Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.

[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.

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.

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.

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.

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.

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.

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.

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.

Using a panel of monoclonal antibodies to detect Maedi virus (MV) in chronic pulmonary distress of sheep.

A selected panel of six monoclonal antibodies (mAbs) against Maedi-Visna virus (MVV), recognising the core proteins (p27 and p15) and the envelope protein (gp105) of MVV, was tested using different unmasking techniques on paraffin embedded lung samples of a seropositive sheep. Only three mAbs were chosen, according to their strong reactivity. mAbs 1A7, 1B6 and 4B3 were employed in an immunohistochemical trial focused on the diagnosis of the lungs of 26 sheep with progressive pulmonary distress. These mAbs demonstrated MVV in 21 out of 26 cases including lymphoid interstitial pneumonia (LIP) and pulmonary adenomatosis. In only nine cases did all three mAbs react positively with the same sample. The sensitivity of immunohistochemical diagnosis of Maedi pneumonia can be increased by using mAbs 1A7, 4B3 and 1B6 together; that is a panel of mAbs direct against the envelope (gp105) and capsid (p27) viral proteins. The positive signal was focal and confined to the cytoplasm of bronchoalveolar epithelial cells and alveolar-interstitial macrophages. The results suggest that this panel of mAbs is useful to confirm severe LIP lesions such as Maedi pneumonia, to demonstrate Maedi infections in mild LIP, to demonstrate MVV in mixed pulmonary changes, and to investigate the pathogenesis of Maedi-Visna.

Maedi-visna control in sheep II. Half-yearly serological testing with culling of positive ewes and progeny.

In 1979 a field trial was started to study the feasibility of maedi-visna control in sheep by half-yearly serological testing (by ELISA) with culling of sero-positive ewes and their progeny. In 13 commercial flocks, with a mean initial incidence of serological reactors of 17%, the sero-positive ewes and all their progeny, those of preceding years included, were culled after each half-yearly test. The percentage of sero-positive sheep decreased gradually and at the end of the second year, at the 5th test, all flocks were sero-negative. Also the 6th and 7th test did not yield sero-positive sheep. At the 8th test, however, 3 sero-positive ewes were detected in one of the flocks. A definite conclusion as to the source of infection could not be drawn. The following flock test was negative. In 2 other commercial flocks, which had a mean initial incidence of sero-positive sheep of 53%, those sero-positive and only their suckling lambs were culled. Here too, a gradual decrease in the incidence of sero-positive sheep was observed at the 2nd and 3rd test, but at the 4th test a sharp increase occurred. The programme was continued and a decrease followed until 0% was reached at the 7th test (end of third year). Age analysis of the sero-positive sheep which caused this peak revealed that the majority had been born before the start of the trial. This suggests that a 'second wave' of sero-positive sheep may be prevented and a quicker result obtained if progeny of preceding years are culled as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.

Evaluation of an ELISA for detection of ovine progressive pneumonia antibodies using a recombinant transmembrane envelope protein.

An enzyme-linked immunosorbent assay (ELISA) was developed using a recombinant protein corresponding to the N'-terminal hydrophilic region of transmembrane glycoprotein (TM) of ovine lentivirus. This assay reproducibly detected antibodies in sera from 207 of 212 ovine progressive pneumonia (OPP) virus-infected sheep, and the recombinant TM ELISA accurately identified 26% (35 vs. 9) more seropositive samples than did the agar gel immunodiffusion test when applied to 100 sera from an infected flock. This assay also yielded no false-positive results in 14 true negative sera. Results of these experiments were further confirmed by the recombinant TM and recombinant p25 Western blot assay. A single recombinant TM antigen, as the coating antigen in ELISA, can be used successfully for the detection of OPP virus-infected animals and can improve the sensitivity and specificity for OPP diagnosis.

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.

An enzyme-linked immunosorbent assay for detection of antibodies to maedi-visna virus in sheep. I. A simple technique for production of antigen using sodium dodecyl sulfate treatment.

We report the efficacy of an anionic detergent, sodium dodecyl sulfate (SDS) for preparing maedi-visna antigens for an indirect enzyme-linked immunosorbent assay (i-ELISA). Ovine maedi-visna virus (MVV) pelleted by differential centrifugation followed by liquid chromatography was treated with SDS or one of three lipid solvents: ethyl ether, chloroform or fluorocarbon. The SDS-treated antigen resulted in higher optical density values with positive serum and better discrimination between positive and negative serum samples from specific-pathogen-free (SPF) sheep experimentally inoculated with the virus. Optimal results were obtained when MVV was treated with concentrations of 0.25% and 0.125% of SDS. A viral antigen prepared by centrifugation and treatment of a viral pellet with SDS was also suitable for the i-ELISA. This latter technique may facilitate the production of MVV antigens for use in the i-ELISA.

An enzyme-linked immunosorbent assay for detection of antibodies to maedi-visna virus in sheep. II. Comparison to conventional agar gel immunodiffusion test.

A study was conducted to compare the indirect enzyme-linked immunosorbent-assay (i-ELISA) test using antigen prepared by a simple technique using sodium dodecyl sulfate (SDS) treatment to the conventional agar gel immunodiffusion test (AGID). Ten specific-pathogen-free (SPF) sheep were inoculated with maedi-visna virus (MVV) and serum antibody titers compared over a period of 14 weeks. All the sheep seroconverted by the i-ELISA compared to 90% by the AGID. The i-ELISA detected antibody at a mean of 2.6 weeks prior to the AGID. In both tests, fluctuations were observed in the serum antibody response of two sheep. The i-ELISA had a specificity of at least 98.8% and an increased relative sensitivity of 15.5% compared to the AGID, based on the analysis of sera from experimental sheep with MVV free status and sera from sheep from various sources. Of the sera from a seronegative flock which had been monitored with the AGID after a "test and remove" eradication program, 10.2% were positive by the i-ELISA. It was concluded that the AGID test may not be adequate to monitor samples for an eradication scheme.

Evaluation of two management procedures for the control of maedi-visna.

Two control programs were evaluated for their efficiency in eradicating the maedi-visna (M-V) virus from a single sheep flock. In both programs, the agar gel immunodiffusion test was used for the detection of M-V infected animals at regular intervals. In program 1, the test and remove program, ewes that were serologically positive for M-V were immediately removed along with their offspring. The prevalence of infected sheep decreased gradually and a seronegative flock was obtained after 30 months of monitoring. Program 2 entailed the removal of replacement ewe lambs at birth prior to the ingestion of colostrum. Maedi-visna antibodies have not been detected in this flock. These results show that under conditions similar to the industry norms, M-V can be expelled. Although the approach of program 1 is more practical for sheep producers, program 2 is more effective because of the earlier development of a M-V seronegative flock. Because of the nature of the humoral response, a longer time period than four years is required to ensure that M-V has been completely eradicated from each flock.