Serological characterization of the new genotype E of small ruminant lentivirus in roccaverano goat flocks

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.     

Maedi-visna virus infection of ovine mammary epithelial cells

The aim of this work was to perform a complete study of maedi-visna virus (MVV) infected mammary glands of naturally-infected sheep, and to determine if cells other than macrophages undergo a productive viral infection in this organ. Fifteen seropositive and two seronegative ewes were selected from MVV-infected flocks on the basis of clinical indurative mastitis and three sheep from an MVV-free flock. Within the mammary gland, MVV-positive cells were located by immunohistochemistry in the stroma and the epithelial alveolar barrier, most likely the ovine mammary epithelial cells (OMEC) of the acini. In situ hybridization confirmed these findings. Ultrastructural studies showed the presence of lentivirus-like particles budding off the cell surface in the alveolar barrier and also free in the acinar lumen. The presence of mammary histopathological lesions and MVV together with clear indications of productive infection (demonstration of a cytopathic effect in OMEC cultures and infection of co-cultures) were observed in the 15 seropositive and one of the seronegative sheep from the infected flock. These findings demonstrate that the OMEC were infected in vivo and probably underwent productive infection when studied ex-vivo. The OMEC of MVV-free sheep, which had subsequently been infected in vitro with MVV, also showed productive infection when challenged in vitro, confirming the replication of MVV in OMEC in vitro. The presence of MVV-infected OMEC in the mammary gland from infected animals, the productive infection in these OMEC and the release of lentiviral particles to the acinar lumen may have relevance in the pathogenesis and transmission of MVV infection.     

Serological characterization of the new genotype E of small ruminant lentivirus in roccaverano goat flocks

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.

     

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.     

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

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

Transmission and control implications of seroconversion to Maedi-Visna virus in Basque dairy-sheep flocks

A retrospective analysis of seroconversion to Maedi-Visna virus (MVV) was carried out for 10 infected semi-intensively reared dairy-sheep flocks that were tested annually between 1994 and 1999. Four of the flocks raised replacement lambs artificially with bovine colostrum and milk replacement to avoid lactogenic MVV infection but did not prevent aerosol contact between replacements and other sheep in the flock. Flock culling percentages ranged between 14 and 25% and in eight flocks the number of sheep that seroconverted was similar to or lower than the number of sheep culled—suggesting that incidence could be reduced by culling seropositive sheep without increasing average culling percentages. Random-effects logistic regression indicated that seroconversion was associated positively with increasing contact with infected sheep and with lifetime MV-serological status of the dam (used as a proxy measure of genetic susceptibility), but not with mode of rearing pre-weaning (artificially or with a seropositive or seronegative dam). Our results indicate that when conditions allow efficient horizontal transmission, there is no evidence that lactogenic infection increases the risk of MV infection and that there is an important inheritable component of disease resistance or susceptibility.     

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.     

Identification of Leptospira spp. carriers among seroreactive goats and sheep by polymerase chain reaction

Few studies were conducted on the diagnosis and control of small ruminants’ leptospirosis. Thirteen goat herds and seven sheep flocks located in the state of Rio de Janeiro, Brazil, were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity by MAT (Microscopic Agglutination Test), 19 and 40 seropositive goats and sheep, respectively, were selected, and urine samples were collected for bacteriology and PCR. For both species of animals, the most prevalent reactions were due to serogroups Sejroe and Shermani. Although leptospires were observed by darkfield microscopy in eight samples, pure isolates were obtained by bacteriological culture from only two samples. However, twelve urine samples (six goats and six sheep) were positive by PCR. Based on these findings, we consider that the combined use of MAT as a screening test followed by urine PCR for the direct detection of Leptospira spp. DNA was adequate for the identification of carrier animals among goats and sheep. These are valuable tools for the control of leptospirosis in small ruminants.     

Prevalence of Bovine viral diarrhoea virus (BVDV) antibodies among sheep and goats in India

The aim of this study was to determine the prevalence of pestivirus antibodies in sheep and goats in India. A total of 2803 serum samples collected between 2004 and 2008 from 1777 sheep in 92 flocks and 1026 goats in 63 flocks belonging to 13 states were tested by competition ELISA for detection of pestivirus antibodies. In sheep, the true prevalence rate was 23.4% (95% confidence interval: 22.9%–27.0%) and in goats it was 16.9% (95% CI: 16.4%–21.3%). The flock level seroprevalence was 66.3% for sheep and 54.0% for goats. Geographical variation in individual and flock prevalence was highly significant. A significant association (p?<?0.05) was found between sheep and goat flocks having cattle contact and the flock level seroprevalence. The seroprevalence was lower in 6 months–1 year age group compared to the 1–2 year and >2 year age groups in both sheep and goats. Cross neutralization studies on 61 seropositive sheep and 34 seropositive goat samples representing all positive flocks, exhibited > four fold higher titre to bovine viral diarrhoea virus type 1 (BVDV-1) in 41 sheep and 23 goat samples and to BVDV-2 in one sheep and goat each. This study for the first time showed serological evidence of wide spread BVDV infections in Indian sheep and goats, with BVDV-1 predominating and BVDV-2 occasionally besides highlighting the potential risk of infection to other species, which needs to be considered whenever BVD control measures are initiated.     

The detection of proviral DNA by semi‐nested polymerase chain reaction and phylogenetic analysis of czech Maedi‐Visna Isolates Based on gag Gene Sequences

A semi‐nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi–Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6 %) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

Molecular characterization of lentiviruses from goats from Poland based on gag gene sequence analysis

Caprine arthritis-encephalitis virus (CAEV) infection in goats is worldwide but with higher prevalence in industrialized countries. While positive serology of CAEV in Polish goats was reported there was no genetic study of this virus. In this study, we described the molecular characterization of lentiviruses isolated from seropositive goats from Poland. We cloned and sequenced a fragment from the gag gene covering part of the coding sequences for the matrix (MA) p17 and for the capsid (CA) p25 proteins. Resulting nucleotide sequences were aligned with those from other ovine/caprine lentivirus isolates. We present data showing that the sequences of most goat lentivirus isolates are closer to the prototypic CAEV-Co isolate, nevertheless from one goat we isolated a virus that is closer to the sheep Maedi Visna virus (MVV) isolate. This might indicate a recent cross-species infection from sheep to goat.     

Phylogenetic analysis of small ruminant lentiviruses detected in Slovenia

Small ruminant lentiviruses (SRLV), which belong to the Retroviridae family, infect goats and sheep worldwide. The aim of this study was to characterize the SRLV strains circulating in Slovenia, by phylogenetic analysis of two genomic regions, 1.8 kb gag-pol fragment and 1.2 kb pol fragment. The results of our study revealed that Slovenian SRLV strains are highly heterogeneous, with ovine strains belonging to genotype A and caprine strains to genotypes A and B. The closest relatives of sheep virus sequences from two flocks that clustered together (SLO 35, 36) were found to be in subtype A5. A cluster composed of four sheep virus sequences (SLO 31) was clearly divergent from all other subtypes in group A and could not be assigned to any of them. The virus sequences from one goat flock belonged solely to subtype B1, whereas virus sequences from more than one genotype were found to circulate within the other two goat flocks, belonging to subtype B1 (SLO 1 and SLO 37) and to genotype A (SLO 2 and 78–88 g). Two goat virus sequences (SLO 2) were found to belong to genotype A and could not be assigned to existing subtypes. One goat virus sequence (37–88 g) from flock 37 was clearly different from other sequences of this flock and was more closely related to genotype A sequences. We propose two new subtypes within genotype A, subtype A14 (SLO 2) and A15 (SLO 31).     

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.     

新疆卡拉库尔羊梅迪/维斯那病毒感染的病理学观察

对来自3个自然感染梅迪/维斯那病毒羊场的7只新疆卡拉库尔羊进行病理学研究。在临床观察期间,试验羊仅出现轻微的与梅迪/维斯那病毒有关的临床症状。病理组织学观察表明,5例血清学阳性羊发生了不同程度的乳腺病理学变化,与其他器官的组织切片观察比较,未见到淋巴组织样间质性肺炎(LIP)、脉管炎、关节炎及滑膜炎病变,表明乳腺是最敏感的靶器官。根据邓普辉等对乳腺炎严重度和分型的描述,新疆卡拉库尔羊乳腺炎病变的严重度均低于其他毒株感染的乳腺炎,从病理学上证实新疆卡拉库尔羊为梅迪/维斯那病毒的低敏感品种。     

Prevention strategies against small ruminant lentiviruses: an update

Small ruminant lentiviruses (SRLVs), including maedi-visna virus (MVV) of sheep and caprine arthritis-encephalitis virus (CAEV), are widespread, cause fatal diseases and are responsible for major production losses in sheep and goats. Seventy years after the legendary maedi-visna sheep epidemic in Iceland, which led to the first isolation of a SRLV and subsequent eradication of the infection, no vaccine or treatment against infection has been fully successful. Research during the last two decades has produced sensitive diagnostic tools, leading to a variety of approaches to control infection. The underlying difficulty is to select the strategies applicable to different epidemiological conditions. This review updates the knowledge on diagnosis, risk of infection, immunisation approaches and criteria for selecting the different strategies to control the spread of SRLVs.     

Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep

Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.     

Detection of contagious ovine ecthyma (orf) and risk factors for infection in small ruminants in Iran

The primary cause of contagious ecthyma is the orf virus, the parapoxvirus prototype. It is a viral problem observed in goat and sheep flocks in Iran, causing economic loss. Orf is a zoonosis with little epidemiological investigation present in Iran. The current research aims at determining the status of this virus, and a PCR was used as a confirmatory instrument. We sampled 668 goats and sheep and various breeding systems. Besides, the orf prevalence was studied, and vaccination efficacy was determined. Moreover, the potential risk factors surveyed for infection with ecthyma were identified. Samples were taken from goat and sheep flocks in the present cross-sectional research, and PCR was used for testing orf DNA. A checklist including animals’ general information was completed. Data were analyzed using univariate tests (chi-square and t-tests) and multivariable binary logistic regression analysis. Three hundred one (45%) goats and sheep detected orf DNA. The age of 70% of positive cases was below one month. Ecthyma infection was significantly higher in imported breeds (87.3%) than indigenous (39.3%). Ninety-six percent of infected goats and sheep in the present work were not vaccinated against ecthyma. The high prevalence of the orf virus was confirmed among goat and sheep flocks in Iran. It is necessary to train ranchers regarding sanitary actions, quarantine, and application of orf vaccination plans.     

The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech Maedi-Visna isolates based on gag gene sequences

A semi-nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi-Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6%) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

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 3–7 years. 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.     

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.