Application of PCR technique in diagnosis of small ruminant lentivirus infection in sheep and goats

Detection of small ruminant lentiviruses (SRLVs) in sheep and goats usually relies on serological testing. In this study, we evaluated semi-nested PCR and nested PCR techniques applied as a diagnostic tool for detection of maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) in naturally infected sheep and goats, respectively. The examination of 193 ovine and 85 caprine serum samples by the ELISA revealed the presence of specific antibodies in 133 (69%) and 18 (21.2%) animals, respectively. Presence of proviral DNA was manifested in 103 (53.4%) sheep and 12 (14.2%) goats. Despite the relatively lower sensitivity of PCR, the fact of detection of proviral DNA in 19 out of 60 ovine samples and 7 out of 67 caprine samples collected from animals previously negative by ELISA was noteworthy. In conclusion, the data demonstrated that combinations of both ELISA and PCR might afford optimal detection of SRLVs infection.     

Genetic characterization of maedi-visna virus (MVV) detected in Finland

The aim of the study was to characterize the small-ruminant lentiviruses (SRLVs) detected in Finland by defining their phylogenetic relationships and by studying the evolution of the virus based on a well-known epidemiology. The study material comprised lung tissue samples of 20 sheep from 5 different farms, a cell-cultured virus from one of the original sheep lung samples, and a blood sample of a goat. The sheep were identified as positive during seroepidemiologic screenings in 1994-1996 and the goat in 2001. Initial classification of a 251 nucleotide sequence within gag gene amplified from the uncultured samples as well as from the cell-cultured virus showed that the SRLVs were genetically close and that they were more closely related to the prototype ovine maedi-visna viruses (MVVs) than to the caprine arthritis-encephalitis virus (CAEV). The lentivirus detected from the goat aligned within the cluster of the Finnish ovine viruses, demonstrating a natural sheep-to-goat transmission. Further phylogenetic analysis of the proviral gag, pol and env sequences confirmed the initial classification and showed that they constituted a new subtype within the diverse MVV group. The sequence analyses also showed that the virus had remained genetically relatively stable, in spite of the time given for virus evolution, an estimated 20 years, and in spite of the virus crossing the host species barrier.     

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.     

Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes

Small ruminant lentiviruses (SRLV = maedi-visna in sheep and caprine arthritis encephalitis in goats) are distributed throughout most countries of the world, particularly Europe. Laboratories from 16 European countries established collaborations within the framework of a COST (CO-operation in the field of Scientific and Technical Research) action sponsored by the European Union in order to (i) better organize their research programmes on SRLVs and (ii) to coordinate efforts to combat these two diseases. After five years, a consensus conference--the first one in the veterinary medicine field--concluded the work of this network of laboratories by reviewing the present position and discussing three important questions in the field of SRLVs: routes of transmission, consequences of infection and potential role of eradication programmes at either a European or local level, according to the situation in each country or region. This paper brings together existing information regarding these questions and identifies areas for future research.     

Impact of natural sheep-goat transmission on detection and control of small ruminant lentivirus group C infections

Dissemination of small ruminant lentivirus (SRLV) infections in Norway is affected by the different control strategies used for maedi-visna virus (MVV) infections in sheep and caprine arthritis-encephalitis virus (CAEV) infections in goats. Here we investigated SRLV phylogenetic group variants in sheep. CAEV-like isolates, belonging to phylogenetic group C, were found among both seropositive sheep and goats in mixed flocks, in which sheep and goats are kept together. Intra-herd clustering confirmed that mixed flock animals were infected by the same virus variant, suggesting ongoing interspecies transmission. Few sheep flocks were found to be infected with the MVV-like phylogenetic group A. The apparent absence of SRLV group A type in goats is probably due to the MVV control programme and animal management practices. SRLV group C targets lungs and mammary glands in sheep, and induces typical SRLV pathological lesions. SRLV group C isolated from the sheep mammary glands suggested a productive infection and potential for transmission to offspring. SRLV group C was most prevalent among goats. A lower PCR sensitivity in seropositive sheep suggested a lower load of SRLV group C provirus in sheep than in goats. Higher genetic divergence of group C than in other SRLV groups and extensive heterogeneity among group C isolates in the matrix C-terminal region demonstrate the need for identifying conserved target regions when developing PCR protocols for SRLV detection. As sheep and goats may serve as reservoirs for all SRLV genogroup types, successful control programmes require inclusion of both species.     

Maedi-Visna virus infection in sheep. History and present knowledge

Briefly the history of maedi-visna and the major clinical symptoms are described. Examples are presented to demonstrate that the genetic composition of a breed determines whether or not sheep become sick after an infection with maedi-visna virus (mvv) or develop solely specific antibodies. The major pathway of transmission is not colostrum and milk, but a cell containing increased nasal discharge in cases of respiratory distress. The role of the environment and prophylactic measures against parasites is stressed, because even sheep of highly susceptible breeds can survive an infection under optimal conditions. The virus and subsequently the disease simply die out. The cooperation between clinicians and laboratories is necessary.     

Serological surveillance for maedi-visna virus and caprine arthritis-encephalitis virus in Northern Ireland

The results of serological testing for maedi-visna virus infection in indigenous and non-indigenous sheep in Northern Ireland, over a five year period (1980 to 1984) are presented. In tests carried out in 1980 and 1981 on pedigree breeds, 10 reactors were identified on seven farms and maedi-visna virus was isolated on three occasions from leucocyte cultures. None of the animals showed clinical signs of maedi-visna and all the reactors were subsequently destroyed. Nine of the reactors were imports from Scotland or the Republic of Ireland and the other was the progeny of an imported ewe. In subsequent tests (1982 to 1984) of exotic and indigenous flocks no further reactors were identified. A survey of goat herds for evidence of caprine arthritis-encephalitis virus infection was also negative.     

Maedi-visna control in sheep. I. Artificial rearing of colostrum-deprived lambs

A field trial to study the practicability and efficacy of maedi-visna control in sheep by artificial rearing of lambs was carried out during the lambing season of 1979. Lambs were immediately separated from the dams at birth, deprived of ovine colostrum, and reared isolated from the parent flock. Bovine colostrum was given instead of maternal colostrum. Eleven farms participated in the experiment. All flocks were severely infected with maedi-visna virus: 63-100% of the ewes were seropositive as demonstrated by ELISA. Artificially reared lambs were serologically tested and positives culled at the age of 6, 12, 18, 24, 30 and 36 months. Only very few positives were found: 1/389, 1/376, 0/337, 1/223, 1/192 and 0/144, respectively. The first two sero-positive lambs occurred in one flock, and it could be ascertained that both had mistakenly been given ovine colostrum probably containing maedi-visna virus. No explanation, other than sub-optimal hygiene and isolation, could be found for the two sero-positive sheep that turned up in another flock at 24 and 30 months of age although, transplacental infection cannot be entirely excluded. It is concluded that artificial rearing of ovine colostrum-deprived lambs is an effective and practicable method for the control of maedi-visna in sheep. The method appears particularly useful when valuable genetic material has to be salvaged.     

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.     

The effects of maedi-visna virus infection on productivity in ewes

A total of five data sets obtained from four separate sheep flocks were used to evaluate the effects of subclinical maedi-visna virus infection (as determined by serological procedures) on productivity in ewes. In general, infection with maedi-visna virus had a detrimental effect on productivity. Infection resulted in a reduction in conception rates, with the odds of an infected ewe conceiving being only approximately 0.67 times those of a non-infected ewe. For ewes which did conceive, there was no detrimental effect of infection on the number of lambs born. The birth weights of lambs born to middle-aged (3–4 years) infected ewes were 3–6% lower than the weights of lambs born to non-infected ewes of the same age. There was very little apparent effect of infection in younger (1–2 years) and older (5 + years) ewes: some factors which may have biased these results are discussed. Lamb weights at 25–50 days of age appeared to be reduced by maedi-visna infection in the ewe but the results were not statistically significant.     

Maedi-visna virus infection in commercial flocks of indigenous sheep in Britain

The recent identification of maedi-visna virus infection in three commercial flocks of indigenous sheep in Britain is described. In one flock the evidence suggested that overt clinical disease was present. The implications of the findings are discussed.     

Importance of ewe/lamb relationship and breed in the epidemiology of maedi-visna virus infections

Sheep from a closed experimental breeding flock containing Finnish Landrace, Ile de France, their F1 crossbreeds, and a new breed were tested for antibodies to maedi-visna virus in 1975 and 1985-86. Over the years, the percentage of seropositive sheep increased: the Ile de France, however, remained virtually negative. Since exposure to the virus was similar, the results indicated a breed-associated difference in susceptibility to maedi-visna virus infection. Analysis of the serological results of 1985-86 yielded a total of 173 dam/progeny pairs. A significant (P less than 0.05) association between the test results of the dams and their progeny was found: seropositive (infected) dams produced 36.6 per cent positive progeny, whereas the seronegative dams had 20.0 per cent positive progeny (odds ratio = 2.3). Further analysis showed that this association was most apparent between dams and their youngest progeny, which is explained by the fact that the chance of infection by horizontal transmission increases with age. These results provide evidence that the ewe/lamb relationship plays a role in the epidemiology of maedi-visna virus infections.     

Clinicopathological investigation of primary, uncomplicated maedi-visna virus infection.

Maedi-visna virus infection in a flock of sheep in Scotland was associated with respiratory disease, neurological disease, mastitis and lameness. The major clinical signs were dyspnoea (particularly on exercise), progressive fore- and hindlimb ataxia and balance defects, mammary induration and multilimb lameness, occasionally with enlarged carpal joints. Pathological examinations revealed lesions in the lungs, central nervous system, mammary glands and joints which were consistent with those induced by maedi-visna virus. The was no clinical or pathological evidence of concurrent sheep pulmonary adenomatosis, and pulmonary bacterial infections, when they occurred, were superimposed on the lesions due to maedi-visna virus.     

In vivo depletion of CD8+ cells does not affect primary maedi visna virus infection in sheep

T-cells have been implicated both, in promoting and reducing viral replication during lentivirus infection. CD8+ lymphocytes are believed to be important in controlling viral load through direct killing of virus-infected cells and by secretion of inhibitory chemokines and cytokines. To evaluate the role of CD8+ T-cells in the induction and control of the primary phase of a lentivirus infection, we have used a non-T-cell tropic lentivirus, maedi-visna virus (MVV), to study the initial pathogenesis and subsequent immune responses in sheep depleted in vivo of CD8+ cells. Sheep were depleted of CD8+ cells in both blood and efferent lymph for up to 14 days. No difference in MVV replication was observed in either the draining efferent lymph or lymph node of these sheep. Surprisingly, these animals displayed a normal induction of pCTL whereas the virus-specific proliferative responses were reduced. This could reflect either that a proportion of functional CD8+ lymphocytes remained in these animals, as suggested by the appearance of pCTLs, or that CD8+ cells are not required for control of primary MVV infection.     

Experimental maedi virus infection in sheep: cellular and humoral immune response during three years following intranasal inoculation

A long-term experiment in sheep inoculated intranasally with 2 strains of Norwegian maedi virus was carried out in 2 groups of Norwegian Dala sheep (7 sheep/group). Virus-specific cellular immune response was assayed in the lymphocyte transformation test sequentially during 3 years after sheep were inoculated in group 1 and 4 times in the 3rd year in group 2. Humoral immune response was assayed by immunodiffusion, complement-fixation, and neutralization tests on sequential serum samples collected from the 2 groups. Attempts to isolate virus were made. All group 1 sheep showed transient and irregularly recurring cellular immune responses. In group 2, 6 of the 7 sheep gave similar responses. The frequency of virus isolations was low compared with that reported by various research workers using other breeds for studying experimental maedi-visna infection. Precipitating antibodies were detected earlier, and in more animals, than were complement-fixing antibodies. Both were, however, detected later and less frequently than were reported by other research workers. There was a marked difference in the capability of the 2 maedi virus strains to induce neutralizing antibodies. The sequential sera usually showed distinct differences in neutralizing capacity of the virus strains, indicating that they are antigenically different.     

Human and ovine lentiviral infections compared

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the “slowness” of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.     

Genome scan for the possibility of identifying candidate resistance genes for goat lentiviral infections in the Italian Garfagnina goat breed

Tropical Animal Health and Production - Small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses of sheep, goat, and wild ruminants responsible of lifelong persistent infection...     

Seroepidemiological survey of maedi-visna virus infection in sheep and goat flocks in Quebec.

Maedi-visna, a chronic viral disease of adult sheep characterized by progressive dyspnoea or neurological manifestations, was first recognized and described clinically in Canada in 1970. Seroepidemiological study was conducted in sheep and goats in various areas of Quebec. Sera of 10% of the animals of selected flocks were collected and specific antibodies against maedi-visna virus were tested by a modified direct complement fixation test. Results show seropositive rate of 67.6% for Sherbrooke sheep; of 40.5, 41.1 and 47.1% for Quebec, Saint-Hyacinthe and Nicolet sheep respectively and only 29.2 and 20.0% positive sera in l'Assomption and Rimouski animals. Prevalence rate of positive goats varied according to geographic areas (0 to 36.8%). Statistical analysis of various factors, e.g. age, breed, mode of raising, origin and size of flock showed no relation between these factors and the geographic areas. But, some clinical problems in the sheep flocks such as cough, rapid breathing, mortality and abortion were associated with high infection rate (greater than or equal to 50%) to maedi-visna virus. In goats, no correlation was demonstrated between these clinical signs and serological results. Our results suggest that it is important to consider this disease in an adequate program of preventive medicine in Quebec.     

Zwoegerziekte virus, the causative agent for progressive interstitial pneumonia (maedi) and meningo-leucoencephalitis (visna) in sheep.

The final results of experimental infections with virus recovered from the lungs of sheep suffering from progressive interstitial pneumonia (=zwoegerziekte=maedi) are reported. The virus could be reisolated from blood samples of all experimentally infected sheep. Every animal produced antibodies against the virus. The neutralising, complement-fixing and precipitating antibodies remained present in the blood for six years. Fourteen out of 21 intrapulmonarily infected sheep developed clinical and/or histopathological lung lesions and in three a meningo-leucoencephalitis was detected in addition. One of these three developed the clinical and pathological signs of 'visna' 14 months after inoculation. Signs of visna were seen in eight of 10 sheep that had been inoculated intracerebrally. Furthermore, nine of these sheep suffered from progressive interstitial pneumonia. Hence the name maedi-visna virus is proposed for the agent which causes both disease entities. Three sheep that yielded virus after infection and in which antibodies were detected, did not develop histopathological lesions.     

Small ruminant lentivirus genetic subgroups associate with sheep TMEM154 genotypes

Small ruminant lentiviruses (SRLVs) are prevalent in North American sheep and a major cause of production losses for the U.S. sheep industry. Sheep susceptibility to SRLV infection is influenced by genetic variation within the ovine transmembrane 154 gene (TMEM154). Animals with either of two distinct TMEM154 haplotypes that both encode glutamate at position 35 of the protein (E35) are at greater risk of SRLV infection than those homozygous with a lysine (K35) haplotype. Prior to this study, it was unknown if TMEM154 associations with infection are influenced by SRLV genetic subgroups. Accordingly, our goals were to characterize SRLVs naturally infecting sheep from a diverse U.S. Midwestern flock and test them for associations with TMEM154 E35K genotypes. Two regions of the SRLV genome were targeted for proviral amplification, cloning, sequence analysis, and association testing with TMEM154 E35K genotypes: gag and the transmembrane region of env. Independent analyses of gag and env sequences showed that they clustered in two subgroups (1 and 2), they were distinct from SRLV subtypes originating from Europe, and that subgroup 1 associated with hemizygous and homozygous TMEM154 K35 genotypes and subgroup 2 with hemi- and homozygous E35 genotypes (gag p < 0.001, env p = 0.01). These results indicate that SRLVs in the U.S. have adapted to infect sheep with specific TMEM154 E35K genotypes. Consequently, both host and SRLV genotypes affect the relative risk of SRLV infection in sheep.