Small Ruminant Lentiviruses in Sheep: Pathology and Tropism of 2 Strains Using the Bone Marrow Route.

The objective of this work was to comparatively study the tissue tropism and the associated pathology of 2 autochthonous small ruminant lentivirus (SRLV) field strains using an experimental infection in sheep through the bone marrow. Fifteen male, SRLV-free lambs of the Rasa Aragonesa breed were inoculated with strain 697 (nervous tissue origin, animals A1-A6), with strain 496 (articular origin, animals B1-B6), or with uninfected culture medium (C1-C3). Clinical, serologic, and polymerase chain reaction (PCR) evaluations were performed periodically. Two lambs from each infected group and a control animal were euthanized at 134, 273, and 319 days postinfection. Tissues were analyzed by gross and histopathologic evaluation; immunohistochemistry for CD3, CD4, CD8, CD68, and FoxP3 cell markers; lung morphometric evaluation; and tissue proviral quantification by PCR. All infected animals became positive either by enzyme-linked immunosorbent assay and/or PCR, with group B lambs showing the highest serologic values and more consistently positive PCR reactions. Group A lambs showed representative lung lesions but only mild histopathologic changes in the central nervous system (CNS) or in carpal joints. Contrarily, group B lambs demonstrated intense carpal arthritis and interstitial pneumonia but an absence of lesions in the CNS. Proviral copies in tissues were detected only in group B lambs. Experimental infection with these SRLV strains indicates that strain 496 is more virulent than strain 697 and more prone to induce arthritis, whereas strain 697 is more likely to reproduce encephalitis in Rasa Aragonesa lambs. Host factors as well as viral factors are responsible for the final clinicopathologic picture during SRLV infections.

Deletion variant near ZNF389 is associated with control of ovine lentivirus in multiple sheep flocks.

Ovine lentivirus (OvLV) is a macrophage-tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker-assisted selective breeding might improve odds of infection and control of OvLV post-infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post-infection in multiple animal sets. Proviral concentration is a live-animal diagnostic measure of OvLV control post-infection related to severity of OvLV-induced lesions. A recent genome-wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/µg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV-positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post-infection in multiple sheep flocks.

Diagnostic performance of ID screen MVV-CAEV Indirect Screening ELISA in identifying small ruminant lentiviruses-infected goats.

Diagnostic performance of ID Screen MVV-CAEV Indirect Screening ELISA in identifying goats infected with small ruminant lentiviruses (SRLV) was evaluated. In total 299 serum samples from the collection of the Laboratory of Veterinary Epidemiology and Economics--109 truly positive and 190 truly negative--were used. To be enrolled in the study a serum sample had to come from at least 2 year-old goat which had reacted identically in two serological surveys preceding sample collection and was kept in a herd of stable serological status confirmed at least twice during preceding 5 years. Moreover, in seropositive herds at least 20% of goats had to be serologically positive at the moment when the serum sample was collected for the study. The test proved to have high accuracy. Area under curve was 98.8% (95% CI: 97.5%, 100%). Diagnostic performance of the test was almost identical (Youlden's index of 90%, sensitivity > 90% and specificity > 95%) within a fairly wide range of cut-off values--between 20% and 60%. At manufacturer's cut-off of 50% sensitivity and specificity were 91.7% (95% CI: 85.0%, 95.6%) and 98.9% (95% CI: 96.2%, 99.7%), respectively. For this cut-off positive likelihood ratio was 87 (95% CI: 22, 346) and negative likelihood ratio was 0.08 (95% CI: 0.04, 0.16). In conclusion, the results of this study indicate that ID Screen MVV-CAEV Indirect Screening ELISA is a highly accurate diagnostic test for SRLV infection.

Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype A and B small ruminant lentivirus strains.

BACKGROUND: Small ruminant lentiviruses (SRLV) are members of the Retroviridae family and infect goats and sheep worldwide. Detection of specific antibodies using AGID and ELISA is the most commonly used means of diagnosing SRLV infection. The most frequent molecular method for detecting the provirus genome is PCR, using peripheral blood leucocytes as target cells. Real time PCR has also recently been used. The aim of this study was to develop a real time PCR for detection of SRLV in order to improve molecular diagnostics of SRLV infections in sheep and goats. RESULTS: Two new real time PCR assays using TaqMan probes for the specific detection of genotype A (MVV assay) and genoptype B (CAEV assay) SRLV strains and differentiation between them were developed and validated at both analytical and diagnostic levels following MIQE guidelines. The validation results showed that the new real time PCR is 100% specific, with a reliable limit of detection of 26 (CAEV assay) and 72 (MVV assay) plasmid DNA copies, while compared to ELISA the diagnostic sensitivity of both assays was 79% when tested with Slovenian SRLV field samples. Intra-assay and inter-assay coefficients of variation showed overall good repeatability and reproducibility of the new real time PCR assays, except for the highest dilutions. CONCLUSIONS: Two new TaqMan probe based real time PCR assays for the specific detection of genotype A and B SRLV strains and differentiation between them were developed and validated. They can serve as an additional tool for confirming infection with SRLV and may also be useful for early detection of infected animals prior to seroconversion.

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.

Update on Small Ruminant Lentiviruses.

Small ruminant lentiviruses (SLRVs) have been recognized throughout the world for decades. SLRVs are a heterogenous group of viruses that can infect sheep, goats, and wild ruminants. Evidence supports cross-species infection. These viruses cause lifelong infections where they target specific organs, which can result in production losses due to diminished milk production, consequential increases in neonatal death and diminished growth, and premature culling of prime age animals. No vaccine or treatments have proved effective. Control programs rely on an understanding of viral transmission and application of highly sensitive, specific, and frequent testing regimens.

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).

Transcriptome Analysis for Genes Associated with Small Ruminant Lentiviruses Infection in Goats of Carpathian Breed.

Small ruminant lentiviruses (SRLV) are economically important viral pathogens of sheep and goats. SRLV infection may interfere in the innate and adaptive immunity of the host, and genes associated with resistance or susceptibility to infection with SRLV have not been fully recognized. The presence of animals with relatively high and low proviral load suggests that some host factors are involved in the control of virus replication. To better understand the role of the genes involved in the host response to SRLV infection, RNA sequencing (RNA-seq) method was used to compare whole gene expression profiles in goats carrying both a high (HPL) and low (LPL) proviral load of SRLV and uninfected animals. Data enabled the identification of 1130 significant differentially expressed genes (DEGs) between control and LPL groups: 411 between control and HPL groups and 1434 DEGs between HPL and LPL groups. DEGs detected between the control group and groups with a proviral load were found to be significantly enriched in several gene ontology (GO) terms, including an integral component of membrane, extracellular region, response to growth factor, inflammatory and innate immune response, transmembrane signaling receptor activity, myeloid differentiation primary response gene 88 (MyD88)-dependent toll-like receptor signaling pathway as well as regulation of cytokine secretion. Our results also demonstrated significant deregulation of selected pathways in response to viral infection. The presence of SRLV proviral load in blood resulted in the modification of gene expression belonging to the toll-like receptor signaling pathway, the tumor necrosis factor (TNF) signaling pathway, the cytokine-cytokine receptor interaction, the phagosome, the Ras signaling pathway, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (PI3K-Akt) signaling pathway and rheumatoid arthritis. It is worth mentioning that the most predominant in all pathways were genes represented by toll-like receptors, tubulins, growth factors as well as interferon gamma receptors. DEGs detected between LPL and HPL groups were found to have significantly enriched regulation of signaling receptor activity, the response to toxic substances, nicotinamide adenine dinucleotide (NADH) dehydrogenase complex assembly, cytokine production, vesicle, and vacuole organization. In turn, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway tool classified DEGs that enrich molecular processes such as B and T-cell receptor signaling pathways, natural killer cell-mediated cytotoxicity, Fc gamma R-mediated phagocytosis, toll-like receptor signaling pathways, TNF, mammalian target of rapamycin (mTOR) signaling and forkhead box O (Foxo) signaling pathways, etc. Our data indicate that changes in SRLV proviral load induced altered expression of genes related to different biological processes such as immune response, inflammation, cell locomotion, and cytokine production. These findings provide significant insights into defense mechanisms against SRLV infection. Furthermore, these data can be useful to develop strategies against SRLV infection by selection of animals with reduced SRLV proviral concentration that may lead to a reduction in the spread of the virus.

Genome analysis of small-ruminant lentivirus genotype E: a caprine lentivirus with natural deletions of the dUTPase subunit, vpr-like accessory gene, and 70-base-pair repeat of the U3 region.

The nucleotide sequence of the highly divergent small-ruminant lentivirus genotype E has been determined. The full genome consists of 8,418 nucleotides and lacks two large portions corresponding nearly to the entire dUTPase subunit of the pol and vpr-like accessory genes. Moreover, the 70-bp repeat of the U3 region of the long terminal repeat was observed to be deleted. Interestingly, this lentivirus genotype is able to persist in a local breed population, and retrospective analysis revealed its presence in milk samples collected in 1999. gag sequences obtained from a flock coinfected with the B1 and E genotypes revealed that the evolutionary rates of the two viruses were quite similar. Since a reduced viral load and/or disease progression was observed for viruses with artificially deleted dUTPase and vpr-like genes, it is proposed that this viral cluster be designated a low-pathogenicity caprine lentivirus.

Lung tumor development and spontaneous regression in lambs coinfected with Jaagsiekte sheep retrovirus and ovine lentivirus.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring and experimentally inducible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The first aim of this study was to monitor the development of OPA with minimally invasive, real-time observations of animals experimentally infected with JSRV as well as ovine lentivirus (maedi-visna virus). Worldwide, simultaneous infection of sheep with these 2 retroviruses is a common occurrence, naturally and experimentally; consequently, the lung tumor homogenates used as inocula contained both viruses. Following inoculation, computed tomography was used to detect tumor nodules early, before the onset of clinical signs, and to monitor tumor advancement. However, not only was OPA disease progression observed, but the apparent spontaneous regression of OPA was witnessed. In fact, regression was more common than progression following JSRV inoculation of neonatal lambs. Immune responses were detected, particularly involving CD3(+) T cells and the production of antibodies against JSRV that may mediate the spontaneous regression of JSRV-induced OPA. The second aim of this study was to determine whether OPA tumors harbor genetic alterations similar to those found in human lung adenocarcinoma. No mutations were found in the tyrosine kinase domain of the epidermal growth factor receptor, KRAS codons 12 and 13, or the DNA-binding domain of p53 in tumor DNA from naturally occurring and experimentally-induced OPA cases. Overall, the genetic profile combined with the disease development data provides further important characterization of OPA and describes, for the first time, spontaneous regression of OPA tumors in experimentally infected sheep.

Ovine progressive pneumonia virus capsid antigen as found in CD163- and CD172a-positive alveolar macrophages of persistently infected sheep.

In situ detection of ovine progressive pneumonia virus (OPPV) and the phenotypic identification of the cells that harbor OPPV have not been described for the OPPV-affected tissues, which include lung, mammary gland, synovial membranes of the carpal joint, and choroid plexus of the brain. In this study, the authors first developed a single enzyme-based automated immunohistochemical (IHC) analysis for detection of OPPV capsid antigen (CA) on OPPV-affected tissues, using 2 anti-CAEV CA monoclonal antibodies, 5A1 and 10A1, and 2 enzyme-based IHC systems. Out of 10 naturally and persistently OPPV-infected ewes, OPPV CA was detected in intercellular regions of the carpal synovial membrane of 1 ewe, in cells resembling alveolar macrophages and pulmonary interstitial macrophages in lung tissue of 3 ewes, and in mammary alveolar cells of 1 ewe. Furthermore, dual enzyme-based automated IHC analyses revealed that OPPV CA was predominantly detected in CD172a- or CD163-positive alveolar macrophages of the lungs and mammary gland. That anti-inflammatory (CD163) and downregulatory (CD172a) types of alveolar macrophage harbor OPPV CA leads to the possibility that during persistent infection with OPPV, the host alveolar macrophage might serve to limit inflammation while OPPV persists undetected by the host adaptive immune response in the lung and mammary gland.

Impact of natural sheep-goat transmission on detection and control of small ruminant lentivirus group C infections.

Dissemination of small ruminant lentivirus (SRLV) infections in Norway is affected by the different control strategies used for maedi-visna virus (MVV) infections in sheep and caprine arthritis-encephalitis virus (CAEV) infections in goats. Here we investigated SRLV phylogenetic group variants in sheep. CAEV-like isolates, belonging to phylogenetic group C, were found among both seropositive sheep and goats in mixed flocks, in which sheep and goats are kept together. Intra-herd clustering confirmed that mixed flock animals were infected by the same virus variant, suggesting ongoing interspecies transmission. Few sheep flocks were found to be infected with the MVV-like phylogenetic group A. The apparent absence of SRLV group A type in goats is probably due to the MVV control programme and animal management practices. SRLV group C targets lungs and mammary glands in sheep, and induces typical SRLV pathological lesions. SRLV group C isolated from the sheep mammary glands suggested a productive infection and potential for transmission to offspring. SRLV group C was most prevalent among goats. A lower PCR sensitivity in seropositive sheep suggested a lower load of SRLV group C provirus in sheep than in goats. Higher genetic divergence of group C than in other SRLV groups and extensive heterogeneity among group C isolates in the matrix C-terminal region demonstrate the need for identifying conserved target regions when developing PCR protocols for SRLV detection. As sheep and goats may serve as reservoirs for all SRLV genogroup types, successful control programmes require inclusion of both species.

Impact of the subclinical small ruminant lentivirus infection of female goats on the litter size and the birth body weight of kids.

A longitudinal study was conducted in a single dairy goat herd to investigate the relationship between subclinical small ruminant lentivirus (SRLV) infection in does and litter size (LS) or birth body weight of kids (BW). Each year kids born to seropositive and seronegative does were weighed before the first consumption of colostrum. LS and BW of each kid were recorded. BW was significantly negatively linked to LS (p = 0.006) - singletons weighed (mean ± SD) 4.20 ± 0.67 kg, twins - 3.75 ± 0.62 kg, and triplets and quadruplets - 3.38 ± 0.47 kg. Male kids were significantly heavier than female kids in twin litters (3.97 ± 0.53 kg vs. 3.52 ± 0.60 kg; p < 0.001) and triplet or quadruplet litters (3.62 ± 0.40 kg vs. 3.17 ± 0.43 kg; p < 0.001). However, BW of male and female kids from singleton litters did not differ (4.31 ± 0.71 kg vs. 4.07 ± 0.65 kg; p = 0.154). Then, two mixed models were developed to assess the relationship between LS (mixed Poisson log linear regression model) or BW (mixed linear model) and SRLV infection in the doe, controlling for potential confounders such as the effect of an individual doe, year in which the parturition took place, parity and kid's sex. Neither LS nor BW proved to be significantly associated with SRLV infection (p = 0.788 and p = 0.214, respectively). On this basis it was concluded that LS and BW were not affected by the subclinical SRLV infection of a doe.

Characterization of an immunodominant epitope of small ruminant lentivirus (SRLV) nucleoprotein.

To extend and complete the epitope mapping of gag-encoded structural proteins, the immunodiagnostic potential of nucleoprotein (NP) of two different small ruminant lentivirus (SRLV) genotypes were antigenically characterized. Respective recombinant counterparts were generated and used in an enzyme-linked immunosorbent assay (ELISA) format to test a panel of sera from infected flocks. Results clearly indicate that a single linear epitope located within the C-terminal is partially cross-reactive among different SRLV genotypes and may complement multiple epitope ELISA for serological diagnosis of infection. However, in contrast to matrix and capsid antigen epitopes, which drive a genotype-specific immunoresponse, a moderate degree of variation was identified in NP independently of the genotype to which it belongs.

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).

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.

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.