A leptospirose é uma zoonose de distribuição global causada por bactérias patogênicas do gênero Leptospira. A doença possui um quadro de manifestações clínicas muito variadas em humanos, que pode apresentar febre, dores de cabeça e muscular, até um quadro clínico mais severo, conhecido como síndrome de Weil, caracterizado por hemorragias, icterícia, falência renal e hepática. Até o presente momento, os mecanismos de patogenicidade da leptospira não são totalmente claros e não existe uma vacina eficaz contra a doença. O sequenciamento de genomas de leptospiras patogênicas possibilitou a identificação de proteínas da membrana externa conservadas entre cepas patogênicas, que são os principais alvos de pesquisa para elucidar os mecanismos de patogenicidade e possivelmente o desenvolvimento de uma vacina. Nesse sentido, foram selecionados por bioinformática os genes LIC11711 e LIC12587 a partir do genoma sequenciado de Leptospira interrogans sorovar Copenhageni, os quais codificam para duas preditas lipoproteínas hipotéticas de funções desconhecidas. Os genes foram amplificados por PCR a partir do DNA genômico da bactéria e clonados no vetor de expressão pAE. As proteínas recombinantes foram expressas em E. coli BL21 DE3 Star pLysS e purificadas por cromatografia de afinidade a metal. As proteínas recombinantes foram inoculadas em camundongos para avaliação da sua atividade imunogênica e obtenção de soros imunes. Ambas as proteínas recombinantes se mostraram reativas com anticorpos em soros de pacientes diagnosticados com a doença, apresentando uma sensibilidade maior em relação ao teste de aglutinação microscópica (MAT), e alta especificidade, diferenciando anticorpos presentes em soros de pacientes diagnosticados com outras doenças não relacionadas. Os resultados de ELISA de bactéria intacta, proteólise por proteinase K e imunofluorescência sugerem que ambas as proteínas estão localizadas na membrana externa bactéria. Com base no ensaio de conservação por western blotting, as proteínas nativas estão presentes em diferentes cepas patogênicas de Leptospira, e ausentes na espécie saprófita L. biflexa. Em ensaios de adesão in vitro, as proteínas recombinantes interagiram com os componentes humanos laminina, e-caderina, plasminogênio, fibrinogênio, fibronectina plasmática, vitronectina, C7, C8 e C9 de forma dose-dependentes, porém com valores de afinidade baixos. Estes resultados sugerem que as proteínas codificadas pelos genes LIC11711 e LIC12587 são expressas durante a patogênese e podem desempenhar múltiplas funções a partir da interação com diferentes componentes, e deste modo auxiliam nas etapas de invasão, disseminação e evasão do sistema imune, auxiliando as leptospiras no processo de infecção. / Leptospirosis is a zoonosis of global distribution caused by pathogenic bacteria of the genus Leptospira. The disease has a wide range of clinical manifestations in humans, which can present with fever, headaches and muscular pain, to a more severe clinical condition, known as Weil\'s syndrome, characterized by hemorrhages, jaundice, renal and hepatic failure. So far, the pathogenicity mechanisms of leptospira are not entirely clear and there is no effective vaccine against a disease. Sequencing of pathogenic leptospires genomes has enabled the identification of conserved outer membrane proteins among pathogenic strains, which are the main research focus to understand the mechanism of pathogenicity and possibly identify a vaccine candidate. In this sense, the genes LIC11711 and LIC12587 were selected from the genome sequence of Leptospira interrogans serovar Copenhageni by bioinformatics, which encode two hypothetical predicted lipoproteins of unknown functions. The genes were amplified by PCR from the genomic DNA of the bacteria and cloned into pAE expression vector. The recombinant proteins were expressed in E. coli BL21 DE3 Star pLysS and purified by metal affinity chromatography. Recombinant proteins were inoculated in mice to evaluate their immunogenic activity and to obtain immune sera. Both recombinant proteins are reactive with antibodies in sera from patients diagnosed with a disease, presenting higher sensitivity than the microscopic agglutination test (MAT), high specificity, differentiating antibodies present in sera from patients diagnosed with other non-specific diseases related. The results of intact bacterial ELISA, proteinase K proteolysis and Immunofluorescence suggest that both proteins are located in the surface of the bacteria. Based on western blotting conservation assay, the coding sequences are present in different pathogenic strains of Leptospira, and absent in the nom-pathogenic L. biflexa. In in vitro adhesion assays, recombinant proteins showed interaction with the human components laminina, e-cadherin, plasminogen, fibrinogen, plasma fibronectina, vitronectin, C7, C8 and C9, in dose-dependent manner, but with low affinity values. These results suggest that the proteins encoded by the genes LIC11711 and LIC12587 are expressed during the infection and may perform multiple tasks and thus assist in the steps of invasion, dissemination and evasion of the immune system, helping leptospires during the establishment of the disease.

Pancreatic cancer is an abhorrent malignancy with limited diagnostics and response to drug therapy. It is believed that noncoding RNAs (ncRNAs) will further the understanding behind the mechanisms of pancreatic cancer development and progression, providing a novel approach for drug development and biomarker discovery. Therefore, a database of pancreatic cancer ncRNAs was established using bioinformatics and text mining approaches. These ncRNAs were characterized for RNA expression, copy number variation, disease association, single nucleotide polymorphisms, secretome analysis, and identification of protein targets. Exosomal proteins and ncRNA identified through this study provide the basis for noninvasive diagnostic potential. Additionally, a secreted microRNA, MIR3620, emerged from this study as a potential prognostic and diagnostic biomarker for pancreatic cancer. By analyzing MIR3620 and its protein targets, a mechanism of regulation for these genes in contributing to the progression and development of pancreatic cancer was established. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

The inflammatory bowel diseases (IBD) are chronic, relapsing and remitting diseases of the gastrointestinal tract. There are two main types of IBD: Crohn’s disease (CD) and ulcerative colitis (UC). The prevalence of IBD is highest in the western world, approximately 100-200 people per 100,000 are affected. In recent years there has been a marked increase in the incidence of CD and UC, in both adults and children (Henderson et al., 2012; Molodecky et al., 2012). This is particularly relevant in Scotland where recent research shows that there has been a 79% increase in the number of cases of paediatric IBD since the 1990’s (Henderson et al., 2012). A yeast 2 hybrid screen identified TLE1as an interacting partner of the known CD susceptibility gene; Nucleotide- binding oligomerisation protein 2 (Nod2). An initial genome wide association study (GWAS) also found an association between the rs6559629 SNP, located in Tle1 and ileal CD (p =3.1 x 10-5) and showed that carriage of the Tle1 risk allele increases the effects of Nod2 mutations in CD. TLE1 functions as a transcriptional co repressor in a variety of different cellular and developmental pathways The work presented in this thesis investigates the potential role of TLE1 in CD. This has been approached using four different strategies: sequencing TLE1 in CD patients and controls, analysing the effects of knocking down TLE1 on genome wide expression, investigating whether the known IBD susceptibility protein XBP1 binds to a predicted binding site in TLE1 and investigating TLE1 levels and localisation in human intestinal samples from CD patients and controls Sequencing TLE1 exons and introns 15/16 and 16/17 in a Scottish cohort of 24 CD patients and healthy controls identified a number of potentially pathogenic exonic and intronic SNPs. Two exonic SNPs and thirteen intronic SNPs were identified and these were further investigated in larger Scottish (203 CD cases, 190 HC) and European cohorts (6,333 CD cases and 15,056 HC) but were not present at statistically significantly different frequencies. Secondly, the effects of TLE1 knock down on genome wide expression were analysed using an Illumina HT12 expression chip. The results showed that TLE1 knock down significantly altered expression of 19 loci (Bonferroni) and 526 loci (FDR). Four of the 19 Bonferroni significant loci are potentially involved in CD: RIOK1 (p=4.3×10-3), SGPL1 (p=4.3×10-3), TUSC3 (p=1.8×10-2) and CCND1 (p=2.7×10-3). Furthermore, expression of SGPL1 and RIOK1 were shown to be differentially expressed at the mRNA level between inflamed patients and controls. The third approach investigates a predicted binding site for the known IBD susceptibility gene, XBP1 in TLE1 which was identified using the Haploreg program. This work shows, using chromatin immunoprecipitation, that exogenous XBP1 does not appear to bind to this predicted binding site. Finally, TLE1 expression was analysed in human intestinal resection samples from patients of known NOD2 status. This work shows that TLE1 and NOD2 are expressed in Paneth cells, however TLE1 expression is not altered in patients carrying CD associated NOD2 variants. In this work TLE1 sequence, expression and potential interacting proteins have been analysed. The results presented suggests multiple mechanisms by which TLE1 may be influencing susceptibility to CD including: the unfolded protein response (TUSC3), S1P signalling and ribosome biogenesis. They also implicate TLE1 in Paneth cell function alongside NOD2. The exact means by which TLE1 may play a role in IBD pathogenesis has yet to be fully elucidated.

O sequenciamento da L. interrogans sorovar Copenhageni e as análises bioinformáticas permitiram a identificação de candidatos vacinais e fatores de virulência. Foram selecionados dois genes, LIC11834 e LIC12253, que foram submetidos a ensaios de presença do DNA genômico e RNA mensageiro em diferentes sorovares de Leptospira. Observamos que o gene LIC12253 foi o mais presente entre os sorovares testados. Os genes foram clonados em vetor de expressão pAE e expressos em E. coli BL21 SI. As proteínas recombinantes foram purificadas e submetidas a ensaio de dicroísmo circular, o qual confirmou que ambas as proteínas estavam estruturadas. Por meio de testes de imunogenicidade em camundongos, ambas as proteínas mostraram-se imunogênicas, apresentando altos títulos de anticorpos, porém não foram capazes de promover resposta imune celular. Em ensaios de localização das proteínas nativas podemos observar a presença destas proteínas na membrana externa de Leptospira. Ensaios de reatividade com soros de pacientes diagnosticados com leptospirose mostraram que há reconhecimento das proteínas por anticorpos presentes nesses soros, sugerindo que as proteínas são expressas durante a infecção. Em ensaios de adesão a componentes de matriz extracelular e componentes do soro e plasma humano, rLIC11834 apresentou ligação à laminina, sendo nomeada de Lsa33, além de ligação ao plasminogênio, ao C4bp e ao fibrinogênio de forma dose-dependente; rLIC12253 apresentou ligação à laminina, sendo chamada de Lsa25, e ao C4bp de forma dose-dependente. Ensaios de desafio demonstraram que as proteínas não apresentam proteção contra infecção letal em hamsters. Assim, acreditamos que estas proteínas multifuncionais possam interagir com proteínas do hospedeiro e ter participação na patogênese da doença. / The genomic sequencing and the advances of bioinformatics analysis allowed the identification of new vaccine candidates and new virulence factors. Therefore, two genes from L. interrogans serovar Copenhageni, LIC11834 and LIC12253 were selected and subjected to assays for the presence of genomic DNA and mRNA in different serovars of Leptospira. These assays found that the gene LIC12253 was the most present among the tested serovars. The genes were then cloned in the expression vector pAE and expressed in E. coli BL21 SI. The recombinant proteins were purified and subjected to circular dichroism, which confirmed that both proteins presented secondary structures. Immunogenicity tests in mice showed that both proteins are immunogenic, with high antibodies titers, but don\'t induce cellular immune response. Localization assays of the native proteins on the leptospiras demonstrated the presence of the proteins on the surface of Leptospira. Reactivity assays with sera of patients diagnosed with leptospirosis showed that the recombinant proteins could be recognized by their antibodies, suggesting that they are expressed during infection. Adhesion assays to the extracellular matrix components, human serum and plasma components, showed that the protein rLIC11834 binds to laminin and was called Lsa33. In addition, Lsa33 interacts to plasminogen, to C4bp and to fibrinogen in a dose-dependent manner. The protein rLIC12253 showed binding to laminina, named Lsa25, and to C4bp in a dose-dependent manner. Challenge assays showed that both recombinant proteins don\'t afford protection against lethal infection in hamsters. Thus, we believe that these multifunctional proteins may interact with host proteins and may play a role in leptospiral pathogenesis.

Candida albicans is an opportunistic pathogen, which is responsible for causing systemic infection in immunocompromised patients in hospital settings (nosocomial infections). 90% of these nosocomial fungal infections are caused by C. albicans, and the estimated annual cost of treating them exceeds $1 billion per year. Despite this expenditure, there is a high mortality rate of 50%. There are two main routes of infections, first a mucosal infection that can spread and invades deeper into the tissues and gets disseminated into the bloodstream. Second, more frequently seen in hospital settings, is when Candida cells dislodge from a biofilm that has formed on intravenous devices or catheters. Treatment of these diseases is difficult due to a dearth of antifungal drugs and new strategies are required to explore mechanisms used by Candida in causing infection. One way of approaching these significant scientific challenges is to identify virulence determinants and mechanisms, which apart from providing insightful knowledge of fungal pathogenesis, can also be used as targets for antifungal drug development. The innate immune responses in humans, which are important for defense against fungal infections, are conserved in Caenorhabditis elegans. In order to identify Candida virulence factors, I have developed a C. elegans based pathogenesis assay, where nematodes were infected with fungi (both S. cerevisiae and C. albicans) and observed for disease phenotypes including death. This assay can be used to study several aspects of disease progression in worms from swelling (inflammation a bio-marker of infection) to colonization in the intestine, leading to intestinal distension and ultimately death of the host worm. The assay offers a fast and simple way of identifying unknown genes, which when established as a virulence determinant in the worm model, can be further studied in mammalian models. I demonstrate the utility of this assay in multiple ways. First as proof of principle using this assay I have identified a fungal mutant cap1, which is susceptible to reactive oxygen species (ROS), and fails to cause disease, except in bli-3 mutant worms that carry a mutation in an oxidase gene and is responsible for the oxidative stress. Second, we screened a library of ~1200 C. albicans mutants, and identified 7 genes, 3 known (CMP1, IFF11 and SAP 8), validating the assay and 4 novel genes (orf19.1219, orf19.6713, DOT4 and ZCF15) that play a role in fungal infection. Third use of this assay is to test potential drugs in a high throughput fashion. Families of related compounds were identified through a screen of 30,000 compounds, for their ability as potential inhibitors of C. albicans adhesion to biological and inert surfaces. These compounds were further tested in this assay for their ability to reduce infection of C. albicans in worms. The assay provides us with a method to test efficacy of antifungals in vivo. Finally, using the survival assay, a test for mortality caused by infection, we can observe disparity in the different C. albicans fluconazole resistant strains isolated from AIDS patients. In addition this assay after small modification can be potentially employed to screen the C. elegans RNAi library to identify the modulators of innate immune responses during fungal infection.

The following dissertation discusses the role of Aβ42 dependent hyperactivation of AMPK mediating synaptic loss through coordinated Mff-dependent mitochondrial fission and Ulk2-dpendent mitophagy in dendrites of PNs. In Chapter 1, I provide a brief background on Alzheimer’s disease and the cellular and molecular mechanisms that have been relevant to the pathogenesis of the disease including disruption on mitochondrial homeostasis and autophagy. In Chapter 2, I discuss the findings of my main project describing the role of Aβ42 induced mitochondrial remodeling leading to synapse loss in vitro and in vivo in part by hyperactivation of CAMKKII-AMPK. Chapter 3 covers a review article that I participated in in examining the role of mitochondria in various ND. In Chapter 4, I discuss about a project I was involved in in examining the mechanism behind maintaining mitochondrial morphology in axon versus dendrite and its functional consequence. In Chapter 5, I end the dissertation by highlighting key findings, potential future studies, and concluding remarks.

Yersinia pseudotuberculosis is a foodborne pathogen that is the ancestral strain to Yersinia pestis, the causative agent of Plague. Y. pseudotuberculosis invades a host through the intestinal epithelium. The bacteria resist mucosal innate immune defenses including antimicrobial chemokines and phagocytic cells, and replicate in local lymph nodes. They cause Tuberculosis-like symptoms, including necrosis of local tissue and granuloma formation. Like all bacteria, Y. pseudotuberculosis has a net negative charge, which contributes to its susceptibility to some cationic antimicrobial peptides. Y. pseudotuberculosis is able to reduce this negative charge by adding 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A portion of lipopolysaccharide. The production and addition of the L-Ara4N is coded for by the pmrHFIJKLM (pmrF) operon. A previous study has shown that the Y. pseudotuberculosis pmrF operon is important for resistance against polymyxin, but is not important for virulence in mice. Several previous reports have shown a strong influence of growth temperature on resistance to antimicrobial peptides and pmrF expression in pathogenic Yersinia species, but these studies also suggest significant variability between species, and even between strains of individual species. In particular, the regulation of the Y. pseudotuberculosis pmrF operon and its effect on bacterial interactions with mucosa-associated antimicrobial chemokines and neutrophils is not understood. In these studies, we investigated the environmental influences on pmrF expression in Y. pseudotuberculosis. We found that the promoter activity of the pmrHFIJKLM operon is increased at lower temperatures (21ºC) and in the presence of human serum. A ΔpmrI mutant strain of Y. pseudotuberculosis defective for addition of L-Ara4N was found to be more susceptible to killing by the antimicrobial chemokine CCL28 compared to wild-type. This suggests that this gene is important in the bacterial defense against antimicrobial chemokines. However, when the ΔpmrI mutant strain was exposed to human neutrophils, there was a decrease in phagocytosis as compared to wild-type bacteria. Our results suggest that the regulation of L-Ara4N modifications in Yersinia is more complex than previously appreciated and varies between species. Addition of L-Ara4N to Y. pseudotuberculosis appears to enhance resistance to some antimicrobial peptides like CCL28 and promote greater phagocytic engulfment by neutrophils. These opposing effects may partly explain why there is no net apparent survival defect in mutants lacking the pmrF operon during infection.

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

Studies presented in this thesis looked at developing new methods for the diagnosis of virulent footrot (VFR) in sheep and identification of serogroups of Dichelobacter nodosus, the principal causative agent of footrot. Earlier studies had shown that immunological memory response in sheep recovered from VFR can be aroused by natural or recurrent infection or by injection of outer membrane protein (OMP) antigens to be used as a retrospective diagnostic test for VFR. But OMP antigen was found to be non-specific in older animals. To overcome this non-specificity of OMP antigen in anamnestic response, pilus antigen was evaluated in a trial at Camden. The results of this trial indicated that antibodies to pilus antigen can be detected over time in a manner similar to OMP antibodies so a retrospective assessment of VFR status can be made by anamnestic test with pilus antigens. The anamnestic response to pilus was similar in character to OMP antigen but unlike OMP was highly specific. The response to anamnestic challenge with pilus was determined by severity of the lesions they had expressed, with severe lesions triggering the greater responses. However, there was variation between individuals, with some (6 of 46 with severe lesions) failing to respond. This individual variation is probably mediated genetically as is response to vaccination. This anamnestic test was tested in flocks of sheep in Nepal that had a history of VFR which had apparently been eradicated. That assessment, based on clinical findings, was confirmed by the uniformly negative results in the pilus anamnestic test applied to a representative sample of the population. This allowed a conclusion that the virulent strains of D. nodosus involved had been eliminated from these flocks. As mentioned in the preceding study pilus antigen was found to be very specific and ideal for retrospective diagnosis of virulent footrot with an anamnestic challenge ELISA test. However, serogroup specificity was seen as a disadvantage of using pilus antigen for the anamnestic test. The possibility of using multivalent pilus antigens was tested in another trial. These animals had been involved in a clinical expression experiment conducted by another research group and had a clinical and bacteriological history extending over more than 12 months. After these initial trials all these animals were treated for footrot and managed for 5 months as a single flock at Camden. These were then challenged with multivalent pilus antigen (serogroup A - I) as a single injection. The results obtained indicate that multivalent pilus anamnestic ELISA is equally effective as monovalent pilus. This has the added advantage that prior knowledge of the serogroups present in the flock is not required. It has the possibility of being used as an indirect test to check the presence of serogroups in a flock without doing the bacterial cultures. This test can identify most animals with pre-existing underrunning lesions (Scores of 3 or higher). However, the sensitivity and specificity of this test need to be tested extensively in flocks of known clinical history before it could be adopted as a routine test. As a key component of a larger study to determine the role of fimbrial genes (fimA and fimB) of D. nodosus in the pathogenesis of footrot using allelic exchange to disrupt these genes of a strain (serogroup G), the study presented in this thesis contributed a detailed characterisation of the resultant mutant and the wild strains and tested these strains for virulence in sheep. The results presented provided the first definitive evidence that the fimA gene is essential for virulence of D.nodosus in sheep. In vivo virulence testing of two fimA mutants showed that they were not able to establish any footrot whereas the wild type of the same strain produced virulent footrot in the same trial conducted under similar conditions. These mutant bacteria were not re-isolated from interdigital skin after in vivo challenge. This indicated that fimA mutant strains could not colonise the ovine foot, and the simplest and most likely explanation for these results was that colonisation of the interdigital skin and subsequent penetration of the stratum corneum requires the adhesive activity of type IV fimbriae. However, since these mutants also had altered ability to secrete extracellular proteases, and perhaps other as yet unknown extracellular proteins, the possibility of the involvement of these factors as major determinants of host colonisation or invasion cannot be excluded. Current methods for the identification of the serogroup of D. nodosus present in the bacterial population requires isolation of the organism and after purification by subculture, antigenic analysis with agglutination tests. This usually takes at least 3 to 4 weeks. With the objective of developing a rapid serogroup specific PCR assay, the basis of serogroup variation in D. nodosus localised in the fimbrial gene region was exploited. A common forward primer and 9 serogroup specific reverse primers were selected from the fimbrial gene sequences of the prototype strains. Analytical sensitivity of the serogroup specific primers on chromosomal DNA was similar to PCR tests in other bacterial species reported before. Immuno-magnetic bead capture PCR method was able to detect 5 to 10 cells in cell lysates. Specificity within and between the serogroups of D. nodosus was tested with all the prototype strains. They were found to be very specific to each serogroup and specific only to D. nodosus when tested with 84 commonly found bacterial strains or strains related to D. nodosus. To overcome the time delay in conducting 9 different amplifications to find out the prevalence of all possible serogroups in a flock multiplex PCR reactions with common forward primer and groups of 3, 4 and 5 reverse primers were successful in reducing the number of reactions to 2 (with groups of 4 and 5) or 3 (with groups of 3) primers. A drawback of the multiplex reaction was that if a template was 1000 times less concentrated that the others in the mixture it was not amplified but the margin for difference is very high. The main aim of developing rapid serogroup specific PCR was to apply these tests directly on footrot lesion samples to make it a rapid diagnostic test for field samples. The sensitivity of the test on lesion samples was found to be very low. To try and improve the sensitivity an overnight or four days old pre-enrichment culture in broth was tested but was found to be no better than direct PCR. The immuno-magnetic capture method which improved the sensitivity of pure culture samples by 10 -100 fold also had very low sensitivity with lesion samples. However, this drawback can be overcome by picking up colonies from 4 days old lesion cultures on hoof agar (HA) plates for serogroup specific multiplex PCR. If the colonies are too small/ too few on the lesion cultures these can be sub cultured onto a quarter of 4 percent HA plates and then used for the PCR test which also reduces the time taken for serogrouping at least by 2 weeks. The other advantage is that individual colonies do not need to be isolated. A PCR test can be done on pooled colonies just as well and can be used to identify all serogroups present in the sample. Serogroup specific PCR is much faster and is more sensitive and accurate than slide agglutination tests which take 3 to 4 weeks to complete. Multiplex PCR makes it easier to detect different serogroups in a sample with a maximum of 3 PCR tests. Serogroup specific multiplex PCR will be a useful tool for footrot control based on specific vaccination. The difficulty in using the test on direct lesion swabs needs to be further looked into. There may be future advances in the application of PCR tests to clinical samples.

The upper respiratory tract (URT) of humans is complex and interconnected region and comprises several major ecosystems including the oral cavity, oropharynx, nasal cavity, sinuses, nasopharynx and middle ear. Most of the anatomical locations within the URT are colonised with a normal bacterial microbiota, within which are often organisms having the potential to cause disease. The diseases of the URT are both varied and frequent in their occurrence, and conditions such as otitis media, rhinosinusitis and pharyngitis are sources of morbidity and mortality in adults and children in both developing and developed countries. The study of diseases of the URT has traditionally been based on application of culture-based methods in which the infection-implicated organisms are first grown in vitro and then studied further. Ongoing advances in DNA-based techniques have led to the development of new molecular tools for the study of infectious diseases. One such technique is PCR-denaturing gradient gel electrophoresis (PCR-DGGE). This is a PCR-based tool that allows the investigation of microbial communities independent of culture. Although this technique has been applied extensively in the study of the gastrointestinal tract, the vagina and endodontic infections in humans, there have been few reports of its application to URT infections. PCR-DGGE was applied in the present study to investigate (a) the bacteria present in the middle ear of children suffering from otitis media with effusion (OME), (b) the microbiota associated with the sinuses in patients with chronic rhinosinusitis (CRS) and (c) perioperative changes in the bacterial population of the middle meatus of patients undergoing nasal or sinus surgery. The analysis of the middle ear fluid samples indicated an increased role in OME for the newly-discovered pathogen Alloiococcus otitidis and also the possible involvement of certain coryneform bacteria and coagulase-negative staphylococci in the aetiology of this condition. PCR-DGGE analysis of patients with CRS revealed a polymicrobial disease with considerable variability in the predominant species detected when multiple, serial samples were evaluated. The perioperative audit showed that when good clinical practice is adhered to, there was no apparent introduction of potentially-harmful organisms into the middle meatus. Streptococcus salivarius is a common, commensal inhabitant of the oral cavity of humans and has also been shown to inhabit the nasopharynx of infants. S. salivarius is also a well known producer of bacteriocins with activity directed against Streptococcus pyogenes. One such strain, S. salivarius K12, is now marketed in New Zealand as the probiotic, K12 Throat Guard[TM]. In the present study, S. salivarius K12 was compared with two additional strongly-inhibitory S. salivarius (strains T18A and T30A) for activity against the common causative pathogens of otitis media. A paediatric formulation of strain K12 was also tested in a pilot clinical trial for its ability to colonise the URT of young children. Although the levels of colonisation of these subjects was not as high as typically obtained with use of the K12 Throat Guard[TM] formulation, it was considered that further development of the paediatric formulation is warranted, particularly with respect to use of a different pre-treatment regimen. In other studies, the molecular basis for the unusual in vitro inhibitory activity of S. salivarius strain T30A was investigated. Although this still remains unresolved, other observations made during the course of this study have led to the introduction of a schema for the division of inhibitory S. salivarius into three groups based on (a) their sensitivity to the lantibiotic salivaricin A and (b) the structure of their salivaricin A genetic locus. This grouping is analogous to the "rock-paper-scissors" system previously described for colicin-producing strains of E. coli. Streptococcus pneumoniae is a major human pathogen responsible for a variety of diseases in humans. There have been very few reports of bacteriocin production by S. pneumoniae when compared to other streptococcal species. In the present study a putative cluster of bacteriocins encoded by the blp locus has been investigated. The distribution of the individual blp determinants within this locus was evaluated in a collection of S. pneumoniae strains using PCR. The blp genes were detected in 92% of 57 tested strains and a variant form (termed the B-form) of the cluster was identified that appeared to have arisen due to a genetic recombination event. In this case an approximately 250 bp portion of the blpMNO cluster appears to have recombined into blpK of the blpIJK cluster. Attempts were made to express the putative bacteriocin peptide genes in an Escherichia coli expression system. Failure to achieve expression was taken to indicate that these bacteriocin-like peptides may be toxic for the host producer cells under these test conditions. Future attempts to achieve expression of the Blp peptides, could explore the use of different fusion proteins, a Gram-positive expression host or a cell-free protein expression system.