Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Richard D. Oberst / Viruses differ in their susceptibility and resistance to disinfectants and their ability to be inactivated based predominantly on structural and size differences. The virucidal methodology followed in this study was consistent with the U.S. Environmental Protection Agency guidelines for such efficacy testing. Various disinfectant chemistries were effective in rapid and complete inactivation of bovine coronavirus (BCV) including 25, 50, and 100% DF-200d, 10% bleach, 1% Virkon® S, and 70% ethanol following 10 min treatments. Efficacy was slightly reduced in the presence of organic challenge material (feces, soil). Treatment with 100% DF-200d or 10% bleach resulted in the degradation of BCV RNA. Treatment with 50 or 100% DF-200d, 10% bleach, or 1% Virkon® S resulted in the degradation of BCV proteins as detected by western blot analysis. Various disinfectants were effective in the complete inactivation of both mammalian and avian influenza test strains and included DF-200d, 10% bleach, 1% Virkon® S, and 70% ethanol following 10 min exposure but was reduced in the presence of organic challenge. Treatment with DF-200d or 10% bleach resulted in significant degradation of influenza RNA. Effective treatments against foot-and-mouth disease virus (FMDV) and bovine enterovirus-2 (BEV-2) included 10% bleach, 4% sodium carbonate, 2% sodium hydroxide, and Sandia DF-200. Treatment with 5% acetic acid was effective against FMDV, but not BEV-2. Treatment with 70% ethanol was not effective for inactivating FMDV or BEV. Additionally, 10 min treatment with 10% bleach, 2% sodium hydroxide, and Sandia DF-200 degraded FMDV and BEV-2 RNA. FMDV inoculated surfaces were exposed to 5% acetic acid, 10% bleach, 70% ethanol, 4% sodium carbonate, 2% sodium hydroxide, Sandia DF-200, 0.4% Oxy-Sept® 333, or 1% Virkon® S. All treatments except 70% ethanol were effective following 10 and 20 min contact on rubber and stainless steel. No treatments were shown to be effective for concrete, due to a low recovery of virus from this surface. Additionally, 10 and 20 min treatments with 10% bleach, 2% sodium hydroxide, and Sandia DF-200 degraded FMDV RNA on the surfaces evaluated.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / George C. Stewart / Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than the vegetative bacterial cells, are the source of anthrax infections. The spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis ΔSterne that carries a deletion of the bclB gene. Immunofluorescence studies indicated that the mutant strain produced spores with increased amounts of the BclA glycoprotein expressed on their surface. Differences in exosporium composition between the mutant and wild-type spores were identified. The mutant was also found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. The resistance properties of the mutant spores were unchanged from that of the wild-type spores. The bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of a rigid and complete exosporium structure in B. anthracis. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

Master of Science / Department of Biology / Sherry D. Fleming / Helicobacter is a common intestinal pathogen of most laboratory mice from both commercial and academic sources worldwide. Not previously thought to have an effect, recent evidence indicates Helicobacter infection alters cytokine, chemokine, and gene expression in the stomach, intestine, and colon. Though the in vivo cell types responsible for these changes are currently unknown, in vitro results suggest macrophages are the likely source. In addition to detection and elimination of pathogens, intestinal macrophages play a role in maintaining homeostasis. By altering gene expression and cytokine production in the microenvironment, we hypothesized that Helicobacter infection altered the phenotype and inflammatory response of submucosal intestinal macrophages. To test this hypothesis, we examined macrophages within whole mounts of intestinal muscle as well as isolated macrophages from Helicobacter-infected or uninfected mouse intestine. Macrophages from the intestinal muscle of Helicobacter-infected mice showed increased expression of F4/80 and CD11b, altered gene expression, and increased phagocytosis when compared to macrophages from uninfected mice. Infection also altered the macrophage response to stimuli. Macrophages from infected mice produced significantly lower concentrations of cytokines, chemokines, and PGE[subscript]2 in response to stimulation with either IFN and LPS or IL-4 and IC. These data support our hypothesis demonstrating that the intestinal muscle macrophage phenotype, function, and response to stimulation are altered by Helicobacter infection both in vivo and in vitro.

Master of Science / Food Science Institute - Animal Science & Industry / Daniel Y.C. Fung / Proper microbiological examination of foods involves proper sample preparation in terms of mixing the solid or liquid food with a suitable sterile diluent (usually a 1:10 dilution) in a sterile bag and homogenizing them manually or by means of an instrument. This thesis addresses the effectiveness of Stomacher®, Pulsifier®, Bagmixer®, and Smasher® instruments in terms of: 1) Number of viable cell counts/g of ten food types, 2) Noise level of the four instruments ascertained by a) human and b) decibel meter at five feet (1.52 meters) from each instrument, 3) Ease in cleaning the instruments after use, and 4) Ergonomics. Following the ISO Method (7218:2007), 25 g each of alfalfa sprouts, spinach, peanuts, ground beef, fish meat, hot dogs, tofu, milk, chicken wing meat, and chicken drum stick meat were placed individually in a sterile sample bag containing 224.5 mL of 0.1% Peptone water plus 0.5 mL of E. coli inoculum diluent. Each food was homogenized for 60 seconds in each of the instruments. During each treatment four laboratory workers standing at five feet (1.52 meters) from the instrument assessed the noise level as: very quiet, quiet, nearly quiet, acceptable noise, and loud. Also the noise level was monitored instrumentally by the use of a decibel meter and recorded as Db. Ease of cleaning and ergonomics were determined with the aid of a subjective scale set with the Stomacher® as a reference point. The results indicate that all four instruments have similar performance in regards to viable cell counts. However, in regards to noise level, the Smasher® and the Bagmixer® are the quietest compared to the Stomacher® and then the Pulsifier®. The Smasher® is also the instrument with the highest ranking in ease of cleaning and ergonomics.

Master of Science / Department of Biology / Peter P. Wong / Contamination of water with various disease causing agents such as bacteria and viruses leads to 4 billion cases of diarrheal disease and 1.8 million deaths per year worldwide. The World Health Organization estimates that 94% of these cases can be prevented by increasing the availability of clean and safe water to those at risk. They also claim that 1.1 billion individuals worldwide do not have access to clean water sources, and suggest the best way to improve this situation is to increase household water treatment and safe storage (HWTS). HWTS can dramatically improve water quality, leading to a significant reduction in diarrheal disease. Being able to produce a small and inexpensive device that can be used in a household to improve water quality will significantly aid in preventing diarrheal disease. Water purification systems have been manufactured in the past in order to turn potentially disease causing water into safe drinking water. The metal iodine has been used in some of these systems. However, iodine is known to leach off and become part of the drinking water, which can lead to non-infectious diseases such as hyperthyroidism. This project shows how the development of new water purification systems utilizing silver and other heavy metal nanoparticles may be used to help purify water and have the potential to prevent diarrheal disease. Various heavy metals, including silver nitrate, magnesium oxide, brominated magnesium oxide and titanium-silicon-manganese dioxide nanoparticles, are used in conjunction with ion-exchange resins to develop a biocidal column to sanitize water. A model virus from each of the following families is used: Reoviridae (rotavirus), Picornaviridae (enterovirus), and Caliciviridae (calicivirus). This research shows not only that some of these metals can be used to purify water by inactivating viruses, but also shows the mechanism of viral inactivation. This includes protein denaturation and destruction of viral RNA.

Master of Science / Food Science Institute / Thomas J. Herald / Food recalls and incidence of foodborne disease are on the rise throughout the world. Food products are recalled in the United States almost daily, and typically a large quantity of food is affected. Pathogenic microorganisms are readily invading the food supply and traditional methods and use of antimicrobials are not performing as well as in the past. The microorganisms that prompt the recalls cause symptoms ranging from mild gastroenteritis to death. All humans eat food, therefore all humans have the potential to be exposed to pathogens in food at some point in their life. There is a need for new, more effective antimicrobials for use on food products in order to ensure that consumers have access to a safe food supply. Any new treatments for prevention of pathogenic growth in the food supply should be researched. Phage preparations used as antimicrobials on food products are a novel idea. Phages are advantageous over traditional antimicrobials such as antibiotics, pesticides, and sanitizers in numerous ways. This report presents the history of phage and phage therapy in humans, advantages and disadvantages of phage use over traditional methods, current phage preparations available or under research, and approvals and objections of phage use in the food supply.

Master of Science / Department of Biology / Helmut Hirt / Enterococcus faecalis, a gram-positive member of the mammalian gastrointestinal flora, emerged as an important contributor to nosocomial infections and antibiotic resistance gene transfer. Lipoteichoic acid (LTA), a vital component of gram-positive cell walls, has been reported to function in numerous cellular processes, ranging from maintenance of cation homeostasis and virulence to modulating function and presentation of wall proteins such as adhesins and autolysins. Interestingly, LTA can be covalently modified by the addition of D-alanyl ester residues, which appear to help regulate its function by altering surface charge. In E. faecalis the process of esterification is catalyzed by four proteins encoded by the dlt operon. Mutants lacking a functional dlt operon display the inability to incorporate D-alanyl residues on LTA and are thus deficient in their ability to regulate the anionic charge of the outer envelope in response to extracellular cues. Recent evidence suggests that two-component systems are responsible for sensing environmental conditions and regulating dlt operon expression. Utilizing a reporter construct with the upstream promoter region of dlt fused to lacZ, we were able to determine how extracellular stimuli affect transcription of this operon by measuring [Beta]-galactosidase activity. Furthermore, we were able to identify specific response regulators important for bile salt, magnesium and polymyxin B signaling.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / Enterococci are gram-positive bacteria typically found as commensals in the gastro-intestinal tracts of most mammals. Enterococci, most notably Enterococcus faecalis and Enterococcus faecium, have become problematic causative agents of several nosocomially acquired infections including urinary tract infections, bacteremia, surgical sight infections, and endocarditis. These bacteria must first overcome the innate immune response in order to establish infection. Many bacteria produce capsular polysaccharides that contribute to pathogenesis by helping the microbe evade the host innate immune response. The capsular polysaccharide produced by E. faecalis has been shown to play a role in pathogenesis; however the mechanisms of innate immune avoidance were unknown. Moreover, the number of capsule serotypes produced by E. faecalis and the genetic differences that contribute to capsule serospecificity were in doubt. In the current study it is made clear that only two capsule serotypes are produced by E. faecalis and that both capsule serotypes contribute to evasion of the host innate immune system. This work shows two mechanisms by which the capsule of E. faecalis contributes to immune evasion. First, the presence of capsule inhibited complement mediated phagocytosis through limiting the detection of opsonic complement protein C3 on the surface of the bacteria. Secondly, the presence of capsule altered cytokine signaling of macrophages by shielding bacterial components from detection. Many pathogenic strains of E. faecalis also produce an extracellular protease known as gelatinase (GelE). This work also shows a novel mechanism involving GelE in innate immune evasion through the degradation of the anaphylatoxin C5a. Degradation of C5a by GelE resulted in decreased neutrophil recruitment in vitro. A rabbit model of endocarditis was employed to assess the effect of GelE production on disease development and progression. Rabbits infected with GelE producing strains had increased bacterial burdens in the heart compared to rabbits infected with strains that were GelE negative. Reduced phagocyte infiltration at primary and secondary infection sites was also observed in rabbits infected with GelE producing strains compared to GelE negative strains. The work presented here demonstrates that both the capsular polysaccharide and GelE play roles in E. faecalis evasion of innate immune responses. Moreover, these pathogenic determinants would be suitable targets for developing alternative therapeutics used to treat E. faecalis infections.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Johann F. Coetzee / The Office International des Epizooties (OIE) Animal Health Code categorizes bovine anaplasmosis as a notifiable disease. Many species of the genus Anaplasma cause anaplasmosis. Co-infections with two or more Anaplasma spp. occur in cattle. A competitive ELISA is regarded as a reliable test for identifying A. marginale-infected cattle. However, cross-reactivity among related Anaplasma spp. has been reported when using cELISA. In the absence of effective treatment strategies and vaccine availability, anaplasmosis control strategies are primarily focused on disease identification and prevention and development of chemosterilization strategies. Four studies were completed to improve the diagnosis, treatment, and control of bovine anaplasmosis. In the first study, a real-time qRT-PCR was developed to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. This detection limit was equitable to the minimum infective unit of one A. marginale bacterium. In the second study, qRT-PCR results determined needle-free injection was superior to needle injection for controlling iatrogenic transmission of A. marginale in cattle. The qRT-PCR demonstrated 100% sensitivity by 21 days post-infection and 21 days prior to 100% sensitivity with cELISA. The third study determined the pharmacokinetic parameters of chlortetracycline in group fed, ruminating Holstein steers: volume of distribution (40.9 L⁄kg); rate constant (0.0478 h-1); dose-normalized area under the curve (0.29 h•µg⁄L); clearance (1.8 L⁄kg⁄h); elimination half-life (16.2 h); maximum concentration/dose (4.5 ng⁄mL); and time of maximum concentration (23.3 h). Dose linearity was confirmed for oral chlortetracycline dosages of 4.4, 11, and 22 mg/kg/day. The final study established an in vivo pharmacokinetic-pharmacodynamic relationship between chlortetracycline and anaplasmosis carrier clearance in bovine plasma (85.3 ng/mL). The qRT-PCR confirmed chemosterilization of all oral chlortetracycline-treated cattle within 49 days of treatment. Furthermore, qRT-PCR was an effective alternative to the subinoculation of splenectomized cattle for accurate and precise disease classification. The diagnosis, treatment, and control of anaplasmosis were enhanced through the application of qRT-PCR. Further studies are necessary for determining the mechanism of action between chlortetracycline binding to the 30S ribosome of A. marginale and carrier clearance.

Master of Science / Department of Biology / Helmut Hirt / Enterococcus faecalis is one of the most frequently encountered enterococcal isolates and accounts for about 80% of enterococcal infections. Treatment of enterococcal infections has become increasingly difficult as this organism has a high incidence of antibiotic resistance. Lipoteichoic acid (LTA) is an essential amphiphilic polymer on the surface of most Gram positive bacteria. While the molecule's exact role is not yet fully understood, a role in cell-cell contact during conjugation enabling the spread of extra chromosomal elements has been discussed. LTA also has implications in regulating autolysis, sequestering cations to the cell surface, adhesion, biofilm formation, antibiotic resistance, UV sensitivity, acid tolerance, and virulence. The gene ebsG was identified in a mutant of E. faecalis with major alterations in LTA structure and decreased ability to act as a recipient in conjugative mating. ebsG codes for a 119 kDa protein with only weak homology to other surface proteins of Gram positive bacteria. Transcriptional linkage analysis indicated ebsG and its downstream genes are organized in an operon. LTA analysis reveals a higher glycosyl content of the molecule in the mutant during stationary phase. Compared to wild type OG1RF, the mutant is more sensitive to nisin, shows higher autolysis activity during stationary phase, and is better able to serve as a recipient in plasmid transfer. Our data indicate ebsG and the members of the operon play a role in LTA structure and may act to degrade LTA.