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1 Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Switzerland
2 Laboratorium Pathologie Oost Nederland, Enschede, The Netherlands
Correspondence
Horst Posthaus
horst.posthaus{at}itpa.unibe.ch
Received November 17, 2008
Accepted February 6, 2009
Clostridium perfringens type C-induced enteritis necroticans is a rare but often fatal disease in humans. A consistent histopathological finding is an acute, deep necrosis of the small intestinal mucosa associated with acute vascular necrosis and massive haemorrhage in the lamina propria and submucosa. Retrospective immunohistochemical investigations of tissues from a diabetic adult who died of enteritis necroticans revealed endothelial localization of C. perfringens β-toxin in small intestinal lesions. Our results indicate that vascular necrosis might be induced by a direct interaction between C. perfringens β-toxin and endothelial cells and that targeted disruption of endothelial cells plays a role in the pathogenesis of enteritis necroticans.
Introduction
Enteritis necroticans (pigbel) is an acute, life-threatening disease caused by Clostridium perfringens type C. It occurs sporadically in Western countries and is endemic in the highland regions of Papua New Guinea (Johnson & Gerding, 1997). The disease is characterized by segmental haemorrhagic necrosis of the jejunum, which rapidly progresses to full-thickness necrosis of small and occasionally large intestinal segments (Lawrence & Walker, 1976; Johnson & Gerding, 1997). Histopathological hallmarks are deep mucosal necrosis with vascular necrosis and extensive haemorrhage (Lawrence & Walker, 1976). Risk factors for developing this disease are diabetes mellitus, malnourishment, low protein diets, sudden intake of proteinaceous meals, ingestion of C. perfringens type C-contaminated pig meat, and high amounts of trypsin inhibitors in the nutrition (Lawrence & Walker, 1976; Johnson & Gerding, 1997; Matsuda et al., 2007). C. perfringens type C strains are defined by the production of 
- (CPA) and β-toxin (CPB). The pathogenesis of type C enteritis is mainly attributable to CPB, a highly trypsin-sensitive exotoxin, causing cellular damage through multimeric pore formation at plasma membranes of susceptible cells (Smedley et al., 2004; Sayeed et al., 2008). The exact role of CPB in the pathogenesis of enteritis necroticans, in particular its target cells, has not been resolved, however. So far, only human endothelial cells (Steinthorsdottir et al., 2000) and a promyeloblastic cell line (HL-60) (Nagahama et al., 2003) have been shown to be susceptible to CPB. Results from immunohistochemical evaluations of C. perfringens type C enteritis in pigs in our laboratory (Miclard et al., 2009) indicated that CPB targets endothelial cells and induces vascular necrosis. The aim of the present study was to evaluate CPB localization in lesions of an archived human case of C. perfringens type C enteritis by immunohistochemistry.
Retrospective case
Archived, paraffin-embedded tissue blocks of small intestinal lesions as well as non-affected colon and stomach were available from a diabetic adult who died from enteritis necroticans in 1983. The clinical presentation and macroscopical, histopathological and microbiological findings have been described previously (Severin et al., 1984). Briefly, the patient was a 24-year-old diabetic male who died of a severe, acute, haemorrhagic enteritis 2 days after initial onset of bloody diarrhoea and 24 h after being admitted to the hospital.
Methods
Histopathology. Tissues were routinely processed for histology and stained with haematoxylin and eosin.
Real-time PCR.
DNA from paraffin-embedded small intestine was extracted as described by Miclard et al. (2009). Simplex real-time PCR amplification of the genes of C. perfringens - (cpa), β- (cpb), β-2- (cpb2), 
- (etx) and 
-toxin (itx) and enterotoxin (cpe) was performed as described by Albini et al. (2008). All reactions were repeated three times.
Immunohistochemistry. Histological sections were deparaffinized with xylene, endogenous peroxidase activity was inhibited by H2O2 (0.1 % in PBS, 15 min), and slides were incubated with 0.1 % proteinase (Sigma-Aldrich; in TBS, pH 7.6, 15 min, 37 °C). Primary antibodies (Center for Veterinary Biologics, Ames, Iowa; 1 : 100 for 2 h at room temperature) were: mouse monoclonal anti-CPB (mAb-CPB; 10A2) and mouse monoclonal anti-CPA (mAb-CPA; 6H7 1F3). Slides were developed using the LSAB and AEC kit (Dako) and counterstained with haemalaun.
Results and Discussion
Histopathological findings are briefly summarized in the legend of Fig. 1
. The absence of a substantial inflammatory reaction in combination with the reported rapid deterioration of the patient's condition (Severin et al., 1984) corresponds to a peracute to acute course of the disease. Real-time PCR on DNA from tissue extracts detected low levels of cpb (Ct value 36.4), cpb2 (Ct value 39) and cpe (Ct value 36.5), but not etx or itx. Although primers and fluorescent probes were designed in highly conserved gene regions of both human and animal isolates (Albini et al., 2008), cpa could not be amplified. Overall, the low levels of cpb and cpb2 indicated low yields of clostridial DNA from the paraffin blocks. Because these genes are usually detectable in higher copy numbers than cpa in C. perfringens isolates (Albini et al., 2008), it is conceivable that cpa was beyond the detection threshold in our samples. Despite this, amplification of cpb and two additional C. perfringens toxin genes reconfirms the initial diagnosis of C. perfringens type C infection achieved through bacteriological culturing and biochemical and guinea pig skin neutralization tests (Severin et al., 1984).
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). Control mAb-CPA did not produce any specific staining (Supplementary Fig. S1 in JMM Online). Non-affected large intestinal segments of the same patient did not exhibit any CPB-specific staining at endothelial cells (Supplementary Fig. S2). Thus we were able to localize CPB at endothelial cells in the necrotizing lesions. Our findings suggest that vascular necrosis, a hallmark of enteritis necroticans (Lawrence & Walker, 1976), results from a direct interaction of CPB with endothelial cells. This conclusion is supported by a previous study (Steinthorsdottir et al., 2000) which demonstrated that CPB forms multimers in plasma membranes of human endothelial cells and induces arachidonic acid and ion release from these cells in vitro. Thus we hypothesize that CPB targeting of endothelial cells could induce vascular necrosis, haemorrhage, and subsequent hypoxic tissue necrosis. A previous report on immunohistochemical localization of CPB in lesions of C. perfringens type C in a human case of enteritis necroticans demonstrated CPB at or around clostridial-like organisms but did not demonstrate binding of CPB to particular cells (Matsuda et al., 2007). Although we also multifocally detected such signals in our samples, it is not clear whether this represents active CPB, which is known to be rapidly inactivated by proteases in the gut content. As a pore-forming toxin, CPB is supposed to cause cellular damage by inserting into plasma membranes of target cells (Steinthorsdottir et al., 2000), thus detection of extracellular CPB does not necessarily confirm a pathogenic role. The difference in results from previous studies and our study could be explained by the use of different antibodies and antigen retrieval methods. Additionally, widespread necrosis of the endothelial cells and proteolytic degradation of CPB could have hampered immunohistochemical detection of CPB at endothelial cells. The detected CPB signals most likely represent the multimeric, membrane-inserted form (Nagahama et al., 2003; Steinthorsdottir et al., 2000), which seems to be present in detectable quantities during this stage of the disease, and coincide with widespread vascular necrosis within the lesions. Our results cannot exclude initial effects of CPB at the intestinal epithelium, because of the limitation to one case which represents the fatal stage of the disease. At this stage, necrosis of enterocytes had already occurred and epithelial cells were largely destroyed and absent in our tissue sections.
Spontaneous C. perfringens type C enteritis rarely occurs in Western countries. Several reported cases were associated with either diabetes mellitus, as in this patient, or diet-induced low levels of proteolytic enzymes in intestinal contents leading to reduced proteolytic inactivation of CPB (Matsuda et al., 2007). Despite different predisposing factors, the recent evidence of CPB as the major virulence factor in inducing necrotizing lesions in an experimental model (Sayeed et al., 2008; Vidal et al., 2008) clearly demonstrates that elevated CPB levels in the intestine play a key role in the pathogenesis of enteritis necroticans. C. perfringens type C enteritis is frequently observed in veterinary medicine, especially in newborn piglets, in contrast to in human medicine, and the diseases share remarkable morphological similarities between human and veterinary cases (Songer, 1996). Our findings in this case in a human patient are consistent with results of our recent larger scale study in neonatal piglets, where we consistently demonstrated endothelial localization of CPB in peracute to acute lesions of C. perfringens type C enteritis (Miclard et al., 2009). Taken together, our findings suggest that targeting and destruction of vascular endothelial cells through CPB might play an important role in the pathogenesis of C. perfringens type C-induced enteritis necroticans.
References
Albini, S., Brodard, I., Jaussi, A., Wollschlaeger, N., Frey, J., Miserez, R. & Abril, C. (2008). Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates. Vet Microbiol 127, 179–185.[CrossRef][Medline]
Johnson, S. & Gerding, D. N. (1997). Enterotoxemic infections. In The Clostridia: Molecular Biology and Pathogenesis, pp. 117–140. Edited by J. I. Rood, B. A. McClaine, J. G. Songer & R. W. Titball. San Diego: Academic Press.
Lawrence, G. & Walker, P. D. (1976). Pathogenesis of enteritis necroticans in Papua New Guinea. Lancet 1, 125–126.[Medline]
Matsuda, T., Okada, Y., Inagi, E., Tanabe, Y., Shimizu, Y., Nagashima, K., Sakurai, J., Nagahama, M. & Tanaka, S. (2007). Enteritis necroticans ‘pigbel’ in a Japanese diabetic adult. Pathol Int 57, 622–626.[CrossRef][Medline]
Miclard, J., Jäggi, M., Sutter, E., Wyder, M., Grabscheid, B. & Posthaus, H. (2009). Clostridium perfringens beta-toxin targets endothelial cells in necrotizing enteritis in piglets. Vet Microbiol 134, (in press)
Nagahama, M., Hayashi, S., Morimitsu, S. & Sakurai, J. (2003). Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells. J Biol Chem 278, 36934–36941.
Sayeed, S., Uzal, F. A., Fisher, D. J., Saputo, J., Vidal, J. E., Chen, Y., Gupta, P., Rood, J. I. & McClane, B. A. (2008). Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model. Mol Microbiol 67, 15–30.[Medline]
Severin, W. P., de la Fuente, A. A. & Stringer, M. F. (1984). Clostridium perfringens type C causing necrotising enteritis. J Clin Pathol 37, 942–944.
Smedley, J. G., Fisher, D. J., Sayeed, S., Chakrabarti, G. & McClane, B. A. (2004). The enteric toxins of Clostridium perfringens. Rev Physiol Biochem Pharmacol 152, 183–204.[CrossRef][Medline]
Songer, J. G. (1996). Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 9, 216–234.
Steinthorsdottir, V., Halldorsson, H. & Andresson, O. S. (2000). Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells. Microb Pathog 28, 45–50.[CrossRef][Medline]
Vidal, J. E., McClane, B. A., Saputo, J., Parker, J. & Uzal, F. A. (2008). Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon. Infect Immun 76, 4396–4404.
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