Border disease in cattle.

Within the family Flaviviridae, viruses within the genus Pestivirus, such as Border disease virus (BDV) of sheep, can cause great economic losses in farm animals. Originally, the taxonomic classification of pestiviruses was based on the host species they were isolated from, but today, it is known that many pestiviruses exhibit a broad species tropism. This review provides an overview of BDV infection in cattle. The clinical, hematological and pathological-anatomical findings in bovines that were transiently or persistently infected with BDV largely resemble those in cattle infected with the closely related pestivirus bovine viral diarrhoea virus (BVDV). Accordingly, the diagnosis of BDV infection can be challenging, as it must be differentiated from various pestiviruses in cattle. The latter is very relevant in countries with control programs to eradicate BVDV in Bovidae, as in most circumstances, pestivirus infections in sheep, which act as reservoir for BDV, are not included in the eradication scheme. Interspecies transmission of BDV between sheep and cattle occurs regularly, but BDV in cattle appears to be of minor general importance. Nevertheless, BDV outbreaks at farm or local level can be very costly.

Insemination with border disease virus-infected semen results in seroconversion in cows but not persistent infection in fetuses.

BACKGROUND: This study examined various health variables in cows after artificial insemination with Border disease virus (BDV)-infected semen and the occurrence of persistent infection in ensuing fetuses. Five cows were inseminated (day 0) with BDV-infected semen as well as with semen from a fertile Eringer bull. One cow, inseminated with virus-free semen only, served as a control. Clinical examination, assessment of eating and rumination activities, measurement of intraruminal temperature and leukocyte count were used to monitor the health of the cows. Blood samples were collected at regular intervals for the detection of viral RNA and antibodies against BDV, and the cows were slaughtered on day 56. The uteri, placentae and fetuses were examined macroscopically, histologically, immunohistochemically and by means of molecular methods for the presence of pestiviruses. RESULTS: The demeanour, eating and rumination activities and intraruminal temperature were not affected by insemination with BDV-infected semen, whereas the total leukocyte and lymphocyte counts dropped transiently and were significantly lower on day 6 than on day 0. Seroconversion occurred by day 28 in the five infected cows but not in the control cow. The uteri, placentae and fetuses had no macroscopic or histological lesions, and immunohistochemical examination and RT-PCR were negative for pestiviruses. CONCLUSIONS: The findings showed that cows inseminated with BDV-infected semen seroconverted and fetuses thus produced were not persistently infected. Transmission of BDV to cattle through infected semen, therefore, seems to be of minor importance.

New insights on pestivirus infections in transhumant sheep and sympatric Pyrenean chamois (Rupicapra p. pyrenaica).

Border Disease Virus (BDV) causes health and economic impact on livestock and is also of importance in wildlife conservation as it causes high mortality outbreaks in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Pastoral practices are known as a main interspecies pathogen transmission. Hence, the presence of pestivirus in transhumant sheep flocks and sympatric chamois was assessed in areas with different epidemiological scenarios of chamois BDV infections. Moreover, the present study had also the goal to identify if inter-specific infections occurred and when they happened. Five sheep flocks grazing in two alpine areas in the Pyrenees with two different BDV epidemiological scenarios in chamois populations were studied during two transhumant seasons. Sheep were sampled before and after transhumance. Pyrenean chamois sera and spleen samples from both areas where also studied during the same period. Antibodies against BDV were assessed by means of ELISA and VNT. A qRT-PCR was used in order to detect the virus. Seroprevalence in sheep ranged between 0 and 91.1% at the flock level. Chamois were found to have high seroprevalences (52.9-77.7%) in both areas, and four new BDV isolates were sequenced. One sheep farm presented persistent BDV circulation and three showed low BDV circulation. The after-transhumance period was identified as the moment when viral transmission occured in the first farm, associated to BDV strains of domestic origin, according to VNT results. However, the BDV isolate was genetical closely related to previous BDV strains from chamois origin. In another farm, antibodies in two of the three positive sera were associated to infection with a chamois-like BDV strain. Altogether indicates that occasional viral transmission from chamois to sheep may occur.

Evidence of circulation of the novel border disease virus genotype 8 in chamois.

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

A novel epidemiological model to better understand and predict the observed seasonal spread of Pestivirus in Pyrenean chamois populations.

Seasonal variations in individual contacts give rise to a complex interplay between host demography and pathogen transmission. This is particularly true for wild populations, which highly depend on their natural habitat. These seasonal cycles induce variations in pathogen transmission. The seasonality of these biological processes should therefore be considered to better represent and predict pathogen spread. In this study, we sought to better understand how the seasonality of both the demography and social contacts of a mountain ungulate population impacts the spread of a pestivirus within, and the dynamics of, this population. We propose a mathematical model to represent this complex biological system. The pestivirus can be transmitted both horizontally through direct contact and vertically in utero. Vertical transmission leads to abortion or to the birth of persistently infected animals with a short life expectancy. Horizontal transmission involves a complex dynamics because of seasonal variations in contact among sexes and age classes. We performed a sensitivity analysis that identified transmission rates and disease-related mortality as key parameters. We then used data from a long-term demographic and epidemiological survey of the studied population to estimate these mostly unknown epidemiological parameters. Our model adequately represents the system dynamics, observations and model predictions showing similar seasonal patterns. We show that the virus has a significant impact on population dynamics, and that persistently infected animals play a major role in the epidemic dynamics. Modeling the seasonal dynamics allowed us to obtain realistic prediction and to identify key parameters of transmission.

The two sides of border disease in Pyrenean chamois (Rupicapra pyrenaica): silent persistence and population collapse.

In 2001, border disease virus (BDV) was identified as the cause of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in Spain. Since then, the disease has caused a dramatic decrease, and in some cases collapse, of chamois populations and has expanded to nearly the entire distribution area in the Pyrenees. Chamois BDV was characterized as BDV-4 genotype and experimental studies confirmed that it was the primary agent of the disease. The infection has become endemic in the Central and Eastern Pyrenees. However, while most Pyrenean chamois populations have been severely affected by the disease, others have not, despite the circulation of BDV in apparently healthy individuals, suggesting the existence of different viral strategies for persisting in the host population. Changes in the interplay of pathogen, host and environmental factors may lead to the formation of different disease patterns. A key factor influencing disease emergence may be pathogen invasiveness through viral mutation. Host factors, such as behavior, immunity at the population level and genetic variability, may also have driven different epidemiological scenarios. Climatic and other ecological factors may have favored secondary infections, such as pneumonia, that under particular circumstances have been major contributing factors in the high mortality observed in some areas.

Transmission of border disease virus from a persistently infected calf to seronegative heifers in early pregnancy.

BACKGROUND: This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection. RESULTS: Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three. CONCLUSIONS: Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs.

Short communication: Transmission of border disease virus to seronegative cows inseminated with infected semen.

The goal of this study was to investigate the transmissibility of border disease (BD) virus to seronegative cows via artificial insemination with cryopreserved semen from a bull persistently infected with BD virus. Five pestivirus naive cows were inseminated with BD virus-infected semen. Blood was collected for detection of pestivirus antibody by means of an ELISA on day 0 (day of insemination) and then every 7 days until day 56, at which time a serum neutralisation test (SNT) for differentiation of BD and BVD virus was carried out. Seroconversion was first noticed in two cows on day 14, in two cows on day 21 and in one cow on day 28. In the SNT, all cows had distinctly positive titres against BD virus. Therefore, BD virus is readily transmitted by infected semen, but none of the cows conceived, most likely because of poor semen quality.

Sheep persistently infected with Border disease readily transmit virus to calves seronegative to BVD virus.

Bovine viral diarrhea- and Border disease viruses of sheep belong to the highly diverse genus pestivirus of the Flaviviridae. Ruminant pestiviruses may infect a wide range of domestic and wild cloven-hooved mammals (artiodactyla). Due to its economic importance, programs to eradicate bovine viral diarrhea are a high priority in the cattle industry. By contrast, Border disease is not a target of eradication, although the Border disease virus is known to be capable of also infecting cattle. In this work, we compared single dose experimental inoculation of calves with Border disease virus with co-mingling of calves with sheep persistently infected with this virus. As indicated by seroconversion, infection was achieved only in one out of seven calves with a dose of Border disease virus that was previously shown to be successful in calves inoculated with BVD virus. By contrast, all calves kept together with persistently infected sheep readily became infected with Border disease virus. The ease of viral transmission from sheep to cattle and the antigenic similarity of bovine and ovine pestiviruses may become a problem for demonstrating freedom of BVD by serology in the cattle population.

Infection of cattle with Border disease virus by sheep on communal alpine pastures.

The purpose of this study was to investigate whether sheep grazing communal alpine pastures with cattle can transmit Border disease virus (BDV) to cattle. A total of 1170 sheep and 923 cattle were tested for BDV using RT-PCR (sheep) and for pestivirus antibodies using an ELISA (cattle), respectively, before being moved to one of 4 pastures (A, B, C and D). Eight sheep from pasture C were viraemic. 396 of 923 cattle examined before the pasture season were seronegative. The latter were re-examined after the pasture season and 99 were seropositive or indeterminate. Antibody specificity was determined in 25 of these using a serum neutralization test (SNT). BDV infection was confirmed in 10 cattle and was considered likely in 8 others. BVDV infection was confirmed in 4 cattle and considered likely in 3 after pasturing. The study has shown that the transmission of BDV from sheep to cattle is possible on communal alpine pastures.

Experimental infection of pigs with Border disease virus isolated from Pyrenean chamois (Rupicapra pyrenaica).

Between 2001 and 2007, several outbreaks of disease associated with Border disease virus (BDV) infection were reported in the central Pyrenees (northeast Spain) and were associated with a major reduction in chamois (Rupicapra pyrenaica) populations. At the same time, wild boars (Sus scrofa) from the same area were found to be seropositive to this pestivirus, without showing clinical signs. The present study examines the susceptibility of domestic swine and the course of the infection with a BDV strain isolated from naturally infected chamois. Twenty pigs were inoculated with 1 x 10(7) TCID(50) (50% tissue culture infective dose) by oronasal route, and 16 control pigs received Eagles sterile Minimal Essential Medium. Serologic (enzyme-linked immunosorbent assay and virus neutralization test) and reverse transcription polymerase chain reaction assays were performed on serum samples obtained at 0, 3, 7, 10, 14, 21, and 31 days postinoculation (dpi). All infected pigs were viremic from 3 to 14 dpi. After 14 dpi, all infected animals developed an antibody response against the homologous virus. Clinical signs or histologic lesions were not observed in inoculated pigs. The present work demonstrates the susceptibility of domestic swine to a BDV strain of chamois origin.

Seroprevalence of antibodies against pestiviruses in small ruminants in The Netherlands.

In this study, a serological survey was performed to determine the prevalence of pestivirus (bovine viral diarrhea virus (BVDV) and border disease virus (BDV)) infected small ruminants herds in the Netherlands. After random selection of sheep farms, a sample size was determined to detect a 5% herd prevalence. 13 out of 29 farms were tested seropositive using an ELISA which detects antibodies directed against the non structural protein 3 (NS3) of pestiviruses. This resulted in a seroprevalence for the Netherlands of 45% [0.36; 0.54]. The within farm prevalence ranged from 4 till 65%. Using a virus neutralization assay, specific anti-BDV antibodies could be detected on two farms, while on one other farm anti-BVDV antibodies were present. On four farms antibodies to both viruses could be detected, on three of these farms antibodies against both viruses were equally present. At five farms that tested positive in the NS3-ELISA we were unable to detect pestivirus neutralizing antibodies in all sera using the VN test. This resulted in an estimated prevalence using the VN for the Netherlands of 28% [0.20; 0.60]. An additional survey in sera from dairy goats revealed that 34 out of 126 farms were serological positive resulting in a seroprevalence of 27% [0.23; 0.31], with a herd prevalence of 32% ranging from 1-100%.

Influence of communal alpine pasturing on the spread of pestiviruses among sheep and goats in Austria: first identification of border disease virus in Austria.

The purpose of this investigation was to determine the influence of communal Alpine pasturing on the spread of pestivirus infections among sheep and goats. The study included 481 sheep from 23 farms and 131 goats from 26 farms pastured on separated Alpine meadows in the western part of Austria. At the starting of pasturing on the sheep meadow, 325 (67.6%) animals were seropositive, on the goat meadows in 16 (12.2%) samples antibodies to pestiviruses were detected. At the end of pasturing, 74 seronegative sheep and two seronegative goats had seroconverted. Between the beginning and the end of pasturing the seroprevalence in sheep increased significantly from 67.6% to 83% (P<0.05). Moreover, in the peripheral blood mononuclear cells of four sheep, pestivirus-specific RNA was detected before as well as after pasturing; these animals remained serologically negative throughout the investigation. They were, therefore, identified as persistently infected. Sequence analysis in the N(pro) region revealed that the detected pestiviruses were the same at genetic level and they were grouped into the Border disease virus (BDV)-3 genotype. No pestivirus RNA was found in goat samples. The results of this survey indicate that communal Alpine pasturing does play a key role in the spread of BDV. Moreover, BDV has been identified and characterized for the first time in sheep in Austria, which until then had been regarded as being free from BD.

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.

[Clinical aspects and diagnosis of border disease]. / Klinik und Diagnostik der Border disease.

The term Border disease (BD) refers to a clinical condition of newborn lambs that results from congenital infection by a pestivirus (family: flaviviridae), occurring during the first one-half of gestation. The most prominent clinical features are tonic-clonic tremors and hairy fleece ("hairy shaker" symptomatology). Moreover early transplacental infection often causes fetal death, abortion or stillbirth and teratogenic disorders. In Germany there are only few reports on clinical cases of BD, however in sheep antibodies have been found frequently in different regions of the country. Similar to the situation with bovine viral diarrhea/mucosal disease (BVD/MD) the existence of persistently infected sheep is proven. As permanent carriers and initiators of clinically apparent as well as more commonly clinically inapparent infections persistently infected animals are of great epidemiological importance. As a new method for the diagnosis of persistently Border disease Virus (BDV) infected sheep flow cytometry is presented. This special procedure of immunofluorescence has the overall advantage of rapid and reliable detection of pestivirus-infected cells by a highly conserved viral epitope (p 80/125). All biotypes of pestiviruses (non cytopathic = ncp and cytopathic = cp isolates) are recognized equally well by monoclonal antibodies (mAb) which provides some advantage compared to conventional techniques because most virus isolates from sheep belong to the ncp biotype.

Border disease virus transmitted to sheep and cattle by a persistently infected ewe: epidemiology and control.

In a Swedish sheep flock comprising 202 ewes and 13 rams, a pair of twin lambs born in the spring of 1990 demonstrated signs of border disease (BD) and were persistently infected (PI) with border disease virus (BDV). Investigation showed that BDV had been introduced in the preceding tupping period 5-6 months earlier by a bought-in ewe which, on the basis of immunoperoxidase- and polymerase chain reaction techniques, was shown to be PI with BDV. Only 7 of the ewes, all of which had been in close contact with the PI ewe, seroconverted during the subsequent gestation. Apart from the PI twin lambs the losses caused by BDV were restricted to 2 barren ewes. The twin lambs, the PI ewe and lambs from the other 4 ewes that seroconverted were removed from the flock. The flock was thereafter free from an ongoing infection with BDV as shown by the absence of seroconversion. In addition, 5 heifers in late pregnancy most probably seroconverted to bovine virus diarrhoea virus (BVDV) when kept in close contact with the same PI ewe during the winter of 1989-90. When these heifers were reintroduced to the BVDV-free dairy herd from which they originated, their serum antibody titres ranged between 1:250 and 1:1250. Neither these heifers--not their calves--caused any spread of the infection in the herd, as indicated by the absence of seroconversion in 70 cows. The present investigation shows that in the control of both BDV in sheep and BVDV in cattle, it is important to ensure that the risk of transmission of pestivirus between the 2 species is minimized.

Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine virus diarrhoea virus.

Two outbreaks of border disease occurred on farms with sheep flocks and breeding cattle. The infection of the pregnant sheep was probably caused by transmission of virus from calves persistently infected with non-cytopathic bovine virus diarrhoea virus (BVDV) which were kept in close confinement with the ewes during mid-pregnancy. Border disease was also induced experimentally in eight lambs by exposing their dams at 38 to 78 days of gestation to a heifer persistently infected with BVDV. Both the natural and the experimental infections were characterised by typical signs such as 'hairy-shaker' lambs and high lamb mortality. The diagnosis was confirmed by virus isolations from live-born lambs, seroconversion and pathology. The study supports the assertion that cattle persistently infected with BVDV and in close contact with pregnant sheep, are an important source of strains of virus capable of causing border disease.

Outbreaks of border disease in goats induced by a pestivirus-contaminated orf vaccine, with virus transmission to sheep and cattle.

Five herds with a total of 276 female goats experienced severe outbreaks of reproductive failure due to a pestivirus-contaminated experimental orf vaccine given early in the breeding season. The reproductive failures comprised barrenness in 42 goats, abortion in 53 and, in 118, the birth of dead or weak kids. The incidence of female goats with such failures was 82 per cent overall, herd incidence rates ranging from 79 to 96 per cent. No progeny showed characteristic signs of border disease (BD). Microscopic lesions in the CNS were moderate, mostly characterized by hypercellularity, cell disorganization and decreased myelin content. Non-cytopathic strains of pestivirus were demonstrated in tissue or serum from two weak-born and two apparently healthy kids. Neutralizing antibodies against a bovine pestivirus were found in all 254 goats examined about 4 months after vaccination. At the end of the breeding season, all kids were removed and 74 young kids were introduced from presumably normal herds. Approximately 8 months later, 64 (86 per cent) of the bought-in kids had pestivirus antibodies. Two years after the outbreaks, such antibodies were found in all but three of 127 vaccinated goats still alive, and another 2 years later, in all 22 vaccinated goats in one of the herds. Sheep also were kept on the same five farms and cattle on one. Sheep in two of the flocks showed reproductive failure probably caused by pestivirus transmitted from infected goat offspring. A pair of twin lambs showed nervous signs and brain lesions characteristic of BD, while 17 other lambs had a very low growth rate. Virus was not isolated from any lamb. The prevalence rates of ewes with pestivirus antibodies in the two affected flocks were 33 and 72 per cent, respectively. One of nine cows aborted a pestivirus-infected foetus, and all were antibody-positive.