The SmpA outer membrane lipoprotein of B. hyodysenteriae has several characteristics that indicate the potential to protect against swine dysentery (SD). It localises to the outer membrane and antibodies directed against SmpA can prevent the growth of B. hyodysenteriae in vitro. There is some variation observed in the distribution and expression of the SmpA lipoprotein, suggesting that vaccination with SmpA may not provide protection against challenge with a heterologous B. hyodysenteriae strain. This study has characterised the variation at the smpA locus, and in the process has identified a novel gene, smpB. There is very low similarity between smpB and smpA, with the exception of an identical lipoprotein signal sequence. This suggests that SmpB may be translocated to the outer membrane of B. hyodysenteriae in a similar fashion to SmpA. The results described in this thesis indicate that strains of B. hyodysenteriae harbour either smpA or smpB, but not both, explaining the earlier results of Turner et al. (1991). The presumed outer membrane location of SmpB lead to further investigations into its potential to protect mice from infection with B. hyodysenteriae. Swine Dysentery is a inflammatory disease of the swine colon. Therefore it is believed that a mucosal immune response may provide increased protection against challenge. In this study, vaccination of mice with recombinant SmpB elicited high levels of serum antibodies, induced the production of Interleukin-4 producing T lymphocytes and decreased the observed histological effects after challenge with virulent B. hyodysenteriae. In efforts to increase the protected conferred by vaccination with SmpB, recombinant Salmonella typhimurium STM-1 vaccines were created to express SmpB or deliver DNA vaccines encoding SmpB. Vaccination with these recombinant Salmonella vectors did not induce a measurable SmpB specific immune response. Macrophage survival and plasmid stability studies indicated that this was due to instability of the expression plasmids in STM-1. Although SmpB will only ever protect against strains of B. hyodysenteriae harbouring smpB, these results indicate that with further research, SmpB (and SmpA) may contribute to protection from SD. Toxin production is an important aspect of the pathogenesis of many pathogenic bacteria. Vaccination with attenuated toxins is commonly used to prevent disease. In this study, the B. hyodysenteriae â-haemolysin HlyA was used to vaccinate mice to determine the protection induced after challenge. Vaccination of mice with recombinant HlyA induced significant levels of serum antibodies and lowered the observed pathological effects after challenge of vaccinated mice with virulent B. hyodysenteriae. In an attempt to increase the mucosal immune response and therefore the protection afforded after vaccination with HlyA, recombinant S. typhimurium STM-1 strains were created to express HlyA or deliver DNA vaccines encoding HlyA. Similar to the recombinant STM-1 vaccines expressing SmpB, a HlyA specific immune response was not observed by ELISA or ELISPOT analysis. Plasmid stability trials revealed that the inability to induce a detectable HlyA specific immune response by recombinant STM-1 vaccination may be due to ins tability of the plasmids. Outer membrane proteins are often important components of vaccines against bacterial and viral pathogens. Considering the variation observed in the smpA locus in this study resulting in the identification of smpB, further investigation into the distribution and conservation of outer membrane encoding genes in B. hyodysenteriae strains was undertaken. In particular, the blpAEFG, vspABCD and vspEFGH clusters were analysed for their distribution. It was demonstrated that genes that are B. hyodysenteriae specific (vspABCD and vspEFGH) displayed higher levels of polymorphism than those that are distributed amongst non-pathogenic species, such as B. innocens (which contains blpAEFG). This suggests that the variation in the vspABCD and vspEFGH clusters amongst B. hyodysenteriae strains may be a result of the exposure to the host immune system. Further investigation was undertaken by PFGE analysis and 2D-gel electrophoresis, to analyse genomic and proteomic variation at a global level. Although strains of B. hyodyse nteriae produced several different electrophoretic types (ET) upon PFGE analysis, only limited correlation between the PFGE ET, the polymorphisms in vspABCD and vspEFGH and the presence of smpA/smpB were observed. 2D-gel electrophoresis analysis of outer membrane preparations of two B. hyodysenteriae strain revealed several distinct differences in the outer membrane between B. hyodysenteriae strains. The observed differences in the proteins contained in the outer membrane of B. hyodysenteriae is important for vaccine design, as the induction of cross protection between strains of B. hyodysenteriae is essential for a effective vaccine.

Salmonella infections remain one of the most common food borne diseases worldwide. Gastroenteritis, which can be caused by many non-typhoidal Salmonella (NTS) serovars, is relatively common in North America. One of the main risk factors of NTS gastroenteritis is travel to endemic areas in the developing world. The current treatment of NTS infections with antibiotics is reserved for severe cases. A growing concern with antibiotic use is that clinical isolates are becoming drug resistant. Although most NTS infections are self-limiting in nature, the burden on the body and recovery can take several months. Thus, it is vital to prevent NTS infections rather than solely rely on treatment. We have previously discovered two novel surface associated polysaccharides in Salmonella: O-Antigen capsule and X-factor. Not only O-Antigen Capsule is considered a common surface antigen, but its’ genes were found to be expressed during in vivo infections in mice. Such an antigen would be a suitable candidate in developing a vaccine against Salmonella induced gastroenteritis. The goal of this research was to evaluate the use of O-Antigen capsule to develop a traveler’s vaccine for NTS associated gastroenteritis. Results and Conclusions: We have developed a purification protocol and purified the capsule and X-factor from Salmonella Typhimurium, Enteritidis, and Heidelberg. Lipopolysaccharide (LPS) was co-isolated with O-Antigen capsule, but removed using Triton extraction. Salmonella LPS is strain-specific and an adaptive immune response against LPS will not provide cross-protection. We generated specific immune sera in rabbits to recognize O-Antigen capsule and X-factor produced by Salmonella Typhimurium and Enteritidis. We used a mouse model to determine the immunization dose of O-Antigen capsule and showed that conjugation is necessary to enhance the immune response in mice. To boost capsule production, we analyzed PyihUTSRQPO activity using a luciferase-based reporter system. Deletion of a putative transcriptional repressor (YihW) resulted in over 100-fold increase in PyihUTSRQPO confirming YihW as a repressor. We have also looked at the effect of growth media, temperature, and sugar precursors on PyihUTSRQPO activity, and were able to show that PyihUTSRQPO has highest activity in Tryptone broth at 30oC in the absence of any additional sugars.

Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 x 10(5) CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 x 10(9) CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, chi 9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity. IMPORTANCE Salmonella enterica is the leading cause of bacterial food-borne infection in the United States. S. Typhimurium is capable of producing up to 13 distinct surface structures called fimbriae that presumably mediate its adherence to surfaces. The roles of most of these fimbriae in disease are unknown. Identifying fimbriae produced during infection will provide important insights into how these bacterial structures contribute to disease and potentially induce protective immunity to Salmonella infection. We identified four fimbriae that are produced during infection. Deletion of all four of these fimbriae results in a significant reduction in virulence. We explored ways in which the expression of these fimbriae may be exploited for use in recombinant Salmonella vaccine strains and found that production of Saf and Stc fimbriae are important for generating a strong immune response against a vectored antigen. This work provides new insight into the role of fimbriae in disease and their potential for improving the efficacy of Salmonella-based vaccines.

Chapter-I Introduction The bacteria known as Salmonellae are gram-negative, rod-shaped intracellular pathogenic bacilli that belong to the family Enterobacteriacea and causes typhoid fever. Enteric fever or typhoid fever is a systemic infection caused by human specific enteric pathogen S.typhi. Another very similar but less severe disease, paratyphoid fever, is caused by another human pathogen S.paratyphi A, B and C and S.sendai. Typhoid fever is estimated to have caused 21.6 million illness and 1-4 % death worldwide in the year of 2000 effecting all ages and 90% of death occurs in Asia. In Asia, the incident of typhoid fever was highest with 274 cases per 100,000 persons worldwide, especially in Southeast Asian countries and the Indian subcontinent, followed by sub-Saharan Africa and Latin America with 50 cases per 100,000 persons. Transmission of the disease occurs through faecal-oral route upon ingestion of contaminated water and food. Salmonella can stay for long in ground and pond water. Typhoid fever can be fatal if left untreated and there are reports of 10-30 fatality in such cases and can persist for weeks. Prevention is better than cure. Same hold true even for typhoid fever also. The important and key preventive measures are clean and safe water, safe food, personal hygiene and appropriate sanitation. There are many antibiotics for typhoid fever but till now there are only two licensed vaccine recommended by the World Health Organization for the typhoid fever, one Vi polysaccharide subunit vaccine (sold as Typhim Vi by Sanofi Pasteur and Typherix by GlaxoSmithKline) which is administered through intramuscular route and another one is live oral attenuated vaccine Ty21a (sold as Vivotif Berna) for oral immunization. Both the vaccines are recommended to be used for the children above the age of 3-5 years. Both are between 50 to 80% protective and are recommended for travelers to areas where typhoid is endemic. Salmonella has evolved many strategies to survive inside host system especially during initial time of infection when bacteria counteract to host AMPs in intestine lumen. Salmonella has many pathogenesis island which help bacteria to invade the host system e.g. SPI-1(Salmonella pathogenicity island -1) and also help in intracellular survival as well proliferation e.g. SPI-2 (Salmonella pathogenicity island -2). Salmonella has many strategies to evade host immune system, one of them which is very important for bacteria is LPS modification. Salmonella is capable to modify its own LPS by increasing the +ve charge and increasing AMPs resistance. This modification and resistance is brought about by PhoP/Q and pmrA/B two different two-component system (TCS). These TCS regulate many genes like pmrD, pmrC, pmrG, pmrH-M operon, pmrE etc, which are important for LPS modification by adding 4-amino-arabinose and provide antimicrobial peptide resistance. Chapter-II Development of live attenuated Salmonella vaccine The superiority of live attenuated vaccines in systemic salmonellosis has been proven over killed and subunit vaccines, because of its ability to induce protective cell mediated immunity by CD8+ T cells. A live attenuated Salmonella enterica serovar Typhimurium vaccine has been developed by systematic site directed deletion of the pmrG-HM-D chromosomal genomic loci. This gene confers involved in antimicrobial peptide resistance and is involved in LPS modification, both of which are the major immune evasive mechanisms in Salmonella. The efficacy of the newly developed strain in inducing protection against mortality after challenge with the virulent wild type Salmonella typhimurium 12023 was evaluated in mice model of typhoid fever. Animals were immunized and then boosted on days 7 and 14. Following challenge with virulent S. typhimurium 12023, organ burden and mortality of vaccinated mice were less compared to non-immunized controls. The vaccine strain also induced elevated CD8+ T cells in the vaccinated mice. This multiple mutant vaccine candidate appears to be safe for use in pregnant mice and provides a model for the development of live vaccine candidates against naturally occurring salmonellosis and typhoid fever. Chapter -III A Safe and Efficient Vaccine against Salmonella Infection During Pregnancy Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain ΔpmrG-HM-D (DV-STM-07) in female mice before onset of pregnancy and followed the immune reaction against challenge with virulent S. typhimurium in pregnant mice. This vaccine strain gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards humoral immunity through Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. Chapter-IV Crosstalk between Salmonella genes involved in antimicrobial peptide resistance (pmrG, pmrD, pmr H-M) The pmr system of Salmonella consists of many genes and they are regulated by two component system (TCS), PmrA/B and PhoP/Q. These two component systems are activated at different Mg 2+and Fe3+ condition, low pH and the presence of antimicrobial peptides. Downstream genes like pmrD, pmrG, pmrH-M operon, pmrE, pmrC ect which are regulated by these TCS are involved in LPS modification and AMPs resistance. When these genes were deleted a highly attenuated strain with good vaccine potential was developed. The high degree of attenuation of the vaccine strain is a combined effect of the deletion of the all genes, when single mutation of the two single genes and the operon were created; the attenuation was not as good as the vaccine strain. When tried checking the cross-talk between these genes in vaccine strain and the single mutants of pmrD, pmrG and pmrH-M operon. In one of the previous report pmrH-M mutant was shown to be attenuated through oral route but not through intra-peritoneal route. However, pmrD-HM-G mutant (DV-STM-07) was attenuated when administered through both the routes of infection. To further explain the cross-talk and regulation of these genes, promoter analysis was done for all genes individually in different mutant background of pmrD, pmrG, pmrH-M and DV-STM-07. We hypothesize that the superior attenuation of the triple mutant is achieved because of transcriptional cross-regulation that exists between these genes which attenuates the bacteria when administered through the intra-peritoneal route.