APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2010, p. 7318–7321 0099-2240/10/$12.00 doi:10.1128/AEM.00511-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
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Isolation of a Novel Campylobacter jejuni Clone Associated with the Bank Vole, Myodes glareolus䌤 N. J. Williams,1,2†* T. R. Jones,1† H. J. Leatherbarrow,3 R. J. Birtles,1 A. Lahuerta-Marin,4 M. Bennett,1 and C. Winstanley1,2 National Centre for Zoonosis Research, School of Veterinary Science, Leahurst Campus, University of Liverpool, Neston, Cheshire CH64 7TE, United Kingdom1; School of Infection and Host Defence, University of Liverpool, Liverpool L69 3BX, United Kingdom2; School of Translational Medicine, University of Manchester, Wythenshawe Hospital, Manchester M23 9LT, United Kingdom3; and European Centre for Disease Prevention and Control (ECDC), Tomtebodavagen, 11A-Solna, SE 17183 Stockholm, Sweden4 Received 25 February 2010/Accepted 4 September 2010
Campylobacter jejuni can be isolated from different animal hosts. Various studies have used multilocus sequence typing to look for associations between particular clones of C. jejuni and specific hosts. Here, we describe the isolation of a novel clone (sequence type 3704 [ST-3704]) of C. jejuni associated with the bank vole (Myodes glareolus).
role of wildlife as reservoirs of zoonotic enteric pathogens for livestock. Farms were sampled in summer/autumn on one occasion and in winter/spring on a second occasion; feces were collected from cattle, from wild mammals opportunistically, and from live-trapped rodents. For the isolation of Campylobacter spp., approximately 0.2 ml of fecal homogenate (1:1 feces in brain heart infusion broth) was added to campylobacter enrichment broth containing 5% (vol/vol) lysed horse blood (Southern Group Labs, Corby, United Kingdom), and samples were incubated at 37°C for 24 h under microaerobic conditions in a variable-atmosphere incubator (Don Whitley Scientific Ltd., Shipley, United Kingdom) before being inoculated onto campylobacter blood-free medium containing cefoperazone and amphotericin. These plates were incubated for up to 96 h at 37°C under microaerobic conditions before being examined for the presence of colonies characteristic of Campylobacter spp. All media were obtained from LabM (IDG), Bury, United Kingdom. Suspect isolates were presumptively identified as C. jejuni by Gram staining, no growth in air, and hippurate hydrolysis (6, 18) tests. For further assignment to species, cell lysates were prepared from isolates and subjected to a number of genus- and species-specific PCR assays (5, 14, 19). The whole genome sequence was obtained from bank vole strain C414 by shotgun sequencing. Of the samples obtained from woodland rodents, 23% (10/ 43) of bank vole samples collected from May to July 2001 and 51% (38/75) collected in January 2003 were positive for Campylobacter spp., whereas Campylobacter spp. were not recovered from any wood mouse samples (40 samples collected from May to July 2001 and 31 samples collected in January 2003). For the farm rodents (samples collected from September 2004 to April 2005), a total of 655 wood mice and 194 bank voles were sampled. In total, 18% (34/194) of bank voles were positive for Campylobacter spp., compared to 1% (6/655) of wood mice (Table 1). In total, 151 isolates were identified as Campylobacter spp. by using a genus-level 16S rRNA gene PCR assay (14). However, of all of these rodent isolates, only
Campylobacter jejuni is one of the most common causes of gastroenteritis in humans, with food (primarily chicken) believed to be the main vehicle for infection (8). Although high prevalences are found in livestock, C. jejuni has also been isolated from wildlife, including wild birds and wild mammals, and from the farm environment (1, 2, 4, 7, 11, 13). Multilocus sequence typing (MLST) has been used to study the distribution of specific clones among isolates from different hosts and the environment (1, 2, 3, 7, 10, 15, 16, 17). Such molecular epidemiological studies have provided evidence for some hostassociated genotypes. For example, some MLST clonal complexes are associated with cattle (sequence type 61 [ST-61] and ST-42 complexes), whereas others are associated with wildlife, such as rabbits and wild birds, and environmental samples (ST-45, ST-177, ST-677, ST-682, and ST-952 complexes) (2, 4, 13). In addition, previously unreported sequence types have been identified in wildlife and environmental samples (4). In this study, we describe the identification of a novel strain of C. jejuni that represents a new sequence type and that is restricted primarily to one wildlife host, namely, bank voles (Myodes glareolus), from which it was isolated over a relatively wide geographic area and time period. We undertook longitudinal studies of feces collected from the sympatric wild rodents bank voles and wood mice (Apodemus sylvaticus) in a private West Cheshire (United Kingdom) woodland habitat. Feces collected from both species were analyzed for the presence of Campylobacter spp. during two sampling periods (May to July 2001 and January 2003). In 2004 (summer/autumn) and 2005 (winter/spring), cross-sectional surveys were conducted on 6 farms (5 dairy and 1 beef farm) in South Cheshire (approximately 30 km away) to investigate the * Corresponding author. Mailing address: National Centre for Zoonosis Research, University of Liverpool, Leahurst, Neston, Cheshire CH64 7TE, United Kingdom. Phone: 44 151-795-6052. Fax: 44 151794-6005. E-mail:
[email protected]. † N.J.W. and T.R.J. contributed equally to this work. 䌤 Published ahead of print on 17 September 2010. 7318
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TABLE 1. Number of samples from rodents captured in the woodland and farm studies, as well as from cattle in the farm study, positive for Campylobacter jejuni and specifically for ST-3704 No. of positive samples/total no. of samples Animal species
Bacterial species or strain
Woodland study May–July 2001
January 2003
Farm study (2004-2005)
Bank voles
C. jejuni C. jejuni ST-3704
10/43 10/43
38/75 38/75
34/194 34/194
Wood mice
C. jejuni C. jejuni ST-3704
0/40 NAa
0/31 NA
6/655 3/655
Cattle
C. jejuni C. jejuni ST-3704
NA NA
NA NA
12/497 1/497
a
NA, not applicable.
three isolates from wood mice from the farm survey could be identified as C. jejuni by species-specific PCR assays. The remaining rodent isolates from both the woodland and the farm study did not give any amplicons using species-specific PCR assays (5, 14, 19).
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Two of these PCR assays targeted the hipO gene (14, 19) and used the same gene sequence (GenBank accession no. z36940) for design of the primers, and this gene shares only 90% homology with the hipO gene of bank vole strain C414 (data not shown). Furthermore, the other PCR assay used to identify C. jejuni targeted the ceuE gene (5), and the ceuE gene of C414 shares only 92% homology with the ceuE gene of NCTC11168 (data not shown). The primer binding sites were also divergent for these targets in C414 (5, 19) or were not present at all (14). By use of the method of Karenlampi et al. (9), which involves PCR amplification and sequencing of a groEL fragment, all of the bank vole isolates from the woodland study and one bank vole isolate from the farm study were found to have the same sequence. This sequence (C414, GenBank accession number HQ213856) was aligned with partial groEL sequences from other Campylobacter isolates, and phylogenetic trees were inferred from this alignment by using algorithms within the MEGA4 software package (12). In these trees, C414 clustered with C. jejuni strains and not with C. coli strains (Fig. 1). Hence, both hippurate hydrolysis (18) testing and partial groEL sequence analysis suggested that the bank vole-associated isolates were C. jejuni. MLST analysis was carried out on 41 bank vole isolates from the woodland study and 34 bank vole isolates and 6 wood
FIG. 1. Evolutionary relationships of strain C414, C. jejuni, C. coli, and C. lari. The evolutionary history was inferred using the minimum evolution (ME) method. The bootstrap consensus tree was inferred from 500 replicates. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those for the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are reported in units of the number of base substitutions per site. The ME tree was searched using a close-neighborinterchange (CNI) algorithm at a search level of 3. A neighbor-joining algorithm was used to generate the initial tree. Codon positions included were first plus second plus third plus noncoding positions. There were a total of 540 positions in the final data set. Phylogenetic analyses were conducted in MEGA4 (12).
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FIG. 2. SmaI PFGE analysis of C. jejuni ST-3704, C. jejuni isolates belonging to other sequence types, and C. fetus isolates from bank voles (BV), wood mice (WM), and cows from four Cheshire farms (F1, F2, F3, and F4). Lane , lambda ladder PFGE marker (New England Biolabs, Hitchin, United Kingdom).
mouse isolates from the farm studies, with each isolate representative of a single sample, using the method of Dingle et al. (3). For each of the seven loci, a new allele was identified (aspA227, glnA297, gltA253, glyA338, pgm-424, tkt-337, and uncA250), generating a new sequence type, ST-3704, for all of the bank vole isolates tested (100% C. jejuni-positive samples). ST-3704 was also identified in 3 wood mouse isolates (3/6 samples), with the remaining isolates representing known sequence types (ST-61 and ST-583). Other C. jejuni isolates (n ⫽ 16) from the farm cross-sectional studies were also subjected to MLST analysis, and 15 of these belonged to known sequence types (ST-45, ST-61, ST-257, and ST-403); however, one calf isolate (1/12 C. jejuni-positive samples from cattle) was identified as ST-3704 (Table 1). Furthermore, more recently (September 2008), we isolated C. jejuni ST-3704, but no other sequence type, from the F1 progeny of a captive colony of bank voles. These animals were fed an artificial diet, which suggests that this clone can be maintained in captive bred animals with no environmental exposure and is therefore strongly associated with the bank vole host. A selection of ST-3704 bank vole isolates (n ⫽ 76) from both the woodland and the farm study, the three ST-3704 wood mouse isolates, and other Campylobacter isolates from cattle were subjected to genotyping using macrorestriction pulsed-field gel electrophoresis (PFGE) and analyzed as described previously (11). Remarkably, all 79 of the ST-3704 isolates examined shared similar macrorestriction patterns (⬍3 bands different) following digestion with SmaI, suggesting that they represent a single clone circulating largely within bank vole populations. Sample macrorestriction patterns for isolates from bank voles from different sources are shown in Fig. 2, which also includes examples of other C. jejuni isolates whose banding patterns are more typical of those normally observed for C. jejuni and for C. fetus isolates from cattle. Thus, we report the identification of a new strain of C. jejuni, ST-3704, isolated from different bank vole populations over a relatively wide geographic area with respect to the home range
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