Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / John C. Reese / Keeping in view the important roles of bacteria in almost every aspect of insect’s life, the current study is the first systemic and intensive work on microbes associated with Hessian fly, a serious pest of wheat crop. A whole body analysis of Hessian fly larvae, pupae, or adults suggested that a remarkable diversity of bacteria is associated with different stages of the insect life cycle. The overriding detection of genera Acinetobacter and Enterobacter throughout the life cycle of Hessian fly suggested a stable and intimate relationship with the insect host. Adult Hessian flies have the most dissimilar bacterial composition from other stages with Bacillus as the most dominant genus. Analysis of 5778 high quality sequence reads obtained from larval gut estimated 187, 142, and 262 operational taxonomic units at 3% distance level from the 1st, 2nd, and 3rd instar respectively. Pseudomonas was the most dominant genus found in the gut of all three instars. The 3rd instar larval gut had the most diverse bacterial composition including genera Stenotrophomonas, Pantoea, Enterobacter, Ensifer, and Achromobacter. The transovarial transmission of major bacterial groups provided evidence of their intimate relationship with the Hessian fly. The Hessian fly is known to manipulate wheat plants to its own advantage. This study demonstrated that the combination of a decrease in carbon compounds and an increase in nitrogen compounds in the feeding tissues of Hessian fly-infested plants results in a C/N ratio of 17:1, nearly 2.5 times less than the C/N ratio (42:1) observed in control plants. We propose that bacteria associated with Hessian fly perform nitrogen fixation in the infested wheat, which was responsible for shifting the C/N ratio. The following findings made in the current study i.e. the presence of bacteria encoding nitrogenase (nifH) genes both in Hessian fly and infested wheat, exclusive expression of nifH in infested wheat, presence of diverse bacteria (including the nitrogen fixing genera) in the Hessian fly larvae, presence of similar bacterial microbiota in Hessian fly larvae and at the feeding site tissues in the infested wheat, and reduction in survival of Hessian fly larvae due to loss of bacteria are consistent with this hypothesis. The reduction in Hessian fly longevity after the loss of Alphaproteobacteria in first instar larvae, highest proportion of Alphaproteobacteria in insects surviving after the antibiotic treatments and the nitrogen fixation ability of associated Alphaproteobacteria strongly implies that Alphaproteobacteria are critical for the survival of Hessian fly larvae. This study provides a foundation for future studies to elucidate the role of associated microbes on Hessian fly virulence and biology. A better understanding of Hessian fly-microbe interactions may lead to new strategies to control this pest.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Copper, as copper sulfate, is often supplemented at elevated concentrations in swine diets, particularly in piglets, to promote growth. Growth promotional effects of copper are believed to be similar to that of antibiotics in that gut microbial flora is altered to reduce loss of nutrients and suppress pathogens. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and E. faecalis, resistance is conferred by a plasmid-borne transferable copper resistance (tcrB) gene. The plasmid also carries macrolide [erm(B)] and glycopeptide (vanA) antibiotics resistance genes. The objectives of the research were to 1) determine the prevalence of tcrB gene in fecal enterococci of piglets in relation to normal (16.5 ppm) and elevated level (125 ppm) of copper supplementation, 2) determine the relationship of tcrB gene and susceptibilities to copper, erythromycin, and vancomycin, and 3) determine the transferability of tcrB gene in enterococci by conjugation. Weaned piglets, housed in pens, fed normal (16.5 ppm; control) or elevated level of copper (125 ppm) were used. Fecal samples were collected weekly for isolation of enterococci. Isolates were speciated by multiplex PCR and sodA gene sequence analysis. The prevalence of tcrB-positive enterococcal isolates was higher (P < 0.05) in the copper supplemented group than the control group. The prevalence of tcrB was affected by sampling days (P < 0.05) with a significant treatment and sampling time interaction (P < 0.05). The tcrB positive isolates were either E. faecium or E. faecalis, and majority of isolates was E. faecium. The mean MIC of copper for tcrB-positive isolates (21.1 mM) was higher (P < 0.001) compared to tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin, tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains suggests the possibility that copper supplementation may exert selection pressure for antibiotic resistance. The positive association between copper supplementation and prevalence of tcrB gene has important implications for antimicrobial resistance and food safety, which warrants further investigation.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen that causes infection in people and several vertebrate animals. One of the striking features of E. chaffeensis infection is the prolonged persistence in its vertebrate and tick hosts. The mechanism of persistent infection and the reasons for the host immune system failure to clear the infection are not well understood. One hypothesis is that differential gene expression serves as an important adaptive mechanism used by E. chaffeensis in support of its continued survival in both tick and vertebrate hosts. One way to test this hypothesis is by performing mutational analysis. However, the methods for introducing mutations in this pathogen have not yet been documented and are challenging, possibly due to its obligate, intraphagosomal growth requirement. Recently, a novel gene mutation method called ‘TargeTron Gene Knockout System’ that is based on the modified group II intron insertion strategy has been developed. This method appears to be effective in creating mutations in a wide range of gram positive and gram negative bacterial organisms. The group II intron can be programmed for insertion into virtually any desired DNA target with possibly high frequency and specificity. In this study, I focus on creating mutations in E. chaffeensis using the TargeTron gene knockout system. I prepared modified group II intron constructs retargeting for insertion into three E. chaffeensis genes: Ech_0126 (a transcriptionally silent gene), macrophage-specific expressed gene (p28-Omp 19, Ech_1143) and tick cell-specific expressed gene (p28-Omp 14, Ech_1136). In support of driving the expression of the modified group II introns in E. chaffeensis, the pathogen- specific high-expressing gene promoter (tuf) was inserted upstream to the transcription start site. In addition, a chloramphenicol acetyltransferase gene with E. chaffeensis rpsl promoter was introduced for use as a selection marker. The constructs were then evaluated by transforming into E. chaffeensis. Transformants with mutations, introduced in two of the three genes (Ech_0126 and Ech_1143), were identified by PCR and Southern blot methods. Although the mutants are detectable for up to 48 hours, establishment of stable transformants remains to be challenging. The outcomes of this project will have important implications in defining the pathogenesis of E. chaffeensis, particularly to assess the differences in the organism in tick and vertebrate hosts.

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.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Acute diarrhea induced by Escherichia coli is an important illness in humans, especially in children under age of two in developing countries. Citrobacter rodentium is used as murine model for E. coli infection in humans because it causes ultrastructural changes in murine colonic epithelium comparable to lesions produced by enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Adult mice of many strains develop self-limiting epithelial hyperplasia when infected, whereas adult C3H and FVB mice are highly susceptible to infection and demonstrate mortality rates between 60 and 100% two weeks after infection. These susceptible strains of mice also have higher bacterial translocation to mesenteric lymph nodes. In mice, the cause of death could be hypovolemia due to dehydration that may occur due to an increase in paracellular permeability as well as dysregulation of apical and basolateral ion transporting proteins. C. rodentium virulence factors resemble those of E. coli and are believed to primarily alter tight junctions of colonic epithelial cells. Effectors delivered via the type III secretory system have been associated with actin condensation and pedestal formation. The exact mechanisms of C. rodentium infection, as well as changes that occur in vitro as well as in the intestine of various strains of mice are not completely understood. This study introduced a new in vitro Ptk6 cell line for C. rodentium infection, which can also serve as a model for EPEC in humans. Effect of C. rodentium on colonic epithelial cells of susceptible and resistant mice was determined in in vivo study. C. rodentium attached to Ptk6 colonic epithelial cells, inducing attaching and effacing (A/E) lesions and loss of monolayer integrity, which charachterizes this cell line as a relevant in vitro model of C. rodentium and EPEC infections. Murine studies revealed that C. rodentium induced more severe disease and 100% mortality in juvenile C3H mice whereas Swiss Webster (SW) mice expressed only moderate morbidity. The colonic lesions and changes in barrier function of colonic epithelium were more prominent in C3H mice. This study determined potential targets in the murine colon that play role the establishment and the outcome of the infection, indicating multifactorial nature of C. rodentium-induced diarrhea. This study identified host factors involved in the initiation of C. rodentium-associated diarrhea and the outcome of infection, which can be useful in developing of novel strategies for preventing and treatment of infectious colitis.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.

Master of Science / Department of Human Nutrition / Tonatiuh Melgarejo / It will be almost impossible to find a household or an informed consumer, completely oblivious to the health risks posed by foods. According to scientific estimates, 48 million cases of foodborne illness occur each year in the United States, causing 128,000 hospitalizations, 3,000 deaths, which costs $6.4-$77.7 billion in expenses by medical care and lost productivity. These illnesses pose a very big constant, perhaps growing, threat to a vast population. Salmonella is the most prevalent foodborne bacteria with more than 1.1 million cases annually in the United States. Increased international trade and distribution, rapid growth in antibiotic resistant bacteria, increase in the number of immunocompromised consumers and changes in agronomic and processing practices poses a very big challenge to monitor, contain and avoid foodborne outbreaks. This can result in contaminated food causing rapid, geographically widespread outbreak. In the wake of numerous recent foodborne illness outbreaks, this report focuses on current safety practices established by research and available to all the consumers. It studies a significant foodborne bacterium (Salmonella), its nature, significance, prevalence, mechanism of action, health risks and preventive safety measures. General food safety practices, to reduce or eliminate the risk, common to all the foodborne bacteria and specially Salmonella, include avoiding cross-contamination, thoroughly cooking foods to right lengths of time and temperature, washing fresh produce and fruits prior to consumption and storing foods at the right temperatures. Strict regulations in safe production, safe processing and consumer awareness is highly recommended. People participation is a must.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Cefovecin is an extended-spectrum long-acting third generation cephalosporin used to treat canine infections. The study objective was to determine the effect of cefovecin on the absolute number and antimicrobial susceptibility of fecal enteric bacteria in healthy dogs. Fourteen Beagles were randomly assigned to a treated (n = 7, 8 mg/kg cefovecin subcutaneously on day 1) or untreated (n = 7) group. LC/MS was used to determine plasma cefovecin concentration on day 14. E. coli, enterococci, and Salmonella were isolated and enumerated from fecal samples collected on days 0, 3, 7, 14, and 28. Antimicrobial resistance was determined using disc diffusion, MIC, and detected using PCR for the bla[subscript CMY-2] gene on select isolates. Mean plasma concentration of cefovecin on day 14 was 9.59 µg/mL in treated dogs; untreated dogs had no measurable plasma cefovecin. The absolute number of E. coli was lower in treated dogs on day 3 (P ≤ 0.0001), and the absolute number of cefovecin-resistant E. coli was higher in treated dogs on days 7 (P = 0.002), 14 (P = 0.004) and 28 (P ≤ 0.0001), compared to untreated dogs. Enterococci increased and were higher in the treatment group on day 7 (P = 0.0226). Isolation of Salmonella was rare. After cefovecin treatment, beta-lactam resistance was more common in fecal E. coli from treated dogs than untreated dogs, while resistance of enterococci was not altered. On day 28, treated dogs were 3.25 times more likely to carry the bla[subscript CMY-2] gene than untreated dogs (95% CI 1.27 – 8.35). The implications of these findings in clinically ill patients require further research.

Master of Science / Division of Biology / Revathi Govind / Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Gary A. Anderson / Brucellosis is one of the major endemic zoonotic diseases worldwide, and it has history dating back to 1937 in Iraq when it was first isolated by an Iraqi physician. In order to establish a solution for the continuous devastating impacts of the disease in humans and livestock, the Brucellosis Control Program was established in 1995. The main responsibilities of this program were setting and implementing the appropriate strategies for controlling the disease. After the war in 2003, the United Nation organization for Food and Agriculture (FAO) developed a strategic plan to control the disease. The main goal of the project was to improve productivity in the livestock sector and reduce the prevalence of disease in small ruminants (sheep and goats) to less than 2%, and less than 0.2% in cattle and buffalo. Achieving such goals ultimately would reduce the disease incidence among the human population from more than 27.2 cases/100,000 persons in 2002, to less than 4 cases/100,000 people within 15 years. A serological surveillance was conducted and revealed the apparent prevalence of the disease in sheep and goats, cattle, buffalo, and camels was 6.51%, 1%, 1.48%, and 0.02%, respectively in Iraqi governorates except the three northern governorates of Kurdistan province . Based on surveillance results, a vaccination policy was the only appropriate strategy that could be chosen to control the disease. Four vaccination campaigns were implemented in 2006, 2007, 2008, and 2009, with a total number of vaccinated animals each year at 10099972, 4698482, 753153, and 1833482 head, respectively. The primary satisfactory outcome of the program was the apparent decline in livestock abortions leading to obvious increases in productivity. Regarding the incidence of brucellosis among the human population, the apparent decline in the middle and south of Iraq began with the vaccination phase of the control program in 2006. The results represented a significant decrease in human cases after only four vaccination campaigns of a program that was intended to continue for 15 years.