EPIDEMIOLOGIA E CONTROLLO DELLE ZOONOSI Survey on The
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Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN EPIDEMIOLOGIA E CONTROLLO DELLE ZOONOSI Ciclo XXII Settore/i scientifico disciplinari di afferenza: VET/05 TITOLO TESI Survey on the spirilar flora of Lagomorphs Presentata da: Dott.ssa Joana Marreiros Cabrita Revez Coordinatore Dottorato Relatore Prof. Giovanni Poglayen Prof. Renato Giulio Zanoni Esame finale anno 2010 To Mirko: “As armas e os barões assinalados, Que da ocidental praia Lusitana, Por mares nunca de antes navegados, Passaram ainda além da Taprobana, Em perigos e guerras esforçados, Mais do que prometia a força humana, E entre gente remota edificaram Novo Reino, que tanto sublimaram;” Luís Vaz de Camões (“Os Lusíadas” – Canto I) “Não me queiram converter a convicção: sou lúcido!” Álvaro de Campos (Fernando Pessoa, “Sou lúcido”) “A scientist in his laboratory is not a mere technician: he is also a child confronting natural phenomena that impress him as though they were fairy tales.” Marie Currie Ad astra per aspera Table of contents Background and objectives 1 Overview of the thesis 5 Part 1: General Introduction – literature review 9 I. Bacterial taxonomy 12 II. Taxonomy of Epsiloproteobacteria 17 Taxonomic history 19 The genus Campylobacter 20 The genus Helicobacter 24 III. Lagomorphs 27 Lagomorpha 27 Production 28 Rabbit intestinal microbiota 30 Epsilonproteobacteria in leporids 30 Zoonoses 31 References 32 Part 2: Experimental work 49 Chapter 1 Campyobacter cuniculorum sp. nov. 51 Isolates 52 DNA extraction and PCR identification 52 16S ribosomal RNA 52 Housekeeping genes rpoB and groEL 54 Whole-cell protein electrophoresis 55 DNA-DNA hybridization 56 Phenotypic characteristics 56 Transmission Electron Microscopy 58 G+C content 58 Conclusion 58 Description of Campylobacter cuniculorum sp. nov. 59 References 59 Chapter 2 Occurrence of Campylobacter and Helicobacter species in leporids 63 Introduction 64 Materials and Methods 66 Rabbit Sampling 66 Hare Sampling 67 Cultural examination 68 DNA extraction 69 Direct PCR analysis of the caecal contents 69 Identification of bacterial isolates 70 Results 71 Direct PCR analysis of the caecal contents 71 Cultural examination and bacterial identification 71 Discussion 73 Conclusion 75 References 75 Chapter 3 Virulence determinants of Campylobacter cuniculorum 79 Introduction 80 Materials and Methods 81 Bacterial strains and grow conditions 81 Detection of cdtB gene from Campylobacter isolates by PCR 82 Preparation of mammalian cell cultures 82 Preparation of the bacteria-free supernatants for toxin assays 82 Detection of the CDT activity by cell culture assays 83 Adhesion and Invasion 84 Statistical analysis 84 Results 85 Detection of cdtB gene and CDT activity 85 Adhesion and Invasion 88 Discussion 90 Conclusion 92 References 93 Chapter 4 Antimicrobial susceptibility 97 Introduction 98 Materials and Methods 100 Campylobacter species isolates 100 Escherichia coli isolates 101 MIC determination of Campylobacter species 101 Disk diffusion 102 Results 102 MIC determination of Campylobacter species 102 Escherichia coli isolation 105 Disk diffusion test of E. coli isolates 105 Discussion 109 Conclusion 112 References 113 Part 3: General discussion and conclusions 117 Part 4 Summaries 123 Acknowledgements 129 Background and objectives 2 Background and objectives Members of the genera Campylobacter and Helicobacter have been in the spotlight in recent decades because of their status as animals and/or humans pathogens, both confirmed and emerging, and because of their association with food-borne and zoonotic diseases. In 1991, the taxonomy of Campylobacter and related organisms was thoroughly revised, since this revision several new Campylobacter and Helicobacter species have been described and well-founded crediting a polyphasic taxonomic approach. There is a paucity of data regarding the occurrence of spiral shaped enteric flora in leporids. The main research aims of the present study were (i) to address to the characterization of Campylobacter-like organisms isolated from rabbits epidemiologically not correlated, by using a polyphasic approach; (ii) the definition of the prevalence of Campylobacter and Helicobacter in leporids; (iii) the study of some virulence determinants and finally (iv) to monitor of antibiotic susceptibility both in Campylobacter isolates and indicator bacteria. 3 4 Overview of the thesis 5 6 Overview of the thesis The objectives laying this thesis focused on the survey of spirilar enteric flora of Lagomorphs, in particular Campylobacter and Helicobacter species in rabbits and hares. The following chapters can be read individually or together. In order to study and understand the significance of the survey, the animals sampled were not correlated epidemiologically. The first part of the thesis presents an overview of the literature relating to the content of this work. It includes a general introduction to bacterial taxonomy (I) and the bacterial species concept and the taxonomy of members of the Epsilon Proteobacteria class (II), giving an overview of the known species and respective methods currently used for classification and identification. Furthermore, a general introduction to the lagomorphs (III) biology and diseases as well as known zoonotic agents is presented. The second part presents an overview of the experimental work performed in the framework of this thesis. Chapter 1 describes a new species, belonging to the group of thermotolerant Campylobacter, i.e. Campylobacter cuniculorum isolated from rabbits. Chapter 2 resolves the prevalence of this new species as well as other genus members and Helicobacter species in leporids, i.e. rabbits and hares, epidemiologically not correlated. Chapter 3 focuses on the study of some virulence determinants of C. cuniculorum, including the effects of Cytolethal Distending Toxin (CDT) on mammalian cells, the presence of the gene ecoding CDT (cdtB), and adhesion and invasion properties. In Chapter 4, results on monitoring antibiotic susceptibilities both in Campylobacter species and indicator bacteria (Escherichia coli) are presented. The third part of the thesis comprises a general discussion and conclusion of the presented work. The fourth part presents the summary of this work in English, Italian and Portuguese. 7 8 Part 1: General Introduction – literature review 9 10 Part 1: General Introduction – literature review This part will whine (I) bacterial taxonomy followed by a short description of the (II) Taxonomy of the ε-Proteobacteria. Within this taxonomic overview, a short history introduction will be presented and members of the Campylobaceriacea and Helicobactereaceae will be described in detail. Subjects regarding the lagomorphs (III), in particular rabbits and hares, will be presented including a brief introduction, production, normal gut microbiota and respective disorders. Moreover, a short synopsis will illustrate the current knowledge on ε-Proteobacteria in leporids and some known zoonoses associated with lagomorphs. 11 I. Bacterial taxonomy Attempts to bring order through categorization to the bewildering variety of organisms have been an ongoing human endeavor (Gevers et al., 2005). Microorganisms are fantastically diverse, and the amount and depth of genetic diversity represented among the named microbial species is greater than that represented among the animals and plants combined (Buckley and Roberts, 2007). In microbiology, bacterial taxonomy (or biosystematics) is an essential discipline that may be defined as the scientific study of the diversity of organisms with the objective of characterizing and arranging them in an orderly scheme. Within biosystematics, three sub disciplines arise: classification, nomenclature and identification (Owen, 2004; Vandamme et al., 1996). Regarding classification, it relates to the orderly arrangements of taxonomic units or taxa into groups on the basis of similarities or defined relationships. Nomenclature instead is referred to the assignment of taxonomic names to the taxa. Finally, identification is naming a distinct taxonomic unit based on the common characteristics or properties that distinguish them from other organisms. Indeed, classification precedes identification, given that it is necessary to describe and characterize the basic taxonomic unit before an isolate can be identified as a member of that unit (Buckley and Roberts, 2007; Gevers et al., 2005; Owen, 2004; Vandamme et al., 1996). In order to completely define modern biosystematics, two additional disciplines are needed: phylogeny (history, origin and evolution of a set of organisms) and population genetics (the variability of populations of bacteria and the formulation of theories that account for its variability) (Buckley and Roberts, 2007; Gevers et al., 2005; Vandamme et al., 1996). Moreover, it is currently a common practice to apply a polyphasic approach in taxonomic studies, incorporating genetic (e.g., DNA-DNA hybridization), phenotypic (e.g., the results of biochemical tests) and phylogenetic data (e.g. rRNA gene sequences), with the aim to create a consensus classification by integrating different kinds of data and information into a classification of the biological entities that contains a minimum of contradictions (Buckley and Roberts, 2007; Gevers et al., 2005; Vandamme et al., 1996). In the twentieth century technical breakthrough changed the field of bacterial taxonomy in a groundbreaking way by defining a species as the basic taxonomic unit (Staley, 2006). For decades, classification and identification