Biomedical subjects
Publications and source records attributed to L B Corbeil.
Bacterial injury in bovine pneumonia may result from bacterial release of exotoxins or from complex interactions between bacterial products such as LPS, proteases, or antigens and host responses. The latter interactions usually result in both protection and tissue damage. The balance between degree of protective functions and injurious functions will determine whether the response is primarily beneficial or damaging to the host.
The ability of the aerobic bacterial flora from the normal bovine respiratory and reproductive tracts to enhance or inhibit the growth of Pasteurella haemolytica, P. multocida, and Haemophilus somnus was tested in vitro. Six strains of each of these pathogens were cross streaked with each isolate of bovine normal flora. Flora which enhanced the growth of these pathogenic bacteria outnumbered inhibitors four to one. An intermediate number of isolates produced no effect on pathogen growth. Most enhancers were gram positive (Micrococcus, Staphylococcus, Corynebacterium, or Rhodococcus isolates), although several isolates of Moraxella and Actinobacter were also good enhancers. For H. somnus, there were proportionally more organisms which produced marked enhancement among the preputial flora than among the nasal flora, which may account for the greater number of genital carriers than nasal carriers. Bacillus isolates were the most significant inhibitors among the nasal flora, whereas no genus or species from the reproductive tract was noted to produce appreciable inhibition. It is proposed that changes in ratios of inhibitors to enhancers may determine, in part, whether a carrier state or disease occurs. Also, suggestions are made for in vitro use of this phenomenon for diagnostic tests.
The serum susceptibility of 64 isolates of Haemophilus somnus from cattle was determined in a bactericidal assay with undiluted fresh or inactivated bovine serum with serial dilutions of bacterial suspension in RPMI 1640 medium. A total of 27 strains isolated from cattle with clinical disease (4 with thromboembolic meningoencephalitis, 13 with pneumonia, and 10 with reproductive failure) were compared with 35 strains from asymptomatic carriers (11 from the vagina and 24 from the prepuce). Essentially, all clinical isolates were serum resistant, whereas approximately 25% of preputial isolates were serum susceptible, as judged after 1 h of incubation in serum; a majority of vaginal isolates showed delayed serum susceptibility. Lysozyme played no role in serum killing, and the alternative complement pathway played only a minor role. Iron saturation, however, appeared to impart greater serum resistance to serum-susceptible strains from the vagina and prepuce. Perhaps the serum-susceptible strains from carriers would be useful vaccine candidates, but resistant strains from carriers may be pathogenic.
Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.
Immunoglobulin (Ig) concentrations in serum and in nasal secretions were correlated with pneumonia and diarrhea during the first 12 weeks of life in 56 calves. The peak onset of pneumonia occurred between 2 and 4 weeks of age when the calves' serum IgG1, IgG2, and IgA concentrations were lowest. As IgG2 concentrations increased, fewer calves developed pneumonia. Peak onset of pneumonia was also correlated with the lowest IgG and IgA concentrations in the calves' nasal secretions. Most calves developed pneumonia when serum concentrations of IgG1 were less than 1.5 g/dl, IgG2 less than 0.3 g/dl, IgA less than 0.1 g/dl, and IgM less than 0.2 g/dl and when the combined IgG and IgA values in nasal secretions were less than 0.2 mg of Ig/mg of protein. In study A, diarrhea preceded pneumonia in 63% of 56 calves. In study B, 38% of 23 calves had diarrhea and/or hemorrhagic feces before pneumonia. Seemingly, there was a relationship between diarrhea and pneumonia. Furthermore, pneumonia occurred at or just after the time when IgG1, IgG2, and IgA concentrations in serum and the combined IgG and IgA concentrations in nasal secretions were lowest. Pneumonia is a common disease of calves between 1 and 5 months of age, a period coinciding with the usual low point in serum immunoglobulin (Ig) concentrations due to catabolism of passively acquired antibodies. Calves that absorb less than adequate amounts of Ig may be susceptible to pneumonia at approximately 2 months of age, when serum Ig concentrations would be lowest.(ABSTRACT TRUNCATED AT 250 WORDS)
The roles of the serum bactericidal system, inflammatory cells, and sex in resisting gonococcal infection were studied in a murine model of gonococcal bacteremia. The role of serum killing in defense was investigated with complement component 5 deficient (C5-deficient) (B1O.D2/OSN) and normal (B1O.D2/NSN) mice. No significant differences were found between LD50's with either murine serum-sensitive or serum-resistant gonococci in those two mouse strains. However, in vitro experiments revealed a heat-stable factor in mouse serum which killed gonococci. Thus it appeared that the C5-deficient mouse is not a good model for the study of the role of C-mediated killing in resistance to gonococcal infection. Mice with Chediak-Higashi disease were used to study the role of phagocytes and natural killer cells. The difference in LD50's between affected mice (C57B1/6J beige J) and controls (C57B1/6J) was significant. The CBA/N mice, which have a B-cell maturation defect, were no more resistant to infection than control mice, which was taken as further evidence that B cells were less important than other leucocytes in innate immunity to gonococcal infection. Finally, male mice were significantly more resistant than female mice to gonococcal bacteremia. Thus, in this study the two most important determinants of resistance to gonococcal infection were inflammatory cells and sex.
Shope fibroma virus (SFV) causes a localized, self-limited, fibroblastic proliferation in adult rabbits. Extracts of Shope fibroma tumors were found to contain a second virus that induces a rapidly progressive disseminated tumor. Dissemination of this malignant fibroma is associated with activation of commensal mucosal infection with Pasteurella multocida, causing purulent conjunctivitis and rhinitis and resulting in death from nasal obstruction. We have isolated this new agent by two cycles of plaque purification. It is a poxvirus that is antigenically virtually identical to SFV as measured by a plaque reduction assay, but behaves differently both in vivo and in vitro. We have called this virus malignant rabbit fibroma virus (MV). Electrophoresis of restriction digests made with HIND III indicates that despite the antigenic similarity of SFV and MV, the locations of HIND III sites in the two viral genomes are quite different. These experiments have enabled us to determine that MV was present in small quantities in our initial uncloned stock of Patuxent strain SFV. Lymphocytes from rabbits bearing MV-induced tumors responded poorly to both B and T lymphocyte mitogens. This nonspecific immunologic dysfunction is evident at or before the time when metastases and Gram-negative infection develop, and it becomes more profound as the disease progresses. MV-induced tumors may provide a model for Gram-negative infection and decreased immunologic responsiveness associated with malignancies.
Pasteurellosis in the rabbit inoculated with a malignant variant of Shope fibroma virus (SFV-MV) is presented as a model for the study of immunosuppression and immunoprophylaxis in pasteurellosis. The rabbits, before the inoculation, were healthy carriers of Pasteurella multocida. They were intradermally inoculated with SFV-MV, and 3 to 6 days later, a primary tumor appeared at the site of inoculation. By postinoculation day (PID) 7 or 8, the rabbits had snuffles, conjunctivitis, and tumor metastases; death occurred on PID 10 to 14. Rabbits given the nonmalignant Patuxent strain of SFV developed local primary tumors, but not pasteurellosis nor metastases. In SFV-MV-inoculated rabbits, there was decreased responsiveness of spleen lymphocytes to B and T cell mitogens by day 6, and of spleen and peripheral blood lymphocytes by day 10. In addition, SFV-MV antigen was detected (by immunofluorescence) in mononuclear phagocytes in all major organs and in epithelial cells of the conjunctiva and nasal mucosa. Both nasal and conjunctival epithelia showed squamous metaplasia as well. These changes did not appear in SFV-infected rabbits. With SFV-MV-inoculated rabbits, we obtained partial protection against pasteurellosis by immunization with heat-killed P multocida or a cross-protective core lipopolysaccharide mutant of Escherichia coli (J5). Rabbits were immunized before the inoculation with SFV-MV which precipitated "spontaneous" pasteurellosis due to impaired defenses. Rabbits immunized with J5 or P multocida had less severe conjunctivitis and snuffles than nonimmunized controls, indicating that immunization with the J5 mutant may be useful as prophylaxis against pasteurellosis in compromised hosts.
Peripheral blood and splenic lymphocytes from rabbits immunized with Pasteurella multocida by various schedules were stimulated to undergo blast transformation in vitro in response to this antigen. Repeated immunizations suppressed this response.
Efforts to prevent Haemophilus influenzae type b (HIB) infections in infancy have been hampered by the low immunogenicity of capsular polysaccharide vaccines in children younger than 18 mos. In searching for alternate immunogens, we have studied the protective potential of polysaccharide-poor, lipid-rich endotoxin (LPS) core in experimental HIB infections. Because all gram-negative bacteria have similar LPS core structures, we were able to use as vaccine the J5 mutant of Escherichia coli 0111, the LPS of which consists only of core components, and thus to avoid problems in interpretation arising from vaccine contamination with non-LPS HIB immunogens. Mice were given graded inocula of HIB and developed lethal infection analogous to human HIB disease when virulence was enhanced with mucin and hemoglobin. After active immunization with heat-killed E. coli J5, 40/50 (80%) of infected mice survived, compared with 14/50 (28%) of saline-immunized controls (P less than 0.005). Passive immunization with rabbit antiserum against E. coli J5 prevented lethal HIB infection when administered 24 or 72 h before or 3 h after infection. This protection was abolished by adsorption of antiserum with purified J5 LPS, with survival reduced from 14/24 to 0/24 (P less than 0.005). Furthermore, rabbit antiserum to purified J5 LPS gave just as potent protection against death as antiserum to whole J5 cells. These studies demonstrate that immunity to core LPS confers protection against experimental murine HIB infection and provide the framework for a new approach to prevention of human disease from HIB.
The role of endotoxin responsiveness in defense against gonococcal infection was studied in endotoxin-resistant (C3H/HeJ) and endotoxin-susceptible (C3H/HeN) mice by using a model of disseminated gonococcal infection (DGI) and a model of gonococcal survival in the female genital tract to determine the ability of the mice to eliminate gonococci. The 50% lethal dose in the DGI model was 10(9.6) for C3H/HeJ mice and 10(5.2) for C3H/HeN mice. Levels of bacteremia during infection indicated the C3H/HeJ mice cleared large numbers of gonococci from their peripheral blood by 24 h post-inoculation but that C3H/HeN mice did not. Additionally, the peritoneal leukocyte response after intraperitoneal inoculation of gonococci was greater in C3H/HeJ mice than in C3H/HeN mice, which suggested that the ability to mount an inflammatory response to endotoxin may be important in defense against DGI. Besides being different in susceptibility to DGI, C3H/HeJ mice were found to be more resistant then C3H/HeN mice to genital colonization by gonococci. The resistance of C3H/HeJ mice to genital colonization by gonococci appeared to be due to both the high numbers of polymorphonuclear leukocytes in the genital secretion and the predominance of inhibitory gram-negative genital flora in that mouse strain.
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Gonococci do not readily cause disseminated infection in mice. To simulate some of the conditions leading to disseminated gonococcal infection in women, we suspended gonococci in mucin plus hemoglobin and studied the development of gonococcal bacteremia. The mucin-hemoglobin mixture was used because the menstruum appears to be involved in dissemination of gonococci from the genital tract during menstruation. Mice did not die after massive inocula of 10(9) gonococci given intraperitoneally in broth, but when gonococci were suspended in mucin (15%) alone, the 50% lethal dose was 10(8.4) and in 15% mucin plus 4% hemoglobin (M/H), the 50% lethal dose fell to 10(6.6). Sublethal doses produced local peritonitis and transient bacteremia. With larger inocula the local peritoneal infection progressed to fatal septicemia. Studies of the mechanism by which M/H lowered the 50% lethal dose showed that systemic clearance mechanisms were compromised, but not enough to account for the total decrease in the 50% lethal dose. If gonococci were given intravenously after intraperitoneal inoculation of M/H, sequestration of gonococci in the peritoneal cavity occurred, suggesting an effect on local peritoneal defenses. The effect on neutrophils appeared most significant, since numbers of neutrophils in the peritoneal fluid were decreased in the presence of M/H and neutrophils were destroyed by M/H in vitro. The serum bactericidal system was not affected. We conclude that M/H promotes gonococcal bacteremia by interference with phagocytosis and intracellular killing of gonococci. The model simulates the disseminated gonococcal infection cases in women which follow pelvic inflammatory disease in its progression from local peritonitis to transient or lethal bacteremia and in factors (mucin and hemoglobin) which enhance infection.
1. Gonococci possess a common heat stable antigen that is not identical to endotoxin. 2. A model of gonococcal peritonitis is described that simulates human disease in its progression from local to disseminated infection and its milieu of mucin and hemoglobin. 3. Vaccination with the heat stable antigen protected against disseminated infection and raised the LD50s from 100 to 1000-fold. 4. Vaccination with antigen from one strain of gonococci conferred protection against heterologous infection. 5. This protection did not require pili antigens or bactericidal antibody. 6. Protection in vaccinated mice resulted from a rapid, intense, and chemotactically-mediated influx of PMNs that eradicates the local infection.
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A new semiquantitative plaque assay was developed to measure killing of gonococci by serum. Results compare well with an established quantitative method, but the plaque assay is faster, simpler, and less expensive.
Suspensions of india ink or gonococci were inoculated into the murine uterus through the cervix with a simple plastic vaginal speculum and a 23 gauge blunted needle. The inoculated suspensions were distributed throughout both uterine horns, cervix, and vagina, but did not flow into the peritoneal cavity. This method avoided surgical stress as well as being simple, fast, and reproducible.