Type B and C diseases originate in the intestines but often later involve sudden death or acute neurological signs resulting from enterotoxemia (absorption of toxins from the intestines into the circulation) (10
). In nearly all livestock and humans the intestinal effects of C. perfringens
type C isolates involve necrotizing enteritis, which is clinically characterized by abundant bloody diarrhea, abdominal pain, and distension (13
). CPB has long been implicated in type B and type C disease, but its contribution to intestinal disease has only recently been demonstrated (19
). Although type B and C isolates usually produce between three and five different lethal toxins (3
), studies with isogenic toxin-null mutants have demonstrated that CPB is necessary to reproduce the intestinal pathology of type C isolate CN3685 in rabbit ileal loops (19
). That study also showed, for the first time, that purified CPB alone is sufficient to cause bloody fluid accumulation and histologic damage in ileal loops (19
). The key to demonstrating the enteric activity of purified CPB in that study was the inclusion of TI in the CPB treatment, which mimicked natural type C disease conditions, where high levels of TI and/or low levels of trypsin are present due to diet or disease.
The present study now significantly extends those initial findings by characterizing more completely the intestinal effects of purified CPB. A trypsin inhibition approach was again used to demonstrate that, in addition to the ileum, purified CPB produces bloody fluid accumulation and tissue damage in the rabbit jejunum and duodenum. However, the duodenum was found to be less CPB-sensitive than the jejunum or ileum. These regional CPB sensitivity differences provide one explanation for gastrointestinal aspects of natural type B or type C diseases, which primarily involve the jejuna and ilea of infected humans and animals (2
). The reduced CPB sensitivity of the duodenum in animal models and natural disease may be attributable, at least in part, to pancreatic secretion producing higher intestinal trypsin levels in the duodenum, which then reduces CPB activity (30
Although purified CPB exhibited activity in all sections of the rabbit small intestines, the present study found that the rabbit colon is not affected by even high CPB doses applied in the presence of TI. These differences in CPB sensitivity between the rabbit small intestine versus the colon provide one explanation for the predominant involvement of the small intestine in type B and C infections of both humans and animals. However, there are also now some emerging reports of colonic damage in human type C disease (2
), which may involve (i) another type C toxin (not CPB) possessing colonic activity, (ii) species-specific differences in colonic CPB susceptibility, or (iii) CPB colonic damage requiring higher CPB doses or longer treatment times than used in the present study. Sorting out these possibilities will require additional experiments, but it is notable that another C. perfringens
toxin, i.e., the enterotoxin, resembles CPB by also failing to damage rabbit colonic loops and yet possessing some limited activity on the human colon in vivo and ex vivo (1
The present study also shows, for the first time, that CPB acts very quickly in the small intestine, with the duodenum, jejunum, or ileum all exhibiting visible tissue damage within the first hour of CPB treatment (Fig. ). However, after 1 h of CPB treatment, no fluid accumulation differences were observed between control versus the toxin-treated duodenum and jejunum, and only modest CPB-induced fluid accumulation was detected in the ileum. Since luminal fluid accumulation developed after the onset of CPB-induced damage and usually included the presence of blood, these results are consistent with the accumulation of bloody luminal fluid in CPB-treated small intestine resulting, at least in part, from severe mucosal necrosis. CPB-induced inflammation may also contribute to this intestinal bleeding since there is a well-established association between hemorrhage and inflammation for other enteric toxins and pathogens (20
). Similar hemorrhaging and intestinal necrosis as seen with purified CPB has also been reported for rabbit ileal loops injected with whole cultures of type C strain CN3685, but not with its isogenic CPB-null mutants (19
), supporting the importance of CPB production in the ability of type C (and possibly type B) isolates to cause enteric effects during natural disease.
The present study also demonstrates that CPB possesses considerable enteric potency. Histologic damage was observed in duodenal, jejunal, and ileal rabbits loops treated for 6 h, in the presence of TI, with only 1 μg of purified CPB. A dose-dependent CPB effect on tissue damage was noted in the relatively CPB-insensitive duodenum. However, the dose dependency of CPB-induced tissue damage in the jejunum or ileum was statistically insignificant, probably because near-maximal effects had already been produced with the lowest CPB dose tested in the present study. Future studies might determine the minimal dose of CPB causing enteric effects in rabbit small-intestinal loops, but the current findings establish that, in the presence of TI, CPB is considerably more potent in rabbit intestinal loops than is C. perfringens
The enteric potency of CPB demonstrated in the present study could help explain, at least in part, the relatively rapid and often fatal progression of type C disease. For example, it is now clear that, in reduced trypsin conditions, even low doses of CPB are sufficient to rapidly induce significant necrosis of the epithelium. Beyond its enteric potency now demonstrated in the present study, CPB possesses the second lowest 50% lethal dose of all C. perfringens
toxins when administered intravenously to mice (5
). This suggests that the absorption of even small amounts of CPB from a damaged small intestine may be sufficient to cause death or damage to internal organs. It is notable in this respect that no rabbits died during the present experiments, which may suggest that longer treatment times or higher CPB doses are required to cause systemic lethality, at least in the rabbit ileal loop model. The present study's observation of extensive CPB-induced enteric damage in the absence of lethality supports the general view that death during type B or type C diseases mainly results from toxemia rather than from intestinal pathology (23
CPB is very sensitive to the action of trypsin, although the molecular basis for this sensitivity is unknown. Endogenous intestinal trypsin is known to play an important role as an innate defense mechanism against type C infection (10
). Lawrence and Cooke showed that C. perfringens
type C could cause a pigbel-like disease in guinea pigs fed with a persistent low protein diet combined with dietary protease inhibitors (7
). Enteric lesions similar to those observed in human pigbel cases have also been successfully reproduced by injecting a type C culture into lambs (14
) or rabbit ileal loops (19
), but only when a TI was given as well. Attempts to reproduce type C disease in animal models injecting CPB alone (no TI) have consistently failed (23
). In this report and a previous study (19
), we have now demonstrated that the presence of TI allows purified CPB to reproduce type C-like pathology in small intestinal loops. Since CPB is very sensitive to trypsin (5
), these results suggest that the presence of even small amounts of endogenous trypsin remaining in washed small intestinal loops can be sufficient to inactivate CPB if a TI is not administered simultaneously with the toxin.
The small-intestine histological alterations observed by using purified CPB in the present study and our previous work (19
) are very similar to those described in type C natural diseases. For example, type C isolates produce in sheep intestinal lesions consisting of diffuse or multifocal hemorrhagic and necrotizing enteritis, mainly in the ileum, with excess of sanguineous serous fluid in the abdominal cavity (27
). In piglets, type C infection also produces necrotizing enteritis with deep mucosal necrosis and emphysema in small intestine, sometimes extending to the colon (24
). The similarity noted in this and our previous studies (3
) between intestinal lesions caused by purified CPB and the lesions of natural type B and C disease, coupled with the inability of isogenic CPB-null mutants to cause intestinal pathology (19
), further support CPB as a critical virulence factor for type C (and likely type B) disease.
In summary, this research shows that, in the presence of TI, CPB induce enteric effects in the duodenum, jejunum, or ileum that resemble those in type C intestinal disease. However, colonic loops were found to be unaffected by similar treatment. Small-intestinal lesions developed quickly and required only a small amount of the purified toxin, indicating that CPB is highly active in the rabbit small intestine. These findings provide new insights into the overall pathogenic mechanism of fatal diseases induced by C. perfringens type B and C isolates and support the importance of CPB immunity for vaccine-induced protection against type B or C disease.