Risk Factors Associated with the Alpine Multispecies Farming System in the Eradication of CAEV in South Tyrol, Italy.

South Tyrol has implemented, in 2007, a mandatory eradication program against Caprine Arthritis Encephalitis Virus (CAEV), a virus known to cause economic losses related to decreases in milk production and milk quality in goats, along with poor animal welfare and premature death. After a great initial decrease in the seroprevalence, the program has reached a tailing phase with scattered positivities. Potential risk factors associated with the multispecies farming system, a traditional approach in South Tyrol, are evaluated in this study, in order to better understand some of the potential causes leading to the tailing phenomenon. A statistically significant number of farms was selected for the present study, based on the risk factors evaluated. Even though there is no statistically significant association between the practices evaluated and the incidence of infection, the authors believe that it is important to highlight potential risks that may threaten the outcome of this eradication program.

Caprine Arthritis Encephalitis Virus Is Associated with Renal Lesions.

Caprine arthritis encephalitis virus (CAEV) is a monocyte/macrophage-tropic lentivirus that primarily infects goats resulting in a well-recognized set of chronic inflammatory syndromes focused on the joint synovium, tissues of the central nervous system, pulmonary interstitium and mammary gland. Clinically affected animals generally manifest with one or more of these classic CAEV-associated tissue lesions; however, CAEV-associated renal inflammation in goats has not been reported in the peer-reviewed literature. Here we describe six goats with chronic, multisystemic CAEV infections in conjunction with CAEV-associated renal lesions. One of the animals had CAEV antigen-associated thrombotic arteritis resulting in infarction of both the kidney and heart. These goats had microscopic evidence of inflammatory renal injury (interstitial nephritis) with detectable renal immunolabeling for CAEV antigen in three of six animals and amplifiable proviral sequences consistent with CAEV in all six animals. Cardiac lesions (vascular, myocardial or endocardial) were also identified in four of six animals. Within the viral promoter (U3) region, known transcription factor binding sites (TFBSs) were generally conserved, although one viral isolate had a duplication of the U3 A region encoding a second gamma-activated site (GAS). Despite the TFBS conservation, the isolates demonstrated a degree of phylogenetic diversity. At present, the clinical consequence of CAEV-associated renal injury is not clear.

Serum epidemiological survey and risk factors investigation for lentivirus in goats from Sisal Region, Bahia, Brazil / Inquérito soroepidemiológico e fatores de risco para lentivírus em rebanhos caprinos da região sisaleira, Bahia

This study aimed to carry out a serum epidemiological survey of goat arthritis encephalitis in the sisal region of Bahia, Brazil, and to evaluate risk factors. We evaluated 831 samples of goat blood serum among males and females older than six months, from 49 farms distributed among the municipalities of Araci, Cansanção, Conceição do Coité, Itiúba, Monte Santo, Nordestina, Queimadas, Santa Luz, São Domingos and Valente. An epidemiological questionnaire for the analysis of possible risk factors was applied. All sera were tested for immune-agar gel. The seroprevalence obtained in surveyed herds was 1.56% (13/831). There was significant difference (p<0.05) for animal racial pattern, type of farming and breeding systems. However, when considering herds with predominantly dairy breeds (Saanen and Alpine Pardo), the seropositivity in animals from Valente, Conceição do Coité and São Domingos amounted to 5.06% (12/237). In these municipalities, from 14 properties analyzed, five (38.5%) had at least one positive-testing animal. This result is extremely worrying when one considers that few control measures are adopted by farmers to prevent the goat arthritis encephalitis in the most important dairy region of Bahia state.(AU)

Este trabalho teve por objetivo realizar um levantamento soroepidemiológico da artrite encefalite caprino na região sisaleira do estado da Bahia e avaliar os fatores de risco. Foram avaliadas 831 amostras de soros sanguíneos de caprinos entre machos e fêmeas, com idade superior a seis meses, provenientes de 49 propriedades rurais distribuídas entre os municípios de Araci, Cansanção, Conceição do Coité, Itiúba, Monte Santo, Nordestina, Queimadas, Santa Luz, São Domingos e Valente. Foi aplicado um questionário epidemiológico destinado à análise de possíveis fatores de risco. Todos os soros foram submetidos ao teste da imunodifusão em gel de ágar. A soroprevalência obtida nos rebanhos pesquisados foi de 1,56% (13/831). Houve diferença significativa (p≤0,05) para padrão racial dos animais, tipo de exploração e sistemas de criação. Quando se consideraram apenas os rebanhos com raças predominantemente leiteiras (Saanen e Pardo alpina), dos municípios de Valente, Conceição do Coité e São Domingos, a soropositividade nos animais elevou-se para 5,06% (12/237). Nesses municípios, de 14 propriedades analisadas, 5 (38,5%) apresentaram pelo menos um animal sororeagente. Esse resultado é extremamente preocupante quando se constata que poucas medidas de controle são adotadas pelos criadores para impedir a disseminação dessa importante enfermidade na bacia leiteira mais relevante do estado da Bahia.(AU)

Impaired Expression of Cytokines as a Result of Viral Infections with an Emphasis on Small Ruminant Lentivirus Infection in Goats.

Knowing about the genes involved in immunity, and being able to identify the factors influencing their expressions, helps in gaining awareness of the immune processes. The qPCR method is a useful gene expression analysis tool, but studies on immune system genes are still limited, especially on the caprine immune system. Caprine arthritis encephalitis, a disease caused by small ruminant lentivirus (SRLV), causes economic losses in goat breeding, and there is no therapy against SRLV. The results of studies on vaccines against other viruses are promising. Moreover, the Marker-Assisted Selection strategy against SRLV is possible, as has been shown in sheep breeding. However, there are still many gaps in our knowledge on the caprine immune response to infection. All types of cytokines play pivotal roles in immunity, and SRLV infection influences the expression of many cytokines in different types of cells. This information encouraged the authors to examine the results of studies conducted on SRLV and other viral infections, with an emphasis on the expression of cytokine genes. This review attempts to summarize the results of studies on the expression of cytokines in the context of the SRLV infection.

Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

Welfare effects of a disease eradication programme for dairy goats.

The Norwegian dairy goat industry has largely succeeded in controlling caprine arthritis encephalitis (CAE), caseous lymphadenitis (CLA) and paratuberculosis through a voluntary disease eradication programme called Healthier Goats (HG). The aim of this study was to apply an on-farm welfare assessment protocol to assess the effects of HG on goat welfare. A total of 30 dairy goat farms were visited, of which 15 had completed disease eradication and 15 had not yet started. Three trained observers assessed the welfare on 10 farms each. The welfare assessment protocol comprised both resource-based and animal-based welfare measures, including a preliminary version of qualitative behavioural assessments with five prefixed terms. A total of 20 goats in each herd were randomly selected for observations of human-animal interactions and physical health. The latter included registering abnormalities of eyes, nostrils, ears, skin, lymph nodes, joints, udder, claws and body condition score. For individual-level data, robust clustered logistic regression analyses with farm as cluster variable were conducted to assess the association with disease eradication. Wilcoxon rank-sum tests were used for comparisons of herd-level data between the two groups. Goats with swollen joints (indicative of CAE) and enlarged lymph nodes (indicative of CLA) were registered on 53% and 93% of the non-HG farms, respectively, but on none of the HG farms. The only other health variables with significantly lower levels in HG herds were skin lesions (P=0.008) and damaged ears due to torn out ear tags (P<0.001). Goats on HG farms showed less fear of unknown humans (P=0.013), and the qualitative behavioural assessments indicated that the animals in these herds were calmer than in non-HG herds. Significantly more space and lower gas concentrations reflected the upgrading of buildings usually done on HG farms. In conclusion, HG has resulted in some welfare improvements beyond the elimination of infectious diseases. The protocol was considered a useful tool to evaluate the welfare consequences of a disease eradication programme. However, larger sample sizes would increase the reliability of prevalence estimates for less common conditions and increase the power to detect differences between the groups. Despite the obvious link between disease and suffering, this aspect is rarely taken into account in the evaluation of disease control programmes. We therefore propose that welfare assessment protocols should be applied to evaluate the merits of disease control or eradication programmes in terms of animal welfare.

Interspecific transmission of small ruminant lentiviruses from goats to sheep.

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

Interspecific transmission of small ruminant lentiviruses from goats to sheep

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND: Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS: Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS: The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS: Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus.

The small ruminant lentiviruses include the prototype for the genus, visna-maedi virus (VMV) as well as caprine arthritis encephalitis virus (CAEV). Infection of sheep or goats with these viruses causes slow, progressive, inflammatory pathology in many tissues, but the most common clinical signs result from pathology in the lung, mammary gland, central nervous system and joints. This review examines replication, immunity to and pathogenesis of these viruses and highlights major differences from and similarities to some of the other lentiviruses.

The presence or absence of the gamma-activated site determines IFN gamma-mediated transcriptional activation in CAEV promoters cloned from the mammary gland and joint synovium of a single CAEV-infected goat.

The caprine arthritis encephalitis virus (CAEV) long terminal repeat promoter was cloned and sequenced from mammary gland and carpal joint synovium isolated from a 15.5 year old, CAEV-infected Toggenburg doe with chronic mastitis and carpal arthritis. A deletion of the CAEV gamma activated site (GAS) was identified in the mammary gland but not the synovial isolate. Subsequent promoter-reporter gene construct experiments indicated that the GAS is necessary for interferon γ-mediated promoter activation. Utilizing a molecular clone of the classic isolate CAEV-CO, these findings were corroborated by a set of GAS mutant promoter-reporter constructs with and without the CAEV GAS. Results of experiments with U937 monocyte cell lines stably transfected with molecular clones of CAEV-CO GAS deletion mutants also indicated the GAS is necessary for IFNγ-mediated promoter activation. The mammary gland CAE viral isolate was propagated in caprine peripheral blood mononuclear cells and was assigned the name CAEV-MA. This is the first report describing two CAE viral isolates cloned from different anatomical locations in the same animal with and without the CAEV GAS, and is the first report detailing cytokine-induced CAEV promoter function in a naturally occurring ΔGAS promoter.

Tissue tropism and promoter sequence variation in caprine arthritis encephalitis virus infected goats.

Caprine arthritis encephalitis virus is a lentivirus that infects goats and is closely related to maedi-visna virus of sheep. Infection with CAEV results in multiple discrete disease manifestations in goats which can include chronic arthritis, mastitis, pneumonia or encephalomyelitis. Presently, no satisfactory mechanistic rationale for viral tropism has been put forward. We propose that specific sequences in the lentiviral promoter (U3 region of the viral long terminal repeat) are associated with viral tissue tropism and subsequent disease expression. A total of 41 distinct CAE viral promoter regions were amplified, sequenced and phylogenetically compared from the tissues of 24 CAEV-infected goats demonstrating a variety of disease manifestations. Phylogenetically, we identified no tendency for clustering of these promoter sequences into tissue-specific groups. These results therefore do not provide evidence for the study hypothesis. However, multiple motifs within the U3 promoter region were highly conserved both within the entire collection of sequences and within tissue-specific groups.

SRLVs: a genetic continuum of lentiviral species in sheep and goats with cumulative evidence of cross species transmission.

Lentiviruses from distinct animal species have in common their genomic organization, the induction of slowly progressive diseases over months or years, the large spectrum of induced symptoms and concerned organs, the frequent inapparent infection without any detectable clinical signs, their ability to persist into their hosts despite an often strong and mature immune response. Lentiviruses are also characterized by their genomic plasticity and the rapid evolution of the viral species. SRLVs infecting goats and sheep follow a genomic evolution pattern similar to that observed in HIV or in other lentiviruses. Based on limited number of complete sequences, they have been initially described as two distinct genetic groups evolving independently in sheep or goats, the ovine strains being closely related to each other and distinct from the caprine ones. Over the last 2 decades, the description of many partial or complete sequences of caprine and ovine field isolates from various geographical regions and their phylogenetic studies clearly evidenced the existence of a genetic continuum with viruses that did not simply clustered according to the animal species they were isolated from. Three classifications have been successively proposed and allowed to refine the SRLV phylogeny over time. Phylogenetic reconstructions support the existence of SRLV cross-species transmission in domestic and wild small ruminants.

Affinity (tropism) of caprine arthritis encephalitis virus for brain cells.

One of the constraints in unraveling the mysteries blurring the advancement of research in the quest to totally put HIV problems under control is getting the appropriate animal model that would truly simulate human cases. This problem is more apparent in studies involving the central nervous system. Consequently, a viable animal model to generate information for the production of drugs and vaccines for the prevention and or control of lentiviral induced dementia in affected host animals is pertinent and vital. In this study, explant cultures prepared from the brain of new-born goat-kid were infected with CaprineArthritis Encephalitis (CAE) virus- a retrovirus affecting goats. The specific brain cell types infected by the (CAE) virus were determined using reverse-transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM techniques). TEM showed that in 85 - 90% cases, microglia were the cells specifically infected by the virus. Amplification of the genomic sequence of the envelope and the gag genes by RT-PCR confirmed the presence of CAEV proviral DNA in the brain cells of affected animals. No productive infection of the astrocytes was observed. The results of this study showed a lot of similarities in the tropism of CAE virus infection of goat brain cells to that of HIV infection in humans thus suggesting the potential usefulness of the caprine model for the study of HIV neuropathology. The goat model system as a non-primate model therefore could be more adaptable as a simple animal model than primate models with their complexity of anthropological, environmental and safety problems.

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway.

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.

Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef.

Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

Aspectos clínicos da artrite-encefalite dos caprinos / Clinical features of caprine arthritis-encephalitis infections

Caprinos de 14 plantéis localizados no estado de São Paulo, naturalmente infectados pelo vírus da artriteencefalite dos caprinos, foram clinicamente avaliados. Demonstrou-se que 17,1 % (64/374) dos caprinos sororreagentes apresentavam a forma clínica articular da enfermidade e que 6,6% (17/249) das cabras sororreagentes apresentavam a forma mamária.

Risk factors for the infection of Swiss goat herds with small ruminant lentivirus: a case-control study.

In the mid-1980s, Switzerland started a programme to eradicate caprine arthritis-encephalitis - an infectious disease of goats caused by the small ruminant lentivirus (srlv). Since 1996, progress towards eradication has slowed down, owing to infections occurring on farms from which the infection had previously been eliminated. To investigate specific risk factors for these new infections and to improve the eradication programme, a case-control study was conducted. Cases consisted of farms that had been officially free of srlv for at least three consecutive years but on which at least one srlv-seropositive animal had been detected during the annual serological surveys of 2001 and 2002. On all the case and control farms where sheep were housed together with goats, a subset of sheep was screened for antibodies to srlv. Potential risk factors were analysed in a logistic regression model; the results indicated that close contact with srlv-seropositive sheep was highly correlated with seroconversion in srlv-seronegative goat herds (odds ratio=26.9), supporting the hypothesis that srlv can be transmitted between sheep and goats, and suggesting that the measures taken so far will not lead to the complete eradication of srlv from Switzerland within the next few years.

Viral expression and leukocyte adhesion after in vitro infection of goat mammary gland cells with caprine arthritis-encephalitis virus.

A characteristic lesion in goats infected by the lentivirus CAEV is mastitis with lymphoid hyperplasia. In order to investigate the mechanism of lesion formation, cultures highly enriched in microvascular endothelial cells, mature and immature luminal epithelial cells, fibroblasts and myoepithelial cells were established from goat mammary gland biopsies. Their susceptibility to in vitro infection with two distinct types of CAEV was investigated by PCR, antigen expression and cytopathy. The capacity of infected mammary gland cells to bind uninfected caprine leukocytes was determined by flow cytometry. All cell types tested were susceptible to CAEV infection in vitro, with different levels of sensitivity according to cell phenotype. Our results suggest that the limited extent of natural infection of mammary gland cells reflects a protective local immune response, and that the myoepithelial cell could act as a reservoir cell. After infection, the mature luminal cell acquires the capacity to bind leukocytes in vitro, which could indicate a facilitation of cellular interactions. The distinct reactions of the different cell types to CAEV infection may be correlated with events leading to progressive lesion development during the natural infection.