- Email: [email protected]
First report of BVDV circulation in sheep in Argentina
Preventive Veterinary Medicine 90 (2009) 274–277
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Short communication
First report of BVDV circulation in sheep in Argentina S. Julia´ *, M.I. Craig, L.S. Jime´nez, G.B. Pinto, E.L. Weber Instituto de Virologı´a, Centro de Investigaciones en Ciencias Veterinarias y Agrono´micas, INTA – Castelar, CC 25 (1712), 1686 Moro´n, Buenos Aires, Argentina
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 January 2009 Received in revised form 8 April 2009 Accepted 12 May 2009
Pestiviruses are capable of infecting a wide range of animals within the order Artyodactila. Currently, the genus Pestivirus includes Bovine Viral Diarrhea Virus 1 (BVDV-1) and 2 (BVDV-2), Border Disease Virus (BDV), and Classical Swine Fever Virus (CSFV). BVDV-1, BVDV-2 and BDV are able to cross species barrier to infect a wide range of hosts, whereas CSFV is restricted to domestic pigs and wild boars. In Argentina, 70% of cattle are seropositive to BVDV. Although there were some serological studies in llamas, alpacas and buffaloes, no reports existed about the circulation of BVDV in sheep in Argentina. Based on these, 54 blood samples of healthy ovines were analysed by serology. The results showed that 46.3% of the analysed sheep were seropositive to BVDV-1, 13% to BVDV-2 and 20.4% for both BVDV-1 and BVDV-2. The molecular analysis confirmed the presence of BVDV-1a in some samples. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Pestivirus BVDV Reservoir Sheep Argentina
1. Introduction According to the International Committee on Taxonomy of Viruses, genus Pestivirus includes four species: Bovine Viral Diarrhea Virus 1 (BVDV-1), Bovine Viral Diarrhea Virus 2 (BVDV-2), Classical Swine Fever Virus (CSFV) and Border Disease Virus (BDV). Also, a tentative fifth species is represented by a Pestivirus of giraffe. In particular for BVDV-1, Vilcek et al. (2004) described several genetic groups (BVDV-1a–j). At first, Pestiviruses were classified according to their host, however, later studies confirmed that, at least, BVDV and BDV are not host specific (Hamblin and Hedger, 1979). While BVDV may infect cattle, sheep, swine, goats and other ungulate species (Becher et al., 1999, 1997; Snowdon and French, 1968; Terpstra and Wensvoort, 1988); some reports confirmed the presence of BDV in sheep, swine and goats (Vilcek and Bela´k, 1996; Vilcek et al., 1997). However CSFV seems to be restricted to pigs and wild boars (Becher et al., 2003; Vilcek and Nettleton, 2006).
* Corresponding author. Tel.: +54 11 4621 1278/1447; fax: +54 11 4621 1743. E-mail address: [email protected] (S. Julia´). 0167-5877/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2009.05.015
An important condition for keeping BVDV in animal populations is the presence of individuals with immunotolerance and persistent infection, which are resulting from transplacental infection of the fetus before the onset of immunological maturity (Toplak et al., 2004). Persistently infected (PI) animals are the main source of virus transmission. These animals spread virus throughout their life. PI animals have been reported for cattle (Duffell and Harkness, 1985), pigs (van Oirschot and Terpstra, 1977; Meyer et al., 1981; Paton et al., 1992), sheep (Nettleton et al., 1992) and eland (Anderson and Rowe, 1998). Particularly in sheep, it was demonstrated that both BVDV-1 as BVDV-2 may infect them naturally (Sullivan and Akkina, 1995; Vilcek et al., 1997) as well as the biology of BVDV-2 infection in pregnant sheep is essentially similar to that of BVDV-1 in pregnant cattle and sheep (Scherer et al., 2001). Moreover, transmission between small ruminants and cattle, in both ways, has been demonstrated (Carlsson, 1991; Løken et al., 1991; Carlsson and Bela´k, 1994; Paton et al., 1995). Infection with BVDV may result in a wide range of clinical conditions, from virtually asymptomatic to fatal disease (Nettleton and Entrican, 1995), causing significant economic losses to the livestock industry. Even though in Argentina the prevalence of BVDV antibodies in adult cattle is around 70% (Rweyemamu
S. Julia´ et al. / Preventive Veterinary Medicine 90 (2009) 274–277
et al., 1990; Pacheco and Lager, 2003), vaccination is not compulsory and it is only intended to livestock. Some authors reported that both BVDV-1 and BVDV-2 are present in cattle in the country (Jones et al., 2001). In addition, there are some serological studies on captive llamas (Puntel et al., 1999) and antigen detection in different organs of water buffaloes by immunohistochemistry (Craig et al., 2008); however, there are no studies on
275
BVDV in ovines. The sheep industry is the second in importance of livestock production in Argentina. Particularly, the Patagonian region has approximately 75% of sheep population. Moreover, in the last 5 years the sheep population is growing up and currently is about 15 million. The most important subproduct is wool and then meat. The aim of this report was to study the viral circulation of BVDV in sheep from several farms of Argentina.
Fig. 1. Cladogram of the 50 UTR sequences of BVDV from Argentinean sheep (2a, 3a, 5a, 8b and 12a) and from different geographical regions.
276
S. Julia´ et al. / Preventive Veterinary Medicine 90 (2009) 274–277
2. Materials and methods Our laboratory received 54 blood samples with EDTA of healthy ovines for genotipification study. Some of the samples (20/54) were taken from a slaughterhouse in Rı´o Gallegos (Santa Cruz) and the others (34/54) from 14 different farms in the provinces of Santa Cruz and Tierra del Fuego (Patagonia Argentina). The samples were fractionated in fresh blood and plasma (obtained by centrifugation) and stored at 80 8C, until use. Virus neutralization test for each of the 54 plasma was determined against BVDV-1 and BVDV-2, using the cytopathic strains NADL and 61380 (AF417986), respectively. For the seroneutralization assay, a fixed dose of virus (100 TCID50) was incubated for 1 h at 37 8C with twofold plasma dilutions in growth medium. Cells were added and cultures were grown in microtitre plates for 4 days. Virus growth or neutralization was monitored by the visualization of the cytopathic effect. Positive and negative control sera were used in each assay. In addition to serological assay, 34 samples obtained from the farms were chosen for RNA extraction, from blood or plasma. The amplification of a 288 bp product by RT-PCR for the 50 UTR was done as described by Vilcek et al. (1994). PCR products were visualized in 1.8% agarose gel stained with ethidium bromide. Once the amplicons were purified, they were directly sequenced on an Applied Biosystems 322 equipment. The obtained sequences were edited with the BioEdit software (BioEdit sequence alignment editor; version 5.0.9) and subsequently, were aligned with CLUSTALX program (Version 1.8.3). The phylogenetic tree was calculated by the Neighbor-Joining method, using the DNADist and Neighbor modules present in the PHYLIP package (Felsenstein, 1989).
polymerase reaction. Taking into account the serological assays, the results show that three positive samples by RTPCR correspond to animals that were seronegative for both BVDV species, another two samples to seropositive animals for BVDV-1, and other two samples to seropositive sheep for both BVDV-1 and BVDV-2. The last two samples showed cytotoxicity to cell culture, so the serological status of these sheep could not be determined. The sequence analysis of five of these samples showed higher identity with BVDV-1. Phylogenetic analysis confirmed that they belonged to subtype BVDV-1a (Fig. 1). It is important to remark that none of the obtained sequences were consistent with Border Disease Virus. This result was the expected one, considering that Argentina is a free country of BDV. The presence of seronegative ovines for both BVDV-1 and BVDV-2 but a positive detection of viral nucleic acid, strongly suggests the existence of PI animals (Saliki and Dubovi, 2004). Although the results indicate that sheep were in contact with BVDV-2, the circulation of this BVDV species could not be confirmed. Although it is known that BVDV infects sheep, this study reports, for first time, the circulation of BVDV in ovines in Argentina. Most farms in Patagonia have sheep but few of them share their habitat with bovines. This fact, coupled with the presence of persistently infected animals suggests that ovines might be considered as a natural reservoir of BVDV in this region. In addition, it is interesting to remark that in one of the farms in which there is no cattle, all sheep sampled (samples 2–5) were positive by PCR, indicating the circulation of the virus among them and without the participation of cattle. 4. Conclusion
3. Results and discussion All the samples belonged to animals without any clinical sign of disease. Serological assays showed that 46.3% of samples (25/54) were positive to BVDV-1, 13% (7/ 54) were positive to BVDV-2, 20.4% (11/54) were positive for both viral species (BVDV-1 and BVDV-2), and 14.8% (8/ 54) were negative for BVDV-1 and BVDV-2. Unfortunately, 5.5% of samples (3/54) showed cytotoxicity to cell culture. These results confirm that ovines were in contact both with BVDV-1 and with BVDV-2, and also show the existence of co-circulation of both viral species in some farms, evidenced by the presence of antibodies against BVDV-1 and BVDV-2. These results are in accordance with those reported by Jones et al. (2001), where the prevalence of BVDV-1 was higher than that found to BVDV-2. Furthermore, the presence of antibodies against BVDV-1 and BVDV-2 in the same animal suggests the sequential exposures to them. Detection of BVDV by RT-PCR was performed on fresh blood samples but unfortunately, some blood samples were not enough to perform both virus neutralization test and RT-PCR. So, RT-PCR was performed on 34 blood samples and nine samples (26.4%) were positive for the detection of viral nucleic acid. Probably, the hemolysis present in most of these samples has inhibited the
We conclude that BVDV-1 circulates in sheep of the Patagonia Argentina and the probable circulation of BVDV2. We also present evidence about the existence of PI sheep with BVDV-1a, acting as natural reservoirs in this region with or without the participation of cattle. From the foregoing the need for a more comprehensive study encompassing the rest of the country is evident. BVDV vaccination in Argentina is not compulsory, however, the results of this work must be taken into account for the knowledge and understanding of virus circulation, and must be considered for future eradication programs. Acknowledgements The authors are grateful to the IMYZA Sequence Service. This work was supported by the FONCyT (Bid 1201/OCAR08-09627). E.L.W. is member of the Scientist Career of CONICET. References Anderson, E.C., Rowe, L.W., 1998. The prevalence of antibody to the viruses of bovine virus diarrhoea, bovine herpes virus 1, rift valley fever, ephemeral fever and bluetongue and to Leptospira sp in freeranging wildlife in Zimbawe. Epidemiol. Infect. 121, 441–449.
S. Julia´ et al. / Preventive Veterinary Medicine 90 (2009) 274–277 Becher, P., Orlich, M., Shannon, A.D., Horner, G., Ko¨nig, M., Thiel, H.-J., 1997. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J. Gen. Virol. 78, 1357–1366. Becher, P., Orlich, M., Kosmidou, A., Ko¨nig, M., Baroth, M., Thiel, H.-J., 1999. Genetic diversity of pestiviruses: identification of novel groups and implications for classification. Virology 262, 64–71. Becher, P., Avalos Ramirez, R., Orlich, M., Cedillo Rosales, S., Konig, M., Schweizer, M., Stalder, H., Schirrmeier, H., Thiel, H.-J., 2003. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311, 96–104. Carlsson, U., 1991. Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine virus diarrhoea virus. Vet. Rec. 128, 145–147. Carlsson, U., Bela´k, K., 1994. Border disease virus transmitted to sheep and cattle by a persistently infected ewe: epidemiology and control. Acta Vet. Scand. 35, 79–88. Craig, M.I., Venzano, A., Ko¨nig, G., Morris, W.E., Jime´nez, L., Julia´, S., Capellino, F., Blanco Viera, J., Weber, E.L., 2008. Detection of bovine viral diarrhoea virus (BVDV) nucleic acid and antigen in different organs of water buffaloes (Bubalus bubalis). Res. Vet. Sci. 85, 194–196. Duffell, S.J., Harkness, J.W., 1985. Bovine virus diarrhoea-mucosal disease infection in cattle. Vet. Rec. 117, 240–245. Felsenstein, J. 1989. PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 5: 164–166. Hamblin, C., Hedger, R.S., 1979. The prevalence of antibodies to bovine viral diarrhoea/mucosal disease virus in African wildlife. Comp. Immunol. Microbiol. Infect. Dis. 2, 295–303. Jones, L.R., Zandomeni, R., Weber, E.L., 2001. Genetic typing of bovine viral diarrhea virus isolates from Argentina. Vet. Microbiol. 81, 367–375. Løken, T., Krogsrud, J., Bjerka´s, I., 1991. Outbreaks of border disease in goats induced by a pestivirus-contaminated orf vaccine, with virus transmission to sheep and cattle. J. Comp. Pathol. 104, 195–209. Meyer, H., Liess, B., Frey, H.R., Hermanns, W., Trautwein, G., 1981. Experimental transplacental transmission of hog colera virus in pigs. IV. Virological and serological studies in newborn piglets. Zentrlbl. Veterinarmed. B 28, 659–668. Nettleton, P.F., Entrican, G., 1995. Ruminant pestiviruses. Br. Vet. J. 151, 615–642. Nettleton, P.F., Gilmour, J.S., Herring, J.A., Sinclair, J.A., 1992. The production and survival of lambs persistently infected with border disease virus. Comp. Immunol. Microbiol. Infect. Dis. 15, 179–188. Pacheco, J.M., Lager, I., 2003. Indirect method ELISA for the detection of antibodies against bovine diarrhea virus in bovine serum. Rev. Argent. Microbiol. 35, 19–23. Paton, D.J., Simpson, V., Done, S.H., 1992. Infection of pigs and cattle with bovine viral diarrhoea virus on a farm in England. Vet. Rec. 131, 185– 188.
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Paton, D.J., Carlsson, U., Lowings, J.P., Sands, J.J., Vilcek, S., Alenius, S., 1995. Identification of herd-specific bovine viral diarrhoea virus isolates from infected cattle and sheep. Vet. Microbiol. 43, 283–294. ´ Donnell, V.K., Marcovecchio, Puntel, M., Fondevila, N.A., Blanco Viera, J., O J.F., Carrillo, B.J., Schudel, A.A., 1999. Serological survey of viral antibodies in llamas (Lama glama) in Argentina. Zentralbl. Veterinarmed. B 46, 157–161. Rweyemamu, M.M., Fernandez, A.A., Espinosa, A.M., Schudel, A.A., Lager, I.A., Mueller, S.B., 1990. Incidence, epidemiology and control of bovine virus diarrhoea virus in South America. Rev. Sci. Tech. 9, 207–221. Saliki, J.T., Dubovi, E.J., 2004. Laboratory diagnosis of bovine viral diarrhea virus infections. Vet. Clin. North Am. Food Anim. Pract. 20, 69–83. Scherer, C.F., Flores, E.F., Weiblen, R., Caron, L., Irigoyen, L.F., Neves, J.P., Maciel, M.N., 2001. Experimental infection of pregnant ewes with bovine viral diarrhea virus type-2 (BVDV-2): effects on the pregnancy and fetus. Vet. Microbiol. 79, 285–299. Snowdon, W.A., French, E.L., 1968. The bovine mucosal disease-swine fever virus complex in pigs. Aust. Vet. J. 44, 179–184. Sullivan, D.G., Akkina, R.K., 1995. A nested polymerase chain reaction assay to differentiate pestiviruses. Virus Res. 38, 231–239. Terpstra, C., Wensvoort, G., 1988. Natural infections of pigs with bovine viral diarrhea virus associated with signs resembling swine fever. Res. Vet. Sci. 45, 137–142. Toplak, I., Sandvik, T., Barlic-Maganja, D., Grom, J., Paton, D.J., 2004. Genetic typing of bovine viral diarrhoea virus: most Slovenian isolates are of genotypes 1d and 1f. Vet. Microbiol. 99, 175–185. van Oirschot, J.T., Terpstra, C., 1977. A congenital persistent swine fever infection. I. Clinical and virological observations. Vet. Microbiol. 2, 121–132. Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309–323. Vilcek, S., Bela´k, S., 1996. Genetic identification of pestivirus strain Frijters as a border disease virus from pigs. J. Virol. Methods 60, 103–108. Vilcek, S., Nettleton, P.F., Paton, D.J., Bela´k, S., 1997. Molecular characterization of ovine pestiviruses. J. Gen. Virol. 78, 725–735. Vilcek, S., Durkovic, B., Kolesa´rova´, M., Greiser-Wilke, I., Paton, D., 2004. Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group. Vet. Res. 35, 609–615. Vilcek, S., Nettleton, P.F., 2006. Pestiviruses in wild animals. Vet. Microbiol. 116, 1–12.
Contents lists available at ScienceDirect
Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed
Short communication
First report of BVDV circulation in sheep in Argentina S. Julia´ *, M.I. Craig, L.S. Jime´nez, G.B. Pinto, E.L. Weber Instituto de Virologı´a, Centro de Investigaciones en Ciencias Veterinarias y Agrono´micas, INTA – Castelar, CC 25 (1712), 1686 Moro´n, Buenos Aires, Argentina
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 January 2009 Received in revised form 8 April 2009 Accepted 12 May 2009
Pestiviruses are capable of infecting a wide range of animals within the order Artyodactila. Currently, the genus Pestivirus includes Bovine Viral Diarrhea Virus 1 (BVDV-1) and 2 (BVDV-2), Border Disease Virus (BDV), and Classical Swine Fever Virus (CSFV). BVDV-1, BVDV-2 and BDV are able to cross species barrier to infect a wide range of hosts, whereas CSFV is restricted to domestic pigs and wild boars. In Argentina, 70% of cattle are seropositive to BVDV. Although there were some serological studies in llamas, alpacas and buffaloes, no reports existed about the circulation of BVDV in sheep in Argentina. Based on these, 54 blood samples of healthy ovines were analysed by serology. The results showed that 46.3% of the analysed sheep were seropositive to BVDV-1, 13% to BVDV-2 and 20.4% for both BVDV-1 and BVDV-2. The molecular analysis confirmed the presence of BVDV-1a in some samples. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Pestivirus BVDV Reservoir Sheep Argentina
1. Introduction According to the International Committee on Taxonomy of Viruses, genus Pestivirus includes four species: Bovine Viral Diarrhea Virus 1 (BVDV-1), Bovine Viral Diarrhea Virus 2 (BVDV-2), Classical Swine Fever Virus (CSFV) and Border Disease Virus (BDV). Also, a tentative fifth species is represented by a Pestivirus of giraffe. In particular for BVDV-1, Vilcek et al. (2004) described several genetic groups (BVDV-1a–j). At first, Pestiviruses were classified according to their host, however, later studies confirmed that, at least, BVDV and BDV are not host specific (Hamblin and Hedger, 1979). While BVDV may infect cattle, sheep, swine, goats and other ungulate species (Becher et al., 1999, 1997; Snowdon and French, 1968; Terpstra and Wensvoort, 1988); some reports confirmed the presence of BDV in sheep, swine and goats (Vilcek and Bela´k, 1996; Vilcek et al., 1997). However CSFV seems to be restricted to pigs and wild boars (Becher et al., 2003; Vilcek and Nettleton, 2006).
* Corresponding author. Tel.: +54 11 4621 1278/1447; fax: +54 11 4621 1743. E-mail address: [email protected] (S. Julia´). 0167-5877/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2009.05.015
An important condition for keeping BVDV in animal populations is the presence of individuals with immunotolerance and persistent infection, which are resulting from transplacental infection of the fetus before the onset of immunological maturity (Toplak et al., 2004). Persistently infected (PI) animals are the main source of virus transmission. These animals spread virus throughout their life. PI animals have been reported for cattle (Duffell and Harkness, 1985), pigs (van Oirschot and Terpstra, 1977; Meyer et al., 1981; Paton et al., 1992), sheep (Nettleton et al., 1992) and eland (Anderson and Rowe, 1998). Particularly in sheep, it was demonstrated that both BVDV-1 as BVDV-2 may infect them naturally (Sullivan and Akkina, 1995; Vilcek et al., 1997) as well as the biology of BVDV-2 infection in pregnant sheep is essentially similar to that of BVDV-1 in pregnant cattle and sheep (Scherer et al., 2001). Moreover, transmission between small ruminants and cattle, in both ways, has been demonstrated (Carlsson, 1991; Løken et al., 1991; Carlsson and Bela´k, 1994; Paton et al., 1995). Infection with BVDV may result in a wide range of clinical conditions, from virtually asymptomatic to fatal disease (Nettleton and Entrican, 1995), causing significant economic losses to the livestock industry. Even though in Argentina the prevalence of BVDV antibodies in adult cattle is around 70% (Rweyemamu
S. Julia´ et al. / Preventive Veterinary Medicine 90 (2009) 274–277
et al., 1990; Pacheco and Lager, 2003), vaccination is not compulsory and it is only intended to livestock. Some authors reported that both BVDV-1 and BVDV-2 are present in cattle in the country (Jones et al., 2001). In addition, there are some serological studies on captive llamas (Puntel et al., 1999) and antigen detection in different organs of water buffaloes by immunohistochemistry (Craig et al., 2008); however, there are no studies on
275
BVDV in ovines. The sheep industry is the second in importance of livestock production in Argentina. Particularly, the Patagonian region has approximately 75% of sheep population. Moreover, in the last 5 years the sheep population is growing up and currently is about 15 million. The most important subproduct is wool and then meat. The aim of this report was to study the viral circulation of BVDV in sheep from several farms of Argentina.
Fig. 1. Cladogram of the 50 UTR sequences of BVDV from Argentinean sheep (2a, 3a, 5a, 8b and 12a) and from different geographical regions.
276
S. Julia´ et al. / Preventive Veterinary Medicine 90 (2009) 274–277
2. Materials and methods Our laboratory received 54 blood samples with EDTA of healthy ovines for genotipification study. Some of the samples (20/54) were taken from a slaughterhouse in Rı´o Gallegos (Santa Cruz) and the others (34/54) from 14 different farms in the provinces of Santa Cruz and Tierra del Fuego (Patagonia Argentina). The samples were fractionated in fresh blood and plasma (obtained by centrifugation) and stored at 80 8C, until use. Virus neutralization test for each of the 54 plasma was determined against BVDV-1 and BVDV-2, using the cytopathic strains NADL and 61380 (AF417986), respectively. For the seroneutralization assay, a fixed dose of virus (100 TCID50) was incubated for 1 h at 37 8C with twofold plasma dilutions in growth medium. Cells were added and cultures were grown in microtitre plates for 4 days. Virus growth or neutralization was monitored by the visualization of the cytopathic effect. Positive and negative control sera were used in each assay. In addition to serological assay, 34 samples obtained from the farms were chosen for RNA extraction, from blood or plasma. The amplification of a 288 bp product by RT-PCR for the 50 UTR was done as described by Vilcek et al. (1994). PCR products were visualized in 1.8% agarose gel stained with ethidium bromide. Once the amplicons were purified, they were directly sequenced on an Applied Biosystems 322 equipment. The obtained sequences were edited with the BioEdit software (BioEdit sequence alignment editor; version 5.0.9) and subsequently, were aligned with CLUSTALX program (Version 1.8.3). The phylogenetic tree was calculated by the Neighbor-Joining method, using the DNADist and Neighbor modules present in the PHYLIP package (Felsenstein, 1989).
polymerase reaction. Taking into account the serological assays, the results show that three positive samples by RTPCR correspond to animals that were seronegative for both BVDV species, another two samples to seropositive animals for BVDV-1, and other two samples to seropositive sheep for both BVDV-1 and BVDV-2. The last two samples showed cytotoxicity to cell culture, so the serological status of these sheep could not be determined. The sequence analysis of five of these samples showed higher identity with BVDV-1. Phylogenetic analysis confirmed that they belonged to subtype BVDV-1a (Fig. 1). It is important to remark that none of the obtained sequences were consistent with Border Disease Virus. This result was the expected one, considering that Argentina is a free country of BDV. The presence of seronegative ovines for both BVDV-1 and BVDV-2 but a positive detection of viral nucleic acid, strongly suggests the existence of PI animals (Saliki and Dubovi, 2004). Although the results indicate that sheep were in contact with BVDV-2, the circulation of this BVDV species could not be confirmed. Although it is known that BVDV infects sheep, this study reports, for first time, the circulation of BVDV in ovines in Argentina. Most farms in Patagonia have sheep but few of them share their habitat with bovines. This fact, coupled with the presence of persistently infected animals suggests that ovines might be considered as a natural reservoir of BVDV in this region. In addition, it is interesting to remark that in one of the farms in which there is no cattle, all sheep sampled (samples 2–5) were positive by PCR, indicating the circulation of the virus among them and without the participation of cattle. 4. Conclusion
3. Results and discussion All the samples belonged to animals without any clinical sign of disease. Serological assays showed that 46.3% of samples (25/54) were positive to BVDV-1, 13% (7/ 54) were positive to BVDV-2, 20.4% (11/54) were positive for both viral species (BVDV-1 and BVDV-2), and 14.8% (8/ 54) were negative for BVDV-1 and BVDV-2. Unfortunately, 5.5% of samples (3/54) showed cytotoxicity to cell culture. These results confirm that ovines were in contact both with BVDV-1 and with BVDV-2, and also show the existence of co-circulation of both viral species in some farms, evidenced by the presence of antibodies against BVDV-1 and BVDV-2. These results are in accordance with those reported by Jones et al. (2001), where the prevalence of BVDV-1 was higher than that found to BVDV-2. Furthermore, the presence of antibodies against BVDV-1 and BVDV-2 in the same animal suggests the sequential exposures to them. Detection of BVDV by RT-PCR was performed on fresh blood samples but unfortunately, some blood samples were not enough to perform both virus neutralization test and RT-PCR. So, RT-PCR was performed on 34 blood samples and nine samples (26.4%) were positive for the detection of viral nucleic acid. Probably, the hemolysis present in most of these samples has inhibited the
We conclude that BVDV-1 circulates in sheep of the Patagonia Argentina and the probable circulation of BVDV2. We also present evidence about the existence of PI sheep with BVDV-1a, acting as natural reservoirs in this region with or without the participation of cattle. From the foregoing the need for a more comprehensive study encompassing the rest of the country is evident. BVDV vaccination in Argentina is not compulsory, however, the results of this work must be taken into account for the knowledge and understanding of virus circulation, and must be considered for future eradication programs. Acknowledgements The authors are grateful to the IMYZA Sequence Service. This work was supported by the FONCyT (Bid 1201/OCAR08-09627). E.L.W. is member of the Scientist Career of CONICET. References Anderson, E.C., Rowe, L.W., 1998. The prevalence of antibody to the viruses of bovine virus diarrhoea, bovine herpes virus 1, rift valley fever, ephemeral fever and bluetongue and to Leptospira sp in freeranging wildlife in Zimbawe. Epidemiol. Infect. 121, 441–449.
S. Julia´ et al. / Preventive Veterinary Medicine 90 (2009) 274–277 Becher, P., Orlich, M., Shannon, A.D., Horner, G., Ko¨nig, M., Thiel, H.-J., 1997. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J. Gen. Virol. 78, 1357–1366. Becher, P., Orlich, M., Kosmidou, A., Ko¨nig, M., Baroth, M., Thiel, H.-J., 1999. Genetic diversity of pestiviruses: identification of novel groups and implications for classification. Virology 262, 64–71. Becher, P., Avalos Ramirez, R., Orlich, M., Cedillo Rosales, S., Konig, M., Schweizer, M., Stalder, H., Schirrmeier, H., Thiel, H.-J., 2003. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311, 96–104. Carlsson, U., 1991. Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine virus diarrhoea virus. Vet. Rec. 128, 145–147. Carlsson, U., Bela´k, K., 1994. Border disease virus transmitted to sheep and cattle by a persistently infected ewe: epidemiology and control. Acta Vet. Scand. 35, 79–88. Craig, M.I., Venzano, A., Ko¨nig, G., Morris, W.E., Jime´nez, L., Julia´, S., Capellino, F., Blanco Viera, J., Weber, E.L., 2008. Detection of bovine viral diarrhoea virus (BVDV) nucleic acid and antigen in different organs of water buffaloes (Bubalus bubalis). Res. Vet. Sci. 85, 194–196. Duffell, S.J., Harkness, J.W., 1985. Bovine virus diarrhoea-mucosal disease infection in cattle. Vet. Rec. 117, 240–245. Felsenstein, J. 1989. PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 5: 164–166. Hamblin, C., Hedger, R.S., 1979. The prevalence of antibodies to bovine viral diarrhoea/mucosal disease virus in African wildlife. Comp. Immunol. Microbiol. Infect. Dis. 2, 295–303. Jones, L.R., Zandomeni, R., Weber, E.L., 2001. Genetic typing of bovine viral diarrhea virus isolates from Argentina. Vet. Microbiol. 81, 367–375. Løken, T., Krogsrud, J., Bjerka´s, I., 1991. Outbreaks of border disease in goats induced by a pestivirus-contaminated orf vaccine, with virus transmission to sheep and cattle. J. Comp. Pathol. 104, 195–209. Meyer, H., Liess, B., Frey, H.R., Hermanns, W., Trautwein, G., 1981. Experimental transplacental transmission of hog colera virus in pigs. IV. Virological and serological studies in newborn piglets. Zentrlbl. Veterinarmed. B 28, 659–668. Nettleton, P.F., Entrican, G., 1995. Ruminant pestiviruses. Br. Vet. J. 151, 615–642. Nettleton, P.F., Gilmour, J.S., Herring, J.A., Sinclair, J.A., 1992. The production and survival of lambs persistently infected with border disease virus. Comp. Immunol. Microbiol. Infect. Dis. 15, 179–188. Pacheco, J.M., Lager, I., 2003. Indirect method ELISA for the detection of antibodies against bovine diarrhea virus in bovine serum. Rev. Argent. Microbiol. 35, 19–23. Paton, D.J., Simpson, V., Done, S.H., 1992. Infection of pigs and cattle with bovine viral diarrhoea virus on a farm in England. Vet. Rec. 131, 185– 188.
277
Paton, D.J., Carlsson, U., Lowings, J.P., Sands, J.J., Vilcek, S., Alenius, S., 1995. Identification of herd-specific bovine viral diarrhoea virus isolates from infected cattle and sheep. Vet. Microbiol. 43, 283–294. ´ Donnell, V.K., Marcovecchio, Puntel, M., Fondevila, N.A., Blanco Viera, J., O J.F., Carrillo, B.J., Schudel, A.A., 1999. Serological survey of viral antibodies in llamas (Lama glama) in Argentina. Zentralbl. Veterinarmed. B 46, 157–161. Rweyemamu, M.M., Fernandez, A.A., Espinosa, A.M., Schudel, A.A., Lager, I.A., Mueller, S.B., 1990. Incidence, epidemiology and control of bovine virus diarrhoea virus in South America. Rev. Sci. Tech. 9, 207–221. Saliki, J.T., Dubovi, E.J., 2004. Laboratory diagnosis of bovine viral diarrhea virus infections. Vet. Clin. North Am. Food Anim. Pract. 20, 69–83. Scherer, C.F., Flores, E.F., Weiblen, R., Caron, L., Irigoyen, L.F., Neves, J.P., Maciel, M.N., 2001. Experimental infection of pregnant ewes with bovine viral diarrhea virus type-2 (BVDV-2): effects on the pregnancy and fetus. Vet. Microbiol. 79, 285–299. Snowdon, W.A., French, E.L., 1968. The bovine mucosal disease-swine fever virus complex in pigs. Aust. Vet. J. 44, 179–184. Sullivan, D.G., Akkina, R.K., 1995. A nested polymerase chain reaction assay to differentiate pestiviruses. Virus Res. 38, 231–239. Terpstra, C., Wensvoort, G., 1988. Natural infections of pigs with bovine viral diarrhea virus associated with signs resembling swine fever. Res. Vet. Sci. 45, 137–142. Toplak, I., Sandvik, T., Barlic-Maganja, D., Grom, J., Paton, D.J., 2004. Genetic typing of bovine viral diarrhoea virus: most Slovenian isolates are of genotypes 1d and 1f. Vet. Microbiol. 99, 175–185. van Oirschot, J.T., Terpstra, C., 1977. A congenital persistent swine fever infection. I. Clinical and virological observations. Vet. Microbiol. 2, 121–132. Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136, 309–323. Vilcek, S., Bela´k, S., 1996. Genetic identification of pestivirus strain Frijters as a border disease virus from pigs. J. Virol. Methods 60, 103–108. Vilcek, S., Nettleton, P.F., Paton, D.J., Bela´k, S., 1997. Molecular characterization of ovine pestiviruses. J. Gen. Virol. 78, 725–735. Vilcek, S., Durkovic, B., Kolesa´rova´, M., Greiser-Wilke, I., Paton, D., 2004. Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group. Vet. Res. 35, 609–615. Vilcek, S., Nettleton, P.F., 2006. Pestiviruses in wild animals. Vet. Microbiol. 116, 1–12.