Campylobacter jejuni and C. coli are the most frequent causes of bacterial gastroenteritis in Finland (www.ktl.fi/ttr). The number of laboratory-confirmed cases has nearly doubled over a 10-year period (2,197 cases in 1995 to 4,002 cases in 2005). Campylobacter species are frequently isolated from a wide variety of sources, including poultry, cattle, pigs, sheep, cats, dogs, wild birds, and water. Most Campylobacter infections are sporadic, and the relative contributions of different sources of infection remain unknown. Risk factors for Campylobacter infection, as identified by case-control studies, include eating or preparing raw or undercooked meat, especially chicken meat, drinking unpasteurized milk or untreated water, contact with domestic animals and pets, and foreign travel (14,18,26). A recent case-control study (19) revealed swimming in natural bodies of water to be a novel risk factor. In addition, eating undercooked meat and drinking from dug wells were independent risk factors for domestically acquired Campylobacter infections in Finland (19).Discriminatory typing methods for use in the study of the molecular epidemiology and population genetics of Campylobacter isolates are crucial to better understand the epidemiology and ecology of the organism. Once such information is obtained, it could be used to develop intervention strategies to limit the numbers of human infections. Dingle et al. (6) developed a multilocus sequence typing (MLST) scheme for C. jejuni, which has been shown to be a valuable tool for studying the diversity and population genetics of Campylobacter isolates.

The association of Penner heat-stable serotypes and pulsed-field gel electrophoresis genotypes of 208 human and 30 chicken Campylobacter jejuni isolates was studied. Overall, 46% of the human strains had overlapping sero-and genotype combinations with chicken strains. The percentage was reduced to 31% for strains that were considered temporally related. This suggests common environmental sources.Campylobacter jejuni is the most frequent bacterial cause of human gastroenteritis in developed countries worldwide (3,11). Case control studies have indicated that improper handling of poultry products and eating raw or undercooked chicken are important risk factors for human campylobacter infections. Yet, most campylobacter infections are sporadic, and the sources of infection remain unidentified. Human campylobacter infections in northern Europe show a peak during the summer months of July, August, and September (9). In Finland, most domestic campylobacter infections occur from July to August, and the number of campylobacter-positive chicken flocks increases during the same time period (6, 11).Serotyping has traditionally been used for typing C. jejuni isolates, but pulsed-field gel electrophoresis (PFGE) using SmaI and SacII/KpnI has been identified as a more highly discriminatory method of studying the epidemiology of C. jejuni infection (4, 6, 7). Overlapping serotypes and PFGE genotypes have been identified in chicken and human isolates (2,6,8), but the geographical and temporal overlap of such strains has not been studied extensively.In an earlier study (15), all domestic-laboratory-confirmed human C. jejuni isolates (533) from sporadic cases were collected from the whole of Finland from July to September 1999. Thirty chicken C. jejuni isolates were collected during the same seasonal peak from every positive flock at three major abattoirs, accounting for 98% of chicken meat production in Finland (10). In the present study 208, previously serotyped, human C. jejuni strains were typed by PFGE and their association with the chicken strains was evaluated. The human strains originated from six hospital districts in Finland (271 isolates in total) and included 10 Penner heat-stable (HS) serotypes, also identified in chicken flocks during the same seasonal peak (HS1/44, HS2, HS4 complex, HS5, HS6/7, HS11, HS12, HS27, HS41, and HS57), as well as nonserotypeable (NS) human strains.The DNA plugs for PFGE analysis were prepared as previously described (5, 7, 12). The DNA was digested by SmaI and KpnI (New England Biolabs Inc.; 20 U per sample), and the restriction fragments were separated with ramped pulses of 1 to 30 s and 1 to 25 s for 19 h, respectively. After computerassisted (BioNumerics, version 3.0; Applied Maths, Kortrijk, Belgium) and visual analyses of the patterns, clusters of closely related and indistinguishable genotypes were identified. PFGE genotypes were considered closely related if they differed by one to three bands and were considered indistinguishable if they had all bands in common (14). The combined ...

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The evolution and taxonomy of enterohepatic Helicobacter species with flexispira morphology were studied by a polyphasic approach including phenotypic characterization, analysis of 16S rRNA and ureB gene sequences and dot-blot DNA–DNA hybridization of whole genomic DNA. In addition, available phylogenetic data on the HSP60 gene were used in the analysis. The study included 14 Finnish canine and feline flexispira strains, the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and Helicobacter bilis ATCC 51630T. Phenotypically, all canine and feline strains were similar to H. bilis. Analysis of 16S rRNA gene sequences of these strains revealed a similarity of 97–99·5 %. Similarity of ureB nucleotide and amino acid sequences within the studied flexispira group was 97–100 % and 99–100 %, respectively, revealing close relatedness. ureB sequences of Helicobacter hepaticus had only 64–66 % similarity to the flexispira group. The similarity to Helicobacter trogontum was 81·5–82·1 %. High levels of DNA–DNA hybridization between the strains were found in dot-blot tests. Polyphasic analysis of the phenotypic and genotypic characteristics of the Finnish flexispira strains and the reference strains of taxa 2, 3 and 8 showed that they differed from other Helicobacter species and are members of the previously described species H. bilis. In addition, canine strain F56 differed in all phylogenetic analyses from the H. bilis group and probably represents a novel Helicobacter species.

Analysis of 16S rRNA gene sequences has been the method generally used to study the evolution and phylogeny of bacteria. Phylogenetic analysis of the 16S rRNA gene has shown the position of the genus Helicobacter in the e-subclass of the Proteobacteria. Because 16S rRNA-based phylogeny does not always correspond to the results of polyphasic taxonomy, and the related species cannot always be separated, new phylogenetic markers for Helicobacter species are needed. In this study, conserved partial (600 bp) 60 kDa heat-shock protein (HSP60) sequences were used to study the phylogeny of 37 strains of gastric and enterohepatic Helicobacter species, including type strains of 15 Helicobacter species with validly published names, reference strains of flexispira taxa and Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis and canine flexispira strains. The partial HSP60 gene sequence proved to be a useful phylogenetic marker for the genus Helicobacter, providing a means of differentiating all 15 Helicobacter species analysed. In the resulting phylogenetic tree, gastric Helicobacter species and enterohepatic species with flexispira morphology formed tight, separate clusters. In general, HSP60 sequence similarities between Helicobacter species were significantly lower than the corresponding 16S rRNA gene sequence similarities, indicating a better resolution for species identification. In addition, a specific PCR method for identifying H. salomonis was developed based on the partial HSP60 sequence. INTRODUCTIONHelicobacter species have been isolated from the gastrointestinal tract of humans and several animal species, including cats, dogs, monkeys, sheep, pigs, rodents, birds, cheetahs and poultry (On, 1996Solnick & Schauer, 2001). At the time of writing (September 2003; http:// www.bacterio.cict.fr), there were 22 Helicobacter species with validly published names and two uncultured 'Candidatus' species (Dewhirst et al., 2000b). Several other Helicobacter taxa remain unnamed, because they have not been properly described according to the internationally accepted rules of nomenclature. Flexispiras constitute one of the unnamed groups of Helicobacter species, sharing spindleshaped cell morphology and periplasmic fibrils (Bryner et al., 1987). The group includes three species with validly published names, Helicobacter bilis (Fox et al., 1995), Helicobacter trogontum (Mendes et al., 1996; Hänninen et al., 2003) and Helicobacter aurati (Patterson et al., 2000), plus several unnamed taxa (on the basis of analysis of 16S rRNA gene sequences) (Dewhirst et al., 2000a).Classification of helicobacters has been hampered by difficulties in growing them in vitro and by their inertness in traditionally used biochemical tests. Culture-independent genetic methods, such as 16S rRNA gene sequence analysis, have therefore been applied to study the phylogeny of the genus as a basis for classification (Dewhirst et al., 2000a; Jalava et al., 1997;). The results from 16S rRNA gene analyses are not always concordant with tho...