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Sample records for clostridium perfringens epsilon

  1. Detection of Clostridium perfringens epsilon toxin by ELISA.

    PubMed

    Naylor, R D; Martin, P K; Sharpe, R T

    1987-03-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed as an alternative to neutralisation tests in mice to detect Clostridium perfringens type D epsilon toxin in the intestinal contents of animals which have died from suspected enterotoxaemia. The test was sensitive and quantitative and gave excellent agreement with the mouse protection test. PMID:2884704

  2. Naturally acquired antibodies against Clostridium perfringens epsilon toxin in goats.

    PubMed

    Veschi, Josir Laine A; Bruzzone, Octavio A; Losada-Eaton, Daniela M; Dutra, Iveraldo S; Fernandez-Miyakawa, Mariano E

    2008-09-15

    Clostridium perfringens type D-producing epsilon toxin is a common cause of death in sheep and goats worldwide. Although anti-epsilon toxin serum antibodies have been detected in healthy non-vaccinated sheep, the information regarding naturally acquired antibodies in ruminants is scanty. The objective of the present report was to characterize the development of naturally acquired antibodies against C. perfringens epsilon toxin in goats. The levels of anti-epsilon toxin antibodies in blood serum of goat kids from two different herds were examined continuously for 14 months. Goats were not vaccinated against any clostridial disease and received heterologous colostrums from cows that were not vaccinated against any clostridial disease. During the survey one of these flocks suffered an unexpectedly severe C. perfringens type D enterotoxemia outbreak. The results showed that natural acquired antibodies against C. perfringens epsilon toxin can appear as early as 6 weeks in young goats and increase with the age without evidence of clinical disease. The enterotoxemia outbreak was coincident with a significant increase in the level of anti-epsilon toxin antibodies. PMID:18538416

  3. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

    PubMed

    Losada-Eaton, D M; Fernandez-Miyakawa, M E

    2010-12-01

    Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia. PMID:20434186

  4. Clostridium perfringens type-D enterotoxaemia in cattle: the diagnostic significance of intestinal epsilon toxin.

    PubMed

    Jones, A L; Dagleish, M P; Caldow, G L

    2015-10-17

    The aims of this study were to describe 42 cases of Clostridium perfringens type-D enterotoxaemia in cattle seen between 2003 and 2014 and to determine the diagnostic value of detecting epsilon toxin in bovine intestinal content. All cases in the series had histological brain changes considered pathognomonic for C. perfringens type-D enterotoxaemia in sheep and goats and the epsilon toxin of C. perfringens was concurrently detected in the intestinal contents of 15 (36 per cent) cases. The data from the case series indicate that intestinal epsilon toxin has a sensitivity of 56 per cent compared with histology of the brain for diagnosis of bovine C. perfringens type-D enterotoxaemia. The diagnostic specificity of detecting epsilon toxin in bovine intestinal content was investigated by screening intestinal contents of 60 bovine carcases submitted for postmortem examination. Epsilon toxin was detected in 11 (18 per cent) carcases but no pathognomonic histological brain change was found in any. The specificity of intestinal epsilon toxin was estimated to be 80.4 per cent. These studies demonstrate that for a definitive diagnosis of C. perfringens type-D enterotoxaemia in cattle histological examination of the brain is essential as the presence of epsilon toxin in the intestinal contents alone is neither sensitive nor specific enough. PMID:26428898

  5. Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?

    PubMed Central

    Stiles, Bradley G.; Barth, Gillian; Barth, Holger; Popoff, Michel R.

    2013-01-01

    Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics). PMID:24284826

  6. Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

    PubMed

    Gonçalves, Luciana A; Lobato, Zélia I P; Silva, Rodrigo O S; Salvarani, Felipe M; Pires, Prhiscylla S; Assis, Ronnie A; Lobato, Francisco C F

    2009-11-01

    Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin. PMID:19779698

  7. Clostridium perfringens epsilon toxin increases the small intestinal permeability in mice and rats.

    PubMed

    Goldstein, Jorge; Morris, Winston E; Loidl, César Fabián; Tironi-Farinati, Carla; Tironi-Farinatti, Carla; McClane, Bruce A; Uzal, Francisco A; Fernandez Miyakawa, Mariano E

    2009-01-01

    Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel. PMID:19763257

  8. Clostridium perfringens Epsilon Toxin Increases the Small Intestinal Permeability in Mice and Rats

    PubMed Central

    Goldstein, Jorge; Morris, Winston E.; Loidl, César Fabián; Tironi-Farinatti, Carla; McClane, Bruce A.; Uzal, Francisco A.; Fernandez Miyakawa, Mariano E.

    2009-01-01

    Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel. PMID:19763257

  9. Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

    PubMed

    Lobato, Francisco C F; Lima, Catarina G R D; Assis, Ronnie A; Pires, Prhiscylla S; Silva, Rodrigo O S; Salvarani, Felipe M; Carmo, Anderson O; Contigli, Christiane; Kalapothakis, Evanguedes

    2010-08-31

    Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia. PMID:20670910

  10. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

    PubMed

    McClain, Mark S; Cover, Timothy L

    2007-04-01

    The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin. PMID:17261609

  11. Binding of epsilon-toxin from Clostridium perfringens in the nervous system.

    PubMed

    Dorca-Arévalo, Jonatan; Soler-Jover, Alex; Gibert, Maryse; Popoff, Michel R; Martín-Satué, Mireia; Blasi, Juan

    2008-09-18

    Epsilon-toxin (epsilon-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. Epsilon-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for epsilon-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An epsilon-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of epsilon-toxin to myelin structures. epsilon-Prototoxin-GFP and epsilon-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by epsilon-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of epsilon-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system. PMID:18406080

  12. Brain lesions associated with clostridium perfringens type D epsilon toxin in a Holstein heifer calf.

    PubMed

    Mete, A; Garcia, J; Ortega, J; Lane, M; Scholes, S; Uzal, F A

    2013-09-01

    A 6-month-old dairy heifer calf with no premonitory signs was acutely down after the morning feeding and could not rise. On presentation, the heifer was in right lateral recumbency and moribund with opisthotonus and left hind limb paddling. Following euthanasia, gross examination of the brain revealed multifocal loss of gray-white matter distinction and extensive petechiae throughout the brainstem. On histopathological examination, there was striking white matter edema and marked perivascular proteinaceous edema surrounding many arterioles and venules (microangiopathy), mainly in the white matter of the internal capsule, thalamus, midbrain, cerebellum, and cerebellar peduncles. The perivascular neuropil was strongly positive for Alzheimer precursor protein A4. Clostridium perfringens epsilon toxin was detected in the intestinal contents. This is the first report of microangiopathy in postneonatal cattle associated with the detection of epsilon toxin in the intestinal contents. PMID:23381925

  13. Pathogenesis of brain damage produced in sheep by Clostridium perfringens type D epsilon toxin: a review.

    PubMed

    Finnie, J W

    2003-04-01

    Microvascular endothelial damage by the epsilon toxin of Clostridium perfringens type D appears to be the fundamental cause of cerebral parenchymal injury and lesions occur in a seemingly dose- and time-dependent manner. Large doses of circulating toxin produce a severe, generalised, vasogenic cerebral oedema and an acute or peracute clinical course to death. With lower doses of toxin, or in partially immune sheep, focal necrosis, often bilaterally symmetrical, occurs in certain selectively vulnerable brain regions, which appear to become fewer as the toxin dose is reduced. These cases follow a more protracted clinical course, but death is the usual outcome. The precise pathogenesis of the focal brain damage found in subacutely intoxicated sheep is unresolved, but several possible mechanisms are discussed. PMID:15080445

  14. Clostridium perfringens

    PubMed Central

    Clifford, Walter J.; Anellis, Abe

    1971-01-01

    A biphasic culture medium suitable for cultivation and sporulation of Clostridium perfringens, C. botulinum, and C. sporogenes was devised. The medium designed for use in a disposable, compartmented, plastic film container contained peptones, yeast extract, minerals, an anion exchange resin, and glucose in 4% agar as the solid phase and (NH4)2SO4 and 0.1% agar as the liquid phase. With the biphasic system, it was not necessary to use active cultures as inocula. Growth was at least equal to that obtained in conventional media, and spore production of 9 out of 12 strains of C. perfringens equalled or usually exceeded that of conventional media. Images PMID:4332043

  15. Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known.

    PubMed

    Alves, Guilherme Guerra; Machado de Ávila, Ricardo Andrez; Chávez-Olórtegui, Carlos Delfin; Lobato, Francisco Carlos Faria

    2014-12-01

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains and causes enterotoxemia, a highly lethal disease with major impacts on the farming of domestic ruminants, particularly sheep. ETX belongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to other toxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this high potency, ETX is considered as a potential bioterrorism agent and has been classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States. The protoxin is converted to an active toxin through proteolytic cleavage performed by specific proteases. ETX is absorbed and acts locally in the intestines then subsequently binds to and causes lesions in other organs, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine and its possible use as a biological weapon have drawn the attention of researchers and have led to a large number of studies investigating ETX. The aim of the present work is to review the existing knowledge on ETX from C. perfringens type B and D. PMID:25234332

  16. Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia.

    PubMed

    Layana, Jorge E; Fernandez Miyakawa, Mariano E; Uzal, Francisco A

    2006-08-01

    Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia. PMID:16857397

  17. Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro.

    PubMed

    Uzal, F A; Rolfe, B E; Smith, N J; Thomas, A C; Kelly, W R

    1999-08-01

    Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro. PMID:10493114

  18. The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep.

    PubMed

    Fernandez Miyakawa, M E; Uzal, F A

    2003-04-01

    Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals. The disease is caused by C. perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine. In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine. Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species. Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin. Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin. Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment: these changes were more marked in goats than in sheep. No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin. These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms. PMID:12777097

  19. Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery.

    PubMed

    Adamson, R H; Ly, J C; Fernandez-Miyakawa, M; Ochi, S; Sakurai, J; Uzal, F; Curry, F E

    2005-08-01

    Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium. PMID:16041001

  20. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide

    PubMed Central

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  1. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

    PubMed

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology. PMID:26452234

  2. Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells.

    PubMed

    Fernandez Miyakawa, Mariano E; Zabal, Osvaldo; Silberstein, Claudia

    2011-04-01

    Clostridium perfringens epsilon toxin (ETX) is responsible for a fatal enterotoxemia in different animal species, producing extensive renal damage, neurological disturbance and edema of lungs, heart and kidneys. However, there is no information about the susceptibility of humans to ETX. Here, we report that primary cultures of human renal tubular epithelial cells (HRTEC) exposed to ETX showed a marked swelling with subsequent large blebs surrounding most cells. The incubation of HRTEC with ETX produced a reduction of cell viability in a dose- and time-dependent manner. The CD(50) after 1-hour and 24-hour incubation were 3 µg/mL and 0.5 µg/mL, respectively. The pulse with ETX for 3 min was enough to produce a significant cytotoxic effect on HRTEC after 1-hour incubation. ETX binds to HRTEC forming a large complex of about 160 kDa similar to what was found in the Madin-Darby canine kidney (MDCK) cell line. The HRTEC could be a useful cell model to improve the understanding of the mechanisms involved on the cell damage mediated by ETX. PMID:20488848

  3. Identification of a Lambda Toxin-Negative Clostridium perfringens Strain that Processes and Activates Epsilon Prototoxin Intracellularly

    PubMed Central

    Harkness, Justine M.; Li, Jihong; McClane, Bruce A.

    2012-01-01

    Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation. PMID:22982043

  4. Solid-phase radioimmunoassays for quantitative antibody determination of bacterial exotoxins. Measurement of Clostridium perfringens type D epsilon antitoxin.

    PubMed

    Bernáth, S

    1976-01-01

    Two reversed solid-phase radioimmunoassays have been developed for quantitative determination of antibodies against Clostridium perfringens type D epsilon toxin. 125I labelled prototoxin was used in the bromoacetyl cellulose-bound antibody method and in the antibody coated tube method. The procedures are based on the competition for 125I labelled prototoxin between the insoluble antibodies and the antibodies present in the unknown sample. The radioactivity bound to solid-phase is in inverse ratio to quantity of measured antibodies. The antibody values which can be detected are in the range of 0.004 IU/ml of investigated serum. The methods allow the rapid and inexpensive screening of large groups of vaccinated sheep, and are very suitable for measuring small amounts of Cl. perfringens D epsilon antibodies with a small experimental error. PMID:187514

  5. Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

    PubMed

    Souza, A M; Reis, J K P; Assis, R A; Horta, C C; Siqueira, F F; Facchin, S; Alvarenga, E R; Castro, C S; Salvarani, F M; Silva, R O S; Pires, P S; Contigli, C; Lobato, F C F; Kalapothakis, E

    2010-01-01

    Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D. PMID:20198582

  6. Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

    PubMed Central

    Bokori-Brown, Monika; Kokkinidou, Maria C; Savva, Christos G; Fernandes da Costa, Sérgio; Naylor, Claire E; Cole, Ambrose R; Moss, David S; Basak, Ajit K; Titball, Richard W

    2013-01-01

    Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity. PMID:23504825

  7. Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination

    PubMed Central

    Linden, Jennifer R.; Ma, Yinghua; Zhao, Baohua; Harris, Jason Michael; Rumah, Kareem Rashid; Schaeren-Wiemers, Nicole

    2015-01-01

    ABSTRACT Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. PMID:26081637

  8. Proteolytic Processing and Activation of Clostridium perfringens Epsilon Toxin by Caprine Small Intestinal Contents

    PubMed Central

    Freedman, John C.; Li, Jihong; Uzal, Francisco A.

    2014-01-01

    ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. PMID:25336460

  9. Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.

    PubMed

    Bokori-Brown, Monika; Kokkinidou, Maria C; Savva, Christos G; Fernandes da Costa, Sérgio; Naylor, Claire E; Cole, Ambrose R; Moss, David S; Basak, Ajit K; Titball, Richard W

    2013-05-01

    Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity. PMID:23504825

  10. A Tripartite Cocktail of Chimeric Monoclonal Antibodies Passively Protects Mice against Ricin, Staphylococcal Enterotoxin B and Clostridium perfringens Epsilon Toxin

    PubMed Central

    Sully, Erin K.; Whaley, Kevin; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael; Velasco, Jesus; Holtsberg, Frederick W.; Stavale, Eric; Aman, M. Javad; Tangudu, Chandra; Uzal, Francisco A.; Mantis, Nicholas J.; Zeitlin, Larry

    2014-01-01

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these toxins are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge. PMID:25260254

  11. Detection of alpha- and epsilon-toxigenic Clostridium perfringens type D in sheep and goats using a DNA amplification technique (PCR).

    PubMed

    Miserez, R; Frey, J; Buogo, C; Capaul, S; Tontis, A; Burnens, A; Nicolet, J

    1998-05-01

    Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the alpha-, beta- and epsilon-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the alpha- and epsilon-toxin genes but were devoid of the beta-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the alpha-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the alpha- and epsilon-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the epsilon-toxin gene, whereas the majority of the colonies were of type A with the alpha-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens. The beta-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation. PMID:9674169

  12. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

    PubMed

    Freedman, John C; Li, Jihong; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural

  13. Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants

    PubMed Central

    Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

    2016-01-01

    Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals. PMID:27004612

  14. Epsilon Toxin Is Essential for the Virulence of Clostridium perfringens Type D Infection in Sheep, Goats, and Mice

    PubMed Central

    Garcia, J. P.; Adams, V.; Beingesser, J.; Hughes, M. L.; Poon, R.; Lyras, D.; Hill, A.; McClane, B. A.; Rood, J. I.

    2013-01-01

    Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis. PMID:23630957

  15. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

    PubMed Central

    Ivie, Susan E.; McClain, Mark S.

    2012-01-01

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  16. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

    PubMed

    Ivie, Susan E; McClain, Mark S

    2012-09-25

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  17. Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

    PubMed

    Alimolaei, Mojtaba; Golchin, Mehdi; Daneshvar, Hamid

    2016-06-01

    Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease. PMID:27012151

  18. CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence of C. perfringens food-poisoning is quite common and costly. Although somewhat fastidious in growth characteristics using synthetic laboratory media, the microorganism is very prolific when found in food products. Despite the pathogen’s ubiquity in the natural environment, foodborne i...

  19. Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model.

    PubMed

    Fernandez-Miyakawa, Mariano E; Jost, B Helen; Billington, Stephen J; Uzal, Francisco A

    2008-03-18

    Epsilon toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice were injected intravenously or intragastrically with CPA or PFO with or without ETX. Sublethal doses of CPA or PFO did not affect the lethality of ETX when either was injected together with the latter intravenously. However, sublethal or lethal doses of CPA or PFO resulted in reduction of the survival time of mice injected simultaneously with ETX when compared with the intravenous effect of ETX injected alone. When PFO was inoculated intragastrically with ETX, a reduction of the survival time was observed. CPA did not alter the survival time when inoculated intragastrically with ETX. The results of the present study suggest that both CPA and PFO have the potential to enhance the ETX lethal effects during enterotoxemia in natural hosts such as sheep and goats. PMID:17997054

  20. Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

    PubMed Central

    Kaushik, Himani; Deshmukh, Sachin; Mathur, Deepika Dayal; Tiwari, Archana; Garg, Lalit C

    2013-01-01

    Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM1 ganglioside receptor of LTB. The rLTB.Etx40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens. Abbreviations aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli. PMID:23904738

  1. Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A.

    2011-01-01

    Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action. PMID:22174687

  2. Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2

    PubMed Central

    Fennessey, Christine M.; Sheng, Jinsong; Rubin, Donald H.; McClain, Mark S.

    2012-01-01

    Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization – an event which is requisite for pore formation and, by extension, cell death. PMID:23056496

  3. Vaccination schedules to raise antibody concentrations against epsilon-toxin of Clostridium perfringens in ewes and their triplet lambs.

    PubMed

    de la Rosa, C; Hogue, D E; Thonney, M L

    1997-09-01

    The objective of this experiment was to compare vaccination schedules for ewes and their lambs to raise antibody concentrations to epsilon-toxin of Clostridium perfringens, the causative agent of enterotoxemia. Half of 200 Finnsheep x Dorset ewes were vaccinated with C. perfringens type D toxoid vaccine 3 wk before lambing. Serum samples were obtained from 20 ewes that were to be vaccinated and 20 ewes that would remain unvaccinated before treatment and at wk 2, 1, and 0 before the start of lambing. Antibody concentrations in sera of unvaccinated ewes remained at 2 IU/mL, but they peaked in vaccinated ewes at 15 IU/mL by wk 1 before lambing. Lambs from each of the first 13 and the first 14 sets of triplets from vaccinated and unvaccinated ewes, respectively, received one of three vaccination treatments: no vaccine (control), vaccination on d 1 and 21 of age, or vaccination on d 21 and 42 of age. Antibody concentrations declined in sera of vaccinated ewes from 8.5 IU/mL immediately after lambing to 3 IU/mL 12 wk later. Vaccination of lambs did not increase sera antibody concentration. However, prepartum vaccination of ewes significantly increased lamb antibody concentrations (19 IU/mL) compared with lambs reared by unvaccinated ewes (2 IU/mL). Vaccination of ewes resulted in lambs with higher antibody concentrations until wk 10 postpartum. Concentrations declined to .6 IU/mL in all lambs at 12 wk. Because concentrations of .2 IU/mL may be protective, these results indicate that vaccination of ewes before lambing imparts passive protection in lambs to 12 wk of age, whereas vaccination of young lambs provides no added protection. PMID:9303449

  4. Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia.

    PubMed

    Bokori-Brown, Monika; Hall, Charlotte A; Vance, Charlotte; Fernandes da Costa, Sérgio P; Savva, Christos G; Naylor, Claire E; Cole, Ambrose R; Basak, Ajit K; Moss, David S; Titball, Richard W

    2014-05-13

    Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia. PMID:24709588

  5. Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

    PubMed Central

    Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R.; Blasi, Juan

    2014-01-01

    Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

  6. Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia

    PubMed Central

    Bokori-Brown, Monika; Hall, Charlotte A.; Vance, Charlotte; Fernandes da Costa, Sérgio P.; Savva, Christos G.; Naylor, Claire E.; Cole, Ambrose R.; Basak, Ajit K.; Moss, David S.; Titball, Richard W.

    2014-01-01

    Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia. PMID:24709588

  7. Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

    PubMed

    Manni, Marco M; Sot, Jesús; Goñi, Félix M

    2015-03-01

    Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes. PMID:25485476

  8. Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

    PubMed

    Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R; Blasi, Juan

    2014-01-01

    Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

  9. ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE RED DEER (CERVUS ELAPHUS) OVER A 13-MONTH PERIOD.

    PubMed

    Scala, Christopher; Duffard, Nicolas; Beauchamp, Guy; Boullier, Séverine; Locatelli, Yann

    2016-03-01

    Deer are sensitive to clostridial diseases, and vaccination with clostridial toxoids is the method of choice to prevent these infections in ruminants. The purpose of this study was to evaluate the serologic responses in red deer (Cervus elaphus) over a 13-mo period after vaccination with a multivalent clostridial vaccine, containing an aluminium hydroxide adjuvant. Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay. Animals from group 1 (9 mo old; n = 6) were naïve and received an initial vaccination with a booster vaccine 4 wk apart and one annual booster. Animals from group 2 (21 mo old; n = 10) had been previously vaccinated 12 mo prior and received a first annual booster at the beginning of this study and a second annual booster 12 mo later. The multivalent clostridial vaccine induced a high antibody response that peaked after each injection and then slowly decreased with time. In group 1, a booster vaccine was required to obtain an initial high humoral response. The annual booster injection induced a strong, rapid, and consistent anamnestic response in both groups. The serologic responses persisted significantly over the baseline value for 9-12 mo in group 1, but more than 12 mo in group 2. It is unknown whether the measured humoral immune responses would have been protective as no challenge studies were performed. Further investigation is needed to determine the protective antibody titers to challenge and how long this immunity might persist after vaccination. PMID:27010263

  10. Bacteriophages of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The specific aims of the book chapter are to: (1) Briefly review the nomenclature of bacteriophages and how these agents are classified. (2) Discuss the problems associated with addition/removal of antibiotics in commercial animal feeds. (3) Provide a brief overview of Clostridium perfringens biolog...

  11. Reverse phase passive haemagglutination and single radial immunodiffusion to detect epsilon antigen of Clostridium perfringens type D.

    PubMed

    Beh, K J; Buttery, S H

    1978-11-01

    Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl. perfringens type D. It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml. When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen. However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult. No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep. The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem. PMID:223537

  12. Comparison of four techniques for the detection of Clostridium perfringens type D epsilon toxin in intestinal contents and other body fluids of sheep and goats.

    PubMed

    Uzal, F A; Kelly, W R; Thomas, R; Hornitzky, M; Galea, F

    2003-03-01

    Polyclonal capture enzyme-linked immunosorbent assay (PC-ELISA), monoclonal capture ELISA (MC-ELISA), mouse neutralization test (MNT), and counterimmunoelectrophoresis (CIEP), were compared for their ability to detect epsilon toxin in intestinal contents and body fluids of sheep and goats. When used to evaluate intestinal contents of sheep artificially spiked with epsilon prototoxin, PC-ELISA detected 0.075 mouse lethal dose (MLD)50/ml, whereas the MNT, MC-ELISA, and CIEP detected 6, 25, and 50 MLD50/ml, respectively. Amounts of epsilon toxin detected by PC-ELISA, MC-ELISA, MNT, and CIEP in sheep pericardial fluid artificially spiked with epsilon prototoxin were 0.075, 0.75, 6, and 200 MLD50/ml, respectively. For assaying epsilon toxin in aqueous humor, PC-ELISA and MC-ELISA detected 0.075 MLD50/ml, whereas CIEP detected 200 MLD50/ml (MNT was not evaluated). When 51 samples of intestinal contents of sheep and goats (32 positive and 19 negative to MNT) were analyzed by the other 3 techniques, the relative sensitivity of PC-ELISA, MC-ELISA, and CIEP was 93.75, 84.37, and 37.50%, respectively. The specificity of PC-ELISA, MC-ELISA, and CIEP was 31.57, 57.89, and 84.21%, respectively. The absolute sensitivity of PC-ELISA, MC-ELISA, CIEP, and MNT was 90.90, 69.69, 15.15, and 54.54%. The absolute specificity of the 4 techniques was 100%. These results show that there is a marked inconsistency among techniques routinely used to detect Clostridium perfringens epsilon toxin. Until more consistent results are achieved, the diagnosis of enterotoxemia should not only be based solely on epsilon toxin detection, but also on clinical and pathological data. PMID:12661718

  13. [Toxins of Clostridium perfringens].

    PubMed

    Morris, W E; Fernández-Miyakawa, M E

    2009-01-01

    Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present. PMID:20085190

  14. Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana.

    PubMed

    Garcia, J P; Beingesser, J; Bohorov, O; Bohorova, N; Goodman, C; Kim, D; Pauly, M; Velasco, J; Whaley, K; Zeitlin, L; Roy, C J; Uzal, F A

    2014-09-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 = 1.6 μg/kg) and resulted in a mean time from inoculation to death of 18 and 180 min when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 min post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication. PMID:24950050

  15. Changes in ganglioside content affect the binding of Clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells.

    PubMed

    Shimamoto, Seiko; Tamai, Eiji; Matsushita, Osamu; Minami, Junzaburo; Okabe, Akinobu; Miyata, Shigeru

    2005-01-01

    Epsilon-toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent-resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside G(M1) dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to G(M1) or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, G(M3), a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane-anchored sialic acid of G(M3) within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells. PMID:15781998

  16. Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana

    PubMed Central

    Garcia, J.P.; Beingesser, J.; Bohorov, O.; Bohorova, N.; Goodman, C.; Kim, D.; Pauly, M.; Velasco, J.; Whaley, K.; Zeitlin, L.; Roy, C.J.; Uzal, F.A.

    2014-01-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 =1.6 μg/kg) and resulted in a mean time from inoculation to death of 18 and 180 minutes when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 minutes post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication. PMID:24950050

  17. Clostridium perfringens in retail chicken.

    PubMed

    Nowell, Victoria J; Poppe, Cornelis; Parreira, Valeria R; Jiang, Yan-Fen; Reid-Smith, Richard; Prescott, John F

    2010-06-01

    Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% "atypical" genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken. PMID:19961943

  18. Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX(Y196E)-C confers strong protection in mice.

    PubMed

    Yao, Wenwu; Kang, Jingjing; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Xin, Wenwen; Wang, Jinglin

    2016-01-01

    Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETX(Y196E)-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:10(5) in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 μg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETX(Y196E)-C is a potential vaccine candidate for future applied researches. PMID:27048879

  19. Immunization with a novel Clostridium perfringens epsilon toxin mutant rETXY196E-C confers strong protection in mice

    PubMed Central

    Yao, Wenwu; Kang, Jingjing; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Xin, Wenwen; Wang, Jinglin

    2016-01-01

    Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETXY196E-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:105 in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 μg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETXY196E-C is a potential vaccine candidate for future applied researches. PMID:27048879

  20. Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid▿ †

    PubMed Central

    Miyamoto, Kazuaki; Li, Jihong; Sayeed, Sameera; Akimoto, Shigeru; McClane, Bruce A.

    2008-01-01

    Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid. PMID:18776010

  1. Toxin plasmids of Clostridium perfringens.

    PubMed

    Li, Jihong; Adams, Vicki; Bannam, Trudi L; Miyamoto, Kazuaki; Garcia, Jorge P; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2013-06-01

    In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  2. NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Freedman, John C.

    2015-01-01

    ABSTRACT Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. IMPORTANCE Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and Ccp

  3. Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

    PubMed

    Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

    2015-06-15

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. PMID:25912943

  4. Faecal carriage of Clostridium perfringens.

    PubMed Central

    Stringer, M. F.; Watson, G. N.; Gilbert, R. J.; Wallace, J. G.; Hassall, J. E.; Tanner, E. I.; Webber, P. P.

    1985-01-01

    The numbers and serotypes of Clostridium perfringens present in the faeces of three groups of hospital patients and young healthy laboratory workers were examined in studies lasting between 10 and 13 weeks. In one hospital some long-stay geriatric patients carried relatively high numbers of C. perfringens (greater than 10(7)/g) most of the time and it was not unusual in any one week for the majority of these patients to carry the same serotype(s). However, the numbers of C. perfringens in the faeces of young long-stay patients in the same hospital were in the range of 10(3)-10(4)/g and carriage of common serotypes was not observed. These results were similar to the findings with the young laboratory workers. This investigation indicates that two of the laboratory criteria often used in the investigation of C. perfringens food poisoning, i.e. faecal counts of greater than or equal to 10(5) C. perfringens/g and patients carrying the same serological type need to be interpreted with caution with suspected outbreaks involving some groups of geriatric long-stay hospital patients. PMID:2866214

  5. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  6. Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

    PubMed

    el Idrissi, A H; Ward, G E

    1992-06-15

    Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias. PMID:1496812

  7. Clostridium perfringens Type C Enterotoxemia.

    PubMed

    Niilo, L

    1988-08-01

    Forms of enteric disease caused by Clostridium perfringens type C are critically reviewed with emphasis on practical aspects and recent research findings. Available data indicate that more animal species may be fatally infected by type C of this organism than by any other type of C. perfringens. Fatal cases have been recorded in pigs, cattle, sheep, horses and humans. Newborn animals are typically the most susceptible, possibly related to aspects of bacterial colonization, intestinal digestive functions, and to some other, unexplained, factors. Both beta toxin and the bacterial cells are required to initiate pathogenesis at the tips of jejunal villi, and subsequent massive adherence of these cells to necrotic mucosa is a characteristic feature. Although major lesions occur in the intestine, death is due to toxemia. The disease can be effectively controlled by vaccination of the dam. Epizootiology of this disease is a possible area for further studies. PMID:17423103

  8. [Toxins of Clostridium perfringens as a natural and bioterroristic threats].

    PubMed

    Omernik, Andrzej; Płusa, Tadeusz

    2015-09-01

    Clostridium perfringens is absolutely anaerobic rod-shaped, sporeforming bacterium. The morbidity is connected with producing toxins. Depending on the type of toxin produced Clostridium perfringens can be divided into five serotypes:A-E. Under natural conditions, this bacterium is responsible for local outbreaks of food poisoning associated with eating contaminated food which which was improperly heat treated. Some countries with lower economic level are endemic foci of necrotizing enteritis caused by Clostridium perfringens. The bacterium is also a major cause of gas gangrene. It is a disease, associated with wound infection, with potentially fatal prognosis in the case of treatment's delays. In the absence of early radical surgery, antibiotic therapy and (if available) hyperbaric treatment leads to the spread of toxins in the body causing shock, coma and death. Due to the force of produced toxins is a pathogen that poses a substrate for the production of biological weapons. It could potentially be used to induce outbreaks of food poisoning and by missiles contamination by spore lead to increased morbidity of gas gangrene in injured soldiers. C. perfringens types B and D produce epsilon toxin considered to be the third most powerful bacterial toxin. Because of the ability to disperse the toxin as an aerosol and a lack of methods of treatment and prevention of poisoning possible factors it is a potential tool for bioterrorism It is advisable to continue research into vaccines and treatments for poisoning toxins of C. perfringens. PMID:26449576

  9. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Following limit dilution cloning and three rounds of plaque purification lytic phage preparations ...

  10. Epsilon-Toxin Production by Clostridium perfringens Type D Strain CN3718 Is Dependent upon the agr Operon but Not the VirS/VirR Two-Component Regulatory System

    PubMed Central

    Chen, Jianming; Rood, Julian I.; McClane, Bruce A.

    2011-01-01

    ABSTRACT Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. PMID:22167225

  11. Improved Medium for Enumeration of Clostridium perfringens

    PubMed Central

    Harmon, Stanley M.; Kautter, Donald A.; Peeler, James T.

    1971-01-01

    An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 μg of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective. PMID:4331774

  12. Improved medium for enumeration of Clostridium perfringens.

    PubMed

    Harmon, S M; Kautter, D A; Peeler, J T

    1971-10-01

    An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 mug of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective. PMID:4331774

  13. Regulation of Toxin Production in Clostridium perfringens

    PubMed Central

    Ohtani, Kaori; Shimizu, Tohru

    2016-01-01

    The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

  14. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enteritis infection among chickens ...

  15. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease and gas gangrene among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enterit...

  16. Quantitation of Clostridium perfringens in foods.

    PubMed

    ANGELOTTI, R; HALL, H E; FOTER, M J; LEWIS, K H

    1962-05-01

    A procedure is described for identifying and enumerating Clostridium perfringens in foods by means of a simplified agar plating method, followed by confirmation of black colonies in tubes of motility-nitrate medium and sporulation broth. The test is routinely completed within 48 hr. Under experimental conditions, the procedure has been used to quantitatively recover various levels of C. perfringens contamination in a variety of foods and has recovered as few as ten C. perfringens per g without interference from food constituents and associated flora. Under practical conditions of field application, the method has been used to investigate five food-poisoning outbreaks, and C. perfringens was implicated as the etiological agent in two of these outbreaks. PMID:13861594

  17. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats.

    PubMed

    Uzal, Francisco A; Songer, J Glenn

    2008-05-01

    Clostridium perfringens produces enteric diseases, generically called enterotoxemias, in sheep, goats, and other animals. This microorganism can be a normal inhabitant of the intestine of most animal species, including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates and produces potent toxins that act locally or are absorbed into the general circulation with usually devastating effects on the host. History, clinical signs, and gross postmortem findings are useful tools for establishing a presumptive diagnosis of clostridial enterotoxemia in sheep and goats. Definitive diagnosis requires laboratory confirmation. Isolation of some types of C. perfringens (e.g., B and C) can be of diagnostic value, but other types (e.g., A) are so commonly found in the intestine of normal animals that isolation is meaningless from a diagnostic point of view. The most accepted criterion in establishing a definitive diagnosis of enterotoxemia is detection of C. perfringens toxins in intestinal contents. Also, histopathological examination of brain is very useful for diagnosis of type D disease, as lesions produced by epsilon toxin in the brains of sheep and goats are pathognomonic for type D enterotoxemia. Ancillary tests, such as measuring urine glucose or observing Gram-stained smears of intestinal mucosa, can be used. However, although such tests have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxemia when negative. PMID:18460610

  18. Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

    PubMed Central

    Uzal, F. A.; Vidal, J. E.; McClane, B. A.; Gurjar, A. A.

    2013-01-01

    Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C

  19. Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases.

    PubMed

    Uzal, F A; Vidal, J E; McClane, B A; Gurjar, A A

    2010-01-01

    Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C

  20. Clostridium perfringens in Meat and Meat Products

    PubMed Central

    Hall, Herbert E.; Angelotti, Robert

    1965-01-01

    A total of 262 specimens of meat and meat dishes were examined for the presence of Clostridium perfringens. Of this total, 161 were raw, unprocessed beef, veal, lamb, pork, or chicken; 101 were processed meats and meat dishes. C. perfringens was isolated from 113 (43.1%) of these specimens. The highest percentage of contamination (82%) was found in veal cuts, and the lowest (4.7%) in sliced sandwich meats and spreads. Only 2 of the 113 isolates were shown to produce heat-resistant spores, which indicates a very low incidence (0.8%) of contamination. These findings indicate that outbreaks of C. perfringens food-borne disease in the Cincinnati area are caused principally by the contamination of the food with vegetative cells or spores of the organism after cooking. Studies of the effects of various holding temperatures on the growth of C. perfringens indicated that, in the range of 5 to 15 C, no multiplication would occur, but that viable cells would still be present at the end of a 5-day holding period. Extremely rapid growth occurred at temperatures around 45 C, and complete inhibition of growth was accomplished between 49 and 52 C. PMID:14325274

  1. Alternative medium for Clostridium perfringens sporulation.

    PubMed Central

    Tórtora, J C

    1984-01-01

    A medium containing 0.50 g of thiotone peptone, 0.30 g of soluble starch, 0.02 g of MgSO4 X 7H2O, 0.90 g of Na2HPO4 X 2H2O, 100.00 ml of distilled water, and optionally , 166 micrograms of dichloridric thiamine supported sporulation of 138 out of 141 Clostridium perfringens strains. Comparatively this medium gave a greater percentage of sporulation than five other media described previously. PMID:6331307

  2. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

    PubMed Central

    Uzal, Francisco A; Freedman, John C; Shrestha, Archana; Theoret, James R; Garcia, Jorge; Awad, Milena M; Adams, Vicki; Moore, Robert J; Rood, Julian I; McClane, Bruce A

    2014-01-01

    Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases. PMID:24762309

  3. Clostridium perfringens infection after transarterial chemoembolization for large hepatocellular carcinoma

    PubMed Central

    Li, Jing-Huan; Yao, Rong-Rong; Shen, Hu-Jia; Zhang, Lan; Xie, Xiao-Ying; Chen, Rong-Xin; Wang, Yan-Hong; Ren, Zheng-Gang

    2015-01-01

    We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2nd day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2nd TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found. PMID:25892893

  4. Clostridium perfringens infection after transarterial chemoembolization for large hepatocellular carcinoma.

    PubMed

    Li, Jing-Huan; Yao, Rong-Rong; Shen, Hu-Jia; Zhang, Lan; Xie, Xiao-Ying; Chen, Rong-Xin; Wang, Yan-Hong; Ren, Zheng-Gang

    2015-04-14

    We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2(nd) day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2(nd) TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found. PMID:25892893

  5. Evaluation and Modifications of Media for Enumeration of Clostridium perfringens

    PubMed Central

    Hauschild, A. H. W.; Hilsheimer, R.

    1974-01-01

    The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. PMID:4358863

  6. Evaluation and modifications of media for enumeration of Clostridium perfringens.

    PubMed

    Hauschild, A H; Hilsheimer, R

    1974-01-01

    The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. PMID:4358863

  7. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    PubMed Central

    Verherstraeten, Stefanie; Goossens, Evy; Valgaeren, Bonnie; Pardon, Bart; Timbermont, Leen; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet; Wade, Kristin R.; Tweten, Rodney; Van Immerseel, Filip

    2015-01-01

    The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis. PMID:26008232

  8. Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test.

    PubMed

    Rosskopf-Streicher, Ute; Volkers, Peter; Noeske, Kerstin; Werner, Esther

    2004-01-01

    Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium. Disease caused by C. perfringens infection is called enterotoxaemia. C. perfringens strains are classified on the basis of the lethal exotoxins formed by the bacteria. Epsilon toxin is one of the major lethal toxins and is formed by C. perfringens types B and D. C. perfringens is an ubiquitous bacterium. Infection occurs via food, water, animal litter or soil. Affected animals include mainly sheep, pigs and cattle. C. perfringens infection manifests as pulpy kidney disease and diarrhoea in suckling lambs. Enterotoxaemia development is peracute in most cases. Animals die suddenly while grazing on the pasture, without any prior signs of disease. Therefore, treatment is possible only in very rare cases. Suitable immunoprophylactic measures are the treatment of choice to combat the disease: Vaccines and immunosera have therefore been used extensively for a long time. The requirements for quality, efficacy and safety testing of the inactivated vaccines are laid down in the Ph. Eur. in the monograph: Clostridium perfringens vaccines for veterinary use. After a marketing authorisation is attained, the product batches must be tested in laboratory animal models for their potency against all vaccine components (Pharmeuropa, 1997). For potency testing (batch control) of C. perfringens types B and D, the induction of specific antibodies against epsilon toxin in rabbits must be verified. For this purpose, 10 rabbits are immunised twice with the product to be tested. Their blood is taken 14 days after the last immunisation and the serum is pooled. The pooled serum is then tested for its protective effect. This is done by means of the toxin neutralisation test in mice (optionally also in guinea pigs) in comparison with an international reference serum. The evaluation criterion is the death rate of the mice in the test and reference groups after administration of lethal doses of epsilon toxin. The

  9. Prevalence of Clostridium perfringens, Clostridium perfringens enterotoxin and dysbiosis in fecal samples of dogs with diarrhea.

    PubMed

    Minamoto, Yasushi; Dhanani, Naila; Markel, Melissa E; Steiner, Jörg M; Suchodolski, Jan S

    2014-12-01

    Clostridium perfringens has been suspected as an enteropathogen in dogs. However, its exact role in gastrointestinal (GI) disorders in dogs remains unknown. Recent studies suggest the importance of an altered intestinal microbiota in the activation of virulence factors of enteropathogens. The aim of this study was to evaluate the relationship between diarrhea, dysbiosis, and the presence of C. perfringens and its enterotoxin (CPE). Fecal samples were collected prospectively from 95 healthy control dogs and 104 dogs with GI disease and assessed for bacterial abundances and the presence of CPE using quantitative PCR and ELISA, respectively. C. perfringens was detected in all dogs. Potentially enterotoxigenic C. perfringens were detected in 33.7% (32/95) of healthy control dogs and 48.1% (50/104) diseased dogs, respectively. CPE was detected by ELISA in 1.0% (1/95) of control dogs and 16.3% (17/104) of diseased dogs. Abundances of Fusobacteria, Ruminococcaceae, Blautia, and Faecalibacterium were significantly decreased in diseased dogs, while abundances of Bifidobacterium, Lactobacillus, and Escherichia coli were significantly increased compared to control dogs. The microbial dysbiosis was independent of the presence of the enterotoxigenic C. perfringens or CPE. In conclusion, the presence of CPE as well as fecal dysbiosis was associated with GI disease. However, the presence of C. perfringens was not indicative of GI disease in all cases of diarrhea, and the observed increased abundance of enterotoxigenic C. perfringens may be part of intestinal dysbiosis occurring in GI disease. The significance of an intestinal dysbiosis in dogs with GI disease deserves further attention. PMID:25458422

  10. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

    PubMed Central

    Gohari, Iman Mehdizadeh; Arroyo, Luis; MacInnes, Janet I.; Timoney, John F.; Parreira, Valeria R.; Prescott, John F.

    2014-01-01

    Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in

  11. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis.

    PubMed

    Gohari, Iman Mehdizadeh; Arroyo, Luis; Macinnes, Janet I; Timoney, John F; Parreira, Valeria R; Prescott, John F

    2014-01-01

    Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in

  12. Clostridium perfringens and other anaerobes isolated from bile.

    PubMed Central

    Sakaguchi, Y; Murata, K; Kimura, M

    1983-01-01

    Clostridium perfringens was isolated from bile in 13 cases of 150 patients examined. The serotypes of C perfringens strains isolated from bile and faeces were investigated using antisera to Hobbs' type 1-17. Two or more serological types were often found in a single specimen, but in the same patient the serotypes of C perfringens strains isolated from the bile were identical with those from the faeces. Beta-glucuronidase production in these C perfringens serotypes was tested with the API-Strep system. Strains agglutinated with Hobbs' antisera produced beta-glucuronidase, but non-agglutinated strains did not. PMID:6298284

  13. Clostridium perfringens in animal disease: a review of current knowledge.

    PubMed

    Niilo, L

    1980-05-01

    The diseases caused by various types of Clostridium perfringens are critically reviewed in the light of current knowledge. Particular emphasis is placed on information concerning these diseases in Canadian livestock. There are two etiologically clearly-defined acute C. perfringens diseases recognized in Canada: hemorrhagic enteritis of the new born calf, caused by C. perfringens type C, and enterotoxemia of sheep, caused by type D. Clostridium perfringens type A may play a role as a secondary pathological agent in various disease conditions, such as necrotic enteritis of chickens. It may also cause wound infections and may provide a source for human food poisoning outbreaks. There appears to be a considerable lack of knowledge regarding the distribution of C. perfringens types, their pathogenesis, diagnosis and the incidence of diseases caused by this organism. PMID:6253040

  14. Detection and characterization of Clostridium perfringens in the feces of healthy and diarrheic dogs

    PubMed Central

    Goldstein, Michael R.; Kruth, Stephen A.; Bersenas, Alexa M.E.; Holowaychuk, Marie K.; Weese, J. Scott

    2012-01-01

    Clostridium perfringens has been implicated as a cause of diarrhea in dogs. The objectives of this study were to compare 2 culture methods and to evaluate a multiplex polymerase chain reaction (PCR) assay to detect C. perfringens toxin genes alpha (α), beta (β ), beta 2 (β2), epsilon (ɛ), iota (ι), and C. perfringens enterotoxin (cpe) from canine isolates. Fecal samples were collected from clinically normal non-diarrheic (ND) dogs, (n = 105) and diarrheic dogs (DD, n = 54). Clostridium perfringens was isolated by directly inoculating stool onto 5% sheep blood agar (SBA) and enrichment in brain-heart infusion (BHI) broth, followed by inoculation onto SBA. Isolates were tested by multiplex PCR for the presence of α, β, β2, ɛ, ι, and cpe genes. C. perfringens was isolated from 84% of ND samples using direct culture and from 87.6% with enrichment (P = 0.79). In the DD group, corresponding isolation rates were 90.7% and 93.8% (P = 0.65). All isolates possessed the α toxin gene. Beta (β), β2, ɛ, ι, and cpe toxin genes were identified in 4.5%, 1.1%, 3.4%, 1.1%, and 14.8% of ND isolates, respectively. In the DD group, β and β2 were identified in 5%, ɛ and ι were not identified, and the cpe gene was identified in 16.9% of isolates. Enrichment with BHI broth did not significantly increase the yield of C. perfringens, but it did increase the time and cost of the procedure. C. perfringens toxin genes were present in equal proportions in both the ND and DD groups (P ≤ 0.15 to 0.6). Within the parameters of this study, culture of C. perfringens and PCR for toxin genes is of limited diagnostic usefulness due to its high prevalence in normal dogs and the lack of apparent difference in the distribution of toxin genes between normal and diarrheic dogs. PMID:23277693

  15. Rapid Technique for the Enumeration of Clostridium perfringens

    PubMed Central

    Marshall, Robert S.; Steenbergen, J. Frank; McClung, L. S.

    1965-01-01

    A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar. PMID:14339262

  16. Suspected neurotoxicity due to Clostridium perfringens type B in a tiger (Panthera tigris).

    PubMed

    Zeira, Offer; Briola, Chiara; Konar, Martin; Dumas, Maria Pia; Wrzosek, Marcin Adam; Papa, Valentina

    2012-09-01

    A 4-yr-old tiger (Panthera tigris) was referred with acute onset of severe abnormal consciousness. Neurological evaluation showed normal palpebral and corneal reflexes, normal pupil diameter with normal direct and consensual papillary light reflex, and absent menace response bilaterally. Diffuse forebrain lesion or focal lesion affecting the ascending reticular activating system was suspected. Complete blood examination and cerebrospinal fluid analysis were normal. Magnetic resonance imaging of the brain showed an empty sella as the only result. Clostridium perfringens 10(4) to 10(7) colony-forming units/g were detected in fecal flora samples. Multiplex polymerase chain reaction assay identified serotype B counts with production of epsilon toxin. This toxin specifically accumulates in the central nervous system, where it causes acute neurological signs in humans, domestic animals, and wildlife. In this communication, the acute onset of neurological signs without evidence of trauma, vascular, metabolic, or inflammatory diseases may be caused by neurotoxicity due to C. perfringens. PMID:23082539

  17. Typing of Clostridium perfringens by in vitro amplification of toxin genes.

    PubMed

    Daube, G; China, B; Simon, P; Hvala, K; Mainil, J

    1994-12-01

    The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of alpha-toxin, beta-toxin, epsilon-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the l-toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the alpha-toxin gene was obtained from direct enrichments of animal intestinal contents. PMID:7822224

  18. Development and application of an oral challenge mouse model for studying Clostridium perfringens type D infection.

    PubMed

    Fernandez-Miyakawa, Mariano E; Sayeed, Sameera; Fisher, Derek J; Poon, Rachael; Adams, Vicki; Rood, Julian I; McClane, Bruce A; Saputo, Julian; Uzal, Francisco A

    2007-09-01

    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. PMID:17562765

  19. Tips to Prevent Illness from Clostridium Perfringens

    MedlinePlus

    ... that is often found on raw meat and poultry, and is one of the most common causes ... are common food sources of C. perfringens ? Beef, poultry, gravies, and dried or precooked foods are common ...

  20. [Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica].

    PubMed

    Gamboa-Coronado, María del Mar; Mau-Inchaustegui, Silvia; Rodríguez-Cavallini, Evelyn

    2011-12-01

    Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins alpha, beta, epsilon and iota, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80 degrees C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils, 20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only alpha toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments. PMID:22208067

  1. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens

    PubMed Central

    Silva, R. O. S.; Salvarani, F.M.; Assis, R.A.; Martins, N.R.S.; Pires, P.S.; Lobato, F.C.F.

    2009-01-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  2. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens.

    PubMed

    Silva, R O S; Salvarani, F M; Assis, R A; Martins, N R S; Pires, P S; Lobato, F C F

    2009-04-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  3. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  4. Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production.

    PubMed

    Li, Jihong; Paredes-Sabja, Daniel; Sarker, Mahfuzur R; McClane, Bruce A

    2016-06-01

    The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold, and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involve their production of a variant small acid-soluble protein-4 that binds more tightly to spore DNA than to the small acid-soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the United States. During this foodborne disease, C. perfringens is ingested with food and then, by using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines. PMID:27337447

  5. Genomic analyses of Clostridium perfringens isolates from five toxinotypes.

    PubMed

    Hassan, Karl A; Elbourne, Liam D H; Tetu, Sasha G; Melville, Stephen B; Rood, Julian I; Paulsen, Ian T

    2015-05-01

    Clostridium perfringens can be isolated from a range of environments, including soil, marine and fresh water sediments, and the gastrointestinal tracts of animals and humans. Some C. perfringens strains have attractive industrial applications, e.g., in the degradation of waste products or the production of useful chemicals. However, C. perfringens has been most studied as the causative agent of a range of enteric and soft tissue infections of varying severities in humans and animals. Host preference and disease type in C. perfringens are intimately linked to the production of key extracellular toxins and on this basis toxigenic C. perfringens strains have been classified into five toxinotypes (A-E). To date, twelve genome sequences have been generated for a diverse collection of C. perfringens isolates, including strains associated with human and animal infections, a human commensal strain, and a strain with potential industrial utility. Most of the sequenced strains are classified as toxinotype A. However, genome sequences of representative strains from each of the other four toxinotypes have also been determined. Analysis of this collection of sequences has highlighted a lack of features differentiating toxinotype A strains from the other isolates, indicating that the primary defining characteristic of toxinotype A strains is their lack of key plasmid-encoded extracellular toxin genes associated with toxinotype B to E strains. The representative B-E strains sequenced to date each harbour many unique genes. Additional genome sequences are needed to determine if these genes are characteristic of their respective toxinotypes. PMID:25445567

  6. Growth potential of Clostridium perfringens during cooling of cooked meats.

    PubMed

    Taormina, Peter J; Dorsa, Warren J

    2004-07-01

    Many meat-based food products are cooked to temperatures sufficient to inactivate vegetative cells of Clostridium perfringens, but spores of this bacterium can survive, germinate, and grow in these products if sufficient time, temperature, and other variables exist. Because ingestion of large numbers of vegetative cells can lead to concomitant sporulation, enterotoxin release in the gastrointestinal tract, and diarrhea-like illness, a necessary food safety objective is to ensure that not more than acceptable levels of C. perfringens are in finished products. As cooked meat items cool they will pass through the growth temperature range of C. perfringens (50 to 15 degrees C). Therefore, an important step in determining the likely level of C. perfringens in the final product is the estimation of growth of the pathogen during cooling of the cooked product. Numerous studies exist dealing with just such estimations, yet consensual methodologies, results, and conclusions are lacking. There is a need to consider the bulk of C. perfringens work relating to cooling of cooked meat-based products and attempt to move toward a better understanding of the true growth potential of the organism. This review attempts to summarize observations made by researchers and highlight variations in experimental approach as possible explanations for different outcomes. An attempt is also made here to identify and justify optimal procedures for conducting C. perfringens growth estimation in meat-based cooked food products during cooling. PMID:15270517

  7. Hazard analysis of Clostridium perfringens in the Skylab Food System

    NASA Technical Reports Server (NTRS)

    Bourland, C. T.; Huber, C. S.; Kiser, P. R.; Heidelbaugh, N. D.; Rowley, D. B.

    1974-01-01

    The Skylab Food System presented unique microbiological problems because food was warmed in null-gravity and because the heat source was limited to 69.4 C (to prevent boiling in null-gravity). For these reasons, the foods were manufactured using critical control point techniques of quality control coupled with appropriate hazard analyses. One of these hazard analyses evaluated the threat from Clostridium perfringens. Samples of food were inoculated with C. perfringens and incubated for 2 h at temperatures ranging from 25 to 55 C. Generation times were determined for the foods at various temperatures. Results of these tests were evaluated taking into consideration: food-borne disease epidemiology, the Skylab food manufacturing procedures, and the performance requirements of the Skylab Food System. Based on this hazard analysis, a limit for C. perfringens of 100/g was established for Skylab foods.

  8. Clostridium perfringens Sepsis and Fetal Demise after Genetic Amniocentesis

    PubMed Central

    Hendrix, Nancy W.; Mackeen, A. Dhanya; Weiner, Stuart

    2011-01-01

    Clostridium perfringens is a rare cause of intrauterine infection. There have been five case reports concerning infection associated with invasive procedures. We report a woman who underwent a genetic amniocentesis due to her history of chronic granulomatous disease. She presented to the hospital ∼38 hours after the amniocentesis complaining of fever and chills. Due to acute decompensation, she underwent an emergent dilatation and evacuation. During her stay, blood cultures came back positive for C. perfringens. Gradual improvement with intensive monitoring led to hospital discharge 4 days after the procedure. Uterine infection due to C. perfringens leading to maternal sepsis is associated with a high morbidity and mortality rate. Our patient was able to survive without a hysterectomy due to the rapid administration of antibiotics and surgical intervention while being evaluated. PMID:23705080

  9. Expression and delivery of an endolysin to combat Clostridium perfringens.

    PubMed

    Gervasi, Teresa; Horn, Nikki; Wegmann, Udo; Dugo, Giacomo; Narbad, Arjan; Mayer, Melinda J

    2014-03-01

    Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract. PMID:23942878

  10. Comparison of media for enumeration of Clostridium perfringens from foods.

    PubMed

    de Jong, A E I; Eijhusen, G P; Brouwer-Post, E J F; Grand, M; Johansson, T; Kärkkäinen, T; Marugg, J; in't Veld, P H; Warmerdam, F H M; Wörner, G; Zicavo, A; Rombouts, F M; Beumer, R R

    2003-09-01

    Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin polymyxin sulfadiazine perfringens (OPSP) agar] were compared in a prestudy, of which four (IS, TSC, SCA, and DCA) were selected for an international collaborative trial. Recovery of 15 pure strains was tested in the prestudy and recovery of one strain from foodstuffs was tested in the collaborative trial. Results from the prestudy did reveal statistical difference of the media but recoveries on all media were within the microbiological limits (+/-30%) of IS, which was set as a reference medium. Recoveries on the media tested in the collaborative trial were statistically different as well, but these differences were of no microbiological-analytical relevance. Food matrices did not affect the recovery of C. perfringens in general. DCA and SCA, in particular, are labor-intensive to prepare and DCA frequently failed to produce black colonies; gray colonies were quite common. Since IS medium is nonselective, it was concluded that TSC was the most favorable medium for the enumeration of C. perfringens from foods. PMID:12842482

  11. Method for Estimating the Presence of Clostridium perfringens in Food

    PubMed Central

    Harmon, S. M.; Kautter, D. A.

    1970-01-01

    The methods currently used for the enumeration of Clostridium perfringens in food are often inadequate because of the rapid loss of viability of this organism when the sample is frozen or refrigerated. A method for estimating the presence of C. perfringens in food which utilizes the hemolytic and lecithinase activities of alpha toxin was developed. The hemolytic activity was measured in hemolysin indicator plates. Lecithinase activity of the extract was determined by the lecithovitellin test. Of 34 strains of C. perfringens associated with foodborne disease outbreaks, 32 produced sufficient alpha toxin in roast beef with gravy and in chicken broth to permit a reliable estimate of growth in these foods. Alpha toxin was extracted from food with 0.4 m saline buffered (at pH 8.0) with 0.05 mN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and concentrated by dialysis against 30% polyethylene glycol. A detectable quantity of alpha toxin was produced by approximately 106C. perfringens cells per g of substrate, and the amount increased in proportion to the cell population. Results obtained with food samples responsible for gastroenteritis in humans indicate that a correlation can be made between the amount of alpha toxin present and previous growth of C. perfringens in food regardless of whether the organisms are viable when the examination is performed. Images PMID:4321712

  12. Modeling growth of Clostridium perfringens in pea soup during cooling.

    PubMed

    de Jong, Aarieke E I; Beumer, Rijkel R; Zwietering, Marcel H

    2005-02-01

    Clostridium perfringens is a pathogen that mainly causes food poisoning outbreaks when large quantities of food are prepared. Therefore, a model was developed to predict the effect of different cooling procedures on the growth of this pathogen during cooling of food: Dutch pea soup. First, a growth rate model based on interpretable parameters was used to predict growth during linear cooling of pea soup. Second, a temperature model for cooling pea soup was constructed by fitting the model to experimental data published earlier. This cooling model was used to estimate the effect of various cooling environments on average cooling times, taking into account the effect of stirring and product volume. The growth model systematically overestimated growth of C. perfringens during cooling in air, but this effect was limited to less than 0.5 log N/ml and this was considered to be acceptable for practical purposes. It was demonstrated that the growth model for C. perfringens combined with the cooling model for pea soup could be used to sufficiently predict growth of C. perfringens in different volume sizes of pea soup during cooling in air as well as the effect of stirring, different cooling temperatures, and various cooling environments on the growth of C. perfringens in pea soup. Although fine-tuning may be needed to eliminate inaccuracies, it was concluded that the combined model could be a useful tool for designing good manufacturing practices (GMP) procedures. PMID:15787757

  13. Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease

    PubMed Central

    Rumah, Kareem Rashid; Linden, Jennifer; Fischetti, Vincent A.; Vartanian, Timothy

    2013-01-01

    We have isolated Clostridium perfringens type B, an epsilon toxin-secreting bacillus, from a young woman at clinical presentation of Multiple Sclerosis (MS) with actively enhancing lesions on brain MRI. This finding represents the first time that C. perfringens type B has been detected in a human. Epsilon toxin’s tropism for the blood-brain barrier (BBB) and binding to oligodendrocytes/myelin makes it a provocative candidate for nascent lesion formation in MS. We examined a well-characterized population of MS patients and healthy controls for carriage of C. perfringens toxinotypes in the gastrointestinal tract. The human commensal Clostridium perfringens type A was present in approximately 50% of healthy human controls compared to only 23% in MS patients. We examined sera and CSF obtained from two tissue banks and found that immunoreactivity to ETX is 10 times more prevalent in people with MS than in healthy controls, indicating prior exposure to ETX in the MS population. C. perfringens epsilon toxin fits mechanistically with nascent MS lesion formation since these lesions are characterized by BBB permeability and oligodendrocyte cell death in the absence of an adaptive immune infiltrate. PMID:24146858

  14. Intravascular Hemolysis and Septicemia due to Clostridium perfringens Emphysematous Cholecystitis and Hepatic Abscesses

    PubMed Central

    Cochrane, Justin; Bland, Lacie; Noble, Mary

    2015-01-01

    Context. Clostridium perfringens septicemia is often associated with translocation from the gastrointestinal or gastrourinary tract and occurs in patients who have malignancy or are immunocompromised. Clostridium perfringens septicemia is usually fatal without early identification, source control, and antibiotics. Case. We present a case of a 65-year-old female with Clostridium perfringens septicemia secondary to emphysematous cholecystitis, with progression to hepatic abscesses. Conclusion. Septicemia secondary to Clostridium perfringens is generally fatal if not detected early. Source control with surgery or percutaneous drainage and early antibiotic therapy is imperative. Hyperbaric oxygen therapy may reduce mortality. Clinicians caring for patients with sepsis and intravascular hemolysis must have Clostridium perfringens septicemia on their differential diagnosis with a low threshold for starting antibiotics and pursuing source of infection. PMID:26229537

  15. Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat.

    PubMed

    Khan, Madiha; Nazir, Jawad; Anjum, Aftab Ahmad; Ahmad, Mansur-Ud-Din; Nawaz, Muhammad; Shabbir, Muhammad Zubair

    2015-08-01

    A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates. PMID:26243960

  16. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    PubMed Central

    Radhika, B.; Kumar, N. Vinod; Sreenivasulu, D.

    2016-01-01

    Aim: The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases. PMID:27051186

  17. Hydrolyzable and condensed tannins resistance in Clostridium perfringens.

    PubMed

    Redondo, L M; Dominguez, J E; Rabinovitz, B C; Redondo, E A; Fernández Miyakawa, M E

    2015-08-01

    Tannins added in the diet are being used to improve nutrition and health in farm animals as an alternative to antibiotic growth promoters and to control enteric clostridial diseases. However, the capacity of Clostridium perfringens to develop resistance under the selective pressure of tannins is unknown. The purpose of this study was to determine if C. perfringens possess the ability to develop resistance against tannins in comparison with antimicrobial agents. Susceptibility for 7 AGPs (antimicrobial growth promoters), 9 therapeutic antimicrobials and 2 tannin based extracts was determined for 30 C. perfringens strains isolated from poultry and cattle. Two susceptible strains were selected and cultured in presence of sub-inhibitory concentrations of tannins and AGPs for resistant sub-populations selection. Tannin resistance of C. perfringens isolates from both animal species revealed no statistically significant differences in MICs (minimum inhibitory concentration). Poultry isolates showed higher MICs to several AGPs compared with cattle isolates. All isolates were susceptible to the therapeutic antimicrobials tested, but avian isolates showed a significantly lower susceptibility to these antimicrobials which was highly correlated with an increased resistance to bacitracin and others AGPs. In-vitro selection of resistant clones suggests that C. perfringens was unable to develop resistance against tannins at least compared to AGPs like bacitracin and avilamycin. Avian origin strains, which were previously exposed to antibiotics showed higher resistance, compared to cattle origin strains. These results suggest that the evolution of resistance against tannins in C. perfringens would be more difficult and slower than to the determined AGPs. PMID:26037239

  18. Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs.

    PubMed

    Marks, Stanley L; Kather, Elizabeth J

    2003-06-24

    Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of perfringens isolates tested had an MIC for ampicillin of perfringens isolates had an MIC of >or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution. PMID:12742714

  19. Membrane filter enumeration method for Clostridium perfringens.

    PubMed Central

    Bisson, J W; Cabelli, V J

    1979-01-01

    A membrane filter procedure has been developed for the rapid quantitation of C. perfringens in the aquatic environment. Background growth is inhibited by the use of D-cycloserine, polymyxin B sulfate, and incubation at 45 degrees C. Differential characteristics include the fermentation of sucrose, production of acid phosphatase, and the absence of beta-D-glucosidase activity. The medium is prepared as follows (in grams per 100 ml of distilled water): tryptose, 3.0; yeast extract, 2.0; sucrose, 0.5; L-cysteine, 0.1; MgSO4. 7H2O, 0.01; bromocresol purple, 0.004; and agar, 1.5. The ingredients are dissolved, and the pH is adjusted to 7.6. After autoclaving at 121 degrees C for 15 min, the medium is allowed to cool at 50 degrees C, and the following are added per 100 ml: D-cycloserine, 40 mg; polymyxin B sulfate, 2.5 mg; indoxyl-beta-D-glucoside, 60 mg; 2.0 ml of a filter-sterilized 0.5% phenolpthalein diphosphate solution; and 0.2 ml of a filter-sterilized 4.5% FeCl3.6H2O solution. Enumeration of C. perfringens in a water sample is completed within 18 to 24 h. The verification of typical colonies was 93%. The average recovery from peptone-water spore suspensions of five strains was 79%, and that from filter-sterilized seawater suspensions was 90%. The precision of the method was approximately equal to that expected from random error alone. Confirmed recoveries of C. perfringens from water and sewage samples generally were greater than those by the Bonde pour tube method. PMID:216310

  20. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

  1. Growth of Clostridium perfringens in cooked chili during cooling.

    PubMed Central

    Blankenship, L C; Craven, S E; Leffler, R G; Custer, C

    1988-01-01

    U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili. PMID:2898919

  2. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications

    PubMed Central

    Freedman, John C.; Shrestha, Archana; McClane, Bruce A.

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. PMID:26999202

  3. Beneficial effect of catalase treatment on growth of Clostridium perfringens.

    PubMed Central

    Harmon, S M; Kautter, D A

    1976-01-01

    Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media. PMID:185958

  4. Occurrence of Clostridium perfringens from different cultivated soils.

    PubMed

    Voidarou, C; Bezirtzoglou, E; Alexopoulos, A; Plessas, S; Stefanis, C; Papadopoulos, I; Vavias, S; Stavropoulou, E; Fotou, K; Tzora, A; Skoufos, I

    2011-12-01

    The occurrence of Clostridium perfringens was estimated in 750 samples originated from a variety of soils bearing various bulb crops: Brawnica oderacea (vegetable), Olea europaea, Daucus carota (carote), Solanum tuberosum (potato), Phaseolus vulgaris (green haricot), Beta vulgaris var. rapaceum (beetroot), Cucurbita pepo (squash), Allium cepa (onion), Cucumis sativus (cucumber) and Capsicum annum (pepper). All isolated strains were tested for their antimicrobial activities to amoxicillin, penicillin G, kanamycin, tetracycline, streptomycin, erythromycin, chloramphenicol and metronidazole. When considering the type of the bulb production, it was observed increased number of C. perfringens spore densities in the most undersurface bulb soils. Moreover, C. perfringens spore are likely to occur in particularly large numbers in soil contaminated by fecal matter. Additionally, there is a close relationship between the spore amount and nature of organic content. Presence of C. perfringens was associated with acidic soil. Most of our strains showed resistance to the studied antibiotics applied usually for human and veterinary care. A systematic monitoring of the cultivated soil ecosystems must include bacteriological parameters together with chemical indices of organic pollution in order to obtain information adequate for assessing their overall quality. PMID:21621626

  5. Pathology of Clostridium perfringens type C enterotoxemia in horses.

    PubMed

    Diab, S S; Kinde, H; Moore, J; Shahriar, M F; Odani, J; Anthenill, L; Songer, G; Uzal, F A

    2012-03-01

    Clostridium perfringens type C is an important cause of enteritis and enterocolitis in foals and occasionally in adult horses. The disease is a classic enterotoxemia, and the enteric lesions and systemic effects are caused primarily by beta toxin, 1 of 2 major toxins produced by C. perfringens type C. Until now, only sporadic cases of C. perfringens type C equine enterotoxemia have been reported. We present a comprehensive description of the lesions in 8 confirmed cases of type C enterotoxemia in foals and adult horses. Grossly, multifocal to segmental hemorrhage and thickening of the intestinal wall were most common in the small intestine, although the colon and cecum were also frequently affected. All horses had variable amounts of fluid, often hemorrhagic intestinal contents. The most characteristic microscopic lesion was necrotizing or necrohemorrhagic enteritis, with mucosal and/or submucosal thrombosis. Numerous gram-positive rods were occasionally seen in affected mucosa. A definitive diagnosis of C. perfringens type C enterotoxemia in all 8 cases was based on the clinical history, gross and histologic lesions, and detection of the beta toxin in intestinal contents. PMID:21502373

  6. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications.

    PubMed

    Freedman, John C; Shrestha, Archana; McClane, Bruce A

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. PMID:26999202

  7. Ulcerative enterocolitis in two goats associated with enterotoxin- and beta2 toxin-positive Clostridium perfringens type D.

    PubMed

    Uzal, Francisco A; Fisher, Derek J; Saputo, Juliann; Sayeed, Sameera; McClane, Bruce A; Songer, Glenn; Trinh, Hien T; Fernandez Miyakawa, Mariano E; Gard, Sharon

    2008-09-01

    Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa. PMID:18776108

  8. Activation and injury of Clostridium perfringens spores by alcohols.

    PubMed Central

    Craven, S E; Blankenship, L C

    1985-01-01

    The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation. PMID:2864897

  9. Recent insights into Clostridium perfringens beta-toxin.

    PubMed

    Nagahama, Masahiro; Ochi, Sadayuki; Oda, Masataka; Miyamoto, Kazuaki; Takehara, Masaya; Kobayashi, Keiko

    2015-02-01

    Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin. PMID:25654787

  10. Recent Insights into Clostridium perfringens Beta-Toxin

    PubMed Central

    Nagahama, Masahiro; Ochi, Sadayuki; Oda, Masataka; Miyamoto, Kazuaki; Takehara, Masaya; Kobayashi, Keiko

    2015-01-01

    Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin. PMID:25654787

  11. Prevalence and characterization of Clostridium perfringens from spices in Argentina.

    PubMed

    Aguilera, Milton Osmar; Stagnitta, Patricia Virginia; Micalizzi, Blas; de Guzmán, Ana María Stefanini

    2005-12-01

    Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high

  12. [Massive intravascular hemolysis secondary to sepsis due to Clostridium perfringens].

    PubMed

    Pita Zapata, E; Sarmiento Penide, A; Bautista Guillén, A; González Cabano, M; Agulla Budiño, J A; Camba Rodríguez, M A

    2010-05-01

    Massive hemolysis secondary to sepsis caused by Clostridium perfringens is a rare entity but appears fairly often in the literature. In nearly all published reports, the clinical course is rapid and fatal. We describe the case of a 75-year-old woman with diabetes who was admitted with symptoms consistent with acute cholecystitis. Deteriorating hemodynamics and laboratory findings were consistent with intravascular hemolysis, coagulation disorder, and renal failure. Gram-positive bacilli of the Clostridium species were detected in blood along with worsening indicators of hemolysis. In spite of antibiotic and surgical treatment, hemodynamic support and infusion of blood products, the patient continued to decline and died in the postoperative recovery unit 14 hours after admission. Mortality ranges from 70% to 100% in sepsis due to Clostridium perfringens, and risk of death is greater if massive hemolysis is present, as in the case we report. Only a high degree of clinical suspicion leading to early diagnosis and treatment can improve the prognosis. This bacterium should therefore be considered whenever severe sepsis and hemolysis coincide. PMID:20527348

  13. Host cell-induced signaling causes Clostridium perfringens to upregulate production of toxins important for intestinal infections

    PubMed Central

    Chen, Jianming; Ma, Menglin; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    Clostridium perfringens causes enteritis and enterotoxemia in humans and livestock due to prolific toxin production. In broth culture, C. perfringens uses the Agr-like quorum sensing (QS) system to regulate production of toxins important for enteritis/enterotoxemia, including beta toxin (CPB), enterotoxin, and epsilon toxin (ETX). The VirS/VirR two-component regulatory system (TCRS) also controls CPB production in broth cultures. Both the Agr-like QS and VirS/VirR systems are important when C. perfringens senses enterocyte-like Caco-2 cells and responds by upregulating CPB production; however, only the Agr-like QS system is needed for host cell-induced ETX production. These in vitro observations have pathophysiologic relevance since both the VirS/VirR and Agr-like QS signaling systems are required for C. perfringens strain CN3685 to produce CPB in vivo and to cause enteritis or enterotoxemia. Thus, apparently upon sensing its presence in the intestines, C. perfringens utilizes QS and TCRS signaling to produce toxins necessary for intestinal virulence. PMID:24061146

  14. Host cell-induced signaling causes Clostridium perfringens to upregulate production of toxins important for intestinal infections.

    PubMed

    Chen, Jianming; Ma, Menglin; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    Clostridium perfringens causes enteritis and enterotoxemia in humans and livestock due to prolific toxin production. In broth culture, C. perfringens uses the Agr-like quorum sensing (QS) system to regulate production of toxins important for enteritis/enterotoxemia, including beta toxin (CPB), enterotoxin, and epsilon toxin (ETX). The VirS/VirR two-component regulatory system (TCRS) also controls CPB production in broth cultures. Both the Agr-like QS and VirS/VirR systems are important when C. perfringens senses enterocyte-like Caco-2 cells and responds by upregulating CPB production; however, only the Agr-like QS system is needed for host cell-induced ETX production. These in vitro observations have pathophysiologic relevance since both the VirS/VirR and Agr-like QS signaling systems are required for C. perfringens strain CN3685 to produce CPB in vivo and to cause enteritis or enterotoxemia. Thus, apparently upon sensing its presence in the intestines, C. perfringens utilizes QS and TCRS signaling to produce toxins necessary for intestinal virulence. PMID:24061146

  15. Clostridium perfringens enterotoxicosis in two Amur leopards (Panthera pardus orientalis).

    PubMed

    Neiffer, D L

    2001-03-01

    Two 6-yr-old male sibling Amur leopards (Panthera pardus orientalis) housed together at the Pittsburgh Zoo presented for acute onset of diarrhea with no changes in appetite or behavior. Heat-fixed modified Wright-stained and Gram-stained fecal smears revealed a mixed bacterial population with a large number of gram-positive Clostridium perfringens-like spores (>20 per high-power oil immersion field). In addition, C. perfringens enterotoxin was isolated from one leopard at 1:256, confirming the presence of C. perfringens enterotoxicosis. Treatment with oral metronidazole, tylosin tartrate, and psyllium fiber was prescribed, with return of more normal stool by the third day of treatment. Fecal consistency steadily improved and was considered normal by the time all prescribed treatments were complete. Diarrhea has not recurred. Partially thawed meat in the leopards' diet may have precipitated the production of an endogenous clostridial enterotoxicosis by disrupting digestive tract flora with resultant clostridial overgrowth and sporulation. PMID:12790411

  16. Effect of cooling on Clostridium perfringens in pea soup.

    PubMed

    de Jong, A E I; Rombouts, F M; Beumer, R R

    2004-02-01

    Foods associated with Clostridium perfringens outbreaks are usually abused after cooking. Because of their short generation times, C. perfringens spores and cells can grow out to high levels during improper cooling. Therefore, the potential of C. perfringens to multiply in Dutch pea soup during different cooling times was investigated. Tubes of preheated pea soup (50 degrees C) were inoculated with cocktails of cells or heat-activated spores of this pathogen. The tubes were linearly cooled to 15 degrees C in time spans of 3, 5, 7.5, and 10 h and were subsequently stored in a refrigerator at 3 or 7 degrees C for up to 84 h. Cell numbers increased by 1-log cycle during the 3-h cooling period and reached their maximum after 10 h of cooling. Subsequent refrigeration hardly reduced cell numbers. Cooling of 3.75 liters of pea soup in an open pan showed that this amount of pea soup cooled from 50 to 15 degrees C in 5 h, which will allow a more than 10-fold increase in cell numbers. These findings emphasize the need of good hygienic practices and quick cooling of heated foods after preparation. PMID:14968969

  17. Comparison of Media for the Enumeration of Clostridium perfringens

    PubMed Central

    Harmon, Stanley M.; Kautter, Donald A.; Peeler, James T.

    1971-01-01

    For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 104 organisms per g, but with 106 organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested. PMID:4324885

  18. Case of Clostridium perfringens bacteremia after routine colonoscopy and polypectomy.

    PubMed

    Kunz, Anjali N; Riera, Diana; Hickey, Patrick

    2009-10-01

    Bacteremia is an uncommon complication after polypectomy and colonoscopy. We report one of the first cases of Clostridium perfringens bacteremia after polypectomy. Our patient was a four years old boy with congenital polyposis, who underwent colonoscopy and polypectomy without complication. Approximately 12h later he developed a fever and tachycardia with no other clinical symptoms. His blood cultures grew out penicillin susceptible C. perfringens and Enterococcus faecalis. He responded to antibiotic therapy and remained clinically asymptomatic for the duration of his course. There are a few reports of bacteremia after routine polypectomy, but no reported cases of C. perfringens bacteremia in the pediatric population. Clostridial sp. bacteremia can be fatal with devastating consequences if appropriate antibiotics and/or surgical debridement are delayed. Polymicrobial infection, as illustrated in our patient, is also common and can be a poor prognostic risk factor. Therefore, for patients with a history of polypectomy and new onset fever, anaerobic infections should be considered and empiric antibiotic therapy should include coverage for these organisms. PMID:19324098

  19. Clostridium perfringens Type E Virulence Traits Involved in Gut Colonization

    PubMed Central

    Redondo, Leandro M.; Carrasco, Juan M. Díaz; Redondo, Enzo A.; Delgado, Fernando; Miyakawa, Mariano E. Fernández

    2015-01-01

    Clostridium perfringens type E disease in ruminants has been characterized by hemorrhagic enteritis or sudden death. Although type E isolates are defined by the production of alpha and iota toxin, little is known about the pathogenesis of C. perfringens type E infections. Thus far, the role of iota toxin as a virulence factor is unknown. In this report, iota toxin showed positive effects on adherence and colonization of C. perfringens type E while having negative effect on the adherence of type A cells. In-vitro and in-vivo models suggest that toxinotype E would be particularly adapted to exploit the changes induced by iota toxin in the surface of epithelial cells. In addition, type E strains produce metabolites that affected the growth of potential intra-specific competitors. These results suggest that the alteration of the enterocyte morphology induced by iota toxin concomitantly with the specific increase of type E cell adhesion and the strong intra-specific growth inhibition of other strains could be competitive traits inherent to type E isolates that improve its fitness within the bovine gut environment. PMID:25799452

  20. Regulation of toxin gene expression in Clostridium perfringens.

    PubMed

    Ohtani, Kaori; Shimizu, Tohru

    2015-05-01

    The Gram-positive, anaerobic, spore-forming, rod-shaped Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tract of humans and animals. C. perfringens causes clostridial myonecrosis (or gas gangrene), enteritis and enterotoxemia in humans and livestock by producing numerous extracellular toxins and enzymes. The toxin gene expression is regulated by a two-component regulatory system and regulatory RNA VirR/VirS-VR-RNA cascade. The VirR/VirS system was originally found in a type A strain, but a recent report showed that it is also important for the toxin gene regulation in other types of strains. Two types of cell-cell signaling, i.e., agr-system and AI-2 signaling, are also important for the regulation of toxin genes. Several regulatory systems independent from the VirR/VirS system, including virX, the orphan histidine kinase ReeS and orphan response regulator RevR, are also involved in the regulation of toxin genes. In addition, the expression of toxin genes is upregulated after contact with Caco-2 cells. C. perfringens has a complex regulatory network for toxin gene expression and thus the coordination of toxin gene expression is important for the process of infection. PMID:25303832

  1. Effect of heat treatment on the performance of tryptose-sulfite-cycloserine agar for enumeration of Clostridium perfringens.

    PubMed

    Brodsky, M H; Ciebin, B W

    1979-05-01

    Dissolving dehydrated tryptose-sulfite-cycloserine agar by only boiling or microwaving was found to inhibit Clostridium perfringens colony development in pour plates when compared with C. perfringens recovery in tryptose-sulfite-cycloserine agar prepared by autoclaving. PMID:225988

  2. The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep.

    PubMed

    Uzal, F A; Kelly, W R; Morris, W E; Bermudez, J; Baisón, M

    2004-09-01

    The pathological findings in sheep with peracute experimental Clostridium perfringens type D enterotoxemia are described. Of 16 animals inoculated intraduodenally with a whole culture of this microorganism and a starch solution in the abomasum, 12 developed clinical signs including increased respiratory efforts, recumbency, paddling, bleating, convulsions, blindness, and opisthotonus. Diarrhea was not observed in any of the animals. The time lapse between the beginning of intraduodenal infusion and onset of clinical signs varied between 30 minutes and 26 hours, and the clinical course varied between 1 and 9 hours. Gross postmortem changes were observed in these 12 animals and included pulmonary edema; excess pericardial, peritoneal, or pleural fluid with or without strands of fibrin; liquid small intestinal contents; leptomeningeal edema; cerebellar coning; and subcapsular petechiae on kidneys. Histological changes consisted of severe edema of pleura and interlobular septa and around blood vessels and airways and acidophilic, homogeneous, proteinaceous perivascular edema in the brain. Five of 12 animals (42%) with clinical signs consistent with enterotoxemia lacked specific histological lesions in the brain. None of the intoxicated or control animals developed nephrosis. Glucose was detected in the urine of 3 of 6 animals that were tested for this analyte. These results stress the importance of the use of histological examination of the brain, coupled with epsilon toxin detection, for a definitive diagnosis of C. perfringens type D enterotoxemia in sheep. PMID:15460322

  3. Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef.

    PubMed

    Emswiler, B S; Pierson, C J; Kotula, A W

    1977-03-01

    The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef. PMID:16345236

  4. Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef

    PubMed Central

    Emswiler, B. S.; Pierson, C. J.; Kotula, A. W.

    1977-01-01

    The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef. PMID:16345236

  5. Effect of Lysozyme on Ionic Forms of Spores of Clostridium perfringens Type A

    PubMed Central

    Ando, Yoshiaki

    1975-01-01

    H spores of Clostridium perfringens type A (two strains) were more sensitive to germination by lysozyme than native spores. Resistance to lysozyme of H spores was restored by calcium loading. PMID:236284

  6. Meningoencephalitis with Subdural Empyema Caused by Toxigenic Clostridium perfringens Type A

    PubMed Central

    Achermann, Yvonne; Kovari, Helen; Dent, Wolfgang; Hombach, Michael; Bloemberg, Guido

    2012-01-01

    We report a clinical case of meningoencephalitis with subdural empyema in an immunocompromised farmer caused by toxigenic Clostridium perfringens type A, which was identified by 16S RNA gene analysis of cerebrospinal fluid and subdural empyema. In immunocompromised patients, C. perfringens should be considered a potential pathogen of sepsis. PMID:22895036

  7. Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining tra...

  8. Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in C. perfringens isolates ...

  9. Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef.

    PubMed Central

    Baez, L A; Juneja, V K

    1995-01-01

    A DNA probe endolabeled with digoxigenin by PCR was developed to detect and enumerate enterotoxigenic Clostridium perfringens in raw beef. After 2 h of hybridization, membranes were developed by using an anti-digoxigenin-alkaline phosphatase conjugated antibody. The resulting chromogenic reaction allowed us to detect and enumerate < or = 10 CFU of C. perfringens per g. PMID:7574619

  10. The Genome Sequence of Bacteriophage CPV1 Virulent for Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents. P...

  11. Potential for growth of Clostridium perfringens from spores in pork scrapple during cooling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We conducted stabilization studies to determine the ability of Clostridium perfringens spores to germinate and grow during exponential cooling of a commercial formulation of pork scrapple. Scrapple was inoculated with a mixture of three strains of C. perfringens spores (NTCC 8238, NCTC 8239, and AT...

  12. THE GENOME SEQUENCE OF BACTERIOPHAGE CpV1 LYTIC FOR CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of bacteriophages and their lytic enzymes to control Clostri-dium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. We have established a collection of 30 bacteriophages lytic for C. perfringens. These were isolated from s...

  13. Clostridium perfringens in Long Island Sound sediments: An urban sedimentary record

    USGS Publications Warehouse

    Buchholtz ten Brink, M. R.; Mecray, E.L.; Galvin, E.L.

    2000-01-01

    Clostridium perfringens is a conservative tracer and an indicator of sewage-derived pollution in the marine environment. The distribution of Clostridium perfringens spores was measured in sediments from Long Island Sound, USA, as part of a regional study designed to: (1) map the distribution of contaminated sediments; (2) determine transport and dispersal paths; (3) identify the locations of sediment and contaminant focusing; and (4) constrain predictive models. In 1996, sediment cores were collected at 58 stations, and surface sediments were collected at 219 locations throughout the Sound. Elevated concentrations of Clostridium perfringens in the sediments indicate that sewage pollution is present throughout Long Island Sound and has persisted for more than a century. Concentrations range from undetectable amounts to 15,000 spores/g dry sediment and are above background levels in the upper 30 cm at nearly all core locations. Sediment focusing strongly impacts the accumulation of Clostridium perfringens spores. Inventories in the cores range from 28 to 70,000 spores/cm2, and elevated concentrations can extend to depths of 50 cm. The steep gradients in Clostridium perfringens profiles in muddier cores contrast with concentrations that are generally constant with depth in sandier cores. Clostridium perfringens concentrations rarely decrease in the uppermost sediment, unlike those reported for metal contaminants. Concentrations in surface sediments are highest in the western end of the Sound, very low in the eastern region, and intermediate in the central part. This pattern reflects winnowing and focusing of Clostridium perfringens spores and fine-grained sediment by the hydrodynamic regime; however, the proximity of sewage sources to the westernmost Sound locally enhances the Clostridium perfringens signals.

  14. Effect of tannins on the in vitro growth of Clostridium perfringens.

    PubMed

    Elizondo, Ana M; Mercado, Elsa C; Rabinovitz, Bettina C; Fernandez-Miyakawa, Mariano E

    2010-10-26

    Vegetable tannins are water-soluble polyphenolic compounds of varying molecular weights that occur abundantly in nature. The diet of many free-ranging wild animals contains significant amounts of tannins. Also, commercial tannins are used in animal industry as food additives to improve animal performance. In order to further determine the capacity of tannins to inhibit the development of intestinal diseases produced by Clostridium pefringens, we evaluated here the effect of tannins from quebracho, chestnut or combinations of both on C. perfringens and their toxins. The C. perfringens (types A, B, C, D and E) growth obtained from the intestine of healthy and diseased animals was reduced in a dose-dependent manner in the presence of quebracho tannins, chestnut tannins, combinations of both or a commercial formula based in these tannins. Although the minimal inhibitory concentration of both tannins varied between isolates, no statistically significant differences were observed between isolates from healthy or sick animals. Comparative analysis showed that the concentrations of quebracho tannin inhibiting the growth of C. perfringens were higher than chestnut tannin. In fact, antibacterial effect of quebracho tannin was increased up to 20 times with the addition of 25% of chestnut tannin and 85 times with 75% of chestnut tannin. Antibacterial activity of the commercial product was up to ~50 times higher than quebracho tannin alone. Quebracho tannin showed partial bactericidal activity, whereas chestnut tannin activity was stronger. Both tannins were able to reduce the alpha toxin lecithinase activity and epsilon toxin cytotoxicity in MDCK cells. These results suggest that tannin-supplemented diet could be useful to prevent some clostridial diseases. PMID:20471759

  15. Clear, defined medium for the sporulation of Clostridium perfringens.

    PubMed Central

    Sacks, L E; Thompson, P A

    1978-01-01

    A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated. Images PMID:25045

  16. Cytology of Spore Formation in Clostridium perfringens1

    PubMed Central

    Hoeniger, Judith F. M.; Stuart, Philip F.; Holt, Stanley C.

    1968-01-01

    The sequential morphological events in spore formation by Clostridium perfringens type D were observed in Ellner's medium where 80 to 100% of the cells formed spores. Gross structural changes were studied with the light microscope under phase-contrast, and in fixed cells by the use of both nigrosin and Giemsa preparations. Fine structure was examined with the electron microscope in both thin sections and frozen-etched preparations. During the first 3 hr of incubation, the original rod-shaped cells became ellipsoid to ovoid in shape; by 5 to 6 hr, subterminal spores had developed within these enlarged cells. The fine structural sequence was in most respects identical to that in other Bacillaceae, although some stages were illustrated with particular clarity. A unique feature was the development of a convoluted, membranous exosporium which adhered to the outer surface of the two coats and had an unusual fine structure resembling a rectangular array of subunits. Images PMID:4302300

  17. Rabbit Ileal Loop Response to Strains of Clostridium perfringens1

    PubMed Central

    Duncan, Charles L.; Sugiyama, H.; Strong, Dorothy H.

    1968-01-01

    The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops. Images PMID:4297020

  18. Cattle enterotoxaemia and Clostridium perfringens: description, diagnosis and prophylaxis.

    PubMed

    Lebrun, M; Mainil, J G; Linden, A

    2010-07-01

    Cattle enterotoxaemia is one of numerous pathologies caused by Clostridium perfringens. These anaerobic Gram-positive bacteria are naturally present in the intestinal flora of mammals, but their uncontrolled multiplication under certain conditions results in the overproduction of toxins in the intestinal tract. Major clinical signs are induced by the systemic spread of these toxins in the blood and tissues. Enterotoxaemia may be acute or peracute, and sudden death is often reported in rapidly growing, apparently healthy cattle. Enterotoxaemia can be prevented only with better understanding of its risk factors and pathogenesis. This paper provides an up-to-date overview of knowledge concerning the aetiology of the syndrome, its epidemiological context, pathogenesis, clinical signs and lesions, the diagnostic procedures and prophylactic tools, with specific attention to field aspects that are directly relevant to practitioners and clinical researchers. PMID:20605954

  19. Characterization of toxin plasmids in Clostridium perfringens type C isolates.

    PubMed

    Gurjar, Abhijit; Li, Jihong; McClane, Bruce A

    2010-11-01

    Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence. PMID:20823204

  20. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis

    PubMed Central

    Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko; Takehara, Masaya; Nagahama, Masahiro

    2016-01-01

    Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2). All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis. PMID:26807591

  1. Structural Basis of Clostridium perfringens Toxin Complex Formation

    SciTech Connect

    Adams,J.; Gregg, K.; Bayer, E.; Boraston, A.; Smith, S.

    2008-01-01

    The virulent properties of the common human and livestock pathogen Clostridium perfringens are attributable to a formidable battery of toxins. Among these are a number of large and highly modular carbohydrate-active enzymes, including the {mu}-toxin and sialidases, whose catalytic properties are consistent with degradation of the mucosal layer of the human gut, glycosaminoglycans, and other cellular glycans found throughout the body. The conservation of noncatalytic ancillary modules among these enzymes suggests they make significant contributions to the overall functionality of the toxins. Here, we describe the structural basis of an ultra-tight interaction (Ka = 1.44 x 1011 M-1) between the X82 and dockerin modules, which are found throughout numerous C. perfringens carbohydrate-active enzymes. Extensive hydrogen-bonding and van der Waals contacts between the X82 and dockerin modules give rise to the observed high affinity. The {mu}-toxin dockerin module in this complex is positioned {approx}180 relative to the orientation of the dockerin modules on the cohesin module surface within cellulolytic complexes. These observations represent a unique property of these clostridial toxins whereby they can associate into large, noncovalent multitoxin complexes that allow potentiation of the activities of the individual toxins by combining complementary toxin specificities.

  2. Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

    PubMed Central

    Nishida, S.; Seo, N.; Nakagawa, M.

    1969-01-01

    A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting. Images PMID:4304763

  3. Heat treatment adaptations in Clostridium perfringens vegetative cells.

    PubMed

    Novak, J S; Tunick, M H; Juneja, V K

    2001-10-01

    Vegetative cells of Clostridium perfringens enterotoxigenic strains NCTC 8679, NCTC 8238. and H6 were grown at 37 degrees C followed by a 60-min exposure to 28 degrees C or 46 degrees C. D10-values, as a measure of thermal resistance at 60 degrees C, were significantly lower for 28 degrees C exposures as compared with cultures given 37 and 46 degrees C exposures. Following refrigeration at 4 degrees C for 24 h, D10-values for the 37 and 46 degrees C samples could not be differentiated from 28 degrees C samples. Western immunoblot analyses of lysates from heat-adapted cells also detected the increased expression of proteins reacting with antiserum directed against the molecular chaperonins from Escherichia coli; GroEL, DnaJ, and the small acid soluble protein from Bacillus subtilis, SspC. Differential scanning calorimetry (DSC) identified thermal transitions corresponding to ribosomal protein denaturations at 72.1 +/- 0.5 degrees C. Any cellular heat adaptations in the DSC profiles were lost following refrigeration for several days to simulate minimally processed food storage conditions. Further analyses of high-speed pellets from crude cell extract fractions using two-dimensional gel electrophoresis detected the differential gene expression of at least four major proteins in heat-adapted vegetative cells of C. perfringens. N-terminal amino acid analyses identified two of the proteins as glyceraldehyde 3-phosphate dehydrogenase and rubrerythrin. Both appear to have roles in this anaerobe under stressful conditions. PMID:11601701

  4. Membrane-Binding Mechanism of Clostridium perfringens Alpha-Toxin

    PubMed Central

    Oda, Masataka; Terao, Yutaka; Sakurai, Jun; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1–250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251–370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55–93 aa), especially H….SW84Y85….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail. PMID:26633512

  5. Diarrhea associated with enterotoxigenic Clostridium perfringens in a red-footed tortoise (Geochelone carbonaria).

    PubMed

    Weese, J S; Staempfli, H R

    2000-06-01

    Enterotoxigenic Clostridium perfringens was associated with diarrhea in a 4-yr-old female captive-bred red-footed tortoise (Geochelone carbonaria). Diagnosis was based on bacterial culture, detection of C. perfringens enterotoxin in feces, and exclusion of commonly recognized pathogens. After treatment with metronidazole, normal feces were passed and C. perfringens enterotoxin was no longer detected in the feces. Although the role of C. perfringens cannot be determined definitively from this case, this pathogen should be considered in cases of diarrhea in tortoises and, perhaps, other reptiles. PMID:10982148

  6. ICMSF methods studies. VIII. Comparative study for the enumeration of Clostridium perfringens in foods.

    PubMed

    Hauschild, A H; Gilbert, R J; Harmon, S M; O'Keeffe, M F; Vahlefeld, R

    1977-07-01

    Four methods were compared in an international comparative study for the enumeration of Clostridium perfringens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserin) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods. The confirmed C. perfringens counts were slightly lower for D than for A-C. The percentages of presumptive colonies confirmed as C. perfringens were essentially the same in each method. The relative numbers of nonspecific colonies were the lowest in C, followed by B, D, and A. The methods were also compared for simplicity and for aspects associated with the recognition and selection of presumptive colonies. PMID:195698

  7. Enumeration of fecal Clostridium perfringens spores in egg yolk-free tryptose-sulfite-cycloserine agar.

    PubMed

    Hauschild, A H; Hilsheimer, R; Griffith, D W

    1974-03-01

    The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin. PMID:4363369

  8. Enumeration of Fecal Clostridium perfringens Spores in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

    PubMed Central

    Hauschild, A. H. W.; Hilsheimer, R.; Griffith, D. W.

    1974-01-01

    The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin. PMID:4363369

  9. Detection of beta2 and major toxin genes by PCR in Clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia.

    PubMed

    Sting, Reinhard

    2009-01-01

    The production of Clostridium (C.) perfringens toxins in the intestine is an important cause of enteritis and enterotoxaemia in livestock. In the present study, the alpha toxin and the genes encoding beta2 and epsilon toxin could be frequently detected by means of phenotypical and PCR examinations in these bacteria. The C. perfringens isolates originated from 1213 field samples taken from diseased or perished livestock located in the north-eastern administrative districts of Baden-Württemberg (Germany) from 2005 to 2008. The beta2 toxin gene of C perfringens was detected in all animal species examined, comprising pigs, the small ruminants sheep and goats, cattle, horses, rabbits, alpacas and lamas, and fallow deer. Among all the animal species included in this study, pigs attracted attention by a high quota of 74.2% (610 of 822) cpb2-positive C. perfringens isolates in comparison to the other animal species tested, revealing a quota of 20.8% (72 of 346). Beta2 toxigenic isolates could be predominantly cultivated from the faeces of young piglets. The beta toxin gene was detected in isolates from piglets and small ruminants only, amounting to 82.5% (33 of 40) in piglets in combination with the cpb2 gene. In this context, cpb2/cpb-positive C. perfringens isolates of piglets could be clearly detected more often in the intestine of perished animals (18 of 158) than in faeces (15 of 629). Furthermore, cpb2-bearing C. perfringens isolates were detected in cattle, horses, rabbits, alpacas and lamas, and fallow deer to a notable degree. The detection of C. perfringens isolates carrying the epsilon toxin gene was restricted to sheep and goats. Of a total of 242 small ruminants that succumbed to sudden death, 71 (29.3%) harboured epsilon toxin-positive C. perfringens isolates in their intestines. These cases clustered seasonally in the second quarter (April, May, and June) of the year. Neither the isolates bearing the beta2 nor beta toxin gene nor those carrying the epsilon

  10. Enumeration and Isolation of cpe-Positive Clostridium perfringens Spores from Feces

    PubMed Central

    Heikinheimo, Annamari; Lindström, Miia; Korkeala, Hannu

    2004-01-01

    A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying (cpe-positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe-positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe-positive C. perfringens was successfully isolated from both of these samples. The detection of cpe-positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe-positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe-positive C. perfringens could be isolated if the ratio of cpe-positive C. perfringens spores to total C. perfringens spores was 6 × 10−5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe-positive C. perfringens. The method also offers a new approach in the investigation of the epidemiology of cpe-positive C. perfringens strains. PMID:15364981

  11. Enumeration and isolation of cpe-positive Clostridium perfringens spores from feces.

    PubMed

    Heikinheimo, Annamari; Lindström, Miia; Korkeala, Hannu

    2004-09-01

    A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying (cpe-positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe-positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe-positive C. perfringens was successfully isolated from both of these samples. The detection of cpe-positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe-positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe-positive C. perfringens could be isolated if the ratio of cpe-positive C. perfringens spores to total C. perfringens spores was 6 x 10(-5) or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe-positive C. perfringens. The method also offers a new approach in the investigation of the epidemiology of cpe-positive C. perfringens strains. PMID:15364981

  12. Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

    PubMed

    Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

    2013-10-01

    The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. PMID:23816139

  13. Isolation and characterization of Clostridium perfringens from apparently healthy animals of the Shandong province of China.

    PubMed

    Chai, T; Wang, L; Wang, H; Duan, H; Müller, W; Zucker, B A

    2007-10-01

    In a pilot study the presence and frequency of Clostridium (C.) perfringens was investigated among apparently healthy farm animals in the Shandong province of China. 748 faecal samples were collected from 9 pig-, 4 sheep-, 7 cattle- and 5 rabbit farms. C. perfringens was isolated from 124 samples (16.6%). The isolates were classified into major toxin types by using PCR analysis detecting the genes encoding these toxins. All isolates were identified as C perfringens toxin type A. There are also some reports from different regions in China linking C. perfringens toxin type A strains to gastrointestinal diseases. Therefore further investigations about the epidemiologic role of C perfringens toxin type A strains in the Shandong region are necessary. Currently, cases of enterotoxemia from this region are investigated for the presence of C perfringens. PMID:17970339

  14. Genotyping of Clostridium perfringens isolated from broiler meat in northeastern of Iran

    PubMed Central

    Afshari, Asma; Jamshidi, Abdollah; Razmyar, Jamshid; Rad, Mehrnaz

    2015-01-01

    Clostridium perfringens (C. perfringens) is an important cause of bacterial food poisoning worldwide. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe gene. The aim of this research was to identify the different types of C. perfringens and the presence of cpe gene in isolated bacteria from broilers’ meat marketed in retail meat shops of Mashhad city in Northeastern of Iran. After isolation of C. perfringens using conventional culture method and confirmation by specific 16S rDNA gene, a multiplex polymerase chain reaction assay with specific primers, were performed for toxin typing of isolates. Clostridium perfringens was isolated from 31 broilers’ meat samples (15.50%) out of 200 samples and for toxin typing the results showed 9 isolates as type A (29.03%) and 22 isolates as type C (70.96%). In this study, cpe-positive C. perfringens were detected in eight isolates of type C (25.00%). Our results indicated that C. perfringens type C is the most common type in broiler chicken carcasses. PMID:26973762

  15. Clostridium perfringens Type A Enterotoxin Damages the Rabbit Colon

    PubMed Central

    Garcia, Jorge P.; Li, Jihong; Shrestha, Archana; Freedman, John C.; Beingesser, Juliann; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease. PMID:24643537

  16. Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.

    PubMed

    Albini, S; Brodard, I; Jaussi, A; Wollschlaeger, N; Frey, J; Miserez, R; Abril, C

    2008-02-01

    Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene. PMID:17855025

  17. Enterotoxemia associated with beta2 toxin-producing Clostridium perfringens type A in two Asiatic black bears (Selenarctos thibetanus).

    PubMed

    Greco, Grazia; Madio, Anna; Martella, Vito; Campolo, Marco; Corrente, Marialaura; Buonavoglia, Domenico; Buonavoglia, Canio

    2005-03-01

    Beta2 (beta2) toxin-producing Clostridium perfringens type A strains were found to be associated with necrotic and hemorrhagic intestinal lesions in 2 Asiatic black bears (Selenarctos thibetanus) that died suddenly. Ten isolates were obtained from the liver, lungs, heart, and small and large intestine of the animals and were examined by multiplex polymerase chain reaction for the genes encoding the 4 lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types as well as for the genes encoding enterotoxin and the novel beta2-toxin for subclassification. In addition, the cpb2 sequence of the 10 isolates was different from the published sequence of cpb2 of pig type C isolate CWC245, whereas it was highly similar to the cpb2 sequence of the C. perfringens type A strain 13. This finding suggests the existence of 2 cpb2 subtypes. This is the first report of enterotoxemia associated with the presence of C. perfringens producing beta2-toxin in the tissues and intestinal content of Asiatic black bears. PMID:15825503

  18. Rapid Confirmation of Clostridium perfringens by Using Chromogenic and Fluorogenic Substrates

    PubMed Central

    Adcock, Philip W.; Saint, Christopher P.

    2001-01-01

    The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-β-d-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life. PMID:11526053

  19. Comparison of methods for the enumeration of Clostridium perfringens spores in water.

    PubMed

    Junqueira, Valéria Christina Amstalden; Neto, Romeu Cantúsio; da Silva, Neusely; Terra, Juliana Hirata

    2012-01-01

    Four methods for enumerating Clostridium perfringens spores in water were evaluated: (1) the IMM (Iron Milk Medium) method (MPN); (2) the LS (Lactose Sulfite Broth) method (MPN); (3) the m-CP (membrane filtration Clostridium perfringens Agar) method (membrane filtration); and (4) the TSC (Tryptose Sulfite Cycloserine Agar) method (membrane filtration). The performance of these methods was compared with that of the DRCM (Differential Reinforced Clostridium Medium) method (MPN) as adopted by CETESB (Brazil's Environmental Sanitation Technology Company) for the analysis of C. perfringens spores in water. Statistical analysis was performed according to ISO 17994:2004 (Water Quality - Criteria for Establishing Equivalence between Microbiological Methods). The LS, m-CP, and TSC methods were considered not equivalent to the DRCM method, as they gave significantly lower results. The IMM showed inconclusive results and, according to ISO 17994:2004, analysis of a greater number of samples is needed to draw definitive conclusions comparing IMM and DRCM. PMID:22233899

  20. Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens*

    PubMed Central

    Jongkees, Seino A. K.; Yoo, Hayoung; Withers, Stephen G.

    2014-01-01

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

  1. Mechanistic investigations of unsaturated glucuronyl hydrolase from Clostridium perfringens.

    PubMed

    Jongkees, Seino A K; Yoo, Hayoung; Withers, Stephen G

    2014-04-18

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct (1)H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

  2. Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    PubMed Central

    Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

    2012-01-01

    Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

  3. Clostridium perfringens Iota-Toxin b Induces Rapid Cell Necrosis▿

    PubMed Central

    Nagahama, Masahiro; Umezaki, Mariko; Oda, Masataka; Kobayashi, Keiko; Tone, Shigenobu; Suda, Taiji; Ishidoh, Kazumi; Sakurai, Jun

    2011-01-01

    Clostridium perfringens iota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-β-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis. PMID:21911469

  4. Diagnostic importance of Clostridium perfringens enterotoxin analysis in recurring enteritis among elderly, chronic care psychiatric patients.

    PubMed Central

    Jackson, S G; Yip-Chuck, D A; Clark, J B; Brodsky, M H

    1986-01-01

    A series of Clostridium perfringens-related gastrointestinal outbreaks occurred over a period of several months among elderly, chronic care patients in a psychiatric hospital. Several serotypes of C. perfringens and many nontypeable isolates were found. The distribution of certain serotypes and the incidence of detection of enterotoxin in fecal extracts were related to wards on which patients were resident (six wards were involved). Several patients were reported to have chronic or recurring fecal incontinence or diarrhea or both. With a background of elevated spore counts of several serotypes and chronic diarrhea, only detection of enterotoxin could provide definitive evidence of C. perfringens etiology in gastoenteritis cases. PMID:2871043

  5. A middle-aged lady with a pyogenic liver abscess caused by Clostridium perfringens

    PubMed Central

    Law, Siu-Tong; Lee, Ming Kai

    2012-01-01

    The pyogenic liver abscess caused by Clostridium perfringens (C. perfringens) is a rare, but rapidly fatal infection. It is usually associated with malignancy and immunosuppression. We report the case of 50-year-old lady with the secondary liver metastases from rectal cancer presented with fever and epigastric pain. The identification of Gram-positive bacilli septicaemia, the presence of gas-forming liver abscess and massive intravascular hemolysis should lead to the suspicion of C. perfringens infection. Here we review twenty cases published since 1990 and their clinical features are discussed. The importance of ”an aggressive treatment policy” with multidisciplinary team approach is emphasized. PMID:22993668

  6. Antimicrobial susceptibility of Clostridium perfringens isolated from piglets with or without diarrhea in Brazil

    PubMed Central

    Salvarani, Felipe Masiero; Silveira Silva, Rodrigo Otávio; Pires, Prhiscylla Sadanã; da Costa Cruz Júnior, Eduardo Coulaud; Albefaro, Isabella Silva; de Carvalho Guedes, Roberto Maurício; Faria Lobato, Francisco Carlos

    2012-01-01

    The minimum inhibitory concentration (MIC) was determined for 13 antibiotics against Clostridium perfringens isolated from Brazilian piglets. The collection of isolates was performed in June to October 2010. All isolates were susceptible to amoxicillin and ceftiofur, whereas most were resistant to tetracycline and lincomycin. Avilamycin and narasin were more effective against isolates from non-diarrheic than from diarrheic piglets. The other antimicrobials were less active in need of high concentrations to inhibit the growth of the C. perfringens type A. These results suggest the need for further studies evaluating molecular factors related to the antimicrobial resistance of C. perfringens. PMID:24031924

  7. Sudden death syndrome in adult cows associated with Clostridium perfringens type E.

    PubMed

    Redondo, L M; Farber, M; Venzano, A; Jost, B H; Parma, Y R; Fernandez-Miyakawa, M E

    2013-04-01

    Clostridium perfringens type E is considered a rare toxinotype and an infrequent cause of enterotoxemia of lambs, calves, and rabbits. Until now, only cases of young animal of C. perfringens type E bovine enterotoxemia, characterized by hemorrhagic enteritis and sudden death, have been reported. The present report details the genotypic characterization of C. perfringens type E isolates obtained from intestinal samples of adult cattle during an outbreak of enterotoxemia in Argentina. The sequences of several housekeeping genes of these isolates were analyzed and compared with those obtained from calves in North America showing a clonal unique lineage. PMID:23354004

  8. Clostridium Perfringens Infection in a Febrile Patient with Severe Hemolytic Anemia

    PubMed Central

    Hashiba, Masamitsu; Tomino, Atsutoshi; Takenaka, Nobuyoshi; Hattori, Tomonori; Kano, Hideki; Tsuda, Masanobu; Takeyama, Naoshi

    2016-01-01

    Patient: Male, 82 Final Diagnosis: Clostridium perfringens infection Symptoms: Anemia • fever • shock Medication: — Clinical Procedure: Antimicrobial chemotherapy Specialty: Infectious Diseases Objective: Rare disease Background: Clostridium perfringens (C. perfringens) can cause various infections, including gas gangrene, crepitant cellulitis, and fasciitis. While C. perfringens sepsis is uncommon, it is often rapidly fatal because the alpha toxin of this bacterium induces massive intravascular hemolysis by disrupting red blood cell membranes. Case Report: We present the case of a male patient with diabetes who developed a fatal liver abscess with massive intravascular hemolysis and septic shock caused by toxigenic C. perfringens. The peripheral blood smear showed loss of central pallor, with numerous spherocytes. Multiplex PCR only detected expression of the cpa gene, indicating that the pathogen was C. perfringens type A. Conclusions: C. perfringens infection should be considered in a febrile patient who has severe hemolytic anemia with a very low MCV, hemolyzed blood sample, and negative Coombs test. The characteristic peripheral blood smear findings may facilitate rapid diagnosis. PMID:27049736

  9. Clostridium perfringens type A enteritis in blue and yellow macaw (Ara ararauna).

    PubMed

    Guimarães, Marta Brito; Torres, Luciana Neves; Mesquita, Ramon Gomes; Ampuero, Fernanda; Cunha, Marcos Paulo Vieira; Ferreira, Thais Sebastiana Porfida; Ferreira, Antonio José Piantino; Catão-Dias, José Luiz; Moreno, Andrea Micke; Knöbl, Terezinha

    2014-12-01

    This study describes an outbreak of necrotic enteritis caused by Clostridium perfringens type A in captive macaws (Ara ararauna). Two psittacine birds presented a history of prostration and died 18 hr after manifestation of clinical signs. The necropsy findings and histopathologic lesions were indicative of necrotic enteritis. Microbiologic assays resulted in the growth of large gram-positive bacilli that were identified as C. perfringens. PCR was used to identify clostridium toxinotypes and confirmed the identification of isolated strains as C pefringens type A, positive to gene codifying beta 2 toxin. The infection source and predisposing factors could not be ascertained. PMID:25619013

  10. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    PubMed Central

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  11. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

    PubMed

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  12. Non-Clostridium perfringens infectious agents producing necrotic enteritis-like lesions in poultry.

    PubMed

    Uzal, F A; Sentíes-Cué, C G; Rimoldi, G; Shivaprasad, H L

    2016-06-01

    Necrotic enteritis (NE) produced by Clostridium perfringens is amongst the most prevalent enteric diseases of chickens and turkeys. However, several other bacterial, parasitic and viral agents can cause clinical signs, gross and microscopic lesions in poultry very similar to those of NE and the diseases produced by those agents need to be differentiated from NE. The main differential diagnoses for C. perfringens NE include bacterial (Clostridium colinum, Clostridium sordellii, Clostridium difficile, Pasteurella multocida, Brachyspira spp.), parasitic (Eimeria spp., Histomonas meleagridis) and viral (Duck Herpesvirus type 1, Avian Paramyxovirus type 1) diseases. Confirmation of the diagnosis of these diseases requires identification of the aetiological agents by morphological, cultural and/or molecular methods. PMID:27009483

  13. ICMSF methods studies. XII. Comparative study for the enumeration of Clostridium perfringens in feces.

    PubMed

    Hauschild, A H; Desmarchelier, P; Gilbert, R J; Harmon, S M; Vahlefeld, R

    1979-09-01

    As the second phase of an international comparative study for the enumeration of Clostridium perfringens, four methods were compared for "total" and spore counts of C. perfringens in fecal specimens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserine) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods. In both the total and spore count procedures, the confirmed C. perfringens counts in method D were lower than in methods A, B, and C. Little differences among methods were found in the percentages of presumptive colonies confirmed as C. perfringens. The nonspecific counts in methods A and D were generally greater than in B and C, but nonspecific microorganisms did not interfere in the enumeration of C. perfringens spores by any of the four methods. In overall performance, methods B and C were superior to A and D. The mean C. perfringens spore count was only 0.17 log lower than the mean total count. Spore counts alone are, therefore, adequate in investigations of C. perfringens outbreaks. PMID:232005

  14. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

  15. Enterotoxin formation by different toxigenic types of Clostridium perfringens.

    PubMed Central

    Skjelkvålé, R; Duncan, C L

    1975-01-01

    Sixty-nine strains of Clostridium perfringens of different toxigenic types were investigated for enterotoxin production. Enterotoxin was definitively detected only in strains of types A and C. This is the first report where enterotoxin production has been demonstrated in a toxigenic type other than type A. The exterotoxin-positive type C strains were isolated from cases of enteritis necroticans ("pig bel+) in New Guinea. The major enterotoxin from type C showed a reaction of complete identity with enterotoxin from type A in immunodiffusion using anti-enterotoxin serum prepared against the latter; it induced erythema when injected intradermally into depilated guinea pigs and caused fluid accumulation in the rabbit ileal loop. The results indicate that the major enterotoxin from type C was serologically and biologically similar to enterotoxin from type A. In some C was serologically and biologically similar to enterotoxin from type A. In some type C strains, an enterotoxin was detected that showed a reaction of partial serological identity. Spore coat proteins were extracted from 14-strains by alkaline dithiothreitol, and the extracts were assayed for enterotoxin-like spore protein. Enterotoxin could be extracted from type A and type C spores, and all positive strains showed a reaction of complete identity in immunodiffusion with enterotoxin obtained from cell extracts of type A. Disc immunoelectrophoresis demonstrated that two distinct components that reacted serologically with anti-enterotoxin serum were present in both the cell extract and in extracted spore protein from one type C strain. These distinct components differed in molecular weight. Images PMID:163799

  16. Improved Medium for Sporulation of Clostridium perfringens1

    PubMed Central

    Duncan, Charles L.; Strong, Dorothy H.

    1968-01-01

    An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na2HPO4. 7H2O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C. PMID:4295179

  17. Application of Lactobacillus johnsonii expressing phage endolysin for control of Clostridium perfringens.

    PubMed

    Gervasi, T; Lo Curto, R; Minniti, E; Narbad, A; Mayer, M J

    2014-10-01

    Clostridium perfringens is frequently found in food and the environment and produces potent toxins that have a negative impact on both human and animal health and particularly on the poultry industry. Lactobacillus johnsonii FI9785, isolated from the chicken gastrointestinal tract, has been demonstrated to exclude Cl. perfringens in poultry. We have investigated the interaction of wild-type Lact. johnsonii FI9785 or an engineered strain expressing a cell wall-hydrolysing endolysin with Cl. perfringens in vitro, using a batch culture designed to simulate human gastrointestinal tract conditions. Co-culture experiments indicated that acid production by Lact. johnsonii is important in pathogen control. The co-culture of the endolysin-secreting Lact. johnsonii with Cl. perfringens showed that the engineered strain had the potential to control the pathogen, but the ability to reduce Cl. perfringens numbers was not consistent. Results obtained indicate that survival of high numbers of Lact. johnsonii will be essential for effective pathogen control. Significance and impact of the study: The bacterium Lactobacillus johnsonii FI9785 reduces numbers of the pathogen Clostridium perfringens in vitro. Biocontrol was improved by engineering the strain to produce and export a cell wall-hydrolysing endolysin, but good survival of the producer strain is essential. The production of bacteriophage endolysins by commensal bacteria has the potential to improve competitive exclusion of pathogens in the gastrointestinal tract. PMID:24961379

  18. Rapid detection of Clostridium perfringens: comparison of lactose sulfite broth with tryptose-sulfite-cycloserine agar.

    PubMed

    Neut, C; Pathak, J; Romond, C; Beerens, H

    1985-01-01

    The lactose sulfite (LS) medium recommended for the detection and identification of Clostridium perfringens in foods was compared with a reference method using tryptose-sulfite-cycloserine (TSC) agar for the enumeration of this organism in a variety of foods and food ingredients. C. perfringens was detected and enumerated in 17 of the 54 samples examined with LS broth, but its presence could be confirmed in only 9 of the samples with TSC agar. In only 2 instances, C. perfringens was detected on TSC agar but not in LS broth. A positive response (FeS + and gas +) in LS broth incubated at 46 degrees C always corresponded to the presence of C. perfringens; whereas the black colonies formed on TSC agar incubated at 37 degrees C were frequently found to be Clostridium species other than C. perfringens. Thus, because of its highly selective nature, LS broth was superior to TSC agar for enumerating and confirming the small numbers of C. perfringens that were present in a majority of the samples. This was especially true when other clostridia were also present. Besides its greater selectivity and sensitivity, LS broth had the additional advantages of requiring less work and giving confirmed results within 24-48 h compared with 3 days for the TSC agar method. PMID:2865247

  19. Characterization of anti-Clostridium perfringens bacteriophages isolated on poultry farms in Central Russia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a main food-borne pathogen causing human diseases. Besides, these Gram-positive anaerobes are responsible for the development of avian necrotic enteritidis, the wide-spread disease in countries engaged in the poultry breeding. For minimization followed by complete exclu...

  20. ESTIMATATION OF GROWTH OF CLOSTRIDIUM PERFRINGENS IN COOKED BEEF UNDER FLUCTUATING TEMPERATURE CONDITIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new concept for estimating the bacterial growth under temperature fluctuations was hypothesized and validated using Clostridium perfringens as a test organism. This new methodology was based on the Gompertz models to calculate the equivalent growth times under different temperatures, and estimate...

  1. Bacteriophages of the family siphoviridae contain amidase enzymes that lyse Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    *Agtech-Danisco, current address In chickens Clostridium perfringens (Cp) is the etiologic agent of necrotic enteritis and causes gas gangrene along with being the third leading cause of bacterial food-borne gastroenteritis in humans. While the disease in poultry can be controlled by antibiotics, th...

  2. Complete genome sequence of the podoviral bacteriophage CP24R virulent for Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage 'CP24R was isolated from raw sewage of a waste treatment plant and lytic activity was observed against a type C Clostridium perfringens isolate. Electron microscopy revealed a small virion (44nm diameter icosahedral capsid) with a short, non-contractile tail, indicative of the family ...

  3. CLOSTRIDIUM PERFRINGENS: STATUS OF A FOOD-BORNE SPORE-FORMER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is responsible for the third most common cause of food-borne illness in the U.S. today, resulting in an estimated 0.25 million cases annually and an associated economic loss of 12.5 billion dollars. The increased production of minimally-processed, extended shelf-life, refrig...

  4. BACTERIOPHAGES OF THE FAMILY SIPHOVIRIDAE CONTAIN AMIDASE ENZYMES THAT LYSE CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In chickens Clostridium perfringens (Cp) is the etiologic agent of necrotic enteritis and causes gas gangrene along with being the third leading cause of bacterial food-borne gastroenteritis in humans. While the disease in poultry can be controlled by antibiotics, there is increasing pressure to ban...

  5. Innate immune response to Clostridium perfringens and Eimeria maxima in necrotic enteritis model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have investigated various aspects of host immune responses using a disease model for necrotic enteritis (NE) in which the severity of lesions produced by Clostridium perfringens was increased, and the growth performance of broiler chickens was decreased by prior infection with Eimeria maxima. Qu...

  6. Molecular Characterization of Podoviridae Bacteriophages Virulent for Clostridium perfringens and Comparison of Their Predicted Lytic Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control ba...

  7. Control of Clostridium perfringens spores by plant-derived antimicrobials during cooling of cooked ground beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens spore germination and outgrowth by carvacrol, cinnamaldehyde, thymol, oregano oil and two green tea extracts with low (green tea leaf powder (GTL); 141 mg of total catechins/g of green tea extract) and high (green tea leaf extract (GTE); 697 mg of total catechin...

  8. Clostridium perfringens and Clostridium difficile in cooked beef sold in Côte d'Ivoire and their antimicrobial susceptibility.

    PubMed

    Kouassi, Kra Athanase; Dadie, Adjéhi Thomas; N'Guessan, Kouadio Florent; Dje, Koffi Marcellin; Loukou, Yao Guillaume

    2014-08-01

    The aim of this study was to evaluate the prevalence of Clostridium difficile and Clostridium perfringens in cooked beef sold in the streets in Côte d'Ivoire and their antimicrobial susceptibility. A total of 395 kidney and flesh samples of cooked beef were collected from vendors at Abidjan and subjected to C. difficile and C. perfringens isolation and identification by using biochemical tests, API 20A system and PCR detection. Subsequently, the antimicrobial susceptibility test was performed for confirmed isolates. Our results showed the prevalence of 12.4% for C. difficile (11.04% in kidney and 13.45% in flesh) and 5.06% for C. perfringens (2.32% in kidney and 7.17% in flesh). Metronidazole and vancomycin remained the most potent antimicrobial agents against C. difficile while metronidazole and penicillin G were the most potent agents against C. perfringens. The resistance rates to tetracycline, doxycycline, chloramphenicol and erythromycin against C. difficile and C. perfringens isolates ranged from 2.05% to 8.16% and from 20% to 50%, respectively. Among all antimicrobial agents tested against C. difficile, percentages of resistance to quinolones ciprofloxacin, norfloxacin and nalidixic acid as well as to gentamicin and cefotaxime were the highest. Eight resistant phenotypes were defined for C. difficile isolates and eleven resistant phenotypes for C. perfringens isolates. Clindamycin/gentamicin/cefotaxime/ciprofloxacin/norfloxacin/nalidixic acid resistance was the most common phenotype for C. difficile (55.10% of isolates) while norfloxacin/nalidixic acid resistance was the most common phenotype for C. perfringens (20% of isolates). PMID:24944124

  9. Fusion of a thermophilic phage endolysin to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  10. A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  11. Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable rates of evolution within a core genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context. We sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricu...

  12. Clostridium perfringens bacteriophages FCP39O and FCP26F: genomic organization and proteomic analysis of the virions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Initial screening for bacteriophages lytic for Clostridium perfringens was performed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Lytic phage preparations were initially characterized by transmission electron microscopy ...

  13. Growth promoting effects of prebiotic yeast cell wall products in starter broilers under an immune stress and Clostridium perfringens challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was designed to investigate the growth promoting effects of supplementing different sources and concentrations of prebiotic yeast cell wall (YCW) products containing mannanoligosaccharides in starter broilers under an immune stress and Clostridium perfringens challenge. Through a series ...

  14. BACTERIOCIN E1073 PRODUCED BY ENTEROCOCCUS FAECIUM LWP1073 IS EFFECTIVE FOR TREATING COMMENSAL CLOSTRIDIUM PERFRINGENS INFECTION IN BROILERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterotoxin-producing Clostridium perfringens type A bacteria occupy a significant place in the etiological structure of food-borne infections in humans. One potential approach to minimize infections associated with food-borne pathogens is to control the carriage of C. perfringens in broilers. For ...

  15. Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens-induced necrotic enteritis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illnesses with an estimated economic burden of $342M USD per year. In addition to being a foodborne pathogen, C. perfringens is also an economically important poultry pathogen and is one of the known etiologic agents...

  16. Predictive model for growth of Clostridium perfringens at temperatures applicable to cooling of cooked uncured beef and chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this investigation was to develop and validate a model for predicting the relative growth of Clostridium perfringens from spore inocula in uncured chicken and beef meat during cooling. Isothermal growth curves of C. perfringens at various temperatures from 10-48.9C were evaluated, ...

  17. Effect of heat treatment on the performance of tryptose-sulfite-cycloserine agar for enumeration of Clostridium perfringens.

    PubMed Central

    Brodsky, M H; Ciebin, B W

    1979-01-01

    Dissolving dehydrated tryptose-sulfite-cycloserine agar by only boiling or microwaving was found to inhibit Clostridium perfringens colony development in pour plates when compared with C. perfringens recovery in tryptose-sulfite-cycloserine agar prepared by autoclaving. Images PMID:225988

  18. The molecular-genetic analysis of Clostridium perfringens strains isolated from broilers on farms in Central Russia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the research was to perform phenotypic and molecular-genetic typing of Clostridium perfringens strains commonly spread on poultry farms in Central Russia. Samples of homogenized iliac and cecal contents from 760 broilers were assayed and 325 C. perfringens strains (42.8 %) were isol...

  19. Importance of histamine-producing Clostridium perfringens in scombrotoxin-forming fish.

    PubMed

    Bjornsdottir-Butler, Kristin; McCarthy, Susan; Burkhardt, William; Benner, Ronald A

    2013-07-01

    It has been suggested that anaerobic histamine-producing bacteria (HPB) are important contributors to scombrotoxin fish poisoning (SFP). In order to assess the role of Clostridium perfringens in SFP, we developed a real-time PCR method for rapid detection of histamine-producing (HP) C. perfringens. The real-time PCR assay was 100% inclusive for detecting 23 HP C. perfringens and did not detect any of the other 116 HP or non-HP isolates examined. The efficiency of the assay with or without internal amplification control DNA was 102%; in the presence of background flora and inhibitory matrices, it was 90 to 99%. To investigate the importance of HP C. perfringens in SFP, we examined histamine production by C. perfringens in inoculated fish samples incubated under anaerobic conditions. C. perfringens produced low histamine levels in tuna (19 ppm) and Spanish mackerel (3 ppm), whereas gram-negative HPB produced high histamine levels (6,345 ppm in tuna; 1,223 ppm in Spanish mackerel) under the same conditions. When one bonito, two bigeye tuna, nine mahi-mahi, and five yellowfin tuna were examined for the presence of HPB, none (0 of 17) of the samples contained HP C. perfringens or other gram-positive HPB, whereas 86% of the samples contained gram-negative HPB. Our study indicates that histamine production by C. perfringens in scombrotoxin-forming fish was minimal compared with that by gram-negative HPB and that C. perfringens may not be an important bacterial species associated with SFP. PMID:23834808

  20. New Quantitative, Qualitative, and Confirmatory Media for Rapid Analysis of Food for Clostridium perfringens1

    PubMed Central

    Shahidi, Syed A.; Ferguson, Alphonza R.

    1971-01-01

    A selective and differential medium, Shahidi-Ferguson Perfringens agar (SFP agar), and a confirmatory medium, lactose-motility agar (LM agar), were developed for the enumeration and identification of Clostridium perfringens in foods. These media provide a rapid, specific, and direct diagnosis of C. perfringens. SFP agar contains sodium metabisulfite and ferric ammonium citrate to demonstrate H2S production and egg yolk to demonstrate lecithinase production by C. perfringens. On SFP agar, C. perfringens produces black colonies, 2 to 3 mm in diameter, surrounded by zones of opaque precipitate. The typical colonies are confirmed on LM agar. Enumeration and identification are completed within 48 hr. All of the ingredients of SFP agar are stable to heat and storage conditions. SFP agar also contains two antibiotics, kanamycin and polymyxin B, which are inhibitory to many bacteria commonly occurring in foods. A comparative study of SFP agar and noninhibitory media showed that SFP agar did not inhibit any of the 16 strains of C. perfringens tested. Recovery of C. perfringens added to foods averaged 90.6% for SFP agar as compared with 69.8% for sulfite polymyxin-sulfadiazine (SPS) agar (BBL) and 60.2% for SPS agar (Difco). The colonies on the SFP agar, were much larger and were consistently black. Of 464 food samples tested, C. perfringens was found in 27 samples with SFP agar and in 5 samples with SPS agar (Difco), with a recovery ratio considerably higher on SFP agar. SFP agar is a more specific presumptive medium for the enumeration of C. perfringens and in conjunction with LM agar should save considerable time, effort, and materials toward the final identification of the species. Images PMID:4324195

  1. Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

    PubMed Central

    Wise, Mark G.; Siragusa, Gregory R.

    2005-01-01

    Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract. PMID:16000804

  2. Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage

    PubMed Central

    Tompkin, R. B.; Christiansen, L. N.; Shaparis, A. B.; Bolin, H.

    1974-01-01

    Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. PMID:4368631

  3. Clostridium perfringens type A fatal acute hemorrhagic gastroenteritis in a dog.

    PubMed

    Schlegel, Ben J; Van Dreumel, Tony; Slavić, Durda; Prescott, John F

    2012-05-01

    The morning after participating in a dog show, a 2-year-old Pomeranian dog was found dead in a pool of bloody feces. Necropsy revealed hemorrhagic gastroenteritis of the entire gastrointestinal tract, with many Gram-positive bacilli on the surface and in the lumen and crypts of the intestine. Enterotoxin-positive type A Clostridium perfringens were isolated in large numbers. This dramatic case of fatal C. perfringens gastroenteritis highlights the need to better understand the role of this bacterium in enteric disease of dogs. PMID:23115371

  4. Hemorrhagic enteritis associated with Clostridium perfringens type A in a dog.

    PubMed

    Sasaki, J; Goryo, M; Asahina, M; Makara, M; Shishido, S; Okada, K

    1999-02-01

    A female Shetland sheep dog died suddenly with hemorrhagic diarrhea and vomitting, and was examined pathologically and microbiologically. Gross pathological change was restricted to the intestinal tract. The intestine contained watery, blood-stained fluid. Histopathologically, the principal intestinal lesion was superficial mucosal hemorrhagic necrosis at the jejunoileum. Many Gram-positive bacilli were found adhering to the necrotic mucosal surface in parts of the intestinal tract. Clostridium perfringens in pure culture were isolated from jejunal contents by anaerobic culture. These results suggested that the typical lesion of this case coincided with canine hemorrhagic enteritis and enterotoxemia due to C. perfringens infection could be the cause of sudden death. PMID:10081759

  5. CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Ma, Menglin; Sarker, Mahfuzur R.; McClane, Bruce A.

    2013-01-01

    ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. PMID:24105766

  6. Mechanism of action of a novel recombinant peptide, MP1102, against Clostridium perfringens type C.

    PubMed

    Zong, Lifen; Teng, Da; Wang, Xiumin; Mao, Ruoyu; Yang, Na; Hao, Ya; Wang, Jianhua

    2016-06-01

    This work is the first to report the antibacterial characteristics and antibacterial mechanisms of MP1102, which is a variant of NZ2114, against pathogenic Clostridium perfringens. MP1102 exhibited strong antimicrobial activity against C. perfringens strains CVCC 61, CVCC 1163, and CVCC 2032 at a low minimal inhibitory concentration (MIC) of 0.91 μM. MP1102 showed anti-C. perfringens activity over a wide pH range of 2.0 and 10.0, high thermal stability from 20 to 80 °C, and remarkable resistance to pepsin. The fractional inhibitory concentration index (FICI) indicated an additive or synergic effect between MP1102 and bacitracin zinc, nisin, vancomycin, virginiamycin, aureomycin, and ampicillin against C. perfringens (FICI = 0.3125-1.0). To further elucidate the antibacterial mechanism of MP1102, its effect on the C. perfringens CVCC 61 cell membrane and intracellular DNA was studied. Flow cytometry and scanning electron microscopy (SEM) indicated that MP1102 treatment resulted in the release of cellular contents by damaging the membrane. A DNA gel retardation and circular dichroism analysis demonstrated that MP1102 interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that MP1102 affected cellular functions, such as DNA synthesis. These results suggested that MP1102 exhibited potential as a new antimicrobial agent against C. perfringens infections. PMID:26921181

  7. Clostridium perfringens food poisoning: use of serotyping in an outbreak setting.

    PubMed Central

    Gross, T P; Kamara, L B; Hatheway, C L; Powers, P; Libonati, J P; Harmon, S M; Israel, E

    1989-01-01

    An outbreak of Clostridium perfringens food poisoning occurred among attendees of a firehouse luncheon. The predominant symptoms of diarrhea (100%) and abdominal pain (81%) among case-patients, the mean incubation period (13.4 h), and the mean duration of illness (21.2 h) were all characteristic of C. perfringens enteritis. Roast beef, although not epidemiologically implicated, was the most likely vehicle of transmission. Fecal specimens from case-patients contained a median C. perfringens spore count of greater than 10(6) and yielded isolates that were heat sensitive and predominantly nonhemolytic, produced C. perfringens enterotoxin A, and, in the majority of specimens (four of five), were identical in serotype. Food samples were negative. This outbreak demonstrates that following enumeration of C. perfringens from a suitable number of fecal specimens from case-patients, serotyping of the isolates may be helpful in implicating C. perfringens as the cause of foodborne illness. This is especially true when implicated food items test negative or are no longer available for testing. PMID:2542360

  8. The inhibitory effects of sorbate and benzoate against Clostridium perfringens type A isolates.

    PubMed

    Alnoman, Maryam; Udompijitkul, Pathima; Paredes-Sabja, Daniel; Sarker, Mahfuzur R

    2015-06-01

    This study evaluated the inhibitory effects of sorbate and benzoate against Clostridium perfringens type A food poisoning (FP) and non-food-borne (NFB) disease isolates. No significant inhibition of germination of spores of both FP and NFB isolates was observed in rich medium (pH 7.0) supplemented with permissive level of sodium sorbate (0.3% ≈ 0.13 mM undissociated sorbic acid) or sodium benzoate (0.1% ≈ 0.01 mM undissociated benzoic acid) used in foods. However, these levels of sorbate and benzoate effectively arrested outgrowth of germinated C. perfringens spores in rich medium. Lowering the pH of the medium increases the inhibitory effects of sorbate and benzoate against germination of spores of NFB isolates, and outgrowth of spores of both FP and NFB isolates. Furthermore, sorbate and benzoate inhibited vegetative growth of C. perfringens isolates. However, the permissible levels of these organic salts could not control the growth of C. perfringens spores in chicken meat stored under extremely abusive conditions. In summary, although sorbate and benzoate showed inhibitory activities against C. perfringens in the rich medium, no such effect was observed in cooked chicken meat. Therefore, caution should be taken when applying these organic salts into meat products to reduce or eliminate C. perfringens spores. PMID:25790996

  9. Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in Thailand.

    PubMed

    Ngamwongsatit, Bhinyada; Tanomsridachchai, Wimonrat; Suthienkul, Orasa; Urairong, Supanee; Navasakuljinda, Wichian; Janvilisri, Tavan

    2016-04-01

    Clostridium perfringens causes diarrhea in neonatal piglets, thereby affecting commercial swine farming. The objective of this study was to determine the prevalence and characterize antimicrobial resistance in C. perfringens isolated from diarrheal neonatal piglets in Thailand. A total of 260 rectal swab samples were collected from 13 farms and were subjected to C. perfringens isolation. A total of 148 samples were PCR-positive for C. perfringens toxin genes, from which 122 were recovered. All isolates were cpb2-encoding C. perfringens type A and enterotoxin gene negative. Most of the isolates were susceptible to ampicillin, bacitracin, chlorotetracycline, doxycycline, and oxytetracycline with MIC50 values ranging from 0.32 to 8 μg/ml. The high resistance rates were observed for ceftiofur, enrofloxacin, erythromycin, lincomycin, and tylosin. Among resistant isolates, 82% were resistant to more than one type of antibiotics. The distinct pattern of multiple drug resistance in C. perfringens was observed in different regions, potentially reflecting the farm specific usage of these agents. PMID:26752714

  10. Feasibility of using food-grade additives to control the growth of Clostridium perfringens.

    PubMed

    Sikes, A; Ehioba, R

    1999-02-18

    Previously, it was demonstrated that the combination of sucrose laurate (SL) ethylenediaminetetraacetate (E) and butylated hydroxyl anisole (B) (SLEB) was an effective antimicrobial agent against both gram-negative (aerobes) and gram-positive (facultative anaerobes) foodborne bacteria. This investigation examines the sensitivity of Clostridium perfringens to SLEB relative to: (1) the minimum inhibitory concentration (MIC) of SLEB required to inhibit the growth of C. perfringens and (2) the antibacterial effectiveness of different combination ratios of SLEB in fluid thioglycollate medium (FTM). Results indicated that the MIC of SLEB (1:1:1, v/v/v) against C. perfringens on tryptose sulfite cycloserine (TSC) agar was > 150 ppm at 37 degrees C. However, in FTM, a SLEB (1:1:1, v/v/v) concentration of > 100 ppm inhibited C. perfringens during an incubation (anaerobic) period of 196 h at 37 degrees C. The sensitivity of C. perfringens to different combination ratios was also investigated in FTM. The results showed that, when the concentrations of SL and E were held at 75 ppm in the SLEB combination, and the concentration of B increased from 0 to 75 ppm, C. perfringens growth increased initially during the first 24 h of incubation (37 degrees C) but remained constant during the next 48 h. Similarly, when concentrations of SL and E were held constant at 150 ppm in the SLEB combination and the B ratio increased from 50 to 150 ppm in FTM, C. perfringens viability decreased in all of the treated samples during 72-h incubation at 37 degrees C. The results indicated that SLEB was an effective inhibitor of C. perfringens growth activities, and the ratios of the components of SLEB can be adjusted to meet specific preservation needs. PMID:10100897

  11. Antibiotic Sensitivity of Clostridium perfringens Isolated From Faeces in Tabriz, Iran

    PubMed Central

    Akhi, Mohammad Taghi; Bidar Asl, Saeid; Pirzadeh, Tahereh; Naghili, Behruz; Yeganeh, Fatemeh; Memar, Yousef; Mohammadzadeh, Yalda

    2015-01-01

    Background: Clostridium perfringens, a Gram-positive, anaerobic bacterium that produces at least 16 virulence factors including 12 toxins (α-ν), enterotoxin, hemolysin and neuraminidase, can create variable pathogenic condition, ranging from a food poisoning to life-threatening myonecrosis. Among C. perfringens strains, resistance to the drug choices such as penicillin as well as to alternatives of penicillin like metronidazole and clindamycin has also been observed. Objectives: The aim of this study was to determine the resistance of isolated toxigenic and non-toxigenic C. perfringens strains against common antimicrobial agents. Materials and Methods: In this descriptive study, a total of 136 stool specimens were collected. At first, cooked meat medium enrichment method was performed on samples at 45°C. Thereafter, a loopful of the enriched culture was transferred to blood agar and incubated anaerobically at 37°C for 24-72 hours. Colonies with double zone of hemolysis were identified by different biochemical tests such as phospholipase C (lecithinase) test, indole and urease production. The Minimum Inhibitory Concentration (MIC) for common antibiotics was determined by Etests (Epsilometer) and duplex Polymerase Chain Reaction (PCR) reaction was performed with specific primers for amplification of cpe (426 bp) and plc (283 bp) Genes. Results: Of 136 stool samples including diarrhea [48] and non-diarrhea [88] ones, 83 (61.02%) C. perfringens were cultured. Of these 83, 79 C. perfringens isolates showed the alpha-toxin (phospholipase C) production gene by PCR. Respectively, 3 (9.09%) and 2 (4.34%) cpe genes were present in diarrhea and non-diarrhea samples. Of 79 isolates of C. perfringens, 34 (43.03%) cases showed no resistance, 18 (22.78%) had one resistance and 27 (34.17%) isolates had multiple resistance to imipenem, metronidazole, ceftriaxone, clindamycin, chloramphenicol, and penicillin. Conclusions: Periodic evaluation of antimicrobial susceptibility for C

  12. Clostridium perfringens type A toxin production in 3 commonly used culture media.

    PubMed

    Fernandez-Miyakawa, Mariano E; Marcellino, Romanella; Uzal, Francisco A

    2007-03-01

    In vitro toxin production is an important tool not only for diagnostic purposes but also for the study of pathogenesis of Clostridium perfringens infections. The present study was carried out to compare the level of toxin production by several strains of C. perfringens type A, isolated from the intestine of animals, when cultured in 3 different conventional culture media. Six strains of C. perfringens type A isolated from the small intestine of healthy sheep were cultured in commercial cooked meat medium (CMM), brain heart infusion (BHI), and tryptone glucose yeast (TGY). Intravenous lethality in mice and phospholipase C (PLC) activity were measured in filtered culture supernatants. Lethality of culture supernatants was highest for all isolates when grown in BHI, followed by CMM. No supernatants from any isolates grown in TGY produced lethality in mice. Phospholipase C activity was highest when the isolates were grown in BHI and CMM and significantly lower when grown in TGY. PMID:17402614

  13. Transcriptional Analysis of the Rubrerythrin and Superoxide Dismutase Genes of Clostridium perfringens

    PubMed Central

    Geissmann, Thomas A.; Teuber, Michael; Meile, Leo

    1999-01-01

    We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from Clostridium perfringens containing the superoxide dismutase-encoding gene, sod. Previously, rubrerythrin from C. perfringens had been isolated and its gene (rbr) had been cloned (Y. Lehmann, L. Meile, and M. Teuber, J. Bacteriol. 178:7152–7158, 1996). Northern blot experiments revealed a length of approximately 800 bases for each transcript of rbr and sod of C. perfringens. Thus, rbr and sod each represent a monocistronic operon. Their transcription start points were located by primer extension analyses. sod transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase. Neither sod nor rbr transcription was influenced by oxidative stress. PMID:10559182

  14. Oxidation-Reduction Potential and Growth of Clostridium perfringens and Pseudomonas fluorescens1

    PubMed Central

    Tabatabai, L. B.; Walker, H. W.

    1970-01-01

    A new apparatus was developed for measuring changes in Eh, pH, and cell numbers. With this apparatus, the relationships of these parameters were studied at initial Eh levels of 200 and 40 mv (pH 7.0), by using Clostridium perfringens and Pseudomonas fluorescens. One of the strains of C. perfringens grew more luxuriantly at the higher Eh, in the presence of small quantities of oxygen, than at the lower one in the absence of oxygen. P. fluorescens could grow at a relatively low Eh (40 mv, pH 7.0) in pure culture but not in the presence of C. perfringens under the same conditions. PMID:4320922

  15. The pathology of enterotoxemia by Clostridium perfringens type C in calves.

    PubMed

    Garcia, Jorge P; Anderson, Mark; Blanchard, Patricia; Mete, Asli; Uzal, Francisco A

    2013-05-01

    The pathology of Clostridium perfringens type C infection has been described with detail only in foals and piglets. The current report describes the diagnostic workup and detailed pathology of 3 cases of C. perfringens type C infection in calves. A 2-day-old Jersey calf and fresh and fixed tissues from a 4-week-old Angus calf and from a 1-week-old Jersey calf were received at the California Animal Health and Food Safety Laboratory System with a history of digestive disease and death. The gross changes in the gastrointestinal tract of 1 calf consisted of multifocal subserosal hemorrhages of the rumen, diffuse congestion and multifocal hemorrhages of the small intestinal mucosa, and dilated cecum with bloody liquid contents. In a second calf, a large segment of small intestine was hemorrhagic. The small intestine of the third calf was dilated and filled with abundant yellow fluid content. Microscopically, all 3 calves had diffuse coagulation necrosis of the intestinal mucosa. Clostridium perfringens type A was isolated from the intestinal content of 2 calves. In addition, enzyme-linked immunosorbent assay for Bovine rotavirus was positive on colonic content of 1 calf. Small numbers of cryptosporidia were seen in smears of colonic content of 2 calves, and Salmonella sp. group E was detected in the small intestinal content of another calf. Clostridium perfringens beta toxin was detected in the intestinal content of the 3 animals. A diagnosis of C. perfringens type C infection was confirmed based on pathological findings and detection of beta toxin in the intestinal content of the 3 animals. PMID:23592750

  16. Unique regulatory mechanism of sporulation and enterotoxin production in Clostridium perfringens.

    PubMed

    Ohtani, Kaori; Hirakawa, Hideki; Paredes-Sabja, Daniel; Tashiro, Kosuke; Kuhara, Satoru; Sarker, Mahfuzur R; Shimizu, Tohru

    2013-06-01

    Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning). PMID:23585540

  17. Use of allicin as feed additive to enhance vaccination capacity of Clostridium perfringens toxoid in rabbits.

    PubMed

    Abu El Hammed, Waleed; Soufy, Hamdy; El-Shemy, Ahmed; Nasr, Soad M; Dessouky, Mohamed I

    2016-04-12

    The present study assessed the efficacy of Clostridium perfringens (C. perfringens) toxoid and/or allicin - as feed additive - in rabbits for preventing or minimizing the severity of infection with locally isolated strain of C. perfringens type A. Serum biochemical, immunological and pathological investigations were also done. One hundred rabbits of 6 weeks of age were divided into five equal groups (G1-G5). G1 were kept as normal control. G2 was allocated for C. perfringens type A infection. G3 was vaccinated with C. perfringens toxoid at zero time and then with a booster dose at the 3rd week of the experimental period. G4 was treated with allicin 20% added to the ration (200mg/kg ration) all over the experimental period. G5 was vaccinated with C. perfringens toxoid at the zero time then with a booster dose at the 3rd week of the experiment period, and treated with allicin 20% from the zero time till the end of the experiment. At the 4th week, G2, G3, G4 and G5 were challenged orally (5 ml) and subcutaneously (2 ml) with 24h cooked meat broth containing 1 × 10(7) colony-forming units/ml of C. perfringens type A strain. Blood and tissue samples were collected from all groups po st-vaccination then post-challenge for biochemical analysis, serum neutralization test and histopathological examinations. Results revealed that rabbits treated with both allicin and toxoid vaccine demonstrated high level of antitoxin titre post-challenge, improved liver and kidney functions, and reduced morbidity and mortality rates and the severity of histopathological changes associated with challenge of rabbits with C. perfringens type A strain. In conclusion, vaccination of rabbits with C. perfringens toxoid combined with allicin 20% gave better protection, enhanced immune response and had no adverse effects on the general health conditions against C. perfringens type A infection compared to rabbits vaccinated with C. perfringens toxoid only. PMID:26973070

  18. Quantitative Microbial Risk Assessment for Clostridium perfringens in Natural and Processed Cheeses.

    PubMed

    Lee, Heeyoung; Lee, Soomin; Kim, Sejeong; Lee, Jeeyeon; Ha, Jimyeong; Yoon, Yohan

    2016-08-01

    This study evaluated the risk of Clostridium perfringens (C. perfringens) foodborne illness from natural and processed cheeses. Microbial risk assessment in this study was conducted according to four steps: hazard identification, hazard characterization, exposure assessment, and risk characterization. The hazard identification of C. perfringens on cheese was identified through literature, and dose response models were utilized for hazard characterization of the pathogen. For exposure assessment, the prevalence of C. perfringens, storage temperatures, storage time, and annual amounts of cheese consumption were surveyed. Eventually, a simulation model was developed using the collected data and the simulation result was used to estimate the probability of C. perfringens foodborne illness by cheese consumption with @RISK. C. perfringens was determined to be low risk on cheese based on hazard identification, and the exponential model (r = 1.82×10(-11)) was deemed appropriate for hazard characterization. Annual amounts of natural and processed cheese consumption were 12.40±19.43 g and 19.46±14.39 g, respectively. Since the contamination levels of C. perfringens on natural (0.30 Log CFU/g) and processed cheeses (0.45 Log CFU/g) were below the detection limit, the initial contamination levels of natural and processed cheeses were estimated by beta distribution (α 1 = 1, α 2 = 91; α 1 = 1, α 2 = 309)×uniform distribution (a = 0, b = 2; a = 0, b = 2.8) to be -2.35 and -2.73 Log CFU/g, respectively. Moreover, no growth of C. perfringens was observed for exposure assessment to simulated conditions of distribution and storage. These data were used for risk characterization by a simulation model, and the mean values of the probability of C. perfringens foodborne illness by cheese consumption per person per day for natural and processed cheeses were 9.57×10(-14) and 3.58×10(-14), respectively. These results indicate that probability of C. perfringens foodborne illness

  19. Quantitative Microbial Risk Assessment for Clostridium perfringens in Natural and Processed Cheeses

    PubMed Central

    Lee, Heeyoung; Lee, Soomin; Kim, Sejeong; Lee, Jeeyeon; Ha, Jimyeong; Yoon, Yohan

    2016-01-01

    This study evaluated the risk of Clostridium perfringens (C. perfringens) foodborne illness from natural and processed cheeses. Microbial risk assessment in this study was conducted according to four steps: hazard identification, hazard characterization, exposure assessment, and risk characterization. The hazard identification of C. perfringens on cheese was identified through literature, and dose response models were utilized for hazard characterization of the pathogen. For exposure assessment, the prevalence of C. perfringens, storage temperatures, storage time, and annual amounts of cheese consumption were surveyed. Eventually, a simulation model was developed using the collected data and the simulation result was used to estimate the probability of C. perfringens foodborne illness by cheese consumption with @RISK. C. perfringens was determined to be low risk on cheese based on hazard identification, and the exponential model (r = 1.82×10−11) was deemed appropriate for hazard characterization. Annual amounts of natural and processed cheese consumption were 12.40±19.43 g and 19.46±14.39 g, respectively. Since the contamination levels of C. perfringens on natural (0.30 Log CFU/g) and processed cheeses (0.45 Log CFU/g) were below the detection limit, the initial contamination levels of natural and processed cheeses were estimated by beta distribution (α1 = 1, α2 = 91; α1 = 1, α2 = 309)×uniform distribution (a = 0, b = 2; a = 0, b = 2.8) to be −2.35 and −2.73 Log CFU/g, respectively. Moreover, no growth of C. perfringens was observed for exposure assessment to simulated conditions of distribution and storage. These data were used for risk characterization by a simulation model, and the mean values of the probability of C. perfringens foodborne illness by cheese consumption per person per day for natural and processed cheeses were 9.57×10−14 and 3.58×10−14, respectively. These results indicate that probability of C. perfringens foodborne illness

  20. Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens β2-toxin

    SciTech Connect

    Gurjar, Abhijit A.; Yennawar, Neela H.; Yennawar, Hemant P.; Rajashankar, Kanagalaghatta R.; Hegde, Narasimha V.; Jayarao, Bhushan M.

    2007-06-01

    The cloning, expression, purification and crystallization of recombinant Clostridium perfringens β2-toxin is described. The crystals diffracted to 2.9 Å resolution. Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. β2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of β2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 Å resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a = b = 103.71, c = 193.48 Å, α = β = 90, γ = 120° using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles ω = 90.0, ϕ = 210.3°.

  1. Severe Sepsis due to Clostridium perfringens Bacteremia of Urinary Origin: A Case Report and Systematic Review

    PubMed Central

    Millard, Michael A.; McManus, Kathleen A.; Wispelwey, Brian

    2016-01-01

    Clostridium perfringens bacteremia is an uncommon yet serious clinical syndrome that typically arises from a gastrointestinal source. However, clinicians should consider nongastrointestinal sources as well. We present a rare case of C. perfringens bacteremia of urinary origin that required surgical intervention for definitive treatment. A 61-year-old male presented with acute nausea and vomiting, altered mental status, and chronic diarrhea. His physical exam revealed right costovertebral tenderness and his laboratory work-up revealed acute renal failure. Percutaneous blood cultures grew C. perfringens. Cross-sectional imaging revealed a right-sided ureteral stone with hydronephrosis, which required nephrostomy placement. On placement of the nephrostomy tube, purulent drainage was identified and Gram stain of the drainage revealed Gram-variable rods. A urinary source of C. perfringens was clinically supported. Although it is not a common presentation, nongastrointestinal sources such as a urinary source should be considered in C. perfringens bacteremia because failure to recognize a nongastrointestinal source can delay appropriate treatment, which may include surgical intervention. PMID:26998370

  2. Enumeration of Clostridium perfringens spores in groundwater samples: comparison of six culture media.

    PubMed

    Araujo, M; Sueiro, R A; Gómez, M J; Garrido, M J

    2004-05-01

    In order to investigate the ability of Fluorocult-supplemented TSC agar (TSCF (Fluorocult supplemented TSC-agar): prepared from Tryptose Sulfite Cycloserine Agar Base (Merck), D-cycloserine (Fluka Chemika, USA), and fluorocult TSC-Agar supplement (Merck)) for detecting spores of Clostridium perfringens in water, we analyzed groundwater samples, pretreated by heating to 80 degrees C/5 min, using this fluorogenic medium together with five other media: mCP agar (Panreac; Cultimed), TSC agar (Merck, Germany), TSN agar (Merck), and SPS agar (BBL, USA) by the membrane filtration technique, and Wilson-Blair agar (WB) following the still-in-force Spanish official method. Variance analysis of the data obtained shows statistically significant differences in the counts obtained between media employed in this work. The C. perfringens spore counts on mCP agar were significantly lower (P<0.05) than the corresponding values of TSC, TSCF, SPS, and WB media. No statistically significant differences were found between C. perfringens spore counts on TSCF compared with those of other methods used. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for C. perfringens spore recovery in groundwater samples. Additionally, the results obtained indicate that mCP agar, which is the reference method in the European Union, is not suitable medium for recovering C. perfringens spores from groundwater samples. PMID:15063057

  3. New medium for rapid screening and enumeration of Clostridium perfringens in foods.

    PubMed Central

    Erickson, J E; Deibel, R H

    1978-01-01

    A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM. PMID:213019

  4. Prevalence of Enterotoxigenic Clostridium perfringens Isolates in Pittsburgh (Pennsylvania) Area Soils and Home Kitchens▿ †

    PubMed Central

    Li, Jihong; Sayeed, Sameera; McClane, Bruce A.

    2007-01-01

    In the United States and Europe, food poisoning due to Clostridium perfringens type A is predominantly caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe). Neither the reservoir for these isolates nor the point in the food chain where these bacteria contaminate foods is currently understood. Therefore, the current study investigated whether type A isolates carrying a chromosomal cpe gene are present in two potential reservoirs, i.e., soil and home kitchen surfaces. No C. perfringens isolates were recovered from home kitchen surfaces, but most surveyed soil samples contained C. perfringens. The recovered soil isolates were predominantly type A, but some type C, D, and E soil isolates were also identified. All cpe-positive isolates recovered from soil were genotyped as type A, with their cpe genes on cpe plasmids rather than the chromosome. However, two cpe-positive soil isolates did not carry a classical cpe plasmid. Both of those atypical cpe-positive soil isolates were sporulation capable yet failed to produce C. perfringens enterotoxin, possibly because of differences in their upstream promoter regions. Collectively these results suggest that neither soil nor home kitchen surfaces represent major reservoirs for type A isolates with chromosomal cpe that cause food poisoning, although soil does appear to be a reservoir for cpe-positive isolates causing non-food-borne gastrointestinal diseases. PMID:17905877

  5. New medium for rapid screening and enumeration of Clostridium perfringens in foods.

    PubMed

    Erickson, J E; Deibel, R H

    1978-10-01

    A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM. PMID:213019

  6. Non-classical azoreductase secretion in Clostridium perfringens in response to sulfonated azo dye exposure.

    PubMed

    Morrison, Jessica M; John, Gilbert H

    2015-08-01

    Clostridium perfringens, a strictly anaerobic microorganism and inhabitant of the human intestine, has been shown to produce an azoreductase enzyme (AzoC), an NADH-dependent flavin oxidoreductase. This enzyme reduces azo dyes into aromatic amines, which can be carcinogenic. A significant amount of work has been completed on the activity of AzoC. Despite this, much is still unknown, including whether azoreduction of these dyes occurs intracellularly or extracellulary. A physiological study of C. perfringens involving the effect of azo dye exposure was completed to answer this question. Through exposure studies, azo dyes were found to cause cytoplasmic protein release, including AzoC, from C. perfringens in dividing and non-dividing cells. Sulfonation (negative charge) of azo dyes proved to be the key to facilitating protein release of AzoC and was found to be azo-dye-concentration-dependent. Additionally, AzoC was found to localize to the Gram-positive periplasmic region. Using a ΔazoC knockout mutant, the presence of additional azoreductases in C. perfringens was suggested. These results support the notion that the azoreduction of these dyes may occur extracellularly for the commensal C. perfringens in the intestine. PMID:25881497

  7. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity. PMID:18154441

  8. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater.

    PubMed

    Shimizu, Tohru; Ohtani, Kaori; Hirakawa, Hideki; Ohshima, Kenshiro; Yamashita, Atsushi; Shiba, Tadayoshi; Ogasawara, Naotake; Hattori, Masahira; Kuhara, Satoru; Hayashi, Hideo

    2002-01-22

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that causes life-threatening gas gangrene and mild enterotoxaemia in humans, although it colonizes as normal intestinal flora of humans and animals. The organism is known to produce a variety of toxins and enzymes that are responsible for the severe myonecrotic lesions. Here we report the complete 3,031,430-bp sequence of C. perfringens strain 13 that comprises 2,660 protein coding regions and 10 rRNA genes, showing pronounced low overall G + C content (28.6%). The genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfringens, and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component VirR/VirS regulon that coordinately regulates the pathogenicity of C. perfringens. Clearly, C. perfringens obtains various essential materials from the host by producing several degradative enzymes and toxins, resulting in massive destruction of the host tissues. PMID:11792842

  9. Severe Sepsis due to Clostridium perfringens Bacteremia of Urinary Origin: A Case Report and Systematic Review.

    PubMed

    Millard, Michael A; McManus, Kathleen A; Wispelwey, Brian

    2016-01-01

    Clostridium perfringens bacteremia is an uncommon yet serious clinical syndrome that typically arises from a gastrointestinal source. However, clinicians should consider nongastrointestinal sources as well. We present a rare case of C. perfringens bacteremia of urinary origin that required surgical intervention for definitive treatment. A 61-year-old male presented with acute nausea and vomiting, altered mental status, and chronic diarrhea. His physical exam revealed right costovertebral tenderness and his laboratory work-up revealed acute renal failure. Percutaneous blood cultures grew C. perfringens. Cross-sectional imaging revealed a right-sided ureteral stone with hydronephrosis, which required nephrostomy placement. On placement of the nephrostomy tube, purulent drainage was identified and Gram stain of the drainage revealed Gram-variable rods. A urinary source of C. perfringens was clinically supported. Although it is not a common presentation, nongastrointestinal sources such as a urinary source should be considered in C. perfringens bacteremia because failure to recognize a nongastrointestinal source can delay appropriate treatment, which may include surgical intervention. PMID:26998370

  10. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens.

    PubMed

    Sawires, Youhanna S; Songer, J Glenn

    2005-10-01

    Clostridium perfringens is ubiquitous in the environment and causes diseases in man and animals, with syndromes ranging from enteritis, enterotoxemia, and sudden death to food poisoning and gas gangrene. Understanding the epidemiology of these infections and of the evolution of virulence in C. perfringens necessitate an efficient, time and cost effective strain typing method. Multiple-locus variable-number tandem repeat analysis (MLVA) has been applied to typing of other pathogens and we describe here the development of a MLVA scheme for C. perfringens. We characterized five variable tandem repeat (VNTR) loci, four of which are contained within protein encoding genes and screened 112 C. perfringens isolates to evaluate typability, reproducibility, and discriminatory power of the scheme. All the isolates were assigned a MLVA genotype and the technique has excellent reproducibility, with a numerical index of discrimination for the five VNTR loci of 0.995. Thus MLVA is an efficient tool for C. perfringens strain typing, and being PCR based makes it rapid, easy, and cost effective. In addition, it can be employed in epidemiological, ecological, and evolutionary investigations of the organism. PMID:16701582