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.     

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 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.     

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.     

A comparative study of bovine and ovine Haemophilus somnus isolates.

Bacterial isolates (including 17 Haemophilus somnus isolates and an H. somnus-like isolate) from asymptomatic or diseased cattle and sheep, were evaluated for markers associated with virulence and host predilection. The isolates were separated into 6 distinct biovariants, 3 for sheep and 3 for cattle, based on reactions in a battery of 21 test media. Three bovine isolates associated with disease caused hemolysis of bovine blood. The rest of the isolates did not hemolyze either bovine or ovine erythrocytes. Protein profiles of all H. somnus isolates were similar with the exception of the major outer membrane proteins (MOMPs). The MOMPs of isolates associated with disease in cattle had a relative molecular weight of approximately 41 kDa compared with 33 kDa for the MOMPs of isolates from asymptomatic cattle. The MOMPs from sheep isolates were either slightly higher or lower than the 41 kDa MOMPs of bovine isolates. Major antigens detected by Western blotting were similar in all isolates except the H. somnus-like isolate. An immunodominant 40 kDa antigen was conserved in all H. somnus isolates. Antibodies to this antigen have previously been found to be protective in cattle and may also be protective for sheep. Marked differences between cattle and sheep isolates were revealed by use of restriction enzyme analysis, which separated the isolates into 12 ribotypes and 15 unique DNA profiles. Thus, cattle and sheep isolates in this collection had distinctive differences in biochemical reactions, MOMP profiles, and DNA analyses. Such differences have potential value for epidemiological studies and may also be used to evaluate host specificity of H. somnus isolates.     

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.     

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)     

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).     

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)     

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.     

Experimental Haemophilus somnus infection in pregnant cattle

Of five pregnant cows inoculated intravenously with 5 X 10(8) viable 'Haemophilus somnus', one aborted within 5 days and excreted 'H. somnus' from the vagina for a further 7 weeks. A second cow proceeded to full term parturition but both it and its apparently healthy calf persistently excreted 'H. somnus'. The other animals underwent normal full term calvings and 'H. somnus' was not isolated from them or their calves. Lesions attributable to 'H. somnus' were detected only in the aborted fetus which showed an acute generalized inflammatory cell response consistent with a systemic Gram-negative bacterial infection. 'H. somnus' was isolated from all fetal tissues, including the placenta. The fetus and placenta also showed evidence of damage prior to inoculation. The placental damage may have predisposed the fetus and placenta to infection with 'H. somnus'. The placental epithelial cells contained intracytoplasmic organisms with the morphological and antigenic properties of 'H. somnus'.     

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 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 role of virus infections in bovine mastitis

Mastitis is an often occurring disease in dairy cattle with an enormous economic impact for milk producers worldwide. Despite intensive research, which is historically based on the detection of bacterial udder pathogens, still around 20-35% of clinical cases of bovine mastitis have an unknown aetiology. Due to the high number of unknown causes of clinical mastitis, studies were undertaken to gain more insight into the role of viruses in this important disease. This review deals with the role of viruses in the aetiology of bovine mastitis, including the results of the recently performed study on the role of bovine herpesvirus 4 (BHV4) in this aetiology. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore their importance and their economic impact needs further attention.