Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Michael D. Apley / The discovery of modern antimicrobials in the early 20th century revolutionized treatment of infectious diseases. Less than 100 years later, antimicrobial resistance has become a global threat to public health. With the rise of antimicrobial resistance, the question that remains to be answered is: Can dosing regimens provide maximal clinical efficacy, yet minimize the development of antimicrobial resistance? A pharmacokinetic / pharmacodynamic approach was utilized to investigate oxytetracycline regimens that would impart efficacy while minimizing the potential for resistance development due to plasmid transfer. An in vitro pharmacodynamic model was used to quantify the response of a Pasteurella multocida isolate to two oxytetracycline dosing regimens. The PK/PD index most closely related to efficacy was the Cmax:MIC. The in vitro pharmacodynamic model was then used to investigate the effects of antimicrobial exposure on plasmid transfer. A mixed population of oxytetracycline-susceptible and resistant bacteria was exposed to two dosing regimens and plasmid transfer was quantified. When oxytetracycline concentrations exceeded the MIC of the recipient, development of resistance was suppressed. The same donor and recipient bacteria were used in an in situ swine model to validate the in vitro findings. Following surgical implantation of porous membrane straws containing the mixed bacterial population, animal subjects in the treatment groups received one of two oxytetracycline treatments. Oxytetracycline concentrations in the plasma and interstitial fluid were quantified. Plasmid transfer within the implant membranes was quantified and correlated to pharmacokinetic measures in the animal. Plasmid transfer rates in the implant membranes did not correlate to the investigated pharmacokinetic parameters. The study methodologies in this dissertation should serve as a foundation for future studies in antimicrobial pharmacokinetic/pharmacodynamic research. The results presented here show that the bacterial response to oxytetracycline can be optimized in a concentration dependent manner and that antimicrobial resistance development through plasmid transfer can be suppressed in vitro when oxytetracycline concentrations exceed the MIC of the recipient bacteria. These results suggest that a proper balance between clinical efficacy and minimizing antimicrobial resistance can be achieved for oxytetracycline through appropriate dosing regimens and drug formulations.

Master of Public Health / Department of Diagnostic Medicine/Pathobiology / Justin J. Kastner / David G. Renter / The history of veterinary medicine is intimately intertwined with duties to public health. This has remained true over centuries, and is becoming even more important with recent significant threats to public health. Despite this, the veterinary profession is failing to meet increasing needs for veterinarians trained in population medicine and public health, nationally and internationally. Current accreditation requirements for veterinary schools and colleges are vague with regard to public health education, leaving each college or school of veterinary medicine to implement its own perception of veterinary public health education. Is the public health education in veterinary curricula adequate among U.S. veterinary colleges and schools? Our inventory of the veterinary curricula in the 28 U.S. veterinary schools and colleges revealed inadequacies in veterinary public health education delivery. We found that most colleges and schools are lacking in the major veterinary public health subjects as recommended by the American College of Veterinary Preventive Medicine and the World Health Organization. The issue of inadequacies, even deficiencies, within health professional curricula is not unique to the veterinary profession. What have other health professions proposed to correct their own perceived deficiencies within their educational curricula? We identified deficiencies and proposed solutions from three health professions and discussed their solutions as potential approaches to remedy the inconsistency in public health delivery in veterinary curricula. The dental profession addressed lack of faculty effectiveness with faculty development programs. The medical profession identified an outdated and irrelevant pre-medical curriculum and is currently considering reforming it. The chiropractic profession identified a lack of public health education in its curriculum and organized a standardized public health course. Health professions are similar in content of curricula and length of professional program. In addition, the health professions have similar pre-requisites. They also share similar challenges: faculty shortages, high student debt loads, and rising educational costs. Because of these similarities, solutions to perceived curricular deficiencies proposed in one health profession can be used to address deficiencies in other health professions. Therefore, the dental, medical, and chiropractic professions have proposed solutions that should be considered in addressing the veterinary profession's curricular deficiency of inadequacy in public health education.

Master of Science / Department of Clinical Sciences / Robert L. Larson / Bradley J. White / We performed research that investigated the ability of three dimensional accelerometers to classify cattle behavior and also describe the circadian patterns within that behavior. The first of three studies (validation study) tested a decision tree classification system and its ability to describe behaviors of lying, standing, and walking. Classification accuracies for lying, standing, and walking behaviors were 99.2%, 98.0%, and 67.8% respectively, with walking behavior having significantly lower accuracy (P<0.01). This study also tested the accuracy of classifying the above behaviors using different device reporting intervals, or epochs. Reporting intervals of 3, 5, and 10 seconds (s) were evaluated in their ability to describe cattle behaviors of lying, standing, and walking. Classification accuracies for the 3s, 5s, and 10s reporting interval were 98.1%, 97.7%, and 85.4% respectively, with no difference in classification accuracy of the 3s and 5s epochs (P=0.73) while the 10s epoch exhibited significantly lower overall accuracy (P<0.01). This validated accelerometer monitoring system was then implemented in two studies (Winter 2007 and Spring 2008) where the devices were used to describe behavior patterns of beef calves in a drylot production setting. Lying behavior of the cattle was analyzed and found to be significantly associated (P<0.001) with hour of the day. Calves in these studies spent most (> 55%) of the nighttime hours (2000 to 0400) involved in lying behavior and spent the least percentage of time lying (<30%) during periods of time where feed was presented at the bunk (0700 and 1700). Mean lying time was also associated with trial day (P<0.01) and most trial days (67.5%) calves spending between 45% and 55% of time lying. Variation of lying behavior was found between individuals (range 29% to 66%); however, consistency in lying behavior was found within individual calves across study periods. The accelerometer monitoring system studies presented here provide evidence these devices have utility in recording behaviors (lying, standing, and walking) of individual beef calves raised in typical production settings.

Le souffle équin est une maladie inflammatoire chronique des petites voies respiratoires, très fréquente chez les chevaux gardés à l’intérieur avec de la paille et du foin moisi et poussiéreux. Les signes cliniques peuvent être prévenus par le contrôle de l’environnement et soulagés par l’administration de corticostéroïdes systémiques et inhalés. L’objectif de cette étude était de déceler les effets secondaires présents sur des chevaux atteints de souffle traités à la fluticasone (Flovent 250 μg HFA®, 2000 μg BID, pendant six mois, et puis 2000 μg SID, pendant six autres mois) par le cortisol sérique et la présence d’ulcères gastriques. Cinq chevaux exempts de maladie respiratoire et onze chevaux atteints du souffle ont été gardés à l’intérieur d’une écurie avec du foin moisi et de la paille dans le but de provoquer la maladie chez le groupe atteints du souffle. Une fois les chevaux atteints de souffle devenus symptomatique, ils ont été divisés en deux groupes : un premier groupe traité avec de la fluticasone, nourri avec du foin et gardé sur une litière de paille, et un deuxième groupe non traité nourrie avec de la moulée et gardé sur une litière de ripe, pendant six mois. Par la suite, les deux groupes ont été mis au pâturage. Le cortisol a été mesuré par Immunoessai enzymatique par chimiluminescence (CEIA, Immunolite 1000, Siemmens®) les 12e et 10e jours avant et les 7e, 28e, 80e, 160e, 200e, 250e, 290e et 320e jours après le début du traitement afin de déterminer le degré de suppression du cortisol sérique. On a également fait une suivi de la présence d`ulcères gastriques à l`aide de vidéo endoscopique. La fluticasone inhalée deux fois par jour cause une diminution du cortisol sérique les 28e, 80e et 160e jours, mais elle n’entraîne pas d’effets sur le score des ulcères gastriques. Les pellets de luzerne causent quant à elles, une augmentation du score des ulcères gastrique chez les animaux exempts de maladie respiratoire. / Recurrent Airways Obstruction (RAO) is a small airways inflammatory disease, very common in horses stabled in mouldy-dusty hay and straw environments. The clinical signs are prevented by environmental control, relieved by systemic and inhaled corticosteroids. Our objectives were to determine whether inhaled corticosteroids cause a suppression of cortisol levels and gastric ulceration in RAO horses treated with fluticasone 2000μg (Flovant HFA®) BID for 6 months and 2000μg SID for another 6 months. Five (5) healthy horses were used as controls and eleven (11) RAO affected horses were stabled in a moldy-dusty environment to induce disease exacerbation. Once they were symptomatic, they were divided into two groups, the treated group was kept on hay/straw and the untreated group was fed with pellets food and bedded on wood shavings six months. Afterwards, all horses were pasture for the next 6 months. Serum cortisol was mesured by Immuno-essai enzymatique par chimiluminescence (CEIA, Immunolite 1000®, Siemmens) 12, 10 days before and 7, 28, 80, 160, 200, 250, 290, 320 days after treatment initiation, in order to determine cortisol suppression. Fluticasone administered twice a day reduces blood cortisol levels after 28, 80 and 160 days, but did not cause any change in gastric ulcers. However, pellets slightly increased gastric ulcer scores in healthy horses.

Chez le chien, les thromboses représentent une complication majeure de nombreuses conditions qui sont revues dans ce manuscrit. L’arsenal thérapeutique actuel présente certaines limites: des effets anticoagulants variables d’un patient à l’autre, des hémorragies et une administration par voie sous-cutanée pour l’héparine. Le rivaroxaban est un nouvel anticoagulant oral approuvé pour la prévention et le traitement des thromboses chez l’humain. C’est un inhibiteur direct du facteur Xa. La présente étude a pour objectif d’évaluer les effets hémostatiques du rivaroxaban chez des chiens en santé, en utilisant les tests de coagulation suivants: temps de prothrombine (PT), temps partiel de thromboplastine (aPTT), activité anti-facteur X, génération de thrombine (GT) et thromboélastographie (TEG®). Tout d’abord, l’effet anticoagulant du rivaroxaban a été évalué in vitro : le plasma citraté pauvre en plaquettes provenant de 20 Beagle en santé a été aliquoté et enrichi avec des solutions de rivaroxaban à des concentrations de 0 à 1000 mg/L d’anticoagulant. Une prolongation concentration-dépendante de tous les tests de coagulation a été notée. Les concentrations de 0.024 et 0.053 mg/L diminuent respectivement de 50% la vitesse de propagation de la GT et la densité optique de l’activité anti-facteur X. Ces derniers tests sont les plus sensibles et précis pour détecter l’effet anticoagulant du rivaroxaban. Ensuite, 24 Beagle en santé ont été répartis aléatoirement en 3 groupes (n=8). Chaque groupe a reçu par voie orale un placebo, ou 20 mg de rivaroxaban une ou deux fois à 8h d’intervalle. Quinze échantillons sanguins ont été prélevés pour chaque chien sur 30 heures. Pour tous les tests de coagulation excepté la TEG®, une différence significative a été notée dans les résultats entre les groupes traités et le groupe placebo (p<0.0001). La durée de l’effet anticoagulant du rivaroxaban était de 7.9-18.7h dans le groupe traité une fois; et de 17.5-26.8h dans le groupe traité deux fois. Le pic d’action de l’effet anticoagulant était d’environ 2h. Seul le paramètre R de la TEG® était significativement affecté dans les groupes traités. En conclusion, le rivaroxaban exerce un effet anticoagulant chez le chien à la dose de 2 mg/kg. Une administration biquotidienne semble appropriée pour un effet de 24h. / In dogs, thrombosis is a major complication detected in many conditions. The limits of the current available anticoagulants in veterinary medicine are their variable effects from one patient to another, bleeding complications and subcutaneous injections for heparin administration. Rivaroxaban is a novel oral anticoagulant approved for the prevention and treatment of thrombosis in humans. It is a direct factor Xa inhibitor. The objectives of the study were to determine the haemostatic effects of rivaroxaban in healthy dogs by evaluating the following coagulation assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), anti-factor X activity, thrombin generation (TG) and thromboelastography (TEG®). An in vitro study was conducted: citrated platelet poor plasma from 20 healthy Beagles was aliquoted and mixed with rivaroxaban to obtain solutions ranging from 0 to 1000 mg/L of the anticoagulant. Rivaroxaban exerted a concentration-dependent anticoagulant effect. Rivaroxaban solutions at 0.024 and 0.053 mg/L cause 50% inhibition of the propagation of TG and of the optical density of anti-factor X activity respectively. These assays were the most sensitive to detect the anticoagulant effect of rivaroxaban. Secondly, 24 healthy Beagles were randomly divided in 3 groups (n=8) and received one placebo pill orally, or 20 mg rivaroxaban once or twice at 8h interval. Fifteen citrated blood samples were collected from each dog over 30h. For each coagulation assay except for TEG®, there was a significant difference in assay results between placebo and rivaroxaban groups (p<0.0001). The duration of the rivaroxaban anticoagulant effect was 7.9-18.7h in the group receiving rivaroxaban once, and 17.5-26.8h in the group receiving rivaroxaban twice. The peak of action of rivaroxaban appeared 2h after the dose. Only R parameter of TEG® was significantly affected by rivaroxaban administration. To conclude, rivaroxaban is an efficient anticoagulant in healthy dogs at 2 mg/kg. A twice daily administration seems appropriate to exert a 24h anticoagulation.

Master of Science / Department of Anatomy and Physiology / Daniel C. Marcus / Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner’s membrane may play a role in the control of luminal Cl-. We tested for the presence of Cl- transport pathways in isolated mouse Reissner’s membrane using whole-cell patch clamp recordings and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl- channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+, and intracellular ClC-2 antibody. Channel transcripts expressed in Reissner’s membrane include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This thesis incorporates the publication by K.X. Kim and D.C. Marcus, Inward-rectifier chloride currents in Reissner’s membrane epithelial cells. Biochem. Biophys. Res. Commun. 394 (2010) 434-438, with permission of the publisher Elsevier, and is the first report of conductive Cl- transport in epithelial cells of Reissner’s membrane and is consistent with an important role in endolymph anion homeostasis.

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

Avian leukosis/sarcoma retroviruses can infect chickens and cause various diseases, malignant tumor formations and mortality in infected chickens. The ALSV group of viruses consists of the endogenous virus of subgroup E which occurs in all chickens and does not cause disease but can interact with exogenous viruses of subgroup A,B,C, and D and cause disease in chickens. Avian leukosis viruses cause high economic losses in the poultry industry since infected chickens not only develop neoplasia but also have decreased production efficiency. Besides chicken to chicken horizontal transmission of ALSV viruses, infected chickens can pass the virus congenitally at a high frequency to the egg. To prevent transmission, the poultry industry must effectively identify infected hens and eggs, and remove them from the breeding populations. Current testing methods for ALSV identification are time consuming, lack sensitivity or specificity, or are ineffective in clearing ALSV infection from potentially infected chicken flocks. The objective of this investigation is to develop an RT-PCR detection system that could effectively screen for viral presence in chicken eggs. To achieve this goal, the aims of this research is as follows: (1) to develop a reverse transcription and polymerase chain reaction assay to detect avian leukosis/sarcoma retroviruses in egg albumen, (2) to perform a survey of the handling and storage conditions of commercial chicken eggs in retail stores in New Orleans, LA., and (3) to determine the molecular prevalence of avian leukosis virus in commercial chicken eggs using the RT-PCR detection system A RT-PCR detection system was developed to detect avian leukosis/sarcoma virus in egg albumen obtained from two strains of White Leghorn chickens naturally exposed to these viruses. A modified RNA isolation procedure using the Promega kit for RNA isolation was employed to isolate viral RNA from egg albumen extracted from eggs obtained from two commercial stocks of White Leghorn chickens. In the samples tested, compared to the 45% sensitivity of the IFA method which is commonly used in the poultry industry for ALSV detection, the RT-PCR technique has a sensitivity of 97% and a specificity of 100%. Sequence analyses confirmed avian leukosis viral presence in the RT-PCR generated product. The survey involving the handling and storage conditions of eggs in New Orleans retail store indicated that 75% of the retail stores hold eggs at a temperature compatible with ALSV survival in the eggs. In addition, 22% of participating retail stores reported using cracked eggs for preparing food for self or consumer use. The survey provided a sampling frame for conducting a study of avian leukosis/sarcoma virus prevalence in commercial chicken eggs. Based on a 20% estimate of avian leukosis viral prevalence, 240 eggs were randomly sampled from 20 retail stores in New Orleans, LA. RT-PCR analyses of the eggs indicate that there is at least a 14% prevalence of avian leukosis virus in commercial chicken eggs. In addition, 80% of the egg cartons in the New Orleans area contain an average of two eggs infected with ALV. Therefore, individuals consuming eggs are at risk of exposure to these poultry oncogenic retroviruses / acase@tulane.edu

In the research reported here, the pathology, virulence, and strain differences of Taura syndrome virus (TSV) was studied. Initial studies on TSV pathogenesis compared the survival of juveniles of a highly Taura syndrome (TS) susceptible line of Penaeus vannamei, a line of TS resistant P. vannamei, and an innately TS resistant P. stylirostris line following TSV challenge by feeding (per os) or injection methods, in the absence of horizontal transmission via cannibalism and/or absorption from the water. Per os_TSV challenge resulted in I00% survival in P. stylirostris, but challenge by per os exposure produced significant mortality commencing on about the same post-exposure day in both SPF and SPR P. vannamei (P < 0.001), suggesting that P. stylirostris is significantly (P < 0.001) more resistant toper os TSV infection and presentation of TS disease than either SPF or SPR P. vannamei. The potential roles of the cuticular lining of the stomach and hindgut and unlined portions of the gut in TSV resistance in penaeid shrimp are discussed as factors where an innate resistance mechanism was postulated to explain the observed differences between the different species and populations of shrimp in TSV susceptibility. To investigate apparent TSV strain differences, three geographic and year isolates of TSV from naturally occurring TS epizootics of cultured penaeid shrimp were obtained from Mexico (SIN98TSV and MX99TSV from P. vannamei and SON2KTSV from P. stylirostris) and one TSV isolate from Belize, Central America (BLZ02TSV from P. vannamei) were analyzed and compared to the reference TSV isolate (HI94TSV) by selected TSV diagnostic and genetic analysis methods. The results show that screening of penaeid shrimp broodstock and postlarvae by MAb I Al testing will not detect all TSV isolates, possibly leading to false negative results, further spread of TSV and re-emergence of TS in regions where it has been eradicated. The putative VP1 antigenic epitope recognized by TSV MAb 1A1 is identified, with SIN98TSV and BLZ02TSV having 70.0% and 80.0% AA homology, respectively, within the 10 AA region. There are three distinct electropherotypes and 'serotypes' of TSV, with electropherotype A (TSV Etype-A) and serotype A (TSV-A) representing those TSV isolates conforming to VP1 properties of the Hawaiian 1994 TSV isolate, electropherotype B (TSV Etype-B) and serotype B (TSV-B) representing those TSV isolates conforming to the VP1 properties of the Sinaloan 1998 TSV isolate, and electropherotype C (TSV Etype-C) and TSV serotype C (TSV-C), representing those TSV isolates conforming to the VP1 properties of the Belize 2002 TSV isolate. In a parallel activity, the University of Arizona (UAZ) Aquaculture Pathology Diagnostic Laboratory (APL) Case Database (DB) and the UAZ Aquaculture Pathology Diagnostic Laboratory Client Address Book Database (AB), relational databases, were created using FileMaker Pro software, are used to keep an up to date and accurate record of all UAZAPL diagnostic and research case and client information and may be searched and sorted to find case data and/or client information of interest.

Clostridium difficile-associated disease (CDAD) in neonatal pigs has emerged as a serious economic concern for swine producers throughout North America. The disease has been diagnosed clinically and reproduced in experimental inoculation trials in pigs, but little is known of the epidemiology or pathogenesis of the disease in pigs. Strain characteristics and distribution of C. difficile isolates from pigs, calves, dogs, horses, and humans were assessed by PCR-ribotyping. Porcine and bovine isolates were dominated by a single ribotype. This ribotype was uncommon among isolates from other host species; it was particularly uncommon from humans, suggesting there is little transfer of isolates between humans and calves or pigs. The reason for a single common ribotype circulating among distinct bovine and porcine populations is unknown. The intragastric inoculation of newborn pigs demonstrated their sensitivity to C. diffcile toxins. Toxin B (TcdB) surprisingly resulted in more severe lesions than Toxin A (TcdA). The two toxins together acted synergistically. Colon explants were sensitive to TcdA in a dose-dependent manner. However, TcdB did not cause significant lesions in the explants, nor was there any synergism with TcdA. Electron microscopy of colon explants treated with TcdA revealed severe, ultrastructural lesions that accrued in a dose-dependent manner by two h post infection. Direct immunohistochemistry assays demonstrated specific binding of biotinylated TcdA throughout the gastrointestinal tract of neonatal pigs. The density of bound toxin in different segments correlated with the severity of lesions in those segments from pigs gavaged with TcdA. TcdB did not bind any tissues, though it was fully active in cell-culture assays. A monoclonal antibody to Galalpha1-3beta1-4GlcNAc-R (alpha-Gal epitope), a putative receptor for TcdA in pigs, specifically bound the brush border of enterocytes, but the distribution of binding did not correlate with the distribution of TcdA binding. Specific TcdA binding to the plasmallema of microvilli was also confirmed by immunoelectron microscopy. By five min post inoculation some toxin was already visible in endosomes or free in the cytoplasm. TcdA localized to the mitochondria of epithelial cells and, less frequently, to the nuclei. Endothelial cells and leucocytes in the superficial lamina propria were similarly labeled by toxin.