Haemophilus somnus as a cause of bronchopneumonia in calves

A bacteriological examination of the pulmonary tissues of calves suffering from bronchopneumonia was complemented by a microaerophilic or anaerobic cultivation. Thirty-two strains (23.7%) of a microorganism hitherto not described in Czechoslovakia were isolated during the examination of 135 calves with disorders of the respiratory system. On the basis of the growth, morphological, biochemical and serological properties, the microorganism was identified as Haemophilus somnus. Chocolate agar supplemented with yeast extract, and blood agar were used for the cultivation. Incubation took place in a microaerophilic medium with 10% carbon dioxide or in an anaerobic medium. Serums against the collection strain of Haemophilus somnus were prepared in rabbits for the serological diagnosis. These antiserums reacted with all tested strains during test tube agglutination but did not enter cross reactions with other bacterial species. The coagglutination test, which was verified, did not provide reliable results.     

Effects of vaccination of calves against induced Haemophilus somnus pneumonia

The efficacy of a killed whole-cell Haemophilus somnus bacterin against induced H somnus pneumonia was examined in 10-week-old male calves. Twenty calves were assigned to 1 of the 3 following groups: group 1, nonvaccinated controls (n = 4); group 2, vaccinated once (n = 8); and group 3, vaccinated twice 14 days apart (n = 8). The serum antibody response to vaccination and challenge exposure was evaluated by the bacterial agglutination test and solid-phase immunoassay (SPIA). Vaccinating calves twice, 14 days apart, significantly (P less than 0.05) reduced the severity of clinical signs of pneumonia and gross lesions. Deaths occurred in 1 of 4 nonvaccinated controls, 1 calf vaccinated once, and none of the calves vaccinated twice, 14 days apart. Postvaccination bacterial agglutination titers measured 14 days after the final vaccination were not significantly different between groups 2 and 3, but SPIA titers were significantly (P less than 0.05) higher in groups 2 and 3, compared with those in group 1. The less severe clinical signs of pneumonia observed in group-3 calves, compared with those in calves in groups 1 and 2, were significantly (P less than 0.01) correlated to higher SPIA titers, indicating the protective value of vaccinating twice.     

Experimental Haemophilus somnus pneumonia in calves and immunoperoxidase localization of bacteria

Pneumonia was produced in nine, conventionally reared calves by intrabronchial inoculation with Haemophilus somnus. Volumes of pneumonic lung were determined stereologically, following serial slicing of lungs fixed by vascular perfusion. Twenty-four hours after inoculation, consistent findings were: neutrophilic to fibrinoid vasculitis, degeneration of alveolar macrophages, necrotizing bronchiolitis, suppurative bronchopneumonia, lobular necrosis, and dilation and thrombosis of lymphatics. Bacteria were identified histologically by an immunoperoxidase technic and were either free in alveoli or associated with degenerative alveolar macrophages. The latter suggests that macrophage degeneration may be a result of bacteria/macrophage interaction. Immune complex deposition is unlikely to be the principal mechanism for the vasculitis because bacterial antigen was not generally found in necrotic vessel walls, and two colostrum-deprived, H. somnus antibody-negative calves also had neutrophilic vasculitis 12 to 24 hours after inoculation with the lowest dose of H. somnus used in the above experiment.     

Efficacy of immunization of feedlot calves with a commercial Haemophilus somnus bacterin.

Two cohorts, consisting of 10,723 calves total, were identified in this prospective follow-up study to investigate whether immunization of auction market beef calves immediately upon arrival at the feedlot with a commercial Haemophilus somnus whole cell killed bacterin would reduce subsequent mortality. In addition to mortality rate, the use of incidence rate of fatal disease is introduced as an effect measure to examine vaccine efficacy in the feedlot. The Haemophilus somnus bacterin had no significant effect on the overall crude mortality rate; however, the bacterin appeared to significantly (p less than 0.05) reduce the incidence rate of fatal disease and the mortality rate during the first two months in the feedlot, when risk of fatal disease onset was highest. Once mortalities likely not associated with hemophilosis (for example, a fractured femoral neck) were removed from the analysis, steer mortality rate, but not heifer mortality rate, was reduced significantly (p less than 0.05) in the vaccinated group. The attributable percent overall for steers was 17.4%; this suggests that 17.4% of fatal respiratory disease in the unvaccinated steers could have been prevented by vaccination with the H. somnus bacterin. Heifer calves demonstrated a significantly (p less than 0.01) higher incidence rate of fatal disease during the first week than did steer calves, indicating that a different pattern of fatal disease existed for the two sexes. Use of a second vaccination two weeks after arrival did little to decrease mortality risk.     

Influence of recombinant bovine interferon gamma and dexamethasone on pneumonia attributable to Haemophilus somnus in calves

The influence of recombinant bovine interferon gamma (rBoIFN-gamma) treatment on resistance of clinically normal and dexamethasone-treated calves to Haemophilus somnus infection was evaluated. Four groups of 6 calves each were treated with saline solution (controls), dexamethasone (0.04 mg/kg of body weight/for 3 days), rBoIFN-gamma (2 micrograms/kg for 2 days), or dexamethasone and rBoIFN-gamma (aforementioned dosages). All treatments were started 24 hours before intrabronchial challenge exposure with 5 x 10(9) colony-forming units of H somnus. Rectal temperature and WBC count were monitored daily. Two of the dexamethasone-treated calves died of pneumonia 4 days after challenge exposure and were necropsied. All other calves were euthanatized and necropsied 7 days after challenge exposure. All calves had pneumonia of variable intensity. Dexamethasone-treated calves had increased volume of pneumonic lung (P less than 0.05) and increased severity of pneumonia, compared with control calves. Recombinant bovine interferon gamma treatment resulted in reduction in pneumonic lung volume and severity of pneumonia in dexamethasone-treated calves (P less than 0.05), although it did not influence severity of pneumonia in nondexamethasone-treated calves.     

Haemophilus somnus-induced IgE in calves vaccinated with commercial monovalent H. somnus bacterins

The ability of commercially available Haemophilus somnus bacterins to elicit an immunoglobulin E (IgE) response was examined in healthy calves using enzyme-linked immunosorbent assay (ELISA) and western blotting techniques. Thirty five calves were utilized in this study. Calves in Group 1 (n=7) did not receive any H. somnus vaccination and served as negative controls. Calves in each of Groups 2-5 (n=7 each) were vaccinated on days 0 (primary) and 14 (booster) with one of four commercially available H. somnus bacterins. Sera were harvested on days 0 and 14 and at weekly intervals for a total of 45 days. Sera were tested for the presence of IgE antibodies using a bovine IgE-specific ELISA.Low levels of H. somnus-specific IgE were detected by ELISA in all animals prior to the initiation of the study. All bacterins induced IgE levels that were significantly higher than control levels. Two bacterins elicited higher IgE levels at all time points. Sera were adsorbed against washed whole cells of either Salmonella typhimurium, P. multocida, or H. somnus or extracts of H. somnus. ELISA absorbance values were significantly decreased by adsorption with washed whole cells or extracts of H. somnus, whereas adsorption with other gram-negative bacteria only minimally decreased ELISA absorbance values. These results indicate that commercially available H. somnus bacterins can induce IgE antibody as early as 14 days post-vaccination. This IgE can be detected 45 days after the primary vaccination. Results also indicate that H. somnus-specific IgE antibodies can be found in serum of some cattle, possibly induced by existing or previous sensitization.     

Aerosol challenge of calves with Haemophilus somnus and Mycoplasma dispar

The aim of the study was to examine the ability of Haemophilus somnus and Mycoplasma dispar to induce pneumonia in healthy calves under conditions closely resembling the supposed natural way of infection, viz. by inhalation of aerosol droplets containing the microorganisms. The infections were investigated by recording clinical data, cytokine expression of peripheral blood cells and pathology. Twelve calves were included in the study: Three animals were exposed to H. somnus only, and two to M. dispar only, whereas five were challenged to M. dispar followed by exposure to H. somnus 11-14 days later. Also, one calf was exposed to M. dispar followed by exposure to a sterile saline solution 11 days later, and one calf was only exposed to a sterile saline solution. Just one animal, only challenged with H. somnus, developed a focal necrotizing pneumonia, from which H. somnus was isolated. Thus, the ability of H. somnus and M. dispar to act as primary pathogens under these conditions were minimal and inconsistent.However, a transient rise in body temperature, a marked granulocytosis and increased levels of interleukin-8 in peripheral blood after inoculation with H. somnus indicated a clear systemic response, probably as a consequence of the natural non-specific local and systemic defence mechanisms acting in healthy calves.     

Haemophilus somnus complex: antigenicity and specificity of fractions of Haemophilus somnus.

Five Haemophilus somnus type 8025 preparations (whole cell, sonicate, crude polysaccharide, purified polysaccharide, and protein) were produced for studies of their antigenicity in rabbits. Bacterial agglutination and passive hemagglutination tests were used to assess the level of antibody produced in rabbits inoculated with the different antigenic preparations. Cross-reactions were seen between the antiserums against the H sumnus 8025 antigens and a variety of related and unrelated bovine pathogens. The strongest cross-reaction occurred between antiserums against H somnus 8025 whole cell and crude polysaccharide antigens and Haemophilus agni and Actinobacillus lignieresii cell suspensions.     

Initial lung lesions in two calves experimentally infected with Haemophilus somnus

The initial lung lesions in two calves intrabronchially inoculated with Haemophilus somnus are described. The animals were euthanized within 7 h after challenge. The in situ location of H. somnus and accompanying lesions were examined by light microscopy, immunohistochemistry and transmission electron microscopy (TEM). Inoculation with H. somnus resulted in the development of acute pulmonary lesions within 3.5 h. H. somnus antigen was demonstrated only within the luminal spaces of the airways and in one area of bronchio-associated lymphoid tissue (BALT). As observed by TEM, the bacteria were phagocytized by both neutrophils and alveolar macrophages. Antigen was never demonstrated in the pulmonary intravascular macrophages.     

Virulence differences among three strains of Haemophilus somnus following intratracheal inoculation of calves.

Pneumonia was induced in four month old Holstein calves by intratracheal inoculation of 1 x 10(9) colony forming units of Haemophilus somnus. Twenty calves were divided into four groups of five and challenged with a pneumonic strain (Group 1), an encephalitic strain (Group 2), a preputial strain (Group 3), or a placebo (Group 4). The clinical score, neutrophil count, respiratory rate, and temperature were significantly increased in group 1 by day 1 postinoculation (P less than 0.05) and maintained until day 6 postinoculation (P less than 0.05). The macroscopic pathological changes were significantly greater in group 1 (P less than 0.05). Haemophilus somnus was consistently isolated from pneumonic tissue of group 1 only. Groups 2 and 3 had mild transient increases in all parameters measured and macroscopically only small focal lesions were present. It is concluded that virulence differences exist between H. somnus strains following intratracheal challenge of bovine lungs.     

Comparison of bacterial cultivation, PCR, in situ hybridization and immunohistochemistry as tools for diagnosis of Haemophilus somnus pneumonia in cattle

The aim of the present study was to compare the potential of bacterial cultivation (BC), PCR, in situ hybridisation (ISH), and immunohistochemistry (IHC) in the diagnosis of Haemophilus somnus, when applied to pneumonic bovine tissue. Lungs from 65 field cases submitted for bacteriological examination were included in the study. The PCR-detection was performed on three different samples: plate-PCR (detection on plate washes after incubation of lung tissue on agar plates); swab-PCR (direct detection on a swab from the cut surface); and, whenever possible, a bronchus-PCR (direct detection on a swab from the main bronchus of the right cranial lung lobe). In order to examine the pathological significance of the findings, a histopathological examination of the cases was performed. H. somnus was detected by one or more techniques in 33 cases in total. By BC the bacterium was isolated from 10 cases, IHC and ISH were positive in 17 and 19 cases, and plate- and swab-PCR were positive in 21 and 29 cases, respectively. The bronchus-PCR was positive in 30 out of 61 cases examined. The PCR-technique was the most sensitive method, and as this technique is fast and relatively inexpensive, it should be considered as a supplementary tool in the diagnosis of H. somnus induced calf pneumonia.     

Ovine Haemophilus somnus: experimental intracisternal infection and antigenic comparison with bovine Haemophilus somnus.

Experimental infection was produced by two of four isolates of ovine Haemophilus somnus given by intracisternal inoculation into two to three-month-old lambs. Isolate 2041 (originally obtained from a septicemic lamb in Alberta) caused lethal infection in eight of nine lambs, isolate 67p from the prepuce of a normal lamb produced less acute disease in four of nine lambs, and the other two isolates (93p and 1190) caused no detectable disease. Significant lesions were limited to the brain and spinal cord. Purulent meningitis was characteristic but vasculitis or septicemia were not detected, perhaps due to the route of inoculation. Since a difference in virulence was noted among strains, we analyzed surface proteins thought to be virulence factors of bovine H. somnus. Protein profiles of bovine and ovine H. somnus done by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed similar patterns for virulent bovine isolates and ovine septicemic isolates. Preputial isolates showed a lower molecular mass major outer membrane protein than septicemic isolates. Antigenic analysis revealed that outer membrane proteins p270, p78, p76, p40, and p39 were detected in both ovine and bovine isolates except for 1190, which was probably not a true H. somnus isolate. Thus the preputial and septicemic isolates of ovine H. somnus were similar to bovine H. somnus in pathogenicity and in surface antigens.     

Specificity of IgG and IgE antibody responses to Haemophilus somnus infection of calves

Haemophilus somnus is an important cause of bovine respiratory disease and septicemia with all it's sequelae. The role of immune responses in protection and immunopathogenesis is not well understood. We showed that infection with bovine respiratory syncytial virus (BRSV) 6 days before H. somnus increased clinical scores and levels of IgE antibody to H. somnus over that of infection with H. somnus alone. To determine whether antigenic specificity of IgE responses differed from IgG responses, Western blots were done with sera from the infected calves, at 0 time and at 21 days post infection. Thus each calf was its own control. IgG antibodies recognized primarily a 40 kDa outer membrane protein (OMP) in whole cell H. somnus preparations and a 270 kDa immunoglobulin binding protein (IgBPs) in culture supernatants but generally not the 41 kDa major OMP (MOMP). IgE antibodies recognized primarily the 41 kDa MOMP in whole cell pellet preparations. Results were consistent among calves. With culture supernatants, IgE antibodies recognized both the 270 kDa IgBPs and the MOMP. Since some H. somnus strains from asymptomatic carriers (including strain 129Pt), do not have IgBPs and express a truncated MOMP (33 kDa rather than 41 kDa), reaction of strain 129Pt cells with serum from calves infected with H. somnus or BRSV and H. somnus was studied. IgE did not react with the truncated MOMP even at much lower (1:100) dilutions than in Western blots with virulent strain 2336 (serum dilution of 1:500). Reactions of IgE with the 40 and 78 kDa antigens in strain 129Pt were noted but since the major reactivities with the IgBPs and the MOMP were not detected, this strain may be useful for inducing protective rather than immunopathogenic responses.     

Haemophilus somnus Mastitis in a Dairy Cow

A clinical case of peracute bovine mastitis is described in which the most severely affected quarter yielded a heavy growth of Haemophilus somnus on culture.     

The occurrence of Haemophilus somnus in feedlot calves and its control by postarrival prophylactic mass medication.

Three field trials were conducted in a large commercial feedlot in Saskatchewan to determine the prevalence of Haemophilus somnus in calves and to evaluate the efficacy of prophylactic mass medication with long-acting oxytetracycline on day 17 (1990, n = 1336), day 11 (1991, n = 4372), or day 8 (1992, n = 5632) postarrival. Hemophilosis accounted for > 40% of the mortality in feedlot calves each year. Haemophilus somnus was cultured from the blood of one febrile calf on day 1 (0.1%, n = 895), but it was not cultured from nasal swabs on day 1 or day 11 (n = 881) or from blood samples on day 11 (n = 883). Similarly, it was not cultured from nasal swabs or blood samples from sick calves first treated for bovine respiratory disease (BRD) (n = 219). Serological titers to H. somnus increased (p < 0.05) in unvaccinated calves from day 1 (Geometric mean titer = 11,846) to day 96 (Geometric mean titer = 63,712), indicating natural infection following feedlot entry. Calves that relapsed twice with BRD or died from BRD +/- hemophilosis had significantly (p < 0.06) lower titers to H. somnus on days 1 and 96 than those that did not relapse twice or die. Postarrival mass medication with long-acting oxytetracycline did not reduce (p > 0.05) the risk of hemophilosis mortality. However, it reduced (p < 0.05) the risk of BRD treatment by 14% and the risk of BRD mortality by 71%. Additional epidemiological studies of H. somnus are needed so that we can develop strategic medication and vaccination programs to reduce losses from hemophilosis.