[Indurative mastitis in a herd of Dorper sheep caused by an infection with Maedi Visna virus]. / Indurative Mastitis in einer Herde Dorperschafe als Folge einer Infektion mit dem Maedi-Visna-Virus.

This case report describes indurative mastitis in a herd of sheep caused by Maedi Visna virus (MVV) infection. Reduced udder formation after delivery, small, indurated udders and increased losses of lambs were observed in a herd of Dorper sheep. Examination of the mammary gland and milk did not reveal findings characteristic of chronic bacterial mastitis. The protein supply was insufficient which may have contributed to reduced milk yield, but was considered unlikely as cause for the induration of the mammary gland. Nineteen of the 21 mothers were positive for MVV by serology. Mammary gland and supramammary lymph nodes were collected in a sheep with indurated udder at the time of slaughter. Meat inspection did not reveal lesions in any other organs. One part of the mammary gland showed a mild to moderate multifocal lymphohistiocytic mastitis, the other exhibited a severe diffuse lymphohistiocytic mastitis with atrophy of the glandular acini, vasculopathy, fibrosis and calcification. MVV antigen was visualized by immunohistochemistry in macrophages, dendritic cells, epithelial cells and endothelial cells in the mammary gland, and macrophages and dendritic cells in the supramammary lymph nodes. A large amount of MVV provirus was detected in the supramammary lymph nodes and the severely indurated part of the mammary gland by PCR. In conclusion, indurative mastitis as a result of a systemic infection may occur independently of the commonly known manifestations of Maedi Visna in the lung and central nervous system. MVV should be considered as differential diagnosis in mastitis of sheep. The MVV status of the herd can be tested by serological detection of specific antibodies. Additionally, characteristic histological lesions are present in the mammary gland. MVV antigen can also be detected by immunohistochemistry and MVV provirus by PCR in the altered mammary gland and regional lymph nodes.

Evaluation of three commercial ELISA tests for serological detection of maedi-visna virus using Bayesian latent class analysis.

Early and accurate diagnosis is fundamental for successful surveillance and control of maedi-visna virus (MVV). MVV was detected in Norway in 2019, almost 14 years after the previous outbreak. Genetic analysis indicates persistence of the virus in the sheep population since 2005. The virus was not detected despite continuous serological surveillance. This emphasises the need for improved surveillance, which relies on an understanding of both diagnostic test performance, sampling strategy and the prevalence of the disease. This study therefore aims to evaluate three commercial ELISA tests for MVV antibodies. We conducted a retrospective study using 615 samples from six flocks diagnosed with MVV in 2019. We ran all samples with the following three tests: ID Screen® MVV/CAEV Indirect (IDvet, Grabels, France), IDEXX MVV/CAEV p28 Ab Verification Test (IDEXX Laboratories, Maine, USA) and Elitest MVV/CAEV (Hyphen Biomed, Neuville-sur-Oise, France), hereinafter referred to as ID Screen, IDEXXp28 and Elitest respectively. Without a perfect reference test, we used Bayesian latent class analysis, including conditional dependence between tests, to estimate diagnostic accuracy and true prevalence in the flocks. Using recommended cut-off values, we found that ID Screen and Elitest had significantly higher sensitivity (Se) estimates (99.3 % [97.4-100.0, 95 % Posterior Credible Interval] and 97.4 % [94.1-99.7 %], respectively) than IDEXXp28 (79.5 % [72.3-86.0 %]), while IDEXXp28 and ID Screen had significantly higher specificity (Sp) estimates than Elitest (99.7 % [99.1-100.0], 99.1 % [98.0-99.8 %] and 93.7 % [91.4-95.7 %], respectively). The estimated true prevalence in the six flocks ranged from a median of 0.8-93.5 %. Combining ID Screen and Elitest in serial interpretation showed the highest median Se and Sp (96.7 % [92.0-99.1] and 100.0 % [99.9-100.0], respectively), as well as the highest median positive predictive value (PPV) for the population with the lowest prevalence. Our study supports the use of ID Screen for screening. Further verification with Elitest in serial interpretation will enhance the PPV.

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.

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.

Maedi in slaughtered sheep: a pathology and polymerase chain reaction study in southwestern Iran.

Maedi-visna (MV) is an important slow viral disease of sheep leading to a progressive lymphoproliferative disease. It affects multiple organs primarily the lungs, where it causes interstitial pneumonia (maedi). In this study, the lungs of 1,000 sheep carcasses were grossly inspected and those suspected to have maedi were studied at histopathological and molecular levels. A polymerase chain reaction (PCR) technique that amplified a 291-base pair DNA in the long terminal repeat (LTR) sequence of MV provirus was conducted on all the 50 suspected lungs together with 10 normal appearing lungs as controls. Amplicons of the expected size were detected in 11 (n=11/50) suspected sheep, and one of the 10 control sheep. Histopathologic study of the pulmonary lesions of all 11 (n=11/11) positive sheep showed MV lesions, including hyperplasia of the perivascular and peribronchiolar lymphoid cells, interstitial lymphoplasmacytic infiltration and smooth muscle hyperplasia and the histopathologic findings were correlated with PCR results. In contrast, the tissue sections of control animals were almost normal at histopathological level; however, PCR technique demonstrated that one of them was affected by maedi. This study showed that the LTR-PCR had high specificity and sensitivity in diagnosis of this viral infection. This study is the first to evaluate the prevalence of MV virus infection in sheep in Iran.

Diagnostic performance of PCR and ELISA on blood and milk samples and serological survey for small ruminant lentiviruses in central Spain.

The diagnostic performance of an ELISA for the detection of antibodies to the small ruminant lentiviruses (SRLVs) maedi-visna virus and caprine arthritis-encephalitis virus in milk and corresponding blood samples was evaluated in 50 sheep. The agreement between ELISA results in blood and milk was 90 per cent, and the κ value was 0.79. In addition, a serological survey in the central zone of Spain was performed using milk samples from 413 animals (250 sheep and 163 goats) from 12 flocks/herds. All flocks/herds had some animals that were positive for SRLV. Among the animals, 60.0 per cent of the sheep and 8.0 per cent of the goats tested were seropositive. Each sample was also tested using a PCR technique, which increased the percentage of positive animals detected. Using a combination of ELISA and PCR gave a total of 72.2 per cent of sheep and 28.8 per cent of goats positive for SRLV.

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.

Diagnostic performance of ELISA and PCR in identifying SRLV-infected sheep and goats using serum, plasma and milk samples and in early detection of infection in dairy flocks through bulk milk testing.

The objective of the study was to evaluate the diagnostic performances of the ELITEST-MVV ELISA for detection of antibodies against small ruminant lentiviruses and of two recently published PCRs for the detection of proviral DNA of SRLV in blood and corresponding individual milk samples. In addition, the feasibility of bulk milk testing was investigated by titrating ELISA positive pooled milk samples in negative milk, and by investigating bulk milk samples by ELISA and PCR in relation to the SRLV-status of the flocks. The results show that plasma and milk are suitable replacements for serum. For sheep, both PCRs showed a better diagnostic performance than for goats. ELISA results for bulk milk samples were promising with a putative detection limit of <3% within-herd prevalence using 1/10 pre-diluted samples and even <1% within-herd prevalence when samples were tested undiluted. In a panel of 249 bulk milk samples, all samples from SRLV free flocks (n=138) tested negative in the ELISA, while 50% of the samples from flocks with an unknown SRLV-status (n=111) were positive. For a subset of 59 bulk milk samples, agreement between ELISA results and leader-gag PCR results was almost 100%. These results demonstrate the potential of bulk milk testing as a cost effective tool for early detection of infection in dairy flocks, which is essential for SRLV-monitoring programs.

Evaluation of a caprine arthritis-encephalitis virus/maedi-visna virus indirect enzyme-linked immunosorbent assay in the serological diagnosis of ovine progressive pneumonia virus in U.S. sheep.

A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (+ or - 5.8%) and 95.9% (+ or - 2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.

Development and validation of an ovine progressive pneumonia virus quantitative PCR.

Ovine progressive pneumonia virus (OPPV) infects at least one sheep in 81% of U.S. sheep flocks, as determined by serology, and can cause viral mastitis, arthritis, dyspnea, and cachexia. Diagnostic tests that quantify OPPV proviral load in peripheral blood leukocytes (PBL) provide an additional method for identification of infected sheep and may help to further understanding of the pathogenesis of OPPV-induced disease. In this study, we compared a new OPPV real-time quantitative PCR (qPCR) assay specific for the transmembrane region of the envelope gene (tm) with a competitive inhibition enzyme-linked immunosorbent assay (cELISA) using 396 PBL samples and sera from Idaho sheep. The OPPV qPCR had a positive concordance of 96.2% +/- 2.3% and a negative concordance of 97.7% +/- 2.5% compared to the cELISA, with a kappa value of 0.93, indicating excellent agreement between the two tests. In addition, the presence of tm in the three OPPV qPCR-positive and cELISA-negative sheep and in 15 sheep with different OPPV proviral loads was confirmed by cloning and sequencing. These data indicate that the OPPV qPCR may be used as a supplemental diagnostic tool for OPPV infection and for measurement of viral load in PBLs of infected sheep.

Detection of maedi-visna virus in the liver and heart of naturally infected sheep.

Maedi-visna virus (MVV) in sheep, which infects mainly cells of the monocyte/macrophage lineage, produces changes in the lung, mammary gland, brain and joints. In this study, however, the liver and heart of six naturally infected sheep were examined for the presence of the virus. MVV proviral DNA was demonstrated by polymerase chain reaction (PCR) analysis, and immunohistochemical examination revealed viral antigens in the cytoplasm of hepatocytes and cardiac myocytes. Although histopathological examination showed mild to moderate, chronic lymphocytic cholangiohepatitis and myocarditis and the presence of small lymphoid aggregates, the typical maedi lymphoproliferative lesions (lymphoid follicle-like structures of considerable size with germinal centres) were not seen in the liver and heart. These novel findings suggest that, although the macrophage is the main cell for productive viral replication, the liver and heart represent additional MVV targets.

Diagnosis of clinical cases of the nervous form of Maedi-Visna in 4- and 6-month-old lambs.

The nervous form of Maedi-Visna (MV) infection was diagnosed in four lambs aged 4 and 6 months, belonging to three different Assaf flocks that were managed intensively for milk and meat production. The animals presented with hindleg ataxia that rapidly progressed to complete recumbency. Lesions consisted of a moderate to severe non-purulent encephalitis affecting mainly the cerebellar peduncles. MV virus was demonstrated in the damaged tissues by immunohistochemistry and polymerase chain reaction. The investigation demonstrated that the clinical presentation of the nervous form of MV which is reported to occur in adult sheep can also be observed in young animals.

Evaluation of three serological tests for diagnosis of Maedi-Visna virus infection using latent class analysis.

Maedi-Visna virus (MVV) infection in sheep is present in several European countries, including Norway. The current Norwegian surveillance and control programme for MVV infection uses three serological tests: an agar gel immunodiffusion test (AGID) and two commercially available indirect ELISAs (Institut Pourquier, P-ELISA and HYPHEN BioMed, H-ELISA). From 18 flocks with suspected or confirmed MVV infection, sera from naturally infected sheep were obtained, and sensitivity (Se) and specificity (Sp) of the three tests were estimated in absence of a perfect reference test using latent class models in a Bayesian analysis. The AGID had higher Sp (95% posterior credibility interval (PCI) [98.4; 99.9]) than either ELISA (95% PCI: P-ELISA, [95.1; 99.0]; H-ELISA, [91.4; 96.6]), but much lower Se (95% PCI: AGID, [41.4; 59.8]; P-ELISA, [92.7; 100.0]; H-ELISA, [90.9; 99.4]). Currently the P-ELISA is used for screening and positive samples are subsequently confirmed by a setup using all three tests in a serial reading. The Se and Sp of the serial interpretations with and without the H-ELISA were estimated. The results suggested that the H-ELISA could be dropped as a confirmatory test as the Se of the three test serial reading was reduced significantly without adding a significant improvement of the Sp compared to the serial reading of the P-ELISA and AGID alone. However, the perceived cost of false positives versus false negatives will influence this decision. Estimates of the predictive values for the tests and combinations suggested that the P-ELISA is a good choice of screening, but confirmatory tests are needed to achieve acceptable levels of positive predictive values.

Effects of fixative type and fixation time on the detection of Maedi Visna virus by PCR and immunohistochemistry in paraffin-embedded ovine lung samples.

In doubtful cases, the histopathological diagnosis of lesions induced by Maedi Visna virus (MVV), a chronic multisystemic lentiviral disease of sheep, needs to be confirmed by the demonstration of MVV in the tissues. The influence of fixatives and the duration of fixation on the detection of MVV by immunohistochemistry (IHC) and PCR in paraffin-embedded tissues was assessed in lung samples with lesions in different degree, from five sheep serologically positive. Samples were fixed in 10% neutral buffered formalin (NBF), Bouin's solution (BS) and a zinc salts-based fixative (ZSF), for different periods of time between 24 h and 30 days. The three fixatives preserved the morphology of the tissues, although in ZSF-fixed samples an increase in the number of desquamated cells was seen in the alveoli. Tissues showed a similar degree of immunolabelling, irrespective of the duration of fixation using ZSF and NBF fixatives. MVV nucleic acids could be detected in samples fixed up to 14 days in NBF and 30 days in ZSF. However, in BS fixed tissues, immunostaining was weak and non-specific signals were observed after 4 days of fixation. Amplification of proviral DNA could not be obtained by PCR in these samples. IHC detected viral antigens in all sheep whereas one sheep with mild lesions was always negative by PCR.

Prevalence of maedi-visna infection in culled ewes in Alberta.

Maedi-visna (MV) is a relatively common chronic infection of sheep in North America resulting in economic loss to the sheep industry. The objectives of this study were to: 1) measure the prevalence of MV infection in culled ewes in Alberta, by histologic examination (lungs and udder) and serologic testing using an agar gel immunodiffusion (AGID) test, 2) examine any geographic differences in its prevalence in the province, 3) evaluate the level of agreement between histopathologic examination and serologic testing, 4) grade the lesions and correlate the serologic results with the presence of severe histological lesions, and 5) correlate the presence of histological lesions in the lungs and udder in the same animal. Based on histologic findings, the prevalence of MV was 26.8%, compared with 13.0% using serologic testing. There were no significant geographical differences in prevalence, fair agreement (kappa = 42.0%) between histopathologic and serologic results, and poor agreement (kappa = 11.5%) between the presence of lung and udder histological lesions within the same animal. This study indicates that MV is relatively common in culled ewes in Alberta, with no significant geographic variation. The poor sensitivity of the AGID test, compared with histologic examination, should be taken into consideration when interpreting serologic results.

Evaluation of an ELISA to detect antibodies to maedi-visna virus in individual and pooled samples of milk from sheep.

An elisa was used to detect antibodies to maedi-visna virus in samples of serum and milk from individual sheep; the results obtained indicated that the elisa can be used to detect antibodies in milk. The assay was also applied to samples of bulk-tank milk; a standard curve was created and used to calculate the seroprevalence of maedi-visna in 11 flocks of sheep and the results were compared with the results obtained by applying the elisa to individual serum samples. There was good agreement between the seroprevalences calculated from the standard curve for bulk-tank milk and from the individual serum samples.

Diagnostic tests for small ruminant lentiviruses.

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.

Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (K value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animal's life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.