THE CLOSTRIDIUM-PERFRINGENS ENTEROTOXIN FROM EQUINE ISOLATES - ITS CHARACTERIZATION, SEQUENCE AND ROLE IN FOAL DIARRHEA

During a survey of foal diarrhoea between 1991 and 1994, Clostridium p erfringens was significantly associated with disease with 56% of cases infected [1]. The contribution of enterotoxigenic C. perfringens to t his association, was assessed by use of the reverse passive latex aggl utination test for enterotoxin (RPLA; Oxoid Unipath) and vero cell tox icity neutralized by antitoxin on stored faecal samples and sporulated faecal isolates of C. perfringens. Polymerase chain reaction (PCR1) b ased on the DNA sequence for the whole enterotoxin gene [2] yielded a fragment from an equine isolate of the anticipated size which, cloned into plasmid M13 phage, had a sequence essentially identical to the pu blished sequence. Consequently, all faecal isolates were also tested b y PCR1 and for a part of the enterotoxin gene (PCR2). Significant asso ciation with diarrhoea (controls not in contact with cases) was found with positive RPLA tests on faeces (OR = 13, P = 0.002) and isolates ( OR = 4.57, P = < 0.0001), vero cell toxicity of isolates (OR = 1.78, P = 0.026), and PCR1 (OR = nd, P = 0.029) but not PCR2 or vero cell tox icity of faeces. Significant association with diarrhoea was also found for isolates negative by RPLA (OR = 3.91; CI 2.05-7.57; P < 0.0001) o r PCR1 (OR = 4.81; CI 2.84-8.20; P < 0.0001). Many of the isolates fro m RPLA positive faeces and verotoxic isolates were PCR negative and no evidence could be found for the presence of the enterotoxin gene in a random selection of RPLA positive/PCR negative isolates by gene probe on chromosomal DNA and PCR reaction product or vero cell toxicity neu tralized by specific antiserum. Failure of the vero cell toxicity on f aeces to be associated with diarrhoea or for cytotoxicity of cultures and RPLA on cultures to agree with the PCRs was believed to be related to the presence of other cytotoxins, the inherent cytotoxicity of equ ine faeces and to the poor specificity of the commercial antiserum use d in the test. Enterotoxigenic C. perfringens could not account for th e overall association of C. perfringens with foal diarrhoea because (a ) cultures positive by PCR, RPLA or cytotoxicity were not significantl y more common amongst isolates from cases than controls; and (b) the p roportion of isolates from cases positive by PCR (PCR1 or PCR2) was to o small at 9.7%.