Comparison of genetic profiles of Campylobacter strains isolated from poultry, pig and Campylobacter human infections in Brittany, France

Pathologie Biologie 57 (2009) 23–29

Comparison of genetic profiles of Campylobacter strains isolated from poultry, pig and Campylobacter human infections in Brittany, France Comparaison ge´ne´tique de Campylobacter spp. issus de volailles et de porcs avec des isolats issus de campylobacte´rioses humaines en Bretagne, France M. Denis a,*, B. Chidaine a, M.-J. Laisney a, I. Kempf a, K. Rivoal a, F. Mégraud b, P. Fravalo a a

Laboratoire d’étude et de recherche avicole, porcine et piscicole, Agence française de sécurité sanitaire des aliments (Afssa), B.P. 53, 22440 Ploufragan, France b Laboratoire de bactériologie, Centre national de référence des campylobacters et hélicobacters, Inserm U853, CHU Pellegrin, place Amélie-Raba-Léon, 33076 Bordeaux cedex, France Received 7 April 2008; accepted 11 April 2008 Available online 4 June 2008

Abstract Five hundred eighty-two Campylobacter isolates (177 from humans, 319 from poultry and 86 from pig) collected in Brittany, France, in 2003 and 2004 were typed by pulsed-field gel electrophoresis. The number of human cases increased during the hot season, particularly for C. jejuni. Twelve genetic groups out of 27 contained human isolates collected over the two years. These groups had 21,3 and 17,0% of the isolates obtained in 2003 and 2004, respectively. In four cases, isolates from 2003 have the same Pulsed-field gel electrophoresis (PFGE) profile as isolates from 2004. Six PFGE profiles common to poultry and human isolates were identified. Poultry isolates were found in 47 clusters containing human isolates. Caeca from farms and slaughterhouses accounted for 66% of these isolates, with chicken legs obtained from supermarkets accounting for the other 34%. Pig isolates never clustered with poultry and human isolates. In conclusion, the analysis of the genetic profiles of Campylobacter resulting from human cases showed that there were few identical or genetically close isolates between the human cases declared in 2003 and those declared in 2004. This highlighted a great genetic diversity in the isolates and indicated that it should be difficult to bind the human infections with groups of Campylobacter isolates presenting particular genetic profiles. The Campylobacter isolates obtained from the two animal production systems had different genotypes, and isolates from pigs differed genetically from isolates obtained from humans. We found that 44.6% of human Campylobacter isolates were genetically related to genotypes found in poultry and a part of these campylobacteriosis are due to contact with poultry. This is not particularly surprising in Brittany, a farming area with many animal-rearing farms and slaughterhouses. This work highlights the implication of the poultry in the French human cases and that handling of poultry is also an important risk for Campylobacter infection in humans. # 2008 Elsevier Masson SAS. All rights reserved. Résumé Cinq cent quatre-vingt-deux isolats de Campylobacter (177 d’humains, 319 de volaille et 86 de porc) isolés en Bretagne, France, en 2003 et 2004 ont été typés par électrophorèse en champs pulsés. Le nombre de cas humains augmente pendant la saison chaude et est plus prononcé pour C. jejuni. Douze groupes génétiques sur 27 contiennent des isolats humains récoltés sur les deux années. Ces groupes contiennent 21,3 et 17,0 % des isolats obtenus en 2003 et 2004, respectivement. Dans quatre cas, des isolats 2003 ont les mêmes profiles PFGE que des isolats 2004. Six profils génétiques identiques ont été trouvés entre la volaille et les cas humains. Des campylobacters de la volaille ont été trouvés dans 47 groupes génétiques contenant également des isolats d’humains. Soixante-six pour cent de ces isolats proviennent de caeca pris à la ferme, et en abattoir et 34 % de cuisses de volaille achetées en grande surface. Les campylobacters de porc sont toujours dans des groupes génétiques différents de ceux de la volaille et d’humains. En conclusion ; l’analyse des profils génétiques de Campylobacter issus de cas humains montre qu’il y a peu d’isolats identiques ou génétiquement proches entre les cas humains déclarés en 2003 et ceux déclarés en 2004. Cela met en évidence une grande diversité

* Corresponding author. E-mail address: [email protected] (M. Denis). 0369-8114/$ – see front matter # 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.patbio.2008.04.007

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génétique dans les isolats de Campylobacter et indique qu’il sera difficile de lier ces infections humaines à des groupes d’isolats présentant des profils génétiques particuliers. Les deux filières animales ont leurs propres génotypes en Campylobacter et les campylobacters de porc sont génétiquement distants de ceux trouvés chez les humains. Nous avons montré que 44,6 % des campylobactérioses humaines en Bretagne sont dues à la volaille et qu’une part de celles-ci est due à la manipulation de ces animaux. Cela est cohérent pour la Bretagne, une région caractérisée par de nombreuses fermes et abattoirs. # 2008 Elsevier Masson SAS. All rights reserved. Keywords: Campylobacter; PFGE; Molecular epidemiology; Poultry; Pig; Human Mots clés : Campylobacter ; PFGE ; Épidémiologie moléculaire ; Volaille ; Porc ; Humain

1. Introduction Campylobacter is the bacterium most frequently implicated in acute gastroenteritis in the industrialized world. The main form of campylobacteriosis of importance for public health is Campylobacter enteritis due to Campylobacter jejuni and Campylobacter coli [1]. In France, C. jejuni and C. coli account for 76.2 and 17.2%, respectively, of the isolates collected from human specimens [2].These two species are commonly found in French poultry (66% C. jejuni and 34% C. coli) [3,4] and farmed pigs (100% C. coli) [5]. We therefore carried out molecular typing to analyze the C. jejuni and C. coli strains originating from these animals and from human populations. Several genetic typing methods have been developed for epidemiological studies of Campylobacter infections, to differentiate between isolates below the level of the species [6]. Macrorestriction profiling by pulsed-field gel electrophoresis has proved useful for this purpose [7,8]. We used pulsed-field gel electrophoresis to investigate the genetic relationship between Campylobacter isolates from poultry, pig and sporadic human cases of campylobacteriosis, in a defined geographic area of France over a defined time period. 2. Materials and methods 2.1. Campylobacter isolates The isolates analyzed in this study were collected in Brittany (France) during 2003 and 2004. In total, 582 isolates were analyzed. These isolates were obtained from poultries (182 C. jejuni, 137 C. coli), pigs (86 C. coli) and from human cases (151 C. jejuni and 26 C. coli). The human isolates were obtained from the French National Reference Center for Campylobacter. They were initially isolated by 16 clinical laboratories spread throughout Brittany, and this collection represents all the Campylobacter strains collected in Brittany in 2003 and 2004. Each isolate corresponded to a patient with gastroenteritis. The 405 isolates of animal origin came from:  poultry samples, from caeca collected from farms (52 C. jejuni, 68 C. coli) or slaughterhouses (42 C. jejuni, 47 C. coli), and from chicken legs obtained from supermarkets (88 C. jejuni, 22 C. coli), and;

 pig samples, from caeca collected in slaughterhouses (86 C. coli). Only one isolate per poultry flock, per chicken leg or per pig batch was studied. 2.2. Identification at the species level C. jejuni and C. coli were identified by real-time PCR for the human isolates [9] and m-PCR for the animal isolates [10]. 2.3. Pulsed-field gel electrophoresis (PFGE) and electrophoretic pattern analysis DNA preparation, restriction endonuclease digestion and PFGE were carried out as described by Rivoal et al. [4]. Two profiles, corresponding to the restriction profiles obtained with SmaI and KpnI, were obtained for each isolate. Electrophoretic patterns were compared using BioNumerics1 (Applied Maths, Sint-Martens-Latem, Belgium). Similarities between profiles, based on band positions, were determined by calculating the Dice correlation coefficient, with a maximum position tolerance of 1%. A dendrogram based on the combined results for Kpn1- and Sma1-digested DNA (KS) was constructed, to reflect the similarities between the strains in the matrix. Strains were clustered by the Unweighted Pair-Group Method using the Arithmetic Mean (UPGMA) [11]. Isolates with high similarity levels were considered to be derived from the same parental strain and were clustered using a threshold of 80% [12]. The Simpson’s index (D) determined as follows [13] to assess the genetic diversity of the Campylobacter populations: D¼1

S X 1 n jðn j  1Þ NðN  1Þ j¼1

N: number of isolates tested; S: number of different genotypes; nj: number of isolates belonging to type j. 3. Results 3.1. Characteristics of Campylobacter human infections in Brittany From January 2003 to December 2004, the French National Reference Center for Campylobacter received 151 C. jejuni and 26 C. coli isolates from Brittany. A seasonal increase, with a

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Fig. 1. Number of human cases, by month, from January 2003 to December 2004.

higher incidence during the warmer months, was noted and was more pronounced for C. jejuni (Fig. 1). A significant peak in the number of cases was observed in August 2003 (18 human cases). The median age of patients was 28 years; 25% of the human cases concerned children under the age of five years and

the number of cases decreased with age (Fig. 2), with a second small peak between the ages of 45 and 54 years for 2003 and 2004. A significant difference was observed between male and female subjects for the five to 14 years and 35 to 54 years age groups, for which Campylobacter infection occurred

Fig. 2. Distribution of human cases of Campylobacter as a function of age in 2003 and 2004.

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Table 1 Genetic diversity of Campylobacter isolates collected in Brittany, France, in 2003 and 2004

3.2. Genetic relationship between human and animal Campylobacter isolates

Campylobacter species

Typing by pulsed-field gel electrophoresis generated 165, 126 and 84 genetic KS profiles for the 182 poultry C. jejuni, the 137 poultry C. coli and the 86 pig C. coli, respectively (Table 1). Simpson’s index was high (0.998, 0.998 and 0.999, respectively) indicating considerably diversity in each population of Campylobacter isolates from animals. Campylobacter isolates of animal origin were then compared with human isolates. We identified six C. jejuni KS profiles as common to poultry and human isolates (Fig. 3). An analysis of the genetic similarity of C. jejuni isolates (animal and human) resulted in the construction of 69 clusters, containing 66.3% of the C. jejuni isolates. Overall, isolates from poultry belonged to 37 clusters that also contained human isolates. These isolates originated from samples collected from farms (24.2%), slaughterhouses (42.3%) and supermarkets (33.5%). For the C. coli isolates (animal and human), no PFGE profile common to the three sources was detected. Analysis of the genetic similarity of the isolates resulted in the construction of 37 clusters, containing 62.2% of the C. coli isolates. Poultry isolates were found in 10 clusters containing human isolates. These isolates were obtained from farms (17.6%), slaughterhouses (55.2%) and supermarkets (27.2%). The pig isolates were systematically found in clusters containing no poultry or human isolates. Sixty-five of the 151 human C. jejuni isolates and 14 of the 26 human C. coli isolates clustered with poultry isolates. Thus, 44.6% of human Campylobacter isolates were genetically related to genotypes found in poultry. The monthly distribution of these human isolates is illustrated in Fig. 4. No significant

C. jejuni

C. coli

Origin of Campylobacter isolates

Isolate number KS number Simpson’s index Isolate number KS number Simpson’s index

Human

Poultry

Pig

151 123 0.997 26 25 0.996

182 165 0.998 137 126 0.998

– – – 86 84 0.999

principally in male subjects (Pearson’s chi-squared, p = 0.003). The ratio of C. jejuni to C. coli isolates did not vary significantly with season, age or sex (Pearson’s chi-squared, p > 0.05). Typing by pulsed-field gel electrophoresis generated 123 genetic KS profiles for the 151 C. jejuni isolates and 25 such profiles for the 26 C. coli isolates (Table 1). Simpson’s index was high for both species (0.997 and 0.996, respectively), indicating a high level of genetic diversity among human Campylobacter strains. Ten isolates in 2003 and five in 2004 had identical KS profiles. In three of these cases, familial Campylobacter infection was reported. The analysis of genetic similarity resulted in 24 clusters for C. jejuni and three clusters for C. coli, encompassing 52.8 and 38.6% of the human isolates obtained in 2003 and 2004, respectively. Clustering did not depend on patient age, time of year or location within Brittany (data not shown). Twelve clusters contained isolates from both years. These clusters comprised 21.3 and 17.0% of the isolates collected in 2003 and 2004, respectively. In four cases, isolates, in both 2003 and 2004, had the same KS.

Fig. 3. C. jejuni isolates with identical electrophoretic patterns from poultry and humans...FM. . ..: human isolates; ..FF. . .. and ..MJL. . . poultry isolates.

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Fig. 4. Distribution of human isolates by month and presence in clusters containing poultry isolates. H + P: human isolates in clusters with poultry isolates; H: other human isolates.

difference was found between human isolates clustered and not clustered with poultry isolates as a function of month in the year (Pearson’s chi-squared test, p > 0.05). However, in the August 2003 peak, 12 of the 18 human isolates were clustered with poultry isolates. Of the 106 clusters in total (69 C. jejuni clusters, 37 C. coli clusters), only 15 included both isolates from farms or slaughterhouses and isolates from supermarkets 4. Discussion The data collected for Campylobacter infections in Brittany, a region of France, in this study, are consistent with the data collected throughout France and reported by Gallay et al. [2]. The ratio of C. jejuni to C. coli isolates was also similar. An increase in human cases during the warmer months was noted and was more pronounced for C. jejuni. The isolation rate was also highest for children under the age of five years. These findings indicate that the human isolate sample used in this study is representative of French human Campylobacter isolates in general. In 2003 and 2004, few human isolates had identical KS profiles, and only 21.3 and 17.0% of the isolates collected in 2003 and 2004, respectively, belonged to the same clusters. This suggests that it would be difficult to link human Campylobacter infections to group of isolates with a particular profile. The main source of human Campylobacter infections highlighted by many epidemiological studies is the consumption of contaminated food — particularly raw or insufficiently cooked poultry products [14,15]. Pork meat has also been implicated in human Campylobacter infection. Gillepsie et al. [16] showed in 2002 that individuals presenting C. coli infections in the UK were more likely to have eaten pork pâté. Friedman et al. [1] in the USA identified the consumption of non poultry meats, such as hamburgers, pork roasts and

sausages, as an important risk factor for sporadic Campylobacter infections. In our PFGE study, the genetic comparison of Campylobacter isolates from poultry and pig production systems with isolates from human cases highlighted the presence of identical isolates in poultry and humans. In addition, 47 clusters contained isolates from both poultry and humans. These results confirm those of other studies implicating poultry in human cases of campylobacteriosis. A number of studies based on PFGE have confirmed the existence of a genetic relationship between human and chicken isolates of Campylobacter. In Canada, Nadeau et al. [17] showed that 20% of the genotypes of human isolates were genetically related to genotypes from poultry isolates. In 2005, Michaud et al. [18] reported that 19 of 41 Canadian poultry isolates had PFGE profiles similar to those of 41 human isolates (of a total of 183). Kärenlampi et al. [19] found that 31% of genotypes were common to poultry and human isolates in Finland. In Sweden, Lindmark et al. [8] described a cluster containing Campylobacter isolates from humans and chicken meat. Cardinale et al. [20] also identified common genotypes between human and poultry isolates of C. jejuni in Senegal, as did Saito et al. [21] in Japan. Other genotyping methods have also demonstrated the existence of a genetic link between isolates from poultry and from humans. Genotypes common to human and poultry isolates and clusters containing both human and poultry isolates were identified by Steinhauserova et al. [22], by PCR-RFLP, in the Czech Republic, by Wieland et al. [23], by AFLP, in Switzerland and by Ishihara et al. [24] by flaA typing, in Japan. We found that 44.6% of human campylobacteriosis cases were related to poultry in Brittany, France. This percentage is similar to that reported in Belgium (40%) in 2002, in a study carried out during the dioxin crisis [25]. The increase in the number of cases in August 2003 concerned human isolates clustered with poultry isolates, in

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particular. This increase may be associated with the seasonal effect reported for the colonization of broiler chickens by Campylobacter in two French studies [26,27] We found that C. coli isolates from pigs were systematically found in clusters different from those including C. coli isolates from poultry and/or humans. In the United Kingdom, Dingle et al. [28], using the multilocus sequence typing (MLST) method, observed that chickens and pigs on the same farm were colonized with different STs indicating the existence of a host preference in certain C. coli genotypes. Host-associated alleles were also identified by MLST of C. coli in the USA by Miller et al. [29]. The genetic separation of C. coli from poultry and C. coli from pigs was demonstrated by Hopkins et al. [30] by AFLP in the United Kingdom and by Siemer et al. [31] in Denmark. Siemer also showed that the C. coli isolates obtained from poultry belonged to the same genetic groups as the isolates from humans with campylobacteriosis. For the same period and the same geographic area in Canada, Guévremont et al. [32] used PFGE to compare 660 isolates from pig caeca with 24 isolates from cases of human diarrhea. No genetically identical isolates common to the two sources was observed. Our results indicate that, in France, these two animal production systems seem to be associated with different genotypes, and that Campylobacter isolates from pigs are not genetically related to those from humans. Sixty-six percent of poultry isolates from clusters containing human isolates were isolated from caeca obtained from farms and slaughterhouses, and 34% were obtained from chicken legs purchased in supermarkets. These findings suggest that cases of campylobacteriosis are due to contact with poultry. Ethelberg et al. [33] identified living in a rural area and contact with animals as risk factors for Campylobacter infection. This hypothesis is supported by our findings for Brittany, an area in France with a large number of animal-rearing farms and slaughterhouses. Only a few clusters included isolates from both farms or slaughterhouses and supermarkets. There was thus little similarity between the genotypes found at farms and slaughterhouses and those found in supermarkets. This is entirely logical, given that the isolates obtained from supermarkets in Brittany were isolated from chicken legs originating from different areas of France. Macrorestriction profiling by PFGE has proved useful in this study for evaluating the genetic relationship between Campylobacter isolates from poultry, pig and sporadic cases of human campylobacteriosis.

[17]

Acknowledgments

[18]

This work was financially supported by the ‘‘Région Bretagne’’ and the ‘‘Syndicat mixte du zoopole’’ of Ploufragan. The autors thank G. Hellard for technical assistance.

[2]

[3]

[4]

[5]

[6] [7]

[8]

[9]

[10]

[11]

[12]

[13] [14] [15]

[16]

[19]

References [1] Friedman CR, Hoekstra RM, Samuel M, Marcus R, Bender J, Shiferaw B, et al. and Emerging Infections Program FoodNet Working Group. Risk

[20]

factors for sporadic Campylobacter infection in the United States: a case-control study in FoodNet sites. Clin Infect Dis 2004; 15;38 Suppl. 3:S285-96. Gallay A, Prouzet-Mauléon V, Kempf I, Lehours P, Labadi L, Camou C, et al. Campylobacter antimicrobial drug resistance among humans, broiler chickens, and pigs. France Emerg Infect Dis 2007;13:259–601. Avrain L, Humbert F, L’Hospitalier R, Sanders P, Vernozy-Rozand C, Kempf I. Antimicrobial resistance in Campylobacter from broilers: association with production type and antimicrobial use. Vet Microbiol 2003;96:267–376. Rivoal K, Ragimbeau C, Salvat G, Colin P, Ermel G. Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free range broiler farms. Comparison with isolates of various origins. Appl Environ Microbiol 2005;71:6216–27. Payot S, Dridi S, Laroche M, Federighi M, Magras C. Prevalence and antimicrobial resistance of Campylobacter coli isolated from fattening pigs in France. Vet Microbiol 2004;101:91–9. Wassenaar TM, Newell DG. Genotyping of Campylobacter spp. Appl Environ Microbiol 2000;66:1–9. On SL, Nielsen EM, Engberg J, Madsen M. Validity of SmaI-defined genotypes of Campylobacter jejuni examined by SalI, KpnI and BamHI polymorphisms: evidence of identical clones infecting humans, poultry, and cattle. Epidemiol Infect 1998;120:213–37. Lindmark H, Harbom B, Thebo L, Andersson L, Hedin G, Osterman B, et al. Genetic characterization and antibiotic resistance of Campylobacter jejuni isolated from meats, water, and humans in Sweden. J Clin Microbiol 2004;42:700–6. Menard A, Dachet F, Prouzet-Mauleon V, Oleastro M, Mégraud F. Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp. Clin Microbiol Infect 2005;11:281–7. Denis M, Soumet C, Rivoal K, Ermel G, Blivet D, Salvat G, et al. Development of a m-PCR for simultaneous identification of Campylobacter jejuni and Campylobacter coli. Lett Appl Microbiol 1999;29: 406–10. Struelens MJ. Members of the European Study Group on Epidemiological Markers (ESGEM). Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems. Clin Microbiol Infect 1996;2:2–11. Denis M, Rose V., Huneau-Salaün A, Balaine L, Salvat G. Diversity of Pulsed-Field Gel Electrophoresis profiles of Campylobacter jejuni and Campylobacter coli from broiler chickens in France. Poultry Sciences 2008; in press. Hunter P. Reproducibility and indices of discriminatory power of microbial typing methods. J Clin Microbiol 1990;28:1903–5. Moore JE, Corcoran D, Dooley JSG, Fanning S, Lucey B, Matsuda M, et al. Campylobacter. Vet Research 2005;36:351–82. Mazick A, Ethelberg S, Moller Nielsen E, Molbak K, Lisby M. An outbreak of Campylobacter jejuni associated with consumption of chicken, Copenhagen, 2005. Euro Surveill 2005;11:137–9. Gillespie IA, O’Brien SJO, Frost JA, Adak JK, Horby P, Swan AV, et al. the Campylobacter Sentinel Surveillance Scheme Collaborators. A case-case comparison of Campylobacter coli and Campylobacter jejuni infections: a tool for generating hypotheses. Emerg Infect Dis 2002;8:937–42. Nadeau E, Messier S, Quessy S. Prevalence and comparison of genetic profiles of Campylobacter strains isolated from poultry and sporadic cases of campylobacteriosis in humans. J Food Protect 2002;65:73–8. Michaud S, Ménard S, Arbeit RD. Role of real-time molecular typing in the surveillance of Campylobacter enteritidis and comparison of pulsedfield gel electrophoresis profiles from chicken and human isolates. J Clin Microbiol 2005;43:1105–11. Kärenlampi R, Rautelin H, Hakkinen M, Hänninen ML. Temporal and geographical distribution and overlap of Penner heat-stable serotypes and pulsed-field gel electrophoresis genotypes of Camplylobacter jejuni isolates collected from humans and chickens in Finland during a seasonal peak. J Clin Microbiol 2003;41:4870–2. Cardinale E, Rose V, Perrier Gros-Claude JD, Tall F, Rivoal K, Mead G, et al. Genetic characterization and antibiotic resistance of Campylobacter

M. Denis et al. / Pathologie Biologie 57 (2009) 23–29

[21]

[22]

[23]

[24]

[25] [26]

[27]

spp. Isolated from poultry and humans in Senegal. J Appl Microbiol 2006;100:209–17. Saito S, Yatsuyanagi J, Harata S, Ito Y, Shinagawa K, Suzuki N, et al. Campylobacter jejuni isolated from retail poultry meat, bovine feces and bile, and human diarrheal samples in Japan: comparison of serotypes and genotypes. FEMS 2005;45:311–9. Steinhauserova I, Ceskova J, Nebola M. PCR/Restriction fragment length polymorphism (RFLP) typing of human and poultry Campylobacter jejuni strains. Lett Appl Microbiol 2002;34:354–8. Wieland B, Wittwer M, Regula G, Wassenaar TM, Burnens AP, Keller J, et al. Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni. J Appl Microbiol 2005;100:316–24. Ishihara K, Yamamoto T, Satake S, Takayama S, Kubota S, Negishi H, et al. Comparison of Campylobacter isolated from humans and foodproducing animals in Japan. J Appl Microbiol 2006;100:153–60. Vellinga A, Van Loock F. The dioxin crisis as experiment to determine poultry-related Campylobacter enteritis. Emerg Infect Dis 2002;8:19–22. Refrégier-Petton J, Rose N, Denis M, Salvat G. Risk factors for Campylobacter jejuni and Campylobacter coli contamination in French broilerchicken flocks at the end of the rearing period. Prev Vet Med 2001;50: 89–100. Huneau-Salaün A, Denis M, Balaine L, Salvat G. Risk factors for Campylobacter spp. colonization in French free range broiler-chicken

[28]

[29]

[30]

[31]

[32]

[33]

29

flocks at the end of the indoor rearing period. Prev Vet Med 2007;80: 34–48. Dingle KA, Colles FM, Falush D, Maiden MCJ. Sequence typing and comparison of population biology of Campylobacter coli and Campylobacter jejuni. J Clin Microbiol 2005;43:340–7. Miller WG, Englen MD, Kathariou S, Wesley IV, Wang G, Pittenger-Allay L, et al. Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals. Microbiol 2006;152:245–55. Hopkins KL, Desai M, Frost JA, Stanley J, Logan JMJ. Fluorescent amplified fragment length polymorphism genotyping of Campylobacter jejuni and Campylobacter coli strains and its relationship with host specificity, serotyping, and phage typing. J Clin Microbiol 2004;42: 229–35. Siemer BL, Nielsen EM, On SLW. Identification and molecular epidemiology of Campylobacter coli isolates from humans gastroenteritis, food and animal sources by amplified fragment length polymorphism analysis and Penner serotyping. Appl Environ Microbiol 2005;71:1953–8. Guévremont E, Higgins R, Quessy S. Characterization of Camplylobacter isolates recovered from clinically healthy pigs and from sporadic cases of campylobacteriosis in humans. J Food Prot 2004;67:228–34. Ethelberg S, Simonsen J, Gerner-Smidt P, Olsen KEP, Molbak K. Spatial distribution and registry-based case-control analysis of Campylobacter infections in Denmark, 1991–2001. Am J Epidemiol 2005;162:1008–15.