Doctor of Philosophy / Department of Food Science / Daniel Y.C. Fung / In the food industry, coliform testing is traditionally done by the time consuming and labor intensive plate count method or tube enumeration methods. The TEMPO® system (bioMérieux, Inc.) was developed to improve laboratory efficiency and to replace traditional methods. It uses a miniaturization of the Most Probable Number (MPN) method with 16 tubes with 3 dilutions in one single disposable card. It utilizes two stations: the TEMPO® Preparation station and the TEMPO® Reading station. In this study, the Oxyase® (Oxyase®, Inc.) enzyme was added to TEMPO® CC (Coliforms Count), TEMPO® AC (aerobic colony count) and TEMPO® EC (E. coli Count) methods. Water samples of 1 ml with 0.1 ml of Oxyase® enzyme were compared to samples without the Oxyase® enzyme using the TEMPO® system. Samples were spiked with different levels of coliforms (10, 102, 103 and 104 CFU/ml), stomached (20 sec), and pipetted into the three different TEMPO® media reagents (4 ml) in duplicate and then automatically transferred into the corresponding TEMPO® cards by the TEMPO® preparation station. Counts were obtained using the TEMPO® reading station after 8, 12, 16, 22 and 24 hours at an incubation temperature of 35°C. Results from 20 replicates were compared statistically. Using TEMPO® tests, high counts in food samples (>6 log 10 CFU/ml) can be read in 6±2 hours of incubation using the time-to-detection calibration curve. The TEMPO® system reduces reading time (reading protocol should be changed). There is no need to wait for 22 hours of incubation only 12 hours is required. Oxyrase® enzyme is not needed for the TEMPO® system.

Doctor of Philosophy / Department of Plant Pathology / Richard B. Todd / Fungi can be found living in a range of environments, including soil and the ocean, and as pathogens of plants and animals. The ability of fungi to adapt to diverse and changing environments is dependent on their ability to sense and respond to an array of signals, including the presence or absence of nitrogen nutrients. Fungi can utilize a diverse array of nitrogen nutrients and do so in a regulated and preferential manner. When preferred nitrogen nutrients such as ammonium and glutamine are present (nitrogen sufficiency), genes required for the utilization of alternative nitrogen sources are not expressed. In the absence of a preferred nitrogen source (nitrogen limitation) the genes for utilization of alternative nitrogen sources are transcriptionally derepressed and can be induced by the presence of a particular nitrogen nutrient, such as nitrate or proline. In the absence of any nitrogen nutrient (nitrogen starvation) the expression of some genes is further elevated. In filamentous fungi the expression of genes required for the utilization of nitrogen nutrients is coordinated by the orthologs of the conserved Aspergillus nidulans GATA transcription factor AreA, which activates transcription of nitrogen utilization genes. AreA activity is controlled by autogenous transcriptional activation, mRNA transcript stability, regulated nucleo-cytoplasmic distribution, and interactions with NmrA, AreB and TamA. The combined effect of these regulatory mechanisms generally results in AreA being inactive during nitrogen sufficiency and active during nitrogen limitation and nitrogen starvation. However, during nitrogen sufficiency AreA remains active at the promoters of some genes, including gdhA, which encodes the key nitrogen assimilation enzyme NADP-dependent glutamate dehydrogenase. In this work we have used both classical genetics and next generation sequencing approaches to examine regulated gene expression and how AreA activity is modulated, primarily during nitrogen sufficiency. We have studied regulation of gdhA to characterize how AreA evades nitrogen metabolite repression. We identify leucine biosynthesis as being a key regulatory signal involved in gdhA expression and characterize the genes required for leucine biosynthesis. We also show that TamA regulates the gdhA promoter by direct DNA binding, which requires interaction with AreA. We have also characterized repression of AreA to identify a potential mode of NmrA corepressor action. Finally, we have characterized the AreA nuclear export signal and explored mechanisms that control regulated nuclear export of AreA.

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.

Master of Science / Food Science / Kelly J.K. Getty / James L. Marsden / Escherichia coli O157:H7 outbreaks have been linked to leafy green produce and bagged spinach. The objective of this study was to evaluate a Photohydroionization (PHI) panel (novel advanced oxidation technology) and varying concentrations of lactic acid washes for controlling E. coli O157:H7 on baby spinach. Leaves were dip inoculated with a five-strain cocktail of E. coli O157:H7 inoculum having a concentration between 5-6 log CFU/ml. Leaves were submerged in inoculum for 30 s and dried for 1 h. Non-inoculated and inoculated leaves were washed for 30 s in food grade lactic acid diluted to concentrations of 0.5, 1.0, or 2.0% and allowed to dry for 10 min. For PHI treatment, leaves were placed under the PHI panel and treated for 1, 2, or 5 min on both sides for total treatment times of 2, 4 or 10 min. Following treatments, leaves were either sealed in low-density polyethylene bags or enumerated. Samples were enumerated at 0, 3, 7, 10, and 14 days following inoculation. Ten gram samples were diluted with sterile peptone and stomached for one min, and then 0.1 ml was plated onto sorbitol MacConkey agar with cefixime and tellurite plates that were incubated at 37°C for 24 h. For lactic acid treatments, E. coli O157:H7 populations were different (P < 0.05) compared to the control. There was no difference (P > 0.05) due to sampling time so sampling times where pooled together for each lactic acid concentration of 0.5, 1.0, and 2.0% and resulted in 2.01, 2.78, and 3.67 log CFU/g reductions, respectively. Leaves treated with 1.0% and 2.0% lactic acid had color degradation and were organoleptically unacceptable by day 14. When leaves were treated with PHI for 1, 2, or 5 min per side, E. coli O157:H7 populations were reduced 1.6, 1.49, or 1.95 log CFU/g, respectively. Leaves treated with PHI were not different from one another, but were different (P < 0.05) from the positive control. No color change occurred in leaves treated with PHI. The PHI panel and lactic acid washes of 0.5% or higher are effective in reducing E. coli O157:H7 in baby spinach.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Harvey Morgan Scott / The use of antibiotics in food animals is of major concern as a purported cause of antimicrobial resistance (AMR) in human pathogens; as a result, alternatives to in-feed antibiotics such as heavy metals have been proposed. The effect of copper and CTC supplementation in weaned pigs on AMR in the gut microbiota was evaluated. Four treatment groups: control, copper, chlortetracycline (CTC), and copper plus CTC were randomly allocated to 32 pens with five pigs per pen. Fecal samples (n = 576) were collected weekly from three pigs per pen over six weeks and two Escherichia coli isolates per sample were tested phenotypically for antimicrobial and copper susceptibilities and genotypically for the presence of tetracycline (tet), copper (pcoD) and ceftiofur bla[subscript]C[subscript]M[subscript]Y₋₂) resistance genes. CTC-supplementation significantly increased tetracycline resistance and susceptibility to copper when compared with the control group. Copper supplementation decreased resistance to most of the antibiotics, including cephalosporins, over all treatment periods. However, copper supplementation did not affect minimum inhibitory concentrations of copper or detection of pcoD. While tetA and bla[subscript]C[subscript]M[subscript]Y₋₂ genes were associated with a higher multi-drug resistance (MDR), tetB and pcoD were associated with lower MDR. Supplementations of CTC or copper alone were associated with increased tetB prevalence; however, their combination was paradoxically associated with reduced prevalence. These studies indicate that E. coli isolates from the weaned pigs studied exhibit high levels of antibiotic resistance with diverse multi-resistant phenotypic profiles. In a related study, total fecal community DNA (n = 569) was used to detect 14 tet genes and to quantify gene copies of tetA, tetB, pcoD and bla[subscript]C[subscript]M[subscript]Y₋₂. CTC and copper plus CTC supplementation increased both the prevalence and gene copies of tetA, while decreasing both the prevalence and gene copies of tetB, when compared with the control group. The diversity of tet genes were reduced over time in the gut bacterial community. The roles of copper supplementation in pig production and pco-mediated copper resistance in E. coli need to be further explored since a strong negative association of pcoD, with both tetA and bla[subscript]C[subscript]M[subscript]Y₋₂, suggests there exist opportunities to select for a more innocuous resistance profile.

Master of Science / Department of Biomedical Science / H. Morgan Scott / The modern phenomenon of increasing prevalence of antibiotic resistance in clinically relevant bacteria threatens humanity’s ability to use antibiotics to treat infection in both humans and animals. Despite the marked complexity of bacterial evolution, there is tremendous importance in unfolding the process by which antibiotic resistance genes emerge, disperse, and persist in the natural world. This thesis investigates certain aspects of this process in two experimental studies that differ primarily by scale but also by methodology. The first study examined the long-term annual prevalence of ceftiofur and tetracycline resistance in Canadian beef cattle from 2002 to 2011 at both phenotypic and genotypic levels. Ceftiofur was present at a very low prevalence (<4%) that did not statistically increase over the decade (p<0.05). Relative proportions of tetracycline genes tet(A), tet(B), and tet(C) also did not significantly change over the observation period. However, it was surprising that almost 20% of isolates recovered from nonselective agar harbored tet(C) given that current literature generally indicates that tet(C) is significantly less prevalent than tet(A) or tet(B). The usage of historical samples in addition to parallel selective plating using agar supplemented with antibiotics provided insight into systemic bias present in common microbial approaches. Long-term sample freezing significantly diminished the recoverability of E. coli over time. Additionally the usage of selective MacConkey agar containing tetracycline biased the proportions of tetracycline genes to over-represent the tet(B) gene in commensal E. coli compared to nonselective MacConkey agar. The second study attempted to explain the short-term selection effects of antibiotic treatment on the overall ecological fitness of commensal E. coli using bacterial growth parameters estimated from spectrophotometric growth curves as a simple surrogate of general fitness. Treating cattle with either tetracycline or ceftiofur was found to not only select in favor of tetracycline resistant bacteria, but also increased the overall fitness among the tetracycline resistant population. However, growth curves were unable able to explain why transiently selected resistant bacteria were eventually replaced by susceptible bacteria once the selection pressure was removed.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ari Jumpponen / Traditional fallow periods in the Bolivian highlands are being shortened in an effort to increase short-term crop yields, with potential long-term impacts on soil communities. Using 454-pyrosequencing, we characterized fungal and bacterial community responses to (1) the length of fallow period and (2) the presence of the plants Parasthrephia sp. or Baccharis sp. (both locally known as ‘thola’). Thola is widely considered by farmers as beneficial to soil health, although it is also frequently harvested as a source of fuel by farmers. Soils in one study area, Ancoraimes, had higher levels of organic matter, nitrogen and other macronutrients compared to the other study area, Umala. In our analyses, Ancoraimes soils supported more diverse fungal communities, whereas Umala had more diverse bacterial communities. Unexpectedly, the longer fallow periods were associated with lower fungal diversity in Umala and lower bacterial diversity in Ancoraimes. Fungi assigned to genera Verticillium, Didymella, and Alternaria, and bacteria assigned to genera Paenibacillus, Segetibacter, and Bacillariophyta decreased in abundance with longer fallow period. The presence of thola did not significantly affect overall soil fungal or bacterial diversity, but did increase the frequency of some genera such as Fusarium and Bradyrhizobium. Our results suggest that fallow period has a range of effects on microbial communities, and that the removal of thola from the fields impacts the dynamics of the soil microbial communities.