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.     

The pulmonary clearance of Pasteurella haemolytica in calves given Corynebacterium parvum and infected with parainfluenza-3 virus.

Four control calves were aerosolized with parainfluenza-3 and one week later with Pasteurella haemolytica. Three calves were given Corynebacterium parvum at a dose of 15 mg/m2 body surface area, infected with parainfluenza-3 virus one week later, and aerosolized with P. haemolytica two weeks after C. parvum injection. All calves were killed four hours after P. haemolytica exposure and the bacterial retention in the lung was determined. Parainfluenza-3 viral infection did not exert any suppressive effect on pulmonary clearance of P. haemolytica in six out of seven calves used. However, the bacterial colony counts in the lungs of control calves were higher (P less than 0.05) than those in calves given C. parvum. Hence, C. parvum appeared to enhance bacterial clearance. Despite the marked influx of neutrophils into the lungs after the bacterial inoculation, the neutrophil:macrophage ratio in lavage samples was less in calves given C. parvum than in the control calves. The alveolar macrophages in C. parvum treated calves were generally larger but did not differ significantly (P less than 0.05) from those in the controls. There was no significant (P less than 0.05) correlation between the percentages of alveolar macrophages and the bacterial clearance. In calves given C. parvum, bacterial clearance was enhanced in those calves which had larger macrophages.     

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.     

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.     

Colonization of the nasal passages of calves with Pasteurella haemolytica serotype 1 and regeneration of colonization after experimentally induced viral infection of the respiratory tract

Healthy nonstressed calves were inoculated intranasally with or subjected to aerosol exposure to Pasteurella haemolytica serotype 1. Only 4 of 28 calves harbored the bacterium in enough numbers to be isolated from the nasal passages for more than 7 days. After apparent clearing from the nasal passages, 8 calves were inoculated intranasally with infectious bovine rhinotracheitis virus; 2 of these calves shed the P haemolytica during clinical illness due to the virus. The remaining 20 calves were aerosol-exposed to parainfluenza-3 virus; 6 of these calves shed P haemolytica during clinical illness due to the parainfluenza-3 virus.     

Cellular inflammatory response in the lungs of calves exposed to bovine viral diarrhea virus, Mycoplasma bovis, and Pasteurella haemolytica

Three, 5, or 7 days after inoculation with bovine viral diarrhea (BVD) virus (n = 12) or Mycoplasma bovis (n = 12), groups of calves were exposed to aerosols of Pasteurella haemolytica and were euthanatized 4 hours later. Histologic lesions in the lungs and the ratios of neutrophils to alveolar macrophages, collected by bronchoalveolar lavage, were compared with those of clinically healthy calves (n = 8) and calves inoculated with BVD virus only (n = 4), M bovis only (n = 4), or P haemolytica only (n = 2). Inoculation with BVD virus or M bovis did not have a significant (P greater than 0.05) effect on the neutrophil/macrophage ratio in the bronchoalveolar lavage. Aerosol exposure to P haemolytica induced a marked and significant (P less than 0.01) change in the neutrophil/macrophage ratio (from less than 1:9 to greater than 9:1). The reversed neutrophil/macrophage ratio in calves exposed to P haemolytica correlated well with the histologic changes in which small bronchi and bronchioles were plugged with purulent exudate. Inoculation with BVD virus did not induce gross or microscopic lesions in the lungs. Inoculation with M bovis resulted in a severe peribronchial lymphoid hyperplasia with mild exudation of neutrophils and macrophages into the cranioventral parts of the lungs.     

Pasteurella haemolytica serotype 1 colonization of the nasal passages of virus-infected calves

Nasal passages of calves with a virus-induced respiratory tract disease became colonized by Pasteurella haemolytica serotype 1 after they were inoculated intranasally with P haemolytica. Inoculation with infectious bovine rhinotracheitis virus caused a more severe clinical illness and resulted in a greater degree of colonization with P haemolytica than developed after inoculation with parainfluenza-3 virus. Nasal passages of parainfluenza-3 virus-inoculated calves were colonized to a greater degree with P haemolytica than were those of healthy, nonstressed calves. Calves were susceptible to P haemolytica colonization during or shortly after virus-induced illness, even though they had been previously exposed to P haemolytica and had serum antibody and nasal secretion antibody to P haemolytica.     

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.

     

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica after intrapulmonary inoculation.

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica were evaluated by measuring immunoglobulin production in blood for 9 days and in pulmonary lavage fluid for 7 days after intrapulmonary inoculation. Clinical signs, pulmonary lesions, pulmonary and systemic inflammatory response, and amount of antigen in lavage fluid were used to evaluate the response of calves to challenge with P haemolytica. The pulmonary response consisted of production of IgG, IgE, and IgM antibodies to P haemolytica antigens and a 17- to 68-fold increase of cells in lavage fluid 8 hours after inoculation, with a gradual decrease toward normal. Antibodies of the IgM isotype to P haemolytica were demonstrated as early as 8 hours through 7 days after inoculation in 3 of 3 calves. Of the anti-P haemolytica isotypes, IgM was found in the highest concentration. In all of the inoculated calves, IgE was found 1 to 2 days after inoculation, and IgG was found in 2 of 3 inoculated calves from day 1 through 7 after inoculation. Detection of IgG correlated with smaller pulmonary lesions. Immunoglobulin A was not detected in lavage fluid. Serum was evaluated for IgG and IgM antibody response to P haemolytica. Specific IgM was detectable 5 days after inoculation, and IgG was detectable 7 days after inoculation. Pasteurella haemolytica antigens were not detected in serum or plasma. A transient increase in neutrophil count was found 8 hours after inoculation, with return to baseline values by 24 hours after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Experimental production of bovine respiratory tract disease with bovine viral diarrhea virus

Five 6-month-old calves were inoculated with bovine viral diarrhea (BVD) virus (n = 3) or Pasteurella haemolytica (n = 2) endobronchially with a fiberoptic bronchoscope. Five additional calves were inoculated sequentially with BVD virus followed by P haemolytica at a 5-day interval. Blood samples were collected daily from the calves for bacterial isolation. Clinical signs of respiratory tract disease in calves were recorded daily. If the calves survived, they were killed for necropsy 3 or 4 days after inoculation with P haemolytica (or 8 days after inoculation with BVD virus). The extent and nature of pulmonary lesions in the calves were determined, and the lower portion of the respiratory tract (lungs and trachea) was examined for both these organisms. The 3 calves, inoculated with BVD virus only, developed mild clinical signs mainly manifested as fever, nasal discharge, and occasional cough. Approximately 2% to 7% of the total lung capacity of these calves was pneumonic. Mild clinical signs and localized lesions involving about 15% of the lung volume developed in the 2 calves exposed to P haemolytica only. However, severe fibrinopurulent bronchopneumonia and pleuritis involving 40% to 75% of lung volume developed in the 5 calves inoculated sequentially with BVD virus and P haemolytica. The possible role BVD virus may have in bovine respiratory tract disease is discussed.     

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.     

Vaccination studies against experimental bovine Pasteurella pneumonia.

Vaccination-challenge experiments were conducted in colostrum-deprived calves to evaluate the efficacy of Pasteurella bacterins and vaccines against experimental pneumonic pasteurellosis. Calves were vaccinated with formalin-killed bacterins and live vaccines, then challenge exposed intratracheally with P. haemolytica or P. multocida. Infectious bovine rhinotracheitis virus was inoculated intranasally three to four days prior to P. haemolytica challenge-exposure. All calves were examined for macroscopic and microscopic lesions after being found dead or following euthanasia four to seven days after challenge exposure with the bacterial pathogen. Clinical, hematological, and pathological responses to challenge exposure in aluminum hydroxide absorbed P. haemolytica and P. multocida bacterin-treated calves were consistent with the pneumonic lesions of pulmonary pasteurellosis in the control calves. An oil-adjuvanted P. haemolytica bacterin limited clinical and pathological responses in the affected calves whereas a P. multocida oil-adjuvanted bacterin did not. Both clinical and pathological responses to challenge exposure in calves vaccinated with live Pasteurella vaccines were less severe than those of the control calves. Vaccine effectiveness appeared to be dose dependent.     

A live Pasteurella haemolytica vaccine efficacy trial

A live Pasteurella haemolytica serotype 1 vaccine was used in an efficacy trial conducted on 100 lightweight feeder calves purchased from a Florida ranch. Forty-one calves were inoculated with the vaccine intradermally in the neck. Fifty-nine calves served as nonvaccinated controls. Fourteen days later, the calves were shipped to an order buyer in eastern Tennessee, where the calves were mixed with 60 local calves in a community sale barn for 72 hours. After 3 additional days, the calves were shipped to a research feedlot in Bushland, Tex. They remained in the feedlot for 56 days, and the test was concluded 76 days after vaccination. The P haemolytica vaccine had no significant effect on performance, morbidity, or mortality. There was no significant difference between the vaccinated and nonvaccinated calves in the number of times Pasteurella was isolated. The calves became seropositive to bovine viral diarrhea virus, respiratory syncytial virus, and infectious bovine rhinotracheitis (IBR) virus during the 76-day experiment. All calves initially were seropositive to parainfluenza-3 virus. A virulent outbreak of IBR occurred 30 days after the calves arrived at the feedlot. Before the onset of IBR, the isolation of P haemolytica serotype 1 from nasal turbinates was rare (2 of 500 nasal swabs). After the IBR outbreak, P haemolytica serotype 1 was isolated from 40 of 92 calves.     

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.     

Effect of vaccination with live or killed Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis

Using 6- to 8-month-old beef calves, 3 experiments were conducted to compare the effect of vaccination with live or killed Pasteurella haemolytica on resistance to a transthoracic challenge exposure with the organism and to correlate serum antibody response with resistance. In each experiment, calves were vaccinated twice at 1-week intervals and were challenge exposed 21 days after the first inoculation. Lung lesions were evaluated by a system, such that higher scores indicated the more severe lesions. In each experiment, calves immunized with live P haemolytica had lower lesion scores than calves vaccinated with saline solution or bacterin. In 2 of the experiments, the differences were significant (P less than 0.05). In all experiments, calves vaccinated parenterally with a commercial P haemolytica/P multocida bacterin or with a formalin-killed P haemolytica bacterin had lesion scores that were not significantly different (P greater than 0.05) than for control calves vaccinated with saline solution. Live and killed bacterial preparations induced a significant serum antibody response to P haemolytica as measured by a quantitative fluorometric immunoassay. The antibody response to vaccination was not affected by preexisting titers to P haemolytica. Serum antibody titers were not consistently as high for calves vaccinated with bacterins as for calves vaccinated with live organisms. Although high antibody titers correlated with low lesion scores when calves vaccinated with saline solution or live organisms were analyzed collectively, there was not a significant correlation between the 2 variables when calves, vaccinated with saline solution or with bacterin, were analyzed collectively. These data indicate that, although bacterins may induce a detectable serum antibody response, they do not induce protection against transthoracic challenge exposure to P haemolytica.     

Clinical and immunologic responses of calves with colostrally acquired maternal antibody against parainfluenza-3 virus to homologous viral infection.

Exposure of colostrum-deprived calves and calves with colostrally acquired maternal antibody to aerosols of parainfluenza-3 (PI-3) virus resulted in signs of infection, leukopenia, and shedding of virus from the nasal passages. However, infection was not as severe in calves with colostrally acquired maternal antibody as it was in colostrum-deprived calves which did not have antibody to PI-3 virus before they were exposed. All calves responded immunologically to PI-3 virus, as indicated by resistance to challenge exposure and subsequent development of virus-neutralizing antibody. However, levels of serum and nasal secretion (NS) antibody at 30 days after viral exposure were lower in calves with colostrally acquired maternal antibody than in colostrum-deprived calves, and a serum antibody response in the former was primarily indicated by an anamnestic response after challenge exposure. After calves were challenge exposed to PI-3 virus, serum and NS antibodies were increased in all calves, but antibody titers were generally lower for calves that had colostrally acquired maternal antibody before their exposure than for those that acquired antibody only after PI-3 viral infection.     

Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung

The potential synergistic effect of bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica in the production of pneumonia after aerosol/intranasal infection of conventionally reared lambs was evaluated. A mild clinical response was observed in lambs given virus and/or bacteria. Gross pulmonary lesions were seen in 3 of 6 lambs given RSV and then P haemolytica 3 or 6 days later, respectively (groups D and E), and in 1 lamb of 5 given virus and bacteria simultaneously (group G). Gross lesions were not seen in control sheep (group A), in lambs given virus or bacteria alone (groups B and C), or in lambs exposed to bacteria and then virus 3 days later (group F). Bovine RSV and P haemolytica were recovered from the lungs of 5 of 7 lambs with macroscopic lesions. Gross pulmonary lesions were cranioventral firm areas of red consolidation. Microscopically, the predominant lesion was a suppurative bronchopneumonia. Bovine RSV was recovered from the nasal cavity of 8 of 27 (30%) lambs given RSV during days 3 to 6 after viral inoculation, including 1 lamb in group B, 2 in groups D, E, and F, and 1 in group G. Pasteurella haemolytica was recovered from the nasal cavity of 9 of 28 (32%) inoculated lambs, including 2 lambs from groups C and E, 3 in group D, and 1 in groups F and G. Viral antigen, as determined by immunofluorescence, was concentrated mainly in individual cells in alveolar walls, some alveolar macrophages, and a few bronchiolar epithelial cells. In vitro alveolar macrophage assays indicated decreased numbers of Fc receptors on those macrophages collected from lambs given RSV 6 days before P haemolytica infection, as compared with that in the other groups. These cellular defects disappeared after 24 hours of culture. Seemingly, bovine RSV does facilitate P haemolytica pulmonary infection in conventional, immuno-competent lambs and provides evidence for decreased Fc receptors on alveolar macrophages.     

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.     

Comparison of the pneumopathogenicity of two strains of bovine viral diarrhea virus

The pneumopathogenicity in calves of 2 strains of bovine viral diarrhea (BVD) virus, isolate 2724 (a noncytopathogenic virus) and isolate 72 (a cytopathogenic virus), was compared. All calves were inoculated endobronchially, using fiberoptic bronchoscopy. Two calves were given Pasteurella haemolytica, 2 calves were given the noncytopathogenic BVD virus, and 2 calves were given cytopathogenic BVD virus. Five calves were inoculated sequentially with BVD virus and, 5 days later, with P haemolytica. Two of these calves were inoculated with the noncytopathogenic BVD virus and the other 3 with the cytopathogenic strain. Both BVD virus strains caused marked respiratory tract disease in the calves sequentially inoculated with P haemolytica and also impaired pulmonary clearance of P haemolytica. However, the effect of the cytopathogenic strain was more severe than the noncytopathogenic strain, indicating that strains of BVD virus may vary in their pneumopathogenicity for calves.