Effect of viral dose on experimental pneumonia caused by aerosol exposure of calves to bovine herpesvirus 1 and Pasteurella haemolytica.

The effect of various aerosol doses of bovine herpesvirus 1, followed four days later by aerosol exposure to a constant level of Pasteurella haemolytica, was studied in 16 crossbred Hereford range calves. A Collision nebulizer was used to generate aerosols from virus suspensions with concentrations of 10(8.2) (high), 10(5.2) (moderate) or 10(2.2) (low) TCID50/mL. The bacterial suspension contained 10(7) colony forming units/mL. Control calves exposed only to P. haemolytica developed no pulmonary lesions. Calves in the low, moderate and high virus exposure groups developed lobular areas of atelectasis; in addition, one calf in the moderate and all four in the high virus exposure group developed fibrinous pneumonia. One of the latter calves died. The 50% effective dose for fibrinous pneumonia under these experimental conditions was 10(6.0) TCID50 bovine herpesvirus 1/mL of suspension in the nebulizer reservoir, and approximately 10(4.0) infectious units inhaled per calf.     

Respiratory disease in calves produced with aerosols of parainfluenza-3 virus and Pasteurella haemolytica.

In four experiments, 22 calves were exposed to aerosols of parainfluenza-3 virus, followed by Pasteurella haemolytica at intervals of three to 13 days. The purpose of each experiment was to study viral-bacterial interactions in the respiratory tracts. Two experiments, in which the viral aerosols were diluted by the addition of air, produced sporadic temperature elevations while two experiments with undiluted viral aerosols produced consistent temperature elevations. Diluted viral aerosols produced lobular sized lesions in the lungs and hemagglutinating inhibition antibodies in sera, whilst undiluted aerosols produced a synergistic effect in the form of purulent pneumonia in ten of 14 calves when the interval between viral and bacterial aerosols was from three to ten days. Histopathological changes attributable to the virus only were seen in all experiments, and the histopathological changes due to mixed infection of parainfluenza-3 virus and P. haemolytica are described in detail. This is the first report of extensive purulent pneumonia in calves after parainfluenza-3 virus and P. haemolytica exposure. This was achieved using much smaller inocula than in experiments previously reported.     

Pneumonia in calves produced with aerosols of Pasteurella multocida alone and in combination with bovine herpesvirus 1.

Pathological changes in respiratory tracts were studied in 30 calves following exposure to aerosols of Pasteurella multocida or to bovine herpesvirus 1 and P. multocida. Two groups of five calves were exposed to aerosols of one of two types of P. multocida only, which produced lobar pneumonia in one calf of each group. Another five groups of four calves were exposed to aerosols of bovine herpesvirus 1 and four to seven days later to one of the two types or one sub-type of P. multocida. Extensive necropurulent lesions were produced throughout the respiratory tract with each type of P. multocida in all four calves in three groups but none in the remaining two groups. The pathological changes differed from those produced following similar exposures to bovine herpesvirus 1 and P. haemolytica, in that the exudate in air passages and alveoli was more purulent and streaming (oat) cells and large mononuclear eosinophilic granulocytes were absent. This is the first report of experimental respiratory disease in cattle as a result of aerosol exposure to P. multocida alone or in combination with bovine herpesvirus 1.     

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.     

Serum complement-fixing antibody to Pasteurella haemolytica A1 and its effect on experimentally induced pneumonic pasteurellosis in calves

Ninety-three calves comprising 16 experimental groups were exposed to viral (bovine herpesvirus-1 or parainfluenza-3 virus) and Pasteurella haemolytica aerosols. Serum samples from these calves were tested before and after exposure for antibodies to P haemolytica by a modified direct complement-fixation test. At slaughter of the calves, the extent of pneumonia produced was estimated for each calf and compared with the results of the modified direct complement-fixation tests. The extent of pneumonia was not related (P greater than 0.05) to the amount of anti-P haemolytica antibody produced by either naturally occurring or experimentally induced infection.     

Experimental Bovine Pneumonic Pasteurellosis II. Genesis and Prevention

Two experiments were conducted. In the first, 16 crossbred Hereford calves were divided into two equal groups. The first group was vaccinated intranasally with a commercial vaccine against bovid herpesvirus 1 and the second group was unvaccinated. The calves were later exposed to an aerosol of bovid herpesvirus 1 (strain 108) for five minutes. Four calves from each group were subjected to transportation and four calves from each group were kept in an environmental chamber for four days. Four days after viral aerosol all calves were exposed to an aerosol of Pasteurella haemolytica and the same subgroups were again transported or held in the chamber for a further four days.

The calves that did not die from pneumonia were necropsied ten days after the final day of transport. Pulmonary lesions were present in both vaccinated and control animals but were less extensive in the vaccinated calves. Six of eight vaccinated but none of the eight control calves survived.

In the second experiment, eight crossbred Hereford calves were divided into two equal groups. One group was vaccinated with bovid herpesvirus 1 (strain 108) and the other acted as controls. Four weeks later all calves were sequentially exposed to aerosols of bovid herpesvirus 1 (strain 108) and P. haemolytica four days apart. Three of the four controls but none of the vaccinates died from pneumonia. Every lobe of the lungs in all the controls was affected by pneumonia while no pulmonary lesions were found in the vaccinated calves. The differences in efficacy of the modes of vaccination and the possible role of transport stress are discussed.

     

Viral-bacterial pneumonia in calves: duration of the interaction between bovine herpesvirus 1 and Pasteurella haemolytica.

Sixteen six to eight month old beef calves were exposed individually to a five minute aerosol of bovine herpesvirus 1, isolate 108. Aerosol exposure to Pasteurella haemolytica (biotype A, serotype 1) was administered individually for five minutes at either four, ten, 20 or 30 days after the virus. Fibrinous pneumonia and pleuritis occurred in all four groups but were most extensive and severe in those exposed to the virus and bacterium four days apart (the positive controls). Fibrinous pneumonia was associated with persistence of bovine herpesvirus 1 in the respiratory tract despite resolution of virus-induced necrotic lesions of the respiratory mucosa. The results presented here suggest that, although the severity of viral-bacterial synergism may be influenced by virus-induced morphological changes, the continued presence of viral antigens after the resolution of respiratory mucosal lesions may continue to exert some effect on host defenses and disease processes.     

Aerosol vaccination of calves with pasteurella haemolytica against experimental respiratory disease.

Three experiments were conducted on calves in which the efficacy of vaccination with live Pasteurella haemolytica in aerosol was tested by challenge with sequential aerosol exposure to bovine herpesvirus 1 and P. haemolytica. Neither single nor multiple aerosol vaccinations protected against the experimental disease. Macroscopically recognizable rhinitis, tonsillitis, tracheitis and pneumonia occurred in both controls and vaccinates. In one experiment as many as three aerosol vaccinations with live P. haemolytica for up to 20 minutes failed to elicit clinical signs in exposed calves. Pasteurella haemolytica was isolated less frequently from tissues of vaccinated calves than from those of nonvaccinated calves. Pasteurella haemolytica was isolated from deep nasal swabs of 4/14 vaccinated calves five and six days after viral exposure. It was concluded that although bovine herpesvirus 1 vaccination has been shown previously to prevent the experimental disease produced by bovine herpesvirus 1-P. haemolytica, live P. haemolytica vaccination by aerosol will not provide the same protection.     

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.     

The Effect of Edema, Hydrocortisone Acetate, Concurrent Viral Infection and Immunization on the Clearance of Pasteurella hemolytica from the Bovine Lung

The influence of pulmonary edema, hydrocortisone, immunization against Pasteurella hemolytica and concurrent infection with parainfluenza-3 virus upon pulmonary clearance of aerosolized P. hemolytica was studied in 31 calves. Following the various treatments calves were challenged with an aerosol of P. hemolytica. One control calf was killed immediately after the aerosol and the numbers of bacteria in the lung taken as 100%. Two calves were killed four hours after challenge and the numbers of bacteria in the lungs were compared to the 100% of the control calf. The result was the percentage clearance of bacteria at four hours.

Pulmonary edema was induced by three different methods: by an aerosol of histamine, by intravenous injection of endotoxin and by intravenous injection of croton oil emulsion. The edema impaired the clearance of P. hemolytica, which was reflected in high numbers of P. hemolytica present in the lungs at four hours after challenge: 260% after histamine, 300% and 400% after endotoxin and 92% after croton oil.

Six days of treatment of four calves with high doses of hydrocortisone acetate produced inconsistent results: two calves treated with a higher daily dose (36 mg/kg) had normal clearance whereas two calves treated with a lower dose had pulmonary edema and displayed lowered clearance with 111% and 31% respectively of P. hemolytica retained in the lungs four hours after challenge.

Immunization of calves by three different methods, a subcutaneously injected bacterin of P. hemolytica (2 calves), single aerosol (2 calves) and four aerosols (4 calves) of live P. hemolytica was reflected in an accelerated pulmonary clearance of P. hemolytica (with a mean of 1.55% of bacteria retained at four hours).

Concurrent infection with parainfluenza-3 virus did not lower the clearance of P. hemolytica in the lungs of 12 calves over 15 days except on the first day following the exposure to parainfluenza-3 virus. These calves had hemagglutinating antibodies against P. hemolytica before exposure.

     

Response of the respiratory tract of calves kept at controlled climatic conditions to bovine Herpesvirus 1 in aerosol.

Seven experiments with four calves each were conducted in which the calves spent at least four days of adaptation in an environmental chamber and then were subjected to climatic stress in the form of a number of constant ambient temperature and humidity combinations. On the second day of climatic stress the calves were individually exposed to measured numbers of infectious units of bovine herpesvirus 1 (BHV1, virus of infectious bovine rhinotrachetis) in aerosol. The calves were killed seven or eight days later. Mycoplasma were found in some nasal swabs and in one lung. Certain bacteria but no Pasteurella were often isolated from the lungs. Bovine herpesvirus 1 was isolated from chamber air and from most postinoculation nasal swabs, tracheas and lungs. The number of macro- and microscopic lesions did not appear to be influenced by the climatic conditions of the experiments. The histopathological changes in epithelium at all levels of the respiratory tract were described in detail.     

Deposition in the Respiratory Tract of Cattle of Spores of Bacillus subtilis var niger by Inhalation and by Nasal Instillation

Spores of Bacillus subtilis var niger were deposited in the lungs, tracheae and nasal cavities of four calves by aerosol inhalation and in three calves by intranasal instillation. From each calf 20 specimens of lung tissue, each weighing one gm, three of trachea and three of nasal mucosa were examined for spore content. The average numbers of spores per gm of lung tissue from animals exposed to aerosols were 3.05 and 4.84, 2.35 and 2.02 x 10⁴. Lungs from animals exposed intranasally contained only 747, 62 and 1424 spores per gm of tissue respectively. Animals exposed intranasally had a hundred to a thousand fold more spores on nasal mucosa than animals exposed by aerosol and the latter had a thousand fold more spores on tracheal mucosa than calves exposed intranasally. Aerosol inhalation exposed the lung and trachea more densely and uniformly than did intranasal instillation.     

Some lesions observed in calves born to cows exposed to the virus of infectious bovine rhinotracheitis in the last trimester of gestation.

Sixteen pregnant cows were challenged with infectious bovine rhinotracheitis virus intranasally. One had a mummified fetus, four aborted, one calf was stillborn, two live fetuses were taken at the abattoir and eight calves were born alive. Of the eight born alive, five were dead by 12 days of age. Four of these had the usual lesions of infectious bovine rhinotracheitis as well as lesions in the intestine and peritoneum and two of the four had a fibrinous pneumonia thought to be caused by aspiration of milk. The lesions, results of virus isolation and fluorescent antibody testing are recorded in these four calves. Attention is drawn to the intestinal lesions, the peritonitis and fibrinous pneumonia and the ease with which the underlying infectious bovine rhinotracheitis infection may be overlooked.     

Immunogenicity of a soluble antigen against Pasteurella haemolytica-associated pneumonia in calves

Three experiments were performed to evaluate the immunogenic potency of a soluble fraction of Pasteurella haemolytica against pneumonic pasteurellosis in calves. A soluble antigen was extracted by a 2.5% saline solution from P. haemolytica. Weaned Holstein bull calves, seronegative for infectious bovine rhinotracheitis virus ( IBRV ) and the pasteurella antigen, were vaccinated either by repeated subcutaneous (SC) vaccination, or by exposure 3 times to the aerosol of P. haemolytica antigen. Challenge exposure to aerosol of P. haemolytica was preceded by infection with IBRV , or in experiments 2 and 3, the virus exposures were combined with a stress treatment. The lung lesions were examined at necropsy 3 to 8 days post infection. In the first experiment, all the vaccinated calves produced specific antibody response to the pasteurella antigen, and none of the calves including controls showed significant lesions in the lung. In the second experiment 2 aerogenically vaccinated calves had no lesions. One of the two SC-vaccinated calves had mild consolidated lesions. Two control calves, one of which died 3 days following the challenge, developed severe fibrinous pneumonia with consolidation of 50% or more of the lung surfaces. P. haemolytica was isolated only from the 2 control animals. In the third experiment, 2 of the 3 control calves developed moderate to severe consolidation, but P. haemolytica was isolated only from one of them. Two of the three aerosol-vaccinated calves also developed significant lesions and one of them yielded the bacteria from the lung. Three SC-vaccinated calves had slight lesions and the organism was not isolated from their lungs. The results did not consistently indicate an immunogenic potential of the soluble antigen against P. haemolytica-related pneumonia. The effect of stress on the pathogenesis of bovine viral pneumonia and correlation between pneumonic lesions and antibacterial resistance in situ are discussed.     

Bovine Viral Diarrhea Virus and Escherichia Coli in Neonatal Calf Enteritis

This study was initiated to determine the etiologic and pathogenic significance of an American strain of bovine viral diarrhea (BVD) virus (strain NADL-MD) in enteritis of neonatal calves (calf scours).

Three colostrum-fed calves from dams exposed intravenously to BVD virus at 6, 16 and 25 days prepartum, respectively, had moderate diarrhea persisting until the eighth day of life. The BVD virus was isolated from all 3 calves and persisted up to 93 days in 1 calf, indicating either that BVD was transmitted in utero or via the dam's milk.

Three specific pathogen free (SPF) calves permitted dams' colostrum for the first 4 feedings and then given milk replacer were exposed orally on the day of birth to BVD virus. One calf died of neonatal enteritis 28 hours post-exposure and at necropsy the BVD virus was isolated from several of its organs. The remaining 2 calves had a mild diarrhea persisting to the eighth day of age.

Two calves permitted dams' colostrum ad lib. for 72 hours, and then weaned, were exposed orally to BVD virus. Both calves had a mild persistent diarrhea and BVD virus was isolated from their blood for 56 days post-exposure.

Of 13 SPF, colostrum-deprived calves exposed orally or intranasally at birth to the BVD virus, 4 had severe diarrhea and died of neonatal enteritis from 38 hours to 13 days postexposure. Isolations of BVD virus were made from several of the organs of the calves at necropsy. All of the 9 surviving calves had a moderate to severe diarrhea frequently persisting for 7 to 10 days, and BVD virus was isolated from the survivors up to 103 days postexposure.

Several strains of Escherichia coli were isolated from calves after the second day of life, but were neither pathogenic for mice, nor serologically related to strains of E. coli usually associated with outbreaks of calf scours. Four colostrum-deprived SPF calves were exposed orally at birth to a strain of E. coli isolated from the intestine of the calf with the most acute symptoms and fatal neonatal enteritis. None of the four calves receiving the E. coli had diarrhea. One calf, however, had respiratory distress and died on day 5.

Two SPF colostrum-deprived control calves had neither diarrhea nor respiratory distress.

The above findings support the conclusion that BVD virus should not be overlooked as a primary cause of the neonatal calf enteritis complex.

     

Protective effect of inactivated bovine herpesvirus-1 in calves experimentally infected with bovine herpesvirus-1 and Pasteurella haemolytica.

The protective effect of an inactivated whole-virion bovine herpesvirus-1 (BHV-1) immunising inoculum, without adjuvant, against viral-bacterial respiratory disease was studied in three experimental treatment groups of five calves each. One group was boosted 14 days after the first vaccination and at this time the second group received their initial inoculation. Seven days later, calves were challenged with BHV-1 in aerosol and four days after this challenge all calves were exposed to Pasteurella haemolytica A1 in aerosol. Among the three groups, differences in rectal temperature responses four days after viral challenge (P less than 0.01) did not relate to protection. However the main response variable, viral-bacterial pneumonia, was reduced in boosted calves (P less than 0.05).     

Experimental infectious respiratory disease in groups of calves:Lobar distribution, variance, and sample-size requirements for vaccine evaluation

The distribution and variance of respiratory disease produced with aerosols of bovine herpesvirus 1 (BHV-1) and Mannheimia haemolytica in control (183 calves in 44 experiments) and vaccinated calves were studied in experiments conducted at the Animal Diseases Research Institute, Lethbridge, Alberta, from 1975 to 1989. All calves had been born and raised at this institute and exposed similarly for 5 min by means of a face mask to viral and bacterial aerosols produced by a Collison atomizer (particles < 3 microm in diameter). We summarized the macroscopic pathological responses of pneumonia (main end point), tonsillitis, tracheitis, and other microbiologic and experimental variables. We also summarized the lobar distribution of pneumonia in 202 control and 192 vaccinated calves with this disease model and in calves similarly exposed to parainfluenza 3 virus/M. haemolytica or BHV-1/Pasteurella multocida. Pneumonia in control calves began in ventral tissues of all lobes, with lobar preferences, and progressed dorsally, the dorsal parts of both large caudal lobes being least affected. A high variance of pneumonia was evident within and among experiments. From the magnitude of variance observed in the control groups, the number of calves per group required in vaccine-challenge studies using this BHV-1/M. haemolytica disease model was estimated. Such estimates are required for any disease model used in vaccine-challenge studies.     

Induction of immunity against pneumonic pasteurellosis following experimental infection in calves.

Immunity against pneumonic pasteurellosis was studied in calves after recovery from experimental respiratory disease with Pasteurella haemolytica. Nine calves were exposed to aerosols of parainfluenza-3 virus and Pasteurella haemolytica A1 six days apart to produce respiratory disease. After recovery from the disease, these nine principal and four control calves were challenged with aerosols of bovine herpesvirus 1 and P. haemolytica A1 four days apart. With this viral-bacterial challenge, the nine principal animals failed to develop clinical responses to this bacterial challenge and their lungs did not show the growth of P. haemolytica on cultures, whereas two of four control calves had elevated temperatures and developed necropurulent pneumonia with the isolation of P. haemolytica from the lungs. The principal calves had developed high levels of cytotoxin neutralizing antibodies in their sera following parainfluenza-3 virus-P. haemolytica infection. This demonstrated that immunity against pneumonic pasteurellosis can be achieved, with a suggestion that further search for an effective vaccine for P. haemolytica is warranted.     

Systemic and secretory antibody responses to sequential bovine respiratory syncytial virus infections in vaccinated and nonvaccinated calves

To investigate the influence of humoral immunity on the severity of disease caused by infection with bovine respiratory syncytial virus (BRSV), an experimentally induced infection study was performed on vaccinated and nonvaccinated calves. Fifteen weanling calves were allotted to 3 groups: 1 group of 6 calves was exposed to 2 live virus aerosols, 35 days apart; another group of 6 calves was vaccinated prior to the same aerosol exposures; and the remaining 3 calves served as controls. Clinical signs of infection were converted to a numerical score for evaluating disease severity. For 14 days after each virus exposure, BRSV-specific IgG and IgM concentrations in serum and BRSV-specific IgA concentration in nasopharyngeal exudate and lung lavage fluid were measured by ELISA. Serum BRSV-specific IgG and IgM and secretory BRSV-specific IgA concentrations did not correlate with disease sign expression. There was a strong correlation between viral isolation and disease scores. Vaccination prior to virus exposure appeared to have little or no effect on severity of the disease, but it did appear to affect disease persistence. Findings indicate that the immunoglobulins evaluated may be primarily protective in nature and do not contribute to disease severity.     

Changes in leukocyte populations in pulmonary lavage fluids of calves after inhalation of Pasteurella haemolytica

The distribution of leukocytes in bovine bronchoalveolar lavage fluids was determined in 15 calves at various times after aerosol exposure to Pasteurella haemolytica. For comparison, 10 calves were exposed to aerosols of phosphate-buffered saline solution; 15 calves, to Staphylococcus epidermidis; and 10 calves, to Salmonella typhimurium endotoxin. At 10 minutes after inhalation exposure for each group, the predominant cell type was the macrophage. Macrophages remained the predominant cell type throughout each lavage interval for calves exposed to phosphate-buffered saline solution and Staph epidermidis. For calves exposed to P haemolytica, there was a decrease in the percentage of macrophages detectable by 30 minutes after exposure, with a corresponding increase in the percentage of neutrophils. Sixty minutes after the inhalation exposure to P haemolytica, the percentages of macrophages and neutrophils in the lavage fluid were equal. By 240 minutes after exposure to P haemolytica, greater than 90% of the cells in the lavage fluids was neutrophils. The increase in the percentage of neutrophils in lavage fluids from calves exposed to S typhimurium endotoxin was similar to that seen for the calves exposed to P haemolytica.