Maedi-visna virus infection in Karayaka and Amasya Herik breed sheep from provinces in northern Turkey.

Maedi-visna is an important virus infection of sheep having prolonged incubation period (slow disease) and reflecting two distinct forms clinically and pathologically. In this study, the presence of MVV was investigated serologically in 58 Amasya Herik sheep breed and 525 Karayaka sheep breed. Seropositivity rates in Amasya Herik sheep breed and Karayaka sheep breed were detected as 69.0% and 18.5%, respectively. MVV antibodies were found in 137 of 583 serum samples (23.5%). Positivity rates for the provinces varied and were as follows: Samsun 19.4%, Sinop 15.4%, Ordu 25.8%, Trabzon 26.7%, Rize 36.7%, Amasya 69.0% and Tokat 35.0%, however no antibody response was detected in all of the sheep in Giresun province.

Epidemiological survey for visna-maedi among sheep in northern prefectures of Japan.

Ovine sera collected from the northern Prefectures of Hokkaido, Iwate and Aomori in Japan, were examined for the presence of antibodies against visna-maedi virus using the agar gel immunodiffusion test and enzyme-linked immunosorbent assays. Three animals (1.12%), out of 267 samples tested, were found to be seropositive to the visna-maedi antigens in both tests. Levels of infection were found in flocks from Hokkaido and Iwate Prefectures, but not in the Aomori Prefecture. Nucleic acid detection by polymerase chain reaction on serum samples did not give positive results. Although no diagnostic measures were in place, the infection could not be related to losses in sheep production or to reduced survival rates. The very limited visna-maedi distribution indicates a highly favourable condition for the application of eradication strategies in this area.

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.

Effects of housing on the incidence of visna/maedi virus infection in sheep flocks.

The incidence of seroconversion to visna/maedi virus (VMV) infection and its relationship with management and sheep building structure was investigated in 15 dairy sheep flocks in Spain during 3-7years. Incidence rates were 0.09 per sheep-year at risk in semi-intensive Latxa flocks and 0.44 per sheep-year at risk in intensive Assaf flocks and was greatest for the one year old Assaf replacement flock. Separate multivariable models developed for replacement and adult flocks indicated that in both cases seroconversion was strongly associated to direct contact exposure to infected sheep and to being born to a seropositive dam. The latter effect was independent of the mode of rearing preweaning and the risk of seroconversion was similar for sheep fed colostrum and milk from a seropositive or a seronegative dam. These results are further evidence of the efficiency of horizontal VMV transmission by close contact between sheep and also suggest a inheritable component of susceptibility and resistance to infection. In contrast, indirect aerogenous contact with seropositive sheep was not associated with seroconversion as evidenced in replacement sheep housed in separate pens in the same building as adult infected sheep for one year. Consequently, VMV may not be efficiently airborne over short distances and this is important for control of infection. Moreover, there was no relationship between seroconversion and shed open areas. The latter could be related to having examined few flocks in which high infection prevalence dominated the transmission process while ventilation, may depend on a variety of unrecorded factors whose relationship to infection needs to be further investigated.

Experimental infection of sheep with visna/maedi virus via the conjunctival space.

Experiments were performed to determine whether visna/maedi virus (VMV), a small ruminant lentivirus (SRLV), could infect sheep via ocular tissues. The EV1 strain of VMV was administered into the conjunctival space of uninfected sheep, and the animals monitored for the presence of provirus DNA and anti-VMV antibodies in blood. The results showed that provirus DNA appeared in peripheral blood mononuclear cells of all animals within a few weeks of receiving either 10(6) TCID50 or 10(3) TCID50 of VMV. Of the animals receiving the higher dose of virus via the conjunctival space, two seroconverted by 7 and 10 weeks post-infection, one seroconverted 8 months post-infection, and one had not seroconverted by 15 months post-infection. With the lower virus dose, the animals infected via the trachea seroconverted by 4 and 14 weeks, respectively. After ocular infection with this dose, one animal showed a transitory seroconversion with low levels of antibody, peaking at 2 weeks post-administration. The remaining three of the animals infected via the eyes did not seroconvert over a period of 13 months. At post-mortem, evidence for the presence of proviral DNA was obtained from ocular tissue, lungs or mediastinal lymph node in both groups of animals. Histological analysis of lung tissue from animals receiving the lower dose of virus showed the presence of early inflammatory lesions. The results thus show for the first time that transmission of VMV can occur via ocular tissues, suggesting that the conjunctival space may be an additional route of natural transmission.

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.

Extensive rearing hinders Maedi-Visna Virus (MVV) infection in sheep.

Maedi-Visna Virus (MVV) seroprevalence and its relationship with housing and mode of rearing of replacement ewe-lambs was investigated in 38 non-randomly selected sheep-flocks in Spain. They included extensive lamb-producing Manchega cross-bred flocks raised almost permanently at pasture, semi-intensive Latxa dairy flocks housed 2-8 months/year and intensively raised Assaf dairy flocks housed most time and at higher stocking density in less ventilated buildings than other flocks. Most flocks raised replacement lambs naturally with their dams until weaning and as a separate flock thereafter until lambing at one year of age. Seroprevalence (95% confidence intervals) was 77%, 25% and 5% (4-6) in intensive, semi-intensive and extensive flocks, respectively and the median (interquartile range) flock-seroprevalence was 82% (66-94) in intensive flocks, 31% (14-31) in semi-intensive flocks and 4% (0-7) in extensive flocks. Seroprevalence was lowest in one year-old sheep and increased to flock levels during the year after introduction into the adult flock in most intensive flocks and more gradually in other flocks. Adult flock seroprevalence was associated with housing time but this relationship was not evident within a particular rearing system, indicating that other unknown factors are critical in horizontal MVV-transmission. Low seroprevalence in extensive flocks further supports previous indications that lactogenic MVV-infection is relatively inefficient and horizontal transmission is necessary to ensure long-term maintenance of MVV and this could explain that MVV has not been reported from countries with mainly extensively reared sheep such as Australia and New Zealand. Moreover, it indicates that MVV-control in extensive and semi-intensive flocks can be simple and inexpensive.

Serum containing ovine IgG2 antibody specific for maedi visna virus envelope glycoprotein mediates antibody dependent cellular cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for maedi visna virus (MVV) has never been described. The IgG antibody response to MVV is restricted to an IgG1 response whilst MVV specific IgG2 is never seen in persistently infected sheep. To determine whether the isotypic restriction of the antibody response is responsible for the lack of ADCC, an ADCC assay was developed using polyclonal serum raised to recombinant MVV ENV protein. Sheep immunised with a recombinant GST:SUenv fusion protein in complete Freund's adjuvant produced an antibody response which contained IgG1 and IgG2 antibodies. The activity of this serum in an ADCC assay was compared to serum from persistently infected sheep. Serum from immunised sheep mediated ADCC reactions whilst no activity was ever seen in persistently infected sheep serum. IgG2 may therefore be the possible effector isotype for ADCC reactions against MVV. Failure of the IgG2 dependent ADCC system in vivo may contribute to the persistence of MVV-infected macrophages in vivo.

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.

Detection of maedi-visna virus in the kidneys of naturally infected sheep.

Infections with maedi-visna virus (MVV) cause progressive inflammation in different organs, mainly the lung, mammary gland, brain and joints. The aim of the present study was to investigate whether the kidney represents a viral target in natural MVV infection. For this, kidney samples from 13 sheep naturally infected with MVV were examined by histology, polymerase chain reaction (PCR), and immunohistochemistry. The kidneys of nine animals showed membranoproliferative glomerulonephritis and interstitial nephritis. The inflammatory infiltrate consisted of lymphocytes, plasma cells and macrophages. Interestingly, lymphoid follicles resembling those known to occur in other MVV-infected tissues were observed. Lung tissue from the same animals had typical MVV lesions, such as lymphofollicular hyperplasia and interstitial pneumonia. Maedi-visna proviral DNA sequences were detected in renal and lung tissue samples from these nine sheep by PCR, and the specificity of the amplified products was further verified by DNA sequencing. Moreover, MVV-specific immunohistochemistry revealed viral antigen in affected kidneys and lungs. These results suggest that the kidney may be a common target in natural MVV infection, and raise the issue of the role of this organ in the disease.

PCR detection of colostrum-associated Maedi-Visna virus (MVV) infection and relationship with ELISA-antibody status in lambs.

A recent large-scale experimental study showed that bottle-feeding ovine colostrum from seropositive ewes results in high MVV-seroconversion in lambs. In contrast, relatively few lambs that naturally suckled colostrum from seropositive dams seroconverted as a result of it. Furthermore, lambs fed uninfected bovine colostrum readily seroconverted when mixed with ovine-colostrum lambs indicating that horizontal MVV transmission between lambs was efficient. MVV-infection was further investigated in the same samples using two PCR tests targeting sequences in the long-terminal repeats (LTR) and POL MVV genes. PCR-tests confirmed previous serological findings. However, the LTR-PCR was more sensitive and allowed detecting infection earlier than the other tests, including 5-8% of new-born lambs from seropositive dams, providing more evidence that prenatal MVV-infection may be more important than considered. The degree of agreement between PCR and antibody tests in individual samples was low up to 6 months of age and moderate at 10 months-old. Nine percent of lambs were always PCR-negative but seroconverted and 19% of lambs were PCR-positive at least once and did not seroconvert. However, seroconversion was associated with increasing number of times lambs were PCR-positive and ovine colostrum-fed lambs were more frequently PCR-positive than other lambs. The significance of these findings in terms of MVV-infection, epidemiology and control is discussed.

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.

Establishment of a maedi-visna-free flock after the purchase of infected sheep.

Maedi-visna (MV) infection was detected in a cohort of 68 purchased ewes, one of several groups of sheep introduced to a farm after the previous stock had been culled with suspected foot-and-mouth disease in 2001. Except for short periods totalling six to seven weeks when the sheep co-grazed with 13 ewe lambs and ram lambs, the infected cohort was kept separate from other sheep on the farm over a total of 21 months. During this period two crops of lambs were reared from the infected ewes. All the lambs were fattened and killed, and all ewes were culled after the second crop of lambs had been weaned. Subsequent serological testing of the remaining sheep on the farm confirmed the elimination of MV infection from the flock, leading to its acceptance in the Maedi Visna Accreditation Scheme of the Scottish Agricultural College's Sheep and Goat Health Schemes.

Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.

Intratracheal inoculation as an efficient route of experimental infection with maedi-visna virus.

Maedi-visna virus (MVV) spreads horizontally via the respiratory route. In order to establish an experimental mucosal infection route, we compared intranasal and intratracheal inoculation using the infectious MVV molecular clone KV1772-kv72/67. For intranasal infection 0.5 x 10(3)-0.5 x 10(7) TCID50 of virus was sprayed into the nostrils of the sheep. For the intratracheal infection 10(0)-10(6) TCID50 of virus was injected into the trachea. Successful infection was indicated by development of MVV specific antibodies and virus isolation over a period of 6 months. In the intranasal infection, only the sheep receiving the highest dose i.e., 0.5 x 10(7) TCID50, became infected, suggesting that intranasal application was not an efficient mode of infection. In the intratracheal infection, the sheep infectious dose 50% was 10(1) TCID50 and virus could be isolated from the central nervous system 4 months post infection with 10(4) TCID50. Therefore it is concluded that intratracheal infection is a very efficient route for experimental inoculation with MVV.

Prevalence of and carcass condemnation from maedi-visna, paratuberculosis and caseous lymphadenitis in culled sheep from Quebec, Canada.

We determined the prevalence of lung and mammary gland lesions associated with maedi-visna (MV) infection, the prevalence of paratuberculosis (PTB), and the prevalence and lesions distribution of caseous lymphadenitis (CL) in culled sheep. Total of 451 ewes and 34 rams were selected randomly from two slaughterhouses in Quebec, Canada. MV serostatus was determined by recombinant ELISA test. PTB diagnosis was based on characteristic histological lesions in the terminal ileum, ileocecal lymph node and/or ileocecal valve and CL by gross detection of abscesses and isolation of Corynebacterium pseudotuberculosis. Seroprevalence of MV was 44% (95% CI: 40, 48). Seropositivity increased with age and was higher in ewes than in rams. The percentages of lung and mammary gland lesions in seropositive sheep were 14 and 40%, respectively, but mammary gland lesions lack specificity. The prevalence of PTB was 3% (95% CI: 2, 5). PTB increased with age and was lower among sheep with abscesses. The prevalence of CL was >/=21% (95% CI: 17, 24). The most-prevalent site of caseous lymphadenitis lesions was the thoracic cavity. The risk of carcass condemnation was significantly associated with region, body score and abscesses. Only the presence of abscesses was associated with an increase in trimming of carcasses.

Acute-phase proteins and hematologic values in ovine lentivirus-infected lambs treated with recombinant ovine IFN-tau.

To evaluate changes in complete blood cell (CBC) counts, haptoglobin and fibrinogen in ovine lentivirus (OvLV)-infected lambs treated with recombinant ovine interferon-tau (rOVIFN-tau), 24 lambs were allocated to one of four groups (n = 6 per group): (1) virus + rOvIFN-tau, VI, (2) virus + placebo, VP, (3) no virus + rOVIFN-tau, NVI, and (4) no virus + placebo, NVP. Three lambs in each group were treated once a day for 12 weeks, and the remaining 3 lambs were treated for 33 weeks. Blood was collected at days 0, 7, and 10 and at weeks 2-10, 12, 32, and 33 to determine CBC counts, as well as haptoglobin and fibrinogen levels. Hematologic values remained within normal limits in all groups. However, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and packed cell volume (PCV) values decreased (p < 0.05) in the two rOvIFN-tau-treated groups (VI and NVI) compared with the placebo-treated (VP and NVP) groups. Both rOvIFN-upsilon and OvLV had a mild negative effect on neutrophil numbers. Although Hb, MCV, MCHC, PCV, and neutrophil values declined in the rOvIFN-tau-treated lambs compared with the placebo-treated lambs, these values remained within the reference range for sheep. Experimental lambs did not show adverse clinical signs associated with OvLV infection or as a result of rOvIFN-tau treatment. The lack of significant side effects of high-dose rOvIFN-tau in sheep and previous reports of broad-spectrum and cross-species antiviral activity suggest that rOvIFN-tau warrants further investigation as an antiviral therapeutic agent.

Laboratory diagnostic tests for retrovirus infections of small ruminants.

The most practical and reliable approach to confirming a diagnosis of OPPV or CAEV infection is a combination of serology and clinical evaluation. Although serology represents the most cost effective method of diagnosing persistently infected, clinically normal animals, testing errors occur; the frequency of error depends on the performance data of the particular serologic assay being used. When PCR detection of OPPV and CAEV becomes routinely available, this detection method can be used in rigorous eradication programs to determine the infection status of animals that cannot be definitively diagnosed by serology. The important aspects of OPPV and CAEV infection that must be considered in designing programs to prevent transmission are (1) OPPV and CAEV persist for life in the infected host, (2) a major route of transmission is to lambs and kids via colostrum and milk during nursing, (3) contact transmission among adults can occur, and (4) time variability can exist among individual sheep and goats from infection to the appearance of detectable antibodies.

Quantitation of transforming growth factor-beta in plasma and pulmonary epithelial lining fluid of sheep experimentally infected with maedi-visna virus.

A model of experimental infection with EV1, a lytic British isolate of maedi-visna virus (MVV), was developed. Ten Texel sheep were allocated to two groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) TCID50 (tissue culture infective dose) of EV1 strain, while four sheep were sham-inoculated with identically prepared virus-free buffer solution. Experimental infection was followed for 8 weeks post-inoculation (PI), with development of precipitating antibodies to MVV developed in the MVV-inoculated animals during the first 4 weeks PI. Transforming growth factor-beta (TGF-beta) levels, in both bronchoalveolar lavage fluid supernatant and plasma samples, were measured. Concentrations of pulmonary epithelial lining fluid (PELF) TGF-beta were calculated. TGF-beta concentrations in PELF were approximately 165-fold higher than in plasma. No significant differences in the concentrations of plasma or PELF TGF-beta, either within or between groups, were observed.