Reproductive difficulties in cattle with antibody titers to Haemophilus somnus

A herd of Holstein cows was examined because of suspected embryonic death. Four cows had embryonic loss, and 2 cows had aborted. Paired serum samples were tested for antibodies to infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, and Haemophilus somnus. Of 16 cows, 8 had antibody titers to H somnus greater than 1:1,024, and 3 had greater than or equal to four-fold changes in antibody titers to H somnus. Haemophilus somnus infection was active in this herd and may have been responsible for the herd's reproductive problems.     

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'.     

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.     

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.     

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.     

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.     

Secretory antibody responses in cattle infected with foot-and-mouth disease virus.

Antibody responses in serum, saliva, nasal secretions, or esophageal-pharyngeal fluid of foot-and-mouth disease virus-infected steers were examined by single radial immunodiffusion and mouse-neutralization tests. In steers infected with type O foot-and-mouth disease virus, high serum antibody titers were detected within 10 days after infection. Antibody was first detected in saliva at 30 days and gradually increased to a plateau at about 90 days. Small amounts of antibody continued to be secreted in saliva and in nasal secretions for at least 6 months. Antibody was not detected in esophageal-pharyngeal fluid. The major antibody activity in secretions was due to secretory immunoglobulin A as revealed by radioimmunoelectrophoresis.     

Epidemiology of Haemophilus somnus infection in dairy cattle in Quebec.

Serological studies on Haemophilus somnus infection were carried out on 1795 cattle from 231 dairy herds in the province of Quebec. An epidemiological investigation was done in each of the dairy operations. Seroreactivity rate and mean log2 titer for all the sera were 55.4% and 4.1620 respectively. Cattle from eastern regions of Quebec demonstrated the lowest prevalence of H. somnus agglutinins. The percentage of seroreactor animals was 60.3 in herds of 100 cattle or more in comparison to 53.2 in herds of smaller size. About 75% of the animals from 16 herds in which one or more cattle showed nervous manifestations of undetermined origin over a one year period had antibodies to H. somnus. Herds in which respiratory diseases occurred had 59.6% seroreactor animals and herds in which weak calf syndrome was diagnosed over a one year period had 61.4% seroreactor animals. In 87 herds located within 20 km of feedlots, 61.8% of the sera had titers and the mean log2 titer was 4.4813.     

Development of a defined medium for Haemophilus somnus isolated from cattle

Nutritional factors that influence the growth of Haemophilus somnus were examined, and a defined medium was developed. Optimal growth of H somnus in broth occurred under conditions of maximum aeration. Nutritional components required for or enhanced growth of most H somnus isolates in the defined medium included uracil, D-glucose, isotonic NaCl, Na2HPO4, nicotinamide, flavin mononucleotide, pantothenic acid, pyridoxine, and a variety of salts and amino acids. The defined medium supported optimum growth of 18 of 21 isolates of H somnus from cattle.     

Selective medium for isolation of Haemophilus somnus from cattle and sheep

Incorporation of vancomycin (5 micrograms/ml), neomycin (5 micrograms/ml), sodium azide (50 micrograms/ml), nystatin (100 iu/ml) and cyclohexamide (100 micrograms/ml) into 5 per cent horse blood agar results in a selective medium for the primary isolation of Haemophilus somnus from cattle and sheep. Addition of thiamine monophosphate (1 microgram/ml) to the medium enhanced growth of this bacterium. Gram-positive bacteria did not grow on the medium and colonies of many Gram-negative bacteria were eliminated or reduced in numbers and size. Colonies of H somnus were larger on the selective medium than on sheep blood agar but retained typical morphology. Recovery of 18 laboratory strains was 73 to 166 per cent (mean 112) on selective medium compared to sheep blood agar. H somnus was isolated from the vagina of a total of 136 (28.6 per cent) of 476 cows surveyed, 79 (16.6 per cent) on sheep blood agar and 129 (27.1 per cent) on selective medium. The selective agents and thiamine were stable indefinitely as a freeze dried mixture while prepared plates were stable for two weeks.     

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.     

Histamine production by Haemophilus somnus

Ten Haemophilus somnus isolates were grown on blood agar plates under a 5% CO2 atmosphere for 48 h. Harvested whole cells were washed and evaluated for the presence of histamine by ELISA. All H. somnus isolates had cell-associated histamine concentrations of between 18.5 and 200 ng/ml. In a separate study, the ability of H. somnus to secrete histamine into BHI growth medium was evaluated using H. somnus strains 8025 and 156A as well as a recent 156A respiratory isolate. Each strain or isolate was grown under various concentrations of CO2 to approximate the CO2 concentration in the bronchi. The histamine content of washed whole cells and medium supernatant were determined at various stages of incubation. Highest histamine concentrations were detected in the recent respiratory isolate; whole cells (225 ng/ml) after 120 h incubation in 15% CO2 and supernatant (1721 ng/ml) after incubation for 41 h in 25% CO2. This study indicates that different H. somnus isolates can produce and secrete histamine which may be enhanced by CO2 concentrations which approximate those in the bronchial tree. Results of this study may partially explain some of the post-vaccination reactions occasionally observed with H. somnus bacterins. Additional studies are needed to determine the actual role of H. somnus-derived histamine in the pathogenesis of bovine respiratory disease and airway hyperresponsiveness.     

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.