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Item Phenotypic characterization and genome analysis of a novel Salmonella Typhimurium phage having unique tail fiber genes.(Springer Nature Limited, 2022-04-06) Sattar S; Ullah I; Khanum S; Bailie M; Shamsi B; Ahmed I; Shah STA; Javed S; Ghafoor A; Pervaiz A; Sohail F; Shah NA; Imdad K; Bostan N; Altermann ESalmonella enterica serovar Typhimurium is a foodborne pathogen causing occasional outbreaks of enteric infections in humans. Salmonella has one of the largest pools of temperate phages in its genome that possess evolutionary significance for pathogen. In this study, we characterized a novel temperate phage Salmonella phage BIS20 (BIS20) with unique tail fiber genes. It belongs to the subfamily Peduovirinae genus Eganvirus and infects Salmonella Typhimurium strain (SE-BS17; Acc. NO MZ503545) of poultry origin. Phage BIS20 was viable only at biological pH and temperature ranges (pH7 and 37 °C). Despite being temperate BIS20 significantly slowed down the growth of host strain for 24 h as compared to control (P < 0.009). Phage BIS20 features 29,477-base pair (bp) linear DNA genome with 53% GC content and encodes for 37 putative ORFs. These ORFs have mosaic arrangement as indicated by its ORF similarity to various phages and prophages in NCBI. Genome analysis indicates its similarity to Salmonella enterica serovar Senftenberg prophage (SEStP) sequence (Nucleotide similarity 87.7%) and Escherichia virus 186 (~ 82.4% nucleotide similarity). Capsid genes were conserved however those associated with tail fiber formation and assembly were unique to all members of genus Eganvirus. We found strong evidence of recombination hotspot in tail fiber gene. Our study identifies BIS20 as a new species of genus Eganvirus temperate phages as its maximum nucleotide similarity is 82.4% with any phage in NCBI. Our findings may contribute to understanding of origin of new temperate phages.Item Spread of Nontyphoidal Salmonella in the Beef Supply Chain in Northern Tanzania: Sensitivity in a Probabilistic Model Integrating Microbiological Data and Data from Stakeholder Interviews(Wiley Periodicals LLC on behalf of Society for Risk Analysis, 2022-05) Zadoks RN; Barker GC; Benschop J; Allan KJ; Chaters G; Cleaveland S; Crump JA; Davis MA; Mmbaga BT; Prinsen G; Thomas KM; Waldman L; French NPEast Africa is a hotspot for foodborne diseases, including infection by nontyphoidal Salmonella (NTS), a zoonotic pathogen that may originate from livestock. Urbanization and increased demand for animal protein drive intensification of livestock production and food processing, creating risks and opportunities for food safety. We built a probabilistic mathematical model, informed by prior beliefs and dedicated stakeholder interviews and microbiological research, to describe sources and prevalence of NTS along the beef supply chain in Moshi, Tanzania. The supply chain was conceptualized using a bow tie model, with terminal livestock markets as pinch point, and a forked pathway postmarket to compare traditional and emerging supply chains. NTS was detected in 36 (7.7%) of 467 samples throughout the supply chain. After combining prior belief and observational data, marginal estimates of true NTS prevalence were 4% in feces of cattle entering the beef supply and 20% in raw meat at butcheries. Based on our model and sensitivity analyses, true NTS prevalence was not significantly different between supply chains. Environmental contamination, associated with butchers and vendors, was estimated to be the most likely source of NTS in meat for human consumption. The model provides a framework for assessing the origin and propagation of NTS along meat supply chains. It can be used to inform decision making when economic factors cause changes in beef production and consumption, such as where to target interventions to reduce risks to consumers. Through sensitivity and value of information analyses, the model also helps to prioritize investment in additional research.Item The prevalence and public health implications of Salmonella brandenburg, an emerging pathogen of sheep in New Zealand : a thesis presented in partial fulfilment (50%) of the requirements for the degree of Master of Veterinary Science in Veterinary Public Health at Massey University, Palmerston North, New Zealand(Massey University, 2001) Motsamai, Obadiah Steven Obakeng MmatsebeSince the first case of Salmonella brandenburg abortion was recorded from a single sheep farm in the South Island of New Zealand in 1996, the outbreak had spread to more than 300 farms by the lambing season of 2000. This study, which was funded by Meat New Zealand and other stakeholders, is a pilot project to estimate the prevalence of Salmonella brandenburg and consider its potential foodborne and occupational health risk implications. Eight farms, four control farms and four affected farms were chosen from known infected areas. Control farms were those which had not experienced Salmonella brandenburg outbreaks, while the four affected properties had clinical outbreaks during the 2000 lambing season which had either been laboratory confirmed or not. At each farm faecal samples were collected from 50 lambs and 50 ewes at drafting and at slaughter. Therefore a total of 200 samples were taken from each group of animals sent for slaughter. The sampling was done in two phases to determine the effect of time interval on the prevalence of Salmonella brandenburg in sheep between abortion outbreaks and slaughter. The same sampling routine was followed for the November-December (Phase I) and February-March (Phase II) periods. The isolates from faecal cultures that had been confirmed as Salmonella spp. by slide agglutination test were sent to the Institute of Environmental Science and Research (ESR) for serotyping. All the 133 samples sent for serotyping turned out as S. brandenburg. The primary comparison of the study was done between the prevalence of Salmonella brandenburg in animals from affected farms and control farms. In addition the study was also interested in comparing S. brandenburg prevalence within class i.e. in lambs or ewes from the same farm at drafting and at slaughter. Comparison was also made between classes i.e. the prevalence of the organism in lambs and ewes from the same farm at drafting and at slaughter. During phase I of the on-farm sampling the prevalence of Salmonella brandenburg in tested lambs and ewes from affected farms was 12.0.% and 18.7% respectively. The on-farm prevalence for the control farms was 4.0% for lambs and 3.5% for ewes. During phase I of abattoir sampling the overall prevalence for the affected farms was 9.0 % for lambs and 22.0 % for ewes compared to 0.0% for lambs and 1.0% for ewes from control farms. The high prevalence of Salmonella brandenburg in animals from affected farms as compared to control farms showed that affected farms were associated with high excretion rates and therefore high levels of environmental contamination. During phase II of the on-farm sampling the prevalence of S. brandenburg in tested lambs and ewes from affected farms was 2.5% and 2.7% respectively. The phase II on-farm prevalence of S. brandenburg from control farms was 0.0% for lambs and 0.8% for ewes. During the same phase, abattoir prevalence of S. brandenburg in lambs and ewes from affected farms was 0.0% and 2.7% respectively compared to 0.5% for lambs and 0.0% for ewes from control farms. Like in phase I the overall prevalence of S. brandenburg was higher in animals from affected farms as compared to animals from control farms. The study also showed that the prevalence of the organism was very high during phase I compared to phase II irrespective of class of animal or site of sampling. This could have been due to the high number of animals still excreting the organisms closer to the outbreak period or the high level of environmental contamination. Both factors would have contributed to a higher prevalence of positive cultures. The higher prevalence of positive cultures during the November-December period as compared to the February-March period showed that the risk of infection and product contamination was greatest at commencement of the season (November-December) but was greatly reduced by February. Therefore further research is required to find the production and processing methods that might reduce the risk of infection and product contamination during the period of November-December. However the positive cultures of Salmonella brandeburg in control farms suggested a spreading disease outbreak and that the absence of clinical outbreaks of the disease did not mean an absence of infected animals on-farm. It is very important to do further investigations to find on-farm risk factors that might result in the absence or presence of clinical outbreaks. The study did not show any obvious differences in the S. brandenburg prevalence within class between on-farm and slaughter samples. There was also no obvious difference in the prevalence of the organism between ewes and lambs from the same farm, either during on-farm or abattoir sampling. Pulsed-field gel electrophoresis (PFGE) of the 24 isolates, which were a representative sample of the study, gave an identical profile. The PFGE and the serotyping suggested that the outbreak strain had become the dominant serotype in the sampled farms in the outbreak regions of the South Island of New Zealand. Therefore factors that gave rise to this dominance should be further investigated.Item Salmonella Brandenburg in sheep meat in New Zealand : preliminary studies to support a risk assessment approach : a thesis presented in partial fulfillment of the requirements for the degree of Masters of Veterinary Sciences in Veterinary Public Health at Massey University, Palmerston North, New Zealand(Massey University, 2002) Sabirovic, MirzetAbortion and death of ewes caused by a particular strain of Salmonella Brandenburg is an animal disease problem that is unique to the South Island of New Zealand. Like most Salmonella serovars, this organism is zoonotic and has caused cases in occupationally exposed people. As Salmonella are primarily recognised as agents of foodborne disease, the potential for foodborne transmission must be acknowledged, although human cases attributed to consumption of sheep meat have not yet been reported. Salmonella Brandenburg has an additional concern for New Zealand’s sheep meat industry owing to the possibility that contamination of sheep meat products could compromise market access. In 1995, the Sanitary Phytosanitary Agreement of the World Trade Organisation specified that scientific risk analysis was required before countries could refuse to import animal or plant materials on the basis of risks to animal, plant, or human health. This thesis presents initial microbiological studies of the prevalence and concentration of Salmonella Brandenburg on sheep meat carcasses that were conducted in conjunction with other projects designed to address the Salmonella Brandenburg issue using a modern risk assessment approach. The microbiological studies (Chapters 3 and 4) are preceded by two introductory discussions that provide the context for the project. Chapter 1 presents an overview of national and international regulatory approaches to food safety, foodborne diseases and protection of consumer health relevant to meat and meat products. A selective review of literature on Salmonella focuses on Salmonella in sheep and on aspects most relevant to food safety. Chapter 2 summarises information on published quantitative microbiological risk assessments (QRA) conducted using the guidelines developed by the Codex Alimentarius Commission to apply QRA to microbiological foodborne hazards. A conceptual framework is presented for developing a QRA for Salmonella Brandenburg in sheep meat that covers all sectors of the food supply chain from animal production to the point of consumption. Following the precedent of previous QRA efforts, the food supply chain is divided into a series of five modules: animal production; transport and lairage; slaughter and processing; retail and distribution; and consumer. For each module, key outputs (prevalence and concentration of Salmonella in animals or product at various points in the supply chain), and their likely determinants, are identified. The specific objective of the microbiological studies conducted was to estimate the prevalence and i concentration of Salmonella on sheep carcasses from animals originating from farms that had experienced Salmonella Brandenburg disease and other farms from the same region that had no history of this disease. Prior to undertaking the field studies, it was necessary to conduct some methodological studies to evaluate the effect of sample handling procedures on the results obtained with quantitative bacteriology. Chapter 3 presents three controlled laboratory experiments with swab samples taken from meat contaminated experimentally with the epidemic strain of Salmonella Brandenburg. The Most Probably Number (MPN) method was used to quantify counts of Salmonella Brandenburg per 100cm2 area of meat swabbed. In each experiment, control samples were processed immediately, and treatment samples were subjected to different periods and conditions of storage. Treatments were chosen to emulate anticipated conditions that would be required for the field studies due to logistic constraints. The three storage protocols evaluated were: Experiment 1: Storage of swabs diluted in buffered peptone water (BPW) for 48h at 40C Experiment 2: Storage of swabs diluted in BPW for 5 days at 40C Experiment 3: Storage of swabs for 24h at 40C before dilution in BPW, followed by storage for a further 48h at 40C. Differences in counts between control and treatment samples were not tested statistically, owing to the small samples sizes, but were numerically less than one log difference in all experiments. In 2 of the 3 experiments, counts for stored samples were in fact numerically greater than for samples processed immediately. These results suggested that carcass swabs contaminated with Salmonella could be stored under the specified conditions without affecting the results of quantitative bacteriology using the MPN method. Chapter 4 presents a study undertaken to obtain initial qualitative and quantitative estimates of the presence of Salmonella organisms on sheep carcasses sampled at 3 points in the processing chain (i.e. slaughter floor, cooler, and boning room). Slaughtered sheep (ewes and lambs) were sourced from six farms in the Central Otago/Southland region of the South Island where Salmonella Brandenburg disease is endemic. Three farms (case farms) were selected based on the occurrence of an outbreak of Salmonella Brandenburg ii disease during the spring of 2000. Three non-case farms from the same region were also sampled. As the disease epidemics are temporally clustered in July and August, well before lambs are sent for slaughter, sampling was replicated after an interval of approximately 2 months to assess likely temporal variation in risk of carcass contamination. For comparative purposes, samples from sheep carcasses were also collected from 6 groups of sheep slaughtered at 2 plants in the North Island where salmonellosis due to Salmonella Brandenburg infection in sheep has not been reported. A total of 1417 carcasses were sampled in the study and initially tested by BAX® test. Of these, 1214 samples were sourced from the 3 case and 3 non-case farms supplying the South Island plant. The remaining 203 carcasses were sampled at the 2 North Island plants. A total of 138 (11.3%) of the 1214 samples collected in the South Island plant tested positive for the presence of Salmonella Brandenburg. No positive findings were obtained from the samples collected in the North Island plants. The vast majority (130 or 94%) of the 138 positive samples was obtained in the first period of sampling, indicating a substantial decline in risk of carcass contamination in the period between the first and second sampling. These findings indicated that the prevalence of carcass contamination with Salmonella Brandenburg was markedly elevated in the region where sheep flocks experienced abortion outbreaks caused by the organism. Although clinical Salmonella Brandenburg enteric disease has not been reported in lambs, the first sampling revealed that overall prevalence of contamination was higher (33%) for lamb carcasses than ewe carcasses (10%) from the same farms. While the prevalence of lamb carcass contamination was comparable for both case and non-case farms, the prevalence of ewe carcass contamination was strongly clustered and only 2 samples were positive from non-case farms. Estimates of the prevalence of contamination were influenced by the location of sampling carcasses (e.g. slaughter floor, cooler), but estimates of bacterial numbers on positive carcasses were generally similar regardless of class of stock, time of sampling, or sampling location in the plant. No positive samples were obtained from swabs of primary cuts in the boning room. Collectively these findings suggest that the emergence of Salmonella Brandenburg infection of sheep in the South Island may have considerable implications for product safety and public health. A strong case can be made for more research to better characterise the potential risks and to explore potential risk mitigation strategies. While the data obtained in this study have provided valuable insights into several important aspects of the issue, due to logistic and other constraints they have considerable shortcomings with respect to the requirements of the formal QRA. These shortcomings were discussed and evaluated in terms of representativeness and suitability for quantitative risk assessment. Chapter 5 presents an extension of the conceptual framework for a QRA outlined in Chapter 2, by integrating the data obtained from the bacteriological study, as well as data from other sources. Major data gaps are identified and suggestions are presented with respect to options for ongoing research to advance understanding and management of Salmonella Brandenburg in New Zealand sheep meat. More extensive and representative surveys are required to obtain more reliable data on farm, and within-farm, prevalence of infection as well as more extensive and representative longitudinal studies of the prevalence and concentration of the organism during slaughter and processing. It is considered that more systematic surveys at the time of apparent highest risk would be a more reliable means of assessing potential exposure of consumers than predictive microbiology.Item Salmonella Brandenburg in New Zealand sheep : the development of a serological diagnostic test and a case control study : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies at Massey University(Massey University, 2003) Kerslake, Joanne IsabelSalmonella Brandenburg causes acute diarrhoea and severe illness in a variety of animals and was first isolated in New Zealand in 1986. Since 1996 Salmonella Brandenburg has been associated with an emerging epidemic of abortions and deaths in sheep in the southern regions of the South Island. Little is known about the specific epidemiology of Salmonella Brandenburg in sheep and as a result control to date has been largely based on anecdotal evidence and general principles. This study focused on the following aims: • To develop a serological test for use in epidemiological studies and for monitoring future control efforts targeting Salmonella Brandenburg in New Zealand sheep. • To identify factors associated with the occurrence and severity of Salmonella Brandenburg outbreaks in New Zealand sheep. Traditionally Salmonella diagnosis has depended on bacteriological culture. Such tests are time consuming, labour and equipment intensive, and may lack sensitivity. ELISA (Enzyme-Linked Immunosorbent Assay) methodologies offer an alternative for the diagnosis of Salmonella infection. Therefore the development of an ELISA test for detecting antibodies to Salmonella Brandenburg organism in sheep plasma was undertaken. Expression of common antigens has resulted in a high level of antibody cross-reactivity between different serovars in serological tests. Lipopolysaccharides (LPS) (O Antigens) are the primary cause of these cross-reactions. Cross-reactivity with two common sheep serovars (Salmonella Typhimurium and Salmonella Brandenburg) was of major concern for the development of a Salmonella Brandenburg ELISA. This was overcome by preparing an antigen mainly composed of flagella and fimbria proteins (LPS free). The antigen preparation was of a relatively crude and non-characterised nature and could only produce a reasonable optical density response at a high concentration. Unfortunately, while the ELISA was responsive, the specificity of the ELISA for Salmonella Brandenburg antibodies remained poor. Further investigation of the specificity of the antigen preparation, through the use of different sera, or through the development of a more pure and specific antigen, is needed for the successful development of a sensitive and specific serological test for determining Salmonella Brandenburg exposure in New Zealand sheep. A case control study was performed as part of a large-scale ongoing investigation aimed at identifying factors associated with Salmonella Brandenburg disease in New Zealand sheep. Details of disease prevalence and farm management methods were collected from two affected regions in southern New Zealand. Associations between possible risk factors and Salmonella Brandenburg were evaluated using odds ratios, with analyses being performed at two different levels: • farm level analysis to compare affected vs. unaffected farms using a case-control approach. • within farm analysis restricted only to affected farms to evaluate risk factors associated with severity of reported disease on affected farms. Data were collected from 405 farms containing a total of 1, 170,737 ewes. Of the 175 case farms, 97% had diseased mixed age (MA) ewes, 45% had diseased two-tooth (TT) ewes, and 5% had diseased hogget (H) ewes. Salmonella Brandenburg appeared to occur in better performing flocks, which are often associated with intensive farming methods. At the farm level, factors such as increased total number of ewes, feeding of hay, and controlled winter grazing appeared to increase the risk of disease. Farming methods such as controlled winter grazing may result in higher stress levels and increase the shedding of Salmonella Brandenburg organisms. This may create a higher risk of exposure in sheep yards and on pasture, resulting in a higher risk of disease. Feeding crop and having hilly terrain decreased the risk of a farm having disease. A protective effect of hilly terrain could be due to less intensive farm management, with a subsequent reduction in stress associated disease risk. Within affected farms, disease appeared to be more severe with the removal of rams after July, feeding of hay, and the practices of strop grazing. Shearing after July, increasing the total number of pre-lamb yardings, and vaccinating for Salmonella appeared to be protective. Therefore reducing stress and vaccinating ewes appear to reduce the risk of a Salmonella Brandenburg outbreak.Item Estimating the contribution of different sources to the burden of human campylobacteriosis and salmonellosis : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2009) Mullner, PetraThis thesis is concerned with the molecular epidemiology of Campylobacter jejuni and Salmonella in New Zealand and the development of source attribution tools for these pathogens. Although campylobacteriosis is the leading enteric zoonosis worldwide, the pathogen's complex epidemiology and di culties with existing typing schemes, have posed challenges for the control of this disease. The rst study of this thesis gives an overview of existing approaches to microbial risk assessment and source attribution, with particular respect to campylobacteriosis, and describes their advantages and shortcomings. Further, the chapter discusses phenoand genotyping techniques for Campylobacter spp. and the value of including microbial typing data in risk assessments. In the second study, data from a sentinel surveillance site in the Manawatu region was used to investigate the molecular epidemiology of human campylobacteriosis cases. This analysis revealed the presence of a dominant C. jejuni clone, namely sequence type (ST) 474, which accounted for 30.7 % of human cases in the study and identi ed risk factors for infection with ruminant and poultry associated STs. The third study investigated the link between C. jejuni in human cases and samples taken from poultry. By applying epidemiological and population genetic techniques this part of the thesis provided further evidence that poultry is a major contributor to human infection. In the fourth study an existing Bayesian source attribution model was modi ed and consecutively applied to New Zealand's major foodborne zoonoses: campylobacteriosis and salmonellosis. The majority (80 %) of human campylobacteriosis cases attributable to C. jejuni were estimated to have been acquired from poultry sources, whereas wildlife source were estimated to contribute only a minor proportion of cases. In the fth study the Salmonella dataset was descriptively analysed and a large proportion of human cases was found to be caused by `exotic' Salmonella types. In the nal study of this thesis four di erent genetic and epidemiological source attribution methodologies were applied to the same dataset in a comparative modelling framework. iv The studies in this thesis show that epidemiological studies combined with molecular tools and modeling can provide valuable risk-based tools to inform the surveillance and control of zoonotic pathogens. Methods from these studies may be readily applied to the control of other (food borne) zoonoses and provide new opportunities for epidemiological investigations and source attribution modelling of major pathogens.