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.     

The production and survival of lambs persistently infected with border disease virus.

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.     

Arachidonic acid immunoregulation in lambs persistently infected with border disease virus

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.     

Study of cattle persistently infected with bovine viral diarrhea virus that lack detectable virus in serum

OBJECTIVE: To determine whether cattle persistently infected with bovine viral diarrhea virus (BVDV) that lack virus detectable in serum by use of the immunoperoxidase microtiter assay (IPMA) can transmit the virus to susceptible herdmates and determine prevalence of these cattle. DESIGN: Clinical trial and serologic survey. SAMPLE POPULATION: 2 cattle and 1,952 blood samples. PROCEDURE: A persistently infected cow in which virus could not be detected in serum was housed with a BVDV-seronegative steer. Blood and nasal swab specimens were tested via virus isolation and serum virus neutralization. Parallel WBC preparations and sera from blood samples of 1,952 adult cows were screened for BVDV by use of IPMA. RESULTS: The steer seroconverted to BVDV within 4 weeks of contact with the cow. Virus was detected in sera and WBC of 5 adult cows that were verified as persistently infected by retest 3 weeks later. Cattle persistently infected with BVDV in which virus could not be detected in both serum and WBC by use of IPMA were not found. CONCLUSION AND CLINICAL RELEVANCE: Cattle persistently infected with BVDV in which virus cannot be detected in serum by use of IPMA may serve as virus reservoirs for infecting susceptible cattle. Persistent infection was detected at a prevalence of 0.26%. Screening adult cattle by use of IPMA on serum samples appears to be a reliable means of detecting persistent infection with BVDV. Prevalence of cattle persistently infected with BVDV that have negative results of IPMA on serum is extremely low.     

Alterations in lymphocyte subpopulations in peripheral blood of sheep persistently infected with border disease virus.

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).     

Enhanced mucosal immune response in cattle persistently infected with foot-and-mouth disease virus

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.     

Peripheral and intracerebral T cell immune response in cats naturally infected with Borna disease virus.

Borna disease virus (BDV) is a neurotropic agent with capacity to cause encephalomyelitis in a wide range of animal species, including horses and cats. Recent studies also point to a link between BDV and human neuropsychiatric disorders. The pathogenesis of Borna disease (BD) has been proposed to be immune-mediated, mainly through the effects of cytotoxic T cells. We used flow cytometric analysis in order to characterize the peripheral and intracerebral T cell immune response in cats naturally infected with BDV. Our results show the presence of two different CD8+ cell populations (CD8+low and CD8+high) in the blood, spleen and brain of these cats. In the brain, CD8+low cells predominated over CD8+high cells. Since CD8+low cells have been suggested to represent a non-MHC-restricted T cell population, the recruitment of such cells to the brains of BDV-infected cats could possibly be of importance for the clearance of virus from neurones.     

Morphometric analysis of the retina from horses infected with the Borna disease virus

Borna disease (BD) is a fatal disorder of horses, often characterized by blindness. Although degeneration of retinal neurons has been demonstrated in a rat model, there are controversial data concerning whether a similar degeneration occurs in the retina of infected horses. To investigate whether BD may cause degeneration of photoreceptors and possibly of other neuronal cells at least at later stages of the disease, we performed a detailed quantitative morphologic study of retinal tissue from Borna-diseased horses. BD was diagnosed by detection of pathognomonic Joest-Degen inclusion bodies in the postmortem brains. Paraffin sections of paraformaldehyde-fixed retinae were used for histologic and immunohistochemical stainings. Numbers of neurons and Müller glial cells were counted, and neuron-to-Müller cell ratios were calculated. Among tissues from 9 horses with BD, we found retinae with strongly altered histologic appearance as well as retinae with only minor changes. The neuron-to-Müller cell ratio for the whole retina was significantly smaller in diseased animals (8.5 +/- 0.4; P < .01) as compared with controls (17.6 +/- 0.8). It can be concluded that BD in horses causes alterations of the retinal histology of a variable degree. The study provides new data about the pathogenesis of BD concerning the retina and demonstrates that a loss of photoreceptors may explain the observed blindness in infected horses.     

Ultrastructure of porcine circovirus in persistently infected PK-15 cells.

The ultrastructure of porcine circovirus was examined in persistently infected porcine kidney (PK)-15 cells. Virus-infected PK-15 cells had large numbers of intracytoplasmic inclusions, and a few cells had intranuclear inclusions. Intracytoplasmic inclusions were dispersed throughout the cytoplasm but were most numerous in the perinuclear cytoplasm. Inclusion were of various sizes, round to oval, and electron dense and were of two general types. Inclusions of the first type were small (0.1-0.5 microm diameter), not surrounded by trilaminar membranes, and granular with indistinct margins that blended with surrounding cytoplasm. Some contained 12+/-2-nm-diameter icosahedral virions in loose aggregates or rarely forming paracrystalline arrays. Small inclusions could be sites of viral assembly or maturation. Intracytoplasmic inclusions of the second type were larger (0.5-5.0 microm diameter) and more numerous and had abrupt margins surrounded by trilaminar membranes. They were more electron dense than small inclusions and were heterogeneous, containing various proportions of aggregated virions, electron-dense crystalline lamellae of 5 nm periodicity, and/or whorls of myelinoid membranes. Virions usually formed paracrystalline arrays and occasionally were loosely aggregated. Larger inclusions were typical of autophagolysosomes. Intranuclear inclusions were not membrane bound and were often associated with reticulated nucleoli or aggregates of heterochromatin. Some inclusions were irregularly shaped aggregates of indistinct, circular 10-12-nm-diameter viruslike particles. Others were 0.1-1.0 microm in diameter, round or ring shaped, dense, and finely granular, with sharply demarcated margins.     

Superinfection in ducks persistently infected with duck plague virus

Superinfections with homologous or heterologous strains of duck plague virus resulted in the deaths of birds persistently infected with duck plague virus. Not all birds that were superinfected died. Protection against mortality depended on the route of exposure, strain of the initial duck plague virus, and strain of the superinfecting virus.     

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.     

Intranuclear inclusions in cells infected with Newcastle disease virus.

Cells infected by Newcastle Disease Virus were observed to contain both intracytoplasmic and intranuclear inclusion bodies. Ultrastructurally, they consisted of twisted strands of about 18-20 nm diameter resembling nucleocapsids. The presence of these inclusions was detected irrespective of host cell or pathogenicity of the virus. In immunofluorescence and immunogold labelling experiments, these structures were tagged by an anti-P protein monoclonal antibody. In summary, we show that intracytoplasmic and intranuclear inclusion bodies, hitherto used as a taxonomic characteristic for the genus Morbillivirus of the Paramyxoviridae, also occur in a member of the genus Rubulavirus.     

Cohabitation of pregnant white-tailed deer and cattle persistently infected with Bovine viral diarrhea virus results in persistently infected fawns

Economic losses due to infection with Bovine viral diarrhea virus (BVDV) have prompted introduction of organized control programs. These programs primarily focus on the removal of persistently infected (PI) animals, the main source of BVDV transmission. Recently, persistent BVDV infection was demonstrated experimentally in white-tailed deer, the most abundant wild ruminant in North America. Contact of cattle and white-tailed deer may result in interspecific BVDV transmission and birth of persistently infected offspring that could be a threat to control programs. The objective of this study was to assess the potential for interspecific BVDV transmission from persistently infected cattle cohabitated with pregnant white-tailed deer. Seven female and one male white-tailed deer were captured and bred in captivity. At approximately 50 days of gestation, two cattle persistently infected with BVDV 1 were cohabitated with the deer. In a pen of approximately 0.8 ha, both species shared food and water sources for a period of 60 days. Transmission of BVDV as indicated by seroconversion was demonstrated in all exposed adult deer. Of the seven pregnancies, four resulted in offspring that were infected with BVDV. Persistent infection was demonstrated in three singlet fawns by immunohistochemistry and ELISA on skin samples, PCR, and virus isolation procedures. Furthermore, two stillborn fetuses were apparently persistently infected. This is the first report of BVDV transmission from cattle to white-tailed deer using a model of natural challenge. Under appropriate circumstances, BVDV may efficiently cross the species barrier to cause transplacental infection and persistently infected offspring in a wildlife species.     

Transformed phenotypes in long-term cultures persistently infected with bovine leukemia virus.

Two cell lines, 3178 and FLK, were established respectively from calf form bovine lymphosarcoma and from fetal lamb kidney cells inoculated in vitro with bovine leukemia virus. These two cell lines persistently infected with bovine leukemia virus were maintained for more than 150 passages over three years. They exhibited the characteristics of transformed cell lines in vitro: 1) anchorage independence, 2) increased saturation density and decreased population doubling time, 3) increased uptake of 2-deoxy-D-glucose and 4) tumorigenicity in athymic nude mice.     

Nosological study of Borna disease virus infection in race horses.

To investigate the prevalence of diseases in the Borna disease virus (BDV) antibody positive race horses, we undertook seroepidemiological studies of BDV infection on 125 culled race horses in Hokkaido, Japan. The serological study disclosed the presence of antibodies only to BDV-p40 or -p24 in 19.2% (24/125) and 3.2% (4/125) of culled horses, respectively. Antibodies to both BDV-p40 and -p24 were found in 24.0% (30/125) of these horses. Of particular note was the finding that locomotorium disorders were detectable at a significantly higher rate in BDV antibody positive horses than that in the seronegative horses. These results imply that BDV infection may possibly contribute to an increase in the incidence rate of locomotorium disorders in race horses.     

Humoral immune response against Borna disease virus (BDV) in experimentally and naturally infected cats

In order to investigate the peripheral and intracerebral humoral immune response against Borna disease virus (BDV) in cats, serum and cerebrospinal fluid (CSF) samples from experimentally and naturally BDV-infected cats were analysed in two different test systems (indirect enzyme-linked immunosorbent assay and indirect immunofluorescent test). The experimentally infected cats developed high antibody titres against the major immunogenic BDV-proteins, p24 and p40. In contrast, the naturally infected cats showed a comparatively weak humoral immune response. The experimentally infected cats were inoculated with either BDV laboratory strain V or a feline BDV-isolate. Some differences existed between the two groups of cats. The former group developed a higher response against p40, whereas the latter group showed, beside the p40-response, a more pronounced p24-response, similar to the situation in the naturally infected cats.     

Feline calicivirus infection in kittens borne by cats persistently infected with the virus

On the basis of repeated isolation of feline calicivirus (FCV) from oropharyngeal swabs four to eight months after exposure to FCV strain 255, four carrier queen cats were identified. These cats gave birth to 16 kittens. Litters were individually housed with their mothers until nine weeks of age and were monitored virologically and serologically from birth until 15 weeks old. All kittens became infected between three and nine weeks old and shed FCV consistently for periods of three to 11 weeks. Clinical signs of FCV were observed in 11 kittens but none developed severe respiratory disease. At the time of initial infection maternal antibody titres in the kittens ranged from 1:4 to 1:24. Within one to three weeks of infection titres began to rise. The results indicated that kittens of queen cats persistently infected with FCV frequently experience mild or subclinical immunising infections.     

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.