The Pennsylvania State University The Graduate School Department of Veterinary and Biomedical Sciences A COMPREHENSIVE STUDY OF THE HEALTH OF FARM-RAISED WHITE- TAILED DEER (ODOCOILEUS VIRGINIANUS) WITH EMPHASIS ON RESPIRATORY TRACT INFECTION BY FUSOBACTERIUM SPP. A Dissertation in Pathobiology by Jason W. Brooks © 2010 Jason W. Brooks Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2010 ii The dissertation of Jason W. Brooks was reviewed and approved* by the following: Bhushan Jayarao Professor of Veterinary Science Dissertation Advisor Chair of Committee Arthur Hattel Senior Research Associate Avery August Professor of Immunology Gary San Julian Professor of Wildlife Resources Sanjeev Narayanan Assistant Professor College of Veterinary Medicine, Kansas State University Special Member Vivek Kapur Professor and Head of the Department of Veterinary and Biomedical Sciences *Signatures are on file in the Graduate School. iii ABSTRACT White-tailed deer farming is an established and growing industry in Pennsylvania. Managers of deer farming operations often struggle with animal health problems, the most common of which is pneumonia associated with Fusobacterium sp. infection. Fusobacterium is a genus of anaerobic, gram negative, rod-shaped bacteria that have been associated with many infectious disease processes in humans and animals. It is important to the deer industry, as well as the cattle and sheep industries, to more clearly understand fusobacterial disease pathogenesis and determine effective treatment and prevention strategies. The objectives of the following series of studies were: 1) to determine the current management practices and animal health problems on white-tailed deer farms in Pennsylvania, 2) to phenotypically and genotypically characterize a set of Fusobacterium isolates from white-tailed deer and evaluate their association with disease in the host, and 3) to determine the pathogenicity of Fusobacterium varium to the respiratory tract of mice and to evaluate this system as a model for pulmonary fusobacterial infection of deer. In order to determine the current management practices used by white-tailed deer farms and identify animal health problems in these herds, a self-administered questionnaire was mailed to managers of 235 farms in Pennsylvania that raise white- tailed deer. Herds ranged in size from 1 to 350 deer. Land holdings ranged from 0.07 to 607 hectares (0.17 to 1,500 acres). Stocking density ranged from 0.1 to 118.6 deer/hectare (0.04 to 48 deer/acre). Most (84%) respondents raised deer for breeding or hunting stock; 13% raised deer exclusively as pets or for hobby purposes, and purpose iv varied by herd size. Multiple associations were identified between management or disease factors and herd size. The use of vaccines, veterinary and diagnostic services, pasture, and artificial insemination increased as herd size increased. The most common disease conditions in herds of all sizes were respiratory tract disease, diarrhea, parasitism, and sudden death. The prevalence of respiratory tract disease increased as herd size increased. Results suggested that many aspects of herd management for white-tailed deer farms in Pennsylvania were associated with herd size, but that regardless of herd size, many preventive medicine practices were improperly used or underused in many herds. A total of 28 clinical strains of Fusobacterium spp. were isolated at necropsy over a two-year period from the respiratory tract of cervids, including white-tailed deer, elk, and reindeer. Isolates were identified as F. varium (21/28, 75%), F. necrophorum subsp. funduliforme (5/28, 17.9%), and F. necrophorum subsp. necrophorum (2/28, 7.1%). Using PCR-based detection of virulence genes, all F. varium isolates were negative for the promoter region of the leukotoxin operon of F. necrophorum and the hemagglutinin- related protein gene of F. necrophorum. In the necropsy population no significant differences in gross or microscopic lesions were detected across Fusobacterium species, suggesting similar potential for virulence, however, toxicity to bovine polymorphonuclear leukocytes was not observed in any F. varium strains, perhaps indicating that a virulence factor other than leukotoxin is involved in the pathogenesis of F. varium infection. F. varium was less susceptible to many antimicrobials than were the F. necrophorum subspecies. These data suggest that F. varium may be a significant pathogen in deer and may require different treatment and prevention methods than F. necrophorum. v C57BL/6 mice and BALB/c mice were inoculated intranasally with various strains and varying dosages of F. varium to evaluate the pathogenicity of F. varium to the respiratory tract of mice and to determine the utility of this system as model of fusobacterial pneumonia. Prior to inoculation, mice were pre-treated with either 0.2 mg dexamethasone intraperitoneally once daily for four days, 10 µg of lipopolysaccharide (LPS) of E. coli 055:B5 intranasally one time, or no pre-treatment. Following inoculation mice were observed for morbidity and mortality for fourteen days. No mice infected with F. varium showed clinical illness or died. No mice infected with F. varium developed gross or microscopic lesions. The bacterium was recovered from the blood of one mouse, but was not recovered from blood or lung of any other mice. Anti- fusobacterial IgM or IgG were not produced in serum by 14 days in response to infection. Pre-existing antibodies detected in pooled serum bound similarly with proteins of two F. necrophorum subspecies and three F. varium strains, but did not bind to proteins of similar size from other common bacterial pathogens including Escherichia coli, Pasteurella pneumotropica, Arcanobacterium pyogenes, and Clostridium perfringens. No serum antibodies were detected in BALB/c mice. These results suggest that F. varium is not highly pathogenic to the respiratory tract of mice, and does not result in a humoral immune response following intranasal inoculation. In conclusion, the results of this series of studies suggest that fusobacterial species, specifically F. varium, are not highly pathogenic to the respiratory tract and are unable to establish lung infection alone without some severe predisposing condition. Management changes on deer farms should be instituted in an effort to minimize stress, overcrowding, nutritional imbalance, and environmental contamination, while vi maximizing immunity through colostrum and vaccination, and developing effective preventive health and biosecurity programs with routine veterinary consultation. vii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….. x LIST OF TABLES………………………………………………………………… xii ACKNOWLEDGEMENTS………………………………………………………. xiv Chapter 1. INTRODUCTION…………………………………………………….. 1 1.1 Statement of the Problem……………………………………………… 2 1.2 Initial Observations……………………………………………………. 2 1.3 Retrospective Study of Deer Mortality………………………………... 3 1.4 Evaluation of Viral Etiologies with emphasis on BVDV and BRSV… 5 1.5 Objectives……………………………………………………………... 7 1.6 References…………………………………………………………….. 8 Chapter 2. REVIEW OF LITERATURE…………………………………………. 9 2.1 Deer Farming Industry………………………………………………… 10 2.2 Animal Health in Deer Populations…………………………………… 11 2.3 Anaerobic Bacteria……………………………………………………. 13 2.3.1 The Fusobacteria…………………………………………….. 15 2.3.2 Fusobacterium necrophorum………………………………... 16 2.3.3 Fusobacterium varium………………………………………. 21 2.4 Lung Infection and Immunity…………………………………………. 22 2.5 Animal Models of Bacterial Pulmonary Infection…………………….. 25 2.6 Animal Models of Anaerobic Bacterial Infection…………………….. 27 2.7 References…………………………………………………………….. 31 viii Chapter 3. MANAGEMENT PRACTICES USED BY WHITE-TAILED DEER FARMS IN PENNSYLVANIA AND HERD HEALTH PROBLEMS……………. 53 3.1 Acknowledgements…………………………………………………… 54 3.2 Abstract……………………………………………………………….. 54 3.3 Introduction…………………………………………………………… 54 3.4 Materials and Methods………………………………………………… 54 3.5 Results…………………………………………………………………. 55 3.6 Discussion…………………………………………………………….. 58 3.7 References…………………………………………………………….. 60 Chapter 4. PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF FUSOBACTERIUM ISOLATES FROM THE RESPIRATORY TRACT OF DEER………………………………………………………………………. 61 4.1 Abstract……………………………………………………………….. 62 4.2 Introduction……………………………………………………………. 62 4.3 Materials and Methods………………………………………………… 64 4.4 Results…………………………………………………………………. 69 4.5 Discussion…………………………………………………………….. 74 4.6 Acknowledgements…………………………………………………… 76 4.7 References…………………………………………………………….. 77 Chapter 5. EFFECTS OF FUSOBACTERIUM VARIUM ON THE RESPIRATORY TRACT OF MICE FOLLOWING INTRANASAL INOCULATION…………… 88 5.1 Abstract……………………………………………………………….. 89 5.2 Introduction…………………………………………………………… 90 ix 5.3 Materials and Methods………………………………………………… 91 5.4 Results…………………………………………………………………. 96 5.5 Discussion…………………………………………………………….. 99 5.6 Acknowledgements…………………………………………………… 103 5.7 References……………………………………………………………... 103 Chapter 6. SUMMARY AND CONCLUSIONS………………………………….. 117 Appendix A. SURVEY QUESTIONNAIRE……………………………………… 122 Appendix B. SUPPLEMENTAL PHENOTYPIC DATA………………………… 126 Appendix C. MOUSE INFECTIVITY TRIALS RAW DATA…………………… 132 x LIST OF FIGURES Figure 2.1. F. necrophorum subsp. necrophorum growth on blood agar.................. 46 Figure 2.2. F. necrophorum subsp. funduliforme growth on blood agar.………….. 47 Figure 2.3. F. varium growth on blood agar……………………………………….. 48 Figure 2.4. Fusobacterium spp. growth in PRAS-BHI broth……………………… 49 Figure 2.5.