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.     

Isolation of pathogenic strains of Haemophilus somnus from the female bovine reproductive tract.

The prevalence of Haemophilus somnus in the genital tract of slaughtered and live cows in southern Ontario was investigated. The vagina and uterus of slaughtered cows were swabbed separately. Live cows were examined and sampled in two field surveys: Centre A and Centre B. In the former, aspirated mucus secretions and in the latter, specimens obtained by guarded swabbing were examined bacteriologically. Haemophilus somnus was isolated from 28 genital tracts of 461 slaughtered (6.1%), and seven of 199 live (3.5%) cows during the centre B survey. The isolates were recovered from both normal and diseased reproductive tracts. Fourteen strains isolated from genital organs were examined for pathogenicity in vivo to test the occurrence of pathogenic isolates. In the initial stage of the in vivo study on pathogenicity, each of the fourteen isolates was examined on one calf using an intracisternal inoculation. Subsequently, one pathogenic and one nonpathogenic strain were inoculated into five calves each to statistically confirm their pathogenic potential. Of 14 genital isolates of H. somnus examined in an intracisternal calf assay, six (43%) caused a fatal peracute neurological disease, while eight were nonpathogenic. A comparative pathological study of pathogenic and nonpathogenic isolates showed that the former caused a severe fatal suppurative meningoencephalitis whereas the latter caused no lesions whatsoever or a mild leukocytic leptomeningitis. The salient data obtained in this study indicate that there are pathogenic strains of H. somnus in the genital tract of apparently normal cows as well as of those with inflammatory disease.     

Experimental bovine respiratory tract disease with Haemophilus somnus

Eight calves were inoculated into the bronchus with H. somnus. Thirteen calves were inoculated with bovine respiratory syncytial virus (BRSV) and 8 days later with H. somnus. All calves developed necrotizing, suppurative, lobular bronchopneumonia and pleuritis. Clinical signs of disease and pneumonic lesions were significantly more severe in calves that were sequentially inoculated with BRSV followed by H. somnus. Pneumonic lesions in the inoculated calves were similar to those described for naturally occurring H. somnus-associated respiratory tract disease. Control calves inoculated with BRSV alone or sham-inoculated with medium did not develop clinical signs of respiratory tract disease. The BRSV-inoculated control calves developed minimal pneumonic lesions.     

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.     

Microscopic lesions associated with the isolation of Haemophilus somnus from pneumonic bovine lungs

Sixty-one of 68 sets of bovine lungs from which only Haemophilus somnus was isolated had microscopic lesions of purulent bronchiolitis and bronchopneumonia. In 37 of 61 lungs, the bronchiolar exudates were markedly necrotic with accompanying necrosis of the adjacent bronchiolar epithelium. Bronchiolitis obliterans was prominent in 23 of 28 lungs affected with chronic lesions with abscesses present in seven. Alveolar filling with inflammatory cells (neutrophils with fewer macrophages) was limited to peribronchiolar alveoli in 25 of 61 lungs and was multifocal to diffuse in the other 36. Lesions in the remaining lungs (7 of 68) were classified as fibrinous pneumonia with bronchiolitis (2), fibrinous pleuritis (2), suppurative interstitial pneumonia with vasculitis (2), and diffuse congestion (1).     

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.     

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.     

Isotypic antibody responses in cattle infected with Haemophilus somnus

Bovine antibody responses to Haemophilus somnus were compared on the basis of clinical and bacteriological findings. Serum IgG1 and IgM antibody titres were significantly increased in clinically normal cattle that were bacteriologically positive for H somnus from the nasal or vaginal mucosae compared with clinically normal, negative cows. IgG2 titres did not differ significantly between these two groups. However, IgG2 antibody was significantly higher in animals with H somnus disease (pneumonia or abortion) than in clinically normal cattle (whether bacteriologically positive or negative), while IgG1 and IgM titres did not differ between diseased and bacteriologically positive, clinically normal cattle. These antibody trends were duplicated in experimental H somnus abortion or pneumonia, with the greatest response occurring within the IgG2 subclass. Cattle vaccinated systemically with killed whole H somnus produced a predominant IgG2 response with minimal IgG1 and IgM responses. These results demonstrate that IgG2 antibody is consistently elevated in H somnus disease, and suggest that this response may be useful in discriminating diseased from asymptomatic cattle.     

Monoclonal antibody in the identification of Haemophilus somnus

Electrophoretic comparisons of outer membrane proteins of Haemophilus somnus isolates revealed 2 major protein bands (46 and 14 kilodaltons [kD]) common to all isolates tested. A monoclonal antibody raised against H. somnus reacted to the 46-kD band. Coagglutination tests were performed using a monoclonal antibody coagglutination assay. The monoclonal reagent was produced by incubating Cowan strain Staphylococcus aureus suspension, used as a source of crude protein A, with mouse ascitic fluid monoclonal antibody or goat anti-H. somnus hyperimmune serum. Bacteria to be tested were suspended at a concentration of 4.5 x 10(9) cells/ml. The coagglutination test was performed by the addition of 50 microliters of the monoclonal reagent to 50 microliters of the bacterial suspension on a glass plate and manual rotation for 2-3 minutes. The coagglutination assay using Cowan strain Staphylococcus aureus protein A, coupled with the monoclonal antibody, agglutinated 10 different H. somnus isolates. The antibody reagent did not coagglutinate with Actinobacillus suis, A. equuli, Pasteurella haemolytica, P. multocida, or P. pneumotropica under similar test conditions.     

Isolation of Haemophilus somnus antigens and their use as vaccines for prevention of bovine thromboembolic meningoencephalitis

Antigens extracted from Haemophilus somnus were examined for their suitability as vaccines for prevention of thromboembolic meningoencephalitis and as antigens in immunologic tests for detection of susceptible cattle. Saline extraction of whole H somnus cells produced an outer membrane complex (OMC) that contained 2 major antigens when tested against antiserum to intact cells by immunoelectrophoresis. Anion exchange chromatography was used to separate an anionic antigen (AA) from the more cationic antigen (CA). According to chemical analysis and polyacrylamide gel electrophoresis, both AA and CA were complex mixtures--probably, outer membrane fragments. Enzyme-linked immunosorbent assays were used to measure serum levels of immunoglobulin (Ig) G and IgM against AA and CA in cattle vaccinated with whole cells, OMC, CA, or AA. Protection of vaccinated cattle was assessed after IV challenge exposure to H somnus. Moderate IgG and IgM responses occurred when cattle were given 2 vaccinal doses of 0.1 mg or 1.0 mg of whole cells, OMC, or AA. Two of 10 cattle given 2 vaccinal doses (1.0 mg) of OMC died after IV challenge exposure, whereas 8 of the 10 controls died, indicating significant protection (P less than 0.05). All of the 10 cattle given 2 vaccinal doses (1.0 mg) of AA were protected from IV challenge exposure, whereas 5 controls died (P less than 0.05). Two vaccinal doses of either 0.1 mg or 1.0 mg of CA produced high IgG and IgM responses. However, 3 of 10 cattle given 2 vaccinal doses (1.0 mg) of CA died after IV challenge exposure, as did 3 of the 10 controls, indicating that CA was not protective.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA sequence of a small, unidentified plasmid isolated from a Haemophilus somnus strain: short communication

One of the plasmids present in a Haemophilus somnus strain isolated from nasal discharge of a cattle with respiratory disease was purified and cloned for DNA sequencing. The plasmid was found to be 1065 base pairs long with 39.2% G + C content, and showed no homology to any DNA sequenced so far. It has no capacity to code any protein longer than 43 residues. It is not clear yet if this plasmid codes Haemophilus somnus specific factors.     

Atypical nervous lesion in Haemophilus somnus infection of cattle

Two cows affected with Haemophilus somnus were examined pathologically. Although manifested lesions were composed of fibrinopurulent meningitis without the usual parenchymatous lesions in the central nervous system, Haemophilus somnus was isolated in pure culture from the cerebral cortex of both cases. Presence of this atypical lesion in the nervous system should be kept in mind for the differential diagnosis of the disease.     

Adherence of Haemophilus somnus to bovine embryos after in vitro exposure

Preimplantation bovine embryos were exposed in vitro to Haemophilus somnus to determine whether the bacteria would adhere to zona pellucida-intact embryos or would adhere to or infect zona pellucida-free embryos. The effect of H somnus on in vitro embryonic development also was investigated. After exposure to H somnus and before washing, some of the zona pellucida-intact embryos were held in antibiotic-containing medium. Haemophilus somnus was isolated from 10 to 42 zona pellucida-intact embryos and none of the zona pellucida-free embryos. Haemophilus somnus was not recovered from any of the 32 antibiotic-treated embryos. The coculture system was not compatible with normal embryonic development, and all embryos had begun to degenerate by the end of the 18-hour exposure period.     

Immunological characterization of the major outer membrane protein of Haemophilus somnus

Antigens and molecular mass diversity of the Haemophilus somnus major outer membrane protein (MOMP) were investigated. The molecular mass of the MOMP of 53 strains of H. somnus varied from 43 to 33 kDa and four MOMP MAb reactivity patterns were detected in immunoblot analysis and immunodot assay. The molecular mass and MAb reactivity data were used for preliminary grouping of H. somnus strains. Disease strains fell into groups 1 and 3, including two of three Group 3 subgroups, whereas strains from asymptomatic carriers were found in all the four groups and three subgroups. Immunoblot analysis with convalescent phase serum showed strain specific reactivity with MOMPs from three isolates used to reproduce disease in cattle. The reaction with the MOMP was only detectable at dilutions of 1:100 or less, whereas the same convalescent sera showed strong reactivity at dilutions of 1:1000 (or more) with other H. somnus antigens. The data suggest that the bovine immune response to the MOMP during infection is weak and is directed to antigenically variable determinants in a strain-specific manner. This may be important in evaluating the role of the antibody response to MOMPs in protective immunity.