SNPs in candidate genes MX dynamin-like GTPase and chemokine (C-C motif) receptor-5 are associated with ovine pulmonary adenocarcinoma progression in Latxa sheep.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer in sheep caused by Jaagsiekte sheep retrovirus (JSRV). OPA is present in many sheep-rearing countries causing economic and welfare issues, as currently no efficient vaccines or treatments are available. Breed differences suggest a host genetic component may influence the pathogenesis of OPA, but so far few genes have been identified. In this work, a genetic association study was carried out in Latxa dairy sheep which were classified as cases/controls based on the presence/absence of OPA lung tumours. Candidate genes included cytokines and a receptor and innate immunity genes. After SNPs in the candidate genes were identified, the distribution of alleles in cases and controls was compared by means of logistic regression analyses at the allelic, genotypic and haplotypic levels. The association analysis showed that several candidate genes were significantly associated with resistance or susceptibility to OPA; two of the candidates, CCR5 and MX1, remained significantly associated with resistance and susceptibility respectively, even after Bonferroni correction.

Small ruminant lentivirus infections and diseases.

Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats and that display a great genetic variability. These characteristics stress the need to consider the whole host range and to perform local surveillance of the viruses to opt for optimum diagnostic tests, in order to establish control programmes. In the absence of effective vaccines, a comprehensive knowledge of the epidemiology of these infections is of major importance to limit their spread. This article intends to cover these aspects and to summarise information related to characteristics of the viruses, pathogenesis of the infection and description of the various syndromes produced, as well as the diagnostic tools available, the mechanisms involved in transmission of the pathogens and, finally, the control strategies that have been designed until now, with remarks on the drawbacks and the advantages of each one. We conclude that there are many variables influencing the expected cost and benefits of control programs that must be evaluated, in order to put into practice measures that might lead to control of these infections.

SNPs in APOBEC3 cytosine deaminases and their association with Visna/Maedi disease progression.

The Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) genes are able to inhibit the replication of a wide range of exogenous retroviruses, as well as endogenous retroviruses and retrotransposons. Three APOBEC3 genes, named APOBEC3Z1, APOBEC3Z2 and APOBEC3Z3, have been described in sheep. In this work the three genes have been screened in order to identify polymorphisms. No polymorphism was detected for the A3Z2 and A3Z3 genes but 16 SNPs and a 3-bp deletion were found in the A3Z1 gene. A thermoestability prediction analysis was applied to the detected amino acidic SNPs by three different programs. This analysis revealed a number of polymorphisms that could affect the protein stability. The SNPs of the 3'UTR were tested to detect alterations on the predicted microRNA target sites. Two new microRNA target sites were discovered for one of the alleles. Two SNPs were selected for association studies in relation with the retroviral disease Visna/Maedi in Latxa and Assaf sheep breeds. Although association analyses resulted unconclusive, probably due to the unsuitability of the SNP allele frequency distribution of the selected polymorphisms in the analyzed breeds, these genes remain good candidates for association studies.

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Pathological and aetiological studies in sheep exhibiting extrathoracic metastasis of ovine pulmonary adenocarcinoma (Jaagsiekte).

Seven sheep with a histopathological diagnosis of pulmonary adenocarcinoma with extrathoracic metastases were included in this retrospective study aiming to describe the pathological findings and to establish their relationship with Jaagsiekte sheep retrovirus (JSRV). In order of frequency, extrathoracic metastases were found in the liver, kidneys, skeletal muscle, digestive tract, spleen, skin and adrenal glands. Intrathoracic metastases involved the chest wall, regional lymph nodes, diaphragm and heart. Immunohistochemistry and polymerase chain reaction allowed detection of JSRV-related protein and nucleic acid, respectively, in the extrathoracic tumours of all cases. It is concluded that extrathoracic metastases constitute a pathological event of ovine pulmonary adenocarcinoma and confirm the malignant character of this virus-induced neoplasia.

Microsatellites in immune-relevant regions and their associations with Maedi-Visna and ovine pulmonary adenocarcinoma viral diseases.

Maedi-Visna (MV) and ovine pulmonary adenocarcinoma (OPA) are two retroviral diseases occurring worldwide that affect adult sheep. Differences in incidence, which may be related to sheep-rearing and housing choices, as well as to genetics, and disease progression have been reported for both diseases. In this work four microsatellites located in immune-relevant regions, the major histocompatibility complex (MHC) region, interferon-γ and interleukin-12p35, were genotyped to determine their association with disease progression. The analysed sample included Latxa sheep with and without OPA and MV-characteristic lesions in their lungs. The microsatellites in the MHC were the most diverse, while the ones located in the cytokines were the less polymorphic. In the case of IFN-γ the results suggested the presence of null alleles. Significant results were detected for several microsatellite alleles in the association analysis carried out by logistic regression. All statistical analyses included a flock effect adjustment to avoid false positives due to genetic structuration. MHC Class I microsatellite alleles OMHC1*205 and OMHC1*193 were associated with disease progression for Maedi and OPA, respectively. Moreover, MHC Class II microsatellite allele DRB2*275 was associated with presence of lesions in Maedi. Furthermore, the MHC microsatellites were combined for a bioinformatic haplotype inference with the PHASE software. In total, 73 haplotypes were detected, 18 of them in more than 6 animals. After standard and weighted logistic regression analysis, two of them were significantly associated with susceptibility: OMHC1*205-DRB2*271 for Maedi and OMHC1*193-DRB2*271 for OPA, both with the Class I microsatellite alleles associated in the marker by marker study. Although more extensive analyses are needed to disentangle the relationship between host genetics and disease, as far as we know this is the first study demonstrating a significant association between sheep MHC Class I microsatellite alleles and susceptibility to Maedi-Visna and OPA viral diseases.

Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves.

In this study, we characterized 8 new isolates obtained from healthy but congenitally infected calves using a BALB/c mouse model. Neospora caninum-infected mice survived without exhibiting any clinical signs of disease. Nevertheless, differences among isolates in parasite organ distribution, parasite burden and the severity of histopathological lesions were determined. Mice infected with the Nc-Spain 5H, Nc-Spain 7 and Nc-Spain 9 isolates showed higher parasite burdens and more severe brain lesions during the late phase of infection compared to mice infected with the Nc-Spain 2H, Nc-Spain 3H or Nc-Spain 6 isolates. Furthermore, differences in the immunoglobulin IgG1 and IgG2a isotype kinetics induced by these isolates were observed, with a more rapid IgG2a response seen in mice infected with the Nc-Spain 2H and Nc-Spain 3H isolates. These results confirm the intra-species variability of N. caninum pathogenicity.

Experimental infection of Eurasian wild boar with Mycobacterium avium subsp. avium.

The Eurasian wild boar (Sus scrofa) is increasingly relevant as a host for several pathogenic mycobacteria. We aimed to characterize the first experimental Mycobacterium avium subsp. avium (MAA) infection in wild boar in order to describe the lesions and the immune response as compared to uninfected controls. Twelve 1-4-month-old wild boar piglets were housed in class III bio-containment facilities. Four concentrations of MAA suspension were used: 10, 10(2) and 10(4) mycobacteria (2 animals each, oropharyngeal route) and 2.5 x 10(6) mycobacteria (2 animals each by the oropharyngeal and nasal routes). No clinical signs were observed and pathology evidenced a low pathogenicity of this MAA strain for this particular host. Bacteriological and pathological evidence of successful infection after experimental inoculation was found for the group challenged with 2.5 x 10(6) mycobacteria. These four wild boar showed a positive IFN-gamma response to the avian PPD and the real-time RT-PCR data revealed that three genes, complement component C3, IFN-gamma and RANTES, were significantly down regulated in infected animals. These results were similar to those found in naturally and experimentally M. bovis-infected wild boar and may constitute biomarkers of mycobacterial infection in this species.

Effects of housing on the incidence of visna/maedi virus infection in sheep flocks.

The incidence of seroconversion to visna/maedi virus (VMV) infection and its relationship with management and sheep building structure was investigated in 15 dairy sheep flocks in Spain during 3-7years. Incidence rates were 0.09 per sheep-year at risk in semi-intensive Latxa flocks and 0.44 per sheep-year at risk in intensive Assaf flocks and was greatest for the one year old Assaf replacement flock. Separate multivariable models developed for replacement and adult flocks indicated that in both cases seroconversion was strongly associated to direct contact exposure to infected sheep and to being born to a seropositive dam. The latter effect was independent of the mode of rearing preweaning and the risk of seroconversion was similar for sheep fed colostrum and milk from a seropositive or a seronegative dam. These results are further evidence of the efficiency of horizontal VMV transmission by close contact between sheep and also suggest a inheritable component of susceptibility and resistance to infection. In contrast, indirect aerogenous contact with seropositive sheep was not associated with seroconversion as evidenced in replacement sheep housed in separate pens in the same building as adult infected sheep for one year. Consequently, VMV may not be efficiently airborne over short distances and this is important for control of infection. Moreover, there was no relationship between seroconversion and shed open areas. The latter could be related to having examined few flocks in which high infection prevalence dominated the transmission process while ventilation, may depend on a variety of unrecorded factors whose relationship to infection needs to be further investigated.

First data on Eurasian wild boar response to oral immunization with BCG and challenge with a Mycobacterium bovis field strain.

The Eurasian wild boar (Sus scrofa) is considered a reservoir for bovine tuberculosis (bTB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex in south-central Spain. The vaccination of wildlife with BCG offers an alternative to culling and to movement restriction for the control of bTB among wildlife reservoirs. In this study, we hypothesized that oral BCG immunization of wild boar would affect the expression of immunoregulatory genes and confer protection against M. bovis. Three groups were used to describe the infection, pathological findings and gene expression profiles in wild boar: BCG-vaccinated and M. bovis-challenged (vaccinated challenged group; N=6), non-vaccinated and M. bovis-challenged (non-vaccinated challenged group; N=4), and non-vaccinated and mock-infected (control group; N=2) animals. M. bovis was isolated from 50% (3/6) and 75% (3/4) of vaccinated challenged and non-vaccinated challenged animals, respectively. All four wild boar from the non-vaccinated challenged group developed bTB-compatible lesions 114 days after challenge. In contrast, only 50% of vaccinated challenged wild boar developed lesions. The PBMC mRNA levels of IL4, RANTES, C3, IFN-gamma and methylmalonyl-CoA mutase (MUT) were analyzed at several days post-vaccination (dpi). When vaccinated challenged animals were compared to controls, all five genes were significantly upregulated at the time of M. bovis infection at 186dpi but IFN-gamma levels were also upregulated at 11 and 46dpi. The C3 and MUT mRNA levels were higher at 46dpi, and 11 and 186dpi, respectively, in vaccinated protected wild boar when compared to non-vaccinated challenged animals. At the end of the experiment (300dpi), the mRNA levels of selected genes were lower in non-vaccinated challenged animals when compared to control wild boar. Exposing wild boar to a dose of 10(4)cfu of M. bovis by the oropharyngeal route is an adequate protocol to produce an infection model in this species. Our results suggested that oral BCG immunization of wild boar results in the upregulation of immunoregulatory genes that may be associated with protective response to M. bovis infection in this species. More studies on vaccine efficacy, delivery, and safety will be needed to confirm if oral vaccination with BCG could be used in bTB control programs for reducing M. bovis infection and clinical disease in wild boar.

Improvements in the detection of small ruminant lentivirus infection in the blood of sheep by PCR.

The polymerase chain reaction on blood samples has been considered a complement to serological methods for the detection of small ruminant lentiviruses (SRLV) infections in sheep and goats. This is a report on the results of a study to evaluate the use of the same blood sample for the detection of infected animals by ELISA and PCR. A comparison between the results obtained by applying PCRs targeting LTR and gag sequences on blood clot, serum and peripheral blood leucocytes was made. In addition to simplifying sampling and laboratory work, the use of blood clot samples with the gag-PCR improved remarkably the detection of infected animals. Finally, this study has shown the existence of a cell-free viremia in the serum of SRLV-infected sheep.

Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype).

To evaluate the pathogenicity of local isolates of ovine pestiviruses (BDV-4 genotype), 13 virus- and antibody-negative, artificially inseminated pregnant ewes were challenged on days 108 (5 ewes), 76 (5 ewes) and 55 of pregnancy (3 ewes) with 2 ml of ovine pestivirus containing 10(6) TCID(50). Viraemia was detected by RT-PCR from 2 to 15 days pi in most ewes. No abortion due to the infection was observed but the number of stillbirths was high (32%), and bodyweight at lambing was significantly reduced compared to the experimental flock of origin used as control. Clinical symptoms in live lambs consisted on tremors, gait anomalies and inability to stand unaided. Skeletal abnormalities (brachygnathia, prognathia, arthrogryposis) were present in 44% of the lambs. Only 20% of the lambs were clinically normal. RT-PCR was a very sensitive technique compared to antigen ELISA in detecting viral presence in experimentally infected ewes and their progeny.

Coexistence of enzootic nasal adenocarcinoma and jaagsiekte retrovirus infection in sheep.

Ten sheep naturally affected with enzootic nasal adenocarcinoma (ENA), a disease associated with ovine enzootic nasal tumour virus (ENTV-1), were found also to be infected with jaagsiekte sheep retrovirus (JSRV), the causal agent of ovine pulmonary adenocarcinoma (OPA). Only one of the sheep showed OPA lung lesions. The animals belonged to 10 flocks located in a geographical area in which OPA is frequently seen. ENTV-1 was found in all the ENA tumours but only occasionally in extra-tumoral sites, confirming the results of a previous study. In contrast, JSRV had a disseminated tissue distribution, similar to that previously reported for animals infected with JSRV. However, the occurrence of JSRV in lymphoid tissues was clearly greater than in sheep infected with JSRV but with no lesions of ENA. The data suggested a synergistic relationship between ENTV-1 and JSRV, resulting in increased proliferation of JSRV.

Enzootic nasal adenocarcinoma of sheep and goats.

Enzootic nasal adenocarcinoma is a contagious tumour of the mucosal nasal glands affecting young adult sheep or goats. The disease occurs naturally in all continents except Australia and New Zealand. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos and skull deformations. The tumour is classified histologically as a low-grade adenocarcinoma. Nasal glands of both respiratory and olfactory muosal glands seem to be the origin of the neoplasia. It has been experimentally transmitted in sheep and goats using either tumour extracts or concentrated nasal fluids. Two distinct retroviruses are implicated in the aetiology of the neoplasia one in sheep (ONAV) and one in goats (CNAV). We suggest that jaagsiekte sheep retrovirus (JSRV), ONAV, CNAV, and their endogenous counterparts represent a unique family of retroviruses. The similarities between these viruses suggests that any control strategies, including vaccination, may be appropriate to both diseases. The differences, however, represent a unique resource for delineating the function of individual regions of the virus. It is intriguing that whilst ONAV and CNAV appear to be as different to each other as they are to JSRV, that they have very similar disease pathologies, distinct from that of OPA. Additionally, all three exogenous viruses manage to avoid instigating any apparent immune response. Whether this is indeed a result of tolerance induced by the endogenous counterparts or whether the viruses themselves have unique immunosuppressive properties will be an important finding.

Sheep pulmonary adenomatosis: characterization of two pathological forms associated with jaagsiekte retrovirus.

Pathological and immunohistochemical studies were performed on the lungs of 10 sheep with lesions of "classical" sheep pulmonary adenomatosis (SPA) and six sheep with "atypical" lung tumours. Lung tumour samples and other tissues from the same 16 animals were tested for the presence of jaagsiekte retrovirus (JSRV) by a polymerase chain reaction (PCR) that amplified a portion of the U3 long terminal repeat. The differences in the gross appearance of the classical and atypical forms paralleled the histopathological differences. The latter mainly concerned the stroma of the tumours which in the atypical cases was more heavily infiltrated by inflammatory cells and connective tissue fibres. JSRV major capsid protein was detected immunohistochemically in the epithelial transformed cells of both classical and atypical tumours, but the immune reactivity was slightly milder in atypical SPA. Proviral U3 sequences of JSRV were detected by specific PCR in all the tumour samples. Furthermore, the sequences of amplimers obtained from the two different pathological forms of the tumour were very similar. However, the dissemination of JSRV to other organs was greater in sheep with classical SPA than in those with atypical SPA. The pathological and virological features of these two forms of tumour are compared in an attempt to clarify whether classical and atypical SPA are two separate diseases or different expressions of a single disease spectrum.

Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.

The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses.

Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis.

Enzootic nasal tumour (ENT) and sheep pulmonary adenomatosis (SPA) are two contagious adenocarcinomas of the respiratory tract of sheep and goats. Both diseases are associated with related, but distinct, type-D-retroviruses (ENTV and JSRV respectively). No evidence of circulating antibodies has been described in animals affected by either ENT or SPA using antigens from natural sources. We evaluated the usefulness of a recombinant JSRV capsid protein (JSRV-CA) as antigen to study the antibody responses of animals naturally affected by ENT or SPA, using immunoblotting. Positive reactions were detected in the sera of both affected and unaffected sheep and goats. The reactivity was abolished completely by absorption with the GST fusion partner but not by JSRV-CA, suggesting that it was not specific. The results support prior observations indicating that sheep and goats infected by JSRV and ENTV do not develop specific humoral responses to these retroviruses.

PCR-based detection and partial characterization of a retrovirus associated with contagious intranasal tumors of sheep and goats.

A type D-related retrovirus has been demonstrated in enzootic nasal tumors (ENTs) of sheep and goats. This retrovirus, ENT virus (ENTV), has antigenic cross-reactivity with the jaagsiekte sheep retrovirus (JSRV), which is associated with a contagious lung tumor of sheep (sheep pulmonary adenomatosis). Here, we present the first report of nucleic acid sequence from ENTV which confirms, at the nucleic acid level, that this retrovirus is related to JSRV yet apparently distinct from it. Reverse transcription-PCR followed by restriction enzyme digestion specifically identified ENTV. By this technique, ENTV was demonstrated exclusively in tumor tissues and exudates of animals with ENT. Thus, there is a unique and consistent association between ENT and the retrovirus, just as there is between JSRV and sheep pulmonary adenomatosis. This gives further weight to the hypothesis that these retroviruses are the etiologic agents of the tumors.

Experimental transmission of enzootic intranasal tumors of goats.

The successful experimental transmission of enzootic intranasal tumor (EIT) from goat to goat is described. Ten kids, less than 48 hours old, from a flock free of the disease and seronegative for ruminant lentiviruses were inoculated intranasally or intrasinusally with either nasal fluid from goats with naturally occurring EIT or EIT retrovirus concentrated from such fluids. EIT was induced in three kids after 12-24 months. The EIT retrovirus was demonstrated in tumor material from each of the three kids by western blotting and electron microscopy. All kids were seronegative for ruminant lentiviruses.