The vaccination and challenge with bovine viral diarrhea virus (BVDV) of calves previously infected with a non-cytopathic BVDV.

Four calves were infected with noncytopathic (NCP) New York-1 strain of bovine viral diarrhea virus (BVDV). During the observation period of one month the calves remained clinically normal but the virus was repeatedly recovered from their pharyngeal swabbings and blood. Thirty days following infection the four calves were vaccinated, together with two uninfected calves, with a modified-live vaccine containing cytopathic (CP) BVDV, infectious bovine rhinotracheitis virus and parainfluenza-3 virus. No detrimental effects were observed after vaccination. Forty-three days after vaccination the calves were challenged by exposure either with the CP TVM-2 strain or the NCP New York-1 strain of BVDV. The vaccinated calves remained healthy throughout the 60-day observation period.     

THE SUSCEPTIBILITY OF CALVES TO INFECTION WITH A STRAIN OF BOVINE EPHEMERAL FEVER VIRUS INOCULATED INTRACEREBRALLY

Three newborn calves were inoculated intracerebrally with bovine ephemeral fever virus strain 525. The 2 that lacked detectable neutralising antibody to bovine ephemeral fever vaccine developed fatal encephalitis after 4 and 7 days respectively. The third calf which had a low level of maternal antibody remained healthy and developed antibody that became undetectable after 6 months. Bovine ephemeral fever virus strain 525 was reisolated from the brains of both dead calves by intracerebral inoculation of suckling mice. Homogenates that were prepared from the brains of the calves failed to produce disease or to induce antibody formation in susceptible calves when inoculated intravenously. Strain 525 of BEF virus has been shown to possess a degree of neurovirulence for laboratory animals that has not been reported for other strains (Tzipori and Spradbrow 1974). Although this strain is unable to produce viraemia in calves after I/V inoculation, the present study shows that strain 525 can multiply in the brain tissues of calves and cause death after I/C inoculation.     

Further observations on the characteristics of a bovine parainfluenza-3 variant.

Calves were inoculated with a bovine para-influenza-3 variant to determine its pathogenicity and the stability of its cytopathic feature and its inability to agglutinate chicken erythrocytes. The inoculated calves and one contact animal developed an immune response without significant clinical illness. The clinical response in calves was similar to that induced by the parent virus strain. The variant was shown to retain its characteristic cytopathic effect for Madin Darby bovine kidney cells and its property of hemagglutination following one passage in the natural host.     

Studies on the occurrence and significance of bovine respiratory syncytial virus in Saskatchewan.

Seventy-six percent of 49 blood samples from Saskatchewan cattle had serum antibodies against bovine respiratory syncytial virus. Experimental infection of one week and seven month old calves with bovine respiratory syncytial virus (Iowa strain) caused transient fever, mucopurulent nasal discharge and coughing but no macroscopic or microscopic lesions attributable to bovine respiratory syncytial virus.     

Potential use of hematological and acute phase protein parameters in the diagnosis of acute Schmallenberg virus infection in experimentally infected calves

The initial viraemic phase of Schmallenberg virus (SBV) infection in bovine animals is characterized by the non-specific and inconspicuous clinical signs of pyrexia (>40 °C), drop in milk yield and sometimes diarrhea. As a result, the early detection of SBV epizootics can difficult, and typically only become apparent when the congenital form of the disease is observed. The aim of the study was to describe the course of the acute phase response and haematological findings in bovine calves following experimental SBV infection. No clinical signs except for increase in rectal temperature were observed in the calves inoculated subcutaneously with a Polish strain of SBV. Viral RNA was detected in serum at 2 and 4 days post inoculation (dpi). SBV antibodies were first detected by ELISA (9–21 dpi), and subsequently by virus neutralization test (14–32 dpi). The hematological parameters showed a reduction in mid-size leucocytes (MID), and also in red blood cell count (RBC). An increase in mean corpuscular hemoglobin was also observed in SBV infected calves. No significant difference in acute phase proteins (APP) was observed between experimentally infected and control calves, suggesting limited potential as diagnostic biomarker of acute SBV infection.     

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen.     

Experimental Infection of Calves with Bovine Viral Diarrhoea Virus Type-2 (BVDV-2) Isolated from a Contaminated Vaccine

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100% homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.     

A study of some pathogenetic aspects of bovine viral diarrhea virus infection

The cytopathic (CP) TVM-2 strain of bovine viral diarrhea virus (BVDV) induced in calves a severe disease, characterized by the clinical picture which is usually reported for the acute primary infection observed under natural conditions. In contrast, the calves inoculated with a different biotype of BVDV, the non-cytopathic (NCP) New York-1 strain, remained clinically normal with the only evidence of virus replication in these calves being the recovery of the virus from their pharyngeal swabbings and blood and also the detection of specific neutralizing antibody in their serums. When calves were immunosuppressed with dexamethasone (DMS), they underwent an overt systemic disease of such a severity that in most of the cases it ended with the death of the animals. This result was obtained with either the CP and the NCP strain of BVDV. Finally, the mixed infection that was obtained in the calves with the CP and the NCP BVDV did not result in any particular unexpected pathological situation. It was speculated that the immunosuppressive activity of BVDV could be a property peculiar to certain isolates of the virus.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretions

Six calves inoculated intranasally with a vaccinal strain of infectious bovine rhinotracheitis (IBR) virus and 6 control calves were given a placebo. All calves were subsequently challenge exposed (by aerosol) with rhinovirus--3 of the calves from each group at 2 days after they were inoculated with IBR virus or with placebo and the remaining calves at 6 days. Nasal excretion of viruses, interferon (IFN) concentrations in nasal secretions (NS), and neutralizing antibody in sera and NS were determined. All calves given the vaccinal IBR virus subsequently had IFN in their NS. Interferon was detected as early as 1 day, reached maximal titers at 2 to 4 days, and persisted in individual calves for 5 to 10 days after inoculation. Rhinovirus shedding was not detected from IBR virus-inoculated calves whose NS contained both rhinovirus antibody and IFN at the time of challenge exposure; such calves were protected at either 2 or 6 days after IBR virus inoculation. The outcome of rhinovirus challenge exposure of calves whose NS contained IFN, but not rhinovirus antibody, varied with the day of challenge exposure. Rhinovirus excretion was detected from 2 of these calves challenge exposed 2 days after IBR virus inoculation, but was not detected from a calf challenge exposed 6 days after inoculation. However, while IFN was present in NS from the former 2 calves, rhinovirus shedding was markedly reduced as compared with that from control calves without IFN or NS antibody at the time of challenge exposure. Consistent relationship was not observed between the rhinovirus neutralizing antibody titer of calves' sera and NS. The antibody titer of NS more closely correlated with protective immunity to rhinovirus infection than did the serum antibody titer.     

Effects in calves of mixed infections with bovine viral diarrhea virus and several other bovine viruses

The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced.

Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus.

From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.     

Identification and eradication of bovine viral diarrhea virus in a persistently infected dairy herd.

A milking herd consisting of 55 Holstein cows had experienced abortions in several cows, as well as congenital malformations in 1 newborn calf. Bovine viral diarrhea virus was isolated from blood mononuclear cell samples obtained from several cattle, documenting 1 acute infection and 8 persistently infected carriers identified by clinical appearance and laboratory testing. Initial suspicion of persistently infected status in some, but not all animals, was facilitated by poor growth rates in some calves. Virus isolation was performed on transtracheal wash fluid obtained from acutely and persistently infected cattle with respiratory tract infection. We describe the measures taken to identify and characterize the infecting virus strain, and the series of actions taken to identify and eliminate persistently infected carriers in a herd experiencing several related problems that were shown to be caused by bovine viral diarrhea virus.     

Recrudescence of infectious bovine rhinotracheitis virus and associated neural changes in calves treated with dexamethasone

Reactivation of infection bovine rhinotracheitis (IBR) virus in calves administered dexamethasone (DM) was studied in 2 experiments. At 2, 3, 5, 15, or 30 months after inoculation of the Los Angeles strain of IBR virus, IV injections of DM were given for 5 consecutive days to induce a recurrent infection (experiment 1). Three months after the 1st treatment, a 2nd recurrent infection was induced, using DM with the same doses as used in experiment 1. The virus was excreted from nasal secretions from the 4th to the 10th day after initial treatment with DM, and from the 6th to the 9th day after the 2nd treatment. On pathologic examination, trigeminal ganglionitis, consisting of many proliferated microglia and inflammatory cells, was observed in all DM-treated calves. Moreover, degeneration of the ganglion cells and neuronophagia were prominent features in the calves after the 2nd recurrent infection. These observations indicated that the trigeminal ganglion may be one of the latent sites of IBR virus in calves after intranasal infection and that calves can develop a recrudescent infection after DM treatment several times during their lifetime.     

A comparison of the virulence of three strains of infectious bovine rhinotracheitis virus

The virulence of three strains of infectious bovine rhinotracheitis (IBR) virus was compared in six-month-old Ayrshire-cross calves. The strains were an isolate from a recent severe outbreak of IBR in Scotland (Strichen strain), the prototype British strain (Oxford strain) and a North American isolate (Colorado strain). The Colorado and Strichen strains produced the characteristic clinical signs and pathological lesions of severe IBR three to four days post infection (p.i.). The Strichen strain was slightly more virulent, possibly as a result of its having been passaged fewer times in tissue culture. In contrast, the Oxford strain produced a mild clinical response with minimal pathological lesions. Virus was recovered from nasal swabs for a longer period from the calves infected with the Strichen strain (up to 13 days p.i.) and Colorado strain (up to 12 days p.i.) than from the animals infected with the Oxford strain (up to 10 days p.i.). These findings support the suggestion that the recent epidemic of severe IBR in Britain had resulted from the importation of a “new” strain of virus.     

Neural changes in vaccinated calves challenge exposed with virulent infectious bovine rhinotracheitis virus

Recurrent infection in calves vaccinated with infectious bovine rhinotracheitis-(IBR) modified live virus was induced by dexamethasone (DM) treatment given 49 days after challenge exposure with virulent IBR virus. Nonchallenge-exposed IM and intranasally vaccinated calves did not excrete the virus after DM treatment; however, IM and intranasally vaccinated and subsequently challenge-exposed calves excreted the challenge-exposure virus into the nasal secretions 5 to 11 days and 6 to 10 days after the DM treatment, respectively. The calves were killed 15 to 18 days (experiment 1) and 14 days (experiment 2) and DM treatment was started and then were examined by histopathologic and fluorescent antibody techniques. All DM-treated calves that were inoculated with the vaccinal virus and challenge exposed with the virulent virus developed nonsuppurative trigeminal ganglionitis and encephalitis. On the contrary, the DM-treated nonchallenge-exposed vaccinated calves did not have lesions in the peripheral nervous system and CNS. Infectious bovine rhinotracheitis virus antigens were not observed in tissues of any of the calves examined (experiments 1 and 2) by fluorescent antibody techniques. These observations indicated that the modified live IBR virus neither produced lesions nor induced latent infection and that modified live IBR virus vaccination did not protect the calves against the establishment of a latent infection after their exposure to large doses of the virulent IBR virus.     

Clinical study of the disease of calves associated with Mycoplasma bovis infection

Clinical, bacteriological and serological examination of 35 calves from the age of 5 to 26 days was performed in a Holstein-Friesian dairy herd endemically infected with Mycoplasma bovis. M. bovis was isolated from 48.6% of nasal swabs taken from the calves at the age of 5 days, and from 91.4% of the same calves at the age of 26 days, indicating the gradual spread of infection. The isolation rate of Pasteurella multocida did not change much, and varied from 28.6 to 25.7%. No P. haemolytica could be detected. In addition to M. bovis and P. multocida, the herd was also infected with different viruses (including bovine viral diarrhoea virus, infectious bovine rhinotracheitis virus, bovine adenoviruses, parainfluenza-3 virus, and bovine respiratory syncytial virus) as a large proportion of the sera of newborn calves contained colostral antibodies against these viruses. In most of the newborn calves severe clinical signs (fever, depression, inappetence, hyperventilation, dyspnoea, nasal discharge and coughing) due to M. bovis infection developed. The clinical signs appeared already on the fifth day of life, and their incidence was the highest at the age of 10 to 15 days. Three calves (8.6%) died as a result of severe serofibrinous pneumonia. The surviving calves showed very poor weight gain (ranging from 1.5 to 3.5 kg) during the first two weeks of life.     

Level and duration of serum antibodies in cattle infected experimentally and naturally with bovine virus diarrhoea virus

Neutralising serum antibodies against bovine virus diarrhoea virus (BVDV) were monitored for three years in 35 cattle that were infected with the virus as calves; 24 of the calves were inoculated intramuscularly or intranasally, and 11 contracted the infection naturally. All the experimentally infected calves seroconverted within 14 to 28 days after inoculation, and all the animals still had high serum levels of antibodies to BVDV three years after infection. Determinations of antibody levels in milk and blood samples excluded the possibility that the calves had been reinfected with BVDV during the study.     

Platelet aggregation responses and virus isolation from platelets in calves experimentally infected with type I or type II bovine viral diarrhea virus.

Altered platelet function has been reported in calves experimentally infected with type II bovine viral diarrhea virus (BVDV). The purpose of the present study was to further evaluate the ability of BVDV isolates to alter platelet function and to examine for the presence of a virus-platelet interaction during BVDV infection. Colostrum-deprived Holstein calves were obtained immediately after birth, housed in isolation, and assigned to 1 of 4 groups (1 control and 3 treatment groups). Control calves (n = 4) were sham inoculated, while calves in the infected groups (n = 4 for each group) were inoculated by intranasal instillation with 10(7) TCID50 of either BVDV 890 (type II), BVDV 7937 (type II), or BVDV TGAN (type I). Whole blood was collected prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation for platelet function testing by optical aggregometry by using adenosine diphosphate and platelet activating factor. The maximum percentage aggregation and the slope of the aggregation curve decreased over time in BVDV-infected calves; however, statistically significant differences (Freidman repeated measures ANOVA on ranks, P < 0.05) were only observed in calves infected with the type II BVDV isolates. Bovine viral diarrhea virus was not isolated from control calves, but was isolated from all calves infected with both type II BVDV isolates from days 4 through 12 after inoculation. In calves infected with type I BVDV, virus was isolated from 1 of 4 calves on days 4 and 12 after inoculation and from all calves on days 6 and 8 after inoculation. Altered platelet function was observed in calves infected with both type II BVDV isolates, but was not observed in calves infected with type I BVDV. Altered platelet function may be important as a difference in virulence between type I and type II BVDV infection.     

Immunofluorescent Studies of Bovine Para-Influenza 3 Virus II. Experimentally Infected Calves

Fluorescent antibody (FA) studies of tissues from three colostrum deprived calves inoculated intranasally with the SF-4 strain of bovine para-influenza 3 (PI-3) virus indicated that these calves developed a mild upper respiratory infection but infected cells were not identified in the lower respiratory tract. Three other calves inoculated intranasally and intratracheally with PI-3 virus developed more severe clinical signs of infection and virus was identified, by FA techniques, in the upper and lower respiratory tract of all three calves and in the spleen of one calf. PI-3 virus was detected in smears of nasal epithelium from five of six calves at some time during the observation period.     

THE SUSCEPTIBILITY OF YOUNG AND NEWBORN CALVES TO BOVINE EPHEMERAL FEVER VIRUS

The pathogenicity of a field strain, 417, of bovine ephemeral fever (BEF) virus for newborn and young calves was investigated. Three colostrum-deprived newborn calves inoculated intravenously developed severe clinical disease and viraemia, and produced long-lasting neutralising antibody. The incubation period in these animals was 10 and 11 days, compared with 5 to 7 days for older calves. Two newborn calves which received colostrum from immune dams and 2 which received colostrum from non-immune dams failed to respond clinically to intravenous inoculation with strain 417. The neutralising antibody response of these calves was of short duration. Four calves, 7 to 8 weeks old and lacking detectable neutralising antibody to BEF virus, or having low levels of antibody, did not develop clinical disease when inoculated intravenously. Four calves 12 to 14 weeks of age and free of detectable neutralising antibody to BEF virus developed clinical disease when inoculated with strain 417.