Molecular Epidemiology of Salmonella and Campylobacter Contamination of Poultry During Transport and Slaughter
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Molecular epidemiology of Salmonella and Campylobacter contamination of poultry during transport and slaughter Geertrui Rasschaert Thesis submitted in the fulfillment of the requirements for the degree of Doctor in Veterinary Science (PhD), Faculty of Veterinary Medicine, Ghent University, March 2007 Promotor: Prof. Dr. De Zutter Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety Image cover: Photo by Stephen Ausmus, ARS Print: DCL Print & Sign, www.dclsigns.be ISBN: 9789058641120 EAN: 9789058641120 To refer to this thesis: Rasschaert, G. (2007). Molecular epidemiology of Salmonella and Campylobacter contamination of poultry during transport and slaughter. Thesis submitted in the fulfillment of the requirements for the degree of Doctor in Veterinary Science (PhD), Faculty of Veterinary Medicine, Ghent University. The author and the promotor give the permission to consult and to copy parts of this work for personal use only. Every other use is subject to the copy write laws. Permission to reproduce any material should be obtained from the author. TABLE OF CONTENT ABBREVIATION LIST LITERATURE REVIEW 1. Campylobacter 1 1.1. Introduction 1 1.2. Taxonomy and characteristics 1 1.3. Clinical aspects 3 1.4. Epidemiology 4 1.5. Sources of infection 7 2. Salmonella 9 2.1. Introduction 9 2.2. Taxonomy and characteristics 9 2.3. Clinical aspects 10 2.4. Epidemiology 12 2.5. Sources of infection 14 3. Flock colonization 16 3.1. Prevalence at farm level 16 3.2. Vertical transmission 20 3.3. Horizontal transmission 21 4. Transport and slaughter of broiler flocks 26 4.1. Transport 26 4.2. Description of a Belgian poultry slaughterhouse 27 4.3. Contamination of carcasses during slaughter 28 5. Identification and characterization 32 5.1. Campylobacter 32 5.2. Salmonella 35 AIMS OF THE STUDY 37 CHAPTER I: Comparison of five repetitive-sequence-based PCR typing methods for molecular discrimination of Salmonella enterica isolates 39 1. Introduction 40 2. Materials and methods 41 3. Results 45 4. Discussion 53 CHAPTER II: The contribution of gastrointestinal colonization and cross-contamination to Salmonella carcass contamination during poultry slaughter 57 1. Introduction 58 2. Materials and methods 59 3. Results 61 4. Discussion 68 CHAPTER III: The contribution of gastrointestinal colonization and cross-contamination to Campylobacter carcass contamination during poultry slaughter 71 1. Introduction 72 2. Materials and methods 73 3. Results 76 4. Discussion 81 CHAPTER IV: Investigation of the concurrent colonization of poultry flocks with Campylobacter and Salmonella and assessment of the sampling site for status determination at the slaughterhouse 85 1. Introduction 86 2. Materials and methods 87 3. Results 89 4. Discussion 91 CHAPTER V: Impact of the slaughter line contamination on the presence of Salmonella on broiler carcasses 95 1. Introduction 96 2. Materials and methods 97 3. Results 101 4. Discussion 106 CHAPTER VI: External contamination of Campylobacter-free flocks after transport in cleaned and disinfected containers 109 1. Introduction 110 2. Materials and methods 111 3. Results 115 4. Discussion 119 GENERAL DISCUSSION 123 REFERENCES 131 SUMMARY 151 SAMENVATTING 155 CURRICULUM VITAE 159 DANKWOORD 163 ABBREVIATION LIST A absent AFLP amplified fragment length polymorphism bp base pairs BPW buffered peptone water CDC Center for Disease Control and Prevention CFU colony-forming units CI confidence interval DI differentiation index Diassalm diagnostic semi-solid Salmonella agar DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid EFSA European Food Safety Authority ERIC enterobacterial repetitive intergenic consensus EU European Union flaA flagellin gene A PCR/restriction fragment length polymorphism GBS Guillain-Barré syndrome IU international unit LPS lipopolysaccharide M molar mCCDA modified cefoperazone charcoal deoxycholate agar MLST multilocus sequence typing NC not countable ND not done, not determined NRSS National Reference Centre for Salmonella and Shigella NT not typeable OD600 optical density at 600 nm P present PCR polymerase chain reaction PFGE pulsed-field gel electrophoresis RA reactive arthritis RAPD random amplification of polymorphic DNA RDNC routine dilution no conformity rep-PCR repetitive extragenic palindromic PCR RFLP restriction fragment length polymorphism rpm revolutions per minute RV rappaport-vassiliadis bouillon SI similarity index SIPH Scientific Institute of Public Health spp. species (plural) TBE Tris-borate-EDTA TSA tryptone soya agar U unit UK United Kingdom UPGMA unweighted-pair group method using arithmetic averages US United States USA United States of America UV ultra violet VBNC viable but non-culturable vol/vol volume/volume WHO World Health Organisation wt/vol weight/volume XLD xylose lysine deoxycholate LITERATURE REVIEW Literature Review 1. CAMPYLOBACTER 1.1. Introduction Although campylobacters were not recognized as enteric pathogens in humans until the late 1970s, they have probably caused illness in men for centuries. These spiral bacteria have been known in veterinary medicine since the beginning of the 20th century. They were associated with abortion, sterility and dysentery in sheep, cattle or swine (Butzler, 2004). However, it took until 1968 to isolate Campylobacter from the stool of a patient with severe diarrhea and fever by a filtration technique (Dekeyser et al., 1972). The later development of selective media, obviating the need to filter suspensions, made it possible for routine microbiological laboratories to isolate campylobacters from fecal samples. Reports from countries all over the world made it in the mid 1980s clear that Campylobacter is the most frequent cause of human bacterial enteritis (Butzler, 2004). 1.2. Taxonomy and characteristics The genus Campylobacter was first proposed in 1963 by Sebald and Véron (1963) and included only two species, formerly classified as Vibrio spp. At present, the genus Campylobacter contains 17 species and 6 subspecies (Figure 1; On, 2001; Foster et al., 2004). The genus Campylobacter is a member of the family of the Campylobacteraceae (Vandamme and De Ley, 1991) together with the genera Arcobacter and Sulfurospirillum (Vandamme et al., 2005). Campylobacters are Gram-negative spiral shaped rods, 0.2 to 0.8 µm wide and 0.5 to 5 µm long. Most species are motile with a characteristic corkscrew-like motion by a flagellum at one or both ends of the cell. Energy is obtained from amino acids or tricarboxylic acid cycle intermediates, not from carbohydrates. Carbohydrates are neither fermented nor oxidized. In general, campylobacters grow under micro-aerobic conditions with low levels of oxygen (ca. 5%) for energy production. Campylobacters are sensitive to several environmental conditions and are generally less resistant to environmental stress than other foodborne pathogens. Campylobacters are sensitive to drying and do not survive well on dry surfaces (Fernandez et al., 1985). They are also sensitive to osmotic stress and will not grow at concentrations of 2% 1 Literature Review NaCl (Doyle and Roman, 1982), whereas Salmonella Typhimurium for example, is able to grow at 4.5% NaCl (ICMSF, 1996). They are sensitive to acidic conditions and are not able to grow below pH 4.9 (Blaser et al., 1980). Under less favorable conditions, campylobacters may form coccoid cells which are associated with a loss of culturability using traditional culture methods (Rollins and Colwell, 1986). It is still debated if this coccoid form is a non- viable, degenerative form or a dormant state that is non-culturable, but metabolically active and recoverable in suitable animal hosts (VBNC, Viable But Non-Culturable) (Moore, 2001). Figure 1. Unrooted tree, based on the 16S rRNA gene sequences, showing the phylogenetic relationships of Campylobacter spp., including the newest species Campylobacter insulaenigrae. Bar, 2% sequence divergence (adapted from Foster et al., 2004). Most campylobacters are not able to grow below 30°C and above 45°C. Campylobacter jejuni, C. coli and C. lari are the so-called thermophilic campylobacters because they are able to grow at a temperature of 37°C as the other Campylobacter species, but also at a temperature of 42°C. C. upsaliensis is also often described as thermophilic, but as not all strains are able to grow at a temperature of 42°C, it might be more appropriate to refer to this species as thermotolerant (Vandamme et al., 2000). The pH for Campylobacter growth ranges from 4.9 to 9.0 with an optimal pH between 6.5 and 7.5 (ICMSF, 1996). 2 Literature Review 1.3. Clinical aspects Campylobacteriosis is an acute diarrheal disease with clinical manifestations such as diarrhea, fever and abdominal pain. A definitive diagnosis can only be made by detecting campylobacters in the feces, since these symptoms are not distinctive from symptoms displayed by other organisms. In Belgium, C. jejuni subsp. jejuni (hereafter called C. jejuni) and C. coli account for 80% and 12% of human Campylobacter infections, respectively (Vandenberg et al., 2004). There does not seem to be any clear difference in the clinical manifestations between infections caused by C. jejuni or C. coli. In one study C. coli was found to cause milder symptoms whereas in another the opposite was reported (Figura and Guglielmetti, 1988; Popovic-Uroic et al., 1988). In general, the infective dose is low. Infection has been induced with doses of 500-800 bacteria (Robinson, 1981; Black et al., 1988). There are influencing factors such