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.

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.

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.

Visna/Maedi virus genetic characterization and serological diagnosis of infection in sheep from a neurological outbreak.

An extensive outbreak characterized by the appearance of neurological symptoms in small ruminant lentivirus (SRLV) infected sheep has been identified in Spain, but the genetic characteristics of the strain involved and differential diagnostic tools for this outbreak remain unexplored. In this work, 23 Visna-affected naturally infected animals from the outbreak, 11 arthritic animals (both groups presenting anti-Visna/Maedi virus serum antibodies), and 100 seronegative animals were used. Eight of the Visna-affected animals were further studied post-mortem by immunohistochemistry. All had lesions in spinal cord, being the most affected part of the central nervous system in six of them. A representative strain of the outbreak was isolated. Together with other proviral sequences from the outbreak the virus was assigned to genotype A2/A3. In vitro culture of the isolate revealed that viral production was slow/low in fibroblast-like cells but it was high in blood monocyte-derived macrophages. The long terminal repeat (LTR) of the viral genome of this isolate lacked an U3-duplication, but its promoter activity in fibroblast-like cells was normal compared to other strains. Thus, viral production could not be inferred from the LTR promoter activity in this isolate. Analysis of the viral immunodominant epitopes among SRLV sequences of the outbreak and other known sequences allowed the design of a synthetic SU peptide ELISA that detected the Visna affected animals, representing a tool of epidemiological interest to control viral spread of this highly pathogenic strain.

Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus.

The aim of this study was the development of gag and pol dual labelled probe real-time PCR and RT PCR assays to quantify the proviral load and the transcripts of the British Visna/maedi virus EV1 strain. Primers and probes were chosen based on the consensus sequences of gag and pol clones representative of EV1 genetic variants. Both PCRs had a detection limit of 3 copies of target gene, with a linearity over 6 orders of magnitude. The performances of the two PCRs in vivo were evaluated and compared on a panel of DNAs extracted from blood of sheep infected experimentally with EV1. The pol assay detected in most cases lower numbers of viral molecules than gag assay, yielding some false negative results. The gag real-time RT PCR had a detection limit of 100 RNA molecules with a linearity over 5 orders of magnitude. This did not result in a lower performance of the RT PCR compared to the PCR in cells permissive for virus replication, which contain higher numbers of viral transcripts than proviral genomes. The real-time assays developed in this study, particularly the gag assay, provide a sensitive tool which can be used to quantify the viral load in experimental infections.

Diagnosis of the nervous form of Maedi-Visna infection with a high frequency in sheep in Castilla y Leon, Spain.

Between 1997 and March 2004, the nervous form, or visna, of maedi-visna infection was diagnosed in 71 of 1631 sheep (4.35 per cent) examined in the Castilla y León region of Spain, of which 634 had shown nervous signs. The presence of the virus was confirmed by immunohistochemistry and in some cases by pcr on frozen-thawed or paraffin-embedded tissue samples. The main clinical signs were hindleg ataxia and paresis, but blindness or nystagmus were also observed. Thirty-three of the affected sheep (46.5 per cent) were two years old or younger. The affected sheep showed variable degrees of a non-suppurative meningoencephalitis, and immunohistochemistry identified positive cells in all cases, with no relation to the intensity of the inflammatory lesion.

[Clinical findings and diagnostic procedure in a dairy sheep with visna]. / Klinische Befunde und diagnostisches Vorgehen bei einem Milchschaf mit Visna.

The goal of this report was to describe the clinical signs and diagnosis of Visna in a seven-year-old East Friesian milk sheep. A striking feature was that the ewe's behaviour changed frequently. At one time, the ewe was somnolent. A few minutes later, the sheep was alert and eating hay. The ewe was thin. It had a slight head tilt and a severe generalised ataxia. Based on the neurological symptoms and chronic weight loss, a tentative diagnosis of visna was made. Serological testing for maedi-visna was positive, and the ewe was euthanised. A postmortem examination was performed, and lung and brain samples were collected aseptically. Cell cultures from these organs were positive for viral enzymatic reverse transcriptase and for maedi-visna RNA.

Use of a recombinant maedi-visna virus protein ELISA for the serologic diagnosis of lentivirus infections in small ruminants.

Highly purified recombinant gag and env proteins derived from Icelandic strain 1514 of maedi-visna virus were used in an indirect enzyme immunoassay (ELISA) to detect antibodies to small ruminant lentiviruses in sheep and goat sera. The recombinant protein-based ELISA performed very well relative to whole maedi-visna virus and whole caprine arthritis-encephalitis-virus-based ELISAs in its ability to detect anti-maedi visna virus and anti-caprine arthritis-encephalitis virus antibodies, despite the antigenic and genomic variability that is known to exist within and between these two small ruminant lentiviruses. The data suggest that these recombinant maedi-visna virus proteins can be reliably used in an ELISA for the routine serodiagnosis of lentiviral infections in sheep and goats.

PCR detection of lentiviral GAG segment DNA in the white blood cells of sheep and goats.

A PCR assay for the detection of small ruminant lentiviral gag DNA (provirus) in the white blood cells of sheep and goats was developed and compared with a serological test (AGIDT). A sample of the DNA prepared from the white blood cells in 3 ml of blood from 208 sheep and goats from 18 different flocks was subjected to PCR assay. One of 85 animals from flocks accredited under the Dutch national MVV/CAEV control programme was positive by PCR while none was positive by AGIDT. In infected flocks, the AGIDT appeared slightly more sensitive, but preliminary results show that the sensitivity of the PCR assay may be further improved by increasing the number of monocytes tested. The PCR assay, however, was clearly more sensitive in detecting animals in the early stages of infection. With the use of a set of mixed primers and probes, the assay was able to detect the variety of CAEV and MVV strains occurring in the field.

A comparative study on the diagnosis of maedi-visna infection in serum and colostrum samples using agar gel immunodiffusion (AGID) technique.

Serum/colostrum pairs were collected from 245 ewes in 6 sheep herds which had been determined previously to be infected with MV virus and were tested against maedi-visna infection using AGID test. Positive rates were detected as 3.8-41.2% in tested flocks. Serum and colostrum samples obtained from 53 sheep were positive for MV virus specific antibodies by AGID test. 16 colostrum samples were negative although serum samples obtained from the same animals were found to be positive for MV antibodies. Of the 245 sera and colostrum pairs tested, there was total agreement of results (+ or -) in 229 and disagreement in the results with the other 16 serum/colostrum pairs. Of the latter, all serum samples were positive and all colostrum samples were negative for MV antibodies. This study compared colostrum and serum samples for the determination of MV antibodies using AGID test under field conditions on naturally infected animals and on healthy animals. The results show that colostrum antibodies can be detected using AGID test and that colostrum is a reliable material to determine anti-MV virus antibodies. The procedure can be used for herd diagnosis.

[Continuation of the observation and serological investigation of a Maedi-Visna virus infected sheep flock from January 1990 to June 1996]. / Fortsetzung der Beobachtung und der serologischen Untersuchung einer Maedi-Visna-Virus-infizierten Schafherde von Jänner 1990 bis Juni 1996.

This paper describes the second part of a longtime-study, started in 1987. Serologic investigations for detecting antibodies against Maedi Visna-virus (MVV) were performed, involving an institute own sheep flock. The method used was the immunodiffusiontest. The flock consisted of different breeds and their offsprings. So far, the virus seems to persist in the herd. This work also shows the importance of the central role of the does for spreading the virus. Seroconversion was detected in a sheep at the age of 32 months. The mother of this sheep was a thoroughbred and MVV-negative mountain sheep. After removal of the animals with high antibody (ab)-titers, until the end of 1991, the percentage of seronegative sheep increased. Then seropositive sheep didn't show high ab-titers anymore. Since 1990 only offsprings increased the size of the herd. The health status of the flock was clinically inconspicuous. It can be concluded that in spite of good food quality, good hygiene, without culling positive animals and just giving away accidentally some sheep, no elimination of MVV was registered in the flock over a period of more than six years. There was only seen a reduction of seropositive animals. Single results of serological tests, without knowing the sheep and the serological status of the herd, could pretend a false negative status.