A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2017 / In South Africa, Salmonella Enteritidis has become a significant pathogen and the numbers of cases reported to the Centre for Enteric Diseases (CED) have increased. Pulsed-field gel electrophoresis (PFGE) is a primary for molecular subtyping of Salmonella. However, this technique has poor discrimination for serotypes with high homogeneity such as Salmonella Enteritidis. Multi-locus variable-number tandem-repeats analysis (MLVA) has shown higher discriminatory power for Salmonella Enteritidis compared to PFGE. In this study, MLVA was used to investigate the molecular epidemiology and relatedness of human Salmonella Enteritidis strains from Gauteng and Western Cape, South Africa. Furthermore, MLVA was also used to investigate the relatedness of human and non-human Salmonella Enteritidis strains. MLVA included analysis of five VNTR loci, with varying degrees of diversity. A total of 1221 human isolates and 43 non-human isolates were included in the study. Eighty-six MLVA profiles were obtained; MLVA profiles 7, 21, 22 and 28 were the predominant MLVA profiles. MLVA profile 28 was the most common MLVA profile amongst both the human and non-human isolates. Isolates had low prevalence of antimicrobial resistance, however sulfamethoxazole resistance was notable amongst both the human (348; 29%) and non-human (10; 23%) isolates. During the study period, seven Salmonella Enteritidis outbreaks were investigated from six provinces and isolates from each individual outbreak showed an identical MLVA profile. MLVA was shown to be a successful molecular subtyping tool for Salmonella Enteritidis, for both surveillance purposes and outbreak investigations. Salmonella Enteritidis strains circulating within the human and non-human population were clonal. The study emphasizes the need for the one health approach, in order to curb the spread of Salmonella Enteritidis in South Africa. / MT2017

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of requirements of the degree of Master of Science. Johannesburg, 2017 / The typhoid fever causing Salmonella Typhi remains an important public health problem in Africa. More importantly, the emergence of the highly antimicrobial resistant H58 Salmonella Typhi haplotype is of greater concern. Rapid and highly discriminatory molecular methods are essential for prompt and effective epidemiological investigation of typhoid fever outbreaks. Traditional methods, such as pulsed-field gel electrophoresis (PFGE) are time-consuming and offer subjective discrimination of highly homologous isolates. On the contrary, molecular subtyping based on multiple-locus variable-number tandem-repeats (VNTR) analysis (MLVA) is a rapid, PCR-based method which has been successfully used for subtyping homogenous isolates of the Salmonella genus. This study describes the development and application of a MLVA assay for molecular characterization of Salmonella Typhi isolates from sub-Saharan Africa (SSA). This involved evaluation of thirteen VNTR loci using a validation panel consisting of 50 diverse Salmonella Typhi isolates. A MLVA assay consisting of five highly variable VNTR loci was adopted. The developed MLVA assay was used, along with PFGE, to characterize 316 Salmonella Typhi isolates from SSA. A total of 226 MLVA types were identified as compared to 143 PFGE fingerprint types. MLVA typing results indicated intracontinental spread of Salmonella Typhi. For the rapid identification of H58 Salmonella Typhi, a conventional PCR targeting a mutation that is exclusive to the H58 haplotype was employed on 105 isolates from South Africa as well as 121 isolates from other SSA countries. Approximately 54% (105/214) of the Salmonella Typhi isolates from South Africa and 62% (75/121) of the isolates from other SSA countries were identified as H58 Salmonella Typhi. The MLVA tool was able to discriminate among H58 Salmonella Typhi isolates. MLVA is viable alternative to PFGE for subtyping Salmonella Typhi and can be used as first-line assay for routine screening of Salmonella Typhi isolates in SSA, providing excellent discrimination of isolates. / MT2017

by Norman Wai-sing Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 145-160). / Chapter Chapter 1 --- Introduction --- p.1 / Chapter A. --- Classification of Salmonella --- p.3 / Chapter B. --- Enteric fever --- p.4 / Chapter C. --- Chloramphenicol and multiple resistance in S. Typhi --- p.7 / Chapter D. --- Resistance plasmids in the typhoid bacillus --- p.11 / Chapter E. --- In vivo acquisition of resistance plasmids by S. Typhi --- p.18 / Chapter F. --- Worldwide distribution of typhoid fever --- p.23 / Chapter G. --- Epidemiology of typhoid fever in Hong Kong --- p.27 / Chapter H. --- Principles of methods used in the epidemiological typing of S. Typhi --- p.29 / Chapter 1. --- Phage typing --- p.29 / Chapter 2. --- Antibiotics sensitivity pattern --- p.32 / Chapter 3. --- Plasmid analyses --- p.32 / Chapter a. --- Genetic methods --- p.33 / Chapter i. --- Phenotypic expression --- p.33 / Chapter ii. --- Transferability of plasmids --- p.33 / Chapter iii. --- Incompatibility --- p.34 / Chapter b. --- Molecular methods --- p.34 / Chapter i. --- Estimation of molecular size by agarose gel electrophoresis --- p.34 / Chapter ii. --- Plasmid fingerprinting --- p.35 / Chapter iii. --- Localization of resistance genes by DNA-DNA hybridization --- p.35 / Chapter 4. --- Ribotyping --- p.36 / Chapter 5. --- Total DNA fingerprinting --- p.37 / Chapter 6. --- Chromosomal insertion sequence IS200 profile analysis --- p.38 / Chapter 7. --- Others --- p.39 / Chapter a. --- Multilocus enzyme electrophoresis --- p.39 / Chapter b. --- Phenotype of lipopolysaccharide (LPS) --- p.39 / Chapter c. --- Envelope protein profiles and immunoblotting --- p.40 / Chapter I. --- Epidemiological typing of S. Typhi --- p.40 / Chapter J. --- Objectives --- p.46 / Chapter Chapter 2 --- Materials and Methods --- p.48 / Chapter A. --- Materials --- p.48 / Chapter I. --- Bacterial strains --- p.48 / Chapter II. --- Materials --- p.51 / Chapter B. --- Methods --- p.56 / Chapter I. --- Sensitivity testing --- p.56 / Chapter II. --- Characterization of plasmids --- p.58 / Chapter 1. --- Genetic studies --- p.58 / Chapter a. --- Transferability of resistance plasmids --- p.58 / Chapter b. --- Mobilization of resistances --- p.59 / Chapter c. --- Incompatibility grouping --- p.59 / Chapter 2. --- Molecular studies --- p.60 / Chapter a. --- Plasmid profile analysis --- p.60 / Chapter i. --- Plasmid extraction --- p.60 / Chapter ii. --- Agarose gel electrophoresis --- p.61 / Chapter b. --- Molecular characterization of plasmid --- p.61 / Chapter i. --- Extraction of purified plasmid DNA --- p.62 / Chapter ii. --- Restriction endonuclease digestion of plasmid DNA --- p.63 / Chapter III. --- Characterization β-lactamases --- p.63 / Chapter 1. --- Extraction of β-lactamases --- p.63 / Chapter 2. --- Determination of isoelectric points (pIs) --- p.64 / Chapter IV. --- Localization of resistance genes --- p.64 / Chapter 1. --- TEM-1 and TEM-2 genes --- p.65 / Chapter a. --- Oligonucleotide probes --- p.65 / Chapter b. --- Total DNA preparation --- p.66 / Chapter c. --- DNA-DNA-hybridization --- p.66 / Chapter i. --- Transfer of DNA to membrane filters --- p.66 / Chapter ii. --- Labelling of oligonucleotide probes (TEM-1 and TEM-2) --- p.67 / Chapter iii. --- Hybridization --- p.68 / Chapter 2. --- Chloramphenicol- and tetracycline-resistance genes --- p.69 / Chapter a. --- Preparation of probes --- p.69 / Chapter b. --- Hybridization --- p.73 / Chapter i. --- Transfer of DNA to membrane filters --- p.73 / Chapter ii. --- Labelling of chloramphenicol- and tetracycline- resistance probes --- p.73 / Chapter V. --- Epidemiological typing --- p.75 / Chapter 1. --- Ribotyping --- p.75 / Chapter 2. --- DNA fingerprinting --- p.77 / Chapter C. --- Plan for achieving objectives --- p.77 / Chapter Chapter 3 --- Results --- p.79 / Chapter A. --- Antimicrobial susceptibilities --- p.79 / Chapter B. --- Characterization of resistance plasmids --- p.83 / Chapter C. --- β-lactamases produced by ampicillin-resistant S. Typhi --- p.85 / Chapter D. --- Localization of resistance genes --- p.85 / Chapter E. --- Plasmid profile analysis --- p.87 / Chapter F. --- Plasmid fingerprinting --- p.90 / Chapter G. --- Ribotyping --- p.93 / Chapter H. --- Chromosomal DNA fingerprinting --- p.107 / Chapter I. --- "Correlation of PstI, ClaI and KpnI ribotypes and NarI, EcoRV and MluI chromosomal types" --- p.124 / Chapter J. --- Correlation of PstI ribotypes and NarI chromosomal types --- p.126 / Chapter K. --- Epidemiology of S. Typhi in Hong Kong --- p.129 / Chapter Chapter 4 --- Discussion --- p.131 / Chapter A. --- Antimicrobial susceptibilities --- p.131 / Chapter B. --- Characterization of resistances --- p.132 / Chapter C. --- Plasmid profile analysis --- p.135 / Chapter D. --- Epidemiological analysis of S. Typhi --- p.136 / Chapter E. --- Area for future research --- p.143 / References --- p.145 / Appendix --- p.161 / Chapter A. --- Buffer and Stock solutions --- p.161 / Chapter B. --- Epidemiological information of S. Typhi isolates --- p.167

by Ka-ming Pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 95-100. / Abstract --- p.i / Introduction --- p.1 / Literature review --- p.2 / Chapter 1. --- Mode of Respiration in Enteric Bacteria --- p.2 / Chapter 2. --- Nitrate Respiration --- p.4 / Chapter 3 . --- Biochemistry of Nitrate Reductase --- p.7 / Chapter 4 . --- Regulation of Nitrate Reductase --- p.8 / Chapter 5. --- Genetics and Regulation of chlC (nitrate Reductase) --- p.10 / Chapter 6. --- chlC in Salmonella typhimurium --- p.14 / Chapter 7. --- Purpose of study --- p.16 / Materials and Methods / Chapter 1 . --- Strains --- p.18 / Chapter 2. --- Media --- p.18 / Chapter 3 . --- Solution --- p.20 / Chapter 4. --- β-galactosidase assay --- p.23 / Chapter 5. --- Preparation of chromosome DNA --- p.24 / Chapter 6. --- Small scale plasmid preparation --- p.25 / Chapter 7. --- Large scale plasmid preparation --- p.26 / Chapter 8. --- Preparation of lambda DNA --- p.27 / Chapter 9. --- Preparation of M13 ssDNA for sequencing --- p.30 / Chapter 10. --- Digestion of DNA with restriction enzymes --- p.31 / Chapter 11 . --- Agarose gel electrophoresis --- p.31 / Chapter 12. --- Ligation --- p.31 / Chapter 13. --- Transformation --- p.32 / Chapter 14. --- Direct gel test and C-test of ssDNA of M13 clones --- p.34 / Chapter 15. --- DNA sequencing --- p.34 / Results / Chapter 1 . --- β-galactosidase assay of HSK1001 --- p.37 / Chapter 2. --- Preparation of HSK1001 DNA --- p.37 / Chapter 3. --- Sau3A partial digestion of HSK1001 chromosomal DNA --- p.37 / Chapter 4 . --- Ligation of HSK1001 DNA to lambda EMBL3 --- p.40 / Chapter 5. --- In vitro packaging and screening of lambda clones --- p.40 / Chapter 6. --- Restriction mapping of lambda clones --- p.43 / Chapter 6.1 --- Restriction mapping of HSK4001 to HSK4006 --- p.43 / Chapter 6.2 --- Orientation of Mu dA fusion clones --- p.43 / Chapter 7 . --- "Sub-cloning of SalI-HindIII fragments of HSK4002 HSK4003, HSK4005 and HSK4006 into pFZYl" --- p.66 / Chapter 8. --- Sub-cloning of 2.4 kb HindIII fragment of HSK4005 and HSK4006 into pFZY1 --- p.67 / Chapter 9. --- Sub-cloning of 2.4 kb HindIII fragment of HSK4006 into M13mpl9 --- p.67 / Chapter 10. --- Sub-cloning of 2.4 kb HindIII fragment of HSK4005 into M13mp19 --- p.69 / Chapter 11. --- Sub-cloning of 2.5 kb SalI-HindIII fragment of HSK4006 into M13mpl8 --- p.72 / Chapter 12. --- Sub-cloning of 2.2 kb SalI-HindIII fragment of HSK4005 into M13mp19 --- p.72 / Chapter 13. --- Complementation test of M13 clones --- p.74 / Chapter 14. --- Sub-cloning of 4.3 kb HindIII-EcoRI fragment and 3.8 kb EcoRI-BamHI fragments of HSK4006 into M13 mp18 and mp19 --- p.74 / Chapter 15. --- DNA sequences of M13 clones --- p.77 / Dicussion / Chapter 1 . --- Cloning of Mu dA operon fusion to EMBL3 vector --- p.88 / Chapter 2 . --- Sequences of SalI-HindIII fragments of HSK4005 to HSK4006 --- p.92 / Reference --- p.95

by Kwong-kwok Wong. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1987. / Bibliography: leaves 158-164.

by Yuen-Shan Chan. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1987. / Bibliography: leaves 142-150.

acase@tulane.edu / Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, which are known to differ in how they functionally contribute to the naïve T cell pool. This distinctive cell population is known to contribute to the maintenance of T cell receptor diversity in the peripheral T cell pool, but their involvement in combating chronic bacterial infections, such as typhoid fever, has not been explored. In the present study, we hypothesized that RTEs are essential contributors to the CD4 T cell response during persistent Salmonella Typhimurium infection, which predominantly relies on helper T cell immunity to control bacteria. To test this hypothesis we performed thymectomy or sham surgical treatment on mice, either prior to or during, an established, chronic Salmonella infection and measured disease outcomes. We found that thymic output is crucial in the formation of protective immune responses during the establishment of a Salmonella infection, but appears to be dispensable once the Salmonella infection is established. We found that thymectomy prior to infection resulted in increased infection-associated mortality, increased bacterial burdens and increased numbers of antigen-specific CD4+ T cells. Furthermore, it appears that thymectomized mice may be impaired in their ability to produce effector cytokines at early time points of infection, compared to thymically intact mice. In contrast, when thymectomy was performed 30 days after the initiation of Salmonella infection, there were no observable differences in survival, bacterial burdens or antigen-specific CD4+ T cell numbers through out infection. To directly study the contribution of RTEs to the Salmonella immune response, we developed an adoptive transfer model where purified RTEs could be transferred to a congenically marked host immediately prior to Salmonella infection and their responses could be tracked throughout infection. We found that RTEs are capable of proliferating and upregulating maturation markers in response to antigen; comparable to other mature, naïve CD4+ T cells. Together, these results may imply a unique role for thymic output and or recent thymic emigrants in the formation of early immune responses against a chronic, enteric pathogen like Salmonella. However, once the pathogen has disseminated systemically, thymic function does not appear to play a vital role in protective immune response. / 1 / James Alan Goggins

Despite its high isolation frequency in Australian chickens, S. II Sofia is rarely associated with animals or human salmonellosis as this serovar is avirulent in nature. The reason for its persistence and avirulence is unknown as very few studies have been conducted on the epidemiology and pathogenicity of this strain. This study details the various experimental methods utilised to investigate the genetic relatedness and molecular mechanisms involved in S. II Sofia pathogenesis. Using PFGE and Rep-PCR, the Australian S. II Sofia isolates were found to show limited genetic diversity and probably share a clonal relationship. A majority of the S. II Sofia isolates were not geographically restricted with the predominant pattern subtype spread out among the isolates from various states. Distribution and variation of the SPI-associated virulence genes within S. II Sofia was also examined. Based on RFLP and sequence analysis, most of the differences observed in SPI1 to SPI5 of S. II Sofia could be attributed to a loss or gain of restriction cleavage sites within these regions. However, a number of genes in SPI1, SPI2, SPI3 and SPI5 were found to have accumulated changes (mutations, insertions and deletions) that could have affected gene transcription and/or protein translation - these genes have been shown to be involved in different aspects of the virulence process. The avirulence of S. II Sofia is probably not the result of a single genetic change but rather a series of alterations to a large number of its virulence-associated genes. Plasmid-mediated virulence was also assessed in S. II Sofia isolates. Southern hybridisation with probes derived from the virulence plasmid of S. Typhimurium indicated either the total absence of the virulence plasmid or possible presence of a virulence plasmid containing major deletions. Clones were constructed with the missing spv operon using high-copy pCR®2.1 and low-copy pWSK29 plasmids and the adherence, invasion and intracellular survival of the mutant strain was evaluated in vitro. The presence of spvRABCD was shown to have no effect on intracellular survival and replication. Although the cloning of spv with pCR®2.1 was observed to significantly increase invasiveness of S. II Sofia, it was not capable of restoring the invasive ability of S. II Sofia to the level of pathogenic S. Typhimurium 82/6915. On the other hand, the uneven adherence and invasion ability of the other mutant strains appeared to be linked to the presence of pWSK29 and this observation is further supported by RT-PCR analysis of the clones - indicating that perhaps pWSK29 is not a suitable vector for this study. Wild-type S. II Sofia isolates are unlikely to regain full pathogenicity because of the numerous mutations in many important virulence genes: even the chance acquisition of a virulence factor (e.g. spvRABCD) is not sufficient to completely restore S. II Sofia virulence. Therefore, S. II Sofia should not be considered similar to other Salmonella spp. when monitoring Salmonellae in food samples.

The purpose of this study was to describe the herd antibody status to Salmonella Typhimurium as estimated from co-mingled milk samples and to describe the resulting geographical patterns found in Texas dairy herds. Bulk tank milk samples were collected from 852 Grade A dairies throughout Texas during the summer of 2001. An indirect enzyme-linked immunosorbent assay (ELISA) using S. Typhimurium lipopolysaccharide was performed with signal to noise ratios calculated for each sample. The ELISA ratio was used in fitting a theoretical variogram and kriging was used to develop a predicted surface for these ratios in Texas. A spatial process with areas of higher risk located in the panhandle and near Waller County was apparent. Lower risk areas included Atascosa, Cooke, Collin, Titus, Comanche and Cherokee Counties. Subsets representing large dairy sheds in northeast Texas, the Erath County area, and the Hopkins County area were also evaluated individually. Each result illustrated a spatial process with areas of low and high ELISA ratio predictions. Cluster analysis was performed for the entire state with cases defined as herds having milk ELISA ratios greater than or equal to 1.8. Using this cutoff, the prevalence of herds with positive bulk tank milk ELISAs was 4.3%. Significant clustering of cases was demonstrated by the Cuzick and Edward's test. The spatial scan statistic then identified the two most likely clusters located in and near the Texas Panhandle. This study demonstrated that the distribution of S. Typhimurium antibodies in bulk tank milk in Texas is describable by a spatial process. Knowledge of this process will help elucidate geospatial influences on the presence of S. Typhimurium in dairy herds and enhance our understanding of the epidemiology of salmonellosis.

Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.