Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.     

Interferon, fluorescent antibody, and neutralizing antibody responses in sera of calves inoculated with bovine respiratory syncytial virus

Interferon, fluorescent antibody, and neutralizing antibody responses were studied in sera of 9 calves inoculated with bovine respiratory syncytial virus, in relation to viral shedding and clinical signs of disease. The calves (5.5 to 6.5 weeks of age) were assigned to 3 groups. Group I was inoculated once with the virus, and groups II and III were challenge exposed at postinoculation day (PID) 15 or 37. Serum-neutralizing and indirect fluorescent antibody techniques were used to measure antibody responses. The plaque-inhibition technique, using vesicular stomatitis virus, was applied to measure serum interferon titers. The virus was recovered by inoculation of nasal secretions onto cell cultures. Fluorescent antibody was detected in all calves on PID 3, with maximum titers appearing approximately on PID 10. Low neutralizing antibody was detected in most animals on PID 3, and titers peaked approximately 4.5 weeks after inoculation and then decreased. Interferon titers were high in all calves during the early stage of infection, dropped to undetectable amounts by PID 6, and reappeared in low amounts at least 1 week later. All infected calves manifested clinical signs of disease by PID 4 to 9. Clinical signs of disease were not observed after challenge exposure at PID 15 or 37, and anamnestic responses were not detected. Virus was recovered after challenge exposure at PID 15, but not at PID 37.     

A comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens.

Virus shedding was monitored in nasal secretions of 12 calves experimentally infected with bovine respiratory syncytial virus (BRSV) using an antigen capture enzyme-linked immunosorbent assay (ELISA) detecting the nucleoprotein (NP) antigen of BRSV, by a polymerase chain reaction (PCR) amplifying the fusion protein of BRSV, and by a microisolation assay combined with immunoperoxidase staining for the F protein of BRSV. Under the conditions of this study, similar limits of detection and quantitative results were obtained from all three assays. BRSV was detected in nasal secretions of all calves for a minimum of 4 d. Virus shedding began on Day 2 after infection, peaked on Days 3-5, and was cleared in most calves by Day 8. The PCR, and to a lesser extent the ELISA, may detect virus shedding for a longer period after infection than virus isolation, possibly due to neutralization of the virus by rising mucosal antibody. Simulated environmental conditions likely to be experienced during transport of clinical field specimens markedly reduced the sensitivity of virus isolation but had a minimal effect on the results of the NP ELISA. Actual field transport conditions (overnight on ice) had minimal apparent effect on the results of the PCR assay. The less stringent specimen handling requirements, combined with low limits of detection, of both the nucleoprotein ELISA and PCR, indicate either of these assays are more suitable for diagnostic applications than virus isolation.     

Experimental infection of calves with strains of Bovid herpesvirus-4

Fourteen calves were inoculated intranasally (i.n.) with the viral isolates as follows: 5 with 85/BH 16TV, 1 with 85/BH 17TV, 1 with 85/BH 18TV, 2 with 85/BH 231TN and 5 with 85/BH 232TN. Strain 85/BH 16TV was the only one which caused overt respiratory-like disease in all inoculated calves. Onset of the disease was observed after 7-8 days of incubation and was characterized by fever, depression, nasal discharge and coughing. Virus was isolated from the nasal swabbings of calves obtained from post-infection day (PID) 2-10. The other viral strains did not cause any sign of disease although virus was isolated regularly from the nasal swabbings of the inoculated calves. Virus was recovered from central nervous system tissues of calves that were infected with 85/BH 16TV or 85/BH 232TN strains and were killed on PID 4 or 8. Virus was also isolated from other tissues, such as lymph node, nasal mucosa (PID 8), or lung (PID 4). It was speculated that the nervous system could be one of the target areas of the virus of the naturally occurring infection by BHV-4. This might indicate a possible role of the nervous system (site of latency?) in the pathogenesis of BHV-4 as is the case in certain herpesviral infections of man and the lower animals.     

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.     

Exposure of calves to aerosols of parainfluenza-3 virus and Pasteurella haemolytica.

The present study was undertaken to investigate whether sequential exposure to aerosols of parainfluenza-3 virus followed by Pasteurella haemolytica, or P. haemolytica followed by parainfluenza-3 virus, could lead to the production of pulmonary lesions in conventionally-raised calves. Twenty male calves with low serum antibody titres to both organisms were placed in five equal groups. Synergism of parainfluenza-3 virus and P. haemolytica was not demonstrated in any of the sequentially infected groups and pulmonary lesions were mild in all challenged calves. Clinical signs of disease were not present after exposure to parainfluenza-3 virus although the virus was repeatedly isolated from nasal secretions of all inoculated calves. Exposure to P. haemolytica produced a transient response which consisted of increased rectal temperatures and respiratory rates, with a mild neutrophilic leukocytosis and a mild left shift present six hours postinoculation and returning to normal within 24 hours. Results from this study suggest, although do not confirm, that reduced pulmonary clearance of inhaled P. haemolytica in parainfluenza-3 virus infected calves does not necessarily lead to production of severe pulmonary lesions and that previous exposure to aerosols of P. haemolytica may not enhance secondary parainfluenza-3 virus infection.     

Efficacy of an inactivated respiratory syncytial virus vaccine in calves.

OBJECTIVE: To determine whether an inactivated bovine respiratory syncytial virus (BRSV) vaccine would protect calves from infection with virulent BRSV. DESIGN: Randomized controlled trial. ANIMALS: 27 nine-week-old calves seronegative for BRSV exposure. PROCEDURE: Group-1 calves (n = 9) were not vaccinated. Group-2 calves (n = 9) were vaccinated on days 0 and 21 with an inactivated BRSV vaccine containing a minimum immunizing dose of antigen. Group-3 calves (n = 9) were vaccinated on days 0 and 21 with an inactivated BRSV vaccine containing an amount of antigen similar to that in a commercial vaccine. All calves were challenged with virulent BRSV on day 42. Clinical signs and immune responses were monitored for 8 days after challenge. Calves were euthanatized on day 50, and lungs were examined for lesions. RESULTS: Vaccination elicited increases in BRSV-specific IgG and virus neutralizing antibody titers and in production of interferon-gamma. Virus neutralizing antibody titers were consistently less than IgG titers. Challenge with BRSV resulted in severe respiratory tract disease and extensive pulmonary lesions in control calves, whereas vaccinated calves had less severe signs of clinical disease and less extensive pulmonary lesions. The percentage of vaccinated calves that shed virus in nasal secretions was significantly lower than the percentage of control calves that did, and peak viral titer was lower for vaccinated than for control calves. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the inactivated BRSV vaccine provided clinical protection from experimental infection with virulent virus and decreased the severity of pulmonary lesions. Efficacy was similar to that reported for modified-live BRSV vaccines.     

Bovine herpesvirus-1 vaccination against experimental bovine herpesvirus-1 and Pasteurella haemolytica respiratory tract infection: onset of protection

The onset of protection offered by intranasal vaccination with attenuated bovine herpesvirus-1 (BHV-1) was studied in 18 calves given a virulent BHV-1 aerosol challenge inoculum and an aerosol challenge exposure to Pasteurella haemolytica. Calves challenge exposed with virus 3, 7, 11, 15, or 19 days after vaccination and challenge exposed 4 days later with Pasteurella haemolytica did not develop viral-bacterial pneumonia, whereas 2 of 3 control calves died of fibrinous bronchopneumonia 40 and 60 hours after the bacterial aerosol and the 3rd control calf had similar lesions. All vaccinated and control calves had detectable amounts of interferon at the time of viral challenge exposure. Protection was observed before detection of neutralizing antibodies to BHV-1 in nasal secretions or in serum. Protection was therefore present from day 3 through day 19 after vaccination, but the mechanism could not be explained completely by neutralizing antibody or interferon.     

Horizontal transmission of pseudorabies virus in cattle

Pseudorabies virus (PRV) was not transmitted horizontally from 3 PRV-infected calves to 2 contact control calves during 4 days of comingling in experiment 1. Although these contact control calves developed clinical signs of pseudorabies when infected intranasally with PRV in experiment 2, they did not transmit PRV to a second pair of contact control calves. However, 1 of 2 pigs comingled with these 4 calves seroconverted. During both experiments, moderate amounts (10(2) to 10(5) TCID50) of PRV were present in the nasal secretions of the infected calves during the contact periods. All infected calves traumatized their nares or periorbital tissue. Infected calves developed a nonsuppurative meningoencephalitis mainly involving the brain stem. Four of the 5 infected calves had nonsuppurative ganglioneuritis and acute lymphoid necrosis of germinal centers. Virus could not be recovered from nasal and tonsillar swab samples from contact-control calves and pigs.     

Neural changes in recurrent infection of infectious bovine rhinotracheitis virus in calves treated with dexamethasone.

Recurrent infection by infectious bovine rhinotracheitis (IBR) virus was induced in calves by dexamethasone (DM) treatment (given 5 days) at 5 months after primary infection. The virus appeared in nasal secretions of the calves on the 4th day after initiation of DM treatment and continued until the 9th day. The calves were killed on the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, and 11th days after DM treatment was started for examination by histopathologic and immunofluorescent antibody techniques. The most significant neural change was trigeminal ganglionitis with neuronophagia, which was observed from the 3rd to the 11th day. Significantly, the extent of changes in the trigeminal ganglion and medulla oblongata corresponded to the amount of DM treatment administered. The IBR virus antigen was first observed in the trigeminal ganglion cells, and thereafter, it was detected in the Schwann cells, satellite cells, neuroglia cells, and nasal mucosa until the 10th day. These observations indicate that the IBR virus is capalbe of producing a persistent infection in the trigeminal ganglion and that trigeminal ganglionitis may be a characteristic lesion for inducing the reactivation of lagent IBR virus.     

Association between route of inoculation with infectious bovine rhinotracheitis virus and site of recrudescence after dexamethasone treatment

Four calves latently infected with infectious bovine rhinotracheitis virus (IBRV) were used to compare the ease of isolation of virus from neuronal ganglia and from mucosal surfaces. Two calves were slaughtered, and neuronal ganglia (cranial cervical, trigeminal, and 3rd and 4th sacral) were cocultivated on bovine fetal kidney cells. Virus was not isolated. Two calves given dexamethasone for 4 days were slaughtered on the 5th day. Virus was not isolated from cocultivated or macerated neuronal ganglia, but virus was isolated from nasal secretions taken from both calves on the day of slaughter. Eleven calves were inoculated with IBRV via different routes and were treated with dexamethasone 3 to 4 months after inoculation. virus was isolated from the nasal cavities, but not the vaginas of 6 heifers inoculated intranasally, and was isolated from the vaginas, but not the nasal cavities of 2 heifers inoculated intravaginally. Of 3 calves inoculated IV, virus was isolated from the nasal cavities of 3, from the oropharynxes of 2, and from the prepuce of 1.     

Virus isolation and immune responses in susceptible swine exposed with pseudorabies virus (Shope strain).

Eighteen seronegative swine weighing from 9 to 11 kg were exposed intranasally with the Shope strain of pseudorabies virus (PRV) and were observed for 21 days in an experiment to detect virus shedding and immune responses. All swine had PRV in their nasal passages at 7 days after exposure; they also had precipitating antibodies to PRV as determined by the microimmunodiffusion test (MIDT) and very low levels of virus-neutralizing (VN) antibodies. The PRV was isolated from only 2 swine at postexposure day 14; all swine were MIDT positive, and VN titers ranged from 4 to 128. Virus was not isolated from the swine at 21 days after exposure, but all were MIDT positive; VN titers ranged between 8 and greater than or equal to 256.     

EXPERIMENTAL ADENOVIRUS PNEUMONIA IN CALVES

A mild interstitial pneumonia with mononuclear cell infiltration was produced in calves following intra-tracheal inoculation of the BIL adenovirus. Viral antigen appeared early in regional lymph node. An interstitial reaction developed early in the lungs, and pneumonic lesions and infective virus were present by the third day after inoculation. Virus spread rapidly trachea, tonsils and nasal mucosa, and was creted from the third to eighth day. Upper and lower respiratory tissues continually harboured virus until the 12th day, when the experiment was terminated. The virus was isolated from the livers of two calves. All tissues yielded low concentrations of virus, and only the lung showed pathological reactions. The reactions were qualitatively similar to known adenovirus pneumonias, but considerably milder, and lacked the characteristic necrotic and proliferative bronchiolar changes.     

OBSERVATIONS OF SW AINSONA GALEGIFOLIA POISONING IN CATTLE IN NORTHERN NEW SOUTH WALES

A mild interstitial pneumonia with mononuclear cell infiltration was produced in calves following intra-tracheal inoculation of the BIL adenovirus. Viral antigen appeared early in the regional lymph node. An interstitial reaction developed early in the lungs, and pneumonic lesions and infective virus were present by the third day after inoculation. Virus spread rapidly to trachea, tonsils and nasal mucosa, and was excreted from the third to eighth day. Upper and lower respiratory tissues continually harboured virus until the 12th day, when the experiment was terminated. The virus was isolated from the livers of two calves. All tissues yielded low concentrations of virus, and only the lung showed pathological reactions. The reactions were qualitatively similar to known adenovirus pneumonias, but considerably milder, and lacked the characteristic necrotic and proliferative bronchiolar changes.     

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.     

Experimental placental transfer of Akabane virus in the hamster

Transplacental infection of hamster fetuses was produced by inoculation of pregnant hamsters with 10(6.3) plaque-forming units (PFU) of Akabane virus by either the intraperitoneal or the subcutaneous route. Virus with titers as high as 10(7.5) PFU/g of tissue was detected first in the placenta and later in the fetus. Virus could also be readily isolated from blood, lung, spleen, and liver of both pregnant and nonpregnant hamsters, but it reached higher titers and persisted longer in the placenta and fetus. Young dying at birth had Akabane virus titers as high as 10(7.3) PFU/g of brain tissue. Litter size was reduced by inoculation of the pregnant hamster at gestational day 11 or earlier, and survival of the newborn to 1 week of age was decreased by inoculation at gestational day 9 or later.     

Humoral immune response of calves to bluetongue virus infection

Humoral immune responses of 7 calves to bluetongue virus (BTV) infection were evaluated by plaque-reduction assay and immunoblotting. Most readily interpretable results were obtained with the immunoblot assay when colostrum-deprived calves were used, and sera were reacted with proteins in partially purified extracts of BTV. Viremia persisted in calves for 35 to 56 days, and BTV coexisted in blood for several weeks with virus-specific neutralizing antibody. Calves developed antibody to virus protein 2, the major determinant of virus neutralization, at 14 to 28 days after inoculation; this time interval also coincided with the appearance of neutralizing antibody in serum. Virus clearance in BTV-infected calves did not coincide with humoral immune responses to protein 2 or other virion proteins.     

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.     

North American Strain of Viral Hemorrhagic Septicemia Virus is Highly Pathogenic for Laboratory-Reared Pacific Herring

Abstract

Specific-pathogen-free Pacific herring Clupea pallasi were reared in the laboratory from eggs and then challenged at 5, 9, and 13 months of age by waterborne exposure to low (10^1.5–2.5 plaque-forming units [PFU] per milliliter), medium (10^3.5–4.5 PFU/mL), or high (10^5.5–6.5 PFU/mL) levels of a North American isolate of viral hemorrhagic septicemia virus (VHSV). The fish were extremely susceptible to the virus, showing clinical disease, mortality approaching 100%, and only a limited increase in resistance with age. Mortality began 4–6 d after exposure and peaked at approximately day 7 in fish exposed to high levels of virus. Whereas the mean time to death showed a significant dose response (P < 0.001), the percent mortality and virus titers in dead fish were generally high in all groups regardless of initial challenge dose. External signs of disease were usually limited to 1–2-mm hemorrhagic areas on the lower jaw and isthmus and around the eye, but 2 of 130 infected fish exhibited extensive cutaneous hemorrhaging. Histopathologic examination of tissues from moribund fish sampled at 2–8 d after exposure revealed multifocal coagulative necrosis of hepatocytes, diffuse necrosis of interstitial hematopoietic tissues in the kidney, diffuse necrosis of the spleen, epidermis, and subcutis, and occasional necrosis of pancreatic acinar cells. Virus titers in tissues of experimentally infected herring were first detected 48 h after exposure and peaked 6-8 d after exposure at 10^7.7 PFU/g. Fish began shedding virus at 48 h after exposure with titers in the flow-through aquaria reaching 10^2.5 PFU/mL at 4–5 d after exposure, just before peak mortality. When the water flow was turned off for 3 h, titers in the water rose to 10^3.5 PFU/mL, and the amount of virus shed by infected fish (on average, greater than 10^6.5 PFU/h per fish) appeared sufficient to sustain a natural epizootic among schooling herring. Taken together, these data suggest that VHSV could be a significant limiting factor for populations of Pacific herring.     

Detection of pseudorabies virus infection in subunit-vaccinated and nonvaccinated pigs using a nucleocapsid-based enzyme-linked immunosorbent assay.

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 10(4) PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid-specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (10(2.3) PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs.