-requiring taxa by tests that measure growth responses to choles-terol or to a number of serum-free broth preparations (Edward, 1971; Razin and Tully, 1970; Rose et al., 1993; Tully, 1995). For instance, the Acholeplasmatales grow through end-point dilutions in serum-containing medium and in serum-free preparations, indicating the absence of a growth requirement for cholesterol.

Analyses of rRNA and other genes have shown that a large group of uncultured, plant-pathogenic organisms referred to by the trivial name phytoplasmas (Sears and Kirkpatrick, 1994) are closely related to acholeplasmas (Lim and Sears, 1992; Toth et al., 1994). The 16S rRNA gene sequences for members of the genus Acholeplasma that have been determined so far show that the acholeplasmas form two clades, one of which is a sister lineage to the phytoplasmas, although the formal taxonomic assignment of “Candidatus Phytoplasma” proposed gen. nov. (IRPCM Phyto-plasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004) currently remains incertae sedis.

References


Edward, D.G. 1971. Determination of sterol requirement for Mycoplas-matales. J. Gen. Microbiol. 69 : 205–210.

Freundt, E.A., R.F. Whitcomb, M.F. Barile, S. Razin and J.G. Tully. 1984. Proposal for elevation of the family Acholeplasmataceae to ordinal rank: Acholeplasmatales. Int. J. Syst. Bacteriol. 34: 346–349.

IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group. 2004. Description of the genus ‘Candidatus Phy-toplasma’, a taxon for the wall-less non-helical prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol. 54: 1243–1255.

Lim, P.O. and B.B. Sears. 1992. Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences. J. Bacteriol. 174: 2606–2611.

Razin, S. and J.G. Tully. 1970. Cholesterol requirement of mycoplasmas. J. Bacteriol. 102: 306–310.

Rose, D.L., J.G. Tully, J.M. Bové and R.F. Whitcomb. 1993. A test for measuring growth responses of Mollicutes to serum and polyoxyethyl-ene sorbitan. Int. J. Syst. Bacteriol. 43: 527–532.

Sears, B.B. and B.C. Kirkpatrick. 1994. Unveiling the evolutionary rela-tionships of plant pathogenic mycoplasmalike organisms. ASM News 60 : 307–312.

Toth, K.F., N. Harrison and B.B. Sears. 1994. Phylogenetic relation-ships among members of the class Mollicutes deduced from rps3 gene sequences. Int. J. Syst. Bacteriol. 44: 119–124.

Tully, J.G., J.M. Bové, F. Laigret and R.F. Whitcomb. 1993. Revised taxonomy of the class Mollicutes - proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiro-plasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378–385.


Family i. Acholeplasmataceae edward and Freundt 1970, 1al


a.cho.le.plas.ma.ta.ce¢ae. N.l. neut. n. Acholeplasma, -atos type genus of the family; -aceae ending to denote a family; N.l. fem. pl. n. Acholeplasmataceae the Acholeplasma family.

Type genus: Acholeplasma Edward and Freundt 1970, 1AL.


This family is monotypic, so its properties are essentially those of the genus Acholeplasma.

687


Genus i. Acholeplasma edward and Freundt 1970, 1al


a.cho.le.plas¢ma. Gr. pref. a not; Gr. n. chole bile; Gr. neut. n. plasma something formed or molded, a form; N.l. neut. n. Acholeplasma name intended to indicate that cholesterol, a constituent of bile, is not required.

Cells are spherical, with a diameter of about 300 nm, or fila-mentous, 2–5 µm long. Nonmotile. Colonies have a “fried-egg” appearance and may reach 2–3 mm in diameter. Facultatively anaerobic; most strains grow readily in simple media. All mem-bers lack a sterol requirement for growth. Chemo- organotrophic, most species utilizing glucose and other sugars as the major energy sources. Many strains are capable of fatty acid biosyn-thesis from acetate. Arginine and urea are not hydrolyzed. Pigmented carotenoids occur in some species. All species are resistant, or only slightly susceptible, to 1.5% digitonin. Sapro-phytes found in soil, compost, wastewaters, or commensals of vertebrates, insects, or plants. None are known to be a primary pathogen, but they may cause cytopathic effects in tissue cul-tures. The genome sizes range from about 1500 to 2100 kbp. All species examined utilize the universal genetic code in which UGA is a stop codon.

DNA G+C content (mol%): 27–38.Type species: Acholeplasma laidlawii (Sabin 1941) Edward and

Freundt 1970, 1AL (Sapromyces laidlawi Sabin 1941, 334).


Cells of acholeplasmas typically appear as pleomorphic coc-coid, coccobacillary, or short filamentous forms when grown in mycoplasma broth containing 20% horse serum or 1% bovine serum fraction. Viable spherical cells usually have a minimum diameter of about 300 nm. Filaments may be as much as 500 nm in length, but some longer filaments and branching filaments occur in some strains. Filaments often show beading with even-tual development of coccoid forms. Cellular morphology may also depend upon the ratio of unsaturated to saturated fatty acids in the medium. Adjustment of preparative materials to the osmolarity of the culture medium is necessary for proper morphological examination.

Most acholeplasmas exhibit heavy turbidity when grown aer-obically in broth containing 5–20% serum, usually of horse or fetal bovine origin, or when grown in 1% bovine serum frac-tion broth at 37°C. Less turbidity is evident when most achole-plasmas are cultured in serum-free broth and some species may be inhibited in media containing 20% horse serum. Strains of some acholeplasmas (Acholeplasma morum, Acholeplasma modi-cum, and Acholeplasma axanthum) may not grow well in serum-free medium unless glucose and some fatty acids (Tween 80 and palmitic acid) are included. Colonies on solid medium contain-ing serum or bovine serum fraction are usually large (100–200 nm in diameter) with the classical “fried-egg” appearance after 24–72 h at 37°C (Figure 114). Colonies of Acholeplasma axanthum and several other acholeplasmas may show only cen-tral zones of growth into the agar or other unusual colony forms, such as mulberry-like colonies. Most acholeplasmas display optimum growth at 37°C. Growth is much slower at 25–27°C and strains may require 7–10 d to reach the turbidity observed after 24 h at 37°C. Species of plant origin (Acholeplasma brassicae and Acholeplasma palmae) have an optimum growth temperature of 30°C.

Most species in the genus are strong fermenters and produce acid from glucose metabolism, although a few species such as Acholeplasma parvum may not ferment glucose or other carbohy-drates (Table 143). Fermentation of mannose is usually nega-tive, although several species do catabolize this carbohydrate. All Acholeplasma species examined possess a fructose 1,6-diphos-phate-activated lactate dehydrogenase, which is a property shared with certain streptococci.

Gourlay (1970) found that a fresh isolate of Acholeplasma laidlawii from a bovine source was infected with a filamen-tous, single-stranded DNA virus designated L1 (Bruce et al., 1972; Maniloff, 1992). Later, L2 and L3 viruses were also iso-lated from Acholeplasma laidlawii (Gourlay, 1971, 1972, 1973; Gourlay et al., 1973). L2 virus is a quasi-spherical, double-stranded DNA virus (Maniloff et al., 1977), and L3 is a short-tailed phage with double-stranded DNA (Garwes et al., 1975; Gourlay, 1974; Haberer et al., 1979; Maniloff et al., 1977). Another virus isolated from Acholeplasma laidlawii is L172, a single-stranded DNA, quasi-spherical virus that is different from L1 (Liska, 1972). Two viruses have been isolated from other acholeplasmas, including one from Acholeplasma modi-cum, designated M1 (Congdon et al., 1979), and from Achole-plasma oculi strain PG49 (designated O1) (Ichimaru and Nakamura, 1983). The nucleic acid structure of the last two viruses has not been defined.

Antisera to filter-cloned whole-cell antigens are utilized in sev-eral serological techniques to assess the antigenic structure of acholeplasmas and to provide identification of the organism to the species level (Tully, 1979). The three most useful techniques are growth inhibition (Clyde, 1983), plate immunofluorescence

FIGURE 114. Colonies of Acholeplasma laidlawii PG8T (=NCTC 10116T; diameter 0.15–0.25 mm) after 3 d growth on Mycoplasma Experience Solid Medium at 36°C in 95% nitrogen/5% carbon dioxide. Original magnification = 25×. Image provided by Helena Windsor and David Windsor.

688

GeNus i. acholeplasma

(Gardella et al., 1983; Tully, 1973), and metabolism inhibition (Taylor-Robinson, 1983).

Acholeplasmas may be the most common mollicutes in ver-tebrate animals and they are found frequently in the upper respiratory tract and urogenital tract of such hosts (Tully, 1979, 1996). Eukaryotic cells in continuous culture are frequently con-taminated with acholeplasmas, primarily from the occurrence of acholeplasmas in animal serum used in tissue culture media. At least five Acholeplasma species have been identified on plant sur-faces (Acholeplasma axanthum, Acholeplasma brassicae, Acholeplasma laidlawii, Acholeplasma oculi, and Acholeplasma palmae), possibly representing contamination from insects. However, with the exception of Acholeplasma pleciae (Knight, 2004), the only achole-plasmas identified from insects have been from mosquitoes. Acholeplasma laidlawii was identified in a pool of Anopheles sinensis, and a strain of Acholeplasma morum was present in a pool of Armig-eres subalbatus (D.L. Williamson and J.G. Tully, unpublished).

Little evidence exists for a pathogenic role of acholeplasmas in natural diseases. The widespread distribution of acholeplasmas in both healthy and diseased animal tissues and of antibodies against acholeplasmas in most animal sera complicates experi-mental pathogenicity studies. However, Acholeplasma axanthum was pathogenic for goslings and young goose embryos (Kisary et al., 1975, 1976). Inoculation into leafhoppers, including those known to be vectors of plant mycoplasma diseases, shows multiplication and prolonged persistence of acholeplasmas in host tissues (Eden-Green and Markham, 1987; Whitcomb et al., 1973; Whitcomb and Williamson, 1975), but there is no evi-dence that the few Acholeplasma species found on plant surfaces play any role in plant or insect disease.

A few recent reports are available on the antibiotic sensitivity of acholeplasmas and whether the actions of these drugs are inhibitory to growth or kill cells. Acholeplasmas are sensitive to the following antibiotics (minimum inhibitory concentration range in µg/ml): tetracycline, 0.5–25.0; erythromycin, 0.03–1.0; lincomycin, 0.25–1.0; tylosin tartarate, 0.1–12.5; and kanamy-cin, 20–200 (Kato et al., 1972; Lewis and Poland, 1978; Ogata et al., 1971).


Typical steps in isolation of all mollicutes were outlined in the recently revised minimal standards for descriptions of novel

species (Brown et al., 2007). Techniques for isolation of achole-plasmas from animal tissues and from cell cultures have been described (Tully, 1983). Although colonies are occasionally first detected on blood agar, complex undefined media such as American Type Culture Collection medium 988 (SP-4) are usually required for primary isolation and maintenance. Cell wall-targeting antibiotics are included to discourage growth of other bacteria. Phenol red facilitates detection of species that excrete acidic or alkaline metabolites. Commonly used alterna-tives such as Frey’s, Hayflick’s and Friis’ media differ from SP-4 mainly in the proportions of inorganic salts, amino acids, serum sources, and types of antibiotics included. Broths are incubated aerobically at 37°C for 14 d and examined periodically for tur-bidity or pH changes, either to acid or alkaline levels. Tubes showing turbidity are plated to agar prepared from the same medium formulation, and the plates are incubated at 37°C in an atmosphere of 95% N2, 5% CO2, as in the GasPak system. Tubes without obvious turbidity should be plated at the end of the 14-d incubation period. Initial isolates may contain a mix-ture of species, so cloning by repeated filtration through mem-brane filters with a pore size of 450 or 220 nm is essential. The initial filtrate and dilutions of it are cultured on solid medium and an isolated colony is subsequently picked from a plate on which only a few colonies have developed. This colony is used to found a new cultural line, which is then expanded, filtered, plated, and picked two additional times. Identification is con-firmed by additional biochemical and serological tests.


Stock acholeplasma cultures can be maintained in either mycoplasma broth medium containing 5–20% serum or in the serum-fraction broth formulation at room temperature (25–30°C) with only weekly transfer (Tully, 1995). Maintenance is best in broth medium devoid of glucose, since excess acid production reduces viability. Stock cultures can also be main-tained indefinitely when frozen at −70°C. Agar colonies can also be maintained for 1–2 weeks at 25°C if plates are sealed to prevent drying. For optimum preservation, acholeplasmas should be lyophilized directly in the culture medium when the broth cultures reach a mid-exponential phase, usually 1–2 d at 37°C. Lyophilized cultures should be sealed under vacuum and stored at 4°C (Leach, 1983).

TABLE 143. Differential characteristics of the species of the genus Acholeplasma a

Characteristic A. l

aidl

awii

A. a

xant

hum

A. b

rass

icae

A. c

avig

enita

lium

A. e

quife

tale

A. g

ranu

laru

m

A. h

ippi

kon

A. m

odic

um

A. m

orum

A. m

ultil

ocal

e

A. o

culi

A. p

alm

ae

A. p

arvu

m

A. p

leci

ae

A. v

ituli

Glucose fermentation + + + + + + + + + + + + − + +Mannose fermentation − − − − + − + − − + − − nd nd +Arbutin hydrolysis + + − − nd − nd − + nd + − nd nd −Esculin hydrolysis + + nd nd nd − nd − + nd + nd − nd −Film and spots + − nd − + − + − − + − nd nd nd −Benzyl viologen

reduction+ + + + + + + + + − + + + nd nd


31–36 31 35.5 36 30.5 30–32 33 29 34 31 27 30 29 31.6 37.6–38.3

aSymbols: +, >85% positive; −, 0–15% positive; nd, not determined.

689


Differentiation of the genus Acholeplasma from other genera

Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. They usually possess two 16S rRNA operons. Aerobic or facultative anaerobic growth in artificial media and the absence of a requirement for sterols or cholesterol for growth exclude assignment to the genera Anaeroplasma, Asteroleplasma, “Candidatus Phytoplasma”, Mycoplasma, or Ure-aplasma. Absence of a spiral cellular morphology, regular asso-ciation with a vertebrate host or fluids of vertebrate origin, and regular use of the codon UGG to encode tryptophan (Knight, 2004) and UGA as a stop codon (Tanaka et al., 1989, 1991) support exclusion from the genera Spiroplasma, Entomoplasma, or Mesoplasma. Reduction of the redox indicator benzyl violo-gen has been reported to be fairly specific for differentiation of the genus Acholeplasma from other mollicutes (Pollack et al., 1996a). Only Acholeplasma multilocale failed to give a positive reaction, although several Mesoplasma and Entomoplasma spe-cies yielded variable responses to the test (Pollack et al., 1996a). Most acholeplasmas have membrane-localized NADH oxidase activity, in comparison to the NADH oxidase activity located in the cytoplasm of other genera within the class. Another special characteristic is the occurrence in most acholeplasmas of unique pyrimidine enzymic activities, especially a dUTPase enzyme, with the possible exception again of Acholeplasma mul-tilocale (Pollack et al., 1996b). Acholeplasmas may possess a number of other biological characteristics that may distinguish them from other genera within the class Mollicutes, including polyterpenol synthesis (Smith and Langworthy, 1979), posi-tional distribution of fatty acids (Rottem and Markowitz, 1979), the presence of superoxide dismutase (Kirby et al., 1980; Lee and Kenny, 1984; Lynch and Cole, 1980; O’Brien et al., 1981), and the presence of spacer tRNA ( Nakagawa et al., 1992). However, most of these features have not been established for even a majority of Acholeplasma species.

taxonomic comments

Acholeplasma genome sizes range from 1215 to 2095 kbp by pulsed-field gel electrophoresis or complete DNA sequencing, but most are in a more narrow range of 1215–1610 kbp (Carle et al., 1993; Neimark et al., 1992) that overlaps with genome sizes of many Spiroplasma species. Tests of eight Acholeplasma species showed less than 8% DNA–DNA hybridization between type strains and surprisingly extensive genomic heterogene-ity within species (Aulakh et al., 1983; Stephens et al., 1983a, b). The highest level of relatedness, 21% DNA–DNA hybrid-ization, was between the type strains of Acholeplasma laidlawii and Acholeplasma granularum. Some strain pairs, such as within Acholeplasma laidlawii, shared as little as 40% DNA–DNA hybrid-ization, differences that in other genera would have justified subdivision of an apparently diverse strain complex into com-ponent species. However, no polyphasic taxonomic basis was found to support such designations. Restriction endonuclease digest patterns also reflect heterogeneity within some species (Razin et al., 1983). The DNA–DNA hybridization and restric-tion digest patterns of eight Acholeplasma axanthum strains iso-lated from a variety of hosts and habitats differed markedly

from each other and some heterogeneity occurred among six different Acholeplasma oculi strains.

Mesoplasma pleciae was first isolated from the hemolymph of a larva of a Plecia corn root maggot and assigned to the genus Mesoplasma because sustained growth occurred in serum-free mycoplasma broth only when the medium contained 0.04% Tween 80 fatty acid mixture (Tully et al., 1994a). However, 16S rRNA gene sequence similarities and its preferred use of UGG rather than UGA to encode tryptophan support proper reclas-sification as Acholeplasma pleciae comb. nov. (Knight, 2004); the type strain is PS-1T (Tully et al., 1994a).

Mycoplasma feliminutum was first described during a time when the only named genus of mollicutes was Mycoplasma. Its publication coincided with the first proposal of the genus Acholeplasma (Edward and Freundt, 1969, 1970), with which Mycoplasma feliminutum is properly affiliated through estab-lished phenotypic (Heyward et al., 1969) and 16S rRNA gene sequence (Brown et al., 1995; Johansson and Pettersson, 2002) similarities. This explains the apparent inconsistencies with its assignment to the genus Mycoplasma. The name Mycoplasma feliminutum should therefore be revised to Acholeplasma felim-inutum comb. nov.; the type strain is BenT (=ATCC 25749T; Hey-ward et al., 1969).

The lack of signature enzymic activities cast serious doubt on the status of Acholeplasma multilocale PN525T as an authentic member of the genus Acholeplasma (Pollack et al., 1996b). It may be affiliated with an unrecognized metabolic subgroup, but it seems more likely to be a strain of Mycoplasma or Ento-moplasma.

acknowledgements

The major contributions to the foundation of this material by Joseph G. Tully are gratefully acknowledged.

Further reading

Taylor-Robinson, D. and J.G. Tully. 1998. Mycoplasmas, ure-aplasmas, spiroplasmas, and related organisms. In Topley and Wilson, Principles and Practice of Microbiology, 9th edn, vol. 2 (edited by Balows and Duerden). Arnold Publishers, London, pp. 799–827.

Tully, J.G. 1989. Class Mollicutes: new perspectives from plant and arthropod studies. In The Mycoplasmas (edited by Whit-comb and Tully). Academic Press, San Diego, pp. 1–31.

Differentiation of the species of the genus Acholeplasma

Esculin hydrolysis by a b-d-glucosidase and arbutin hydrolysis are sometimes useful diagnostic tests for differentiation of some acholeplasmas (Bradbury, 1977; Rose and Tully, 1983). The production of carotenoid pigments, principally neuro-sporene, has been used to differentiate some acholeplasmas, especially Acholeplasma axanthum and Acholeplasma modicum (Mayberry et al., 1974; Smith and Langworthy, 1979; Tully and Razin, 1970). Carotenoids are also synthesized in some strains of Acholeplasma laidlawii under certain growth condi-tions (Johansson, 1974). The “film and spots” reaction, which occurs in a number of Mycoplasma and several Acholeplasma spe-cies, relates to the production of crystallized calcium soaps of fatty acids on the surface of agar plates (Edward, 1954; Fabricant and Freundt, 1967). Fatty acids are liberated from the serum or

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1. Acholeplasma laidlawii (Sabin 1941) Edward and Freundt 1970, 1AL (Sapromyces laidlawi Sabin 1941, 334)

laid.law¢i.i. N.L. gen. masc. n. laidlawii of Laidlaw, named after Patrick P. Laidlaw, one of the microbiologists who first isolated this species.

This is the type species of the genus Acholeplasma. Fila-ments, usually relatively short, although much longer branched filaments may develop in media with certain ratios of saturated to unsaturated fatty acids. Coccoid forms may predominate in certain cultures including co-culture with eukaryotic cells. Agar colonies are large for a mollicute and exhibit well-developed central zones and peripheral growth on horse serum agar. On serum-free agar, colonies are smaller and may show only the central zone of growth into the agar. Relatively strong turbidity is produced during growth in broth containing serum. Temperature range for growth is 20–41°C with optimum at 37°C, even for strains recovered from plant or non-animal sources. Usually pro-duces large amounts of carotenoids when cultivated in the presence of PPLO serum fraction (Difco).

Serologically distinct from most established species in the genus, but partial cross-reactions may occur with Achole-plasma granularum strains. DNA–DNA hybridization between strains of this species range from 40 to >80%. Acholeplasma granularum strain BTS-39T showed 20% hybridization with Acholeplasma laidlawii strain PG8T. Pathogenicity has not been established.

Source: isolated from sewage, manure, humus, soil, and many animal hosts and their tissues, including some isolates from the human oral cavity, vagina, and wounds. Has been recovered from the surfaces of some plants, although few isolations have been reported from insect hosts. Frequent contaminant of eukaryotic cell cultures.

DNA G+C content (mol%): 31.7–35.7 (Bd, Tm).Type strain: ATCC 23206, PG8, NCTC 10116, CIP 75.27,

NBRC 14400.Sequence accession no. (16S rRNA gene): U14905.Further comment: on the Approved Lists of Bacterial Names

and on the Approved Lists of Bacterial Names (Amended Edition), this taxon is incorrectly cited as Acholeplasma laid-lawii [Freundt 1955 (sic)] Edward and Freundt (1970).

2. Acholeplasma axanthum Tully and Razin 1970, 754AL

a.xan¢thum. Gr. pref. a not, without; Gr. adj. xanthos -ê -on yellow; N.L. neut. adj. axanthum without yellow (pigment).

Predominantly coccobacillary and coccoid with a few short myceloid elements. Large colonies with clearly marked centers form on horse serum agar; colonies on serum-free agar are smaller and usually lack the peripheral

growth around their center. Agar colonies produce zones of b-hemolysis by the overlay technique. Growth in media devoid of serum or serum fraction is much poorer than for other acholeplasmas. Minimal nutritional requirements are poorly defined, but marked stimulation of growth with poly-oxyethylene sorbitan (Tween 80) suggests a requirement for fatty acids. Temperature range for growth is 22–37°C with optimum growth at 37°C. Synthesis of carotenoid pig-ments can be demonstrated only when large volume cul-tures are tested. Produces sphingolipids. No evidence for pathogenicity.

Source: originally isolated from murine leukemia tissue cul-ture cells, but numerous subsequent isolations of the organ-ism from bovine serum and a variety of bovine tissue sites (nasal cavity, lymph nodes, kidney) suggest cell-culture con-tamination was of bovine serum origin. Also isolated from variety of other animals and surfaces of some plants.

DNA G+C content (mol%): 31 (Bd).Type strain: S-743, ATCC 25176, NCTC 10138.Sequence accession no. (16S rRNA gene): AF412968.

3. Acholeplasma brassicae Tully, Whitcomb, Rose, Bové, Carle, Somerson, Williamson and Eden-Green 1994b, 683VP

bras.si¢cae. L. fem. gen. n. brassicae of cabbage, referring to the plant origin of the organism.

Cells are primarily coccoid. Temperature range for growth is 18–37°C. Optimal growth occurs at 30°C. No evi-dence for pathogenicity.

Source: isolated as a surface contaminant from broccoli (Brassica oleracea var. italica).

DNA G+C content (mol%): 35.5 (Bd, Tm, HPLC).Type strain: 0502, ATCC 49388.Sequence accession no. (16S rRNA gene): AY538163.

4. Acholeplasma cavigenitalium Hill 1992, 591VP

ca.vi.ge.ni.ta¢li.um. N.L. n. cavia guinea pig (Cavia cobaya); L. pl. n. genitalia -ium the genitals; N.L. pl. gen. n. cavigenita-lium of guinea pig genitals.

Pleomorphic cells, mostly coccoid. Grows on broth or agar medium under aerobic conditions, with optimum tem-perature between 35 and 37°C. Colonies on agar medium have typical fried-egg appearance. Originally described as a non-fermenter, but the type strain ferments glucose. Does not grow well on SP-4 broth or in horse serum broth, but grows well on simple base medium with additions of 10–15% fetal bovine serum. No evidence for pathogenicity.

Source: isolated from the vagina of guinea pigs.DNA G+C content (mol%): 36 (Bd).Type strain: GP3, NCTC 11727, ATCC 49901.Sequence accession no. (16S rRNA gene): AY538164.

list of species of the genus Acholeplasma

supplemental egg yolk (Fabricant and Freundt, 1967; Thorns and Boughton, 1978) in the agar medium by the lipolytic activ-ity of the organisms. Failure to cross-react with antisera against previously recognized species provides evidence for species novelty. For this reason, deposition of antiserum against a novel type strain into a recognized collection is still manda-tory for new species descriptions (Brown et al., 2007). Prelimi-

nary differentiation can be by PCR and DNA sequencing using primers specific for bacterial 16S rRNA genes or the 16S–23S intergenic region. A similarity matrix relating the candidate strain to its closest neighbors, usually species with >0.94 16S rRNA gene sequence similarity, will suggest an assemblage of related species that should be examined for serological cross-reactivities.

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5. Acholeplasma entomophilum Tully, Rose, Carle, Bové, Hackett and Whitcomb 1988, 166VP

en.to.mo.phi¢lum. Gr. n. entomon insect; N.L. neut. adj. phi-lum (from Gr. neut. adj. philon) friend, loving; N.L. neut. adj. entomophilum insect-loving.

Cells are pleomorphic, but primarily coccoid. Colonies on solid medium usually have a fried-egg appearance. Acid is produced from glucose, but not mannose. Carotenoids are not produced. “Film and spot” reaction is negative. Agar colonies hemadsorb guinea pig erythrocytes. Strains require 0.4% Tween 80 or fatty acid supplements for growth in serum-free media. Temperature range for growth is 23–32°C, with optimum growth at about 30°C. Pathogenic-ity has not been established.

Source: isolated from gut contents of tabanid flies, beetles, butterflies, honey bees, and moths, and from flowers.

DNA G+C content (mol%): 30 (Bd).Type strain: TAC, ATCC 43706.Sequence accession no. (16S rRNA gene): M23931.Further comment: with the proposal of the order Ento-

moplasmatales (Tully et al., 1993), Acholeplasma entomophi-lum was transferred to the family Entomoplasmataceae. The name Acholeplasma entomophilum was therefore revised to Mesoplasma entomophilum comb. nov. The type strain is TACT (=ATCC 43706T; Tully et al., 1988).

6. Acholeplasma equifetale Kirchhoff 1978, 81AL

eq.ui.fe.ta¢le. L. n. equus horse; N.L. adj. fetalis -is -e pertain-ing to the fetus; N.L. neut. adj. equifetale pertaining to the horse fetus.

Cells are pleomorphic, but predominantly coccoid. Col-onies on solid medium containing serum usually have a fried-egg appearance; on serum-free medium, colonies are similar, but usually smaller. Growth temperature range is 22–37°C. Pathogenicity has not been established.

Source: isolated from the lung and liver of aborted horse fetuses. Also recovered from the respiratory tract of appar-ently normal horses and the respiratory tract and cloacae of broiler chickens (Bradbury, 1978).

DNA G+C content (mol%): 30.5 (Bd).Type strain: C112, ATCC 29724, NCTC 10171.Sequence accession no. (16S rRNA gene): AY538165.Further comment: Kirchhoff is incorrectly cited as “ Kirchoff”

on the Approved Lists of Bacterial Names.

7. Acholeplasma florum McCoy, Basham, Tully, Rose, Carle and Bové 1984, 14VP

flo¢rum. L. gen. p1. n. florum of flowers, indicating the recovery site of the organism.

Cells are ovoid. Colonies on agar are umbonate. Films and spots are produced on serum-containing media. Glu-cose is utilized, but mannose is not. Carotenes are not pro-duced, nor is b-d-glucosidase. Pathogenicity has not been established.

Source: the known strains were isolated from flower sur-faces.

DNA G+C content (mol%): 27.3 (Bd).Type strain: L1, ATCC 33453.Sequence accession nos: AF300327 (16S rRNA gene),

NC_006055 (strain L1T complete genome).

Further comment: with the proposal of the order Ento-moplasmatales (Tully et al., 1993), Acholeplasma florum was transferred to the family Entomoplasmataceae. The name Acholeplasma florum was therefore revised to Mesoplasma florum comb. nov. The type strain is L1T (=ATCC 33453T; McCoy et al., 1984).

8. Acholeplasma granularum (Switzer 1964) Edward and Fre-undt 1970, 2AL (Mycoplasma granularum Switzer 1964, 504)

gra.nu.la¢rum. N.L. fem. n. granula (from L. neut. n. granu-lum) a small grain, a granule; N.L. gen. pl. n. granularum of small grains, made up of granules, granular.

Cells are pleomorphic, with short filaments and coc-coid cells. Colonies on solid medium are large with clearly marked central zones and a fried-egg appearance. Colonies on serum-free medium are smaller and may lack the periph-eral zone of growth around central core. Temperature range for growth is 22–37°C, with optimum around 37°C. Agar colonies produce a zone of b-hemolysis by the overlay technique using sheep erythrocytes. DNA–DNA hybridiza-tion studies showed 20–22% hybridization with Acholeplasma laidlawii, but none with other acholeplasmas. Pathogenic-ity has not been established. Aerosol challenge of specific pathogen-free pigs did not induce clinical or histological evidence of disease.

Source: isolated frequently from the nasal cavity of swine, with occasional isolates from swine lung and feces. Also isolated from the conjunctivae and nasopharynx of horses, and the genital tract of guinea pigs. Occasional contami-nant of eukaryotic cell cultures.

DNA G+C content (mol%): 30.5–32.4 (Tm, Bd).Type strain: BTS-39, ATCC 19168, NCTC 10128.Sequence accession no. (16S rRNA gene): AY538166.

9. Acholeplasma hippikon Kirchhoff 1978, 81AL

hip.pi¢kon. Gr. neut. adj. hippikon pertaining to the horse.

Cells are pleomorphic with predominantly coccoid forms. Colonies on solid medium containing horse serum typically have a fried-egg appearance, with smaller colonies on serum-free agar medium. Growth occurs over a tempera-ture range of 22–37°C, with optimal growth at 35–37°C. Agar colonies produce b-hemolysis with the overlay technique, using a variety of animal red blood cells. Pathogenicity has not been established.

Source: isolated from the lung of aborted horse fetuses.DNA G+C content (mol%): 33.1 (Bd).Type strain: C1, ATCC 29725, NCTC 10172.Sequence accession no. (16S rRNA gene): AY538167.Further comment: Kirchhoff is incorrectly cited as “ Kirchoff”

on the Approved Lists of Bacterial Names.

10. Acholeplasma modicum Leach 1973, 147AL

mo¢di.cum. L. neut. adj. modicum moderate, referring to moderate growth.

Cells are pleomorphic, with spherical, ring-shaped, and coccobacillary forms. Colonies on solid medium are dis-tinctly smaller than those of most other acholeplasmas. Very small colonies without peripheral zones of growth are noted on serum-free solid medium. Very light turbidity is observed in serum-free broth, but more turbidity is found

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in broth containing serum. Growth temperature range is 22–37°C, with optimum growth around 35–37°C. Can be shown to produce carotenoids when large volumes of cells are examined. Agar colonies produce a- or b-hemolysis by the overlay technique using sheep, ox, or guinea pig red blood cells. Pathogenicity has not been established.

Source: isolated from various tissues of cattle, including blood, bronchial lymph nodes, thoracic fluids, lungs, and semen. Also isolated from nasal secretions of pigs, and occa-sionally from chickens, turkeys, and ducks.

DNA G+C content (mol%): 29.3 (Tm).Type strain: PG49, ATCC 29102, NCTC 10134.Sequence accession no. (16S rRNA gene): M23933.

11. Acholeplasma morum Rose, Tully and Del Giudice 1980, 653VP

mor¢um. L. n. morum a mulberry, denoting the mulberry-like appearance of agar colonies of the organism.

Cells are pleomorphic, predominantly coccoid or cocco-bacillary forms, but with some beaded filaments. Colonies on solid medium without serum supplements are very small in size and have only central zones without any peripheral growth. Optimal growth on solid medium occurs with a 10% serum concentration and colony growth appears to be suppressed in a medium with 20% serum. Optimal growth in broth is apparent when 5–10% serum is added or when 1% bovine serum fraction supplements are added, but poor growth occurs in broth containing 20% horse serum. Growth in serum-free broth usually requires some fatty acid supplements, such as palmitic acid or polyoxyethylene sorbi-tan (Tween 80). Temperature range for growth is 23–37°C, with optimum growth at about 35–37°C.

Pathogenicity has not been established. Calf kidney cell cul-tures containing the organism show cytopathogenic effects.

Source: originally recovered from commercial fetal bovine serum and from calf kidney cultures containing fetal bovine serum. One isolation, in broth containing horse serum, was from a pool of Armigeres subalbatus mosquitoes collected by Leon Rosen in Taiwan in 1978 (strain SP7; D.L. Williamson and J.G. Tully, unpublished).

DNA G+C content (mol%): 34.0 (Tm).Type strain: 72-043, ATCC 33211, NCTC 10188.Sequence accession no. (16S rRNA gene): AY538168.

12. Acholeplasma multilocale Hill, Polak-Vogelzang and Angulo 1992, 516VP

mul.ti.lo.ca¢le. L. adj. multus many, numerous; L. adj. localis -is -e of or belonging to a place, local; N.L. neut. adj. multilo-cale referring to more than one location.

Pleomorphic cells. Colonies on agar medium have a typical fried-egg appearance. Organisms grow well in broth medium at 35–37°C. No evidence for pathogenicity.

Source: isolated from the nasopharynx of a horse and the feces of a rabbit.

DNA G+C content (mol%): 31 (Bd).Type strain: PN525, NCTC 11723, ATCC 49900.Sequence accession no. (16S rRNA gene): AY538169.

13. Acholeplasma oculi corrig. al-Aubaidi, Dardiri, Muscoplatt and McCauley 1973, 126AL

o¢cu.li. L. n. oculus the eye; L. gen. n. oculi of the eye.

Cells are pleomorphic, including spherical, ring-shaped, and coccobacillary forms. Medium-sized colonies with typi-cal fried-egg appearance are formed on horse serum agar. Colonies on serum-free agar are smaller and may lack the peripheral growth around the central core. Growth occurs at temperatures of 25–37°C. Agar colonies produce zones of hemolysis by the overlay technique using sheep red blood cells.

Pathogenicity is not well established. Intravenous inoc-ulation of goats produced signs of pneumonia and death within 6 d. Conjunctival inoculation of goats produced mild conjunctivitis.

Source: isolated from the conjunctiva of goats with kera-toconjunctivitis; porcine nasal secretions; equine nasophar-ynx, lung, spinal fluid, joint, and semen; the urogenital tract of cattle; and the external genitalia of guinea pigs. Present in amniotic fluid of pregnant women (Waites et al., 1987). Occasionally isolated from ducks and turkeys, with unreported isolations from an ostrich. Also several isola-tions from palm trees and other plants (Eden-Green and Tully, 1979; Somerson et al., 1982). Isolations from eukary-otic cell cultures may represent contamination of bovine origin.

DNA G+C content (mol%): 27 (Tm).Type strain: 19-L, ATCC 27350, NCTC 10150.Sequence accession no. (16S rRNA gene): U14904.Further comment: originally named Acholeplasma oculusi

by al-Aubaidi et al. (1973); the orthographic error was cor-rected by al-Aubaidi (1975).

14. Acholeplasma palmae Tully, Whitcomb, Rose, Bové, Carle, Somerson, Williamson and Eden-Green 1994b, 683VP

pal¢mae. L. fem. gen. n. palmae of a palm tree, referring to the plant from which the organism was isolated.

Cells are primarily coccoid. Colonies on solid medium usually have a fried-egg appearance. The temperature range for growth is 18–37°C, with optimal growth occurring at 30°C. No evidence for pathogenicity. It is one of the clos-est phylogenetic relatives of the phytoplasmas.

Source: isolated from the crown tissues of a palm tree (Cocos nucifera) with lethal yellowing disease.

DNA G+C content (mol%): 30 (Bd, Tm, HPLC).Type strain: J233, ATCC 49389.Sequence accession no. (16S rRNA gene): L33734.

15. Acholeplasma parvum Atobe, Watabe and Ogata 1983, 348VP

par¢vum. L. neut. adj. parvum small, intended to refer to the poor biochemical activities and tiny agar colonies of the organism.

Pleomorphic coccobacillary cells. Colonies on agar medium present a typical fried-egg appearance under both aerobic and anaerobic conditions. Initial reports of growth in the absence of cholesterol or serum have been made, but growth on serum-free medium is not well confirmed. The organism does not grow in most standard media for achole-plasmas or in most other medium formulations for sterol-requiring mycoplasmas. Needs special growth factor of 1% phytone or soytone peptone supplements; growth is some-times better with the addition of 15% fetal bovine serum. Organisms grow on agar better than in broth; growth is

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better under aerobic conditions than under anaerobic conditions and better at 22–30°C than at 37°C. No evidence of fermentation of any carbohydrate, including glucose, salicin, and esculin. No evidence for pathogenicity.

Source: isolated from the oral cavities and vagina of healthy horses.

DNA G+C content (mol%): 29.1 (Tm).Type strain: H23M, ATCC 29892, NCTC 10198.Sequence accession no. (16S rRNA gene): AY538170.

16. Acholeplasma pleciae (Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a) Knight 2004, 1952VP (Mesoplasma pleciae Tully, Whitcomb, Hackett, Rose, Henegar, Bové, Carle, Williamson and Clark 1994a, 690)

ple.ci¢ae. N.L. gen. n. pleciae of Plecia, referring to the genus of corn maggot (Plecia sp.) from which the organism was first isolated.

Cells are primarily coccoid. Colonies on solid media incubated under anaerobic conditions at 30°C have a fried-egg appearance. Supplements of 0.04% polyoxyethylene sorbitan (Tween 80) are required for growth in serum-free media. Temperature range for growth is 18–32°C, with optimal growth at 30°C. Agar colonies do not hemadsorb guinea pig erythrocytes. No evidence for pathogenicity.

Source: originally isolated from the hemolymph of a larva of the corn root maggot (Plecia sp.).

DNA G+C content (mol%): 31.6 (Bd, Tm, HPLC).Type strain: PS-1, ATCC 49582.Sequence accession no. (16S rRNA gene): AY257485.

17. Acholeplasma seiffertii Bonnet, Saillard, Vignault, Garnier, Carle, Bové, Rose, Tully and Whitcomb 1991, 48VP

seif.fer¢ti.i. N.L. gen. masc. n. seiffertii of Seiffert, in honor of Gustav Seiffert, a German microbiologist who performed

pioneering studies of sterol-nonrequiring mollicutes that occur in soil and compost.

Cells are primarily coccoid. Colonies on solid medium usually have the appearance of fried-eggs. Acid produced from glucose and mannose. Colonies on agar hemadsorb guinea pig erythrocytes. Temperature range for growth is 20–35°C; optimum growth occurs at 28°C. No evidence for pathogenicity.

Source: isolated from floral surfaces of a sweet orange (Citrus sinensis) and wild angelica (Angelica sylvestris).

DNA G+C content (mol%): 30 (Bd).Type strain: F7, ATCC 49495.Sequence accession no. (16S rRNA gene): AY351331.Further comment: with the proposal of the order Ento-

moplasmatales (Tully et al., 1993), Acholeplasma seiffertii was transferred to the family Entomoplasmataceae. The name Acholeplasma seiffertii was therefore revised to Mesoplasma seiffertii comb. nov. The type strain is F7T (=ATCC 49495T; Bonnet et al., 1991).

18. Acholeplasma vituli Angulo, Reijgers, Brugman, Kroesen, Hekkens, Carle, Bové, Tully, Hill, Schouls, Schot, Roholl and Polak-Vogelzang 2000, 1130VP

vi.tu¢li. L. n. vitulus calf; L. gen. n. vituli of calf, referring to the provenance or occurrence of the organism in fetal calf serum.

Cells are predominantly coccoid in shape. Colonies on solid media demonstrate a fried-egg appearance under both aerobic and anaerobic conditions. Temperature range for growth is 25–37°C. No evidence for pathogenicity.

Source: isolated from fetal bovine serum or contaminated eukaryotic cell cultures containing serum.

DNA G+C content (mol%): 38.3 (Bd), 37.6 (Tm).Type strain: FC 097-2, ATCC 700667, CIP 107001.Sequence accession no. (16S rRNA gene): AF031479.

References

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al-Aubaidi, J.M., A.H. Dardiri, C.C. Muscoplatt and E.H. McCauley. 1973. Identification and characterization of Acholeplasma oculusi spec. nov. from the eyes of goats with keratoconjunctivitis. Cornell Vet. 63: 117–129.

Angulo, A.F., R. Reijgers, J. Brugman, I. Kroesen, F.E.N. Hekkens, P. Carle, J.M. Bové, J.G. Tully, A.C. Hill, L.M. Schouls, C.S. Schot, P.J.M. Roholl and A.A. Polak-Vogelzang. 2000. Acholeplasma vituli sp. nov., from bovine serum and cell cultures. Int. J. Syst. Evol. Micro-biol. 50: 1125–1131.

Atobe, H., J. Watabe and M. Ogata. 1983. Acholeplasma parvum, a new species from horses. Int. J. Syst. Bacteriol. 33: 344–349.

Aulakh, G.S., E.B. Stephens, D.L. Rose, J.G. Tully and M.F. Barile. 1983. Nucleic acid relationships among Acholeplasma species. J. Bacteriol. 153: 1338–1341.

Bonnet, F., C. Saillard, J.C. Vignault, M. Garnier, P. Carle, J.M. Bové, D.L. Rose, J.G. Tully and R.F. Whitcomb. 1991. Acholeplasma seiffertii sp. nov., a mollicute from plant surfaces. Int. J. Syst. Bacteriol. 41: 45–49.

Bradbury, J. 1977. Rapid biochemical tests for characterization of the Mycoplasmatales. J. Clin. Microbiol. 5: 531–534.

Bradbury, J.M. 1978. Acholeplasma equifetale in broiler chickens. Vet. Rec. 102: 516.

Brown, D., G. McLaughlin and M. Brown. 1995. Taxonomy of the feline mycoplasmas Mycoplasma felifaucium, Mycoplasma feliminutum, Myco-plasma felis, Mycoplasma gateae, Mycoplasma leocaptivus, Mycoplasma leopharyngis, and Mycoplasma simbae by 16S rRNA gene sequence com-parisons. Int. J. Syst. Bacteriol. 45: 560–564.


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Family ii. Incertae sedis

Phytoplasmas (Sears and Kirkpatrick, 1994) are wall-less, nutri-tionally fastidious, phytopathogenic prokaryotes 0.2–0.8 µm in diameter that morphologically resemble members of the Mollicutes. Sequencing of nearly full-length PCR-amplified 16S rRNA genes (Gundersen et al., 1994; Namba et al., 1993; Seemüller et al., 1994), combined with earlier studies (Kuske and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first comprehensive phylogeny of the organisms and showed that they constitute a unique, monophyletic clade within the Mollicutes. These organisms are most closely related to mem-bers of the genus Acholeplasma within the Anaeroplasma clade as defined by Weisburg et al. (1989). Sustained culture in cell-free

media has not yet been demonstrated for any phytoplasma. Their genome sizes have been estimated to range from 530 to 1350 kb, and the G+C content of phytoplasma DNA is about 23–30 mol%. The presence of a characteristic oligonucleotide sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG CYG GDC T, and standard codon usage indicate that phytoplas-mas represent a distinct taxon for which the name “Candidatus Phytoplasma” has been adopted by specialists in the molecular biology and pathogenicity of these and similar phytopatho-genic organisms (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). At present, the designation “Candidatus” must still be used for new types.

This family includes the phytoplasma strains of the order Acholeplasmatales. Although never cultured in cell-free media,

these plant pathogens and symbionts have been well studied by culture- independent methods.

Genus i. “Candidatus Phytoplasma” gen. nov. iRpcm phytoplasma/spiroplasma Working team 2004, 1244

nigel a. haRRison, Dawn gunDeRsen-RinDal anD RoBeRt e. Davis

phy.to.plas¢ma. Gr. masc. n. phytos a plant; Gr. neut. n. plasma something formed or molded, a form.

696

Genus I. “CandIdatus PhytoPlasma”


Phytoplasma cells typically have a diameter less than 1 µm and are polymorphic. Viewed in ultra-thin section by electron microscopy Figure 115, they appear ovoid, oblong, or filamen-tous in plant and insect hosts (Doi et al., 1967; Hearon et al., 1976). Transmission electron microscopy of semi-thick (0.3 µm) sections (Thomas, 1979) and serial sections (Chen and Hiruki, 1978; Florance and Cameron, 1978; Waters and P. Hunt., 1980), and scanning electron microscopy studies (Bertaccini et al., 1999; Haggis and Sinha, 1978; Marcone et al., 1996) have done much to clarify the gross cellular morphology of phytoplasmas. They range from spherical to filamentous, often with exten-sive branching reminiscent of that seen in Mycoplasma mycoides. Small dense rounded forms ~0.1 µm in diameter, formerly con-sidered to be “elementary bodies” when seen in thin section, were shown to represent constrictions in filamentous forms. Dumbbell-shaped forms once thought to be “dividing cells” are actually branch points of filamentous forms, whereas forms thought to have internal vesicles have been shown to have invo-luted membranes oriented such that the plane of the sections cut through the cell membrane twice (McCoy, 1979). Phyto-plasma cell membranes are resistant to digitonin and sensitive to hypotonic salt solutions, and, as such, are similar to those of non-sterol requiring mollicutes (Lim et al., 1992).

Phytoplasmas are consistently observed within phloem sieve elements (Christensen et al., 2004; McCoy et al., 1989; Oshima et al., 2001b; Webb et al., 1999) and occasionally have been reported in both companion cells (Rudzinska-Langwald and Kaminska., 1999; Sears and Klomparens, 1989) and paren-chyma cells (Esau et al., 1976; Siller et al., 1987) of infected plants. Sieve elements are specialized living cells that lack nuclei when mature and transport photosynthate from leaves not only to growing tissues, but also to other tissues unable to photo-synthesize (Oparka and Turgeon, 1999; Sjölund, 1997). This applies particularly to roots that require considerable energy

for the uptake of water and nutrients (Flores et al., 1999). Phloem sap is unique in that it contains from 12 to 30% sucrose and is under high hydrostatic (turgor) pressure (Evert, 1977). Sieve elements have pores in their end plates and lateral walls, allowing passage of photosynthate to adjacent sieve tube ele-ments. The sieve pores, which have an average diameter of ~0.2 µm, are of sufficient size to allow passage of spherical and fila-mentous phytoplasma cells from one sieve element to another (McCoy, 1979). The chemical composition of sieve sap is com-plex, containing sugars, minerals, free amino acids, proteins, and ATP (Van Helden et al., 1994). This rich milieu, with its high osmotic and hydrostatic pressures, serves to support exten-sive multiplication of phytoplasmas in planta. Phytoplasmas also multiply in the internal tissues and organs of their insect vectors (Kirkpatrick et al., 1987; Lefol et al., 1994; Marzachi et al., 2004; Nasu et al., 1970), which are primarily leafhoppers, planthop-pers, and psyllids (D’Arcy and Nault, 1982; Jones, 2002; Wein-traub and Beanland, 2006). In many respects, the composition of insect hemolymph is similar to that of plant phloem sap, as both contain high levels of complex and simple organic com-pounds (Moriwaki et al., 2003; Saglio and Whitcomb, 1979).

Physical maps of several phytoplasma genomes have been constructed (Firrao et al., 1996; Lauer and Seemüller, 2000; Marcone and Seemüller, 2001; Padovan et al., 2000). The pres-ence of extrachromosomal DNAs or plasmids in numerous phytoplasmas has also been reported (Davis et al., 1988; Denes and Sinha, 1991; Kuboyama et al., 1998; Kuske and Kirkpat-rick, 1990; Liefting et al., 2004; Lin et al., 2009; Nakashima and Hayashi, 1997; Nishigawa et al., 2003; Oshima et al., 2001a; Tran-Nguyen and Gibb, 2006) and suggested as a potential means of intermolecular recombination (Nishigawa et al., 2002b). Phyto-plasma-associated extrachromosomal DNAs have been shown to contain genes encoding a putative geminivirus-related rep-lication (Rep) protein (Liefting et al., 2006; Nishigawa et al., 2001; Rekab et al., 1999) and a single-stranded DNA-binding

FIGURE 115. Electron micrographs of ultrathin sections of leaf petiole from a sunnhemp (Crotalaria juncea L.) plant displaying Crotalaria phyllody disease symptoms. (a) Polymorphic phytoplasma cells occluding the lumen of adjacent leaf phloem sieve tube elements. Bar = 2 µm. (b) Ultratructural morphology indicates phytoplasma cells are bounded by a unit membrane and contain DNA fibrils and ribosomes. Bar = 200 nm. Images provided by Phil Jones.

697

FamIly II. InCertae sedIs

protein (Nishigawa et al., 2002a), as well as a putative gene simi-lar to DNA primase of other bacterial chromosomes (Liefting et al., 2004) and still other genes of as yet unknown identity. Moreover, heterogeneity in extrachromosomal DNAs has been associated with reduced pathogenicity and loss of insect vector transmissibility (Denes and Sinha, 1992; Nishigawa et al., 2002a, 2003).

Onion yellows mild strain (OY-M) was the first phytoplasma genome to be completely sequenced. The genome of this aster yellows group strain consists of a circular chromosome of 860,632 bp. It also contains two extrachromosomal DNAs, EcOYM (5025 bp) and plasmid pOYM (3932 bp) (Nishigawa et al., 2003; Oshima et al., 2002), representing two different classes based on the type of replication protein encoded. While EcOYM contains a rep gene homologous to that of the gemi-niviruses, pOYM has a rep gene that encodes a unique protein with characteristics of both viral-rep and plasmid-rep (Namba, 2002). The chromosome is a circular DNA molecule with a G+C content of 28 mol% and contains 754 open reading frames (ORFs), comprising 73% of the chromosome. Of these, 66% of ORFs exhibit significant homology to gene sequences currently archived in the GenBank database. Putative proteins encoded by ORFs could be assigned to one of six different functional categories: (1) information storage and processing (260 ORFs); (2) metabolism (107 ORFs); (3) cellular processes (77 ORFs); (4) poorly characterized, i.e., with homology to uncharacter-ized proteins of other organisms (50 ORFs); or (5) others, i.e., without homology to any known proteins (260 ORFs). Like mycoplasmal genomes, the OY-M phytoplasma genome lacks many genes related to amino acid and fatty acid biosynthesis, the tricarboxylic acid cycle, and oxidative phosphorylation. However, OY-M phytoplasma differs from mycoplasma in that it lacks genes for the phosphotransferase system and for metabo-lizing UDP-galactose to glucose 1-phosphate, suggesting that it possesses a unique sugar intake and metabolic system. Fur-thermore, OY-M phytoplasma lacks most of the genes needed to synthesize nucleotides and ATP suggesting that it probably assimilates these and other necessary metabolites from host cytoplasm. Many genes, such as those for glycolysis, are pres-ent as multiple redundant copies representing 18% of the total genome. Twenty-seven genes encoding transporter systems such as malate, metal-ion and amino acid transporters, some of which have multiple copies, were identified, suggesting that phytoplasmas aggressively import many metabolites from the host cell. Other than genes encoding glucanase and hemolysin-like proteins, no other genes presently known to be related to bacterial pathogenicity were evident in the OY-M phytoplasma genome, suggesting novel mechanisms for virulence.

Annotation of the OY-M phytoplasma genome has been followed by three other phytoplasma genome annotations. Aster yellows witches’-broom phytoplasma (“Candidatus Phy-toplasma asteris”-related strain AY-WB) possesses a circu-lar 706,569 nucleotide chromosome and plasmids AYWB-pI (3872 bp), -pII (4009 bp), -pIII (5104 bp), and -pIV (4316 bp) (Bai et al., 2006). Australian tomato big bud phytoplasma (“ Candidatus Phytoplasma australiense”-related strain TBB) has a circular 879,324 bp chromosome and a 3700 bp plas-mid (Tran-Nguyen et al., 2008), whereas apple proliferation phytoplasma (“Candidatus Phytoplasma mali”-related strain AT) has a linear 601,943 bp chromosome (Kube et al., 2008).

The chromosome of “ Candidatus Phytoplasma mali” is charac-terized by large terminal inverted repeats and covalently closed hairpin ends. Analysis of protein-coding genes revealed that gly-colysis, the major energy-yielding pathway supposed for OY-M phytoplasma, is incomplete in AT phytoplasma. It also differs from OY-M and AY-WB phytoplasmas by a lower G+C content (21.4 mol%), fewer paralogous genes, a strongly reduced num-ber of ABC transporters for amino acids, and an extended set of genes for homologous recombination, excision repair, and SOS response.

Comparative genomics have also recently identified ORFs shared by AY-WB phytoplasma and the distantly-related corn stunt pathogen Spiroplasma kunkelii that are absent from obligate animal and human pathogenic mollicutes. These proteins were identified as polynucleotide phosphorylase (PNPase), cmp-binding factor (CBF), cytosine deaminase, and Y1xR protein and could be important for insect transmission or plant patho-genicity. Also identified were four additional proteins, ppGpp synthetase, HAD hydrolase, AtA (AAA type ATPase), and P-type Mg2+ transport ATPase, that seemed to be more closely related between AY-WB and Spiroplasma kunkelii than to their mycoplas-mal counterparts (Bai et al., 2004).

Phytoplasmas possess a unique genome architecture that is characterized by multiple, nonrandomly distributed sequence-variable mosaics (SVMs) of clustered genes, originally rec-ognized in a study of closely related “Candidatus Phytoplasma asteris”-related strains CPh and OY-M (Jomantiene and Davis, 2006). Targeted genome sequencing and comparative genomics indicated that this genome architecture is a common charac-teristic among phytoplasmas, leading to the proposal that the origin of SVMs was an ancient event in the evolution of the phy-toplasma clade (Jomantiene et al., 2007), perhaps as a result of recurrent targeted attacks by mobile elements such as phages (Wei et al., 2008a). Jomantiene and Davis (2006) proposed that sizes and numbers of SVMs could account in part for the known variation in genome size among phytoplasma strains; this con-cept was independently suggested by Bai et al. (2006) on the basis of results from a comparative study of two completely sequenced phytoplasma genomes. Nucleotide sequences within SVMs included full length or pseudogene forms of fliA, an ATP- dependent Zn protease gene, tra5, smc, himA, tmk, and ssb (encod-ing single-stranded DNA-binding protein), genes potentially encoding hypothetical proteins of unknown function, genes exhibiting similarities to transposase, and a phage-related gene ( Jomantiene et al., 2007). A similar set of nucleotide sequences occurs in AY-WB genomic regions termed potential mobile units (PMUs) by Bai et al. (2006). The presence of sequences encod-ing putatively secreted and/or transmembrane, cell surface-interacting proteins indicates that these genomic features are likely to be significant for phytoplasma/host interactions (Bai et al., 2006; Jomantiene and Davis, 2006; Jomantiene et al., 2007).

Short (17–35 bases) conserved, imperfect palindromic DNA sequences (PhREPs) that are present in SVMs possibly play a role in phytoplasma genome plasticity and targeting of mobile genetic elements. SVMs can be viewed as composites formed by the acquisition of genes through horizontal transfer, recom-bination, and rearrangement, and capture of mobile elements recurrently targeted to SVMs, leading Jomantiene et al. (2007) to suggest that SVMs provide loci for acquisition of new genes

698


and targeting of mobile genetic elements to specific regions in phytoplasma chromosomes.

The chromosomes of avirulent, mildly, moderately, and highly virulent strains of “Candidatus Phytoplasma mali” ( Seemüller and Schneider, 2007) differ from one another in size and exhibit distinct restriction endonuclease patterns when cleaved with rare cutting enzymes. PCR-based DNA ampli-fications, primed separately by eight primer pairs, revealed target sequence heterogeneity among all “Candidatus Phyto-plasma mali”-related strains tested, but no correlations linked molecular markers with strain virulence or the maximum titer obtained upon infection of apple trees. In a separate study, a comparison of mild (OY-M) and severe (OY-W) strains of onion yellows (OY) phytoplasma indicated that severe symptoms were associated with higher populations of OY-W in infected host plants (Oshima et al., 2001b). A cluster of eight genes, consid-ered essential for glycolysis, were subsequently identified within a similar 30 kb genomic region of both strains (Oshima et al., 2007). Of these, five genes (smtA, greA, osmC, eno, and pfkA) were randomly duplicated in OY-W, possibly influencing glycolytic activitiy. A higher consumption of metabolites such as sugars in the intracellular environment of the phloem may explain dif-ferences between OY-W and OY-M in growth rate, which in turn may be linked, directly or indirectly, to symptom severity.

Cloned fragments of phytoplasma DNA have been widely employed as probes in dot and Southern blot hybridization assays to identify and characterize phytoplasmas (reviewed by Lee and Davis, 1992; Lee et al. (2000). Southern blot restriction fragment length polymorphism (RFLP) analysis has enabled investigations of genetic relationships among phytoplasmas associated with similar hosts or with symptomatologically similar diseases (Kison et al., 1997, 1994; Kuske et al., 1991; Schneider and Seemüller, 1994b). Several discrete phytoplasma groups, each comprising strains that shared extensive sequence homol-ogy and little or no apparent homology with other phytoplas-mas, were identified by this type of analysis. Lee and co-workers (1992) coined the term “genomic strain cluster” to denote each of seven discrete genotypic groups resolved by employing a selection of phytoplasma genomic DNA probes (reviewed by Lee and Davis, 1992). Of these, aster yellows (AY) was the largest group, represented by 15 genetically variable strains that were further delineated into three distinct genomic types (types I, II, and III) or subclusters (Lee et al., 1992). Significantly, major groupings later revealed by RFLP analysis of 16S rRNA genes were consistent with those defined by monoclonal antibody typing (Lee et al., 1993a) and other molecular methods (Lee et al., 1998b), but differed from distinctions made in traditional classification based solely on biological properties such as plant host range, symptomatology, and insect vector specificity ( Chiykowski and Sinha, 1990).

Polyclonal antibodies (PAbs) have been produced against phytoplasma-enriched extracts (intact organisms or membrane fractions) partially purified from plants (reviewed by Chen et al., 1989) and against vector leafhopper-derived immunogens (Errampelli and Fletcher, 1993; Kirkpatrick et al., 1987). Most PAbs exhibit relatively high background reactions with healthy host antigens; thus, generation of useful polyclonal antisera has been limited so far to a few phytoplasmas maintained at high titer in host tissues. Phytoplasmas can be differentiated on the basis of their antigenic properties through the use of PAbs in

enzyme-linked immunosorbent (ELISA), immunofluorescence, or Western blot assays. Antigenic similarity revealed among phy-toplasmas by these assays is often in agreement with relationships demonstrated by vector transmission studies. Detection of anti-genically distinct phytoplasmas in plants exhibiting very similar disease symptoms attests to the unreliability of symptom expres-sion alone as a means of differentiating phytoplasmas.

Improvements in phytoplasma extraction methods have provided a source of immunogens for monoclonal antibody (MAb) production (Chang et al., 1995; Hsu et al., 1990; Jiang et al., 1989; Loi et al., 2002, 1998; Shen and Lin, 1993; Tanne et al., 2001). Used in ELISA, dot or tissue blot immunoassay, immunocapture PCR (Rajan et al., 1995), immunofluorescence microscopy, or immunosorbent electron microscopy (ISEM) (Clark, 1992; Shen and Lin, 1994), MAbs have demonstrated considerable promise for detection and differentiation of phy-toplasmas infecting a broad range of host plants, including woody perennials (Guo et al., 1998). Due to their high degree of specificity, monoclonals seem most suited for differentiating very closely related strains (Lee et al., 1993a).

Isolation, cloning, and expression of immunodominant pro-tein genes have identified putative proteins that account for a major portion of the membrane proteins of several phytoplas-mas (Arashida et al., 2008; Barbara et al., 2002; Berg et al., 1999; Blomquist et al., 2001; Galetto et al., 2008; Kakizawa et al., 2004, 2009; Morton et al., 2003; Suzuki et al., 2006; Yu et al., 1998). When these purified proteins were used as immunogens, the resulting polyclonal antisera exhibited high specific titers and low background reactions in ELISA and Western blot analyses that were designed to detect phytoplasma proteins in infected hosts. Similarly, the secA gene was cloned from an onion yellows (OY-M) strain of aster yellows phytoplasma (Kakizawa et al., 2001) and used to raise an anti-SecA rabbit antibody against a purified par-tial SecA protein expressed in Escherichia coli. Light microscopy of thin sections of garland chrysanthemum (Chrysanthemum coro-narium) treated by immunohistochemical straining revealed that the SecA protein was present in phloem of OY-M-infected but not healthy host plants. In addition, antisera against both OY-M phy-toplasma SecA protein and GyrA protein of Acholeplasma laidlawii reacted with proteins of several unrelated phytoplasmas extracted from plant tissues (Koui et al., 2002; Wei et al., 2004a).

Phytoplasmas are the apparent etiological agents of diseases of at least 1000 plant species worldwide (McCoy et al., 1989; Seemüller et al., 1998). Although they can be transmitted from infected to healthy plants by scion or root grafts, most plant to plant spread occurs naturally via phloem-feeding insect vec-tors primarily of the family Cicadellidae (leafhoppers) and, less commonly, by planthoppers (Fulgoroidea) of the family Ciixidae and psyllids (Psylloidea) (D’Arcy and Nault, 1982; Tsai, 1979; Weintraub and Beanland, 2006). Phytoplasmas are transmit-ted in a circulative-propagative manner that typically involves a transmission latent period from 2 to 8 weeks (Carraro et al., 2001; Webb et al., 1999). The insect vector becomes infected upon ingesting phytoplasmas in phloem sap of infected plants. After an incubation period of one to several weeks, the phyto-plasma multiplies to high titer in the salivary glands and the insect becomes capable of infecting the phloem of the healthy plants on which it feeds (Kunkel, 1926; Lee et al., 1998a; Nasu et al., 1970). Generally, phytoplasma infection does not appear to significantly affect the activity, weight, longevity, or fecundity

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of vector insects (Garnier et al., 2001). Some phytoplasmas can be vectored by many species of leafhoppers (McCoy et al., 1989; Nielson, 1979) and different insect species may serve as vectors in different geographic regions. Several vectors also have the ability to transmit more than one type of phytoplasma, whereas other phytoplasmas are transmitted by one or a few vector spe-cies to a narrow range of plant species (Lee et al., 1998a). There is mounting evidence also for transovarial transmission of some phytoplasmas (Alma et al., 1997; Hanboonsong et al., 2002; Kawakita et al., 2000; Tedeschi et al., 2006).

Plants may serve as both natural and experimental hosts to several different phytoplasmas. Dual or mixed infections involv-ing related or unrelated phytoplasmas are known to occur naturally in plants and appear to be more common in peren-nial than annual plants (Bianco et al., 1993; Lee et al., 1995). Also, closely related phytoplasma strains are capable of induc-ing dissimilar symptoms on the same plant species (White et al., 1998), whereas similar symptoms on the same host plant may be induced by unrelated phytoplasmas (Harrison et al., 2003). The ability to accurately identify phytoplasmas by using DNA-based methods has shown that these organisms are more genetically diverse than was once thought (Davis and Sinclair, 1998). The geographic occurrence of phytoplasmas is determined largely by geographic ranges and feeding behavior (mono-, oligo-, or polyphagous) of the vector species, the relative susceptibility of the preferred host plant species, and the native host ranges of plant and insect hosts (Lee et al., 1998a). Phytoplasmas can be introduced into new geographic regions by long-distance dis-persal of infectious vectors (Lee et al., 2003) and by movement of infected plants or vegetative plant parts. Most recently, phy-toplasma DNA has been detected in embryos of aborted seed from diseased plants (Cordova et al., 2003; Nipah et al., 2007) and seed transmission of phytoplasmas infecting alfalfa (Medi-cago sativa L.) has been demonstrated (Khan et al., 2002).

An array of characteristic symptoms is associated with phy-toplasma infection of several hundred plant species world-wide. Symptoms vary according to the particular host species, stage of host infection and the associated phytoplasma strain (reviewed by Davis and Lee, 1992; Hogenhout et al., 2008; Kirkpatrick, 1989, 1992; Lee et al., 2000; McCoy et al., 1989; Seemüller et al., 2002; Sinclair et al., 1994). Some symptoms indicative of profound disturbances in the normal balance of growth regulators in plants include virescence (greening of pet-als), phyllody (conversion of floral organs into leafy structures), big bud, floral proliferation, sterility of flowers, proliferation of adventitious or axillary shoots, internode elongation and etiolation, generalized stunting (small flowers, leaves and fruits or shortened internodes), unseasonal discoloration of leaves or shoots (yellow to purple discoloration), leaf curling, cupping or crinkling, witches’-brooms (bunchy growth at stem apices), vein clearing, vein enlargement, phloem discoloration, and gen-eral plant decline such as die-back of twigs, branches and trunks (Lee and Davis, 1992; Lee et al., 2000; McCoy et al., 1989).

Infection of herbaceous host plants is followed by rapid intraphloemic spread of phytoplasma from leaves to roots, often accompanied by six-fold increases in phytoplasma populations in these tissues between 14 and 28 d post-inoculation (Kuske and Kirkpatrick, 1992a; Wei et al., 2004b). Phytoplasma con-centrations ranging from 2.2 × 108 to 1.5 × 109 cells per gram of tissue have been measured in high titer herbaceous hosts such

as periwinkle (Catharanthus roseus) and in certain woody peren-nial hosts such as alder (Alnus) and most poplar (Populus) spe-cies. Lowest phytoplasma concentrations, from 370 to 34,000 cells per gram of tissue, were detected in apple trees that were grafted on resistant rootstocks and in oak (Quercus robur) or hornbeam (Carpinus betulus) trees exhibiting nonspecific leaf yellowing symptoms (Berg and Seemüller, 1999).

Colonization is usually marked by phloem dysfunction and a reduction in photosynthetic capacity. Alterations in phloem function have been correlated with structural degeneration of sieve elements due possibly to physical blockage by colonizing phytoplasma or the action of a phytotoxin (Guthrie et al., 2001; Siddique et al., 1998). The onset of symptoms may be accom-panied by substantial impairment of the photosynthetic rate of mature leaves and by fluctuations in carbohydrate and amino acid levels in source versus sink leaves (Lepka et al., 1999; Tan and Whitlow, 2001). Leaf yellowing is associated with: decreases in chlorophyll content, carotenoids, and soluble proteins (Ber-tamini and Nedunchezhian, 2001); abnormal stomatal func-tion (Martinez et al., 2000); histopathological changes such the amount of total polyphenols; loss of cellular integrity (Musetti et al., 2000); fluctuations in hydrogen peroxide; peroxidase activity and glutathione content in diseased versus healthy plant tissues (Musetti et al., 2004); and increases in calcium (Ca2+) ions in cells (Musetti and Favali, 2003; Rudzinska-Langwald and Kaminska, 2003). Such adverse changes are accompanied by differential regulation of genes encoding proteins involved in floral development (Pracros et al., 2006), photosynthesis, sugar transport, and response to stress or in pathways of lipid and phenylpropanoid or phytosterol synthesis (Albertazzi et al., 2009; Carginale et al., 2004; Hren et al., 2009; Jagoueix- Eveillard et al., 2001).

The organisms degenerate and lose their cellular contents following treatment of infected plants with tetracycline antibi-otics (Kaminska and Sliwa, 2003; Sinha and Peterson, 1972). Tetracycline sensitivity and the lack of sensitivity to cell wall-inhibiting antibiotics such as penicillin (Davis and Whitcomb, 1970; Ishii et al., 1967) also support their inclusion in the Mol-licutes. Protective or therapeutic treatments with tetracycline antibiotics for phytoplasma disease control have been extended to a few high-value crop plants such as coconut for control of palm lethal yellowing, and to cherries and peaches for control of X-disease (McCoy, 1982; Nyland, 1971; Raju and Nyland, 1988). Administered by trunk injection, treatment of each tree with 1.0 g (protective dose) or 3.0 g (therapeutic dose) three times per year was sufficient for control of coconut lethal yel-lowing disease (McCoy, 1982).


Isolation of phytoplasma-enriched fractions from plant and insect host tissues is possible by differential centrifugation and filtration after first disrupting tissues in osmotically-augmented buffers (Kirkpatrick et al., 1995; Lee et al., 1988; Sinha, 1979; Thomas and Balasundaran, 2001). Further purification of phy-toplasmas is possible by centrifugation of enriched preparations in discontinuous Percoll density gradients (Davis et al., 1988; Gomez et al., 1996; Jiang and Chen, 1987) or by affinity chro-matography using phytoplasma-specific antibodies coupled to Protein A-Sepharose columns (Jiang et al., 1988; Seddas et al., 1995). Viability of these enriched preparations may be assessed

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by infectivity tests in which aliquots of the phytoplasma prepa-rations are micro-injected into vector insects, which are then fed on healthy indicator plants (Nasu et al., 1974; Sinha, 1979; Whitcomb et al., 1966a, b). Separation of enriched phytoplasma DNA from mixtures with host DNA is also possible by use of cesium chloride-bisbenzimide buoyant density gradient centrif-ugation (Kollar and Seemüller, 1989). Present as an uppermost band in final gradients, phytoplasma DNA fractionated by this means was suitable for endonuclease digestion and cloning for DNA probe development (Harrison et al., 1992, 1991; Kollar and Seemüller, 1990).


Viable phytoplasmas have been maintained for at least 6 years in intact vector insects frozen at −70°C (Chiykowski, 1983). Viable X-disease phytoplasmas have been maintained for 2 weeks in salivary glands suspended in a tissue culture medium (Nasu et al., 1974). Extracts of phytoplasma-infected insects prepared in a MgCl2/glycine buffer, osmotically adjusted to 800 milliosmoles/kg with sucrose, retained their infectivity for up to 3 d (Smith et al., 1981). Phytoplasma strains have been routinely maintained in diseased plants kept in an insect-proof greenhouse or in plantlets grown in tissue culture (Bertaccini et al., 1992; Davis and Lee, 1992; Jarausch et al., 1996; Sears and Klomparens, 1989; Wongkaew and Fletcher, 2004). While plant to plant transmission is accomplished naturally by vector insects and, in some cases, through grafts, experimental trans-missions commonly include the use of plant parasitic dodders (Cuscuta sp.) (Marcone et al., 1999a). Although phytoplasma strains are commonly maintained in plants by periodic graft inoculation, maintenance of phytoplasmas exclusively in plants can result in strain attenuation over time and an associated loss of transmissibility by vector insects (Chiykowski, 1988; Denes and Sinha, 1992).

differentiation of the genus “Candidatus Phytoplasma” from other genera

Phytoplasma-specific nucleic acid probes and PCR technol-ogy have largely supplanted traditional methods of electron microscopy and biological criteria for sensitive detection, iden-tification, and genetic characterization of phytoplasmas. Molec-ular-based analyses have shown phytoplasma genomes to be A+T rich (Kollar and Seemuller, 1989; Oshima et al., 2004) and to range from 530 to 1350 kbp in size (Marcone et al., 1999b, 2001; Neimark and Kirkpatrick, 1993). Before any phytoplasma genomes were sequenced, phytoplasmas were shown to con-tain two rRNA operons (Davis, 2003a; Harrison et al., 2002; Ho et al., 2001; Jomantiene et al., 2002; Jung et al., 2003a; Lee et al., 1998b; Liefting et al., 1996; Marcone et al., 2000; Schneider and Seemüller, 1994a). Other genes that have been identified include ribosomal protein genes (Gundersen et al., 1994; Lee et al., 1998b; Lim and Sears, 1992; Martini et al., 2007; Miyata et al., 2002a; Toth et al., 1994) of the S10-spc operon (Miyata et al., 2002a), a nitroreductase gene (Jarausch et al., 1994), DNA gyrase genes (Chuang and Lin, 2000), genes encoding elongation factors G and Tu (An et al., 2006; Berg and Seemül-ler, 1999; Koui et al., 2003; Marcone et al., 2000; Miyata et al., 2002b; Schneider et al., 1997), secA, secY, and secE genes of a functional Sec protein translocation system (Kakizawa et al., 2001, 2004), gidA, potB, potC, and potD (Mounsey et al., 2006),

a gene encoding an RNase P ribozyme (Wagner et al., 2001), recA (Chu et al., 2006), rpoC (Lin et al. 2006), polC (Chi and Lin., 2005), and insertion sequence (IS)-like elements (Lee et al., 2005). Numerous other putative genes or pseudogenes have been identified recently after partially or fully sequencing random fragments of genomic DNA cloned from phytoplasmas by various methods (Bai et al., 2004; Cimerman et al., 2006, 2009; Davis et al., 2003b, 2005; Garcia-Chapa et al., 2004; Lieft-ing and Kirkpatrick, 2003; Melamed et al., 2003; Miyata et al., 2003; Streten and Gibb, 2003).

Development of phytoplasma-specific rRNA gene primers has permitted PCR-mediated amplification of various regions of the rRNA operons (Ahrens and Seemüller, 1992; Baric and Dalla-Via, 2004; Davis and Lee, 1993; Deng and Hiruki, 1991; Gundersen and Lee, 1996; Lee et al., 1993b) (Namba et al., 1993; Smart et al., 1996). RFLP analysis of PCR-amplified rDNA provided a practical solution to the problem of phytoplasma identification and classification (Lee et al., 2000, 1998b). Pair-wise comparisons of disparate strains were marked by consid-erable differences in RFLP patterns, whereas strains that were considered closely related on the basis of similar biological properties were often, although not always, indistinguishable on the basis of RFLP patterns. Alternatively, heteroduplex mobility analysis has demonstrated greater sensitivity than RFLP analy-sis for detecting minor variability in 16S rRNA genes of closely related phytoplasma strains (Cousin et al., 1998; Wang and Hiruki, 2000), since RFLP analysis is limited to detection of rec-ognition sites for restriction endonucleases. Cluster analysis of rDNA RFLP patterns provided the first means to differentiate between known and unknown phytoplasmas from a wide range of plant hosts and geographic locations, and to resolve phyto-plasmas into well-defined phylogenetic groups and subgroups (Ahrens and Seemüller, 1992; Lee et al., 1993b; Schneider et al., 1993, 1995).

taxonomic comments

The inability to cultivate phytoplasmas outside of their plant and insect hosts has thus far rendered traditional methods impractical as aids for taxonomy of these organisms. Unlike their culturable Mollicute relatives, which were originally clas-sified based only upon biological and phenotypic properties in pure culture, phytoplasmas cannot be classified by these criteria. Through application of DNA-based methods, it is now possible to accurately identify and characterize phytoplasmas and to assess their genetic interrelationships. These capabilities have assisted development of classification systems, first based on hybridization data, later based on 16S rDNA RFLPs, and ulti-mately on phylogenetic analysis of 16S rRNA genes and other conserved gene sequences. Classification schemes founded upon these molecular criteria have been refined and expanded upon over time, with the goal of defining a taxonomy for these unique organisms. In a phytoplasma classification scheme pro-posed by Lee et al. (1993b), based on analyses of rDNA RFLPs, a total of nine primary 16S rDNA groups (termed 16Sr groups) and 14 subgroups were initially recognized. Phytoplasma groups delineated by these analyses were consistent with genomic strain clusters previously identified by DNA hybridization analy-sis (Lee et al., 1992), although a greater diversity among strains comprising group 16SrI (aster yellows and related strains) was indicated by the earlier hybridization data. Subgroups within a

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given 16Sr group were distinguished by the presence of one or more restriction sites in a phytoplasma strain that differed from those in all existing members of a given subgroup. For those strains in which intra-rRNA operon heterogeneity was detected, subgroup designations were assigned according to the com-bined patterns of both 16S rRNA genes.

RFLP analysis of more variable ribosomal protein genes (Gundersen et al., 1994; Lee et al., 2004a, b) or tuf genes (Marcone et al., 2000; Schneider et al., 1997) has provided a means for more detailed subdivision of phytoplasma primary groups delineated by 16S rDNA RFLP data. This strategy for finer subgroup differentiation has been used to modify and expand upon earlier classifications and to incorporate many newly identified phytoplasma strains. Based on RFLP analy-sis of nearly full-length 16S rRNAs, at least 15 primary 16Sr groups have been recognized (Lee et al., 1998b; Montano et al., 2001): 16SrI, Aster yellows; 16SrII, Peanut witches’-broom; 16SrIII, X-disease; 16SrIV, Coconut lethal yellows; 16SrV, Elm yellows; 16SrVI, Clover proliferation; 16SrVII, Ash yellows; 16SrVIII, Loofah witches’-broom; 16SrIX, Pigeonpea witches’-broom; 16SrX, Apple proliferation; 16SrXI, Rice yellow dwarf; 16SrXII, Stolbur; 16SrXIII, Mexican periwinkle virescence; 16SrXIV, Bermuda grass white leaf; and 16SrXV Hibiscus witches’-broom. A total of 45 subgroups were identified when ribosomal protein gene RFLP data was also considered in the analyses.

Sequencing of 30 nearly full-length amplified 16S rRNA genes was undertaken by Namba et al. (1993), Gundersen et al. (1994), and Seemüller et al. (1994) from a diversity of strains previously characterized by rDNA RFLP analysis. These collective efforts, combined with earlier studies (Kuske and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first comprehensive phytoplasma phylogeny. In recognition of their unique phylogenetic status, the trivial name “phytoplasma” was initially proposed (Sears and Kirkpatrick, 1994) and has since been adopted formally (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004) to col-lectively name these fastidious, phytopathogenic mollicutes previously known as mycoplasma-like organisms. Within the phytoplasma clade, major subclades (primary groups repre-senting “Candidatus” species) include: (1) Stolbur; (2) Aster yellows; (3) Apple proliferation; (4) Coconut lethal yellowing; (5) Pigeonpea witches’-broom; (6) X-disease; (7) Rice yellow dwarf; (8) Elm yellows; (9) Ash yellows; (10) Sunnhemp witch-es’-broom; (11) Loofah witches’-broom; (12) Clover prolifera-tion; and (13) Peanut witches’-broom (Kirkpatrick et al., 1995; Schneider et al., 1995; White et al., 1998). Primary phytoplasma groups including 19 novel groups, namely Australian grapevine yellows (AUSGY), Italian bindweed stolbur (IBS), Buckthorn witches’-broom (BWB), Spartium witches’-broom (SpaWB), Galactia little leaf (GaLL), Vigna little leaf (ViLL), Clover yellow edge (CYE), Hibiscus witches’-broom (HibWB), Pear decline (PD), European stone fruit yellows (ESFY), Japanese hydran-gea phyllody (JHP), Psammotettix cephalotes-borne (BVK), Italian alfalfa witches’-broom (IAWB), Cirsium phyllody (CirP), Bermuda grass white leaf (BGWL), Sugarcane white leaf (SCWL), Tanzanian lethal decline (TLD), Stylosanthes little leaf (StLL), and Pinus sylvestris yellows (PinP), that were absent from previous classification schemes have been subsequently defined. These new taxonomic entities were delineated on the basis of phylogenetic tree branching patterns, differences in 16S rRNA

gene sequence similarities that were 1.2–2.3% or greater and, in some instances, by additional considerations such as plant host and vector specificity, primer specificity, and RFLP com-parisons of ribosomal and nonribosomal DNA, as well as sero-logical comparisons (Seemüller et al., 2002, 1998).

Most recently, Wei et al. (2007) applied computer-simulated RFLP analysis for classification of phytoplasma strains. Through comparisons of virtual RFLP patterns of 16S rRNA genes and calculations of coefficients of RFLP similarity, the authors classified all available 16S rRNA gene sequences, includ-ing sequences from 250 previously unclassified phytoplasma strains, into a total of 28 16Sr RFLP groups. These included ten new groups and dozens of new subgroup lineages (Cai et al., 2008; Wei et al., 2008b). Each new group represents a potential “Candidatus Phytoplasma” species level taxon. This informa-tion was used to augment the 16Sr RFLP classification system (Lee et al., 2000, 1998b, 1993b) with the following additional groups: 16SrXVI, Sugarcane yellow leaf syndrome; 16SrXVII, Papaya bunchy top group; 16SrXVIII, American potato purple top wilt group; 16SrXIX, Japanese chestnut witches’-broom group; 16SrXX, Buckthorn witches’-broom group; 16SrXXI, Pine shoot proliferation group; 16SrXXI, Nigerian coconut lethal decline (LDN) group; 16SrXXIII, Buckland valley grape-vine yellows group; 16SrXXIV, Sorghum bunchy shoot group; 16SrXXV, Weeping tea witches’-broom group; 16SrXXVI, Mau-ritius sugarcane yellow D3T1 group; 16SrXXVII, Mauritius sug-arcane yellow D3T2 group; and 16SrXXVIII, Havana derbid phytoplasma group. The virtual RFLP patterns are available for online use as reference patterns at http://www.ba.ars.usda.gov/data/mppl/virtualgel.html.

The spacer region (SR) separating the 16S from the 23S rRNA gene of phytoplasmas was also shown to be a reliable phylogenetic marker. Phylogenetic trees derived from the entire 16S–23S SR (Gibb et al., 1998; Kenyon et al., 1998) or variable regions flanking the tRNAile gene (Kirkpatrick et al., 1995; Schneider et al., 1995) differentiated phytoplasmas into groups that were concordant with the major groups established previously from analyses of 16S rRNA genes. Phytoplasmas col-lectively differ in their 16S rRNA gene sequence by no more than 14%, whereas their respective 16S–23S SR sequences dif-fer by as much as 22%. This added variation has contributed to improved accuracy of phytoplasma classification at the sub-group level. Similarly, phylogenetic analysis of ribosomal pro-tein genes, secY, secA, or 23S rRNA genes has been employed to differentiate closely related phytoplasma strains, as well as to aid the group and subgroup classification of diverse phytoplas-mas (Daire, 1993; Hodgetts et al., 2008; Lee et al., 1998b, 2004a, 2006b; Martini et al., 2007; Reinert, 1999). Such studies have led to finer differentiation among phytoplasma subgroups and to enriched descriptions of “Candidatus Phytoplasma” species (Lee et al., 2004a, 2006a, b).

A polyphasic system for taxonomy based on integration of genotypic, phenotypic, and phylogenetic information employed for bacterial classification (Murray et al., 1990; Stackebrandt and Goebel, 1994) has proved problematic for nonculturable phytoplasmas. In response to a rapidly growing database of phylogenetic markers, even in the absence of species-defining biological or phenotypic characters, the Working Team on Phy-toplasmas of the International Research Programme of Com-parative Mycoplasmology (IRPCM Phytoplasma/Spiroplasma

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http://www.ba.ars.usda.gov/data/mppl/virtualgel.html

http://www.ba.ars.usda.gov/data/mppl/virtualgel.html


Working Team – Phytoplasma Taxonomy Group, 2004) pro-posed that taxonomy of phytoplasmas be based primarily upon phylogenetic analyses. This proposal was agreed to and adopted as policy by the ICSB Subcommittee on the Taxonomy of Mollicutes (1993, 1997), which also recommended that the pro-visional taxonomic status of “Candidatus”, originally proposed by Murray and Schleifer (1994), be used for assigning genera names as follows: “Candidatus Phytoplasma” (from phytos, Greek for plant; plasma, Greek for thing molded) [(Mollicutes) NC; NA; O; NAS (GenBank no. M30790); oligonucleotide sequence of unique region of the 16S rRNA gene is CAA GAY BAT KAT GTK TAG CYG GDC T; P (Plant, phloem; Insect, salivary gland); M]. (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). By this same approach, major groups within the genus also delineated by phylogenetic analysis of near full-length 16S rRNA gene sequences were con-sidered to represent one or more distinct species.

Current guidelines for “Candidatus Phytoplasma” species descriptions (Anonymous, 2000; Firrao et al., 2005; IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Tax-onomy Group, 2004) are based upon identification of a signifi-cantly unique 16S rRNA gene sequence >1200 bp in length. The strain from which the sequence is obtained should be designated as the reference strain. Strains with minimal differ-ences in the 16S rRNA sequence, relative to the reference strain, should be referred to as related strains. In general, a strain can be described as a new “Candidatus Phytoplasma” species if its 16S rRNA gene sequence has less than 97% identity to any pre-viously described “Candidatus Phytoplasma” species (ICSB Sub-committee on the Taxonomy of Mollicutes, 2001). There are cases in which phytoplasmas may share more than 97% of their 16S rRNA gene sequence, but clearly represent ecologically distinct populations and, thus, they may warrant description as

separate species. In such cases, the description of two different species is recommended when all of the following conditions apply: (1) the two phytoplasmas are transmitted by different vector species; (2) the two phytoplasmas have a different natu-ral plant host, or at least their symptomatology is significantly different in the same plant host; (3) there is evidence of signifi-cant molecular diversity between phytoplasmas as determined by DNA hybridization assays with cloned nonribosomal DNA markers, serological reactions, or by PCR-based assays. The taxonomic rank of subspecies should not be used. Reference strains should be available to the scientific community in graft-inoculated or in vitro micropropagated host plants or as DNA if perpetuation of strains in infected host plants is not feasible. Descriptions of “Candidatus Phytoplasma” species should be preferably submitted to the International Journal of Systematic and Evolutionary Microbiology (http://ijs. sgmjournals.org/).

Recent phylogenetic investigations, including the pres-ent analyses (Figure 116), suggest 97.5% 16S rRNA gene sequence similarity may represent a more suitable upper threshold for “Candidatus Phytoplasma” species separation, in that taxonomic subgroups designated based on 16S rRNA gene sequence similarities of £97.5% more consistently define species that are phylogenetically distinct from nearest related species. Regardless of homology criteria, a taxonomy is emerg-ing for the phytoplasmas in the absence of cultivability where species and related strains of a species are clearly recognized with due consideration of the genetic, ecological, and environ-mental constraints unique to this group of plant- and insect-associated Mollicutes. To a large extent, the present taxonomy employs vernacular names based on associated diseases, but is constantly shifting towards a traditional taxonomy as more and more “Candidatus Phytoplasma” species continue to be recognized and proposed.

list of species of the genus “Candidatus Phytoplasma”

In accordance with the current guidelines for “Candidatus Phytoplasma” species descriptions, the following species have been designated. Proposed assignments to the class Mollicutes are based on nucleic acid sequences. None of these species have been cultivated independently of their host, and their metabo-lism and growth temperatures are unknown.

1. “Candidatus Phytoplasma allocasuarinae” Marcone, Gibb, Streten and Schneider 2004a, 1028

Vernacular epithet: Allocasuarina yellows phytoplasma, strain AlloYR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AY135523.Unique region of 16S rRNA gene: 5¢-TTTATTCGAGAG-

GGCG-3¢.Habitat, association, or host: phloem of Allocasuarina muel-

leriana (Slaty she-oak).

2. “Candidatus Phytoplasma americanum” Lee, Bottner, Secor and Rivera-Varas 2006a, 1596

Vernacular epithet: Potato purple top, strain APPTW12-NER.

Gram reaction: not applicable.

Morphology: other.Sequence accession no. (16S rRNA gene): DQ174122.Unique regions of 16S rRNA gene: 5¢-GTTTCTTCGGAAA-3¢

(68–80), 5¢-GTTAGAAATGACT-3¢ (142–153), 5¢-GCTGGT-GGCTT-3¢ (1438–1448).

Habitat, association, or host: Solanum tuberosum phloem.

3. “Candidatus Phytoplasma asteris” Lee, Gundersen-Rindal, Davis, Bottner, Marcone and Seemüller 2004a, 1046

Vernacular epithet: Aster yellows (AY) phytoplasma, strain OAY R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): M30790.Unique regions of 16S rRNA gene: 5¢-GGGAGGA-3¢,

5¢-CTGACGGTACC-3¢, and 5¢-CACAGTGGAGGTTAT-CAGTTG-3¢.

Habitat, association, or host: phloem of Oenothera hookeri (Evening primrose).

4. “Candidatus Phytoplasma aurantifolia” Zreik, Carle, Bové and Garnier 1995, 452

Vernacular epithet: Witches’-broom disease of lime phyto-plasma, strain WBDLR.

703

http://ijs.sgmjournals.org/


FIGURE 116. Phylogenetic analysis of the phytoplasmas. Phylogenetic trees were constructed by parsimony analy-ses of phytoplasma 16S rRNA gene sequences using the computer program PAUP (Swofford, 1998). The closely related culturable Acholeplasma palmae was employed as the outgroup. Because phytoplasma taxa are too numerous to present in a single inclusive tree, a global phylogeny of representative phytoplasmas is first presented. The global tree is divided into lower (a) and upper (b) regions. Each region of the global phylogeny is then expanded into inclusive trees, a and b, which collectively include 145 phytoplasmas from diverse geographic origins. Taxonomic subgroups, representing phytoplasmas sharing at least 97.5% 16S rRNA gene sequence similarity, are identified on each inclusive tree. Each phylogenetically distinct subgroup is equivalent to a subclade (or putative species) within the genus “Candidatus Phytoplasma”. In all trees, branch lengths are proportional to the number of inferred char-acter state transformations. Bootstrap (confidence) values greater than or equal to 50 are shown on the branches. Phytoplasmas for which 16S rRNA gene sequences of at least 1200 bp in length have been determined (312 total) are listed by subgroup in Table 144 along with their sequence accession numbers.

P.trifoli (CP)

P. fraxini (AshY1)P. ulmi (EY1)

P. ziziphi (JWB-G1)

LfWBStLL

LDGLDT

LY-c2

SBSP. castaneae (CnWB)

P. pini (PinP)

CIRPGaLL

BVK

P. oryzae (RYD)SCWL

P. cynodontis (BGWL)P. phoenecium (AlmWB-A4)

ViLL

WXGLL-eth

P. brasiliense (HibWB)

IAWBSPLL

P. australasia (PpYC)P. aurantifolia (WBDL)

WTWB

P. mali (AP15)P. prunorum (ESFY-G1)

P. pyri (PD1)

P. spartii (SPAR)P. allocasuarinae (AlloY)

P. rhamni (BWB)

STOLP. australiense (AusGY)

P. japonicum (JHP)IBS

P. asteris (MIAY)

MPVA. palmae

A. laidlawii

10 changes

100

100

59

91

98

98

100

100

100

100

72

100

77

79

100

56

88 b

a

93

704


Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): U15442.Unique region of 16S rRNA gene: 5¢-GCAAGTGGTGAAC-

CATTTGTTT-3¢.Habitat, association, or host: phloem of Citrus; hemolymph

and salivary glands of Hishimonus phycitis (Cicadellidae).

5. “Candidatus Phytoplasma australasia” White, Blackall, Scott and Walsh 1998, 949

Vernacular epithet: Papaya yellow crinkle phytoplasma, strain PpYCR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): Y10097.Unique regions of 16S rRNA gene: 5¢-TAAAAGGCATCTTT-

TATC-3¢ and 5¢-CAAGGAAGAAAAGCAAATGGCGAAC-CATTTGTTT-3¢.

Habitat, association, or host: phloem of Carica papaya and Lycopersicon esculentum.

6. “Candidatus Phytoplasma australiense” Davis, Dally, Gun-dersen, Lee and Habili 1997, 268

Vernacular epithet: Australian grapevine yellows phyto-plasma, strain AUSGY

R.Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): L76865.Unique regions of 16S rRNA gene: 5¢-CGGTAGAAATAT-

CGT-3¢ and 5¢-TTTATCTTTAAAAGACCTCGCAAGA-3¢.Habitat, association, or host: Vitis phloem.

7. “Candidatus Phytoplasma brasiliense” Montano, Davis, Dally, Hogenhout, Pimentel and Brioso 2001, 1117

Vernacular epithet: Hibiscus witches’-broom (HibWB) phytoplasma, strain HibWB26R.

FIGURE 116. (Continued)

A. palmae

5 changes

SUNHPSPWB

PnWBAlWBGPhP. australasia (PpYC)

CoAWBIAWB

PEPSPLL

GLL-ethP. brasiliense (HibWB)

CaMCaWB1FBP

P. aurantifolia (WBDL)BoLL

WTWBP. mali (AP15)AP1/93AP2AT

P. pyri (PD1)PDPPER

P. prunorum (ESFY-G1)ESFY-142ESFY4ESFY5

P. spartii (Spar)P. allocasuarinae (AlloY)

P. rhamni (BWB)P. asteris (MIAY)

PPTMBS

PaWBMD

Bstv2M.f12HyPHAPWBIOWBPRIVCRPh

ApSLAAY

SAYValYWcWBOY-MCabD3

AY-WBBB

HYDPCPh

STRAWB2BBS

ACLRCWLSYPY

VKSTOL

STOL2PpDB

PYLSLYSV3101P. australiense (AusGY)

P. japonicum (JHP)IBS

MPVSTRAWB1

CbY1

52

100

100

51

95

100

100

100

100

82

100

100

65

80

100

87

84

P. australiense

P. australasia

P. aurantifolia

GLL-ethP. brasiliense

IAWBSPLL

WTWB

P. spartii

P. rhamni

P. asteris

STOL

IBSP. japonicum

MPV

P. mali

P. pyri

P. allocasuarinae

P. prunorum

BoLL

a

705


Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AF147708.Unique regions of 16S rRNA gene: 5¢-GAAAAAGAAAG-3¢,

5¢-TCTTTCTTT-3¢, 5¢-CAG-3¢, 5¢-ACTTTG-3¢, and 5¢-GTCA AAAC-3¢.

Habitat, association, or host: Hibiscus phloem.

8. “Candidatus Phytoplasma caricae” Arocha, López, Piñol, Fernández, Picornell, Almeida, Palenzuela, Wilson and Jones 2005, 2462

Vernacular epithet: Cuban papaya phytoplasma, strain PAYR.Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AY725234.

LfWBLfWB-t

StLLCY

LY-c2PanD

ScYLDYLfY1

LDGLDN

LDTSBS

SGS-v1SCWL

BVKCIRP

GaLLP. oryzae (RYD-J)RYD-Th

BGWL-2CWLP. cynodontis (BGWL-C1)

BGWLP. casteneae (CnWB)

P. pini (Pin127)PinG

KAPPPWB-f

P. phoenecium (AlmWB-A4)ViLL

BBPTWBVAC

CYE-CDanVir-a

GDIIICbY18

WWB-aBLWBPoiBI

VGYIIICXScYPI-AfrWX

LPA. palmae

5 changes

ALYFD

HD1SpaWB229

VCRuS

P. ulmi (EY1)ULW

JWB-chP. ziziphi (JWB-G1)CLY-5

NecY-In1JWB-Ka

P. trifolii (CP)PWB

BLL-bdBLLCSV

BLTVAVR

ArAWBEriWB

P. fraxini (AshY1)ASHYLWB3

AshY3

100

100

97

51100

88

100

b

88

100

100

86

100

100

72

81

56

WX

P. trifolii

P. ulmi

LfWB

LY

SBS

CIRPBVK

SCWL

P. fraxini

P. ziziphi

StLL

LDG

LDT

GaLL

P. casteneae

ViLL

P. oryzae

P. phoenecium

P. pini

P. cynodontis

EriWB

FIGURE 116. (Continued)

706


TablE 144. Provisional groupings, strain designations, associated plant disease, geographic origin and accession numbers of 16S rRNA gene sequences derived from phytoplasmasa

Subgroup Strain Associated plant disease“Candidatus

Phytoplasma species” Geographic origin 16S accession no.a

AlloY AlloY Allocasuarina yellows P. allocasuarinae Australia AY135523*AP AP15R Apple proliferation P. mali Italy AJ542541* AT Apple proliferation Germany X68375* AP2 Apple proliferation Germany AF248958* AP1/93 Apple proliferation France AJ542542* D365/04 Apple proliferation Slovenia EF025917 APSb Apple proliferation Italy EF193361ESFY ESFY-G1R European stone fruit yellows P. prunorum Germany AJ542544* ESFY5 European stone fruit yellows Austria AY029540* ESFY4 European stone fruit yellows Czech Republic Y11933* PPER European stone fruit yellows Germany X68374 ESFY-142 European stone fruit yellows Spain AJ575108* ESFY-173 European stone fruit yellows Spain AJ575106 ESFY-215 European stone fruit yellows Spain AJ575105AshY AshY1 Ash yellows P. fraxini USA, New York AF092209* AshY3 Ash yellows USA, Utah AF105315* ASHY Ash yellows Germany X68339* LWB3 Lilac witches’-broom USA, Massachusetts AF105317*EriWB EriWB Erigeron witches’-broom Brazil AY034608 ArAWB Argentinian alfalfa witches’-

broom Argentina AY147038

AusGY AusGY Australian grapevine yellows P. australiense Australia L76865 PpDB Papaya die-back Australia Y10095* PYL Phormium yellow leaf, rrnA New Zealand U43569 PYLb Phormium yellow leaf New Zealand U43570* SLY Strawberry lethal yellows Australia AJ243045* SV3101 Strawberry virescence Tonga AY377868*AY MIAY Oenothera virescence P. asteris USA, Michigan M30790* OY-M Onion yellows Japan NC005303* MBS Maize bushy stunt Mexico AY265208* APWB Aphanamixis polystachya

witches’-broom Bangladesh AY495702*

IOWB Ipomoea obscura witches’-broom

Taiwan AY265205*

HyPH Hydrangea phyllody Italy AY265207* HYPh Hydrangea phyllody France AY265219 RPh Oilseed rape phyllody Czech Republic U89378* MD Mulberry dwarf South Korea AY075038* GDS DQ112021 AYWB_ro4 Aster yellows witches’-

broom Ohio, USA NC007716

PaWB Paulownia witches’-broom Korea AF279271* PY1 Periwinkle yellows China AF453328 BVGY AY083605 AAY American aster yellows Southern USA X68373* CabD4 Cabbage proliferation USA, Texas AY180932 AY-BW Aster yellows USA, Ohio AY389820 ApSL Apple sessile leaf Lithuania AY734454 SAY Severe aster yellows USA, California M86340* ValY Valeriana yellows, rrnA Lithuania AY102274* PRIVC Primrose virescence Germany AY265210* WcWB Watercress witches’-broom USA, Hawaii AY665676* CabD3 Cabbage proliferation USA, Texas AY180947* AY-sb Sugar beet aster yellows Hungary AF245439 Bstv2Mf12 AY180951 ACLR-AY Apricot chlorotic leafroll Spain AY265211 ACLR Apricot chlorotic leafroll Europe X68338* BBS3 Blueberry stunt USA, Michigan AY265213 STRAWB2 Strawberry green petal USA, Florida U96616* PoY Populus yellows Croatia AF503568 KVG Clover phyllody Germany X83870

(continued)

707




CPh Clover phyllody Canada AF222066* HYDP Hydrangea phyllody Belgium AY265215* AY-WB Aster yellows USA, Ohio AY389827* BB Tomato big bud USA, Arkansas AY180955* PPT Potato purple top Mexico AF217247*THP THP Tomato ‘hoja de perejil’ P. lycopersici Bolivia AY787136 Derbid Derbid phytoplasma Cuba AY744945BWB BWB Buckthorn witches’-broom P. rhamni Germany X76431*BGWL BGWL-C1 Bermudagrass white leaf P. cynodontis Italy AJ550984* BGWL Bermudagrass white leaf Italy Y16388* BGWL-2 Bermudagrass white leaf Thailand AF248961* CWL Cynodon white leaf Australia AF509321*BVK BVK Psammotettic cephalotes-

borne Germany X76429*

CIRP CIRP Cirsium phyllody Germany X83438*CnWB CnWB Chestnut witches’- broom P. castaneae Korea AB054986*CP CPR Clover proliferation P. trifolii Canada AY390261* BLL Brinjal little leaf India X83431* BLTVA Columbia basin potato

purple top USA, Washington AY692280*

VR Vinca virescence USA, California AY500817* PWB Potato witches’-broom Canada AY500818* CSV Centauria stolstitialis

virescence Italy AY270156*

EY EY1 Elm yellows P. ulmi USA, New York AY197655* ULW Ulmus witches’-broom Italy X68376* FD Flavescence doree Italy X76560* RuS Rubus stunt Italy AY197648* HD1 Hemp dogbane yellows USA, New York AY197654* VC Asymptomatic Virginia

creeper USA, Florida AF305198*

SpaWB SpaWB229 Spartium witches’-broom P. spartii Italy AY197652*JWB JWB-G1T Jujube witches’-broom Gifu

isolate 1P. ziziphi Japan AB052876*

JWB-Ka Jujube witches’-broom Korea isolate 1

Korea AB052879*

JWB-ch Ziziphus jujube witches’-broom

China AF305240

NecY-In1 Nectarine yellows India AY332659* CLY-5 Cherry lethal yellows China AY197659*FBP WBDL Witches’-broom disease of

limeP. aurantifolia Oman U15442*

CaWB-YNO1 Cactus witches’-broom China AJ293216 FBP Faba bean phyllody Sudan X83432* PPLL Pigeon pea little leaf Australia AJ289191BoLL BoLL Bonamia little leaf Australia Y15863*GaLL GaLL Galactia little leaf Australia Y15865*GLL-eth GLL-eth Gliricidia little leaf Ethiopia AF361018*HibWB HibWB Hibiscus witches’-broom P. brasiliense Brazil AF147708*IAWB IAWB Alfalfa witches’-broom Italy Y16390* PEP Pichris echioides phyllody Italy Y16393*IBS IBS Italian bindweed yellows Southern Italy Y16391*StrawY StrawY Strawberry lethal yellows P. fragariae Lithuania DQ086423JHP JHP Japanese hydrangea

phyllodyP. japonicum Japan AB010425

LDG LDG Cape St Paul wilt Ghana Y13912* LDN Awka disease of coconut Nigeria Y14175*LDT LDT Coconut lethal disease Tanzania X80177*LfWB LfWB Loofah WB Taiwan L33764* LfWB-t Loofah WB Taiwan AF086621*LY CPY Carludovica palmata yellows Mexico AF237615 LDY Yucatan coconut decline Mexico U18753*

(continued)

TablE 144. (continued)

708




LfY1 Coconut leaf yellowing Mexico AF500329* LfY5(PE65) Coconut leaf yellowing Mexico AF500334 LY-c2 Coconut lethal yellows USA, Florida AF498309* LY-JC8 Coconut lethal yellows Jamaica AF498307 PanD Pandanus decline USA, Florida AF361020* ScY Sugarcane yellows, group 4 Mauritius AJ539178*ScY SCD3T2 Sugarcane yellows, group 3 Mauritius AJ539180 SCD3T1 Sugarcane yellows, group 3 Mauritius AJ539179MPV MPV Mexican periwinkle

virescence Mexico AF248960*

PerWB-FL Periwinkle witches’-broom USA, Florida AY204549 CbY1 Chinaberry yellows Bolivia AF495882* STRAWB1 Strawberry green petal USA, Florida U96614*PD PD1 Pear decline P. pyri Italy AJ542543* PD Pear decline Germany X76425* PYLR Peach yellow leafroll USA, California Y16394 EPC Pear decline Iran DQ471321PinP Pin127R Pinus halepensis yellows P. pini Spain AJ632155* PinG Pinus sylvestris yellows Germany AJ310849*PPWB AlmWB-A4 Almond witches’-broom P. phoenecium Lebanon AF515636* KAP Knautia arvensis phyllody Italy Y18052* PPWB-f Pigeonpea witches’-broom USA, Florida AF248957*RYD RYD-J Rice yellow dwarf P. oryzae Japan D12581 RYD-Th Rice yellow dwarf Thailand AB052873*SBS SBS Sorghum bunchy shoot Australia AF509322*SCWL SCWL Sugarcane white leaf Thailand X76432* SGS-v1 Sorghum grassy shoot,

variant 1 Australia AF509324*

SpaWB Spar Spartium witches’-broom P. spartii Italy X92869*SPLL SPLL Sweet potato little leaf Australia X90591*SPWB PpYC Papaya yellow crinkle P. australasia Australia Y10097* GPh Gerbera phyllody Japan? AB026155* PnWB Peanut witches’ broom Taiwan L33765* CoAWB Cocky apple witches’-broom Australia AJ295330* SPLL Sweet potato little leaf Australia AJ289193 SUNHP Sunnhemp phyllody Thailand X76433* AlWB Alfalfa witches’-broom Oman AY169322* TBB Australian tomato big bud Australia Y08173StLL StLL Stylosanthes little leaf Australia AJ289192*STOL STOL Stolbur of Capsicum annum Europe X76427* VK Grapevine yellows Europe X76428* 2642BN Grapevine yellows P. solani France AJ964960ViLL ViLL Vigna little leaf Australia Y15866*CIWB IM-3 Cassia italica witches’-broom P. omaniense Oman EF666051WTWB WTWB Weeping tea witches’-broom Australia AF521672*WX BBP Blueberry proliferation Lithuania AY034090* BLWB Black locust witches’-broom USA, Maryland AF244363* CbY18 Chinaberry yellows Bolivia AF495657* CX Canadian peach X Canada L33733* CYE Clover yellow edge Canada AF175304* DanVir-a Dandelion virescence, rrnA Lithuania AF370119* LP Little peach USA, S. Carolina AF236122* PoiBI Poinsettia branch-inducing Southern USA AF190223* ScYP I-Afr Sugarcane yellows Africa AF056095* TWB Tsuwabuki WB Japan D12580* VAC Vaccinium witches’-broom Germany X76430* VGYIII Virginia grapevine yellows USA, Virginia AF060875* WWB-a Walnut witches’-broom,

rrnA USA, Georgia AF190226*

WX Western X USA, California L04682*

aAccession numbers denoted by an asterisk were used as sources of 16S rRNA gene sequences for comprehensive phylogenetic analysis of subgroup phytoplasmas from diverse geographic origins.

TablE 144. (continued)

709


Unique regions of 16S rRNA gene: 5¢-AAA-3¢ (196–198), 5¢-ATT-3¢ (600–603), 5¢-AGGCGCC-3¢ (1089–1095), 5¢-GCG-GATTTAGTCACTTTTCAGGC-3¢ (1379–1401).

Habitat, association, or host: Carica papaya phloem.

9. “Candidatus Phytoplasma castaneae” Jung, Sawayanagi, Kakizawa, Nishigawa, Miyata, Oshima, Ugaki, Lee, Hibi and Namba 2002, 1548

Vernacular epithet: Chestnut witches’ broom phytoplasma, strain CnWBR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AB054986.Unique regions of 16S rRNA gene: 5¢-CTAGTTTAAAAA-

CAATGCTC-3¢ and 5¢-CTCATCTTCCTCCAATTC-3¢.Habitat, association, or host: Castanea crenata phloem.

10. “Candidatus Phytoplasma cynodontis” Marcone, Schneider and Seemüller 2004b, 1081

Vernacular epithet: Bermuda grass white leaf (BGWL) phy-toplasma, strain BGWl-C1R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AJ550984.Unique region of 16S rRNA gene: 5¢-AATTAGAAGGCAT-

CTTTTAAT-3¢.Habitat, association, or host: phloem of Cynodon dactylon

(Bermuda grass).

11. “Candidatus Phytoplasma fragariae” Valiunas, Staniulis and Davis 2006, 280

Vernacular epithet: Strawberry yellows phytoplasma, strain StrawY R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): DQ086423.Unique regions of 16S rRNA gene: 5¢-GTGCAATGCT-

CAACGTTGTGAT-3¢, 5¢-AATTGCA-3¢, and 5¢-TGAGTAAT-CAAGAGGGAG-3¢.

Habitat, association, or host: phloem of Fragaria x ananassa.

12. “Candidatus Phytoplasma fraxini” Griffiths, Sinclair, Smart and Davis 1999, 1613

Vernacular epithet: Ash yellows phytoplasma, strain AshYR and lilac witches’-broom (LWB) phytoplasma.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AF092209.Unique regions of 16S rRNA gene: 5¢-CGGAAACCCCT-

CAAAAGGTTT-3¢ and 5¢-AGGAAAGTC-3¢.Habitat, association, or host: phloem of Fraxinus and

Syringa.

13. “Candidatus Phytoplasma graminis” Arocha, López, Piñol, Fernández, Picornell, Almeida, Palenzuela, Wilson and Jones 2005, 2462

Vernacular epithet: Sugarcane yellow leaf phytoplasma, strain SCYPR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AY725228.Unique regions of 16S rRNA gene: 5¢-TTTG-3¢ (465–468),

5¢-TTG-3¢ (478–480), 5¢-GGG-3¢ (1552–1554), 5¢-TAA-3¢

(1381–1383), and 5¢-ATTTACGTTTCTG-3¢ (1392–1404).Habitat, association, or host: Saccharum officinarum phloem.

14. “Candidatus Phytoplasma japonicum” Sawayanagi, Horikoshi, Kanehira, Shinohara, Bertaccini, Cousin, Hiruki and Namba 1999, 1284

Vernacular epithet: Japanese Hydrangea phyllody phyto-plasma, strain JHPR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AB010425.Unique regions of 16S rRNA gene: 5¢-GTGTAGCCG-

GGCTGAGAGGTCA-3¢ and 5¢-TCCAACTCTAGCTAAA-CAGTTTCTG-3¢.

Habitat, association, or host: Hydrangea phloem.

15. “Candidatus Phytoplasma lycopersici” Arocha, Antesana, Montellano, Franco, Plata and Jones 2007, 1709

Vernacular epithet: Tomato “hoja de perejil” phytoplasma, strain THPR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AY787136.Unique regions of 16S rRNA gene: 5¢-CTTA-3¢ (positions

175–178), 5¢-AATGGT-3¢ (198–203), 5¢-ATA-3¢ (229–231), 5¢-TGGAGGAA-3¢ (234–242), 5¢-CACG-3¢ (302–305), 5¢-TCT-3¢ (315–317), 5¢-GCT-3¢ (334–336), 5¢-TAT-3¢ (336–338), 5¢-TAC-3¢ (413–415), and 5¢-AGC-3¢ (434–436).

Habitat, association, or host: Lycopersicon esculentum phloem.

16. “Candidatus Phytoplasma mali” Seemüller and Schneider 2004, 1224

Vernacular epithet: Apple proliferation (AP) phytoplasma, strain AP15R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AJ542541.Unique region of 16S rRNA gene: 5¢-AATACTCGAAACCA-

GTA-3¢.Habitat, association, or host: Malus phloem.

17. “Candidatus Phytoplasma omanense” Al-Saady, Khan, Calari, Al-Subhi and Bertaccini 2008, 464

Vernacular epithet: Cassia witches’-broom (CWB) phyto-plasma, strain IM-1R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): EF666051.Unique regions of 16S rRNA gene: 5¢-AAAAAACAGT-3¢ (467–

474), 5¢-TTGC-3¢ (642–645), 5¢-GTTAAAG-3¢ (853–861), 5¢-TAATT-3¢ (1010–1014), and 5¢-AAATT-3¢ (1052–1056).

Habitat, association, or host: Cassia italica phloem.

18. “Candidatus Phytoplasma oryzae” Jung, Sawayanagi, Wong-kaew, Kakizawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi and Namba 2003c, 1928

Vernacular epithet: Rice yellow dwarf (RYD) phytoplasma, strain RYD-ThR.

Gram reaction: not applicable.Morphology: other.Sequence accession nos (16S rRNA gene): D12581, AB052873

(RYD-Th).

710


Unique regions of 16S rRNA gene: 5¢-AACTGGATAGGAAAT-TAAAAGGT-3¢ and 5¢-ATGAGACTGCCAATA-3¢.

Habitat, association, or host: Oryza sativa phloem.

19. “Candidatus Phytoplasma phoenicium” Verdin, Salar, Danet, Choueiri, Jreijiri, El Zammar, Gélie, Bové and Garnier 2003, 837

Vernacular epithet: Almond witches’-broom (AlmWB) phyto plasma, strain AlmWB-A4R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AF515636.Unique region of 16S rRNA gene: 5¢-CCTTTTTCGGAAGG-

TATG-3¢.Habitat, association, or host: Prunus amygdalus phloem.

20. “Candidatus Phytoplasma pini” Schneider, Torres, Martín, Schröder, Behnke and Seemüller 2005, 306

Vernacular epithet: Pinus halepensis yellows (Pin) phyto-plasma, strain Pin127SR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AJ632155.Unique regions of 16S rRNA gene: 5¢-GGAAATCTTTCG-

GGATTTTAGT-3¢ and 5¢-TCTCAGTGCTTAACGCTGT-TCT-3¢.

Habitat, association, or host: Pinus phloem.

21. “Candidatus Phytoplasma prunorum” Seemüller and Sch-neider 2004, 1224

Vernacular epithet: European stone fruit yellows (ESFY) phytoplasma, strain ESFY-G1R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AJ542544.Unique regions of 16S rRNA gene: 5¢-AATACCCGAAACCA-

GTA-3¢ and 5¢-TGAAGTTTTGAGGCATCTCGAA-3¢.Habitat, association, or host: Prunus phloem.

22. “Candidatus Phytoplasma pyri” Seemüller and Schneider 2004, 1224

Vernacular epithet: Pear decline (PD) phytoplasma, strain PD1R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AJ542543.Unique regions of 16S rRNA gene: 5¢-AATACTCAAAACCA-

GTA-3¢ and 5¢-ATACGGCCCAAACTCATACGGA-3¢.Habitat, association, or host: Pyrus phloem.

23. “Candidatus Phytoplasma rhamni” Marcone, Gibb, Streten and Schneider 2004a, 1028

Vernacular epithet: Buckthorn witches’-broom phyto-plasma, strain BWBR.

Gram reaction: not applicable.Morphology: other.Sequence accession nos (16S rRNA gene): X76431, AJ583009.Unique regions of 16S rRNA gene: 5¢-CGAAGTATTTCGA-

TAC-3¢.Habitat, association, or host: phloem of Rhamnus catharticus

(buckthorn).

24. “Candidatus Phytoplasma solani” Firrao, Gibb and Streton 2005, 251

Vernacular epithet: Stolbur phytoplasma; subgroup A ref-erence type of the stolbur phytoplasma taxonomic group 16SrXII (Lee et al., 2000).

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AJ970609 (strain

PO; Cimerman et al., 2006).Unique region of 16S rRNA gene: not reported.Habitat, association, or host: many species of Solanaceae plus

several species in other plant families, and Fulguromorpha spp. planthopper vectors.

25. “Candidatus Phytoplasma spartii” Marcone, Gibb, Streten and Schneider 2004a, 1028

Vernacular epithet: Spartium witches’-broom phytoplasma, strain SpaWBR.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): X92869.Unique region of 16S rRNA gene: 5¢-TTATCCGCGTTAC-3¢.Habitat, association, or host: phloem of Spartium junceum

(Spanish broom).

26. “Candidatus Phytoplasma tamaricis” Zhao, Sun, Wei, Davis, Wu and Liu 2009, 2496

Vernacular epithet: Salt cedar witches’-broom phytoplasma, strain SCWB1R.

Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): FJ432664.Unique regions of 16S rRNA gene: 5¢-ATTAGGCATCTAG-

TAACTTTG-3¢, 5¢-TGCTCAACATTGTTGC-3¢, 5¢-AGCTTT-GCAAAGTTG-3¢, and 5¢-TAACAGAGGTTATCAGAGTT-3¢.

Habitat, association, or host: phloem of Tamarix chinensis (salt cedar).

27. “Candidatus Phytoplasma trifolii” Hiruki and Wang 2004, 1352Vernacular epithet: Clover proliferation phytoplasma,

strain CPR.Gram reaction: not applicable.Morphology: other.Sequence accession no. (16S rRNA gene): AY390261.Unique regions of 16S rRNA gene: 5¢-TTCTTACGA-3¢ and

5¢-TAGAGTTAAAAGCC-3¢.Habitat, association, or host: Trifolium phloem.

28. “Candidatus Phytoplasma ulmi” Lee, Martini, Marcone and Zhu 2004b, 345

Vernacular epithet: Elm yellows phytoplasma (EY) phyto-plasma, strain EY1R.

Gram reaction: not applicable.Morphology: other.Sequence accession nos (16S rRNA gene): AY197655,

AY197675, and AY197690.Unique regions of 16S rRNA gene: 5¢-GGAAA-3¢ and 5¢-CGT-

TAGTTGCC-3¢.Habitat, association, or host: Ulmus americana phloem.

29. “Candidatus Phytoplasma vitis” Firrao, Gibb and Streton 2005, 251

Vernacular epithet: Flavescence dorée phytoplasma; strains are genetically heterogenous and vary in degree of virulence, but all are referable to subgroups C or D of the elm yellows

711


phytoplasma taxonomic group 16SrV (Lee et al., 2000).Gram reaction: not applicable.Morphology: other.Sequence accession nos (16S rRNA gene): AY197645 (16SrV

subgroup C), AY197644 (16SrV subgroup D1).Unique regions of 16S rRNA gene: not reported.Habitat, association, or host: grapevines (Vitis vinifera) and

the leafhopper vector Scaphoideus titanus.

30. “Candidatus Phytoplasma ziziphi” Jung, Sawayanagi, Kak-izawa, Nishigawa, Wei, Oshima, Miyata, Ugaki, Hibi and Namba 2003b, 1041

Vernacular epithet: Jujube witches’-broom phytoplasma, strain JWBR.

Gram reaction: not applicable.Morphology: other.Sequence accession nos (16S rRNA gene):AB052875–

AB052879.Unique regions of 16S rRNA gene: 5¢-TAAAAAGGCATCTT-

TTTGTT-3¢ and 5¢-AATCCGGACTAAGACTGT-3¢.Habitat, association, or host: Ziziphyus jujube phloem.

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order IV. anaeroPlasmatales

order IV. anaeroplasmatales robinson and Freundt 1987, 81VP


a.na.e.ro.plas.ma.ta¢les. n.l. neut. n. Anaeroplasma, -atos type genus of the order; -ales ending to denote an order; n.l. fem. pl. n. Anaeroplasmatales the Anaeroplasma order.

This order in the class Mollicutes represents a unique group of strictly anaerobic, wall-less prokaryotes (trivial name, anaero-plasmas) first isolated from the bovine and ovine rumen. Other than their anaerobiosis, the description of organisms in the order is essentially the same as for the class. A single family, Anaeroplasmataceae, with two genera, was proposed to recognize the two most prominent characteristics of the organisms: a requirement of sterol supplements for growth by those strictly anaerobic organisms now assigned to the genus Anaeroplasma; and strictly anaerobic growth in the absence of sterol supplements by those now assigned to the genus Aster-oleplasma. Genome sizes range from 1542 to 1794 kbp as esti-mated by renaturation kinetics. The DNA G+C content ranges from 29 to 40 mol%. All species examined utilize the univer-sal genetic code in which UGA is a stop codon. Phylogenetic studies indicate that members of the Anaeroplasmatales are much more closely related to the Acholeplasmatales than to the Mycoplasmatales or Entomoplasmatales (Weisburg et al., 1989).

Type genus: Anaeroplasma Robinson, Allison and Hartman 1975, 179AL.


The initial proposal for elevation of the anaeroplasmas to an order of the class Mollicutes (Robinson and Freundt, 1987) was based upon the description of three novel species and the observation that some anaeroplasmas did not have a sterol requirement for growth.

The obligate requirement for anaerobic growth condi-tions is the single most important property in distinguishing members of the Anaeroplasmatales from other mollicutes. The

anaeroplasmas exist in a natural environment where the oxi-dation potential is maintained at a low level by the metabo-lism of associated micro-organisms. Anaerobic methods for preparing media and culture techniques for the organisms are essentially those described by Hungate (1969), with media and inocula maintained in closed vessels and expo-sure to air avoided during inoculation and incubation. A primary isolation medium and clarified rumen fluid broth have been described (Bryant and Robinson, 1961; Robinson, 1983; Robinson et al., 1975).

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Zreik, L., P. Carle, J.M. Bové and M. Garnier. 1995. Characterization of the mycoplasmalike organism associated with witches’ broom dis-ease of lime and proposition of a Candidatus taxon for the organ-ism, “Candidatus Phytoplasma aurantifolia”. Int. J. Syst. Bacteriol. 45: 449–453.

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Family i. anaeroplasmataceae

Family i. Anaeroplasmataceae robinson and Freundt 1987, 80Vp


a.na.e.ro.plas.ma.ta.ce¢ae. n.l. neut. n. Anaeroplasma, -atos type genus of the family; -aceae ending to denote a family; n.l. fem. pl. n. Anaeroplasmataceae the Anaeroplasma family.

All members have an obligate requirement for anaerobiosis. Organisms assigned to the genus Anaeroplasma require sterol supplements for growth. Organisms assigned to the genus Aster-oleplasma grow in the absence of sterol supplements. Other characteristics are as described for the type genus.

Type genus: Anaeroplasma Robinson, Allison and Hartman 1975, 179AL.


The obligate requirement for anaerobic growth conditions and for growth only in media containing cholesterol is established with the methods described by Hungate (1969), with media and inocula maintained in closed vessels and exposure to air avoided during inoculation and incubation. The primary isola-tion medium and the clarified rumen fluid broth supplemented with cholesterol have been described (Robinson, 1983).

Genus i. Anaeroplasma robinson, allison and Hartman 1975, 179al


a.na.e.ro.plas¢ma. Gr. prefix an without; Gr. masc. n. aer air; Gr. neut. n. plasma a form; n.l. neut. n. Anaero-plasma intended to denote “anaerobic mycoplasma”.

Cells are predominantly coccoid, about 500 nm in diameter; clusters of up to ten coccoid cells may be joined by short fila-ments. Older cells have a variety of pleomorphic forms. Cells lack a cell wall and are bound by a single plasma membrane. Gram-stain-negative due to absence of cell wall. Obligately anaerobic; the inhibitory effect of oxygen on growth is not alleviated during repeated subcultures. Require sterol supple-ments for growth. Nonmotile. Optimal temperature, 37°C; no growth at 26 or 47°C. Optimal pH, 6.5–7.0. Surface colonies have a dense center with a translucent periphery, or “fried-egg” appearance. Subsurface colonies are golden, irregular, and often multilobed. Strains vary in their ability to ferment vari-ous carbohydrates. The products of carbohydrate fermentation include acids (generally acetic, formic, propionic, lactic, and succinic), ethanol, and gases (primarily CO2, but some strains also produce H2). Bacteriolytic and nonbacteriolytic strains have been described. Commensals in the bovine and ovine rumen.

DNA G+C content (mol%): 29–34 (Tm, Bd).Type species: Anaeroplasma abactoclasticum Robinson, Allison

and Hartman 1975, 179AL.


Cells of Anaeroplasma examined by phase-contrast microscopy appear as single cells, clumps, dumbbell forms, and clusters of coccoid forms joined by short filaments. In electron micro-graphs of negatively stained preparations, pleomorphic forms are observed; these include filamentous cells, budding cells, and cells with bleb-like structures.

All species examined have similar fermentation products of acetate, formate, lactate, ethanol, and carbon dioxide (Robin-son et al., 1975). Anaeroplasma abactoclasticum is the only species known not to digest casein. Anaeroplasma abactoclasticum strains are the only ones known to produce succinate through fermen-tation. Anaeroplasma bactoclasticum, Anaeroplasma intermedium, and Anaeroplasma varium are the only species known to produce hydrogen and propionate during their fermentation.

The roll-tube anaerobic culture technique (Hungate, 1969), with pre-reduced medium maintained in a system for exclusion of oxygen, is used to culture the organisms (Robin-son, 1983; Robinson and Allison, 1975; Robinson et al., 1975;

Robinson and Hungate, 1973). Anaerobic mollicutes in a sew-age sludge digester were cultured in an anaerobic cabinet (Rose and Pirt, 1981). Although it is possible that other types of anaerobic culture techniques might be acceptable (anaero-bic culture jar or GasPak system), the effective use of such equipment has not been demonstrated (Robinson, 1983). Strains with bacteriolytic activity are detected with the addi-tion of autoclaved Escherichia coli cells to the Primary Isolation Medium (PIM) described below. Clear zones around colonies of anaeroplasmas, when viewed by a stereoscopic microscope, are suggestive of bacteriolytic anaeroplasmas. Colonies can be subcultured to clarified rumen fluid broth (CRFB) medium described below.

A slide agglutination test was first used to show that the anti-gens of anaerobic mollicutes were not related to established Mycoplasma or Acholeplasma species found in cattle (Robinson and Hungate, 1973). Later, the agglutination test was adapted to either a plate or tube test and combined with an agar gel diffusion test and a modified growth inhibition procedure to examine the antigenic interrelationships among the anaero-bic mollicutes (Robinson and Rhoades, 1977). On the basis of these tests, a serological grouping of anaerobic mollicutes appeared compatible with the group separations based upon cultural, biochemical, and biophysical properties of the organ-isms (Robinson, 1979; Robinson and Rhoades, 1977).

There is no current evidence for the pathogenicity of any of the Anaeroplasma species described so far. Obligately anaerobic mollicutes appear to be a heterogeneous group that has been found so far only in the rumen of cattle and sheep (Robinson, 1979; Robinson et al., 1975). Each new isolated group of these organisms seems to have different properties, suggesting that additional undescribed species are likely to exist. The ecological role of these organisms in the rumen has not been determined. Although the titer of these organisms in the rumen appears to be low when compared to titers of other rumen organisms, the mollicutes probably contribute to the pool of microbial fermentation products at that site. Growth of anaeroplasmas is inhibited by thallium acetate (0.2%), bacitracin (1000 mg/ml), streptomycin (200 mg/ml), and d-cycloserine (500 mg/ml), but not by benzylpenicillic acid (1000 U/ml).

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Genus i. anaeroplasma


The PIM medium used to grow and detect anaerobic mycoplas-mas (Robinson, 1983; Robinson et al., 1975; Robinson and Hun-gate, 1973) contains: 40% (v/v) rumen fluid strained through cheesecloth, autoclaved, and clarified by centrifugation; 0.05% (w/v) glucose; 0.05% (w/v) cellobiose; 0.05% (w/v) starch; 3.75% (v/v) of a mineral solution consisting of 1.7 × 10−3 M K2HPO4, 1.3 × 10−3 M KH2PO4, 7.6 × 10−4 M NaCl, 3.4 × 10−3 M (NH4)2SO4, 4.1 × 10−4 M CaCl2, and 3.8 × 10−4 M MgSO4·7H2O; 0.2% (w/v) trypticase; 0.1% (w/v) yeast extract; 0.0001% (w/v) resazurin; 0.5% (w/v) autoclaved Escherichia coli cells; 0.4% (w/v) Na2CO3; 0.05% (w/v) cysteine hydrochloride; 1.5% (w/v) agar; and 0.0006% (w/v) benzylpenicillic acid. Pure cultures are estab-lished by picking individual colonies from PIM roll tubes and sub-culturing into CRFB medium. CRFB medium contains the same ingredients and concentrations as PIM, except: glucose, cello-biose, and starch concentrations are 0.2% (w/v), and autoclaved Escherichia coli cells, agar, and benzylpenicillic acid are omitted. Growth also occurs in a rumen fluid-free medium (Medium D) in which growth factors supplied in rumen fluid are replaced by lipopolysaccharide (Boivin; Difco) and cholesterol (Robinson, 1983; Robinson et al., 1975), or in a completely defined medium in which the trypticase, yeast extract, and lipopolysaccharide of Medium D are replaced by amino acids, vitamins, and phosphati-dylcholine esterified with unsaturated fatty acids (Robinson, 1979, 1983). An agar-overlay plating technique carried out in an anaerobic hood has also been reported to be an effective isola-tion procedure (Robinson, 1979). Anaerobic mollicutes grow only in a prereduced medium maintained in a system for exclu-sion of oxygen. When resazurin is used in the test medium and becomes oxidized, mollicutes will fail to grow.


Cultures are viable after storage for as long as 5 years at −40°C in CRFB medium. They may also be preserved by lyophilization using standard techniques for other mollicutes (Leach, 1983). However, the type strains of several species of Anaeroplasma are no longer available from the American Type Culture Collection because it was impossible to revive the cultures sent by the depositors.

Differentiation of the genus Anaeroplasma from other genera

Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. The obligately anaerobic nature of Anaeroplasma spe-cies is a distinctive and stable characteristic among these organ-isms. Strictly anaerobic growth plus the requirement for sterol supplements for growth exclude assignment to any other taxon in the class. Moreover, the bacteriolytic capability possessed by some of the anaeroplasmas has not been reported for other mycoplasmas. Plasmalogens (alkenyl-glycerol ethers), which are found in various anaerobic bacteria but not in aerobic bac-teria, are major components of polar lipids from anaeroplasmas (Langworthy et al., 1975); this further supports the contention that anaeroplasmas are distinct from other mollicutes.

taxonomic comments

The first organism in the group to be described was referred to as Acholeplasma bactoclasticum (type strain JRT = ATCC 27112T; Robinson and Hungate, 1973) because the organism was

thought to lack a sterol requirement for growth. Later, when other obligately anaerobic mollicutes were isolated, these and the JRT strain were found to require sterol for growth. A pro-posal was then made to form the new genus Anaeroplasma to accommodate strain JRT (as Anaeroplasma bactoclasticum; Rob-inson and Allison, 1975) and a second anaerobic mollicute designated Anaeroplasma abactoclasticum (Robinson et al., 1975). These developments prompted a proposal for an amended clas-sification of anaerobic mollicutes, which included descriptions of Anaeroplasma varium and Anaeroplasma intermedium, the family Anaeroplasmataceae, and the order Anaeroplasmatales within the class Mollicutes (Robinson and Freundt, 1987).

Early serological studies suggested the existence of several distinct species of anaeroplasmas. Subsequent reports on DNA–DNA hybridization, DNA base composition, and genome size comparisons of organisms in the group also indicated the exis-tence of a number of species in two distinct genera of anaero-bic mollicutes (Christiansen et al., 1986; Stephens et al., 1985). Strains initially assigned to Acholeplasma abactoclasticum [serovar 3, type strain 6-1T (=ATCC 27879T)] were found to be a single spe-cies with about 80% interstrain DNA–DNA hybridization. How-ever, strains A-2T (serovar 1) and 7LAT (serovar 2), previously included in the description of Acholeplasma bactoclasticum, are the type strains of separate species designated Anaeroplasma varium and Anaeroplasma intermedium, respectively (Robinson and Fre-undt, 1987). Strains of Anaeroplasma all have DNA G+C contents in the range 29.3–33.7 mol%, whereas the base composition of serovar 4 strains 161T, 162, and 163 clustered above 40 mol%. These were assigned to the new genus Asteroleplasma [type strain 161T (=ATCC 27880T)] whose members are anaerobic, but do not require sterol for growth. Genome sizes ranged from 1542 to 1715 kbp for Anaeroplasma species, as determined by renatur-ation kinetics (Christiansen et al., 1986). Although the genome sizes reported were in the expected range for members of the class Mollicutes, no data are currently available on genome sizes estimated by the more accurate pulsed-field gel electrophoresis technique. A phylogenetic analysis of members of the Anaero-plasmatales, based upon 16S rRNA gene sequence comparison, was carried out by Weisburg et al. (1989). Anaeroplasma and Acholeplasma are sister genera basal on the mollicute tree.

acknowledgements


Further reading


Differentiation of the species of the genus Anaeroplasma

The technical challenges of cultivating these anaerobic molli-cutes have led to a current reliance principally on the combina-tion of 16S rRNA gene sequencing and reciprocal serology for species differentiation. Serological characterization of anaero-plasmas has been performed with agglutination, modified metabolism inhibition, and immunodiffusion tests (Robinson and Rhoades, 1977). Failure to cross-react with antisera against previously recognized species provides substantial evidence for species novelty. DNA–DNA hybridization values between species

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Family i. anaeroplasmataceae

examined are less than 5%. Bacteriolytic and nonbacteriolytic organisms occur within the genus. When grown on agar media containing a suspension of autoclaved Escherichia coli cells, bacteriolytic strains form colonies surrounded by a clear zone

due to lysis of the suspended cells by a diffusible enzyme(s). On media lacking suspended cells, bacteriolytic and nonbac-teriolytic strains of Anaeroplasma cannot be distinguished from each other on the basis of colonial or cellular morphology.

list of species of the genus Anaeroplasma

1. Anaeroplasma abactoclasticum Robinson, Allison and Hart-man 1975, 179AL

a.bac.to.clas¢ti.cum. Gr. pref. a without; Gr. bakt- (L. trans-literation bact-) part of the stem of the Gr. dim. n. bakterion (L. transliteration bacterium) a small rod; N.L. adj. clasticus, -a, um (from Gr. adj. klastos, -ê, -on broken in pieces) break-ing; N.L. neut. adj. abactoclasticum intended to denote “not bacteriolytic”.

This is the type species of the genus Anaeroplasma. Cells are coccoid, about 500 nm in diameter, sometimes joined into short chains by filaments. Colonies on solid medium are subsurface, but nevertheless present a typical fried-egg appearance. Growth is inhibited by 20 mg/ml digitonin. A major distinguishing factor is the lack of the extracellular bacterioclastic and proteolytic enzymes that characterize the lytic species.

No evidence of a role in pathogenicity.Source: occurs primarily in the bovine and ovine rumen.DNA G+C content (mol%): 29.3 (Bd).Type strain: 6-1, ATCC 27879.Sequence accession no. (16S rRNA gene): M25050.

2. Anaeroplasma bactoclasticum (Robinson and Hungate 1973) Robinson and Allison 1975, 186AL (Acholeplasma bacto-clasticum Robinson and Hungate 1973, 180)

bac.to.clas¢ti.cum. Gr. bakt- (L. transliteration bact-) part of the stem of the Gr. dim. n. bakterion (L. transliteration bac-terium) a small rod; N.L. adj. clasticus, -a, um (from Gr. adj. klastos, -ê, -on broken in pieces) breaking; N.L. neut. adj. bac-toclasticum bacteria-breaking.

Pleomorphic and coccoid cells ranging in size from 550 to 2000 nm in diameter. Cells cluster and sometimes form short chains. Colonies on solid medium have a typical fried-egg appearance. Optimal temperature for growth is between 30 and 47°C. Growth is inhibited by 20 mg/ml digitonin. Skim milk is cleared by a proteolytic, extracellular enzyme and certain bacteria are lysed by an extracellular enzyme

that attacks the peptidoglycan layer of the cell wall. Shares some serological relationship to other established species in the genus, but can be distinguished by agglutination, modi-fied metabolism inhibition, and agar gel immunodiffusion precipitation tests.

No evidence of pathogenicity.Source: occurs in the bovine and ovine rumen.DNA G+C content (mol%): 32.5 to 33.7 (Tm, Bd).Type strain: JR, ATCC 27112.Sequence accession no. (16S rRNA gene): M25049.

3. Anaeroplasma intermedium Robinson and Freundt 1987, 79VP

in.ter.me¢di.um. L. neut. adj. intermedium intermediate.

Cellular morphology and colonial features are similar to those of Acholeplasma bactoclasticum. Serologically distinct from other species in the genus by agglutination, metabo-lism inhibition, and agar gel immunodiffusion precipitation tests (Robinson and Rhoades, 1977).

No evidence of pathogenicity.Source: occurs in the bovine and ovine rumen.DNA G+C content (mol%): 32.5 (Bd).Type strain: 7LA, ATCC 43166.Sequence accession no. (16S rRNA gene): not available.

4. Anaeroplasma varium Robinson and Freundt 1987, 79VP

va¢ri.um. L. neut. adj. varium diverse, varied, intended to mean different from Anaeroplasma bactoclasticum.

Cellular morphology and colonial features are similar to those of Acholeplasma bactoclasticum. Serologically distinct from other species in the genus by agglutination, metabo-lism inhibition, and agar gel immunodiffusion precipitation tests (Robinson and Rhoades, 1977).

No evidence of pathogenicity.Source: occurs in the bovine and ovine rumen.DNA G+C content (mol%): 33.4 (Tm).Type strain: A-2, ATCC 43167.Sequence accession no. (16S rRNA gene): M23934.

Genus ii. Asteroleplasma robinson and Freundt 1987, 79Vp


a.ste.rol.e.plas¢ma. Gr. pref. a not; n.l. neut. n. sterolum sterol; e combining vowel; Gr. neut. n. plasma something formed or molded, a form; n.l. neut. n. Asteroleplasma name intended to indicate that sterol is not required for growth.

Cellular and colonial morphology similar to species of the genus Anaeroplasma. Nonmotile. The three strains that form the new genus and species are obligately anaerobic and capable of growth in the absence of cholesterol or serum supplements. Temperature optimum for growth is 37°C. No evidence of bac-teriolytic activity. The organisms are serologically distinct from other members in the family Anaeroplasmataceae. Occur in the ovine rumen.

DNA G+C content (mol%): about 40 (Tm, Bd).

Type species: Asteroleplasma anaerobium Robinson and Fre-undt 1987, 79VP.


The most prominent characteristics of the organisms are strictly anaerobic growth and growth in the absence of sterol supple-ments. The G+C contents of strains analyzed to date are higher than the values for Anaeroplasma species (Stephens et al., 1985). DNA–DNA reassociation values clearly show that strains 161T,

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Genus ii. asteroleplasma

162, and 163 of Asteroleplasma anaerobium are genetically related and distinct from established species in the genera Acholeplasma or Anaeroplasma (Stephens et al., 1985). Tube agglutination tests and gel diffusion precipitation tests showed that strains assigned to Asteroleplasma anaerobium are serologically distinct from Anaeroplasma species (Robinson and Rhoades, 1977). No data have been reported on antibiotic sensitivity or pathoge-nicity of asteroleplasmas. Strains have been isolated only from sheep rumen. Isolation and maintenance techniques are simi-lar to those reported for Anaeroplasma species.

Differentiation of the genus Asteroleplasma from other genera

Properties that partially fulfill criteria for assignment to the class Mollicutes (Brown et al., 2007) include absence of a cell wall, filterability, and the presence of conserved 16S rRNA gene sequences. The obligately anaerobic nature of Asteroleplasma is a distinctive and stable characteristic. Strictly anaerobic growth plus growth in the absence of sterol supplements exclude assign-ment to any other taxon in the class. Extracellular bacteriolytic and proteolytic enzymes are absent. Growth is not inhibited by 20 mg/ml digitonin.

taxonomic comments

The taxonomic position of strains 161T, 162, and 163 of obligately anaerobic mollicute serovar 4 was delineated through obser-vations that they did not require sterol for growth (Robinson

et al., 1975), were serologically distinct (Robinson and Rhoades, 1977), and had G+C contents that were much higher than those of Anaeroplasma species (Christiansen et al., 1986). Less than 5% DNA–DNA relatedness existed between these strains and spe-cies assigned to the genus Anaeroplasma (Stephens et al., 1985). Lastly, the significance of the group and the need to clarify its taxonomic status was emphasized when it was demonstrated that a significant proportion of the anaerobic mollicute popula-tion in the bovine and ovine rumen does not require sterol for growth (Robinson and Rhoades, 1982). The phylogenetic analy-sis of Weisburg et al. (1989) indicated that Asteroleplasma anaero-bium had branched from the Firmicutes lineage independently of Acholeplasma and Anaeroplasma. Further, Asteroleplasma shared two of three important synapomorphies that united the Myco-plasma and Spiroplasma lineages. Thus, the question of possible monophyly and the true phylogenetic position of Asteroleplasma with respect to other mollicutes remains open.

acknowledgements


Further reading


list of species of the genus Asteroleplasma

1. Asteroleplasma anaerobium Robinson and Freundt 1987, 79VP

a.na.e.ro¢bi.um. Gr. pref. an not; Gr. n. aer air; Gr. n. bios life; N.L. neut. adj. anaerobium not living in air.

This is the type species of the genus Asteroleplasma. Cell morphology and colonial characteristics are similar to those of other members of the order Anaeroplasmatales. Strains 161T, 162, and 163 form a homogeneous and distinct serological group, as judged by about 80% DNA–DNA hybridization and

serological agglutination, metabolism inhibition, and agar gel immunodiffusion precipitation tests.

No evidence of pathogenicity.Source: all isolates have been identified from the bovine or

ovine rumen.DNA G+C content (mol%): 40.2–40.5 (Bd, Tm).Type strain: 161 (the type strain ATCC 27880 no longer

exists).Sequence accession no. (16S rRNA gene): M22351.

references


Christiansen, C., E.A. Freundt and I.M. Robinson. 1986. Genome size and deoxyribonucleic acid base composition of Anaeroplasma abac-toclasticum, Anaeroplasma bactoclasticum, and a sterol-nonrequiring anaerobic mollicute. Int. J. Syst. Bacteriol. 36: 483–485.

Hungate, R.E. 1969. A roll tube method for cultivation of strict anaer-obes. In Methods in Microbiology, vol. 3B (edited by Norris and Ribbons). Academic Press, London, pp. 117–132.

Langworthy, T., W. Mayberry, P. Smith and I. Robinson. 1975. Plasmalo-gen composition of Anaeroplasma. J. Bacteriol. 122: 785–787.


Robinson, I.M. and M.J. Allison. 1975. Transfer of Acholeplasma bactoclas-ticum Robinson and Hungate to genus Anaeroplasma (Anaeroplasma bactoclasticum Robinson and Hungate comb. nov.), emended descrip-tion of species. Int. J. Syst. Bacteriol. 25: 182–186.


Robinson, I.M. and K.R. Rhoades. 1977. Serological relationships between strains of anaerobic mycoplasmas. Int. J. Syst. Bacteriol. 27: 200–203.

Robinson, I.M. 1979. Special features of anaeroplasmas. In The Myco-plasmas, vol. 1 (edited by Barile and Razin). Academic Press, New York, pp. 515–528.

Robinson, I.M. and K.R. Rhoades. 1982. Serologic relationships between strains of anaerobic mycoplasmas. Rev. Infect. Dis. 4: S271.

Robinson, I.M. 1983. Culture media for anaeroplasmas. In Methods in Mycoplasmology, vol. 1 (edited by Razin and Tully). Academic Press, New York, pp. 159–162.

Robinson, I.M. and E.A. Freundt. 1987. Proposal for an amended classification of anaerobic mollicutes. Int. J. Syst. Bacteriol. 37: 78–81.

Robinson, J.P. and R.E. Hungate. 1973. Acholeplasma bactoclasticum sp. n., an anaerobic mycoplasma from the bovine rumen. Int. J. Syst. Bacteriol. 23: 171–181.

Rose, C. and S. Pirt. 1981. Conversion of glucose to fatty acids and meth-ane: roles of two mycoplasmal agents. J. Bacteriol. 147: 248–254.

Stephens, E., I. Robinson and M. Barile. 1985. Nucleic acid relationships among the anaerobic mycoplasmas. J. Gen. Microbiol. 131: 1223–1227.

Weisburg, W., J. Tully, D. Rose, J. Petzel, H. Oyaizu, D. Yang, L. Mandelco, J. Sechrest, T. Lawrence and J. Van Etten. 1989. A phylogenetic analy-sis of the mycoplasmas: basis for their classification. J. Bacteriol. 171: 6455–6467.

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