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Diagnostics and Epidemiology of Yersinia Pseudotuberculosis

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Diagnostics and Epidemiology of Yersinia Pseudotuberculosis DIAGNOSTICS AND EPIDEMIOLOGY OF YERSINIA PSEUDOTUBERCULOSIS Taina Niskanen Department of Food and Environmental Hygiene Faculty of Veterinary Medicine University of Helsinki, Finland Helsinki 2010 Department of Food and Environmental Hygiene Faculty of Veterinary Medicine University of Helsinki Helsinki, Finland DIAGNOSTICS AND EPIDEMIOLOGY OF YERSINIA PSEUDOTUBERCULOSIS Taina Niskanen ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Veterinary Medicine, University of Helsinki, for public examination in Auditorium Arppeanum, Snellmaninkatu 3, Helsinki, on 29 January 2010, at 12 noon. Supervising Professor Professor Hannu Korkeala, DVM, PhD, MSocSc Department of Food and Environmental Hygiene Faculty of Veterinary Medicine University of Helsinki Helsinki, Finland Supervised by Professor Hannu Korkeala, DVM, PhD, MSocSc Department of Food and Environmental Hygiene Faculty of Veterinary Medicine University of Helsinki Helsinki, Finland Maria Fredriksson-Ahomaa, DVM, PhD Institute of Food Hygiene and Technology Faculty of Veterinary Medicine Ludwig-Maximilians-Universität Munich Germany Reviewed by Professor Dr. Lieven De Zutter Faculty of Veterinary Medicine Ghent University, Belgium Professor Stan Fenwick, BVMS, PhD Faculty of Health Sciences Murdoch University, Australia Opponent Docent Kaisa Granfors, PhD National Institute for Health and Welfare (THL) Turku, Finland ISBN 978-952-92-6589-3 (Paperback) ISBN 978-952-10-5921-6 (PDF) Helsinki University Print Helsinki 2009 Contents ACKNOWLEDGEMENTS ...........................................................................................................................6 ABBREVIATIONS.........................................................................................................................................6 ABSTRACT.....................................................................................................................................................9 LIST OF ORIGINAL PUBLICATIONS....................................................................................................11 1. INTRODUCTION ....................................................................................................................................12 2. REVIEW OF THE LITERATURE.........................................................................................................13 2.1. YERSINIA PSEUDOTUBERCULOSIS ....................................................................................................13 2.1.1. Classification and characteristics..........................................................................................13 2.1.2. Pathogenicity ........................................................................................................................14 2.2. ISOLATION AND IDENTIFICATION OF Y. PSEUDOTUBERCULOSIS ......................................................18 2.2.1. Isolation ................................................................................................................................18 2.2.2. Identification.........................................................................................................................20 2.2.3. Confirmation of pathogenicity..............................................................................................21 2.3. DETECTION OF Y. PSEUDOTUBERCULOSIS WITH PCR......................................................................22 2.4. CHARACTERIZATION OF Y. PSEUDOTUBERCULOSIS.........................................................................23 2.4.1. Phenotyping..........................................................................................................................23 2.4.2. Genotyping ...........................................................................................................................25 2.5. RESERVOIRS OF Y. PSEUDOTUBERCULOSIS .....................................................................................28 2.5.1. Environment .........................................................................................................................28 2.5.2. Animals.................................................................................................................................30 2.5.3. Food and drinking water.......................................................................................................34 2.6. HUMAN YERSINIOSIS .....................................................................................................................35 2.6.1. Occurrence............................................................................................................................36 2.6.2. Clinical symptoms ................................................................................................................38 2.6.3. Pathogenesis .........................................................................................................................40 2.6.4. Y. pseudotuberculosis outbreaks...........................................................................................41 2.7. TRANSMISSION ROUTES OF Y. PSEUDOTUBERCULOSIS INFECTIONS .................................................42 3. AIMS OF THE STUDY ...........................................................................................................................44 4. MATERIALS AND METHODS .............................................................................................................45 4.1. SAMPLING (I-IV)...........................................................................................................................45 4.2. STRAINS OF YERSINIA SPP. (V) .......................................................................................................46 4.3. ISOLATION OF Y. PSEUDOTUBERCULOSIS (I-IV)..............................................................................47 4.3.1. Sample preparation (I-IV).....................................................................................................47 4.3.2. Isolation methods (I-IV) .......................................................................................................47 4.4. IDENTIFICATION OF Y. PSEUDOTUBERCULOSIS (I-V).......................................................................48 4.4.1. Identification with biochemical reactions (I-V)....................................................................48 4.4.2. PCR analysis (II, V)..............................................................................................................48 4.5. BIO- AND SEROTYPING (I-V) .........................................................................................................50 4.6. VIRULENCE TESTS (I-III, V) ..........................................................................................................51 4.6.1. Phenotypic methods (I-III, V) .............................................................................................. 51 4.6.2. Detection of virF gene by PCR (I-III, V) ............................................................................. 51 4.7. PFGE ANALYSIS (I-V)................................................................................................................... 52 4.7.1. DNA preparation (I-V)......................................................................................................... 52 4.7.2. Macrorestriction analysis (I-V) ............................................................................................ 52 4.8. RIBOTYPING (V)............................................................................................................................ 53 4.9. CHARACTERIZATION OF THE O-ANTIGEN GENE CLUSTER (V)........................................................ 54 4.9.1. Long-range PCR (V) ............................................................................................................ 54 4.9.2. Nucleotide sequencing and sequence analysis (V)............................................................... 54 4.10. EPIDEMIOLOGICAL AND TRACEBACK INVESTIGATIONS (IV)........................................................ 55 5. RESULTS.................................................................................................................................................. 56 5.1. ISOLATION OF Y. PSEUDOTUBERCULOSIS (I-IV).............................................................................. 56 5.2. PREVALENCE OF VIRF-POSITIVE Y. PSEUDOTUBERCULOSIS IN PIG TONSILS AND FAECES AND IN THE PIG HOUSE ENVIRONMENT (I, II) ................................................................................................... 57 5.3. PREVALENCE OF VIRF-POSITIVE Y. PSEUDOTUBERCULOSIS IN WILD BIRDS (III).............................. 58 5.4. ISOLATION OF VIRF -POSITIVE Y. PSEUDOTUBERCULOSIS FROM FARMS PRODUCING ICEBERG LETTUCE (IV)................................................................................................................................ 59 5.5. GENOTYPING OF Y. PSEUDOTUBERCULOSIS (I-IV).......................................................................... 59 5.6. INVESTIGATION AND TRACE-BACK OF THE SOURCE OF A Y. PSEUDOTUBERCULOSIS OUTBREAK (IV) ..................................................................................................................................................... 62 5.7. CHARACTERIZATION OF Y. PSEUDOTUBERCULOSIS-LIKE ISOLATES (V).......................................... 62 6. DISCUSSION...........................................................................................................................................
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