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Prevalence of 11 pathogenic genes of Campylobacter jejuni by PCR in strains isolated from humans, poultry meat and broiler and bovine faeces

Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Correspondence Kikuji Itoh akikuji{at}mail.ecc.u-tokyo.ac.jp

Received 19 August 2002 Accepted 5 December 2002

Although many genes related to the pathogenicity of Campylobacter jejuni have been reported, the relationships between these genes and the sources of strains are not clear. In this study, the presence of 11 pathogenic genes responsible for the expression of adherence, invasion, colonization and cytotoxin production was examined in 111 C. jejuni isolated from human clinical samples, poultry meat, broiler faeces and bovine faeces. For most of the pathogenic genes, no difference in their presence in C. jejuni was found among the sources, but, for racR, wlaN and virB11, there were some variations among sources. The racR gene was present at rates of 98.2 (human clinical samples), 90.5 (poultry meat), 85.7 (broiler faeces) and 76.7 % (bovine faeces). Detection rates for the wlaN gene were 25.0, 23.8, 4.7 and 7.7 % and those for the virB11 gene were 10.7, 9.5, 9.5 and 15.4 % in human clinical samples, poultry meat, broiler faeces and bovine faeces, respectively. One hundred and seven of 111 strains (96.4 %) carried from eight to 10 of the pathogenic genes. These data did not show remarkable differences in the presence of pathogenic genes carried by C. jejuni from various sources.

Campylobacter jejuni is one of the most common causes of bacterial enteritis. Campylobacteriosis is mainly a food-borne infection, and poultry products may play an important role in transmission to humans (Park, 2002). C. jejuni contamination of poultry meat during processing has been reported (Kazwala et al., 1990; Chuma et al., 1994, 1997; Berrang et al., 2001; Hartnett et al., 2001). Adherence, invasion and cytotoxin production appear to be possible virulence factors. The clinical and epidemiological characteristics of the disease provide clues to the molecular mechanisms that play a role in C. jejuni infection. Recently, some genes have been recognized as responsible for the expression of pathogenicity. In this study, flaA (Nuijten et al., 2000), cadF (Ziprin et al., 2001), racR (Bras et al., 1999) and dnaJ (Ziprin et al., 2001) were selected as pathogenic genes responsible for the expression of adherence and colonization, virB11 (Bacon et al., 2000), ciaB (Konkel et al., 1999a; Rivera-Amill et al., 2001) and pldA (Ziprin et al., 2001) as pathogenic genes responsible for the expression of invasion and cdtA, cdtB and cdtC as pathogenic genes responsible for the expression of cytotoxin production (Purdy et al., 2000; Lara-Tejero & Galan, 2001), while wlaN (Linton et al., 2000) was selected as a pathogenic gene responsible for the expression of Guillain–Barré syndrome. In this study, PCR was applied to analyse the relationship between pathogenic genes and sources of C. jejuni.

Methods

Bacterial strains and growth conditions.

One hundred and eleven strains in total were tested in this study, including 56 isolates from human clinical samples, 21 from poultry meat, 21 from broiler faeces and 13 from bovine faeces. All strains were incubated on Müller–Hinton agar (Oxoid) at 42 °C under microaerobic conditions (85 % N₂, 5 % O₂, 10 % CO₂) for 24 h.

Preparation of DNA.

Template DNAs for PCR were extracted by the conventional boiling method. Fresh cultures of C. jejuni were suspended in 300 µl TE buffer and the suspensions were boiled at 95 °C for 10 min. After centrifugation at 15 000 r.p.m. for 2 min, the supernatants were stored at –20 °C and used as template DNA.

PCR primer design and amplification.

Three sets of primers were described by Konkel et al. (1999b) (cadF), Hickey et al. (2000) (cdtA) and Linton et al. (2000) (wlaN). Eight further sets of primers were designed based on sequences from GenBank accession numbers AF345999 (flaA), AF053960 (racR), AF052661 (dnaJ), AF226280 (virB11), AF114831 (ciaB), NC_002163 (pldA), AF289020 (cdtB) and AJ000859 (cdtC). Primer sequences, annealing temperatures and lengths of products are listed in Table 1.

All PCR amplifications were performed in a mixture (30 µl) consisting of 3 µl 10x ExTaq buffer, 0.6 U ExTaq (Takara), 2.4 µl dNTPs (dATP, dCTP, dGTP, TTP, each at 2.5 mM; Takara), 0.6 µl extracted template DNA and 0.3 µl of a 20 pM solution of each primer. Distilled water was added to make 30 µl. The reaction mixture was overlaid with mineral oil and subjected to 30 cycles of amplification in a DNA thermal cycler 480 (Takara). The cycling was as follows: denaturation at 94 °C for 1 min, annealing at a temperature specific to the primer pair for 1 min and extension at 72 °C for 1 min. Part of this amplified sample was analysed by electrophoresis on 1.5 % NuSieve agarose gels (Rockland). DNA bands were stained with ethidium bromide, visualized with a UV transilluminator (ATTO) and photographed (Fig. 1).

View larger version (112K): [in this window] [in a new window]   Fig. 1. Agarose gel electrophoresis of PCR products of 11 pathogenic genes of C. jejuni. Lanes: M, 100 bp marker; 1, flaA; 2, cadF; 3, racR; 4, dnaJ; 5, virB11; 6, ciaB; 7, pldA; 8, cdtA; 9, cdtB; 10, cdtC; 11, wlaN.

Results and Discussion

Five of the 11 pathogenic genes, flaA, cadF, cdtA, cdtB and cdtC, were detected from all strains, and the detection rates of three genes, racR, wlaN and virB11, varied among sources of isolates. Rates of three other genes, dnaJ, ciaB and pldA, also varied among sources, but not so markedly (Table 2).

Only two strains from human clinical samples had all 11 pathogenic genes, and two strains from bovine faeces had six genes. The other 107 strains carried from eight to 10 genes related to pathogenicity, independent of source (Table 3).

C. jejuni strains from all sources tested in this study had most of the pathogenic genes examined. However, racR, wlaN and virB11 showed some variation in prevalence according to source. The importance of these three genes in the pathogenicity of C. jejuni compared with other pathogenic genes is not clear. Interestingly, the detection rate of the wlaN gene from human clinical samples (25.0 %) was similar to that from poultry meat (23.8 %), but different from that from broiler and bovine faeces. We could not distinguish C. jejuni strains on the basis of source using data for the pathogenic genes carried by C. jejuni. Further studies are needed to reveal the relationship between each pathogenic gene or combinations of these genes and the pathogenicity of C. jejuni.

ACKNOWLEDGEMENTS

Acknowledgements

We thank Dr D. G. Newell (Central Veterinary Laboratory, UK), Dr T. Itoh (Tokyo Metropolitan Research Laboratory of Public Health, Japan), Dr H. Lior (Laboratory Centre for Disease Control, Canada), Dr G. B. Lindblom (University of Göteborg, Sweden), Dr A. S. Greef (Medical University of Southern Africa, South Africa), Dr S. Yamai (Kanagawa Institute of Public Health, Japan) and Dr T. Chuma (Kagoshima University, Japan) for kindly providing C. jejuni strains used in this study.

References