Equine Clinic Head: Ao.Univ.-Prof. Dr.med.vet. Christine Aurich Department for Companion Animals and Horses University of Veterinary Medicine, Vienna Subject: Equine Surgery POSSIBLE INVOLVEMENT OF HERPESVIRUS AND TREPONEMA IN PERIODONTAL DISEASE OF THE ANTERIOR DENTITION IN HORSES INAUGURAL-DOCTORAL THESIS for promotion to DOCTOR MEDICINAE VETERINARIAE at the University of Veterinary Medicine, Vienna Presented by Katharina Pieber, Mag.med.vet. Vienna, October 2012 Academic Supervisor: O. Univ. Prof. Dr. med. vet. Christian Stanek Assistent Supervisor: Dipl.-Ing. Dr. Sabine Brandt Assistent Supervisor: Ass.Prof. Dr. Hubert Simhofer Reviewer: Prof. Dr. Carsten Staszyk Keywords: horse, periodontitis, EOTRH, hypercementosis, resorption Contents Contents List of Abbreviations vi I. Review of literature 1 1. The microbiome of the human oral cavity 1 1.1. Normal bacterial flora in humans .............................. 1 1.2. Saliva as sampling fluid ................................... 2 2. Periodontal disease 2 2.1. Definition .......................................... 2 2.2. Pathogenesis of periodontal disease ............................ 2 2.3. Classification of periodontal disease ............................ 3 2.4. Progression of periodontal disease ............................. 3 2.5. Risk factors ......................................... 5 2.6. Diagnosing periodontal disease ............................... 6 2.7. Measuring periodontal indices ............................... 6 2.8. Other factors leading to diagnosis ............................. 7 2.9. Prevalence of periodontal disease .............................. 7 2.10. Therapy of periodontal disease ............................... 8 3. Pathogens in the oral cavity of periodontitis-affected patients 8 3.1. Bacteria ........................................... 8 3.2. Viruses ........................................... 11 4. The normal oral microbiome in horses 12 5. Periodontal disease in horses 13 5.1. Risk factors for periodontal disease in equines ....................... 13 5.2. Clinical symptoms of periodontal disease ......................... 13 5.3. Grading of periodontal disease in horses .......................... 13 iii Contents 5.4. Comparing human and equine periodontal disease ..................... 14 5.5. Bacteria in the equine oral cavity with periodontal disease ................. 15 5.6. Treatment of periodontal disease in horses ......................... 15 6. EOTRH 16 6.1. Definition .......................................... 16 6.2. Risk factors for EOTRH .................................. 16 6.3. Clinical symptoms ..................................... 16 6.4. Radiological signs ...................................... 17 6.5. Aetiology of EOTRH .................................... 18 6.6. Histological features of EOTRH .............................. 20 6.7. Treatment of EOTRH .................................... 20 7. Treponemes 22 7.1. Characterisation of Treponemes .............................. 22 7.2. Diseases caused by treponemes ............................... 22 7.3. Treponemes in human periodontal disease ......................... 23 II. Hypotheses and objectives 25 III. Material and Methods 26 8. Clinical evaluation 26 8.1. General examination of horses ............................... 26 8.2. Oral examination ...................................... 26 8.3. Radiological examination .................................. 27 8.4. Animals ........................................... 27 8.5. Sampling of crevicular fluid/saliva............................. 27 9. Molecular genetic methods 28 9.1. Sample processing ..................................... 28 iv Contents 9.2. PCR for detection of herpesviruses ............................. 31 9.3. Detection of Treponema sp. ................................. 32 9.4. Sequence analysis ...................................... 32 IV. Results 33 10. Information provided by owners 33 11. Clinical findings 34 11.1. General findings ....................................... 34 11.2. Dental disease-related findings ............................... 35 11.3. Radiological findings .................................... 37 12. Molecular biological findings 40 V. Discussion 45 VI. Summary 52 VII. Zusammenfassung 53 Bibliography 54 List of Figures 63 List of Tables 64 VIII. Acknowledgements 65 v List of Abbreviations List of Abbreviations ACTH ........................ Adrenocorticotropic hormone AST.......................... Aspartate aminotransferase AT ........................... Annealing temperatures BHV ......................... Bovine herpesvirus BLAST ....................... Basic Local Alignment Search Tool BOP.......................... Bleeding on probing bp ............................ Basepairs BVH-1 ........................ Bovine herpesvirus type 1 BVH-2 ........................ Bovine herpesvirus type 3 BVH-4 ........................ Bovine herpesvirus type 4 BVH-5 ........................ Bovine herpesvirus type 5 C ............................. Control horse CAL.......................... Clinical attachment loss CMV ......................... Cytomegalovirus D ............................. Dentine DD ........................... Digital dermatitis DNA ......................... Deoxyribonucleic acid DNS.......................... German: Desoxyribonukleinsaure¨ e.g. ........................... Forexample EBV.......................... Epstein-Barr-Virus EDTA ........................ Ethylenediaminetetraacetic acid EGUS ........................ Equine Gastric Ulcer Syndrome EHV.......................... Equine herpesvirus EMS.......................... Equine Metabolic Syndrome EOTRH....................... Equine odontoclastic tooth resorption and hypercementosis ET ........................... Elongation temperatures EVH-1 ........................ Equine herpesvirus type 1 EVH-21 ....................... Equine herpesvirus type 2 vi List of Abbreviations EVH-3 ........................ Equine herpesvirus type 3 EVH-4 ........................ Equine herpesvirus type 4 EVH-5 ........................ Equine herpesvirus type 5 F ............................. Further EOTRH-affected horse GCF.......................... Gingival crevicular fluid H2S .......................... Hydrogen sulphide HIV .......................... Human immunodeficiency virus HIV-1......................... Human immunodeficiency virus type 1 i.e. ........................... That is i.v. ........................... Intravenous ID............................ Identification IL-1α ......................... Interleukin 1 alpha IL-1β ......................... Interleukin 1 beta irC ........................... Irregular Cement LLD.......................... Lamina dura dentis M ............................ Moderate m ............................ Mild mm........................... Millimetre MMP......................... Matrix metalloproteinases mRNA........................ Messenger RNA n ............................. No nC............................ Normal Cement nr ............................ Number nT............................ Necrotic Tissue ntc ........................... No-template control P ............................. Patient horse PCR.......................... Polymerase chain reaction PD ........................... Pocket depth PDL.......................... Periodontal ligament vii List of Abbreviations PGE2 ......................... Prostaglandin E2 rads .......................... Radiographs RAO.......................... Recurrent airway obstruction RNA.......................... Ribonucleic acid RT-PCR ....................... Reverse transcription PCR S ............................. Severe SD ........................... Standard deviation sIg ........................... Secretory Immunoglobulin sIgA .......................... Secretory Immunoglobulin A sIgM ......................... Secretory Immunoglobulin M T. ............................ Treponema TAE .......................... Tris-Acetate-EDTA TNF-α ........................ Tumour necrosis factor-alpha vc ............................ Vascular channel Vetmeduni .................... Veterinary University WNL ......................... Within normal limits y ............................. Yes viii Part I. Review of literature Part I. Review of literature 1. The microbiome of the human oral cavity 1.1. Normal bacterial flora in humans The healthy oral cavity is a habitat for numerous microorganisms. Bacteria and viruses, but also fungi and archaes can be found. Over the past few decades, knowledge on these microorganisms has dramat- ically improved. To date, about 700 different microbial species are known to reside in the human oral cavity (AAS et al., 2005), consisting of mainly anaerobic bacteria, fungi and protozoa. It is estimated that 34 to 72 different species can be found in the oral cavity of one individual. Their distribution is variable, depending on the localization within the oral cavity (AAS et al., 2005). Most of the microorganisms live in subgingival pockets; others are compounds of the supragingival bacterial biofilm (FABIAN et al., 2008). Some of the individual variations regarding compositions of the microflora can by explained by cultural factors and age; other factors still remain to be elucidated. Little is known about the microbiome of the healthy oral cavity because most studies focus on pa- tients suffering from various dental/gingival oral diseases (SAKAMOTO et al., 2000; AAS et al., 2005; FABIAN et al., 2008). AAS et al. (2005) have detected 141 different bacterial taxa representing six dif-