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.

Vibrio cholerae is the cholera causing agent, divided into two biotypes, including the classical biotype and ElTor biotype. Both of these biotypes caused cholera epidemics in the world. The classical biotype caused 6th cholera pandemic (from 1921 to 1961), and ElTor biotype caused 7th cholera pandemic (from 1961 to the 70s). Haemolysin A, a hemolytic protein of V. cholerae ElTor biotype, is encoded by the hlyA gene. This gene is often used for analyzing genetic relationship between strains in the same species or between species in the same Vibrio genus. Results of analyzing nucleotide and amino acid sequences of hlyA gene of V. cholerae strain causing cholera in Vietnam (named hlyA.VN) showed that: the hlyA.VN gene sequence was similar to the hlyA gene sequences of V. cholerae strains of the 6thand 7thcholera epidemics. The hlyA gene of the 6th cholera epidemic strain was deficient in 11 nuleotides (this deficiency leading to the loss of 4 amino acids in the haemolysin A protein) comparing to hlyA.VN gene and hlyA gene of the 7th cholera epidemic strain. The results of genetic distance analysis as well as phylogenetic tree construction also confirmed V. cholerae causing cholera in Vietnam was closely relationship to the strains causing cholera pandemics in the world. It is great significance for the surveillance of molecular epidemiology to prevent cholera effectively. / Vibrio cholerae là tác nhân gây bệnh tả, được chia thành hai typ sinh học, đó là typ sinh học cổ điển và typ sinh học ElTor. Cả hai typ này đã từng gây ra các đại dịch tả trên thế giới. Typ sinh học cổ điển đã từng gây ra đại dịch tả lần thứ 6 (từ năm 1921 đến 1961), còn typ sinh học ElTor đã từng gây ra đại dịch tả lần thứ 7 (từ 1961 đến những năm 70). Haemolysin A, một protein có chức năng làm tan máu của V. cholerae typ sinh học ElTor, được mã hóa bởi gen hlyA. Gene này thường được sử dụng cho các phân tích quan hệ di truyền giữa các chủng trong cùng một loài V. cholerae hay giữa các loài trong cùng một chi Vibrio. Kết quả phân tích trình tự nucleotide và axit amin gen hlyA của chủng V. cholerae gâybệnh ở Việt Nam (hlyA.VN) cho thấy: trình tự gen hlyA.VN có sự tương đồng lớn với trình tự gen hlyA của chủng gây đại dịch tả 6 và 7. Gen hlyA của chủng gây đại dịch tả 6 bị thiếu hụt 11 nuleotide (sự thiếu hụt này dẫn tới sự mất đi 4 axit amin trong phân tử haemolysin A) so với gen hlyA.VN và gene hlyA của chủng gây đại dịch tả 7. Kết quả phân tích khoảng cách di truyền cũng như xây dựng cây phát sinh chủng loại cũng đã khẳng định: chủng gây bệnh ở Việt Nam có quan hệ rất gần với các chủng gây đại dịch tả trên thế giới. Nhận định này có ý nghĩa rất lớn đối với công tác giám sát dịch tễ học phân tử để ngăn chặn bệnh tả hiệu quả.

La toxina alfa hemolisina (HlyA) es un importante factor de virulencia producido por cepas uropatogénicas de Escherichia coli. Su expresión se correlaciona con una mayor severidad de las infecciones producidas por estas cepas con mayor prevalencia de daño renal y bacteriemia. HlyA es secretada por la bacteria en una conformación soluble en medio acuoso e interacciona con la membrana plasmática de la célula blanco produciendo poros proteolipídicos que conducen a la lisis celular. Este proceso es complejo y altamente dependiente de las características físicas de la membrana. El objetivo de este trabajo de tesis fue profundizar en el conocimiento del mecanismo de acción de HlyA, específicamente en la interacción de HlyA con la membrana y con los lípidos de membrana.