Infection with Foamy Virus in Wild Ruminants-Evidence for a New Virus Reservoir?

Foamy viruses (FVs) are widely distributed and infect many animal species including non-human primates, horses, cattle, and cats. Several reports also suggest that other species can be FV hosts. Since most of such studies involved livestock or companion animals, we aimed to test blood samples from wild ruminants for the presence of FV-specific antibodies and, subsequently, genetic material. Out of 269 serum samples tested by ELISA with the bovine foamy virus (BFV) Gag and Bet antigens, 23 sera showed increased reactivity to at least one of them. High reactive sera represented 30% of bison samples and 7.5% of deer specimens. Eleven of the ELISA-positives were also strongly positive in immunoblot analyses. The peripheral blood DNA of seroreactive animals was tested by semi-nested PCR. The specific 275 bp fragment of the pol gene was amplified only in one sample collected from a red deer and the analysis of its sequence showed the highest homology for European BFV isolates. Such results may suggest the existence of a new FV reservoir in bison as well as in deer populations. Whether the origin of such infections stems from a new FV or is the result of BFV inter-species transmission remains to be clarified.

EPIDEMIOLOGY OF SCHMALLENBERG VIRUS IN EUROPEAN BISON ( BISON BONASUS) IN POLAND.

Schmallenberg virus (SBV), an emerging arbovirus in Europe, is an important pathogen in domestic ruminants; however, its impact on free-ranging wild ruminants is not well studied. Three hundred and forty-seven serum samples collected between 2011 and 2016 from 302 European bison ( Bison bonasus) from 12 different sites in Poland were tested for the presence of SBV antibodies. In addition, 86 sera were collected between 2013 and 2016 from three species of cervids for testing for SBV antibodies. After the first detection of the virus in Poland in October 2012, the proportion of SBV-seropositive European bison reached 81% (95% confidence interval [CI]: 77.1-85.8%), whereas in cervids seroprevalence was 34% (95% CI: 23.5-43.9%). There was an increase in seroprevalence in European bison from 2012 to 2014. Biting midges ( Culicoides spp.), the primary vectors of SBV, were monitored entomologically for the identification of the biting midge populations and virologically for SBV infections in the Bialowieza Forest region, which contains the world's largest European bison population. We detected SBV by PCR in 3% of Culicoides pools from 2015. In addition, seven fetal brain samples from European bison or cervids were tested and were negative for SBV RNA. Our results indicate a high seroprevalence with reduced transmission of SBV in subsequent years in the European bison populations and lower seroprevalence in cervids.

Cross-sectional study of Schmallenberg virus seroprevalence in wild ruminants in Poland at the end of the vector season of 2013.

BACKGROUND: In view of recurrent Schmallenberg virus (SBV) infections all over Europe between 2011 and 2013, a lively scientific debate over the importance of the sylvatic transmission cycle of the virus has emerged. The study presents results of serosurvey which included wild ruminants representing species of red deer (Cervus elaphus), roe deer (Capreolus capreolus), European bison (Bison bonasus), fallow deer (Dama dama), mouflon (Ovis orientalis musimon) hunted or immobilized at 34 different locations of Poland in the autumn/winter 2013. RESULTS: Out of 580 sera, 145 (25%) were considered positive for SBV antibodies. The overall SBV seroprevalence calculated using district probability weights was estimated at 27.7% (95% CI: 24.0-31.4). The seroprevalences at the district level varied between 0 and 80.0% (95% CI: 24.5-135.0%) with the mean within-district prevalence of 24.0% (95% CI: 16.5-31.4). Significantly higher seroprevalence was observed in animals from the Eastern provinces (36.6%) compared to the Western provinces (22.8%). SBV infection impact varied significantly between different species (higher SBV seroprevalence in larger species such as European bison), population type (free-ranging; captive), age, body weight, percent of the district forest area, part of Poland, and the densities of wild and domestic ruminants at the district and province level. Using statistical multivariable logistic model, population type, age, part of Poland and domestic ruminant density were identified as the main risk factors for SBV infection in wild ruminants in Poland. CONCLUSIONS: SBV seroprevalence in wild ruminants, similarly to the epizootic situation in domestic ruminants in the country, varied significantly between districts and provinces. Association between SBV seropositivity, species, animal body weight and age group expressed by a higher prevalence in larger ruminants may be explained by more frequent exposure to midge-vector bites of the latter, however it might also be related to the different species susceptibility to SBV infection. The positive effect of higher domestic ruminant density on the risk of SBV infection in wildlife and lower SBV seroprevalences in the latter suggested that the sylvatic cycle of SBV transmission is an effect of the pathogen spillover from the domestic animals.

Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2.

Malignant catarrhal fever (MCF), due to ovine herpesvirus 2 (OvHV-2), causes appreciable death loss in ranched bison (Bison bison) throughout North America. No vaccine exists to protect animals from disease. Since OvHV-2 has not been propagated in vitro, one strategy to develop a modified live vaccine is to use a closely related, non-pathogenic member of the malignant catarrhal fever virus family as a vector expressing potentially protective OvHV-2 epitopes. To date, no controlled experimental challenge studies with alcelaphine herpesvirus 2 (AlHV-2) derived from topi (Damaliscus lunatus jimela) have been reported The unique or light DNA segment of the AlHV-2 genome was sequenced and annotated and the virus was tested for its ability to infect and induce disease in American bison. Yearling bison were inoculated intranasally (n=4) or intramuscularly (n=3) with 2 × 10(-4.7) TCID50 of AlHV-2, and monitored for infection and the development of disease. Six inoculated bison became infected with AlHV-2. Two of the six animals developed clinical signs and had gross and histological lesions consistent with terminal MCF, which differed in distribution from those in bison with MCF due to OvHV-2. One other animal developed minor clinical signs and had gross and histological pulmonary lesions consistent with early (pre-clinical) stages of MCF. Unmodified low cell culture passage AlHV-2 derived from topi is an unsuitable vaccine vector for the prevention of MCF. However, the annotated genome might be useful in identifying genes which could be deleted to potentially attenuate the virus for bison.

Fibroblasts express OvHV-2 capsid protein in vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever.

American bison (Bison bison) are particularly susceptible to developing fatal sheep-associated malignant catarrhal fever (SA-MCF) caused by ovine herpesvirus-2 (OvHV-2), a γ-herpesvirus in the Macavirus genus. This generally fatal disease is characterized by lymphoproliferation, vasculitis, and mucosal ulceration in American bison, domestic cattle (Bos taurus), and other clinically susceptible species which are considered non-adapted, dead-end hosts. The pathogenesis and cellular tropism of OvHV-2 infection have not been fully defined. An earlier study detected OvHV-2 open reading frame 25 (ORF25) transcripts encoding the viral major capsid protein in tissues of bison with SA-MCF, and levels of viral transcript expression positively correlated with lesion severity. To further define the cellular tropism and replication of OvHV-2 infection in vascular lesions of bison, immunofluorescence studies were performed to identify cell type(s) expressing ORF25 protein within tissues. Cytoplasmic and not nuclear ORF25 protein was demonstrated in predominantly perivascular fibroblasts in six bison with experimentally-induced SA-MCF, and there was no evidence of immunoreactivity in vascular endothelium, smooth muscle, or infiltrating leukocytes. The cytoplasmic distribution of viral major capsid protein suggests that viral replication in perivascular fibroblasts may be abortive in this dead-end host. These findings provide a novel foundation for defining the pathogenesis of vasculitis in non-adapted hosts with SA-MCF.

CD8(+)/perforin(+)/WC1(-) gammadelta T cells, not CD8(+) alphabeta T cells, infiltrate vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever.

Sheep-associated malignant catarrhal fever (SA-MCF) caused by ovine herpesvirus-2 (OvHV-2), a gamma-herpesvirus in the Macavirus genus, is a fatal disease associated with lymphoproliferation, lymphocytic vasculitis, and mucosal ulceration in clinically susceptible species. SA-MCF is an important threat to American bison (Bison bison) due to their high susceptibility to this disease. Currently, the pathogenesis of disease in SA-MCF is poorly understood, and the immunophenotype of lymphocytes that infiltrate the vascular lesions of bison and cattle with SA-MCF has been only partially defined. Previous single-color immunohistochemistry studies have demonstrated that CD8(+) cells and CD4(+) cells predominate within vascular infiltrates in cattle and bison. The CD8(+) cells detected in the vascular lesions of cattle and bison were assumed to be cytotoxic alphabeta T lymphocytes. However, polychromatic immunophenotyping analyses in this study showed that CD8(+)/perforin(+) gammadelta T cells, CD4(+)/perforin(-) alphabeta T cells, and B cells infiltrate vascular lesions in the urinary bladder, kidney, and liver of six bison with experimentally-induced SA-MCF. CD8(+) alphabeta T cells and WC1(+) gammadelta T cell cells were only infrequently and inconsistently identified. This study confirmed our hypothesis that the predominant CD8(+) lymphocytes infiltrating the vascular lesions of bison with SA-MCF are cytotoxic lymphocytes of the innate immune system, not CD8(+) alphabeta T cells. Results of the present study support the previous suggestions that MCF is fundamentally a disease of immune dysregulation.

DNA genotyping suggests that recent brucellosis outbreaks in the Greater Yellowstone Area originated from elk.

Identifying the source of infectious disease outbreaks is difficult, especially for pathogens that infect multiple wildlife species. Brucella spp. are among the most problematic zoonotic agents worldwide, and they are notoriously difficult to detect and identify. We genotyped 10 variable number of tandem repeat (VNTR) DNA loci in 56 Brucella abortus isolates from bison (Bos bison), elk (Cervus elaphus), and cattle (Bos taurus) to test the wildlife species most likely to be the origin of recent outbreaks of brucellosis in cattle in the Greater Yellowstone Area. Isolates from cattle and elk were nearly identical but highly divergent from bison isolates. These data suggest elk, not bison, are the reservoir species of origin for these cattle infections. This study illustrates the potential power of VNTR genotyping to assess the origin of disease outbreaks, which are increasing worldwide following habitat fragmentation, climate change, and expansion of human and livestock populations.

Malignant catarrhal fever in a captive American bison (Bison bison) in Italy.

Malignant catarrhal fever (MCF) is a fatal, systemic disease of cattle and other domestic and wild ruminants that, in Europe, is caused by Ovine herpesvirus 2 (OvHV-2). American bison (Bison bison) are highly susceptible to the disease. An adult American bison, housed in a zoo in southern Italy in close cohabitation with a group of domestic sheep (Ovis aries aries) displayed clinical signs that resembled the acute form of MCF. By real-time polymerase chain reaction, OvHV-2 DNA was detected intravitam in blood, in nasal and ocular swabs, and postmortem in tissue samples of the bison. By indirect fluorescent antibody test, high MCF antibody titers were found in the bison serum. Ovine herpesvirus 2 DNA and antibodies were also found in blood samples from the domestic sheep, thus suggesting a potential role of these animals as a source of the infection. To the authors' knowledge, this is the first MCF case in captive ruminants in Italy and the second confirmed case in captive bison of European zoos.

Long distance spread of malignant catarrhal fever virus from feedlot lambs to ranch bison.

Malignant catarrhal fever (MCF) caused by OvHV-2 occurred in ranch bison herds separated by significant distances from feedlot lambs. Mortality rates correlated with distances: 17.5%, 6.1%, and 0.43% at approximately 1.6, 4.2, and 5.1 km, respectively. The study further defines the importance of distance of species separation for MCF control.

Intra-nasal inoculation of American bison (Bison bison) with ovine herpesvirus-2 (OvHV-2) reliably reproduces malignant catarrhal fever.

Sheep-associated malignant catarrhal fever (MCF) due to infection with ovine herpesvirus 2 (OvHV-2) is common in commercial herds of American bison ( Bison bison). Inability to propagate OvHV-2 in vitro has been a constraint on experimental studies of the disease. We sought to establish whether nasal secretions from sheep that shed OvHV-2 might induce the disease in bison and to define a minimum challenge dose. Fourteen bison were nebulized with sheep nasal sections containing 10(3)-10(7) OvHV-2 deoxyribonucleic acid (DNA) copies. Most challenged bison (11/14, 78.6%) developed clinical signs at 29-52 days postnebulization (DPN). The mean incubation time was 42.18 (+/-7.33 SD) DPN. Using real-time polymerase chain reaction, we detected OvHV-2 DNA in peripheral blood leukocytes at 21-31 DPN. All bison that developed MCF had antibodies against the MCF group viruses. Gross and histologic lesions were typical of the acute disease. There was no morphologic evidence of a dose-related difference in the severity or distribution of lesions. This is the first successful reproduction of MCF in bison using a nasal route of exposure. Experimentally challenged bison are more susceptible to MCF, compared with experimentally challenged domestic cattle in a previous experiment. Bison are a pertinent ruminant species in which the pathogenesis of the disease can be investigated.

Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

Seroprevalence of selected viral and bacterial pathogens in free-ranging European bison from the Bialowieza Primeval Forest (Poland).

Blood was collected from bison that were selected between 1991 and 2001 for poor body condition, cachexia, lameness and balanoposthitis. Sera were tested for antibodies to bovine herpes virus type 1 (BHV-1), bovine viral diarrhoea virus (BVDV), foot-and-mouth disease virus (FMDV), parainfluenza virus type 3 (PI-3), Brucella abortus, Chlamydophila abortus, Coxiella burnetti, and Leptospira interrogans. The results of serological tests showed a prevalence of low titers of a serological reaction to Chlamydophila abortus (45.1%), bovine viral diarrhoea virus (29.5%), Leptospira interrogans (21.3%) and parainfluenza virus type 3 (13.9%). There were differences in the prevalence of antibodies to Ch. abortus between female and male bison. Futhermore, a relationship between age and antibodies to BVDV was observed in older bison. These results suggest that there is some transmission of bacterial and viral pathogens occurring between domestic and wild ruminants grazing in the same pastures in the peripherial areas of Bialowieza Primeval Forest.

Complete genomes and phylogenetic positions of bovine papillomavirus type 8 and a variant type from a European bison.

The DNA of bovine papillomavirus (BPV) type 8 was extracted from papillomas on cattle kept in Japan, and DNA of bovine papillomavirus BPV-8-EB was extracted from a European bison (Bison bonasus) born in Italy and released into the wild in Slovakia. The DNA genomes of these BPVs were amplified using multiply primed rolling circle amplification and polymerase chain reaction, then characterized by direct sequencing method. The BPV-8 and BPV-8-EB genomes consisted of 7,791 base pairs (bp) and 7,773 bp, respectively (GenBank accession numbers DQ098913 and DQ098917). The nucleotide sequence similarity of these BPVs indicated that BPV-8-EB was a variant of BPV-8. In the genome of BPV-8-EB, one nucleotide substitution was found in the E2 and E5 open reading frame (ORF) and upstream regulatory region (URR), and a short deletion and addition were found in the URR. The high similarity of sequences between the BPV-8 to BPV-5 in total genome (70%) and L1 ORF (75%) as well as a phylogenetic analysis were the bases for classifying BPV-8 in the genus Epsilon papillomavirus. The BPV-8 E6 and E7 ORFs/proteins also showed some characteristic features of genus Epsilon papillomavirus. However, BPV-8 contained E4 ORF, which was not found in BPV-5. In addition, the secondary structure of E5 proteins of BPV-5 and BPV-8 suggested that these proteins may have cell-transforming ability.

Papillomatosis in a European bison.

Five European bison (Bison bonasus) from three European zoos were shipped to the Bukovské Vrchy Hills (Slovakia) in June 2004 and kept together in an acclimatization enclosure. The European bison were released into the wild in December 2004. At that time, papillomas were found at the medial canthus of the left eye of a 12-yr-old female bison. Cutaneous papillomatosis was confirmed histologically. Negative stain transmission electron microscopic examination revealed papillomavirus in the papillomas, and papillomavirus DNA also was detected using the polymerase chain reaction with FAP59 and FAP64 primers. The amplified 413 bp DNA sequence was identical to that of BAPV2 bovine papillomavirus. This paper is the first report of papillomatosis in European bison.

An outbreak of sheep-associated malignant catarrhal fever in bison (Bison bison) after exposure to sheep at a public auction sale.

An outbreak of malignant catarrhal fever (MCF) among bison sold at an auction market was studied for an 18-month period. Forty-five of 163 bison submitted for sale from 8 different bison farms died on 7 other destination farms. The outbreak began on day 50 after the sale, peaked between days 60 and 70, and ended on day 220. Twenty-one dead bison were confirmed to be MCF cases by clinical histories, pathology, and detection of ovine herpesvirus-2 DNA in their tissues with polymerase chain reaction assays. Twenty-four dead bison were classified as suspect MCF cases from clinical histories. No cases of MCF were observed among bison remaining on originating farms or resident bison mixed with sale bison on destination farms. There were no sheep reported within 3 km of originating or destination farms, limiting bison exposure to sheep to the auction facility, where sheep were present for less than 1 day. The outbreak provides an illustration of the temporal distribution of MCF mortality expected in bison and an estimate of the time from exposure until death from MCF after a single short exposure to sheep. The study provides evidence that bison with MCF do not transmit MCF to other bison.

Virologic investigations of free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland.

We conducted virologic investigations on postmortem specimens from 261 free-living European bison (Bison bonasus) from the Bialowieza Primeval Forest, Poland collected between 1990 and 2000. Fifty-four of 94 males had balanoposthitis; none of the 167 female bison examined had reproductive tract lesions. Peripheral blood, swabs, and various tissues were analyzed for bovine viruses as well as for viral DNA by bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 4 (BoHV-4) specific polymerase chain reaction (PCR) technique. An infectious bovine rhinotracheitis like BoHV-1 strain was isolated from the spleen of a female bison calf and additionally was detected by nested PCR from splenic tissue. None of the bison had significant antibody titers against BoHV-1, bovine herpesvirus 2, BoHV-4, caprine herpesvirus 1, cervid herpesvirus 1, or bovine viral diarrhea (BVD) virus-1. However, low antibody titers in two animals indicate that this European bison population has been exposed to BVD virus or BVD-like viruses and BoHV-2.

Malignant catarrhal fever in a bison (Bison bison) feedlot, 1993-2000.

A fatal enteric syndrome was identified in American bison (Bison bison) at a large feedlot in the American Midwest in early 1998. An estimated 150 bison died of the syndrome between January 1998 and December 1999. The syndrome was identified as malignant catarrhal fever (MCF), primarily the alimentary form. Clinical onset was acute, and most affected bison died within 1-3 days; none recovered. Consistent lesions were hemorrhagic cystitis, ulcerative enterotyphlocolitis, and arteritis-phlebitis. Vasculitis was milder and more localized than that in cattle with MCF, and in contrast to the situation in cattle, lymphadenomegaly was minimal. Virtually all affected bison examined were positive for ovine herpesvirus 2 (OvHV-2) by polymerase chain reaction (PCR) assay. A retrospective study of archived tissues established that MCF occurred in the yard as early as 1993. A prospective study was undertaken to establish the importance of MCF relative to other fatal diseases at the feedlot. The fate of a group of 300 healthy male bison in a consignment of 1,101 animals was followed for up to 7 months to slaughter. At entry, 23% (71/300) of bison were seropositive for MCF viruses, and 11% (8/71) of these seropositive bison were PCR positive for OvHV-2. Forty seronegative bison were selected at random from the group, and all were PCR negative for OvHV-2. There was no change in seroprevalence in the group during the investigation. The minimum infection rate for MCF virus was 36.3% (93/256). Twenty-two (7.3%) of the 300 bison in the feedlot died. Of these, 15 had MCF, 4 had acute or chronic pneumonia, and 3 were unexamined. Losses in the entire consignment were higher (98/1,101; 8.8% death loss); 76% of deaths were attributable to MCF. The study failed to reveal a relationship between subclinical infection and development of clinical disease.

Polymerase chain reaction and DNA sequencing for detection of ovine herpesvirus 2 in American bison (Bison bison).

Serum samples were collected at slaughter from 226 24-30-month-old ranch-raised, clinically normal American bison (Bison bison) bulls from North Dakota, Minnesota, Kansas, and Manitoba to assess the presence of antibodies to ovine herpesvirus 2 (OHV-2). Antibodies to OHV-2 were detected by competitive inhibition enzyme-linked immunosorbent assay in 10 of 226 (4.40%) samples. Polymerase chain reaction (PCR) analysis of sera positive for OHV-2 DNA demonstrated a 238 kilobase fragment. The nucleotide sequence of the PCR-positive samples in comparison to the reported OHV-2 nucleotide sequence resulted in a homology range of 82.8-95.4%.