Five strains of Actinobacillus pleuropneumoniae serotype 1 were used to intranasally infect 5 groups of pigs. Using each bacterial strain, infected pigs (termed seeder pigs) were commingled for 48 hours with 5 groups of noninfected test pigs, then were removed. Seeder and test pigs were maintained in isolation and were observed for 14 days. Seeder pigs had mortality that was threefold greater than that of test pigs (24% vs 8%). Rectal temperature in excess of 40.3 C was achieved for 84% of test pigs and 88% of seeder pigs. Neither of these 2 variables was statistically different between the 2 groups of pigs. Clinical impression scores greater than or equal to 2 (on a 0 to 3 scale) were three-fold (64% vs 20%) greater for seeder than for test pigs (P < 0.05). The total number of bacterial isolations or nonrecoverable isolates was tabulated for test and seeder pigs' lungs at necropsy, irrespective of the amount of lesions. The number of A pleuropneumoniae isolations was not statistically different between test and seeder pig populations. Recovery of Pasteurella multocida or other bacteria was greater from the seeder pigs (P < 0.05), whereas the number of non-recoverable isolates was greater from test pigs than from seeder pigs (P < 0.05). Assessment of lung lesions at necropsy by either visual estimation or on a weight basis were in agreement. Fewer test pigs had lung lesions in excess of 5% of total lung volume than did seeder pigs (40% vs 84%) and, according to the odds ratio estimation, seeder pigs were 7 times more likely than test pigs to have such lesions. These results indicate a predictable, moderate intensity, natural exposure model for use in the study of Actinobacillus pleuropneumoniae-induced pneumonia. The seeder pig model appears to mimic field infection in development of clinical illness, febrile response, lung lesions, mortality, and low potential for secondary pneumonic bacterial involvement, thus providing a useful tool for preliminary evaluation of anti-infective modalities.

Nine CNS bovine herpesvirus type 1 (BHV-1) isolates, recovered from bovine brain samples submitted to the Texas Veterinary Medical Diagnostic Laboratories from 1974-1989, were compared by analyzing their DNA restriction endonuclease (RE) fragment migration pattern. Seven had pattern similar to that of the respiratory BHV-1 Cooper strain. The remaining 2 isolates, however, had variant patterns, similar to that of each other, but completely different from patterns for the other 7. The RE patterns of these 2 variants were similar to published RE patterns for 2 encephalitic or neuropathogenic BHV- 1 strains--the Australian N-569 strain and the Argentine A-663 strain. One of the Texas encephalitic variants (No. 30326) was isolated from the CNS of a calf that died during an epizootic of encephalitis in 1974. The other, designated TX-89, was isolated in 1989 from the CNS of a 7-month-old feedlot steer with acute fatal encephalitis. Microscopic lesions of encephalitis with neuronal degeneration and intranuclear inclusions were observed for 3 of the 9 isolates, the 2 variant isolates (No. 30326 and TX-89), and a respiratory isolate. The remaining 6 CNS isolates, all respiratory subtypes, were recovered from cattle that did not have clinical CNS disease or gross or microscopic CNS lesions; in 5 of these cattle, virus was recovered from at least 1 other organ (lungs) besides the CNS. We conclude that the CNS of calves can be naturally infected with 2 distinct BHV-1 subtypes, the respiratory and the encephalitic, and that the encephalitic subtype (subtype 3 or BHV-1.3) has been present in Texas cattle since at least 1974.

Hemagglutinins HA) of H1N1 swine influenza viruses isolated in the United States have remained antigenically and genetically conserved for many years. In contrast to such conservation, the RA of A/Swine/Nebraska/1/92 (Sw/Neb) could readily be distinguished from those of contemporary porcine viruses. Twenty-eight amino acid mutations differentiated the HA of Sw/Neb and A/Swine/Indiana/1726/88, the most recent H1N1 swine influenza virus for which HA sequence data were available. Among these differences were mutations at potential asparagine-linked glycosylation sites and charge changes at many residues. The Sw/Neb virus also could be differentiated from other swine influenza viruses in hemagglutination-inhibition assays with monoclonal antibodies to recent H1 swine HA. Nonetheless, overall sequence analysis of the HA and the nucleoprotein genes of Sw/Neb indicated that this virus was more closely related genetically to classic H1N1 swine influenza viruses than to H1N1 avian or human viruses. Infection of swine with Sw/Neb under experimental conditions induced clinical signs and lesions typical of swine influenza. However, affected swine in the field had high, persistent fevers, but relatively mild signs of respiratory tract disease. This study indicated that an antigenically and genetically novel variant of swine influenza virus was detected in the United States.

Twenty-four 10-month-old Polled Hereford heifers were inoculated sc with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus, live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

Chorioallantoic membranae (CAM) explants were used to determine the in vitro growth and cytotoxic potential of 3 strains of Brucella abortus. Bovine CAM explants were inoculated with 2 X 10(7) colony-forming units of the pathonic strain 2308, attenuated strain 19, or the rough strain RB51 of B abortus. After inoculation, the explants were harvested and examined at 2 or 4 hours, 12 or 14 hours, and 24 or 26 hours of incubation. Bacterial growth associated with each explant was determined by counting colony-forming units. The degree of cellular damage in each explant associated with bacterial growth or bacterial toxins were evaluated by morphometric analysis after trypan blue staining. Significant differences were not detected in the numbers of bacteria of any strain of B abortus in the CAM explants at comparable time intervals. The rate of growth of the bacteria in CAM explants was higher between 2 and 12 hours after inoculation than between 12 and 24 hours after inoculation. Cytotoxic effects associated with strain 2308 were significantly (P < 0.05) greater than that caused by other strains. Cytotoxic effects associated with strain 19 and rough strain RB51 were similar, and both were significantly (P < 0.05) greater than the phosphate buffer solution control. Chorioallantoic membrane explants inoculated with a filtrate of heat-killed strain 2308 induced minimal cellular damage, compared with that caused by the viable bacteria. These results indicated that the number of B abortus in trophoblasts was independent of the degree of cellular damage.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-3 (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-3, 7 groups of 3 calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2308 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51, 19 delta 31K, and 19 delta SOD. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase. In experiment 4, designed to compare the effects of age, 4 groups (n = 4) of 10-month-old heifers were given 1 B abortus strain each (19, RB51, 19 delta 31K, or 19 deltaSOD), using the same methods. Results of bacteriologic culturing and antibody responses were similar to those in the calves, except that strain RB51 persisted longer in heifers. Results of these studies indicated that, in cattle, the genetically engineered deletion mutants of B abortus do not cause unusual lesions, do have characteristics that closely resemble the parental strain, and could be candidates for use in a live vaccine.

Campylobacter jejuni A74/O and A74/C are congenic strains. An oral dose of 10(5) organisms of strain A74/C colonizes chicken intestines. Strain A74/O, from which A74/C is derived, does not colonize the chicken intestines with an oral dose of 10(5) organisms. In this study, the congenic bacteria were compared to identify possible colonization mechanisms. Differences were not observed in plasmid content or by HindIII, Pst I, Acc I, HincII, Ava I, Ava II, Xba I, and BamHI restriction enzyme digestion of total DNA. Transmission electron microscopy of negatively stained samples revealed no differences between the strains. Sections of cecal tissue from nonfed day-of-hatch chicks were cultured with each strain for 2 hours and then examined by light and electron microscopy. Both strains caused necrosis of villus epithelial cells. Immunofluorescent or silver staining revealed strain A74/C located deep in numerous epithelial crypts, but strain A74/O only was present in one sample mixed with sloughed necrotic cells. Similarly, organisms were detected by transmission electron microscopy deep in crypts in tissues cultured with A74/C, but not A74/O. Cells of A74/C detected in crypts did not appear to associate with epithelial cells. The strains did not differ in chemotactic behavior to mucin or fucose.

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 X 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as betweeen vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.

Objective-To evaluate the ability of bovine complement to kill a variety of field isolates and laboratory strains of Brucella abortus. Design-The experimental approach was to determine the sensitivity of B abortus isolates to killing by bovine serum, and to document the role of complement in brucellacidal activity. Sample population-Six laboratory isolates and 12 field isolates of B abortus were tested. Procedure-The ability of B abortus to survive exposure to undiluted bovine serum for 2 hours at 37 C was assessed. The role of complement in killing was determined by examining the ability of heat (56 C for 60 minutes) and cobra venom factor to obliterate the activity in serum, and by detecting binding of the ninth component of bovine complement to serum-sensitive target cells. Results-Isolates of B abortus that were resistant to the bactericidal activity of normal bovine serum were revealed. These included field isolates and laboratory strains. Furthermore, the study confirmed earlier reports that bovine serum-mediated killing of B abortus is caused by the complement cascade. Conclusions-Some isolates of B abortus, like other gram-negative bacteria, were resistant to complement-mediated killing. Resistance was associated with smooth colony morphology. Isolates lacking detectable O antigen were serum sensitive.