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.     

Indurative lymphocytic mastitis in sheep after experimental infection with maedivisna virus

Summary

In order to provide further evidence for the association of an indurative lymphocytic mastitis in sheep with MVV (maedi‐visna virus) infection, an experimental study was performed. Fourteen MVV‐free pregnant ewes, 2 years of age, were divided into two groups. Eight were intravenously inoculated with MVV (strain ZZV‐1050); six ewes served as sham‐inoculated controls. Post‐mortem exat. inations were carried out at 8, 16 and 28 months.

After 8 months, the 3 infected ewes had indurated udders with extensive lymphoid proliferation around lactiferous ducts and in the acinar tissue. The ducts were often partially obliterated. After 16 months, one of the two infected ewes suffered from indurative lymphocytic mastitis. The other was free of specific udder lesions. After 28 months only one of three infected ewes had mild lymphocytic infiltration in the udder. None of the controls, two in each post‐mortem session, had lesions typical of this form of mastitis. The lesions were most severe 8 months after infection. At 16 and 28 months lesions were of a lesser degree or were absent.

The lung lesions in the infected ewes 8 months after inoculation were similar to the changes in the udder regarding the lymphoid accumulation, although the proliferation around bronchial tree and blood vessels was less pronounced. After 16 and 28 months all infected ewes had peribronchial and perivascular lymphocytic infiltration though of a lesser degree than after 8 months.

From these resutls it is concluded that in addition to the lung and brain lesions MVV infections may cause a specific indurative lymphocytic mastitis.     

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.     

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.     

Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes

Two groups of sheep were experimentally infected by intratracheal route with two small ruminant lentivirus (SRLV) isolates belonging to different genotypes (It-561 genotype A3 and It-Pi1 genotype B2). Seroconversion was evaluated using recombinant homologous and heterologous matrix protein/capsid antigen fusion protein. Results clearly indicate that seroconversion against homologous antigen was detected well in advance as regards heterologous antigen in both groups, although the advantage of using homologous antigen was less evident in detecting seroconversion against the caprine arthritis encephalitis virus (CAEV)-like strain, compared with the maedi-visna virus (MVV)-like infection. Commercially available ELISAs detect CAEV-like seroconversion earlier than MVV-like infection suggesting a closer relationship between CAEV-like isolate and the antigen used in the latter ELISA tests. Seven recombinant subunits developed from matrix protein and capsid antigen of strain K1514 (prototype A1) were used to better define the antibody response in sheep infected with It-561 isolate. Two animals clearly reacted against type specific epitopes in the early stage of infection. This study highlights the relative insensitivity of gag encoded cross-reacting epitopes during the early stage of infection and suggests the development of novel diagnostic tests based on both genotype specific antigens.     

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.     

Infection of primary cultures of mammary epithelial cells by small ruminant lentiviruses.

The caprine arthritis-encephalitis virus (CAEV) and Visna Maedi virus cause persistent infections with long latent periods and induce degenerative and chronic inflammatory lesions of the central nervous system, joints, lungs and udder. Monocyte/macrophage lineage is the main target cell for CAEV and Visna Maedi virus but we speculate that mammary epithelial cells may also be infected. Primary cultures of milk cells, mammary tissues of experimentally and naturally infected goats and ewes were used. Primary cultures of mammary tissue from ewes and goats were infected with the CAEV Cork strain. The lentiviral infection of the primary culture was demonstrated by a typical cytopathic effect in mammary epithelial cells and the presence of an infectious virus in coculture with permissive fibroblasts. To identify the epithelial cells in explants and demonstrate the antigenic expression of CAEV, primary cultures were immunostained with polyclonal anti-keratin and monoclonal anti-CAEV p30. Colocalisation studies under a UV fluorescence microscope and by epifluorescence microscopy showed the expression of specific viral antigens in mammary epithelial cells from the eight animals used. Infected mammary epithelial cells may act as a reservoir for the virus which may play an important role in the virus dissemination and in the pathogenesis of the mammary lentiviral disease.     

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.     

Prevalence, spread and control of caprine arthritis-encephalitis virus in dairy goat herds in New South Wales

SUMMARY A study of the prevalence, spread and control of caprine arthritis-encephalitis virus (CAEV) in dairy goat herds in New South Wales (NSW) during 1986–1988 found that 56.8% of 1484 goats in 14 dairy herds were infected with CAEV. The prevalence of CAEV infection within most herds not implementing control measures increased during the study. At the end of the study, 59.7% of 1322 goats were infected. The prevalence of CAEV increased with age. Differences between breeds were less apparent. Within seven herds with a high standard of identification of goats, 149 of 812 goats seroconverted in an ELISA. Of these newly infected goats, 142 (95.3%) were > 1 yr of age and 96 (64.4%) were > 2 yr suggesting lateral spread of the virus. Most of the goats > 2 yr of age had been in the milking herd for a minimum of 3 to 6 months. The high seroconversion rate within the milking herd suggested that factors other than the ingestion of infected colostrum and milk before weaning were important for the spread of CAEV. Observations indicated that behaviour of goats, particularly reproductive behaviour among lactating does, and milking herd management practices are important in the spread of CAEV. A high density of livestock, poor livestock control and contamination of feed, water, equipment and personnel were implicated in transmission. Poorly functioning milking machines may also be involved. CAEV was eradicated from 3 herds by the implementation of strict control measures.     

绵羊进行性肺炎病毒与山羊关节炎一脑炎病毒的血清学交叉反应的研究

本实验用琼脂凝胶免疫扩散试验（ＡＧＩＤＴ）和免疫印迹试验（ＩＢＡ）对实验感染绵羊进行性肺炎病毒（ＯＰＰＶ）的山羊血清与山羊关节炎—脑炎病毒（ＣＡＥＶ）抗原以及实验感染ＣＡＥＶ的绵羊血清与ＯＰＰＶ抗原的交叉反应进行了研究。４只接种ＯＰＰＶ的山羊中有一只山羊的血清可与ＣＡＥＶ琼扩抗原发生交叉反应,并在免疫印迹试验中可识别ＣＡＥＶ的ｇｐ４４、ｐ３５和ｐ２８。２只接种ＣＡＥＶ的绵羊中有一只绵羊的血清可与ＯＰＰＶ琼扩抗原发生交叉反应,并在免疫印迹试验中可识别ＯＰＰＶ的ｇｐ４４和ｐ２８。以上的交叉反应结果表明ＯＰＰＶ与ＣＡＥＶ的抗原之间具有密切的相关性,这对于ＯＰＰＶ通过山羊和ＣＡＥＶ通过绵羊的传代研究是非常重要的,并对将来的免疫预防策略具有重要的指导意义。     

Immunomodulation of caprine lentiviral infection by interleukin-16

Interleukin-16 (IL-16) is a proinflammatory cytokine produced by a variety of cells including lymphocytes, macrophages, mast cells, and eosinophils. We have shown in our previous studies increased expression of IL-16 mRNA and protein in caprine arthritis-encephalitis virus (CAEV)-infected goats blood. In this study, we determined the immunomodulatory effects of IL-16 in vitro using cells derived from CAEV infected and uninfected goats. Human recombinant IL-16 (rhIL-16) significantly increased chemotaxis of peripheral blood mononuclear cells (PBMCs) of both control and CAEV-infected goats. Pretreatment of PBMC with anti-goat CD4 monoclonal antibody inhibited IL-16-induced chemotaxis of PBMC of control and infected goats suggesting that IL-16 exerts its action in goats primarily by binding to CD4. The CAEV proviral DNA was less in caprine monocytes treated with rhIL-16 infected in vitro with CAEV. These data suggest inhibitory effect of IL-16 on viral integration. Flow cytometric studies indicated a trend toward IL-16-induced increased expression of lymphocyte activation markers. Combined with our previously reported data, these experiments suggest that increased IL-16 expression during CAEV infection may inhibit viral integration.     

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.     

Experimental infection of Mouflon-domestic sheep hybrids with caprine arthritis-encephalitis virus

OBJECTIVE: To determine whether monocyte-derived macrophages from Mouflon-domestic sheep hybrids (Ovis musimon X Ovis spp) were susceptible to productive infection with caprine arthritis-encephalitis virus (CAEV) in vitro and whether experimental inoculation of Mouflon-domestic sheep hybrids with a molecularly cloned CAEV would result in persistent infection. ANIMALS: 5 Mouflon hybrids. PROCEDURE: Macrophage monolayers were inoculated with virus in vitro. Three animals were inoculated with virus intratracheally. RESULTS: Productive replication of CAEV was demonstrated in monocyte-derived macrophages following in vitro and in vivo inoculation. Titer of infectious cytopathic CAEV produced by macrophages from the Mouflon hybrids was similar to titers produced by macrophages from an infected goat or by synovial membrane cells. Isolation of virus from monocyte-derived macrophages and use of a semiquantitative polymerase chain reaction assay to amplify a portion of the viral genome demonstrated persistent virus replication in all 3 inoculated animals. Two weeks after inoculation of sheep, approximately 1 of 5,000 monocytes was harboring the virus. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that Mouflon-domestic sheep hybrids are susceptible to infection with isolates of CAEV that cause infection in domestic small ruminants.     

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.     

实验感染山羊关节炎—脑炎病毒的绵羊抗体应答反应的研究

本实验用琼脂凝胶免疫扩散试验和免疫印迹试验对实验感染山羊关节炎—脑炎病毒（ＣＡＥＶ）的绵羊抗体应答反应进行了研究，用两种方法都可在接毒绵羊的血清中检测到ＣＡＥＶ的抗体。琼脂凝胶免疫扩散试验最早可于接毒后的第７周时检测到抗体，免疫印迹试验最早可于接毒后的第６周时检测到抗ＣＡＥＶ的ｇｐ１２５、ｇｐ４４、ｐ３５、ｐ２８和ｐ１４的抗体，这说明免疫印迹试验更为敏感一些。本实验的结果表明ＣＡＥＶ可在绵羊体内诱生明显的体液免疫应答反应，因此用ＣＡＥＶ通过绵羊体传代的方法可能会得到具有良好的抗原性的ＣＡＥＶ毒株，这对于人工培养ＣＡＥＶ强毒是非常重要的。此外，本实验还为ＣＡＥＶ通过绵羊体传代的研究提供了非常实用的检测手段     

A deletion in the R region of long terminal repeats in small ruminant lentiviruses is associated with decreased pathology in the lung

A particular variant of the maedi visna virus (MVV) that although present in blood causes no clinical signs in infected sheep has been described. This variant carries a 13-14 nucleotide deletion in the R region of the proviral long terminal repeats. The hypothesis that this specific deletion may be associated with low pathogenicity has been investigated by comparing the distribution of proviral sequences, the histopathological lesions and the expression of viral proteins in the brain, lungs and udders of sheep naturally infected with viral strains carrying the deletion. Provirus could be demonstrated in most of the tissues examined from sheep infected with either type of virus, and the tissue-derived virus carried the typical deletion in the study flock animals. Histopathological analysis revealed that the lungs were significantly less affected in the animals infected with virus carrying the deletion. Concomitantly, viral expression was significantly reduced in the lungs of these animals. The findings suggest that the reduced pathogenicity of MVV with the specific deletion in the R region is not due to a restriction in the availability of specific tissues to infection, but is associated with a reduced capacity for viral expression in the lungs.     

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.     

绵羊梅迪-维斯纳病毒CA蛋白可溶性表达、多克隆抗体制备及鉴定

[目的] 制备绵羊梅迪-维斯纳病毒（maedi-visna virus,MVV）衣壳蛋白（capsid protein,CA）多克隆抗体,并鉴定其特异性。[方法] 根据MVV内蒙古分离株CA基因序列设计特异性引物,扩增CA基因,构建重组质粒;对 CA重组蛋白进行原核表达及纯化,制备兔源MVV CA重组蛋白多克隆抗体,采用间接ELISA方法测定其抗体效价,利用Western blot和免疫组化方法对其进行特异性鉴定。[结果] 成功构建MVV CA重组蛋白原核表达系统,纯化后的目的蛋白大小约27 kDa;间接ELISA方法测定制备的多克隆抗体效价为1:8 192;Western blot检测感染MVV绵羊的病肺组织,在25 kDa处出现特异性条带;免疫组化结果显示感染MVV绵羊的病肺中巨噬细胞的胞浆内有明显棕黄色阳性信号。[结论] 利用获得的可溶性重组MVV CA蛋白制备的多克隆抗体具有较好特异性,可为MVV血清学诊断技术提供检测抗体。     

Goat uterine epithelial cells are susceptible to infection with Caprine Arthritis Encephalitis Virus (CAEV) in vivo

ABSTRACT: The aim of this study was to determine, using immunofluorescence and in situ hybridization, whether CAEV is capable of infecting goat uterine epithelial cells in vivo. Five CAEV seropositive goats confirmed as infected using double nested polymerase chain reaction (dnPCR) on leucocytes and on vaginal secretions were used as CAEV positive goats. Five CAEV-free goats were used as controls. Samples from the uterine horn were prepared for dnPCR, in situ hybridization, and immunofluorescence. The results from dnPCR confirmed the presence of CAEV proviral DNA in the uterine horn samples of infected goats whereas no CAEV proviral DNA was detected in samples taken from the uninfected control goats. The in situ hybridization probe was complementary to part of the CAEV gag gene and confirmed the presence of CAEV nucleic acids in uterine samples. The positively staining cells were seen concentrated in the mucosa of the lamina propria of uterine sections. Finally, laser confocal analysis of double p28/cytokeratin immunolabelled transverse sections of CAEV infected goat uterus, demonstrated that the virus was localized in glandular and epithelial cells. This study clearly demonstrates that goat uterine epithelial cells are susceptible to CAEV infection in vivo. This finding could help to further our understanding of the epidemiology of CAEV, and in particular the possibility of vertical transmission.     

Pathogenic mechanisms of caprine arthritis-encephalitis virus

Goats infected with caprine arthritis-encephalitis virus (CAEV) show chronic arthritis and cachexia, which are progressive in nature. The immunopathogenic mechanisms responsible for these progressive clinical symptoms have not been fully elucidated. Various haematological and immunological parameters were evaluated in experimentally-infected goats showing typical signs of CAEV-induced disease. Infected goats showed recurrent lymphocytosis that may be due to constant presentation of antigen by infected cells of a monocyte/macrophage lineage. The serum alkaline phosphatase and -glutamyl transferase concentrations were elevated in infected goats, a characteristic of hepatic and bone disorders. All other serum chemistry parameters were similar between infected and control goats. Importantly, the serum tumour necrosis factor- (TNF-) levels were higher in infected goats. The cachexia seen in infected goats may be at least partly due to altered metabolism as a result of prolonged elevation of serum TNF- levels. Depressed natural killer cell activity was observed in infected goats and may contribute towards the establishment of a persistent infection with CAEV.Abbreviations AIDS acquired immunodeficiency syndrome - CAEV caprine arthritis-encephalitis virus - GGT -glutamyl transferase - HBSS Hanks' balanced salt solution - HIV human immunodeficiency virus - NK natural killer - PBMC peripheral blood mononuclear cells - PCR polymerase chain reaction - SAP serum alkaline phosphatase - TNF tumour necrosis factor