Experimental infection of calves with two strains of bovine virus diarrhoea virus: virus recovery and clinical reactions

Fifteen calves were inoculated with a mixture of two strains of bovine virus diarrhoea virus (BVDV), the cytopathogenic NADL strain which had been passaged over 20 times n vitro, and the non-cytopathogenic FCS strain, passaged only once after isolation from fetal calf serum. In a second experiment, seven calves received the NADL strain, and eight the FCS strain. The clinical and virological results of the two experiments were compared. In dual infections, the NADL strain interfered with the replication of the FCS strain resulting in less severe disease than the FCS strain alone. The FCS-BVDV was recovered from nasopharyngeal swabs and buffy coat cells whereas the NADL-BVDV was recovered only from nasopharyngeal swabs. The cytopathogenicity of the two strains did not change after passage in vivo. The differences observed are discussed in relation to cultural history and cytopathogenicity.     

Failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus

Previous reports on the spread of bovine virus diarrhoea virus (BVDV) from animals primarily infected with the agent are contradictory. In this study, the possibility of transmission of BVDV from calves simultaneously subjected to acute BVDV and bovine coronavirus (BCV) infection was investigated. Ten calves were inoculated intranasally with BVDV Type 1. Each of the 10 calves was then randomly allocated to one of two groups. In each group there were four additional calves, resulting in five infected and four susceptible calves per group. Virulent BCV was actively introduced in one of the groups by means of a transmitter calf. Two calves, susceptible to both BVDV and BCV, were kept in a separate group, as controls. All ten calves actively inoculated with BVDV became infected as shown by seroconversions, and six of them also shed the virus in nasal secretions. However, none of the other eight calves in the two groups (four in each) seroconverted to this agent. In contrast, it proved impossible to prevent the spread of BCV infection between the experimental groups and consequently all 20 study calves became infected with the virus. Following infection, BCV was detected in nasal secretions and in faeces of the calves and, after three weeks in the study, all had seroconverted to this virus. All calves, including the controls, showed at least one of the following clinical signs during days 3-15 after the trial started: fever (> or =40 degrees C), depressed general condition, diarrhoea, and cough. The study showed that BVDV primarily infected cattle, even when co-infected with an enteric and respiratory pathogen, are inefficient transmitters of BVDV. This finding supports the principle of the Scandinavian BVDV control programmes that elimination of BVDV infection from cattle populations can be achieved by identifying and removing persistently infected (PI) animals, i.e. that long-term circulation of the virus without the presence of PI animals is highly unlikely.     

Experimental infection of Nigerian sheep and goats with bovine virus diarrhoea virus

Apart from leucopenia and a low grade pyrexia Nigerian goats showed no ill effects following inoculation with bovine virus diarrhoea virus. Sheep showed a variable leucopenia without pyrexia. No difficulty should arise in differentiating BVD infections from peste des petits ruminants.     

Experimental inoculation of calves with laboratory strains of bovine viral diarrhea virus

Diarrhea, erosions and ulcers of the oral mucosa, with conjunctival and nasal discharges, were observed in six calves inoculated with a mixture of two laboratory cytopathic reference strains of bovine viral diarrhea virus (BVDV)-Oregon C24 V and NADL. The clinical picture was accompanied by biphasic body temperature elevation, transient leukopenia and a decrease in the number of lymphocytes. High dose of viruses and multiple routes of inoculation promoted the development of clinical and hematological changes typical for BVDV infection although laboratory strains were used.     

The pathology of concurrent bovine viral diarrhoea and infectious bovine rhinotracheitis virus infection in newborn calves

Concurrent bovine viral diarrhoea (BVD) and systemic infectious bovine rhinotracheitis (IBR) are reported from two neonatal (11 and 15 days old) calves. The diseases occurred sporadically in a large-scale herd which may have been due to the calves' heterogeneous immunobiological status. Gross pathological and histopathological examinations revealed focal interstitial pneumonia with acidophilic intranuclear inclusions in the alveolar epithelial cells and necrotic foci in the liver with a few intranuclear inclusions in the hepatocytes. There were subserous haemorrhages in the forestomachs and intestine, necrotic changes in the rumen, enteritis, lymphocytic necrosis in the Peyer's patches, and fibrinoid necrosis in the wall of some of the neighbouring blood vessels. BVD virus was demonstrated by immunofluorescence (IF), whereas IBR virus by electron microscopy, immunofluorescence and virus isolation.     

Hepatic immune response in calves during acute subclinical infection with bovine viral diarrhoea virus type 1

Eight colostrum-deprived calves aged 8-12 weeks were inoculated intranasally with a non-cytopathic strain of bovine viral diarrhoea virus (BVDV) genotype-1 and the effects on the hepatic immune response were studied. Two calves were sacrificed at each of 3, 6, 9 and 14 days post-inoculation (dpi) and two uninoculated animals were used as negative controls. BVDV was detected in hepatic macrophages and monocytes from 3 to 14dpi and in Küpffer cells (KCs) from 6 to 14dpi. Increases in the numbers of MAC387(+) KCs and monocytes, but not interstitial macrophages, differentiated by morphological features, were evident in the liver following inoculation with BVDV. There was a substantial increase in the number of monocytes positive for tumour necrosis factor (TNF)-α, but only small increases in the numbers of TNF-α(+) KCs and interstitial macrophages and interleukin (IL)-6(+) monocytes, KCs and interstitial macrophages. There was an increase in the number of interstitial CD3(+) T lymphocytes in the liver, but no substantial changes in the numbers of circulating CD3(+) T lymphocytes, interstitial or circulating CD4(+) or CD8(+) T lymphocytes, or CD79αcy(+) B lymphocytes. Serum haptoglobin and serum amyloid A increased transiently at 12dpi. Upregulation of some pro-inflammatory cytokines by hepatic macrophages is evident in subclinical acute BVDV type 1 infection in calves.     

Experimental infection and cross protection tests in calves with cytopathic strains of bovine rotavirus

Four cytopathic strains (81/32F, 81/36F, 81/40F, 82/80F) of bovine rotavirus were shown to be pathogenic for conventionally reared newborn calves. Calves were infected orally, using 3 calves for each isolate. All became febrile, were depressed and diarrhoeic. Two calves, one of which in the group of those infected with 81/36F isolate, and the other infected with strain 81/40F, were killed when moribund. A 3rd calf from the 81/36F infected group, died. At necropsy localized lesions of the small intestines, which are considered to be typical of rotavirus infection, were found. Virus was consistently isolated from the fecal samples of the inoculated calves up to 13 days post-inoculation. It was speculated that some differences existed in the virulence of the bovine rotaviruses tested. The cross protection tests revealed that 1 strain (81/36F) might be antigenically more complex than the others.     

Masking of two in vitro immunological assays for Mycobacterium bovis (BCG) in calves acutely infected with non-cytopathic bovine viral diarrhoea virus.

Acute infection of calves, previously vaccinated with bacille Calmette-Guerin (BCG), with non-cytopathic viral diarrhoea virus (BVDV) resulted in the temporary suppression of two in vitro assays used to monitor Mycobacterium bovis infection. Lymphocyte proliferation and interferon-gamma production by whole blood cultures containing purified protein derivatives prepared from Mycobacterium avium (PPD-A) and M bovis (PPD-B) were markedly suppressed. The implication is that acute infections of cattle with non-cytopathic BVDV may temporarily compromise diagnostic tests for M. bovis infections and result in a failure to identify cattle with tuberculosis.     

Experimental infection of calves with bovine respiratory syncytial virus (Quebec strain).

An experiment was designed to evaluate the clinical, haematological, viral and serological aspects of bovine respiratory syncytial virus infection in calves. Eleven calves were inoculated intranasally with bovine respiratory syncytial virus (Quebec strain) in aerosol. Clinical, haemotological and serological responses of the calves and virus shedding were monitored. The experimentally infected animals manifested moderate to severe signs of respiratory disease. The parameters used to evaluate the severity of the disease included ocular discharge, conjunctivitis, lung sounds, nasal discharge, pyrexia and leukopenia. The animals were scored accordingly (scale infected 70.8-148.5, control 22-29.3). Highest disease scores were observed between day 6-9 after infection. Virological and serological assessment demonstrated that the observed clinical picture was due to bovine respiratory syncytial virus infection.     

Experimental infection of cattle in early pregnancy with a cytopathic strain of bovine virus diarrhoea virus

Nine pregnant heifers, in early gestation (63 to 107 days), were infected intranasally or in utero with cytopathic bovine virus diarrhoea virus (BVDV) and each dam seroconverted. All nine calves developed to full term; four were stillborn, of which one had seroconverted but virus was not recovered from their tissues. One of the five liveborn calves appeared to have seroconverted in utero to an adventitious BVDV infection in late pregnancy but the remaining four were not viraemic and showed a normal secondary antibody response to BVDV infection at about six months old. Thus, in contrast to results with noncytopathic virus there was no evidence that infection in utero with cytopathic virus could result in a persistent viraemia or immunotolerance. It is suggested that cells able to support a persistent viraemia with cytopathic virus may not be developed in the young fetus.     

Primary bovine viral diarrhoea virus infection in calves following direct contact with a persistently viraemic calf.

Six calves, aged 24 to 58 days and not previously exposed to bovine viral diarrhoea virus (BVDV), were infected with this agent by nose-to-nose contact with a persistently BVDV viraemic calf. The study was conducted in two trials, using 3 calves in each. All 6 calves showed a peak interferon level in serum at 4 days post infection (dpi), and they seroconverted to BVDV at 16-21 dpi. The calves in trial 1 had diarrhoea for 2 or 3 days between 2 and 6 dpi and one calf again from 9 to 11 dpi. During the periods of fever, the calves were slightly depressed. Those in trial 2 were more depressed and their oral and nasal mucous membranes were reddened but they never had diarrhoea. In both trials, fever (up to 41.3 degrees C) was a prominent symptom at 8 to 9 dpi and 2 calves showed a diphasic fever course. Respiratory affection was mild and no medical treatment was required. Haematological assessment demonstrated a transient but significant leukopenia and lymphopenia at 4 dpi (P less than 0.01 and P less than 0.05 respectively) and 11 dpi (P less than 0.05 and P less than 0.01 respectively). A significant decrease in thrombocyte count was seen at 4 dpi (P less than 0.05, n = 3). This study has demonstrated that nose-to-nose contact is an effective way of transmitting BVDV from persistently infected to susceptible cattle.     

Experimental bovine respiratory syncytial virus infection in conventional calves: ultrastructural respiratory lesions

The morphogenesis and repair of airway and alveolar injury induced by bovine respiratory syncytial virus (BRSV) was studied ultrastructurally in conventional calves to characterize pulmonary cell types susceptible to viral infection and cytopathologic changes associated with infection. Viral nucleocapsids and budding virions were present in tracheal and bronchial ciliated and nonciliated epithelial cells and mucous cells 3, 5, and 7 days after inoculation and in bronchiolar ciliated and nonciliated epithelial cells 5 days after inoculation. Mild interstitial pneumonia was observed 5 days after inoculation and was characterized by swelling of type 1 and type 2 alveolar epithelial cells, interstitial edema, and infiltration by lymphocytes and macrophages. Viral assembly and release in tracheal and bronchial epithelial cells was associated with loss of cilia from ciliated cells, formation of syncytial epithelial cells, swelling of mitochondria and endoplasmic reticulum, and cell necrosis. Neutrophils, lymphocytes, and macrophages were present in close association with the viral-infected and damaged epithelial cells. There was intercurrent hyperplasia of basal epithelial cells that, in association with other epithelial lesions, resulted in the loss of normal ciliated epithelium in these airways 5 and 7 days after inoculation. Regeneration of airway epithelium was largely completed by 10 days after inoculation, except in 1 of 4 calves that had failure of epithelial repair and that developed secondary bacterial pneumonia. Pulmonary ultrastructure in BRSV-inoculated calves 30 days after inoculation was indistinguishable from that in controls. The results demonstrated that BRSV can induce reversible alterations in airway epithelium, which may cause depression of mucociliary clearance and thereby enhance susceptibility to bacterial infection.     

Experimental infection of rabbits with bovine viral diarrhoea virus by a natural route of exposure

Bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle that can naturally infect a wide range of even-toed ungulates. Non-bovine hosts may represent reservoirs for the virus that have the potential to hamper BVDV eradication programs usually focused on cattle. Rabbits are very abundant in countries such as the United Kingdom or Australia and are often living on or near livestock pastures. Earlier reports indicated that rabbits can propagate BVDV upon intravenous exposure and that natural infection of rabbits with BVDV may occur but experimental proof of infection of rabbits by a natural route is lacking. Therefore, New Zealand White rabbits were exposed to a Scottish BVDV field strain intravenously, oro-nasally and by contaminating their hay with virus. None of the animals showed any clinical signs. However, the lymphoid organs from animals sacrificed at day five after exposure showed histological changes typical of transient infection with pestivirus. Most organ samples and some buffy coat samples were virus positive at day five but saliva samples remained negative. Development of antibodies was observed in all intravenously challenged animals, in all of the nebulised group and in four of six animals exposed to contaminated hay. To our knowledge this is the first report of BVDV propagation in a species other than ruminants or pigs after exposure to the virus by a natural route. However, to assess the role of rabbits as a potential reservoir for BVDV it remains to be determined whether persistent infection caused by intra-uterine infection is possible and whether BVDV is circulating in wild rabbit populations.     

Persistent bovine virus diarrhoea virus infection in a bull

Investigation of a sight defect in a pedigree bull, born as a result of artificial insemination and ovum transplantation, led to the finding that the animal was persistently infected with bovine virus diarrhoea virus. Virus was cultured from blood and from nasal and ocular swabs and was present in semen in high titre. At necropsy, virus was cultured from a wide range of tissues. The pathological findings are described and discussed as are the potential hazards of such infections.     

Spread of bovine virus diarrhoea virus in a herd of heifer calves.

A calf persistently infected and immunotolerant to Bovine Virus Diarrhoea virus (BVD virus) was, on purpose, introduced to a herd of heifer calves over 4 months of age that had been reared as recipients for embryo transplantation. All calves were brought in contact with the persistently infected animal. In total, 240 calves were involved in this experiment, 22 of which were serologically negative when introduced. These serologically negative animals developed antibodies against BVD virus within 5 months after introduction. At short distances from the persistently infected BVD virus shedder, negative calves seroconverted within 2 months, but at greater distances the moment of seroconversion was unpredictable. The calves that had undergone a natural infection with BVD virus received embryos after transportation to an allied farm. In total, 14 calves were born after embryo transplantation, all of which were free of BVD virus, in spite of the presence of BVD-virus on the latter farm.     

Experimental infection of calves with bovine leukemia virus (BLV): an applicable model of a retroviral infection

An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.     

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.