Test of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis.

Aromatic compound-dependent (aro) derivatives of three mouse-virulent strains of Salmonella choleraesuis (Salmonella cholerae-suis) were constructed and shown to be nonvirulent for mice (intraperitoneal [i.p.] 50% lethal dose [LD50], greater than 5 X 10(6) CFU). A pur derivative, and a thy derivative, each of a different virulent parent, remained moderately virulent (i.p. LD50S for BALB/c mice, ca. 10(5) and 5 X 10(4) CFU, respectively). Tested as live vaccines i.p., the aro strains were ineffective in salmonella-susceptible BALB/c and C57BL/6 mice but were somewhat effective in salmonella-resistant CBA/J mice and in outbred CD-1 mice. The pur and thy strains were effective as live vaccines in BALB/c mice when given in sublethal doses. Two previously isolated nonvirulent galE derivatives of S. choleraesuis (i.p. LD50 in BALB/c mice, greater than 10(6) CFU) were also ineffective as live vaccines in BALB/c and C57BL/6 mice. The main antigenic difference between S. choleraesuis (O-6,7) and S. typhimurium (O-4,12) is in O-antigen character, thought to largely determine the specificity of protection in salmonellosis. Paired, nearly isogenic O-6,7 and O-4,12 derivatives were constructed from an aro S. typhimurium strain of proven efficacy as a live vaccine. Used as live vaccines, the O-4,12 member protected BALB/c mice against challenge with virulent S. typhimurium, whereas the O-6,7 member did not protect against virulent S. choleraesuis. However, BALB/c mice vaccinated with the O-6,7 member and mice vaccinated with an aro S. choleraesuis strain were protected against challenge with a moderately virulent (LD50, 5 X 10(4) CFU) O-6,7 derivative of an S. typhimurium strain.     

Immune responses in BALB/c mice following immunization with aromatic compound or purine-dependent Salmonella typhimurium strains.

Two near isogenic strains of Salmonella typhimurium HWSH, stably mutated in either the aroA gene affecting the biosynthesis of aromatic compounds, or the purA gene affecting the biosynthesis of purines, were administered intravenously as live attenuated vaccines to BALB/c mice. HWSH aroA-immunized mice were well protected against intravenous (i.v.) challenge with wild-type virulent HWSH for at least 10 weeks, whereas HWSH purA-immunized mice were unprotected. Furthermore, HWSH aroA-immunized mice could also control a heterologous challenge with virulent Listeria monocytogenes at 7 and 14 days post-immunization, whereas mice receiving a similar dose of HWSH purA could not. Increasing the i.v. dose of HWSH purA compared to HWSH aroA induced some resistance to L. monocytogenes. Induction of early anti-S. typhimurium resistance by HWSH aroA immunization appeared slightly later than the anti-L. monocytogenes resistance. Mice immunized with either vaccine were able to mount S. typhimurium-specific T-cell proliferative responses and produced anti-S. typhimurium humoral antibodies in their serum. The antibody titre was greater in those mice immunized with the aroA mutant.     

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).     

Development and evaluation of an experimental vaccination program using a live avirulent Salmonella typhimurium strain to protect immunized chickens against challenge with homologous and heterologous Salmonella serotypes.

A stable live avirulent, genetically modified delta cya delta crp Salmonella typhimurium vaccine strain, chi 3985, was used in several vaccination strategies to evaluate its use in the control of Salmonella infection in chickens. Oral vaccination of chickens at 1 and at 14 days of age with 10(8) CFU of chi 3985 protected against invasion of spleen, ovary, and bursa of Fabricius and colonization of the ileum and cecum in chickens challenged with 10(6) CFU of virulent homologous Salmonella strains from group B. Chickens challenged with heterologous Salmonella strains from groups C, D, and E were protected against visceral invasion of spleen and ovary, while invasion of the bursa of Fabricius and colonization of ileum and cecum was reduced in vaccinated chickens. Oral vaccination at 2 and at 4 weeks of age induced an excellent protection against challenge with virulent group B Salmonella serotypes and very good protection against challenge with group D or E Salmonella serotypes, while protection against challenge with group C Salmonella serotypes was marginal but significant. Vaccination at 2 and at 4 weeks of age also protected vaccinated chickens against challenge with 10(8) CFU of highly invasive S. typhimurium or S. enteritidis strains. The protection of chickens vaccinated with chi 3985 against challenge with homologous and heterologous Salmonella serotypes is outstanding, and the complete protection against ovarian invasion in chickens challenged with 10(8) CFU of highly invasive S. typhimurium or S. enteritidis strains suggests that vaccination of chickens with chi 3985 can complement the present hygiene- and sanitation-based Salmonella control measures. This paper reports a breakthrough in the use of live avirulent vaccine to control Salmonella carriers in chickens.     

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).     

Comparison of the abilities ofSalmonella typhimurium rpoS,aroAandrpoS aroAstrains to elicit humoral immune responses in BALB/c mice and to cause lethal infection in athymic BALB/c mice

Salmonella typhimurium rpoS and rpoS aroA mutants are effective live vaccines in the murine model of salmonellosis (Coynault et al., Mol. Microbiol. 1996; 22: 149-60). Here, we further investigate the characteristics of these vaccines. The systemic humoral response induced by S. typhimurium rpoS, aroA and rpoS aroA vaccine candidates against S. typhimurium LPS was studied by ELISA. In BALB/c mice, the rpoS aroA strain induced a systemic anti-LPS humoral response similar to that induced by the rpoS and aroA strains. The virulence of aroA and rpoS aroA vaccines in nude (nu/nu) BALB/c mice was also compared. Salmonella typhimurium aroA and rpoS aroA vaccines both produced slowly progressing lethal infections in athymic mice inoculated i.p. but the rpoS aroA strain was more attenuated than the aroA strain, as determined by time to death and bacterial counts in spleens. Finally, a rpoS mutant of Salmonella dublin conferred protection in mice against an oral challenge with a wild-type strain of S. dublin whereas a rpoS mutant of S. typhimurium did not. This suggests that the protection provided by the S. typhimurium rpoS vaccine is serotype-dependent.     

Relevance of inoculation route to virulence of three Salmonella spp. strains in mice.

The virulence of three Salmonella species strains was compared by the i.p. and i.v. routes in BALB/c mice. Salmonella choleraesuis, SL2824 (serogroup C1, O-6,7), was more virulent by the i.v. than i.p. route. A strain of S. typhimurium, SL1260 (serogroup B; O-1,4,12) was more virulent i.p. than i.v. while another strain, SL3201 (O-4,5,12) was equally virulent i.p. or i.v. The LD50 of each strain by either route correlated with the number of bacteria in the liver and spleen on day one after inoculation and thus seems determined mainly by initial bactericidal mechanisms. The rate of bacterial growth in the liver and spleen was independent of inoculation route but differed between the three strains. Salmonella choleraesuis multiplied faster than either strain of S. typhimurium. Non-virulent aromatic-dependent (aro) derivatives of these strains were tested, instead of their virulent ancestors, for survival within peritoneal macrophages in vitro. Salmonella choleraesuis SL 2824 aro and S. typhimurium SL1260 aro were much more readily killed intracellularly than S. typhimurium SL3201 aro. The data indicate that the survival and multiplication of different Salmonella serotypes or strains in vivo may depend on different critical properties or mechanisms to overcome host defenses.     

Vaccination of chickens with a Salmonella enteritidis aroA live oral Salmonella vaccine

A mouse-virulent strain of Salmonella enteritidis, Se795 (LD50 less than 10 organisms for mice), was non-virulent for 12-day-old chickens given 10(6) cfu intravenously; the organisms were cleared from liver and spleen by day 14 as measured by direct plating and by day 21 by enrichment. An Se795aroA mutant, CU58, was also cleared from liver and spleen by day 14 after intravenous inoculation of 10(7) cfu. Day-old chicks vaccinated orally with either one dose of 10(9) CU58 at 1 day of age, 10(7) at 1 and 14 days, or 10(5) at 1 and 7 days followed by 10(9) at 14 and 21 days of age, were challenged orally with a nalidixic acid resistant variant of the virulent phage type 4 S. enteritidis strain 109. All vaccinated groups showed a reduction in faecal shedding of the challenge. Chickens given four doses of CU58 showed a significant reduction of cfu in liver, spleen and faeces following intravenous challenge with virulent strain 109. Intramuscular vaccination with 10(9) cfu of Aro strain CU58 at 1 day of age gave no protection against oral challenge with virulent strain 109. Serum antibody production to LPS (ELISA) was minimal in all vaccinated birds. The results indicate that oral vaccination with Aro- S. enteritidis can confer protection to day old chicks against virulent S. enteritidis.     

Antibody response and protection against challenge in mice vaccinated intraperitoneally with a live aroA O4-O9 hybrid Salmonella dublin strain.

An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12). By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium. Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S. dublin and S. typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts. The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S. dublin (50% lethal dose of ca. 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S. typhimurium (50% lethal dose of ca. 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice). Compared with the protection elicited in homologous systems (S. dublin SL5631 against S. dublin and S. typhimurium SL1479 against S. typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S. dublin challenge and 100-fold against S. typhimurium challenge. Vaccination with S. typhimurium SL1479 conferred no protection against S. dublin challenge, and vaccination with S. dublin SL5631 conferred no protection against S. typhimurium challenge. The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.     

Identification of p60 antibodies in human sera and presentation of this listerial antigen on the surface of attenuated salmonellae by the HlyB-HlyD secretion system.

Antibodies directed against the major secreted protein of Listeria monocytogenes, termed p60, were found more frequently than antilisteriolysin antibodies in sera of listeriosis patients. Anti-p60 antibodies were also identified in all tested sera from healthy individuals. To test whether p60 provides protection against L. monocytogenes, we constructed an attenuated Salmonella typhimurium aroA strain which secretes p60 via the Escherichia coli hemolysin secretion pathway. Application of this Salmonella strain to BALB/c mice prior to an L. monocytogenes infection induced p60 antibodies in these mice and led to a significantly reduced number of viable bacteria in the spleen compared with that in control animals which were primed with the S. typhimurium aroA strain alone.     

Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium.

Hybridomas producing monoclonal immunoglobulin A (IgA) antibodies against Salmonella typhimurium were generated by mucosal immunization of BALB/c mice with attenuated strains of S. typhimurium and subsequent fusion of Peyer's patch lymphoblasts with myeloma cells. To test the role of secretory IgA (sIgA) in protection against Salmonella sp., we analyzed in detail the protective capacity of a monoclonal IgA, Sal4, produced in polymeric as well as monomeric forms, that is directed against a carbohydrate epitope exposed on the surface of S. typhimurium. BALB/c mice bearing subcutaneous Sal4 hybridoma tumors and secreting monoclonal sIgA into their gastrointestinal tracts were protected against oral challenge with S. typhimurium. This protection was directly dependent on specific recognition by the monoclonal IgA, since mice secreting Sal4 IgA from hybridoma tumors were not protected against a fully virulent mutant that lacks the Sal4 epitope. Although monoclonal Sal4 IgA was present in the bloodstreams and tissues of tumor-bearing mice, it did not protect against intraperitoneal challenge and did not possess complement-fixing or bacteriocidal activity in vitro. Taken together, these results indicate that secretion of sIgA alone can prevent infection by an invasive enteric pathogen, presumably by immune exclusion at the mucosal surface.     

Vaccination route, infectivity and thioglycollate broth administration: effects on live vaccine efficacy of auxotrophic derivatives of Salmonella choleraesuis

An aromatic-dependent, therefore non-virulent, derivative of a mouse-virulent strain of Salmonella choleraesuis previously shown not to be effective as a live vaccine when given intraperitoneally (i.p.) to Itys mice, was administered to BALB/c mice. Two doses given i.p. or by feeding did not protect against i.p. or oral challenge with 50 to 5000 LD50 of the virulent ancestor strain. By contrast two doses given intravenously (i.v.) gave almost complete protection against i.p. or oral challenge with 500 LD50 and some protection against larger doses. The number of live bacteria (cfu) in the liver and spleen 24 h after administration of the live vaccine was less than 1% of the number inoculated i.p., but c. 25% of the number injected i.v. The number of cfu in the gut 24 h after oral vaccine administration was only c 10(-5) of the number fed. Administration of thioglycollate broth i.p. 5 days before i.p. vaccination increased recovery of live vaccine cfu in the liver and spleen and its protective efficacy. In each case the live vaccine did not multiply extensively in vivo. We have previously shown that a purine- and a thymine-requiring derivative of S. choleraesuis were each considerably attenuated but unlike the aro derivative were effective as i.p. live vaccines in mice. Doses of these strains (c. 10(4) cfu) found protective were administered i.p. to BALB/c mice. Each strain multiplied extensively in the liver and spleen to c. 10(7) cfu by day 6. All these results are in agreement with a correlation of protective efficacy of a live vaccine with the persistence of a large number of the vaccine bacteria in the liver and spleen for several days.     

Salmonella typhimurium aroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune systems

We investigated the ability of Salmonella typhimurium vaccines to deliver heterologous antigens to the systemic and secretory immune systems of the mouse, while retaining their immunogenicity against salmonellosis. S. typhimurium SL3261, an avirulent aroA mutant, or SL3261 carrying plasmid pBRD026, a pBR322 derivative encoding the gene for Escherichia coli LT-B were used to immunize BALB/c mice orally. Both immunizing strains invaded the mononuclear phagocyte system of the mice, grew slowly until approximately day 14 post-infection, and then were rapidly cleared. No salmonellae were detected in livers, spleens, mesenteric lymph nodes or Peyer's patches by day 42. Mice immunized with either strain and challenged orally with the virulent parent strain, SL1344, several weeks after clearing the immunizing organism, were protected against the lethal S. typhimurium infection. Mice infected with SL3261 (pBRD026) developed substantial levels of IgG and IgA anti-LT-B antibodies 14 days post-infection in both serum and gut samples. The sera neutralized the effects of LT in an in vitro Vero cell assay. Thus, aroA mutants of S. typhimurium can deliver a heterologous antigen from a different enteric pathogen to the murine systemic and secretory immune systems without altering their efficacy against salmonellosis.     

Genetic manipulation of Salmonella serotype Bovismorbificans to aromatic-dependence and evaluation of its vaccine potential in mice

The generation of smooth aromatic-dependent Salmonella serotype Bovismorbificans (Group C2, O6, 8) from a smooth wild-type parent strain by transduction with phage P1, and conjugation with Salmonella serotype Typhimurium carrying F'-8gal is described. The smooth aromatic-dependent S. serotype Bovismorbificans was non-lethal for mice at an oral challenge dose of 2 x 10(9) cfu (equivalent to 200 LD50 of the parent, wild-type strain). The safety of the auxotrophic mutant was further substantiated by comparing its multiplication kinetics in vivo with that of its virulent parent organisms. Mice immunised with live, smooth aromatic-dependent S. serotype Bovismorbificans by either the oral or intraperitoneal (i.p.) route were protected against oral challenge with virulent S. serotype Bovismorbificans; the degree of protection was significantly better (p less than 0.05) at a challenge dose of 100 or 200 LD50 in mice receiving two rather than one vaccination. In contrast, mice immunised with three doses of the formalin-killed virulent, parent organisms by the i.p. route were not protected, in spite of high antibody titres. Only those mice immunised with the live, smooth aromatic-dependent S. serotype Bovismorbificans i.p. developed significant (p less than 0.01-0.05) delayed-type hypersensitivity.     

Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains

We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice. Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses. We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S. typhimurium strains such as SR11. To test this hypothesis, we have constructed S. typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice. We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice. These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain. Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S. typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent. Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S. typhimurium outer membrane proteins. Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain. This work demonstrates that S. typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system.     

Protection of C3H/HeJ mice from lethal Salmonella typhimurium LT2 infection by immunization with lipopolysaccharide-lipid A-associated protein complexes.

C3H/HeJ mice were immunized intraperitoneally (i.p.) with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes or with purified protein-free LPS prior to lethal i.p. or intravenous Salmonella typhimurium LT2 challenge. Our results demonstrated that these Salmonella-hypersusceptible mice can be effectively protected against 1,000 100% lethal doses of S. typhimurium LT2 (i.e., 1,000 viable bacteria) administered by intravenous challenge when previously immunized with LAP-LPS complexes. In contrast to these results, immunization with LPS afforded markedly less protection regardless of the route of challenge, thus suggesting that the LAP portion of LAP-LPS complexes may be necessary for inducing protection against Salmonella infections. For most experiments, antigens were emulsified in complete Freund adjuvant (CFA); however, the CFA portion of the vaccine was suggested not to be an essential component for the induction of immunity to Salmonella infections, since equivalent levels of protection were obtained when it was omitted from the vaccine. The induction of immunity to murine salmonellosis by prior immunization with CFA-LAP-LPS was demonstrated not to be a transient phenomenon, since C3H/HeJ mice were still protected against lethal S. typhimurium LT2 challenge as late as 225 days postimmunization.     

Characterization and protective properties of attenuated mutants of Salmonella choleraesuis.

We have constructed crp::Tn10 and cya::Tn10 Salmonella choleraesuis mutants and their fusaric acid-resistant derivatives with deletions (delta) of the Tn10 and adjacent DNA sequences and found them to be avirulent and able to induce protection against a wild-type challenge in 8-week-old BALB/c mice. Mice survived infection with the crp and cya mutants at doses of more than 7 x 10(3) times the oral (p.o.) 50% lethal dose (LD50) and more than 8 x 10(2) times the intraperitoneal LD50 of the wild-type S. choleraesuis parent. Mice vaccinated with attenuated strains were protected against challenge with more than 1.6 x 10(4) times the p.o. LD50 and more than 80 times the intraperitoneal LD50 of the wild-type virulent S. choleraesuis parent. One deletion mutation isolated in the crp region extends to an adjacent gene(s) that was shown to be associated with avirulence. This gene or operon has been designated cdt (colonization of deep tissues). A delta (crp-cdt)19 strain, when complemented with the wild-type crp gene and promoter on a pBR-derived plasmid, had p.o. LD50 values 10(3) times higher than those for the wild type. A delta cya delta (crp-cdt)19 double mutant was less virulent than and afforded more complete protection against a challenge with the wild-type strain than a delta crp-11 delta cya double mutant or the individual cya, crp, or crp+/cdt mutants. The deletion derivatives exhibited reduced invasion of CHO cells in vitro, and the numbers of the mutants recovered from mouse tissues were less than that of the parent strain. These studies suggest that one or more of the genes involved in cell attachment to and/or invasion of S. choleraesuis may be under catabolite repression. In addition, we describe a new deletion of a gene(s) located in the crp region between cysG and argD that is associated with virulence in S. choleraesuis.     

Role of T cells, TNF alpha and IFN gamma in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro- Salmonella vaccines.

The SL3261 Salmonella typhimurium aroA live vaccine strain confers solid protection against oral challenge with virulent salmonellae, immunity persisting long after the vaccine has been cleared from the tissues. BALB/c mice immunized with SL3261 and later subjected to in vivo depletion of both CD4+ and CD8+ T cells had impaired recall of immunity to oral challenge with the virulent S. typhimurium C5, with increased mortality and higher bacterial loads in the reticuloendothelial system (RES). Selective depletion of CD4+ cells alone significantly impaired resistance both 8 and 14 weeks after vaccination as determined by estimation of bacterial numbers in organ homogenates. Depletion of CD8+ cells alone had less effect on immunity when performed at 8 weeks than at 14 weeks after immunization. Administration of anti-IFN gamma or anti-TNF alpha antibodies also impaired recall of immunity, exacerbating a secondary infection in vaccinated mice. Challenge of T cell-depleted immune mice with virulent salmonellae caused hepatosplenomegaly with minute grossly visible focal lesions, and a marked increase in the number and severity of necrotic foci in spleen, liver and lymph nodes. A widespread mononuclear cell infiltrate was present. The histopathology in anti-IFN gamma-treated mice was qualitatively similar to that seen in T-cell depleted mice. In contrast, in the anti-TNF alpha-treated mice splenomegaly was much less than in T cell-depleted mice. Granulomas were absent, no mononuclear infiltration was observed and there was severe necrosis; the lesions appeared similar to or worse than those seen in na?ve mice. Surprisingly, IFN gamma was detectable in sera of both controls and T cell-depleted mice on day 8 of the secondary infection, as well as in sera of anti-TNF alpha-treated mice on day 6 of infection. The results indicate that T cells, IFN gamma and TNF alpha are all important in the specific recall of immunity to virulent salmonellae conferred by immunization with live vaccines, with the effect of T cell and IFN gamma depletion (marked macrophage infiltration) being qualitatively very different from that of TNF alpha neutralization (no mononuclear infiltrate or granuloma formation).     

The effects of O-antigen character and enterobacterial common antigen content on the in vivo persistence of aromatic-dependent Salmonella sp. live-vaccine strains

Aromatic-dependent (aro) derivatives of Salmonella choleraesuis like aro S. typhimurium are non-virulent but, unlike them, are ineffective as live vaccines in mice, given i.p. An aro derivative of S. choleraesuis did not persist in the liver and spleen (RES) of mice after i.p. inoculation whereas a similar derivative of S. typhimurium persisted. S. choleraesuis (O group C1; O-6,7) and S. typhimurium [O group B; O-(1),4(5),12] differ in O antigen of LPS, determined by chromosomal locus, rfb. Three pairs of nearly-isogenic aro derivatives, one member O-6,7 and the other O-(1),4,(5),12, were constructed in two lines of S. typhimurium by replacement of their B-rfb genes with the C1-rfb genes of S. choleraesuis. In tests for persistence after mixed or separate i.p. inoculation of equal doses into BALB/c mice the O-(1),4,(5),12 member of each pair was recovered as CFU in the RES at ca. 100-fold greater number than the O-6,7 member at 24 hours post-inoculation and subsequently. O-6,7 derivatives of S. typhimurium constructed as described above by a simple replacement of group B with group C-rfb locus synthesise only trace (tr) amounts of enterobacterial common antigen (ECA). An ECA+ (able to make normal levels of ECA) derivative of one aro, O-6,7 S. typhimurium strain was constructed by replacement of its B-rfe locus with the C-rfe locus of S. choleraesuis. Tested by mixed inoculation i.p. this strain persisted in the RES in numbers 10-fold greater than its O-6,7 ECAtr but 5-10-fold lesser than its O-(1),4,(5),12 cousins. Thus both O-specificity and ECA contribute to the survival of salmonella species in mice as determined by in vivo persistence of non-multiplying aro derivatives.     

Protection against experimental salmonellosis in mice and sheep by immunisation with aromatic-dependent Salmonella typhimurium

Mice immunised by the oral or intraperitoneal route with a live aromatic-dependent strain of Salmonella typhimurium exhibited significantly less protection against oral challenge with 50 LD50 of an ovine isolate of S. typhimurium (12313) than when a bovine isolate with the same O antigens and phage-type as strain 12313 was used as the challenge organism. When challenged with 10 LD50, however, protection against both strains was significantly better than that obtained when mice were vaccinated with killed vaccines (heat-killed, acetone-killed or irradiated) even when the antigenic mass of the killed vaccine was increased by up to 500-fold in an attempt to compensate for the expected limited multiplication of the mutant organism. Sheep immunised with the live mutant strain by either the intramuscular or oral route were protected against oral challenge with the virulent ovine isolate of S. typhimurium; unimmunised sheep died of acute enteritis within 7 days, although there was no evidence of systemic invasion by the challenge organism. After challenge, immunised animals ate more food than the unimmunised controls and suffered only transient, mild diarrhoea. Serum antibody titres against O and H antigens measured by direct or antiglobulin tests were significantly higher in sheep immunised by the intramuscular route than in those immunised orally. Sheep in both immunised groups developed skin swellings within 30 min after intradermal inoculation with purified homologous lipopolysaccharide indicating development of immediate-type hypersensitivity, but only those immunised by the intramuscular route showed significant indurated skin swellings characteristic of delayed-type hypersensitivity 48 and 72 h post-inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)