An outbreak of visna-maedi in a flock of sheep in Southern Brazil.

Visna-maedi is a multisystemic and progressive inflammatory disease caused by a non-oncogenic retrovirus (Visna-maedi virus, VMV). An outbreak of visna-maedi occurred in Southern Brazil in sheep with clinical signs of blindness and stumbling gait. At post-mortem examination, all animals had similar lesions, including heavy non-collapsed lungs and multifocal yellow areas in the cerebral white matter, affecting mainly the periventricular region. These lesions corresponded histologically to lymphocytic interstitial pneumonia and histiocytic periventricular encephalitis surrounding areas of necrosis, in addition to significant demyelination in the brain. Serology was performed in all the sheep from the flock and 14% were seropositive for VMV. The presence of VMV was confirmed through PCR and partial sequencing of the 5'LTR. Sequencing demonstrated that the virus had 89.7 to 90.0% of nucleotide identity with VMV strains reported in the USA. This is the first description of clinical disease related to VMV in Brazil leading to economic losses. This study calls for the need to implement control measures to prevent the spread of small ruminant lentiviruses in Brazil.

Clearance of Maedi-visna infection in a longitudinal study of naturally infected rams is associated with homozygosity for the TMEM154 resistance allele.

Maedi-visna (MV) is a lentiviral disease of sheep responsible for severe production losses in affected flocks. There are no vaccination or treatment options with control reliant on test and cull strategies. The most common diagnostic methods used at present are combination ELISAs for Gag and Env proteins with virus variability making PCR diagnostics still largely an experimental tool. To assess variability in viral loads and diagnostic tests results, serology, DNA and RNA viral loads were measured in the blood of 12 naturally infected rams repeatedly blood sampled over 16 months. Six animals tested negative in one or more tests at one or more time points and would have been missed on screening programmes reliant on one test method or a single time point. In addition the one animal homozygous for the 'K' allele of the TMEM154 E35K SNP maintained very low viral loads in all assays and apparently cleared infection to below detectable limits at the final time point it was sampled. This adds crucial data to the strong epidemiological evidence that this locus represents a genuine resistance marker for MV infection and is a strong candidate for selective breeding of sheep for resistance to disease.

A novel 2 bp deletion variant in Ovine-DRB1 gene is associated with increased Visna/maedi susceptibility in Turkish sheep.

Visna/maedi (VM) is a multisystemic lentivirus infection of sheep that affecting sheep industry across the globe. TMEM154 gene has been identified to be a major VM-associated host gene, nevertheless, a recent study showed that the frequency of the VM-resistant TMEM154 haplotypes was very low or absent in indigenous sheep. Thus, the present study was designed to determine other possible co-receptors associated with VM. For this purpose, DRB1 gene, which is renowned for its role in host immune response against various diseases was targeted. A total number of 151 case-control matched pairs were constructed from 2266 serologically tested sheep. A broad range of DRB1 haplotype diversity was detected by sequence-based genotyping. Moreover, a novel 2 bp deletion (del) in the DRB1 intron 1 was identified. For the final statistic, the sheep carrying VM-resistant TMEM154 diplotypes were removed and a McNemar's test with a matched pairs experimental design was conducted. Consequently, it was identified for the first time that the 2 bp del variant is a genetic risk factor for VM (p value 0.002; chi-square 8.31; odds ratio 2.9; statistical power 0.90) in the dominant model. Thus, negative selection for 2 bp del variant could decrease VM infection risk in Turkish sheep.

Test positivity for Maedi-Visna virus and Mycobacterium avium ssp. paratuberculosis in Sarda ewes: Effects on milk composition and coagulation traits and heritability estimates for susceptibility.

Maedi-Visna virus (MVV) and Mycobacterium avium ssp. paratuberculosis (MAP) are two pathogens that cause chronic, production-limiting diseases in dairy sheep. Although they are present worldwide, there are no detailed reports on their actual effects on milk traits in the literature. This study was designed to investigate the effects of test positivity to MVV and MAP on ovine milk yield, composition and coagulation properties, and curd-firming over time (CFt) variables in clinically healthy animals at the field level. The additive genetic variation and heritabilities of MVV and MAP positivity were also estimated. Milk samples were collected from 1,079 Sarda sheep kept on 23 farms, and pedigree information was obtained from the flock book. Milk yield was also recorded on the sampling date. Positivity for MVV and MAP was determined from milk samples using indirect ELISA test kits. Milk composition traits were measured by spectroscopy, milk coagulation properties were measured with a Formagraph (Foss Italia, Padua, Italy), and CFt traits were calculated using the data from the Formagraph diagram. The effects of MVV and MAP positivity on milk traits were determined through a set of mixed linear models, which took into account various sources of variation, such as days in milk, parity, and flock effects, and included the effects (positive or negative) of the 2 pathogens. A Bayesian threshold sire model with sire relationship was used to estimate genetic variation and heritability. The overall animal prevalence of MVV-positive ewes was 43.6%; on only 1 farm of the 23 tested were all sampled ewes negative. An overall animal prevalence of 10.6% was recorded for MAP, with 4 farms at 0%. Positivity for MVV significantly affected the logarithmic score of the bacterial count, curd firmness after 30 min and 45 min, and the curd-firming instant rate constant. We found significant effects of MAP infection on milk composition, pH, and rennet coagulation time. The mean of the posterior distributions of heritability estimates on the liability scale was 0.15 for MAP and 0.07 for MVV. Our results demonstrate that only a few traits are negatively affected by MVV and MAP positivity, and that there is exploitable genetic variation in MVV and MAP susceptibility in dairy sheep.

Characterization of minimal lesions related to the presence of visna/maedi virus in the mammary gland and milk of dairy sheep.

BACKGROUND: In order to characterize the complete range of lesions, especially minimal, affecting mammary gland and viral antigen distribution and target cells using immunohistochemistry in naturally Visna/maedi (VM) 84 infected sheep were studied, forty-four from flocks with clinical cases (A) and 35 randomly sampled from two abattoirs (B) together with five negative controls (C). An immunocytochemistry technique was developed and further milk samples (n = 39) were used to study viral excretion, carrier cells and the role of milk and colostrum in the transmission of the disease. RESULTS: All sheep from group C and three sheep from group B were negative to VM in tissue sections by histopathology, immunohistochemistry and PCR, and also in serum using ELISA. Several degrees of CD3 + lymphocytic interstitial mastitis were observed in groups A and B: minimal (+) n = 26 sheep; moderate (++), n = 32 and severe (+++), n = 12. No differences in lesion distribution were observed between groups A and B. Viral presence was confirmed by immunohistochemistry using two different antibodies and/or PCR in every tissue with lesions while serology was negative in six sheep with lesions. Two milk samples taken from milk tanks from two flocks from group A and fourteen milk samples from 29 infected sheep from group B were positive to VM (most of them from animals with moderate and severe lesions). Positivity was only found in macrophages, even in focal and minimal lesions, while no positivity was observed in epithelial or any other cells in either tissue and milk samples. CONCLUSIONS: This new observation of the minimal lesions described in this work increased the prevalence of VM lesions in mammary gland up to 90.9% and VM should be considered as a differential diagnosis when minimal interstitial lesions are detected. A high prevalence of VM was observed in intensive milk-producing sheep, ELISA serology did not detect as positivity all infected animals, while histology, IHC or PCR showed higher sensitivity. The cytological technique developed was very useful in milk-cell studies using hematoxylin and eosin and immunocytochemistry. Viral detection in milk samples (16/39) confirms a potential but limited role of milk/colostrum in viral transmission.

First survey on association of TMEM154 and CCR5 variants with serological maedi-visna status of sheep in German flocks.

Maedi-visna, a disease caused by small ruminant lentiviruses (SRLVs), is present in sheep from many countries, also including Germany. An amino acid substitution (E/K) at position 35 of the transmembrane protein 154 (TMEM154) as well as a deletion in the chemokine (C-C motif) receptor type 5 gene (CCR5) were reported to be associated with the serological MV status and/or the SRLV provirus concentration in North American sheep populations. The aim of this study was to test if those two gene variants might be useful markers for MV susceptibility in Germany. For this purpose, more than 500 sheep from 17 serologically MV positive German sheep flocks with different breed backgrounds were genotyped applying PCR-based methods. Both, crosstab and non-parametric analyses showed significant associations of the amino acid substitution at position 35 of TMEM154 with the serological MV status (cut-off-based classification) and the median MV ELISA S/P value in all samples and in two of the four analyzed breed subsets. The deletion in the CCR5 promoter did not show a consistent association with serological MV status or median ELISA S/P value. It can be concluded that the amino acid substitution at position 35 of TMEM154 is a promising marker for breeding towards a lower number of serologically MV positive sheep in German flocks, at least in flocks of the Texel breed, while this remains questionable for the deletion in the CCR5 promoter. The findings of this study still need to be verified in additional sheep breeds.

Inflammatory Lesion Patterns in Target Organs of Visna/Maedi in Sheep and their Significance in the Pathogenesis and Diagnosis of the Infection.

Ovine visna/maedi (VM) infection is characterized by the development of chronic inflammatory lesions in different organs, mainly in the lung, mammary gland and central nervous system (CNS), with either histiocytic or lymphocytic pattern predominance being described in the CNS. To help to understand the role of host immune response in the development of these patterns, 50 naturally-infected sheep and eight non-infected sheep from intensive milk-producing flocks were studied. The histological lesion patterns in the three main target organs in each sheep were characterized. Lesion severity was determined, including minimal lesions. A histiocytic pattern was observed in 23 sheep (46%), a lymphocytic inflammatory pattern in 19 sheep (38%) and a mixed inflammatory pattern in eight sheep (16%). Forty animals showed moderate or severe lesions (80%), while 10 had minimal lesions (20%). Moderate or severe lesions affected only one target organ in 20 sheep (50%), two organs in 14 sheep (35%) and all three target organs in six sheep (15%). Infection was confirmed by immunohistochemistry (IHC) using an antibody specific for p28 of VM virus/caprine arthritis and encephalitis virus and by polymerase chain reaction (PCR) in all sheep. Minimal inflammatory lesions associated with positive IHC and PCR were observed. The results suggest that the development of a predominant inflammatory pattern in different organs within the same animal may be related to the host immune response. Minimal and focal lesions, not considered previously, should be taken into account when formulating a differential diagnosis in affected sheep.

Molecular characteristics and prevalence of small ruminant lentiviruses in goats in Japan.

Small ruminant lentiviruses (SRLVs), which comprise caprine arthritis-encephalitis virus (CAEV) and maedi-visna virus (MVV), are prevalent in goats and sheep worldwide, including in Japan. However, little is known about the molecular characteristics of goat lentiviruses in Japan. In this study, a molecular and phylogenetic analysis of the long gag region was performed. The phylogenic tree demonstrated that all samples belonged to SRLV subtype B1. Two clusters were identified, with one cluster distinct from previously reported strains of subtype B1. In addition, several alterations in the amino acid sequence were detected in immunodominant epitopes of the gag region. To gain a deeper understanding of the genetic diversity of SRLVs in Japan, it will be necessary to increase the sample size and conduct a broader survey. The present report is important for establishing baseline information on the prevalence of SRLV in Japan and providing data to develop a new, more sensitive diagnostic test for effective control of SRLV.

SNPs in APOBEC3 cytosine deaminases and their association with Visna/Maedi disease progression.

The Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) genes are able to inhibit the replication of a wide range of exogenous retroviruses, as well as endogenous retroviruses and retrotransposons. Three APOBEC3 genes, named APOBEC3Z1, APOBEC3Z2 and APOBEC3Z3, have been described in sheep. In this work the three genes have been screened in order to identify polymorphisms. No polymorphism was detected for the A3Z2 and A3Z3 genes but 16 SNPs and a 3-bp deletion were found in the A3Z1 gene. A thermoestability prediction analysis was applied to the detected amino acidic SNPs by three different programs. This analysis revealed a number of polymorphisms that could affect the protein stability. The SNPs of the 3'UTR were tested to detect alterations on the predicted microRNA target sites. Two new microRNA target sites were discovered for one of the alleles. Two SNPs were selected for association studies in relation with the retroviral disease Visna/Maedi in Latxa and Assaf sheep breeds. Although association analyses resulted unconclusive, probably due to the unsuitability of the SNP allele frequency distribution of the selected polymorphisms in the analyzed breeds, these genes remain good candidates for association studies.

Modulation of the long terminal repeat promoter activity of small ruminant lentiviruses by steroids.

Production and excretion of small ruminant lentiviruses (SRLVs) varies with the stage of the host reproductive cycle, suggesting hormonal involvement in this variation. Stress may also affect viral expression. To determine if hormones affect SRLV transcriptional activity, the expression of green fluorescent protein (GFP) driven by the promoters in the U3-cap region of the long terminal repeats (LTRs) of different strains of SRLV was assessed in cell culture. High concentrations of steroids (progesterone, cortisol and dehydroepiandrosterone) inhibited expression of GFP driven by SRLV promoters. This effect decreased in a dose-dependent manner with decreasing concentrations of steroids. In some strains, physiological concentrations of cortisol or dehydroepiandrosterone (DHEA) induced the expression of GFP above the baseline. There was strain variation in sensitivity to hormones, but this differed for different hormones. The presence of deletions and a 43 base repeat in the U3 region upstream of the TATA box of the LTR made strain EV1 less sensitive to DHEA. However, no clear tendencies or patterns were observed when comparing strains of different genotypes and/or subtypes, or those triggering different forms of disease.

Successful Visna/maedi control in a highly infected ovine dairy flock using serologic segregation and management strategies.

A control system for Visna/maedi virus (VMV) infection based on serologic segregation and management strategies was applied in an infected Spanish dairy Manchega breed sheep flock (n=670) that was affected by a severe respiratory process associated to VMV. The control started in 2004 and consisted on the serological study of animals, segregation in two different flocks (seropositive and seronegative), separate management of flocks, selection of young female lambs for replacement only from seronegative ewes offspring, immediate removal of seropositive animals detected in the seronegative flock and a management tending toward the reduction and final culling of the seropositive flock. The serological control was repeated yearly or twice a year, approximately. Initial VMV seroprevalence of the undivided flock was 66.4% (January 2004) that descended to 47.3%, 12.8%, 2.2% and 0.2% between July 2004 and May 2006. Residual seroprevalence fluctuated slightly thereafter with a peak of 2.2% in April 2008. After segregation, number of animals in the seronegative flock was 378 that descended to 323 in October 2005. Since then, this number has increased steadily reaching 650 sheep in December 2011. The seropositive flock was progressively reduced by culling until its total disappearance in June 2010. This work presents the detailed results obtained in the control strategy against VMV in a single dairy sheep flock by implementing a segregation system based on serologic testing. The system is highly successful, as it reduces to residual levels VMV infection in about two years without the need of culling a high number of animals, as required by other methods. Moreover, the original size flock was been recovered within 8 years and has led to a subjective improvement of animal health and welfare in the flock. The residual seroprevalence could be eliminated at this stage by applying more sensitive molecular or other serological techniques to reach eradication.

Small ruminant lentiviruses in Jordan: evaluation of sheep and goat serological response using recombinant and peptide antigens.

Small ruminant lentiviruses infect sheep and goats worldwide, causing chronic progressive diseases and relevant economic losses. Disease eradication and prevention is mostly based on serological testing. The goal of this research was to investigate the presence of the small ruminant lentiviruses (SRLVs) in Jordan and to characterize the serological response in sheep and goat populations. A panel of sera were collected from flocks located in Northern Jordan and Jordan Valley. The samples were tested using three ELISA assays: a commercially available ELISA based on p25 recombinant protein and transmembrane peptide derived from British maedi-visna virus (MVV) EV1 strain, an ELISA based on P16-P25 recombinant protein derived from two Italian strains representative of MVV- and caprine arthritis encephalitis virus (CAEV)-like SRLVs, and an ELISA based on SU5 peptide from the same two Italian isolates. The results indicate that both MVV- and CAEV-like strains are present in Jordan and that the majority of the viruses circulating among sheep and goat populations belong to the MVV-like genotype.

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Vaccination delays Maedi-Visna lentivirus infection in a naturally-infected sheep flock.

BACKGROUND: The Maedi-Visna (MV) lentivirus causes two slowly progressive eventually fatal diseases of sheep, Maedi, a progressive interstitial pneumonia, and Visna, a progressive demyelinating disease of the central nervous system. Other lentiviruses also cause fatal slow infections in their natural hosts, e.g. the HIV virus in humans. Results of experimental vaccination against any lentivirus where vaccinees are challenged by natural routes, may therefore be of general interest. From 1991-1998 experiments with formalin-inactivated whole Maedi-Visna virus vaccine were carried out in the Department of Microbiology at the University of Iceland. Western Blot tests showed good immune response to all major proteins of the virus. When aluminium hydroxide was added to the vaccine all vaccinees developed neutralizing antibodies to the vaccine strain at titers 1/8 - 1/256. After housing 5 twin pairs, one twin in each pair vaccinated, the other unvaccinated, with infected sheep for 4 years, all the unvaccinated twins became infected, but only 2 of their vaccinated siblings as confirmed by virus cultivation experiments on tissues from their lungs spleens lymph nodes and choroid plexuses. RESULTS: One twin in each of 40 female twin pairs, born into a Maedi-Visna-infected sheep flock and kept under natural farming conditions in Cyprus, was vaccinated at birth, 3 weeks and 3 months, with formalin-inactivated whole Maedi-Visna lentivirus vaccine adjuvanted with aluminium hydroxide. 17 mothers of the twins were seronegative, 13 seroconverting and 10 had old infection. Of 17 vaccinees born to seronegative mothers 9 were uninfected at 28 months, but only 2 of their unvaccinated siblings. Of 13 unvaccinated twins born to seroconverting mothers, 12 caught infection during their first 10 weeks, but only 4 of their vaccinated siblings. Vaccination had no effects on 10 vaccinees born to mothers with long-standing Maedi-Visna infections and broad andibody response at birth of their lambs. CONCLUSION: Compared with their unvaccinated siblings, natural infection was delayed in significant number of vaccinated twins born by seronegative and seroconverting mothers and vaccinated at birth, 3 weeks and 3 months with formalin inactivated whole MV vaccine adjuvanted with aluminium hydroxide. Maternal antibodies interfered with vaccination so early in life if the mother had old infection.

The origin of lentivirus research: Maedi-visna virus.

Maedi and visna are contagious sheep diseases which were introduced into Iceland in 1933 by imported sheep of Karakul breed. Maedi, a slowly progressing pneumonia, and the central nervous system disease visna were shown to be transmissible in sheep and most likely caused by a virus. In 1957, visna virus was isolated in tissue culture from sheep brain and maedi virus was isolated the following year from sheep lungs. Both viruses showed similar cytopathic effect in tissue culture. Electron microscope studies of ultrathin sections from visna virus infected cells demonstrated spherical particles, 70-100 nm in diameter, which were formed by budding from the cell membrane. Later studies showed identical particles in maedi virus infected cultures. These, and several other comparative studies, strongly indicated that maedi and visna were caused by strains of the same virus, later named maedi-visna virus (MVV). Comparative studies in tissue culture suggested that MVV was related to RNA tumor viruses of animals, the oncornaviruses. This was later supported by the finding that MVV is an RNA virus. A few months after reverse transcriptase was demonstrated in oncornaviruses, the enzyme was also found in MVV virions. Thus, MVV was classified as a retrovirus together with the oncornaviruses. However, MVV is not oncogenic in vivo or in vitro and was in 1975 placed in a subgroup of retroviruses named lentiviruses, which cause cytopathic effect in vitro and slowly progressing inflammatory disease in animals, but are nononcogenic. In the early 1980s, the causative agent of AIDS was found to be a non-oncogenic retrovirus and was classified as a lentivirus. Thus, HIV became the first human lentivirus.

Perivascular inflammatory cells in ovine Visna/maedi encephalitis and their possible role in virus infection and lesion progression.

We examined the distribution in the perivascular spaces of Visna/maedi antigen, T cells (CD3+, CD4+ and CD8+), B cells and macrophages by immunohistochemistry in 22 natural cases of Visna/maedi encephalitis. Sheep showed lymphocytic or histiocytic lesions. In mild lymphocytic lesions, the viral antigen was detected in perivascular cuffs where CD8+ T cells predominated, but in severe lymphocytic lesions, sparse antigen was identified, and CD8+/CD4+ T cells appeared in a similar proportion in multilayer perivascular sleeves. In histiocytic lesions, vessels were surrounded by macrophages with abundant viral antigen, with CD8+/CD4+ T cells and B cells in the periphery. These results could reflect different stages of virus neuroinvasion and clarify the neuropathogenesis of Visna/maedi encephalitis.

Ovine TRIM5α can restrict visna/maedi virus.

The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.