Regional Histological Variations of the Nasal Mucosa of Cattle

Aspects of the histology of the nasal mucosa of calves from four to six months of age have been described.

The functional significance of the type of epithelium, its thickness, the degree of neutrophil invasion, and its relationship to the numbers of Pasteurella haemolytica in the nasal cavity have been discussed. In addition, the arrangement and depth of glands in the lamina propria and the presence of lymph follicles have been described.

     

A Quantitative Flurometric Assay for the Measurement of Antibody to Pasteurella haemolytica in Cattle

A rapid, simple fluorometric method is described for measuring antibody to Pasteurella haemolytica in sera of cattle. Various antigen preparations were compared for the test including live, formalin-killed and phenol-killed P. haemolytica. A preparation composed of formalin-killed organisms from a 22 hour culture gave consistent results and was used in the studies. The test was reproduciable with percent coefficients of variation for fluorescent signal unit values on ten or more replicate samples ranging from 5.7 to 28.0. Sera from calves vaccinated by aerosol exposure to live P. haemolytica had up to a five-fold increase in antibody titer as measured by the flurometric method test during a 21 day period. Fluorometric method titers were comparable to those obtained by the indirect bacterial agglutination test. There was no seroconversion to P. haemolytica in calves vaccinated by aerosol exposure of P. multocida. The major advantages of the fluorometric method test over conventional methods are that the assay does not require serial dilutions of serum samples and thus limits time and effort to determine antibody titers.     

Pasteurella haemolytica of Cattle: Serotype, Production of Bete-galactosidase and Antibacterial Sensitivity

Two hundred and three isolates of Pasteurella haemolytica from cattle were studied. They originated from the nasal cavity of cattle in housed herds; the nasal cavity and pneumonic lungs of experimental feedlot calves and from pneumonic bovine lungs submitted for bacteriological diagnosis.

To determine whether a single characteristic or combination of characteristics might be a feature of isolates collected from animals with pneumonic pasteurellosis (Shipping Fever), the following tests were made. Cultures were serotyped by indirect haemagglutination; the ability to produce beta-galactosidase was examined in the ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) test and antibacterial sensitivity tests were done. None of these factors could be directly related to the role of P. haemolytica in “Shipping Fever”.

     

The effect of Pasteurella haemolytica and the leukotoxin of Pasteurella haemolytica on bovine lung explants.

Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neisseria subflava, or the crude leukotoxin of P. haemolytica on alveolar macrophages and lung parenchymal cells. Concentrated, purified peripheral blood neutrophil suspensions were added with the bacteria to some explants. Duplicate pairs of cultures from each treatment group were fixed at regular intervals up to 24 hours after seeding and morphological changes were assessed by light and electron microscopy. Pasteurella haemolytica caused deterioration of alveolar macrophages within one hour but did not affect parenchymal cells for more than 12 hours. Neisseria subflava did not affect alveolar macrophages initially, but caused an accelerated deterioration after four hours. After 24 hours, bacterial overgrowth caused similar deterioration of all cells in explants seeded with either bacterium. Alveolar macrophages phagocytosed large numbers of N. subflava but rarely ingested P. haemolytica. Added neutrophils did not have any discernible effect on any of the explants and did not potentiate bacterial effects. Addition of crude leukotoxin of P. haemolytica to the culture medium significantly accelerated alveolar macrophage deterioration without apparent effect on parenchymal cell survival. These results support the hypothesis that the severe tissue destruction of fulminant pneumonic pasteurellosis is not a direct result of bacterial infection.     

Long-chain fatty acids of Pasteurella multocida nad Pasteurella haemolytica

The relative contents of long-chain fatty acids in P. multocida and P. haemolytica were investigated. A dependence on the composition of the broth was established. Accordingly, comparative quantitative studies on fatty acid contents have to be conducted using bacteria grown with the same lot of broth medium. As for P. multocida, there were significant differences between the serovars (C14 in TDHM and C16, delta 2C18 in BPL). These differences are, however, not significant to replace serotyping. Highly significant differences were also detected between P. multocida isolates from nasal swabs and pneumonic lungs (interims of C14, delta C16 on BPL and BRU). The largest differences were measured for strains grown on BRU, which is interpreted as an expression of virulence. Significant differences were found between biotypes A and T of P. haemolytica, namely for C14, C16 in TDHM, and C14, delta C16, C16, C18 in BPL medium.     

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.     

Pasteurella haemolytica A1 and Bovine Respiratory Disease: Pathogenesis

The severe fibrinonecrotic pneumonia associated with pneumonic pasteurellosis usually results from colonization of the lower respiratory tract by Pasteurella haemolytica biotype A, serotype 1(A1). Despite recent research efforts, the authors lack a detailed understanding of the interactions and host response to P. haemolytica in the respiratory tract. The authors hypothesize that management and environmental stress factors or viral infection alters the upper respiratory tract (URT) epithelium allowing P. haemolytica to colonize the epithelium. Once the URT is colonized, large numbers of organisms enter the lung where they interact with alveolar macrophages. Endotoxin, released from the bacteria, crosses the alveolar wall where it activates pulmonary intravascular macrophages, endothelium, neutrophils, lymphocytes, platelets, complement, and Hageman factor leading to complex interactions of cells and mediators. It is the progression of this inflammatory response with neutrophil influx that is ultimately responsible for the pulmonary injury. Leukotoxin is a major virulence factor of P. haemolytica that allows it to survive by destroying phagocytic cells. At subcytolytic concentrations it may also enhance the inflammatory response by activating cells to produce mediators and release reactive oxygen metabolites and proteases.     

Efficacy of a streptomycin-dependent, live Pasteurella haemolytica vaccine against challenge exposure to Pasteurella haemolytica in cattle

A streptomycin-dependent, live Pasteurella haemolytica vaccine was given in 1 or 2 doses to 2 groups of weaned calves; 2 other groups of calves were not vaccinated. All calves in the vaccinated groups and calves in 1 of the nonvaccinated groups were stressed by transport, intratracheally inoculated with bovine herpesvirus type-1 (Cooper strain), and then intratracheally inoculated with P haemolytica type A1. The 4th group of calves (nonvaccinated controls) was not stressed and were not intratracheally inoculated with virus or bacteria. Mean daily weight gains, total clinical sign scores, lung lesion scores, plasma fibrinogen concentrations, and antibody titers against P haemolytica were determined at various intervals. Calves that had been vaccinated twice had greater mean daily weight gains and lower total clinical sign scores and lung lesion scores than did nonvaccinated, challenge-exposed calves, but the difference was not significant (P greater than 0.05). Calves vaccinated once had the greatest mean daily weight gains, the lowest total clinical sign scores, and the lowest lung lesion scores when compared with the other 2 challenge-exposed groups of calves. Mean daily weight gains and total clinical sign scores of calves vaccinated once were significantly different (P less than 0.05) than those of calves vaccinated twice. Nonvaccinated, nonchallenge-exposed control calves did not develop clinical signs of disease, did not develop lung lesions, and had consistently positive daily weight gains, and had scores in these areas that were significantly different (P less than 0.05) from those of all challenge-exposed groups of calves. Increases in plasma fibrinogen concentrations corresponded to infection with P haemolytica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pasteurella haemolytica in the Tracheal Air of Calves

Pasteurella haemolytica was shown to be present in the tracheal air of calves and was likely transported in droplet nuclei formed in the nasal passages. The number of colonies of P. haemolytica found in the tracheal air of the calves ranged from 1.9 to 12.5 colonies per cu ft of air. As long as P. haemolytica colonized the nasal passage in numbers detectable in nasal swabs it could be found in the tracheal air but there was no direct correlation between the numbers in the nasal flora and the numbers found in the tracheal air. Of the P. haemolytica which travel via the tracheal air 47.8% were in droplets of the aerodynamic size of from less than one to five microns, the size range which is considered hazardous for lung penetration in man.

The technique used demonstrated the presence of P. haemolytica in the tracheal air of calves and provides a useful tool for monitoring and determining the phase in the colonization of the respiratory tract in which the majority of the potential pathogen P. haemolytica pass from the nose to the tracheal air and presumably to the lung.

     

Vaccination of neonatal colostrum-deprived calves against Pasteurella haemolytica A1.

Colostrum-deprived Holstein calves were vaccinated at 2 and 4 wk of age with a Pasteurella haemolytica A1 culture supernatant vaccine to determine whether active immune responses and protection could be induced in this age group in the absence of maternal antibodies. All calves responded to vaccination with high titers of IgM antibodies to capsular polysaccharide within 1 wk of primary vaccination. Mean titers of IgG1 and IgG2 antibodies to this antigen increased significantly by 2 wk after secondary vaccination, but peak antibody titers were low. All of the vaccinated calves seroconverted with production of leukotoxin-neutralizing antibodies, but peak antibody titers were low. Vaccinated calves experienced considerable lung damage after experimental challenge, but survival rate, clinical scores, and percent lung involvement were significantly better than those of control (placebo-injected) calves.     

Infection of the middle nasal meatus of calves with Pasteurella haemolytica serotype 1

Eight healthy nonstressed calves were inoculated with Pasteurella haemolytica serotype 1, by instilling a broth culture into the middle nasal meatus of the left nostril. The inoculated left nostrils shed P haemolytica from the ventral nasal meatus at a steady rate for a mean of 7 days, whereas the uninoculated right nostrils of the same calves shed P haemolytica sporadically and in lower concentrations. The duration, frequency, and concentration of P haemolytica shed from the inoculated nostrils was significantly (P less than 0.05) greater than from the nostrils of other healthy calves that had been exposed by instilling the culture into the ventral nasal meatus of both nostrils in a previous study. The concentration of antibodies (IgG, IgA, and IgM) to P haemolytica increased significantly (P less than 0.05) in serum and nasal secretions after exposure. Four weeks after initial P haemolytica exposure, calves were exposed to infectious bovine rhinotracheitis virus and became clinically ill. Four calves were induced to shed P haemolytica from both nostrils by the virus infection; thus, they were harboring the bacterium and were susceptible to active recolonization. Four calves were not induced to shed P haemolytica. The apparent reason was not that they were resistant to active colonization, but that they were no longer harboring the bacterium, because they became active shedders after they were reinfected with P haemolytica.     

Serological types of Pasteurella haemolytica in Kenya

The 12 known serotypes of P. haemolytica and two biotypes A and T were found to occur in Kenya. Biotype T was more common in disease conditions than biotype A, which was more common in the nasal passages of healthy animals. Only biotypes A strains were recovered from cattle and the majority were serotypes 1 and 2, but serotypes 4 and 11 were also isolated. All serotypes were found to occur in sheep and goats, but serotypes 3, 4, 6, 8 and 10 were more commonly associated with pneumonia. It was observed that chickens could harbour both biotypes A and T in pathological conditions. Biotype A serotype 2 was isolated from an adult wildebeest, but the prevalence of P. haemolytica in wild animals needs furter investigation.     

Bovine herpesvirus-1 and Pasteurella haemolytica aerobiology in experimentally infected calves

Eight calves (2 calves in each of 4 groups) were exposed to an aerosol of bovine herpesvirus-1 (BHV-1) and 4 days later to an aerosol of Pasteurella haemolytica. Samples of tracheal and exhaled air were taken simultaneously beginning 1 day before viral exposure and once a day up to 3 to 4 days after the bacterial exposure. Samples were also taken during the period of aerosol exposure. Only 0.04% to 0.42% of P haemolytica-carrying droplets of the bacterial aerosol passed beyond the cranial part of the respiratory tract to the trachea. Nevertheless, numbers of bacteria as few as 1 bacterium/L of tracheal air were sufficient to produce fatal disease in the lungs of BHV-1-infected calves. In 1 of 4 groups, BHV-1 was isolated from most daily samples of exhaled and tracheal air. Pasteurella haemolytica was isolated 7 times more frequently from air when calves were kept at 1 C than when calves were kept at 23 C. The number of P haemolytica-carrying droplets in exhaled air was low (less than 1/L of air); however, samples obtained during the time that calves were coughing contained up to 10 P haemolytica-carrying droplets/L of air. It was learned that the cranial part of the respiratory tract serves as an efficient filter on inhalation and exhalation, but this filter is deficient in the animal when coughing occurs. This process expels infective droplets of size suitable for inhalation by other cattle in close proximity.     

Protein electrophoretic pattern of Pasteurella haemolytica.

Electrophoresis in polyacrylamide gels of the constituent proteins of the 12 serotypes and an untypable strain of Pasteurella haemolytica showed a pattern of bands that divided the group into two. This division conformed to the A and T biotype groupings of Smith (1959) although the serotype A9 showed only minor band difference from the three T serotypes 3, 4 and 10. It was not possible by this method to separate all the type strains from each other by the specific recognition of the patterns of protein mobilities produced.     

Factors affecting the immunogenicity of Pasteurella haemolytica in mice

An appreciable level of immunity from intraperitoneal infection with Pasteurella haemolytica was established in mice by using a vaccine prepared in a conventional bacteriological culture medium, with aluminium hydroxide gel as adjuvant. The level of immunity could not be elevated by using bacteria grown in tissue culture media, enriched brain heart infusion broth, the addition of serum to the media or by using bacteria that had been harvested in the logarithmic growth phase. Although various extracts of the bacteria elicited a distinct immunity, the immunogenicity of vaccines containing bacteria could not be enhanced by augmentation with those products. The potential application of the vaccine in cattle and sheep is discussed.     

Antigenic analysis of Pasteurella haemolytica serovars 1 through 15 by crossed immunoelectrophoresis

Pasteurella haemolytica serovars 1 through 12, grown in broth and on agar plates, and 2 field isolates (types A1 and T10) were used to develop polyvalent crossed immunoelectrophoresis (XIE) reference systems. The maximal number of antigens was revealed by XIE when sonicates of agar plate-grown organisms were used as the immunogen (to produce antibodies) and as the soluble antigen for XIE. Antigens produced from agar plate-grown organisms were less contaminated (by antigenic components of the medium) than were those produced from organisms grown in broth. Seventy-two antigens were detected in sonicated preparations of agar plate-grown P haemolytica. The common antigen of gram-negative bacteria was identified in the P haemolytica XIE reference system; precipitation was observed with rabbit antiserum to the common antigen of gram-negative bacteria isolated from Escherichia coli, as well as with rabbit immunoglobulins (obtained from unvaccinated rabbits). Most preimmune sera from our vaccinated rabbits also precipitated the common antigen. Serovar-specific antigens in the P haemolytica XIE reference system were defined and presumptively identified as part of the bacterial lipopolysaccharide complex by use of the limulus amebocyte lysate test. Partial cross-reactions were found between serovar-specific antigens within each biovar (A and T). Pasteurella haemolytica biovar A-specific and biovar T-specific antigens were defined by crossed-line immunoelectrophoresis. When serovars A13, A14, and T15 were tested in the P haemolytica XIE reference system, they gave high matching coefficient values of 0.98, 0.98, and 0.87, respectively. The proposal to separate P haemolytica biovars A and T into 2 different species was supported by immunotaxonomic data obtained from crossed immunoelectrophoresis, but more extensive studies will be necessary to establish the appropriate taxonomic position of these 2 groups of organisms.