The development of protein A-gold electron microscopy for immunological studies of Haemophilus somnus.

Two separate experiments were conducted using modifications of protein A-gold immunoelectron microscopy (PAGIEM) to evaluate the ability of sera from calves vaccinated against Haemophilus somnus to bind virulent organisms (experiment I) and to detect differences in the antibody accessible antigenic sites on the outer membrane of selected strains of H. somnus having different virulence attributes using a high IgG2 titre specific bovine hyperimmune serum (experiment II). The results of experiment I demonstrated that the direct opsonisation of H. somnus by specific antisera was related to its IgG2 titre. In experiment II, strain-dependent differences in the labelling of antigenic sites by specific IgG2 antibodies were observed. The virulent strains of both septicaemic and genital isolates of H. somnus showed higher protein A-gold labelling than their non-virulent counterparts. The results from a comparison of pathogenic and non-pathogenic respiratory isolates did not reveal the same difference in labelling intensity. The studies demonstrated the PAGIEM technique to be a sensitive, versatile and a reliable laboratory method to analyse antigen-antibody interactions of H. somnus.

Differences in protein expression of Haemophilus somnus grown under conditions of iron-restriction.

Outer membrane protein profiles were compared in 14 H. somnus strains isolated from brain and lung lesions as well as from the genital tract of asymptomatic carriers during in vitro growth under iron-restricted conditions. Ethylenediamine-di-O-hydroxyphenyl acetic acid (EDDA) was used to obtain iron-restricted conditions in media used for this study. The outer membrane protein profiles were studied by the discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoretic system (SDS-PAGE), and the proteins were stained with silver or transferred to nitrocellulose sheets and western blots conducted. Growth under iron-restricted conditions resulted in the induction of outer membrane proteins in most H. somnus strains examined. Studies also indicated differences among H. somnus strains in the number of induced proteins and their molecular weights but the results did not indicate a specific relationship between these strain-dependent differences and tissue trophism. Western blot analysis revealed a high degree of immunological relatedness among strains of H. somnus in their iron-regulated proteins. However, hyperimmune serum used in these assays failed to recognize certain iron-regulated proteins expressed by some H. somnus strains, a finding which may have important implications for the induction of protective immunity in cattle against this bovine pathogen.

Endothelial cytotoxicity of Actinobacillus pleuropneumoniae.

The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin.

The protective effect of vaccination against experimental pneumonia in cattle with Haemophilus somnus outer membrane antigens and interference by lipopolysaccharide.

A semi-purified outer membrane anionic antigen (AA) fraction was isolated from Haemophilus somnus by a modified procedure of anion exchange chromatography to yield a protein fraction free of lipopolysaccharides (LPS). The AA fraction (1 mg) was administered with or without the homologous lipopolysaccharide (10 micrograms/kg body weight) as vaccines to groups of cattle twice, three weeks apart. A control group which did not receive any antigen was included in the trial. Six weeks after the first vaccination, the animals were challenged intratracheally with a virulent pneumonic strain of H. somnus (70986) and observed for clinical signs of respiratory disease. The cattle were euthanized six days later and the lungs were evaluated for the severity of lesions macroscopically as well as histopathologically. Vaccination with AA alone provided the best protection against pneumonia as indicated by significantly lower clinical scores, less extensive gross lung lesions and mild histopathological lesions with immune cell infiltration. However, when AA was combined with LPS in the vaccination, this protective effect was negated and the animals showed more detrimental histopathological lesions than the controls.

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.

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.

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)

An evaluation of the API ZYM system as a means of identifying Haemophilus somnus and related taxa.

The commercially available API ZYM microbiological identification system was evaluated for the rapid identification of Haemophilus somnus. Eighty-seven isolates of the organism had API ZYM profiles which were characteristic. The API ZYM profiles demonstrate clear differences between H. somnus and other genera but suggest a close association to three related organisms. Enzyme activity of H. somnus isolates were similar to organisms identified as Histophilus ovis, Haemophilus agni and strains UQV of Actinobacillus actinoides and Actinobacillus seminis but was clearly different from isolates of Pasteurella haemolytica, Pasteurella multocida, Bordetella bronchiseptica and group EF4. The API ZYM system allowed more rapid identification of H. somnus than conventional biochemical tests and may be a useful adjunct to conventional methods used for identification of H. somnus isolates. The test did not reveal obvious differences between isolates from various anatomic locations.

Caprine beta-D-mannosidosis: characterization of a model lysosomal storage disorder.

Interest in using caprine beta-D-mannosidosis as a model to evaluate bone marrow transplantation in the treatment of human lysosomal storage disorders provided the stimulus for characterization of beta-D-mannosidase in selected goat tissues and induction of hemopoietic chimerism in the goat. Total beta-D-mannosidase activity was measured with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. Residual activity in mutant liver was 52% of control but no activity was detectable in mutant kidney or brain tissue. Normal adult goat liver contained two forms of beta-D-mannosidase, a nonlysosomal form (52%) with a broad pH range for optimum activity (4.5-8.0) and a lysosomal form (48%) with a pH optimum of 5.5. Residual enzyme in mutant liver consisted entirely of the nonlysosomal form. Normal adult thyroid, kidney and brain contained two major lysosomal isoenzymes with pIs 5.5 and 5.9 and traces of a minor isoenzyme with pI 5.0. Normal liver contained three isoenzymes with similar pIs; however, an isoenzyme with pI 5.0 predominated. In 60-day fetal liver lysosomal isoenzymes predominated and only trace amounts of nonlysosomal isoenzyme were detectable. Total hepatic beta-D-mannosidase activity increased towards adult levels during the last 90 days of gestation as a result of increasing nonlysosomal isoenzyme activity. Intraperitoneal injection of fetal liver cells into 60-day goat fetuses resulted in sustained hemopoietic chimerism in surviving kids without evidence of graft-versus-host-disease. These results suggest that transplantation of normal fetal liver cells into preimmunocompetent goat fetuses affected with beta-D-mannosidosis is feasible and may provide an alternative strategy for evaluation of postnatal bone marrow transplantation in the treatment of human lysosomal storage disorders.

Agents of equine viral encephalomyelitis: correlation of serum and cerebrospinal fluid antibodies.

A survey was conducted by testing 115 paired equine serum and cerebrospinal fluid samples by hemagglutination-inhibition for antibodies to Powassan and snowshoe hare viruses, and by virus neutralization for antibodies to equine herpesvirus type 1. Twenty-five samples were from horses with spontaneous neurological disease and the remainder from horses euthanized because of various nonneurological disorders. All sera and cerebrospinal fluids were negative for antibodies to Powassan virus. Fifty-one sera (44.3%) and 15 cerebrospinal fluids (13.0%) had antibodies to snowshoe hare virus. Ninety-eight sera (85.2%) and four cerebrospinal fluids (3.5%) were positive for antibodies to equine herpesvirus type 1. Powassan virus was inoculated intracerebrally into one, and intravenously into four ponies. Neurological signs associated with a nonsuppurative encephalomyelitis occurred in three ponies. Antibodies to Powassan virus were detected in sera of all animals but in cerebrospinal fluids of only two. Powassan virus was isolated from brain and spinal cord of only the intracerebrally inoculated animal.

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.

Effect of Haemophilus somnus on bovine endothelial cell in organ culture.

The effect of 2 strains of Haemophilus somnus on bovine endothelial cells in cultured arterial segments was investigated and compared with the effects of Escherichia coli and Salmonella typhimurium. In cultures inoculated with either strain of H somnus, there was widespread contraction and desquamation of endothelial cells, exposing large areas of subendothelial collagen. Many bacteria were adherent to endothelial cells and some were in phagosomes within cells. Endothelial changes were milder in arterial cultures inoculated with E coli or S typhimurium than in those inoculated with either strain of H somnus. Adhesion of H somnus to vascular endothelial cells followed by exposure of subendothelial collagen may initiate the thrombosis, vasculitis, and ischemic necrosis characteristic of infectious thromboembolic meningoencephalitis in cattle. Arterial cultures might be useful in assaying the virulences of different strains of H somnus, and could be used to investigate the mechanism of their action on endothelial cells.

Humoral immunity in experimental thromboembolic meningoencephalitis in cattle caused by Haemophilus somnus.

Of 23 cattle inoculated IV with Haemophilus somnus, 16 (70%) died of thromboembolic meningoencephalitis. The inoculum was prepared from a minimally subcultured isolate of H somnus that was passaged through a calf by intracisternal inoculation immediately before use. Serum antibody titers, as measured by 7 serologic tests, were not correlated with the animal's susceptibility to infection. All cattle that died had a mean 4-fold increase in agglutination titer during the acute phase of the disease, 2 to 4 days after inoculation. Similarly, high acute-phase titers were demonstrated in 15 cattle with naturally occurring disease. Haemophilus somnus was isolated more frequently and in greater numbers from the CNS and urinary tract than from other organs of cattle that died.

Vaccination of cattle against experimentally induced thromboembolic meningoencephalitis with a Haemophilus somnus bacterin.

The capability of a commercial Haemophilus somnus bacterin to protect cattle against experimentally induced thromboembolic meningoencephalitis was examined. Eighteen cattle were vaccinated twice, 8 were vaccinated once, and 14 were nonvaccinated controls. Serum antibody responses to vaccination were measured by gel immunodiffusion, bacterial agglutination test, and complement-fixation test. Deaths occurred in 8 of the 14 controls, 3 of the cattle vaccinated once, and 3 of the cattle vaccinated twice. Two vaccinations were found to give significant protection against challenge exposure (P less than 0.05). There were no cattle which gave positive reactions in the gel immunodiffusion test, and significant changes in bacterial agglutination test titers were not seen in the cattle after vaccination. There was a significant (P less than 0.01) increase in the complement-fixation test titers of cattle vaccinated twice. Serum antibody titers were unrelated to the outcome of challenge infection, regardless of vaccination status, in any of the serotests.

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)

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.

Immunocytochemical localization of adrenocorticotropic hormone-immunoreactive cells of the pars intermedia in thoroughbreds.

OBJECTIVE: To analyze and characterize adenoma and hyperplasia of the pars intermedia (PI) of Thoroughbred mares. ANIMALS: 165 Thoroughbred mares, without clinical signs of hyperadrenocorticism that had been euthanatized or had died, of causes such as sudden death, colic, pneumonia, or trauma, and were necropsied. Five of those horses, 17 to 25 years old, had a large pituitary gland at necropsy. Eight mares, 5 to 15 years old with normal-size pituitary gland, were selected at random for comparison. PROCEDURE: A morphologic comparison of the pituitary gland between horses with and without tumors of the PI was conducted by use of immunocytochemistry and morphometry. RESULTS: In horses with normal pituitary gland, the PI was supplied by a vast capillary or sinusoidal plexus, which connected that in the pars distalis (PD) with that in the pars nervosa (PN). Cells of the PI stained slightly with ACTH antiserum, but some cells in the border region, which is contiguous to the PD, were strongly ACTH immunoreactive. At necrospy, horses with an enlarged pituitary gland also had adenoma of the pituitary gland involving the PI. Cells of the border region were hypertrophied and stained strongly with ACTH antiserum. The area and number of individual tumor cells of the border region of the PI of horses with adenoma were significantly increased, compared with those in horses with normal pituitary gland. CONCLUSIONS: Cells of the PI-PD border region may secrete substantial quantities of ACTH, owing to stimulation by corticotropin-releasing factor. Adenoma and hyperplasia of the PI in Thoroughbred mares may be associated with hyperadrenocorticism.

Prevalence and distribution of Haemophilus somnus in the male bovine reproductive tract.

"Haemophilus somnus' was isolated from 77% of 31 reproductive tracts of bulls from an Ontario slaughterhouse. Identification of H somnus was based on morphologic and cultural characteristics and on fluorescent antibody and immunodiffusion tests, using antisera prepared against a known pathogenic encephalitic isolate of H somnus. The infection rate and distribution of H somnus within the tract were as follows: preputial orifice--71% preputial cavity--71%; urinary bladder--26%; accessory sex glands--19% and ampulla of ductus deferens--10%. Isolates were not obtained from the testes or epididymides. On 2 occasions, H somnus was isolated in pure culture from the preputial cavity. A higher prevalence of infection was found in young bulls. There were no differences found in infection rates between breeds. Differences in hemolytic activity and minor antigenic variation between isolates indicated that a series of biotypes within the species H somnus may exist. The study indicates that organisms presently identified as H somnus may normally form part of the flora of the bovine prepuce and that dissemination from the male bovine reproductive tract is one possible means of infection in H somnus-associated diseases. The pathogenic significance of genital isolates of H somnus awaits further study.

The gross and histopathologic lesions of maignant catarrhal fever in three captive sika deer (Cervus nippon) in southern Ontario.

The gross and histopathologic lesions of three captive sika deer (Cervus nippon) with malignant catarrhal fever are described. Lesions included those of the head and eye form and the more commonly described peracute form. One deer had been exposed to a wildebeeste (Connochaetes gnou) and the other two to domestic sheep.