First description of enhanced expression of transforming growth factor-alpha (TGF-α) and glia maturation factor-beta (GMF-ß) correlate with severity of neuropathology in border disease virus-infected small ruminants.

Border disease (BD) is caused by Pestivirus and characterized by severe neuropathology, and histopathologically observed severe hypomyelination. We have previously shown that small ruminants infected with border disease virus (BDV) play an important role for neuropathology and pathogenesis of severe oxidative damage in brain tissue, neuronal mtDNA; in the production of high pathologic levels of nitric oxide; in glial cell activation and stimulation of intrinsic apoptosis pathway. This study aimed to investigate the relationship between glia maturation factor beta (GMF-ß) and transforming growth factor alpha (TGF-α) expressions and the causes of BDV-induced neuropathology and to investigate their role in neuropathogenesis in a way that was not presented before. Expression levels of GMF-ß and TGF-α were investigated. Results of the study revealed that the levels of GMF-ß (P < 0.005) and TGF-α (P < 0.005) expression in the brain tissue markedly increased in the BDV-infected animals compared to the non-infected healthy control group. While TGF-α expressions were predominantly observed in neurons, GMF-ß expressions were found in astrocytes, glial cells and neurons. These results were reasonable to suggest that BDV-mediated increased GMF-ß might play a pivotal role neuropathogenesis and a different type of role in the mechanism of neurodegeneration/neuropathology in the process of BD. The results also indicated that increased levels of GMF up-regulation in glial cells and neurons causes neuronal destruction, suggesting pathological pathway involving GMF-mediated brain cell cytotoxicity. It is clearly indicated that the cause of astrogliosis is due to severe TGF-a expression. This is the first study to demonstrate the expression of GMF-ß and TGF-α in neurons and reactive glial cells and its association with neuropathology in BD.

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.

Chinese border disease virus strain JSLS12-01 infects piglets and down-regulates the antibody responses of classical swine fever virus C strain vaccination.

During 2012 and 2013, several border disease virus (BDV) strains were identified from Chinese goat and sheep herds. At the same time, pigs from the same areas were found to be seropositive to BDV by ELISA, without showing clinical signs (unpublished data). To examine the susceptibility of pigs to the Chinese BDV strains, BDV isolate JSLS12-01, isolated from naturally infected sheep, was used to infect pigs. Antibody responses, viremia, clinical signs and pathological changes of the infected animals were examined. It confirmed that the current BDV strain could infect the domestic pigs, the animals showed viremia during 4 to 14 days post infection (dpi) and sero-conversion from 14dpi; no clinical and pathological changes were observed. In addition, CSFV maternal antibody did not influence BDV infection. Subsequently, pigs were infected with the BDV isolate and vaccinated with Hog cholera lapinized virus (HCLV) 21 days later to determine the effect of BDV infection on antibody induction of CSFV vaccination. The specific CSFV antibody and neutralizing antibody titers of the BDV infected group remained negative after the primary vaccination. Even after the boost vaccination, they were still significantly lower than those of the uninfected groups (p<0.05). These results indicated that BDV infection could down-regulate the antibody responses of CSFV C-strain vaccination. It should be paid attention that BDV prevalence in pig herds and in live vaccines might hamper the vaccination of CSF.

Approved and experimental countermeasures against pestiviral diseases: Bovine viral diarrhea, classical swine fever and border disease.

The pestiviruses, bovine viral diarrhea virus (BVDV), classical swine fever (CSFV) and border disease virus, are important livestock pathogens in many countries, but current vaccines do not completely prevent the spread of infection. Control of pestiviral diseases is especially difficult due to the constant viremia and viral shedding of persistently infected (PI) animals, which must be identified and eliminated to prevent disease transmission. Existing vaccines are limited by the delay between vaccination and the onset of protection, the difficulty of differentiating serologically between vaccinated and naturally infected animals and the need for broad vaccine cross-protection against diverse virus strains. Antiviral therapy could potentially supplement vaccination by providing immediate protection in the case of an outbreak. Numerous compounds with in vitro antiviral activity against BVDV have been identified through its role as a surrogate for hepatitis C virus. Fewer drugs active against CSFV have been identified, but many compounds that are effective against BVDV will likely inhibit CSFV, given their similar genomic sequences. While in vitro research has been promising, the paucity of efficacy studies in animals has hindered the commercial development of effective antiviral drugs against the pestiviruses. In this article, we summarize the clinical syndromes and routes of transmission of BVD, CSF and border disease, discuss currently approved vaccines, review efforts to develop antiviral therapies for use in outbreak control and suggest promising directions for future research.

Two different epidemiological scenarios of border disease in the populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the first disease outbreaks.

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.

Development and evaluation of indirect enzyme-linked immunosorbent assay for a screening test to detect antibodies against classical swine fever virus.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for a screening test to detect antibodies against classical swine fever virus (CSFV). Viral glycoproteins, which were purified from swine kidney cells infected with CSFV ALD/A76 strain by the immunoaffinity purification using monoclonal antibody against E2 protein, were adsorbed on a microtiter plate as the antigen for the antibody detection. Each antibody titer of serum sample was expressed as a sample per positive value calculated with optical absorbance of each sample and that of a positive control. The advantage of this ELISA is its higher sensitivity: most sera containing more than 4 neutralization titers were determined to be positive. This ELISA is unable to discriminate between antibodies against CSFV and those against other ruminant pestiviruses, therefore positive sera in this ELISA should be evaluated by a cross-neutralization test using CSFV, bovine viral diarrhea virus, and border disease virus. Taken together, the indirect ELISA developed in this study is useful screening tool to detect antibodies against CSFV for the large-scale monitoring of classical swine fever.

Experimental infection with chamois border disease virus causes long-lasting viraemia and disease in Pyrenean chamois (Rupicapra pyrenaica).

Since 2001, severe outbreaks of disease associated with border disease virus (BDV) infection have been reported in Pyrenean chamois. The disease is characterized by variable degrees of cachexia, alopecia and neurological manifestations prior to death. The aim of this study was to investigate this disease under experimental conditions. To assess viral virulence, humoral immune response, dissemination and probable routes of transmission, seven chamois (five seronegative and two seropositive for BDV) were inoculated with a BDV isolated from a naturally infected chamois. A group of three chamois were maintained as uninfected controls. The five seronegative chamois became viraemic from day 2 post-inoculation (p.i.) until their death (three animals) or the end of the experiment (on day 34 p.i.) and developed neutralizing antibodies from day 18 p.i. until the end of the study. Continuous shedding of the virus was detected by RT-PCR in oral, nasal and rectal swabs in viraemic chamois from day 5 p.i. Despite none of the viraemic chamois showing obvious neurological signs, all of them had a non-suppurative meningoencephalitis as seen in naturally infected chamois. The two inoculated BDV-seropositive chamois did not become viraemic. This study confirms that BDV is the primary agent of the disease that has been affecting chamois populations in recent years in the Pyrenees and that previously acquired humoral immunity is protective.

A serological investigation of pestiviruses in sheep in eastern border of Turkey.

All pestiviruses are important veterinary pathogens causing economic losses in cattle, sheep, and pigs. In this study, blood samples randomly collected from 465 sheep were analysed for the presence of antibodies to pestiviruses (bovine viral diarrhea virus, border disease virus) using an enzyme-linked immunosorbent assay in the province of Van and their towns. The seroprevalance were estimated as 75.9% and 60.0-82.5% in the sampled animals and sampled towns, respectively. The results revealed that pestiviruses are important abort pathogens in the province of Van and their towns.

Comparison of hematological and biochemical parameters in sheep naturally and persistently infected with a border disease virus.

In this study, we investigated the changes occurring in the activities of determining the biochemical and hematological parameters in persistently infected sheep with border disease virus (BDV) and control sheep. While cholesterol, aspartate aminotransferase, lactate dehydrogenase, high-density lipoprotein, and glucose parameters were found to be statistically different between control and BDV positive groups (p<0.01), total protein, alkaline phosphotase, creatine kinase, amylase, glucose, and high-density lipoprotein were found to be statistically different between control and persistently infected group (p<0.01). Interestingly, all groups were shown only mean corpuscular volume parameter was different (p<0.01). It was found that cholesterol, aspartate aminotransferase, amylase, high-density lipoprotein, and low-density lipoprotein parameters were different between PI and infected sheep (p<0.01). It was speculated that BDV might effect also the expression of low-density lipoprotein receptor and determination of the changes in BD and its clinical importance might contribute to the veterinarians and scientists studying in this area.

Border disease virus seroprevalence correlates to antibodies in bulk-tank milk and reproductive performance of dairy sheep flocks.

There is a great need to establish effective tools to control border disease virus (BDV) in European dairy sheep flocks. Hence, our main aim was to investigate the accuracy of analyzing anti-BDV antibodies in bulk-tank milk (BTM) in detecting the real BDV seroprevalence in dairy sheep flocks. Furthermore, the relevance of BDV to reproductive performance of dairy sheep flocks prompted us to search for the association between BDV seroprevalence and reproductive parameters. For these purposes, 34 flocks were selected based on different percentages of antibody inhibition (AIP) values in BTM as estimated by ELISA. Serum samples from 10 replacement lambs older than 6 mo, 10 ewes 1 to 2 yr old, and 10 ewes > 2 yr old were collected and analyzed for the presence of anti-BDV antibodies by ELISA. A negative relationship between BDV AIP in BTM and within-flock seroprevalence was observed. Flocks with a high AIP (> 80%) had an average of 2.5% seropositive animals; flocks with a moderate AIP (46-79%) had 11.4% seropositive animals; and finally, flocks with an AIP < or = 45% showed a high flock seroprevalence (57.2%). Ten out of 34 flocks showed a high BDV seroprevalence in lambs, suggesting the presence of persistently infected animals in the flock. The observed AIP values in BTM from these likely BDV-infected flocks were indicative of a high seroprevalence. The analysis of reproductive-parameters data collected from these flocks showed no differences in fertility or prolificacy in relation to BDV circulation rates. Nonetheless, lamb mortality was significantly greater in flocks with low-moderate seroprevalence (10-30%), probably as a result of a first-time contact with BDV of previously naïve ewes. These findings suggest that testing of BTM samples may be useful in inferring the BDV seroprevalence in a flock.

Seroprevalence and risk factors for the presence of ruminant pestiviruses in the Dutch swine population.

Swine can be infected with classical swine fever virus (CSFV), as well as ruminant pestiviruses: bovine viral diarrhoea virus (BVDV), and Border disease virus (BDV). Cross-reactions between pestiviruses occur, both regarding protective immunity and in diagnostic tests. The presence of BVDV and BDV in a swine population may thus affect the transmission of CSFV, but also the diagnosis of a CSFV infection. In this study, the seroprevalence against BVDV and BDV in two categories of swine, sows and finishing pigs, in the Netherlands was determined. Furthermore, several risk factors, associated with the presence of swine and ruminants on the same farm or in the immediate surroundings, were evaluated. In sows, the seroprevalence against BVDV was 2.5% on the animal level, and 11.0% on herd level. In finishing pigs these prevalences were 0.42% and 3.2%, respectively. Antibodies against BDV were found in three sows only. Risk factors, associated with a BVDV-seropositive status in breeding pigs, were the presence of cattle on the same premises and a high density of sheep and/or goats herds in a radius of 3km. While BVDV and BDV hardly pose any threat to the swine population themselves, knowledge, and therefore regular monitoring, on the presence of these viruses in the swine population is important with respect to CSF eradication. It will allow for a better interpretation of diagnostic test results, both in terms of possible false positives and false negatives, but may also bring about additional measures or surveillance protocols in times of CSF outbreaks to avoid surprises caused by cross-reactivity with ruminant pestiviruses.

Associations of veterinary services and farmer characteristics with the prevalences of brucellosis and border disease in small ruminants in Spain.

We investigated the farm factors associated with the prevalences of brucellosis and border disease (BD) in small-ruminant herds in the Madrid region of Spain. These infections were used as models of diseases of well-known and totally unknown distribution, respectively, to assess the association between the perception of the importance of a given disease on the relative contributions of veterinary services and the farmer's attitudes to its prevention. Sera, farming-management information and data concerning veterinary assistance and farmer characteristics were collected from 60 sheep or goat herds. The overall sero-prevalence of brucellosis was 5.7% (complement fixation) and for BD was 17.9% (ELISA test). The relationship between sero-positivity and the variables in the questionnaire was assessed by multivariable analysis using random-effects logistic-normal regression. 'Availability of veterinary services' was a major protective factor for brucellosis. In contrast, no association with veterinary services was observed for BD, whereas 'membership in a farmers' organization' (a variable associated with good farming practice and animal care) was a protective factor. 'Membership of a farmers' organisation' and two other farmer variables indicative of good husbandry ('youth' and 'schooling') were associated with a lower sero-prevalence of brucellosis in univariable analysis but they did not remain significant in the multivariable model. Our observations suggest that veterinary-activity variables predominate over non-specific protective farm factors related to good husbandry in the case the disease is subject to disease surveillance. This underscores the importance of organized control programs for veterinary services to be effective in terms of animal disease prevention.

Morphometric analysis of growth retardation in fetal lambs following experimental infection of pregnant ewes with border disease virus.

The onset of growth retardation was investigated in fetal lambs following experimental infection of pregnant ewes with Border Disease virus (BDV) on day 53 of pregnancy. Fetuses from control and infected ewes were harvested at weekly intervals between day 60 and day 95 of gestation and morphometric studies were completed on tibial radiographs and tibial growth cartilage metaphyseal junctions. Mean tibial areas were significantly reduced (P < 0.01) in fetuses from infected ewes at 35 and 42 days after infection and growth cartilage metaphyseal junctions were less mature in fetuses from infected ewes at 42 days after infection. Positive immunostaining for BDV antigen was demonstrated in the brains of all fetuses from infected ewes between 14 and 42 days after infection. Attempts to demonstrate BDV antigen in bone proved unsuccessful. It is concluded that intrauterine growth retardation is an early manifestation of BDV infection in lambs and that the process is initiated shortly following infection of the fetus.

A double monoclonal antibody ELISA for detecting pestivirus antigen in the blood of viraemic cattle and sheep.

A panel of monoclonal antibodies (mAbs) has been produced to the p125/p80 non-structural polypeptide of border disease virus (BDV) and bovine virus diarrhoea virus (BVDV). This polypeptide appears to be highly conserved among BDV and BVDV isolates and consequently the mAbs directed against it have a broad cross-reactivity with pestivirus isolates. The epitope specificities of these mAbs were determined by competitive binding and four of the mAbs with mutually exclusive epitope specificities were selected for the development of a diagnostic ELISA. Two mAbs were used to capture virus antigen prepared from the blood of infected cattle and sheep, then two different mAbs used to detect the captured antigen. This double mAb ELISA was compared to existing ELISAs which rely on polyclonal antibodies (pAbs) for detecting captured antigen. The mAb detection ELISA was more sensitive than the pAb detection ELISAs for both cattle and sheep and resulted in higher optical densities for positive samples without an increase in background readings of negative controls.

Distribution of Border disease virus antigen in lymphocyte subpopulations in the peripheral blood of experimentally infected lambs.

Ten lambs were experimentally infected with Border disease virus and the distribution of viral antigen in lymphocyte subpopulations studied by flow cytometry. The virus was isolated in culture from the peripheral blood mononuclear cells (MNC) of all experimentally infected lambs for a mean period of 9.8 +/- 1.03 days. The peak virus titre of 3.26 log10 TCID50 per 10(6) MNC was attained Day 9 post-inoculation (pi). Viral antigen was present in peripheral blood lymphocytes of experimentally infected lambs as early as 24 h pi and continued to be detected up to Day 10 pi. The number of lymphocytes expressing viral antigen rose from 12.38 +/- 1.22% in samples taken Day 3 pi to 23.21 +/- 2.82% on those collected Day 7 pi, dropping gradually thereafter. During the peak period of infection, 12.46 +/- 2.09% of B cells, 37.71 +/- 10.96% of T cells and 52.33 +/- 8.27% of lymphocytes which were neither B nor T lymphocytes expressed viral antigen. Most of the infected lymphocytes expressed the OvCD5 (T cell) molecule. The virus affected all T cell subsets but the suppressor/cytotoxic (OvCD8+) cells appeared to be the main targets. During the peak period of infection, 54.20 +/- 6.16% of the infected T cells expressed the OvCD8 molecule, 31.58 +/- 7.12% were OvCD4+, and 12.67 +/- 6.50% were OvWC1+ (T-19+, gamma/delta).

Border disease virus antigens in lymphocyte subpopulations in the peripheral blood of persistently infected sheep.

Biotinylated virus-specific antibodies were used to detect Border disease virus antigen by flow cytometry in the mononuclear cells of the peripheral blood of 15 sheep persistently infected with Border disease virus. The viral antigen was present in 12.86 +/- 4.65% (mean +/- SD) of mononuclear cells (MNC). The percentage of MNC that contained viral antigen was higher in lambs than in adult sheep, with mean rates of 22.68 +/- 5.02% and 11.65 +/- 4.39%, respectively. Depletion methods were used to estimate the distribution of viral antigen in peripheral blood lymphocyte subsets. The viral antigen was present in T-cells (OvCD5+), B-cells (LCAp220+) and non-T- and non-B-cells. Depletion studies revealed that 5.39 +/- 2.47% of the cells expressing the LCAp220 epitope (B-cells), 23.38 +/- 11.38% of those expressing the OvCD5 epitope (T-cells) and 55.07 +/- 10.93% of those which were neither B- nor T-cells were positive for viral antigen. Most (57.18 +/- 5.41%) of the T-cells containing viral antigen were cytotoxic/suppressor (OvCD8), 25.63 +/- 2.97% were helper (OvCD4) cells and 12.24 +/- 3.21% expressed the gamma/delta (OvWC1) epitope.

Cytotoxic T cell responses in lambs experimentally infected with Border disease virus.

Mononuclear cells cytotoxic to a noncytopathic strain of Border disease virus (BDV) but not to uninfected cells were detected in the peripheral blood of experimentally infected lambs, 10 days after experimental infection, reaching peak levels of cytotoxicity 15 days post-inoculation. The specificity of the cytotoxic activity to BDV-infected cells was tested by including autologous targets infected with another virus (bovine respiratory syncytial virus, BRSV) and normal uninfected autologous targets. The cytotoxic T cells were virus-specific, as only autologous BDV-infected targets were lysed, whereas autologous targets infected with BRSV and uninfected targets were not lysed. The present results also suggest that the cytotoxic activity was largely MHC-restricted as the specific release from BDV-infected autologous targets was significantly higher than that of BDV-infected heterologous targets (P < 0.001) and the cytotoxicity against BDV-infected homologous targets was significantly reduced by the selective depletion of OvCD8+ T cells (P < 0.001).

Lymphocyte responses to viral antigens and phytohaemagglutinin in persistently viraemic sheep and lambs experimentally infected with Border disease virus.

Lymphocytes obtained from lambs 5 to 21 days after experimental infection with Border disease virus (BDV) showed significant blastogenic responses to live or inactivated BDV. Live virus stimulated significantly higher lymphocyte transformation (LT) responses than inactivated virus. Lymphocytes obtained from uninfected control and persistently infected lambs had no significant response to live or inactivated antigen. Lymphocyte responses to phytohaemagglutinin were significantly higher in control lambs than in experimentally infected lambs in samples obtained 5 to 10 days post-inoculation.

Effects of experimental infection with border disease virus on lymphocyte subpopulations in the peripheral blood of lambs.

Experimental infection with Border disease virus was characterised by significant changes in the total numbers of leucocytes and neutrophils, and in the proportions and numbers of the different lymphocyte subpopulations. Three days after experimental infection there was significant leucopenia due to lymphocytopenia and neutropenia (P < 0.001). The lymphocytopenia and neutropenia lasted for up to seven days after inoculation. The lymphocytopenia was due to a reduction in the number of both T cells and B cells. During the early period of infection, the reduction in T cells was mainly due to a reduction in the number of OvCD4+ and T-19+ lymphocytes as the number of circulating OvCD8+ cells was not significantly affected. The cells expressing the OvCD4 and OvWC1 epitopes returned to pre-inoculation values 10 and 14 days after inoculation, respectively. In contrast, during the same period, the number of T cells expressing the OvCD8 molecule became significantly higher than the corresponding pre-inoculation values. There were no significant changes in all the T cell subsets in the control lambs.