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Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine
clostridial abomasitis and jejunal hemorrhage syndrome
- Benjamin Schlegel
Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the
environment and in the intestines of warm-blooded animals (1). A classification system for C.
perfringens strains divides them into types A through E based on toxin production, and there are
several important toxins that fall outside of the traditional typing system. C. perfringens type A
is defined as strains producing α toxin (cpa gene), type B as strains producing α, β (cpb gene),
and ε (cpe gene) toxins, type C as strains producing α and β toxins, type D as strains producing α
and ε toxins, and type E as strains producing α and ι (itx gene) toxins (1). Enterotoxin (cpe gene)
is associated with human food poisoning strains and domestic animal diarrheal strains and may
be produced by any C. perfringens type (1). β2 toxin (cpb2 gene) positive C. perfringens strains
are associated with the occurrence of enteric disease in domestic animals (11). Atypical β2 toxin
(atypical cpb2 gene) has 62-80% similarity to consensus β2 toxin and is far more common in
non-porcine animal isolates than consensus β2 toxin, although the atypical β2 toxin is often not
expressed (11). NetB and TpeL are recently discovered toxins that have been shown to be a part
of the pathogenesis of necrotic enteritis in broiler chickens (12,13).
In young calves, severe abomasitis associated with C. perfringens type A is
characterized by sudden onset of disease, abomasal tympany, abdominal pain, and hemorrhagic
diarrhea (2-4). Pathologically, the disease is characterized by acute emphysematous and
necrotizing hemorrhagic inflammation of the mucosa of the abomasum, with marked edema in
the lamina propria and submucosa (2-4). The disease may produce similar lesions in the rumen,
reticulum, and duodenum. In these cases, Gram-positive bacilli typical of C. perfringens are
often found on the mucosa and in the submucosa (3), and C. perfringens is invariably isolated.
The disease may be very acute; calves with a normal appearance may be found dead 12 hours
later with abomasal necrosis and bloat (4). To describe this disease the term bovine clostridial
abomasitis (BCA) will be used. Bovine clostridial abomasitis has been reproduced by
experimental infection with a C. perfringens BCA isolate in calves (5). This disease of calves
can be persistent in a herd, hard to diagnose, and hard to treat (4). Diagnosis of C. perfringens
type A enteritis is however complicated by the fact that C. perfringens type A is found in the
gastrointestinal tract of healthy animals. C. perfringens type A BCA disease is identified by the
presence of characteristic pathological and histopathological changes of hemorrhage and
necrosis, including gas production and the involvement of the abomasum, as well as the isolation
of C. perfringens in high numbers from the intestinal contents. Traditionally, the diagnosis of
enteric disease caused by C. perfringens is based on the detection of specific toxins in the
intestinal contents (6), but no specific toxin has been associated with BCA. Laboratory diagnosis
would be simpler if the infection was linked to a specific toxin (6) or other C. perfringens
“virulence signature” (7), but no such characteristic has been identified to date (8.9).
Jejunal hemorrhage syndrome (JHS) is characterized by mechanical obstruction of some
length of the jejunum with sloughed mucosa and clotted blood in mature cattle (9). Affected
animals have a mean age of 4 years (9,10); it occurs in both male and female beef and dairy
cattle, and the syndrome is associated with the winter season (9). C. perfringens type A has been
isolated from the intestinal contents of many affected cattle and may have a causative role in the
syndrome (9,10).
This study examined known or possible virulence-associated genes in C. perfringens type
A from cases of BCA and from cases of JHS, and compared these to isolates from calves which
were healthy or had undifferentiated diarrheal illness. A real time PCR assay was used to
genotype the 218 C. perfringens isolates examined. Isolates were sourced from healthy and
diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n =
22), and from mature cattle with JHS (n = 5). Two hundred and eight of 216 (96%) isolates were
positive for the cpa gene, and 13% (29/218) for atypical cpb2. Three of 8 (37.5%) confirmed
BCA isolates, 2 of 13 (15.4%) suspected CA isolates and no JHS isolates tested positive for
atypical cpb2. All isolates were negative for cpb, cpb2, cpe, etx, itx, netB, and tpeL, so that the
results of the present study do not support a role for these genes in BCA or JHS. A subset of
unique genes identified in one bovine clostridial abomasitis isolate (F262), for which a genome
sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes were
consistently present in all or even in a majority of BCA isolates. Many of these genes were also
variably and inconsistently present in type A isolates from calves that did not have BCA.
C. perfringens type A has been previously isolated from cases of BCA and JHS and
genotyped, however the present study looked at additional C. perfringens toxin genes such as
netB, tpeL, and atypical cpb2 that have not been examined before (2-4). Since all BCA and
suspected BCA isolates, and all JHS isolates, were negative for cpb, consensus cpb2, cpe, etx,
itx, netB, and tpeL genes, the present study does not support a role for these genes in BCA or
JHS of cattle. The data from PCR amplification failed to identify novel genes found in the F262
isolate sequence common to the BCA and JHS isolates examined. The unique virulence
characteristics, if any, of BCA and JHS isolates therefore remain to be determined.
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