Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias

ABSTRACT Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.

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Development and Application of an Oral Challenge Mouse Model for Studying Clostridium perfringens Type D Infection

Infection and Immunity ◽

10.1128/iai.00562-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4282-4288 ◽

Cited By ~ 25

Author(s):

Mariano E. Fernandez-Miyakawa ◽

Sameera Sayeed ◽

Derek J. Fisher ◽

Rachael Poon ◽

Vicki Adams ◽

...

Keyword(s):

Mouse Model ◽

Clostridium Perfringens ◽

Clinical Signs ◽

Passive Immunization ◽

Small Animal ◽

Toxin Production ◽

Oral Challenge ◽

Type D ◽

Epsilon Toxin

ABSTRACT Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.

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Both Epsilon-Toxin and Beta-Toxin Are Important for the Lethal Properties of Clostridium perfringens Type B Isolates in the Mouse Intravenous Injection Model

Infection and Immunity ◽

10.1128/iai.01672-06 ◽

2007 ◽

Vol 75 (3) ◽

pp. 1443-1452 ◽

Cited By ~ 38

Author(s):

Mariano E. Fernandez-Miyakawa ◽

Derek J. Fisher ◽

Rachael Poon ◽

Sameera Sayeed ◽

Vicki Adams ◽

...

Keyword(s):

Clostridium Perfringens ◽

Clinical Signs ◽

Toxin Production ◽

Intestinal Damage ◽

Type B ◽

Trypsin Treatment ◽

Epsilon Toxin ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

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Effects of Clostridium perfringens Beta-Toxin on the Rabbit Small Intestine and Colon

Infection and Immunity ◽

10.1128/iai.00547-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4396-4404 ◽

Cited By ~ 54

Author(s):

Jorge E. Vidal ◽

Bruce A. McClane ◽

Juliann Saputo ◽

Jaquelyn Parker ◽

Francisco A. Uzal

Keyword(s):

Small Intestine ◽

Clostridium Perfringens ◽

Time Course ◽

Defense Mechanism ◽

Intestinal Damage ◽

Loop Model ◽

Type B ◽

Ileal Loop ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.

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Endothelial Binding of Beta Toxin to Small Intestinal Mucosal Endothelial Cells in Early Stages of Experimentally Induced Clostridium Perfringens Type C Enteritis in Pigs

Veterinary Pathology ◽

10.1177/0300985812461362 ◽

2012 ◽

Vol 50 (4) ◽

pp. 626-629 ◽

Cited By ~ 18

Author(s):

V. L. Schumacher ◽

A. Martel ◽

F. Pasmans ◽

F. Van Immerseel ◽

H. Posthaus

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Early Stages ◽

Type C ◽

Small Intestinal ◽

Beta Toxin ◽

Experimentally Induced

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Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany

Infection and Immunity ◽

10.1128/iai.00818-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4354-4363 ◽

Cited By ~ 25

Author(s):

Menglin Ma ◽

Jihong Li ◽

Bruce A. McClane

Keyword(s):

World War Ii ◽

Heat Resistance ◽

Clostridium Perfringens ◽

Type A ◽

World War ◽

Content Type ◽

Post World War Ii ◽

Heat Resistant ◽

Type C ◽

Beta Toxin

ABSTRACTClostridium perfringenstype C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates arecpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores withD100values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomalcpegene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both thecpeandcpbgenes can be mobilized in Darmbrand isolates. These results suggest thatC. perfringenstype A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomalcpegene.

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The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽

10.1128/mbio.00338-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 24

Author(s):

Menglin Ma ◽

Jorge Vidal ◽

Juliann Saputo ◽

Bruce A. McClane ◽

Francisco Uzal

Keyword(s):

Clostridium Perfringens ◽

Regulatory System ◽

Gas Gangrene ◽

Necrotic Enteritis ◽

Vegetative Cells ◽

Content Type ◽

Two Component ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

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Clostridial Enteric Infections in Pigs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870501700602 ◽

2005 ◽

Vol 17 (6) ◽

pp. 528-536 ◽

Cited By ~ 135

Author(s):

J. Glenn Songer ◽

Francisco A. Uzal

Keyword(s):

Clostridium Difficile ◽

Clinical Signs ◽

Type A ◽

Enteric Infection ◽

Gene Encoding ◽

Presumptive Diagnosis ◽

Large Numbers ◽

Intestinal Contents ◽

Type C ◽

Beta Toxin

Clostridium perfringens types A and C and Clostridium difficile are the principal enteric clostridial pathogens of swine. History, clinical signs of disease, and gross and microscopic findings form the basis for a presumptive diagnosis of C. perfringens type-C enteritis. Confirmation is based on isolation of large numbers of type-C C. perfringens and/or detection of beta toxin in intestinal contents. Diagnosis of C. perfringens type-A infection, however, remains controversial, mostly because the condition has not been well defined and because type-A organisms and their most important major (alpha) toxin can be found in intestinal contents of healthy and diseased pigs. Isolation of large numbers of C. perfringens type A from intestinal contents, in the absence of other enteric pathogens, is the most reliable criterion on which to base a diagnosis. Recently, beta2 (CPB2) toxin-producing C. perfringens type A has been linked to disease in piglets and other animals. However, implication of CPB2 in pathogenesis of porcine infections is based principally on isolation of C. perfringens carrying cpb2, the gene encoding CPB2, and the specific role of CPB2 in enteric disease of pigs remains to be fully defined. Clostridium difficile can also be a normal inhabitant of the intestine of healthy pigs, and diagnosis of enteric infection with this microorganism is based on detection of its toxins in feces or intestinal contents.

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Rapid Cytopathic Effects of Clostridium perfringens Beta-Toxin on Porcine Endothelial Cells

Infection and Immunity ◽

10.1128/iai.01284-09 ◽

2010 ◽

Vol 78 (7) ◽

pp. 2966-2973 ◽

Cited By ~ 27

Author(s):

Corinne Gurtner ◽

Francesca Popescu ◽

Marianne Wyder ◽

Esther Sutter ◽

Friederike Zeeh ◽

...

Keyword(s):

Endothelial Cells ◽

Clostridium Perfringens ◽

Proximal Jejunum ◽

Endothelial Lining ◽

Aortic Endothelial Cells ◽

Type C ◽

Mucosal Necrosis ◽

Beta Toxin ◽

Immunohistochemical Studies

ABSTRACT Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C β-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

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