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Provencher, MicheleKinematic magnetic resonance imaging for evaluation of cervical spondylomyelopathy in dogs
Master of Science, The Ohio State University, 2016, Comparative and Veterinary Medicine
The pathogenesis of cervical spondylomyelopathy (CSM) is similar to cervical spondylotic myelopathy in humans (hCSM). In patients with hCSM, kinematic MRI (kMRI) is used, which evaluates how flexion and extension of the cervical vertebral column affects spinal cord compression. The purposes of this study were to (1) assess how cervical flexion and extension affect spinal cord compression in Doberman Pinschers and Great Danes with CSM, and (2) evaluate an MRI compatible positioning device that allowed controlled flexion and extension of the cervical vertebral column. Nine Doberman Pinschers with disc-associated CSM (DA-CSM) and 12 dogs with osseous-associated CSM (OA-CSM) were prospectively studied. An MRI was performed with the cervical vertebral column positioned in neutral, traction (DA-CSM only), flexion, and extension. Morphologic and morphometric assessments comparing neutral, flexion and extension were then performed. In dogs with DA-CSM, flexion was associated with improvement or resolution of spinal cord compression and extension caused worsening of compressions. Extension also identified new compressive lesions and was significantly associated with dorsal and ventral compression at C5-C6 (p=0.021) and C6-C7 (p=0.031). In dogs with OA-CSM, extension identified compressions that were not present with standard positioning, especially at C4-C5 (p = 0.02). Neurologic deterioration did not occur in the patient population evaluated here. We concluded that kMRI can be safely performed in dogs with DA-CSM and OA-CSM. Specifically, imaging in extension may reveal sites of spinal cord compression that are not present with standard positioning.

Committee:

Ronaldo da Costa (Advisor)

Subjects:

Veterinary Services

Brash, Breanna MThe Effect of Trypan Blue on Posterior Capsule Opacification in an Ex Vivo Canine Model
Master of Science, The Ohio State University, 2016, Comparative and Veterinary Medicine
Purpose. To determine if trypan blue (TB) reduces lens epithelial (LEC) or corneal endothelial cell viability. Methods. Tissue was harvested from canine cadavers. Cultured LECs were treated with TB at 0, 0.05, 0.1, 0.2, or 0.3% for 30, 60, or 120 seconds. Cell morphology was evaluated and an LDH viability assay performed. Cultured LECs were treated with 0 and 0.3% TB for 120 seconds and an apoptosis assay was performed to assess caspase-3 activity. To evaluate the effects of TB on ex vivo PCO, following mock cataract surgery, lens capsules were treated with 0 and 0.3% TB at the above times and maintained in culture for two weeks. Capsules were monitored for changes in cell density and morphology; histology was performed at experimental completion. Corneal endothelial cells were treated with 0 and 0.3% TB for 120 seconds and an LDH viability assay performed. Results. TB did not significantly reduce LEC density. While TB-treated LECs demonstrate higher rates of cell death compared to vehicle control, the difference was not significant. Induction of apoptotic signaling was found in TB-treated LEC cultures. Ex vivo PCO formation was not significantly different in any treatment group. Endothelial cells treated with TB or vehicle showed no significant differences in cell death. Conclusions. TB induced low levels of LEC death via apoptotic signaling cascades but was not effective at reducing ex vivo PCO formation. TB did not induce endothelial cell death. Funded by ACVO Vision for Animals Foundation grant (VAF2014-01). Trypan blue provided by Acrivet.

Committee:

David Wilkie, DVM, MS, DACVO (Advisor); Anne Gemensky-Metzler, DVM, MS, DACVO (Committee Member); Eric Miller, DVM, MS, DACVO (Committee Member); Heather Chandler, PHD (Committee Member)

Subjects:

Medicine; Ophthalmology; Veterinary Services

Keywords:

Canine; Lens; Posterior Capsule Opacification; Trypan Blue; Cataract Surgery

Weeman, Matthew FChanges in Antimicrobial Susceptibility of Fecal Escherichia Coli Recovered From Dairy Cattle on 16 Farms in Ohio 2001-2011
Master of Science, The Ohio State University, 2016, Comparative and Veterinary Medicine
It has been hypothesized that the use of veterinary antimicrobials in livestock populations may lead to an increase in bacterial resistance to these antimicrobials among both animals and humans. Additionally, the transfer of resistance genes among bacterial pathogens may result in increased risk of food-borne disease resulting from pathogens with reduced susceptibility to the antimicrobials commonly used to treat them. Our objective is to measure changes in antimicrobial susceptibility of fecal Escherichia coli between 2001 and 2011 on 16 Ohio dairy farms. We assessed the reduced susceptibility proportions (RSP) of the 17 select antimicrobials, excluding apramycin, included in the National Antimicrobial Resistance Monitoring System surveillance program. We also examined the difference in the reduced susceptibility index (RSI) for these 16 herds. Over a 2 year period (2001-2002) 9,253 fecal samples were collected via rectal palpation from cows on 42 Ohio dairy farms. In 2011 we returned to 16 of these same 42 herds and collected a composite sample of 400 fecal samples (25 samples from each of the 16 herds) using the same methods of collection as in the previous study period. Results of the RSP data shows an increase in resistance to 4 antimicrobials (Ampicillin, Cephalothin, Chloramphenicol and Sulfamethoxazole) and no significant change in resistance to the remaining 12 antimicrobials included in our study.

Committee:

Thomas Wittum, Dr. (Advisor); Andrew Niehaus, Dr. (Committee Member); Gustavo Schuenemann, Dr. (Committee Member)

Subjects:

Comparative; Epidemiology; Health Sciences; Veterinary Services

Keywords:

E coli Resistance; Escherichia coli; dairy cattle; NARMS; fecal flora resistance in dairy cattle; Antimicrobial Susceptibility

Binkley, Laura ElysePrevalence of Cryptosporidium, Giardia, Salmonella, and Cephalosporin-Resistant E. coli Strains in Canada goose Feces Urban and Peri-Urban Sites in Central Ohio
Master of Science, The Ohio State University, 2015, Environment and Natural Resources
Large populations of resident geese can pose a pathogen exposure hazard and disease risk to humans and animals in urban areas. Evidence suggests that waterfowl play a role in pathogen dissemination and disease transmission to humans, however, more definitive data are often needed. This exploratory study sought to identify potential exposure hazards, the first step in risk assessment. This research also discusses dose-response for protozoan organisms and initiated the exposure assessment process by measuring environmental variables that may be associated with exposure. A total of 199 Canada goose fecal samples were collected from 5 peri-urban and 7 urban sites throughout the Greater Columbus, Ohio area. Samples were collected during two time periods: during 4-11 June, 2013 when geese had just begun their molt, and 16-30 August, 2013 after geese regained flight. Juveniles were distinguished from adults only during the first sample period. Antigen capture enzyme-linked immunosorbent assay (ELISA) was used to detect presence of Giardia and Cryptosporidium. Selective media were used to culture Salmonella and cephalosporin-resistant E. coli strains. Cryptosporidium was the most prevalent pathogen with 44.7% of samples testing positive. Feces collected from urban sites during the first period were 1.86 times more likely to be positive for Cryptosporidium than peri-urban sites (P = 0.10). Forward model selection methods determined that prevalence was positively associated with human population density surrounding collection sites, proportion of each site defined as pasture by the National Land Cover Database (NLCD), and distance of each site from nearest wastewater treatment plant (WWTP). Feces collected from urban sites during the second period were no more likely to be positive for Cryptosporidium than peri-urban sites (P = 1.00, Odds Ratio= 1.00). Forward stepwise model selection determined that prevalence was positively associated with human population density within study sites, distance of each site from nearest livestock farm, and distance of each site from nearest wastewater treatment plant. Giardia was present in only 3.5% of samples. None were positive for Giardia in the first period. Feces collected from urban sites during the second period were 1.9 times more likely to be positive for Giardia than peri-urban sites (P = 0.74). Prevalence of cephalosporin-resistant E. coli strains was 10.8%. Only one positive was detected during the first period. Feces collected from urban sites during the second period were 6.7 times more likely to be positive for cephalosporin-resistant E.coli than peri-urban sites (P = 0.10). No Salmonella was detected in the fecal samples. All pathogens tested, with the exception of Salmonella, showed a similar trend where a greater percentage of positives were detected at urban sites than peri-urban sites. The exposure potential of goose feces to the human population appears to be high for Cryptosporidium but low for Giardia and Salmonella. The discovery of cephalosporin-resistant strains of E.coli in fecal samples poses a potential exposure hazard because antibiotic-resistant strains are difficult to treat if infection occurs.

Committee:

Robert Gates, Dr. (Advisor); Michael Bisesi, Dr. (Committee Member); Stephen Matthews, Dr. (Committee Member)

Subjects:

Animal Diseases; Biology; Ecology; Environmental Studies; Epidemiology; Molecular Biology; Veterinary Services; Wildlife Management

Keywords:

zoonotic disease; canada goose; hazard identification; disease ecology

Yoak, Andrew JamesDisease Control through Fertility Control: Explorations in Two Urban Systems
Doctor of Philosophy, The Ohio State University, 2015, Evolution, Ecology and Organismal Biology
In many areas, wildlife populations have increased substantially in their local density because of a loss of natural controls or some artificially supplemented resource. These populations are often managed to avoid harmful effects on other wildlife species and human-wildlife conflicts. Many species are managed using lethal population reduction, but in those that are resistant to these means or where the method is unpalatable due to public concern, fertility control is becoming increasingly common. This method seeks to reduce the population size of some target problem species by capturing, sterilizing, and releasing individuals back into their habitat. Fertility control is often paired with vaccination programs because each has synergistic effects. Sterilization reduces the population size, making it easier to achieve a higher vaccination proportions for herd immunity. However, these programs have uncertain effects on both the basic biology, population demographics, and disease epidemiology. The current literature makes strongly countered species-specific conclusions. It is also unclear if fertility control is an effective method at reducing the population size in an economically viable way, compared to lethal removal. Here I use computer simulations, cross sectional surveys, and long-term monitoring of two populations, the street dogs (Canis lupus familiaris) of Rajasthan, India, and the raccoons (Procyon lotor) of the Columbus Zoo and Aquarium, to investigate what impact fertility control makes on the populations it targets. In Chapter 2, I exposed replicate simulated populations to various control schemes to see which most lowered the population size and increased vaccination coverage. In Chapter 3, I report the results of surveys of dogs from several real world Indian cities with varied histories of fertility control for several diseases. In Chapters 4 and 5, I report the results of a randomized control study on raccoons, which measured differences in parasite load and survival among control, vaccinated and vaccinated/sterilized individuals. My work demonstrates that fertility control programs can be more effective than lethal control, although the methods used to locate sexually intact individuals for treatment can significantly affect the results. In Chapter 3, I found that intact dogs living in cities with more fertility control had significantly lower prevalence of several diseases compared to those dogs living in cities with less fertility control. This is especially significant because the interventions only vaccinated against rabies, meaning that the fertility control affected local disease epidemiology. This indicates that the sterilization program buffered treated individuals’ ability to resist or spread disease enough to lower exposure to non-treated individuals. I found that sterilization and vaccination in raccoons did not affect the apparent monthly survival rates, but lowered parasite prevalence in males. However, female raccoon parasite prevalence was negatively affected by sterilization. I suggest that the sterilization method used does not eliminate hormone production, causing females to increase the length or intensity of their reproductive seasons. As a whole, this work highlights the importance of understanding the secondary effects of intervention policies. I show that altering reproductive behavior can cause dramatic changes to population dynamics and epidemiology.

Committee:

Ian Hamilton, Dr (Advisor); Stanley Gehrt, Dr. (Advisor); Rebecca Garabed, Dr. (Committee Member); Liza Comita, Dr. (Committee Member)

Subjects:

Animal Diseases; Ecology; Organismal Biology; Parasitology; Veterinary Services; Wildlife Management; Zoology

Keywords:

lethal control; fertility control; procyon lotor; raccoon; domestic dog; free roaming dog; street dog; canis lupus familiaris; agent based modeling; individual based modeling; dog population management;

Nichols, SylvainUse of a polycaprolactone polymer graft to repair a teat cistern mucosal defect in a dairy cow model
Master of Science, The Ohio State University, 2007, Veterinary Clinical Sciences

Teat injuries are frequent in dairy cattle. Removal of a large internal (covered) lesion precludes the closure of the teat mucosa. If left to heal by second intention, collapse of the teat cistern and adhesions between the teat walls will most likely occur. Different tissues and grafting materials have been used to reconstruct teat mucosa. At this time, each technique developed has many post¬operative complications and carries a guard prognosis for return to normal function of the teat.

In our study we hypothesized that polycaprolactone (PCL), a new tissue engineered polymer, will promote better healing when grafted over an induced teat mucosa defect if compared with a defect left to heal by second intention. The objectives of our study were to evaluate the biocompatibility of the polymer with cow’s mammary tissue and to compare the healing of an induced teat mucosa defect covered by PCL with the healing of a defect left to heal by second intention.

Four cows allowing for a total of 16 teats were used in this study. Each teat was randomly assigned to a treatment group (group l=defect left to heal by second intention and group 2=defect cover by a PCL polymer graft). With the cow in lateral recumbency, a 3 cm incision was made to expose the teat cistern mucosa where a circular 8mm defect was created. The defect was covered or not with the PCL graft and the incision was closed. The healing of the defect was followed weekly by ultrasound and bi-weekly by theloscopic evaluation. The cows were euthanized 28 days after surgery and the teats were retrieved for histopathologic evaluation.

No statistically significant differences were found between groups regarding the degree of inflammation, fibroplasia and epithelialisation. The defects left uncovered were smaller 28 days after surgery and were covered mostly by a stratified epithelium. The defects covered by the PCL polymer graft were mostly covered by a squamous epithelium. Adhesions and exuberant granulations tissues were present over the defect for both treatment groups. No statistically significant differences were found between groups regarding the ultrasound and theloscopic findings. However, in both groups, the teat wall and the defect sizes during ultrasonic evaluation were statistically significantly thicker at 21 and 28 days if compared with measurements obtained at Day 0, 7 and 14.

The biocompatibility of the PCL polymer with cow’s mammary tissue is considered to be poor. However, many factors other than the material itself can explain the results obtained in this study. The exposure to milk secretions and the surgical technique might have predisposed the surgery site to inflammation and adhesion formation. The small number of animal used in this study might have influenced negatively our results. Finally, the processing of the polymer into 8 mm sheet might not have been adequate.

In conclusion, in a dry cow model, it is not recommended to cover a 8 mm mucosal defect with a 5 µm of thickness PCL sheet. Pre-seeding the polymer with epithelial cells and using it for larger defects in lactating cows needs to be further evaluated to determine if PCL polymer can be of any use in teat and udder surgery.

Committee:

D. Micheal Rings (Advisor)

Subjects:

Veterinary Services

Van Balen Rubio, Joany ChristinaMethicillin-resistant Staphylococcus aureus: ecology and molecular epidemiology of environmental contamination in veterinary and human healthcare settings during non-outbreak periods
Doctor of Philosophy, The Ohio State University, 2015, Veterinary Preventive Medicine
Within healthcare settings, patients, healthcare workers (HCWs) and the environment interact in the transmission cycle of MRSA. In particular, the role that inanimate surfaces might play in this cycle is unclear, even though a plausible involvement of the environment has been described during outbreaks. Therefore, the main goal of this dissertation was to establish the ecology (presence, distribution and maintenance) and molecular characteristics of MRSA strains present in human and animal hospitals during non-outbreak periods. Yearlong active MRSA surveillances were established in two veterinary hospitals where contact surfaces and incoming patients were sampled every month. Similarly, monthly surveillance was performed for one year in two human general medicine wards, in addition to the collection of aggregated data of MRSA patient infection burden within these wards. The first two studies (Chapter 2 & 3) were performed in a small animal veterinary hospital. Overall, 13.5% of surfaces and 5.7% of incoming dogs were positive for MRSA. The majority of the isolates (=80%) were SCCmec typeII/USA100. Gurneys, doors, and examination tables/floors were the most frequently contaminated surfaces. The third study (Chapter 4), performed in an equine hospital, found that 8.6% of the surfaces and 5.8% of the horses sampled were positive for MRSA. The majority of the isolates (=60%) were classified as SCCmec typeIV/USA500. The most common contaminated surfaces were: computers, feed/water buckets, and surgery tables/mats. Molecular analysis performed in both veterinary hospitals showed that new pulsotypes were constantly introduced into the hospital and circulated throughout several areas and surfaces. Furthermore, maintenance of strains in the environment was also observed when unique clones were detected for 2-3 consecutive months on the same surfaces. The fourth study (Chapter 5) performed at the human hospital demonstrated that 23.7% of the surfaces were positive for MRSA. The majority of the isolates (=46%) were SCCmec typeII/USA100. Chart holders, medicine carts, elevators and doors were the most common contaminated surfaces. Similarly to the veterinary settings, analysis of contamination patterns showed the introduction and/or reintroduction of clones into the environment. Conversely, the frequent contamination of surfaces for 2-3 consecutive months did occur but with different pulsotypes. Over 85% of the strains recovered from the human and animal hospitals were resistant to three or more classes of antimicrobials, which represent a clinical and public health concern. Finally, the original results presented in this dissertation demonstrated that MRSA is frequently found in the environments of human and animal hospitals in the absence of an outbreak. It was shown that MRSA is constantly introduced and/or reintroduced into the hospital likely by patients, visitors and hospital personnel. Such influx of MRSA strains, along with lack or inadequate cleaning and disinfection protocols, promoted the persistence over time of this bacterium within the hospital environment. Most importantly, strains were moved across different areas of the hospitals (by HCWs and/or patients) allowing the spread of MRSA throughout each facility. Therefore, it was concluded that the hospital environment plays a key role as a reservoir and as a plausible source of MRSA within human and veterinary hospitals.

Committee:

Armando Hoet (Advisor); Thomas Wittum (Committee Member); Paivi Rajala-Schultz (Committee Member); Shu-Hua Wang (Committee Member)

Subjects:

Epidemiology; Molecular Biology; Public Health; Veterinary Services

Keywords:

MRSA, Hospital environment, Surveillance, Molecular Epidemiology

Sahin, OrhanDevelopment of a Selective Medium for Rhodococcus Equi
Master of Science, The Ohio State University, 1997, Veterinary Clinical Sciences
Rhodococcus equi is a frequent cause of chronic suppurative bronchopneumonia, lymphadenitis, and enteritis in foals, in most cases during the first four months of life. This study was carried out to develop a selective medium for the organism for its isolation from highly contaminated sources such as soil and feces of grazing herbivores. Twenty-eight clinical isolates of R. equi recovered from lesions of infected foals were tested for susceptibility to antimicrobial agents by disk diffusion and microbroth dilution susceptibility testing methods. In-vitro the most active antibiotics were gentamicin, amikacin, netilmicin, erythromycin and trimethoprim/sulphonamide combination whereas oxacillin, cloxacillin, ceftiofur, ceftazidime, aztreonam, trimethoprim, novobiocin, and nystatin were routinely impotent to tested isolates. Based on these susceptibility patterns of R. equi isolates a selective agar medium was developed. The medium contained aztreonam 100 (µg/ml, novobiocin 25 (µg/ml and nystatin 100 Units/ml with (herein referred to as IST-ANYT) or without (herein called IST-ANY) 0.005 % potassium tellurite. After the medium was proved for its selectivity for R. equi, it was tested with a number of soil and fecal samples for isolation of the organism from a Quarter Horse breeding farm in Columbus, OH. All soil samples (n=10) and fecal specimens from the rectum of healthy mares (n=12) yielded R. equi on either medium after 48 hours of incubation while the organism was not isolates from feces of any yearlings (n=4). These results show the satisfactory capacity of the new selective medium for isolation of R. equi from contaminated sources, and confirm the findings of others that the organism is widespread in equine feces and their soil environment.

Committee:

Joseph J. Kowalski (Advisor); Stephen M. Reed (Committee Member); Kenneth W. Hinchcliff (Committee Member)

Subjects:

Veterinary Services

Pina-Mimbela, Ruby MelisaAssociation of Polyphosphate (poly P) Kinases with Campylobacter jejuni Invasion and Survival in Human Epithelial Cells
Master of Science, The Ohio State University, 2013, Veterinary Preventive Medicine
The foodborne and zoonotic bacterium Campylobacter jejuni, is one of the major causes of gastrointestinal disorders in humans worldwide. This human pathogen expresses a variety of carbohydrate, protein and lipid structures decorating its outer material (OM) which have been associated with virulence. On the other hand, inorganic polyphosphate (poly P), in many bacteria, is essential for stringent survival response and adaptation, and is considered as a virulence factor. Two kinases in C. jejuni have been associated with virulence: poly phosphate kinase 1 (PPK1) and poly phosphate kinase 2 (PPK2). PPK1 is responsible for poly P generation and PPK2 for GTP biosynthesis from poly P. GTP is a molecule involved in signaling processes as well as protein and capsule synthesis. In previous studies, we demonstrated that C. jejuni delta ppk1 shows reduced stringent survival response, adaptation, and in vivo colonization in chickens, whereas, delta ppk2, in addition to its stress tolerance defect, also displays reduced virulence properties. In the present study, the contribution of component(s) present in the outer material (OM) from the wild-type, delta ppk1 and delta ppk2 to C. jejuni invasion and intracellular survival in human epithelial cells in vitro was investigated. OM from C. jejuni wild type 81-176, delta ppk1 and delta ppk2 was extracted and fractionated into carbohydrates (lipoglycans, poly-oligosaccharides), lipids, and proteins. These fractions were tested for their contribution to C. jejuni invasion, intracellular survival and IL-8 production in INT-407 human embryonic intestinal cells. Different fractions from C. jejuni 81-176 wild type, delta ppk1 and delta ppk2 were pre-incubated with INT-407 human epithelial cells prior to infection with C. jejuni wild type. Results obtained imply that components present in the C. jejuni OM are associated to invasion and intracellular survival in INT-407 cells. Additionally, poly P kinases play a role in intracellular survival in epithelial cells. Alterations in OM from delta ppk1 and delta ppk2 are suggested to be important in C. jejuni intracellular survival in INT-407 cells. Our results suggested that OM proteins mediate C. jejuni invasion and intracellular survival to human epithelial cells whereas lipoglycans are associated with intracellular survival. In addition, alterations present in delta ppk2 lipids are associated with C. jejuni intracellular survival in INT-407 cells. On the other hand, poly- and oligo- saccharides fraction from delta ppk1 and delta ppk2 are likely to play a role in C. jejuni survival within epithelial cells. Further, we evaluated production of IL-8 in INT-407 cells by OM and its fractions from wild type, delta ppk1 and delta ppk2 strains. C. jejuni OM from wild type was not able to induce IL-8 in INT-407 cells, whereas IL-8 secretion was observed when these epithelial cells were exposed to OM from delta ppk1 and delta ppk2. Our results demonstrate that proteins, lipids, and lipoglycans present in C. jejuni OM are associated with its invasion and intracellular survival in human epithelial cells. Additionally, this study provides insights about the role of poly P in modulation of C. jejuni OM composition, thereby contributing to invasion and intracellular survival.

Committee:

Gireesh Rajashekara, Dr. (Advisor); Jordi B. Torrelles, Dr. (Committee Member); Chang-Won Lee, Dr. (Committee Member)

Subjects:

Biochemistry; Cellular Biology; Microbiology; Veterinary Services

Keywords:

Campylobacter jejuni; Poly P kinases; invasion; survival; epithelial cells; INT-407 cells

Lim, Kelvin ZEFFECT OF FLAVOMYCIN (FLAVOPHOSPHOLIPOL) ON THE ACQUISITION AND LOSS OF ANTIMICROBIAL RESISTANCE IN MULTIDRUG-RESISTANT SALMONELLA ENTERICA SEROVAR ENTERITIDIS IN BROILER CHICKENS
Master of Public Health, The Ohio State University, 2015, Public Health
Multidrug-resistant nontyphoidal Salmonella is a major zoonotic and foodborne pathogen which is commonly associated with poultry and poultry products. It threatens to increase the severity of diarrheal diseases and limit options for previously treatable infections. It was hypothesized that flavophospholipol, a veterinary antimicrobial with no human analog has an anti-conjugative and plasmid curing effect on multidrug-resistant bacteria of family Enterobacteriaceae such as Salmonella. Therefore, a controlled study was designed to determine if flavophospholipol given in-feed at concentrations of 10 ppm and 64 ppm alone and in the presence of subtherapeutic levels of ampicillin could exert these effects in vivo on plasmid-mediated multidrug-resistant Salmonella enterica serovar Enteritidis in broiler chickens. 270 female day-old chicks were housed in 90 cages and divided into 8 treatment groups with allocated flavophospholipol diet concentrations. 4 treatment groups were allocated to test anti-conjugative and plasmid curing effects of flavophospholipol each. Chicks were inoculated with cocktails of challenge strains of plasmid-mediated multidrug-resistant S. Enteritidis with nalidixic acid resistance markers on day 3. Individual cloacal swabs were taken on days 7, 14 and 23 and individual ceca were removed on day 35 to be cultured for the presence of Salmonella. 69 selected isolates were genotyped and 2,137 isolates were tested for antimicrobial susceptibility. Pulsed field gel electrophoresis (PFGE) of selected isolates showed clonal relatedness to the transconjugant and recipient Salmonella strains. There were significantly higher proportions of ampicillin-resistant recipient strain isolates harvested from the chickens in the control group compared to the treatment group given in-feed flavophospholipol 64 ppm at day 14 and at day 35 in the cecal samples, indicating a reduction of in vivo conjugation events with flavophospholipol 64 ppm. When the resistance profiles of the transconjugant isolates across treatment groups were compared, reduced acquisition of streptomycin and tetracycline by transconjugant isolates from the treatment group given in-feed flavophospholipol 64 ppm was observed. Differences in proportions of transconjugant isolates acquiring resistance to extended-spectrum cephalosporins across treatment groups were not observable. No differences in resistance phenotypes were observed between the treatment group given flavophospholipol 10 ppm and the treatment group given basal diet with ampicillin compared with the control with basal diet only. There were no significant differences in the loss of plasmid-mediated ampicillin resistance by transconjugant isolates between the three treatment groups, flavophospholipol 10 ppm with ampicillin, flavophospholipol 64 ppm alone and flavophospholipol 64 ppm with ampicillin against the control group with basal diet testing the plasmid curing effects of flavophospholipol. There were also no differences in resistance phenotypes for streptomycin and tetracycline resistances of isolates across the treatment groups and study period. Significantly lower proportions of chickens in treatment groups given flavophospholipol 64 ppm alone and with subtherapeutic levels of ampicillin acquired resistance to extended-spectrum cephalosporins compared with the control group. The study provides sufficient evidence that flavophospholipol 64 ppm could reduce plasmid conjugation in vivo in the chicken gut but there was insufficient evidence that flavophospholipol 10 ppm and 64 ppm given in-feed alone and with ampicillin exert any plasmid curing effects.

Committee:

Wondwossen Gebreyes (Committee Chair); Michael Pennell (Committee Member); Stephanie Lewis (Committee Member); Mohamed El-Gazzar (Committee Member)

Subjects:

Agriculture; Epidemiology; Public Health; Veterinary Services

Keywords:

Flavomycin; Flavophospholipol; Salmonella; Antimicrobial Resistance; Chickens

Liepman, Rachel SarahAlterations in the Fecal Microbiome of Healthy Horses in Response to Antibiotic Treatment
Master of Science, The Ohio State University, 2015, Comparative and Veterinary Medicine
Acute colitis is the most common and devastating complication of antibiotic therapy in horses. Fecal culture often fails to identify etiologic agents and has low sensitivity in detecting organisms that are difficult to cultivate. Metagenomic studies utilize 16S ribosomal RNA (rRNA) sequence for bacterial identification and classification and can produce profiles of genetic diversity from microbial communities. There are limited data to explain how antimicrobials affect the fecal microbiome of horses over time. The purpose of this study was to provide a comprehensive description of bacterial phylum structures within feces of normal horses in central Ohio during the winter and summer, to determine how commonly used antibiotics affect these communities over time and if these changes persisted over longer time periods. Healthy horses were acclimated to the same diet, environment and husbandry and were treated intravenously with different classes of antibiotics (ceftiofur sodium, enrofloxacin and oxytetracycline) or saline for 3 or 5 days consecutively and fecal microbiota evaluated. Fecal samples were collected and frozen, bacterial DNA was extracted and PCR amplified, and analyzed by 454 pyrosequencing of the 16S rRNA gene from baseline (before treatment), and at various time points. We demonstrated that the fecal microbiome of healthy horses in central Ohio was highly diverse both within and between subjects before and after treatment. Major phyla present in greatest abundance in the feces of all subjects included Firmicutes and Bacteroidetes, while minor phyla included Proteobacteria, Spirochaetes, Tenericutes and Verrucomicrobia. Enrofloxacin and ceftiofur lead to the greatest shifts in the fecal microbiome over time, however none of the treated horses developed diarrhea. Many of these changes persisted over longer time periods, while some returned to baseline. These variations were specific to the antibiotic used and may represent repeatable trends. Additionally, 3 and 5 days of treatment were sufficient to identify alterations in the fecal microbiome induced by antibiotic treatment.

Committee:

Ramiro Toribio (Advisor); Prosper Boyaka (Committee Member); Teresa Burns (Committee Member)

Subjects:

Veterinary Services

Keywords:

fecal microbiome; equine diarrhea

Titler, Mallory LAssessment of Daily Behavioral Activity Patterns using Electronic Data Loggers as Predictor of Parturition, Dystocia and Metritis in Lactating Holstein Cows
Master of Science, The Ohio State University, 2014, Veterinary Clinical Sciences
Dystocia and metritis in dairy cows increase the risk for health disorders and mortality, and reduce performance (milk yield, and reproductive performance). The objectives of the present study were to assess the effect of (1) parturition and (2) two calving-related events (dystocia and metritis) on behavioral activity beginning 4 d before calving and clinical diagnosis, respectively. Activity data were collected from 147 Holstein cows housed in free-stall barns from 3 dairy herds. All cows were housed in similar facilities using a close-up pen 21 d prior to the expected calving date and moved into a contiguous individual maternity pen for parturition. Electronic data loggers (IceQubeTM, IceRobotics, Edinburgh, Scotland, UK) were placed on the hind leg of periparturient dairy cows at 7 ± 3 d prior to the expected calving date and removed at 14 ± 3 DIM. The number of steps (n/d), standing time (min/d), number of laying bouts (n/d), and mean duration of LB (min/b) were recorded. Calving ease (CE; scale 1-4), parity, calving date and time, and stillbirth (born dead or died within 24 h) were recorded. For partuririton and dystocia analyses, unassisted cows (n = 132; CE score of 1) were compared to assisted cows (n = 15; CE scores of 2-3). For the metritis analysis, cows with metritis (n = 15) were matched by parity with non-metritis cows (n = 15). Data were analyzed using MIXED (activity patterns) and GLIMMIX (stillbirth) procedures of SAS. Activity patterns were adjusted for the effect of herd, parity, and CE. Cows with unassisted birth had increased number of steps (P < 0.05) and decreased standing time (P < 0.05) with more LB of shorter duration (P < 0.05) 24 h prior to calving. Additionally, cows with assisted births had increased number of steps and LB (P > 0.05), but LB of longer duration (P < 0.05) 24 h prior to birth compared to unassisted cows. Metritis cows spent more time standing, had fewer steps and LB, and LB of longer duration 1-3 d prior to diagnosis compared to non-metritis cows (P < 0.05). These findings provided evidence that cows experiencing parturition as well as dystocia or metirits showed distinct behavioral activity patterns at least 24 h prior to calving or diagnosis. The potential benefits of electronic data loggers (as precision management tool around the time of calving) to predict parturition, difficult births or cows at risk of metritis in real-time and around-the-clock would allow dairy producers and their personnel to improve the overall survival and welfare of cows and calves.

Committee:

Gustavo Schuenemann (Advisor); Paivi Rajala-Schultz (Committee Member); Eric Gordon (Committee Member)

Subjects:

Animal Sciences; Veterinary Services

Murphy, Jamie EllenDevelopment and evaluation of a sarcocystis neurona-specific IgM capture enzyme linked immunosorbent assay
Master of Science, The Ohio State University, 2005, Veterinary Clinical Sciences

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. The parasite causes asymmetric neurologic deficits in horses in both North and South America. EPM has a significant economic impact on the US horse industry, with estimated costs from $55.4 to $110.8 million per year.

Currently, antemortem testing is limited in its specificity in exposed and diseased horses. This paper reports on the development of an IgM capture enzyme linked immunoassay (ELISA) for the identification of acute infection with S. neurona. The ELISA was based on the S. neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S. neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S. neurona sporocysts, all of whose histopathology results were available.

The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S. neurona would have an increased S. neurona-specific IgM (Sn-IgM) concentration relative to their pre-infected state. For horses challenged with 1.5 million sporocysts, the results indicate a significant increase in serum Sn-IgM concentrations relative to pre-inoculation for weeks 2 through 6 post-inoculation.

For horses inoculated with lower doses of S. neurona, there were significant increases in Sn-IgM concentration at various points in time depending on the inoculation dose. Finally, there was a significant increase between the pre- and post-inoculation CSF Sn-IgM concentrations. Our results support the assay as a valuable addition as a diagnostic tool during the acute phase of EPM.

Committee:

Stephen M. Reed (Advisor)

Subjects:

Veterinary Services

Zaldivar-Lopez, SaraBlood Gases and Cooximetry in Retired Racing Greyhounds: Unique Hemoglobin Physiology and Oxygen Carrying Properties
Master of Science, The Ohio State University, 2010, Veterinary Clinical Sciences
Greyhounds have differences in many hematological parameters compared to other breeds [i.e. higher PCV and red blood cell counts], attributed to selective breeding, and to a compensatory mechanism for their high oxygen affinity hemoglobin (Hb). The purpose of this study was to evaluate this oxygen affinity of Hb in retired racing Greyhounds (RRGs) using a blood gas analyzer with cooximeter (Nova CCX), and to establish reference intervals in this breed. Venous blood samples from 57 RRGs (G) and 30 non-Greyhounds (NG) were analyzed, and groups were compared using T-test. The G group had significantly higher pH, partial pressure of oxygen (PO2), oxygen saturation (SO2), oxyhemoglobin (O2Hb), total Hb (tHb), oxygen content (O2Ct), and oxygen capacity (O2Cap) and significantly lower deoxyhemoglobin (HHb) and P50 when compared to NG, supporting the higher oxygen-carrying capacity in this breed. Consistent with previous reports, P50 was lower (high oxygen affinity). Current studies on Hb-based oxygen carriers have revealed that in tissues which need more oxygen, a high-affinity oxygen carrier is beneficial (i.e. strenuous exercise), potentially explaining the benefits of having a high-affinity Hb. Given the narrow range found in the P50 value, and Greyhounds’ high mean Hct, we postulated that the high viscosity had affected the results by impairing a constant blood flow through the analyzer. Venous samples from 13 RRGs were obtained, and divided in two heparinized tubes, diluting one to 20% with PBS. Both undiluted and diluted samples were analyzed simultaneously, and P50 was also calculated manually (formula in the user’s manual). No significant differences were found among undiluted and diluted samples, but when calculated manually, mean P50 was significantly lower than the one generated by the instrument in both undiluted and diluted samples. In conclusion, the high viscosity did not affect the results obtained in the previous study. In order to evaluate the P50 variation during storage, venous blood from 19 RRGs was analyzed and then stored at 4°C for 3 weeks. At this time, the analysis was repeated and the two time points were compared. The mean P50 value decreased, but unexpectedly, almost half of the values remained the same after 3 weeks. The Hemox-Analyzer is an instrument that accurately records and plots the oxyhemoglobin dissociation curve (ODC) during deoxygenation, providing information about the delivery of oxygen to the tissues. We evaluated venous samples from two adult dogs (one RRG and one mixed breed); both dogs had blood gas and cooximetry analysis performed too. Although both dogs had the same P50 using the Nova CCX, the Greyhound had lower P50 than the mixed breed dog using the Hemox-Analyzer. Overall, we conclude that Greyhounds have high affinity Hb, as reflected by a low P50, compared to NG. However, this is not accurately assessed by the Nova CCX analyzer, probably because it is not designed for animals (uses human ODC algorithms). Based on limitations imposed by the machine’s calculation of P50, higher SO2 in Greyhounds (>80%) could also make the analyzer assign a default value, explaining such a narrow range in P50 values.

Committee:

C. Guillermo Couto, DVM (Committee Chair); Edward Cooper, VMD, MS (Committee Member); Maxey Wellman, DVM, PhD (Committee Member)

Subjects:

Sports Medicine; Veterinary Services

Keywords:

hemoglobin; oxygen affinity; Greyhound

Sams, Lisa MichelleThe Effect of Morphine-Lidocaine-Ketamine-Dexmedetomidine Co-infusion on Minimum Alveolar Concentration of Isoflurane in Dogs
Master of Science, The Ohio State University, 2011, Veterinary Clinical Sciences

The purpose of this study was to determine the effects of an infusion of dexmedetomidine, a co-infusion of morphine-lidocaine-ketamine (MLK), and a co-infusion of dexmedetomidine-morphine-lidocaine-ketamine (alpha-MLK) on minimum alveolar concentration (MAC) of isoflurane in dogs. The MAC of an inhalant anesthetic required to prohibit purposeful movement is a measure of anesthetic potency (Eger et al 1965). Isoflurane is the most commonly used inhalant anesthetic in veterinary practice (Lozano et al 2009), but has potent vasodilatory effects and causes a dose-dependent decrease in mean arterial pressure in anesthetized dogs (Steffey and Howland 1977). Additional drugs are used during anesthesia to decrease the inhalant anesthetic requirement, a concept referred to as balanced anesthesia. Each of the drugs we infused has a different central nervous system receptor mechanism of action. Co-infusion of MLK, as well as the infusion of each drug separately, has been shown to reduce MAC in isoflurane-anesthetized dogs (Muir et al 2003). Dexmedetomidine, an alpha-2 agonist, has been shown to reduce isoflurane MAC in dogs (Pascoe et al 2006). Dexmedetomidine is a commonly used sedative with analgesic and muscle relaxant properties. The effect of a co-infusion of alpha-MLK on isoflurane anesthesia has not been critically evaluated in the dog. Our hypothesis was that infusion of alpha-MLK would significantly decrease isoflurane MAC (MAC-Iso) compared to dexmedetomidine or MLK infusions.

For this study, six dogs were anesthetized to determine MAC-Iso (baseline). Following MAC-Iso determination, each dog was anesthetized on three separate occasions with isoflurane, with a minimum of 7 days between anesthetic episodes. On each occasion, and in random order, dexmedetomidine, MLK, or alpha-MLK infusions were administered, and MAC was determined. Additionally, hemodynamic and metabolic parameters as well as bispectral index (BIS) were measured during each experiment. The investigator was blinded to the infusion given. Data were analyzed using ANOVA for repeated measures. Dunnett and Tukey posttests were performed to identify differences within and among groups, respectively, when differences were detected.

The MAC-Iso was 1.3 ± 0.15%. Dexmedetomidine, MLK, and alpha-MLK significantly lowered MAC-Iso by 30, 55, and 90%, respectively. Heart rate was significantly decreased from baseline in all infusion groups. Heart rate did not differ significantly between dexmedetomidine and alpha-MLK infusion groups. Mean arterial pressure for alpha-MLK increased significantly from baseline. Systemic vascular resistance was significantly increased for the dexmedetomidine and alpha-MLK infusion groups. The BIS values increased significantly compared to baseline for the MLK and alpha-MLK infusion groups. There was no significant difference for time to extubation and sternal recumbency among groups.

Infusions of dexmedetomidine, MLK, and alpha-MLK reduced MAC-Iso in dogs. Changes in cardiovascular parameters were within clinically acceptable ranges for healthy dogs. BIS can be an effective monitoring tool for measuring the depth of anesthesia.

Committee:

Phillip Lerche, BVSc, MS (Advisor); Richard Bednarski, DVM, MS (Committee Member); John Hubbell, DVM, MS (Committee Member)

Subjects:

Veterinary Services

Keywords:

MAC; isoflurane; multimodal anesthesia; dogs; dexmedetomidine; BIS

Leise, Britta S.Laminar Inflammation and the Equine Epidermal Epithelial Cell: Determining the Role in Laminar Failure
Doctor of Philosophy, The Ohio State University, 2010, Comparative and Veterinary Medicine

Laminitis is a devastatingly painful, life-threatening disease of the equine digit. Initiating causes include sepsis/systemic inflammatory response syndrome (SIRS) related conditions, metabolic syndrome/endocrine dysfunction, and severe contralateral limb lameness. The pathogenesis of laminitis has yet to be completely elucidated; however, several theories on the predominate mechanism exist. Recent evidence shows a prominent proinflammatory response very early in the developmental phases of laminitis induced experimentally by administration of black walnut extract (BWE). As dysregulation of the inflammatory response is suspected to play an important role in sepsis/SIRS and subsequent multiple organ failure in humans, a similar mechanism has been proposed to occur in the horse, with the hoof being the predominate organ that fails. Laminar failure occurs when there is dysadhesion between the epidermal and dermal lamina at the basement membrane.

The studies conducted were performed to determine if inflammation in the front and hind limb lamina occur after administration of a carbohydrate overload (CHO), which is a model that more closely resembles clinical cases of laminitis occurring from sepsis, to determine if phosphorylation of STAT proteins play a role in the inflammatory response of the lamina, and to determine if other organs have a similar inflammatory response as the lamina after CHO administration. Additional studies were performed to develop an understanding of how the equine epidermal cell responds to bacterial ligands known to be produced in the cecum after CHO administration and to develop a technique for isolation of RNA specifically from the equine laminar basal epithelial cell (LBEC) using laser capture microdissection (LCM).

Results of these studies demonstrate that inflammation does occur in the front and hind lamina after CHO administration; however, the response occurs at the onset of lameness in these horses which is different from the BWE model where it occurs at the early developmental phase. STAT3, but not STAT1, phosphorylation occurs in the lamina and is most likely a direct result of the profound IL-6 response that has been documented to occur in the lamina from horses receiving both BWE and CHO. Inflammation does occur in the liver, lung and kidney after CHO administration; however, a different pattern of inflammatory gene expression occurs in each tissue with the lamina overall having the greatest response of any organ evaluated. Equine epidermal epithelial cells in culture respond to the gram negative bacterial components, LPS and flagellin, by producing proinflammatory cytokines, but do not response to the bacterial component of gram positive organisms, which is similar to what is observed clinically where horses with sepsis resulting from a gram negative source are much more likely to develop laminitis.

Overall, inflammation appears to be involved in the development of laminitis. In addition, it is likely the laminar epithelial cell plays a much more active role, not only structurally but also producing and responding to inflammatory mediators. The methods developed to isolate RNA from the LCM technique will allow for extensive study of these cells in context of disease, thereby providing essential information from future studies.

Committee:

James Belknap, PhD (Advisor); Rustin Moore, PhD (Committee Member); Prosper Boyaka, PhD (Committee Member); Sashwati Roy, PhD (Committee Member); Elliott Crouser, MD (Committee Member)

Subjects:

Veterinary Services

Keywords:

laminitis; horse; equine; inflammation; keratinocyte; laminar epithelial cell; gene expression

Burns, Colby GailInfluence of Locking Bolt Location on the Mechanical Properties of an Interlocking Nail in the Canine Femur
Master of Science, The Ohio State University, 2010, Veterinary Clinical Sciences
Long bone fractures are common injuries in the canine patient. The aims of this study were to determine whether the fatigue properties of an interlocking nail construct are influenced by metaphyseal or diaphyseal location of the locking bolt and to evaluate fatigue properties of locking bolts in metaphyseal and diaphyseal bone under axial and torsional loading. Paired femora from 20 skeletally mature dogs were implanted with a 6-mm diameter, model 11, interlocking nail (ILN) and locked with a 2.7 mm bolt placed in either the diaphysis or metaphysis. Constructs were tested in axial loading (10 pairs) or torsion (10 pairs) to failure (defined as displacement >2 mm or a total of 500,000 cycles for axial loading, and rotation >45° degrees for torsional loading.) Outcome measures included initial construct stiffness, number of cycles to failure, peak load and peak torque. Microradiography and histology were used to determine the location and nature of construct failure. Metaphyseal bolts failed at higher axial loads than diaphyseal bolts, with bolt failure due to bending at the nail-bolt interface. All metaphyseal constructs were intact after torsional loading with no evidence of fracture of the bone or the bolt whereas 9 of 10 diaphyseal constructs failed catastrophically due to spiral fracture through the adjacent cortical bone. Placement of a locking bolt in metaphyseal bone extends fatigue life under axial loading and decreases the incidence of catastrophic failure under torsional loading. Therefore when inserting an interlocking nail for repair of long bone fractures, efforts should be made to obtain firm seating of at least one locking bolt in metaphyseal bone.

Committee:

Matthew Allen, Vet MB, PhD (Advisor); Alan Litsky, MD, ScD (Committee Member); Kenneth Johnson, MVSc, PhD, FACVSc (Committee Member)

Subjects:

Animals; Mechanics; Surgery; Veterinary Services

Dryburgh, Elizabeth LilaIDENTIFICATION OF DIFFERING STRAINS OF SARCOCYSTIS NEURONA MEROZOITES
Master of Science, The Ohio State University, 2012, Veterinary Preventive Medicine
Sarcocystis neurona is considered the major etiologic agent of equine protozoal myeloencephalitis (EPM), a neurological disease in horses. Raccoons (Procyon lotor) act as an intermediate host in the life cycle of S. neurona. The focus of this study was to determine if sarcocysts would develop in raccoons experimentally inoculated with different host-derived strains of in vitro cultivated S. neurona merozoites. Four raccoons were experimentally inoculated with merozoites from the isolates Sn-OT-1 (sea otter derived), Sn-37-R (raccoon derived), Sn-UCD 1 (equine derived) and Sn-Mucat2 (cat derived). Two raccoons were orally inoculated with sporocysts to act as positive controls. Raccoon tissues were then fed to laboratory raised opossums (Didelphis virginiana), the definitive host of S. neurona. Gastrointestinal scraping revealed sporocysts in two of the opossums who received muscle from the raccoons that were inoculated with the raccoon-derived or the sea otter derived isolates. These results suggest that raccoons can form tissue cysts from infection with in vitro derived S. neurona merozoites. In contrast, the equine and cat-derived isolates did not produce microscopically or biologically detected sarcocysts. All raccoons seroconverted to S. neurona as detected via immunoblot analysis. Further western blot analysis revealed antigenic differences when tested with the raccoon sera. Immunohistochemical tests also indicated antigenic differences between the merozoite and sarcocyst stages. These overall results demonstrated antigenic and biological differences between isolates and life-cycle stages. The successful infections achieved in this study indicates that the life-cycle can be manipulated in the laboratory without affecting subsequent stage development, thereby allowing further purification of strains and artificial maintenance of the life cycle.

Committee:

William Saville, Dr (Advisor); Michael Oglesbee, Dr. (Committee Member); Lawrence Capitini, Dr. (Committee Member)

Subjects:

Veterinary Services

Keywords:

Sarcocystis neurona; EPM

Drozd, Mary R.Campylobacter jejuni Survival Strategies and Counter-Attack: An investigation of Campylobacter phosphate mediated biofilms and the design of a high-throughput small-molecule screen for TAT inhibition
Doctor of Philosophy, The Ohio State University, 2012, Veterinary Preventive Medicine

In these investigations we studied 1) the ability of Campylobacter to modulate its behavior in response to phosphate actuated signals, 2) the modulation of biofilm in response to phosphate related stressors, and 3) we designed and carried out a high-throughput small-molecule screen that targets protein transport via the Twin Arginine Translocation (TAT) system. We identified that the phoX , ppk1 and ppk2 genes were key components of the phosphate response that manifested increased biofilm phenotypes, and were modulated in the presence of inorganic phosphate. We used several molecular and microbiological techniques to investigate the effect of polyP, phosphate uptake inactivation, and inorganic phosphate availability on Campylobacter’s response to phosphate stress. Additionally, we counted and measured attached biofilms, as well as measured pellicle size, biofilm shedding over the course of three days, and changes in the expression of genes known to be involved in biofilm formation phenotypes. By resolving biofilm components such as pellicles, attached cells, and shed cells we found that not only did ppk1, phoX, and ppk2 deletion affect the ability of Campylobacter to form biofilms, but biofilm components were not congruently and equally affected in each mutant. Additionally, the presence of phosphate modulated those effects both independently of and additively to gene knockouts.

Furthermore, we observed that biofilm components were additionally affected by biofilm age: where some components had their most robust growth on day 2, biofilm shedding and pellicle growth increased the most on day 3. This growth was not uniform for all mutants, as ppk1 biofilms generally matured more quickly than wild-type cells, but the ppk2 mutant in the presence of phosphate matured more slowly.

In our high-throughput small-molecule screen we designed and carried out a primary screen of small molecules to identify compounds that had anti-Campylobacter activity in the presence of 1mM CuSO4. To screen a greater number of compounds, this study was streamlined from a dual-plate study where each chemical was tested both in the presence and absence of copper sulfate. Our screen resulted in the identification of 680 small-molecule primary hits from the NSRB small-molecule library. These hits were identified from 11 different small-molecule libraries containing more than 50,000 compounds. Using database bioactivity results from past trials, the primary small-molecule positive hits were reduced to 476 targeted hits through in silica primary screens.

We used Golden Triangle in silica medicinal chemistry methods to identify molecules that were likely to be less suitable due to low molecular weight, interactions with solutes, and compound stability. From there, common chemical motifs were identified among the remaining 350 molecules. From these ‘chemical families’ a representative sample of each group was chosen as likely having similar chemical activity. We chose 54 chemicals as representative of 4 chemical motifs: thiourea, benzimidazoles, oxadiazoles, and acylhydrazones. The rest of the molecules were selected for greatest diversity. Using these techniques, 149 compounds have been chosen that will be used as ‘cherry pick’ hits for secondary screens in the near future.

Committee:

Gireesh Rajashekara, PhD/DVM (Advisor); Yehia Saif, PhD/DvM (Advisor); Armando Hoet, PhD/DVM (Committee Member); Daral Jackwood, MS/PhD (Committee Member)

Subjects:

Cellular Biology; Microbiology; Molecular Biology; Veterinary Services

Keywords:

Campylobacter; C. jejuni; small molecule; Twin Arginine Translocation; TAT; Biofilm; Phosphate Metabolim; phoX; ppk1;ppk2

Grundmann, Ilva Nena MariaDigital Radiographic and Magnetic Resonance Imaging of the Normal Equine Foot: a Focus on the Soft Tissue Structures of the Hoof Wall and Sole
Master of Science, The Ohio State University, 2012, Veterinary Clinical Sciences
The equine foot is an anatomically complex structure, in which soft tissue attachments between the distal phalanx (DP) and the hoof capsule support the entire musculoskeletal system. Equine laminitis affects these soft tissues of the foot and commonly leads to its structural collapse. Radiography is commonly used to diagnose laminitis and guide treatment and therapy. Several measurements to assess the position of the DP in relation to the hoof capsule have been described. Many of those have not been fully validated and the soft tissues in the sole area have not been the focus of previous diagnostic imaging evaluations. Our objectives were to establish normal hoof wall and sole measurements for Digital Radiography (DR) and Magnetic Resonance Imaging (MRI), to correlate and compare DR measurements with those made on MR images, to compare DR measurements before and after barium application to the surface of the sole, and to evaluate inter- and intra-observer correlation. We also aimed to distinguish if the two soft tissue layers seen on DR correspond to the epidermal and dermal layers as suggested previously and if these structures can also be imaged in the sole region. Fifty cadaver front feet of 25 adult horses of various breeds were imaged with DR and a 3 Tesla MR and various measurements were performed. Normal DR and MRI measurements are presented and statistically different (P < 0.0001). However, the difference is small (< 2mm). Measurements with barium applied to the surface of the sole were consistently smaller, than measurements without barium on the sole (P < 0.0001). There was good overall inter- and intra-observer correlation between DR (0.98/0.98) and MR (0.99/0.99) measurements. MRI measurements of the deep/sublamellar dermis and interdigitating epidermal and dermal layer corresponded to the lucent soft tissue opaque band surrounding the DP on DR images. Our measurements not only support established measurements, but also offer new approaches to quantitatively assess the anatomy of the equine foot.

Committee:

Tod Drost (Committee Chair); Lisa Zekas (Committee Member); James Belknap, PhD (Committee Member)

Subjects:

Veterinary Services

Keywords:

horse; foot; digital radiography; laminitis; MRI; 3T

Marin, Liliana MarcelaAMINOCAPROIC ACID FOR THE PREVENTION OF POSTOPERATIVE BLEEDING IN GREYHOUNDS
Master of Science, The Ohio State University, 2011, Veterinary Clinical Sciences
Delayed postoperative bleeding is common in retired racing Greyhounds (RRGs), despite normal results of routine hemostasis assays. The excessive postoperative bleeding in the RRGs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. Providing a method to prevent or minimize the severity of postoperative bleeding in RRGs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. Epsilon aminocaproic acid (EACA) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. The objective of this double-blinded, prospective, randomized study was to evaluate the effects of EACA versus placebo on the prevalence of bleeding in RRGs, and to investigate its mechanism of action by using TEG. We compared the effects of EACA and placebo in 100 RRGs that underwent elective ovariohysterectomy or orchiectomy at the Veterinary Medical Center, The Ohio State University during 2 years. The main endpoint was bleeding (prevalence and severity); minor endpoints included most TEG parameters. Thirty percent (15/50) of the RRGs in the placebo group had delayed postoperative bleeding starting 36 to 48 hours after surgery, compared to 10% (5/50) in the EACA group (P= 0.0124). On the TEG parameters, the slopes for R and K time were significantly different between treatment groups (P=0.050); the R and K time decreased over time in the EACA group after surgery, while they increased in the placebo group. The angle, MA, and G slopes were also significantly different between treatment groups (P=0.001, 0.001, and 0.006, respectively). The angle, MA, and G increased postoperatively over time in the EACA group, while they decreased in the placebo group. All these changes are supportive of hypercoagulability associated with EACA administration. Therefore, the postoperative administration of EACA significantly decreased the prevalence of postoperative bleeding in RRGs by increasing the clot strength.

Committee:

Guillermo Couto, DVM (Advisor); Mary McLoughlin, DVM (Committee Member); Julien Guillaumin, DVM (Committee Member)

Subjects:

Veterinary Services

Keywords:

Hemostasis; dog; fibrinolysis; antifibrinolytics; surgery.

Karnik, KetakiAccuracy of Computed Tomography in Determining Lesion Size in Canine Osteosarcoma of the Appendicular Skeleton
Master of Science, The Ohio State University, 2011, Comparative and Veterinary Medicine
Multidetector contrast enhanced computed tomography with acquisition of 0.625 mm thick axial transverse images was used to measure the extent of appendicular osteosarcoma (OSA) in 10 dogs. The measured length of tumor based on CT was compared to the true length of tumor using histopathology. There was good correlation of the true length of OSA compared to the length of intramedullary/endosteal abnormalities on CT with a mean overestimation of 1.8% (SD = 15%). There was poor correlation of the true length of OSA compared to the length of periosteal proliferation on CT with a mean overestimation of 9.7% (SD = 30.3%). There was poor correlation of the true length of OSA compared to the length of abnormal contrast enhancement by 9.6% (SD = 34.8%). The measured extent of intramedullary/endosteal abnormalities using sub-millimeter thick axial transverse acquisition of images with multidetector CT should be of value in assessing patient candidacy and surgical margins for limb spare surgery. It may also be useful for evaluating response to therapy in dogs that receive chemotherapy or radiation therapy when surgery is not performed.

Committee:

E Green, DVM (Advisor); Steven Weisbrode, DVM (Committee Member); Cheryl London, DVM (Committee Member)

Subjects:

Animal Diseases; Animal Sciences; Animals; Medical Imaging; Medicine; Veterinary Services

Keywords:

CT; computed tomography; OSA; osteosarcoma; multidetector; appendicular; canine; dog; neoplasia; bone

Burns, Teresa AThe equine metabolic syndrome: studies on the pathophysiology of obesity, insulin resistance, and laminitis in equids
Doctor of Philosophy, The Ohio State University, 2013, Comparative and Veterinary Medicine
Insulin resistance has been associated with predisposition to laminitis in horses and ponies; in humans, adipose tissue (AT) inflammation is associated with IR. The first series of studies reported here characterized inflammatory gene expression and leukocyte infiltration in several AT depots of insulin-resistant (IR) and insulin-sensitive (IS) horses. No differences in expression of TNF-a, IL-1ß, IL-6, PAI-1, or MCP-1 were noted between IR and IS groups for each depot. However, when data from IR and IS groups were combined for each depot, the expression of IL-1ß (p=0.009) and IL-6 (p=0.023) was significantly higher in nuchal ligament AT, suggesting that this depot is more likely to adopt an inflammatory phenotype than others. The results also suggested a role for MCP-2 as a macrophage chemoattractant in equine AT; however, there was no difference in total leukocyte content between depots. Within the digital laminae, vascular dysfunction and inflammation have been reported to play roles in laminitis associated with equine IR. The purpose of the next series of studies was to characterize laminar insulin receptor (IRc) expression and inflammation in ponies subjected to a dietary carbohydrate challenge. Laminar keratinocytes did not show significant expression of IRc under basal or fed conditions. Up-regulation of IRc expression in the laminar vasculature was observed acutely in response to carbohydrate challenge and accompanied hyperinsulinemia. Primary inflammatory events (including laminar inflammatory gene expression and leukocyte infiltration) were not associated with dietary carbohydrate challenge and do not appear central to EMS-associated laminitis. The purpose of the next study was to characterize the cellular localization and activation state of AMPK in liver, skeletal muscle, and digital laminae of ponies subjected to a dietary carbohydrate challenge. Western blot analysis for phospho(P)-AMPK demonstrated decreased laminar P-AMPK concentrations upon challenge with dietary carbohydrate (p = 0.01). In contrast, P-AMPK concentrations were unchanged in skeletal muscle (p = 0.33), and there was a trend for increased AMPK activation in the liver in obese ponies in response to dietary carbohydrate (p = 0.13). Unchanged or increased P-AMPK concentrations with increased caloric intake suggest IR in skeletal muscle and liver; the decreased laminar P-AMPK concentrations with CHO challenge indicate that the laminae remain IS. Laminar dysfunction in EMS is more likely due to local effects of hyperinsulinemia, not local IR/energy failure. The purpose of the final study was to characterize the morphology of the equine endocrine pancreas in response to dietary carbohydrate challenge. No effect of dietary carbohydrate was noted on ß-cell surface area (p = 0.12). The change in serum insulin concentration was significantly greater in the high carbohydrate-fed ponies than in controls (403.8 +/- 317.1 mIU/L vs. 1.00 +/- 4.03 mIU/L; p = 0.002); however, this variable was not correlated with total islet surface area (r = 0.32; p = 0.17) or ß-cell surface area (r= 0.25; p = 0.3). It will be important to assess both ß-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in EMS.

Committee:

James Belknap (Advisor); Ray Geor (Committee Member); Jilll McCutcheon (Committee Member); Ramiro Toribio (Committee Member); Chris Adin (Committee Member); Martha Belury (Committee Member)

Subjects:

Endocrinology; Veterinary Services

Blass, Keith AndrewEffect of ivabradine, a novel If current inhibitor, on dynamic obstruction of the left ventricular outflow tract in cats with preclinical hypertrophic cardiomyopathy: a single-dose study
Master of Science, The Ohio State University, 2013, Veterinary Clinical Sciences
A relevant subset of cats with hypertrophic cardiomyopathy (HCM) develop dynamic obstruction of the left ventricular outflow tract and mitral regurgitation secondary to hypertrophy of the left ventricle and systolic anterior motion (SAM) of the mitral valve. This leads to an increase in left ventricular systolic pressure, and thus wall stress, that may ultimately lead to progression of the disease. While prospective studies documenting the morbidity and mortality associated with dynamic obstruction have not been performed in feline HCM, it is possible that the results would be similar to human studies, in which SAM has been proven repeatedly to be a negative prognostic indicator in HCM, as well as an independent predictor of disease progression and sudden death. Beta-blockers are the primary medication used to reduce heart rate and relieve dynamic left ventricular outflow tract obstruction in cats with HCM, however, side effects or contraindications sometimes limit their use. Ivabradine is a highly selective If current inhibitor that exerts negative chronotropic effects without significant effects on inotropy, lusitropy, or dromotropy as documented in multiple species. Ivabradine has been studied in cats, and has been shown to have unique heart rate lowering properties. The drug is clinically well tolerated without any discernable side effects, has only minimal effects on central hemodynamics, has pharmacokinetic and pharmacodynamic properties that allow for twice daily dosing, and enhances left atrial function. The purpose of this study was to determine whether ivabradine, at a single dose of 0.3 mg/kg PO, could reduce or eliminate dynamic obstruction of the left ventricular outflow tract in cats with HCM. In this prospective, randomized, double-blind, active-control single dose study, 21 cats with preclinical HCM and dynamic left ventricular outflow tract obstruction received one dose of atenolol at approximately 2 mg/kg PO or one dose of ivabradine at 0.3 mg/kg PO. Baseline and 3-hour post-treatment heart rate, echocardiographic variables, and blood pressure were recorded. Statistical comparisons were made using analysis of covariance (ANCOVA). Peak velocity in the left ventricular outflow tract was significantly decreased compared to baseline for both drugs, however the mean change in velocity was more reduced for atenolol (2.54 m/s; 95% confidence interval 1.83 m/s to 3.25 m/s) compared to ivabradine (0.51 m/s; 95% confidence interval 0.01 m/s to 1.01 m/s; P<0.0001). Echocardiographic indices of systolic function were largely unchanged by ivabradine, but reduced by atenolol. Doppler systolic blood pressure was not affected by either drug. Predictably, heart rate was reduced by both drugs. These findings indicate that a single dose of ivabradine at 0.3 mg/kg mildly reduces dynamic left ventricular outflow tract obstruction in cats with HCM while preserving systolic function, but this reduction is inferior compared to that achieved by atenolol and is likely not clinically relevant. Further studies evaluating the utility of ivabradine in reducing dynamic obstruction over a longer time period are needed.

Committee:

Karsten Schober (Advisor); John Bonagura (Committee Member); Brian Scansen (Committee Member)

Subjects:

Veterinary Services

Keywords:

Ivabradine; Feline; Hypertrophic Cardiomyopathy; Systolic Anterior Motion; Dynamic Left Ventricular Outflow Tract Obstruction; Amyl Nitrite

Jugan, Maria ChristineEffects of Akkermansia muciniphila Supplementation on Markers of Intestinal Permeability in Dogs Following Antibiotic Treatment
Master of Science, The Ohio State University, 2017, Comparative and Veterinary Medicine
Diarrhea and other sequelae of gastrointestinal hyperpermeability are common complications of antibiotic therapy. Akkermansia muciniphila is a mucin-degrading bacterium, positively associated with gastrointestinal epithelial health and decreased permeability. The objectives of this study were to measure effects of oral Akkermansia administration on markers of gastrointestinal permeability following antibiotic administration. Eight healthy, purpose-bred dogs were randomized to receive either Akkermansia (109 CFU/kg; N=4) or vehicle (N=4) for 6 days following a 7-day course of metronidazole. After a 20-day washout, dogs were crossed-over to the alternate treatment. After an additional 20-day washout, the experiment was repeated with amoxicillin-clavulanate. Fecal Akkermansia qPCR and plasma concentrations (measured by ELISA) of cytokeratin-18, lipopolysaccharide, and glucagon-like peptides (GLP-1, GLP-2) were measured at baseline (T0), post-antibiotic (T1), and post-treatment (vehicle or Akkermansia; T2). For each antibiotic, absolute or delta concentrations were compared between time-points using paired samples t test. Akkermansia was detected in feces in 7/8 dogs following supplementation (T2) but not at T0 or T1. Delta (T2-T1) cytokeratin-18 after metronidazole was significantly lower on vehicle (-0.27 ng/ml) versus Akkermansia (2.4 ng/ml; p=0.03). Cytokeratin-18 concentrations tended to decrease from T0 to T1 on amoxicillin-clavulanate (p=0.05). Post-prandial GLP-1 concentrations (38.2 pM) were higher than pre-prandial (15.5 pM) concentrations. No adverse side-effects or other significant biomarker alterations were noted. Akkermansia muciniphila PCR detection suggested successful gastrointestinal transit following oral supplementation in dogs, with an effect on gastrointestinal epithelium based on plasma cytokeratin-18 alterations. Further study is needed to determine impact in dogs with naturally-occurring disease.

Committee:

Prosper Boyaka, PhD (Advisor); Chen Gilor, DVM, PhD (Committee Member); Joshua Daniels, DVM, PhD (Committee Member)

Subjects:

Veterinary Services

Keywords:

Akkermansia muciniphila; gastrointestinal permeability; antibiotics; canine; fecal score

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