Master of Science / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Escherichia coli O157:H7 is a human food-borne pathogen and cattle feces are a major source of contamination. Immunization against E. coli O157 may be a practical pre-harvest intervention strategy. A siderophore receptor/porin proteins (SRP) based vaccine has been developed to decrease the prevalence of E. coli O157 in cattle. Two studies were conducted to determine the efficacy of the SRP vaccine. In the first study, thirty calves were randomly assigned to one of two groups: control or SRP vaccine. Two weeks after the second vaccination, calves were orally inoculated with nalidixic acid-resistant (Nal[superscriptR]) E. coli O157. Fecal samples were collected for five weeks. Calves were necropsied on day 35 to collect gut contents and tissue swabs to determine Nal[superscriptR] E. coli O157:H7. The number of calves that were culture positive for E. coli O157 were lower (P= 0.07) in vaccinated group compared to the control. In the second study, cattle in two feedlots were randomized to SRP vaccine or control. Cattle were vaccinated on days 0 and 21. Rectal fecal samples were collected on day 0, and pen floor samples were collected on days 21, 35, and 70. Rectal fecal samples, RAMS, and hide swab samples were collected on d 85. Cattle were weighed on days 0, 21, and 85. Vaccination significantly reduced (P = 0.04) fecal E. coli O157 prevalence. There was also a decrease (P < 0.05) in E. coli O157 prevalence on hides and in fecal samples on day 85 in vaccinated cattle compared to the control.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157:H7 is a food-borne pathogen that causes hemorrhagic enteritis in humans. Cattle are asymptomatic carriers and their feces are the major source of infection. The objective of the first study was to determine the prevalence of E. coli O157:H7 in the gut of cattle at slaughter. Gut contents (rumen, cecum, colon and rectum) were collected from slaughtered cattle (n=815) at a packing plant and prevalence of E. coli O157:H7 was determined. The overall prevalence of E. coli O157:H7 in cattle was 20.6%. The prevalence (%) in the rumen, cecum, colon, and rectum was 4.9, 9.1, 7.7, and 10.3, respectively. Prevalence in rectal content was positively associated (P < 0.01) with that of the rumen or colon and not of the cecum. Pulsed-field gel electrophoresis typing showed that the majority of isolates obtained within the same animal shared a clonal similarity. There was no significant difference in the acid tolerance of ruminal compared to hindgut isolates. It was concluded that hindgut was the major site of prevalence of E. coli O157:H7 in cattle at slaughter. Neomycin, an aminoglycoside, is approved as a feed additive and for use in water to cattle. The objective of the second study was to determine the efficacy of feeding neomycin on fecal shedding of E. coli O157:H7 in cattle. Cattle were randomly assigned to control (n=14) or neomycin (n=10) supplemented group and orally inoculated with nalidixic acid-resistant (NalR) E. coli O157:H7. Neomycin was fed at 10 mg/0.45 Kg body weight for 15 days. Fecal samples and rectoanal mucosal swab (RAMS) samples were collected day before (d -1), on days 1, 3, 5, 10, 13, 17, 20, 24, 27, 31, 34, 38, 41, 44, and 48, and then approximately weekly through day 111. Fecal shedding of NalR E. coli O157:H7 was quantified and prevalence in RAMS was determined. Neomycin significantly reduced prevalence and concentration of E. coli O157:H7 compared to the control. Following two weeks of neomycin feeding, concentration and prevalence were similar between the two groups. Short term neomycin feeding before slaughter may reduce the E. coli O157:H7 load in cattle.

Rapid detection of foodborne pathogen is one of the most urgent problems in the world, because foodborne pathogen could cause serious illness, such as nausea, vomiting and diarrhea. We have developed a sensitive microfluidic system based on dendrimers and aptamers for rapid detection of Escherichia coli O157:H7 at very low cells concentration. Dendrimers, with high level of functional groups and homogeneous spherical shape, are prefect nanoscale polymers used as a template material by increasing sensitivity and specificity of analytes detection in microfluidics. In this work, we develop a sensitive microfluidic system based on dendrimers and aptamers for detecting Escherichia coli O157:H7 at very low cell concentrations. Carboxyl functionalized G7-polyamidoamine (PAMAM-COOH) dendrimers are immobilized on (3-aminopropyl)-trimethoxysilane (APTMS) pretreated microfluidic channels. The aptamers are subsequently conjugated on the immobilized dendrimes through chemicals. The sensitivity and specificity are validated by injecting fluorescein isothiocyanate (FITC) labelled Escherichia coli O157:H7 at various cells concentration into the resulting microchannels, indicating that the detectable cells concentration can be reached as low as 100 (cells/ml) and the detection time is 10 hours. To further exploit and improve the work efficiency our microfluidic device, the microfluidic channel is designed into a staggered herringbone microchannel (SHM) to create the chaotic dynamics inside the microfluidic device, and the SHM is then simulated by a COMSOL software showing that the staggered herringbone structures can improve chaotic dynamics of designed microchannel and will enhance the probability of particles to attach on the surface of microdevice. All the results show that our approach has the potential to develop the field of rapid and accurate detection on foodborne pathogens.

The house fly, Musca domestica can transmit human pathogens including Escherichia coli O157:H7 through regurgitation of ingested bacteria from the crop which is a foregut organ of house fly and stores the excess ingested nutrients. Interactions between the ingested bacteria and the crop have a direct influence on bacteria persistence, survival and ultimately fly vector competence. In this research, in situ crop vessel assay was developed to investigate bacterial growth within fly crops up to 48 hours post-ingestion. Flies were fasted for 12 h prior to feeding E. coli O157:H7 pEGFP and then fed bacteria with red food color which was added to confirm that flies had consumed the bacteria. After feeding, flies with red abdomens were aseptically dissected and crops were removed and maintained in sterile phosphate buffered saline in microtiter plates held at 32˚C. For each time point (0, 24 and 48 hours post-ingestion), five crops were homogenized individually using a tissue grinder and bacterial levels (CFU/crop) were monitored using plate counts. Confocal microscopy of intact crops was used to monitor biofilm development. There was no statistical difference in cell numbers (CFU/crop) over the 48 h incubation period. Microscopy showed that upon prolonged incubation, GFP-expressing E. coli within the crop produced biofilms. This method showed greater reproducibility in studying crop bacteria level than using a live fly feeding study. But this system was not recommended to study the interaction between bacteria and the crop of housefly.

Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease in the developed countries. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein, intimin, is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and thought to function as a receptor for intimin. In this thesis, we characterized A/E lesion formation by in vivo and in vitro-grown EHEC, aimed at testing whether bacterial adaptation to the mammalian host included up regulation of A/E lesion formation. Our results showed that actin signaling by EHEC was induced upon bacterial growth in vivo, and this induction was likely due to the up regulation of multiple activities by in vivo-grown EHEC. We also focused on the interaction between intimin and the host cell, an interaction that triggers actin condensation of A/E lesion formation. We evaluated the role of β1 integrins, one of the proposed receptors of intimin, in A/E lesion formation, and demonstrated that β1 integrins are not essential for intimin-mediated cell binding and actin condensation. To better understand intimin function, we mapped the functional domains of intimin, showed that the minimal cell binding domain of intimin correlates with the minimal Tir-binding domain. This minimal Tir-binding domain, when purified and coated on latex beads, was sufficient to trigger actin condensation on preinfected mammalian cells, suggesting that Tir-binding by intimin is critical in the final step of A/E lesion formation. To further demonstrate the significance of the interaction between intimin and Tir in A/E lesion formation, we developed a yeast two-hybrid system to identify intimin mutants diminished in Tir-binding, and then characterized those mutants for the ability to trigger actin condensation, the final step of A/E lesion formation. Finally, as a first step to study the downstream actin signaling pathway after Tir-binding, we mapped the domain of Tir involved in intimin-binding, and showed that the N-terminus and C-terminus of Tir are likely to be localized in the host cell cytoplasm, available to interact with downstream effectors in actin signaling.

Understanding the composition of camel milk coupled with studying the effects of thermal and non-thermal treatments on its components and bacterial inactivation were the general objectives of this dissertation. In the first study (Chapter 2), the gross composition of camel milk including milk protein, fat, casein, total solids, lactose, ash, and mineral content were analyzed. In addition, fatty acid profile, amino acid profile, protein fractions, and volatile compounds were evaluated as well. Our results revealed that camel milk has its unique nutrients profile. These findings make it easier for the researchers and consumers to understand some of the nutritional attributes of camel milk.The impact of non-thermal ultrasound treatment (900 W, 20 kHz, 100% power level) on some milk-borne microorganisms and the components of camel milk was studied in Chapter3. We reported that continuous ultrasound processing was efficient in inactivating Escherichia coli (E.coli) O157: H7 and Salmonella Typhimurium (S. Typhimurium) in camel milk without detrimental effects on milk fatty acids profile, lipid peroxides, and protein fractions except for some changes in milk volatile compounds (VC). In Chapter 4, another non-thermal technique, ultraviolet-C (UV-C) light, was applied to camel milk to study the effects of different UV-C light doses on the viability of E. coli O157:H7 and S. Typhimurium and the chemical changes to milk components. The main findings of this study were: (i) UV-C treatment at a dose of 12.45 mJ/cm2 resulted in only 3.9-log10 for both bacterial strains which did not meet the Food and Drug Administration (FDA) requirements for the 5-log pathogen reduction; (ii) the UV-C treatment at the above dose, had limited effects on camel milk components. Thermal pasteurization of milk was first introduced to prevent milk-borne infectious diseases, however, its effects on camel milk components and quality are still unknown. Therefore, in Chapter 5, we investigated the efficacy of three previously reported thermal methods: PAST-1 (65ºC/30 min), PAST-2 (72ºC/5 min), and PAST-3 (80ºC/5 min) on bacterial inactivation and some camel milk components such as the fatty acid profile, lipid peroxidation, VC, and milk protein fractions. Complete elimination (6 log10 CFU/ml reduction) of E. coli O157: H7 was achieved using all pasteurization methods, however, only 3.4 log10 CFU/ml reduction of the total viable counts was reported using PAST-1 and PAST-3 methods. We also reported that the PAST-1 and PAST-3 methods did not affect the chemical composition of camel milk. In conclusion, we assessed the main components of camel milk along with the amino fatty acid profile, acid profile, volatile compounds, and protein fractions. Thermal methods were more effective than the non-thermal methods in terms of microbial inactivation and most camel milk components were not significantly influenced by thermal and non-thermal methods.

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Bacteriocins are antimicrobial protein produced by certain Gram positive and negative bacteria as a defense mechanism against closely related bacteria competing for the same nutrient or in the same niche. The competition for the same nutrient is supported by the fact that bacteriocins have narrow range of effect and only likely to be effective against closely related bacteria for the same scares resources hence a bacteriocin produced by a Gram positive bacteria will be active against a Gram positive pathogens and a bacteriocin produced by a Gram negative bacteria will be active against Gram negative pathogens. This is due to the difference in cell wall composition, they are either bacteriocidal or bacteriostatic Bacteriocins have been used for thousands of years for food preservation unknowingly to man, they are considered advantageous not only to the producing bacteria, but it's now been used by the food industry as a tool to control both spoilage and pathogenic bacteria in food, in a natural manner which is acceptable to the consumer. With a lot of research been carried out on bacteriocins produced by Gram positive bacteria, antagonist to Gram positive food borne pathogens, little is known about bacteriocins produced by Gram negative bacteria which would be active against Gram negative food borne pathogens that predominate in produce. The objective of my research therefore is to screen for antimicrobial antagonist to Gram negative food borne pathogens (Escherichia coli O157:H7 and Salmonella Enteritidis) from produce, to determine an appropriate screening method, to carry out a preliminary characterization of antagonist discovered and also to determine antimicrobial spectrum of antagonist found. Lettuce was screened for antimicrobial antagonist against Gram negative pathogen (Escherichia coli O157:H7 and Salmonella Enteritidis) which were used as indicator strains With over 5000 colonies screen, 1 colony (Serratia plymuthica) was discovered to be antagonistic against these indicator strain. Further screening of cell free extract using the spot test method showed that extract from Serratia plymuthica grown alone in TSBYE showed antagonist activity against indicator strain with a little clearing on the spot of extract dropped. But extract of a co-culture of Serratia plymuthica and either Escherichia coli O157:H7 or Salmonella Enteritidis showed a more obvious clearing around spotted zone, which further indicates antagonism against indicator strains. Preliminary heat test indicates antagonist compound to be heat stable at 60oC for 30mins, 100oC for 30minutes and 60mins and 121oC for 20minites, and antagonist compound possessed antagonist activity against other strains of Escherichia coli when tested.

Verotoxin-producing Escherichia coli (VTEC) of serotype O157:H7 is a pathogen causing illness in humans worldwide. The path and nature of transmission from and among cattle is important knowledge when it comes to preventing cases of disease in humans. Two concepts potentially playing an important role in transmission of VTEC O157:H7 are super-shedding and super-spreading. Super-shedders are individuals (here calves) shedding a high amount of bacteria. Super-spreaders are individuals (here calves) spreading the disease in a higher extent compared to the rest of the population investigated. Little is known about these phenomenons’ effect on transmission as well as the relation between them. Therefore, it is important to investigate this further. The purpose of this master thesis was to get a better understanding of how super-spreaders can be identified. One way to identify super-spreaders and explore the transmission of a pathogen is to investigate molecular data using computational methods. Here, a literature study with a systematic approach was conducted in order to scan the literature for such methods. In this first phase of the master thesis three methods, all constructing transmission trees, were identified as relevant methods for the second phase. These methods are called outbreaker2, phybreak and TransPhylo. In the second phase of the master thesis, 32 whole genome sequences of VTEC O157:H7 collected from four different cattle farms were investigated using the methods outbreaker2 and phybreak. Both methods were able to identify samples infecting more secondary cases compared to the rest of the investigated population. Some of these samples came from the environment, possibly shedding light on the importance of the pathogen's ability to survive outside of the host, and therefore playing an important role in transmission of the disease. The rest of the samples infecting more secondary cases were from calves, and a minority of these were super-shedders. From this the importance of the relation between super-shedders and super-spreaders can neither be confirmed nor denied. Outbreaker2 suggested that the spread of the pathogen is frequently occurring between the four neighbouring farms, while phybreak instead suggested that the spread mostly occurs within the farms. From this, a scenario explaining that the transmission possibly occurs within farms is presented.