M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Water shortages, water pollution and climate changes are highly interrelated global issues. These have raised immense concerns about serious adverse effects on the quality, treatment and re-use of wastewater. A major role of water is for vitality of life on earth. Water is recognized as source of evolution from origin to degree of civilization, since it is an essential resource its treatment becomes a necessity for day to day for life. Nanoparticles and their application in treatment of wastewater is becoming a major area of research. It is mainly applicable to the removal of major contaminants like microorganisms. This study was carried out with an objective to investigate the antibacterial and antifungal potentials of nanoparticles. Cobalt and cobalt complexes of urea and thiourea were synthesized and characterized using UV-Vs, PL, FTIR, TEM, SEM, XRD and TGA techniques. The Co particles are in a mixture of rod, agglomerates with irregular shape around 50 – 100 nm in diameter. The Co/Thiourea particles appear to be around 10 – 30nm in size. The Co complexed with urea images showed spherical to hexagonal shape with 50 nm size in diameter. The antimicrobial activity was determined using Minimum Inhibitory and bactericidal concentration and the well diffusion method. The antibacterial and antifungal activities of ratios (1:1, 1:2, 1:3, 2:1 μg/mL) of doped cobalt nanoparticles were tested against a panel of five Gramnegative bacteria - (Escherichia coli, Pseudomonas aeruginosa, Shigella enterica, Salmonella typhi and Salmonella sonnei) human pathogenic bacteria; and two fungal strains - Aspergillus niger and Candida albicans. Zones of inhibition as a consequence of nanoparticles were compared with that of different standards like Neomycin for antibacterial activity and Amphotericin B for antifungal activity. The results showed a remarkable inhibition of the bacterial growth against the tested organisms. The most striking feature of this study is that Cobalt, Urea and Thiourea nanoparticles have antifungal activity comparable or more effective (as in case of Thiourea on A. niger) than Amphotericin B and nearly promising antibacterial activity although not comparable to Neomycin.

Le genre bactérien Salmonella regroupe plus de 2500 sérovars, mais peu sont responsables de pathologies humaines. Salmonella enterica sérovar Typhi (S. Typhi) est reconnu pour son importance médicale à travers le globe. S. Typhi cause la fièvre typhoïde chez l’Homme, une maladie infectieuse létale caractérisée par la dissémination systémique de la bactérie vers des organes du système réticulo-endothélial. La fièvre typhoïde représente un fardeau pour la santé mondiale, notamment auprès des pays en développement où les conditions sanitaires sont désuètes. La situation se complique davantage par l’apparition de souches résistantes aux antibiotiques. De plus, les deux vaccins licenciés sont d’efficacité modérée, présentent certaines contraintes techniques et ne sont pas appropriés pour les jeunes enfants et nourrissons. La phase systémique de l’infection par Salmonella repose sur sa survie dans les macrophages du système immunitaire. Dans ce compartiment intracellulaire, la bactérie module les défenses antimicrobiennes grâce à de multiples facteurs de virulence encodés dans son génome. Les mécanismes moléculaires sollicités sont complexes et finement régulés. Malgré les progrès scientifiques réalisés précédemment, plusieurs incompréhensions persistent au sujet de l’adaptation de ce pathogène dans les macrophages de l’hôte. Pour mieux concevoir les déterminants génétiques de S. Typhi impliqués dans l’interaction avec ces cellules, une stratégie de sélection négative a été appliquée afin de vérifier systématiquement l’effet direct des gènes pendant l’infection. En premier temps, une librairie de mutants par transposon chez S. Typhi a été créée pour l’infection de macrophages humains en culture. Après 24 heures d’infection, la présence des mutants fut évaluée simultanément par analyse sur des biopuces de Salmonella. Au total, 130 gènes ont été sélectionnés pour leur contribution potentielle auprès des macrophages infectés. Ces gènes comptaient des composantes d’enveloppe bactérienne, des éléments fimbriaires, des portions du flagelle, des régulateurs, des facteurs de pathogenèse et plusieurs protéines sans fonction connue. En deuxième temps, cette collection de gènes a dirigé la création de 28 mutants de délétion définie chez S. Typhi. Les capacités d’entrée et de réplication intracellulaire de ces mutants au sein des macrophages humains ont été caractérisées. D’abord, les macrophages ont été co-infectés avec les mutants en présence de la souche sauvage, pour vérifier la compétitivité de chacun d’eux envers cette dernière. Ensuite, les mutants ont été inoculés individuellement chez les macrophages et leur infectivité fut mesurée comparativement à celle de la souche sauvage. Sommairement, 26 mutants ont présenté des défauts lorsqu’en compétition, tandis que 14 mutants se sont montrés défectueux lorsque testés seuls. Par ailleurs, 12 mutants ont exposé une déficience lors de l’infection mixte et individuelle, incluant les mutants acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, STY1867-68, STY2346 et SPI-4. Notamment, 35 nouveaux phénotypes défectueux d’entrée ou de survie intracellulaire chez Salmonella ont été révélés par cette étude. Les données générées ici offrent plusieurs nouvelles pistes pour élucider comment S. Typhi manipule sa niche intracellulaire, menant à l’infection systémique. Les gènes décrits représentent des cibles potentielles pour atténuer la bactérie chez l’humain et pourraient contribuer au développement de meilleures souches vaccinales pour immuniser contre la fièvre typhoïde. / The bacterial genus Salmonella holds over 2500 serovars, but few are responsible for human pathologies. Salmonella enterica serovar Typhi (S. Typhi) is recognized across the globe for its medical importance. S. Typhi causes typhoid fever in humans, a lethal infectious disease characterized by systemic dissemination of the bacteria to organs of the reticulo-endothelial system. Typhoid fever represents a burden for public health, notably in developing countries where sanitary conditions are obsolete. The situation is further complicated by the appearance of strains resistant to antibiotics. Moreover, both of the licensed vaccines are of moderate efficiency, present certain technical constraints and are not appropriate for young children and newborns. The systemic phase of infection by Salmonella relies on its survival within macrophages of the immune system. In this intracellular compartment, the bacterium modulates antimicrobial defenses thanks to multiple virulence factors encoded within its genome. Molecular mechanisms taking place are complex and finely regulated. Despite scientific advances made previously, many misunderstandings persist concerning the adaptation of this pathogen within host macrophages. To better conceive the genetic determinants of S. Typhi involved in interaction with these cells, a negative selection strategy was applied to systematically verify the direct effect of genes during infection. Firstly, a library of transposon insertion mutants in S. Typhi was created for infection of cultured human macrophages. After 24 hours of infection, the presence of mutants was evaluated simultaneously by analysis on Salmonella microarrays. In total, 130 genes were selected for their potential contribution within infected macrophages. These genes included bacterial envelope components, fimbrial elements, portions of the flagellum, regulators, pathogenesis factors, and many proteins of unknown function. Secondly, this collection of genes led to the creation of 28 defined deletion mutants in S. Typhi. The ability of entry and intracellular replication of these mutants within human macrophages were characterized. To start, macrophages were coinfected with mutants in the presence of the wild-type strain, in order to verify the competitiveness of each of them against the latter. Then, mutants were inoculated individually into macrophages and their infectiveness was measured in comparison with the wild-type strain. In summary, 26 mutants presented defects when in competition, whereas 14 mutants were shown defective when tested alone. Furthermore, 12 mutants exposed a deficiency during mixed and individual infection experiments, including mutants acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, STY1867-68, STY2346, and SPI-4. In particular, 35 new defective phenotypes of Salmonella entry or intracellular survival were revealed in this study. Data generated here provides significant novel insight for elucidating how S. Typhi manipulates its intracellular niche, leading to systemic infection. Genes described represent potential targets for attenuating the bacteria in the human host and could contribute to the development of better vaccine strains to immunize against typhoid fever.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

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I performed two randomised controlled trials (RCTs) to determine the best treatments for enteric fever in Kathmandu, Nepal, an area with a high proportion of nalidixic acid resistant S. Typhi and S. Paratyphi A isolates. I recruited 844 patients with suspected enteric fever to compare chloramphenicol versus gatifloxacin. 352 patients were culture confirmed. 14/175 patients treated with chloramphenicol and 12/177 patients treated with gatifloxacin experienced treatment failure (HR=0.86 (95% CI 0.40 to 1.86), p=0.70). The median times to fever clearance were 3.95 and 3.90 days, respectively (HR=1.06 [CI 0.86 to 1.32], p=0.59). The second RCT compared ofloxacin versus gatifloxacin and recruited 627 patients. Of the 170 patients infected with nalidixic acid resistant strains, the number of patients with treatment failure was 6/83 in the ofloxacin group and 5/87 in the gatifloxacin group (Hazard Ratio, HR=0.81, 95% CI 0.25 to 2.65; p=0.73); the median times to fever clearance were 4.7 and 3.3 days respectively (HR=1.59 [CI 1.16 to 2.18], p=0.004). I compared conventional blood culture against an electricity free culture approach. 66 of 304 patients with suspected enteric fever were positive for S. Typhi or S. Paratyphi A, 55 (85%) isolates were identified by the conventional blood culture and 60 (92%) isolates were identified by the experimental method. The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% and 96.0%, respectively. This electricity free blood culture system may have utility in resource-limited settings or potentially in disaster relief and refugee camps. I performed a literature review of RCTs of enteric fever which showed that trial design varied greatly. I was interested in the perspective of patients and what they regarded as cure. 1,481 patients were interviewed at the start of treatment, 860 (58%) reported that the resolution of fever would mean cure to them. At the completion of treatment, 877/1,448 (60.6%) reported that they felt cured when fever was completely gone. We suggest that fever clearance time is the best surrogate for clinical cure in patients with enteric fever and should be used as the primary outcome in future RCTs for the treatment of enteric fever.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-05-06T22:21:44Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_FebreTifoideExperiencia.pdf: 1402656 bytes, checksum: 7a17b4a720b4331aeaeaa8105af844d0 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-05-08T14:07:15Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_FebreTifoideExperiencia.pdf: 1402656 bytes, checksum: 7a17b4a720b4331aeaeaa8105af844d0 (MD5) / Made available in DSpace on 2013-05-08T14:07:15Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_FebreTifoideExperiencia.pdf: 1402656 bytes, checksum: 7a17b4a720b4331aeaeaa8105af844d0 (MD5) Previous issue date: 2005 / A febre tifóide é doença infecciosa de distribuição mundial e, estando estreitamente relacionada com baixos níveis sócio-econômicos, ocorre com maior freqüência nos países em desenvolvimento. Neste trabalho é apresentada a experiência do Instituto Evandro Chagas (IEe) com essa doença desde 1987 até 2004, período no qual se construiu uma casuística de 443 casos, todos diagnosticados por meio do isolamento da Salmonella Typhi no sangue e/ou nas fezes, sendo alguns deles complementados com a reação soro lógica de Widal. Os casos foram procedentes de Belém e do interior do Estado, ora encaminhados ao IEC pelas respectivas unidades do Sistema Único de Saúde para esclarecimento diagnóstico de síndrome febril, a maioria de curso prolongado, aqui designados como "demanda espontânea", ora detectados por ocasião da investigação de surtos ocorridos em localidades do interior. Discutiram-se aspectos relacionados à apresentação clínica, com ênfase às manifestações atípicas; à distribuição por gênero e faixa etária; à sazonalidade; e à distribuição por área de procedência, identificando-se os municípios de maior prevalência, e a distribuição por bairros, em relação aos casos procedentes de Belém. Foram discutidos, também, aspectos relacionados ao diagnóstico laboratorial com ênfase à aplicação dos métodos de cultivo (coprocultura e hemocultura) comparando o rendimento delas em relação ao tempo de doença e a relação dessas provas com a reação de Widal, comparando o valor desta como método auxiliar ou complementar no diagnóstico da doença. As análises estatísticas foram realizadas por meio do programa Bio Estat versão 3.0, aplicando-se o teste do qui-quadrado e teste "G" e a significância estatística foi aceita ao nível de 95%. Os resultados nos permitiram tirar as seguintes conclusões: 1) em situações de normalidade epidemiológica a febre tifóide acomete mais a faixa etária do adulto jovem, enquanto que, nas situações epidêmicas, a faixa etária mais atingida é a infantil; 2) existem homogeneidade entre as amostras provenientes dos casos de surtos e da demanda espontânea quanto à distribuição dos gêneros, sendo que o masculino está mais exposta à febre tifóide; 3) no interior do Estado a doença se mostrou mais freqüente nas regiões onde se concentra maior número de populações ribeirinhas e, em relação à capital, ela mostra a maior prevalência no bairro do Jurunas; 4) a enfermidade tem um perfil sazonal, que mostra a maior ocorrência na segunda metade do ano, favorecida provavelmente por fatores geo-climáticos e culturais; 5) a febre tifóide constitui sério problema de saúde pública no Estado do Pará, confirmando sua estreita relação com os elevados níveis de pobreza; 6) a fórmula leucocitária revelou padrão normal ou leucocitose em maior percentual em relação à leucopenia; 7) na abordagem laboratorial não se deve prescindir dos ensaios que visam ao isolamento em detrimento do teste sorológico, e a coprocultura e a hemocultura devem ser solicitadas em todos os casos sem se levar em conta o tempo de evolução do quadro clínico; 8) em nossa região, nas áreas onde não há disponibilidade das provas de cultivo a reação de Widal pode ser uma alternativa de valor diagnóstico; 9) é enfermidade de curso clínico prolongado e com manifestações clínicas atípicas (pneumonias e hepatite colestática) cujos sinais/sintomas, quando presentes, devem suscitar a suspeita por parte do investigador; 10) nas regiões de elevada endemicidade, a febre tifóide pode causar um impacto negativo na economia tanto pelos custos gerados com exames laboratoriais, tratamento, hospitalizações e eventuais intervenções cirúrgicas, como por afastar o trabalhador do seu posto de trabalho por um período de tempo prolongado. / Typhoid fever is an infection disease of world occurrence, but as it is strictly related with low socioeconomic levels it occurs most frequently in developing countries. In this work we present the experience of Instituto Evandro Chagas (IEC) with that disease since 1987 up to 2004, period in which a number of 443 cases was accumulated, all diagnosed by Salmonella Typhi’s isolation from blood or feces, being some of them complemented with Widal serologic reaction. The case were proceeding from Belém and the municipal districts of Pará state conduced to IEC by the respective Units from Health System for etiologic diagnosis of fever syndrome, most of them of persistent course, here designated as “spontaneous” demand, at that time detected during outbreak investigations occurred in the municipal districts. We discussed aspects related to the clinical presentation, with emphasis to the atypical manifestations, the distribution according to sex and age group, the seasoning, the distribution by origin area, identifying the municipal districts of larger prevalence, and the distribution by districts, in relation to the cases coming from Belém. We also discussed aspects related to the laboratorial diagnosis emphasizing the application of the cultivation methods (coproculture and hemoculture) comparing their performance concerning the time of disease and the relationship of those tests with Widal reaction, by measuring the value of these as auxiliary or complementary method in the diagnosis of the disease. Clinical, epidemiological and laboratorial analyses were made, using Bio Estat version 3.0 program by applying qui-square and G test and the statistic significance was accepted at 95% level. The results allowed us to have the following conclusions: 1) in situations of epidemic normality the typhoid fever affects more the young adult group, while, in the epidemic situations, the affected age group is the infantile; 2) there is homogeneity among the samples from cases of outbreaks and the spontaneous demand according to the gender distribution, but males are more exposed to typhoid fever; 3) in municipal districts of the State the disease was shown to be more frequent in the areas where it concentrates larger number of populations who live near rivers and, in relation to the capital, it showed larger prevalence in jurunas district; 4) the illness has a seasonal profile, which shows the largest occurrence in the second half of the year, probably favored by geo-climatic and cultural factors; 5) the typhoid fever constitutes serious problem of public health en the State do Pará, confirming its narrow relationship with the high poverty levels; 6) the leukocytic formula revealed normal pattern or leucocytosis compared to leucopenia; 7) in the laboratorial approach it should not dispense the assays that seek to the isolation in detriment of the serological test, and the coproculture and the hemoculture should be requested in all the cases without considering the time of evolution of the clinical situation; 8) in our area, where there is not availability of cultivation tests, Widal reaction can be an alternative of value diagnosis; 9) it is illness of persistent clinical course and with atypical clinical manifestations (pneumonias and cholestatic hepatitis) whose sings/symptoms, when presented, should raise the suspicion by the investigator; 10) in the areas high endemicity, the typhoid fever can cause a negative impact in the economy not only for the costs generated with laboratorial exams, treatment, hospitalizations and eventual surgical interventions, but also because it can let the worker to be away from work for a prolonged period of time.

Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

Typhoid disease, caused by infection with S. Typhi, is a significant cause of mortality and morbidity in resource–poor countries. Efforts have been made to generate a new generation of vaccines that are efficacious and can be given to infants, but have been hindered by a poor understanding of the protective immune response to S. Typhi infection, and in particular by the absence of a correlate of protection. Controlled human infection studies (‘challenge studies’) provide a model for investigating infectious diseases and appraising novel vaccines, including in typhoid disease. This DPhil described the development of a human challenge model of typhoid fever using <en>S. Typhi Quailes strain administered to healthy adults in a sodium bicarbonate buffer. The careful characterisation and manufactured of the strain is described. Following ingestion of 10³ CFU of S. Typhi 55% of participants developed typhoid disease, whilst ingestion of 10⁴ CFU gave a higher attack rate of 65%. At this attack rate vaccine efficacy against human challenge should be demonstrable with a modest sample size. Validity of the model in the appraisal of vaccines was demonstrated using Ty21a, a live, oral, attenuated vaccine. Protective efficacy of Ty21a compared to placebo against challenge was 35%, comparable to that observed in some endemic settings, and the estimated protection in the first year after vaccination in Cochrane meta-analysis. Clinical, microbiological and humoral immune responses were investigated in participants challenged during model development. Typhoid disease was associated with a high fever in most, but not all participants, and a range of symptoms. Severity of disease was variable, and included asymptomatic bacteraemia, as well as fever and symptoms in participants in whom bacteraemia could not be demonstrated. Typhoid disease was associated with a strong humoral immune response to the flagellin and lipopolysaccharide antigens of S. Typhi but not the Vi polysaccharide capsule. Humoral immune responses were not demonstrated in participants without typhoid fever. There was a dose-response relationship to the clinical, microbiological and humoral responses with participants challenged with 10⁴ CFU having more marked responses than those challenged with 10³ CFU. Future success of challenge studies relies on the willing participation of healthy adult volunteers. The motivations for participation, and experiences of participants, were appraised by questionnaire. Whilst financial compensation was an important motivator, it was not the sole motivator. Participants were positive about their experiences, and most would participate again.