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In Switzerland, viruses belonging to two different phylogenetic groups of small ruminant lentiviruses (SRLV) are currently circulating: the caprine arthritis-encephalitis virus (CAEV) and visna/maedi virus (VMV). In the past two decades, a mandatory national control program has led to a very low prevalence of seropositivity, while completely eliminating CAE as a clinical manifestation. However, in order to reduce the high costs and effort associated with this program, adjustments based on the most recent epidemiological knowledge are needed. The purpose of this study was to estimate the seroprevalence of CAEV and VMV using the newest diagnostic tools available, and to identify potential risk factors for infection with these viruses in Switzerland. For the prevalence estimation, a census was carried out including 10,696 farms with a total of 85,454 goats. Blood samples were analysed using a 3-step serological testing algorithm consisting of Chekit ELISA, Western Blot and SU5 ELISA. A risk factor analysis was conducted using logistic regression models built with data obtained from a mail questionnaire, and serological results from the census. The apparent herd-level prevalences were 0.38%, 2.77%, and 3.04% for CAEV, VMV and SRLV, respectively. Animal-level prevalences were 0.06% for CAEV, 0.55% for VMV, and 0.61% for SRLV. No statistically significant risk factors associated with CAEV or VMV infection were identified. However, the proportional high number of CAEV seropositive dwarf goats, in relation to their population size, could indicate that these hobby breeds may slip through some of the official controls. For an infection with SRLV, a medium herd size (7-40 goats) was found to be protective, compared with smaller (OR=1.90, p=0.034) and larger herds (OR=1.95, p=0.038). In conclusion, considering that all CAEV positive animals were culled, these results imply that CAEV is no longer actively spreading and has successfully been controlled in Switzerland. However, given the uncertain pathogenic potential of VMV in goats, future surveillance should also be taking into account the not insignificant number of VMV circulating in the Swiss goat population., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pestiviruses infect a wide variety of animals of the order Artiodactyla, with bovine viral diarrhea virus (BVDV) being an economically important pathogen of livestock globally. BVDV is maintained in the cattle population by infecting fetuses early in gestation and, thus, by generating persistently infected (PI) animals that efficiently transmit the virus throughout their lifetime. In 2008, Switzerland started a national control campaign with the aim to eradicate BVDV from all bovines in the country by searching for and eliminating every PI cattle. Different from previous eradication programs, all animals of the entire population were tested for virus within one year, followed by testing each newborn calf in the subsequent four years. Overall, 3,855,814 animals were tested from 2008 through 2011, 20,553 of which returned an initial BVDV-positive result. We were able to obtain samples from at least 36% of all initially positive tested animals. We sequenced the 5' untranslated region (UTR) of more than 7400 pestiviral strains and compiled the sequence data in a database together with an array of information on the PI animals, among others, the location of the farm in which they were born, their dams, and the locations where the animals had lived. To our knowledge, this is the largest database combining viral sequences with animal data of an endemic viral disease. Using unique identification tags, the different datasets within the database were connected to run diverse molecular epidemiological analyses. The large sets of animal and sequence data made it possible to run analyses in both directions, i.e., starting from a likely epidemiological link, or starting from related sequences. We present the results of three epidemiological investigations in detail and a compilation of 122 individual investigations that show the usefulness of such a database in a country-wide BVD eradication program., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

Bovine viral diarrhea virus (BVDV) is endemic worldwide. Together with classical swine fever and border disease viruses, it belongs to the genus Pestivirus of the family Flaviviridae. Most infections with BVDV take a transient, acute, course. Only rarely BVDV persists in its hosts. Due to the early time point of infection in utero, persistently infected (PI) animals are immunotolerant to the infecting non-cytopathic BVDV. In such animals the virus may mutate to a cytopathic biotype, causing lethal mucosal disease. In BVD-endemic regions, approximately 1% of the animals are PI. Removal of all PI animals leads to extinction of BVD. This approach to BVD eradication has been vindicated in Scandinavia. Following the same principles, regional and country-wide eradication programs are run in different parts of the world. These programs differ in the way PI animals are detected and in the role of vaccines. The Scandinavian two-step method of detecting PI animals is based on (i) the high level of seroprevalence in herds where PI animals are present and (ii) on testing all animals for virus in such herds. However, the high average herd seroprevalence in Switzerland made it impossible to define a reasonable threshold for virus testing. Therefore, all animals were directly tested for virus in the year 2008 and all newborn calves until the end of 2012, when the PI prevalence had dropped to 0.02%. Vaccination remains prohibited. Since 2013, surveillance for BVD is accomplished by serology. As a unique consequence of eradication, over 7500 viral strains are available to us for genetic studies.

Background: In 2006, bluetongue virus serotype 8 (BTV-8) was detected for the first time in central Europe. Measures to control the infection in livestock were implemented in Switzerland but the question was raised whether free-ranging wildlife could be a maintenance host for BTV-8. Furthermore Toggenburg orbivirus (TOV), considered as a potential 25th BTV serotype, was detected in 2007 in domestic goats in Switzerland and wild ruminants were considered a potential source of infection. To assess prevalences of BTV-8 and TOV infections in wildlife, we conducted a serological and virological survey in red deer, roe deer, Alpine chamois and Alpine ibex between 2009 and 2011. Because samples originating from wildlife carcasses are often of poor quality, we also documented the influence of hemolysis on test results, and evaluated the usefulness of confirmatory tests., Results: Ten out of 1,898 animals (0.5%, 95% confidence interval 0.3-1.0%) had detectable antibodies against BTV-8 and BTV-8 RNA was found in two chamois and one roe deer (0.3%, 0.1-0.8%). Seroprevalence was highest among red deer, and the majority of positive wild animals were sampled close to areas where outbreaks had been reported in livestock. Most samples were hemolytic and the range of the optical density percentage values obtained in the screening test increased with increasing hemolysis. Confirmatory tests significantly increased specificity of the testing procedure and proved to be applicable even on poor quality samples. Nearly all samples confirmed as positive had an optical density percentage value greater than 50% in the ELISA screening., Conclusions: Prevalence of BTV-8 infection was low, and none of the tested animals were positive for TOV. Currently, wild ruminants are apparently not a reservoir for these viruses in Switzerland. However, we report for the first time BTV-8 RNA in Alpine chamois. This animal was found at high altitude and far from a domestic outbreak, which suggests that the virus could spread into/through the Alps. Regarding testing procedures, hemolysis did not significantly affect test results but confirmatory tests proved to be necessary to obtain reliable prevalence estimates. The cut-off value recommended by the manufacturer for the screening test was applicable for wildlife samples.

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle worldwide. Infection of a pregnant animal may lead to persistent infection of the foetus and birth of a persistently infected (PI) calf that sheds the virus throughout its life. However, BVD viruses are not strictly species specific. BVDV has been isolated from many domesticated and wild ruminants. This is of practical importance as virus reservoirs in non-bovine hosts may hamper BVDV control in cattle. A goat given as a social companion to a BVDV PI calf gave birth to a PI goat kid. In order to test if goat to goat infections were possible, seronegative pregnant goats were exposed to the PI goat. In parallel, seronegative pregnant goats were kept together with the PI calf. Only the goat to goat transmission resulted in the birth of a next generation of BVDV PI kids whereas all goats kept together with the PI calf aborted. To our knowledge, this is the first report which shows that a PI goat cannot only transmit BVD virus to other goats but that such transmission may indeed lead to the birth of a second generation of PI goats. Genetic analyses indicated that establishment in the new host species may be associated with step-wise adaptations in the viral genome. Thus, goats have the potential to be a reservoir for BVDV. However, the PI goats showed growth retardation and anaemia and their survival under natural conditions remains questionable.

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Streptomycin is used in arboriculture to control fire blight. Using sheep as a model, multidrug-resistant bacteria in mammals were found to be selected after the intentional release of streptomycin into the environment. Escherichia coli and Staphylococcus spp. were isolated from the faeces and nasal cavities, respectively, of sheep grazing on a field sprayed with streptomycin at concentrations used in orchards (test group) and on a field without streptomycin (control group). Before the application of streptomycin, the percentage of streptomycin-resistant E. coli isolates in faeces was 15.8% in the control group and 14.7% in the test group. After the application of streptomycin, the overall number of streptomycin-resistant E. coli isolates was significantly higher in the test group (39.9%) than in the control group (22.3%). Streptomycin-resistant Staphylococcus isolates were only detected after the application of streptomycin. Streptomycin resistance was frequently associated with resistance to sulfamethoxazole, ampicillin, tetracycline and chloramphenicol and less frequently to cefotaxime in E. coli, and to tetracycline, fusidic acid and tiamulin in Staphylococcus spp. This study shows that the application of low concentrations of streptomycin on grass, as occurs during the spraying of orchards, selects for multidrug-resistant nasal and enteric bacterial flora, including extended-spectrum beta-lactamase-producing E. coli., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Background: In the frame of an eradication program for bovine viral diarrhea (BVD) in Swiss livestock, the question was raised whether free-ranging wildlife could threaten the success of this sanitary measure. Therefore, we conducted serological and virological investigations on BVD virus (BVDV) infections in the four indigenous wild ruminant species (roe deer, red deer, Alpine chamois and Alpine ibex) from 2009 to 2011, and gathered information on interactions between wild and domestic ruminants in an alpine environment by questionnaire survey., Results: Thirty-two sera out of 1'877 (1.7%, 95% confidence interval [CI] 1.2-2.4) were seropositive for BVDV, and a BVDV1 sub genotype h virus was found in a seropositive chamois (0.05%, 95% CI 0.001-0.3). The seropositive animals originated from sub-alpine or alpine regions and significantly more seropositive red deer, chamois and ibex than roe deer were found. There were no statistically significant differences between sampling units, age classes, genders, and sampling years. The obtained prevalences were significantly lower than those documented in livestock, and most positive wild ruminants were found in proximity of domestic outbreaks. Additionally, BVDV seroprevalence in ibex was significantly lower than previously reported from Switzerland. The survey on interspecific interactions revealed that interactions expected to allow BVDV transmission, from physical contacts to non-simultaneous use of the same areas, regularly occur on pastures among all investigated ruminant species. Interactions involving cervids were more often observed with cattle than with small ruminants, chamois were observed with all three domestic species, and ibex interacted mostly with small ruminants. Interactions related to the use of anthropogenic food sources were frequently observed, especially between red deer and cattle in wintertime., Conclusions: To our knowledge, this is the first report of BVDV RNA isolated from an Alpine chamois. Nevertheless, our results suggest that BVDV infections are only sporadic in Swiss wild ruminants, despite regular occurrence of interactions with potentially infected livestock. Overall, serological, virological and ethological data indicate that wildlife is currently an incidental spill-over host and not a reservoir for BVDV in Switzerland.

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems due to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0-11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Short synthetic peptides are important tools in biomedical research permitting to generate hapten specific polyclonal sera for analytical purposes or functional studies. In this paper we provide proof of principle that a peptide located in a highly conserved portion of the Gag protein of the caprine arthritis encephalitis virus and carrying an immunodominant T helper cell epitope functions as an efficient carrier peptide, mediating a strong antibody response to a peptidic hapten encompassing a well-characterized B cell epitope of Env. The carrier and hapten peptides were collinearly synthesized permutating their molecular arrangement. While the antibody response to the hapten was similar for both constructs, the antibody response to a B cell epitope overlapping the T helper cell epitope of the Gag carrier peptide was considerably different. This permits a modular use of the carrier peptide to generate antibody directed exclusively to the hapten peptide or a strong humoral response to both carrier- and hapten-peptide. Finally, we have mapped the epitopes involved in this polarized antibody response and discussed the potential immunological implications.

The seroprevalence of pestivirus infections in small ruminants and new world camelids in Switzerland was determined. In 5'059 sera of sheep from 382 herds, 503 sera of goats from 54 herds and 109 sera of alpacas and lamas from 53 herds, population prevalences of 16.1% (sheep), 25.4% (goats) and 4.6% (new world camelids), respectively, were found. In order to determine the source of infection, the serological reactions were further characterized by cross-neutralization against two pestiviruses representing the genotypes BVDV (Bovine Virus Diarrhea Virus)-1 and BDV (Border Disease Virus)-1. Based on the ratio of respective antibody titres, 56.1% of the infections in sheep were induced by a BDV-1, 12.9% by a BVDV-1 and 31.0% by an unresolved pestivirus. In goats, the corresponding proportions were 23.4%, 10.2% and 66.4%, respectively. In Alpacas and Lamas, the source of infection of 1 animal was BDV-1 and that of 4 seropositive animals remained unresolved. In view of the phylogenetic relationship between pestiviruses, the unresolved source of infection is most probably attributable to other pestivirus genotypes circulating in small ruminants and new world camelids. Due to the predominance of pestiviral genotypes other than BVDV-1, the risk of transmission of BVDV from persistently infected small ruminants and new world camelids to cattle appears to be moderate, apart from close direct contact in mixed animal husbandry, communal pasturing and grazing in the Alps.

The immunization of goats with a synthetic peptide encompassing the G5 antigenic site of the rabies virus surface glycoprotein induces a strong humoral immune response in the absence of a carrier protein. The immunized animals mounted high antibody titers and showed a strong avidity maturation of the B cell immune response to both the G5-peptide and purified surface glycoprotein G. This antibody weakly neutralized rabies virus carrying the G5 epitope but failed to neutralize escape mutants carrying a single point mutation in this epitope. A putative T helper cell epitope, functional in the context of different caprine MHC haplotypes, was identified by structure analysis of the G5-peptide. This striking dichotomy between high titers and antibody of high avidity to the glycoprotein G and poor neutralizing activity strongly suggests that antibody binding assays such as ELISA cannot always reliably predict the neutralizing activity of sera as measured in functional assays.

CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte-macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.

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.

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.

We have genetically analyzed ruminant pestiviruses. All >150 bovine viral diarrhea (BVD) viruses isolated from cattle in Switzerland belonged to genotype 1, with subgenogroups e, h, k and b found in decreasing frequency. To date, representatives of subgenogroup k have been detected in Switzerland only. Despite serological evidence of Border disease in sheep, only few Border disease viruses have been isolated, all of which belong to the novel group 3. Serological evidence suggested that pestivirus infections may occur also in wild ruminants in Switzerland but no isolates are available for analysis. In addition, we describe two pestiviruses, one a cell culture contaminant and the other isolated from a buffalo, that cluster with a recently proposed novel pestivirus species.

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.

We performed a phylogenetic analysis of caprine and ovine lentiviruses using long sequences in gag and pol of 104 new Swiss isolates and six available corresponding database sequences. Forty-five isolates, forming five sequence clusters, were unclassifiable by the present classification. Pairwise DNA distance analysis indicated different categories of relatedness, requiring a new classification system. We propose four principal sequence groups, A-D, which differ by 25-37%. Groups A and B are further divided into subtypes which differ by 15-27%. Group D and four of the seven group A subtypes, A3, A4, A5 and A7, are formed by new Swiss isolates. Molecular epidemiology revealed that Swiss B1 strains differed no more from French, Brazilian or US strains than from each other, suggesting virus propagation through international livestock trade. Furthermore, infection of goats by subtypes A3 or A4 was significantly associated with documented contact with sheep, which also harbor these subtypes, thus indicating regularly occurring sheep-to-goat transmission.

Caprine arthritis encephalitis virus (CAEV)-infected goats develop high titers of nonneutralizing antibody to several immunodominant epitopes of the viral envelope glycoprotein. Antibodies directed to these structures, and especially the principal immunodominant domain (PID) of the transmembrane portion of the envelope glycoprotein, have been implicated in the immunopathological mechanisms leading to the development of arthritis. We previously mapped the PID and additional epitopes of CAEV gp38 and showed an association between the development of clinical arthritis in infected animals and the antibody response to these epitopes. The development of clinical arthritis is associated with a higher rate of viral expression, especially in the affected tissue, indicating that antibody may favorably modulate virus replication. To test this hypothesis, we immunized goats with synthetic peptides corresponding to the mapped epitopes. The immunized animals developed high titers of nonneutralizing antibody to the immunizing peptides. In radioimmunoprecipitation experiments these antibodies were shown to react to the envelope glycoproteins in extracts obtained under nondenaturing conditions. Finally, these sera were tested in cultures of primary macrophages infected at low multiplicity without showing any (either positive or negative) modulatory activity., (Copyright 2002 S. Karger AG, Basel)

Goats infected with caprine arthritis-encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera. In addition to the four previously described epitopes, seven novel antigenic sites were identified, of which five were located on the surface (SU) and two in the transmembrane (TM) subunits of Env. The SU antibody-binding domains located in the variable regions of the C-terminal part of the molecule (SU3 to SU5) showed the strongest reactivity and induced a rapid seroconversion in six experimentally infected goats. However, the response to these immunodominant epitopes did not appear to be associated with any neutralizing activity. The pattern of serum reactivity of naturally infected goats with these epitopes was restricted, suggesting a type-specific reaction. Interestingly, the reactivity of peptides representing SU5 sequences derived from CAEV field isolates varied with the geographical and/or breeding origin of the animals. This suggests that peptides corresponding to the immunodominant SU epitopes may well be useful in the serotyping of CAEV isolates. Furthermore, the identification of the CAEV Env epitopes will permit us to functionally dissect the antibody response and to address the role of anti-Env antibodies either in the protection from or in the pathogenesis of CAEV infection.

3866 sheep from 226 flocks of breeding associations and 1218 sheep from 15 independent sheep owners were tested for the presence of serum antibodies against Maedi-Visna and Border Disease viruses. The flocks were randomly selected based on the relative proportion and the geographical distribution of the 4 predominant Swiss sheep breeds (Braunköpfiges Fleischschaf, Schwarzbraunes Berg- und Juraschaf, Walliser Schwarznasenschaf, Weisses Alpenschaf). Additionally two smaller breeds were included in the study (Charollais Suisse, Milchschafe). Sera of all sheep older than 1 year were collected together with data characterizing host and management factors. The sera were tested using established ELISAs for detection of antibodies to Maedi-Visna and Bovine Virus Diarrhea/Border Disease viruses. ELISA results of Maedi-Visna serology were confirmed by immunoblotting. 9% of the sheep of breeding associations were antibody-positive for Maedi-Visna virus. The results of the different breeds varied between 0.4% and 36%. A multiple logistic regression procedure identified breed, age, airing in barns, herd size, pasturing on alps and way of keeping the animals during winter as associated factors with individual serostatus. The prevalence of antibodies to Border Disease was 20% in sheep of breeding associations and 65% in those of independent sheep owners.

Maedi-Visna is a lentiviral disease of sheep with a worldwide distribution. The transmission of the virus occurs primarily via colostrum and milk from the infected ewe to its newborn lamb but also horizontally between sheep. The most obvious clinical symptoms are progressive dyspnea and emaciation. In this prospective study an eradication based on serological testing and removing of seropositive animals was performed in 24 flocks of sheep of the breed "Walliser Schwarznasenschafe" leading to a reduction of the seroprevalence from 36% to 1% within two years. The control group consisted of 21 flocks of sheep. Lambs of seropositive ewes had a 7.6 times higher risk to seroconvert within their first two years of life compared to those of seronegative ewes. The dynamics of the spread of the infection were studied in birth cohort groups. Cohort animals of seropositive ewes showed an obvious trend to seroconvert slowly. Seropositive ewes had a significantly lower reproduction rate and their lambs suffered from significantly higher death and lower growth rates, probably due to a reduced milk production, resulting in economic losses.

Caprine arthritis encephalitis (CAE) is a lentiviral infection of goats characterized by mononuclear cell infiltration of various tissues, most prominently the joints, mammary glands and, in young animals, the brain. We have investigated the early stages of arthritis induced by intracarpal and intravenous infection with molecularly cloned CAE virus. Analysis of the synovial membranes by immunohistological methods showed that the proportion of CD8+ T cells peaked around day 12 post-infection. CD4+ T cells increased to a lesser degree. The relative proportion of B cells rose steadily post-infection. At 33 days post-infection, plasma cells accounted for over one third of all inflammatory cells in the inflamed synovium. Histopathologically, the arthritic lesions in the synovial membranes closely resembled those in membranes of animals with a 2-year history of chronic arthritis. Our observations indicate that this type of short-term experimental infection is particularly suitable for studying the pathogenesis of goat lentiviral infection. In addition, our observations support the view that a predominantly humoral (type 2) immune response may contribute to the pathogenesis of CAE.

Infection of goats with the lentivirus caprine arthritis encephalitis virus (CAEV) leads to persistent infection and development of chronic arthritis. We analyzed the expression of cytokines and viral RNA in the joints of goats at early time points after experimental infection with CAEV and in those of animals suffering from chronic arthritis as a result of natural infection. In situ hybridization experiments showed that the pattern of cytokine expression in caprine arthritis was similar to that found in rheumatoid arthritis (RA), with a few cells expressing the lymphocyte-derived cytokines interferon (IFN)-gamma and interleukin (IL)-2 and rather more cells expressing monocyte chemoattractant protein (MCP)-1, IL-6, and tumor necrosis factor (TNF)-alpha. IFN-gamma mRNA expression in experimentally infected joints peaked at day 12 and was mostly detected in areas containing viral RNA. At later time points, no IFN-gamma- or virus-expressing cells were found in inflamed joints but both were again detected in goats with severe arthritis. Interestingly, at the clinical stage of arthritis reflecting the chronic stage of infection, the inflammatory lesion was found to be immunologically compartmentalized. Humoral immune responses and cell-mediated immune responses appeared to concurrently occur in distinct areas of the synovial membrane.

Goats infected with caprine arthritis encephalitis virus (CAEV) develop chronic arthritis sharing many features with human rheumatoid arthritis (RA). TNF is thought to be a key mediator contributing to the formation of the arthritic lesion in RA. We studied this cytokine in goats suffering from chronic arthritis. TNF alpha expressing cells were detected by in situ hybridization in synovial membranes of arthritic carpal joints. Expression of TNF alpha did not correlate with the degree of viral replication as assessed by in situ hybridization for viral RNA. In line with the lack of correlation between the degree of viral replication and TNF expression in vivo, we failed to detect increased cytokine RNA in goat macrophages infected with CAE virus in vitro and no TNF protein was found in culture supernatants. In addition, virus infection failed to prime goat macrophages for enhanced TNF expression in response to lipopolysaccharide. Taken together, these findings argue against a direct role of CAE virus in increasing the expression of TNF alpha in caprine arthritis.

Agalactosyl IgG [Gal(0)] was first discovered in patients with rheumatoid arthritis (RA). However, the proportion of this glycoform is also raised in tuberculosis and leprosy. This has helped reinforce the suggestion that RA may be triggered by a mycobacterium-like slow bacterial infection. On the other hand, arthritis can occur in mycobacterial diseases, so raised Gal(0) could be associated with a tendency to arthritis, rather than with a particular type of infection. Therefore, we wished to find out whether the percentage of Gal(0) [%Gal(0)] is increased in sheep and goats following infection with maedi visna virus or caprine arthritis encephalitis virus (CAEV), both of which can lead to inflammatory synovitis. We found that the normal level of Gal(0) in these species is much lower than in humans. Goats infected with CAEV or Mycobacterium paratuberculosis (used as a control mycobacterial infection) had a significant increase in %Gal(0), though it was still below the level seen in normal humans. Studies by Western blot confirmed the presence of terminal N-acetylglucosamine on heavy chains, and percentages of Gal(0) comparable to those seen in human RA could be generated by exposing goat IgG to streptococcal beta-galactosidase. The rise in %Gal(0) was greatest in members of infected herds that were just starting to manifest arthritis, and tended to be lower in those in which severe carpitis had developed at the time of bleeding, implying the possibility that raise %Gal(0) may be an early or predisposing event for the development of arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

A new ELISA kit was developed, based on highly purified whole-virus antigen derived from the Swiss maedi-visna virus strain OLV. The sensitivity, specificity and accuracy of this assay were compared with that of an established ELISA based on recombinant GAG (group-specific antigens)-GST (glutathione S-transferase) fusion protein expressed in E. coli (GAG-GST ELISA). The whole-virus ELISA exhibits at least comparable specificity (99.3%) but higher sensitivity (98.6 versus 86.3%) and agreement with the 'true' status beyond chance in the detection of antiviral antibodies in serum from goats. Antibodies in milk samples are detected with higher specificity (98.9 versus 97.8%) but lower sensitivity (91.4 versus 98.2%) than in GAG-GST ELISA. The specificity of the new ELISA in the detection of antibodies in serum might be superior, since a set of 40 samples falsely rated positive in GAG-GST ELISA in routine diagnostic work was negative in the new ELISA. In both assays, milk samples can be tested instead of serum, although with slightly reduced sensitivity in the new ELISA. The major advantage of the new test kit is the low number of equivocal samples needing confirmation in a supplementary test. Results obtained with sheep sera indicate that the new ELISA kit is also suitable for the detection of antibodies to maedi-visna virus.

High titers of antibodies to caprine arthritis-encephalitis virus (CAEV) envelope (Env) glycoproteins are found in infected goats developing a progressive arthritis. In order to identify linear B epitopes of the CAEV Env, which may be involved in the immunopathology of arthritis, we constructed a lambda gt11 Env expression library. By combining library screening with sera from naturally infected Swiss goats with an enzyme immunoassay with overlapping peptides (pepscan), four group-specific epitopes could be precisely defined in the transmembrane envelope proteins: TM1 to TM4, including a conserved structure (TM3) that corresponds to the immunodominant epitope of human immunodeficiency virus type 1 and other lentiviruses. A panel of 190 CAEV naturally infected goat serum samples, obtained from animals with defined clinical status, was tested for reactivity to synthetic peptides corresponding to the TM epitopes in an enzyme-linked immunosorbent assay. Antibody reactivity to two epitopes was highly associated (TM3, P = 0.002, and TM4, P < 0.001) with the presence of clinically detectable arthritis. Such an association is absent for anti-Gag antibody. Antibodies to the immunodominant structures of the TM glycoprotein could thus have an important role in the immunopathogenic process leading to disease.

Infections with the Bovine Viral Diarrhea/Mucosal Disease Virus (BVDV) are widespread and cause a variety of diseases including reproductive disorders, abortion and malformation, pneumoenteritis, thrombocytopenia and mucosal disease. Together with the closely related border disease virus of sheep (BDV) and European Swine fever virus (CSFV), also referred to as Hog Cholera virus, BVDV is now classified in the genus pestivirus of the Flaviviridae family. The BVDV exists in two biotypes, noncytopathic and cytopathic, the latter differing in structural proteins from the noncytopathic biotypes. In virus-free animals infection is transient and mostly subclinical or mild but may also lead to an array of diverse symptoms such as pneumoenteritis (often in combination with other microorganisms). Infection of the developing fetus early in gestation with a noncytopathic biotype of BVDV may result in persistent infection and birth of apparently healthy calves. Such calves may later in their lives develop Mucosal Disease, a lethal course of infection associated with a mutation to the cytopathic biotype or superinfection with a cytopathic BVDV antigenically similar to the non-cytopathic virus already present in these animals. Diagnosis of infections with BVDV is based on the clinical symptoms and demonstration of virus. Paired serum samples allow the detection of seroconversion in acute infections while persistently infected animals are immunotolerant and generally lack antiviral antibody. Although generally found in their respective host species, pestiviruses of cattle, sheep and pigs are capable of crossing the species barrier into the other species. The existence of pestiviruses in wild ruminants and boars may complicate control strategies that are aimed at removing virus carriers and the control of animal movements.