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.     

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.     

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.     

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.     

Adherence of Haemophilus somnus to tumor necrosis factor-alpha-stimulated bovine endothelial cells in culture.

Vascular thrombosis and tissue infarction is a principal lesion in Haemophilus somnus septicemia known also as thrombotic meningoencephalitis. This study was undertaken to examine whether tumor necrosis factor-alpha (TNF-alpha) can influence the adherence of H. somnus to cultured bovine aortic endothelial cells (BAEC). Confluent BAEC were exposed to 0-100 nM of human recombinant TNF-alpha for 12-48 h. Suspensions of different strains of H. somnus (approximately 1.5-3 x 10(8) labelled with [methyl-3H]-thymidine, were added to BAEC and incubated for 1.5 h. Initial studies with one pathogenic (P) strain and one non-pathogenic (NP) strain revealed that both strains adhered to normal endothelial cells but minimally to subendothelial matrix remaining after removal of BAEC. Adherence to BAEC was reduced by an excess of unlabelled H. somnus of the same strain. Adherence was enhanced for both strains by exposure of BAEC to TNF-alpha in a manner that increased with TNF-alpha concentration and with duration of exposure to TNF-alpha prior to addition of bacteria. A survey of adherence of six live P strains and six NP strains demonstrated considerable variation but no difference in adherence between P and NP strains to normal or to TNF-alpha-stimulated BAEC. However, TNF-alpha consistently increased adhesion of each strain to BAEC. Both P and NP strains caused more severe cytotoxic changes in TNF-alpha-treated BAEC. Tumor necrosis factor-alpha also increased adhesion of formalin-killed bacteria of P and NP strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Haemophilus somnus: investigations of its potential role in bovine mastitis

Mammary glands of 6 lactating Holstein cows were inoculated with Haemophilus somnus strain 43826. Three cows developed chronic mastitis and shed bacteria for up to 1 year. Three cows developed acute gangrenous mastitis, with evidence of bacteremia and endotoxemia. Cows with gangrenous mastitis had lower somatic cell counts early after inoculation in affected quarter secretions compared with those in cows that developed chronic mastitis. Cows with gangrenous mastitis developed hypocalcemia, hypoalbuminemia, azotemia, hyperbilirubinemia, mildly increased serum aspartate aminotransferase and creatine kinase activity, and a marked neutropenia with a degenerative left shift. Histopathologic examination of gangrenous quarters revealed edema, necrosis, and vascular thrombosis with few inflammatory cells. A limited survey failed to recover H somnus from dairy cows with clinical mastitis or from mammary secretions from 41 beef cattle at an abattoir.     

A Comparison of Various Haemophilus somnus Strains

Sixty-eight Haemophilus somnus strains isolated from the bovine in Canada and the U.S.A. were compared. In media enriched with 5% ovine serum, 5% bovine serum and 10% yeast extract, H. somnus fermented glucose, levulose, maltose, mannitol, mannose, sorbitol, trehalose and xylose, but failed to ferment arabinose, dulcitol, galactose, inositol, lactose, raffinose, rhamnose, salicin and sucrose. The organisms acidified litmus milk, produced cytochrome oxidase, indole and hydrogen sulfide (H₂S) and reduced nitrates to nitrites. The motility, methyl-red, acetylmethyl-carbinol urease catalase, citrate, malonate, lysine, ornithine and arginine tests were negative. Haemophilus somnus was resistant to lincomycin, neomycin and triple sulfa, but susceptible to ampicillin, chloramphenicol, streptomycin, penicillin and tetracycline. No antigenic differences were noted between strains when tested against rabbit antisera of eight strains using agglutination, complement-fixation, immunodiffusion and counterimmunoelectrophoresis tests. Low titre cross-reactions were found in the agglutination tests with some of the anti-H. somnus rabbit sera with Actinobacillus lignieresi and Moraxella bovis. No distinct antigenic similarities to nine other species of pathogenic bacteria of animal origin were found. No difference was observed between H. somnus isolates from Ontario and those from western Canada and the U.S.A.     

Tumor necrosis factor-alpha enhances Haemophilus somnus lipooligosaccharide-induced apoptosis of bovine endothelial cells

Haemophilus somnus lipooligosaccharide (LOS)-induced apoptosis of bovine pulmonary artery endothelial cells has been shown previously to be dependent on capsase-8 activation. Activation of caspase-8 can occur via a death receptor-dependent mechanism (e.g., TNF- binding to TNF- receptor 1 (TNF-R1)). In this study, we tested the hypothesis that TNF- can enhance LOS-induced apoptosis of bovine endothelial cells. Addition of exogenous recombinant human TNF- alone failed to cause apoptosis, or enhance LOS-induced apoptosis, of bovine endothelial cells. However, blocking de novo protein synthesis by addition of cycloheximide significantly enhanced apoptosis of bovine endothelial cells by TNF-, LOS or TNF- and LOS in combination. Conversely, addition of soluble recombinant human (sTNF-R1) diminished LOS-induced apoptosis. Overall, these data suggest that LOS-mediated apoptosis may be due, in part, to activation of a TNR-R1-dependent death pathway.     

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).     

Immune mechanisms of pathogenetic synergy in concurrent bovine pulmonary infection with Haemophilus somnus and bovine respiratory syncytial virus

Bovine respiratory syncytial virus (BRSV) and Haemophilus somnus are two bovine respiratory pathogens that cause disease singly or as part of a polymicrobial infection. BRSV infection is often associated with a predisposition towards production of a T helper type 2 (Th2) response and IgE production. In contrast, an IgG2 response to H. somnus has been shown to be most important for recovery. An experiment was performed to evaluate the hypothesis that infection with H. somnus on day 6 of experimental BRSV infection would result in disease enhancement and potentially an altered immune response when compared with single infection. Three groups of calves were either dually infected or singly infected with H. somnus or BRSV. Serum and bronchoalveolar lavage fluid (BALF) pathogen specific IgG1, IgG2, IgE, and IgA responses were evaluated by ELISA. TaqMan RT-PCR was used to examine cytokine gene expression by PBMC and BAL cells. Clinical signs were evaluated for 28 days after BRSV infection, followed by necropsy and histological examination of the lungs. In dually infected calves, disease was significantly more severe, H. somnus was isolated from the lungs at necropsy, and high IgE and IgG responses were detected to H. somnus antigens. Cytokine profiles on day 27 were elevated in dually infected calves, but did not reflect a skewed profile. These results contrasted with singly infected calves that were essentially normal by day 10 of infection and lacked both lung pathology and the presence of H. somnus in the lung at necropsy. The increase in IgE antibodies specific for antigens of H. somnus presents a possible mechanism for pathogenesis of the disease enhancement.     

Reactive oxygen and nitrogen intermediates contribute to Haemophilus somnus lipooligosaccharide-mediated apoptosis of bovine endothelial cells

Although Haemophilus somnus causes septicemia and vasculitis in cattle, relatively little is known about how H. somnus affects endothelial cells in vitro. We previously reported that H. somnus lipooligosaccharide (LOS)-induced activation of caspases-3, -8 and -9, and apoptosis of bovine pulmonary artery endothelial cells (BPAEC) in vitro. Previous reports indicate that the generation of reactive oxygen species (ROS) or reactive nitrogen intermediates (RNI) can contribute to the induction of apoptosis. In the present study, we sought to determine whether ROS and RNI are involved in LOS-mediated apoptosis of BPAEC. We found that H. somnus LOS induced the generation of ROS in BPAEC, which was blocked by pretreatment with membrane permeable ROS scavengers, such as dimethylsulfoxide (DMSO) and allopurinol (AP). Addition of DMSO or AP significantly reduced H. somnus LOS-mediated caspase-3 activation. Addition of membrane impermeable ROS scavengers (e.g. catalase and superoxide dismutase), failed to block LOS-mediated caspase-3 activation, suggesting a role for intracellular generation of ROS in LOS-induced apoptosis of BPAEC. Addition of N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, which are selective inhibitors of nitric oxide synthase, blocked NO release and significantly reduced caspase-3 activation in LOS treated BPAEC. These data suggest H. somnus LOS triggers endogenous ROS and RNI production by endothelial cells, which contributes to apoptosis.     

Characterization of putative Haemophilus somnus isolates from tonsils of American bison (Bison bison).

Three Haemophilus somnus isolates (2a, 3a, and 27b) and one H. somnus-like (13b) isolate from tonsils of commercially reared American bison were compared with 2 known H. somnus isolates from cattle, namely, 2336, shown to cause respiratory disease, and 129Pt, from the prepuce of an asymptomatic bull. All H. somnus isolates, but not the H. somnus-like isolate, required CO2 for growth. Biochemical utilization profiles were identical for bison and bovine H. somnus isolates with the exception of alpha-fucosidase production by isolate 3a. Isolate 27a varied from 2a, 2336 and 129Pt by hemolysis of bovine erythrocytes. Isolate 13b hemolyzed sheep but not bovine or bison erythrocytes and varied from other isolates in biochemical utilization tests. Outer membrane protein profiles of 2a, 3a and 27a were almost identical with those of bovine isolate 2336 and similar to that of 129Pt, but quite different from that of 13b. Western blots of bison isolates were similar to that of the virulent bovine 2336 isolate, including detection of high molecular mass antigens above 100 kDa and the 76 kDa antigens associated with bovine IgG2 Fc binding characteristic of virulent strains, as well as antigens of approximately 78, 60 and 40 kDa. Producers and veterinarians should be aware that H. somnus may be carried by bison and may have potential for causing diseases in bison similar to those described in cattle and sheep.     

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)     

Epidemiology of Haemophilus somnus in young rams

The prevalence of Haemophilus somnus in the prepuce of young rams was examined. Of 473 rams entering Record of Performance (ROP) stations at 50 days of age, 43 (9.1%) were positive. Average daily gain was not affected by Haemophilus status, but was influenced by breed of ram. Suffolks were predicted to gain 0.515 kg daily compared to 0.427 kg for a group combining all other breeds. Using logistic regression to identify risk factors for individual H. somnus infection, rams in 1989 were 0.382 times as likely to be infected as rams in 1988, and Suffolks were 0.314 times as likely to be infected as the other breeds group, but these factors were not significant at the flock level. Of 80 eligible flocks of origin, 22 (27.5%) were classified as infected with H. somnus, based on rams submitted to the ROP station. Infected flocks contributed 133 rams, 43 (32.3%) of which were positive. There was no association between H. somnus status and lambing percent of the percent of abortions and stillbirths, but there was a statistically significant association with the percent of ewes which failed to lamb. In the model developed, 6% of the bred ewes in noninfected flocks failed to lamb, compared to a rate of 12% in infected flocks. These results suggest H. somnus may influence ewe fertility earlier, rather than later in gestation. Purchasing replacement animals and having cattle on the farm were risk factors for Haemophilus infection in the flock. Where replacements had been purchased within the previous year, the risk of flock infection rose 8.5 times, and on farms having cattle as well as sheep, the risk rose 13.2 times.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Environmental survival of Haemophilus somnus and influence of secretions and excretions.

Environmental survival of the Haemophilus somnus virulent strain 43826 was examined by mixing it with bovine secretions and excretions and observing viability after storage at -70 degrees C, 3 degrees C, 23.5 degrees C and 37 degrees C at one day, five days, 12 days, 19 days and intermittently up to 75 days. Survival of the organism beyond 70 days occurred when it was mixed with cerebrospinal fluid, whole blood, blood plasma, vaginal mucus and milk and frozen at -70 degrees C. At 3 degrees C the organism in these fluids survived for five days or less. At 23.5 degrees C the organism survived beyond 70 days when mixed with whole blood and nasal mucus. The viability of H. somnus in urine at all temperatures was less than 24 hours and less than 15 minutes at 20 degrees C and 37 degrees C. Infective cerebrospinal fluid frozen alone in liquid nitrogen and with the addition of various cryopreservatives allowed the organism to survive and maintain virulence for at least 56 days. The implications of these studies to disease transmission and experimental studies is discussed.