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 / Food Science Institute / Michael E. Dikeman / The objective was to determine the effects of needle-free injection (NF) compared with traditional needle injection (N) on microbial translocation of generic E. coli in beef strip loins. Longissimus muscles (LM) (n=5) from USDA Select carcasses were used in preliminary research to determine the optimal injection pressure required for NF injections. Seven treatments with sterile colored saline solution were administered: 1) 90 psi ; 2) 55 psi ; 3) 50 psi ; 4) 45 psi ; 5) 30 psi ; 6) 25 psi ; or 7) 20 psi . For the second portion of the experiment 15 LM were obtained and halved; the surfaces were inoculated with generic E. coli at a level of 106 CFU/cm2 (three replications of five loins). Matching halves were allocated to NF or N injection treatments with a phosphate, salt solution. Immediately after injection, two cores, 23 cm2 in area, were taken aseptically from each half. A 2-mm thick cross-sectional slice was removed from the inoculated surface of the core and labeled “surface”. Using sterile technique, the two cores from each half were sliced into cross-sectional strips at depths of 1, 3, and 5 cm. Corresponding depth measurements were combined in stomacher bags with 99 ml of peptone water and stomached. Serial dilutions were then plated. From the preliminary study, it was determined that 25 psi was the optimal pressure for NF injection based on dispersion, visual appraisal, and solution retention. Samples taken from the surface of N injected LM had lower (P < 0.05) microbial counts than NF-injected muscles (2.79 versus 3.23 log CFU/g, respectively). The 3 and 5 cm depth samples from N injection had the least (P < 0.05) microbial contamination (1.69 and 2.12 log CFU/g) compared to NF injections. Samples from 1 cm deep of N injected LM had lower (P < 0.05) (2.53 log CFU/g) microbial counts than the 1 cm samples of NF injected LM (3.04 log CFU/g). Traditional N injection resulted in approximately 0.5 log CFU/g less microbial contamination at all depths. N injection posed fewer microbial risks when compared with NF injection using these defined application settings.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Ludek Zurek / Feed samples and live stored-product insects from feed mills and swine farms were collected and cultured for Enterococcus spp. The mean concentration of enterococci in insect and feed were 2.7 ± 0.5 × 101 cfu/insect and 6.3 ± 0.7 × 103 cfu/g respectively. A total of 362 isolates of enterococci collected from 89 feed samples and 228 stored-product insects were identified to the species level using PCR. These isolates were represented by Enterococcus casseliflavus (53.0%), E. gallinarum (20.4%), E. faecium (16.2%), E. hirae (5.2%), and E. faecalis (5.0%). Enterococci were phenotypically resistant to tetracycline (48.0%), erythromycin (14.3%), streptomycin (16.8%), kanamycin (12.1%), ciprofloxacin (11.0%), ampicillin (3.3%), and chloramphenicol (1.1%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM (50.0%), tetO (15.1%), tetK (0.5%), tetS (0.2%) and other unknown tetracycline determinants. Enterococci carried virulence genes including gelatinase (gelE; 21.5%), an enterococcus surface protein (esp; 1.9%), and cytolysin (cylA; 2.2%). An aggregation substance (asa1) gene was detected in 61.0% of E. faecalis isolates. Fifty perncet of E. faecalis isolates were phenotipically tested positive for aggregation substances. Enterococci with cylA genes were hemolytic (52.0%) and with gelE genes were gelatinolytic (18.5%). The ermB gene, encoding erythromycin resistance was detected in 8.8% of the total isolates. The Tn916/1545 family of conjugative transposons was detected in 10.7% of the isolates. Laboratory experiments showed that adults of the red flour beetle, Tribolium castaneum (Herbst), fed on poultry and cattle feeds inoculated with E. faecalis OG1RF:pCF10, were able to successfully acquire enterococci and contaminate sterile poultry and cattle feeds. To assess the potential of horizontal gene transfer, conjugation assays were carried out with E. faecalis using a donor (wild strains) and recipient (E. faecalis OG1SSP) in ratio of 1:10. Only one isolate (1 out of 18 E. faecalis) could transfer tetM to a recipient using broth mating. However, filter mating assay, followed by PCR confirmation revealed that 89.0% (16 out of 18 E. faecalis) of isolates could transfer tetM to E. faecalis. Transfer ratios of transconjugant per recipients ranged from 2.6 × 10-4 to 1 × 10-9. In summary, feed (52.0%) and stored-product insects (41.6%) collected from feed mills and swine farms carried antibiotic-resistant and potentially virulent enterococci. Our study showed that T. castaneum, a pest commonly associated with feed, served as a potential vector for enterococci in the feed environment. Conjugation assays followed by PCR confirmed presence of the tetM gene on a mobile genetic element(s) such as Tn916 and may be horizontally transferred to other Enterococcus species and to other bacteria of clinical significance.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Bovine viral diarrhea virus (BVDV) has emerged as one of the most important infectious diseases in cattle. One particular important manifestation, after successfully establishing an in utero infection of the fetus during the first trimester, is the development of a persistently-infected BVDV (PI-BVDV) calf. Persistently infected BVDV animals are a continuous source of virus and can shed the virus in virtually all secretions and excretions, including nasal discharges, saliva, semen, urine, tears, milk, and, to a lesser extent, feces. The objectives of this research were to determine: 1) the effects of short term exposure (13 – 18 days on feed (DOF)) to PI-BVDV feeder cattle; 2) the outcome of testing and removing PI-BVDV feeder calves at time of feedlot arrival on health, performance, and carcass characteristics; 3) the survival of BVDV on materials associated with livestock production; and 4) characterization of testing and longitudinal prevalences for PI-BVDV beef cattle. Testing and removing PI-BVDV calves at 13 to 18 DOF was too late to remove a morbidity effect due to PI-BVDV exposure. However, mortality, performance, and carcass characteristics were not different in cattle exposed to PI-BVDV cattle. Additionally, there were no harmful outcomes when newly arrived feeder cattle were exposed to a PI-BVDV animal for one to two days following feedlot entry. A non-cytopathic, Type 1b, BVDV was capable of surviving after application to various materials used in livestock production. BVDV tended to survive longer on non-porous materials than porous materials. When in the presence of mucus, BVDV was protected from degradation for longer periods of time than when not in the presence of mucus. There was no difference in overall PI-BVDV prevalence within cattle sampled in 2006 and 2007. Cattle that weighed less than 300 lbs. had a greater likelihood of being PI-positive than cattle with increased weights. Several months of the year had a greater likelihood of having PI-positive animals. Based on operation, cow-calf and stocker operations had a greater likelihood of having PI-positive animals than did feedlot operations.

Master of Science / Department of Biology / Peter P. Wong / Abstract Water purification methods that remove pathogens and harmful or distasting molecules make water potable. Recently, silver loaded ion-exchange resins have demonstrated a strong role in removing microbes. The goal is to make an effective silver ion-based water purifier that is portable, environmentally stable, and cost efficient. The project was conducted as a collaborative effort with Safewater A/S, an up and coming entrepreneurial business located in Denmark that is interested in developing novel water purifiers for developing nations, adventurers and military personnel. Purolite, a prominent business in ion-exchange resins located in Whales, designed and provided Safewater A/S and our research team with experimental resins for water purification, which will be discussed in the body of this thesis. The data reveals critical issues that may render this tool unavailable for commercial production in some countries due to the mode of action for killing the bacteria and the amount of silver leaching. Tests were conducted using Escherichia coli K12 and Enterococcus faecalis OG1SSp as model fecal organisms using different silver ion-exchange resins. Surveillance of leached silver ions, pH changes, and total dissolved solids (TDS) were also monitored to find correlations with capacity (liters of purified water produced) and effectiveness of microbicidal action. Overall, one resin was found to contain properties consistent with the stated objectives; however its use in some countries as a water purifier for human consumption will be nullified due to extensive silver leaching. Although this resin could be used in the United States of America since it passes the Environmental Protection Agency (EPA) standards, Safewater A/S is interested in further developing it for countries with stricter regulatory constraints before mass production. The goal of the present thesis report is to address the stated objectives in the development of a water purifier.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / George C. Stewart / Fusobacterium necrophorum, a gram negative anaerobe and an opportunistic pathogen, causes necrotic infections in humans and animals. Two subspecies of F. necrophorum, subsp. necrophorum and subsp. funduliforme are described. Leukotoxin (Lkt), a secreted protein encoded by a tricistronic operon (lktBAC), is the major virulence factor of F. necrophorum. The concentration of Lkt produced by subsp. necrophorum is higher than that of subsp. funduliforme. Quantitative-PCR was used to determine the relative expression of lktA by the two subspecies of bovine origin. The mRNA transcript of lktA was detectable in early-log phase of growth in subsp. necrophorum, whereas in subsp. funduliforme, the lktA transcript was detected only in the mid-log phase. Q-PCR analysis revealed that subsp. necrophorum had 20-fold more lktA transcript than subsp. funduliforme. The amount of lktA transcript declined by late-log phase in both subspecies; but lktA mRNA levels in subsp. necrophorum was 8-fold higher than in subsp. funduliforme. Leukotoxin protein stability assays showed the Lkt to be stable in both subspecies despite the decrease in the concentration of the protein during late-log phase. The subspecies identity of human F. necrophorum strains and whether they possess lktA and leukotoxin activity are not known. Human clinical isolates (n = 4) of F. necrophorum were identified as subsp. funduliforme based on 16S rRNA sequence and absence of hemagglutinin gene. Four human strains had the lkt promoter, lktB, and lktC similar to that of subsp. funduliforme. One strain had full length lktA, while other three strains exhibited considerable heterogeneity. All four strains secreted Lkt that was toxic to human leukocytes. Fusobacterium equinum, formerly F. necrophorum, is a newly recognized species. It is associated with infections of the respiratory tract in horses. Little is known about the virulence factors of F. equinum. Southern hybridization revealed that F. equinum strains had lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The toxicity of culture supernatants of isolates to equine leukocytes was variable. Our data indicate that F. equinum isolates possess lktA gene and exhibit leukotoxin activity. The importance of leukotoxin as a virulence factor in human and equine fusobacterial infections needs to be investigated.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, an emerging tick-borne pathogen, causes human monocytic ehrlichiosis (HME). The relationship between E. chaffeensis and its target cells in ticks and vertebrates is critical as the organism must persist in them. We hypothesize that E. chaffeensis alters gene expression in support of adapting to dual hosts. In support of testing this hypothesis, we developed an ORF-based microarray and performed global transcriptional analysis on the pathogen grown in macrophage and tick cells. The analysis revealed the expression of about 30% of all the predicted E. chaffeensis genes, in macrophages or tick cell. Two-thirds of the transcribed genes are common for both host cell backgrounds. About 20% of the commonly expressed genes also varied in expression levels which ranged from two to five fold. Microarray data was verified by RT-PCR for a subset of randomly selected genes. Together, this is the first report describing the global host cell-specific gene expression patterns in E. chaffeensis. Differential gene expression may be an important adaptive mechanism used by E. chaffeensis for its continued survival in dual hosts. To test this hypothesis, we established many basic protocols and tools needed for performing mutational analysis in E. chaffeensis. Four antibiotic selection markers; gentamicin, chloramphenicol, spectinomycin and rifampin, and two promoters constitutively expressed in E. chaffeensis, genes rpsL and tr, were identified. Two regions of the genome were also identified for performing initial mutational analysis. Several plasmid constructs were also made. The optimal conditions for introducing these plasmids into host cell-free viable E. chaffeensis organisms were also established. The molecular evaluation of several E. chaffeensis transformants using these plasmids suggested that the plasmids gained entry, but failed to get integrated into the genome or remain in the bacteria for longer periods of time. In summary, we demonstrated global host cell-specific differential gene expression in E. chaffeensis by employing microarray analysis. Numerous host-specific expressed genes will be important for studies leading to effective methods of control. We also established several basic protocols and tools needed for performing mutational analysis useful in evaluating the impact of the loss of expression of uniquely expressed genes.

Master of Science / Food Science Institute, Animal Science and Industry / Daniel Y.C. Fung / Within the United States, it has been estimated that 60 deaths and 73,000 illnesses are caused by Escherichia coli O157:H7 infection annually (Gavin et al., 2004). Multiple effects have been known to occur with the onset of infection from E. coli O157:H7 in which some of these can become life-threatening. Escherichia coli O157:H7 is defined as a Shiga-toxin-producing E. coli strain (STEC). This microbial pathogen is a gram-negative bacillus organism that is motile, non-sorbitol fermenting, and β-glucuronidase negative. The infectious dose of E. coli O157:H7 can be as low as ten cells (Food and Drug Administration, 2009). Consumption of contaminated food, mainly undercooked ground beef and non or incorrectly pasteurized milk, are the primary sources of E. coli O157:H7 infection in human. Cattle, in particular, are considered chief asymptomatic reservoirs for this pathogen. Carried in their gut, feces, and milk, cattle carry this Shiga toxin-producing E. coli in ranges from 10[superscript]2 to 10[superscript]5 CFU/g. Although colonized with E. coli O157:H7, cattle and other ruminants show no adverse side effects from the pathogenic bacteria. There is also a difference in the prevalence of this pathogen between human and cattle. There has been a low incidence of illness caused by E. coli O157:H7 in humans when compared to the high prevalence of E. coli 057:H7 found in cattle and their environment. It has been discovered, through population genetic analysis, that E. coli O157:H7 and other O157:H- isolates make up a clone complex. In spite of the clonal nature of E. coli O157:H7 and other O157:H[superscript]- isolates, there are significant characteristics showing variability between the clone complex. These variability aspects can possibly account for the rapid divergence of E. coli strains including the recently discovered divergence of E. coli O157:H7 in to two separate lineages. Other possible reasons for a non-linear relationship between cattle prevalence and human infection include diversity of the Shiga Toxin-Encoding bacteriophage and receptors in cattle verses human, and finally the difference between the production of Locus of Enterocyte Effacement (LEE) in both human and cattle lineages.

Doctor of Philosophy / Food Science Program / Daniel Y.C. Fung / Curtis L. Kastner / This dissertation was designed to evaluate antimicrobial, quality, and shelf life effects of controlled phase carbon dioxide (CPCO2) on beef trimmings destined for ground beef. Critical parameters included pressure, temperature, exposure times, modified atmosphere conditions, and days of simulated retail display. 1500 psi CPCO2 for 15 min achieved 0.83, 0.96, 1.00, and 1.06 log reductions for Total Plate Count (TPC), Generic E. coli (GEC), E. coli O157:H7 (O157), and Salmonella spp. (SS), respectively. Bacterial reductions in ground beef and beef trimmings were similar (P≥0.05). CIE L*, a*, and b* values in raw patties showed no differences (P≥0.05) immediately after CPCO2 application on beef trimmings. Nevertheless, significant (P<0.05) interactions were found in pressure by packaging for L*, in pressure by packaging by days of simulated retail display for a*, and in packaging by days of simulated retail display for b* scores. Nevertheless, after 5 days of simulated retail display, L*, a*, and reflectance (630/580nm) ratios were similar for all treatments (P≥0.05), and b* scores were most acceptable with 1500 CPCO2 (P≥0.05), regardless of the packaging conditions. After 5 days of display, cooked patties showed similar (P≥0.05) values for crude protein (%CP) and crude fat (%CF), the extent of lipid oxidation (TBARS), was higher (P0.05) in aerobic trays than flushed packages with 100% CO2. Ground beef patties manufactured from beef trimmings treated with CPCO2scored higher values for tenderness (P0.05) than other treatments. In addition, no differences (P0.05) for juiciness, beef flavor intensity, or off flavor intensity were found between non-treated and the 1500 psi CPCO2 treated patties. Microbial control of spoilage organisms and foodborne pathogens in ground beef patties with CPCO2 application in beef trimmings was effective (0.6 to 1.2 logs). Lethality levels are comparable to other intervention strategies. Discoloration of beef trimmings after CPCO2 application may not be a concern for grinding purposes. Further packaging with 100% CO2 is viable for controlling spoilage and pathogenic microorganisms after packaging and during refrigerated storage, although discoloration of raw ground beef patties packaged with 100% CO2 may be a concern for product marketing.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Sanjeevkumar Narayanan / Antimicrobials are included in finishing cattle diets for growth promotion, feed efficiency, and protection against liver abscesses. The inclusion of in-feed antimicrobials at or below therapeutic concentrations may provide a selective pressure for antimicrobial resistant microorganisms. Additionally, heavy metals such as copper and zinc may be included in cattle diets because of growth-promoting effects. Heavy metal resistance genes are on transferable plasmids that also contain antimicrobial resistance genes. The objectives of this research were to 1) determine the prevalence of food-borne pathogens, Salmonella and E. coli O157, in cattle fed diets with or without monensin and tylosin and 0 or 25% wet corn distiller's grains (WDGS), 2) determine the prevalence of food-borne pathogens in cattle fed elevated concentrations of copper and zinc 3) evaluate the effect of antimicrobials on antimicrobial susceptibility of food-borne pathogens and commensal fecal bacteria, and 4) determine a possible association between in-feed antimicrobials and the concentration of antimicrobial resistance genes in the feces of cattle. Inclusion of 25% WDGS was associated with a higher prevalence of E. coli O157 on one of two sample collection days; however, there was no association between the use of monensin and tylosin, or copper and zinc on the prevalence of food-borne pathogens. Including monensin and tylosin in cattle diets was associated with an increased resistance of enterococci to macrolides, but was not related to concentration of the common macrolide resistance gene, ermB. In cattle fed diets with copper and/or zinc, no differences were observed in antimicrobial susceptibility or the concentration of antimicrobial resistance genes. In conclusion, results indicate that including growth-promoting antimicrobials in cattle diets at below therapeutic concentrations only limitedly impacted antimicrobial susceptibility and concentration of fecal antimicrobial resistance genes; however, this research encompassed only a select number of microorganisms. The positive association between WDGS and E. coli O157 prevalence in cattle has important implications for food safety, and warrants further investigation.