Immunity conferred by Aro- Salmonella live vaccines.

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).

Oxygen derived free radicals and the course of Eimeria vermiformis infection in inbred strains of mice.

Free radical generation by peritoneal leukocytes from BALB/c and C57BL/6 mice was monitored for 18 days following infection with Eimeria vermiformis. Free radical generation occurred earlier and was quantitatively much greater in resistant BALB/c mice than in susceptible C57BL/6 mice, resistance being indicated by a much lower oocyst production and a shorter patent period of E. vermiformis. Plasma greatly enhanced free radical generation in response to a soluble antigen prepared from sporulated oocysts indicating the presence of plasma-borne factor(s) which enhance free radical generation in response to E. vermiformis.

Immunity induced by live attenuated Salmonella vaccines.

Studies on the degree and specificity of protection conferred by immunization with aroA salmonella live vaccines in BALB/c mice are described. Animals were immunized i.v. and challenged orally 3 months later to ensure that the vaccine had been cleared from the tissues. Vaccination with Salmonella typhimurium aroA SL3261 conferred very good protection against virulent S. typhimurium C5 (over 10,000 x LD50). The specificity of cross protection was studied using S. typhimurium, Salmonella enteritidis and Salmonella dublin for vaccination and challenge, including challenge with variants of S. typhimurium and S. enteritidis of similar virulence which differed in the main LPS (lipopolysaccharide) antigen (0-4 or 0-9). S. typhimurium SL3261 gave very good protection against S. typhimurium C5 (0-4), but no protection against S. enteritidis Se795 (0-9). However, challenge with strains differing in the main 0 antigens showed that, although protection was generally better to strains expressing the same LPS type as the vaccine, specificity of protection was determined more by the background (S. typhimurium or S. enteritidis) of the parent strain used for the challenge than by 0 factors 4 or 9, suggesting that other factors could be involved. The nature of the antigen(s) responsible for protection in this model is unclear, but it would not appear to be the main 0-specific antigen. An S. enteritidis Se795 aroA vaccine was far less effective than S. typhimurium SL3261; it conferred good protection against the homologous wild type at 2 weeks post-vaccination, but far less at three months (approx 10-200 x LD50). This was unexpected, as the persistence of the S. enteritidis vaccine in the liver and spleen was similar to that of S. typhimurium SL3261, and the S. enteritidis and S. typhimurium challenge strains were of similar virulence. An S. dublin aroA vaccine conferred similar protection against wild type S. dublin (approx 300 x LD50).

Moderate immunodeficiency does not increase susceptibility to Salmonella typhimurium aroA live vaccines in mice.

Salmonellae carrying appropriate mutations in genes of the aromatic biosynthesis pathway are effective as live vaccines in animals, and they are candidate typhoid vaccines for human use. They are also very effective as carriers of recombinant antigens from other pathogens to the immune system, eliciting circulatory, secretory, and cell-mediated immunity to foreign antigens. Their attenuation is believed to be due to their requirement for the metabolites p-aminobenzoic acid and 2,3-dihydroxybenzoate, which are not available in mammalian tissues. Immunosuppression (e.g., acquired immunodeficiency syndrome) is a major contraindication to the use of live vaccines. If the avirulence of Aro mutants is largely due to their auxotrophy, they should not be markedly more invasive in immunosuppressed animals. We report that wild-type Salmonella typhimurium M525 of intermediate virulence was much more invasive in sublethally irradiated BALB/c mice than in normal BALB/c mice, whereas sublethal irradiation had little if any effect on the invasiveness of an S. typhimurium aorA vaccine strain apart from a delay in its clearance from the reticuloendothelial system. xid mutant CBA/N mice carry an X-linked B-cell functional defect which results in immunoglobulin G3 agammaglobulinemia, and they are known to be more susceptible to salmonellae in late stages of the infection. We found that whereas male (CBA/N x BALB/c)F1 mice (immunodefective) were more susceptible to wild-type S. typhimurium C5 than female littermates (immunocompetent), there was no difference in the response to the S. typhimurium aroA vaccine strain. The results indicate that moderate immunosuppression does not markedly enhance susceptibility to S. typhimurium aroA live vaccines.

T cells do not mediate the initial suppression of a Salmonella infection in the RES.

The course of a sublethal salmonella infection was followed in mice rendered susceptible by irradiation, reconstituted with T-cell-depleted bone marrow from normal donors and given a synergistic pool of CD4 and CD8 monoclonal antibodies. The results indicate that the host response causing the early plateau and suppression of bacterial growth at the end of the first week of the infection, which is essential for survival, does not require T cells.

Beta-lactam antibiotics (aztreonam, ampicillin, cefazolin and ceftazidime) in the control and eradication of Salmonella typhimurium in naturally resistant and susceptible mice.

This study was undertaken to investigate the efficacy of different beta-lactam antibiotics in the treatment of systemic salmonella infections in the mouse typhoid model. Innately susceptible BALB/c (Itys) and resistant CBA (Ityr) mice were used to investigate the efficacy of one monocyclic (aztreonam) and three bicyclic (ampicillin, cefazolin, ceftazidime) beta-lactam antibiotics in controlling systemic salmonella infections when given for brief or prolonged periods. The present study confirms and amplifies earlier reports on ampicillin therapy, demonstrates marked differences in the efficacy of the different antibiotics and shows that aztreonam is not only very effective but can completely eradicate the salmonellae from the RES when given early in the infection.

Immunity to coccidiosis: T-cell control of infection with Eimeria vermiformis in mice does not require co-operation with inflammatory cells.

The necessity for co-operation between lymphocytes and myeloid-derived inflammatory cells in the mediation of anti-coccidial immunity was investigated using mice infected with Eimeria vermiformis. Reciprocal exchange of immune lymphocytes between H-2 compatible strains of contrasting susceptibility to infection (resistant BALB/B and susceptible C57BL/10) resulted in successful transfer of immunity in both homologous and heterologous exchanges. Recipients of immune cells, whatever their original response phenotype, expressed a high degree of immunity to infection, indicating that the differential susceptibility of the strains is a property of their lymphoid cells and is not attributable to their capacity to mount inflammatory responses. This conclusion was confirmed by the successful adoptive transfer of immunity into NIH mice previously exposed to 600 rad X-irradiation; at this level of irradiation inflammatory responsiveness is severely depressed. Additional confirmation that strain-response phenotype is lymphocyte dependent and that immune lymphocytes can mediate their effects against E. vermiformis without the intervention of inflammatory cells was obtained from studies on the mucosal mast cell response to infection. No correlation existed between the development of intestinal mastocytosis, an index of T-cell-mediated inflammatory responsiveness, and the expression of resistance to E. vermiformis in BALB/c (resistant), C57BL/10 (susceptible) and NIH (susceptible) mice.

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains.

A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB lambda receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.

Mediation of immunity to Eimeria vermiformis in mice by L3T4+ T cells.

Immunity to infection with Eimeria vermiformis was transferred in NIH mice by both the nylon wool-adherent (B-cell-enriched) and nonadherent (T-cell-enriched) fractions of lymphocytes (spleen and mesenteric lymph node) taken from infected donors. Transfer was more variable with the adherent fraction, and when contaminating T cells were removed by treatment with anti-Thy1 monoclonal antibody (MAb) and complement, this fraction lost all protective activity. The protective effect of T-cell-enriched populations of mesenteric lymphocytes was abrogated by treatment with anti-L3T4 MAb and complement in vitro before transfer or by opsonization with this MAb in vitro before intravenous inoculation into recipients. Similar treatments of cells with anti-Lyt2 MAb did not have this effect, confirming that Thy1+ L3T4+ cells mediate the adoptive transfer of immunity to E. vermiformis. Thy1+ L3T4+ cells were also shown to limit the replication of E. vermiformis in primary infections: mice depleted of this subset (by thymectomy followed by intravenous injection of anti-L3T4 MAb) passed greater numbers of oocysts over a longer period of time than did mice similarly depleted of Lyt2+ cells.

Resistance to infection with Eimeria vermiformis in mouse radiation chimeras is determined by donor bone-marrow cells.

The course of infection with Eimeria vermiformis was determined in BALB/b, BALB/c, and C57BL/10ScSn (B10) mice and in radiation chimeras prepared from the H-2-compatible BALB/b and B10 mice. The BALB strains, irrespective of H-2 haplotype, were resistant, the B10 mice were susceptible, and in the chimeras infection was characterized by the genotype of the donated bone-marrow cells and not by the phenotype of the recipient. Thus, the genetic control of relative resistance or susceptibility to infection with this parasite is expressed through bone-marrow-derived cells.

Immunity to coccidiosis: adoptive transfer in NIH mice challenged with Eimeria vermiformis.

The development of a reliable model for the adoptive transfer of immunity to coccidiosis (infection with Eimeria vermiformis in NIH mice) is described. More than 10(8) of a mixture of spleen and mesenteric lymph node (MLN) cells, given either intravenously or intraperitoneally, were required to transfer a significant degree of protection. Dividing cells, present in the donors at 10 or 14 days after priming, but not at 5 or 19 days, were shown to be the effectors. When examined separately, MLN cells were found to be superior to spleen cells, and the injection of as few as 5 x 10(7) was capable of substantially reducing the oocyst output from a challenge inoculum. The recipients of cells from primed mice had earlier, and sometimes higher, titres of specific antibodies in the serum but, overall, there was no correlation between these titres and protection. Further characterization of the cells responsible for adoptively transferring immunity to this infection should now be possible.

The initial suppression of bacterial growth in a salmonella infection is mediated by a localized rather than a systemic response.

Mice were infected intravenously with two antibiotic resistance tagged variants of the same S. typhimurium strain given in close succession, or simultaneously with strains of different virulence. The first manifestation of acquired resistance--suppression of exponential bacterial growth in liver and spleen--occurred independently for the different strains in the same individuals, implying that it is due to localized rather than systemic events. This early suppression of bacterial growth was ablated by whole body X-irradiation (800R), whereas the immediately preceding phase of exponential growth (Ity controlled innate resistance) was not affected. Transfer of spleen cell suspensions from infected mice into syngeneic recipients conferred protection by suppressing the growth of an intravenous challenge. Pre-treatment of the suspensions to deplete them of macrophages abolished their protective capacity, while depletion of T-cells did not. Mice deficient in T-cells by adult thymectomy and anti-T-cell monoclonal antibody treatment were able to suppress the growth of an intravenous challenge. Taken collectively, the present data show that the very early phase of acquired resistance to salmonellae, essential for survival, is not the result of systemically developing resistance but a localized event at the site of infection.

Suppression of Taenia crassiceps during concurrent infections with Mesocestoides corti in mice.

Parasite burden (by volume) was measured in female mice of three strains given single or concurrent infections of Mesocestoides corti and Taenia crassiceps. Significant suppression of T. crassiceps volume was observed over a range of M. corti inocula and occurred irrespective of whether M. corti was introduced before or after infection with T. crassiceps. Suppression of T. crassiceps volume was greatest with larger inocula of M. corti and with increased duration of M. corti infection. No significant difference was found in the intraperitoneal volume of M. corti from mice given single or simultaneous concurrent infections of M. corti and T. crassiceps. When M. corti were inoculated into mice with an established infection of T. crassiceps a significantly smaller volume of M. corti was retrieved at post-mortem from the peritoneal cavities of these mice, than from mice given just M. corti.

Natural resistance to salmonellae in mice: control by genes within the major histocompatibility complex.

Determinations of 50% lethal dose (LD50) values in H-2 congenic B10 lines showed that late-emerging resistance (postimmune response phase) to salmonellae of intermediate virulence was less in H-2b and H-2d than in H-2a, H-2k, and H-2f mice. Association of resistance to H-2 was confirmed by backcross analysis, and LD50 determinations on H-2 recombinant haplotype strains showed that resistance maps to the I-E subregion. Bacterial growth curves in liver and spleen showed that susceptible mice carried bacteria for longer in the reticuloendothelial system than did resistant mice and that susceptible mice showed greater splenomegaly. Association of resistance and susceptibility to H-2 was not different when sister transductant salmonellae expressing somatic antigens O4 and O9 were used. Thus a gene(s) within the major histocompatibility complex controls natural resistance to salmonellae in mice by influencing the ability to clear bacteria from the reticuloendothelial system in the later phase of the infection, and the immunodominant O antigen cannot be solely involved.