Retrospective study of pestivirus infection in Pyrenean chamois (Rupicapra pyrenaica) and other ungulates in the Pyrenees (NE Spain)

In 2001 a new Pestivirus (Family Flaviviridae) was associated with an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in the Pyrenees (NE Spain). Molecular characterization assigned this virus to the Border Disease Virus (BDV) cluster, BDV-4 genotype. A retrospective study was performed in archived sera and spleen of 74 Pyrenean chamois and in archived sera of 28 mouflon (Ovis ammon), 56 red deer (Cervus elaphus), 43 roe deer (Capreolus capreolus) and 29 fallow deer (Dama dama) from the Pyrenees between the years 1990 and 2000. Thirty six of 74 (48.6%) sera of Pyrenean chamois, one of mouflon and one of red deer were positive by an ELISA antibody test. Comparative virus neutralization tests were performed on 26 seropositive chamois, one mouflon and one red deer, using five pestivirus strains. An ELISA antigen test was performed on 37 seronegative chamois and yielded positive results in one chamois and inconclusive result in two. RT-PCR and virus isolation performed on spleen samples from these three animals gave positive results in the positive and one inconclusive animal. Sequence analysis in the 5' unstranslated region revealed that they were grouped into the BDV-4 genotype. Virological and serological data of the present study indicate that BDV infection has been present in the chamois population since at least 1990, 11 years before the first outbreak of disease. Therefore, the emergence of the disease in 2001 is apparently due to other factors rather than the introduction of a new virus in the chamois population.     

Epidemiological study of border disease virus infection in Southern chamois (Rupicapra pyrenaica) after an outbreak of disease in the Pyrenees (NE Spain)

In 2001 and 2002, an outbreak of a previously unreported disease, associated with a border disease virus (BDV), caused high mortality in the Southern chamois (Rupicapra pyrenaica) population in the Alt Pallars-Aran National Hunting Reserve in the Catalan Pyrenees (NE Spain). Between 2002 and 2006, sera and/or tissue samples taken from 116 healthy chamois shot during the hunting season, plus 42 from chamois affected by different diseases, were studied. A blocking enzyme-immunosorbent assay (ELISA) was used to study pestivirus seroprevalence in 114 healthy hunted and 31 diseased chamois, yielding positive results in 73.7 and 22.6% of the chamois, respectively. Comparative virus neutralization tests (VNT) performed on 42 seropositive samples with 6 pestivirus strains yielded statistically higher titres to BDV Spain 97, followed by BDV chamois, BDV 137/4, BDV Moredun, Bovine Diarrhoea virus-1 (BVDV-1) NADL and BVDV-2 atypical. Virological investigations for pestivirus detection were performed using an antigen ELISA test in 82 healthy and 18 diseased chamois, RT-PCR in 16 healthy and in all diseased chamois, and virus isolation in 14 diseased chamois. No viral antigen was detected in any of the healthy animals. A pestivirus, characterized as BDV by monoclonal antibodies, was detected in the 10 chamois showing clinical signs consistent with BDV infection. Sequence analysis in the 5' untranslated region (5'-UTR) revealed that they were grouped into the BDV-4 genotype. In the remaining chamois, infectious keratoconjunctivitis, pneumonia, trauma and contagious ecthyma were diagnosed. The cause of death was unknown in five chamois. The results suggest that the infection has become endemic in the population and that it could have a significant impact on chamois population dynamics.     

Identification and molecular characterization of border disease virus (BDV) from sheep in India

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5′-UTR, N^pro and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N^pro and entire region coding structural proteins showed that the N^pro (168), C (100 aa), E^rns (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.     

Heterogeneity of ruminant pestiviruses: academic interest or important basis for the development of vaccines and diagnostics?

Pestiviruses cause economically important diseases of farm animals. Members of the Pestiviruses are bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, classical swine fever virus (CSFV) and border disease virus (BDV). Phylogenetic analyses based on the entire nucleic acid sequence encoding the Npro allow a statistically significant segregation of established species and of subgroups within the species. BVDV-1 strains isolated in Germany can be associated with at least five different subgroups. In contrast all BVDV-2 isolates detected in Germany so far are closely related, belonging to one subgroup. A group of virus isolates from sheep and zoo animals is clearly different from established pestivirus species and can be designated as BDV-2. Antigenetic relatedness of pestiviruses was studied using defined virus isolates and antisera in cross-neutralization assays. Six antigenic groups were distinguished corresponding to the genetic clusters BVDV-1, BVDV-2, CSFV, BDV-1, BDV-2 and Giraffe-1. A significant antigenic difference was also observed between members of subgroups 1a and 1b of BVDV-1. Studies on the genetic and antigenic heterogeneity of pestiviruses are important for the development of new vaccines, diagnostic tests and for eradication programs.     

Age-specific seroprevalence of Border disease virus and presence of persistently infected sheep in Basque dairy-sheep flocks

Using p125/p80 antibody and antigen-ELISA tests, age-specific seroprevalence and presence of persistently infected (PI) sheep were investigated in six commercial latxa dairy-flocks, housed for variable periods. The flocks all had a recent history of Border disease (BD). Every flock included seropositive sheep and seven 0.5-3-year-old PI sheep were detected in two of four flocks tested. Age-specific antibody patterns differed according to the presence or absence of PI sheep in the flock. In flocks free of PI sheep, seroprevalence was 6-13% in 1-year-old sheep and 42-93% in older sheep. In contrast, seroprevalence was 67-99% in sheep raised with PI sheep for at least 1 year and 29-33% in replacement 0.5-0.6-year-old sheep (including a PI sheep) indicating that Border disease virus (BDV) transmission in Basque dairy-flocks can be relatively slow. Moderate seroprevalence in young replacement sheep should not discourage further testing to detect PI sheep, and our results highlight the risk of failing to achieve "natural vaccination" prior to pregnancy by mixing PI sheep with BDV-unexposed ewes.     

Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm.]

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.     

Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: identification of a new subgroup in BVDV-1

Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete N(pro) encoding nucleotide sequences. A total of 30 virus isolates obtained from 15 provinces around the country between 1997 and 2005 were included in the phylogenetic analysis. Virus isolates mostly originated from cattle with one isolate from sheep. The bovine isolates all belonged to BVDV-1, the sheep isolate to BVDV-2. Fifteen isolates formed a new subgroup within BVDV-1, tentatively named BVDV-1l. The remaining bovine isolates were typed as BVDV-1a (n=4), BVDV-1b (n=4), BVDV-1d (n=3), BVDV-1f (n=2) and BVDV-1h (n=1). The isolates allocated to BVDV-1l originated from various geographical regions in different years. There was no correlation between genetic grouping and locations where isolates were obtained. Viruses originating from one farm in most cases belonged to the same subgroup (n=5). This study indicates that the newly detected subgroup BVDV-1l is predominant and widespread in Turkey. Moreover, an ovine virus isolate was identified as the first member of BVDV-2 reported in Turkey. A serological survey using samples from western Turkey indicated that BVDV-2 is also present in cattle.     

Haematology and serum chemistry of Pyrenean chamois (Rupicapra pyrenaica) naturally infected with a border disease virus

In 2005 and 2006 an outbreak of disease associated with border disease virus (BDV) infection caused high mortality in the Pyrenean chamois (Rupicapra pyrenaica) in the Catalan Pyrenees (NE Spain). The aim of this study was to determine values for different haematological and serum biochemical analytes in 32 free-ranging Pyrenean chamois affected by the disease and to compare them with those obtained from healthy chamois.In the affected chamois red blood cell counts, haemoglobin concentrations, packed cell volumes, mean corpuscular volumes and lymphocyte counts were all lower, while the neutrophil and platelet counts were higher. Glucose, lactate, triglycerides, creatinine, total protein concentrations and alkaline phosphatase activity were also lower, in contrast to the concentrations of total bilirubin, urea and aspartate aminotransferase activity, which were higher.Most of the observed changes could be associated with cachexia and inflammation in the affected chamois. Lymphopenia could be directly related to the BDV, which would lead to immunosuppression and explain the high rate of secondary infection observed in these animals.     

Serological survey for antibodies against pestiviruses in sheep in Austria

The prevalence of antibodies to pestiviruses was investigated in 4931 sheep, in 377 flocks, in four federal states of Austria, by means of an indirect elisa that detected antibodies to Border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The mean flock prevalence was 62.9 per cent and the mean individual prevalence was 29.4 per cent. Comparative neutralisation studies on the elisa-positive samples with BVDV type 1 (BVDV-1), BVDV type 2 (BVDV-2) and BDV recorded 336 samples with higher titres (more than four times average) to BVDV-1, three samples with higher titres to BVDV-2 and 55 samples with higher titres to BDV. The other samples did not show clear differences in antibody titres against the strains of pestivirus tested because of cross-reactions. The seroprevalence of pestiviruses in sheep was significantly higher on farms with cattle. There were significant regional differences between the prevalences in flocks and individual sheep, the highest prevalences being in the region of Austria where communal alpine pasturing of sheep, goats and cattle is an important part of farming.     

Toxoplasmosis and border disease in 54 Swedish sheep flocks. Seroprevalence and incidence during one gestation period.

Serum samples from 704 animals from 54 Swedish sheep flocks were analysed by ELISA twice during 1 breeding season for antibodies to Toxoplasma gondii and border disease virus (BDV). An ELISA, originally developed for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle, was assessed on sheep sera and the results were compared with those obtained in a virus neutralization test. The correlation between the 2 assays proved good. Before breeding, 132 (19%) sheep in 42 flocks had antibodies to T. gondii and 7 (1%) sheep in 5 flocks were seropositive to BDV. During the observation period 4 sheep seroconverted to T. gondii and 13 to BDV, giving an incidence rate of 0.7% and 1.9% respectively. No clinical signs due to the infections were observed. In 5 flocks the frequency of barrenness, abortion or stillbirths exceeded 5%, 5% and 8%, respectively, but there was no evidence that this was attributable to the agents studied. The proportion of BDV-positive flocks was significantly higher among flocks that had been in contact with cattle than among those that had not.     

Flock-prevalence of border disease virus infection in Basque dairy-sheep estimated by bulk-tank milk analysis

Bulk-tank milk (BTM) samples from 154 sheep flocks were used to estimate BDV prevalence in the Basque Country in Spain using an ELISA and a RT-PCR test. The proportion of antibody-positive flocks was 68% but varied significantly between provinces and was 93% in Araba and 54-55% in Bizkaia and Gipuzkoa. Most ELISA-positive flocks had very low antibody inhibition percentage (AIP) indicating high seroprevalence and recent BDV exposure. However, only 9% flocks were PCR-positive suggesting few infected ewes were being milked at the time of sampling. Phylogenetic analysis of the 5' NCR sequences of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study in the region, whereas the remaining isolate was closest to BDV type A. These results suggest that BDV strains in most Basque flocks have a common origin and differences in prevalence between provinces are associated to recent events affecting BDV spread such as use of communal pastures and sheep trading. The widespread distribution of BDV in the region, advocates for the implementation of BDV control strategies and highlights the potential risk of sheep as a pestivirus reservoir for other species.     

Prevalence of border disease virus in Spanish lambs

The prevalence of border disease virus (BDV) viraemia in Spanish lambs was determined from 2089 sera randomly collected at two slaughterhouses in 2001 and 2003, as well as in 126 sera obtained in 2004 from a fattening unit with an acute disease problem. BDV was detected with an indirect peroxidase monolayer assay (IPMA), and for the fattening unit sera also by an antigen ELISA. A subset of sera was additionally tested for BDV antibodies. The BDV prevalence in the slaughterhouse sera was 0.24%, whereas 7.1% of randomly selected and 38.6% of sera from clinically affected lambs in the fattening unit were virus positive. Pestivirus antibodies were found in 17.6% of the slaughterhouse sera and 28.6% of those from randomly selected lambs in the fattening unit. In total, 33 virus isolates and 3 antigen positive samples were identified. Genetic typing of 5'-UTR sequences classified all 36 pestiviruses as of BDV type 4. This shows that from a low BDV prevalence in apparently healthy lambs in the entire sheep population, clinical problems associated with BDV can develop when viraemic sheep are brought into intense rearing units.     

Effects of sarcoptic mange on serum proteins and immunoglobulin G levels in chamois (Rupicapra pyrenaica) and Spanish ibex (Capra pyrenaica)

Three groups of chamois (Rupicapra pyrenaica) and three groups of Spanish ibex (Capra pyrenaica) were established to study the effects of sarcoptic mange on serum proteins and immunoglobulin G (IgG) levels. The first group of chamois consisted of 22 healthy Pyrenean chamois (R. pyrenaica pyrenaica) from a non-infested area, the second group consisted of 20 healthy Cantabrian chamois (R. p. parva) from an area where sarcoptic mange has been reported since 1994 and the third group consisted of 16 Cantabrian chamois from the same area but naturally infested by Sarcoptes scabiei. The first group of Spanish ibex was 39 healthy animals from a sarcoptic mange non-infested area, the second group was 23 healthy animals from a sarcoptic mange infested area and the third group consisted of 20 animals from the same area but naturally infested with the parasite. Blood samples were taken after killing the animals as part of hunting programmes. Values for total proteins, gamma-globulin and IgG were higher in infested and healthy chamois from the infested area compared to healthy chamois from the non-infested area, and IgG levels were higher in infested chamois compared to healthy-exposed chamois. Values for alpha2-globulin were higher in healthy Cantabrian chamois. In Spanish ibex, albumin, alpha2-globulin and IgG levels were lower in the healthy Spanish ibex from the non-infested area than in healthy animals from an infested area. The differences found in the chamois were indicative of the establishment of a humoral antibody response in the animals in contact with the disease. As the IgG levels were not significantly different between healthy and infested Spanish ibex from the same area, a different pattern of chronic infection with humoral response to the disease was suggested.     

Pestivirus and alphaherpesvirus infections in Swedish reindeer (Rangifer tarandus tarandus L.)

Herding semi-domesticated reindeer has economic and social value for Sami people in the northern territories of Fennoscandia. However, with the intensification of reindeer husbandry, interspecies transmission of pathogens between reindeer and domestic animals may become a problem, especially for countries such as Sweden, Norway, and Finland where pestivirus and alphaherpesvirus have been eradicated in domestic ruminants. This study, which included 1158 Swedish reindeer, showed relatively high prevalence of antibodies against bovine viral diarrhoea virus (BVDV) (32%) and bovine herpesvirus-1 (BoHV-1) (53%). Adult animals were more often seropositive for BVDV and BoHV-1 (50% and 78%, respectively) than were calves (18 and 11%, respectively). While the seroprevalence of alphaherpesvirus was similar in different herding districts, pestivirus seropositivity was highest in the South and diminished towards the North of the Swedish reindeer herding area. High correlation of the seropositivity against both pathogens at both individual and herd levels may indicate possible mutual synergetic effects and may be explained by the immunosuppressive nature of the viruses. While alphaherpesvirus seroprevalence was probably related to putative cervid herpesvirus 2 (CvHV-2), the pestivirus infecting reindeer remains undefined. The virus neutralisation test of reindeer sera using different pestivirus strains, revealed higher titres against Border disease virus strains like 137/4 (BDV-1) and Reindeer-1 (BDV-2) than against BVDV-1. However, the virus was not identified by real time RT-PCR in any of the samples (n=276) from seronegative reindeers. The study showed that pestivirus and alphaherpesvirus infections are endemic in the Swedish reindeer population.     

Isolation and preliminary characterization of chicken anemia virus from chickens in Nigeria

Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.