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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 jejuni and from Patients with 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 with acute 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 and from enterotoxigenic 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 , Shigella, and Yersinia MATERIALS AND METHODS species combined (5, 30, 33). or hens are known to be one of the most common sources of (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 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 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 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 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). After the plates were rewashed three times in place of infection. In the CHO cell test 32.2% were positive, PBS-0.05% Tween 20 (PBS-Tween), 100 ,ul of the undiluted and in the GM1 ELISA 19.3% were positive (X2 = 7.8; P < Campylobacter supernatant per well was added in duplicate. 0.01). There was no significant difference regarding the The plates were incubated in a moist chamber at 20°C for 2 frequency of enterotoxin production in C. jejuni or C. coli h and rewashed three times in PBS-Tween 20. Crude rabbit strains from patients infected in Sweden or abroad, as anti-E. coli heat-labile enterotoxin was used as antiserum evaluated by the CHO cell test or the GM1 ELISA. (kindly provided by A. M. Svennerholm, Goteborg, Swe- In 140 of the 202 strains (69.3%), there were identical den) and was diluted 1/500 in PBS-Tween-0. 1% bovine results for the CHO cell test and the GM1 ELISA. A total of serum albumin. One hundred microliters was added to each 21 strains were positive (10.4%) and 119 strains were nega- well. The reaction time for antiserum was 1 h in a moist tive (58.9%). chamber, at 20°C; thereafter, the well was washed three HS antigen 1 was the most common HS antigen, followed times with PBS-Tween. A 1:1,000 dilution (PBS-Tween and by HS antigens 2, 4, 8, 13, and 18; but in no case was there 0.1% bovine serum albumin) of alkaline phosphatase-conju- a significant difference regarding enterotoxigenic and nonen- gated goat anti-rabbit immunoglobulin G antiserum (no. terotoxigenic strains for C. jejuni strains or C. coli strains A-8025; Sigma) was added at 100 ,ul per well and was kept for (Table 1). 2 h in a moist chamber at 20°C. Washing was performed as Of the HL antigens 9, 2, 6, and 17 dominated; HL antigens described above, and 200 ,ul of the substrate alkaline phos- 9 and 17 occurred relatively more frequently among the phatase (stock no. 104-105; Sigma), at 1 mg/ml in diethanol- enterotoxigenic strains, and antigens 2 and 6 occurred rela- amine buffer, was added to each well and incubated at room tively more frequently among the nonenterotoxigenic ones temperature for 100 min. As a positive control, we used (Table 2). crude E. coli heat-labile enterotoxin diluted 1:200 to 1:3,200 Healthy laying hens. Of the 28 Campylobacter strains in GC medium (kindly provided by A. M. Svennerholm, tested (26 C. jejuni and 2 C. coli) from hens bred at a chicken Goteborg, Sweden). GC medium was used as a negative farm, 32.1% were positive by the CHO cell test and 14.1% control (32). were positive by the GM1 ELISA. Of these strains, 75.0% The enzyme substrate reaction in the GM1 ELISA was gave the same results in the two tests, 10.7% were positive in recorded with a spectrophotometer (Titertek Multiscan; both tests, and 64.3% were negative in both tests. Flow Laboratories Ef-lab., Helsinki, Finland) at 405 nm. Ail As observed for isolates, HS antigen 1 occurred supernatants were tested twice within 1 week. The samples most frequently among the strains from hens (n = 12) and 1274 LINDBLOM ET AL. J. CLIN. MICROBIOL.

TABLE 1. Enterotoxigenic and nonenterotoxigenic C. jejuni and TABLE 3. Enterotoxigenic and nonenterotoxigenic C. jejuni and C. coli strains from humans with acute diarrhea in relation to C. coli strains from healthy laying hens in relation to the the presence of HS antigens presence of HS and HL antigens Enterotoxigenic Nonenterotoxigenic Total Enterotoxigenic Nonenterotoxigenic Total HS antigen Antigen n % n % n % n % n % n % 1 13 (1) 20.0 18 (3) 13.1 31 (4) 15.3 HS antigen 2 5 (1) 7.7 il (1) 8.0 16 (2) 7.9 1 5 55.6 7 36.8 12 42.8 3 2 3.1 5 (1) 3.6 7 (1) 3.5 2 0 1 5.3 1 3.6 4 5 7.7 8 (1) 5.8 13 (1) 6.4 4 2 22.2 1 5.3 3 10.7 5 3 (1) 4.6 7 (1) 5.1 10 (2) 4.9 5 0 1 5.3 1 3.6 6 1 1.5 6 4.4 7 3.5 8 0 1 5.3 1 3.6 8 4 (1) 6.2 7 (2) 5.1 11 (3) 5.5 13 1 11.1 3 15.8 4 14.3 9 1 1.5 0 1 0.5 14 0 1 5.3 1 3.6 10 1 1.5 2(1) 1.5 3(1) 1.5 18 0 1 5.3 1 3.6 il 0 2(1) 1.5 2(1) 1.0 21 0 1 (1) 5.3 1 3.6 13 3 4.6 8 (3) 5.8 11 (3) 5.5 23 0 1 5.3 1 3.6 14 0 5(1) 3.6 5(1) 2.5 NT' 1 11.1 1 (1) 5.3 2 7.1 15 2 3.1 1 0.7 3 1.5 Total n 9 19 (2) 28 16 2 3.1 7 5.1 9 4.5 Total % 32.1 67.9 100.0 17 0 1 0.7 1 0.5 18 3 (1) 4.6 8 5.8 il (1) 5.5 HL antigen 23 0 1 0.7 1 0.5 2 0 1 5.3 1 3.6 37 3 (1) 4.6 6 (2) 4.4 9 (3) 4.5 4 0 6 31.6 6 21.4 NTb 17 (1) 26.2 34 (8) 24.8 51 (9) 25.2 10 0 1 5.3 1 3.6 Total no. 65 (7) 137 (25) 202 (32) 18 2 22.2 1 5.3 3 10.7 Total % 32.2 67.8 100.0 21 4 44.4 6 31.6 10 35.7 36 2 22.2 0 2 7.1 a A total of 170 C. jejuni and 32 C. coli strains from 202 humans with acute NT 1 11.1 4 (2) 1.1 5 17.9 diarrhea were tested. The number of C. coli strains is given in parentheses. Total n 9 19 (2) 28 b NT, Nontypable with our antisera. Total % 32.1 67.9 100.0 " A total of 26 C. jejuni and 2 C. coli strains from 28 healthy laying hens. was relatively more common among the enterotoxigenic The number of C. coli strains is given in parentheses. strains (Table 3). b NT, Nontypable with our antisera. Of the HL antigens, antigens 21, 4, and 18 dominated. HL antigens 18 and 21 occurred relatively more often among the enterotoxigenic strains and HL antigen 4 occurred relatively serotype HS2:HL4. Of these, 14 (87.5%) gave the same more often among the nonenterotoxigenic strains (Table 3). results in the two toxin tests, 8 (50%) were positive, and 6 The 16 isolates from hens (all C. jejuni) kept at the animal (37.5%) were negative. Of the two remaining strains, one facilities associated with our laboratory all belonged to was positive in the CHO cell test and negative in the GM1 ELISA and the other strain gave the opposite results. TABLE 2. Enterotoxigenic and nonenterotoxigenic C. jejuni and C. coli strains from humans with acute diarrhea in relation to DISCUSSION the presence of HL antigens The pathogenicity of C. jejuni has been discussed for Enterotoxigenic Nonenterotoxigenic Total the as HL antigen several years. Invasion into bloodstream, shown, for n % n % n % example, by positive blood cultures, may represent an important virulence function (20). However, a more Shigel- 1 3 4.6 8 (2) 5.8 11 (2) 5.5 la-like invasion of the epithelial cells can be indicated by the 2 9 13.8 27 (7) 19.7 36 (7) 17.8 of and in feces 4 5 7.7 4 2.9 9 4.5 presence blood, mucus, leukocytes (2, 8, 9, 5 1 1.5 3 (1) 2.2 4(1) 2.0 26). The existence of a possible enterotoxigenic toxin has 6 2 3.1 14 (1) 10.2 16 (1) 7.9 been described for several years, but in 1983 it was shown 7 4 6.2 6 (1) 4.4 10 (1) 4.9 that C. jejuni can produce an HL enterotoxin (26). The C. 8 1 (1) 1.5 7 (4) 5.1 8 (5) 4.0 jejuni enterotoxin has been shown to share functional prop- 9 17 (1) 26.2 20 (3) 14.6 37 (4) 18.3 erties and have partial immunological homology with cholera il 0 2 1.5 2 1.0 toxin and E. coli HL enterotoxin (18-20, 26). 12 2 (1) 3.1 4 (2) 2.9 6 (3) 3.0 The significance of the Campylobacter enterotoxin for 13 2 (1) 3.1 3 (1) 2.2 5 (2) 2.5 induction of diarrheal states is not obvious at this time. The 16 2 (1) 3.1 1 0.7 3 (1) 1.5 of strains differs in studies 17 6 9.2 7 5.1 13 6.4 frequency enterotoxin-producing 19 2 (1) 3.1 2 1.5 4(1) 2.0 from different parts of the world where the CHO cell test has 20 0 2 1.5 2 1.0 been used. In Belgium, close to 100% of strains were found 21 0 2(1) 1.5 2 (1) 1.0 to be positive (10), whereas in Mexico this value was NTb 9 (1) 13.9 25 (2) 18.3 34 (3) 16.8 approximately 70% (26). In both studies strains from chil- Total n 65 (7) 137 (25) 202 (32) dren with acute enteritis were used. Mathan et al. (Mathan et Total % 32.2 67.8 100.0 al., Letter, Lancet ii:981, 1984) have reported that an HL was in about 30% of strains from Indian ' A total of 170 C. jejuni and 32 C. coli strains from 202 humans with acute enterotoxin present diarrhea were tested. The number of C. coli strains is given in parentheses. children with acute enteritis, but they also found, in contrast b NT, Nontypable with our antisera. to the Mexican study, the same values for symptom-free VOL. 27, 1989 C. JEJUNI AND C. COLI ENTEROTOXIN AND SEROGROUPS 1275 carriers. In our studies, 32% of the Campylobacter strains tion against enterotoxin (20, 26), we propose that entero- from both patients with acute enteritis and healthy laying toxin production can be an important factor for the induction hens were enterotoxigenic. There were no significant differ- of Campylobacter diarrhea. However, we believe that other ences regarding the enterotoxigenicity between C. jejuni and characteristics may additionally contribute to the pathoge- C. coli strains. nicity of the bacteria, for instance, the ability of campylo- The differences in the frequency of enterotoxigenicity bacters to produce cytotoxin and to adhere to and invade the reported from other parts of the world might be a result of intestinal mucosa. technical variation as well as different patient materials. In India and Mexico, for example, symptomless carriers ap- ACKNOWLEDGMENTS peared. This is very uncommon in Sweden, where almost all humans colonized with C. jejuni have diarrhea (31, 33). This investigation was supported by grant 07169 from the Swedish The most common method for showing the production of Medical Research Council and grant 598 from the Swedish Council Campylobacter HL enterotoxin is the CHO test. The entero- for Forestry and Agricultural Research. toxin raises intracellular We thank G. Ruiz-Palacios and his staff at the Institute Nacional the cyclic AMP level, which causes de la Nutricion, Mexico D.F., Mexico, for personal advice and help cytotonic changes in the CHO cells (12, 18). In the present with the CHO cell technique. We also thank S. Jeanson, lab investigation we adopted the CHO cell test for enterotoxin technicians Krystyna Pawlowska and Gerd Eriksson for providing testing. Some investigators also use the rat ileal loop test. A the CHO cells, and Anne-Bell Ek for the skillful typing of the high similarity between the rat ileal loop and the CHO cell manuscript. tests has been found in comparative studies (20, 26). Fur- thermore, some researchers find a higher rate of positive LITERATURE CITED strains with the CHO cell test compared with that found with 1. Black, R. E., M. H. Merson, M. I. Huq, A. R. M. A. Alim, and the GM1 ELISA, possibly because of a higher sensitivity of M. D. Yunus. 1981. Incidence and severity of rotavirus and the former test (26). Escherichia coli diarrhoeae in rural Bangladesh. Implications Like other gram-negative rods, Campylobacter strains for vaccine development. Lancet i:141-143. have some marked surface antigens which can be used for 2. Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B. Relier, and W.-L. L. Wang. 1979. Campylobacter enteritis: serogrouping. The HS antigen ad modum Penner and col- clinical and epidemiological features. Ann. Intern. Med. 91: leagues (24, 25) and the HL antigen ad modum Lior et al. (22) 179-185. are the most generally accepted. In earlier studies, we have 3. Blaser, M. J., R. I. Glass, M. I. Huq, B. Stoll, G. M. Kibriya, shown that there is a great variability of serogroups among and A. R. M. A. Alim. 1980. Isolation of human strains (16). In the present investigation, there was no subsp. jejuni from Bangladeshi children. J. Clin. Microbiol. correlation between enterotoxin production and serogroup 12:744-747. pattern, which indicates that serogrouping is an inadequate 4. Blaser, M. J., F. M. LaForce, N. A. Wilson, and W.-L. L. Wang. method for detecting enterotoxin-positive Campylobacter 1980. Reservoirs for human campylobacteriosis. J. Infect. Dis. strains. This is in agreement with findings from other gram- 141:665-669. 5. Blaser, M. J., D. N. Taylor, and R. A. Feidman. 1983. Epidemi- negative rods that produce enterotoxin, except for E. coli ology of . Epidemiol. Rev. 06:K15:H16 and 01, which are regularly 5:157-176. enterotoxin producing (17). 6. Brouwer, R., M. J. A. Mertens, T. H. Siem, and J. Katchshi. Chickens and laying hens are generally colonized with 1979. An explosive outbreak of Campylobacter enteritis in Campylobacter species (21). Consequently, they are a very soldiers. Antonie van Leeuwenhoek J. Microbiol. Serol. 45: common source of human infection worldwide (4, 6, 11, 23, 517-519. 28). The birds do not seem to be affected by the presence of 7. Butzler, J. P., P. Dekeyser, M. Detrani, and F. Dehaen. 1973. Campylobacter strains in the , regard- Related Vibrio in stools. J. Pediatr. 82:493-495. less of whether the bacteria are capable of enterotoxin 8. Butzler, J. P., and M. B. Skirrow. 1979. Campylobacter enteri- production. In tis. Clin. Gastroenterol. 8:737-765. the present investigation, we found a similar 9. Drake, A. A., M. J. R. Gilchrist, J. A. Washington, K. A. frequency of Campylobacter enterotoxin producers in chick- Huizenga, and R. E. Van Scoy. 1981. Diarrhea due to Campylo- ens and humans. Furthermore, the serogroups from hens bacter fetus subspecies jejuni. A clinical review of 63 cases. from the chicken farm, in general, corresponded to those Mayo Clin. Proc. 56:414-423. found in humans. However, HL antigens 18 and 21 were 10. Goosens, H., J.-P. Butzler, and Y. Takeda. 1985. Demonstration rarely found in human strains; and HL antigen 9, a common ofcholera-like enterotoxin production by Campylobacterjejuni. human serotype, was not found among hen strains. From the FEMS Microbiol. Lett. 29:73-76. hens kept in our laboratory facility, one serogroup (HS2: 11. Grant, I. H., N. J. Richardson, and V. D. Bokkenheuser. 1980. HL4) was isolated repeatedly. Isolates of this serogroup chickens as potential source of Campylobacter infec- probably tions in humans. J. Clin. Microbiol. 11:508-511. represent one strain that colonized all the hens. 12. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L. I. The results from the two enterotoxin tests of these strains Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophos- showed a high correlation. The results indicate, however, phate and alteration of Chinese hamster ovary cell morphology: that epidemiologically related isolates might give differences a rapid, sensitive in vitro assay for the enterotoxins of Vibrio in enterotoxin production. These data illustrate some of the cholerae and Escherichia coli. Infect. Immun. 10:320-327. difficulties in postulating a role for enterotoxin production in 13. Guerrant, R. L., R. G. Lahita, W. C. Weim, and R. B. Roberts. Campylobacter pathogenesis. Whether the different results 1978. Campylobacteriosis in man: pathogenic mechanisms and are caused by loss of the ability of some isolates to produce review of 91 . Am. J. Med. 65:584-592. enterotoxin after laboratory maintenance or technical differ- 14. Hwang, M.-N., and G. M. Ederer. 1975. Rapid hippurate hy- drolysis. Method for presumptive identification of group B ences regarding the assay or whether this reflects the natural streptococci. J. Clin. Microbiol. 1:114-115. occurrence of enterotoxigenic Campylobacter strains remain 15. Johnson, W. M., and H. Lior. 1986. Cytotoxic and cytotonic to be further investigated. factors produced by Campylobacterjejuni, Campylobacter coli In conclusion, based on other reports of enterotoxin and Campylobacter laridis. J. Clin. Microbiol. 24:275-281. production, clinical signs of infection, and antibody produc- 16. Kaijser, B., and E. Sjogren. 1985. Campylobacter strains in 1276 LINDBLOM ET AL. J. CLIN. MICROBIOL.

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