PCR analysis of the reference strains for the different toxin types of C. perfringens
showed the presence of the α-toxin gene cpa
in all C. perfringens
reference strains as well as in our β2-toxigenic internal reference strain C. perfringens
E 482/97 and in strain S 1040/95. The
-toxin gene etx
was found in the reference strains of type B (ATCC 3626) and type D (NCTC 8346). The β-toxin gene cpb
was found in the reference strain of type B (ATCC 3626). The enterotoxin gene cpe
was found in enterotoxigenic type A strain NCTC 10239. The β2-toxin gene cpb2
was detected by PCR in our β2-toxigenic internal reference strain C. perfringens
E 482/97 and in strain S 1040/95. PCR analysis of C. difficile
ATCC 43255 showed the presence of the two genes for toxins A and B. These results thus confirm the specificity of the analytical methods.
The results of bacteriological and PCR examinations are given in Tables and . C. perfringens
was isolated from 5 of 21 (24%) fecal samples from 21 horses with intestinal diseases. All these isolates proved to harbor the β2-toxin gene cpb2
and the α-toxin gene cpa
(Table ). Feces from 4 of the 21 (19%) horses contained C. difficile
, and 12 (57%) further horses were negative for both C. perfringens
and C. difficile
. All isolated C. perfringens
strains were devoid of the β- and
-toxin and enterotoxin genes, as assessed by PCR. Examination of feces from the 53 control horses without intestinal disease showed that they contained no β2-toxigenic C. perfringens
isolates and no C. difficile
isolates. A C. perfringens
type A strain without an enterotoxin gene was isolated from one of these control animals.
TABLE 2 Bacteriological results for equine fecal samples, samples of intestinal ingesta, or biopsy specimens of the intestinal wall (small intestine and largecolon)
TABLE 3 Results of bacteriological examinations of all equinespecimens
The results of bacteriological examinations of specimens of intestinal ingesta or biopsy specimens of the intestinal wall (small and large intestine) revealed that of 20 specimens from 20 different horses with intestinal disease, 14 specimens proved to be positive for β2-toxigenic C. perfringens (Table ). One horse with typical typhlocolitis which was positive for β2-toxigenic C. perfringens also contained nontoxigenic C. difficile. Both β2-toxigenic C. perfringens and toxigenic C. difficile (containing the genes for toxins A and B) were isolated from one horse with atypical typhlocolitis, and one horse was negative for C. perfringens and C. difficile. C. difficile alone was isolated from two of eight horses with intestinal diseases other than typhlocolitis. All specimens of ingesta from the five horses from the control group (group IV) were bacteriologically negative for C. perfringens and C. difficile.
In total, β2-toxigenic C. perfringens
was isolated from samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis (Table ). Samples from two horses from this group contained C. perfringens
type A and six contained C. difficile
; of the latter, two were found to contain C. difficile
in combination with β2-toxigenic C. perfringens
. In horses with other intestinal diseases, the incidence of β2-toxigenic C. perfringens
was 6 of 16 (37%) animals. C. perfringens
type A was isolated from the specimen from one animal in this group, and C. difficile
was isolated from two samples. β2-Toxigenic C. perfringens
was not isolated from any of the 58 horses in the control group, and C. perfringens
type A was found only once. All C. perfringens
strains analyzed were devoid of the β- and
-toxin and enterotoxin genes.
Among the animals in group I (typical typhlocolitis), 8 of 18 horses died or were subjected to euthanasia due to severe shock or laminitis. Of these, 4 were positive for β2-toxigenic C. perfringens and had been medicated with an NSAID and antibiotics (gentamicin and penicillin). All horses in group II (atypical typhlocolitis) died or were euthanized (n = 7); five of them were positive for β2-toxigenic C. perfringens. Four of these five animals were treated with an NSAID and antibiotics.
Taken together, from a total of 25 horses with typical and atypical typhlocolitis, 14 were treated with antibiotics and 9 harbored β2-toxigenic C. perfringens. Of these animals all eight horses which had received antibiotic treatment and yielded β2-toxigenic C. perfringens died. Among the animals in group III (n = 16), 10 horses were euthanized, and four of these were positive for β2-toxigenic C. perfringens and received antibiotic and anti-inflammatory therapy (gentamicin, penicillin, and NSAID).