Seroprevalence of small ruminant lentivirus infection in goats in Thailand.

Small ruminant lentivirus (SRLV), which causes caprine arthritis encephalitis in goats and ovine progressive pneumonia (maedi-visna disease) in sheep, is classified in genus Lentiviruses belonging to Retroviridae family. It persists in infected goats and sheep, which mostly are sub- clinical. A serological survey was conducted to determine the prevalence of small ruminant lentivirus infection in Thai goat population. Serum samples were taken from 1,925 goats distributed throughout the country, then they were tested for the presence of SRLV antibodies using commercial indirect enzyme-linked immunosorbent assay (ELISA) test kits. Results revealed that a total of 68 goats were found seropositive, representing the apparent prevalence and true prevalence of 3.57% and 2.60%, respectively. The seroprevalence, revealed in this study, was lower than in the previous reports. The decreasing of seroprevalence might be caused by successful control strategies from Department of Livestock Development (DLD).

Bayesian estimation of seroprevalence of small ruminant lentiviruses in sheep from Poland.

In Poland, no systematic survey of ruminant lentiviruses (SRLVs) infection was performed, neither at the national nor at the regional level and only limited knowledge exists on the prevalence of SRLVs among sheep. The aim of the present study was to establish the true prevalence of SRLVs infection in sheep from Poland at the animal and herd-levels. The blood samples used for this study were the fraction of samples collected by Veterinary Inspection during an official sampling for the national monitoring program for brucellosis. Under this program the animals and herds were randomly selected using the data available from ARMA (Agency for Restructuring and Modernisation of Agriculture). The sampling unit was the herd and the target population included at least 5% of sheep, over 6 months old, from each of 16 voievodships (provinces) of Poland. Two-stage cluster sampling design was performed in this study offering the possibility to determine the prevalence of SRLVs infection, when only a fraction of herds and a fraction of animals in the herds are tested. In total, 8233 sheep serum samples coming from 832 herds were tested by indirect ELISA. 1474 (17.9%) samples were positive and 261 (31.4%) herds with at least one seropositive animals were identified. The overall true prevalence estimated by the Bayesian framework was 9.3% (95% CI 6.8, 11.3) and 33.3% (95% CI 26.5, 38.2) on the animal and herd level, respectively. Large variation in the animal and herd prevalence between the voivodships was observed. True prevalence on the herd level varied from 0.0% (95% CI 0.0, 0.0) to 71.6% (95% CI 67.6, 75.9) whereas true prevalence on the animal level ranged from 0.0% (95% CI 0.0, 0.0) to 55.3% (95% CI 50.0, 61.2). The true prevalence of SRLVs infection at animal and herd level increased according to herd size as was proved by posterior probabilities (POPR).

Prevalence of small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis co-infection in Ontario dairy sheep and dairy goats.

Infection with small ruminant lentiviruses (SRLV) causes a variety of chronic inflammatory conditions that limit production. Mycobacterium avium subsp. paratuberculosis (MAP) is also a major production-limiting disease of sheep and goats, which causes severe inflammation of the small intestine. Previous studies have indicated that both SRLV and MAP are widespread in small ruminants in Ontario. This study estimated the prevalence of SRLV and MAP co-infection. Serum samples that were previously tested for MAP infection were re-tested for SRLV. The apparent prevalence of co-infection was low, with 3.4% [95% confidence interval (CI): 1.9 to 5.9] and 14.3% (95% CI: 11.6 to 17.5) of sheep and goats respectively, positive for both infections. However, co-infection is widespread with 36.8% (95% CI: 19.1 to 59.1) and 71.4% (95% CI: 52.8 to 84.9) of sheep and goat farms with 1 or more co-infected animals. A significant association was found between SRLV seropositivity and MAP fecal culture (P = 0.021), suggesting that co-infected goats may be more likely to shed MAP in their feces.

L'infection par lentivirus des petits ruminants (SRLV) provoque une variété d'états inflammatoires chroniques qui limitent la production. Mycobacterium avium subsp. paratuberculose (MAP) est aussi une maladie limitant la production majeure de moutons et de chèvres, ce qui provoque une inflammation grave de l'intestin grêle. Des études antérieures ont indiqué que les deux infections de SRLV et MAP sont très répandues dans l'Ontario petits ruminants. Cette étude a été réalisée pour estimer la prévalence de SRLV et MAP co-infection. Des échantillons de sérum qui avaient été préalablement testés pour l'infection de MAP ont été utilisés pour détecter des anticorps spécifiques SLRV. La prévalence de la co-infection était faible, avec 3,4 % intervalle de confiance (95% IC : 1,9­5,9) et 14,3 % (95% IC : 11,6­17,5) des ovins et caprins, respectivement, positive pour les deux infections. Cependant la co-infection est très répandue avec 36,8 % (95% IC : 19,1­59,1) et 71,4 % (95% IC : 52,8­84,9) des élevages ovins et caprins avec un ou plusieurs animaux co-infecté. Une association significative a été trouvée entre SRLV et séropositivité MAP culture fécale (P = 0,021), ce qui suggère que les chèvres co-infectés peuvent être plus susceptibles de jeter le MAP dans leurs excréments.(Traduit par les auteurs).

The presence of small ruminant lentiviruses in Mexican Pelibuey sheep.

The transmission frequency of small ruminant lentiviruses (SRLVs) through the placenta is controversial and may be associated with breed susceptibility. In Mexico, SRLV infections in sheep have been poorly studied. This work explores the presence of antibodies and proviral DNA in Mexican Pelibuey sheep. Enzyme-linked immunosorbent assays (ELISAs; three commercial kits and two on the basis of synthetic peptides) and polymerase chain reaction (PCR; amplifying the long terminal repeat and gag segments) were performed to diagnose SRLV infection in 25 adult Pelibuey ewes with an average age of 2.5 years and 32 fetuses with gestational ages ranging from 40 to 90 days without clinical signs of SRLV. Two of the three commercial ELISAs and the synthetic peptide-based ones were positive for SRLV antibody detection in 28% and 24% of the ewes, respectively, whereas none of the fetuses were positive by any of the ELISAs. By PCR, 31% of the ewes and, interestingly, two fetuses were positive. Characteristic SRLV lesions were not found in the fetal and/or ewe tissues, including those with positive PCR results. These findings demonstrate the susceptibility of Pelibuey sheep to SRLV infection and the low transmission frequency through the placenta.

Identification and characterization of an emerging small ruminant lentivirus circulating recombinant form (CRF).

The molecular epidemiology of small ruminant lentiviruses (SRLVs) is constantly changing due to animal movements, cross species transmission and because of their rapid evolutionary rate. This study reports a comprehensive genetic and phylogenetic analysis based on consensus gag and pol sequences covering 3kb of the SRLV genome from small ruminants in Québec, Canada. A group of strains obtained from goats originating from different flocks, segregated in a unique clade distinct from currently known SRLV groups. Genetic dissection of the gag gene from these strains revealed that it originated as a result of a recombination event between parental strains currently circulating in small ruminants of the country. Following HIV nomenclature, we propose to call this group of strains, circulating recombinant form 1 SRLV, or CRF01_AB SRLV. In addition, the study confirms the existence of genetically distinct and homogeneous populations of SRLVs infecting sheep and goats housed in single species flocks.

Toll like receptor 9 (TLR9) polymorphism G520R in sheep is associated with seropositivity for Small Ruminant Lentivirus.

Infectious diseases of sheep are of major economic importance causing direct and indirect losses. Among the major sheep infectious agents are Small Ruminant Lentivirus, Chlamydophila abortus and Mycobacterium avium subsp. paratuberculosis infections, mainly due to their worldwide distribution and economic impact that they cause. Based on the differential susceptibility to infectious diseases between and within breeds and on the recent findings regarding the putative involvement of TLR9 in disease susceptibility, the aim of this study was to evaluate the levels of nucleotide variation of TLR9 and its mediator MyD88 in three sheep flocks originated from different breeds and assess their possible association with seropositivity/seronegativity for different infectious agents. The analysis indicated that the change of G to R at codon 520 of TLR9 polypeptide shows a significant association with Small Ruminant Lentivirus seropositivity. This amino-acid substitution, which can result in polarity change, might influence structure and function of LRR17, interfering with ligand binding and thus could be used in studies investigating susceptibility/resistance to Small Ruminant Lentivirus infections in sheep.

Lentivírus em caprinos leiteiros do semiárido paraibano: prevalência de anticorpos, fatores de risco e detecção molecular / Lentivirus in dairy goats from the semiarid region of Paraiba state: seroprevalence, risk factors and molecular detection

Os objetivos do presente trabalho foram determinar a prevalência de caprinos leiteiros soropositivos para a infecção por Lentivirus de pequenos ruminantes no semiárido do Estado da Paraíba, Nordeste do Brasil, identificar fatores de risco associados à prevalência de rebanhos positivos, e realizar a detecção molecular do agente. Foram utilizadas 1047 cabras leiteiras de 110 propriedades selecionadas aleatoriamente no Município de Monteiro, Estado da Paraíba, no período de março de 2009 a dezembro de 2011. Para o diagnóstico da infecção por Lentivirus, foi utilizado o teste de imunodifusão em gel de ágar (AGID). Um ano após foi realizada nova sorologia, e PCR em tempo real foi aplicada em amostras de sangue e leite de 48 cabras procedentes de quatro propriedades com animais soropositivos. As prevalências de propriedades positivas e de animais soropositivos na AGID foram 44,6% (IC 95% = 35,1% - 54,3%) e 8,1% (IC 95% = 5,6% - 16,8%), respectivamente. Realizar corte e desinfecção de umbigo (odds ratio = 2,44; p = 0,048) e condições de aglomeração de animais (odds ratio = 3,45; p = 0,048) foram associadas com a prevalência de propriedades positivas. Um ano após a realização do inquérito sorológico, foi verificada a permanência de animais infectados, detectados por PCR em tempo real a partir de amostras de sangue e leite. A PCR em tempo real das amostras de leucócitos circulantes apresentou boa performance, com sensibilidade de 100%, especificidade de 92,86%, concordância de 93,75% e indicador Kappa de 0,765. Sugere-se que seja realizado um trabalho de educação sanitária junto aos produtores sobre medidas de prevenção com o objetivo de reduzir a disseminação da infecção nos rebanhos.

The aims of this study were to determine the seroprevalence of infection by ruminants Lentivirus in dairy goats in the semiarid of the Paraiba State, Northeastern Brazil, to identify risk factors associated with the herd-level prevalence and to perform molecular detection of the agent. A total of 1,047 dairy goats from 110 herds were randomly selected from the county of Monteiro, Paraiba State, and serum samples were collected from March 2009 to December 2011. For the diagnosis of Lentivirus infection, the agar gel immunodiffusion test (AGID) was used. One year after that a new serology was performed and the real-time PCR assay was applied in blood and milk samples from 48 goats from four herds with seropositive animals. Prevalence of positive herds and seropositive animals at AGID were 44.6% (95% CI=35.1-54.3%) and 8.1% (95% CI =5.6-16.8%), respectively. Umbilical cord cutting and disinfection (odds ratio = 2.44; p = 0.048) and conditions of animal agglomeration (odds ratio=3.45; p=0.048) were associated with herd-level prevalence. One year after the serological profile, the permanence of infected animals detected by real-time PCR in blood and milk samples was verified. Real-time PCR using white blood cells had a good performance, with sensitivity of 100%, specificity of 92.86%, concordance of 93.75% and Kappa index of 0.765. It was suggested to teach sanitary measures to the herd owners in order to encourage them to adopt prevention measures aiming to reduce the spread of the infection in the herds.

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection.

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools.

Risk factors associated with seropositivity to small ruminant lentiviruses in goat herds.

Cross-sectional studies based on serological testing and questionnaires were conducted at 5-yr intervals (1996, 2002 and 2007) in goat breeding herds from Poland to determine true herd-level seroprevalence of caprine arthritis-encephalitis and the herd-level risk factors for seropositivity. Multivariable logistic regression models were developed for data from 1996 and the combined data from 2002 and 2007, separately. True herd-level seroprevalences in 1996, 2002 and 2007 were 30.8% (CI 95%: 20.5-41.0%), 65.7% and 71.9%, respectively. The import of goats from abroad was a risk factor only in 1996 (OR 13.6, CI 95%: 1.14-162). The presence of seropositive bucks in a herd was a risk factor in 1996 (OR 21, CI 95%: 1.89-233) and in 2002-2007 (OR 2.9, CI 95%: 1.04-8.4). Moreover, large herds (>30 does in 1996 or >100 does in 2002-2007) were more likely to be seropositive than smaller herds (OR=10.1, CI 95%: 2.17-46 in 1996 and OR 5.4, CI 95%: 1.11-26 in 2002-2007).

Mannose receptor may be involved in small ruminant lentivirus pathogenesis.

Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV) of known genotype (A or B), and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR) expression (transcript levels) and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints). Control sheep were SRLV-seropositive asymptomatic (n = 3), seronegative (n = 3) or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case). MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively). The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.

Small ruminant lentivirus genotype E is widespread in Sarda goat.

The highly divergent SRLV genotype E has recently been characterized in Italy as a low pathogenic caprine lentivirus in the Roccaverano breed. The availability of a genotype specific diagnostic test based on a comparative assay, using a combination of genotype specific recombinant antigens allows a wide serosurvey in other goat populations. The island of Sardinia still has the highest small ruminant population of any Italian region and crossbreeding has been limited to goats, mainly with the Maltese breed. A serological survey was carried out on sheep flocks and goat herds, using individual sera as well as a bulk milk-adapted procedure. Genotype E was identified in more than 50% of goat herds and none of the sheep flocks thus supporting the idea that this genotype is specifically associated with the goat species. The full-length proviral sequence of a Sardinian isolate revealed and confirmed the deletion of dUTPase subunit and the absence of both vpr gene and the 71bp repeat of the LTR. Genetic similarity of this isolate with the prototype strain Roccaverano was not more than 84%, supporting the designation of two subtypes within genotype E. Nevertheless, in vitro properties of the Sardinian strain were different from those of the Roccaverano strain in terms of ability to infect synovial membrane and produce syncitia. Remarkable differences in the HV1 and HV2 of the env gene were recorded, with the Sardinian isolate displaying sequence motif more similar to arthritic strains. Data presented suggest diffusion of genotype E is wider than previously thought.

Antibody-based diagnosis of small ruminant lentivirus infection in seminal fluid.

Antibody-based diagnosis of small ruminant lentiviruses (SRLVs) has been efficiently achieved using serum and milk, but not semen, for which polymerase chain reaction (PCR) has been proposed as a confirmatory technique. This work, involving 296 ovine (Ovis aries) and caprine (Capra hircus) semen donors, investigates whether seminal fluid (SF) can be reliably used in antibody-based SRLV diagnosis. First, a gold standard was established to assess the infection status and determine the sensitivity and specificity of three commercial enzyme-linked immunosorbent assays (ELISAs) in serum testing using Western blot and PCR as confirmatory tests. For SF testing, both gold standard and serum testing results were used as reference. The performance of SF testing was affected not only by the ELISA assay sensitivity (related to antigen spectrum) compared with that of the gold standard (as it occurred in serum testing) but also by SF sample quality and SF working dilution. Nonturbid SF samples, commonly collected in artificial insemination centers (AICs), were required. Compared with serum, SF testing had a decreased sensitivity in two of the ELISA assays (with original serum working dilutions

Development of specific diagnostic test for small ruminant lentivirus genotype E.

Small ruminant lentivirus (SRLV) belonging to the highly divergent genotype E has recently been identified in the Italian goat breed Roccaverano. In this report we have developed a specific serological test based on recombinant matrix/capsid antigen fusion protein. Performance has been evaluated and compared with a similar test based on genotype B antigen. Herds under study were selected according to the infectious status characterized by blood PCR and sequencing. Results clearly showed that B and E based recombinant ELISA only detected homologous infection and an apparent cross-reactivity was recorded in a herd in which co-infection was present. Three commercially available ELISAs showed different abilities in detecting genotype E infection, being the whole virus-based immunoassay the best choice. Genotype E-recombinant antigen was not detected in ELISA by three commercially available Mabs known to be cross-reactive among CAEV and MVV capsid antigens, further supporting the high divergence of the E genotype from others. Finally, a SRLV-free herd according to commercial ELISA testing, was analysed in the same area where genotype E was identified and few animals belonging to Roccaverano breed were found slightly reactive with the E antigens. Our results suggest that the prevalence of genotype E in other small ruminant populations may be conveniently estimated using a comparative assay based on a combination of genotype specific recombinant antigens and may highlight a wider space in which SRLVs evolve.

Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep.

Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.

Evidence of proviral clearance following postpartum transmission of an ovine lentivirus.

Lentiviral transmission by transfer of infected colostrum and/or milk is considered to be highly efficient. In this study, postpartum transmission of ovine progressive pneumonia virus (OPPV) from 10 naturally infected ewes to their 23 lambs was followed from the perinatal period throughout a four-year period. The lambs were allowed to suckle from their dam from birth through 32 weeks of age. Virus was tracked by virus isolation, quantitative PCR (qPCR), and anti-OPPV antibody responses as measured by cELISA. Cell-associated OPPV was isolated from colostrum/milk cells in 7 out of 10 ewes and provirus envelope (env) loads ranged 8 to 10(5) copies/mug DNA in colostrum/milk cells from the 10 ewes using qPCR. Provirus env loads were also detected in the peripheral circulation of 21 lambs at 8 weeks and two lambs at 22 weeks. The qPCR product at 8 weeks was confirmed as the transmembrane (tm) gene of OPPV by cloning and sequencing. Both cELISA titers ranging from 325 to 3125 and cross-neutralizing antibody titers ranging from 6 to 162 to seven different OPPV strains were found in the colostrum of the 10 ewes. Furthermore, cELISA titers in serum from lambs remained detectable through 32 weeks following the clearance of provirus at 24 weeks. After 32 weeks, both provirus and anti-OPPV antibody responses have subsequently remained undetectable through 4 years of age. These data suggest the clearance of cell-associated lentiviruses from lamb circulation after passive transfer of antibody via colostrum.

Controle da Artrite-Encefalite Caprina, em um capril comercial endemicamente contaminado / Caprine Arthritis-Encephalitis control in an endemically contaminated commercial goat farm

A CAE é provocada por um lentivírus. Os animais infectam-se, principalmente, quando mamam colostro e/ou leite contaminados. Neste trabalho, propôs-se um plano de controle da CAE, sem que se sacrificassem as mães contaminadas. Utilizaram-se 39 cabritas, nascidas de mães soropositivas para a CAE. Após o nascimento, as cabritas foram isoladas das mães e alimentadas com colostro de cabras soronegativas, tratado termicamente, e com leite de cabra pasteurizado, até os dois meses. Submeteram-se todas as cabritas ao teste sorológico, trimestralmente, do nascimento aos 12 meses; segregaram-se as soropositivas do rebanho. O grupo controle consistiu de 12 cabritos, nascidos de cabras soropositivas, os quais permaneceram com suas mães. O procedimento de diagnóstico foi o mesmo, mas não foram segregados os positivos. Ao final de 12 meses, 34 (87%) animais do grupo experimental permaneceram soronegativos, com limites de confiança de 76% a 98%; nos animais do grupo controle, a taxa de negatividade acumulada foi de 17%, com limites de confiança entre 0% e 38%. Com esses resultados, conclui-se que o plano proposto é viável para garantir o controle da enfermidade, em rebanhos contaminados, ou seja, a não-adoção do mesmo pode levar à contaminação dos animais nascidos de cabras infectadas.

CAE is caused by a lentivirus. The animals are mainly infected when taking contaminated colostrums and/ or milk. This study proposed a CAE control strategy without sacrificing contaminated mothers. Thirty-nine female kids, born to CAE seropositive mothers were isolated from their mothers at birth and fed heat-treated colostrum and pasteurized milk from seronegative goats up to two months of age. All kids were submitted to three-monthly serological tests from birth to 12 months; seropositives were segregated from the herd. The control group consisted of 12 kids born to seropositive mothers that remained with their mothers. Diagnosis was the same, but seropositive animals were not segregated. At the end of 12 months, 34 (87%) animals from the experimental group remained seronegative with 76% to 98% confidence limits; in control group animals, the accumulated negativity rate was 17%, with 0% and 38% confidence limits. These results show that the proposed plan is viable to assure disease control in contaminated herds and that without it contamination can pass to animals born to infected goats.

Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA.

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.

Detection of ovine lentivirus in the cumulus cells, but not in the oocytes or follicular fluid, of naturally infected sheep.

The aim of this study was to examine the Maedi-Visna virus (MVV) infection status of oocytes, cumulus cells, and follicular fluid taken from 140 ewes from breeding flocks. MVV proviral-DNA and MVV RNA were detected using nested-PCR and RT-PCR MVV gene amplification, respectively in the gag gene. Nested-PCR analysis for MVV proviral-DNA was positive in peripheral blood mononuclear cells in 37.1% (52/140) of ewes and in 44.6% (125/280) of ovarian cortex samples. The examination of samples taken from ovarian follicles demonstrated that 8/280 batches of cumulus cells contained MVV proviral-DNA, whereas none of the 280 batches of oocytes taken from the same ovaries and whose cumulus cells has been removed, was found to be PCR positive. This was confirmed by RT-PCR analysis showing no MVV-viral RNA detection in all batches of oocytes without cumulus cells (0/280) and follicular fluid samples taken from the last 88 ovaries (0/88). The purity of the oocyte fraction and the efficacy of cumulus cell removal from oocytes was proved by absence of granulosa cell-specific mRNA in all batches of oocytes lacking the cumulus cells, using RT-PCR. This is the first demonstration that ewe cumulus cells harbor MVV genome and despite being in contact with these infected-cumulus cells, the oocytes and follicular fluid remain free from infection. In addition, the enzymatic and mechanical procedures we used to remove infected-cumulus cells surrounding the oocytes, are effective to generate MVV free-oocytes from MVV-infected ewes.

Application of PCR technique in diagnosis of small ruminant lentivirus infection in sheep and goats.

Detection of small ruminant lentiviruses (SRLVs) in sheep and goats usually relies on serological testing. In this study, we evaluated semi-nested PCR and nested PCR techniques applied as a diagnostic tool for detection of maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) in naturally infected sheep and goats, respectively. The examination of 193 ovine and 85 caprine serum samples by the ELISA revealed the presence of specific antibodies in 133 (69%) and 18 (21.2%) animals, respectively. Presence of proviral DNA was manifested in 103 (53.4%) sheep and 12 (14.2%) goats. Despite the relatively lower sensitivity of PCR, the fact of detection of proviral DNA in 19 out of 60 ovine samples and 7 out of 67 caprine samples collected from animals previously negative by ELISA was noteworthy. In conclusion, the data demonstrated that combinations of both ELISA and PCR might afford optimal detection of SRLVs infection.

Possibility of vertical transmission of caprine arthritis-encephalitis virus in neonate kids

Estudou-se a capacidade do vírus da artrite-encefalite dos caprinos (CAEV) infectar o feto ou o cabrito neonato pela via de transmissão transplacentária ou no momento do parto. Foram utilizados 26 cabritos recém-nascidos, filhos de cabras sororreagentes aos antígenos do CAEV e que nasceram de partos eutócicos. Na pesquisa de anticorpos séricos anti-CAEV, foi utilizada a técnica de imunodifusão em gel de ágar. Nenhum cabrito nasceu sororreagente aos antígenos do vírus, indicando que a possibilidade de transmissão vertical transplacentária da infecção foi menor do que 3,8 por cento (< 1:26).