Clonal Nature of Campylobacter fetus as Defined by Multilocus Sequence Typing
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微生物临床杂志 2005年第12期
Animal Sciences Group, Division of Infectious Diseases (OIE Reference Laboratory for Campylobacteriosis), Lelystad, The Netherlands
Nuffield Department of Clinical Sciences, University of Oxford, Department of Microbiology, John Radcliffe Hospital, Oxford, United Kingdom
The Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford, Oxford, United Kingdom
Veterinary Laboratories Agency, Weybridge, United Kingdom
Department of Infectious Diseases and Immunology (OIE Reference Laboratory for Campylobacteriosis), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
ABSTRACT
Campylobacter fetus can be divided into the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. C. fetus subsp. fetus causes sporadic infections in humans and abortion in cattle and sheep and has been isolated from a variety of sites in different hosts. C. fetus subsp. venerealis is host restricted, being isolated mainly from the genital tracts of cattle, and is the causative agent of bovine genital campylobacteriosis. Despite differences in niche preference, microbiological subspecies differentiation has proven difficult. Different typing methods divided C. fetus isolates into different subgroups, depending on the methods used. The relative value of these methods can be assessed by the evolutionary relationship of isolates belonging to the genus; therefore, we developed a multilocus sequence typing (MLST) scheme for C. fetus. This scheme was applied to 140 C. fetus isolates previously typed by amplified fragment length polymorphism (AFLP) analysis. A total of 14 different sequence types (STs) were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. These MLST data indicate that C. fetus is genetically homogeneous compared to the homogeneity of other Campylobacter species. The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis, which represents a "bovine" clone. The C. fetus subsp. fetus isolates studied were more diverse in terms of their STs, and the STs correlated with epidemiological relationships. Congruence was observed among C. fetus subspecies, sap type, and ST; therefore, MLST confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche.
INTRODUCTION
Campylobacter fetus is currently divided into the two subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis (38). C. fetus subsp. venerealis also includes a phenotypic biovar designated C. fetus subsp. venerealis bv. intermedius (8, 12). Both subspecies are primary pathogens; C. fetus subsp. fetus is associated with abortions most often in sheep and to a lesser extent in cattle and humans and can frequently be isolated from the gallbladders and intestinal tracts of a variety of hosts, including ungulates, fowl, reptiles, and humans (12). C. fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, an infectious venereal disease which may lead to reproductive problems, such as sterility and abortion. C. fetus subsp. venerealis is host restricted, being isolated primarily from the bovine genital tract (12, 38), whereas C. fetus subsp. fetus can be isolated from a wider range of species.
The subspecies are conventionally defined with biochemical tests, namely, growth in 1% glycine (32) and H2S production from medium containing 0.02% cysteine (1). Despite these differences and host and niche preferences, studies that have used potentially discriminatory methods, including DNA-DNA hybridization (29, 37), serotyping (27), ribotyping (3), cellular fatty acid analysis (2), and whole-cell protein analysis (37), have been unable to distinguish C. fetus subsp. fetus and C. fetus subsp. venerealis. Therefore, from a taxonomic viewpoint it is questionable whether C. fetus should be divided into subspecies. However, a dichotomy has been detected among C. fetus serotype A and B strains (7, 22, 27). These serotypes reflect differences in both lipopolysaccharide structure and the type of surface layer protein (SLP) and surface array protein (Sap). The two serotypes correlate with the corresponding sap type, sapA or sapB (33). C. fetus subsp. venerealis strains are type A, whereas C. fetus subsp. fetus strains can be either serotype A or serotype B.
Several molecular methods, including pulsed-field gel electrophoresis (PFGE) fingerprinting (25, 31), amplified fragment length polymorphism (AFLP) analysis (39), and PCR amplification (15, 40), have been used for subspecies differentiation; but they give contradictory results. Recently, subspecies-specific random amplification of polymorphic DNA PCRs were described, but they were evaluated with a limited number of strains (34). A new C. fetus subsp. venerealis-specific PCR has been developed based on an AFLP marker (36). Subspecies differentiation is of such statutory importance that additional unambiguous typing methods are still required, preferably those that assist with epidemiological investigations. This may be useful for outbreak control and has been performed by using serotyping and PFGE. The currently available serotyping scheme has a low level of discrimination, as only a few lipopolysaccharide serotypes have been detected. However, PFGE has proven discriminatory and has been applied in epidemiological studies of both human and veterinary infections (11, 20, 23, 24, 28).
Multilocus sequence typing (MLST) is a nucleotide sequence-based approach to bacterial typing in which variations in 500-bp fragments of housekeeping genes (generally seven) are indexed. MLST is useful for studies of bacterial epidemiology and population genetics, including studies of species of the genus Campylobacter (4, 5, 9, 18, 21). MLST has the advantages that variation in the target gene is accessed directly and that the technology employed is both readily disseminated and highly reproducible among laboratories (19). Furthermore, comparisons of the population structures and population genetics of different species can be made by using MLST data, particularly if the same loci are used for different species.
Here we describe an MLST scheme for C. fetus that uses the same loci as a previous scheme for C. jejuni and C. coli (4, 5). We tested this scheme using a diverse collection of isolates and examined the C. fetus population structure and genetic diversity, together with the suitability of MLST for studies of C. fetus subspecies differentiation and epidemiology.
MATERIALS AND METHODS
Bacterial strains. A collection of 140 C. fetus isolates, including 8 reference strains and 132 field isolates (Table 1) were included. This diverse collection contained both epidemiologically unrelated isolates, and isolates from seven outbreaks which occurred at bovine artificial insemination (AI) stations. Isolates were obtained from bovine, ovine, human, equine, and monkey hosts located in different geographical areas: Argentina, Belgium, France, Germany, Hungary, Japan, The Netherlands, South Africa, Spain, Turkey, the United Kingdom, and the United States (Table 1). Isolates were grown under microaerobic conditions (6% O2, 7% CO2, 7% H2, 80% N2; Anoxomat; Mart Microbiology B.V., Lichtenvoorde, The Netherlands) on heart infusion agar supplemented with 5% sheep blood (Biotrading, Mijdrecht, The Netherlands) for 3 days and were stored at –80°C in heart infusion broth containing 15% glycerol.
Preparation of chromosomal DNA. Chromosomal DNA was isolated by using the Puregene kit (Gentra Systems, Minneapolis, MN) and diluted to a final concentration of approximately 5 ng/μl for AFLP analysis and PCR and to 25 ng/μl for PCR amplification during MLST.
Species identification and subspecies differentiation. Isolates were identified genotypically as C. fetus by using a previously described PCR protocol (15), with modifications (39). The subspecies were then identified genotypically by Cf C05 PCR (36); the PCR of Hum et al. (15); AFLP analysis (39); and biochemically, according to growth in the presence of 1% glycine (Merck, Darmstadt, Germany) (32) and H2S production from brucella broth (Difco, Becton Dickinson, Franklin Lakes, NJ) containing 0.02% cysteine (Sigma Aldrich, St. Louis, MO) (1, 17).
sap typing. sap typing was performed with PCR primers based on the sapA and sapB 5' conserved regions, as described by Tu et al. (33).
AFLP analysis. AFLP analysis was performed by a previous method, with modifications (6, 39). These enhancements comprised the use of a capillary-based sequencing system (ABI 3100; Applied Biosystems, Foster City, CA) and data analysis with Bionumerics 3.5 software (Applied Maths, St-Martens-Latem, Belgium). Chromosomal DNA was digested with the restriction enzymes HindIII and HhaI, and site-specific adaptors were ligated. A preselective PCR amplification was performed, followed by selective PCR with primers containing a 3' selective nucleotide and one fluorescently labeled (6-carboxyfluorescein) primer. PCR products underwent electrophoresis through an ABI 3100 capillary sequence system by using a performance optimized polymer-4 polymer matrix (Applied Biosystems). AFLP patterns were analyzed by importing the AFLP data (via the conversion software GeneScan 3.7 [Applied Maths]) into the Bionumerics 3.5 software. Patterns were normalized by referring to the molecular mass of the internal DNA marker (GeneScan-500 [carboxy X rhodamine]; Applied Biosystems). The genetic similarity between the patterns was calculated by using the Pearson product-moment correlation coefficient, and clustering analysis was performed by the unweighted pair group method with arithmetic averages for the region corresponding to 84.6% to 87.1% of the band pattern.
PFGE. PFGE was performed as described previously by using the restriction enzymes SmaI and SalI (36a)
Multilocus sequence typing. (i) Primer design. C. fetus sequences corresponding to the seven loci used in a previously described C. jejuni and C. coli MLST scheme were required: aspA, glnA, gltA, glyA, pgm, tkt, and uncA (4, 5). Various C. jejuni and C. coli MLST primers (Sigma-Genosys, Haverhill, United Kingdom) were employed in multiple combinations to identify primers that would amplify these sequences from C. fetus. A reduced annealing temperature of 48°C was used to improve the binding of the C. jejuni primers to heterologous C. fetus DNA. Of the many primer combinations tested, those that provided amplification products were as follows: for C. jejuni aspA, primers A9 and A10; for C. jejuni glnA, primers A1 and A2; for C. jejuni gltA, primers A1 and A2; for C. jejuni glyA, primers A1 and A2; for C. jejuni tkt, primers A5 and A4; and for C. coli uncA, primers A1 and A2 (the sequences of all primers except C. jejuni tkt A5 and C. coli uncA A1 and A2 are available at http://pubmlst.org/campylobacter/; the sequence of tkt A5 is 5'-TTTAAGTGCTGATATGGTGC-3', the sequence of C. coli uncA A1 is 5'-ATGTAGCCATAGGACAAAAGC-3', and the sequence of C. coli uncA A2 is 5'-CATTCTTGTCGCGTTCAGTTG-3'). The C. fetus sequence corresponding to the pgm locus failed to amplify with any C. jejuni- or C. coli-specific primers; however, this sequence was kindly provided by D. Sanchez, Universidad Nacional de General San Martín, Instituto de Investigaciones Biotecnologicas, San Martín, Argentina. The C. fetus amplicons were sequenced directly by using the same primers used for amplification. Nucleotide sequence extension reactions were carried out by using BigDye ready reaction mix (version 3; Applied Biosystems), in accordance with the manufacturer's instructions. The reaction products were separated with an ABI 3730 automated DNA sequencer (Applied Biosystems).
These C. fetus sequences from the seven loci were used to design seven pairs of C. fetus-specific primers for MLST (Table 2). The MLST allele trimming sites for C. fetus were chosen to correspond to the previous C. jejuni and C. coli sites to assist with future phylogenetic comparisons among species. At least 28 nucleotides separated the MLST trimming site and the 3' end of the C. fetus primers. MLST was performed by using the same primer pair used for both amplification and sequencing.
(ii) PCR amplification and nucleotide sequencing. Seven PCR amplicons were obtained for each isolate by using the primers shown in Table 2. Each 50-μl PCR mixture contained 39.75 μl molecular biology-grade water (Sigma Aldrich), 5 μl 10x PCR buffer (QIAGEN, Crawley, United Kingdom), 1 μl 10 μM each forward and reverse primer, 1 μl 10 mM deoxynucleoside triphosphate mixture (Invitrogen, Paisley, United Kingdom), 0.25 μl HotStar Taq DNA polymerase (QIAGEN), and 2 μl C. fetus chromosomal DNA (approximately 50 ng). The amplification conditions were 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min and storage at 4°C. Nucleotide sequencing (forward and reverse) was performed with the same primers (10 μM diluted 1:15 in water); and the reaction conditions were 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 2 min.
(iii) Allele and ST assignment. For each locus, distinct allele sequences were assigned arbitrary allele numbers in the order of identification. Each genotype was therefore designated by seven numbers (e.g., 1-3-2-4-1-1-2) that constituted an allelic profile or sequence type (ST; e.g., ST-3). The STs were assigned arbitrary numbers in the order of description. All data for newly described C. fetus alleles and STs were deposited in a Campylobacter fetus MLST database (http://pubmlst.org/cfetus/). New sequences were assigned allele numbers, and isolates were assigned their STs by interrogating the database. Allele numbers for new sequences and ST numbers for new allelic profiles are available by submission to the database.
(iv) Data analysis. Phylogenetic analysis was performed with MEGA (version 2.1) software, available at http://www.megasoftware.net, by using the concatenated MLST gene sequence fragments for each isolate. Data were also subjected to split decomposition analysis by using SPLITSTREE (version 3.1) software (16).
RESULTS
C. fetus genotyping and determination of population structure by MLST. The collection of 140 C. fetus isolates was typed by MLST. The C. fetus MLST data were used to (i) assess the level of genetic diversity within this Campylobacter species; (ii) elucidate the C. fetus population structure and compare it to the population structures of other Campylobacter species; (iii) investigate any correlation between ST and cell surface antigen(s), represented by the sap type; and (iv) evaluate the utility of MLST for subspecies differentiation and epidemiological typing compared with those of AFLP analysis and PFGE.
All of the 140 isolates examined were typeable by MLST. The number of alleles per locus ranged from two to four, and the number of variable sites per locus ranged from one to five (Table 3). A total of 14 different STs were identified, some of which shared up to six or seven identical loci. The majority of isolates (91%) were assigned to one of the following STs: ST-2, ST-3, ST-4, ST-5, or ST-6 (Table 1). In this isolate collection, ST-1 was associated with an epidemiologically related group of isolates (n = 5), while the remaining STs (ST-7 to ST-14) each occurred only once.
The level of nucleotide sequence variation within the C. fetus MLST loci was assessed by concatenating the seven sequences to create a continuous 3,312-nucleotide length. There were 22 variable sites in the 14 STs (0.66%) (Table 4). The five most common STs had 12 variable sites and were apparently distributed along the sequence. Three of these mutations (25%) caused a change in the amino acid sequence.
C. fetus is genetically homogeneous with an apparently clonal population structure. In contrast, the five other Campylobacter species for which MLST data are available (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus) (4, 5, 21) show much greater diversity. This was illustrated by comparing data for the three loci used in all six Campylobacter MLST schemes described to date: glnA, glyA, and tkt (data for glnA are shown in Fig. 1). The variation detected in C. fetus is equivalent to a homogeneous C. jejuni clonal complex and is much less than that in many clonal complexes of this species (5).
The extent of association between the C. fetus STs (defined by housekeeping genes) and cell surface antigens was assessed by examining the relationship between the ST and the sap type (sapA or sapB). A clear correlation was observed (Table 1; Fig. 2). SapA was confined to isolates of ST-2, -4, -6, -7, -8, -9, -11, and -12, whereas sapB was associated with isolates of ST-1, -3, -5, -10, -13, and -14. Therefore, the association of this cell surface antigen with the ST demonstrated congruence.
C. fetus sequence type and subspecies. The subspecies was defined by using the classical biochemical approach, PCR (15, 36), and AFLP analysis (39). An AFLP analysis with improved resolution and throughput (described here for the first time) allowed us to define a distinct cluster designated C. fetus subsp. venerealis bv. intermedius. This cluster contained the previously described C. fetus subsp. venerealis bv. intermedius strains 3292 and 3293 (31) and produced H2S, and most isolates were negative for growth in glycine (Table 1). Three subspecies groups were therefore identified: C. fetus subsp. venerealis, C. fetus subsp. venerealis bv. intermedius, and C. fetus subsp. fetus.
The value of MLST for subspecies determination was assessed. Certain STs did correlate with each of the three AFLP groups. A total of 55 of 57 of the C. fetus subsp. venerealis or C. fetus subsp. venerealis bv. intermedius isolates were ST-4 (Table 1; Fig. 2). The remaining 2 of the 57 isolates were ST-7 and ST-12, respectively; ST-12differed by only one nucleotide from ST-4. The C. fetus subsp. fetus AFLP group showed a greater diversity of STs; but notably, ST-4, ST-7, and ST-12 were absent (Table 1; Fig. 2).
Investigation of C. fetus epidemiology by MLST. MLST data were examined for (i) the reliability of outbreak identification, (ii) any correlation of the ST with the isolation source or geographical region, and (iii) any correlation with and discrimination attained compared to the results of PFGE. Such data were available for a subset of epidemiologically related and unrelated isolates (n = 23).
The collection of 140 isolates studied included isolates from seven known outbreaks (with two strains per outbreak) from different bovine AI stations (Table 1). All but one of the outbreaks were associated with STs frequently identified within this data set (ST-1 to ST-4 and ST-6). The remaining outbreak was associated with a single bovine ST-5 isolate from an additional AI station. Epidemiologically related strains shared identical STs and PFGE types (e.g., ST-1 and ST-2; Table 1). However, some epidemiologically unrelated strains had identical STs, but PFGE typing revealed discriminatory patterns (e.g., ST-3, ST-4, ST-5, and ST-6; Table 1), which indicated that PFGE has a greater discriminatory power.
The MLST data and the isolate data on host and geographical region were evaluated for potential associations. Bovine, ovine, and human isolates were the most numerous; but none of these hosts harbored a single ST. However, ST-1 (n = 5) and ST-4 (n = 48) were isolated almost exclusively from bovine hosts, the exceptions being two human isolates. Seven further ST-4 isolates (indicated by a question mark in Table 1) were considered likely (but unconfirmed) bovine isolates (M. Henton, personal communication). Therefore, with the possible exception of ST-4, which is the C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius ST and, therefore, by definition, cattle associated, no association between the ST and the host species was detected. The ST and the geographical region of isolation also lacked association, and all the common STs were found in Europe and North America.
DISCUSSION
An MLST scheme for C. fetus was developed, together with a database for the storage and exchange of MLST data and protocols (http://pubmlst.org/cfetus/). The scheme was evaluated by using 140 C. fetus isolates chosen for their diversity in terms of geography and host species. C. fetus lacked genetic diversity and had a high level of clonality compared to those of other Campylobacter species studied by MLST (4, 5, 21). This could be because mammalian C. fetus isolates evolved from a recent ancestor, or it may be due to a small population size and high-fidelity error-intolerant repair; or the genetic diversity of C. fetus may be limited by a lack of the natural competence found in other Campylobacter species.
C. fetus was originally divided into two subspecies by using host and niche preferences (38), but subsequent taxonomic studies led investigators to question this approach (14, 29). The difficulty of reliable subspecies differentiation reflects the fact that the two subspecies are extremely closely related genetically. For example, it proved impossible to differentiate subspecies by using the nucleotide sequences of two other housekeeping genes, sapD and recA (33), or the 16S rRNA gene (13, 25, 41). Since subspecies differentiation is of high statutory importance, the suitability of MLST for C. fetus subspecies differentiation was investigated. The C. fetus subsp. venerealis strains were all ST-4, although they were geographically and temporally distinct. The C. fetus subsp. venerealis bv. intermedius strains were also mostly ST-4, with two exceptions (one ST-7 strain and another ST-12 strain). Therefore, the MLST scheme did not allow differentiation between C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius strains. However, ST-4 was not detected among the C. fetus subsp. fetus isolates. Extension of the MLST scheme to other loci may reveal differences between C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius strains. The C. fetus subsp. venerealis "ST-4 clone" shows even less diversity within the housekeeping genes than the C. fetus subsp. fetus STs, supporting the theory of Veron and Chatelain, who proposed that C. fetus subsp. venerealis is a mutant of C. fetus subsp. fetus (38).
The data suggest that the MLST approach is reliable for subspecies differentiation since it was performed "blind" without prior knowledge of the subspecies assignments. Although C. fetus subsp. venerealis ST-4 differs from the closest C. fetus subsp. fetus ST (ST-6) by a single nucleotide (Fig. 2), the data generated by this highly reproducible sequencing technique confirm that MLST is an accurate method for the differentiation of the subspecies. Previous studies suggested that differences between C. fetus subsp. venerealis and C. fetus subsp. fetus are plasmid encoded (25, 26), although more recent studies have questioned this proposal (42). This MLST scheme indicates that the differences between the two subspecies are not merely plasmid encoded but are also reflected in the allelic profiles (STs) of the housekeeping genes.
C. fetus isolates from mammals are divided into two sap types, sapA and sapB, on the basis of their SLPs. The MLST data showed a clear correlation among STs and sap types (Fig. 2), supporting the classification of C. fetus into two sap groups. This correlation suggests evolutionary stability and a lack of frequent recombination between the two groups, since in C. jejuni (which recombines frequently) a lack of correlation among STs-clonal complexes and cell surface antigens has been detected.
MLST is very useful for the global epidemiological typing of many bacterial species, and its use is expanding rapidly (18, 35). We demonstrated in the present study that epidemiologically related C. fetus strains had identical STs. However, as a result of the relatively low level of genetic diversity found in C. fetus housekeeping genes, some unrelated isolates had STs identical to those of some of the outbreak strains. Sequencing of additional, more variable loci has proven useful in investigations of outbreaks of Neisseria meningitidis infections and may increase the discriminatory power of the method (10). Whether this approach would also improve C. fetus discrimination requires further investigation. C. fetus has a global distribution; however, no geographically distinct ST cluster could be distinguished by MLST. The major STs were found in multiple host species; however, the C. fetus subsp. venerealis ST-4 was restricted to bovine hosts. It has been suggested that C. fetus subsp. venerealis is a host-restricted mutant clone of C. fetus subsp. fetus that is unable to infect multiple host species (38).
MLST was compared to biochemical and established C. fetus band-based typing methods, AFLP analysis, PFGE, and PCR. Each of these methods measures genotypic characteristics which may change independently; hence, the subtypes that they define may not always be congruent (30). However, MLST subspecies differentiation results showed a high agreement with AFLP, Cf C05 PCR, and H2S results compared to the results of the PCR of Hum et al. (15) and the glycine test. In the present study, a strong correlation was observed between MLST and PFGE data, and both methods gave identical clusters of isolates from known C. fetus outbreaks. PFGE provided a higher level of discrimination than MLST. However, MLST measures variation that accumulates relatively slowly in the absence of selective pressure, and it may be more appropriate for studies of long-term C. fetus epidemiology and phylogenetics. PFGE indexes genome-wide variations and, despite the difficulties of interlaboratory comparisons, may be better suited for short-term investigations of C. fetus epidemiology, such as outbreak investigations, when such investigations are performed by a single laboratory. The level of discrimination of C. fetus isolates was higher by MLST than by the AFLP analysis method described, irrespective of the region of the AFLP pattern analyzed.
In summary, the observations that mammalian C. fetus isolates have a low level of genetic diversity and are genetically homogeneous compared to the homogeneities of other Campylobacter species suggest that C. fetus is young in evolutionary terms and may have arisen from a recent ancestor. In Fig. 1, the long branch length separating C. fetus from the other Campylobacter species suggests that its putative recent ancestor is missing from this tree. This is supported by the lack of any sequences characteristic of these five other Campylobacter species within the C. fetus housekeeping genes. The predominantly stepwise accumulation of point mutations in C. fetus indicates clonal evolution, in which genetic change accumulates slowly only by the vertical transmission of point mutations. The genetic stability of C. fetus is further confirmed by the wide geographic distribution of identical STs and the observation of congruence between the C. fetus subspecies, sap type, and ST. The two C. fetus subspecies were extremely closely genetically related, but ST-4 was associated only with C. fetus subsp. venerealis and was therefore, by definition, a "bovine" subclone. MLST confirms that mammalian C. fetus is genetically stable and clonal, provides a useful additional tool for C. fetus subspecies differentiation and epidemiology, and compares favorably to other bacterial typing methods.
ACKNOWLEDGMENTS
This study was partly funded by COST Action 920.
We are very grateful to D. Sanchez (Universidad Nacional de General San Martín, Instituto de Investigaciones Biotecnologicas, San Martín, Argentina) for comparing the C. jejuni pgm sequence with the sequence of C. fetus by use of the BLAST program and providing us with the C. fetus pgm sequence identified for primer design. Furthermore, we thank the following for kindly supplying C. fetus isolates: C. Campero, Instituto Nacional de Tecnoloíga Agropecuaria, Balcarce, Argentina; J. Godfroid, Veterinary and Agrochemical Research Centre, Brussels, Belgium; J. P. Butzler, University of Brussels, Brussels, Belgium; P. Idigoras, Hospital Donostia, San Sebastián, Spain; W. Kalka-Moll, University of Cologne, Germany; J. Varga, Faculty of Veterinary Science, Budapest, Hungary; S. Fujimoto, School of Health Sciences, Kyushu University, Kyushu, Japan; W. Ang, Erasmus University Medical Centre, Rotterdam, The Netherlands; M. Koene and I. Visser, Animal Health Service, Deventer, The Netherlands; M. Hinton, Onderstepoort Veterinary Institute, Onderstepoort, South Africa; L. Guler, Veteriner Kontrol ve Arastirma Enstitusu, Konyn, Turkey; J. Frost, Health Protection Agency, London, United Kingdom; J. Corry, University of Bristol, Bristol, United Kingdom; M. Toszeghy, Veterinary Laboratories Agency, Weybridge, United Kingdom; P. Fields, Centers for Disease Control and Prevention, Atlanta, GA; L. Schroeder-Tucker, National Veterinary Services Laboratories, Ames, IA; I. Wesley, National Animal Disease Centre, Ames, IA; and Z. Tu and M. Blaser, Vanderbilt University School of Medicine, Nashville, TN.
M. van Bergen and K. Dingle contributed equally to the manuscript, and both are considered the first author.
REFERENCES
Berg, R. L., J. W. Jutila, and B. D. Firehammer. 1971. A revised classification of Vibrio fetus.Am. J. Vet. Res. 32:11-22.
Blaser, M. J., C. W. Moss, and R. E. Weaver.1980 . Cellular fatty acid composition of Campylobacter fetus. J. Clin. Microbiol. 11:448-451.
Denes, A. S., C. L. Lutze-Wallace, M. L. Cormier, and M. M. Garcia. 1997. DNA fingerprinting of Campylobacter fetus using cloned constructs of ribosomal RNA and surface array protein genes. Vet. Microbiol. 54:185-193.
Dingle, K. E., F. M. Colles, D. Falush, and M. C. J. Maiden. 2005. Sequence typing and comparison of population biology of Campylobacter coli and Campylobacter jejuni. J. Clin. Microbiol. 43:340-347.
Dingle, K. E., F. M. Colles, D. R. A. Wareing, R. Ure, A. J. Fox, F. E. Bolton, H. J. Bootsma, R. J. L. Willems, R. Urwin, and M. C. J. Maiden. 2001. Multilocus sequence typing system for Campylobacter jejuni.J. Clin. Microbiol. 39:14-23.
Duim, B., T. M. Wassenaar, A. Rigter, and J. A. Wagenaar. 1999. High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting. Appl. Environ. Microbiol. 65:2369-2375.
Dworkin, J., M. K. Tummuru, and M. J. Blaser.1995 . Segmental conservation of sapA sequences in type B Campylobacter fetus cells. J. Biol. Chem. 270:15093-15101.
Elazhary, M. 1968. An assay of isolation and identification of some animal vibrios and of elucidation of their pathological significance. Meded. Veeartschool. Rijksuniv. Gent 12:1-80.
Enright, M. C., N. P. J. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt.1998 . A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144:3049-3060.
Feavers, I. M., S. J. Gray, R. Urwin, J. E. Russell, J. A. Bygraves, E. B. Kaczmarski, and M. C. Maiden. 1999. Multilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak. J. Clin. Microbiol. 37:3883-3887.
Fujita, M., S. Fujimoto, T. Morooka, and K. Amako. 1995. Analysis of strains of Campylobacter fetus by pulsed-field gel electrophoresis. J. Clin. Microbiol. 33:1676-1678.
Garcia, M. M., M. D. Eaglesome, and C. Rigby.1983 . Campylobacters important in veterinary medicine.Vet. Bull. 53:793-818.
Gorkiewicz, G., G. Feierl, C. Schober, F. Dieber, J. Kfer, R. Zechner, and E. L. Zechner. 2003. Species-specific identification of campylobacters by partial 16S rRNA gene sequencing.J. Clin. Microbiol. 41:2537-2546.
Harvey, S., and J. R. Greenwood. 1983. Relationships among catalase-positive campylobacters determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization. Int. J. Syst. Bacteriol. 33:275-284.
Hum, S., K. Quinn, J. Brunner, and S. L. W. On.1997 . Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies.Aust. Vet. J. 75:827-831.
Huson, D. H. 1998. SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14:68-73.
MacLaren, A. P. C., and G. J. O. Agumbah. 1988. Infertility in cattle in south-west Scotland caused by an "intermediate" strain of Campylobacter fetus subspecies fetus (formerly Campylobacter fetus intestinalis). Br. Vet. J. 144:29-44.
Maiden, M. C., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95:3140-3145.
Maiden, M. C. J. 2000. High-throughput sequencing in the population analysis of bacterial pathogens.Int. J. Med. Microbiol. 290:183-190.
Mannering, S. A., D. M. West, S. G. Fenwick, R. M. Marchant, N. R. Perkins, and K. O'Connell. 2004. Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand. N. Z. Vet. J. 52:358-363.
Miller, W. G., S. L. W. On, G. Wang, S. Fontanoz, A. J. Lastovica, and R. E. Mandrell.2005 . System for extended multilocus sequence typing of strains of Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus.J. Clin. Microbiol. 43:2315-2329.
Moran, A. P., D. T. O'Malley, T. U. Kosunen, and I. M. Helander. 1994. Biochemical characterization of Campylobacter fetus lipopolysaccharides.Infect. Immun. 62:3922-3929.
Morooka, T., A. Umeda, M. Fujita, H. Matano, S. Fujimoto, K. Yukitake, K. Amako, and T. Oda. 1996. Epidemiologic application of pulsed-field gel electrophoresis to an outbreak of Campylobacter fetus meningitis in a neonatal intensive care unit. Scand. J. Infect. Dis. 28:269-270.
Newell, D. G., J. A. Frost, B. Duim, J. A. Wagenaar, R. H. Madden, J. van der Plas, and S. L. W. On. 2000. New developments in the subtyping of Campylobacter species, p.27 -44. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, D.C.
On, S. L. W., and C. S. Harrington.2001 . Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods.J. Appl. Microbiol. 90:285-293.
Panaccio, M., L. Stephens, and P. Widders. 1993. Field evaluation of a PCR-based test for the diagnosis of bovine campylobacteriosis DAV106. Dairy Research and Development Corporation, Melbourne, Australia.
Perez-Perez, G. I., M. J. Blaser, and J. H. Bryner. 1986. Lipopolysaccharide structures of Campylobacter fetus are related to heat-stable serogroups.Infect. Immun. 51:209-212.
Rennie, R. P., D. Strong, D. E. Taylor, S. M. Salama, C. Davidson, and H. Tabor. 1994. Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism.J. Clin. Microbiol. 32:721-724.
Roop, R. M., II, R. M. Smibert, J. L. Johnson, and N. R. Krieg. 1984. Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups. Can. J. Microbiol. 30:938-951.
Sails, A. D., B. Swaminathan, and P. I. Fields.2003 . Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strains associations identified by multilocus enzyme electrophoresis.J. Clin. Microbiol. 41:4058-4067.
Salama, S. M., M. M. Garcia, and D. E. Taylor. 1992. Differentiation of the subspecies of Campylobacter fetus by genomic sizing. Int. J. Syst. Bacteriol. 42:446-450.
Smibert, R. M. 1984. Genus II Campylobacter Sebald and Veron 1963, 907, p.111 -118. In N. R. Krieg and H. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, Md.
Tu, Z.-C., F. E. Dewhirst, and M. J. Blaser.2001 . Evidence that the Campylobacter fetus sap locus is an ancient genomic constituent with origins before mammals and reptiles diverged. Infect. Immun. 69:2237-2244.
Tu, Z.-C., W. Eisner, B. N. Kreiswirth, and M. J. Blaser. 2005. Genetic divergence of Campylobacter fetus strains of mammal and reptile origins. J. Clin. Microbiol. 43:3334-3340.
Urwin, R., and M. C. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 11:479-487.
Van Bergen, M. A. P., G. Simons, L. Van der Graaf-Van Bloois, J. Rombout, J. P. M. Van Putten, and J. A. Wagenaar. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. J. Med. Microbiol., in press.
Van Bergen, M. A. P., L. van der Graaf-van Bloois, I. J. R. Visser, Jos P. M. van Putten, and J. A. Wagenaar. Molecular epidemiology of Campylobacter fetus subsp. fetus on bovine artificial insemination stations using pulsed field gel electrophoresis. Vet. Microbiol., in press.
Vandamme, P., B. Pot, E. Falsen, K. Kersters, and J. De Ley. 1990. Intra- and interspecific relationships of veterinary campylobacters revealed by numerical analysis of electrophoretic protein profiles and DNA:DNA hybridizations. Syst. Appl. Microbiol. 13:295-303.
Veron, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and Veron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) Sebald and Veron. Int. J. Syst. Bacteriol. 23:122-134.
Wagenaar, J. A., M. A. P. Van Bergen, D. G. Newell, R. Grogono-Thomas, and B. Duim. 2001. Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals.J. Clin. Microbiol. 39:2283-2286.
Wang, G., C. G. Clark, T. M. Taylor, C. Pucknell, C. Barton, L. Price, D. L. Woodward, and F. G. Rodgers. 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J. Clin. Microbiol. 40:4744-4747.
Wesley, I. V., R. D. Wesley, M. Cardella, F. E. Dewhirst, and B. J. Paster. 1991. Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.J. Clin. Microbiol. 29:1812-1817.
Willoughby, K., P. F. Nettleton, M. Quirie, M. A. Maley, G. Foster, M. Toszeghy, and D. G. Newell. 2005. A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis: use on UK isolates of Campylobacter fetus and other Campylobacter spp. J. Appl. Microbiol. 99:758-766.(Marcel A. P. van Bergen, )
Nuffield Department of Clinical Sciences, University of Oxford, Department of Microbiology, John Radcliffe Hospital, Oxford, United Kingdom
The Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford, Oxford, United Kingdom
Veterinary Laboratories Agency, Weybridge, United Kingdom
Department of Infectious Diseases and Immunology (OIE Reference Laboratory for Campylobacteriosis), Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
ABSTRACT
Campylobacter fetus can be divided into the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. C. fetus subsp. fetus causes sporadic infections in humans and abortion in cattle and sheep and has been isolated from a variety of sites in different hosts. C. fetus subsp. venerealis is host restricted, being isolated mainly from the genital tracts of cattle, and is the causative agent of bovine genital campylobacteriosis. Despite differences in niche preference, microbiological subspecies differentiation has proven difficult. Different typing methods divided C. fetus isolates into different subgroups, depending on the methods used. The relative value of these methods can be assessed by the evolutionary relationship of isolates belonging to the genus; therefore, we developed a multilocus sequence typing (MLST) scheme for C. fetus. This scheme was applied to 140 C. fetus isolates previously typed by amplified fragment length polymorphism (AFLP) analysis. A total of 14 different sequence types (STs) were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. These MLST data indicate that C. fetus is genetically homogeneous compared to the homogeneity of other Campylobacter species. The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis, which represents a "bovine" clone. The C. fetus subsp. fetus isolates studied were more diverse in terms of their STs, and the STs correlated with epidemiological relationships. Congruence was observed among C. fetus subspecies, sap type, and ST; therefore, MLST confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche.
INTRODUCTION
Campylobacter fetus is currently divided into the two subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis (38). C. fetus subsp. venerealis also includes a phenotypic biovar designated C. fetus subsp. venerealis bv. intermedius (8, 12). Both subspecies are primary pathogens; C. fetus subsp. fetus is associated with abortions most often in sheep and to a lesser extent in cattle and humans and can frequently be isolated from the gallbladders and intestinal tracts of a variety of hosts, including ungulates, fowl, reptiles, and humans (12). C. fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, an infectious venereal disease which may lead to reproductive problems, such as sterility and abortion. C. fetus subsp. venerealis is host restricted, being isolated primarily from the bovine genital tract (12, 38), whereas C. fetus subsp. fetus can be isolated from a wider range of species.
The subspecies are conventionally defined with biochemical tests, namely, growth in 1% glycine (32) and H2S production from medium containing 0.02% cysteine (1). Despite these differences and host and niche preferences, studies that have used potentially discriminatory methods, including DNA-DNA hybridization (29, 37), serotyping (27), ribotyping (3), cellular fatty acid analysis (2), and whole-cell protein analysis (37), have been unable to distinguish C. fetus subsp. fetus and C. fetus subsp. venerealis. Therefore, from a taxonomic viewpoint it is questionable whether C. fetus should be divided into subspecies. However, a dichotomy has been detected among C. fetus serotype A and B strains (7, 22, 27). These serotypes reflect differences in both lipopolysaccharide structure and the type of surface layer protein (SLP) and surface array protein (Sap). The two serotypes correlate with the corresponding sap type, sapA or sapB (33). C. fetus subsp. venerealis strains are type A, whereas C. fetus subsp. fetus strains can be either serotype A or serotype B.
Several molecular methods, including pulsed-field gel electrophoresis (PFGE) fingerprinting (25, 31), amplified fragment length polymorphism (AFLP) analysis (39), and PCR amplification (15, 40), have been used for subspecies differentiation; but they give contradictory results. Recently, subspecies-specific random amplification of polymorphic DNA PCRs were described, but they were evaluated with a limited number of strains (34). A new C. fetus subsp. venerealis-specific PCR has been developed based on an AFLP marker (36). Subspecies differentiation is of such statutory importance that additional unambiguous typing methods are still required, preferably those that assist with epidemiological investigations. This may be useful for outbreak control and has been performed by using serotyping and PFGE. The currently available serotyping scheme has a low level of discrimination, as only a few lipopolysaccharide serotypes have been detected. However, PFGE has proven discriminatory and has been applied in epidemiological studies of both human and veterinary infections (11, 20, 23, 24, 28).
Multilocus sequence typing (MLST) is a nucleotide sequence-based approach to bacterial typing in which variations in 500-bp fragments of housekeeping genes (generally seven) are indexed. MLST is useful for studies of bacterial epidemiology and population genetics, including studies of species of the genus Campylobacter (4, 5, 9, 18, 21). MLST has the advantages that variation in the target gene is accessed directly and that the technology employed is both readily disseminated and highly reproducible among laboratories (19). Furthermore, comparisons of the population structures and population genetics of different species can be made by using MLST data, particularly if the same loci are used for different species.
Here we describe an MLST scheme for C. fetus that uses the same loci as a previous scheme for C. jejuni and C. coli (4, 5). We tested this scheme using a diverse collection of isolates and examined the C. fetus population structure and genetic diversity, together with the suitability of MLST for studies of C. fetus subspecies differentiation and epidemiology.
MATERIALS AND METHODS
Bacterial strains. A collection of 140 C. fetus isolates, including 8 reference strains and 132 field isolates (Table 1) were included. This diverse collection contained both epidemiologically unrelated isolates, and isolates from seven outbreaks which occurred at bovine artificial insemination (AI) stations. Isolates were obtained from bovine, ovine, human, equine, and monkey hosts located in different geographical areas: Argentina, Belgium, France, Germany, Hungary, Japan, The Netherlands, South Africa, Spain, Turkey, the United Kingdom, and the United States (Table 1). Isolates were grown under microaerobic conditions (6% O2, 7% CO2, 7% H2, 80% N2; Anoxomat; Mart Microbiology B.V., Lichtenvoorde, The Netherlands) on heart infusion agar supplemented with 5% sheep blood (Biotrading, Mijdrecht, The Netherlands) for 3 days and were stored at –80°C in heart infusion broth containing 15% glycerol.
Preparation of chromosomal DNA. Chromosomal DNA was isolated by using the Puregene kit (Gentra Systems, Minneapolis, MN) and diluted to a final concentration of approximately 5 ng/μl for AFLP analysis and PCR and to 25 ng/μl for PCR amplification during MLST.
Species identification and subspecies differentiation. Isolates were identified genotypically as C. fetus by using a previously described PCR protocol (15), with modifications (39). The subspecies were then identified genotypically by Cf C05 PCR (36); the PCR of Hum et al. (15); AFLP analysis (39); and biochemically, according to growth in the presence of 1% glycine (Merck, Darmstadt, Germany) (32) and H2S production from brucella broth (Difco, Becton Dickinson, Franklin Lakes, NJ) containing 0.02% cysteine (Sigma Aldrich, St. Louis, MO) (1, 17).
sap typing. sap typing was performed with PCR primers based on the sapA and sapB 5' conserved regions, as described by Tu et al. (33).
AFLP analysis. AFLP analysis was performed by a previous method, with modifications (6, 39). These enhancements comprised the use of a capillary-based sequencing system (ABI 3100; Applied Biosystems, Foster City, CA) and data analysis with Bionumerics 3.5 software (Applied Maths, St-Martens-Latem, Belgium). Chromosomal DNA was digested with the restriction enzymes HindIII and HhaI, and site-specific adaptors were ligated. A preselective PCR amplification was performed, followed by selective PCR with primers containing a 3' selective nucleotide and one fluorescently labeled (6-carboxyfluorescein) primer. PCR products underwent electrophoresis through an ABI 3100 capillary sequence system by using a performance optimized polymer-4 polymer matrix (Applied Biosystems). AFLP patterns were analyzed by importing the AFLP data (via the conversion software GeneScan 3.7 [Applied Maths]) into the Bionumerics 3.5 software. Patterns were normalized by referring to the molecular mass of the internal DNA marker (GeneScan-500 [carboxy X rhodamine]; Applied Biosystems). The genetic similarity between the patterns was calculated by using the Pearson product-moment correlation coefficient, and clustering analysis was performed by the unweighted pair group method with arithmetic averages for the region corresponding to 84.6% to 87.1% of the band pattern.
PFGE. PFGE was performed as described previously by using the restriction enzymes SmaI and SalI (36a)
Multilocus sequence typing. (i) Primer design. C. fetus sequences corresponding to the seven loci used in a previously described C. jejuni and C. coli MLST scheme were required: aspA, glnA, gltA, glyA, pgm, tkt, and uncA (4, 5). Various C. jejuni and C. coli MLST primers (Sigma-Genosys, Haverhill, United Kingdom) were employed in multiple combinations to identify primers that would amplify these sequences from C. fetus. A reduced annealing temperature of 48°C was used to improve the binding of the C. jejuni primers to heterologous C. fetus DNA. Of the many primer combinations tested, those that provided amplification products were as follows: for C. jejuni aspA, primers A9 and A10; for C. jejuni glnA, primers A1 and A2; for C. jejuni gltA, primers A1 and A2; for C. jejuni glyA, primers A1 and A2; for C. jejuni tkt, primers A5 and A4; and for C. coli uncA, primers A1 and A2 (the sequences of all primers except C. jejuni tkt A5 and C. coli uncA A1 and A2 are available at http://pubmlst.org/campylobacter/; the sequence of tkt A5 is 5'-TTTAAGTGCTGATATGGTGC-3', the sequence of C. coli uncA A1 is 5'-ATGTAGCCATAGGACAAAAGC-3', and the sequence of C. coli uncA A2 is 5'-CATTCTTGTCGCGTTCAGTTG-3'). The C. fetus sequence corresponding to the pgm locus failed to amplify with any C. jejuni- or C. coli-specific primers; however, this sequence was kindly provided by D. Sanchez, Universidad Nacional de General San Martín, Instituto de Investigaciones Biotecnologicas, San Martín, Argentina. The C. fetus amplicons were sequenced directly by using the same primers used for amplification. Nucleotide sequence extension reactions were carried out by using BigDye ready reaction mix (version 3; Applied Biosystems), in accordance with the manufacturer's instructions. The reaction products were separated with an ABI 3730 automated DNA sequencer (Applied Biosystems).
These C. fetus sequences from the seven loci were used to design seven pairs of C. fetus-specific primers for MLST (Table 2). The MLST allele trimming sites for C. fetus were chosen to correspond to the previous C. jejuni and C. coli sites to assist with future phylogenetic comparisons among species. At least 28 nucleotides separated the MLST trimming site and the 3' end of the C. fetus primers. MLST was performed by using the same primer pair used for both amplification and sequencing.
(ii) PCR amplification and nucleotide sequencing. Seven PCR amplicons were obtained for each isolate by using the primers shown in Table 2. Each 50-μl PCR mixture contained 39.75 μl molecular biology-grade water (Sigma Aldrich), 5 μl 10x PCR buffer (QIAGEN, Crawley, United Kingdom), 1 μl 10 μM each forward and reverse primer, 1 μl 10 mM deoxynucleoside triphosphate mixture (Invitrogen, Paisley, United Kingdom), 0.25 μl HotStar Taq DNA polymerase (QIAGEN), and 2 μl C. fetus chromosomal DNA (approximately 50 ng). The amplification conditions were 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min and storage at 4°C. Nucleotide sequencing (forward and reverse) was performed with the same primers (10 μM diluted 1:15 in water); and the reaction conditions were 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 2 min.
(iii) Allele and ST assignment. For each locus, distinct allele sequences were assigned arbitrary allele numbers in the order of identification. Each genotype was therefore designated by seven numbers (e.g., 1-3-2-4-1-1-2) that constituted an allelic profile or sequence type (ST; e.g., ST-3). The STs were assigned arbitrary numbers in the order of description. All data for newly described C. fetus alleles and STs were deposited in a Campylobacter fetus MLST database (http://pubmlst.org/cfetus/). New sequences were assigned allele numbers, and isolates were assigned their STs by interrogating the database. Allele numbers for new sequences and ST numbers for new allelic profiles are available by submission to the database.
(iv) Data analysis. Phylogenetic analysis was performed with MEGA (version 2.1) software, available at http://www.megasoftware.net, by using the concatenated MLST gene sequence fragments for each isolate. Data were also subjected to split decomposition analysis by using SPLITSTREE (version 3.1) software (16).
RESULTS
C. fetus genotyping and determination of population structure by MLST. The collection of 140 C. fetus isolates was typed by MLST. The C. fetus MLST data were used to (i) assess the level of genetic diversity within this Campylobacter species; (ii) elucidate the C. fetus population structure and compare it to the population structures of other Campylobacter species; (iii) investigate any correlation between ST and cell surface antigen(s), represented by the sap type; and (iv) evaluate the utility of MLST for subspecies differentiation and epidemiological typing compared with those of AFLP analysis and PFGE.
All of the 140 isolates examined were typeable by MLST. The number of alleles per locus ranged from two to four, and the number of variable sites per locus ranged from one to five (Table 3). A total of 14 different STs were identified, some of which shared up to six or seven identical loci. The majority of isolates (91%) were assigned to one of the following STs: ST-2, ST-3, ST-4, ST-5, or ST-6 (Table 1). In this isolate collection, ST-1 was associated with an epidemiologically related group of isolates (n = 5), while the remaining STs (ST-7 to ST-14) each occurred only once.
The level of nucleotide sequence variation within the C. fetus MLST loci was assessed by concatenating the seven sequences to create a continuous 3,312-nucleotide length. There were 22 variable sites in the 14 STs (0.66%) (Table 4). The five most common STs had 12 variable sites and were apparently distributed along the sequence. Three of these mutations (25%) caused a change in the amino acid sequence.
C. fetus is genetically homogeneous with an apparently clonal population structure. In contrast, the five other Campylobacter species for which MLST data are available (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus) (4, 5, 21) show much greater diversity. This was illustrated by comparing data for the three loci used in all six Campylobacter MLST schemes described to date: glnA, glyA, and tkt (data for glnA are shown in Fig. 1). The variation detected in C. fetus is equivalent to a homogeneous C. jejuni clonal complex and is much less than that in many clonal complexes of this species (5).
The extent of association between the C. fetus STs (defined by housekeeping genes) and cell surface antigens was assessed by examining the relationship between the ST and the sap type (sapA or sapB). A clear correlation was observed (Table 1; Fig. 2). SapA was confined to isolates of ST-2, -4, -6, -7, -8, -9, -11, and -12, whereas sapB was associated with isolates of ST-1, -3, -5, -10, -13, and -14. Therefore, the association of this cell surface antigen with the ST demonstrated congruence.
C. fetus sequence type and subspecies. The subspecies was defined by using the classical biochemical approach, PCR (15, 36), and AFLP analysis (39). An AFLP analysis with improved resolution and throughput (described here for the first time) allowed us to define a distinct cluster designated C. fetus subsp. venerealis bv. intermedius. This cluster contained the previously described C. fetus subsp. venerealis bv. intermedius strains 3292 and 3293 (31) and produced H2S, and most isolates were negative for growth in glycine (Table 1). Three subspecies groups were therefore identified: C. fetus subsp. venerealis, C. fetus subsp. venerealis bv. intermedius, and C. fetus subsp. fetus.
The value of MLST for subspecies determination was assessed. Certain STs did correlate with each of the three AFLP groups. A total of 55 of 57 of the C. fetus subsp. venerealis or C. fetus subsp. venerealis bv. intermedius isolates were ST-4 (Table 1; Fig. 2). The remaining 2 of the 57 isolates were ST-7 and ST-12, respectively; ST-12differed by only one nucleotide from ST-4. The C. fetus subsp. fetus AFLP group showed a greater diversity of STs; but notably, ST-4, ST-7, and ST-12 were absent (Table 1; Fig. 2).
Investigation of C. fetus epidemiology by MLST. MLST data were examined for (i) the reliability of outbreak identification, (ii) any correlation of the ST with the isolation source or geographical region, and (iii) any correlation with and discrimination attained compared to the results of PFGE. Such data were available for a subset of epidemiologically related and unrelated isolates (n = 23).
The collection of 140 isolates studied included isolates from seven known outbreaks (with two strains per outbreak) from different bovine AI stations (Table 1). All but one of the outbreaks were associated with STs frequently identified within this data set (ST-1 to ST-4 and ST-6). The remaining outbreak was associated with a single bovine ST-5 isolate from an additional AI station. Epidemiologically related strains shared identical STs and PFGE types (e.g., ST-1 and ST-2; Table 1). However, some epidemiologically unrelated strains had identical STs, but PFGE typing revealed discriminatory patterns (e.g., ST-3, ST-4, ST-5, and ST-6; Table 1), which indicated that PFGE has a greater discriminatory power.
The MLST data and the isolate data on host and geographical region were evaluated for potential associations. Bovine, ovine, and human isolates were the most numerous; but none of these hosts harbored a single ST. However, ST-1 (n = 5) and ST-4 (n = 48) were isolated almost exclusively from bovine hosts, the exceptions being two human isolates. Seven further ST-4 isolates (indicated by a question mark in Table 1) were considered likely (but unconfirmed) bovine isolates (M. Henton, personal communication). Therefore, with the possible exception of ST-4, which is the C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius ST and, therefore, by definition, cattle associated, no association between the ST and the host species was detected. The ST and the geographical region of isolation also lacked association, and all the common STs were found in Europe and North America.
DISCUSSION
An MLST scheme for C. fetus was developed, together with a database for the storage and exchange of MLST data and protocols (http://pubmlst.org/cfetus/). The scheme was evaluated by using 140 C. fetus isolates chosen for their diversity in terms of geography and host species. C. fetus lacked genetic diversity and had a high level of clonality compared to those of other Campylobacter species studied by MLST (4, 5, 21). This could be because mammalian C. fetus isolates evolved from a recent ancestor, or it may be due to a small population size and high-fidelity error-intolerant repair; or the genetic diversity of C. fetus may be limited by a lack of the natural competence found in other Campylobacter species.
C. fetus was originally divided into two subspecies by using host and niche preferences (38), but subsequent taxonomic studies led investigators to question this approach (14, 29). The difficulty of reliable subspecies differentiation reflects the fact that the two subspecies are extremely closely related genetically. For example, it proved impossible to differentiate subspecies by using the nucleotide sequences of two other housekeeping genes, sapD and recA (33), or the 16S rRNA gene (13, 25, 41). Since subspecies differentiation is of high statutory importance, the suitability of MLST for C. fetus subspecies differentiation was investigated. The C. fetus subsp. venerealis strains were all ST-4, although they were geographically and temporally distinct. The C. fetus subsp. venerealis bv. intermedius strains were also mostly ST-4, with two exceptions (one ST-7 strain and another ST-12 strain). Therefore, the MLST scheme did not allow differentiation between C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius strains. However, ST-4 was not detected among the C. fetus subsp. fetus isolates. Extension of the MLST scheme to other loci may reveal differences between C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius strains. The C. fetus subsp. venerealis "ST-4 clone" shows even less diversity within the housekeeping genes than the C. fetus subsp. fetus STs, supporting the theory of Veron and Chatelain, who proposed that C. fetus subsp. venerealis is a mutant of C. fetus subsp. fetus (38).
The data suggest that the MLST approach is reliable for subspecies differentiation since it was performed "blind" without prior knowledge of the subspecies assignments. Although C. fetus subsp. venerealis ST-4 differs from the closest C. fetus subsp. fetus ST (ST-6) by a single nucleotide (Fig. 2), the data generated by this highly reproducible sequencing technique confirm that MLST is an accurate method for the differentiation of the subspecies. Previous studies suggested that differences between C. fetus subsp. venerealis and C. fetus subsp. fetus are plasmid encoded (25, 26), although more recent studies have questioned this proposal (42). This MLST scheme indicates that the differences between the two subspecies are not merely plasmid encoded but are also reflected in the allelic profiles (STs) of the housekeeping genes.
C. fetus isolates from mammals are divided into two sap types, sapA and sapB, on the basis of their SLPs. The MLST data showed a clear correlation among STs and sap types (Fig. 2), supporting the classification of C. fetus into two sap groups. This correlation suggests evolutionary stability and a lack of frequent recombination between the two groups, since in C. jejuni (which recombines frequently) a lack of correlation among STs-clonal complexes and cell surface antigens has been detected.
MLST is very useful for the global epidemiological typing of many bacterial species, and its use is expanding rapidly (18, 35). We demonstrated in the present study that epidemiologically related C. fetus strains had identical STs. However, as a result of the relatively low level of genetic diversity found in C. fetus housekeeping genes, some unrelated isolates had STs identical to those of some of the outbreak strains. Sequencing of additional, more variable loci has proven useful in investigations of outbreaks of Neisseria meningitidis infections and may increase the discriminatory power of the method (10). Whether this approach would also improve C. fetus discrimination requires further investigation. C. fetus has a global distribution; however, no geographically distinct ST cluster could be distinguished by MLST. The major STs were found in multiple host species; however, the C. fetus subsp. venerealis ST-4 was restricted to bovine hosts. It has been suggested that C. fetus subsp. venerealis is a host-restricted mutant clone of C. fetus subsp. fetus that is unable to infect multiple host species (38).
MLST was compared to biochemical and established C. fetus band-based typing methods, AFLP analysis, PFGE, and PCR. Each of these methods measures genotypic characteristics which may change independently; hence, the subtypes that they define may not always be congruent (30). However, MLST subspecies differentiation results showed a high agreement with AFLP, Cf C05 PCR, and H2S results compared to the results of the PCR of Hum et al. (15) and the glycine test. In the present study, a strong correlation was observed between MLST and PFGE data, and both methods gave identical clusters of isolates from known C. fetus outbreaks. PFGE provided a higher level of discrimination than MLST. However, MLST measures variation that accumulates relatively slowly in the absence of selective pressure, and it may be more appropriate for studies of long-term C. fetus epidemiology and phylogenetics. PFGE indexes genome-wide variations and, despite the difficulties of interlaboratory comparisons, may be better suited for short-term investigations of C. fetus epidemiology, such as outbreak investigations, when such investigations are performed by a single laboratory. The level of discrimination of C. fetus isolates was higher by MLST than by the AFLP analysis method described, irrespective of the region of the AFLP pattern analyzed.
In summary, the observations that mammalian C. fetus isolates have a low level of genetic diversity and are genetically homogeneous compared to the homogeneities of other Campylobacter species suggest that C. fetus is young in evolutionary terms and may have arisen from a recent ancestor. In Fig. 1, the long branch length separating C. fetus from the other Campylobacter species suggests that its putative recent ancestor is missing from this tree. This is supported by the lack of any sequences characteristic of these five other Campylobacter species within the C. fetus housekeeping genes. The predominantly stepwise accumulation of point mutations in C. fetus indicates clonal evolution, in which genetic change accumulates slowly only by the vertical transmission of point mutations. The genetic stability of C. fetus is further confirmed by the wide geographic distribution of identical STs and the observation of congruence between the C. fetus subspecies, sap type, and ST. The two C. fetus subspecies were extremely closely genetically related, but ST-4 was associated only with C. fetus subsp. venerealis and was therefore, by definition, a "bovine" subclone. MLST confirms that mammalian C. fetus is genetically stable and clonal, provides a useful additional tool for C. fetus subspecies differentiation and epidemiology, and compares favorably to other bacterial typing methods.
ACKNOWLEDGMENTS
This study was partly funded by COST Action 920.
We are very grateful to D. Sanchez (Universidad Nacional de General San Martín, Instituto de Investigaciones Biotecnologicas, San Martín, Argentina) for comparing the C. jejuni pgm sequence with the sequence of C. fetus by use of the BLAST program and providing us with the C. fetus pgm sequence identified for primer design. Furthermore, we thank the following for kindly supplying C. fetus isolates: C. Campero, Instituto Nacional de Tecnoloíga Agropecuaria, Balcarce, Argentina; J. Godfroid, Veterinary and Agrochemical Research Centre, Brussels, Belgium; J. P. Butzler, University of Brussels, Brussels, Belgium; P. Idigoras, Hospital Donostia, San Sebastián, Spain; W. Kalka-Moll, University of Cologne, Germany; J. Varga, Faculty of Veterinary Science, Budapest, Hungary; S. Fujimoto, School of Health Sciences, Kyushu University, Kyushu, Japan; W. Ang, Erasmus University Medical Centre, Rotterdam, The Netherlands; M. Koene and I. Visser, Animal Health Service, Deventer, The Netherlands; M. Hinton, Onderstepoort Veterinary Institute, Onderstepoort, South Africa; L. Guler, Veteriner Kontrol ve Arastirma Enstitusu, Konyn, Turkey; J. Frost, Health Protection Agency, London, United Kingdom; J. Corry, University of Bristol, Bristol, United Kingdom; M. Toszeghy, Veterinary Laboratories Agency, Weybridge, United Kingdom; P. Fields, Centers for Disease Control and Prevention, Atlanta, GA; L. Schroeder-Tucker, National Veterinary Services Laboratories, Ames, IA; I. Wesley, National Animal Disease Centre, Ames, IA; and Z. Tu and M. Blaser, Vanderbilt University School of Medicine, Nashville, TN.
M. van Bergen and K. Dingle contributed equally to the manuscript, and both are considered the first author.
REFERENCES
Berg, R. L., J. W. Jutila, and B. D. Firehammer. 1971. A revised classification of Vibrio fetus.Am. J. Vet. Res. 32:11-22.
Blaser, M. J., C. W. Moss, and R. E. Weaver.1980 . Cellular fatty acid composition of Campylobacter fetus. J. Clin. Microbiol. 11:448-451.
Denes, A. S., C. L. Lutze-Wallace, M. L. Cormier, and M. M. Garcia. 1997. DNA fingerprinting of Campylobacter fetus using cloned constructs of ribosomal RNA and surface array protein genes. Vet. Microbiol. 54:185-193.
Dingle, K. E., F. M. Colles, D. Falush, and M. C. J. Maiden. 2005. Sequence typing and comparison of population biology of Campylobacter coli and Campylobacter jejuni. J. Clin. Microbiol. 43:340-347.
Dingle, K. E., F. M. Colles, D. R. A. Wareing, R. Ure, A. J. Fox, F. E. Bolton, H. J. Bootsma, R. J. L. Willems, R. Urwin, and M. C. J. Maiden. 2001. Multilocus sequence typing system for Campylobacter jejuni.J. Clin. Microbiol. 39:14-23.
Duim, B., T. M. Wassenaar, A. Rigter, and J. A. Wagenaar. 1999. High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting. Appl. Environ. Microbiol. 65:2369-2375.
Dworkin, J., M. K. Tummuru, and M. J. Blaser.1995 . Segmental conservation of sapA sequences in type B Campylobacter fetus cells. J. Biol. Chem. 270:15093-15101.
Elazhary, M. 1968. An assay of isolation and identification of some animal vibrios and of elucidation of their pathological significance. Meded. Veeartschool. Rijksuniv. Gent 12:1-80.
Enright, M. C., N. P. J. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt.1998 . A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144:3049-3060.
Feavers, I. M., S. J. Gray, R. Urwin, J. E. Russell, J. A. Bygraves, E. B. Kaczmarski, and M. C. Maiden. 1999. Multilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak. J. Clin. Microbiol. 37:3883-3887.
Fujita, M., S. Fujimoto, T. Morooka, and K. Amako. 1995. Analysis of strains of Campylobacter fetus by pulsed-field gel electrophoresis. J. Clin. Microbiol. 33:1676-1678.
Garcia, M. M., M. D. Eaglesome, and C. Rigby.1983 . Campylobacters important in veterinary medicine.Vet. Bull. 53:793-818.
Gorkiewicz, G., G. Feierl, C. Schober, F. Dieber, J. Kfer, R. Zechner, and E. L. Zechner. 2003. Species-specific identification of campylobacters by partial 16S rRNA gene sequencing.J. Clin. Microbiol. 41:2537-2546.
Harvey, S., and J. R. Greenwood. 1983. Relationships among catalase-positive campylobacters determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization. Int. J. Syst. Bacteriol. 33:275-284.
Hum, S., K. Quinn, J. Brunner, and S. L. W. On.1997 . Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies.Aust. Vet. J. 75:827-831.
Huson, D. H. 1998. SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14:68-73.
MacLaren, A. P. C., and G. J. O. Agumbah. 1988. Infertility in cattle in south-west Scotland caused by an "intermediate" strain of Campylobacter fetus subspecies fetus (formerly Campylobacter fetus intestinalis). Br. Vet. J. 144:29-44.
Maiden, M. C., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95:3140-3145.
Maiden, M. C. J. 2000. High-throughput sequencing in the population analysis of bacterial pathogens.Int. J. Med. Microbiol. 290:183-190.
Mannering, S. A., D. M. West, S. G. Fenwick, R. M. Marchant, N. R. Perkins, and K. O'Connell. 2004. Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand. N. Z. Vet. J. 52:358-363.
Miller, W. G., S. L. W. On, G. Wang, S. Fontanoz, A. J. Lastovica, and R. E. Mandrell.2005 . System for extended multilocus sequence typing of strains of Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus.J. Clin. Microbiol. 43:2315-2329.
Moran, A. P., D. T. O'Malley, T. U. Kosunen, and I. M. Helander. 1994. Biochemical characterization of Campylobacter fetus lipopolysaccharides.Infect. Immun. 62:3922-3929.
Morooka, T., A. Umeda, M. Fujita, H. Matano, S. Fujimoto, K. Yukitake, K. Amako, and T. Oda. 1996. Epidemiologic application of pulsed-field gel electrophoresis to an outbreak of Campylobacter fetus meningitis in a neonatal intensive care unit. Scand. J. Infect. Dis. 28:269-270.
Newell, D. G., J. A. Frost, B. Duim, J. A. Wagenaar, R. H. Madden, J. van der Plas, and S. L. W. On. 2000. New developments in the subtyping of Campylobacter species, p.27 -44. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, D.C.
On, S. L. W., and C. S. Harrington.2001 . Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods.J. Appl. Microbiol. 90:285-293.
Panaccio, M., L. Stephens, and P. Widders. 1993. Field evaluation of a PCR-based test for the diagnosis of bovine campylobacteriosis DAV106. Dairy Research and Development Corporation, Melbourne, Australia.
Perez-Perez, G. I., M. J. Blaser, and J. H. Bryner. 1986. Lipopolysaccharide structures of Campylobacter fetus are related to heat-stable serogroups.Infect. Immun. 51:209-212.
Rennie, R. P., D. Strong, D. E. Taylor, S. M. Salama, C. Davidson, and H. Tabor. 1994. Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism.J. Clin. Microbiol. 32:721-724.
Roop, R. M., II, R. M. Smibert, J. L. Johnson, and N. R. Krieg. 1984. Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups. Can. J. Microbiol. 30:938-951.
Sails, A. D., B. Swaminathan, and P. I. Fields.2003 . Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strains associations identified by multilocus enzyme electrophoresis.J. Clin. Microbiol. 41:4058-4067.
Salama, S. M., M. M. Garcia, and D. E. Taylor. 1992. Differentiation of the subspecies of Campylobacter fetus by genomic sizing. Int. J. Syst. Bacteriol. 42:446-450.
Smibert, R. M. 1984. Genus II Campylobacter Sebald and Veron 1963, 907, p.111 -118. In N. R. Krieg and H. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, Md.
Tu, Z.-C., F. E. Dewhirst, and M. J. Blaser.2001 . Evidence that the Campylobacter fetus sap locus is an ancient genomic constituent with origins before mammals and reptiles diverged. Infect. Immun. 69:2237-2244.
Tu, Z.-C., W. Eisner, B. N. Kreiswirth, and M. J. Blaser. 2005. Genetic divergence of Campylobacter fetus strains of mammal and reptile origins. J. Clin. Microbiol. 43:3334-3340.
Urwin, R., and M. C. Maiden. 2003. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol. 11:479-487.
Van Bergen, M. A. P., G. Simons, L. Van der Graaf-Van Bloois, J. Rombout, J. P. M. Van Putten, and J. A. Wagenaar. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. J. Med. Microbiol., in press.
Van Bergen, M. A. P., L. van der Graaf-van Bloois, I. J. R. Visser, Jos P. M. van Putten, and J. A. Wagenaar. Molecular epidemiology of Campylobacter fetus subsp. fetus on bovine artificial insemination stations using pulsed field gel electrophoresis. Vet. Microbiol., in press.
Vandamme, P., B. Pot, E. Falsen, K. Kersters, and J. De Ley. 1990. Intra- and interspecific relationships of veterinary campylobacters revealed by numerical analysis of electrophoretic protein profiles and DNA:DNA hybridizations. Syst. Appl. Microbiol. 13:295-303.
Veron, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and Veron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) Sebald and Veron. Int. J. Syst. Bacteriol. 23:122-134.
Wagenaar, J. A., M. A. P. Van Bergen, D. G. Newell, R. Grogono-Thomas, and B. Duim. 2001. Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals.J. Clin. Microbiol. 39:2283-2286.
Wang, G., C. G. Clark, T. M. Taylor, C. Pucknell, C. Barton, L. Price, D. L. Woodward, and F. G. Rodgers. 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J. Clin. Microbiol. 40:4744-4747.
Wesley, I. V., R. D. Wesley, M. Cardella, F. E. Dewhirst, and B. J. Paster. 1991. Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.J. Clin. Microbiol. 29:1812-1817.
Willoughby, K., P. F. Nettleton, M. Quirie, M. A. Maley, G. Foster, M. Toszeghy, and D. G. Newell. 2005. A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis: use on UK isolates of Campylobacter fetus and other Campylobacter spp. J. Appl. Microbiol. 99:758-766.(Marcel A. P. van Bergen, )