During a survey of foal diarrhoea between 1991 and 1994,
Clostridium perfringens was
significantly associated with disease with 56% of cases infected [1].
The contribution of
enterotoxigenic C. perfringens to this association, was assessed
by
use of the reverse passive
latex agglutination test for enterotoxin (RPLA; Oxoid Unipath) and vero
cell toxicity
neutralized by antitoxin on stored faecal samples and sporulated faecal
isolates of C.
perfringens. Polymerase chain reaction (PCR1) based 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 published sequence.
Consequently, all faecal isolates were also tested by PCR1 and for a part
of the enterotoxin
gene (PCR2).Significant association 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 toxicity 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) or PCR1 (OR=4·81; CI
2·84–8·20; P<0·0001). Many of
the isolates from 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 neutralized by specific
antiserum. Failure
of the vero cell toxicity on faeces 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 equine faeces and to the
poor specificity of the commercial antiserum used in the test.Enterotoxigenic C. perfringens could not account for the
overall association of C. perfringens
with foal diarrhoea because (a) cultures positive by PCR, RPLA or cytotoxicity
were not
significantly more common amongst isolates from cases than controls; and
(b) the proportion
of isolates from cases positive by PCR (PCR1 or PCR2) was too small at
9·7%.
Author response: Selection of chromosomal DNA libraries using a multiplex CRISPR system
