2008 Research Programs Dr. Sue Hudson Duran Development of Transdermal dosage forms in horses and deer. Phenylbutazone+ enhancer formulations will be prepared and placed over joints of horses and deer.  The joints will be tapped under sterile aseptic technique and samples assayed by HPLC for phenylbutazone.  The objection is to teach a student how to prepare and test transdermal delivery drugs under good manufacturing practice guidelines.  The student will learn to use a dermatome, Franz- diffusion apparatus and a HPLC.  The student will also learn about animal welfare protocols and animal testing of the drug which includes blood sampling and synovial fluid sampling under the supervision of Dr. John Schumacher. The student will also learn how to design a drug study and documentation necessary for FDA pre-approval of drugs. The studies will be conducted in the laboratory at the pharmacy school, the multi-purpose large animal research laboratory and in the equine research barn at the Auburn School of Pharmacy and the Auburn Large Animal Clinic. Dr. Betsy Welles          Clinical Pathology Laboratory Projects There are a few projects in which students could participate. 1) Comparison of multiple different methods of determination of fibrinogen in horses.  Summer of 2006 - 20 healthy horse samples were analyzed for reference intervals and 20 sick horse samples were collected.  Summer of 2007 - 40 additional sick horses had samples collected and analyzed.  Statistical analysis and comparison of these data are a great summer project. 2) Comparison of manual reticulocyte determination and values derived from Advia 120 Hematology instrument.  New Methylene Blue stained smears from 100 anemic dogs and >50 anemic cats have been made.  Advia run screens and UVIS CBC reports have been saved on these samples.  The student project would be manual determination of reticulocyte counts and then statistical analysis and comparison of manual data with Advia data. 3) Continued acquisition of cases and images for the web-based histopathology/cytology teaching project would be another project.  The student project would be to select a theme (such as patients with liver disease or renal disease) and compare and contrast histologic and cytologic findings. 4) Further investigation into the use of actual parameter measurement vs. urine dipstick data.  Raw data were analyzed in 2007, but urine parameter:urine creatinine ratios (for glucose and bilirubin) need to be analyzed, compared with serum parameter data and the patient disease process. 5) Urine enzyme activities (alkaline phosphatase and GGT) were determined on all samples collected in 2007.  Our hypothesis is that detection of increased urine enzyme activities would be useful in detection of early renal tubular disease.  Minimal analysis of these data have been done.  A student project would be to analyze the data presently available (of which most samples are from patients without renal disease) and to specifically collect urine samples from patients with renal disease for comparison. Dr. Haroldo Toro Avian Diseases Several projects are available in the area of avian diseases.  In the area of viral diseases students will work with experimentally infected birds and assess disease progression as well as interaction between viruses inducing immunodeficiency in chickens. In the area of vaccine development students will work with vaccinated chickens and assess immune responses in the laboratory. In the area of bacteria students will work with bacteriophages and Salmonella. In the area of avian immunology students will work with mucosal immunity.Work involves inoculation of chickens with avian pathogens or vaccine constructs, and evaluation of gross pathological changes and sampling. In the laboratory students will be involved in serological testing of vaccinated birds, other assays such as ELISPOT and immunohistochemistry, virus isolation, and virus detection by PCR. Dr. Frederik W. van Ginkel The activities of my research project for 2008 will focus on measuring the immune response to avian pathogens or to vaccines specific for avian pathogens in Harderian glands or other relevant tissues. These approaches will help address the role of mucosal immune response in protection of chickens to diseases such as influenza virus and IBV. Dr. Curtis Bird Project 1.  Cloning and Expression of Canine Genes Associated with Mammary Cancer.  Cancer is a genetic disease caused by the accumulation of mutations in those genes which regulate cell proliferation and the death pathway.  Cells that escape these controls can become neoplastic.  Students will clone new genes thought to play important roles in the regulation of these events and that have potential as targets of gene therapy.  Students will design there own polymerase chain reaction assays from the canine genome and optimize such assays to detect mRNAs expressed in canine mammary cancer cells.  The amplicons will then be cloned and sequenced and expression analyzed in cell lines derived from canine mammary tumors. Project 2.  Characterization of Gene Expression in Canine Mammary Cancer Cells by 5-Color Flow Cytometry.  Cancer is a genetic disease caused by the accumulation of mutations in those genes which regulate cell proliferation and the death pathway.  Cells that escape these controls can become neoplastic but this phenotype has been reversed through gene therapy.  Target cells will include canine mammary tumor cells expressing transgenes which our lab has created.  Students will label individual cloned cell lines with up to 5 fluorochrome-conjugated antibodies and perform high speed single cell flow cytometry analysis of gene expression and the changes induced by trans-gene expression. Dr. Anne A. Wooldridge My lab is interested in smooth muscle signaling pathways in normal and diseased equine and bovine laminar vessels.  I am particularly interested in calcium sensitization and desensitization pathways.  Proposed projects in the lab involve using laminar tissue collected from normal horses or cows (euthanized) and investigating calcium sensitization and desensitization pathways using a small vessel wire myograph, and evaluating expression/activation of proteins involved in these pathways.  Enzyme activity assays, immunoblot analysis with phosphospecific antibodies, and real-time PCR are all techniques that will be employed to evaluate these pathways.  Eventually, laminitis will be experimentally induced in and samples collected and compared to the normal samples.    Dr. Dean Schwartz The incidence of diabetes mellitus and obesity are rapidly rising to epidemic levels in the United States and worldwide.  The cardiovascular complications that arise from the diabetic state are the leading cause of death in diabetic patients and persons with diabetes mellitus are at a much greater risk for heart attacks, strokes and high blood pressure. Oxidative stress or the generation of reactive oxygen species (ROS) is a contributing factor to the progression of many disorders and is at the forefront of research in diabetes and cardiovascular disease.  Students enrolled in the summer program will participate in a project looking at the protective effects of agents that decrease reactive oxygen species generation on cardiac oxidative stress, inflammatory response and cardiac mechanical function in diabetes. Dr. Vicky van Santen Avian Virology Our laboratory currently focuses on interaction of avian viruses with their hosts at the cell and organism levels. Projects in which a student might participate include: Project 1. Significance of minor viral subpopulations within infectious bronchitis virus vaccines. Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, and causes disease in commercial chickens in spite of intensive vaccination programs. Our sequence analysis of live-attenuated IBV vaccine strains of a particular serotype (propagated in chicken embryos) show that they are genetically heterogeneous and that a minor subcomponent of the vaccines is selected in chickens. The subpopulation selected in chickens is more similar to the virulent parent of the attenuated vaccines. Different vaccines contain different proportions of the subpopulation selected in chickens. The summer student will participate in a project to explore the consequences of the different population structure of the vaccines for pathogenicity, persistence, and stimulation of immune response in chickens. Activities might include collection of samples from chickens, isolation of RNA, RT-PCR, sequence analysis, and ELISAs. Alternatively, the summer student will participate in a project to explore the selection process in a tracheal organ culture system. Project 2. Molecular determinants of host cell specificity of chicken anemia virus. Chicken anemia virus (CAV) infects and depletes hemoblastocysts in the bone marrow and precursor T cells in the thymus, leading to transient anemia and immunosuppression. Immunosuppression due to this ubiquitous virus is thought to contribute to diseases caused by other infectious agents in commercial chickens. CAV replicates only in certain transformed lymphoblastoid cells in culture. The summer student will participate in a project to explore the basis for the cell specificity of CAV, which is not understood. Activities will include tissue culture, monitoring of viral replication by quantitative PCR, generation of CAV mutants by PCR, and DNA sequencing.  Dr. Barbara Kemppainen The research project involves testing if a medicinal plant (American skullcap) with antioxidant properties is able to protect chickens and goats from the toxic effects of a clinically important fungal toxin (aflatoxin).  The study involves dosing chickens and goats with (1) control feed, (2) skullcap, (3) aflatoxin and (4) aflatoxin and skullcap,and measuring parameters that indicate if the chickens are protected by this medicinal plant.  Procedures will include orally dosing chickens (gavage) daily with treatment for 21 days, measuring antioxidant capacity and total oxidative stress markers in blood and tissues, evaluating gross and microscopic changes in tissues, measuring changes in immune function,  and performance (feed intake, weight gain, feed conversion efficiency).  If time permits, studies will be done to determine if this medicinal plant protects chickens and goats from the toxic effects of another clinically important fungal toxin (fumonisin). Dr. Mahmoud Mansour Proposed project: Central melanocortin activates neural pancreatic MC4R to regulate insulin and glucose levels Project summary: Our laboratory will be involved in a project that addresses the effect of pancreatic neural melanocortin-4 receptor (MC4R) activation on glucose and insulin levels. MC4R, a G protein-coupled receptor, is primarily expressed in the brain and is essential for long-term maintenance of energy balance in rodents and human.  Its activation in the brain by the anorexigenic agonist α–melanocyte-stimulating hormone (-MSH) is under influence of leptin signal transduction. Leptin potentiates neural circuits that decrease food intake (MSH/MC4R as part of the melanocortin system) and inhibits neural circuits that increase feeding behavior (neuropeptide Y and agouti-related protein).  In wild-type rodents, evidence suggests that central MC4R agonists reduce glucose levels but neither the mechanism nor the role of peripheral pancreatic MC4R is known. PCR and immunohistochemical data generated in our laboratory indicated that MC4R mRNA is strongly expressed in the pancreas and its protein is localized at basal level in autonomic nerve axons. We thus hypothesize that central melanocortins activate pancreatic MC4R resulting in reduced glucose levels independent of leptin signaling.  The study will elucidate the role of pancreatic MC4R in coupling of energy intake and peripheral insulin action. Understanding such a link is essential for the development of therapy that targets obesity and type 2 diabetes. Prospective student will be trained in techniques such as nucleic acids isolation, Reverse Transcriptase and Real-time PCR, Immunohistochemistry and Western Blotting. Dr. Robert Judd and Dr. Eric Plaisance Our laboratory is primarily focused on the regulation of adiponectin trafficking and secretion.  Adiponectin is a protein secreted by adipose tissue which has been shown to possess insulin sensitizing and anti-atherogenic properties.  Serum adiponectin concentrations are low in conditions such as obesity, diabetes and cardiovascular disease and have recently been shown to be reduced in cigarette smokers.  However, little is known about the cellular and molecular events responsible for the reduction in adiponectin secretion and/or gene expression.  We hypothesize that each of these conditions is associated with a reduced intracellular adiponectin trafficking capacity leading to reduced serum adiponectin levels.  Therefore, our studies in Summer 2008 will be focused on the mechanisms by which obesity and cigarette smoking and/or nicotine influence the trafficking and secretion of adiponectin. We have previously shown that niacin (nicotinic acid or vitamin B3), effective as a cholesterol lowering agent at pharmacological doses, also increases adiponectin levels dramatically in humans and in adipocyte cell culture.  Another goal for Summer 2008 will be to identify the downstream mediators of adiponectin secretion and/or gene expression following niacin administration.  The prospective student will be exposed to a number of adipocyte cell and molecular biology techniques including: 3T3-L1 and primary adipocyte cell culture, rodent handling and care, electron microscopy, confocal microscopy, real-time polymerase chain reaction, Western blotting, ELISAs, glycerol colorimetric assays. Dr. Dan Givens Understanding the impact of immune response on the development of persistent Testicular Infection with bovine viral diarrhea virus (BVDV).  Bovine viral diarrhea virus causes significant respiratory, gastro-intestinal and reproductive disease in cattle within the United States.  Due to the negative impact of BVDV on animal health, the similarities of clinical signs to foreign animal diseases, and the inconsistency with which vaccines have been able to control infection, there is informed support for development and implementation of eradication programs for BVDV in this country.  Recent research indicates that bulls which slowly develop a humoral immune response to vaccination with modified-live virus may maintain persistent testicular infections with BVDV.  Thus, the purpose of this project is to better understand the impact of immunosuppression and the role of cell-mediated immunity in the development of prolonged testicular infections.  This project will include animal handling, sample collection and bench top laboratory work including cell culture, virus isolation, virus neutralization, reverse transcription nested PCR and quantitative PCR.  The expectation will be for the student to compose an abstract and present a poster at the summer Merck-Merial research forum and a second national meeting.  Further information on Dr. Givens’ research program is available at http://myprofile.cos.com/givens5 Dr. Ya-Xiong Tao My lab works on two melanocortin receptors expressed in the brain. These receptors are critically involved in regulating energy homeostasis. Dysfunctions in these receptors cause obesity. Agonists for these receptors can potentially be used for the treatment of obesity. We have cloned these receptors from cat and are performing pharmacological characterizations on these receptors. The project for the summer scholar will be to perform in situ hybridization and immunohistochemistry to localize the expression of these receptors in the cat brain. We hope to obtain data at both mRNA and protein levels. These data will be very important in elucidating the potential functions of these receptors in the cat. Dr. Douglas R. Martin Therapy Development for Neurodegenerative Disease Dr. Martin’s laboratory focuses on therapy of neurodegenerative disease using gene therapy, stem cell transplantation and/or small molecule therapy. The laboratory’s model of neurodegenerative disease is feline gangliosidosis, a disorder in which abnormal functioning of lysosomes causes progressive nervous system dysfunction and neuronal death.  Although gangliosidosis is fatal in humans, new therapies based on gene and/or stem cell therapy have been tested in mouse models of gangliosidosis with excellent results. Before inclusion in human clinical trials, new therapies are tested in the feline model for safety and therapeutic benefit. The laboratory employs a variety of experimental techniques, ranging from intracranial injection of therapeutic agents to biochemical and molecular biological evaluation of therapeutic effectiveness. Students may participate in both experimental procedures and laboratory research. Dr. Bruce F. Smith Molecular Genetics Of Inherited Disease And Cancer.  Several projects are available in the area of gene therapy for muscular dystrophy and a variety of cancers.  In the area of muscular dystrophy, students will work directly with affected and carrier dogs assessing the disease and its progression.  In addition, the latest gene therapy vectors are now being tested in affected dogs, and students will have the opportunity to assist in evaluating the efficacy of these treatments from the level of the whole dog to the expression of specific genes.  Cancer projects include laboratory studies and pre-clinical and clinical trials for dogs with osteosarcoma, lymphoma, melanoma and breast cancer.  These studies involve the administration of gene therapy vectors, and the assessment of patient progress, as well as detailed laboratory assessments of the impact of the therapy.  Projects involve the use of a wide variety of techniques including RNA and DNA isolation, quantitative PCR amplification, cell culture and flow cytometry as well as animal handling, phlebotomy, bone marrow aspiration, tissue biopsy and necropsy. Dr. Mary K. Boudreaux Inherited Diseases of Platelets. My laboratory is involved with evaluating inherited platelet disorders in dogs, horses, and cows at the functional, biochemical, and molecular level.  Students working in my laboratory would have exposure to a broad array of experiences ranging from blood collection, platelet isolation, platelet function testing, DNA isolation, PCR techniques, and flow cytometry.  Dr. Dawn Boothe The epidemiology and phenotypic and genotypic expression of Escherchia coli resistance associated with antimicrobial therapy in dogs.   We continue to investigate the role that antimicrobial therapy has in the advent of antimicrobial resistance in dogs and cats, using two approaches.  A. Spontaneous disease. Using E coli as our sentinel organism, and in partnership with IDEXX® laboratories (who supplies the isolates and coordinates questionnaires),  we are characterizing the geographical relationship of resistant isolates collected from dogs or cats throughout the United States. E coli  isolates (both resistant and non-resistant) are subjected phenotyping using an  E-test® to determine the level (eg, high or low; single verus multiple step) of susceptibility and a  standard (Viotek®) antibiogram  to determine patterns of resistance (eg, single versus multiple drug; drug “partnerships”, etc). Isolates with similar phenotypes are subjected to genotyping of both chromosomal DNA, and, if present, plasmid DNA. Fingerprinting techniques involve pulse field and standard electrophoresis, and PCR. Molecular techniques will be used to determine the mechanisms by which resistance has developed toward each drug or drug class,or drug “partners”. We are also investigating the relationship between antimicrobial prescribing patterns and the advent of specific mechanisms of resistance. Further, we are investigating the role of patient factors (signalment, disease, etc) in the advent of resistance. Tools for the latter include questionnaire based data collection methods. Finally, using experimental dogs, molecular techniques and antibiograms,, we are evaluating the rapidity with which resistant coliform microbes emerge in the gastrointestinal tract once routine use of common antimiocrobials is instituted, and determining the time that must lapse before the flora returns to normal, after the antimicrobial has been discontinued. B. Experimental Model  We have induced resistant in 100% of fecal Escherichia coli in normal dogs following 3 to 9 days administration of either amoxicillin or enrofloxacin. We continue to characterized the resistance phenotypically and genotypically using techniques described for spontaneous disease. The advantage of the model is the controlled exposure under clinically relevant conditions yielding a limited number of phenotypes and genotype patterns. Student responsibility: The summer student will be asked to generate a testable hypothesis relating to the phenotypic or genotypic expression in E coli resistance. The hypothesis must be one that can be largely completed within the 11 week period of the fellowshipy. Flexibility in the hypothesis is intended to match student exposure to molecular and non-molecular laboratory techniques. Dr. Valery A. Petrenko Phage-derived Diagnostic and Therapeutic Probes for Breast Cancer Cells.  The laboratory is developing phage-derived molecular probes for monitoring, detection and treatment of cancer diseases using drug and gene targeting techniques.  We have constructed phage display libraries, designed protocols for selection of cancer cell-binding phage clones and developed phage-based imaging probes and therapeutics.  Students working on this project will develop phage probes with enhanced specificity and selectivity for breast cancer cells.  They will learn how to select cancer cell-specific binding phages from phage libraries, amplify phage, sequence their DNA, prepare phage probes and study their performance in reactions with the target and non-target cancer cells. Dr. Michael H. Irwin and Dr. Carl A. Pinkert: Genetic engineering and transmitochondrial animal models. Ongoing studies revolve around innovative approaches toward the development of transmitochondrial species including mice, swine and cattle.  Using gene transfer techniques targeting the mitochondrial DNA (mtDNA) genome, we hope to create animal models of: 1) severely debilitating (and often lethal) human mitochondrial disorders caused by mitochondrial mutations and 2) increased production efficiency - harnessing mitochondrial genetics toward greater cellular and metabolic performance.  Such animal models will be used to provide a greater understanding of mitochondrial dynamics and pave the way for new gene therapy approaches in a host of species. Summer projects will focus on the creation and subsequent characterization of mouse and livestock models for targeted human mtDNA diseases and metabolic production / trait modification in domestic species. Dr. Edward E. Morrison Olfactory Systems My laboratory focus is on the main and accessory olfactory systems.  Projects are directed at the neuroanatomy, physiology and neural development of these systems.  Additional projects examine the olfactory neural stem cell population, the role of the olfactory system in diabetes disease process, canine olfaction and pathogen-host interaction at the neuron level.  Students will learn and be exposed to light microscopy techniques (bright field, epifluorescence, confocal) electron microscopy and immunocytochemical techniques. Dr. Tatiana I. Samoylova Targeted delivery to the brain The goals of Dr. Samoylova's laboratory are directed towards development of molecular technologies that can be applied to improved diagnostic procedures and therapies of companion animal diseases. The main emphasis of the group is on brain research. We focus on phage display-based strategies by which diagnostic and therapeutic agents can be targeted directly to specific areas of normal brain and to malignant brain tumors providing the most precise diagnoses, improving drug effectiveness, and eliminating side effects caused by indiscriminant drug delivery. Throughout the years, we have developed targeting peptides for various animal and human brain cell types and tissues, including normal astrocytes, microglia and different glial malignancies. Our current research projects on brain therapies include development of targeting peptides for drug delivery to brain tumors and identification of cell-specific biomarkers for brain tumor stem cells. An additional project was designed to develop methods for brain delivery of protein drugs for treatment of lysosomal or other degenerative brain diseases using transporter peptides that cross blood-brain barrier. Dr. Benson T. Akingbemi There is growing public concern that chemicals in the environment (food, air, water), which have estrogenic properties, may have adverse effects on reproductive health.  Acting via steroid hormone receptors (androgen and estrogen receptors, AR, ER), these compounds alter the endocrine profile and are designated endocrine disruptors.  In this regard, the increasing interest in consumption of soy-based food products as a low-fat source of protein has raised the question of whether the estrogenic properties and potential reproductive toxicity of isoflavones, which are present in soybeans, need further attention. Of interest, approximately 750,000 infants fed soy-based formula in the United States each year are exposed to the phytoestrogens genistein and daidzein, the predominant isoflavones in soybeans. Isoflavones exhibit estrogenic activity (i.e. act as phytoestrogens) and if consumed for extended periods and/or at high levels would be expected to exert reproductive toxicity.  Similarly, the industrial chemical bisphenol A (BPA) is known to have estrogenic properties, and is used in the manufacture of polycarbonate plastics, epoxy resins, and flame retardants.  In mid 2004, U.S. BPA production volume by the six BPA and four polycarbonate plants was 1.024 thousand metric tons, up from 7.26 billion grams in 1991.  Thus, exposure of the population to BPA is significant.  Using the rat model and in vitro assays of primary Leydig cell cultures, experiments will be performed to determine which phytoestrogen doses are compatible with, versus harmful to, testicular steroidogenesis. Dr. Debra Ruffin, Dr. Bill Brawner, and Dr. John Hathcock Characterization of Blood Flow to the Equine Hoof The goal of this study is to fully evaluate the vascular pattern and integrity of horses with different hoof mass and shape.  This study is based on previous studies performed by Dr. Robert Bowker of Michigan State University.  Bowker hypothesizes s that anatomical differences in hoof mass and structure are associated with anatomical and quantitative differences in the perfusion of the hoof.  This study has great implications into the etiology, prevention and treatment of equine navicular disease and laminitis.  The student would be assisting us with evaluation of the perfusion of the equine digit in 18 horses. The following techniques will be performed on each research horse; radiographs of the digit, Doppler ultrasound of the palmar digital artery, electromyography of the deep digital flexor muscle, scintigraphy of the hoof after labeling red blood cells with Tc99, CT angiography of the distal limb and contrast venography of the hoof. This opportunity would expose a student to clinically relevant research.  The techniques that will be learned can be utilized both in clinical veterinary medicine as well as equine research.  This would be a valuable learning opportunity for any student interested in equine clinical practice or equine research.     Dr. Lawrence J. Myers The program for the summer of 2008 will have as its aims the determination of the contribution of veterinary care to the performance of selected dog-handler teams.   A group of volunteer detector dog-handler teams working for law enforcement and/or civilian search and rescue organizations has been identified and will be tested via a realistic double blind design to determine Type I (failure to detect presence) and Type II (alert upon non-target items) rates. Prior to, during and after this, veterinary medical records will be examined to determine frequency of veterinary check-ups, type of heartworm preventative, type of ecto- and endo-parasite control measures, vaccination status, and any current or previous health problems. In addition, each dog will be given a physical exam at the time of the testing.   In addition to the veterinary factors, we will attempt to evaluate training methods and frequency of maintenance training, perform motion analysis on videotapes of the testing, and determine olfactory threshold by behavioral olfactometry. Further we may be able to examine handlers by administration of the Myers-Briggs Personality Preference Inventory.