FEMS Microbiology Letters 36 (1986) 53-55 53 Published by Elsevier

FEM 02499

Serum antibodies reacting with Escherichia coli heat-labile enterotoxin in sera of patients

suffering from Campylobacterjejuni infection

(Campylobacterjejuni; Escherichia coli; serum antibody; heat-labile enterotoxin)

Takeshi Honda a, Toshio Miwatani a, Shin-ichi Mizuno b and Sunao Maki b

a Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka 565, and b Department of Pediatrics, Kinki University School of Medicine, Sayamacho, Minamikawachigun, Osaka 589, Japan

1. SUMMARY

Using an enzyme-linked immunosorbent assay (ELISA) with purified Escherichia coli heat-labile enterotoxin (LT), an increase in serum antibody against LT was demonstrated in 49% of patients infected with Campylobacterjejuni tested. The an- tibody titers, however, were not as high as those in patients with cholera. This finding suggests that some strains of C. jejuni in patients with diseases due to C. jejuni produce a toxin (or substance) immunologically related to LT in the intestine.

that this toxin may be involved in this type of diarrhea. However, the precise role of the toxin in the pathophysiology of C. jejuni-induced diarrhea is unknown. This is partly because antibodies against CJ-T in the sera of patients with C. jejuni have not been found until recently [13]. In this study we investigated these antibodies in the sera of C. jejuni patients using an enzyme-linked im- munosorbent assay (ELISA).

3. MATERIALS AND METHODS

2. INTRODUCTION

C. jejuni is now recognized to be a major cause of acute diarrhea in both developing and devel- oped countries [1-4]. The mechanisms involved in the pathogenesis of C. jejuni diarrhea, however, are not yet clear. The invasiveness of C. jejuni may be the primary reason for the bloody diarrhea [3,4], but although C. jejuni is known to cause bloody diarrhea [4], the illness is more frequently associated with watery diarrhea. Recent studies [5-12] have shown that some strains of C. jejuni produce a heat-labile enterotoxin (C J-T) that is immunologieally and biologically similar to cholera toxin (CT) and E. coli heat-labile toxin (LT), and

3.1. Serum specimens Serum specimens were obtained from the fol-

lowing groups: Group I, control children, 1.5-12 yr old (n = 15) with enteritis not due to C. jejuni; Group II, patients (n = 35, 2-13 yr old) with C. jejuni enteritis diagnosed by isolation of the organism from their stools and by clinical signs and symptoms; and Group III, cholera patients (n = 15, adults of 27-54 yr-old). All sera were obtained 2-8 days after the onset of disease.

3.2. EL IS A ELISA was performed as follows. Purified LT

[14], CT (Sanko Junyaku Co. Tokyo) or bovine serum albumin (BSA) was diluted to 5 ~g/ml with 0.05 M carbonate buffer (pH 9.6), and 150-/~1

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

54

volumes were distributed into the wells of a fiat- bottomed vinyl microtiter plate (Falcon Micro test III, Becton Dickinson, CA, U.S.A.) for coating as described [15,16]. A volume of 200 #1/well of bovine serum albumin (1 m g / m l of phosphate- buffered saline) was used to cover the unbound surfaces of the wells and to reduce non-specific reactions. Test sera were diluted to 1 :500 with PBS-Tween 20 [15,16] and 120-#1 volumes of di- luted sera were introduced into the wells, and the plates were incubated for 1 h at 37 o C. The plates were washed 4 times with PBS-Tween 20. Then 120 #1 of alkaline phosphatase-conjugated anti- body against human immunoglobulin G (Sigma) at 1 : 2000 dilution was added, and the plates were incubated for 1 h at 37°C. The plates were then washed 4 times with PBS-Tween 20, and 0.2 ml of p-nitrophenyl phosphate substrate in diethanola- mine buffer containing 1 mM MgC12 was added [15,16]. The color developed was measured at 405 nm as described [15,16]. The ratio of the reading with CT or LT coating to that with BSA was calculated as the ELISA ratio.

4. RESULTS AND DISCUSSION

To check the ELISA system used, we first examined Group III sera (cholera patients) using both CT and LT as coating antigens, and found that LT-coating gave much higher and clearer reactions than CT-coating (Fig. 1). The reason for this is not clear, but during disease epitopes com- mon to both CT and LT [14] are recognized much better by the human immune system than epitopes specific to CT, and the former epitopes are ex- pressed more by LT molecules on the surface of microplates. Thus we performed ELISA using LT as a coating antigen for analyses of sera in Group II (patients with C. jejuni-enteritis). As shown in Fig. 2, the mean ELISA ratio in Group II was 2.5 + 1.57. All ELISA values of sera in Group I were below 2.0 (mean value, 1.45 + 0.26), but 17 of 35 cases of C. jejuni gave a value of more than 2.00 (mean, 3.89 + 0.39; significantly, P < 0.05, higher than that for Group I). These results sug- gest that 49% (17 of 35) of the cases tested devel- oped antibody-reactive LT during C. jejuni infec- tion.

|

o

Is ~ 15 •

> i0 <> i0 •

A

8

I I I I I I I I

Fig. 1. Ant ibody titers of Group I and IlI sera by ELISA with CT or LT as coating antigens. ELISA value was expressed as the ratio of the reading with CT- or LT-coating to that with BSA. Group III, sera of cholera patients; Group I, sera of control children with enteritis not due to C. jejuni.

The value for Group II sera (mean, 2.50 + 1.57, mean for positive cases, 3.89 + 0.39) was, however, rather lower than that for Group III (cholera patients; mean, 11.83-t-8.38, mean for positive cases, 13.44 + 7.84). The reason for this is not dear , but possible reasons are as follows: (i) The immunological response of children to C. jejuni toxin is not as strong as that of adults to cholera toxin; (ii) C. jejuni produces less toxin than Vibrio cholerae; (iii) The positive response observed in C. jejuni patients was non-specific. The fact that all sera in Group I had a negative ELISA value suggests that the last possibility is unlikely.

Our observations essentially confirm the recent finding of Ruiz-Palacios et al. [13] obtained with a different ELISA system. We used purified LT [14]

4

<

< 2

|

i

GROUP II GROUP I

Fig. 2. Antibody titers of Group I and II sera in ELISA with LT as a coating antigen. Group II, sera of patients with C. jejuni enteritis; Group I, sera of control children with enteritis not due to C. jejuni.

as a coating antigen, whereas they used ganglio- side as a primary coating substance and let crude LT bind to it. They reported seroconversion in all 10 children with enterotoxigenic C. jejuni but in only 1 of 9 children with non-enterotoxigenic C. jejuni. We could not demonstrate production of CJ-T in cultures of our isolates from patients by either ELISA [15] or CHO cell assay (data not shown) [17], so we could not analyse our data in a similar way. The antibody level was elevated in 49% of all our C. jejuni patients, a value which is comparable to that reported by Ruiz-Palacios et al. [13]. However, considering a recent report sug- gesting that all clinical isolates of C. jejuni pro- duce CJ-T when grown in an improved medium

55

[12], the number of cases with antibody seems lower than expected. Further studies are required on this problem.

Present study indicates that at least 49~ of the strains of C jejuni produced a toxin or a sub- stance immunologically related to LT in the hu- man intestine during disease. This appears to be important suggestive evidence that CJ-T, as re- ported by others [5-12], plays a role in inducing diarrhea in human C. jejuni infection similar to the diarrheas induced by LT and CT of enter- otoxigenic E. coli and V. cholerae [18].

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