Doctor of Philosophy / Food Science Institute -- Diagnostic Medicine/Pathobiology / David G. Renter / Salmonella and Escherichia coli O157 are important causes of foodborne illness in humans and have been associated with the consumption of undercooked, contaminated beef. Individual feedlot cattle may shed these organisms in their feces and subsequently contaminate cattle hides and carcasses at harvest. Preharvest and harvest interventions may significantly decrease the risk of beef contamination and subsequent risk of human illness. Previous research suggests that preharvest interventions for Salmonella or E. coli O157 may compliment harvest interventions and reduce the risk of carcass contamination. In my research, I used diverse study designs to develop a better understanding of the epidemiology of Salmonella and E. coli O157 and evaluate the impact of specific preharvest interventions in commercial feedlot cattle. A randomized controlled trial indicated that a commercially available vaccine did not affect the fecal prevalence of Salmonella, or health and performance of cohorts of feedlot cattle. However, the fecal prevalence of Salmonella varied by cohort, suggesting cattle source as a risk factor. In a repeated cross-sectional study, the fecal prevalence of Salmonella in cattle at feedlot arrival was not associated with the prevalence immediately prior to harvest, yet specific Salmonella subtypes, as defined by pulsed-field gel electrophoresis (PFGE), persisted throughout the feeding period. Another of my studies defined and compared PFGE subtypes of E. coli O157 isolated from cattle feces and carcass samples at harvest to determine relationships between fecal shedding and carcass contamination. Truckload appeared to be an important factor, and feces from cattle shedding both high- and low-concentrations of E. coli O157 posed a risk for carcass contamination. A stochastic Monte-Carlo modeling framework was later used to assess the impact of seasonal fecal prevalence and combinations of preharvest interventions on the risk of carcass contamination with E. coli O157. Results indicated that it may be important to incorporate multiple preharvest interventions, especially during periods of high fecal prevalence of E. coli O157. Overall, the research described in this dissertation demonstrates that multiple risk factors and interventions at the cohort level must be considered in order to mitigate the risks associated with Salmonella and E. coli O157 in beef production systems.

Master of Science / Department of Diagnostic Medicine/Pathobiology / T. G. Nagaraja / Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157 is a major foodborne pathogen that causes enteritis in humans ranging in severity from mild to bloody diarrhea to hemolytic uremic syndrome and even death. Cattle are asymptomatic carriers and fecal shedding of the organism is the major source of contamination of food and water for human infections. Distiller’s grains (DG) are ethanol fermentation co-products that are valuable feed ingredients for use in cattle diets. Previous research suggests an association between feeding DG and an increased fecal shedding of Escherichia coli O157:H7. The objectives of the research were to evaluate fecal E. coli O157:H7 prevalence and concentration in cattle fed diets with and without DG, determine if the association was dependent on inclusion level or form (wet or dried), evaluate the association in populations of cattle at harvest, and evaluate a potential intervention strategy. Our results indicated that cattle fed DG had a higher prevalence and shed a higher concentration of E. coli O157 than cattle fed diets without DG. The relationship was not dependent on the DG form, however, it was affected by the inclusion level of DG in the diet. Cattle that were fed 40% DG had a higher E. coli O157:H7 prevalence than cattle fed control or 20% DG diets and cattle fed 20% DG had a prevalence that was not statistically different from control cattle. The same response was observed in a subpopulation of cattle, termed super-shedders, which shed E. coli O157:H7 at higher concentrations than the general population. At harvest, we did not find differences in E. coli O157:H7 or super-shedder prevalence between cattle fed diets with or without DG, however, study design limitations affected the power of the study. Finally, previous work had shown that cattle fed dry-rolled grains had a decreased prevalence of E. coli O157:H7 when compared to cattle fed steam-flaked grains. We evaluated the effect of feeding DG and dry-rolled corn (DRC), alone or in combination, and observed no difference in E. coli O157 prevalence between cattle fed either DG or DRC diets. In conclusion, DG supplementation increased the prevalence and concentration of E. coli O157:H7 in cattle.

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Orientador: Manoel Victor Franco Lemos / Banca: Fernando Antonio de Ávila / Banca: Felipe Perecin / Resumo: Escherichia coli O157:H7 é uma cepa de importância crescente por estar associada a vários surtos graves de doença em humanos, a maioria derivada do consumo de carne bovina crua ou mal cozida. Os bovinos constituem seu reservatório mais importante, aventando-se a hipótese de que mudanças do regime alimentar em confinamentos atuariam favoravelmente ao aparecimento de cepas shigatoxigênicas. Neste estudo objetivou-se verificar, comparativamente durante o abate, a prevalência desse sorotipo e o comportamento de métodos indicadores como a contagem total de microrganismos viáveis (CTMV) e de contaminação fecal - coliformes totais e E. coli, em amostras de fezes e em carcaças de bovinos terminados a pasto e em confinamento, possibilitando a disponibilização de subsídios necessários aos programas de Análise de Perigos e Pontos Críticos de Controle (APPCC) e de Análise de Risco (RA), empregados na redução do risco de doenças transmitidas por alimentos. Identificados os lotes de acordo com a terminação (dez de cada tipo), desses foram aleatoriamente colhidas e analisadas 100 amostras de suabe retal, 100 amostras de carcaças e 67 amostras de "recortes" da desossa (carne industrial) utilizando-se, para a E. coli O157- H7, técnica automatizada de PCR. À exceção de uma única amostra de recortes (0,37%), as demais, tanto de fezes quanto de carcaças, foram negativas para a cepa pesquisada. Além de contatar-se uma prevalência muito baixa, não se evidenciou diferenças entre os tipos de terminação dos animais. Os resultados dos indicadores - CTMV, de coliformes totais e E. coli, foram considerados aceitáveis em 91%, 85% e 93% das amostras, respectivamente, oferecendo suporte e concordância com a baixa prevalência encontrada. / Abstract: Escherichia coli O157:H7 is an important strain that has been associated with outbreaks of serious disease in humans, most being derived from consumption of raw or poorly cooked beef. It is likely that cattle are an important reservoir, suggesting the possibility that changes in feedlot diet favor the emergence of shigatoxigenic strains of E. coli. This study is intended to verify, comparatively during bovine slaughter, the occurrence of E. coli O157:H7 associated with the sampling results obtained by means of general indicator methods (total viable count) and fecal contamination indicators (coliforms and E. coli). Samples will be taken from both excreta and carcasses of cattle finished either on pasture or feedlot, allowing the provision of subsidies necessary for Hazard Analysis and Critical Control Points (HACCP) and Risk Analysis (RA) programs and applied in the reduction of the risk of foodborne diseases. After identification of batches according to the type of finishing (feedlot or pasture), samples were randomly collected and analyzed. 100 rectal swabs, 100 samples from carcasses sponging, and 67 samples of "sliced meat" from the boning room (industrial meat). An automatic PCR technique for detection of E. coli O157:H7 was used. Except for one sample of sliced meat (0.37%), all others, both for excreta and carcasses, were negative for the O157:H7 E. coli strain. There were no significant differences in prevalence between the types of cattle finishing of the animals. The results of the indicators methods (TVC, coliforms and E. coli); were considered acceptable in 91%, 85% and 93% of tested samples, respectively, supporting and in agreement with low prevalence of O157:H7 found. / Mestre

Shiga producing E. coli (STEC) O157 is a zoonotic pathogen. In humans STEC O157 causes bloody diarrhoea and potentially fatal renal failure. Cattle are the major reservoir, where bacteria are limited to the intestinal tract and do not cause clinical signs of disease. Previous studies indicate that shiga toxins produced by STEC O157, suppress STEC-specific cellular immune responses in vivo. This study aimed to initially examine the humoral immune response in cattle following natural challenge and the effects of a toxoid vaccination on this humoral STEC specific-immune response. We determined a statistically significant suppression in Tir specific IgA in STEC O157 positive cattle compared to O157 negative cattle but not in super shedding cattle. Following toxoid vaccination we determined a significant increase in flagellin specific IgG1 antibody levels in toxoid vaccinated animals despite lower numbers of positive faecal samples compared to placebo vaccinated controls. These results suggest that shiga toxins produced by STEC O157 are actively suppressing the STEC specific immune response in natural colonisation. To clarify this suppression further calves were orally challenged with STEC O157 (either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain) and their STEC specific immune responses monitored. STEC specific systemic antibody responses were variable and weak in some cases. STEC specific local antibody responses were only significantly increased following challenge with the PT21/28 Stx2a+Stx2c+ challenge. Transcripts for genes associated with immune responses, and in particular B cell activation, at the terminal rectum were analysed by reverse transcriptase quantitative PCR. Suppression of IL2RA transcripts was observed in calves challenged with PT21/28 Stx2a+Stx2c+ compared to control calves but not with the other two STEC O157 strains tested. This study also aimed to determine the effects of cattle colonisation with STEC O157 on the immune response to a non-bacterial T-cell dependent antigen, ovalbumin (OVA). Cattle were orally challenged with either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain or unchallenged. Calves were subcutaneously immunised with OVA five days post challenge, on two separate occasions with a two week interval. Lymphocytes from lymph nodes local to the immunisation site demonstrated significantly increased OVA-specific proliferation and OVA-specific activation of CD4+ and CD8+ cells in calves that were challenged with the PT21/28 Stx2c+ strain (but not with the other two challenge strains), compared to unchallenged controls. These results indicate that colonisation with STEC O157 can alter local adaptive immune responses to non-bacterial antigens in a strain dependent manner, unexpectedly enhancing the immune response rather than suppressing it. Circulating T cell responses were unaffected. In conclusion this study provides some further evidence of adaption of the host immune response by STEC O157, which is strain dependent, and variable. It seems unlikely from the data in this study that STEC O157 colonisation is having a major impact on the responses of cattle to other vaccines or infections in the field.

Escherichia coli O157:H7 can contaminate dropped apples used for juicing via contact with manure or fecally tainted irrigation water and attach to the flesh of the apple through bruises and wounds where surface sanitizers are not effective. The goal of this project was to determine the efficacy of gamma irradiation at the maximum allowed dose of 1000 Gy to inactivate Escherichia coli O157: H7 in whole apples used for juicing. Whole apples were punctured to simulate wounds which were then inoculated with an outbreak strain of E.coli O157:H7 and subjected to gamma irradiation at doses upto 1000 Gy. The D-value of the E.coli O157:H7 strain was 334 Gy indicating that irradiation at 1000 Gy would result in a 3-log reduction of this pathogen. Contaminated apples were also stored for 3 weeks at refrigerated temperature during which time E.coli O157:H7 survived but did not grow. The inoculated apples were juiced, and the juice was stored up to 72 h. There was no change in counts of E.coli O157:H7 in the juice from the control apples, but irradiation at >600 Gy reduced counts by >3 logs, and survivors were not detected after 72 h storage. Sensory testing of juice treated at 652 Gy indicated consumers could tell the difference from control juice, due mostly to greater sweetness of the juice from irradiated apples. These results show that E.coli O157:H7 can easily survive in bruised apples and the juice made from them. Irradiation at 1000 Gy can provide significant lethality of E.coli O157:H7 in apples and juice conferring a greater level of safety without negative effects on sensory quality.

Le travail de thèse a eu pour but de contribuer à une meilleure connaissance de la dangerosité pour l'homme des souches STEC d'origine bovine, en explorant la corrélation pouvant exister entre la présence du gène stx chez de telles souches et la réalité de son expression. La quantification de l'expression du gène stx2 présent chez 46 souches STEC bovines a été réalisée à l'aide d'un test ELISA commercial détectant spécifiquement les shiga toxines, le test ProSpecT® Shiga toxin (OXOID). L'ensemble des résultats de validation préalable obtenus pour ce test a permis de considérer qu'il pouvait être valablement appliqué à l'étude du panel de souches d'E. coli O157 :H7 bovines collectées au laboratoire, tout en déterminant les limites méthodologiques, et donc d'interprétation. Utilisé comme outil de quantification de la production de Stx2 par les souches du panel choisi, et dans deux conditions expérimentales différentes (présence ou absence d'induction par l'enrofloxacine), ce test a permis de mettre en évidence que seulement 15,2% des souches d'E.coli O157:H7/H- étudiés produisent des quantités significatives de Stx2 détectables sans induction, et ce à des niveaux variables. En revanche, la majorité de ces isolats, bien que n'exprimant pas la protéine Stx2 de manière constitutive, produit des quantités significatives de Stx2 en présence de concentrations subinhibitrices d'enrofloxacine, antibiotique de la famille des fluoroquinolones et utilisé en médecine vétérinaire. Enfin, des mutants résistants à l'enrofloxacine sélectionnés à partir de certaines souches d'E. coli O157:H7, produisent, après induction par l'enrofloxacine, 3 fois plus de toxine Stx2 que les souches sauvages. Les mutants sont également inductibles en utilisant des doses d'enrofloxacine 100 fois supérieures à celles utilisables pour les souches sauvages. L'ensemble de ces résultats montre (i) la corrélation, ou non, qui peut exister entre la présence du gène stx2 et son expression, (ii) que la proportion inductible des souches STEC bovines est potentiellement importante, (iii) que l'enrofloxacine induit fortement l'expression du gène stx2 chez les souches STEC bovines et que (iv) l'induction par l'enrofloxacine conduit à des taux d'expression du gène stx2 supérieurs chez des souches résistantes aux fluoroquinolones que chez les souches sensibles. Au final, cette étude contribue à documenter la variabilité des niveaux d'expression des gènes stx et illustre le risque que des STEC issus de bovins puissent devenir plus fréquemment pathogènes pour l'homme suite à l'usage croissant des fluoroquinolones vétérinaires.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (Feb. 23, 2007). Includes bibliographical references.