University of Veterinary Medicine Hannover Investigations on the taxonomy of the genus Riemerella and diagnosis of Riemerella infections in domestic poultry and pigeons Thesis Submitted in partial fulfilment of the requirements for the degree - Doctor of Veterinary Medicine - Doctor medicinae veterinariae (Dr. med. vet.) by Dennis Rubbenstroth, PhD Bielefeld Hannover 2012 Academic supervision Prof. S. Rautenschlein (Clinic for Poultry, University of Veterinary Medicine Hannover, Germany) 1st Referee Prof. S. Rautenschlein 2nd Referee Prof. P. Valentin-Weigand (Institute of Microbiology, University of Veterinary Medicine Hannover, Germany) Date of oral exam: November 7 th , 2012 Meinen beiden Großmüttern in dankbarer Erinnerung Table of contents v Table of contents Table of contents....................................................................................................................... v List of abbreviations ...............................................................................................................vii Manuscripts and participation of this author ........................................................................viii 1. Introduction .................................................................................................................. 1 2. Literature review .......................................................................................................... 3 2.1. Taxonomy of the genus Riemerella ...................................................................... 3 2.2. Morphology, identification and characterization of Riemerella spp.................... 4 2.3. Epidemiology of Riemerella infections................................................................. 8 2.4. Riemerella -induced disease in avian species ......................................................10 2.5. Diagnosis of Riemerella infections .....................................................................15 3. Goals and objectives ..................................................................................................19 4. Isolation and characterization of atypical Riemerella columbina strains from pigeons and their differentiation from Riemerella anatipestifer ..............................21 5. Description of Riemerella columbipharyngis sp. nov., isolated from the pharynx of healthy domestic pigeons ( Columba livia f. domestica ), and emended description of the genus Riemerella , Riemerella anatipestifer and Riemerella columbina ................................................................................................33 6. Evaluation of different diagnostic tools for the detection and identification of Riemerella anatipestifer .............................................................................................55 7. Discussion ..................................................................................................................85 7.1. Differentiation of Riemerella spp. by biochemical and morphological characteristics.......................................................................................................86 7.2. Identification and detection of RA by a new PCR assay....................................87 7.3. Identification of Riemerella spp. based on whole cell mass spectrometry ........88 7.4. Riemerella serotyping..........................................................................................90 7.5. Role of Riemerella spp. as pathogens for domestic poultry and pigeons ..........91 7.6. Future perspectives for Riemerella diagnosis and research................................92 8. Summary ....................................................................................................................93 9. Zusammenfassung......................................................................................................96 10. Literature ..................................................................................................................101 Acknowledgements ..............................................................................................................111 Table of contents vi Manuscripts VII List of abbreviations aMPV Avian Metapneumovirus MG Mycoplasma gallisepticum ATCC American type culture MS mass spectrometry collection NDV Newcastle disease virus CSB Columbia sheep blood NGB Neomycin-Gentamycin-Blood dnaB DnaB helicase ompA outer membrane protein A DNA desoxy-ribonucleic acid PCR polymerase chain reaction ELISA enzyme linked immunosorbent PFGE pulse field gel electrophoresis assay pPMV-1 pigeon-type Paramyxovirus 1 ERIC enterobacterial repetitive intergenic consensus sequence RA Riemerella anatipestifer GLC gas-liquid chromatography RC Riemerella columbina IFT immunofluorescence test RCP Riemerella columbipharyngis IgG immunoglobulin G RFLP restriction fragment length polymorphism IgY immunoglobulin Y RNA ribonucleic acid kDa kilo Dalton rpoB RNA polymerase beta subunit LAMP loop-mediated isothermal amplification rRNA ribosomal RNA LD 50 median lethal dose SDS-PAGE sodium dodecyl sulfate polyacrylamide gel MALDI-TOF matrix assisted laser electrophoresis desorption/ionisation - time of flight vapD virulence associated protein D Manuscripts VIII Manuscripts and major contributions of this author Chapter 4: Rubbenstroth, D. , Hotzel, H., Knobloch, J., Teske, L., Rautenschlein, S. & Ryll, M. (2011). Isolation and characterization of atypical Riemerella columbina strains from pigeons and their differentiation from Riemerella anatipestifer . Veterinary Microbiology, 147 , 103-112. • sample collection and preparation • cultivation, phenotypical and biochemical characterization of RC isolates • MALDI-TOF MS analysis • preparation of manuscript and corresponding author Chapter 5: Rubbenstroth, D. , Ryll, M., Hotzel, H., Christensen, H., Knobloch, J. K., Rautenschlein, S. & Bisgaard, M. (2013). Description of Riemerella columbipharyngis sp. nov., isolated from the pharynx of healthy domestic pigeons ( Columba livia f. domestica ), and emended descriptions of the genus Riemerella , Riemerella anatipestifer and Riemerella columbina . International Journal of Systematic and Evolutonary Microbiology, 63 , 280-287. • sample collection and preparation • cultivation, phenotypical and biochemical characterization of RCP isolates • analysis of MALDI-TOF MS results • preparation of manuscript and corresponding author Manuscripts IX Chapter 6: Rubbenstroth, D. , Ryll, M., Knobloch, J. K., Kohler, B. & Rautenschlein, S. (2013). Evaluation of different diagnostic tools for the detection and identification of Riemerella anatipestifer . Avian Pathology, 42 , 17-26. • sample collection and preparation • cultivation and identification of RA isolates • analysis of RA sequences • design and validation of a new RA-specific PCR • serological examination of RA strains • analysis of MALDI-TOF MS results • preparation of manuscript Introduction 1 1. Introduction Riemerella anatipestifer (RA) and Riemerella columbina (RC) constitute the genus Riemerella within the family Flavobacteriaceae . RA is known to be an economically important pathogen of domestic poultry. It is considered to be a primary pathogen of waterfowl and may cause high mortality in ducklings and goslings. In addition, gallinaceous birds, particularly turkeys, may also be affected by RA infections. RA- induced gross pathology is dominated by polyserositis, but lesions may also affect joints and the central nervous system. Clinical disease is characterized by respiratory signs, lameness and central nervous symptoms. Its considerable economic impact is caused by increased mortality, reduction of growth rates and enhanced condemnation rates at slaughter. While RA is well characterized as an avian pathogen, only little is known about RC. So far it has been isolated almost exclusively from domestic pigeons. Its isolation from tissues showing gross lesions from clinically diseased pigeons suggests a pathogenic potential of RC for this host. However, epidemiological or experimental data to support this assumption is scarce. Differentiation of RA and RC from each other, as well as from other related species, by biochemical characteristics is often difficult due to the low biochemical activity of many Flavobacteriaceae members. Molecular-biological methods, such as DNA-DNA hybridization or sequence analysis of certain well-conserved genes, may compensate this drawback under research conditions, but since they are time- and labour-consuming, they are not feasible for routine diagnostic purposes. Polymerase chain reaction (PCR) and matrix assisted laser desorption/ionisation - time of flight (MALDI-TOF) based protein profiling have proven to be useful tools for the rapid detection and identification of bacterial pathogens. However, their value in the diagnosis of Riemerella infections has not been evaluated thoroughly to date. The goal of this study was therefore to gain further knowledge about phenotypic and genotypic characteristics of the at present poorly described species RC, as well as a potential new Riemerella sp. isolated from domestic pigeons ( Columba livia f. domestica ). Diagnostic methods to be used for detection, identification and differentiation of Riemerella spp. were Introduction 2 evaluated and compared with the aim of developing improved strategies for the diagnosis of Riemerella infections in domestic poultry and other avian species. Literature review 3 2. Literature review 2.1. Taxonomy of the genus Riemerella Riemerella anatipestifer (RA), the type species