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Veterinary Parasitology 216 (2016) 52–58

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l h om epa ge: www.elsev ier .com/ locate /vetpar

esearch paper

cute, fatal Sarcocystis calchasi-associated hepatitis in Roller pigeonsColumba livia f. dom.) at Philadelphia Zoo

.G. Trupkiewicza, R. Calero-Bernalb, S.K. Vermab, J. Moweryc, S. Davisond, P. Habeckerd,.A. Georoff a, D.M. Ialeggioa, J.P. Dubeyb,∗

Philadelphia Zoo, 3400 W. Girard Ave, Philadelphia, PA 19104, USAUnited States Department of Agriculture, Agricultural Research Service, Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center,uilding 1001, Beltsville, MD 20705-2350, USAUnited States Department of Agriculture, Agricultural Research Service, Electron and Confocal Microscopy Unit, Beltsville Agricultural Research Center,uilding 12, Beltsville, MD 20705, USAPennsylvania Animal Diagnostic Laboratory System, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, 382 West Street Road,ennett Square, PA 19348, USA

r t i c l e i n f o

rticle history:eceived 8 September 2015eceived in revised form 6 November 2015

a b s t r a c t

Four Roller pigeons (Columba livia f. dom.) at the Philadelphia Zoo died suddenly. Necropsy examinationrevealed macroscopic hepatitis. Microscopically, the predominant lesions were in liver, characterizedwith necrosis and mixed cell inflammatory response. Sarcocystis calchasi-like schizonts and free mero-

ccepted 16 November 2015

eywords:arcocystis calchasioller pigeons (Columba livia)chizonts

zoites were identified in liver. Transmission electron microscopy confirmed that schizonts were inhepatocytes. A few schizonts were in spleen. PCR using S. calchasi-specific primers confirmed the diagno-sis. Neither lesions nor protozoa were found in brain and muscles. This is the first report of acute visceralS. calchasi-associated sarcocystosis in naturally infected avian hosts.

Published by Elsevier B.V.

epatitis

. Introduction

A new clinical syndrome, called pigeon protozoal encephalitisPPE) was recognized by Olias et al. (2009) in Germany. In threeocks of racing pigeons 47 of 244 pigeons were affected. Thir-een of these 47 pigeons were necropsied; sarcocysts were foundssociated with encephalitis and myositis (Table 1). They namedhe parasite, Sarcocystis calchasi, and described its life cycle (Oliast al., 2010a). The Northern goshawk (Accipiter gentilis) was foundo be its definitive host. Feeding of sporulated sporocysts from anxperimentally infected goshawk induced fatal disease in pigeons.chizonts were found in the liver, spleen, and lung 7–12 daysost inoculation (p.i.). Schizonts were present in hepatocytes andndothelial cells. In pigeons fed with higher doses of sporocysts,chizonts were seen associated with macroscopic hepatitis (Oliast al., 2010e; Maier et al., 2015a). Sarcocysts were first seen 20 day

.i. and had matured by 30 day p.i. Encephalitis was first detected0 day p.i., coincident with the sarcocysts. Sarcocysts had becomeacroscopic by 51 day p.i. The encephalitis, sometimes developing

∗ Corresponding author. Fax: +1 301 504 9222.E-mail address: jitender.dubey@ars.usda.gov (J.P. Dubey).

ttp://dx.doi.org/10.1016/j.vetpar.2015.11.008304-4017/Published by Elsevier B.V.

in the absence of schizonts, was considered to be immune medi-ated (Olias et al., 2013). The disease was considered diphasic, withpolyurea and apathy during the acute phase and neurological signsduring the later (chronic) phase (Olias et al., 2010c). Similar diseasewas subsequently induced in cockatiels (Nymphicus hollandicus)(Olias et al., 2014). In the cockatiels fed 3 million sporocysts, sch-izonts were seen in liver and spleen at 7 days p.i. and were alsopresent at 57 day p.i. in one cockatiel. Sarcocysts were first seen at13 days p.i.

Sarcocysts and severe encephalitis were seen in five of 60 feralpigeons from Minnesota and Missouri, USA (Olias et al., 2014). Inthree of the encephalitic pigeons schizonts were associated withneural lesions. Treatment with the anticoccidial toltrazuril had noeffect on S. calchasi in experimentally infected pigeons (Maier et al.,2015b).

The PPE associated with S. calchasi has been reported fromGermany, Japan and USA (Table 1). The objective of the presentreport is to document sudden death due to hepatitis in Rollerpigeons from Pennsylvania.

J.G. Trupkiewicz et al. / Veterinary Parasitology 216 (2016) 52–58 53

Table 1Reports of S. calchasi-associated mortality in avian species.

Avian host Country No Main clinicalsigns

Diagnosis Reference

Lesions Schizonts Sarcocysts PCR

Domestic pigeon(Columbia livia f.domestica)

Germany 13 Neurological Encephalitis, myositis,meningitis

Brain of 1 Myocardium,skeletal muscle(LM, TEM)

ITS-1, 28S Olias et al. (2009)

Rock pigeon (Columbialivia)

Japan 1 Neurological Encephalitis Brain and spinalcord

Brain,myocardium,skeletal muscle

ITS-1 Ushio et al. (2015)

Domestic pigeon(Columbia livia f.domestica)

MN, USA 1 Neurological Brain, severe meningitis No Skeletal muscle,heart (TEM)

ITS-1 Wünschmann et al. (2011)

Domestic feral pigeon(Columbia livia f.domestica)

MN, USA 4 Neurological Brain, severemeningoencephalitis

Yes in 3 Skeletal muscleof 4

NE Olias et al. (2014)

MO, USA 1 Negative Yes NEPrincess parrot (Polytelisalexandrae)

CA, USA 2 Neurological Encephalitis Brain of both Skeletal musclein both (TEM in1)

18S, ITS-1 Rimoldi et al. (2013)

Rose-breasted cockatoo(Eolophus roseicapilla)

2 Brain of both Skeletal musclein both

NE

Long-billed corella 1 Brain NE 18S, ITS-1

N

2

2

Ppsihartb(er1sn(a

wfimowPWpVPea

2

(Cacatua tenuirostris)

E: not examined.

. Materials and methods

.1. Clinical history and antemortem investigation

In March 2015, three Roller pigeons (Columba livia) at thehiladelphia Zoo died over a two day period. The birds wereart of a large colony housed in an outdoor aviary. Following theudden deaths of the three birds, the entire colony was movedndoors for supportive care and monitoring. One additional birdad hyperpnea and lethargy, however, the remaining 14 birdsppeared clinically normal. A complete blood count was withineference range for the species, however lymphocytes appearedo be reactive, and numerous erythroplastids (non-nucleated redlood cells) were noted. Serum chemistry showed elevation of AST892 U/L; reference range 18–162 U/L), uric acid (35.1 mg/dL; refer-nce range 1.5–16.2 mg/dL), and potassium (7.1 mmol/L; referenceange 0.9–4.9 mmol/L) (International Species Inventory System,997). Fecal culture, Chlamydophila sp. PCR (cloacal and choanalwabs), and Highly Pathogenic Avian Influenza Virus PCR were allegative. The bird was treated with a single dose of doxycycline1 mL/10 mg, oral), but the animal’s condition continued to decline,nd it died a short time later.

The remaining birds in the colony were hospitalized and treatedith Trimethoprim-Sulfadiazine (67 mg trimethoprim, 333 mg sul-

adiazine/g, Uniprim®, Neogen Corporation, Lexington, KY, USA)n the drinking water (480 mg/L) for 28 days, with no additional

ortalities. Five birds from the colony developed clinical signsf weight loss and lethargy and recovered following treatmentith Trimethoprim-Sulfamethoxazole (60 mg/kg PO s.i.d., Hi-Tech

harmacal, Amityville, NY, USA) and Ponazuril (10 mg/kg PO s.i.d.,edgewood Pharmacy, Swedesboro, NJ, USA) for 30 days. Multi-

le tissue samples were submitted for ancillary testing, includingetScan Antigen ELISA for Avian Influenza A (Tracheal Swab),aramyxovirus (Newcastle Disease) Hemagglutination assay onmbryonated chicken egg inoculated with pooled tissue samples,nd Salmonella PCR (Liver) were all negative.

.2. Postmortem investigation

All four pigeons were necropsied.

2.2.1. Virus isolationSwab samples from the lungs, trachea and cloaca were pooled

in 5.0 mL of Brain Heart Infusion (BHI) broth and submitted tothe National Veterinary Services Laboratory (NVSL) for Paramyx-ovirus detection by inoculation of embryonating chicken embryosfollowed by a hemagglutination assay (HA).

2.2.2. Antigen capture ELISATracheal swabs were pooled in 1 mL of BHI and tested for avian

influenza using VetScan® Avian Influenza Virus Type A AntigenTest, (Abaxis, Union City, CA, USA).

2.3. Histological examination

Samples of liver, spleen, heart, kidneys, pancreas, lung, trachea,skeletal muscle, crop, proventriculus, ventriculus, ovary, oviduct,brain and small intestine (Table 2) were fixed in 10% buffered for-malin, and paraffin-embedded histological sections were examinedafter hematoxylin and eosin staining. Pieces of liver were embed-ded in glycol methylate for histological sections.

2.4. Transmission electron microscopy (TEM)

Pieces of liver from one pigeon (#38158) were processed forTEM. Formalin-fixed pieces were fixed for 2 h at room temperaturein 2.5% glutaraldehyde, 0.05 M NaCacodylate, 0.005 M CaCl2 (pH7.0), then refrigerated at 4 ◦C overnight, rinsed 6 times with 0.05 MNaCacodylate, 0.005 M CaCl2 buffer and post-fixed in 1% bufferedosmium tetroxide for 2 h at room temperature. The tissue was thenrinsed 6 times in the same buffer, dehydrated in a graded ethanolseries followed by 2 exchanges of propylene oxide, infiltrated in agraded series of LX-112 resin/propylene oxide and polymerized inLX-112 resin at 65 ◦C for 24 h, 60–90 nm silver–gold sections werecut on a Reichert/AO Ultracut ultramicrotome with a Diatome dia-

mond knife and mounted onto 200 mesh formvar-coated coppergrids. Grids were stained with 4% uranyl acetate and 3% lead citrateand imaged at 80 kV with a Hitachi HT-7700 transmission electronmicroscope.

54 J.G. Trupkiewicz et al. / Veterinary Pa

Tab

le

2Sa

rcoc

ysti

s

calc

hasi

-ass

ocia

ted

hep

atit

is

in

Rol

ler

pig

eon

s

in

Phil

adep

hia

Zoo.

Pige

on

ID

His

tolo

gica

l exa

min

atio

n

TEM

ITS-

1

of

S.

calc

hasi

(PC

R

amp

lifi

cati

on)

28S

rRN

A

(am

pli

fica

tion

/seq

uen

cin

g)

Cox1

(am

pli

fica

tion

/seq

uen

cin

g)

3815

3

I,

Li

NA

Posi

tive

Am

pli

fied

/not

sequ

ence

d

NA

3815

6

B, H

, I, K

, Li,

Lu, P

, Sp

NA

Posi

tive

Am

pli

fied

/seq

uen

ced

(acc

essi

on

nu

mbe

r

KU

2209

51)

Am

pli

fied

/seq

uen

ced

(acc

essi

on

nu

mbe

r

KU

2209

52)

3815

7

B, H

, K, L

i,

Lu

NA

Posi

tive

Am

pli

fied

/not

sequ

ence

d

Am

pli

fied

/seq

uen

ced

(acc

essi

on

nu

mbe

r

KU

2209

53)

3815

8

H, K

, Li,

Lu, P

, Sp

Sch

izon

ts

in

live

r

Posi

tive

Am

pli

fied

/not

sequ

ence

d

Am

pli

fied

/not

sequ

ence

d

B:

brai

n, H

:

hea

rt, I

:

smal

l in

test

ine,

K:

kid

ney

s,

Li:

live

r,

Lu:

lun

g,

P:

pan

crea

s,

Sp:

sple

en. S

k:

skel

etal

mu

scle

. NA

:

not

avai

labl

e.

In

bold

ind

icat

es

lesi

ons

and

sch

izon

ts.

rasitology 216 (2016) 52–58

2.5. Immunohistochemical examination

For immunohistochemical (IHC) staining, sections from all fourpigeon tissues were stained with anti-Sarcocystis neurona serumprepared in a rabbit injected with culture-derived S. neurona mero-zoites as described (Dubey et al., 1999). The specificity of the S.neurona antiserum has been described (Dubey and Hamir, 2000). Inaddition, IHC staining for antigens of Chlamydophila was conductedon the liver, small intestine, heart, lung, trachea, skeletal musclecrop, proventriculus, ventriculus, ovary, oviduct and kidney.

2.6. DNA extractions and PCR amplification

Genomic DNA was extracted from frozen liver of all four pigeons(#38153, #38156, #38157, #38158) using DNeasy Blood and Tis-sue Kit (Qiagen, Inc., Valencia, CA, USA) according to manufacturerinstructions. DNA quantification and quality were determined byThermo Scientific NanoDrop Lite Spectrophotometer (Thermo Sci-entific, Waltham, MA, USA). PCR amplification and sequencingwere done for the nuclear ribosomal DNA unit; 28S rRNA, and themitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Thepartial sequences of 28S rRNA and cox1 loci were amplified usingprimer pairs; KL1/LS2R, and SF1/SR5 respectively as described pre-viously (Gjerde and Josefsen, 2014). The PCR amplifications wereperformed in 50 �L total reaction volume containing 20 pmol ofeach primer and 1x Taq PCR Master Mix Kit. The thermal cyclerconditions were set at initial denaturation at 95 ◦C for 10 min; 40cycles of amplification (95 ◦C for 45 s, 52–56 ◦C for 45 s, and 72 ◦Cfor 1 min) and final extension at 72 ◦C for 10 min. Both, the posi-tive (DNA from S. neurona isolate) and the negative (H2O) controlswere included in all the batches respectively. S. neurona DNA fromculture-derived merozoites was used as positive control becausethese primers were designed to amplify DNA of Sarcocystidae.

The species of Sarcocystis was detected using previ-ously published semi-nested primer pairs; SCa1/SCa2/SNCa3,SCo1/SCo2/SNCo3 and SNi1/SNi2/SNNi3 that amplifies specificallyan ITS-1 region of S. calchasi (136 bp), Sarcocystis columbae (129 bp)and Sarcocystis sp. ex Accipiter nisus (124 bp) (Olias et al., 2011).Briefly, the primary PCR amplifications were performed in 50 �Ltotal reaction volume containing 3 �L of template DNA, 20 pmolof each primer pairs; SCa1/SCa2, SCo1/SCo2 and SNi1/SNi2, and1x Taq PCR Master Mix Kit (Qiagen, Inc., Valencia, CA, USA) in athermal cycler (Veriti® Thermal Cycler, Applied Biosystems, FosterCity, CA, USA). The thermal cycler conditions were set at initialdenaturation at 95 ◦C for 5 min; 35 cycles of amplification (95 ◦Cfor 30 s, 55 ◦C for 30 s, and 72 ◦C for 45 s) and final extension at72 ◦C for 10 min. Semi-nested PCR was carried out using primerpairs: SCa1/SNCa3, SCo1/SNCo3 and SNi1/SNNi3, and 2 �L of1:100 diluted product of primary PCR as a template in 50 �L totalreaction volume. Same thermal cycler conditions were used inboth PCR reactions. The amplified PCR products were run on 2.5%(w/v) agarose gel with ethidium bromide stain and visualized byusing Gel Logic 212 Imaging Systems (Eastman Kodak Company,Rochester, NY).

The single PCR amplicons of 28S rRNA, and cox1 were excisedfrom the gel and purified using QIAquick Gel Extraction (Qiagen,Inc., Valencia, CA, USA) according to the manufacturer’s recom-mendation. The purified PCR products were sent to MacrogenCorporation (Rockville, MD, USA) for direct sequencing using thesame primer pair used in PCR amplification to obtain the bothstrands reads. The resulting sequences were imported, read, edited

manually if necessary, and analyzed using the software Geneiousversion 8.0.4 (Biomatters Ltd., Auckland, NZ). The sequences werealigned and compared with the previously published sequences inNCBI database by BLASTn analysis.

J.G. Trupkiewicz et al. / Veterinary Parasitology 216 (2016) 52–58 55

d mul

3

iwta

Fig. 1. Viscera of a roller pigeon. Note enlarged liver with depressed areas, an

. Results

At necropsy, all four birds had similar gross lesions. Birds were

n good post mortem and nutritional condition and the hen birds

ere in production. Subcutaneous hemorrhage was noted beneathhe skin of the cervical region. The coelomic cavity contained a largemount of clear gelatinous fluid. The liver was pale, with miliary

tiple foci of pale areas, indicative of necrosis. The adipose tissue is jaundiced.

pinpoint gray–white foci throughout the parenchyma. Some liverlesions had depressed centers (Fig. 1). The spleen was also enlarged.The kidneys were also pale.

Impression smears of the liver showed protozoal merozoites(Fig. 2A). Microscopic lesions were confined mainly to the liverand intestines in all birds. The liver had multifocal and coalescing,marked foci of subacute hepatic necrosis with marked infiltrates

56 J.G. Trupkiewicz et al. / Veterinary Parasitology 216 (2016) 52–58

Fig. 2. Schizogonic stages of S. calchasi in liver of a pigeon. A, impression smear stained with Giemsa. B–E, 2 �m plastic-embedded sections stained with hematoxylin ande imagen eads).a a res

oeh

osin. F, 1 �m resin embedded section stained with Toluidine blue. Bar applies to all

ucleus is lobulated. (B) Immature schizont with lobulation of the nucleus (arrowht the periphery. A few merozoites are in the center of the schizont that appear like

f macrophages, lymphocytes, and fewer plasma cells and het-rophils. In these foci, there were numerous protozoa. Intestinesad moderate lymphoplasmacytic infiltrates in the lamina propria,

s. (A) An immature schizont with merozoites (arrow) budding from the surface. The (C and D) Multinucleated schizonts. (E) A nearly mature schizont with merozoitesidual body. (F) Mature schizont with merozoites.

in some cases associated with large ascaridoid nematodes, and coc-cidian stages.

Protozoal schizonts and merozoites were seen in liver of all fourbirds (Table 2). A few schizonts were present in spleens of two of

J.G. Trupkiewicz et al. / Veterinary Parasitology 216 (2016) 52–58 57

Fig. 3. Transmission electron microscopy image of a longitudinally cut merozoite.Aws

tmwzmtet(

far(wKtTls4Sa

scecwa

4

ofi

Fig. 4. PCR amplification of DNA extracted from liver of four pigeons to distinguishinfections among Sarcocystis calchasi, S. columbae, and Sarcocystis sp. ex Accipiternisus. Lane M: 50 bp DNA ladder (New England Biolabs), Lanes 1–4: S. calchasi specificprimer pairs. Lanes 5–8: S. columbae specific primer pairs. Lanes 9–12: Sarcocystissp. ex A. nisus specific primer pairs. The semi-nested PCR resulted in amplificationof ∼136 bp long, single band from the DNA of all four pigeons by the S. calchasispecific primer pairs. There was no PCR amplification visible in gel by S. columbae

lthough the tissue was degenerating the organelles in the merozoite are fairlyell preserved. Note, a conoid (co), a nucleus (nu), and several micronemes (mn)

cattered in the cytoplasma that has no rhoptries.

he pigeons examined. Developing and mature schizonts, and freeerozoites were present in liver (Fig. 2B–F). By TEM, the schizontsere upto 40 �m in diameter and contained up to 25 mature mero-

oites. The merozoites were up to 6 �m long. The schizonts anderozoites stained faintly in 5 �m histological sections with hema-

oxylin and eosin. Images seen in 2–3 �m sections methyl glycolatembedded sections are shown in Fig. 2. By TEM, the merozoites con-ained a conoid, a nucleus, several micronemes, but no rhoptriesFig. 3). The organisms did not react with S. neurona antibody.

The 28S rRNA locus was amplified by PCR using DNA extractedrom the liver of one pigeon (#38156). Direct sequencing of PCRmplicons resulted the unambiguous sequences of the nuclear DNAegion; 28S rRNA (915 bp), and the mitochondrial DNA locus; cox1626 bp, and 484 bp). These sequences were submitted to GenBankith accession numbers KU220951—(28S rRNA), and—KU220952,U220953 (cox1). The 28S rRNA sequence shared the highest iden-

ity with that of Sarcocystis sp. ex Columba livia (FJ232949) 100.0%.he cox1 locus was amplified by PCR using DNA extracted from theiver of 3 of 4 pigeons (#38156, #38157, #38158). The partial cox1equence was obtained from two pigeons (626 bp for #38156, and84 bp for #38157). The both sequences shared 99% identity witharcocystis lutrae (KM657808), and Sarcocystis arctica (KF601321),nd 95% and 98% with Sarcocystis rileyi (KJ396582).

The semi-nested PCR resulted in amplification of 136 bp long,ingle band from the DNA (Fig. 4) of all four pigeons by the S.alchasi specific primer pairs; SCa1/SCa2 and SCa1/SNCa3 (Oliast al., 2011). There was no PCR amplification visible in gel by S.olumbae and Sarcocystis sp. ex A. nisus specific primers. The birdsere negative for avian influenza, paramyxovirus, Salmonella spp.

nd Chlamydia sp.

. Discussion

The diagnosis of S. calchasi in the present study was basedn histological and molecular data. Parasite identity was con-rmed by species specific amplification of ITS-1 region in all four

and Sarcocystis sp. ex A. nisus specific primers. DNA amplification by PCR confirmedS. calchasi infection in all four pigeons.

pigeons by previously designed primers, and 100.0% identity of28S rRNA sequence obtained from one pigeon with previously pub-lished sequence of S. calchasi [FJ232949 (Sarcocystis sp. ex Columbalivia)]. Amplification of ITS-1 region has been used in several stud-ies to identify Sarcocystis species because of higher intra speciesvariation in this region of DNA (Dubey et al., 2015a). The cox1sequences obtained from two pigeons were not compared withS. calchasi sequences due to unavailability of reference sequencesin the database. The sample which was examined by TEM, didnot give unambiguous sequences of 28S rRNA, and cox1 loci byPCR-DNA sequencing. It may be related to quality, and quantityof DNA; DNA was extracted from tissue of pigeons not from cul-tured parasites or individual sarcocyst. There are several aspectsof the diagnosis that warrant comments. Before the discovery ofS. calchasi and PPE in pigeons (Olias et al., 2009, 2010b,d), mostof the Sarcocystis-associated mortalities in avian species, partic-ularly psittacines, were attributed to Sarcocystis falcatula (Dubeyet al., 2015a). In S. falcatula infections respiratory distress dueto pneumonia is the major clinical sign. In pigeons experimen-tally infected with S. falcatula, more than 99% of schizonts werein lung, and they developed exclusively in vascular endothelia(Neill et al., 1989; Smith et al., 1990). Only a few (<0.1%) schizontswere in liver and spleen. Schizonts in liver were in Kupffer cells.In additions to passerine birds, S. falcatula infections have beendiagnosed in a free ranging bald eagle (Haliaeetus leucocephalus),and a Great horned owl (Bubo virginianus) (Wünschmann et al.,2009, 2010). Additionally, Sarcocystis neurona—like infections havebeen reported in a bald eagle (Olson et al., 2007). However, S. neu-rona has not been identified definitively in tissues of any avianspecies (Dubey et al., 2015b). Unlike S. falcatula, S. neurona sch-izonts occur mainly in neural cells and not in vascular endothelium.Opossums (Didelphis spp.) are definitive hosts for S. falcatula and S.neurona, S. lindsayi, and other related species and infections areconfined to the Americas (Dubey et al., 2015a). Unlike opossums,hawks, the definitive hosts for S. calchasi, have a worldwide distri-bution.

In addition to the type of cell for the development of S. calchasi,

S. neurona, and S. falcatula schizonts, reactivity to antibodiesin immunohistochemistry is useful in diagnosis. An immunotestbased on S. calchasi antibodies has been described by Olias et al.

5 ary Pa

(cgSwacuwSror

atnsInstsceSowoaowpspi

A

laU

R

D

D

D

D

S.B., Barr, B.C., 2010. Natural fatal Sarcocystis falcatula infections in free-ranging

8 J.G. Trupkiewicz et al. / Veterin

2013). Two rabbits were immunized with formalin-killed cellulture-derived merozoites of a S. calchasi isolate from Northernoshawk. The antibodies reacted with schizonts and sarcocysts of. calchasi (Olias et al., 2013). However, specificity of this antibodyas not published with respect to cross reactivity to S. neurona

nd S. falcatula. In the present study, there was no reactivity of S.alchasi schizonts to the S. neurona polyclonal rabbit antibodies wesed. We can confirm the results reported by Rimoldi et al. (2013)ho found that S. calchasi schizonts and sarcocysts did not react to

. neurona polyclonal and monoclonal antibodies. We did not testeactivity to our S. falcatula antibody (Dubey et al., 2001a,b) becausef concern of specificity, but Rimoldi et al. (2013) reported no crosseactivity between S. calchasi and S. falcatula.

In the present study most schizonts were present in the hep-tocytes of the pigeons. Electron microscopy helped to locate theypes of host cell parasitized. It is noteworthy that neither schizontsor lesions were seen in the brains and muscles. We searched forarcocysts but none were found in the examined muscles (Table 2).n all previously reported cases of S. calchasi infections in pigeons,eurological signs were the main presenting signs (Table 1) andarcocysts were seen in muscles. Considering the life cycle eventshat followed in pigeons experimentally infected with sporocysts,chizonts were first seen in liver (around 7–10 days p.i.), and sar-ocysts were first detected 20 day p.i. (Maier et al., 2015a; Oliast al., 2010b). It appears that the pigeons ingested a heavy dose of. calchasi sporocyst-contaminated water or feed within 2 weeksf their death. The pigeon colony is housed in outdoor enclosures,hich are partially covered, however portions of the roof consist

f open mesh. Periodically, feral hawks are seen on zoo grounds,nd occasional losses of collection birds, primarily waterfowl haveccurred due to hawk predation. Prior to the pigeon deaths, a hawkas seen roosting in a tree near the pigeon enclosure, and this isresumed to be the source of the parasitic infection. The enclo-ure has since been covered completely, and other managementractices have been instituted to prevent contact with feral birds,

ncluding hawks.

cknowledgments

Mention of trade names or commercial products in this pub-ication is solely for the purpose of providing specific informationnd does not imply recommendation or endorsement by the USDA;SDA is an equal opportunity provider and employer.

eferences

ubey, J.P., Mattson, D.E., Speer, C.A., Baker, R.J., Mulrooney, D.M., Tornquist, S.J.,Hamir, A.N., Gerros, T.C., 1999. Characterization of Sarcocystis neurona isolate(SN6) from a naturally infected horse from Oregon. J. Eukaryot. Microbiol. 46,500–506.

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