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Virology 339 (20

Rapid Communication

Phylogenetic analysis of small-ruminant lentivirus subtype B1 in mixed

flocks: Evidence for natural transmission from goats to sheep

Giuliano Pisonia,*, Antonio Quassob, Paolo Moronia

aDepartment of Animal Pathology, Hygiene and Veterinary Public Health, University of Milano, via Celoria 10, 20133 Milano, ItalybDepartment of Prevention, Veterinary Services, Animal Health Division, ASL n. 19 Asti, Italy

Received 26 April 2005; returned to author for revision 16 May 2005; accepted 8 June 2005

Available online 7 July 2005

Abstract

Small-ruminant lentiviruses (SRLV), consisting of the caprine arthritis–encephalitis virus (CAEV) and the maedi–visna virus (MVV),

cause chronic multisystemic infections in goats and sheep. The SRLV subtype B1, characterized by the prototypic strain CAEV-CO, has a

worldwide distribution and, remarkably, has been isolated exclusively from goats, suggesting potential host specificity. To test this

hypothesis, SRLV pol sequences were obtained by PCR amplification from blood samples of seropositive dairy goats and sheep living in

mixed flocks. Phylogenetic analysis of these sequences demonstrates that SRLV subtype B1 does cross the species barrier under field

conditions through direct contact between adult animals. This implies that SRLV control programs targeting only sheep or goats can no

longer be proposed (based on a putative species specificity of the SRLV subtype B1).

D 2005 Elsevier Inc. All rights reserved.

Keywords: Small-ruminant lentivirus; Phylogeny; Interspecies transmission

Introduction

Caprine arthritis–encephalitis virus (CAEV) and maedi–

visna virus (MVV) are small-ruminant lentiviruses (SRLV)

that infect goats and sheep (Narayan et al., 1980; Pasick,

1998; Pepin et al., 1998; Sundquist, 1981). Recent work

based on long sequences in gag and pol of isolates with

worldwide distribution has demonstrated that the SRLV can

be divided into four principal sequence groups A to D,

which differ by 25 to 37% in gag and pol sequences.

Sequence groups A and B are further divided into different

subtypes that differ from each other by 15 to 27%. Group A

contains at least 7 subtypes, A1 to A7, and group B contains

two subtypes, B1 and B2 (Shah et al., 2004a). To date,

subtypes A1 and A2 have been isolated only from sheep,

and subtypes A5, A7, and B1 and groups C and D have

0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2005.06.013

* Corresponding author. Fax: +39 02 50318079.

E-mail address: giuliano.pisoni@unimi.it (G. Pisoni).

been isolated only from goats. In contrast, subtypes A3, A4,

A6, and B2 have been isolated from both sheep and goats.

The fact that some SRLV subtypes have been isolated

from both sheep and goats indicates that interspecies

transmission must have occurred at least once in each of

these subtypes, although the frequency and direction of

transmission remain unknown (Leroux et al., 1997; Rava-

zzolo et al., 2001; Rolland et al., 2002; Zanoni, 1998).

Recently, direct evidence for natural interspecies trans-

mission of SRLV subtype A4 has been observed (Shah et

al., 2004b).

Most transmissions of SRLVamong goats or sheep occur

through the ingestion of virus-infected colostrum or milk by

the newborn (De Boer et al., 1979; Greenwood et al., 1995;

McGuire et al., 1990; Rowe and East, 1997). Less efficient

transmission has been associated with prolonged contact

with infected animals (East et al., 1987; Greenwood et al.,

1995; Rowe and East, 1997). Contact transmission is seen

particularly with the maedi form of MVV infection, in

which respiratory exudates may play an important role

(Narayan and Cork, 1985). The widespread distribution of

05) 147 – 152

Rapid Communication148

these viruses in certain regions and the resulting economic

losses have led to segregation-based virus eradication

programs for both goats and sheep in several countries

(Greenwood et al., 1995; Houwers et al., 1987; Peretz et al.,

1994; Rowe and East, 1997; Scheer-Czechowski et al.,

2000). No regulated or compulsory SRLV eradication

programs currently exist in Italy, but many attempts at

eradication have been started on a voluntary basis in single

farms (Turin et al., 2005) and in small geographic regions

(Quasso, 2000, data unpublished). Complete eradication of

lentivirus infection in the goat and sheep population will be

possible only when detailed information about viral

variability within geographical regions, infected flocks,

and individual animals becomes widely available. In this

study, we demonstrate for the first time the interspecies

transmission of SRLV subtype B1 from goats to sheep in

mixed flocks.

Fig. 1. Serology and epidemiology of goats and selected sheep from farm

A. Each horizontal line identifies an animal. The +, �, and +/� symbols

indicate the serological status of an animal at a given time (negative (�),positive (+), and indeterminate (�/+)). Thirteen goats were sampled four

times between 2000 and 2004, while the other goats were sampled 1 to 3

times because they were culled or newborns. In spring 2001, sixteen goats

were purchased for genetic improvement from a farm with unknown CAEV

serological status. Goats and sheep selected for this investigation are boxed.

Results

In autumn 2001, we investigated 49 dairy goats from

farm A which had been certified CAEV-free from 1995 to

spring 2000 after a voluntary eradication program but had

become infected through 16 goats that were purchased from

another farm (not certified CAEV-free) for genetic improve-

ment. Nine goats were found to be seropositive, and five

were seroindeterminate and, although all seropositive or

seroindeterminate animals were culled, the farm has been

unable to regain the status of certified CAEV negativity. In

the annual testing of the following year (2002), 11 goats

were found seropositive, and all of the goats were sero-

positive at the end of 2004. Of the seropositive goats, 15%

developed clinical symptoms of arthritis. In the same farm, a

group of dairy sheep (eleven animals) was kept separated

from the goats in another building and was not subjected to

annual serological control. When the 11 dairy sheep of the

farm were tested in spring 2000, none were seropositive.

Later in the same year and for the first time ever on the farm,

goats and sheep were mixed during grazing on a common

pasture, and in the following serological test, 50% of sheep

were seropositive. At the end of 2004, all sheep were

seropositive (Fig. 1).

Evidence for interspecies transmission involving SRLV

subtype B1 was also found when other groups of sero-

positive goats kept together with sheep were investigated

(farms B and C). Neither farm had been subjected to an

eradication program, so serological information was not as

detailed as for farm A. In May 1999, goats from farm C

were investigated after introduction of a new stock of sheep;

80% of goats were found to be seropositive, while all sheep

were seronegative. The following year, one sheep was found

to be seropositive. The flock was tested again in 2004, and

the level of seropositivity had increased to 50%. Goats and

sheep from farm B were investigated only during 2004, with

a seroprevalence of 100% in both goats and sheep. Little

information is available on the route of infection in this

interspecies transmission. In both farms, the dairy goats and

sheep were housed in the same large barn, sharing a

common milking parlor and grazing together on the same

pastures.

We amplified a 512 bp fragment comprising of a portion

of the sequence of the pol gene (the proximal one-third of

Rapid Communication 149

RT) from five seropositive goats and five seropositive sheep

collected from the three mixed flocks. Provirus amplifica-

tion was successful in all goats and sheep. Analysis of the

pol sequences demonstrated that all proviruses isolated from

goats and sheep belonged to SRLVof subtype B1 (according

to the classification by Shah et al., 2004a; Fig. 2). Subtype

B1 contains low divergent strains isolated from France,

Brazil, and Switzerland and is related to the prototype

isolate CAEV-CO. The Italian isolates formed three

separated clusters, according to the farm of origin, and

were characterized by low divergence, with a mean

similarity degree (Tstandard deviation) of 92.3% T 0.8%

(Fig. 3). In comparison, the mean divergence (TSD) betweenviruses belonging to subtype B1 that were isolated from

animals originating from epidemiologically unlinked

regions was 82.1% T 1% (Shah et al., 2004a). The different

SRLV isolates that we examined do not cluster according to

ovine or caprine host species. None of the genetic groupings

appeared to correlate with clinical symptoms in affected

animals. Strains isolated from farm A were closely related,

exhibiting an intra-farm variation of 0.6 to 2.0% nucleotide

substitutions. The strains isolated from goats and sheep

Fig. 2. Phylogenetic relationship of goat and sheep lentiviruses isolated from Italia

constructed by the joining method and is based on the distances calculated wit

Bootstrap values are based on 1000 repetitions. SRLV isolates of the present inve

from different geographical areas (Brazil, France, Iceland, Norway, South Africa, a

from the present study are labeled with AT representing the province of origin

respectively. Database-derived sequences are denoted with their GenBank accessio

groups A, B, and C (Shah et al., 2004a).

within the two clusters (B and C) were closely related,

exhibiting an intra-farm variation of 1.4% to 5% (farm B)

and 0.4% to 3.5% (farm C) nucleotide substitutions.

Discussion

Analysis of selected sequences in the pol coding region

of naturally occurring lentivirus in goats and sheep from

mixed flocks indicates that these viruses have a complex

population structure. Some subtypes (A3, A4, A6, and B2)

of the large and diverse SRLV groups are found among both

goats and sheep, thus presenting evidence for more than one

event of interspecies transmission in the past (Leroux et al.,

1997; Zanoni, 1998; Shah et al., 2004a), while SRLV

subtype B1 strains were found only in goats in different

studies (Ravazzolo et al., 2001; Valas et al., 1997; Zanoni,

1998) and related to the prototype isolate CAEV-CO.

Although interspecies transmission has been achieved

experimentally (Banks et al., 1983; Smith et al., 1985),

the natural transmission of the SRLV subtype B1 across the

species barrier has never been documented directly, suggest-

n mixed dairy flocks (farm A, farm B, and farm C). The unrooted tree was

h the F84 substitution model, as described under Materials and methods.

stigation are shown together with available database sequences originating

nd United States). Farms A, B, and C are denoted by gray areas. Sequences

, a number representing animal, and S or G representing sheep or goat,

n number. Tree branches are labeled according to the classification based on

Fig. 3. Phylogenetic analysis of goat and sheep lentiviruses isolated from

Italian mixed dairy flocks (A, B, and C). The unrooted tree was constructed

by the neighbor joining method and is based on the distances calculated

with the F84 substitution model, as described under Materials and methods.

Bootstrap values are based on 1000 repetitions. Sequences from the present

study are labeled with AT representing the province of origin, a number

representing animal, and S–G representing sheep or goat, respectively.

Rapid Communication150

ing potential host specificity. In this study, we have

phylogenetically analyzed viruses isolated from goats and

sheep located in the same flocks and exposed to natural

SRLV infection. We thus present the first direct evidence

that the SRLV subtype B1 is transmitted from goats to sheep

by natural horizontal infection between adult animals. While

the direction of transmission is unclear in small ruminants

on farm B, phylogenetic analysis and serological epidemi-

ology of strains isolated from farm A and farm C

demonstrate goat-to-sheep transmission. With regard to

goat-to-sheep transmission, farm A had been always

certified CAEV-free prior to the purchase of new animals,

and this therefore strongly suggests that the virus was newly

introduced into the farm by these 16 goats and that it was

later transmitted to the sheep. Farm C was never subjected

to an eradication program or to annual serological control,

so it is not possible to know when the virus was initially

introduced in the flock, however, the goats represented the

only possible source of infection for the initially sero-

negative sheep brought into the herd. Direct contact between

goats and sheep represents an important risk factor in viral

transmission as demonstrated by the fact that the first time

goats and sheep from farm A shared the same pasture, 50%

of sheep seroconverted in the following 6 months. This

result may be explained by aerosol transmission from the

respiratory tract. Even though the lungs are not the major

target organ for SRLV in goat species, SRLV infection in

goats may also manifest itself as a chronic progressive

interstitial pneumonia, so it is conceivable that a similar

mechanism may be involved in horizontal transmission

from goats to sheep. Originally, MVV and CAEV were

considered two distinct yet closely related viruses that infect

sheep and goats, respectively. As has been confirmed, these

viruses are not strictly host-specific. It is unclear at what rate

these viruses spread within the small ruminant host

population, but now the findings that SRLV subtype B1

cross the species barrier must be taken into account in

control programs. Specifically, the same regulations should

apply to both MVV and CAEV, and control programs that

only target sheep and goats alone are no longer acceptable.

Materials and methods

Animal specimens

Goats and sheep from mixed flocks were identified by

the Local Sanitary Association (ASL) of Asti province.

Serological testing was performed by the Istituto Zoopro-

filattico Sperimentale of Piedmont (MVV/CAEV elisa test,

Institut Pourquier, Montpellier, France). Ten milliliter of

blood from each animals was collected in 2 mM EDTA

vacutainers. Peripheral blood mononuclear cells (PBMCs)

were isolated from the blood samples and purified by

centrifugation through Ficoll\ gradients, as previously

described (Narayan et al., 1983). DNA was extracted from

pellets containing >5 � 106 cells using commercial silica-

gel spin-columns selective for genomic DNA (QIAamp

DNA Blood Mini Kit, QIAGEN), according to the

manufacturer’s instructions. Purified DNA stocks were

stored at �70 -C until use.

Sequence amplification and sequence analysis

Proviral sequences from 523 bp region in the pol gene

were amplified by nested PCR by using amplification

primers specific for the pol gene of CAEV_CO (M33677):

POLEX5 (1863–1882) 5V-AGCACCACCTATGGTT-

CAGG-3V and POLEX3 (2828–2847) 5V-TCCTAGC-

CATTTTGCTGGAT-3V for the first round, and POLIN5

(2189–2212) 5V-AATTGAAAGAGGGATGTACGGGTC-3V and POLIN3 (2689–2712) 5V-ATCATCCATATATATGC-CAAATTG-3V for the second round. Specific primers were

also chosen to amplify the caprine glyceraldehyde 3-

phosphate dehydrogenase (GAPDH) as an internal control

for the quality and quantity of the DNA samples. These

Rapid Communication 151

primers were as follows, according to the partial caprine

GAPDH sequence (Harmache et al., 1996): CGAP1 (sense

primer), 5V-GTTCCACTATGATTCCACCC-3V (25–44 nt);

CGAPR1 (antisense primer), 5V-TCCCTCCACGATGC-CAAA-3V (406–386 nt). After the amplification, PCR

products from each sample were separated by electro-

phoresis on a 2% agarose gel with ethidium bromide (1 Ag/ml). Specific products were purified with Perfectprep Gel

Cleanup Kit (Eppendorf), according to the supplier’s

instruction, and sequenced by CRIBI Services (CRIBI,

Padova, Italy) on an ABI377 sequencer by using the ABI

PRISM dye-terminator cycle sequencing ready reaction kit

with Amplitaq DNA polymerase (Perkin-Elmer, Applied

Biosystems). Multiple alignment of the sequences was

generated with ClustalW (Thompson et al., 1994) and

edited with BioEdit 7.0 (Hall, 1999).

Phylogenetic analysis

Pairwise genetic distances were calculated by using

MEGA version 2.1 (Kumar et al., 2001) with the Tamura-

Nei substitution model, applying the default setting and with

the exception that all sites with ambiguous codes and gaps

were ignored. Phylogeny construction was carried out using

the neighbor joining (NJ) method (Saitou and Nei, 1987)

implemented in MEGA with the Tamura-Nei gamma

distance (Tamura and Nei, 1993). The statistical confidence

of the topologies was assessed with 1000 bootstrap

replicates (Felsenstein, 1985). The shape parameter alpha

for a discrete gamma distribution of substitution rates,

which accommodates for substitution rate variation across

sites (e.g. higher substitution rates in mutational hot spots;

Chang, 1996; Yang, 1995) and the transition/transversion

rate ratio parameter kappa were estimated simultaneously by

maximum likelihood using Yang’s BASEML program

implemented in the PAML (Phylogenetic Analysis by

Maximum Likelihood) program package (Yang, 1996a,

1996b). The substitution model assumed was Felsenstein’s

F84 model, which accommodates unequal base frequencies

and transition/transversion rate bias (Felsenstein and

Churchill, 1996). In order to reduce the computing time of

PAML and to get standard errors for the parameter

estimates, all calculations were performed with user trees,

estimated with Felsenstein’s DNAML (DNA Maximum

Likelihood program) implemented in the Phyliph program

package, version 3.6b (Felsenstein, 1981). To improve the

tree search efficiency, the input order of each data set was

jumbled (randomized) up to ten times, and local rearrange-

ments were allowed.

Nucleotide sequence accession numbers

All new nucleotide sequences were deposited in the

GenBank database and are available under accession

number DQ013234 to DQ013243 for the sequences from

farm A, under accession number DQ013224 to DQ013233

for farm B and under accession number DQ013214 to

DQ013223 for farm C.

Acknowledgments

We are thankful to G. Bertoni and P. Boettcher for critical

reading of the manuscript. This work was supported partly

by the CRAFT Project ‘‘S.R.L.V.’’ No QLK2-CT-2001-

70356 and by the Italian FIRST 2001 (G. Ruffo).

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