JOURNAL OF CLINICAL MICROBIOLOGY, June 1989, p. 1272-1276 Vol. 27, No. 6 0095-1137/89/061272-05$02.00/0 Copyright C 1989, American Society for Microbiology Enterotoxin Production and Serogroups of Campylobacter jejuni and Campylobacter coli from Patients with Diarrhea and from Healthy Laying Hens GUN-BRITT LINDBLOM,* BERTIL KAIJSER, AND EVA SJOGREN Department of Clinical Bacteriology, Institute of Clinical Bacteriology, Clinical Immunology and Clinical Virology, University of Goteborg, S-413 46 Goteborg, Sweden Received 27 October 1988/Accepted 21 February 1989 Enterotoxin production, a possible virulence factor, was determined in Campylobacterjejuni and Campylo- bacter coli by two different techniques, the CHO cell test and the GM1 enzyme-linked immunosorbent assay. The frequency of enterotoxigenic Campylobacter strains was 32% in strains from both humans with acute enteritis and healthy laying hens, as measured by the CHO cell test. The CHO cell test was significantly more sensitive than the GM1 enzyme-linked immunosorbent assay in the detection of enterotoxigenic strains. Enterotoxin production was compared with the presence of heat-stable and heat-labile antigens. There was no significant correlation between enterotoxin production and serogroups for C. jejuni or C. coli. The difference in enterotoxigenicity between C. jejuni (34.1%) and C. coli (21.9%) was not significant. Campylobacter jejuni-C. coli is the third most common different assays enterotoxin production in strains from adult cause of diarrhea in children in developing countries, after patients with Campylobacter gastroenteritis and from enterotoxigenic Escherichia coli and rotavirus (1, 3, 27; P. healthy laying hens, with respect to the different serogroups DeMol and E. Bosmans, Letter, Lancet ii:604, 1978). In for each strain. adults in developed countries, C. jejuni is known to cause diarrhea as often as Salmonella, Shigella, and Yersinia MATERIALS AND METHODS species combined (5, 30, 33). Chickens or hens are known to be one of the most common sources of infection (4, 6, 11, 23, Bacterial strains. Included in the study were 202 Campylo- 28). bacter strains from the same number of patients; 84.2% were The mechanism(s) for induction of diarrhea caused by C. C. jejuni and 15.8% were C. coli. Differentiation between C. jejuni-C. coli is not known (2, 7, 8, 13, 29). C. jejuni-C. coli jejuni and C. coli strains was performed by hippurate hydro- is believed to have the capacity to invade the bloodstream (2, lysis, as described by Hwang and Ederer (14). The patients, 13). It has also been shown that 20 to 80% of the patients all of whom were more than 15 years of age, had sought have blood, mucus, and leukocytes in the stools, suggesting medical care for diarrhea at the Hospital for Infectious invasion of the epithelial cells as a virulence mechanism (2, Diseases, Gôteborg, Sweden, during a 12-month period. 8, 9, 29). Most cases of Campylobacter enteritis, however, Forty-four Campylobacter strains from as many healthy give secretory diarrhea, which suggests an involvement of laying hens were included in the study. The birds were 7 to enterotoxin. 65 weeks of age and came from a single breeder, but they Originally, no toxins from C. jejuni-C. coli were detected were housed and raised at two different locations (21). A (8, 9; T. Wadstrom, S. B. Baloda, K. Krovacek, A. Fairs, S. total of 28 strains (26 C. jejuni and 2 C. coli strains) came Bengtsson, and M. Walder, Letter, Lancet ii:911, 1983), but from hens raised at a chicken farm, and 16 strains (all C. in 1983 Ruiz-Palacios and colleagues (26) reported that 24 of jejuni) came from hens housed in the institute facility. This 32 strains isolated from children with acute secretory diar- latter group was housed under hygienic conditions estab- rhea produced a heat-labile (HL) enterotoxin, while only 1 of lished for our animal facility. 6 strains cultured from symptom-free children were entero- All the strains were lyophilized before use. toxigenic. The presence of an enterotoxin has been con- Enterotoxin production. The lyophilized bacteria were firmed by other researchers (10, 15, 18-20; V. J. Mathan, grown for 24 h on blood agar plates in a microaerobic D. P. Rajan, F. A. Klipstein, and R. F. Engert, Letter, atmosphere (5% 02, 10% C02, 85% N2) at 420C. Lancet ii:981, 1984; B. A. McCardell, J. M. Madden, and A loopful of bacteria was inoculated into 25 ml of GC E. C. Lee, Letter, Lancet i:448-449, 1984). medium broth (0289; Difco Laboratories, Detroit, Mich.) The relationship of serogroups to clinical symptoms has plus 0.1% IsoVitaleX (BBL Microbiology Systems, Cock- been investigated by Kaijser and Sjogren (16). There is no eysville, Md.). evidence that a specific serogroup surface antigen specifi- The cultures were incubated at 42°C in a microaerobic cally contributes to virulence. The relation between sero- atmosphere for 24 h and were then centrifugated at 20,000 x groups and enterotoxin production has, however, not yet g for 10 min at 4°C. The supernatant was tested for entero- been reported. toxin production within 0 to 2 days and stored at 40C. The The aim of the present investigation was to analyze by two titers of the positive supernatant varied between 1/1 and 1/4. Enterotoxin assays. (i) CHO assay. CHO cells were used for the detection of the enterotoxin (12, 20, 26). CHO-Ki * Corresponding author. cells were obtained from the American Type Culture Collec- 1272 VOL. 27, 1989 C. JEJUNI AND C. COLI ENTEROTOXIN AND SEROGROUPS 1273 tion (Rockville, Md.). Cell monolayers were grown in F12 were regarded as positive (Campylobacter enterotoxin con- medium (GIBCO Laboratories, Grand Island, N.Y.) supple- taining) when an A405 of .0.2 above the background value mented with 8% fetal bovine serum in 5% C02 at 37°C and was obtained. were passaged by trypsinization. The medium used for the Serogrouping assays. (i) HS antigen. Heat-stable (HS) enterotoxin assay was Eagle minimum essential medium antigen was identified by using the indirect hemagglutination supplemented with 1% fetal bovine serum. In both media, technique, with a heated supernatant from the bacteria used gentamicin (10 ,ug/ml; Sigma Chemical Co., St. Louis, Mo.) as the antigen (24, 25). The hyperimmune antisera were was used. The cell suspension (103 cells per ml) was sus- produced by immunization of rabbits with whole, live bac- pended in 96-well microtiter plates (Nunc, Roskilde, Den- teria (24). Typing was performed for HS antigens 1 to 23 and mark) at 200 ,uI per well. The cells were kept in the wells for 37, excluding antigens 7 and 12. The HS antisera used have 30 min in 5% C02 at 37°C to allow adherence to the plate previously been shown to have a 75% typability of C. before adding the samples. All samples were tested in jejuni-C. coli strains from Swedish patients (16) and are duplicate with working volumes of 100 ,ul per well and antigens that are common in Canada (24). incubated in 5% C02 at 37°C (20). The assay was registered (ii) HL antigen. HL antigen was identified by the direct by using an inverted microscope and was examined indepen- slide agglutination technique with whole, live bacteria (22). dently by two people after 24, 48, and 72 h. The results were The hyperimmune antisera used were prepared by immuniz- based on the registration at 48 h. The observations at 48 and ing rabbits initially with Formalin-killed bacteria and later 72 h were similar, while that at 24 h gave a somewhat lower with whole, live bacteria (22). Serogrouping was performed positive result. One hundred cells were counted per well. for HL antigens 1 to 22 and 36, excluding antigens 3, 14, and Elongation of 50% or more of the cells in a well was 15. The HL antisera used earlier gave a 90% typability in a considered as a positive response, e.g., the presence of an Swedish study (16) and are the antisera that correspond to enterotoxin. Ail supernatants were retested within 1 week. the most common antigens in Canada (22). We used purified cholera toxin (List Biological Laboratories, The nontypable strains in our study were defined as strains Inc., Campbell, Calif.) as a positive control and GC medium that were not typable with the HS or HL antisera used in this as a negative control. The background elongation of cells study. was never more than 5 to 10%. The definition of an enterotoxigenic strain in this study is (ûi) ELISA GM,. Microtiter enzyme-linked immunosor- a strain that was positive in the CHO cell test. bent assay (ELISA) plates (Dynatech Laboratories, Sussex, Statistical calculations. Differences of probabilities were England) were coated with ganglioside GM1 (Supelco Inc., tested by using the chi-square test with the Yates correction. Supelco Park, Bellefonte, Pa.; or Seromed, Munich, Federal Republic of Germany) by incubating the plates with 0.1 ml of 1.5 FM GM1 diluted in phosphate-buffered saline (PBS; 0.05 RESULTS M phosphate, 0.15 M NaCI [pH 7.2]) at 20°C overnight (32). The coated plates could be kept at 4°C for up to 3 weeks Humans with acute diarrhea. Of 202 strains from humans before use. with acute diarrhea tested (84.2% C. jejuni and 15.8% C. The plates were washed twice in PBS, and the remaining coli), 31.2% were found in patients infected in Sweden and binding sites were blocked by incubating the plates with 1% 68.8% were found in patients infected abroad. There was no bovine serum albumin-PBS solution at 37°C for 30 min (200 significant difference between the two species regarding the ,ul per well).