Differentiation of Campylobacter jejuni and Campylobacter coli. Strains by ..... analysis of isolates of Campylobacter fetus subsp. venerealis recovered from.
JOURNAL OF CLINICAL MICROBIOLOGY, May 1995, p. 1136–1140 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 33, No. 5
Differentiation of Campylobacter jejuni and Campylobacter coli Strains by Using Restriction Endonuclease DNA Profiles and DNA Fragment Polymorphisms VICTORIA KOROLIK,* LISA MOORTHY,
AND
PETER J. COLOE
Department of Applied Biology and Biotechnology, Royal Melbourne Institute of Technology, Melbourne, Victoria 3001, Australia Received 7 June 1994/Returned for modification 1 September 1994/Accepted 25 January 1995
The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolates from poultry and humans were analyzed by using DNA restriction endonucleases ClaI and EcoRV. The DNA restriction patterns produced by ClaI and EcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains of C. jejuni tested. The DNA restriction patterns were further examined by Southern blot analysis with a previously constructed DNA probe, pMO2005, which is also able to distinguish between C. jejuni and C. coli spp. (5). Two types of patterns were produced by hybridization with the ClaI-cleaved DNA of C. jejuni strains, one of a single 18.5-kb genomic fragment and the other of 14.5- and 4.0-kb fragments. This indicated the presence of an extra ClaI site in this genomic fragment in the strains with the duplex pattern. The Southern blot analysis of 169 C. jejuni and C. coli isolates from poultry and from humans with DNA probe pMO2005 demonstrated that 78% of C. jejuni strains isolated from chickens hybridized with DNA probe pMO2005 with a characteristic 14.5- and 4.0-kb banding pattern and 22% hybridized with a single 18.5-kb fragment, whereas 71% of human isolates hybridized with the single 18.5-kb fragment and only 29% hybridized with 14.5- and 4.0-kb fragments. These findings suggest that only a small proportion of C. jejuni strains that colonize chickens may cause disease in humans. commercial chicken flocks in six isolated farms in Australia were analyzed and compared, in terms of their genetic diversity, with C. jejuni and C. coli strains isolated from human patients with acute enteritis. Comparison was based on RE profiles and restriction fragment length polymorphisms in Southern blots with a DNA probe, pMO2005, that we have developed previously. This probe can distinguish between C. jejuni and C. coli on the basis of the hybridization patterns obtained by Southern blotting. The probe hybridizes with an
Campylobacter jejuni and Campylobacter coli are well recognized as causes of acute enteritis in humans, and the source of infection is frequently considered to be consumption of contaminated poultry since it has been well documented that Campylobacter species commonly colonize the chicken intestine (10). Many attempts have been made to differentiate between Campylobacter strains from animals and people. C. jejuni and C. coli strains isolated from animals and humans have been subjected to various serological, biochemical, and molecular analyses including multilocus enzyme electrophoresis (2, 9). Various patterns of enzymatic electrophoretic profiles have been described indicating that animal and human strains form a single group. However, disease in humans caused by Campylobacter spp. greatly varies in severity, ranging from mild discomfort to bloody pyretic diarrhea (3). The study of Campylobacter spp. has been hampered by their genetic diversity. This diversity has been shown by the DNA fragment patterns obtained with restriction endonuclease cleavage of chromosomal DNAs from a variety of strains. However, the diversity of Campylobacter genomes has some advantages in that restriction endonuclease (RE) fragment profiles of chromosomal DNA is an effective tool for discriminating between the strains that cannot be distinguished biochemically or serologically (4, 8, 12). In addition, DNA probes to specific genomic regions may be helpful in elucidating genetic differences in Campylobacter spp. that are otherwise undetectable. Such differences in RE profiles in Southern blots with DNA probes were previously used successfully to group strains of Campylobacter mucosalis (6). In the study described here C. jejuni and C. coli strains from
FIG. 1. DNA RE analysis of ClaI-restricted C. jejuni and C. coli DNAs. Lanes: 1, bacteriophage l (cleaved with HindIII); 2, C. jejuni 3015; 3, C. jejuni 8001; 4, C. coli 8009; 5, C. jejuni 8005; 6, C. jejuni 8006; 7, C. jejuni 8007; 8, C. jejuni 8010; 9, C. jejuni 9003; 10, C. jejuni 9014.
* Corresponding author. 1136
DIFFERENTIATION OF C. JEJUNI AND C. COLI STRAINS
VOL. 33, 1995
1137
FIG. 2. (a) DNA RE ClaI analysis of C. jejuni and C. coli strains isolated from chickens and humans. Lanes: 1, bacteriophage l (cleaved with HindIII); 2, C. jejuni 301 (chicken); 3, C. jejuni 311 (chicken); 4, C. jejuni 413 (chicken); 5, C. jejuni 902 (chicken); 6, C. jejuni 831 (chicken); 7, C. jejuni 860 (human); 8, C. jejuni 957 (human); 9, C. jejuni 410 (human); 10, C. jejuni FF3 (human); 11, C. jejuni p7 (human); 12, C. coli 424 (chicken); 13, C. coli 324 (chicken); 14, C. coli 7006 (chicken); 15, C. coli 966 (chicken); 16, C. coli 388 (human); 17, C. coli 900 (human); 18, C. coli 832 (human); 19, C. coli D126 (human); 20, bacteriophage l (cleaved with HindIII). (b) DNA RE EcoRV analysis of C. jejuni and C. coli strains isolated from chickens and humans. Lanes: 1, bacteriophage l (cleaved with HindIII); 2, C. jejuni 301 (chicken); 3, C. jejuni 311 (chicken); 4, C. jejuni 413 (chicken); 5, C. jejuni 831 (chicken); 6, C. jejuni 860 (human); 7, C. jejuni 957 (human); 8, C. jejuni 410 (human); 9, C. jejuni FF3 (human); 10, C. jejuni p7 (human); 11, C. coli 424 (chicken); 12, C. coli 7006 (chicken); 13, C. coli 966 (chicken); 14, C. coli 388 (human); 15, C. coli 832 (human); 16, C. coli 900 (human); 17, C. coli D126 (human), 18, bacteriophage l (cleaved with HindIII).
18.5-kb genomic fragment in C. jejuni strains and a 9.0-kb fragment in C. coli strains (5). MATERIALS AND METHODS Isolation of Campylobacter strains from chickens. The bacterial strains described here were isolated from chicken fecal material as follows. Samples from the cloaca were collected with swabs and were swabbed onto Oxoid Columbia agar supplemented with 7% defibrinated horse blood and Skirrow Campylobacter isolation medium supplement (Oxoid SR69) (Skirrow medium). The plates were incubated in a microaerophilic atmosphere of 90% N2–5% CO2–5% O2 for 4 to 5 days at 378C. Campylobacter isolates were then selected on the basis of colony morphology, Gram stained, and characterized biochemically (7). Antibiotics. The antibiotic susceptibilities of the isolates were determined with antibiotic-impregnated disks (Oxoid): kanamycin, 30 and 50 mg/ml; cephalothin, 30 mg/ml; naladixic acid, 30 mg/ml, tetracycline, 10 and 30 mg/ml; ampicillin, 25 mg/ml; and streptomycin, 10 and 25 mg/ml. Approximately 5 3 107 C. jejuni or C.
coli cells were plated onto Oxoid Columbia agar supplemented with 7% defibrinated horse blood and were allowed to dry. Antibiotic disks were placed on the plate surface, and the plates were incubated in a microaerophilic atmosphere of 90% N2–5% CO2–5% O2 for 2 days at 378C. Susceptibility status was assessed by measuring the diameter of the growth inhibition zone around the antibiotic disk as described by the manufacturer. Western blotting (immunoblotting). The proteins resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were transblotted onto a nitrocellulose membrane (Bio-Rad) electrophoretically (11) and were probed with anti-C. jejuni serum raised to C. jejuni total cell antigen as described previously (5). Reacting antigens were detected with 35S-labelled staphylococcal protein A (Amersham). Genomic DNA extraction and purification. A lawn culture of an organism growing on an agar plate (equivalent to 6 h of growth for Escherichia coli or 36 h for Campylobacter spp.) was flooded with 3 ml of sucrose buffer (25% sucrose in 50 mM Tris-HCl [pH 8.3]), 2 ml of cells was harvested from the plate surface, and 0.5 ml of 0.25 M EDTA (pH 8.0) containing 20 mg of lysozyme (freshly made) per ml was added. This mixture was incubated for 15 min at 378C, and
1138
KOROLIK ET AL.
FIG. 3. Autoradiograph of the Western blot analysis of the total membrane preparations of C. jejuni FF3 (lane 1), E. coli DHI carrying a cloning vector pBR322 (lane 2), and E. coli DHI carrying recombinant plasmid pMO2005 (lane 3). These were probed with an antiserum raised to whole cells of C. jejuni (5). K, molecular size (in kilodaltons).
then 0.5 ml of 10% SDS was added to the samples to assist in the cell lysis. A 150-ml volume of pronase at 10 mg/ml was added, and the sample was incubated for 30 min at 378C, and then an additional 50 ml of pronase was added and the sample was incubated for an additional 15 min. The density of the sample was reduced by the addition of 0.5 to 1.0 ml of TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]), and the samples were extracted with an equal volume of phenol by vigorous shaking for 1 h on a rotary shaker at ambient temperature. The aqueous phase was separated by centrifugation at 3,000 rpm in an Eppendorf centrifuge, and 2 ml of the aqueous phase was collected and extracted twice with phenolchloroform-isoamyl alcohol (25:24:1) with vigorous shaking for 15 min. Duplicate 700-ml volumes of the aqueous phase were collected into tubes, and the DNA was precipitated with an equal volume of isopropanol at 2208C for 30 min. The DNA pellet was collected by centrifugation. The duplicate samples were resuspended in 250 ml of TE buffer each, pooled, and incubated with 20 ml of 10 mg of RNase per ml for 15 min at 378C. Residual protein was removed by incubating with 20 ml of 10 mg of pronase per ml for 30 min at 378C and was then extracted once with phenol, twice with phenol-chloroform, and once with chloroform. A 400-ml aliquot of the aqueous phase was collected, and the DNA was precipitated with 1/5 volume of 10 M ammonium acetate and 2.5 volumes of absolute ethanol at 2208C. The DNA was collected by centrifugation and was resuspended in 200 to 400 ml of TE. DNA manipulation. RE cleavage, agarose gel electrophoresis, and Southern blotting were performed as described previously (5). The enzymes ClaI, EcoRV, and BglII (Promega Biotech) were used at 20 U/mg of DNA. Preparation of radiolabelled probe DNA. The DNA probe was oligoradiolabelled with a random primer system (GIGA prime DNA labelling kit [Bresatec, Adelaide, Australia]) according to the manufacturer’s instructions.
RESULTS Use of DNA fingerprinting for epidemiological identification of Campylobacter strains from humans and chicken flocks. Total genomic DNAs of C. jejuni and C. coli strains were purified from 169 strains; 120 of the strains were isolated from chicken fecal material and 49 were isolated from humans. The DNA was cleaved with ClaI, and the fragments were resolved by agarose gel electrophoresis. Examples of the profiles obtained from nine such strains are shown in Fig. 1. The DNA of each strain cleaved with ClaI produces a RE profile, or fingerprint, that is unique to the strain. Each strain was thus genetically compared with the other strains by using the DNA fingerprints. Strains such as 8001 (Fig. 1, lane 3) and 8005 (Fig. 1, lane 5) showing identical fingerprints were isolated from the same flock of chickens. These two isolates showing identical ClaI RE profiles were further tested by examining additional profiles produced by EcoRV and BglII. Identical DNA RE profiles were produced by cleavage of DNA with the three enzymes for each pair of isolates, proving that they were multiple isolates of the same strain (data not shown). This was confirmed by biochemical and antibiotic profile anal-
J. CLIN. MICROBIOL.
FIG. 4. Southern blot analysis of ClaI-restricted C. jejuni and C. coli DNAs. Lanes: 1, bacteriophage l (cleaved with HindIII); 2, C. jejuni 3015; 3, C. jejuni 8001; 4, C. coli 8009; 5, C. jejuni 8005; 6, C. jejuni 8006; 7, C. jejuni 8007; 8, C. jejuni 8010; 9, C. jejuni 9003; 10, C. jejuni 9014.
yses. Thus, on the basis of these analyses any two or more isolates showing identical RE profiles following DNA cleavage with REs were considered to be multiple isolates of the same strain. RE profile screening showed that of a total of 120 isolates from chicken flocks, 89 were unique strains and the remaining strains were 1 of 2 or more isolates of the same strain. Of the multiple isolates there were 14 pairs, three triplets, two quadruplets, and one set of six. One strain from each group of multiple isolates is represented in 89 unique profiles. With the exception of one isolate, all multiple isolates were from the same flock. Use of DNA fingerprints to distinguish Campylobacter strains. The genetic fingerprints of poultry isolates were compared with those of the Campylobacter strains isolated from human patients with severe gastroenteritis. The RE profiles produced by ClaI with the genomic DNAs of all 169 Campylobacter strains were examined. The RE profiles of selected strains of C. jejuni and C. coli from humans and chickens are shown in Fig. 2a, demonstrating that C. jejuni strains of poultry origin cannot be distinguished from those of human origin on the basis of the DNA RE profiles. This also applied to C. coli isolates from chickens and humans. However, there was a combination of two specific bands which were present in all C. jejuni strains but not in the C. coli strains tested. These C. jejuni-specific bands were ClaI fragments of 9 to 9.5 kb present as broad double bands and another strong band (also a doublet) of 3.5 kb, indicated by the arrows in Fig. 2a. C. jejuni subsp. doylei NCTC 11357 also shows this banding pattern (data not shown). The RE EcoRV was also used to compare poultry and human Campylobacter strains (Fig. 2b). The DNA RE profiles of C. jejuni and C. coli strains isolated from poultry cannot be distinguished from those of strains of human origin. EcoRV profiles, however, also feature a unique DNA fragment of 3.0 kb that is present in C. jejuni strains (indicated by an arrow in Fig. 2b) but that is absent from C. coli strains. Expression of C. jejuni antigen by pMO2005 in E. coli. Plasmid pMO2005 is a recombinant carrying a C. jejuni genomic DNA fragment in plasmid vector pBR322 and was selected from a C. jejuni genomic library constructed in E. coli on the basis of its response to anti-C. jejuni serum raised to membrane proteins (5). The C. jejuni antigen expressed in E. coli by pMO2005 was further characterized by Western blot analysis. Figure 3 shows a 31.5-kDa antigen which could be detected in the membrane profile of E. coli DHI carrying recombinant plasmid pMO2005 expressing this antigen, but not in E. coli
VOL. 33, 1995
DIFFERENTIATION OF C. JEJUNI AND C. COLI STRAINS
1139
FIG. 5. (a) Southern blot analysis of ClaI-restricted DNAs from C. jejuni and C. coli strains isolated from chickens and humans hybridized with DNA probe pMO2005. Lanes: 1, bacteriophage l (cleaved with HindIII); 2, C. jejuni 301 (chicken); 3, C. jejuni 311 (chicken); 4, C. jejuni 413 (chicken); 5, C. jejuni 902 (chicken); 6, C. jejuni 831 (chicken); 7, C. jejuni 860 (human); 8, C. jejuni 957 (human); 9, C. jejuni 410 (human); 10, C. jejuni FF3 (human); 11, C. jejuni p7 (human); 12, C. coli 424 (chicken); 13, C. coli 324 (chicken); 14, C. coli 7006 (chicken); 15, C. coli 966 (chicken); 16, C. coli 388 (human); 17, C. coli 900 (human); 18, C. coli 832 (human); 19, C. coli D126 (human); 20, bacteriophage l (cleaved with HindIII). (b) Southern blot analysis of EcoRV-restricted DNA from C. jejuni and C. coli strains isolated from chickens and humans hybridized with DNA probe pMO2005. Lanes: 1, bacteriophage l (cleaved with HindIII). 2, C. jejuni 301 (chicken); 3, C. jejuni 311 (chicken); 4, C. jejuni 413 (chicken); 5, C. jejuni 831 (chicken); 6, C. jejuni 860 (human); 7, C. jejuni 957 (human); 8, C. jejuni 410 (human); 9, C. jejuni FF3 (human); 10, C. jejuni p7 (human); 11, C. coli 424 (chicken); 12, C. coli 7006 (chicken); 13, C. coli 966 (chicken); 14, C. coli 388 (human); 15, C. coli 832 (human); 16, C. coli 900 (human); 17, C. coli D126 (human), 18, bacteriophage l (cleaved with HindIII).
DHI carrying the vector plasmid pBR322. The antigenic profile of C. jejuni FF3, which was used to construct the genomic library, shows an antigen of comparable size. Use of a C. jejuni antigen sequence as a DNA probe for discrimination of Campylobacter isolates. The identities and species of the Campylobacter spp. were further confirmed by Southern blot analysis of the DNA fingerprints by using DNA probe pMO2005, which encodes a C. jejuni membrane antigen. The DNA fingerprints shown in Fig. 1 were Southern blotted and probed with a radiolabelled DNA probe pMO2005 (Fig. 4). The probe hybridized with a C. jejuni genomic fragment of 18.5 kb or with two fragments of 14.5 and 4.0 kb, which together constitute an 18.5-kb fragment, indicating that some C. jejuni strains have a polymorphic ClaI site within this genomic fragment. C. coli strains hybridized strongly to the probe with a 9.0-kb fragment and weakly with a secondary 5.8-kb fragment, which became more apparent with overexposure of the autoradiograph. No polymorphic bands were detected for C. coli.
Use of a DNA probe to distinguish between human and chicken Campylobacter strains. The ClaI DNA polymorphisms of all Campylobacter strains from humans and chickens were examined by using pMO2005 as a probe. Figure 5a shows a selection of C. jejuni isolates (as shown in Fig. 2a) from chickens and humans so analyzed. In C. jejuni strains from humans pMO2005 hybridized with an 18.5-kb fragment, whereas in strains from chickens it hybridized with 14.5- and 4.0-kb fragments. In all C. coli isolates a 9.0-kb DNA fragment hybridized with pMO2005 strongly, and a secondary 5.8-kb fragment hybridized weakly but cannot be clearly observed because of the short exposure time of the autoradiograph. No polymorphisms were detected for C. coli strains. Subsequently, 77 poultry and 37 human C. jejuni isolates and 12 poultry and 12 human C. coli isolates were analyzed by Southern blotting by using ClaI DNA profiles probed with pMO2005. Seventy-eight percent of the isolates from poultry hybridized with pMO2005 with 14.5- and 4.0-kb polymorphic fragments and 22% hybridized with an 18.5-kb ClaI fragment. In the case of C. jejuni strains isolated
1140
KOROLIK ET AL.
from humans with Campylobacter enteritis, 71% of the isolates showed the 18.5-kb ClaI polymorphism and only 29% of the isolates showed the 14.5- and 4.0-kb polymorphisms. In view of these results, EcoRV DNA profiles of C. jejuni and C. coli strains (as shown in Fig. 2b) were also subjected to Southern blot analysis with pMO2005 as a probe. All C. jejuni isolates showed the same hybridizing bands of 5.8 and 1.2 kb, and C. coli hybridized with a single 20-kb fragment. No polymorphic variation could be detected (Fig. 5b). DISCUSSION A total of 120 C. jejuni and C. coli isolates from poultry and 49 isolates from humans were analyzed by DNA fingerprinting with the REs ClaI and EcoRV. Specific bands were detected in ClaI and EcoRV restriction profiles, which are unique to all strains of C. jejuni tested. These comprise a specific combination of ClaI fragments of 9 to 9.5 kb as a broad double band and a strong 3.5-kb band as well as a single specific band of 3.0 kb produced by EcoRV. These bands appear to be characteristic of all C. jejuni isolates and were absent from all C. coli DNA profiles. The DNA fingerprint analysis described in this report offers a powerful tool for identifying isolates of the same strain. In situations in which a patient has multiple bouts of Campylobacter-induced enteritis it is often impossible by current methods to determine whether the disease is caused by reinfection with a different strain of the same serotype or simply reactivation of the original infecting strain (1). The RE profile described in this report will enable the accurate differentiation of individual strains and will facilitate the management of patients with Campylobacter infections as well as provide accurate epidemiological information. The DNA fingerprints of the C. jejuni and C. coli strains were also examined by Southern blotting analysis with a previously constructed DNA probe, pMO2005, which was highly specific for C. jejuni and C. coli and which does not react with other thermophilic Campylobacter species (5). The probe was based on the C. jejuni genomic DNA sequence encoding a 31.5-kDa membrane antigen which was considered to enhance its ability to differentiate between C. jejuni strains. For C. jejuni strains probed with pMO2005, two patterns were produced; one pattern was a single 18.5-kb genomic fragment and the other consisted of 14.5- and 4.0-kb fragments. This indicates the presence of an extra ClaI site in the 18.5-kb genomic fragment in the strains with the latter pattern and can be used to differentiate C. jejuni isolates, especially when it is used in conjunction with the RE profile. The genomic differences in C. jejuni strains isolated from poultry and those isolated from human patients were obvious. Analysis of the results showed that 78% of the C. jejuni strains isolated from poultry showed 14.5- and 4.0-kb banding patterns and 22% showed a single 18.5-kb fragment, whereas 71% of the isolates from humans had the single 18.5-kb fragment and only 29% had the 14.5- and 4.0-kb fragments. There is clearly some overlap between isolates of C. jejuni from chickens and
J. CLIN. MICROBIOL.
humans, but it appears that there is not a direct relationship between isolates of C. jejuni from chickens and human disease. This is the first study that has shown any difference between Campylobacter isolates from poultry and humans and suggests that not all Campylobacter spp. may be pathogenic for humans. This would parallel the situation with many other enteric organisms such as E. coli and some Salmonella spp. Indeed, these findings suggest that there may be significant differences between those C. jejuni isolates that colonize chickens and those that cause disease in humans, indicating that it is indeed possible that only a small proportion of C. jejuni strains found in chicken flocks are capable of causing disease in humans. This is an important finding that will actively stimulate further research into Campylobacter spp. We believe that it is paramount to be able to identify pathogenic strains and trace them epidemiologically from the patient to the source of infection. The DNA probe pMO2005 may provide such a marker for the identification of C. jejuni strains on the basis of restriction fragment length polymorphism profiles in Southern blots, since it has been clearly shown to distinguish C. jejuni from C. coli and to differentiate C. jejuni into two subgroups. ACKNOWLEDGMENTS We thank N. Gerraty for assisting with the collection of samples. This work was supported by the Australian Chicken Meat Research and Development Council. REFERENCES 1. Aeschbacher, M., and J.-C. Piffaretti. 1989. Population genetics of human and animal enteric Campylobacter strains. Infect. Immun. 57:1432–1437. 2. Albert, M. J., A. Leach, V. Asche, J. Hennessy, and J. L. Penner. 1992. Serotype distribution of Campylobacter jejuni and Campylobacter coli isolated from hospitalized patients with diarrhea in Central Australia. J. Clin. Microbiol. 30:207–210. 3. Blaser, M. J., D. N. Taylor, and R. A. Feldman. 1983. Epidemiology of Campylobacter jejuni infections. Epidemiol. Rev. 5:157–173. 4. Fraser, A. D., B. W. Brooks, M. M. Garcia, and H. Lior. 1992. Molecular discrimination of Campylobacter coli serogroup 20 biotype 1 (Lior) strains. Vet. Microbiol. 30:267–280. 5. Korolik, V., V. Krishnapillai, and P. J. Coloe. 1988. A specific DNA probe for the identification of Campylobacter jejuni. J. Gen. Microbiol. 134:521– 529. 6. Lin, G. F., C. J. Gebhart, and M. P. Murtaugh. 1991. Southern blot analysis of strain variation in Campylobacter mucosalis. Vet. Microbiol. 28:279–289. 7. Morris, G. K., and C. M. Patton. 1985. Campylobacter, p. 302–308. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 8. Owen, R. J., and J. Hernandez. 1990. Genotypic variation in Campylobacter upsaliensis from blood and faeces of patients in different countries. FEMS Microbiol. Lett. 60:5–10. 9. Patton, C. M., I. K. Wachsmuth, G. M. Evins, J. A. Kiehlbauch, B. D. Plikaytis, N. Troup, L. Tompkins, and H. Lior. 1991. Evaluation of 10 methods to distinguish epidemic-associated Campylobacter strains. J. Clin. Microbiol. 29:680–688. 10. Shane, S. M. 1992. The significance of Campylobacter jejuni infection in poultry: a review. Avian Pathol. 21:189–213. 11. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354. 12. Wesley, I. V., and J. H. Bryner. 1989. Antigenic and restriction enzyme analysis of isolates of Campylobacter fetus subsp. venerealis recovered from presistently infected cattle. Am. J. Vet. Res. 50:807–813.
