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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-β) 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-β and TGF-α were investigated. Results of the study revealed that the levels of GMF-β (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-β expressions were found in astrocytes, glial cells and neurons. These results were reasonable to suggest that BDV-mediated increased GMF-β 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-β and TGF-α in neurons and reactive glial cells and its association with neuropathology in BD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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., (2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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.

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.

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 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.

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).

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.

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).

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.

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.

To evaluate arachidonic acid-related immunoregulatory mechanisms during long-term persistent pestivirus infection, we measured plasma contents of leukotriene C4 (LTC4), prostaglandin D2 (PGD2) and their plasma fatty acid (FA) precursor, arachidonic acid (AA), in six lambs with congenital border disease (BD). Significantly elevated average plasma LTC4 during the first half year of life was associated with increased PDG2 when compared to uninfected control lambs. Significantly elevated total plasma esterified AA stores suggest an effective BDV-mediated prostenoid immunostimulation. However, at 1 yr old, prostenoid secretion had fallen to normal (LTC4) or below normal (PGD2) levels. In contrast, there remained significantly elevated plasma esterified AA, present as available substrate for formation of these anti-viral immunoregulatory agents. These results suggested that preventing mobilization of AA from lipid stores for effective immune responses may be a viral strategy of BD virus that is associated with long term border disease effects.

From 1985 to 1989 lambs persistently infected with border disease virus (BDV) were produced for comparative immunological studies by infecting 57 susceptible pregnant ewes between 50 and 60 days' gestation with Moredun or Oban strains of BDV. Ewes were infected either by injection with virus grown in cell culture or by housing with lambs excreting BDV. There was no significant difference in the outcomes of these different methods of infection. There was a significant difference in the number of viable lambs born to ewes receiving the two viruses. Of 41 ewes infected with Moredun virus 21 produced 32 live lambs of which 17 were reared to 1 month old (53% viability). Of 16 ewes receiving Oban virus 10 gave birth to 17 live lambs of which 15 were reared to 1 month old (88% viability). All the lambs born to ewes infected with Moredun BDV had varying signs of tremor and increased hairiness ("hairy-shakers") while those born to ewes infected with the Oban virus had no obvious clinical signs. Survival of the lambs was poor. Up until February 1991, 14 Moredun and 10 Oban sheep between the ages of 4 months and 5.5 yr had died from a variety of causes. The two commonest causes were a chronic wasting syndrome and a mucosal disease-like syndrome which was associated with the recovery of cytopathic BDV. Mating of unrelated persistently infected sheep was largely unproductive although 2 lambs were reared.

The prefemoral efferent lymphatics of sheep persistently infected (PI) with Border disease virus (BDV) were cannulated in order to study the effects of the virus on cells of the immune system. Efferent lymphocytes recovered from PI sheep were phenotyped using a panel of monoclonal antibodies (MoAb) specific for ovine cell-surface markers and compared to lymphocytes recovered from normal, healthy controls. PI sheep had an increased percentage of cells expressing the T cell-associated molecules CD5, CD4, CD8 and T19, also an increase in cells expressing CD1 and a population of cells expressing low levels of the T19 molecule which was not present in control sheep. The lymphocytes were examined for the presence of BDV using virus-specific MoAb. On average 8.5% of the efferent lymphocytes from PI sheep carried virus antigen. BDV antigen was also found in the mononuclear cells and connective tissue of lymph nodes indicating widespread virus dissemination within the lymphoid system of PI sheep.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibody to border disease virus (BDV) in sheep serum. A monoclonal antibody bound to 96-well microplates was used to capture antigen from detergent-solubilised BDV-infected cells. Single dilutions of test sera were then added to wells containing bound BDV antigen and control wells containing uninfected cell lysates. Specific antibody to BDV was detected by an anti-ovine IgG antiserum conjugated with horseradish peroxidase and the results expressed as ELISA units with reference to a standard curve. Sequential sera from 16 experimentally infected sheep and single sera from 103 sheep involved in a field outbreak were tested in the ELISA and for neutralising antibody. There was good qualitative correlation between the two tests.

Fifty pregnant goats, inoculated intramuscularly at different gestational stages with a non-cytopathic ovine pestivirus or a cytopathic bovine pestivirus, all developed pestivirus-neutralizing antibodies within 5 weeks of inoculation. The incidence of reproductive failure was similar for the two agents. Parturition at term with only healthy kids occurred in 13 (26 per cent) of the goats. Viable kids were not born to any of the 17 goats inoculated at about day 40 of gestation. Three of the 17 delivered dead or weak kids, seven aborted and three of seven which were necropsied during pregnancy had markedly underdeveloped and autolysed or mummified fetuses in utero, while four were barren. When inoculated at around the 60th day of gestation, two of 18 animals gave birth to only healthy kids, 12 to dead and/or weak kids, two aborted and, at necropsy, a small, decomposed fetus was found in one goat while one other was barren. In this group, one kid was ataxic and seven others had body tremors characteristic of border disease. One of the latter kids was viable. Of 15 goats inoculated at around day 100 of gestation, 11 gave birth to healthy kids only, three to dead and/or weak kids and one aborted. In 23 progeny, histological changes in the central nervous system (CNS) consisted mainly of cerebral white matter necrosis, cerebellar dysplasia, hypercellular areas in white matter and lymphocytic perivascular cuffings. All seven weak-born kids with signs of border disease had CNS lesions, particularly cerebellar dysplasia and/or hypercellular areas. Non-cytopathic pestivirus was isolated from tissues from all eight progeny examined in the 40-day inoculation group, from tissues and/or serum from 10 of 23 progeny in the 60-day group, and from four of 24 in the 100-day group. Persistent infection was demonstrated in a healthy kid, in a viable shaker and in two other kids which appeared normal at birth. Examination of offspring before ingestion of colostrum revealed pestivirus antibodies in one kid in each of the 40- and 60-day inoculation groups and in five kids in the 100-day group.

Fourteen sheep persistently infected with border disease virus were investigated to examine the effects of persistent viraemia on lymphocyte subpopulations in peripheral blood and their responses to the mitogen phytohaemagglutinin (PHA) in vitro. Persistently infected sheep had significantly more CD8+ (cytotoxic-suppressor) T-lymphocytes than uninfected sheep of the same age (P less than 0.001). The total number of CD4+ (helper) T-lymphocytes were not significantly different but there were more T-lymphocytes (CD5+) which were CD4- and CD8- in normal sheep than persistently infected sheep (P less than 0.001). Peripheral lymphocytes obtained from persistently infected sheep showed significantly reduced blastogenesis induced by PHA than those obtained from normal sheep (P less than 0.001).

Lambs congenitally infected with border disease (BD) virus and sheep exposed to BD virus as adults were studied for one year to determine the pathogenesis of congenital exposure compared with adult exposure to the virus. Persistent BD virus was isolated in tissue culture and detected by immunofluorescence of the peripheral white blood cells, urine, and cerebrospinal fluid of lambs with congenital BD up to one year of age. These animals had no detectable serum neutralizing antibody response to the virus for the same interval. BD virus antigen was also detected by immunofluorescence in many central nervous system tissues of these lambs with congenital BD. Dysmyelination and glial proliferation in the central nervous system and microencephaly were noted in the lambs with congenital BD, and these lesions appeared to remain the same over a 12-month period.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.

Study of the virological and serological status of abnormal sheep derived from a single outbreak of Border disease indicated a relationship between infection status and diverse pathological lesions. Persistent infection was associated with typical Border disease whereas an active, even exaggerated, serological response correlated with severe intracranial malformations which probably resulted from fetal infections occurring during the early stages of acquisition of immune responsiveness.

The pathogenesis of BD virus infection of sheep is reviewed briefly. The most serious consequences occur when susceptible pregnant sheep are infected. The virus crosses the placenta readily and can cause foetal death with resorption, mummification or stillbirths. Other lambs survive, however, and are born with varying degrees of tremor and/or hairy fleeces. Many such "hairy-shaker" lambs die shortly after birth but survivors gradually recover. These survivors plus other apparently normal lambs can be persistently infected with virus and excrete it constantly often for the rest of their lives. It is these persistently infected sheep that are the key to the epidemiology of the virus and may be responsible for its introduction into a susceptible flock. In addition, cattle grazed with sheep are potentially an important source of pestiviruses capable of causing BD since in some herds the prevalence of cattle persistently infected with pestivirus is one to three per cent. Results are presented of studies on antigenic relation ships among Scottish isolates of cattle pestiviruses (BVD) and BD viruses, and the conclusion drawn that while a single strain of BVDV may be suitable for use as a cattle vaccine this would not be very efficacious in preventing BD since two antigenically distinguishable strains of BD virus have been identified. An effective vaccine against BD would have to protect sheep against both these strains and ideally a cattle vaccine should also contain both strains.

A flock of sheep in which border disease (BD) was enzootic was studied through a breeding season. At the beginning of the study (August 1981), 125 (82%) of 152 ewes were seropositive to the cross-reacting bovine viral diarrhea virus. Within 7 months, 3 (18%) of 17 seropositive ewes retested had reverted to seronegative. Of the remaining 21 ewes identified as seronegative, 7 (33%) converted to seropositive by the end of the study. Triplet lambs were born, 2 of which exhibited clinical signs of BD. The virus was isolated from blood lymphocytes from both of the affected lambs. The most severely affected lamb shed virus into the urine, saliva, and feces through 10 weeks of age. Lymphocyte stimulation tests indicated that the lymphocytes from the affected lambs had decreased function in months 4 through 7, but returned to normal function by the eighth month. Transmission of BD virus was investigated by exposing 5 seronegative ewes to the BD-infected lambs. Two of the contact ewes developed viremia and 3 converted to seropositive within the 13-week exposure period. Evidence from this and other studies supports a model of BD in gravid, nongravid, and persistently infected adult sheep.

In vitro comparison of the replication cycles of three strains of border disease virus and one strain of bovine viral diarrhoea virus showed similar growth curve patterns and a tendency of cell-free virus to exceed cell-associated virus. Each virus was antigenically distinguishable from the others in cross-neutralisation tests.

Border disease (BD) was experimentally induced in 9 lambs by inoculation of their dams with the BD-31 strain of border disease virus (BDV) at 50 days of gestation. These ewes developed serum-neutralizing antibody titers to BDV. The diagnosis of BD in their lambs was confirmed by hairy birthcoats, intrauterine growth retardation, tremors, and hypomyelination. Three clinically healthy age-matched control lambs, whose dams had been given an inoculum containing only BDV-free cell culture supernatant, were studied in parallel. There were significant differences in birth weights and in the lengths of the right tibiae and radii between the affected and the control lambs. There was a gradient in severity of clinical neurologic signs among the affected lambs, which directly correlated with the severity of hypomyelination in their spinal cords. However, the difference in severity of the neurologic deficits did not correlate with differences either in the precolostral serum-neutralizing antibody titers to BDV in these lambs or in the number of BDV antigen-containing cells in their spinal cords. Only approximately 0.01% to 0.3% of spinal cord cells, both in gray matter and white matter, were BDV antigen positive in the affected lambs. These results indicate that extensive infection of CNS cells with their subsequent destruction or functional alteration may not be a critical part of the pathogenesis of the hypomyelination in BD.

The surface phenotypes of peripheral blood lymphocytes in groups of lambs and adult sheep persistently infected with Border disease virus (P-I BD) were compared with those of healthy controls. The proportion and number of lymphocytes bearing surface immunoglobulin (sIg+) and expressing class II MHC antigen (B cells) were significantly increased. A significant increase in CD1+ lymphocytes was also evident. Conversely, the proportion of T lymphocytes in P-I BD lambs was reduced. A marked reduction in the proportion of circulating lymphocytes expressing class I MHC antigen was also observed. These findings were not affected by differences in the strain of the virus responsible for the persistent infection.