Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic.

Salmonella typhimurium SR-11 mutants with cya::Tn10 or crp::Tn10 mutations were found to be avirulent and immunogenic for BALB/c mice. Fusaric acid-resistant derivatives with deletions of the Tn10 and adjacent DNA sequences were constructed in S. typhimurium SR-11 strains with or without the virulence plasmid pStSR100. These delta cya delta crp strains grew more slowly than wild-type strains. They possessed wild-type ability to attach to, invade, and persist in gut-associated lymphoid tissue for up to a week but exhibited a diminished ability to reach mesenteric lymph nodes and the spleen. Mice 4 to 8 weeks old were resistant to oral infection with 10(9) cells of several different delta cya and delta cya delta crp strains (the equivalent to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11) and 30 days after immunization became resistant to oral challenge with 10(3) to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11.     

Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination.

Avirulent salmonellae expressing foreign genes are attractive for use as oral vaccine carriers. To facilitate the stable expression of heterologous genes without conferring antibiotic resistance, a deletion of the asdA1 gene was introduced into Salmonella typhimurium and S. typhi delta cya delta crp mutant vaccine strains. An asd-complementing plasmid expressing hybrid hepatitis B virus nucleocapsid-pre-S (HBcAg-pre-S) particles was constructed. These hybrid HBcAg-pre-S particle genes were stably expressed in S. typhimurium and S. typhi delta cya delta crp mutant vaccine strains in this balanced, lethal host-vector combination. A single oral immunization of BALB/c mice with a recombinant S. typhimurium delta cya delta crp mutant synthesizing hybrid HBcAg-pre-S elicited potentially virus-neutralizing anti-pre-S serum immunoglobulin G antibodies. In addition, serum immunoglobulin G recognizing S. typhimurium lipopolysaccharide was induced. Distribution in tissue after oral immunization was analyzed in one plasmid-strain combination. The recombinant S. typhimurium colonized the gut-associated lymphoid tissue and the spleen and persisted for over 4 weeks, retaining the HBcAg-pre-S expression plasmid. An isogenic virulence plasmid-cured S. typhimurium delta cya delta crp strain expressing the same HBcAg-pre-S gene had reduced immunogenicity for the carried antigen after oral immunization.     

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.     

Control of colonization by virulent Salmonella typhimurium by oral immunization of chickens with avirulent delta cya delta crp S. typhimurium

Oral immunization with a delta cya delta crp Salmonella typhimurium strain has been shown to preclude colonization by wild-type, virulent S. typhimurium and induces humoral and cellular immune response in chickens. Intestinal tract colonization by the virulent challenge strain was used to determine the level of protection conferred by immunization with the delta cya delta crp mutant. The associated humoral and cellular immune responses were measured by ELISA and delayed-type hypersensitivity (DTH) tests, respectively. The levels of colonization by both Salmonella strains were determined by enumeration of viable cells in the intestinal tract. A reduction in faecal excretion of the wild-type strain was observed with a single oral immunization with the delta cya delta crp mutant, but caecal colonization was not affected. However, double oral immunization with the delta cya delta crp mutant precludes caecal colonization by the virulent strain. IgM, IgA and IgG were detected against sonicated Salmonella whole-cell antigens. Outer membrane and flagella proteins induced DTH responses, whereas lipopolysaccharide failed to do so. The effectiveness of the delta cya delta crp strain in reducing caecal colonization by the highly virulent challenge strain in chickens demonstrates that oral vaccination with the delta cya delta crp S. typhimurium should aid in eliminating Salmonella carriers in chickens. The elimination of these carriers on the poultry farm should help to control Salmonella contamination of poultry products, therapy improving public health.     

表达鼠精子Cyritestin蛋白的减毒沙门氏菌疫苗株的构建及其鉴定

目的 构建表达鼠精子eyritestin蛋白的可口服减毒沙让氏菌活疫苗株。方法 应用质粒TAZ83／13，将之进行双酶切获得含有成熟cyritestin蛋白cDNA序列的酶切片段。将其插入表达载体yA3149中，构建重组质粒pCFL。将该重组质粒转入鼠沙门氏伤寒杆菌疫苗株X4632和X4550中，以获得重组菌株X4632（pCFL)和X4550（pCFL)。结果 该两种无抗药性的重组菌株在体外营养选择压力下，可较稳定地携带重组质粒传代繁殖。X4550(pCFL)在体内可较稳定地害居于Peyer‘s结，肠系膜淋巴结和脾脏；X4632（pCFL)可较稳定地定居于Peyer‘s结。二者均可表达被抗cyritestin单抗（mAb)识别的重组cyritestin蛋白。口服该疫苗菌株无明显的毒性作用。结论 X4632（pCFL),X4550（pCFL)疫苗株的构建，为研究以cyritestin为靶抗原的精子抗原避孕疫苗打下了基础。     

鼠精子表面蛋白cyritestin在沙门氏菌平衡致死系统中的高效表达

目的 松建表达鼠精子cyritestin蛋白的可口服减毒沙门氏菌活疫苗株。方法 将编码成熟cyritestin蛋白cDNA序列的酶切片段插入含pUC复制子的表达载体pYA3339中,构建重组质粒pMCY,将该重组质粒转入鼠沙门氏伤寒杆菌疫苗株X4550,获得杂合菌株X4550（pMCY）。应用 western blot对重组菌所表达的重组cyritestin蛋白进行免疫分析,结果 该无抗药性的杂合菌株在体外营养选择压力下可较稳定携带重组质粒传代繁殖,在体内可较稳定地定居于Peyer‘s结、肠系膜淋巴结和脾脏。该重组菌可高水平表达重组cyritestin蛋白。结论 高效表达cyritestin蛋白的X4550（pMCY）疫苗株可用于免疫性避孕研究。     

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.     

Swine immunity to an attenuated Salmonella typhimurium mutant containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus.

Salmonella typhimurium chi 4064, an attenuated delta cya delta crp mutant of S. typhimurium SR-11, was shown to be avirulent in swine. S. typhimurium chi 4064 was used as a carrier for plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Given orally, S. typhimurium chi 4064(pBA31-R7) colonized the intestine and mesenteric lymph nodes of 5- to 6-week-old crossbred swine. Orally immunized animals developed serum and intestinal antibody responses to the B. abortus 31-kDa protein and to salmonella endotoxin as measured by enzyme-linked immunosorbent assay. Similarly immunized swine did not develop delayed-type hypersensitivity following a subcutaneous injection of recombinant BCSP31. However, swine parenterally immunized with recombinant BCSP31 incorporated in Freund incomplete adjuvant did develop a delayed-type hypersensitivity response to the homologous antigen. The data indicated that oral presentation of antigen to swine in the context of recombinant S. typhimurium effective stimulated mucosal and systemic antibody-mediated immunity but failed to sensitize swine for either an antigen-specific delayed-type hypersensitivity or a blastogenic response to the cloned BCSP31.     

Characterization and immunogenicity of Salmonella typhimurium SL1344 and UK-1 delta crp and delta cdt deletion mutants.

S. typhimurium SL1344 and UK-1 mutants with deletions of the crp (cyclic AMP receptor protein) and cdt (colonization of deep tissues) genes have been constructed and characterized, and their levels of virulence and immunogenicity have been determined for BALB/c mice. All Crp- Cdt- and Crp+ Cdt- mutants were avirulent, as mice survived oral doses of 10(9) cells without illness. All the mutants colonized the gut-associated lymphoid tissue efficiently, but capacities to colonize deeper tissues, such as those of the spleen and liver, and blood were significantly reduced for the Crp- Cdt- and Crp+ Cdt- mutants compared with the Crp- Cdt+ mutant and the wild-type parent strain. The Crp- Cdt- and Crp+ Cdt- SL1344 strains induced complete protection, as all mice immunized with the mutants survived challenge with approximately 10(4) times the 50% lethal dose of the wild-type SL1344 strain. The Crp- UK-1 strain was least attenuated yet induced the highest level of protective immunity against challenge with the wild-type UK-1 strain. The Crp+ Cdt- and Crp- Cdt- strains, although totally attenuated, differed in immunogenicity to challenge with the highly virulent UK-1 parent, with the apparently hyperattenuated Crp- Cdt- strain inducing a lower level of protective immunity than the Crp+ Cdt- strain. Nevertheless, these UK-1 Crp- Cdt- and Crp+ Cdt- strains induced complete protective immunity to challenge with the less-virulent SL1344 wild-type strain. Taken collectively, the results indicate that the attenuation of a highly virulent S. typhimurium strain can yield a vaccine that induces excellent protective immunity to challenge with less-virulent S. typhimurium strains.     

Differential induction of carrier antigen-specific immunity by Salmonella typhimurium live-vaccine strains after single mucosal or intravenous immunization of BALB/c mice.

In this study, we constructed strain KR21 (chi 4550 delta cya delta crp delta asd/pYA292asd(+)-toxC+) and compared it with BRD847 (aroA aroD/pnirB-toxC) for the ability to induce humoral and cellular immunity after a single oral or intravenous immunization in 3- to 4-week-old BALB/c mice. ToxC-specific serum immunoglobulin G (IgG) was detectable in animals orally immunized with either BRD847 or KR21. However, after intravenous immunization, IgG was detected only in BRD847-immunized animals. Measurement of immunoglobin types IgG1 and IgG2a suggests that a Th1 cellular response is prominent after immunizations with either system. ToxC-specific IgA was detected in fecal and vaginal samples of animals immunized orally and intravenously with BRD847, while those immunized with KR21 failed to show fecal or vaginal IgA responses. Delayed-type hypersensitivity was used as a measure of induction of T-cell responses in vivo. Mice immunized either orally or intravenously with BRD847 showed significant ear swelling responses after ToxC injections, while KR21-immunized animals failed to show a cellular response. These data indicate that the aroA aroD/pnirB system holds greater potential for inducing global immunity after a single dose when directly compared with the balanced lethal system (delta cya delta crp delta asd/pYA292asd+).     

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: persistence and induction of humoral responses in rats.

Recombinant Salmonella typhimurium has been used as an oral vaccine for various microbial pathogens. Here we report immune responses in Fischer rats orally immunized with a recombinant S. typhimurium strain encoding surface protein antigen A (SpaA) of Streptococcus sobrinus. The attenuated S. typhimurium chi 4072 delta cya delta crp delta asd mutant used in this study contains the Asd+ plasmid pYA2905 expressing a fragment of the SpaA protein. Salmonella cells were cleared from spleens by 7 days and from Peyer's patches by 14 days in rats receiving a single oral immunization of 10(9) CFU of chi 4072. In animals receiving multiple (i.e., days 0 and 7 or days 0, 7, and 21) immunizations, Salmonella cells were cleared from the Peyer's patches by 25 days following the initial immunization. Antigen-specific systemic and mucosal antibody responses were greater in rats receiving multiple immunizations than in those receiving a single immunization. Serum anti-Salmonella activity was potentiated following boosting on day 21. Mucosal immunoglobulin A antibody responses were also greater in rats receiving multiple immunizations than in rats receiving a single immunization. Anti-Salmonella and anti-Streptococcus immunoglobulin A activity persisted longer in rats boosted on day 21 than in rats immunized on days 0 and 7. These data indicate that oral immunization of rats with the recombinant S. typhimurium chi 4072(pYA2905) vaccine induces systemic as well as mucosal antibody responses specific to the Salmonella cells and to the cloned SpaA protein. This is the first report of the use of an attenuated mutant of the murine pathogen S. typhimurium as an oral vaccine in rats.     

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system.

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.     

Immune responses to Streptococcus sobrinus surface protein antigen A expressed by recombinant Salmonella typhimurium.

In this study, we used a vaccine strain of Salmonella typhimurium to express antigenic determinants of the SpaA antigen of Streptococcus sobrinus, which is involved in the caries-forming process. We cloned either a single repeat (pYA2901) or three tandem repeats (pYA2905) of the 0.48-kb fragment of the spaA gene, which codes for an important component of the SpaA protein, plus a 1.2-kb minor antigenic determinant and measured the resulting immune responses to SpaA in orally immunized BALB/c mice. The single or triple repeat of the spaA gene fragment was inserted into the Asd+ vector pYA292 and was transformed into the S. typhimurium delta cya delta crp vaccine strain chi 4072 containing delta asd in the chromosome. Female BALB/c mice were then orally immunized with two doses of the S. typhimurium containing either of the two SpaA constructs, and the immune responses to the expressed SpaA protein were assessed. Significant serum immunoglobulin G (IgG) anti-SpaA titers were detected in mice immunized with chi 4072(pYA2905) but not chi 4072(pYA2901). Salivary anti-SpaA IgA titers were minimal and were only detected in mice immunized with S. typhimurium expressing the SpaA encoded by pYA2905. Intestinal anti-SpaA IgA titers, however, were detected in both groups of mice, particularly in mice immunized with chi 4072(pYA2905). An oral booster 26 weeks after the initial series of immunizations resulted in increased serum IgG titers in both chi 4072(pYA2901)- and chi 4072(pYA2905)-immunized animals, particularly in the chi 4072(pYA2905)-immunized animals. No anamnestic IgA response was detected in the saliva following the booster immunization.     

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.     

Construction of a recombinant oral vaccine against Salmonella typhi and Salmonella typhimurium.

The viaB gene coding for the Vi antigen of Salmonella typhi Ty2 was subcloned into expression vector pYA248. The recombinant plasmid was termed SMM202 and transformed into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Recombinant S. typimurium Vi4072 had the ability to produce Vi capsular polysaccharide and also to invade and colonize the small intestine, mesenteric lymph nodes, and spleen of BALB/c mice. Mice orally immunized with Vi4072 developed serum and secretory antibody responses to the Vi antigen, as measured by a passive hemagglutination assay. Mice developed a delayed-type hypersensitivity following a footpad injection with Vi antigen after being sensitized orally with a suitable dose of Vi4072. Immunization of mice with Vi4072 afforded complete protection against fatal infection with virulent S. typhi Ty2. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity and for inducing a cell-mediated immune response in BALB/c mice. Thus, S. typhimurium Vi4072 may serve as a vaccine for protection against typhoid fever and salmonellosis caused by S. typhimurium.     

Oral immunization of mice with attenuated Salmonella typhimurium containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus.

Salmonella typhimurium chi 4064, an attenuated delta cya delta crp mutant of S. typhimurium SR-11, was used as a carrier for the plasmid pBA31-R7. This plasmid codes for the expression of a 31-kilodalton (kDa) protein from Brucella abortus (BCSP31). Recombinant S. typhimurium chi 4064(pBA31-R7) expressed BCSP31 in vitro as shown by Western blot (immunoblot) analysis. The plasmid was stable in vitro and in vivo and did not affect the ability of the mutant to invade and colonize the small intestine, mesenteric lymph nodes, liver, or spleen of BALB/cByJ mice. Animals orally immunized with S. typhimurium chi 4064(pBA31-R7) developed serum and intestinal antibody responses to the B. abortus 31-kDa protein and to salmonella endotoxin as measured by enzyme-linked immunosorbent assay. Mice orally immunized with S. typhimurium chi 4064pBA31-R7 did not develop a delayed-type hypersensitivity following a footpad injection with recombinant BCSP31. Antigen-specific blastogenic data also support these in vivo results. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity but that the B. abortus 31-kDa protein is a poor immunogen for inducing a cell-mediated immune response in BALB/cByJ mice.     

Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium.

A 17-kDa lipoprotein, TUL4, of the facultative intracellular bacterium Francisella tularensis is one of several membrane proteins that induce an in vitro response in T cells from F. tularensis-primed humans. A DNA fragment of the live vaccine strain F. tularensis LVS encoding TUL4 was cloned into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Expression of the protein by the recombinant S. typhimurium chi 4072 (pTUL4-15) was maintained after passage in BALB/cJ mice. When mice were immunized with S. typhimurium chi 4072(pTUL4-15), some animals showed an antibody response and a T-cell response to TUL4. When the immunized mice were challenged with the live vaccine strain F. tularensis LVS, bacterial counts in the liver and spleen were lower than in animals immunized with S. typhimurium chi 4072. Immunization with F. tularensis LVS caused a much stronger protection against the challenge than did immunization with S. typhimurium chi 4072(pTUL4-15). The present study demonstrated that the 17-kDa lipoprotein TUL4 of F. tularensis is involved in a protective immunity to tularemia. Possibly, several T-cell-reactive proteins of the organism have to contribute for optimal protection to be achieved.     

Virulent Salmonella typhimurium-induced lymphocyte depletion and immunosuppression in chickens.

The effect of experimental Salmonella infection on chicken lymphoid organs, immune responses, and fecal shedding of salmonellae were assessed following oral inoculation of 1-day-old chicks or intra-air-sac infection of 4-week-old chickens with virulent S. typhimurium wild-type chi 3761 or avirulent S. typhimurium delta cya delta crp vaccine strain chi 3985. Some 4-week-old chickens infected intra-air-sac with chi 3761 or chi 3985 were challenged with Bordetella avium to determine the effect of Salmonella infection on secondary infection by B. avium. S. typhimurium chi 3761 caused lymphocyte depletion, atrophy of lymphoid organs, and immunosuppression 2 days after infection in 1-day-old chicks and 4-week-old chickens. The observed lymphocyte depletion or atrophy of lymphoid organs was transient and dose dependent. Lymphocyte depletion and immunosuppression were associated with prolonged fecal shedding of S. typhimurium chi 3761. No lymphocyte depletion, immunosuppression, or prolonged Salmonella shedding was observed in groups of chickens infected orally or intra-air-sac with chi 3985. Infection of chickens with salmonellae before challenge with B. avium did not suppress the specific antibody response to B. avium. However, B. avium isolation was higher in visceral organs of chickens infected with chi 3761 and challenged with B. avium than in chickens infected with B. avium only. Infection of chickens with chi 3985 reduced B. avium colonization. We report a new factor in Salmonella pathogenesis and reveal a phenomenon which may play a critical role in the development of Salmonella carrier status in chickens. We also showed that 10(8) CFU of chi 3985, which is our established oral vaccination dose for chickens, did not cause immunosuppression or enhance the development of Salmonella carrier status in chickens.     

Essential role for cyclic AMP and its receptor protein in Yersinia enterocolitica virulence

Insertion mutations were isolated in cya and crp of Yersinia enterocolitica, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP). The cya and crp mutants were affected for the production of proteins exported by the Ysc, Ysa, and flagellar type III secretion systems (TTSS). Protein production by each TTSS was restored when the respective mutation was complemented by a plasmid-encoded copy of the wild-type gene. Both cya and crp mutants exhibited reduced virulence for orally infected BALB/c mice in a 50% lethal dose analysis. Examination of bacterial survival in host tissues showed that cya and crp mutants colonized Peyer's patches and, to a lesser extent, mesenteric lymph nodes. However, the mutants did not appear to disseminate to the liver and spleen of infected mice. An initial examination of the effectiveness of Y. enterocolitica cya and crp mutants to stimulate protective immunity against subsequent challenge with virulent bacteria in mice was promising. The results indicate that the cAMP-CRP regulatory system is required for Y. enterocolitica virulence.     

Protein antigens of Mycobacterium leprae

Protein antigens of Mycobacterium leprae have been identified by screening the lambda gt11, pYA626 and pHC79::M. leprae genomic libraries with pooled sera from leprosy patients and with antiserum to M. leprae cell wall protein (CWP) aggregate. Immunological screening of the lambda gt11 library with pooled sera from 21 lepromatous (LL) leprosy patients resulted in the identification of 19 antigens that are apparently different from previously identified M. leprae antigens. Five additional antigens were identified by screening the lambda gt11 library with pooled sera from 30 borderline tuberculoid or tuberculoid patients. Four other antigens were identified by screening the lambda gt11 library with anti-CWP. Two groups of recombinant cosmids were identified by screening the pHC79 library with LL patients' sera: one group specified proteins that reacted with monoclonal antibodies (mAb) against the 65-kDa protein and against the 18-kDa protein; the other group specified a 15-kDa protein that did not react with any of the mAb that were tested. One pYA626 clone also specified a 15-kDa protein that reacted with LL patients' sera, but did not react with any mAb. Genes specifying several of these antigens have been subcloned into the Asd+ plasmid vector pYA292 and have been introduced into a delta cya delta crp delta asd Salmonella typhimurium strain to evaluate the ability of individual M. leprae proteins to elicit immune responses against M. leprae infection.