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Sample records for clostridium perfringens epsilon

  1. 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

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

    PubMed

    Gil, Carles; Dorca-Arvalo, 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

  3. 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

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

  5. Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli.

    PubMed Central

    Hunter, S E; Clarke, I N; Kelly, D C; Titball, R W

    1992-01-01

    The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin. Images PMID:1729175

  6. 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

  7. A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin.

    PubMed

    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

    2014-12-15

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (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 agents 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

  8. 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

  9. 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...

  10. 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

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

    PubMed

    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

  12. 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

  13. Enzyme linked immunosorbent assay for potency testing of vaccines containing Clostridium perfringens type D epsilon-toxoid.

    PubMed

    Pfahler, W; Bisesti, E; Pereyra, J B; Iribarren, F

    1998-04-01

    For ethical, economic and technical reasons in vivo assays need to be replaced in routine laboratory procedures. Based on a method which is already accepted by the British authorities, an indirect ELISA has been developed and evaluated for Clostridium perfringens type D-containing vaccines. Individual and pooled sera of vaccinated rabbits were tested at a single dilution level, the results transferred into IU/ml, and compared with the conventional toxin neutralization test in mice. The ELISA was found to give reproducible estimates of antitoxin levels and showed good correlation with the conventional in vivo test in mice. PMID:9588107

  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?g/kg) and resulted in a mean time from inoculation to death of 18 and 180min 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=0.3mg/kg; ED100=0.8mg/kg) and also provided protection when delivered 15-30min 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. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Clostridium perfringens Type D. Each serial shall be tested as provided in this section. Any serial found unsatisfactory by a prescribed test shall not be released. (a) Each serial shall meet the applicable general... each serial shall be tested using the toxin-neutralization test for Epsilon Antitoxin provided in...

  16. 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

  17. Clostridium perfringens Type C Enterotoxemia

    PubMed Central

    Niilo, Leo

    1988-01-01

    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. ImagesFigure 2.Figure 3. PMID:17423103

  18. 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 ...

  19. [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

  20. 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 ...

  1. 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...

  2. Lumbar discitis caused by Clostridium perfringens.

    PubMed

    Lotte, Romain; Popoff, M R; Degand, Nicolas; Lotte, Laurene; Bouvet, Philippe; Baudin, Guillaume; Cua, Eric; Roger, Pierre-Marie; Ruimy, Raymond

    2014-10-01

    We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of ?-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern. PMID:25056327

  3. Lumbar Discitis Caused by Clostridium perfringens

    PubMed Central

    Popoff, M. R.; Degand, Nicolas; Lotte, Laurene; Bouvet, Philippe; Baudin, Guillaume; Cua, Eric; Roger, Pierre-Marie; Ruimy, Raymond

    2014-01-01

    We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of ?-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern. PMID:25056327

  4. Clostridium perfringens sepsis following a molar pregnancy.

    PubMed

    Adams, Brandi N; Lekovic, Jovana P; Robinson, Suzzette

    2014-01-01

    Clostridium perfringens sepsis is rare since the legalization of abortion in 1973. This is a 49 year old female who developed clostridial sepsis after suction dilation and curettage for a molar pregnancy. A hysterectomy was performed after prompt recognition, and the patient survived. PMID:24096275

  5. Molecular genetics and pathogenesis of Clostridium perfringens.

    PubMed Central

    Rood, J I; Cole, S T

    1991-01-01

    Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals. Recently significant advances have been made in the development of C. perfringens genetics. Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C. perfringens genes and the construction of shuttle plasmids. The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated. A detailed physical map of the C. perfringens chromosome has been prepared, and numerous genes have been located on that map. Reproducible transformation methods for the introduction of plasmids into C. perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned. Now that it is possible to freely move genetic information back and forth between C. perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C. perfringens infections. PMID:1779929

  6. 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. perfringens in animal disease. These research tools are helping us to establish the role of each C. perfringens toxin in animal disease, to investigate the in vivo mechanism of action of these toxins, and to develop more effective vaccines against diseases produced by these microorganisms. PMID:24511335

  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. 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

  9. Animal models to study the pathogenesis of human and animal Clostridium perfringens infections.

    PubMed

    Uzal, Francisco A; McClane, Bruce A; Cheung, Jackie K; Theoret, James; Garcia, Jorge P; Moore, Robert J; Rood, Julian I

    2015-08-31

    The most common animal models used to study Clostridium perfringens infections in humans and animals are reviewed here. The classical C. perfringens-mediated histotoxic disease of humans is clostridial myonecrosis or gas gangrene and the use of a mouse myonecrosis model coupled with genetic studies has contributed greatly to our understanding of disease pathogenesis. Similarly, the use of a chicken model has enhanced our understanding of type A-mediated necrotic enteritis in poultry and has led to the identification of NetB as the primary toxin involved in disease. C. perfringens type A food poisoning is a highly prevalent bacterial illness in the USA and elsewhere. Rabbits and mice are the species most commonly used to study the action of enterotoxin, the causative toxin. Other animal models used to study the effect of this toxin are rats, non-human primates, sheep and cattle. In rabbits and mice, CPE produces severe necrosis of the small intestinal epithelium along with fluid accumulation. C. perfringens type D infection has been studied by inoculating epsilon toxin (ETX) intravenously into mice, rats, sheep, goats and cattle, and by intraduodenal inoculation of whole cultures of this microorganism in mice, sheep, goats and cattle. Molecular Koch's postulates have been fulfilled for enterotoxigenic C. perfringens type A in rabbits and mice, for C. perfringens type A necrotic enteritis and gas gangrene in chickens and mice, respectively, for C. perfringens type C in mice, rabbits and goats, and for C. perfringens type D in mice, sheep and goats. PMID:25770894

  10. Clostridium perfringens endophthalmitis following perforating eye injury

    PubMed Central

    Lee, Helena; Idrees, Zubair; Kinsella, Frank

    2009-01-01

    A 59-year-old man presented with endophthalmitis, following a perforating eye injury from pulling out a wire that was embedded in the ground. On presentation, his vision was perception of light (PL). Tetanus toxoid was given, and he was commenced on ciprofloxacin. A primary repair was performed. Conjunctival swabs, discharge from wound site and anterior chamber aspirate were sent for culture. The eye was tense and the anterior chamber was full of a gelatinous brown substance which precluded performance of vitrectomy. Intravitreal vancomycin and ceftazidime was given. Hourly topical fortified vancomycin and ceftazidime was given. Postoperatively, the patient’s vision remained PL with no evidence of improvement. On day 2, Clostridium perfringens was cultured. The patient was commenced on intravenous benzylpenicillin and clindamycin. Intravitreal clindamycin and vancomycin was administered. The patient was NPL on day 3. There was no evidence of response to treatment and an evisceration was performed on day 6. PMID:21747903

  11. 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 Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered. PMID:24396174

  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 Central

    Niilo, L.

    1980-01-01

    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 (?), beta (? ), beta 2 (?2), epsilon (?), iota (?), 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 ?, ?, ?2, ?, ?, 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 ? toxin gene. Beta (?), ?2, ?, ?, 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, ? and ?2 were identified in 5%, ? and ? 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. [Massive intravascular hemolysis in septicemia caused by Clostridium perfringens].

    PubMed

    Borrego, D; Mara-Tome, P; Cascales, P; Garca Aguayo, J M; Prez Amoros, G; Abad, A

    1991-08-01

    A case of massive haemolytic anaemia in the course of a C. perfringens sepsis of hepatic origin is presented. The diagnosis was strongly suggested by the presence of intragranulocytic capsulated bacilli in a Giemsa stained peripheral blood smear. The patient developed disseminated intravascular coagulation. The outcome was fatal and the patient died eight hours after admission. We review the aetiopathogenesis, diagnosis and therapy of haemolysis in Clostridium perfringens infections. PMID:1776111

  16. Predisposing factors and prevention of Clostridium perfringens-associated enteritis.

    PubMed

    Allaart, Janneke G; van Asten, Alphons J A M; Grne, Andrea

    2013-09-01

    Clostridium perfringens is one of the major causes of intestinal disease in humans and animals. Its pathogenicity is contributed to by the production of a variety of toxins. In addition, predisposing environmental factors are important for the induction of C. perfringens-associated enteritis as shown by infection models. Environmental contamination, gastric and intestinal pH, intestinal microflora, nutrition, concurrent infections, and medical interventions may influence the intestinal colonization, growth, and toxin production by C. perfringens. Prevention of C. perfringens-associated enteritis may be mediated by the use of feed additives like probiotics, prebiotics, organic acids, essential oils, bacteriophages, lysozymes, bacteriocins, and antimicrobial peptides. Here we summarize and discuss published data on the influence of different environmental predisposing factors and preventive measures. Further research should focus on feed composition and feed additives in order to prevent C. perfringens-associated enteritis. PMID:23790636

  17. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Clostridium Perfringens Type C Antitoxin. 113.454 Section 113.454 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Antibody Products §...

  18. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Clostridium Perfringens Type D Antitoxin. 113.455 Section 113.455 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Antibody Products §...

  19. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Clostridium Perfringens Type C Antitoxin. 113.454 Section 113.454 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Antibody Products §...

  20. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Clostridium Perfringens Type D Antitoxin. 113.455 Section 113.455 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Antibody Products §...

  1. MATHEMATICAL MODELING OF GROWTH OF CLOSTRIDIUM PERFRINGENS IN COOKED BEEF

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this work was to study the growth kinetics of Clostridium perfringens spores in thermally processed ground beef and compare the suitability of the Gompertz, logistic, and Baranyi models used to describe the isothermal bacterial growth. Ground beef samples inoculated with the spores ...

  2. 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 ...

  3. 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

  4. 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

  5. Identification of Clostridium Species and DNA Fingerprinting of Clostridium perfringens by Amplified Fragment Length Polymorphism Analysis?

    PubMed Central

    Keto-Timonen, Riikka; Heikinheimo, Annamari; Eerola, Erkki; Korkeala, Hannu

    2006-01-01

    An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species. PMID:16971642

  6. 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.

  7. Characterization of Virulence Plasmid Diversity among Clostridium perfringens Type B Isolates?

    PubMed Central

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

    2010-01-01

    The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to ?90-kb plasmids in most type B isolates, although a few isolates carried a ?65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located ?3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids. PMID:19858300

  8. Method for estimating the presence of Clostridium perfringens in food.

    PubMed

    Harmon, S M; Kautter, D A

    1970-12-01

    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 10(6)C. 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. PMID:4321712

  9. Growth of Clostridium perfringens during cooling of refried beans.

    PubMed

    Cevallos-Cevallos, Juan M; Akins, E Deann; Friedrich, Loretta M; Danyluk, Michelle D; Simonne, Amarat H

    2012-10-01

    Outbreaks of Clostridium perfringens have been associated with dishes containing refried beans from food service establishments. However, growth of C. perfringens in refried beans has not been investigated, and predictive models have not been validated in this food matrix. We investigated the growth of C. perfringens during the cooling of refried beans. Refried beans (pinto and black, with and without salt added) were inoculated with 3 log CFU/g C. perfringens spores and incubated isothermally at 12, 23, 30, 35, 40, 45, and 50C. The levels of C. perfringens were monitored 3, 5, 8, and 10 h after inoculation, and then fitted to the Baranyi primary model and the Rosso secondary model prior to solving the Baranyi differential equation. The final model was validated by dynamic cooling experiments carried out in stockpots, thus mimicking the worst possible food service conditions. All refried beans samples supported the growth of C. perfringens, and all models fit the data with pseudo-R(2) values of 0.95 or greater and mean square errors of 0.3 or lower. The estimated maximum specific growth rates were generally higher in pinto beans, with or without salt added (2.64 and 1.95 h(-1), respectively), when compared with black beans, with or without salt added (1.78 and 1.61 h(-1), respectively). After 10 h of incubation, maximum populations of C. perfringens were significantly higher in samples with no salt added (7.9 log CFU/g for both pinto and black beans) than in samples with salt added (7.3 and 7.2 log CFU/g for pinto and black beans, respectively). The dynamic model predicted the growth of C. perfringens during cooling, with an average root mean squared error of 0.44. The use of large stockpots to cool refried beans led to an observed 1.2-log increase (1.5-log increase predicted by model) in levels of C. perfringens during cooling. The use of shallower pans for cooling is recommended, because they cool faster, therefore limiting the growth of C. perfringens. PMID:23043826

  10. Detection and molecular typing of Clostridium perfringens isolates from beef, chicken and turkey meats.

    PubMed

    Aras, Zeki; Hadimli, Hasan Hseyin

    2015-04-01

    Here we describe a study investigating the presence of Clostridium perfringens strains in meat samples and the toxin genes in the isolates by PCR. This study, for the first time, demonstrated the presence of C. perfringens type E in turkey meats, while C. perfringens type C strains isolated from chicken meats. PMID:25460196

  11. 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

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

    PubMed

    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

  13. Clostridium perfringens type C and Clostridium difficile co-infection in foals.

    PubMed

    Uzal, F A; Diab, S S; Blanchard, P; Moore, J; Anthenill, L; Shahriar, F; Garcia, J P; Songer, J G

    2012-05-01

    Clostridium perfringens type C is one of the most important agents of enteric disease in newborn foals. Clostridium difficile is now recognized as an important cause of enterocolitis in horses of all ages. While infections by C. perfringens type C or C. difficile are frequently seen, we are not aware of any report describing combined infection by these two microorganisms in foals. We present here five cases of foal enterocolitis associated with C. difficile and C. perfringens type C infection. Five foals between one and seven days of age were submitted for necropsy examination to the California Animal Health and Food Safety Laboratory. The five animals had a clinical history of acute hemorrhagic diarrhea followed by death and none had received antimicrobials or been hospitalized. Postmortem examination revealed hemorrhagic and necrotizing entero-typhlo-colitis. Histologically, the mucosa of the small intestine and colon presented diffuse necrosis and hemorrhage and it was often covered by a pseudomembrane. Thrombosis was observed in submucosal and/or mucosal vessels. Immunohistochemistry of intestinal sections of all foals showed that many large bacilli in the sections were C. perfringens. C. perfringens beta toxin was detected by ELISA in intestinal content of all animals and C. difficile toxin A/B was detected in intestinal content of three animals. C. perfringens (identified as type C by PCR) was isolated from the intestinal content of three foals. C. difficile (typed as A(+)/B(+) by PCR) was isolated from the intestinal content in 3 out of the 5 cases. This report suggests a possible synergism of C. perfringens type C and C. difficile in foal enterocolitis. Because none of the foals had received antibiotic therapy, the predisposing factor, if any, for the C. difficile infection remains undetermined; it is possible that the C. perfringens infection acted as a predisposing factor for C. difficile and/or vice versa. This report also stresses the need to perform a complete diagnostic workup in all cases of foal digestive disease. PMID:22177970

  14. Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

    PubMed Central

    Ossiprandi, Maria Cristina; Rumah, Kareem R.; Fischetti, Vincent A.

    2013-01-01

    With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low µg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or nonclostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins. PMID:21085950

  15. 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

  16. 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

  17. 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...

  18. 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

  19. 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 toxins 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

  20. Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

    PubMed Central

    2012-01-01

    Background Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. Results Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, β2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. Conclusions Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain. PMID:22748233

  1. 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

  2. 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

  3. 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

  4. NetB, a Pore-Forming Toxin from Necrotic Enteritis Strains of Clostridium perfringens

    PubMed Central

    Keyburn, Anthony L.; Bannam, Trudi L.; Moore, Robert J.; Rood, Julian I.

    2010-01-01

    The Clostridium perfringens necrotic enteritis B-like toxin (NetB) is a recently discovered member of the ?-barrel pore-forming toxin family and is produced by a subset of avian C. perfringens type A strains. NetB is cytotoxic for avian cells and is associated with avian necrotic enteritis. This review examines the current state of knowledge of NetB: its role in pathogenesis, its distribution and expression in C. perfringens and its vaccine potential. PMID:22069665

  5. Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning.

    PubMed

    Arcieri, R; Dionisi, A M; Caprioli, A; Lopalco, P; Prato, R; Germinario, C; Rizzo, C; Larocca, A M; Barbuti, S; Greco, D; Luzzi, I

    1999-01-01

    An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al rag' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning. PMID:10030546

  6. 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...

  7. 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...

  8. 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...

  9. 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

  10. EFFECT OF OZONE STRESS ON CLOSTRIDIUM PERFRINGENS VIABILITY FOLLOWING THE AQUEOUS TREATMENT OF BEEF SURFACES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antimicrobial efficacy of ozone on the food-borne pathogen, Clostridium perfringens, was evaluated on London Broil top round cut beef surfaces using an aqueous wash system. Current food processing methods do not assure elimination of spores of C. perfringens, thus there is a high likelihood of ...

  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. Comparison of two bacteriophage derived enzymes that lyse strains of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that is the third leading cause of food-borne bacterial disease among humans while in chickens C. perfringens is the presumptive etiology of necrotic enteritis. Although the organism can be controlled by antibiotics, there ...

  13. 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 ...

  14. Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR.

    PubMed Central

    Yoo, H S; Lee, S U; Park, K Y; Park, Y H

    1997-01-01

    Clostridium perfringens has been classified into five toxigenic types (A through E) on the basis of its capability to produce major lethal toxins (alpha, beta, epsilon, and iota toxins). Seroneutralization with mice or guinea pigs has been used to type each toxin, but this conventional method has some disadvantages. Therefore, we used a molecular biological technique to type the bacterium in the present study. A multiplex PCR was developed for this purpose. This method has several advantages in comparison with seroneutralization with mice or guinea pigs. By this method, we also investigated the most prevalent type(s) of the organism in Korean calves, piglets, and chickens showing clinical symptoms such as diarrhea, enterotoxemia, and necrotic enteritis. Only type A was isolated from calves and chickens, while type C (2 of 14 isolates), in addition to type A, was isolated from piglets. These results suggested that seroneutralization could be replaced by our new method and that type A of C. perfringens is the most prevalent type in livestock in Korea. PMID:8968913

  15. 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.

  16. 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

  17. Acute Hemolysis in the Emergency Department: Think about Clostridium perfringens!

    PubMed

    Ccilia, Roustit; Baptiste, Vall; Benjamin, Clouzeau; Virginie, Heydel; Guillaume, Valdenaire; Philippe, Revel; Matthieu, Biais

    2013-01-01

    Clostridium perfringens (CP) gives several clinical settings, from an asymptomatic to a massive intravascular hemolysis. We report a case of fatal intravascular hemolysis due to CP septicemia having a hepatic supposed starting point in the emergency department. Like in many cases, the diagnosis was made when patient had already gone into shock and died. The CP septicemia often complicated the course of the digestive or genital pathologies. The alpha toxin can damage the structural integrity of the red cell membrane by means of a phospholipase activity. Nevertheless, a massive intravascular hemolysis arises only rarely in this septicemia, only from 7 to 15% of the cases. The emergency physician has to think about this complication in case of hemoglobinuria and/or signs of hemolysis associated with a septic syndrome. An immediate antibiotic treatment adapted as well as the symptomatic treatment of the spread intravascular coagulation could improve the survival of these patients. PMID:24151563

  18. 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.

  19. 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

  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. 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

  2. In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains.

    PubMed

    Schoster, A; Kokotovic, B; Permin, A; Pedersen, P D; Dal Bello, F; Guardabassi, L

    2013-04-01

    Probiotics have gained importance in human and veterinary medicine to prevent and control clostridial enteric disease. Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile and Clostridium perfringens. The objective of this study was to examine the in vitro inhibitory effects of selected commercial bacterial strains on pathogenic clostridia and their growth characteristics under simulated gastrointestinal conditions. The inhibitory effects of 17 commercial strains of Lactobacillus (n = 16) and Bifidobacterium (n = 1) on the reference strains of C. difficile and C. perfringens were assessed by an agar well diffusion assay and by a broth culture inhibition assay using cell-free supernatant harvested at different growth phases, with and without pH neutralization. To study growth characteristics, probiotic strains were cultivated in different acid and bile environments, and growth in the modified media was compared to growth in standard medium. In the agar well diffusion assay, supernatant obtained from two probiotic strains inhibited the growth of both reference and clinical strains of C. perfringens. This effect as seen when supernatant was assessed with and without pH neutralization. Supernatants obtained from 10 probiotic strains inhibited C. difficile only when supernatant was added without pH neutralization. In the broth culture inhibition assay, growth of C. perfringens and C. difficile was inhibited by supernatant without pH neutralization from 5 and 10 probiotic strains, respectively. All potential probiotic strains were able to grow at pH 4.0 and in the presence of 0.15% and 0.3% bile but none were able to grow or survive at pH 2.0. Altogether five probiotic strains [Lactobacillus plantarum (n = 2), Lactobacillus rhamnosus (n = 2), Bifidobacterium animalis lactis (n = 1)] were shown to inhibit all strains of C. difficile and C. perfringens. The inhibitory effect was probiotic strain-specific. Two strains showed a pH-independent inhibitory effect likely due to production of either antibiotics or bacteriocins inhibiting C. perfringens only. These strains have favourable growth characteristics for use as probiotics and their efficacy as prophylactic or therapeutic measures against clostridial enteric disease should be further evaluated by clinical trials in animals. PMID:23471038

  3. 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

  4. Detection and Toxin Typing of Clostridium perfringens in Formalin-Fixed, Paraffin-Embedded Tissue Samples by PCR?

    PubMed Central

    Wu, Josephine; Zhang, Wandi; Xie, Boxun; Wu, Maoxin; Tong, Xiaodi; Kalpoe, Jayant; Zhang, David

    2009-01-01

    Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases. PMID:19109478

  5. Four Foodborne Disease Outbreaks Caused by a New Type of Enterotoxin-Producing Clostridium perfringens

    PubMed Central

    Hatakeyama, Kaoru; Obata, Hiromi; Yokoyama, Keiko; Konishi, Noriko; Itoh, Takeshi; Kai, Akemi

    2015-01-01

    The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin. PMID:25568432

  6. Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

    PubMed Central

    Huyet, Jessica; Naylor, Claire E.; Savva, Christos G.; Gibert, Maryse; Popoff, Michel R.; Basak, Ajit K.

    2013-01-01

    Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus ?-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal ?-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins. PMID:23805259

  7. 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

  8. Clostridium perfringens type A netF and netE positive and Clostridium difficile co-infection in two adult dogs.

    PubMed

    Diniz, Amanda Nádia; Silva, Rodrigo Otávio Silveira; Oliveira Junior, Carlos Augusto; Pierezan, Felipe; Lobato, Francisco Carlos Faria

    2016-04-01

    The aim of this study was to report two cases of Clostridium perfringens type A and Clostridium difficile co-infection in adult dogs. Both animals were positive for A/B toxin. Toxigenic C. difficile and C. perfringens type A positive for NetE and NetF-encoding genes were isolated. This report reinforces the necessity of studying a possible synergism of C. difficile and C. perfringens in enteric disorders. PMID:26762654

  9. Purification and characterization of Clostridium perfringens delta-toxin.

    PubMed Central

    Alouf, J E; Jolivet-Reynaud, C

    1981-01-01

    Delta-toxin, an extracellular hemolysin released by Clostridium perfringens type C, was purified from culture supernatant fluid by sequential ammonium sulfate precipitation, thiol-Sepharose gel chromatography, isoelectric focusing, and Sephadex G-75 gel filtration. The purified preparation had a specific activity of 320,000 hemolytic units per mg of protein and was homogeneous, as determined by immunochemical and electrophoretic tests. This toxin was characterized as a single polypeptide chain composed of 391 amino acid residues, 30% of which were hydrophobic. The molecular weight was found to be 42,000, and the isoelectric point was pH 9.1. Delta-toxin appeared to be amphiphilic by charge shift electrophoresis in a three-detergent system. It was immunogenic in rabbits and lethal to mice at a dose of 0.12 micrograms. The lytic activity of delta-toxin was restricted to erythrocytes of even-toed ungulates (sheep, goats, and pigs). This activity was inhibited by GM2 ganglioside but not by other gangliosides, cholesterol, lecithin, or sphingomyelin. Images PMID:6260669

  10. 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

  11. The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens.

    TOXLINE Toxicology Bibliographic Information

    Al-Sheikhly F; Truscott RB

    1977-04-01

    The intraduodenal administration of large numbers of Clostridium perfringens cells harvested from broth cultures and resuspended in PBS or fresh sterile thioglycollate broth produced a very mild form of necrotic enteritis. Administering an appropriate number of cells in culture supernatant, however, produced typical field-type disease. Alpha toxin was shown to be the significant toxin recoverable from broth-culture supernatant fluids. Requirements to produce the disease are minor intestinal damage and sufficient numbers of toxigenic C. perfringens in the intestine.

  12. Clostridium perfringens in stool, intrapartum antibiotics and gastrointestinal signs in a neonatal intensive care unit.

    PubMed

    Ahtonen, P; Lehtonen, O P; Kero, P; Eerola, E; Hartiala, K

    1994-04-01

    In 1989, we observed in our neonatal intensive care unit (NICU), an increased number of infants with gastrointestinal signs, including five cases of necrotizing enterocolitis. Clostridium perfringens was found in 26% of newborns (n = 168) and was associated significantly with the occurrence of flatulence, distended abdomen, foul-smelling stools, diarrhea and blood in stool (all p < 0.001). C. difficile was found in 17% of the newborns (n = 72). Cesarean section, low gestational age and low birth weight were significantly associated with C. perfringens in stools (all p < 0.001). Treatment with antibiotics was not associated with occurrence of C. perfringens. However, in infants with C. perfringens, intrapartum antibiotics were associated with increased appearance of abdominal distension (p < 0.05). Thus the antibiotics, which disturb primary colonization, may also favor the pathogenic role of opportunistic gut bacteria, such as C. perfringens. PMID:8025394

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

    PubMed

    Salvarani, Felipe Masiero; Silveira Silva, Rodrigo Otvio; Pires, Prhiscylla Sadan; da Costa Cruz Jnior, Eduardo Coulaud; Albefaro, Isabella Silva; de Carvalho Guedes, Roberto Maurcio; Faria Lobato, Francisco Carlos

    2012-07-01

    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

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

    PubMed Central

    Salvarani, Felipe Masiero; Silveira Silva, Rodrigo Otvio; Pires, Prhiscylla Sadan; da Costa Cruz Jnior, Eduardo Coulaud; Albefaro, Isabella Silva; de Carvalho Guedes, Roberto Maurcio; 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

  15. 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

  16. Prevalence of ?2-Toxigenic Clostridium perfringens in Horses with Intestinal Disorders

    PubMed Central

    Herholz, Cornelia; Miserez, Raymond; Nicolet, Jacques; Frey, Joachim; Popoff, Michel; Gibert, Maryse; Gerber, Heinz; Straub, Reto

    1999-01-01

    The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the ?-toxin and the recently described ?2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the ?-, ?-, ?2-, and ?-toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the ?2-toxin gene cpb2. ?2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded ?2-toxigenic C. perfringens. No ?2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for ?2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for ?2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the ?- and ?-toxins and enterotoxin. The high incidence of ?2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that ?2-toxigenic C. perfringens play an important role in the pathogenesis of typhlocolitis. PMID:9889218

  17. Influence of Water Activity on the Growth of Clostridium perfringens

    PubMed Central

    Strong, Dorothy H.; Foster, Edith F.; Duncan, Charles L.

    1970-01-01

    Each of four strains of Clostridium perfringens was grown in modified fluid thioglycolate medium which was adjusted to yield selected water activity (aw) levels. The adjustments to secure the desired aw levels were made with NaCl, KCl, or glucose. At each aw level, further modification was effected to produce four pH values. Cultures were incubated at either 37 or 46 C. The solute used to achieve the reduced aw levels appeared to have a definite effect on the magnitude of growth achieved, the rate of growth, and the limiting aw at which growth would occur. Use of glucose as the controlling solute permitted growth at the lowest aw level tested, 0.960, and yielded the greatest magnitude of growth as measured by turbidity values, at all of the aw levels investigated. Cultures grown in the medium with added KCl generally demonstrated the longest lag times and the least amount of growth. Regardless of specific solute used, as the aw level was lowered and the pH value decreased within each aw level, the rate and amount of growth were lessened. It appeared, however, that low pH values had less effect on inhibiting growth at low aw levels than at higher aw levels. Those cultures incubated at 46 C generally exhibited shorter lag periods than those at 37 C, although the maximal growth attained was somewhat less than that achieved at 37 C. The response to all of the investigated conditions was similar for each of the four strains tested. PMID:4318452

  18. Comparison of Virulence Plasmids among Clostridium perfringens Type E Isolates?

    PubMed Central

    Li, Jihong; Miyamoto, Kazuaki; McClane, Bruce A.

    2007-01-01

    Clostridium perfringens type E isolates produce iota-toxin, which is encoded by iap and ibp genes. Using Southern blot analyses, the current study identified iap/ibp plasmids of ?97 or ?135 kb among eight type E isolates. For most of these isolates, their iap/ibp plasmid also encoded urease and lambda-toxin. However, the beta2-toxin gene, if present, was on a different plasmid from the iap/ibp plasmid. For all isolates, the iap/ibp plasmid carried a tcp locus, strongly suggesting that these plasmids are conjugative. Overlapping PCR analyses demonstrated some similarity between the iap/ibp plasmids and enterotoxin-encoding plasmids of type A isolates. Additional PCR analyses demonstrated that the iap/ibp locus is located near dcm sequences, an apparent plasmid hot spot for toxin gene insertion, and that two IS1151-related sequences are present in the iap/ibp locus. To begin testing whether those IS1151-like sequences can mobilize iap/ibp genes, a PCR assay was performed that amplifies a product only from circular DNA forms that could represent transposition intermediates. This PCR assay detected circular forms containing iap/ibp genes and silent enterotoxin gene sequences, with or without an IS1151-like sequence. Collectively, these results suggest that a mobile genetic element carrying iap/ibp has inserted onto a tcp-carrying enterotoxin plasmid in a type A isolate, creating a progenitor iap/ibp plasmid. That plasmid then spread via conjugation to other isolates, converting them to type E. Further iap/ibp plasmid diversity occurred when either the iap/ibp genes later remobilized and inserted onto other conjugative plasmids or some iap/ibp plasmids acquired additional DNA sequences. PMID:17261608

  19. 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...

  20. ISOLATION AND GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA).

    PubMed

    Silva, Rodrigo O S; Almeida, Lara R; Junior, Carlos A Oliveira; Lima, Paula C S; Soares, Danielle F M; Pereira, Pedro L L; Silva, Israel J; Lobato, Francisco C F

    2016-03-01

    The importance of Clostridium perfringens for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens in stool samples from free-living South American coati ( Nasua nasua ) in Brazil. Forty-six free-living N. nasua were trapped and stool samples were collected. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and pre-enrichment in broth followed by plating in selective agar. Clostridium perfringens type A was isolated from 15 (32.6%) animals by direct plating and 36 (78.3%) animals by broth PE, and the rate of isolation was significantly different between these two methods (P < 0.01). Twelve of the 36 (33.3%) isolated strains by the PE protocol were positive for the β-2 toxin-encoding gene (cpb2) whereas the enterotoxin-encoding gene (cpe) and necrotic enteritis like-B toxin gene (netb) were not found. These results suggest that C. perfringens is commonly part of the microbiota of free-living coatis. Additionally, the use of a PE protocol appears to be essential for studies on C. perfringens in this species. PMID:27010297

  1. 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...

  2. 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...

  3. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid. 113.112 Section 113.112 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS...

  4. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid. 113.111 Section 113.111 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS...

  5. 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 ...

  6. 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...

  7. Comparison of two bacteriophage derived enzymes with lytic activity against strains of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins which are responsible for disease symptoms and pathogenesis in a variety of animals, humans and poultry. The organism is the third leading cause of food-borne bacterial disease among ...

  8. 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...

  9. CHITOSAN PROTECTS COOKED GROUND BEEF AND TURKEY AGAINST CLOSTRIDIUM PERFRINGENS SPORES DURING CHILLING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by the biopolymer chitosan during abusive chilling of cooked ground beef (25% fat) and turkey (7% fat) obtained from a retail store. Chitosan was mixed into the thawed beef or turkey at concentrations of 0.5, ...

  10. 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...

  11. 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...

  12. 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...

  13. INHIBITION OF QUORUM SENSING IN CLOSTRIDIUM PERFRINGENS AS A MEANS TOWARD FOOD SAFETY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell density-dependent signaling through the use of autoinducers, classified as quorum sensing, may play a role in the survival and virulence of Clostridium perfringens in foods. The natural 2-(5H)-furanone, ascorbic acid (vitamin C), was chosen for evaluation as a quorum sensing analogue due to it...

  14. 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...

  15. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid. 113.111 Section 113.111 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS...

  16. 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...

  17. 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...

  18. 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 ...

  19. 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 ...

  20. 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...

  1. 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 ...

  2. 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...

  3. 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

  4. Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens

    PubMed Central

    Myers, Garry S.A.; Rasko, David A.; Cheung, Jackie K.; Ravel, Jacques; Seshadri, Rekha; DeBoy, Robert T.; Ren, Qinghu; Varga, John; Awad, Milena M.; Brinkac, Lauren M.; Daugherty, Sean C.; Haft, Daniel H.; Dodson, Robert J.; Madupu, Ramana; Nelson, William C.; Rosovitz, M.J.; Sullivan, Steven A.; Khouri, Hoda; Dimitrov, George I.; Watkins, Kisha L.; Mulligan, Stephanie; Benton, Jonathan; Radune, Diana; Fisher, Derek J.; Atkins, Helen S.; Hiscox, Tom; Jost, B. Helen; Billington, Stephen J.; Songer, J. Glenn; McClane, Bruce A.; Titball, Richard W.; Rood, Julian I.; Melville, Stephen B.; Paulsen, Ian T.

    2006-01-01

    Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique genomic islands identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen. PMID:16825665

  5. The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens.

    PubMed

    Al-Sheikhly, F; Truscott, R B

    1977-01-01

    The intraduodenal administration of large numbers of Clostridium perfringens cells harvested from broth cultures and resuspended in PBS or fresh sterile thioglycollate broth produced a very mild form of necrotic enteritis. Administering an appropriate number of cells in culture supernatant, however, produced typical field-type disease. Alpha toxin was shown to be the significant toxin recoverable from broth-culture supernatant fluids. Requirements to produce the disease are minor intestinal damage and sufficient numbers of toxigenic C. perfringens in the intestine. PMID:194572

  6. 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

  7. Toxinotyping of Clostridium perfringens fecal isolates of reintroduced Pre David's deer (Elaphurus davidianus) in China.

    PubMed

    Qiu, Huiling; Chen, Fu; Leng, Xinyan; Fei, Rongmei; Wang, Libo

    2014-10-01

    Clostridium perfringens is an important pathogen causing sudden death syndrome, necrotic enteritis, and gas gangrene in ruminants, especially some deer species. Pre David's deer (Elaphurus davidianus) is one of the world's rare species and is an endangered and protected species in China. Some Pre David's deer in the Chinese Shishou Pre David's Deer Preserve died due to C. perfringens infection. We investigated the toxin types and C. perfringens enterotoxin-positive (cpe(+)) strains of isolated C. perfringens in Pre David's deer in China. We collected 155 fecal samples from the Beijing Nanhaizi Pre David's Deer Park and the Jiangsu Dafeng Pre David's Deer National Nature Reserve between July 2010 and July 2011. Bacteria isolated using blood agar and mannitol agar plates were identified by Gram staining and nested PCR for 16S rRNA. We isolated C. perfringens from 41 fecal samples and used PCR amplification of five toxin genes to identify the toxinotypes and the cpe(+) strains of C. perfringens. Twenty-one isolates were type A, 15 were type E, and five were type D. Fifteen isolates were cpe(+) strains, including eight that were type A and seven that were type E. PMID:25050802

  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%?0.13mM undissociated sorbic acid) or sodium benzoate (0.1%?0.01mM 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. SPORULATION OF CLOSTRIDIUM BOTULINUM TYPES A, B, AND E, CLOSTRIDIUM PERFRINGENS, AND PUTREFACTIVE ANAEROBE 3679 IN DIALYSIS SACS1

    PubMed Central

    Schneider, Morris D.; Grecz, Nicholas; Anellis, Abe

    1963-01-01

    Schneider, Morris D. (Quartermaster Food and Container Institute for the Armed Forces, U.S. Army, Chicago, Ill.), Nicholas Grecz, and Abe Anellis. Sporulation of Clostridium botulinum types A, B, and E, Clostridium perfringens, and Putrefactive Anaerobe 3679 in dialysis sacs. J. Bacteriol. 85:126133. 1963.Concentrated cultures of spores of Clostridium botulinum type A (33A, 37A), B (41B, 51B), and E (strain VH), C. perfringens (strain E), and Putrefactive Anaerobe 3679 were prepared in intussuscepted cellulose dialysis tubing. The apparatus consisted of a telescoped cellulose bag immersed into a suitable sporulation medium in a large Pyrex tube. The initial inoculum was a heavy suspension in physiological saline solution of either vegetative cells or heat-shocked spores. The seed material was introduced into the interior of the dialysis bag. Maximal spore populations were obtained within 10 to 12 days. Strains of C. botulinum type E and C. perfringens, known for their poor sporulation in conventional cultures, gave good spore crops in the dialysis bag. Some crops were of the order of 1010 and 1011 viable spores per liter of medium. The spores produced in the dialysis bag were conspicuously large, particularly after incubation for 20 to 30 days. Observations of the characteristics of spores formed in telescoped bags indicate that two highly resistant strains of C. botulinum, 33A and 41B, were apparently less resistant to gamma rays than spores of the same strains produced in identical media in conventional cultures. Images PMID:13992169

  10. 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

  11. Clostridium perfringens in poultry: an emerging threat for animal and public health.

    PubMed

    Van Immerseel, Filip; De Buck, Jeroen; Pasmans, Frank; Huyghebaert, Gerard; Haesebrouck, Freddy; Ducatelle, Richard

    2004-12-01

    The incidence of Clostridium perfringens-associated necrotic enteritis in poultry has increased in countries that stopped using antibiotic growth promoters. Necrotic enteritis and the subclinical form of C. perfringens infection in poultry are caused by C. perfringens type A, producing the alpha toxin, and to a lesser extent type C, producing both alpha toxin and beta toxin. Some strains of C. perfringens type A produce an enterotoxin at the moment of sporulation and are responsible for foodborne disease in humans. The mechanisms of colonization of the avian small intestinal tract and the factors involved in toxin production are largely unknown. It is generally accepted, however, that predisposing factors are required for these bacteria to colonize and cause disease in poultry. The best known predisposing factor is mucosal damage, caused by coccidiosis. Diets with high levels of indigestible, water-soluble non-starch polysaccharides, known to increase the viscosity of the intestinal contents, also predispose to necrotic enteritis. Standardized models are being developed for the reproduction of colonization of poultry by C. perfringens and the C. perfringens-associated necrotic enteritis. One such model is a combined infection with Eimeria species and C. perfringens. Few tools and strategies are available for prevention and control of C. perfringens in poultry. Vaccination against the pathogen and the use of probiotic and prebiotic products has been suggested, but are not available for practical use in the field at the present time. The most cost-effective control will probably be achieved by balancing the composition of the feed. PMID:15763720

  12. Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

    PubMed Central

    Löffler, A; Labbé, R

    1986-01-01

    Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts. Images PMID:2867991

  13. 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

  14. 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 (5ml) and subcutaneously (2ml) 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 post-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

  15. Beta2-toxin of Clostridium perfringens in a hamadryas baboon (Papio hamadryas) with enteritis.

    PubMed

    Nikolaou, Georgios N; Kik, Marja J L; van Asten, Alphons J A M; Grne, Andrea

    2009-12-01

    An 11-yr-old female hamadryas baboon (Papio hamadryas) that died with a history of diarrhea and anorexia was submitted for necropsy. Major pathologic changes were restricted to the gastrointestinal tract. The small intestinal contents were watery and sanguinous, with a deepening of the red color in the large intestines. The intestinal mucosa was hyperemic. Microscopically, lesions consisted of surface epithelial cell necrosis in association with numerous rod-shaped bacteria and high numbers of Trichuris cynocephalus nematodes. Culturing of the small intestine yielded Clostridium perfringens. No other pathogenic bacteria were cultured using routine bacteriologic techniques. Polymerase chain reaction (PCR) analysis classified the Clostridium perfringens as type A cpb2-positive. Immunohistochemical examination with anti-beta2-toxin antibodies revealed beta2-toxin in close approximation with the intestinal lesions. PMID:20063832

  16. Spoilage of an acid food product by Clostridium perfringens, C. barati and C. butyricum.

    PubMed

    de Jong, J

    1989-05-01

    Spoilage of canned pasteurized brined mung bean sprouts, acidified with citric acid to pH 4.0-4.5, was found to be caused by acid tolerant Clostridium spp. including the species barati, perfringens and butyricum. The pH limit for growth in the brine used were estimated 3.7, 3.7 and 4.0 respectively. Some of the isolated C. perfringens strains produced enterotoxins in sporulation media. The spores of the isolated anaerobes appeared to originate from mung beans, but C. barati and C. perfringens strains freshly isolated from dry beans, were unable to grow in acidified brine. During germination and sprouting of mung beans, the oxygen concentration decreased, while carbon dioxide concentration increased considerably, due to respiration of the sprouts and actively growing Enterobacteriaceae and lactobacilli. It was assumed that this allowed C. barati and C. perfringens strains to grow and acquire the observed unusual acid tolerance. After increasing aerobicity during sprouting, no growth of Clostridium spp. was observed, substantiating the assumption. PMID:2561952

  17. Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens ?2-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 ?2-toxin is described. The crystals diffracted to 2.9 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. ?2-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 ?2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 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 , ? = ? = 90, ? = 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 ? = 90.0, ? = 210.3.

  18. Time of Enterotoxin Formation and Release During Sporulation of Clostridium perfringens Type A

    PubMed Central

    Duncan, Charles L.

    1973-01-01

    Clostridium perfringens enterotoxin was detected intracellularly about 3 hr after the inoculation of vegetative cells into sporulation medium. The subsequent increase in intracellular enterotoxin concentration roughly paralleled but followed by 2.5 to 5 hr the increase in number of heat-resistant spores. The increase in biologically active toxin coincided with the increase in enterotoxin antigen. Enterotoxin was released from the sporangium with its lysis, concomitantly with the mature spore release. Images PMID:4347930

  19. Effect of Lactobacillus fermentum on beta2 toxin production by Clostridium perfringens.

    PubMed

    Allaart, Janneke G; van Asten, Alphons J A M; Vernooij, Johannes C M; Grne, Andrea

    2011-07-01

    Clostridium perfringens, although a member of the normal gut flora, is also an important cause of intestinal disease in animals and, to a lesser extent, in humans. Disease is associated with the production of one or more toxins, and little is known about environmental influences on the production of these toxins. One of the health-promoting effects of lactic acid bacteria (LAB) is the establishment and maintenance of a low pH in the intestine since an acidic environment inhibits the growth of many potentially harmful bacteria. Here, the effect of the LAB Lactobacillus fermentum on beta2 toxin production by C. perfringens is described. Coculturing of C. perfringens with L. fermentum showed that under in vitro conditions, L. fermentum was capable of silencing beta2 toxin production by C. perfringens without influencing bacterial viability. The reduction in toxin production was shown to be most likely a result of the decline in pH. Quantitative PCR showed that the reduction in beta2 toxin production was due to a decrease in cpb2 mRNA. These results suggest that in the intestine, the production of beta2 toxin by C. perfringens might be regulated by other members of the normal intestinal flora. PMID:21602389

  20. Effect of Lactobacillus fermentum on Beta2 Toxin Production by Clostridium perfringens?

    PubMed Central

    Allaart, Janneke G.; van Asten, Alphons J. A. M.; Vernooij, Johannes C. M.; Grne, Andrea

    2011-01-01

    Clostridium perfringens, although a member of the normal gut flora, is also an important cause of intestinal disease in animals and, to a lesser extent, in humans. Disease is associated with the production of one or more toxins, and little is known about environmental influences on the production of these toxins. One of the health-promoting effects of lactic acid bacteria (LAB) is the establishment and maintenance of a low pH in the intestine since an acidic environment inhibits the growth of many potentially harmful bacteria. Here, the effect of the LAB Lactobacillus fermentum on beta2 toxin production by C. perfringens is described. Coculturing of C. perfringens with L. fermentum showed that under in vitro conditions, L. fermentum was capable of silencing beta2 toxin production by C. perfringens without influencing bacterial viability. The reduction in toxin production was shown to be most likely a result of the decline in pH. Quantitative PCR showed that the reduction in beta2 toxin production was due to a decrease in cpb2 mRNA. These results suggest that in the intestine, the production of beta2 toxin by C. perfringens might be regulated by other members of the normal intestinal flora. PMID:21602389

  1. Benthic distribution of sewage sludge indicated by clostridium perfringens at a deep-ocean dump site

    SciTech Connect

    Hill, R.T.; Knight, I.T.; Anikis, M.S.; Colwell, R.R.

    1993-01-01

    Clostridium perfringens in sediment samples collected at the Deep Water Municipal Sewage Sludge Disposal Site (also called the 106-Mile Site), off the coast of New Jersey, was enumerated. The counts of C. perfringens found in sediment samples collected within and to the southwest of the 106-Mile Site were significantly elevated (P. < 0.01) compared with counts of samples from reference stations of similar depth (2,400 to 2,700 m), topography, and distance from the continental shelf, indicating that the benthic environment was contaminated by sewage dumping at the site. Low counts of C. perfringens in sediment samples collected at stations between the base of the continental shelf and the 106-Mile Site indicated that coastal runoff was not a significant source of contamination. Elevated counts were observed for samples up to 92 km to the southwest, whereas low counts were obtained for samples from stations to the east of the 106-Mile Site. The distribution is consistent with previous model predictions of sludge deposition. In areas heavily impacted by sludge dumping, C. perfringens counts were generally highest in the top 1 cm of sediment and exceeded 9,000 CFU g (dry weight) of sediment. The patterns of C. perfringens dispersal observed in the study have proved useful for selection of heavily impacted areas and control stations for further ecological evaluation by a multidisciplinary research team.

  2. Benthic Distribution of Sewage Sludge Indicated by Clostridium perfringens at a Deep-Ocean Dump Site.

    PubMed

    Hill, R T; Knight, I T; Anikis, M S; Colwell, R R

    1993-01-01

    Clostridium perfringens in sediment samples collected at the Deep Water Municipal Sewage Sludge Disposal Site (also called the 106-Mile Site), off the coast of New Jersey, was enumerated. The counts of C. perfringens found in sediment samples collected within and to the southwest of the 106-Mile Site were significantly elevated (P < 0.01) compared with counts of samples from reference stations of similar depth (2,400 to 2,700 m), topography, and distance from the continental shelf, indicating that the benthic environment was contaminated by sewage dumping at this site. Low counts of C. perfringens in sediment samples collected at stations between the base of the continental shelf and the 106-Mile Site indicated that coastal runoff was not a significant source of contamination. Elevated counts were observed for samples up to 92 km to the southwest, whereas low counts were obtained for samples from stations to the east of the 106-Mile Site. This distribution is consistent with previous model predictions of sludge deposition. In areas heavily impacted by sludge dumping, C. perfringens counts were generally highest in the top 1 cm of sediment and exceeded 9,000 CFU g (dry weight) of sediment. The patterns of C. perfringens dispersal observed in this study have proved useful for selection of heavily impacted areas and control stations for further ecological evaluation by a multidisciplinary research team. PMID:16348859

  3. Evaluation of fluorogenic TSC agar for recovering Clostridium perfringens in groundwater samples.

    PubMed

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

    2001-01-01

    Clostridium perfringens is widely recognised as a reliable water pollution indicator. Since several media can be employed for the membrane filtration enumeration of this microorganism, the main aim of this work was to investigate the ability of fluorocult-supplemented TSC-agar (Merck) for recovering Cl. perfringens from public springs used for direct human consumption. Cl. perfringens recovery was also performed on mCP agar (Cultimed) according to Directive 98/83 as well as on TSC-Agar (Merck), TSN-Agar (Merck) and SPS-Agar (BBL) media. Variance analysis of data obtained showed no statistically significant differences in the counts obtained among all media employed in this work. However, the Cl. perfringens recovery efficiencies with TSC and fluorogenic TSC agars were significantly greater (P = < 0.05) than the corresponding values of mCP and TSN media. 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 Cl. perfringens recovery in groundwater samples (85.3% of typical colonies and 82.8% of atypical colonies confirmed). In summary, the membrane filtration technique with fluorogenic TSC agar showed the best performance characteristics of all the media tested as judged by their recovery efficiency and specificity in these water samples. PMID:11464756

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

    PubMed

    Araujo, M; Sueiro, R A; Gmez, 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

  5. 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.

  6. Membrane vesicles of Clostridium perfringens type A strains induce innate and adaptive immunity.

    PubMed

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L; Curtiss, Roy

    2014-05-01

    Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-?) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-?, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection. PMID:24631214

  7. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

    PubMed

    Collier, C T; Hofacre, C L; Payne, A M; Anderson, D B; Kaiser, P; Mackie, R I; Gaskins, H R

    2008-03-15

    This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE. PMID:18068809

  8. Membrane vesicles of Clostridium perfringens Type A strains induce innate and adaptive immunity

    PubMed Central

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L.; Curtiss, Roy

    2014-01-01

    Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection. PMID:24631214

  9. Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium 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 C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

  10. Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine.

    PubMed Central

    Venczel, L V; Arrowood, M; Hurd, M; Sobsey, M D

    1997-01-01

    Cryptosporidium parvum oocysts and Clostridium perfringens spores are very resistant to chlorine and other drinking-water disinfectants. Clostridium perfringens spores have been suggested as a surrogate indicator of disinfectant activity against Cryptosporidium parvum and other hardy pathogens in water. In this study, an alternative disinfectant system consisting of an electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.) was evaluated for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores. The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine on the basis of equal weight per volume concentrations of total oxidants. Batch inactivation experiments were done on purified oocysts and spores in buffered, oxidant demand-free water at pH 7 an 25 degrees C by using a disinfectant dose of 5 mg/liter and contact times of up to 24 h. The mixed-oxidant solution was considerably more effective than free chlorine in activating both microorganisms. A 5-mg/liter dose of mixed oxidants produced a > 3-log10-unit (> 99.9%) inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores in 4 h. Free chlorine produce no measurable inactivation of Cryptosporidium parvum oocysts by 4 or 24 h, although Clostridium perfringens spores were inactivated by 1.4 log10 units after 4 h. The on-site generation of mixed oxidants may be a practical and cost-effective system of drinking water disinfection protecting against even the most resistant pathogens, including Cryptosporidium oocysts. PMID:9097455

  11. Laboratory diagnosis of antibiotic-associated diarrhea: a Polish pilot study into the clinical relevance of Clostridium difficile and Clostridium perfringens toxins.

    PubMed

    Pituch, Hanna; Obuch-Woszczaty?ski, Piotr; Wulta?ska, Dorota; van Belkum, Alex; Meisel-Miko?ajczyk, Felicja; ?uczak, Miros?aw

    2007-05-01

    In the present study, we investigated the prevalence of the Clostridium perfringens enterotoxin (CPEnt) in stool samples originally submitted for detection of Clostridium difficile toxins. Fifty-two fecal samples from inpatients were screened simultaneously for C. difficile and C. perfringens toxins: 75% of the specimens were positive for TcdA/TcdB toxins, 40% were positive for CPEnt, and 31% gave positive test results for both. It is interesting to note that only a relatively small number of C. perfringens isolates were positive for the cpe gene. All C. difficile strains were susceptible to metronidazole, but intermediate metronidazole resistance was documented for the C. perfringens isolates, which decreased upon in vitro passaging in the absence of metronidazole. We recommend that CPEnt detection should be included when diagnosing patients with presumed antibiotic-associated diarrhea. PMID:17300901

  12. Clostridium perfringens growth from spore inocula in sous-vide processed pork-based Mexican entre.

    PubMed

    Miguel-Garcia, Denise Y; Juneja, Vijay K; Valenzuela-Melendrez, Martin; Daz-Cinco, Martha E; Thippareddi, H; Aida Pea-Ramos, E

    2009-01-01

    The combined effect of Citricidal wih irradiation on Clostridium perfringens growth from spores in a sous-vide processed marinated pork meat Mexican entre was investigated. Citricidal was added at 200 or 800 ppm after mixing pork meat with tomatillo sauce and inoculated with 3 log(10) CFU/g of C. perfringens spores. Samples were irradiated at either 0 or 2 kGy, heated to an internal temperature of 71 degrees C, and stored at 4 degrees C for 28 d, 15 degrees C for 45 d, and 25 degrees C for 26 h. To simulate the conditions that may occur during transportation, distribution, storage, or handling in supermarkets or by consumers, the effect of static temperature abuse on C. perfringens growth was assessed by transferring samples stored at 4 to 25 degrees C for 13 and 15 h. Total C. perfringens populations were determined by plating diluted samples on tryptose-sulfite-cycloserine agar. Growth was not observed up to 45 d of storage at 15 degrees C in samples supplemented with 800 ppm of Citricidal. At 25 degrees C, no significant differences (P > 0.05) on the lag phase duration due to antimicrobial treatments was observed. The temperature abuse of refrigerated products for up to 15 h did not lead to C. perfringens growth to high infective dose levels of 1 million cells required to cause food poisoning. The results suggest that 800 ppm Citricidal can have significant bacteriostatic activity against C. perfringens and may provide a degree of protection against this pathogen in sous-vide processed marinated pork meat Mexican entre, under mild temperature abuse (

  13. Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids.

    PubMed

    Park, Miseon; Deck, Joanna; Foley, Steven L; Nayak, Rajesh; Songer, J Glenn; Seibel, Janice R; Khan, Saeed A; Rooney, Alejandro P; Hecht, David W; Rafii, Fatemeh

    2016-04-01

    Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes. PMID:26608548

  14. Biofilm formation of Clostridium perfringens and its exposure to low-dose antimicrobials

    PubMed Central

    Charlebois, Audrey; Jacques, Mario; Archambault, Marie

    2014-01-01

    Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Very little is known on the biofilm of C. perfringens and its exposure to subminimal inhibitory concentrations of antimicrobials. This study was undertaken to address these issues. Most of the C. perfringens human and animal isolates tested in this study were able to form biofilm (230/277). Porcine clinical isolates formed significantly more biofilm than the porcine commensal isolates. A subgroup of clinical and commensal C. perfringens isolates was randomly selected for further characterization. Biofilm was found to protect C. perfringens bacterial cells from exposure to high concentrations of tested antimicrobials. Exposure to low doses of some of these antimicrobials tended to lead to a diminution of the biofilm formed. However, a few isolates showed an increase in biofilm formation when exposed to low doses of tylosin, bacitracin, virginiamycin, and monensin. Six isolates were randomly selected for biofilm analysis using scanning laser confocal microscopy. Of those, four produced more biofilm in presence of low doses of bacitracin whereas biofilms formed without bacitracin were thinner and less elevated. An increase in the area occupied by bacteria in the biofilm following exposure to low doses of bacitracin was also observed in the majority of isolates. Morphology examination revealed flat biofilms with the exception of one isolate that demonstrated a mushroom-like biofilm. Matrix composition analysis showed the presence of proteins, beta-1,4 linked polysaccharides and extracellular DNA, but no poly-beta-1,6-N-acetyl-D-glucosamine. This study brings new information on the biofilm produced by C. perfringens and its exposure to low doses of antimicrobials. PMID:24795711

  15. Multilocus Sequence Typing Subtypes of Poultry Clostridium perfringens Isolates Demonstrate Disease Niche Partitioning▿

    PubMed Central

    Hibberd, M. C.; Neumann, A. P.; Rehberger, T. G.; Siragusa, G. R.

    2011-01-01

    Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance. PMID:21270221

  16. A recombinant carboxy-terminal domain of alpha-toxin protects mice against Clostridium perfringens.

    PubMed

    Nagahama, Masahiro; Oda, Masataka; Kobayashi, Keiko; Ochi, Sadayuki; Takagishi, Teruhisa; Shibutani, Masahiro; Sakurai, Jun

    2013-05-01

    Clostridium perfringens alpha-toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C-terminal domain (CP251-370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins. Antibodies that cross-reacted with alpha-toxin were produced after immunization with recombinant proteins including GST-CP251-370, GST-CP281-370, GST-CP311-370, CB1-372 and GST-CB251-372. Anti-GST-CP251-370, anti-GST-CP281-370 and anti-GST-CP311-370 sera neutralized both the PLC and hemolytic activities of alpha-toxin, whereas anti-CB1-372 and anti-GST-CB251-372 weakly neutralized these activities. Immunization with GST-CP251-370 and GST-CP281-370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST-CP311-370 and CB1-372. GST-CP251-370 and GST-CP281-370 are promising candidates for vaccines for clostridial-induced gas gangrene. PMID:23668605

  17. Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target

    PubMed Central

    Bhatia, Bharti; Ponia, Sanket Singh; Solanki, Amit Kumar; Dixit, Aparna; Garg, Lalit C

    2014-01-01

    Clostridium perfringens is an anaerobic pathogen known to cause vast number of diseases in mammals and birds. Various toxins and hydrolysing enzymes released by the organism are responsible for the necrosis of soft tissues. Due to serious safety issues associated with current vaccines against C. perfringens, there is a need for new drug or vaccine targets. C. perfringens is extremely dependent on its host for nutrition which can be targeted for vaccine development or drug design. Therefore, it is of interest to identify the unique transport systems used by C. perfringens involved in uptake of essential amino acids that are synthesized by the host, so that therapeutic agents can be designed to target the specific transport systems. Use of bioinformatics tools resulted in the identification of a protein component of the glutamate transport system that is not present in the host. Analysis of the conservation profile of the protein domain indicated it to be a glutamate binding protein which also stimulates the ATPase activity of ATP Binding Cassettes (ABC) transporters. Homology modelling of the protein showed two distinct lobes, which is a characteristic of substrate binding proteins. This suggests that the carboxylates of glutamate might be stabilized by electrostatic interactions with basic residues as is observed with other binding proteins. Hence, the homology model of this potential drug target can be employed for in silico docking studies by suitable inhibitors. PMID:25187678

  18. TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks.

    PubMed

    Coursodon, C F; Glock, R D; Moore, K L; Cooper, K K; Songer, J G

    2012-02-01

    Clostridium perfringens type A and type C are causative agents of necrotic enteritis (NE) in poultry. TpeL, a recently-described novel member of the family of large clostridial cytotoxins, was found in C. perfringens type C. Others have since reported TpeL in type A isolates from NE outbreaks, suggesting that it may contribute to the pathogenesis of NE. The virulence of TpeL-positive and -negative C. perfringens strains from cases of NE was examined by challenge of broiler chicks. Gross lesions typical of NE were observed in all challenged birds, and those inoculated with TpeL(pos) strains had higher average macroscopic lesion scores than those inoculated with a TpeL(neg) strain. Infection with TpeL(pos) strains may yield disease with a more rapid course and higher case fatality rate. Thus, TpeL may potentiate the effect of other virulence attributes of NE strains of C. perfringens. However, TpeL(pos) and Tpel(neg) strains compared here were not isogenic, and definitive results await the production and testing of specific TpeL mutants. PMID:22019986

  19. Use of power ultrasound to enhance the thermal inactivation of Clostridium perfringens spores in beef slurry.

    PubMed

    Evelyn; Silva, Filipa V M

    2015-08-01

    Clostridium perfringens is a pathogen of concern in pasteurised foods. The main objective of this study was to use power ultrasound to enhance the thermal inactivation of C. perfringens spores in beef slurry. The effect of simultaneous ultrasound and heat (TS, thermosonication) on the spore inactivation in beef slurry was first investigated. At 75 C, a 60 min TS process (24 kHz, 0.33 W/g) resulted in a less than 1.5 log reduction for both C. perfringens NZRM 898 and NZRM 2621 spores. Then, the thermal inactivation first order kinetic parameters of C. perfringens spores in beef slurry were estimated for the two strains. The D105 C- and z-values were 2.5 min and 10.6 C for NZRM 898 and 1.8 min and 10.9 C for NZRM 2621. After, the effect of a spore heat shock followed by ultrasound on its thermal inactivation in beef slurry was investigated. This heat shock+ultrasound pretreatment was able to double the spore thermal inactivation rate in beef slurry. For example at 95 C D-value of 20.2 min decreased to 9.8 min, demonstrating that spore exposure to heat shock followed by ultrasonication enhanced its thermal inactivation. PMID:25912313

  20. Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens

    PubMed Central

    Kim, So-Young

    2014-01-01

    This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry. PMID:26761495

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

    PubMed Central

    Gurtner, Corinne; Popescu, Francesca; Wyder, Marianne; Sutter, Esther; Zeeh, Friederike; Frey, Joachim; von Schubert, Conrad; Posthaus, Horst

    2010-01-01

    Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C ?-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis. PMID:20404076

  2. Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens.

    PubMed

    Han, Sun-Kyung; Shin, Myeong-Su; Park, Ho-Eun; Kim, So-Young; Lee, Wan-Kyu

    2014-01-01

    This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry. PMID:26761495

  3. Immunization of Broiler Chickens against Clostridium perfringens-Induced Necrotic Enteritis▿

    PubMed Central

    Kulkarni, R. R.; Parreira, V. R.; Sharif, S.; Prescott, J. F.

    2007-01-01

    Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens. PMID:17634510

  4. Comparison of different methods of cell lysis and protein measurements in Clostridium perfringens: application to the cell volume determination.

    PubMed

    Guerlava, P; Izac, V; Tholozan, J L

    1998-03-01

    Four cell lysis methods (NaOH-SDS solubilization, French press treatment, sonication, mutanolysin treatment) and three methods of protein assays (Lowry, Bradford, Pierce) were studied for their applicability to determination of cell volume in Clostridium perfringens NCTC 8798 cell suspensions. Protein contents were higher after a mechanical disruption of the cells than with the other techniques of lysis. The lowest concentrations of protein were obtained with the Bradford procedure. With each of the three protein assay methods, Clostridium perfringens NCTC 8798 protein cell contents were 45% to 58% of protein. Other factors possibly involved in variations of the intracellular volume measurements were examined. A control of the level of protein concentration in the test sample and the type of silicone oil used for the centrifugation were of prime importance during sample preparation. Under our conditions, an intracellular volume of 4 microl/(mg of protein) was routinely found for Clostridium perfringens NCTC 8798. PMID:9516540

  5. Comparative in vitro bactericidal activity of 24 antimicrobial drugs against Clostridium perfringens.

    PubMed

    Traub, W H

    1990-01-01

    Twenty-four antimicrobial drugs were examined for rapidity of onset and magnitude of bactericidal activity against selected strains of Clostridium perfringens. Ceftriaxone, imipenem, metronidazole, mezlocillin, penicillin G, piperacillin, and teicoplanin reduced colony counts by at least 3 log10 units within 2-4 h after exposure. Clindamycin, fluoroquinolones, josamycin, and tetracycline caused delayed kill (greater than or equal to 99.9% reduction of viable counts at 4-22 h after exposure). Chloramphenicol and rifampin lacked bactericidal activity against 2 of 4 strains, whereas erythromycin, fusidic acid, and fosfomycin (with added glucose-6-phosphate) were merely inhibitory for all 4 strains. Imipenem and penicillin G were combined with 9 and 12 antimicrobial drugs, respectively. Essentially all drug combinations yielded indifferent effects; only penicillin G plus doxycycline resulted in an antagonistic effect against C. perfringens. PMID:2311441

  6. Survival trends of Staphylococcus aureus, Pseudomonas aeruginosa, and Clostridium perfringens in a sandy South Florida beach.

    PubMed

    Mohammed, R L; Echeverry, A; Stinson, C M; Green, M; Bonilla, T D; Hartz, A; McCorquodale, D S; Rogerson, A; Esiobu, N

    2012-06-01

    The search for alternative indicators of disease-risk from non-enteric pathogens at the beach revealed high densities of targeted bacteria. To explain the high numbers of potential non-enteric pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, in beach sand, we investigated factors affecting their survival and distribution, as well as those of a potential fecal indicator, Clostridium perfringens. Results indicated greater S. aureus and P. aeruginosa survival and proliferation in sterile beach sand, than seawater, with diminished numbers upon exposure to natural micro-predators. C. perfringens remained relatively consistent with initial numbers. Intermediate sand particles (850 ?m-2 mm) constituted the major micro-niche; creating implications for beach classification programs. Colonization of sterile sand boxes at the beach by S. aureus and P. aeruginosa confirmed the filtering action (>100) of beach sand. The use of these potential pathogens in periodic sanitary evaluation of beach sand quality is indicated, regardless of the factors influencing their abundance. PMID:22516512

  7. [Studies of necrotizing enteritis of suckling piglets (Clostridium perfringens type C enterotoxemia) in industrialized sow breeding units. 4. Epizootiology].

    PubMed

    Khler, B; Zabke, J; Sondermann, R; Pulst, H; Rummler, H J

    1979-01-01

    Necrotising enteritis had been the cause of death of 4.9 per cent in 5,177 nursed piglets, which was established by pathological examination. The number of piglets, in that context, which had come from industrialised sow breeding units was equivalent to 92 per cent. The nursed piglet held the third position, next to smaller ruminants (19.4 per cent) and fowl (6.0 per cent), with regard to the occurrence of Clostridium perfringens enterotoxemia or necrotising enteritis in 112,218 animals which were pathologically examined after death. Necrotising enteritis so far has been rare in the GDR. No regional accumulation has been observed. Several outbreaks on industrialised sow breeding units actually remained stationary. The occurrence of the disease may be favoured by a number of factors which are conducive to accumulation of Clostridium perfringens Type C in a given stock. Group keeping of pregnant sows, simultaneous farrowing of larger groups of sows, group treatment of nursed piglets, using neomycin, chloramphenicol, oxytetracycline, and other antibiotics to which Clostridium perfringens is primarily resistant or has acquired resistance in the course of time are some of those contributive factors. Transmission of Clostridium perfringens Type C through feedstuff is possible, though it would lead to a real outbreak only by high intensity of the contamination, and it played a minor role in proliferation of the disease. 3479 Clostridium perfringens strains were isolated from 9,481 animals, both clinically intact and after death, with 30 species being included. Type classification revealed 2454 strains of Type A (70 per cent), 204 of Type D (5.88 per cent), 164 of Type C (four per cent), and 48 of Type B (1.34 per cent). There were 688 atoxic strains (17 per cent). Swine is the major carrier of Clostridium perfringens Type C, with 87 per cent of all Clostridium perfringens Type C strains having been isolated from swine. Swine was followed by fowl (four per cent), sheep (four per cent), cattle, rabbit, and dog (1.27 per cent each). Clostridium perfringens Type C was obtained from the faeces of clinically intact sows in seven instances, including two cases with sows (0.46 per cent) from farms with no previous record of necrotising enteritis. PMID:232840

  8. Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks

    PubMed Central

    Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

    2015-01-01

    There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium. PMID:26584048

  9. Multilocus Sequence Typing Analysis of Clostridium perfringens Isolates from Necrotic Enteritis Outbreaks in Broiler Chicken Populations?

    PubMed Central

    Chalmers, G.; Bruce, H. L.; Hunter, D. B.; Parreira, V. R.; Kulkarni, R. R.; Jiang, Y.-F.; Prescott, J. F.; Boerlin, P.

    2008-01-01

    Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds. PMID:18945840

  10. Epidemiology of Foodborne Disease Outbreaks Caused by Clostridium perfringens, United States, 1998–2010

    PubMed Central

    Grass, Julian E.; Gould, L. Hannah; Mahon, Barbara E.

    2015-01-01

    Clostridium perfringens is estimated to be the second most common bacterial cause of foodborne illness in the United States, causing one million illnesses each year. Local, state, and territorial health departments voluntarily report C. perfringens outbreaks to the U.S. Centers for Disease Control and Prevention through the Foodborne Disease Outbreak Surveillance System. Our analysis included outbreaks confirmed by laboratory evidence during 1998–2010. A food item was implicated if C. perfringens was isolated from food or based on epidemiologic evidence. Implicated foods were classified into one of 17 standard food commodities when possible. From 1998 to 2010, 289 confirmed outbreaks of C. perfringens illness were reported with 15,208 illnesses, 83 hospitalizations, and eight deaths. The number of outbreaks reported each year ranged from 16 to 31 with no apparent trend over time. The annual number of outbreak-associated illnesses ranged from 359 to 2,173, and the median outbreak size was 24 illnesses. Outbreaks occurred year round, with the largest number in November and December. Restaurants (43%) were the most common setting of food preparation. Other settings included catering facility (19%), private home (16%), prison or jail (11%), and other (10%). Among the 144 (50%) outbreaks attributed to a single food commodity, beef was the most common commodity (66 outbreaks, 46%), followed by poultry (43 outbreaks, 30%), and pork (23 outbreaks, 16%). Meat and poultry outbreaks accounted for 92% of outbreaks with an identified single food commodity. Outbreaks caused by C. perfringens occur regularly, are often large, and can cause substantial morbidity yet are preventable if contamination of raw meat and poultry products is prevented at the farm or slaughterhouse or, after contamination, if these products are properly handled and prepared, particularly in restaurants and catering facilities. PMID:23379281

  11. Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    PubMed Central

    Charlebois, Audrey; Jalbert, Louis-Alexandre; Harel, Josée; Masson, Luke; Archambault, Marie

    2012-01-01

    Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC90 (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC50 was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens. PMID:22970221

  12. Sequence of Two Plasmids from Clostridium perfringens Chicken Necrotic Enteritis Isolates and Comparison with C. perfringens Conjugative Plasmids

    PubMed Central

    Parreira, Valeria R.; Costa, Marcio; Eikmeyer, Felix; Blom, Jochen; Prescott, John F.

    2012-01-01

    Twenty-six isolates of Clostridium perfringens of different MLST types from chickens with necrotic enteritis (NE) (15 netB-positive) or from healthy chickens (6 netB-positive, 5 netB-negative) were found to contain 1–4 large plasmids, with most netB-positive isolates containing 3 large and variably sized plasmids which were more numerous and larger than plasmids in netB-negative isolates. NetB and cpb2 were found on different plasmids consistent with previous studies. The pathogenicity locus NELoc1, which includes netB, was largely conserved in these plasmids whereas NeLoc3, present in the cpb2 containing plasmids, was less well conserved. A netB-positive and a cpb2-positive plasmid were likely to be conjugative, and the plasmids were completely sequenced. Both plasmids possessed the intact tcp conjugative region characteristic of C. perfringens conjugative plasmids. Comparative genomic analysis of nine CpCPs, including the two plasmids described here, showed extensive gene rearrangements including pathogenicity locus and accessory gene insertions around rather than within the backbone region. The pattern that emerges from this analysis is that the major toxin-containing regions of the variety of virulence-associated CpCPs are organized as complex pathogenicity loci. How these different but related CpCPs can co-exist in the same host has been an unanswered question. Analysis of the replication-partition region of these plasmids suggests that this region controls plasmid incompatibility, and that CpCPs can be grouped into at least four incompatibility groups. PMID:23189158

  13. Clostridium perfringens: a flesh-eating bacterium living in your garden.

    PubMed

    Rothwell, Ann

    2010-10-01

    Gas gangrene is a painful, rapidly developing and potentially fatal infection despite antibiotic treatment. During the First World War thousands of soldiers died from this disease. Dr Alexis Carrel pioneered a controversial method of irrigating wounds with Dakin's solution to destroy Clostridium perfringens, a bacterium found in heavily fertilised soils that causes gas gangrene. Although this method is no longer used due to the discovery of antibiotics, many of his other ideas, such as scientifically determining the type and number of bacteria and delaying the closure of a wound until the bacteria had been eradicated, are still used today. PMID:21049805

  14. [A case of freeze-dried gas gangrene antitoxin for the treatment of Clostridium perfringens sepsis].

    PubMed

    Yoshida, Juichiro; Nakamura, Hideki; Yamada, Shinya; Sekoguchi, Satoru; Suzuki, Takahiro; Tomatsuri, Naoya; Sato, Hideki; Okuyama, Yusuke; Kimura, Hiroyuki; Yoshida, Norimasa

    2015-02-01

    A 66-year-old man was admitted to our hospital with high fever. We diagnosed a gas-containing liver abscess and performed percutaneous abscess drainage. However, 15 hours after admission, he developed massive intravascular hemolysis and acidosis. Sepsis due to Clostridium perfringens was suspected and we treated the patient intensively with multidisciplinary approaches, including antibiotics, mechanical ventilation, and renal replacement therapy. Furthermore, we administered freeze-dried gas gangrene antitoxin. Despite intensive care, the patient died 43 hours after admission. PMID:25748160

  15. Bayesian modeling of Clostridium perfringens growth in beef-in-sauce products.

    PubMed

    Jaloustre, S; Cornu, M; Morelli, E; Nol, V; Delignette-Muller, M L

    2011-04-01

    Models on Clostridium perfringens growth which have been published to date have all been deterministic. A probabilistic model describing growth under non-isothermal conditions was thus proposed for predicting C. perfringens growth in beef-in-sauce products cooked and distributed in a French hospital. Model parameters were estimated from different types of data from various studies. A Bayesian approach was proposed to model the overall uncertainty regarding parameters and potential variability on the 'work to be done' (h(0)) during the germination, outgrowth and lag phase. Three models which differed according to their description of this parameter h(0) were tested. The model with inter-curve variability on h(0) was found to be the best one, on the basis of goodness-of-fit assessment and validation with literature data on results obtained under non-isothermal conditions. This model was used in two-dimensional Monte Carlo simulations to predict C. perfringens growth throughout the preparation of beef-in-sauce products, using temperature profiles recorded in a hospital kitchen. The median predicted growth was 7.810(-2) log(10) cfug(-1) (95% credibility interval [2.410(-2), 0.8]) despite the fact that for more than 50% of the registered temperature profiles cooling steps were longer than those required by French regulations. PMID:21315989

  16. Beta Lactamase Producing Clostridium perfringens Bacteremia in an Elderly Man with Acute Pancreatitis

    PubMed Central

    Mishra, Rashmi; Duncalf, Richard

    2016-01-01

    Clostridium perfringens bacteremia is associated with adverse outcomes. Known risk factors include chronic kidney disease, malignancy, diabetes mellitus, and gastrointestinal disease. We present a 74-year-old man admitted with confusion, vomiting, and abdominal pain. Exam revealed tachycardia, hypotension, lethargy, distended abdomen, and cold extremities. He required intubation and aggressive resuscitation for septic shock. Laboratory data showed leukocytosis, metabolic acidosis, acute kidney injury, and elevated lipase. CT scan of abdomen revealed acute pancreatitis and small bowel ileus. He was started on vancomycin and piperacillin-tazobactam. Initial blood cultures were positive for C. perfringens on day five. Metronidazole and clindamycin were added to the regimen. Repeat CT (day 7) revealed pancreatic necrosis. The patient developed profound circulatory shock requiring multiple vasopressors, renal failure requiring dialysis, and bacteremia with vancomycin-resistant enterococci. Hemodynamic instability precluded surgical intervention and he succumbed to multiorgan failure. Interestingly, our isolate was beta lactamase producing. We review the epidemiology, risk factors, presentation, and management of C. perfringens bacteremia. This case indicates a need for high clinical suspicion for clostridial sepsis and that extended spectrum beta lactam antibiotic coverage may be inadequate and should be supplemented with use of clindamycin or metronidazole if culture is positive, until sensitivities are known. PMID:26904307

  17. Benthic distribution of sewage sludge indicated by Clostridium perfringens at a deep-ocean dump site

    SciTech Connect

    Hill, R.T.; Anikis, M.S.; Colwell, R.R. ); Knight, I.T. )

    1993-01-01

    Since 1986, sewage sludge from New York and northern New Jersey has been dumped 196 km off the coast of New Jersey at the Deep Water Municipal Sewage Sludge Disposal Site. This study determines the distribution of sludge contamination of the benthic environment in the area, by using Clostridium perfringens as an indicator. The counts of C. perfringens confirm a previous report that sewage sludge is reaching the ocean floor at the disposal site as a result of the sludge dumping. C. perfringes counts within the dump site and to the south and west of the dump site are considerably elevated compared to counts east of the site. The distribution pattern of C. perfringes is broadly consistent with the estimates of the sea floor area impacted in the most recent computer model. However, the area of maximum desposition of sludge may be slightly further north than predicted. Use of C. perfringens has proven to be an efficient and reliable method for tracing sewage contamination of deep ocean sediments. 18 refs., 4 figs., 3 tabs.

  18. Beta Lactamase Producing Clostridium perfringens Bacteremia in an Elderly Man with Acute Pancreatitis.

    PubMed

    Mishra, Rashmi; Sinha, Nupur; Duncalf, Richard

    2016-01-01

    Clostridium perfringens bacteremia is associated with adverse outcomes. Known risk factors include chronic kidney disease, malignancy, diabetes mellitus, and gastrointestinal disease. We present a 74-year-old man admitted with confusion, vomiting, and abdominal pain. Exam revealed tachycardia, hypotension, lethargy, distended abdomen, and cold extremities. He required intubation and aggressive resuscitation for septic shock. Laboratory data showed leukocytosis, metabolic acidosis, acute kidney injury, and elevated lipase. CT scan of abdomen revealed acute pancreatitis and small bowel ileus. He was started on vancomycin and piperacillin-tazobactam. Initial blood cultures were positive for C. perfringens on day five. Metronidazole and clindamycin were added to the regimen. Repeat CT (day 7) revealed pancreatic necrosis. The patient developed profound circulatory shock requiring multiple vasopressors, renal failure requiring dialysis, and bacteremia with vancomycin-resistant enterococci. Hemodynamic instability precluded surgical intervention and he succumbed to multiorgan failure. Interestingly, our isolate was beta lactamase producing. We review the epidemiology, risk factors, presentation, and management of C. perfringens bacteremia. This case indicates a need for high clinical suspicion for clostridial sepsis and that extended spectrum beta lactam antibiotic coverage may be inadequate and should be supplemented with use of clindamycin or metronidazole if culture is positive, until sensitivities are known. PMID:26904307

  19. CARVACROL, CINNAMALDEHYDE, OREGANO OIL, AND THYMOL INHIBIT CLOSTRIDIUM PERFRINGENS SPORE GERMINATION AND OUTGROWTH IN GROUND TURKEY DURING CHILLING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens by plant-derived carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground turkey (75% lean) obtained from a local grocery store. Test substances were mixed into thawed turkey product at concentrations of 0.1, 0.5...

  20. Draft Genome Sequence of Clostridium perfringens Strain JJC, a Highly Efficient Hydrogen Producer Isolated from Landfill Leachate Sludge

    PubMed Central

    Wong, Y. M.; Gan, H. M.; Austin, C. M.

    2014-01-01

    Clostridium perfringens strain JJC is an effective biohydrogen and biochemical producer that was isolated from landfill leachate sludge. Here, we present the assembly and annotation of its genome, which may provide further insights into the gene interactions involved in efficient biohydrogen production. PMID:24604637

  1. INFLUENCE OF NAC1 CONTENT AND COOLING RATE ON OUTGROWTH OF CLOSTRIDIUM PERFRINGENS SPORES IN COOKED HAM AND BEEF

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of NaCl concentration and cooling rate on the ability of Clostridium perfringens to grow from spore inocula was studied using a process that simulates the industrial cooking and cooling of smoked boneless ham and beef roast. Sufficient NaCl was added to 2 different ground commercially-ob...

  2. INHIBITION OF GERMINATION AND OUTGROWTH OF CLOSTRIDIUM PERFRINGENS SPORES BY LACTIC ACID SALTS DURING COOLING OF COOKED GROUND TURKEY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens spore germination and outgrowth by lactic acid salts during exponential cooling of cooked ground turkey products was evaluated. Injected turkey containing either calcium lactate, potassium lactate, or sodium lactate (1.0, 2.0, 3.0 or 4.8% w/w) along with a cont...

  3. CONTROL OF CLOSTRIDIUM PERFRINGENS GERMINATION AND OUTGROWTH BY BUFFERED SODIUM CITRATE DURING CHILLING OF ROAST BEEF AND INJECTED PORK

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens germination and outgrowth by buffered sodium citrate and buffered sodium citrate supplemented with sodium diacetate was evaluated during abusive chilling of roast beef and injected pork. Beef top rounds and pork loins were injected with a brine containing NaCl (...

  4. CONTROL OF CLOSTRIDIUM PERFRINGENS GERMINATION AND OUTGROWTH BY BUFFERED SODIUM CITRATE DURING CHILLING OF ROAST BEEF AND INJECTED PORK.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens germination and outgrowth by buffered sodium citrate and buffered sodium citrate supplemented with sodium diacetate was evaluated during abusive chilling of roast beef and injected pork. Beef top rounds and pork loins were injected with a brine containing NaCl (...

  5. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

    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 b...

  6. TRANSLOCATION OF CAMPYLOBACTER, SALMONELLA AND CLOSTRIDIUM PERFRINGENS TO SEVERAL LYMPHOID ORGANS FOLLOWING ORAL OR INTRACLOACAL INOCULATION OF BROILER CHICKS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Day old broiler chicks were either orally or intracloacally inoculated with a 100ul suspension containing 106-109 cells of one of three marker strains of either Campylobacter jejuni, Salmonella spp. or Clostridium perfringens. At one hour, one day and one week following inoculation, five birds from...

  7. Effect of phosphate and meat (pork) types on the germination and outgrowth of Clostridium perfringens spores during abusive chilling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of blends of phosphates and the pork meat type (pale, soft and exudative, PSE; normal; and dark, firm and dry, DFD) on the germination and outgrowth of Clostridium perfringens during abusive exponential chilling times was evaluated. Two different phosphates, tetrasodium pyrophosphate (TSP...

  8. Control of Clostridium perfringens Spores by Green Tea Leaf Extracts During Cooling of Cooked Ground Beef, Chicken, and Pork

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by two green tea extracts with low (GTL; 141 mg total catechins/g of green tea extract) and high (GTE; 697 mg total catechins/g of extract) catechin levels during abusive chilling of retail cooked ground beef, ...

  9. AN EVALUATION OF ASCORBIC ACID AS A QUORUM SENSING ANALOGUE TO CONTROL GROWTH, SPORULATION, AND ENTEROTOXIN PRODUCTION IN CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of quorum sensing by enterotoxin-producing strains of Clostridium perfringens was investigated. Autoinducer-2 (AI-2) activity was measured in the presence and absence of ascorbic acid (vitamin C; concentrations ranging from 10 to 300 mM), an AI-2 analogue. Subsequent effects on AI-2 pro...

  10. Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

    PubMed Central

    2009-01-01

    Background Clostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized. Results Using 2-DE mass spectrometry strategy, we identified major surface (22) and cell envelope (10) proteins from Clostridium perfringens ATCC13124 and those differentially expressed (11) in cells grown on cooked meat medium (CMM) in comparison with cells grown in reference state (tryptose-yeast extract-glucose medium). Riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited 2.19 to 8.5 fold increase in the expression level in cells growing on CMM. Conclusion Ornithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. Based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of C. perfringens from the environment and food. Most of the proteins over-expressed in CMM were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism. PMID:19664283

  11. Epidemiological and pathobiological profiles of Clostridium perfringens infections: review of consecutive series of 33 cases over a 13-year period

    PubMed Central

    Shindo, Yuji; Dobashi, Yoh; Sakai, Toshiyasu; Monma, Chie; Miyatani, Hiroyuki; Yoshida, Yukio

    2015-01-01

    Background: Although Clostridium perfringens (C. perfringens) is well known as the causative agent of several forms of enteric disease, precise epidemiological and pathobiological aspects are still unknown. Methods: We retrospectively reviewed the culture results of samples collected in our hospital from 2001 through 2013. In addition, for the detection and toxinogenic typing of C. perfringens, polymerase-chain-reaction amplification (PCR)-based rapid analysis was performed in 6 cases using DNA extracted from paraffin-embedded tissues. Results: A total of 35 samples from 33 cases were positive for C. perfringens, representing an incidence of 0.017% (35/205, 114). Among 33 patients, 21 patients manifested sepsis and 7 patients had bacteremia. One of the septic cases was complicated by fatal intravascular hemolysis and thus, the prevalence was estimated at 3.0% among C. perfringens infections (1/33). The direct causative disease or state for C. perfringens infection was identified in 18 patients: surgery or intervention for cancers, 8 patients; chemotherapy for cancer, 2 patients; surgery or intervention for non-neoplastic disease, 6 patients; liver cirrhosis, 3 patients, etc. PCR-based toxinogenic typing of C. perfringens detected the alpha-toxin gene only in tissue from a patient who died of massive hemolysis; none of the toxin genes could be amplified in the other 5 cases examined. Conclusions: The prevalence of overt C. perfringens infection is low, but upon detection, infected patients should be carefully monitored for fatal acute hemolysis caused by type A C. perfringens. Furthermore, PCR-based rapid detection of C. perfringens and toxinogenic typing by archival pathological material is applicable as a diagnostic tool. PMID:25755747

  12. Occurrence of anaerobic bacterial, clostridial, and Clostridium perfringens spores in raw goose livers from a poultry processing plant in Hungary.

    PubMed

    Turcsn, J; Varga, L; Turcsn, Z; Szigeti, J; Farkas, L

    2001-08-01

    Anaerobic bacterial, clostridial, and Clostridium perfringens spores were enumerated in raw goose liver samples taken after evisceration of the birds (EB) in the slaughterhouse and after removal of blood vessels from the liver (RBVL) in the cannery. The samples taken after RBVL had significantly higher (P < 0.05) spore counts than did those taken after EB, indicating contamination of livers during processing. The number of C. perfringens spores was one log cycle higher in the samples taken after RBVL than in those taken after EB (P < 0.05). The confirmation of C. perfringens according to the profiles of Rapid ID 32 A tests was carried out by means of the ATB Plus computer program. With an identification percentage of 99.9 and a T-value of 0.65, the suspect colonies proved to be C. perfringens. Therefore, the importance of an appropriate cleaning and sanitation program and of personnel hygiene should be emphasized in the industry. PMID:11510671

  13. Regulation of Sialidase Production in Clostridium perfringens by the Orphan Sensor Histidine Kinase ReeS

    PubMed Central

    Hiscox, Thomas J.; Harrison, Paul F.; Chakravorty, Anjana; Choo, Jocelyn M.; Ohtani, Kaori; Shimizu, Tohru

    2013-01-01

    Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. ?-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, ?-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens. PMID:24023881

  14. Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase

    SciTech Connect

    Suryadinata, Randy Seabrook, Shane A.; Adams, Timothy E.; Nuttall, Stewart D.; Peat, Thomas S.

    2015-06-30

    The structure of C. perfringens sortase D was determined at 1.99 Å resolution. Comparative biochemical and structural analyses revealed that this transpeptidase may represent a new subclass of the sortase D family. The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

  15. Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm.

    PubMed

    Chalmers, G; Martin, S W; Hunter, D B; Prescott, J F; Weber, L J; Boerlin, P

    2008-02-01

    Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study. PMID:17888591

  16. Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin

    PubMed Central

    Keyburn, Anthony L.; Yan, Xu-Xia; Bannam, Trudi L.; Van Immerseel, Filip; Rood, Julian I.; Moore, Robert J.

    2009-01-01

    A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis. PMID:19931005

  17. Production of a bacteriocin by a poultry derived Campylobacter jejuni isolate with antimicrobial activity against Clostridium perfringens and other Gram positive bacteria.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have purified a bacteriocin peptide (termed CUV-3), produced by a poultry cecal isolate of Campylobacter jejuni (strain CUV-3) with inhibitory activity against Gram positive bacteria including Clostridium perfringens (38 strains), Staphylococcus aureus, Staphylococcus epidermidis and Listeria mon...

  18. Novel use of tryptose sulfite cycloserine egg yolk agar for isolation of Clostridium perfringens during an outbreak of necrotizing enterocolitis in a neonatal unit.

    PubMed

    Kotsanas, Despina; Carson, Jolene A; Awad, Milena M; Lyras, Dena; Rood, Julian I; Jenkin, Grant A; Stuart, Rhonda L; Korman, Tony M

    2010-11-01

    Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens. PMID:20826643

  19. Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to iden...

  20. Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

    PubMed

    Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

    2014-09-01

    The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis genes in the jejunum of broilers is modulated by fishmeal inclusion in the diet. Furthermore, we show for the first time suppression of CD36 mRNA levels in the intestine of broilers challenged with Eimeria or C. perfringens. PMID:25518436

  1. Identification of Small Molecule Inhibitors of Clostridium perfringens ?-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

    PubMed Central

    Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

    2010-01-01

    The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ?-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ?-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-?-toxin therapeutics. PMID:20721308

  2. The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review.

    PubMed

    Shojadoost, Bahram; Vince, Andrew R; Prescott, John F

    2012-01-01

    Necrotic enteritis (NE) is one of the most important enteric diseases in poultry and is a high cost to the industry worldwide. It is caused by avian-specific, Necrotic Enteritis Beta toxin (NetB)-producing, strains of Clostridium perfringens that also possess in common other virulence-associated genes. In Europe the disease incidence has increased since the ban on in-feed "growth promoting" antibiotics. Because of this, many recent studies of NE have focused on finding different ways to control the disease, and on understanding its pathogenesis. Frustratingly, reproduction of the disease has proven impossible for some researchers. This review describes and discusses factors known to be important in reproducing the disease experimentally, as well as other considerations in reproducing the disease. The critical bacterial factor is the use of virulent, netB-positive, strains; virulence can be enhanced by using tpeL- positive strains and by the use of young rather than old broth cultures to increase toxin expression. Intestinal damaging factors, notably the use of concurrent or preceding coccidial infection, or administration of coccidial vaccines, combined with netB-positive C. perfringens administration, can also be used to induce NE. Nutritional factors, particularly feeding high percentage of cereals containing non-starch polysaccharides (NSP) (wheat, rye, and barley) enhance disease by increasing digesta viscosity, mucus production and bacterial growth. Animal proteins, especially fish meal, enhance C. perfringens proliferation and toxin production. Other factors are discussed that may affect outcome but for which evidence of their importance is lacking. The review compares the different challenge approaches; depending on the aim of particular studies, the different critical factors can be adjusted to affect the severity of the lesions induced. A standardized scoring system is proposed for international adoption based on gross rather than histopathological lesions; if universally adopted this will allow better comparison between studies done by different researchers. Also a scoring system is provided to assist decisions on humane euthanasia of sick birds. PMID:23101966

  3. Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase

    PubMed Central

    Suryadinata, Randy; Seabrook, Shane A.; Adams, Timothy E.; Nuttall, Stewart D.; Peat, Thomas S.

    2015-01-01

    The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99? resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded ?-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316?K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents. PMID:26143922

  4. Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase.

    PubMed

    Suryadinata, Randy; Seabrook, Shane A; Adams, Timothy E; Nuttall, Stewart D; Peat, Thomas S

    2015-07-01

    The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents. PMID:26143922

  5. Characterization of Clostridium perfringens TpeL toxin gene carriage, production, cytotoxic contributions, and trypsin sensitivity.

    PubMed

    Chen, Jianming; McClane, Bruce A

    2015-06-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  6. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  7. Identification of Novel Pathogenicity Loci in Clostridium perfringens Strains That Cause Avian Necrotic Enteritis

    PubMed Central

    Parreira, Valeria R.; Marri, Pradeep R.; Rosey, Everett L.; Gong, Joshua; Songer, J. Glenn; Vedantam, Gayatri; Prescott, John F.

    2010-01-01

    Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne. PMID:20532244

  8. [New insight from basic research of Clostridium perfringens alpha-toxin].

    PubMed

    Oda, Masataka

    2012-08-01

    Clostridium perfringens causes gas gangrene with inflammatory myopathies and infrequently septicemia associated with massive intravascular hemolysis. The microorganism is known to produce a variety of toxins and enzymes that are responsible for severe myonecrotic lesions. Notably, alpha-toxin, which possesses hemolytic, necrotic and lethal activities, and phospholipase C and sphingomyelinase activities, is an important agent for the diseases. The cytokine storm induced by alpha-toxin, mainly the release of TNF-alpha, plays an important role in the death and massive hemolysis. The toxin-induced release of TNF-alpha from neutrophils and macrophages is dependent on the activation of ERK1/2 signal transduction via TrkA receptor. In addition, 14- and 15-membered macrolides specifically block the toxin-induced events through the activation of neutrophils and macrophages. PMID:22894064

  9. Specificity of Interaction between Clostridium perfringens Enterotoxin and Claudin-Family Tight Junction Proteins

    PubMed Central

    Mitchell, Leslie A.; Koval, Michael

    2010-01-01

    Clostridium perfringens enterotoxin (CPE), a major cause of food poisoning, forms physical pores in the plasma membrane of intestinal epithelial cells. The ability of CPE to recognize the epithelium is due to the C-terminal binding domain, which binds to a specific motif on the second extracellular loop of tight junction proteins known as claudins. The interaction between claudins and CPE plays a key role in mediating CPE toxicity by facilitating pore formation and by promoting tight junction disassembly. Recently, the ability of CPE to distinguish between specific claudins has been used to develop tools for studying roles for claudins in epithelial barrier function. Moreover, the high affinity of CPE to selected claudins makes CPE a useful platform for targeted drug delivery to tumors expressing these claudins. PMID:22069652

  10. Claudins Overexpression in Ovarian Cancer: Potential Targets for Clostridium Perfringens Enterotoxin (CPE) Based Diagnosis and Therapy

    PubMed Central

    English, Diana P.; Santin, Alessandro D.

    2013-01-01

    Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE), a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents. PMID:23685873

  11. Carriage of Clostridium perfringens by benthic crabs in a sewage-polluted estuary.

    PubMed

    La Sala, Luciano F; Redondo, Leandro M; Daz Carrasco, Juan M; Pereyra, Ana Mara; Farber, Marisa; Jost, Helen; Fernndez-Miyakawa, Mariano E

    2015-08-15

    The Estuary of Baha Blanca (EBB), Argentina, is an important wetland under intense sewage pollution. We investigated the occurrence of Clostridium perfringens (CP) in populations of two benthic crabs (Neohelice granulata and Cyrtograpsus angulatus) and in sediment from the EBB. CP was found in 49.1% of the crabs and all of the isolates were identified as type A. The alpha (cpa) and enterotoxin (cpe) encoding genes were identified. Genetic analyses identified 13 novel sequence types, and found no clustering among isolates, suggesting that CP is not part of the crabs' commensal flora. CP carriage was 51 times more likely in crabs from the area nearest sewage outfalls compared with crabs from a reference site. Our in vitro experiments suggest that the carriage of CP in crabs is transient. The use of these benthic crabs as monitoring organisms of sewage pollution in coastal habitats is proposed. PMID:26130524

  12. The Structure of Clostridium perfringens NanI Sialidase and Its Catalytic Intermediates*

    PubMed Central

    Newstead, Simon L.; Potter, Jane A.; Wilson, Jennifer C.; Xu, Guogang; Chien, Chin-Hsiang; Watts, Andrew G.; Withers, Stephen G.; Taylor, Garry L.

    2008-01-01

    Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97Å resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97Å resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5Å resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2Å resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested. PMID:18218621

  13. Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene.

    PubMed

    Flores-Daz, Marietta; Alape-Girn, Alberto

    2003-12-15

    Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfringens and characterized by severe myonecrosis, intravascular leukocyte accumulation, and significant thrombosis. Several lines of evidence indicate that C. perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in this disease. This toxin is a Zn2+ metalloenzyme with lecithinase and sphingomyelinase activities. Its three dimensional structure shows two domains, an N-terminal domain which contains the active site, and a C-terminal domain required for the Ca2+dependent interaction with membranes. Cp-PLC displays several biological activities: it increases capillary permeability, induces platelet aggregation, hemolysis, myonecrosis, decreases cardiac contractility, and is lethal. Experiments with genetically engineered Cp-PLC variants have revealed that the sphingomyelinase activity and the C-terminal domain are required for toxicity. The myotoxicity of Cp-PLC is largely dependent on its membrane damaging effect. In addition, it has been suggested that the alterations in the blood flow induced by this toxin also contribute to muscle damage. In gas gangrene, Cp-PLC dysregulates transduction pathways in endothelial cells, platelets and neutrophils leading to the uncontrolled production of several intercellular mediators and adhesion molecules. Thus, Cp-PLC alters the traffic of neutrophils to the infected tissue and promotes thrombotic events, enhancing the conditions for anaerobic growth. PMID:15019495

  14. Investigation of the pathogenesis of massive hemolysis in a case of Clostridium perfringens septicemia.

    PubMed

    Hbl, W; Mostbeck, B; Hartleb, H; Pointner, H; Kofler, K; Bayer, P M

    1993-09-01

    Massive hemolysis is a rare, usually fatal complication of Clostridium perfringens septicemia. Of all toxins produced by the bacterium, phospholipase C (PLC) is believed to be the most likely cause of hemolysis. An influence of neuraminidase has often been suspected. In the present study, a case of C. perfringens septicemia with acute massive intravascular hemolysis is described. It led to death within 4 h of admission to the hospital. While the course of events was comparable to previously reported cases, we succeeded in gaining deeper insight into the pathogenesis by monitoring serum anti-T titer and quantifying serum PLC activity during the course of the disease. We excluded an effect of neuraminidase by a negative direct antiglobulin test, a negative anti-T lectin test, and a steady serum anti-T titer of 1 in 32. Serum PLC activity, on the other hand, showed a nearly fivefold increase (6.0 to 27.3 U/l), which is consistent with the hypothesized dominant role of this enzyme. PMID:8373904

  15. Biochemistry and Physiology of the ? Class Carbonic Anhydrase (Cpb) from Clostridium perfringens Strain 13

    PubMed Central

    Kumar, R. Siva Sai; Hendrick, William; Correll, Jared B.; Patterson, Andrew D.; Melville, Stephen B.

    2013-01-01

    The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the ? class, the first ? class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO2 and 4.8 106 s?1 M?1, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO2. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO2 in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO2 fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed. PMID:23475974

  16. Clostridium perfringens beta2-toxin in an African elephant (Loxodonta africana) with ulcerative enteritis.

    PubMed

    Bacciarini, L N; Pagan, O; Frey, J; Grne, A

    2001-11-17

    A 22-year-old female African elephant (Loxodonta africana) developed diarrhoea of unknown cause which lasted for two days. The animal was euthanased after it remained recumbent and refused to get up. Gross pathological changes were present mainly in the gastrointestinal tract. The intestinal contents were watery and dark brown. Several areas of the mucosa of the small intestine were covered minimally to moderately with fibrin and had a few 0.1 x 10 to 15 cm linear ulcerations. Microscopical lesions consisted of discrete areas of necrosis of the surface and crypt epithelium without overt inflammatory infiltrates. Culture of the small intestinal contents resulted in a moderate growth of Clostridium perfringens. No salmonella were found in the small or large intestine. PCR of the isolate of C. perfringens revealed the presence of the beta2-toxin gene cpb2 and the alpha-toxin gene cpa but no other known toxin genes. The expression of the beta2-toxin gene in vivo was demonstrated by the immunohistochemical localisation of the beta2-toxin to the microscopical lesions in the small intestine. PMID:11761293

  17. Structure-Function Analysis of Peptide Signaling in the Clostridium perfringens Agr-Like Quorum Sensing System

    PubMed Central

    Ma, Menglin; Li, Jihong

    2015-01-01

    ABSTRACT The accessory growth regulator (Agr)-like quorum sensing (QS) system of Clostridium perfringens controls the production of many toxins, including beta toxin (CPB). We previously showed (J. E. Vidal, M. Ma, J. Saputo, J. Garcia, F. A. Uzal, and B. A. McClane, Mol Microbiol 83:179–194, 2012, http://dx.doi.org/10.1111/j.1365-2958.2011.07925.x) that an 8-amino-acid, AgrD-derived peptide named 8-R upregulates CPB production by this QS system. The current study synthesized a series of small signaling peptides corresponding to sequences within the C. perfringens AgrD polypeptide to investigate the C. perfringens autoinducing peptide (AIP) structure-function relationship. When both linear and cyclic ring forms of these peptides were added to agrB null mutants of type B strain CN1795 or type C strain CN3685, the 5-amino-acid peptides, whether in a linear or ring (thiolactone or lactone) form, induced better signaling (more CPB production) than peptide 8-R for both C. perfringens strains. The 5-mer thiolactone ring peptide induced faster signaling than the 5-mer linear peptide. Strain-related variations in sensing these peptides were detected, with CN3685 sensing the synthetic peptides more strongly than CN1795. Consistent with those synthetic peptide results, Transwell coculture experiments showed that CN3685 exquisitely senses native AIP signals from other isolates (types A, B, C, and D), while CN1795 barely senses even its own AIP. Finally, a C. perfringens AgrD sequence-based peptide with a 6-amino-acid thiolactone ring interfered with CPB production by several C. perfringens strains, suggesting potential therapeutic applications. These results indicate that AIP signaling sensitivity and responsiveness vary among C. perfringens strains and suggest C. perfringens prefers a 5-mer AIP to initiate Agr signaling. IMPORTANCE Clostridium perfringens possesses an Agr-like quorum sensing (QS) system that regulates virulence, sporulation, and toxin production. The current study used synthetic peptides to identify the structure-function relationship for the signaling peptide that activates this QS system. We found that a 5-mer peptide induces optimal signaling. Unlike other Agr systems, a linear version of this peptide (in addition to thiolactone and lactone versions) could induce signaling. Two C. perfringens strains were found to vary in sensitivity to these peptides. We also found that a 6-mer peptide can inhibit toxin production by some strains, suggesting therapeutic applications. PMID:25777675

  18. Inducible Clostridium perfringens bacteriophages ΦS9 and ΦS63

    PubMed Central

    Kim, Kwang-Pyo; Born, Yannick; Lurz, Rudi; Eichenseher, Fritz; Zimmer, Markus; Loessner, Martin J.; Klumpp, Jochen

    2012-01-01

    Two inducible temperate bacteriophages ΦS9 and ΦS63 from Clostridium perfringens were sequenced and analyzed. Isometric heads and long non-contractile tails classify ΦS9 and ΦS63 in the Siphoviridae family, and their genomes consist of 39,457 bp (ΦS9) and 33,609 bp (ΦS63) linear dsDNA, respectively. ΦS63 has 3′-overlapping cohesive genome ends, whereas ΦS9 is the first Clostridium phage featuring an experimentally proven terminally redundant and circularly permuted genome. A total of 50 and 43 coding sequences were predicted for ΦS9 and ΦS63, respectively, organized into 6 distinct lifestyle-associated modules typical for temperate Siphoviruses. Putative functions could be assigned to 26 gene products of ΦS9, and to 25 of ΦS63. The ΦS9 attB attachment and insertion site is located in a non-coding region upstream of a putative phosphorylase gene. Interestingly, ΦS63 integrates into the 3′ part of sigK in C. perfringens, and represents the first functional skin-element-like phage described for this genus. With respect to possible effects of lysogeny, we did not obtain evidence that ΦS9 may influence sporulation of a lysogenized host. In contrast, interruption of sigK, a sporulation associated gene in various bacteria, by the ΦS63 prophage insertion is more likely to affect sporulation of its carrier. PMID:23050219

  19. Comparative neuropathology of ovine enterotoxemia produced by clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives.

    PubMed

    Garcia, J P; Giannitti, F; Finnie, J W; Manavis, J; Beingesser, J; Adams, V; Rood, J I; Uzal, F A

    2015-05-01

    Clostridium perfringens type D causes enterotoxemia in sheep and goats. The disease is mediated by epsilon toxin (ETX), which affects the cerebrovascular endothelium, increasing vascular permeability and leading to cerebral edema. In the present study, we compared the distribution and severity of the cerebrovascular changes induced in lambs by C. perfringens type D strain CN1020, its isogenic etx null mutant, and the ETX-producing complemented mutant. We also applied histochemical and immunohistochemical markers to further characterize the brain lesions induced by ETX. Both ETX-producing strains induced extensive cerebrovascular damage that did not differ significantly between each other in nature, neuroanatomic distribution, or severity. By contrast, lambs inoculated with the etx mutant or sterile, nontoxic culture medium did not develop detectable brain lesions, confirming that the neuropathologic effects observed in these infections are dependent on ETX production. Lambs treated with the wild-type and complemented strains showed perivascular and mural vascular edema, as well as serum albumin extravasation, particularly severe in the cerebral white matter, midbrain, medulla oblongata, and cerebellum. Brains of animals inoculated with the ETX-producing strains showed decreased expression of glial fibrillary acidic protein and increased expression of aquaporin-4 in the end-feet processes of the astrocytes around blood vessels. Early axonal injury was demonstrated with anti-amyloid precursor protein immunohistochemistry. Perivascular accumulation of macrophages/microglia with intracytoplasmic albumin globules was also observed in these animals. This study demonstrates that ETX is responsible for the major cerebrovascular changes in C. perfringens type D-induced disease. PMID:24964921

  20. Genome Sequencing and Analysis of a Type A Clostridium perfringens Isolate from a Case of Bovine Clostridial Abomasitis

    PubMed Central

    Nowell, Victoria J.; Kropinski, Andrew M.; Songer, J. Glenn; MacInnes, Janet I.; Parreira, Valeria R.; Prescott, John F.

    2012-01-01

    Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ∼3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (∼18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified. PMID:22412860

  1. NetB, a New Toxin That Is Associated with Avian Necrotic Enteritis Caused by Clostridium perfringens

    PubMed Central

    Keyburn, Anthony L; Boyce, John D; Vaz, Paola; Bannam, Trudi L; Ford, Mark E; Parker, Dane; Di Rubbo, Antonio; Rood, Julian I; Moore, Robert J

    2008-01-01

    For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential. PMID:18266469

  2. Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens.

    PubMed

    Yan, Xu-Xia; Porter, Corrine J; Hardy, Simon P; Steer, David; Smith, A Ian; Quinsey, Noelene S; Hughes, Victoria; Cheung, Jackie K; Keyburn, Anthony L; Kaldhusdal, Magne; Moore, Robert J; Bannam, Trudi L; Whisstock, James C; Rood, Julian I

    2013-01-01

    Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers. PMID:23386432

  3. Global regulation of gene expression in response to cysteine availability in Clostridium perfringens

    PubMed Central

    2010-01-01

    Background Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail. Results We first performed a tentative reconstruction of sulfur metabolism in C. perfringens and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, C. perfringens can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (cysK-cysE, cysP1 and cysP2) or an S-box riboswitch (metK and metT). We also showed that the ubiG operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA. In addition, we found that expression of pfoA (theta-toxin), nagL (one of the five genes encoding hyaluronidases) and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the ldh gene encoding the lactate dehydrogenase was induced during cysteine limitation. Conclusion Several key functions for the cellular physiology of this anaerobic bacterium were controlled in response to cysteine availability. While most of the genes involved in sulfur metabolism are regulated by premature termination of transcription, other still uncharacterized mechanisms of regulation participated in the induction of gene expression during cysteine starvation. PMID:20822510

  4. Assessing the Performance of Clostridium perfringens Cooling Models for Cooked, Uncured Meat and Poultry Products.

    PubMed

    Mohr, T B; Juneja, V K; Thippareddi, H H; Schaffner, D W; Bronstein, P A; Silverman, M; Cook, L V

    2015-08-01

    Heat-resistant spores of Clostridium perfringens may germinate and multiply in cooked meat and poultry products when the rate and extent of cooling does not occur in a timely manner. Therefore, six cooling models (PMP 7.0 broth model; PMIP uncured beef, chicken, and pork models; Smith-Schaffner version 3; and UK IFR ComBase Perfringens Predictor) were evaluated for relative performance in predicting growth of C. perfringens under dynamic temperature conditions encountered during cooling of cooked, uncured meat and poultry products. The predicted growth responses from the models were extensively compared with those observed in food. Data from 188 time-temperature cooling profiles (176 for single-rate exponential cooling and 12 for dual-rate exponential cooling) were collected from 17 independent sources (16 peer-reviewed publications and one report) for model evaluation. Data were obtained for a variety of cooked products, including meat and poultry slurries, ground meat and poultry products with and without added ingredients (e.g., potato starch, sodium triphosphate, and potassium tetrapyrophosphate), and processed products such as ham and roast beef. Performance of the models was evaluated using three sets of criteria, and accuracy was defined within a 1- to 2-log range. The percentages of accurate, fail-safe, or fail-dangerous predictions for each cooling model differed depending on which criterion was used to evaluate the data set. Nevertheless, the combined percentages of accurate and fail-safe predictions based on the three performance criteria were 34.66 to 42.61% for the PMP 7.0 beef broth model, 100% for the PMIP cooling models for uncured beef, uncured pork and uncured chicken, 80.11 to 93.18% for the Smith-Schaffner cooling model, and 74.43 to 85.23% for the UK IFR ComBase Perfringens Predictor model during single-rate exponential chilling. Except for the PMP 7.0 broth model, the other five cooling models (PMIP, Smith-Schaffner, and UK IFR ComBase) are useful and reliable tools that food processors and regulatory agencies can use to evaluate the safety of cooked or heat-treated uncured meat and poultry products exposed to cooling deviations or to develop customized cooling schedules. PMID:26219365

  5. High-level expression of his-tagged clostridial collagenase in Clostridium perfringens.

    PubMed

    Tamai, Eiji; Miyata, Shigeru; Tanaka, Hiroaki; Nariya, Hirofumi; Suzuki, Motoo; Matsushita, Osamu; Hatano, Naoya; Okabe, Akinobu

    2008-09-01

    Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases. PMID:18629492

  6. The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens.

    PubMed

    Antonissen, Gunther; Van Immerseel, Filip; Pasmans, Frank; Ducatelle, Richard; Haesebrouck, Freddy; Timbermont, Leen; Verlinden, Marc; Janssens, Geert Paul Jules; Eeckhaut, Venessa; Eeckhout, Mia; De Saeger, Sarah; Hessenberger, Sabine; Martel, An; Croubels, Siska

    2014-01-01

    Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 g/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 202.6% to 473.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P?=?0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P?=?0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 g/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens. PMID:25268498

  7. The Mycotoxin Deoxynivalenol Predisposes for the Development of Clostridium perfringens-Induced Necrotic Enteritis in Broiler Chickens

    PubMed Central

    Antonissen, Gunther; Ducatelle, Richard; Haesebrouck, Freddy; Timbermont, Leen; Verlinden, Marc; Janssens, Geert Paul Jules; Eeckhaut, Venessa; Eeckhout, Mia; De Saeger, Sarah; Hessenberger, Sabine; Martel, An; Croubels, Siska

    2014-01-01

    Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens. PMID:25268498

  8. Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes.

    PubMed

    Johansson, A; Greko, C; Engstrm, B E; Karlsson, M

    2004-04-19

    This study was undertaken to determine the in vitro susceptibility of Clostridium perfringens, isolated from poultry to antimicrobials used in poultry production. The minimal inhibitory concentration (MIC) of eight antimicrobials, including the ionophoric coccidiostat narasin, was determined for 102 C. perfringens isolates, 58 from Sweden, 24 from Norway and 20 from Denmark. Susceptibility to each antimicrobial compound was determined by broth microdilution. The isolates were obtained from broilers (89), laying hens (9) and turkeys (4), affected by necrotic enteritis (NE) or by C. perfringens associated hepatitis (CPH), and from healthy broilers. All strains, regardless of origin, proved inherently susceptible to ampicillin, narasin, avilamycin, erythromycin and vancomycin. A low frequency of resistance to virginiamycin and bacitracin was also found. Resistance to tetracycline was found in strains isolated in all three countries; Sweden (76%), Denmark (10%) and Norway (29%). In 80% of the tetracycline-resistant isolates, the two resistance genes tetA(P) and tetB(P) were amplified by PCR whereas in 20% only the tetA(P) gene was detected. No tetM gene amplicon was obtained from any of the tetracycline-resistant isolates. The uniform susceptibility to narasin revealed in this study shows that the substance can still be used to control clostridiosis. In this study, C. perfringens also showed a low degree of resistance to most other antimicrobials tested. Despite the small amounts of tetracycline used in poultry, a considerable degree of resistance to tetracycline was found in C. perfringens isolates from Swedish broilers. PMID:15066727

  9. Bioassay-Guided Chromatographic Isolation and Identification of Antibacterial Compounds from Artemisia annua L. That Inhibit Clostridium perfringens Growth.

    PubMed

    Ivarsen, Elise; Frett, Xavier C; Christensen, Kathrine B; Christensen, Lars P; Engberg, Ricarda M; Grevsen, Kai; Kjaer, Anders

    2014-01-01

    Clostridium perfringens is the causative agent of necrotic enteritis leading to significant losses in the poultry industry. Dichloromethane and n-hexane extracts of aerial parts of Artemisia annua (Asteraceae) exhibited activity against C. perfringens with minimum inhibitory concentrations (MIC) of 185 and 270 ?g/mL, respectively. Bioassay-guided fractionation of the extracts gave several active fractions (MIC between 75 and 600 ?g/mL). Investigations of the most active fractions resulted in the isolation and characterization of the polyacetylene ponticaepoxide (MIC between 100 and 200 ?g/mL) and (+)-threo-(5E)-trideca-1,5-dien-7,9,11-triyne-3,4-diol (MIC between 400 and 800 ?g/mL), the flavonols chrysosplenol D (MIC between 200 and 400 ?g/mL) and casticin (slight inhibition at 800 ?g/mL), and 2,4-dihydroxy-6-methoxyacetophenone (slight inhibition at 800 ?g/mL). Also, the coumarin scopoletin and the sesquiterpene lactone artemisinin were isolated from active fractions but showed no inhibition of C. perfringens growth at 800 and 2000 ?g/mL, respectively. Fractions containing essential oil components with camphor constituting >60% did not show inhibition of C. perfringens up to 1600 ?g/mL. Extracts and some active fractions showed higher antibacterial effect than individual bioactive compounds, suggesting that synergistic effects may underlie the observed antibacterial effect. The present study adds new valuable information on the antibacterial effect of A. annua against C. perfringens. PMID:25902977

  10. Determination of the effect of single abomasal or jejunal inoculation of Clostridium perfringens Type A in dairy cows

    PubMed Central

    2005-01-01

    Abstract A randomized study was conducted to determine if inoculation of the abomasum or jejunum with Clostridium perfringens Type A would induce jejunal hemorrhage syndrome in healthy cows. Twelve adult nonlactating dairy cows were inoculated with 10 mL of pure culture broth of C. perfringens type A (beta2 toxin positive) into the abomasum (n = 6) or jejunum (n = 6). On day 6, the cows were euthanized and samples for culture were taken from the abomasum, jejunum, and feces. No cows developed clinical signs of jejunal hemorrhage syndrome during the course of the study. Five of 6 abomasal samples and 1 of 6 jejunal samples were positive for C. perfringens Type A (beta2 negative) prior to inoculation. Eight of 12 abomasal samples, 11 of 12 fecal samples, and 10 of 12 jejunal samples were positive for C. perfringens Type A (beta2 negative) after inoculation. Intraluminal inoculation of C. perfringens Type A alone at this dose and under these conditions did not induce clinical signs of jejunal hemorrhage syndrome in adult dairy cows. The multifactorial nature of the disease likely contributed to our inability to reproduce the disease in this study. PMID:16231652

  11. Differential responses of cecal microbiota to fishmeal, Eimeria and Clostridium perfringens in a necrotic enteritis challenge model in chickens.

    PubMed

    Stanley, Dragana; Wu, Shu-Biao; Rodgers, Nicholas; Swick, Robert A; Moore, Robert J

    2014-01-01

    Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for progressing towards clinical symptoms of necrotic enteritis. PMID:25167074

  12. Implications of Decreased Nitrite Concentrations on Clostridium perfringens Outgrowth during Cooling of Ready-to-Eat Meats.

    PubMed

    Myers, Megan I; Sebranek, Joseph G; Dickson, James S; Shaw, Angela M; Tarté, Rodrigo; Adams, Kristin R; Neibuhr, Steve

    2016-01-01

    Increased popularity of natural and organic processed meats can be attributed to the growing consumer demand for preservative-free foods, including processed meats. To meet this consumer demand, meat processors have begun using celery juice concentrate in place of sodium nitrite to create products labeled as no-nitrate or no-nitrite-added meat products while maintaining the characteristics unique to conventionally cured processed meats. Because of flavor limitations, natural cures with celery concentrate typically provide lower ingoing nitrite concentrations for ready-to-eat processed meats than do conventional cures, which could allow for increased growth of pathogens, such as Clostridium perfringens, during cooked product cooling such as that required by the U.S. Department of Agriculture. The objective of this study was to investigate the implications associated with reduced nitrite concentrations for preventing C. perfringens outgrowth during a typical cooling cycle used for cooked products. Nitrite treatments of 0, 50, and 100 ppm were tested in a broth system inoculated with a three-strain C. perfringens cocktail and heated with a simulated product thermal process followed by a typical cooling-stabilization process. The nitrite concentration of 50 ppm was more effective for preventing C. perfringens outgrowth than was 0 ppm but was not as effective as 100 ppm. The interaction between nitrite and temperature significantly affected (P < 0.05) C. perfringens outgrowth in both total population and number of vegetative cells. Both temperature and nitrite concentration significantly affected (P < 0.05) C. perfringens spore survival, but the interaction between nitrite and temperature did not have a significant effect (P > 0.05) on spore outgrowth. Results indicate that decreased nitrite concentrations (50 ppm) have increased potential for total C. perfringens population outgrowth during cooling and may require additional protective measures, such as faster chilling rates. PMID:26735043

  13. Differential Responses of Cecal Microbiota to Fishmeal, Eimeria and Clostridium perfringens in a Necrotic Enteritis Challenge Model in Chickens

    PubMed Central

    Rodgers, Nicholas; Swick, Robert A.; Moore, Robert J.

    2014-01-01

    Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for progressing towards clinical symptoms of necrotic enteritis. PMID:25167074

  14. Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis

    PubMed Central

    2014-01-01

    Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers. PMID:24708344

  15. Regulation of Virulence by the RevR Response Regulator in Clostridium perfringens ?

    PubMed Central

    Hiscox, Thomas J.; Chakravorty, Anjana; Choo, Jocelyn M.; Ohtani, Kaori; Shimizu, Tohru; Cheung, Jackie K.; Rood, Julian I.

    2011-01-01

    Clostridium perfringens causes clostridial myonecrosis or gas gangrene and produces several extracellular hydrolytic enzymes and toxins, many of which are regulated by the VirSR signal transduction system. The revR gene encodes a putative orphan response regulator that has similarity to the YycF (WalR), VicR, PhoB, and PhoP proteins from other Gram-positive bacteria. RevR appears to be a classical response regulator, with an N-terminal receiver domain and a C-terminal domain with a putative winged helix-turn-helix DNA binding region. To determine its functional role, a revR mutant was constructed by allelic exchange and compared to the wild type by microarray analysis. The results showed that more than 100 genes were differentially expressed in the mutant, including several genes involved in cell wall metabolism. The revR mutant had an altered cellular morphology; unlike the short rods observed with the wild type, the mutant cells formed long filaments. These changes were reversed upon complementation with a plasmid that carried the wild-type revR gene. Several genes encoding extracellular hydrolytic enzymes (sialidase, hyaluronidase, and ?-clostripain) were differentially expressed in the revR mutant. Quantitative enzyme assays confirmed that these changes led to altered enzyme activity and that complementation restored the wild-type phenotype. Most importantly, the revR mutant was attenuated for virulence in the mouse myonecrosis model compared to the wild type and the complemented strains. These results provide evidence that RevR regulates virulence in C. perfringens; it is the first response regulator other than VirR to be shown to regulate virulence in this important pathogen. PMID:21402758

  16. Virulence for chickens of Clostridium perfringens isolated from poultry and other sources.

    PubMed

    Cooper, Kerry K; Theoret, James R; Stewart, Bernard A; Trinh, Hien T; Glock, Robert D; Songer, J Glenn

    2010-06-01

    Clostridium perfringens type A is the most common cause of poultry necrotic enteritis (NE). Of the four "major" toxins, type A strains produce only alpha toxin (CPA), which has long been considered a major factor in pathogenesis of NE. We investigated the virulence for poultry of type A strains from a variety of enteric sources. Newly-hatched CornishxRock chicks were fed a low protein diet for one week, a high protein diet for a second week, and then challenged with log-phase cultures of C. perfringens, mixed 3:4 (v/v) with high protein feed. Strain JGS4143 [genotype A, beta2 positive (cpb2(pos)), from a field case of NE] produced gross lesions compatible with NE in >85% of challenged birds. However, strains JGS1714 (enterotoxigenic genotype A, cpb2(pos), human food poisoning), JGS1936 (genotype A, cpb2(neg), bovine neonatal enteritis), JGS4142 (genotype A, cpb2(pos), bovine jejunal hemorrhage syndrome), JGS1473 (genotype A, cpb2(pos), chicken normal flora), JGS1070 (genotype C, cpb2(pos), porcine hemorrhagic enteritis), JGS1882 (genotype A, cpb2(pos), porcine neonatal enteritis), JGS1120 (ATCC 13124, genotype A, cpb2(neg), gas gangrene), JGS4151 (strain 13, genotype A, cpb2(pos), canine), and JGS4303 (SM101, enterotoxigenic genotype A, cpb2(neg), human food poisoning) failed to produce disease. In vivo passage failed to increase virulence of the non-NE strains. NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease. PMID:20193771

  17. Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

    PubMed Central

    Yan, Xu-Xia; Porter, Corrine J.; Hardy, Simon P.; Steer, David; Smith, A. Ian; Quinsey, Noelene S.; Hughes, Victoria; Cheung, Jackie K.; Keyburn, Anthony L.; Kaldhusdal, Magne; Moore, Robert J.; Bannam, Trudi L.; Whisstock, James C.; Rood, Julian I.

    2013-01-01

    ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. PMID:23386432

  18. Enterotoxigenicity and Genetic Relatedness of Clostridium perfringens Isolates from Retail Foods in the United States

    PubMed Central

    Lin, Yuan-Tong; Labbe, Ronald

    2003-01-01

    Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of the C. perfringens enterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences. All isolates possessed cpa. None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity of cpe+ strains in retail foods and the genetic diversity among nonoutbreak strains. PMID:12620854

  19. Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

    PubMed Central

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

    2010-01-01

    Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity. PMID:20532170

  20. The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review

    PubMed Central

    2012-01-01

    Necrotic enteritis (NE) is one of the most important enteric diseases in poultry and is a high cost to the industry worldwide. It is caused by avian-specific, Necrotic Enteritis Beta toxin (NetB)-producing, strains of Clostridium perfringens that also possess in common other virulence-associated genes. In Europe the disease incidence has increased since the ban on in-feed “growth promoting” antibiotics. Because of this, many recent studies of NE have focused on finding different ways to control the disease, and on understanding its pathogenesis. Frustratingly, reproduction of the disease has proven impossible for some researchers. This review describes and discusses factors known to be important in reproducing the disease experimentally, as well as other considerations in reproducing the disease. The critical bacterial factor is the use of virulent, netB-positive, strains; virulence can be enhanced by using tpeL- positive strains and by the use of young rather than old broth cultures to increase toxin expression. Intestinal damaging factors, notably the use of concurrent or preceding coccidial infection, or administration of coccidial vaccines, combined with netB-positive C. perfringens administration, can also be used to induce NE. Nutritional factors, particularly feeding high percentage of cereals containing non-starch polysaccharides (NSP) (wheat, rye, and barley) enhance disease by increasing digesta viscosity, mucus production and bacterial growth. Animal proteins, especially fish meal, enhance C. perfringens proliferation and toxin production. Other factors are discussed that may affect outcome but for which evidence of their importance is lacking. The review compares the different challenge approaches; depending on the aim of particular studies, the different critical factors can be adjusted to affect the severity of the lesions induced. A standardized scoring system is proposed for international adoption based on gross rather than histopathological lesions; if universally adopted this will allow better comparison between studies done by different researchers. Also a scoring system is provided to assist decisions on humane euthanasia of sick birds. PMID:23101966

  1. Evaluation of PCR and DNA sequencing for direct detection of Clostridium perfringens in the intestinal tract of broilers.

    PubMed

    Henriksen, Maya; Bisgaard, Magne; Francesch, Maria; Gabriel, Irene; Christensen, Henrik

    2009-09-01

    The aim of this investigation was to determine the presence of the opportunistic pathogen Clostridium perfringens by PCR and DNA sequencing, without previous cultivation. This methodology was then used to investigate how C. perfringens was affected by different preventive measures, such as ionophores and feed additives, for necrotic enteritis in broilers chickens. DNA was extracted from the intestinal content or intestinal tissue by DNA extraction kits. Detection limits for 16S rRNA, alpha-toxin, and cpb2 PCR gene targets were approximately 1 x 10(3), 5 x 10(4), and 1 x 10(6) cells per g of intestinal content or tissue, respectively, as determined with samples spiked with C. perfringens. The method was evaluated with samples from single conventional broilers or from pools of six birds of experimentally reared broilers. Conventional chickens, raised with salinomycin in their feed, showed reduced numbers of C. perfringens-positive samples (P < 0.05) for all three PCR tests. With respect to cpb2, a tendency to detect more samples as positive for C. perfringens was observed with increasing age. The addition of sodium butyrate and lactic acid in the feed for experimental birds had a minor effect (P < 0.10) on positive samples, as detected with the 16S rRNA PCR. For experimental birds fed whole wheat, only three out of six pools of six birds allowed detection of C. perfringens by the 16S rRNA PCR, compared to five for the untreated controls or the Avilamycin- or prebiotic-treated birds. All 16S rRNA partial gene sequences obtained were identical and were 99.5% similar to the rrnB gene of the type strain of C. perfringens. Two types of the partial cpb2 gene sequence were detected with a similarity of 93%. One type was translated into protein, whereas a stop codon was found in the other type. Both types were located in the "atypical" phylogenetic group of the cpb2 gene sequences. The PCR test, based on extraction of DNA from intestinal content, provided rapid screening of poultry for C. perfringens without the need to have access to facilities in order to immediately cultivate and identify bacteria at the location of sampling. Further work is suggested to determine the relationship between the degree of necrotic enteritis, the actual level of C. perfringens in the animal, and the detection achieved by PCR. PMID:19848086

  2. Effects of Tylosin on Bacterial Mucolysis, Clostridium perfringens Colonization, and Intestinal Barrier Function in a Chick Model of Necrotic Enteritis

    PubMed Central

    Collier, C. T.; van der Klis, J. D.; Deplancke, B.; Anderson, D. B.; Gaskins, H. R.

    2003-01-01

    Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota. PMID:14506046

  3. Inflammatory responses to a Clostridium perfringens type A strain and ?-toxin in primary intestinal epithelial cells of chicken embryos.

    PubMed

    Guo, Shuangshuang; Li, Changwu; Liu, Dan; Guo, Yuming

    2015-01-01

    The causative pathogen of necrotic enteritis is the Gram-positive bacterium Clostridium perfringens. Its main cell wall component, peptidoglycan (PGN), can be recognized by Toll-like receptor 2 and nucleotide-binding oligomerization domain (NOD). Consequently, the immune response is initiated via activation of nuclear factor kappa B (NF-?B) signalling pathway. An in vitro study was conducted to investigate chicken intestinal inflammatory responses to C. perfringens type A and one of its virulence factors, ?-toxin. In primary intestinal epithelial cells, C. perfringens as well as commercially available PGN and ?-toxin challenge upregulated mRNA expression of interleukin (IL)-6, IL-8 and inducible nitric oxide synthase (iNOS) with a dosage-dependent manner at 3 h post infection (p.i.; P ? 0.001). Time-course effects of three stimulators at high concentration were further examined. C. perfringens infection elevated IL-6, IL-8 and iNOS levels from 1 h to 9 h p.i., while PGN treatment increased IL-6 and IL-8 expression at 1 h and 3 h p.i. (P < 0.05). Bacterial and PGN treatments induced NOD1 expression at 6 h p.i. and only bacterial infection boosted NF-?B p65 expression at 6 h and 9 h p.i. (P < 0.05). ?-Toxin treatment upregulated IL-6 and IL-8 expression throughout infection, as well as iNOS, TNF-? and NF-?B p65 expression at later hours p.i. (P < 0.05). In conclusion, both C. perfringens and ?-toxin challenge induced intense cytokine expression associated with NF-?B activation in chicken intestinal epithelial cells. The receptors for the recognition of PGN component of C. perfringens need further investigation. PMID:25584964

  4. The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats.

    PubMed

    Garcia, J P; Beingesser, J; Fisher, D J; Sayeed, S; McClane, B A; Posthaus, H; Uzal, F A

    2012-06-15

    Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis. PMID:22296994

  5. Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxin

    PubMed Central

    Tsuge, Hideaki; Nagahama, Masahiro; Oda, Masataka; Iwamoto, Shinobu; Utsunomiya, Hiroko; Marquez, Victor E.; Katunuma, Nobuhiko; Nishizawa, Mugio; Sakurai, Jun

    2008-01-01

    The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens ι-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog βTAD at 2.8 Å resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60–Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn–turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition. PMID:18490658

  6. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

    PubMed

    Swift, Steven M; Seal, Bruce S; Garrish, Johnna K; Oakley, Brian B; Hiett, Kelli; Yeh, Hung-Yueh; Woolsey, Rebekah; Schegg, Kathleen M; Line, John Eric; Donovan, David M

    2015-06-01

    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. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production. PMID:26075507

  7. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

    PubMed Central

    Swift, Steven M.; Seal, Bruce S.; Garrish, Johnna K.; Oakley, Brian B.; Hiett, Kelli; Yeh, Hung-Yueh; Woolsey, Rebekah; Schegg, Kathleen M.; Line, John Eric; Donovan, David M.

    2015-01-01

    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. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-l-alanine amidase domain from the endolysin of the thermophilic bacteriophage ΦGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ΦCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50 °C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production. PMID:26075507

  8. C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine

    PubMed Central

    Suzuki, Hidehiko; Watari, Akihiro; Hashimoto, Eri; Yonemitsu, Miki; Kiyono, Hiroshi; Yagi, Kiyohito; Kondoh, Masuo; Kunisawa, Jun

    2015-01-01

    Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection. PMID:26018248

  9. Clostridium perfringens and sulphite reducing clostridia densities in selected tropical Malaysian rivers.

    PubMed

    Lee, Florence C H; Hakim, S L; Kamaluddin, M A; Thong, Kwai Lin

    2012-01-01

    Clostridium perfringens (CP) and sulphite reducing clostridia (SRC) densities in the Selangor River, Bernam River and Tengi River Canal were examined between April 2007 and January 2008. Water samples were taken from two or three locations along each river, using either depth-integration or grab sampling methods. The downstream sampling site of the Selangor River, Rantau Panjang, reported the highest arithmetic mean of CP and SRC densities (583.45 and 8,120.08 cfu/100 ml, respectively). Both CP and SRC densities in the Selangor River increased further downstream, but the reverse was true in the Bernam River. The SRC densities in these rivers were significantly different from each other (p < 0.05) when comparing upstream and downstream results, but CP densities were not significantly different (p > 0.05). SRC densities were significantly correlated (p < 0.05) in different locations along the Selangor River and the Bernam River. The CP densities did not show such pattern (p > 0.05). River discharge had no significant correlation with SRC or CP densities by study site (p > 0.05). Since the Selangor River has a denser human population along its banks, this study confirms CP as a suitable indicator of human fecal contamination. However, tracing CP distribution along the river is more difficult than SRC. To our knowledge, this is the first study of CP and SRC densities from Malaysian rivers. CP densities found in this study were within the range of general water bodies reported from other countries. PMID:23082563

  10. Effect of Cookery and Holding on Hams and Turkey Rolls Contaminated with Clostridium perfringens1

    PubMed Central

    Strong, Dorothy H.; Ripp, Nancy M.

    1967-01-01

    Canned hams, turkey rolls, and ground-beef casseroles were inoculated with a mixture of vegetative cells and spores of selected strains of Clostridium perfringens, in approximately known numbers. After cooking and holding at different temperatures for various times, samples of the food were plated directly on sulfadiazine-polymixin-sulfite-agar. In all cases, small but measurable percentages of the organisms survived cookery. The number of cells viable after cookery of the ham or turkey was influenced by the position of the slice of meat in the roast as well as by the final temperature to which the product was heated. Plate counts for turkey or beef casserole held at temperatures in the range of 5 to 10 C for 48 hr indicated stabilization of the population or a tendency to decrease. At 24 C, the multiplication of cells was apparent in 4 hr and rapid in 6 hr. When the food was maintained at 68 C, populations remained viable for 6 hr and the counts did not change markedly. In turkey maintained at 37 C, the number of cells increased sharply within 4 hr. PMID:4294821

  11. Clostridium perfringens infection among inmates at a county jail--Wisconsin, August 2008.

    PubMed

    2009-02-20

    On August 8, 2008, employees at a Wisconsin county jail noted nausea, vomiting, and diarrhea among more than 100 inmates during the early morning inspection. Seven inmates were seen by the jail nurse that morning. Following jail protocol, guards gave at least 60 inmates bismuth subsalicylate to relieve symptoms, and the jail nurse notified local health department staff members, who suspected a foodborne outbreak at the jail and initiated an investigation. This report summarizes the findings of an investigation by the Wisconsin Division of Public Health (WDPH) and the local health department, which determined the outbreak was caused by eating casserole containing ground turkey and beef (relative risk [RR] = 25.1) that was served during the evening meal on August 7. Clostridium perfringens enterotoxin was detected in stool samples collected from six ill inmates, and 43,000 CFU/g of the organism were isolated from a remaining sample of casserole. An environmental investigation determined the casserole was made with food items that were prepared and stored improperly. Proper food preparation and storage methods are especially important in large institutions such as jails and prisons, where large amounts of foods are prepared and served at one time. PMID:19229165

  12. Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials.

    PubMed

    Martel, A; Devriese, L A; Cauwerts, K; De Gussem, K; Decostere, A; Haesebrouck, F

    2004-02-01

    Clostridium perfringens strains isolated in 2002 from the intestines of broiler chickens from 31 different farms located in Belgium were tested for susceptibility to 12 antibiotics used for therapy, growth promotion or prevention of coccidiosis. All strains were uniformly sensitive to the ionophore antibiotics monensin, lasalocid, salinomycin, maduramycin and narasin. All were sensitive to avilamycin, tylosin and amoxicillin, while flavomycin (bambermycin) showed low or no activity. Chlortetracycline and oxytetracycline were active at very low concentrations, but low-level acquired resistance was detected in 66% of the strains investigated. Fifty percent of these strains carried the tetP(B) resistance gene, while the tet(Q) gene was detected in only one strain. One strain with high-level resistance against tetracyclines carried the tet(M) gene. Sixty-three percent of the strains showed low-level resistance to lincomycin. The lnu(A) and lnu(B) genes were each only found in one strain. Compared with a similar investigation carried out in 1980, an increase was seen in resistance percentages with lincomycin (63% against 49%) and a slight decrease with tetracycline (66% against 74%). PMID:14681061

  13. Investigating an outbreak of Clostridium perfringens gastroenteritis in a school using smartphone technology, London, March 2013.

    PubMed

    Simone, B; Atchison, C; Ruiz, B; Greenop, P; Dave, J; Ready, D; Maguire, H; Walsh, B; Anderson, S

    2014-01-01

    On 22 March 2013, 150 of 1,255 students (1317 years) and staff at a school in London reported gastrointestinal symptoms; onset peaked 8 to 12 hours after a lunch served in the school on 21 March. We performed a retrospective cohort study of all students and staff. We defined cases as school attenders on 20 and 21 March with onset of gastrointestinal symptoms between 20 and 23 March. We tested food, environmental and stool samples of cases for common pathogens and bacterial toxins. We administered an online questionnaire via email, encouraging the use of smartphones to respond, to measure risk of illness for food items eaten at school on 20 and 21 March. Survey response was 45%. Adjusted risk ratios were generated in a multivariable analysis. Those who ate chicken balti on 21 March were 19.3 times more likely to become ill (95% confidence interval: 7.350.9). Clostridium perfringens was detected in all 19 stool samples collected. Within eight school hours of its launch, 412 of 561 (73%) responders had completed the survey. Hygienic standards in the kitchen were satisfactory. The investigation was done rapidly due to smartphone technology and we recommend considering this technology in future outbreaks. PMID:24852955

  14. Intestinal events and nutritional dynamics predispose Clostridium perfringens virulence in broilers.

    PubMed

    Moran, Edwin T

    2014-12-01

    Clostridium perfringensA (CPA) entering the gastrointestinal system depends on favorable conditions to develop and subsequently extend pathogenicity. Reduction in digestive dynamics progressing from the duodenum decreases lumen oxygen, leading to anaerobic conditions in the distal lumen that favor CPA. When nutritional support is concurrently provided, an expanding population threatens the mucosa. Dietary nonstarch polysaccharides that increase viscosity further impair oxygen transfer from the mucosa, improving the ability of CPA to thrive. Incompletion of feed digestion early in the small intestine along with endogenous N provide additional support for population expansion. Glucosidase versatility with mucin elicited by distal CPA concurrently erodes the villus unstirred water layer at the apex, providing access to underlying binding sites for colonization. Proteolytic destruction within the lamina propria supports colonization to create subclinical necrotic enteritis. Eventual vascular entry of CPA and toxins provides a portal path for instituting cholangiohepatitis. Liver condemnations from inspection detect acute flock infection compared with preceding marginal losses in nutrient absorption that decrease feed efficiency. Enterocyte lysis by coccidia enable CPA access to binding sites, thereby extending villus necrosis and further impairing feed conversion. Loss of BW and increased mortality follow as mucosa involvement proceeds. In practice, supplemental feed hemicellulases that reduce digesta viscosity minimize a favorable environment for CPA, while superimposing a combination of amylase, phytase, and protease avoids nutritional support. Physical dynamics of the small intestine together with characteristics of feed that modify digesta viscosity and nutritional availability are central to establishing transient CPA as a pathogen. PMID:25260526

  15. [Acute intravasal hemolysis in Clostridium perfringens sepsis. Differential diagnosis of hemolytic episodes].

    PubMed

    Strobel, E; Nathrath, M; Peters, J; Abele-Horn, M; Wllenweber, J

    1994-03-18

    A 19-year-old man with acute lymphoblastic leukaemia developed fever, general deterioration and somnolence 3 days after a cycle of cytostatic treatment. He had anaemia (haemoglobin 6.6 g/dl), leukopenia (100/microliters) and thrombocytopenia (7,000/microliters). As an acute septicaemia was suspected he received broad spectrum antibiotic therapy, together with two units of red cell and platelet concentrates. However, his condition worsened rapidly over the next 5 hours (meningism, seizures, fever to 41.1 degrees C, dyspnoea). Another blood count revealed severe haemolysis. Computed tomography of the skull demonstrated multilocular intraparenchymal gas formation. Although the antibiotic treatment was extended the patient died several hours later. Retrospective examination for suspected transfusion mismatch provided no evidence for erythrocyte incompatibility. But there was liberation of T-antigen as sign of a bacterial cause of erythrocyte damage. An anaerobic blood culture grew Clostridium perfringens. This case demonstrates that acute intravascular haemolysis in septicaemia should be considered in the differential diagnosis of transfusion mismatch. PMID:8131716

  16. Clostridium perfringens?-toxin interaction with red cells and model membranes.

    PubMed

    Jewell, S A; Titball, R W; Huyet, J; Naylor, C E; Basak, A K; Gologan, P; Winlove, C P; Petrov, P G

    2015-10-21

    The effects of Clostridium perfringens?-toxin on host cells have previously been studied extensively but the biophysical processes associated with toxicity are poorly understood. The work reported here shows that the initial interaction between the toxin and lipid membrane leads to measurable changes in the physical properties and morphology of the membrane. A Langmuir monolayer technique was used to assess the response of different lipid species to toxin. Sphingomyelin and unsaturated phosphatidylcholine showed the highest susceptibility to toxin lypolitic action, with a two stage response to the toxin (an initial, rapid hydrolysis stage followed by the insertion and/or reorganisation of material in the monolayer). Fluorescence confocal microscopy on unsaturated phosphatidylcholine vesicles shows that the toxin initially aggregates at discrete sites followed by the formation of localised "droplets" accumulating the hydrolysis products. This process is accompanied by local increases in the membrane dipole potential by about 50 (42) mV. In contrast, red blood cells incubated with the toxin suffered a decrease of the membrane dipole potential by 50 (40) mV in areas of high toxin activity (equivalent to a change in electric field strength of 10(7) V m(-1)) which is sufficient to affect the functioning of the cell membrane. Changes in erythrocyte morphology caused by the toxin are presented, and the early stages of interaction between toxin and membrane are characterised using thermal shape fluctuation analysis of red cells which revealed two distinct regimes of membrane-toxin interaction. PMID:26303814

  17. Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A.

    PubMed

    Cooper, Kerry K; Songer, J Glenn

    2009-01-01

    Withdrawal of antimicrobial growth promoters and ionophore coccidiostats has been accompanied by a resurgence in incidence of necrotic enteritis (NE), a severe Clostridium perfringens-induced disease which some consider the most clinically dramatic bacterial enteric disease of poultry. Lesions, in jejunum and ileum, are focal-to-confluent, often with a tightly adhered pseudomembrane, and hemorrhage is uncommon. The key risk factor for development of NE is an intestinal environment that favors growth of the organism. Birds on high energy, protein-rich, wheat- or barley-based diets experience NE at a rate up to ten times greater than do birds on maize-based diets. Specific strains of type A cause NE, although only a few specific virulence attributes are known. The role of alpha toxin (CPA) has been called into question by the finding that an engineered CPA mutant retained full virulence in vivo, although the counterpoint to this is the finding that immunization with CPA toxoids provides substantial protection against NE. A recently described toxin, NetB, seems likely to be involved in pathogenesis of infection by most NE strains. Immunization with CPA, NetB, or other proteins, delivered by conventional means or vectored by recombinant attenuated Salmonella vectors may help the industry deal with NE. Future progress may be based in large part on genomic and proteomic analyses. PMID:19186215

  18. Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A.

    TOXLINE Toxicology Bibliographic Information

    Cooper KK; Songer JG

    2009-02-01

    Withdrawal of antimicrobial growth promoters and ionophore coccidiostats has been accompanied by a resurgence in incidence of necrotic enteritis (NE), a severe Clostridium perfringens-induced disease which some consider the most clinically dramatic bacterial enteric disease of poultry. Lesions, in jejunum and ileum, are focal-to-confluent, often with a tightly adhered pseudomembrane, and hemorrhage is uncommon. The key risk factor for development of NE is an intestinal environment that favors growth of the organism. Birds on high energy, protein-rich, wheat- or barley-based diets experience NE at a rate up to ten times greater than do birds on maize-based diets. Specific strains of type A cause NE, although only a few specific virulence attributes are known. The role of alpha toxin (CPA) has been called into question by the finding that an engineered CPA mutant retained full virulence in vivo, although the counterpoint to this is the finding that immunization with CPA toxoids provides substantial protection against NE. A recently described toxin, NetB, seems likely to be involved in pathogenesis of infection by most NE strains. Immunization with CPA, NetB, or other proteins, delivered by conventional means or vectored by recombinant attenuated Salmonella vectors may help the industry deal with NE. Future progress may be based in large part on genomic and proteomic analyses.

  19. Properties of four temperate bacteriophages active on Clostridium perfringens type A.

    PubMed

    Paquette, G; Fredette, V

    1977-09-01

    Four temperature bacteriophages (designated as PF1, PF2, PF3 and PF4) were isolated from lysogenic strains of Clostridium perfringens type A. On the basis of plaque morphology, pH stability, DNase and RNase resistance, buoyant density, one-step growth parameters and electron microscope phage dimensions, it seems that these phages are different and unrelated. Calcium was required for better phage replication. Bacterial strain S107 appears to be the only UV-inducible strain as compared with the other three lysogenic strains. PF2 has a unique pattern of pH stability showing two optima values: one at pH 5 and the second at pH 8-9. Generally, all four phages have a better resistance in acid than in alkaline pH values. The CsC1 equilibrium centrifugation patterns reveal low figures for phage PF1, PF2 and PF3 and show off the fact that PF4 lysates contain two viral particules different with respect to their densities, a property which other determinations failed to demonstrate. Each phage, except PF4, is well characterized by the parameters of the one-step growth cycle. PMID:22105

  20. TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens.

    PubMed

    Wisniewski, Jessica A; Traore, Daouda A; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I

    2016-03-01

    Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47?kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391?bp intergenic region upstream of tcpM. The oriT site was localized to a 150?bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer. PMID:26560080

  1. Effects of chilling rate on outgrowth of Clostridium perfringens spores in vacuum-packaged cooked beef and pork.

    PubMed

    Danler, Robert J; Boyle, Elizabeth A E; Kastner, Curtis L; Thippareddi, Harshavardhan; Fung, Daniel Y C; Phebus, Randall K

    2003-03-01

    Cooked, chilled beef and cooked, chilled pork were inoculated with three strains of Clostridium perfringens (NCTC 8238 [Hobbs serotype 2], NCTC 8239 [Hobbs serotype 3], and NCTC 10240). Inoculated products were heated to 75 degrees C, held for 10 min in a circulating water bath to heat activate the spores, and then chilled by circulating chilled brine through the water bath. Samples were chilled from 54.4 to 26.6 degrees C in 2 h and from 26.6 to 4.4 degrees C in 5 h. Differences in initial C. perfringens log counts and log counts after chilling were determined and compared with the U.S. Department of Agriculture (USDA) stabilization guidelines requiring that the chilling process allow no more than 1 log total growth of C. perfringens in the finished product. This chilling method resulted in average C. perfringens increases of 0.52 and 0.68 log units in cooked beef and cooked pork, respectively. These log increases were well within the maximum 1-log increase permitted by the USDA, thus meeting the USDA compliance guidelines for the cooling of heat-treated meat and poultry products. PMID:12636309

  2. Expression Profiles of Genes in Toll-Like Receptor-Mediated Signaling of Broilers Infected with Clostridium perfringens?

    PubMed Central

    Lu, Yang; Sarson, Aimie J.; Gong, Joshua; Zhou, Huaijun; Zhu, Weiyun; Kang, Zhumei; Yu, Hai; Sharif, Shayan; Han, Yanming

    2009-01-01

    Toll-like receptors (TLRs) participate in detecting microbial pattern molecules for activation of the host immune response. We investigated possible roles of TLRs in the chicken response to Clostridium perfringens infection by examining the expression of TLR genes and other genes involved in TLR-mediated signaling within the spleens and ilea of C. perfringens-challenged broilers. Upregulation of a tumor necrosis factor alpha-inducing factor homolog in challenged chickens compared to nave chickens was observed, regardless of the incidence of necrotic enteritis. In addition, the members of the TLR2 subfamily were found to be most strongly involved in the host response to C. perfringens challenge, although the expression of TLR4 and TLR7 was also upregulated in spleen tissues. While the combination of TLR1.2, TLR2.1, and TLR15 appeared to play a major role in the splenic response, the expression of TLR2.2 and TLR1.1 was positively correlated to the expression of adaptor molecules MyD88, TRAF6, TRIF, and receptor interacting protein 1 in the ileal tissues, demonstrating a dynamic spatial and temporal innate host response to C. perfringens. PMID:19776194

  3. Clostridium perfringens is not suitable for the indication of fecal pollution from ruminant wildlife but is associated with excreta from nonherbivorous animals and human sewage.

    PubMed

    Vierheilig, J; Frick, C; Mayer, R E; Kirschner, A K T; Reischer, G H; Derx, J; Mach, R L; Sommer, R; Farnleitner, A H

    2013-08-01

    During a 3-year study, Clostridium perfringens was investigated in defined fecal sources from a temperate alluvial backwater area of a large river system. The results reveal that using C. perfringens as a conservative water quality indicator for total fecal pollution monitoring is no longer justified but suggest that it can be used as a tracer for excreta from nonherbivorous wildlife and human sewage. PMID:23747707

  4. Clostridium perfringens Is Not Suitable for the Indication of Fecal Pollution from Ruminant Wildlife but Is Associated with Excreta from Nonherbivorous Animals and Human Sewage

    PubMed Central

    Vierheilig, J.; Frick, C.; Mayer, R. E.; Kirschner, A. K. T.; Reischer, G. H.; Derx, J.; Mach, R. L.; Farnleitner, A. H.

    2013-01-01

    During a 3-year study, Clostridium perfringens was investigated in defined fecal sources from a temperate alluvial backwater area of a large river system. The results reveal that using C. perfringens as a conservative water quality indicator for total fecal pollution monitoring is no longer justified but suggest that it can be used as a tracer for excreta from nonherbivorous wildlife and human sewage. PMID:23747707

  5. Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation, and inflammation cytokines profile of Clostridium perfringens type A strain CP4.

    PubMed

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L; Wolf, Amanda; Curtiss, Roy

    2014-04-01

    Clostridium perfringens is an important Gram-positive pathogen responsible for food poisoning, necrotic enteritis, gas gangrene, and even death. Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic strain with demonstrated benefits. In this study, we evaluated the effects of EcN on growth, toxin production, biofilm formation, and inflammatory cytokine responses of C. perfringens. In vitro co-culture experiments demonstrated that EcN inhibited growth, gas production, and toxin production (α-toxin and NetB) of C. perfringens in a dose-dependent manner. The growth inhibition effect was not observed when C. perfringens was incubated with EcN cell-free supernatants (CFSE), suggesting that growth inhibition was caused by nutrition competition during co-incubation. In vitro studies demonstrated that pre-incubation with EcN did not inhibit C. perfringens attachment to Caco-2 cells, but did reduce C. perfringens total number, toxin production, and cytotoxicity after 24 h. The similar growth inhibition results were also observed during the formation of C. perfringens biofilm. Finally, pre-incubation of EcN with RAW264.7 cells significantly decreased the production of inflammatory cytokines caused by the introduction of C. perfringens. Our results indicate that EcN can inhibit many of the pathological effects of C. perfringens in vitro conditions. PMID:24532573

  6. Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation and inflammation cytokines profile of Clostridium perfringens type A strain CP4

    PubMed Central

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L.; Wolf, Amanda; Curtiss, Roy

    2014-01-01

    Clostridium perfringens is an important Gram-positive pathogen responsible for food poisoning, necrotic enteritis, gas gangrene, and even death. Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic strain with demonstrated benefits. In this study we evaluated the effects of EcN on growth, toxin production, biofilm formation and inflammatory cytokine responses of C. perfringens. In vitro co-culture experiments demonstrated that EcN inhibited growth, gas production and toxin production (α-toxin and NetB) of C. perfringens in a dose dependent manner. The growth inhibition effect was not observed when C. perfringens was incubated with EcN cell free supernatants (CFSE), suggesting that growth inhibition was caused by nutrition competition during co-incubation. In vitro studies demonstrated that pre-incubation with EcN did not inhibit C. perfringens attachment to Caco-2 cells, but did reduce C. perfringens total number, toxin production and cytotoxicity after 24 h. The similar growth inhibition results were also observed during the formation of C. perfringens biofilm. Finally, pre-incubation of EcN with RAW264.7 cells significantly decreased the production of inflammatory cytokines caused by introduction of C. perfringens. Our results indicate that EcN can inhibit many of the pathological effects of C. perfringens in vitro conditions. PMID:24532573

  7. Multi-Organ Failure Secondary to a Clostridium Perfringens Gaseous Liver Abscess following a Self-Limited Episode of Acute Gastroenteritis

    PubMed Central

    Eltawansy, Sherif Ali; Merchant, Chandni; Atluri, Paavani; Dwivedi, Sukrut

    2015-01-01

    Patient: Female, 81 Final Diagnosis: Liver absces Symptoms: Diarrhea jaundice vomiting weakness Medication: Clinical Procedure: CT scan guided drainage Specialty: Gastroenterology and Hepatology Objective: Rare disease Background: Clostridium perfringens is an unusual pathogen responsible for the development of a gas-forming pyogenic liver abscess. Progression to septicemia with this infection has amplified case fatality rates. Case Report: We report a case of an 81-year-old lady with pyogenic liver abscess with gas formation that was preceded by an acute gastroenteritis. The most common precipitating factors are invasive procedures and immunosuppression. Clostridium perfringens was unexpectedly isolated in the drained abscess, as well as blood. It is a normal inhabitant of the human bowel and a common cause of food poisoning, notoriously leading to tissue necrosis and gas gangrene. Conclusions: We report a case of gas-forming pyogenic liver abscess and bacteremia progressing to fatal septic shock, caused by an uncommon Clostridium perfringens isolate. PMID:25807198

  8. The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the beta2-toxin in enteric disease of domestic animals, wild animals and humans.

    PubMed

    van Asten, Alphons J A M; Nikolaou, Georgios N; Grne, Andrea

    2010-02-01

    The virulence of Clostridium perfringens, a bacterium causing enteritis and enterotoxaemia in domestic and wild animals and humans, results largely from its ability to produce toxins. In 1997, an unknown toxin of C. perfringens, the beta2-toxin, and its encoding gene cpb2 were described. Since that time numerous studies have been published dealing with a possible association of cpb2-harbouring strains of C. perfringens and the occurrence of enteric disease in domestic and wild animals and humans. This article offers an overview of the current literature on the spread and pathological significance of cpb2-harbouring C. perfringens. Unambiguous conclusions on the prevalence of cpb2 and the contribution of beta2-toxin to the disease cannot be drawn from existing studies but in some animal species a strong correlation between the presence of cpb2-harbouring C. perfringens, the beta2-toxin and enteric disease has been reported. PMID:19101180

  9. The epidemiology of Clostridium perfringens type A on Ontario swine farms, with special reference to cpb2-positive isolates

    PubMed Central

    2012-01-01

    Background There is poor understanding of most aspects of Clostridium perfringens type A as a possible cause of neonatal diarrhea in piglets, and the prevalence and types of C. perfringens present on Ontario swine farms is unknown. To study the prevalence of fecal C. perfringens and selected toxin genes, 48 Ontario swine farms were visited between August 2010 and May 2011, and 354 fecal samples were collected from suckling pigs, lactating sows, weanling pigs, grower-finisher pigs, and gestating sows, as well as from manure pits. The fecal samples were cultured quantitatively, and toxin genes were detected by real-time multiplex polymerase chain reaction (PCR). Results In mixed multivariable linear analysis, log10C. perfringens in fecal samples from suckling pigs were higher than that of weanling pigs, grower-finisher pigs, and manure pit samples (P <0.05). In mixed multivariable logistic analysis, the C. perfringens isolates recovered from lactating sows (OR = 0.069, P <0.001), gestating sows (OR = 0.020, P <0.001), grower-finishers (OR = 0.017, P <0.001), and manure pits (OR = 0.11, P <0.001) were less likely to be positive for the consensus beta2 toxin gene cpb2 compared to the isolates from suckling pigs. The prevalence of cpb2 in the isolates recovered from weanlings did not differ significantly from suckling pigs. C. perfringens isolates that were positive for cpb2 were more likely to carry the atypical cpb2 gene (atyp-cpb2) (OR = 19, P <0.001) compared to isolates that were negative for cpb2. Multivariable analysis did not identify farm factors affecting the presence of consensus cpb2 and atyp-cpb2 genes. Conclusions This study provides baseline data on the prevalence of C. perfringens and associated toxin genes in healthy pigs at different stages of production on Ontario swine farms. The study suggests that if C. perfringens type A are involved in neonatal enteritis, there may be strains with specific characteristics that cannot be identified by the existing genotyping system. PMID:22947389

  10. Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

    PubMed Central

    Maheux, Andrée F.; Bérubé, Ève; Boudreau, Dominique K.; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc

    2013-01-01

    We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP−/rtPCR+ colonies were identified as C. perfringens, whereas 3 mCP+/rtPCR− colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection. PMID:24077714

  11. Predictive model for growth of Clostridium perfringens during cooling of cooked uncured meat and poultry.

    PubMed

    Juneja, Vijay K; Marks, Harry; Huang, Lihan; Thippareddi, H

    2011-06-01

    Comparison of Clostridium perfringens spore germination and outgrowth in cooked uncured products during cooling for different meat species is presented. Cooked, uncured product was inoculated with C. perfringens spores and vacuum packaged. For the isothermal experiments, all samples were incubated in a water bath stabilized at selected temperatures between 10 and 51C and sampled periodically. For dynamic experiments, the samples were cooled from 54.4 to 27C and subsequently from 27 to 4C for different time periods, designated as x and y hours, respectively. The growth models used were based on a model developed by Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int. J. Food Micro. 23, 277-294), which incorporates a constant, referred to as the physiological state constant, q(0). The value of this constant captures the cells' history before the cooling begins. To estimate specific growth rates, data from isothermal experiments were used, from which a secondary model was developed, based on a form of Ratkowsky's 4-parameter equation. The estimated growth kinetics associated with pork and chicken were similar, but growth appeared to be slightly greater in beef; for beef, the maximum specific growth rates estimated from the Ratkowsky curve was about 2.7 log(10) cfu/h, while for the other two species, chicken and pork, the estimate was about 2.2 log(10) cfu/h. Physiological state constants were estimated by minimizing the mean square error of predictions of the log(10) of the relative increase versus the corresponding observed quantities for the dynamic experiments: for beef the estimate was 0.007, while those for pork and chicken the estimates were about 0.014 and 0.011, respectively. For a hypothetical 1.5h cooling from 54C to 27 and 5h to 4C, corresponding to USDA-FSIS cooling compliance guidelines, the predicted growth (log(10) of the relative increase) for each species was: 1.29 for beef; 1.07 for chicken and 0.95 log(10) for pork. However, it was noticed that for pork in particular, the model using the derived q(0) had a tendency to over-predict relative growth when the observed amount of relative growth was small, and under-predict the relative growth when the observed amount of relative growth was large. To provide more fail-safe estimate, rather than using the derived value of q(0), a value of 0.04 is recommended for pork. PMID:21511140

  12. Efficacy of a lactylate on production performance and intestinal health of broilers during a subclinical Clostridium perfringens infection.

    PubMed

    Lensing, M; van der Klis, J D; Fabri, T; Cazemier, A; Else, A J

    2010-11-01

    Clostridium perfringens, an ?-toxin producing gram-positive bacterium, is an enteric pathogen for poultry. Because subclinical C. perfringens infections often result in damage of the intestinal mucosa, decreased nutrient digestion, and poor performance, efforts should be taken to find an effective strategy that controls overgrowth of C. perfringens. For this purpose, the efficacy of a sodium lauroyl lactylate (LauL) as a feed additive to prevent C. perfringens colonization in broilers was determined. First, the effect of LauL was compared with capric and lauric mono- and diglycerides (MDG) and capric and lauric free fatty acids in Clostridium-infected chickens. Clostridial lesion scoring at d 16 showed that MDG and LauL were both effective in reducing the severity of lesions. When taking into account results on BW gain and mortality, LauL was more effective than MDG. For this reason, a dose response study was made to determine the optimal dietary dosage of LauL. In this experiment, it was shown that a LauL dose higher than 0.15% should be used to expect positive effects on lesion severity and mortality. None of the LauL doses led to a significant better response on growth performance. In a third trial, efficacy of LauL was compared with commercial products that limit bacterial activity in the intestinal tract (Aromabiotic Poul 60) or coccidiosis (chemical coccidiostat, Clinacox). None of the products were able to reduce the number or severity of lesions, and no effect on production performance was observed. Thus, despite the clear positive effect seen in experiment 1, and in experiment 2 with LauL doses higher than 0.15%, supplementing this lactylate to the diet does not consistently reduce C. perfringens colonization in broiler chickens because no such effects were observed in experiment 3. These results, however, provide a scientific basis for future studies to further investigate lactylates as potential additives to reduce the severity of necrotic enteritis in broilers in a C. perfringens challenge model. PMID:20952703

  13. Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

    PubMed

    Siqueira, Flvia F; Almeida, Marcelle O; Barroca, Tatiana M; Horta, Carolina C R; Carmo, Anderson O; Silva, Rodrigo O S; Pires, Prhiscylla S; Lobato, Francisco C F; Kalapothakis, Evanguedes

    2012-10-12

    Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence. PMID:22560738

  14. [Presence of Clostridium perfringens in meat-based preparations in public food services in central San Jose, Costa Rica].

    PubMed

    Gutirrez, A; Gamboa, M M; Rodrguez, E; Arias, M L

    1999-09-01

    In Costa Rica there are a large number of public food services distributed along the country, where a considerable number of people eat daily. Clostridium perfringens is a bacteria associated with foodborne illness related, especially, to meat products kept for long time at temperatures under 70 degrees C. The aim of this study was to evaluate the public food services that use water baths for keeping food hot in order to establish the presence of C. perfringens in cooked bovine meat dishes and to evaluate the enterotoxigenic capacity of the strains isolated. 81 samples of cooked bovine meat plates coming from 27 public food services, located in the Central County of San Jos were analyzed. The methodology described by Labbe & Harmon for the isolation of C. perfringens was used in 10 g of sample. Also, the enterotoxigenic capacity of the strains was evaluated using the passive-reverse-latex-agglutination assay from Oxoid. From the 27 public food services analyzed, eight (30%) were positive in the three samplings done, nine (33%) were positive in one or two occasions, and ten (37%) were negative all times. This implies that in 17 (63%) of the establishments studied, the bacteria was isolated at least once. From the 81 preparations studied, 37 (46%) were positive for the bacteria. The temperatures at which food was kept varied from 56 to 82 degrees C, with an average of 68.7 degrees C. From the 37 strains identified as C. perfringens, 12 (32%) were positive for enterotoxin. In conclusion, the presence of C. perfringens in bovine meat dishes, maintained in water baths, represents an important risk for public health, and the temperature at which the preparation is kept is critical for the multiplication of the bacteria. PMID:10667269

  15. Control of Clostridium perfringens spores by green tea leaf extracts during cooling of cooked ground beef, chicken, and pork.

    PubMed

    Juneja, Vijay K; Bari, M L; Inatsu, Y; Kawamoto, S; Friedman, Mendel

    2007-06-01

    We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by two green tea extracts with low (green tea leaf powder [GTL]; 141 mg of total catechins per g of green tea extract) and high (green tea leaf extract [GTE]; 697 mg of total catechins per g of extract) catechin levels during abusive chilling of retail cooked ground beef, chicken, and pork. Green tea extracts were mixed into the thawed beef, chicken, and pork at concentrations of 0.5, 1.0, and 2.0% (wt/ wt), along with a heat-activated (75 degrees C for 20 min) three-strain spore cocktail to obtain a final concentration of approximately 3 log spores per g. Samples (5 g) of the ground beef, chicken, and pork were then vacuum packaged and cooked to 71 degrees C for 1 h in a temperature-controlled water bath. Thereafter, the products were cooled from 54.4 to 7.2 degrees C in 12, 15, 18, or 21 h, resulting in significant increases (P < 0.05) in the germination and outgrowth of C. perfringens populations in the ground beef, chicken, and pork control samples without GTL or GTE. Supplementation with 0.5 to 2% levels of GTL did not inhibit C. perfringens growth from spores. In contrast, the addition of 0.5 to 2% levels of GTE to beef, chicken, and pork resulted in a concentration-and time-dependent inhibition of C. perfringens growth from spores. At a 2% level of GTE, a significant (P < 0.05) inhibition of growth occurred at all chill rates for cooked ground beef, chicken, and pork. These results suggest that widely consumed catechins from green tea can reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling from 54.4 to 7.2 degrees C in 12, 15, 18, or 21 h of cooling for ground beef, chicken, and pork. PMID:17612073

  16. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

    PubMed Central

    Payment, P; Franco, E

    1993-01-01

    To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8368831

  17. Influence of NaCl content and cooling rate on outgrowth of Clostridium perfringens spores in cooked ham and beef.

    PubMed

    Zaika, Laura L

    2003-09-01

    The effect of NaCl concentration and cooling rate on the ability of Clostridium perfringens to grow from spore inocula was studied with the use of a process that simulates the industrial cooking and cooling of smoked boneless ham and beef roasts. NaCl was added to ground cooked hams A and B (which were commercially obtained) to obtain levels of 2.4, 3.1, 3.6, and 4.1% (wt/wt) and 2.8, 3.3, 3.8, and 4.3% (wt/wt), respectively, and to raw ground beef to obtain levels of 0, 1, 2, 3, and 4% (wt/wt). Ham C, a specially formulated, commercially prepared product, was supplemented with NaCl to obtain levels of 2.0, 2.5, 3.0, and 3.5%. The samples were inoculated with a three-strain mixture of C. perfringens spores to obtain concentrations of ca. 3 log10 CFU/g. Portions of meat (5 g each) were spread into thin layers (1 to 2 mm) in plastic bags, vacuum packaged, and stored at -40 degrees C. Thawed samples were heated at 75 degrees C for 20 min and subsequently cooled in a programmed water bath from 54.4 to < or = 8.5 degrees C in 15, 18, or 21 h. For the enumeration of C. perfringens, samples were plated on tryptose-sulfite-cycloserine agar and incubated in an anaerobic chamber at 37 degrees C for 48 h. Population densities for cooked ham and beef increased as cooling time increased, and NaCl exerted a strong inhibitory effect on the germination and outgrowth of C. perfringens. For beef, while 3% NaCl completely arrested growth, pathogen numbers increased by > or = 3, 5, and 5 log10 CFU/g in 15, 18, and 21 h, respectively, when the NaCl level was <2%. C. perfringens did not grow during cooling for 15, 18, or 21 h in ham samples containing > or = 3.1% NaCl. Results obtained in this study suggest that a 15-h cooling time for cooked ham, which is normally formulated to contain >2% NaCl, would yield an acceptable product (with an increase of <1 log10 CFU/g in the C. perfringens count); however, for beef containing <2% NaCl, C. perfringens populations may reach levels high enough to cause illness. PMID:14503712

  18. The CcpA protein is necessary for efficient sporulation and enterotoxin gene (cpe) regulation in Clostridium perfringens.

    PubMed

    Varga, John; Stirewalt, Veronica L; Melville, Stephen B

    2004-08-01

    Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme beta-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin). PMID:15292123

  19. Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.

    PubMed

    Protze, Jonas; Eichner, Miriam; Piontek, Anna; Dinter, Stefan; Rossa, Jan; Blecharz, Kinga Gra?yna; Vajkoczy, Peter; Piontek, Joerg; Krause, Gerd

    2015-04-01

    Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 3310nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors. PMID:25342221

  20. Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells.

    PubMed

    Nagahama, Masahiro; Hayashi, Shinya; Morimitsu, Shinsuke; Sakurai, Jun

    2003-09-19

    Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxyfluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa. PMID:12851396

  1. Identification of Accessory Genome Regions in Poultry Clostridium perfringens Isolates Carrying the netB Plasmid

    PubMed Central

    Lepp, D.; Songer, J. G.; Boerlin, P.; Parreira, V. R.

    2013-01-01

    Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance. PMID:23292780

  2. The Oncopathic Potency of Clostridium perfringens Is Independent of Its α-Toxin Gene

    PubMed Central

    Li, Zhiyu; Fallon, John; Mandeli, John; Wetmur, James

    2009-01-01

    Abstract Hypoxia in solid tumors is a major obstacle in conventional treatment because of inefficient delivery of therapeutic agents to the lesions, but offers the potential for anaerobic bacterial colonization that can lead to tumor destruction. We have previously reported a recombinant Clostridium perfringens (Cp) strain constructed by deletion of the superoxide dismutase (sod) gene and insertion of the Panton–Valentine leukocidin (PVL) gene, Cp/sod−/PVL, which showed elevated oxygen sensitivity, tumor selectivity, and oncopathic potency in an orthotopic model of pancreatic cancer in immune-competent and syngeneic mice, and that led to substantial prolongation of animal survival. A major limitation to Cp/sod−/PVL in clinical applications is that it expresses phospholipase C (plc), the α-toxin and the major virulence determinant in Cp that is causative in the development of gas gangrene. In this study, the plc gene in Cp/sod−/PVL was knocked out to create Cp/plc−/sod−/PVL, which was shown to be incapable of inducing gas gangrene in mice. Intravenous injection of Cp/plc−/sod−/PVL spores led to a significant survival advantage in tumor-bearing mice with the same efficacy as Cp/sod−/PVL, indicating that the oncopathic potency of Cp is independent of a functional plc gene. The treatment also did not lead to an attenuated immune response to a subsequent pathogen challenge, indicating that a systemic immune-suppressive effect in the host is absent. Consequently, Cp/plc−/sod−/PVL is a novel oncopathic bacterial agent for the effective treatment of pancreatic cancer and other poorly vascularized tumors, with a substantially enhanced safety profile, which is essential for the development of translational studies in the future. PMID:19298132

  3. Effects of Clostridium perfringens Alpha-Toxin (PLC) and Perfringolysin O (PFO) on Cytotoxicity to Macrophages, on Escape from the Phagosomes of Macrophages, and on Persistence of C. perfringens in Host Tissues

    PubMed Central

    O'Brien, David K.; Melville, Stephen B.

    2004-01-01

    Clostridium perfringens is the most common cause of clostridial myonecrosis (gas gangrene). Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results). However, the importance of macrophages in the host defense against C. perfringens infections is still unknown. Two membrane-active toxins produced by the anaerobic C. perfringens, alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection. Therefore, C. perfringens mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues. C. perfringens survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC. PFO was shown to be the primary mediator of C. perfringens-dependent cytotoxicity to macrophages. Escape of C. perfringens cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages. At lethal doses (109) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (106) both PFO and PLC were necessary for survival of C. perfringens in mouse muscle tissue. These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of C. perfringens to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection. PMID:15322015

  4. Clostridium perfringens septicemia in a long-beaked common dolphin Delphinus capensis: an etiology of gas bubble accumulation in cetaceans.

    PubMed

    Danil, Kerri; St Leger, Judy A; Dennison, Sophie; Bernaldo de Quirs, Yara; Scadeng, Miriam; Nilson, Erika; Beaulieu, Nicole

    2014-10-16

    An adult female long-beaked common dolphin Delphinus capensis live-stranded in La Jolla, California, USA, on July 30, 2012 and subsequently died on the beach. Computed tomography and magnetic resonance imaging revealed gas bubble accumulation in the vasculature, organ parenchyma, mandibular fat pads, and subdermal sheath as well as a gas-filled cavity within the liver, mild caudal abdominal effusion, and fluid in the uterus. Gross examination confirmed these findings and also identified mild ulcerations on the palate, ventral skin, and flukes, uterine necrosis, and multifocal parenchymal cavitations in the brain. Histological review demonstrated necrosis and round clear spaces interpreted as gas bubbles with associated bacterial rods within the brain, liver, spleen, and lymph nodes. Anaerobic cultures of the lung, spleen, liver, bone marrow, and abdominal fluid yielded Clostridium perfringens, which was further identified as type A via a multiplex PCR assay. The gas composition of sampled bubbles was typical of putrefaction gases, which is consistent with the by-products of C. perfringens, a gas-producing bacterium. Gas bubble formation in marine mammals due to barotrauma, and peri- or postmortem off-gassing of supersaturated tissues and blood has been previously described. This case study concluded that a systemic infection of C. perfringens likely resulted in production of gas and toxins, causing tissue necrosis. PMID:25320031

  5. Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens

    SciTech Connect

    Ficko-Blean, Elizabeth; Boraston, Alisdair B.

    2005-09-01

    Crystallization of a family 84 glycoside hydrolase, a putative virulence factor, secreted by C. perfringens is reported. Clostridium perfringens is a ubiquitous environmental organism that is capable of causing a variety of diseases in mammals, including gas gangrene and necrotic enteritis in humans. The activity of a secreted hyaluronidase, attributed to the NagH protein, contributes to the pathogenicity of this organism. The family 84 catalytic module of one of the three homologues of NagH found in C. perfringens (ATCC 13124) has been cloned. The 69 kDa catalytic module of NagJ, here called GH84C, was overproduced in Escherichia coli and purified by immobilized metal-affinity chromatography (IMAC). Crystals belonging to space group I222 or I2{sub 1}2{sub 1}2{sub 1} with unit-cell parameters a = 130.39, b = 150.05, c = 155.43 Å were obtained that diffracted to 2.1 Å. Selenomethionyl crystals have also been produced, leading to the possibility of solving the phase problem by MAD using synchrotron radiation.

  6. Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

    PubMed Central

    Ryzinska-Paier, G.; Sommer, R.; Haider, J.M.; Knetsch, S.; Frick, C.; Kirschner, A.K.T.; Farnleitner, A.H.

    2011-01-01

    Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method. PMID:21872622

  7. The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

    PubMed Central

    2013-01-01

    Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells. PMID:23782465

  8. Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene.

    PubMed Central

    Garnier, T; Cole, S T

    1986-01-01

    The bacteriocinogenic plasmid pIP404 from Clostridium perfringens was isolated and cloned in Escherichia coli, and its physical map was deduced. Expression of the bcn gene, encoding bacteriocin BCN5, is inducible by UV irradiation of C. perfringens and thus resembles the SOS-regulated bacteriocin genes of enteric bacteria. The location of bcn on pIP404 was established by a dot-blot procedure, using specific hybridization probes to analyze mRNA samples from induced and uninduced cultures. From the nucleotide sequence of its gene, the molecular weight of BCN5 was deduced to be 96,591, and a protein of this size was secreted by bacteriocin-producing cultures of C. perfringens. The primary structure of the protein suggests that it may function as an ionophore, since a hydrophobic domain, resembling those of the ionophoric colicins, is present at the COOH terminus. No bacteriocin activity could be detected in E. coli harboring plasmids bearing the bcn gene, even when the transcriptional and translational signals were replaced by those of lacZ. A possible explanation may be found in the unusual codon usage of the adenine-thymine-rich bcn gene, as this shows a preference for codons with a high adenine-plus-thymine content, especially in the wobble position. Many of the frequently used codons correspond to those recognized by minor tRNA species in E. coli. Consequently, bcn expression might be limited by tRNA availability in this bacterium. Images PMID:2877971

  9. Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular beta-N-Acetylglucosaminidase from Clostridium perfringens

    SciTech Connect

    Ficko-Blean, E.; Gregg, K; Adams, J; Hehemann, J; Czjzek, M; Smith, S; Boraston, A

    2009-01-01

    Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens ?-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.

  10. DYNAMIC COMPUTER SIMULATION OF THE MULTIPLICATION OF CLOSTRIDIUM PERFRINGENS IN COOKED GROUND BEEF

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to develop a computer simulation algorithm to dynamically estimate and predict the growth of C. perfringens spores in cooked ground beef. The computational algorithm was based on the implicit form of the Gompertz model, the growth kinetics of C. perfringens in cooed ...

  11. Predictive model for growth of Clostridium perfringens during cooling of cooked uncured meat and poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparison of C. perfringens spore germination and outgrowth in cooked uncured products during cooling for different meat species is presented. Cooked, uncured product was inoculated with C. perfringens spores and vacuum packaged. For the isothermal experiments, all samples were incubated in a wat...

  12. Predictive model for growth of Clostridium perfringens during cooling of cooked ground pork

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A predictive dynamic model for C. perfringens spore germination and outgrowth in cooked pork products during cooling is presented. Cooked, ground pork was inoculated with C. perfringens spores and vacuum packaged. For the isothermal experiments, all samples were incubated in a water bath stabilize...

  13. A poultry-intestinal isolate of Campylobacter jejuni produces a bacteriocin (CUV-3) active against a range of Gram positive bacterial pathogens including Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A newly isolated bacteriocin, CUV-3, produced by a poultry cecal isolate of Campylobacter jejuni strain CUV-3 had inhibitory activity against several Gram positive bacteria including Clostridium perfringens (38 strains), Staphylococcus aureus, Staph.epidermidis and Listeria monocytogenes. The pept...

  14. Use of Calcium, Potassium, and Sodium Lactates to Control Germination and Outgrowth of Clostridium perfringens Spores during Chilling of Injected Pork

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens spore germination and outgrowth by calcium (CaL), potassium (KL) or sodium (NaL) lactate in injected pork during abusive chilling regimes was investigated. Lactates (Ca, K, or Na) were incorporated into injected pork at various concentrations (1.0, 2.0, 3.0 and...

  15. INHIBITORY EFFECTS OF ORGANIC ACID SALTS ON GROWTH OF CLOSTRIDIUM PERFRINGENS FROM SPORE INOCULA DURING CHILLING OF MARINATED GROUND TURKEY BREAST

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens germination and outgrowth by salts of organic acids such as sodium lactate, sodium acetate, buffered sodium citrate and buffered sodium citrate supplemented with sodium diacetate was evaluated during continuous chilling of ground turkey. Turkey breast meat was ...

  16. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne di...

  17. Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium.

    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 b...

  18. Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium

    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 b...

  19. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne d...

  20. VIABILITY OF CLOSTRIDIUM PERFRINGENS, ESCHERICHIA COLI, AND LISTERIA MONOCYTOGNES SURVIVING MILD HEAT OR AQUEOUS OZONE TREATMENT ON BEEF FOLLOWED BY HEAT, ALKALI, OR SALT STRESS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The threat of pathogen survival following ozone treatment of meat necessitates careful evaluation of the surviving microorganisms for tolerance to subsequent heat, pH, and NaCl stress. Log reductions in CFU/g of 3-strain cocktails of Clostridium perfringens, Escherichia coli O157:H7, and Listeria m...

  1. ATTEMPTS TO ISOLATE NATURALLY OCCURRING CAMPYLOBACTER, SALMONELLA AND CLOSTRIDIUM PERFRINGENS FROM THE DUCTUS DEFERENS, TESTES AND CECA OF COMMERCIAL BROILER BREEDER ROOSTERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies have shown a significant presence of Campylobacter in the semen of mid-life and late-life roosters. The present study was done to determine if several foodborne pathogens (Campylobacter, Salmonella, Clostridium perfringens) could be isolated from the ductus deferens, testes and ceca ...

  2. Inhibition of clostridium perfringens spore germination and outgrowth by buffered vinegar and lemon juice concentrate during chilling.....of ground turkey road containing minimal ingredients

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens spore germination and outgrowth in ground turkey roast containing minimal ingredients (salt and sugar), by buffered vinegar (MoStatin V) and a blend (buffered) of lemon juice concentrate and vinegar (MoStatin LV) was evaluated. Ground turkey roast was formulat...

  3. Inhibition of Clostridium perfringens spore germination and outgrowth by lemon juice and vinegar product in reduced NaCl roast beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1, 1.5, or 2%, wt/wt), blend of sodium pyro-...

  4. Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5C for 3 or...

  5. Expression of Two Bacteriophage Enzymes that Lyse Clostridium perfringens which Share Sequences in the Cell Binding Domain of the Molecules but are Dissimilar in their Catalytic Enzymatic Domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins which are responsible for disease symptoms and pathogenesis in a variety of animals, humans and poultry. The organism is the third leading cause of food-borne bacterial disease among ...

  6. Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was performed to compare four Clostridium perfringens recombinant proteins as vaccine candidates using the Montanide ISA 71 VG adjuvant in an experimental model of necrotic enteritis. Broiler chickens were immunized with clostridial recombinant proteins with ISA 71 VG, and intestinal le...

  7. Immunopathology and Cytokine Responses in Broiler Chickens Coinfected with Eimeria maxima and Clostridium perfringens Using an Animal Model of Necrotic Enteritis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. While pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP ...

  8. High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens.

    PubMed

    Tanaka, Hiroaki; Nariya, Hirofumi; Suzuki, Motoo; Houchi, Hitoshi; Tamai, Eiji; Miyata, Shigeru; Okabe, Akinobu

    2011-03-01

    Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO. PMID:20940055

  9. Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks.

    PubMed

    Barbara, Angelique J; Trinh, Hien T; Glock, Robert D; Glenn Songer, J

    2008-01-25

    We inoculated broiler chicks with mixtures of Clostridium perfringens strains to investigate the single strain dominance observed in natural cases of necrotic enteritis (NE) [Nauerby, B., Pedersen, K., Madsen, M., 2003. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 94, 257-266]. Pre-inoculation bacteriologic culture of chick intestines yielded up to six pulsed-field gel electrophoresis (PFGE) types of C. perfringens. Birds developed typical NE lesions in response to administration (2x per day for 4 days) of a combined inoculum comprising one NE strain (JGS4143, PFGE pattern 8) and four non-NE strains (from piglet necrotizing enteritis, chicken normal flora, human gas gangrene, and bovine neonatal enteritis). After inoculation commenced, only the NE strain was recovered through the first post-inoculation day, in spite of intense efforts to recover pre-challenge flora strains and the other challenge strains. Thereafter, pre-inoculation and previously undetected PFGE types were found, and JGS4143 became undetectable. Birds inoculated simultaneously with five NE strains (from disease in chickens or turkeys, and including JGS4143) also developed lesions, but again only JGS4143 was recovered through the 1st day post-challenge. At that time, birds began to be repopulated with pre-challenge PFGE types. Two NE strains (JGS4143 and JGS4064) produced bacteriocins, which inhibited each other and normal flora strains (n=17), while normal flora strains inhibited neither NE strains nor each other. Thus, it appears that naturally occurring dominance of the gut by NE strains can be reproduced experimentally. Bacteriocins directed against normal flora could possibly provide the necessary advantage, although inhibition of one NE strain by another suggests that other factors may be partially or completely responsible for the dominance. PMID:17850994

  10. The CpAL Quorum Sensing System Regulates Production of Hemolysins CPA and PFO To Build Clostridium perfringens Biofilms

    PubMed Central

    Shak, Joshua R.; Canizalez-Roman, Adrian

    2015-01-01

    Clostridium perfringens strains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by the C. perfringens Agr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. Although C. perfringens strains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrB mutant strains were not able to produce biofilms, a ΔluxS mutant produced wild-type levels. The transcript levels of CpAL-regulated cpa and pfoA genes, but not cpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpa and ΔpfoA mutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpb made wild-type levels. Biofilm formation was restored in complemented Δcpa/cpa and ΔpfoA/pfoA strains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation in C. perfringens by increasing levels of certain toxins required to build biofilms. PMID:25824838

  11. Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

    PubMed Central

    Liu, Hualan; Bouillaut, Laurent; Sonenshein, Abraham L.

    2013-01-01

    Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility. PMID:23204460

  12. Three-dimensional structure of a putative non-cellulosomal cohesin module from a Clostridium perfringens family 84 glycoside hydrolase.

    PubMed

    Chitayat, Seth; Gregg, Katie; Adams, Jarrett J; Ficko-Blean, Elizabeth; Bayer, Edward A; Boraston, Alisdair B; Smith, Steven P

    2008-01-01

    The genomes of myonecrotic strains of Clostridium perfringens encode a large number of secreted glycoside hydrolases. The activities of these enzymes are consistent with degradation of the mucosal layer of the human gastrointestinal tract, glycosaminoglycans and other cellular glycans found throughout the body. In many cases this is thought to aid in the propagation of the major toxins produced by C. perfringens. One such example is the family 84 glycoside hydrolases, which contains five C. perfringens members (CpGH84A-E), each displaying a unique modular architecture. The smallest and most extensively studied member, CpGH84C, comprises an N-terminal catalytic domain with beta-N-acetylglucosaminidase activity, a family 32 carbohydrate-binding module, a family 82 X-module (X82) of unknown function, and a fibronectin type-III-like module. Here we present the structure of the X82 module from CpGH84C, determined by both NMR spectroscopy and X-ray crystallography. CpGH84C X82 adopts a jell-roll fold comprising two beta-sheets formed by nine beta-strands. CpGH84C X82 displays distant amino acid sequence identity yet close structural similarity to the cohesin modules of cellulolytic anaerobic bacteria. Cohesin modules are responsible for the assembly of numerous hydrolytic enzymes in a cellulose-degrading multi-enzyme complex, termed the cellulosome, through a high-affinity interaction with the calcium-binding dockerin module. A planar surface is located on the face of the CpGH84 X82 structure that corresponds to the dockerin-binding region of cellulolytic cohesin modules and has the approximate dimensions to accommodate a dockerin module. The presence of cohesin-like X82 modules in glycoside hydrolases of C. perfringens is an indication that the formation of novel X82-dockerin mediated multi-enzyme complexes, with potential roles in pathogenesis, is possible. PMID:17999932

  13. The CpAL quorum sensing system regulates production of hemolysins CPA and PFO to build Clostridium perfringens biofilms.

    PubMed

    Vidal, Jorge E; Shak, Joshua R; Canizalez-Roman, Adrian

    2015-06-01

    Clostridium perfringens strains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by the C. perfringens Agr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. Although C. perfringens strains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrB mutant strains were not able to produce biofilms, a ΔluxS mutant produced wild-type levels. The transcript levels of CpAL-regulated cpa and pfoA genes, but not cpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpa and ΔpfoA mutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpb made wild-type levels. Biofilm formation was restored in complemented Δcpa/cpa and ΔpfoA/pfoA strains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation in C. perfringens by increasing levels of certain toxins required to build biofilms. PMID:25824838

  14. Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract.

    PubMed

    Józefiak, D; Kierończyk, B; Rawski, M; Hejdysz, M; Rutkowski, A; Engberg, R M; Højberg, O

    2014-06-01

    The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as dietary fat type significantly affects microbiota activity in the broiler chicken gastrointestinal tract. PMID:24674938

  15. A Wide Variety of Clostridium perfringens Type A Food-Borne Isolates That Carry a Chromosomal cpe Gene Belong to One Multilocus Sequence Typing Cluster

    PubMed Central

    Xiao, Yinghua; Wagendorp, Arjen; Moezelaar, Roy; Abee, Tjakko

    2012-01-01

    Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)5 fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods. PMID:22865060

  16. Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens.

    PubMed

    Nees, S; Schauer, R; Mayer, F

    1976-06-01

    Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented. PMID:182637

  17. Combination chemotherapy with Clostridium perfringens phospholipase C and cytosine antimetabolites: complementary inhibition directed at membrane lipids.

    PubMed

    Lee, M H; Sartorelli, A C

    1983-01-01

    Tumor cell membranes were susceptible to the action of Clostridium perfringens phospholipase C, and this was reflected by inhibition of cellular replication in culture. The differential susceptibility of two neoplastic cell lines to this enzyme was studied in detail. The growth of sarcoma 180 cells cultured in Fischer's medium was markedly inhibited by phospholipase C; whereas, in contrast, cultured L1210 leukemia cells were relatively resistant to the cytotoxic effects of this enzyme. The differential sensitivity of these two neoplastic cell lines to phospholipase C was corroborated by dye-exclusion tests. Thus, leukemia L1210 cells exposed to a concentration of 0.2 mg of phospholipase C per ml of Fischer's medium for 30 min at 37 degrees C were able to exclude Trypan Blue; whereas, only about 21% of sarcoma 180 cells treated under identical conditions were able to exclude the dye. That the cytotoxicity of phospholipase C to sarcoma 180 was the result of hydrolysis of phospholipids of the plasma membrane was supported by measurements of the rate of hydrolysis of radioactivity from the phospholipid of neoplastic cells prelabeled with [3H]choline. Eighty-two percent of incorporated radioactive choline was released from sarcoma 180 cells treated with phospholipase C in Fischer's medium, whereas, only 20% of the label from [3H]choline was solubilized from L1210 leukemia cells treated with the enzyme under similar conditions. Scanning electron microscopy revealed significant damage to sarcoma 180 cells exposed to phospholipase C in Fischer's medium, which was characterized by alterations in size and shape of cells, disappearance of microvilli, and appearance of fistulas in cell membranes; relatively resistant L1210 leukemic cells did not appear to be markedly damaged by comparable enzyme treatment. Exposure of leukemia L1210 cells to phospholipase C in Puck's saline A increased the sensitivity of these cells to enzymatic action. Under these conditions, a comparable amount of phospholipid was hydrolyzed from surface membranes of sarcoma 180 and leukemia L1210 cells, and the degree of membrane damage appeared to be similar, as measured by the capacity of the tumor cell lines to exclude Trypan Blue and by scanning electron microscopy. The extensive damage to membranes by hydrolysis of phospholipids was not accompanied by a change in the degree of specific binding of [3H]concanavalin A(ConA).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:6085755

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

    PubMed

    Swaggerty, C L; McReynolds, J L; Byrd, J A; Pevzner, I Y; Duke, S E; Genovese, K J; He, H; Kogut, M H

    2016-02-01

    We developed a novel selection method based on an inherently high and low phenotype of pro-inflammatory mediators and produced "high" and "low" line chickens. We have shown high line birds are more resistant to Salmonella enterica serovar Enteritidis and Eimeria tenella compared to the low line. Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illness, and is also an economically important poultry pathogen and known etiologic agent of necrotic enteritis (NE). The objective of this study was to determine if high line birds were also more resistant to NE than low line birds using an established model. Birds were reared in floor pens and challenges were conducted twice (high line = 25/trial, 50 birds total; low line = 26/trial, 52 birds total). Day-old chicks were provided a 55% wheat-corn-based un-medicated starter diet. A bursal disease vaccine was administered at 10 the recommended dose via the ocular route at 14-d-of-age. Birds were challenged daily for 3 d beginning at 16-d-of-age by oral gavage (3 mL) with 10(7) colony forming units (cfu) of C. perfringens/mL then necropsied at 21-d-of-age. All birds had sections of the intestine examined and scored for lesions while the first 10 necropsied also had gut content collected for C. perfringens enumeration. Chickens from the high line were more resistant to C. perfringens-induced NE pathology compared to the low line, as indicated by reduced lesion scores. Ninety percent of the high line birds had lesions of zero or one compared to 67% of the low line birds. Wilcoxon rank sum test showed significantly higher lesion scores in the low line birds compared to the high line (P < 0.0001). There were no differences in the C. perfringens recovered (P = 0.83). These data provide additional validation and support selection based on elevated levels of pro-inflammatory mediators produces chickens with increased resistance against foodborne and poultry pathogens. PMID:26706357

  19. Association of Beta2-Positive Clostridium perfringens Type A With Focal Duodenal Necrosis in Egg-Laying Chickens in the United States.

    PubMed

    França, M; Barrios, M A; Stabler, L; Zavala, Guillermo; Shivaprasad, H L; Lee, M D; Villegas, A M; Uzal, Francisco A

    2016-03-01

    Focal duodenal necrosis (FDN) is a poorly understood intestinal disease of egg layers, and has been associated with drops in egg production and decreased egg weights. The etiology of this disease is still unknown, but the condition has been associated with Clostridium colinum and Clostridium perfringens. In order to investigate the etiology, duodenal samples were taken from hens with FDN. The hens originated from table egg layer farms in three states. The samples were examined by histopathology, bacteriology, and immunohistochemistry. Macroscopically, all samples contained focal to multifocal, variably sized, reddened or brownish gray areas of mucosal erosion. Histopathology revealed mild to severe heterophilic and lymphoplasmacytic enteritis with loss of enterocytes at the villous tips, luminal fibrinonecrotic exudate, and variable numbers of Gram-positive and Gram-negative rod-shaped bacteria within the lesions in 16/30 samples. Clostridium perfringens was isolated by anaerobic bacteriology from 4/13 samples that had characteristic microscopic lesions of FDN. Polymerase chain reaction (PCR) revealed that all four isolates were Type A C. perfringens, positive for beta2 gene and negative for necrotic enteritis toxin B and enterotoxin genes. PCR for Clostridium colinum applied to DNA extracted from frozen intestinal samples yielded negative results in 14/14 duodenal samples. Immunohistochemistry (IHC) for 7C. perfringens, alpha and beta2 toxins stained a few to numerous long rod-shaped bacteria present in the lesions. IHC for alpha and beta2 toxins also stained enterocytes at the villous tips, inflammatory cells in the lamina propria, as well as degenerated and sloughed enterocytes present within the luminal exudate. These findings suggest that C. perfringens may play a role in the development of FDN. Experimental challenge studies with these isolates still need to be performed in order to reproduce the disease and fulfill Koch's postulates. PMID:26953942

  20. Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil

    PubMed Central

    Silva, Rodrigo Otvio Silveira; Santos, Renata Lara Resende; Pires, Prhiscylla Sadan; Pereira, Luiz Carlos; Pereira, Silvia Trindade; Duarte, Marina Carvalho; de Assis, Ronnie Antunes; Lobato, Francisco Carlos Faria

    2013-01-01

    The objective of this study was to detect C. difficile A/B toxins and to isolate strains of C. perfringens and C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficile A/B toxins were detected by ELISA, and C. perfringens and C. difficile were identified by multiplex PCR. C. difficile A/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+ and two were A?B?. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtB was found in one strain, which was A+B+ and was derived from a non-diarrheic dog. C. perfringens strains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringens type A and there was an association between the detection of the cpe gene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2 gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficile and C. perfringens in dogs and to better our understanding of C. difficile as a zoonotic agent. This is the first study to report the binary toxin gene in C. difficile strains isolated from dogs in Brazil. PMID:24159295

  1. Direct Dynamic Kinetic Analysis and Computer Simulation of Growth of Clostridium perfringens in Cooked Turkey during Cooling.

    PubMed

    Huang, Lihan; Vinyard, Bryan T

    2016-03-01

    This research applied a new 1-step methodology to directly construct a tertiary model that describes the growth of Clostridium perfringens in cooked turkey meat under dynamically cooling conditions. The kinetic parameters of the growth models were determined by numerical analysis and optimization using multiple dynamic growth curves. The models and kinetic parameters were validated using independent growth curves obtained under various cooling conditions. The results showed that the residual errors (ε) of the predictions followed a Laplace distribution that is symmetric with respect to ε = 0. For residual errors, 90.6% are within ±0.5 Log CFU/g and 73.4% are ±0.25 Log CFU/g for all growth curves used for validation. For relative growth <1.0 Log CFU/g, 88.9% of the residual errors are within ±0.5 Log CFU/g, and 63.0% are within ±0.25 Log CFU/g. For relative growth of <2.0 Log CFU/g, 92.7% of the residual errors are within ±0.5 Log CFU/g, and 70.3% are within ±0.25 Log CFU/g. The scale and distribution of residual errors clearly suggests that the models and estimated kinetic parameters are reasonably accurate in predicting the growth of C. perfringens. Monte Carlo simulation was used to estimate the probabilities of >1.0 and 2.0 Log CFU/g relative growth of C. perfringens in the final products at the end of cooling. This probabilistic process analysis approach provides a new alternative for estimating and managing the risk of a product and can help the food industry and regulatory agencies assess the safety of cooked meat in the event of cooling deviation. PMID:26801359

  2. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

    PubMed Central

    Volozhantsev, Nikolay V.; Oakley, Brian B.; Morales, Cesar A.; Verevkin, Vladimir V.; Bannov, Vasily A.; Krasilnikova, Valentina M.; Popova, Anastasia V.; Zhilenkov, Eugeni L.; Garrish, Johnna K.; Schegg, Kathleen M.; Woolsey, Rebekah; Quilici, David R.; Line, J. Eric; Hiett, Kelli L.; Siragusa, Gregory R.; Svetoch, Edward A.; Seal, Bruce S.

    2012-01-01

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae. PMID:22666499

  3. A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

    PubMed Central

    Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

    2016-01-01

    Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

  4. An unusual necrotic myositis by Clostridium perfringens in a German Shepherd dog: A clinical report, bacteriological and molecular identification

    PubMed Central

    Salari Sedigh, Hamideh; Rajabioun, Masoud; Razmyar, Jamshid; Kazemi Mehrjerdi, Hossein

    2015-01-01

    Clostridial myositis, considered to be rare in pet animals, is an acutely fatal toxaemic condition. Some species of clostridia are responsible for necrotic myositis. A 2-year-old male German shepherd dog was admitted with non-weight bearing lameness and massive swelling of the left hind limb. Clostridium perfringens type A with alpha toxin was diagnosed as a pathogenic agent. Based on the history, the bacteria were introduced inside the tissue via contaminated needle following intramuscular injection. Urgent medical therapy followed by surgical intervention was performed. The dog was discharged completely healthy after hospitalization for four weeks. The objective of this report was to describe necrotic myositis in a dog with an emphasis on clinical signs and treatment as well as bacteriological and molecular identification of the micro-organism. Because of the fatal entity of the disease, prompt diagnosis as well as proper and urgent treatment is very important for successful therapy.

  5. A serotyping system for Clostridium welchii (C. perfringens) type A, and studies on the type-specific antigens.

    PubMed

    Hughes, J A; Turnbull, P C; Stringer, M F

    1976-11-01

    A serotyping scheme for Clostridium welchii (C. perfringens) type A employing 57 antisera has been used to investigate the epidemiology of 153 food-poisoning outbreaks and 32 cases of gas gangrene and other clinical infections. Respectively 65% and 59% of the isolates were typable, and in 55% of the food-poisoning outbreaks the causative serotypes were established. Isolation and reporting methods that would render the typing scheme of even greater epidemiological value are described. The type-specific antigen was shown to reside in the capsule and to be lost from strains that had become rough. Development of roughness and its prevention are described. A great range of antisera and an internationally acceptable serotyping scheme is expected after integration of this set with those developed independently in America and Japan. PMID:63553

  6. Clostridium Perfringens Enterotoxin (CPE) and CPE-Binding Domain (c-CPE) for the Detection and Treatment of Gynecologic Cancers

    PubMed Central

    Black, Jonathan D.; Lopez, Salvatore; Cocco, Emiliano; Schwab, Carlton L.; English, Diana P.; Santin, Alessandro D.

    2015-01-01

    Clostridium perfringens enterotoxin (CPE) is a three-domain polypeptide, which binds to Claudin-3 and Claudin-4 with high affinity. Because these receptors are highly differentially expressed in many human tumors, claudin-3 and claudin-4 may provide an efficient molecular tool to specifically identify and target biologically aggressive human cancer cells for CPE-specific binding and cytolysis. In this review we will discuss these surface proteins as targets for the detection and treatment of chemotherapy-resistant gynecologic malignancies overexpressing claudin-3 and -4 using CPE-based theranostic agents. We will also discuss the use of fluorescent c-CPE peptide in the operative setting for real time detection of micro-metastatic tumors during surgery and review the potential role of CPE in other medical applications. PMID:25835384

  7. A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin.

    PubMed

    Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

    2016-01-01

    Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

  8. Opening of the active site of Clostridium perfringens alpha-toxin may be triggered by membrane binding.

    PubMed

    Titball, R W; Naylor, C E; Miller, J; Moss, D S; Basak, A K

    2000-10-01

    On the basis of amino acid sequence homologies with other phospholipases C, the alpha-toxin of Clostridium perfringens was predicted to be a two-domain protein. Using truncated forms of alpha-toxin the phospholipase C active site was shown to be located in the amino-terminal domain. Crystallographic studies have confirmed this organisation and have also revealed that the carboxy-terminal domain is structurally similar to the phospholipid-binding domains in eukaryotic proteins. This information has been used to devise a model predicting how alpha-toxin interacts with membranes via calcium-mediated recognition of phospholipid head groups and the interaction of hydrophobic amino acids with the phospholipid tail group. The binding of alpha-toxin to membranes appears to result in the opening of the active site allowing hydrolysis of membrane phospholipids. PMID:11111911

  9. Opening of the active site of Clostridium perfringens alpha-toxin may be triggered by membrane binding.

    TOXLINE Toxicology Bibliographic Information

    Titball RW; Naylor CE; Miller J; Moss DS; Basak AK

    2000-10-01

    On the basis of amino acid sequence homologies with other phospholipases C, the alpha-toxin of Clostridium perfringens was predicted to be a two-domain protein. Using truncated forms of alpha-toxin the phospholipase C active site was shown to be located in the amino-terminal domain. Crystallographic studies have confirmed this organisation and have also revealed that the carboxy-terminal domain is structurally similar to the phospholipid-binding domains in eukaryotic proteins. This information has been used to devise a model predicting how alpha-toxin interacts with membranes via calcium-mediated recognition of phospholipid head groups and the interaction of hydrophobic amino acids with the phospholipid tail group. The binding of alpha-toxin to membranes appears to result in the opening of the active site allowing hydrolysis of membrane phospholipids.

  10. [Determination of clostridium perfringens in pork sausages from the Metropolitan area of Costa Rica].

    PubMed

    Morera, J; Rodríguez, E; Gamboa, M M

    1999-09-01

    The presence of C. perfringens was analyzed in 75 samples of pork sausages (chorizo, salchichon and bologna), obtained from five processing plants located in the Metropolitan Area of Costa Rica. Previously and after the biochemical identification of the strains, the most probable number (MPN) of C. perfringens per gram of food was determined and it varied from less than 3 to more than 2.4 x 10(5). There were significant statistical differences (p < 0.005) that support the need of employing biochemical tests for confirming C. perfringens in a given food. C. perfringens was present in 92% of the chorizos, in 28% of the bolognas and in 12% of the salchichon. Every positive sample was tested looking for at least one enterotoxigenic strain, using the reverse passive agglutination latex test; 8% of the tested strains were enterotoxigenic and corresponded to chorizo and bologna from one processing plant and chorizo from another plant. The results obtained in this study show that pork sausages, and not just not processed meats, are important as risk factors for food intoxication by C. perfringens. PMID:10667270

  11. Hypermotility in Clostridium perfringens Strain SM101 Is Due to Spontaneous Mutations in Genes Linked to Cell Division

    PubMed Central

    Liu, Hualan; McCord, Kristin D.; Howarth, Jonathon; Popham, David L.; Jensen, Roderick V.

    2014-01-01

    Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. Although they lack flagella, C. perfringens bacteria can still migrate across surfaces using a type of gliding motility that involves the formation of filaments of bacteria lined up in an end-to-end conformation. In strain SM101, hypermotile variants are often found arising from the edges of colonies on agar plates. Hypermotile cells are longer than wild-type cells, and video microscopy of their gliding motility suggests that they form long, thin filaments that move rapidly away from a colony, analogously to swarmer cells in bacteria with flagella. To identify the cause(s) of the hypermotility phenotype, the genome sequences of normal strains and their direct hypermotile derivatives were determined and compared. Strains SM124 and SM127, hypermotile derivatives of strains SM101 and SM102, respectively, contained 10 and 6 single nucleotide polymorphisms (SNPs) relative to their parent strains. While SNPs were located in different genes in the two sets of strains, one feature in common was mutations in cell division genes, an ftsI homolog in strain SM124 (CPR_1831) and a minE homolog in strain SM127 (CPR_2104). Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model explaining the principles underlying the hypermotility phenotype is presented. PMID:24748614

  12. Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation.

    PubMed

    Zimmer, Markus; Scherer, Siegfried; Loessner, Martin J

    2002-08-01

    Two temperate viruses, phi3626 and phi8533, have been isolated from lysogenic Clostridium perfringens strains. Phage phi3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3'-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of phi3626 with onset of sporulation in C. perfringens. We found that the phi3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3' end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence. PMID:12142405

  13. Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis

    PubMed Central

    Mehdizadeh Gohari, Iman; Kropinski, Andrew M.; Weese, Scott J.; Parreira, Valeria R.; Whitehead, Ashley E.; Boerlin, Patrick; Prescott, John F.

    2016-01-01

    The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus. PMID:26859667

  14. Sporulation and Enterotoxin (CPE) Synthesis Are Controlled by the Sporulation-Specific Sigma Factors SigE and SigK in Clostridium perfringens?

    PubMed Central

    Harry, Kathryn H.; Zhou, Ruanbao; Kroos, Lee; Melville, Stephen B.

    2009-01-01

    Clostridium perfringens is the third most frequent cause of bacterial food poisoning annually in the United States. Ingested C. perfringens vegetative cells sporulate in the intestinal tract and produce an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. Studies of Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors ?F, ?E, ?G, and ?K coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be ?K dependent, while P2 and P3 were proposed to be ?E dependent based on consensus promoter recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. With the sigE and sigK mutants, gusA fusion assays indicated that there was no expression of cpe in either mutant. Results from gusA fusion assays and immunoblotting experiments indicate that ?E-associated RNA polymerase and ?K-associated RNA polymerase coregulate each other's expression. Transcription and translation of the spoIIID gene in C. perfringens were not affected by mutations in sigE and sigK, which differs from B. subtilis, in which spoIIID transcription requires ?E-associated RNA polymerase. The results presented here show that the regulation of developmental events in the mother cell compartment of C. perfringens is not the same as that in B. subtilis and Clostridium acetobutylicum. PMID:19201796

  15. Entérite nécrotique chez le poulet de gril II. Caractères des souches de Clostridium perfringens isolées

    PubMed Central

    Bernier, G.; Filion, R.; Malo, R.; Phaneuf, J.-B.

    1974-01-01

    A Gram positive bacillus, strictly anaerobic, was isolated from the viscera of all diseased birds showing lesions of necrotic enteritis. Its morphology and biochemical reactions, the presence of alpha and thêta hemolysins and the production of a lecithinase-C in vitro, all these characteristics indicated a similarity to those belonging to the group of Clostridium perfringens. The two hemolysins were neutralized in vitro only by the antitoxin A. Broiler chickens injected I.V. with a Viande-Foie (VF) broth culture of Clostridium perfringens together with the antitoxin A survived, whereas those receiving antitoxin C died. These results seem to indicate that this organism belongs to the type A. This bacillus was sensitive to a great variety of antibiotics, except neomycin. PMID:4368193

  16. Assessing the performance of Clostridium perfringens cooling models for cooked, uncured meat and poultry products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat-resistant spores of C. perfringens may germinate and multiply in cooked meat and poultry products if the rate and extent of cooling does not occur in a timely manner. Therefore, six cooling models (PMP 7.0 broth model; PMIP Uncured Beef, Chicken, and Pork Models; Smith-Schaffner (version 3); a...

  17. Evidence of chitinase activity within necrotic enteritis-associated subtypes of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    C. perfringens (Cp) is associated with the necrotic gastrointestinal condition known as necrotic enteritis (NE) in the chicken. rep-PCR subtyping identified subtypes of Cp from the gastrointestinal tracts of broiler chickens afflicted with NE that were distinguished from strains from environmental,...

  18. Dynamic determination of kinetic parameters and computer simulation of growth of Clostridium perfringens in cooked beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this research was to develop a new one-step methodology that uses a dynamic approach to directly construct a tertiary model for prediction of the growth of C. perfringens in cooked beef. This methodology was based on numerical analysis and optimization of both primary and secondary...

  19. Growth potential of Clostridium perfringens from spores in acidified beef, pork and poultry products during chilling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of C. perfringens to germinate and grow in acidified ground beef as well as in ten commercially prepared acidified beef, pork and poultry products was assessed. The pH of ground beef was adjusted using organic vinegar to achieve various pH values between 5.0 and 5.6; the pH of the commer...

  20. NUMERICAL ANALYSIS OF THE GROWTH OF CLOSTRIDIUM PERFRINGENS IN COOKED BEEF UNDER ISOTHERMAL AND DYNAMIC CONDITIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to develop a mathematical methodology to estimate the growth of C. perfringens in cooked beef under dynamic temperature conditions. Two differential equations governing the lag phase development and cell multiplication were proposed and solved using a 4th-order Runge...

  1. Impact of a drug-free program on broiler chicken growth performances, gut health, Clostridium perfringens and Campylobacter jejuni occurrences at the farm level.

    PubMed

    Gaucher, M-L; Quessy, S; Letellier, A; Arsenault, J; Boulianne, M

    2015-08-01

    The use of antimicrobial agents as feed additives in poultry production is a public health concern due to the overall increase in antimicrobial resistance. Although some alternative products are commercially available, little is known on their potential impact on flock health and productivity. A prospective study involving 1.55 million birds was conducted on eight commercial broiler farms in Québec, Canada, to evaluate the impact of replacing antibiotic growth promoters and anticoccidial drugs by a drug-free program including improved brooding conditions, anticoccidial vaccination, essential oil-based feed additives, and water acidification. Various productivity and health parameters were compared between barns allocated to the conventional and the drug-free program. Zootechnical performances were monitored as productivity criteria. Clinical necrotic enteritis and subclinical enteritis occurrences, litter and fecal moistures content were measured, and microscopic gut health was evaluated. Clostridium perfringens and Campylobacter spp. strains were recovered from fecal samples collected during farm visits. Clostridium perfringens counts were used as poultry health indicators and Campylobacter prevalence was noted as well. The drug-free program was associated with a significant increase in feed conversion ratio and a decrease in mean live weight at slaughter and in daily weight gain. An increased incidence of necrotic enteritis outbreaks and subclinical enteritis cases, as well as an increase in litter moisture content at the end of the rearing period were also observed for this program. Mean microscopic intestinal lesion scores and prevalence of Campylobacter colonization were not statistically different between the two groups but the drug-free program was associated with higher Clostridium perfringens isolation rates. According to the current study design, the results suggest that substitution of antibiotic growth promoters and anticoccidial drugs by a drug-free program impacts various broiler chicken production parameters and Clostridium perfringens carriage levels. PMID:26047674

  2. The relationship between the presence of Helicobacter pylori, Clostridium perfringens type A, Campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves.

    PubMed Central

    Jelinski, M D; Ribble, C S; Chirino-Trejo, M; Clark, E G; Janzen, E D

    1995-01-01

    A case-control study involving 30 unweaned beef calves was conducted to determine whether specific species of bacteria or fungi were associated with fatal abomasal ulcer formation. Special microbiological and histological techniques were used to detect Clostridium perfringens type A, Helicobacter pylori, or Campylobacter spp. It has been speculated that these bacteria are potential ulcerogenic agents of unweaned beef calves. Calves were recruited for the study at necropsy, with those dying of either a perforating or a hemorrhagic ulcer representing the cases, and calves of a similar age dying of a disease unrelated to the abomasum representing the controls. Helicobacter pylori was not visualized in or cultured from any of the abomasal tissue samples. Clostridium perfringens type A was isolated from 78.6% of the cases and 75% of the controls. These isolates were further dichotomized into "heavy" and "light" growth; no significant association was found between ulcers and the amount of growth. A light growth of Campylobacter spp. was recovered from 3 cases and 3 controls. There was no compelling evidence to suggest that Clostridium perfringens type A, Helicobacter pylori, or Campylobacter spp. were involved in ulcer formation. PMID:7648542

  3. Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling.

    PubMed

    Redondo-Solano, Mauricio; Valenzuela-Martinez, Carol; Cassada, David A; Snow, Daniel D; Juneja, Vijay K; Burson, Dennis E; Thippareddi, Harshavardhan

    2013-09-01

    The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5 C for 3 or 24 h after preparation) were transferred to a vacuum bag and inoculated with a three-strain C. perfringens spore cocktail to obtain an inoculum of ca. 2.5 log spores/g. The bags were vacuum-sealed, and the meat was heat treated (75 C, 20 min) and cooled within 15 h from 54.4 to 7.2 C. Residual nitrite was determined before and after heat treatment using ion chromatography with colorimetric detection. Cooling of ham (control) stored for 3 and 24 h, resulted in C. perfringens population increases of 1.46 and 4.20 log CFU/g, respectively. For samples that contained low NaNO2 concentrations and were stored for 3 h, C. perfringens populations of 5.22 and 2.83 log CFU/g were observed with or without sodium erythorbate, respectively. Residual nitrite was stable (p > 0.05) for both storage times. Meat processing ingredients (sodium nitrite and sodium erythorbate) and their concentrations, and storage time subsequent to preparation of meat (oxygen content) affect C. perfringens spore germination and outgrowth during abusive cooling of ham. PMID:23664261

  4. Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and In Vitro Cytotoxicity Activity

    PubMed Central

    Theoret, James R.; Uzal, Francisco A.

    2015-01-01

    By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin. PMID:25643999

  5. Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia.

    PubMed

    Das, Shreya; Majumder, Saugata; Kingston, Joseph J; Batra, Harsh V

    2016-02-01

    Clostridium perfringens beta (CPB) and iota (CPI) toxaemias result in some of the most lethal forms of haemorrhagic and necrotic enteritis and sudden death syndrome affecting especially neonates. While CPB enterotoxemia is one of the most common forms of clostridial enterotoxemia, CPI enterotoxemia though putatively considered to be rare is an emerging cause of concern. The similarities in clinical manifestation, gross and histopathology findings of both types of toxaemias coupled to the infrequency of CPI toxaemia might lead to symptomatic misidentification with Type C resulting in therapeutic failure due to habitual administration of CPB anti-toxin which is ineffective against CPI. Therefore in the present study, to generate a composite anti-toxin capable of neutralizing both toxaemias, a novel bivalent chimera r-Cpib was constructed by splicing the non-toxic C terminal binding regions of CPB and CPI, via a flexible glycine linker (G4S) by overlap-extension PCR. The fusion protein was characterized for its therapeutic abilities toward CPI and CPB toxin neutralizations. The r-Cpib was found to be non-toxic and could competitively inhibit binding of CPB to host cell receptors thereby reducing its cytotoxicity. Immunization of mice with r-Cpib generated specific antibodies capable of neutralizing the above toxaemias both in vitro and in vivo. Caco-2 cells exposed to a mixture of anti-r-Cpib sera and native CPI or CPB, displayed significantly superior protection against the respective toxins while passive challenge of mice with a similar mixture resulted in 83 and 91% protection against CPI and CPB respectively. Alternatively, mice exposed to a mixture of sham sera and native toxins died within 2-3 days. This work thus demonstrates r-Cpib as a novel bivalent fusion protein capable of efficient immunotherapy against C. perfringens CPI and CPB toxaemia. PMID:26774054

  6. Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

    PubMed

    Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

    2013-01-01

    Clostridium perfringens ?-toxin (CPB) is a ?-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis"). PMID:23734212

  7. Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

    PubMed Central

    Bannam, Trudi L.; Yan, Xu-Xia; Harrison, Paul F.; Seemann, Torsten; Keyburn, Anthony L.; Stubenrauch, Christopher; Weeramantri, Lakmini H.; Cheung, Jackie K.; McClane, Bruce A.; Boyce, John D.; Moore, Robert J.; Rood, Julian I.

    2011-01-01

    ABSTRACT The pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netB was insertionally inactivated by the chloramphenicol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netB gene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained the netB gene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2 gene, which encodes a different pore-forming toxin, beta2 toxin. PMID:21954306

  8. Human Claudin-8 and -14 Are Receptors Capable of Conveying the Cytotoxic Effects of Clostridium perfringens Enterotoxin

    PubMed Central

    Shrestha, Archana; McClane, Bruce A.

    2013-01-01

    ABSTRACT Clostridium perfringens enterotoxin (CPE) contributes to several important human gastrointestinal (GI) diseases. This toxin and its derivatives are also being explored for translational applications, i.e., cancer therapy or drug delivery. Some, but not all, members of the 24-member claudin (Cldn) family of mammalian tight junction proteins can serve as CPE receptors. Among the human Cldns (hCldns), hCldn-3 and -4 are known to convey CPE sensitivity when expressed by fibroblast transfectants. However, other Cldns are also reportedly expressed in the intestines, where they might contribute to natural CPE-mediated GI disease, and in other organs, where they might react with CPE-based therapeutics. Therefore, the current study assessed whether two additional hCldns beside hCldn-3 and -4 are also functional CPE receptors. Using Cldn-expressing transfectants, hCldn-8 and -14 were shown to convey CPE-mediated cytotoxicity at pathophysiologically relevant concentrations of this toxin, although ~2-to-10-fold less efficiently than hCldn-4. Site-directed mutagenesis then demonstrated that the N146 residue in hCldn-14 and the S151 residue in hCldn-8 are largely responsible for modulating the weaker CPE binding properties of hCldn-8 and -14 versus hCldn-4, which broadens understanding of Cldn:CPE binding interactions. Since Cldn-8 and -14 are reportedly expressed in mammalian intestines, the current results support the possibility that these two hCldns contribute to natural CPE-mediated gastrointestinal disease and could be CPE-based therapeutic targets for cancers overexpressing those claudins. However, these results also suggest caution during therapeutic use of CPE, which might trigger toxic side effects in normal human tissues producing hCldn-8 or -14, as well as in those producing hCldn-3 or -4. IMPORTANCE Clostridium perfringens enterotoxin (CPE) is responsible for the gastrointestinal symptoms of the second-most-common bacterial food-borne illness and is also being explored for use as a cancer therapeutic or for increasing drug delivery. Until now, the only known human CPE receptors were claudin-3 and -4. This work shows that human claudin-8 and -14 can also bind CPE and convey cytotoxicity, although slightly less efficiently than claudin-3 and -4. The claudin-8 and -14 residues responsible for this weaker CPE binding were identified, shedding new light on CPE:claudin interactions. PMID:23322640

  9. NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands.

    PubMed

    Allaart, Janneke G; de Bruijn, Naomi D; van Asten, Alphons J A M; Fabri, Teun H F; Grne, Andrea

    2012-12-01

    Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent. PMID:23237366

  10. Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl–l-alanine amidase as a potential antimicrobial to control the bacterium

    PubMed Central

    Tillman, Glenn E.; Simmons, Mustafa; Garrish, Johnna K.; Seal, Bruce S.

    2014-01-01

    Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal, and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl–l-alanine amidase or MurnAc–lAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and l-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction, and the gene product (PlyCpAmi) was expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact, and injured compared to the untreated cells. Among a variety of C. perfringens strains, there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results further demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens. PMID:23934074

  11. Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

    PubMed

    Labbe, R; Somers, E; Duncan, C

    1976-03-01

    Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters. PMID:180885

  12. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

  13. Factors affecting the incidence of necrotic enteritis, caecal carriage of Clostridium perfringens and bird performance in broiler chicks.

    PubMed

    Elwinger, K; Schneitz, C; Berndtson, E; Fossum, O; Teglf, B; Engstm, B

    1992-01-01

    Two trials were conducted to study the effects of a competitive exclusion (CE) product BROILACT and the anticoccidial narasin on the incidence of necrotic enteritis (NE), the numbers of Clostridium perfringens (CP) in the caeca of broiler chicks and the performance of the birds. In trial 1 the effects of type of protein and partial replacement of a narasin containing diet with whole wheat were also studied. All groups of chicks were studied up to the point of slaughter at 43 days of age and after evisceration in a processing plant to determine slaughter yield. In trial 1, statistically significant results included the following: CE-treatment reduced total mortality, and incidence of NE, on diet containing animal but not vegetable protein. Caecal carriage of CP was also reduced, while slaughter yield increased. Narasin reduced caecal carriage of CP and increased both growth rate and slaughter yield in both trials. Whole wheat replacement improved feed conversion but reduced bird growth rate. In trial 2, both CE-treatment and narasin influenced feed intake, CE-treatment significantly only at days 22 and 44. Narasin improved feed conversion until 5 weeks of age and CE-treatment did so until 22 days of age. In both trials, there was also an interaction effect indicating that CE-treatment increased slaughter yield for birds that were not fed narasin. PMID:1488953

  14. Comparative analysis of extractable proteins from Clostridium perfringens type A and type C strains showing varying degree of virulence.

    PubMed

    Dwivedi, Pratistha; Alam, Syed Imteyaz; Kumar, Om; Kumar, Ravi Bhushan

    2015-10-01

    The prevailing scenario of bioterrorism warrants development of medical countermeasures with expanded coverage of select agents. Clostridium perfringens is a pathogen of medical, veterinary and military importance, and has been listed as Validated Biological Agent. We employed 2DE-MS approach to identify a total of 134 unique proteins (529 protein spot features) from the extractable proteome of four type A and type C strains. Proteins showing altered expression under host-simulated conditions from virulent type A strain (ATCC13124) were also elucidated. Significant among the differentially expressed proteins were elongation factor, molecular chaperones, ribosomal proteins, carbamoyl phosphate synthase, clpB protein, choloylglycine hydrolase, phosphopyruvate hydratase, and trigger factor. Predictive elucidation, of putative virulence associated proteins and sequence conservation pattern of selected candidates, was carried out using homologous proteins from other bacterial select agents to screen for the commonality of putative antigenic determinants. Pathogens (17 select agents) were observed to form three discrete clusters; composition of I and II being consistent in most of the phylogenetic reconstructions. This work provides a basis for further validation of putative candidate proteins as prophylactic agents and for their ability to provide protection against clusters of pathogenic select bacterial agents; aimed at mitigating the shadows of biothreat. PMID:26238688

  15. Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant.

    PubMed

    Coursodon, Christine F; Trinh, Hien T; Mallozzi, Michael; Vedantam, Gayatri; Glock, R D; Songer, J G

    2010-12-01

    Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation. PMID:20934524

  16. Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant.

    TOXLINE Toxicology Bibliographic Information

    Coursodon CF; Trinh HT; Mallozzi M; Vedantam G; Glock RD; Songer JG

    2010-12-01

    Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation.

  17. Molecular Architecture and Functional Analysis of NetB, a Pore-forming Toxin from Clostridium perfringens*

    PubMed Central

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

    2013-01-01

    NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology. PMID:23239883

  18. Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC, MEK1 and NF?B Activation

    PubMed Central

    Monturiol-Gross, Laura; Flores-Daz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called ?-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NF?B signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NF?B pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NF?B inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis. PMID:24466113

  19. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice. (iii... Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard antitoxin. The Epsilon... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  20. Beta2 toxin is not involved in in vitro cell cytotoxicity caused by human and porcine cpb2-harbouring Clostridium perfringens.

    PubMed

    Allaart, Janneke G; van Asten, Alphons J A M; Vernooij, Johannes C M; Grne, Andrea

    2014-06-25

    Clostridium perfringens is a common cause of intestinal disease in animals and humans. Its pathogenicity is attributed to the toxins it can produce, including the beta2 toxin. The presence of cpb2, the gene encoding the beta2 toxin, has been associated with diarrhoea in neonatal piglets and humans. However, the exact role of the beta2 toxin in the development of diarrhoea is still unknown. In this study we investigated the level of cytotoxicity to porcine IPI-21 and human Caco-2 cell-lines caused by porcine and human cpb2-harbouring C. perfringens and the significance of the beta2 toxin for the induction of cell cytotoxicity. Supernatants of porcine cpb2-harbouring C. perfringens strains were cytotoxic to both cell lines. Cell cytotoxicity caused by supernatant of human cpb2-harbouring C. perfringens strains was variable among strains. However, removal of the beta2 toxin by anti-beta2 toxin antibodies or degradation of the beta2 toxin by trypsin did not reduce the cytotoxic effect of any of the supernatants. These data suggest that beta2 toxin does not play a role in the development of cell cytotoxicity in in vitro experiments. In vivo studies are necessary to definitely define the role of beta2 toxin in the development of cell cytotoxicity and subsequent diarrhoea. PMID:24768003

  1. Two Novel Membrane Proteins, TcpD and TcpE, Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

    PubMed Central

    Wisniewski, Jessica A.; Teng, Wee L.; Bannam, Trudi L.

    2014-01-01

    The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus. PMID:25488300

  2. Location and stoichiometry of the protease CspB and the cortex-lytic enzyme SleC in Clostridium perfringens spores.

    PubMed

    Banawas, Saeed; Korza, George; Paredes-Sabja, Daniel; Li, Yunfeng; Hao, Bing; Setlow, Peter; Sarker, Mahfuzur R

    2015-09-01

    The protease CspB and the cortex-lytic enzyme SleC are essential for peptoglycan cortex hydrolysis during germination of spores of the Clostridium perfringens food poisoning isolate SM101. In this study, Western blot analyses were used to demonstrate that CspB and SleC are present exclusively in the C.perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. These results indicate why decoating treatments greatly reduce both germination and apparent viability of C.perfringens spores in the absence of an exogenous lytic enzyme. In addition, quantitative Western blot analyses showed that there are approximately 2000 and 130,000 molecules of CspB and pro-SleC, respectively, per C.perfringens SM101 spore, consistent with CspB's role in acting catalytically on pro-SleC to convert this zymogen to the active enzyme. PMID:25998819

  3. The Interaction of a Carbohydrate-Binding Module from a Clostridium perfringens N-Acetyl-beta-hexosaminidase with its Carbohydrate Receptor

    SciTech Connect

    Ficko-Blean,E.; Boraston, A.

    2006-01-01

    Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-{beta}-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc ({beta}-d-galactosyl-1,4-{beta}-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Angstroms, respectively.

  4. Distribution of Clostridium perfringens and Fecal Sterols in a Benthic Coastal Marine Environment Influenced by the Sewage Outfall from McMurdo Station, Antarctica†

    PubMed Central

    Edwards, Diane D.; McFeters, Gordon A.; Venkatesan, M. Indira

    1998-01-01

    The spatial distribution, movement, and impact of the untreated wastewater outfall from McMurdo Station, Antarctica, were investigated under early austral summer conditions. The benthic environment was examined to determine the distribution of Clostridium perfringens in sediment cores and the intestinal contents of native invertebrates and fish along a transect of stations. These stations extended ca. 411 m south of the outfall. The findings revealed that the concentration of C. perfringens decreased with depth in the sediment and distance from the outfall. High percentages of tunicates and sea urchins were colonized with this bacterium along the transect. Coprostanol concentrations were also measured in sediment samples taken from each of the transect stations, and a similar trend was observed. These results are in agreement with the findings of previous studies performed with the water column and collectively provide evidence that the disposal of domestic wastes deserves special consideration in polar marine environments. PMID:9647835

  5. Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics

    PubMed Central

    Xiao, Yinghua; van Hijum, Sacha A. F. T.; Abee, Tjakko; Wells-Bennik, Marjon H. J.

    2015-01-01

    The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies. PMID:25978838

  6. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium1

    PubMed Central

    Seal, Bruce S.

    2014-01-01

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as nonfoodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria. PMID:23300321

  7. Use of organic acids for the control of Clostridium perfringens in cooked vacuum-packaged restructured roast beef during an alternative cooling procedure.

    PubMed

    Sabah, J R; Thippareddi, H; Marsden, J L; Fung, D Y C

    2003-08-01

    This study was conducted to determine how well Clostridium perfringens spores germinate and grow in restructured roast beef treated with different commercial organic salts during an alternative chilling procedure. The meat was prepared according to an industrial recipe (10% water, 1.5% sodium chloride, and 0.5% sodium triphosphate). The base meat was treated with sodium citrate at 2 or 4.8% (wt/wt), buffered to a pH of 5.6, 5.0, or 4.4 (six treatments); a 60% (wt/wt) solution of sodium lactate at 2 or 4.8% (wt/wt); sodium acetate at 0.25% (wt/wt); or sodium diacetate at 0.25% (wt/wt). Untreated meat was used as a control. Meat samples were inoculated with a three-strain cocktail of C. perfringens spores (strains ATCC 10388, NCTC 8238, and NCTC 8239). Meat was vacuum packaged in bags and cooked in a stirred water bath to an internal temperature of 75 degrees C for 20 min, and then the bags were cooled from 54.4 to 4.4 degrees C within 18 h. Samples were taken after inoculation, after cooking, and after chilling. Spore and vegetative cell counts were obtained after incubation at 37 degrees C for 8 to 10 h in Fung's Double Tubes containing tryptose sulfite agar without egg yolk enrichment. Cooking was not sufficient to eliminate C. perfringens spores. Over the 18-h cooling period, sodium citrate, sodium lactate, and sodium diacetate reduced the growth of C. perfringens to < 1 log unit, a growth level that meets U.S. Department of Agriculture performance standards. The use of sodium citrate or sodium lactate at a concentration of > or = 2% (wt/wt) inhibited C. perfringens growth over the 18-h cooling period. PMID:12929827

  8. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium.

    PubMed

    Seal, Bruce S

    2013-02-01

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as non-foodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria. PMID:23300321

  9. Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway.

    PubMed Central

    Titball, R W; Leslie, D L; Harvey, S; Kelly, D

    1991-01-01

    The N-terminal domain of Clostridium perfringens alpha-toxin, homologous with the nontoxic phospholipase C of Bacillus cereus, was expressed in Escherichia coli and shown to retain all of the phosphatidylcholine hydrolyzing activity of the alpha-toxin, but not the sphingomyelinase, hemolytic, or lethal activities. The C-terminal domain of alpha-toxin showed sequence and predicted structural homologies with the N-terminal region of arachidonate 5-lipoxygenase, an enzyme from the human arachidonic acid pathway which plays a role in inflammatory and cardiovascular diseases in humans. Images PMID:1902199

  10. Influence of enrichment broths on multiplex PCR detection of total coliform bacteria, Escherichia coli and Clostridium perfringens, in spiked water samples.

    PubMed

    Worakhunpiset, S; Tharnpoophasiam, P

    2009-07-01

    Although multiplex PCR amplification condition for simultaneous detection of total coliform bacteria, Escherichia coli and Clostridium perfringens in water sample has been developed, results with high sensitivity are obtained when amplifying purified DNA, but the sensitivity is low when applied to spiked water samples. An enrichment broth culture prior PCR analysis increases sensitivity of the test but the specific nature of enrichment broth can affect the PCR results. Three enrichment broths, lactose broth, reinforced clostridial medium and fluid thioglycollate broth, were compared for their influence on sensitivity and on time required with multiplex PCR assay. Fluid thioglycollate broth was the most effective with shortest enrichment time and lowest detection limit. PMID:19842417

  11. Comparison of the Effect of Curing Ingredients Derived from Purified and Natural Sources on Inhibition of Clostridium perfringens Outgrowth during Cooling of Deli-Style Turkey Breast.

    PubMed

    King, Amanda M; Glass, Kathleen A; Milkowski, Andrew L; Sindelar, Jeffrey J

    2015-08-01

    The antimicrobial impact of purified and natural sources of both nitrite and ascorbate were evaluated against Clostridium perfringens during the postthermal processing cooling period of deli-style turkey breast. The objective of phase I was to assess comparable concentrations of nitrite (0 or 100 ppm) and ascorbate (0 or 547 ppm) from both purified and natural sources. Phase II was conducted to investigate concentrations of nitrite (50, 75, or 100 ppm) from cultured celery juice powder and ascorbate (0, 250, or 500 ppm) from cherry powder to simulate alternative curing formulations. Ground turkey breast (75% moisture, 1.2% salt, pH 6.2) treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1C, and chilled from 54.4 to 26.7C in 5 h and from 26.7 to 7.2C in 10 additional hours. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar; experiments were replicated three times. In phase I, uncured, purified nitrite, and natural nitrite treatments without ascorbate had 5.3-, 4.2-, and 4.4-log increases in C. perfringens, respectively, at 15 h, but <1-log increase was observed at the end of chilling in treatments containing 100 ppm of nitrite and 547 ppm of ascorbate from either source. In phase II, 0, 50, 75, and 100 ppm of nitrite and 50 ppm of nitrite plus 250 ppm of ascorbate supported 4.5-, 3.9-, 3.5-, 2.2-, and 1.5-log increases in C. perfringens, respectively. In contrast, <1-log increase was observed after 15 h in the remaining phase II treatments supplemented with 50 ppm of nitrite and 500 ppm of ascorbate or ?75 ppm of nitrite and ?250 ppm of ascorbate. These results confirm that equivalent concentrations of nitrite, regardless of the source, provide similar inhibition of C. perfringens during chilling and that ascorbate enhances the antimicrobial effect of nitrite on C. perfringens at concentrations commonly used in alternative cured meats. PMID:26219366

  12. Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential

    PubMed Central

    2011-01-01

    Background Because 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 agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products. PMID:21631945

  13. The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin

    PubMed Central

    Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A.; Fischetti, Vincent A.; McClain, Mark S.; Vartanian, Timothy

    2015-01-01

    Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity. PMID:25993478

  14. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

    PubMed Central

    Takagishi, Teruhisa; Oda, Masataka; Kabura, Michiko; Kurosawa, Mie; Tominaga, Kaori; Urano, Shiori; Ueda, Yoshibumi; Kobayashi, Keiko; Kobayashi, Toshihide; Sakurai, Jun; Terao, Yutaka; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-? (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase C?-1 (PLC?-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLC?-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLC?-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA. PMID:25910247

  15. The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens.

    PubMed Central

    Ba-Thein, W; Lyristis, M; Ohtani, K; Nisbet, I T; Hayashi, H; Rood, J I; Shimizu, T

    1996-01-01

    Extracellular toxin production in Clostridium perfringens is positively regulated by the two-component regulatory genes virR and virS. Northern (RNA) blots carried out with RNA preparations from the wild-type strain 13 and the isogenic virR and virS mutants TS133 and JIR4000 showed that the virR and virS genes composed an operon and were transcribed as a single 2.1-kb mRNA molecule. Primer extension analysis led to the identification of two promoters upstream of virR. Hybridization analysis of the mutants and their complemented derivatives showed that the virR/virS system positively regulated the production of alpha-toxin (or phospholipase C, theta-toxin (perfringolysin O), and kappa-toxin (collagenase) at the transcriptional level. However, the modes of regulation of these genes were shown to differ. The theta-toxin structural gene, pfoA, had both a major and a very minor promoter, with the major promoter being virR/virS dependent. The colA gene, which encodes the kappa-toxin, had two major promoters, only one of which was virR/virS-dependent. In contrast, the alpha-toxin structural gene, p1c, had only one promoter, which was shown to be partially regulated by the virR and virS genes. Comparative analysis of the virR/virS-dependent promoters did not reveal any common sequence motifs that could represent VirR-binding sites. It was concluded that either the virR/virS system modulates its effects via secondary regulatory genes that are specific for each toxin structural gene or the VirR protein does not have a single consensus binding sequence. PMID:8626316

  16. B-cell epitope of beta toxin of Clostridium perfringens genetically conjugated to a carrier protein: expression, purification and characterization of the chimeric protein.

    PubMed

    Bhatia, Bharti; Solanki, Amit Kumar; Kaushik, Himani; Dixit, Aparna; Garg, Lalit C

    2014-10-01

    Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens. PMID:24996028

  17. Relative disease susceptibility and clostridial toxin antibody responses in three commercial broiler lines co-infected with Clostridium perfringens and Eimeria maxima using an experimental model of necrotic enteritis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Necrotic enteritis is an enteric disease of poultry resulting from infection by Clostridium perfringens with co-infection by Eimeria spp. constituting a major risk factor for disease pathogenesis. This study compared three commercial broiler chicken lines using an experimental model of necrotic ente...

  18. Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans and poultry. The organism is the third leading cause of human food-borne bacterial disease a...

  19. Differential outgrowth potential of Clostridium perfringens food-borne isolates with various cpe-genotypes in vacuum-packed ground beef during storage at 12C.

    PubMed

    Xiao, Yinghua; Wagendorp, Arjen; Abee, Tjakko; Wells-Bennik, Marjon H J

    2015-02-01

    In the current study, the outgrowth of spores of 15 different food isolates of Clostridium perfringens was evaluated in vacuum-packed ground beef during storage at 12C and 25C. This included enterotoxic strains carrying the gene encoding the CPE enterotoxin on the chromosome (C-cpe), on a plasmid (P-cpe) and cpe-negative strains. The 15 strains were selected from a larger group of strains that were first evaluated for their ability to sporulate in modified Duncan-Strong sporulating medium. Sporulation ability varied greatly between strains but was not associated with a particular cpe genotype. In line with previous studies, the tested C-cpe strains produced spores with significantly higher heat resistance than the cpe-negative and P-cpe strains (both IS1151 and IS1470-like) with the exception of strain VWA009. Following inoculation of vacuum-packed cooked ground beef with spores, the heat-resistant C-cpe strains showed lower outgrowth potential in this model food stored at 12C than the P-cpe and cpe-negative strains, while no significant differences were observed at 25C. These results suggest that the latter strains may have a competitive advantage over C-cpe strains at reduced temperatures during storage of foods that support the growth of C. perfringens. While spores of P-cpe strains are readily inactivated by heat processing, post-processing contamination by food handlers who may carry P-cpe strains that have a better growth potential at lower temperatures must be avoided. The varying responses of C. perfringens spores to heat and the differences in outgrowth capacity at different temperatures are factors to be considered in strain selection for challenge tests, and for predictive modelling of C. perfringens. PMID:25461607

  20. Effects of heat stress on the formation of splenic germinal centres and immunoglobulins in broilers infected by Clostridium perfringens type A.

    PubMed

    Calefi, Atílio Sersun; de Siqueira, Adriana; Namazu, Lilian Bernadete; Costola-de-Souza, Carolina; Honda, Bruno Bueno Takashi; Ferreira, Antonio José Piantino; Quinteiro-Filho, Wanderley Moreno; da Silva Fonseca, Juliana Garcia; Palermo-Neto, João

    2016-03-01

    Avian necrotic enteritis (NE) induced by Clostridium perfringens is a disease that affects mainly the first weeks of poultry's life. The pathogenesis of NE is complex and involves the combination of several factors, such as co-infection with different species of coccidia, immunosuppression and stress. Stress is one of the main limiting factors in poultry production. Although several studies emphasized the effects of stress on immunity, few works analyzed these effects on immunoglobulins and on germinal centres (GCs), which are specialized microenvironments, responsible for generating immune cells with high affinity antibodies and memory B-lymphocytes. Thus, the effects of heat stress associated or not with thioglycolate broth culture medium intake and/or C. perfringens infection on corticosterone serum levels, spleen GCs development and immunoglobulin production in broilers were evaluated. Results showed that heat stress, thioglycolate and C. perfringens per se increased corticosterone serum levels, although this was not observed in heat stressed and thioglycolate and C. perfringens-treated chickens. The serum levels of IgA, IgM and IgY were differently affected by heat stress and/or infection/thioglycolate. Heat stress decreased the duodenal concentrations of sIgA, which was accompanied by a reduction in GCs number in the duodenal lamina propria; a trend to similar findings of sIgA concentrations was observed in the chickens' jejunum. Changes in spleen and Bursa of Fabricius relative weights as well as in spleen morphometry were also noted in heat stressed animals, infected or not. Together, these data suggest that heat stress change GCs formation in chickens infected or not, which that may lead to failures in vaccination protocols as well as in the poultries' host resistance to infectious diseases during periods of exposure to heat stress. PMID:26964716

  1. Molecular Characterization of Clostridium perfringens Isolates from Humans with Sporadic Diarrhea: Evidence for Transcriptional Regulation of the Beta2-Toxin-Encoding Gene

    PubMed Central

    Harrison, Ben; Raju, Deepa; Garmory, Helen S.; Brett, Moira M.; Titball, Richard W.; Sarker, Mahfuzur R.

    2005-01-01

    Clostridium perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused by C. perfringens plasmid cpe isolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14 cpe+ C. perfringens fecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified as cpe+ type A, and 12 of the 14 cpe+ isolates carry their cpe gene on the plasmid and 2 carry it on the chromosome. Interestingly, cpb2 is present in only 12 plasmid cpe isolates; 11 isolates carry cpe and cpb2 on different plasmids, but cpe and cpb2 are located on the same plasmid in one isolate. C. perfringens enterotoxin is produced by all 14 cpe+ SD isolates. However, only 10 of the 12 cpe+/cpb2+ SD isolates produced CPB2, with significant variation in amounts. The levels of cpb2 mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypical cpb2 was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression of cpb2 mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94 cpb2, which overexpressed cpb2 mRNA, restored CPB2 production in F4969 (a cpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin in C. perfringens-associated SD. PMID:16332823

  2. A Live Oral Recombinant Salmonella enterica Serovar Typhimurium Vaccine Expressing Clostridium perfringens Antigens Confers Protection against Necrotic Enteritis in Broiler Chickens▿

    PubMed Central

    Kulkarni, R. R.; Parreira, V. R.; Jiang, Y.-F.; Prescott, J. F.

    2010-01-01

    Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for Salmonella and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated Salmonella enterica serovar Typhimurium χ9352 vaccine vehicle. The expression of these Clostridium perfringens proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with Salmonella expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against Clostridium and Salmonella antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a Salmonella vaccine vehicle. PMID:20007363

  3. Effect of Clostridium perfringens infection and antibiotic administration on microbiota in the small intestine of broiler chickens.

    PubMed

    Fasina, Yewande O; Newman, Molli M; Stough, Joshua M; Liles, Mark R

    2016-02-01

    The etiological agent of necrotic enteritis (NE) is Clostridium perfringens (CP), which is an economically significant problem for broiler chicken producers worldwide. Traditional use of in-feed antibiotic growth promoters to control NE disease have resulted in the emergence of antibiotic resistance in CP strains. Identification of probiotic bacteria strains as an alternative to antibiotics for the control of intestinal CP colonization is crucial. Two experiments were conducted to determine changes in intestinal bacterial assemblages in response to CP infection and in-feed bacitracin methylene disalicylate (BMD) in broiler chickens. In each experiment conducted in battery-cage or floor-pen housing, chicks were randomly assigned to the following treatment groups: 1) BMD-supplemented diet with no CP challenge (CM), 2) BMD-free control diet with no CP challenge (CX), 3) BMD-supplemented diet with CP challenge (PCM), or 4) BMD-free control diet with CP challenge (PCX). The establishment of CP infection was confirmed, with the treatment groups exposed to CP having a 1.5- to 2-fold higher CP levels (P < 0.05) compared to the non-exposed groups. Next-generation sequencing of PCR amplified 16S rRNA genes, was used to perform intestinal bacterial diversity analyses pre-challenge, and at 1, 7, and 21 d post-challenge. The results indicated that the intestinal bacterial assemblage was dominated by members of the phylum Firmicutes in all treatments before and after CP challenge, especially the Lactobacillaceae and Clostridiales families. In addition, we observed post-challenge emergence of members of the Enterobacteriaceae and Streptococcaceae in the non-medicated PCX treatment, and emergence of the Enterococcaceae in the medicated PCM treatment. This study highlights the bacterial interactions that could be important in suppressing or eliminating CP infection within the chicken intestine. Future studies should explore the potential to use commensal strains of unknown Clostridiales, Lactobacillaceae, Enterobacteriaceae, Streptococcaceae, and Enterococcaceae in effective probiotic formulations for the control of CP and NE disease. PMID:26567176

  4. Effects of Clostridium perfringens enterotoxin via claudin-4 on normal human pancreatic duct epithelial cells and cancer cells.

    PubMed

    Yamaguchi, Hiroshi; Kojima, Takashi; Ito, Tatsuya; Kyuno, Daisuke; Kimura, Yasutoshi; Imamura, Masafumi; Hirata, Koichi; Sawada, Norimasa

    2011-09-01

    The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer. PMID:21573709

  5. Effects of Dietary Additives and Early Feeding on Performance, Gut Development and Immune Status of Broiler Chickens Challenged with Clostridium perfringens

    PubMed Central

    Ao, Z.; Kocher, A.; Choct, M.

    2012-01-01

    The effects of dietary additives and holding time on resistance and resilience of broiler chickens to Clostridium perfringens challenge were investigated by offering four dietary treatments. These were a negative control (basal), a positive control (Zn-bacitracin) and two dietary additives, mannanoligosaccharides (MOS), and acidifier. Two holding times included (a) immediate access to feed and water post hatch (FED) and (b) access to both feed and water 48 h post hatch (HELD). Chicks fed Zn-bacitracin had no intestinal lesions attributed to necrotic enteritis (NE), whereas chicks fed both MOS or acidifier showed signs of NE related lesions. All dietary treatments were effective in reducing the numbers of C. perfringens in the ileum post challenge. The FED chicks had heavier body weight and numerically lower mortality. The FED chicks also showed stronger immune responses to NE challenge, showing enhanced (p<0.05) proliferation of T-cells. Early feeding of the MOS supplemented diet increased (p<0.05) IL-6 production. The relative bursa weight of the FED chicks was heavier at d 21 (p<0.05). All the additives increased the relative spleen weight of the HELD chicks at d 14 (p<0.05). The FED chicks had increased villus height and reduced crypt depth, and hence an increased villus/crypt ratio, especially in the jejunum at d 14 (p<0.05). The same was true for the HELD chicks given dietary additives (p<0.05). It may be concluded that the chicks with early access to dietary additives showed enhanced immune response and gut development, under C. perfringens challenge. The findings of this study shed light on managerial and nutritional strategies that could be used to prevent NE in the broiler industry without the use of in-feed antibiotics. PMID:25049595

  6. Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica

    USGS Publications Warehouse

    Lisle, J.T.; Smith, J.J.; Edwards, D.D.; McFeters, G.A.

    2004-01-01

    McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.

  7. Hydrogen production at high Faradaic efficiency by a bio-electrode based on TiO2 adsorption of a new [FeFe]-hydrogenase from Clostridium perfringens.

    PubMed

    Morra, Simone; Valetti, Francesca; Sarasso, Veronica; Castrignan, Silvia; Sadeghi, Sheila J; Gilardi, Gianfranco

    2015-12-01

    The [FeFe]-hydrogenase CpHydA from Clostridium perfringens was immobilized by adsorption on anatase TiO2 electrodes for clean hydrogen production. The immobilized enzyme proved to perform direct electron transfer to and from the electrode surface and catalyses both H2 oxidation (H2 uptake) and H2 production (H2 evolution) with a current density for H2 evolution of about 2 mA cm(-1). The TiO2/CpHydA bioelectrode remained active for several days upon storage and when a reducing potential was set, H2 evolution occurred with a mean Faradaic efficiency of 98%. The high turnover frequency of H2 production and the tight coupling of electron transfer, resulting in a Faradaic efficiency close to 100%, support the exploitation of the novel TiO2/CpHydA stationary electrode as a powerful device for H2 production. PMID:26278509

  8. Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity.

    PubMed Central

    Hanna, P C; Wnek, A P; McClane, B A

    1989-01-01

    Clostridium perfringens type A enterotoxin (CPE) causes the symptoms associated with C. perfringens food poisoning. To determine whether the C-terminal half of CPE contains receptor-binding activity, the 3' half of the cpe structural gene was cloned with an Escherichia coli expression vector system. E. coli lysates containing the expressed C-terminal CPE fragment (CPEfrag) were then assayed for CPE-like serologic, receptor-binding, and cytotoxic activities. CPEfrag was shown to contain an epitope located at or near the receptor-binding domain of the CPE molecule. Competitive-binding studies showed specific competition for CPE receptors between CPE and CPEfrag lysates. CPEfrag lysates did not cause cytotoxicity in Vero (African green monkey kidney) cells. However, preincubation with CPEfrag lysates specifically protected Vero cells from subsequent CPE challenge. This indicates that CPEfrag recognizes the physiologic receptor which mediates CPE cytotoxicity. Collectively, these studies indicate that the C-terminal half of CPE contains a receptor-binding domain but additional amino acid sequences appear to be required for CPE cytotoxicity. Images PMID:2556374

  9. Relative disease susceptibility and clostridial toxin antibody responses in three commercial broiler lines coinfected with Clostridium perfringens and Eimeria maxima using an experimental model of necrotic enteritis.

    PubMed

    Jang, Seung I; Lillehoj, Hyun S; Lee, Sung-Hyen; Lee, Kyung Woo; Lillehoj, Erik P; Hong, Yeong Ho; An, Dong-Jun; Jeoung, D Hye-Young; Chun, Ji-Eun

    2013-09-01

    Necrotic enteritis is an enteric disease of poultry resulting from infection by Clostridium perfringens with coinfection by Eimeria spp. constituting a major risk factor for disease pathogenesis. This study compared three commercial broiler chicken lines using an experimental model of necrotic enteritis. Day-old male Cobb, Ross, and Hubbard broilers were orally infected with viable C. perfringens and E. maxima and fed a high-protein diet to promote the development of experimental disease. Body weight loss, intestinal lesions, and serum antibody levels against alpha-toxin and necrotic enteritis B-like (NetB) toxin were measured as parameters of disease susceptibility and host immune response. Cobb chickens exhibited increased body weight loss compared with Ross and Hubbard breeds and greater gut lesion severity compared with Ross chickens. NetB antibody levels were greater in Cobb chickens compared with the Ross or Hubbard groups. These results suggest that Cobb chickens may be more susceptible to necrotic enteritis in the field compared with the Ross and Hubbard lines. PMID:24283139

  10. [Studies of necrotizing enteritis of suckling piglets (Clostridium perfringens type C enterotoxemia) in industrialized sow breeding units. 5. Control of the disease].

    PubMed

    Khler, B; Zabke, J; Reiher, K; Rummler, H J

    1979-01-01

    Recent methods used and experience obtained in the control of necrotising enteritis are reported in this paper, with reference being made to both the pathogenesis and epizootiology of the disease. Two inoculations of the sows, using "Enterotoxmievakzine Dessau bivalent" five and three weeks before parturition, have worked well for prophylaxis. Oral treatment was applied to nursed piglets, using 40,000 I.U. of "Aviapen" and "V-Tablopen" penicillin per animal and day over periods between two and four days, helped to minimise piglet loss, particularly in the period between a fresh outbreak and full effectiveness of immunoprophylactic action. Such treatment was conducted metaphylactically and therapeutically. The first metaphylactic treatment was given within 24 hours from parturition. Combination of mother animal vaccination with the above therapeutic use of those two penicillin preparations worked extremely well in enzootically contaminated stocks and proved to be the most effective approach, for the time being, to controlling necrotising enteritis of nursed piglets. Yet, all those control measures failed to bring about full stock sanitation on industrialised units. Sow trading was not permitted until at least four weeks had elapsed from full effectiveness of mother animal vaccination, with the view to reducing the proliferation of Clostridium perfringens Type C via sales of breeding animals. All sows were given two "Enterotoxmievakzine Dessau bivalent" vaccinations, prior to sale. The animals were sold only to smaller farms (less than 500 sows for breeding) with concentional keeping patterns which were kept under constant diagnostic supervision. Neomycin, oxytetracycline, chloramphenicol, and other antibiotics against which Clostridium perfringens was resistant or in a position to assume resistance were used on endangered stocks only in conjunction with penicillin or not at all. This programme of control has proved to be efficient through a period of more than three years. PMID:232841

  11. Recombinant Attenuated Salmonella enterica Serovar Typhimurium Expressing the Carboxy-Terminal Domain of Alpha Toxin from Clostridium perfringens Induces Protective Responses against Necrotic Enteritis in Chickens▿

    PubMed Central

    Zekarias, Bereket; Mo, Hua; Curtiss, Roy

    2008-01-01

    Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3′ end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain χ8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 109 CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE. PMID:18337376

  12. Impact of Clean-Label Antimicrobials and Nitrite Derived from Natural Sources on the Outgrowth of Clostridium perfringens during Cooling of Deli-Style Turkey Breast.

    PubMed

    King, Amanda M; Glass, Kathleen A; Milkowski, Andrew L; Sindelar, Jeffrey J

    2015-05-01

    Organic acids and sodium nitrite have long been shown to provide antimicrobial activity during chilling of cured meat products. However, neither purified organic acids nor NaNO2 is permitted in products labeled natural and both are generally avoided in clean-label formulations; efficacy of their replacement is not well understood. Natural and clean-label antimicrobial alternatives were evaluated in both uncured and in alternative cured (a process that uses natural sources of nitrite) deli-style turkey breast to determine inhibition of Clostridium perfringens outgrowth during 15 h of chilling. Ten treatments of ground turkey breast (76% moisture, 1.2% salt) included a control and four antimicrobials: 1.0% tropical fruit extract, 0.7% dried vinegar, 1.0% cultured sugar-vinegar blend, and 2.0% lemon-vinegar blend. Each treatment was formulated without (uncured) and with nitrite (PCN; 50 ppm of NaNO2 from cultured celery juice powder). Treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in an additional 10 h. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar. Uncured control and PCN-only treatments allowed for 4.6- and 4.2-log increases at 15 h, respectively, and although all antimicrobial treatments allowed less outgrowth than uncured and PCN, the degree of inhibition varied. The 1.0% fruit extract and 1.0% cultured sugar-vinegar blend were effective at controlling populations at or below initial levels, whether or not PCN was included. Without PCN, 0.7% dried vinegar and 2.0% lemon-vinegar blend allowed for 2.0- and 2.5-log increases, respectively, and ∼1.5-log increases with PCN. Results suggest using clean-label antimicrobials can provide for safe cooling following the study parameters, and greater inhibition of C. perfringens may exist when antimicrobials are used with nitrite. PMID:25951389

  13. Direct dynamic kinetic analysis and computer simulation of growth of Clostridium perfringens in cooked turkey during cooling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research applied a new one-step methodology to directly construct a tertiary model for describing the growth of C. perfringens in cooked turkey meat under dynamically cooling conditions. The kinetic parameters of the growth models were determined by numerical analysis and optimization using mu...

  14. NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens

    PubMed Central

    Therit, Blair; Cheung, Jackie K.; Rood, Julian I.; Melville, Stephen B.

    2015-01-01

    Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell. PMID:26197388

  15. A Novel Pore-Forming Toxin in Type A Clostridium perfringens Is Associated with Both Fatal Canine Hemorrhagic Gastroenteritis and Fatal Foal Necrotizing Enterocolitis

    PubMed Central

    Nowell, Victoria J.; Nicholson, Vivian M.; Oliphant, Kaitlyn; Prescott, John F.

    2015-01-01

    A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute hemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals. PMID:25853427

  16. Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1.

    PubMed

    Crost, E H; Ajandouz, E H; Villard, C; Geraert, P A; Puigserver, A; Fons, M

    2011-09-01

    When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized. PMID:21586310

  17. Comparison of the alpha-toxin genes of Clostridium perfringens type A and C strains: evidence for extragenic regulation of transcription.

    PubMed Central

    Katayama, S; Matsushita, O; Minami, J; Mizobuchi, S; Okabe, A

    1993-01-01

    The Clostridium perfringens plc gene encoding phospholipase C (alpha-toxin) was cloned from type C NCIB 10662, a strain which produces low levels of phospholipase C activity. The nucleotide sequence of a cloned 3.1-kb HindIII fragment was determined. The same fragment was also cloned from type A NCTC 8237, a phospholipase C-overproducing strain. In this case, an open reading frame (ORF2) truncated in the previously cloned 2-kb fragment was also sequenced. Comparison of the nucleotide sequence between the 3.1-kb fragments of the two type strains shows some differences both in the plc gene and in ORF2. However, when the 3.1-kb fragment was cloned into plasmid pUC19 and expressed in Escherichia coli, the plc genes from both type strains were similarly expressed and the toxins produced showed similar levels of activity. Northern blot analysis revealed that the type A strain produced 16 to 23 times more plc mRNA than the type C strain. These results indicate that in C. perfringens the two plc genes are transcribed at different rates, probably because of a difference in a locus lying outside of the cloned fragments. Gel retardation analysis showed that the type A strain possessed two different proteins that bound different regions of the plc gene. However, one of these proteins, which binds within the plc coding region, was not found in the type C strain, suggesting that it plays a role in the regulation of the plc gene expression. Images PMID:8423073

  18. The effect of Artemisia annua on broiler performance, on intestinal microbiota and on the course of a Clostridium perfringens infection applying a necrotic enteritis disease model.

    PubMed

    Engberg, Ricarda Margarete; Grevsen, Kai; Ivarsen, Elise; Fretté, Xavier; Christensen, Lars Porskjær; Højberg, Ole; Jensen, Bent Borg; Canibe, Nuria

    2012-01-01

    The aerial parts of the plant Artemisia annua contain essential oils having antimicrobial properties against Clostridium perfringens Type A, the causal agent for necrotic enteritis in broilers. In two experiments, the influence of increasing dietary concentrations of dried A. annua leaves (0, 5, 10 and 20 g/kg) and n-hexane extract from fresh A. annua leaves (0, 125, 250 and 500 mg/kg) on broiler performance was investigated. Dried plant material decreased feed intake and body weight in a dose-dependent manner, and 10 and 20 g/kg diet tended to improve the feed conversion ratio. The n-hexane extract also reduced feed intake, but broiler weight tended to decrease only at the highest dietary concentration. The feed conversion ratio tended to improve when birds received 250 and 500 mg/kg n-hexane extract. In a third experiment, a necrotic enteritis disease model was applied to investigate the effect of the dietary addition of dried A. annua leaves (10 g/kg on top) or n-hexane extract of A. annua (250 mg/kg) on the severity of the disease in broilers. The addition of n-hexane extract reduced the intestinal C. perfringens numbers and the severity of the disease-related small intestinal lesions. Over the infection period from day 17 to day 27, birds supplemented with the n-hexane extract gained more weight than both the challenged control birds and birds receiving dried plant material. The results indicate that n-hexane extracts derived from A. annua can modulate the course of necrotic enteritis and compensate to a certain extent for the disease-associated weight losses. PMID:22834551

  19. Complete Sequencing and Diversity Analysis of the Enterotoxin-Encoding Plasmids in Clostridium perfringens Type A Non-Food-Borne Human Gastrointestinal Disease Isolates

    PubMed Central

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

    2006-01-01

    Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an ?35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the ?43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively. PMID:16452442

  20. Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

    PubMed

    Kawase, Jun; Kurosaki, Morito; Kawakami, Yuta; Kashimoto, Takashi; Tsunomori, Yoshie; Sato, Koji; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Etoh, Yoshiki; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2014-01-01

    In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR. PMID:25410559

  1. Release of Glycoprotein (GP1) from the Tegumental Surface of Taenia solium by Phospholipase C from Clostridium perfringens Suggests a Novel Protein-Anchor to Membranes

    PubMed Central

    Landa, Abraham; Willms, Kaethe; Laclette, Juan Pedro

    2010-01-01

    In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC). Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43?kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, ?methyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD), suggesting a novel anchor to the membrane for the glycoprotein GP1. PMID:20130782

  2. Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway*

    PubMed Central

    Plissier, Marie-Ccile; Sebban-Kreuzer, Corinne; Guerlesquin, Franoise; Brannigan, James A.; Bourne, Yves; Vincent, Florence

    2014-01-01

    Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (?/?)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys66 as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. 1H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys66 for the epimerization reaction with participation of neighboring Arg43, Asp126, and Glu180 residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases. PMID:25320079

  3. Ileal Loop Fluid Accumulation and Production of Diarrhea in Rabbits by Cell-free Products of Clostridium perfringens1

    PubMed Central

    Duncan, Charles L.; Strong, Dorothy H.

    1969-01-01

    The ability of cell extracts and culture filtrates of various strains of C. perfringens to produce ileal loop fluid accumulation and overt diarrhea in rabbits was tested. Good correlation was obtained in the ability of whole cells and a toxic factor (present in cell extracts and concentrated culture filtrates) to produce both fluid accumulation in ileal loops and diarrhea when injected into the normal ileum of the rabbit. The toxic factor was present in cell-free preparations when cells were grown in a sporulation medium, but not when they were grown in an asporogenic medium. The factor was shown to be heat labile, nondialyzable, and was inactivated by Pronase but not by trypsin, lipase, or amylase. Loss of activity occurred at pH 1.0, 3.0, 5.0, and 12.0. PMID:4310083

  4. Recombinant Expression of Two Bacteriophage Proteins That Lyse Clostridium perfringens and Share Identical Sequences in the C-Terminal Cell Wall Binding Domain of the Molecules but Are Dissimilar in Their N-Terminal Active Domains

    PubMed Central

    Simmons, Mustafa; Donovan, David M.; Siragusa, Gregory R.; Seal, Bruce S.

    2014-01-01

    Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans, and poultry. The organism is the third leading cause of human foodborne bacterial disease, and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address this issue, new antimicrobial agents, putative lysins from the genomes of bacteriophages, are identified. Two putative phage lysin genes (ply) from the clostridial phages phiCP39O and phiCP26F were cloned and expressed in Escherichia coli, and the resultant proteins were purified to near homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequences of the two recombinant proteins were homologous to N-acetylmuramoyl-L-alanine amidases. The proteins were identical in the C-terminal putative cell-wall binding domain, but only 55% identical to each other in the presumptive N-terminal catalytic domain. Both recombinant lysins were capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays. The observed reduction in turbidity was correlated with up to a 3 log cfu/mL reduction in viable C. perfringens on brain–heart infusion agar plates. However, other member species of the clostridia were resistant to the lytic activity by both assays. PMID:20825156

  5. Selection of Bacillus spp. for Cellulase and Xylanase Production as Direct-Fed Microbials to Reduce Digesta Viscosity and Clostridium perfringens Proliferation Using an in vitro Digestive Model in Different Poultry Diets

    PubMed Central

    Latorre, Juan D.; Hernandez-Velasco, Xochitl; Kuttappan, Vivek A.; Wolfenden, Ross E.; Vicente, Jose L.; Wolfenden, Amanda D.; Bielke, Lisa R.; Prado-Rebolledo, Omar F.; Morales, Eduardo; Hargis, Billy M.; Tellez, Guillermo

    2015-01-01

    Previously, our laboratory has screened and identified Bacillus spp. isolates as direct-fed microbials (DFM). The purpose of the present study was to evaluate the cellulase and xylanase production of these isolates and select the most appropriate Bacillus spp. candidates for DFM. Furthermore, an in vitro digestive model, simulating different compartments of the gastrointestinal tract, was used to determine the effect of these selected candidates on digesta viscosity and Clostridium perfringens proliferation in different poultry diets. Production of cellulase and xylanase were based on their relative enzyme activity. Analysis of 16S rRNA sequence classified two strains as Bacillus amyloliquefaciens and one of the strains as Bacillus subtilis. The DFM was included at a concentration of 108 spores/g of feed in five different sterile soybean-based diets containing corn, wheat, rye, barley, or oat. After digestion time, supernatants from different diets were collected to measure viscosity, and C. perfringens proliferation. Additionally, from each in vitro simulated compartment, samples were taken to enumerate viable Bacillus spores using a plate count method after heat-treatment. Significant (P < 0.05) DFM-associated reductions in supernatant viscosity and C. perfringens proliferation were observed for all non-corn diets. These results suggest that antinutritional factors, such as non-starch polysaccharides from different cereals, can enhance viscosity and C. perfringens growth. Remarkably, dietary inclusion of the DFM that produce cellulase and xylanase reduced both viscosity and C. perfringens proliferation compared with control diets. Regardless of diet composition, 90% of the DFM spores germinated during the first 30 min in the crop compartment of the digestion model, followed by a noteworthy increased in the intestine compartment by ~2log10, suggesting a full-life cycle development. Further studies to evaluate in vivo necrotic enteritis effects are in progress. PMID:26664954

  6. Genetic relatedness and netB prevalence among environmental Clostridium perfringens strains associated with a broiler flock affected by mild necrotic enteritis.

    PubMed

    Engstrm, Bjrn E; Johansson, Anders; Aspan, Anna; Kaldhusdal, Magne

    2012-09-14

    In a previous study we investigated pulsed-field gel electrophoresis (PFGE) genotype diversity and prevalence of the netB toxin gene in Clostridium perfringens (CP) isolates recovered from a broiler flock (flock 1) affected by necrotic enteritis (NE). In this follow-up work, we examined samples collected before placement of flock 1, to see if NE during rearing could be traced back to the cleaned and empty building or the day-old chicks. Litter from the next flock in the same building (flock 2) was also examined. We detected 25 different PFGE genotypes, five of which were found only in litter from flock 2. Six genotypes which had been found in flock 1 during rearing were detected in samples collected before placement. NetB positive isolates belonging to two of these genotypes had been recovered from NE lesions during rearing, suggesting that virulent strains were transmitted from the cleaned and disinfected broiler house. NetB frequency among isolates from the empty building was 45%, indicating that netB positive strains were prevalent in a building that previously had housed a healthy flock offered in-feed narasin (flock 0). NetB frequency among isolates from litter used by flock 2 was 22%, indicating that netB positive strains were present in the environment of a 14-days-old healthy flock offered in-feed narasin. Two prevalent genotypes were consistently either netB negative or netB positive. However, the presence of genotypes represented by both negative and positive isolates may suggest that the gene can spread horizontally among different CP strains. PMID:22516191

  7. Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis.

    PubMed

    Park, Soon S; Lillehoj, Hyun S; Allen, Patricia C; Park, Dong Woon; FitzCoy, Steve; Bautista, Daniel A; Lillehoje, Erik P

    2008-03-01

    The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-alpha, IFN-gamma, IL-1beta, IL-2, IL-12, IL-13, IL-17, and TGF-beta4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens. PMID:18459290

  8. Structural Requirement in Clostridium perfringens Collagenase mRNA 5′ Leader Sequence for Translational Induction through Small RNA-mRNA Base Pairing

    PubMed Central

    Nomura, Nobuhiko; Nakamura, Kouji

    2013-01-01

    The Gram-positive anaerobic bacterium Clostridium perfringens is pathogenic to humans and animals, and the production of its toxins is strictly regulated during the exponential phase. We recently found that the 5′ leader sequence of the colA transcript encoding collagenase, which is a major toxin of this organism, is processed and stabilized in the presence of the small RNA VR-RNA. The primary colA 5′-untranslated region (5′UTR) forms a long stem-loop structure containing an internal bulge and masks its own ribosomal binding site. Here we found that VR-RNA directly regulates colA expression through base pairing with colA mRNA in vivo. However, when the internal bulge structure was closed by point mutations in colA mRNA, translation ceased despite the presence of VR-RNA. In addition, a mutation disrupting the colA stem-loop structure induced mRNA processing and ColA-FLAG translational activation in the absence of VR-RNA, indicating that the stem-loop and internal bulge structure of the colA 5′ leader sequence is important for regulation by VR-RNA. On the other hand, processing was required for maximal ColA expression but was not essential for VR-RNA-dependent colA regulation. Finally, colA processing and translational activation were induced at a high temperature without VR-RNA. These results suggest that inhibition of the colA 5′ leader structure through base pairing is the primary role of VR-RNA in colA regulation and that the colA 5′ leader structure is a possible thermosensor. PMID:23585542

  9. Transport of MS2 phage, Escherichia coli, Clostridium perfringens, Cryptosporidium parvum, and Giardia intestinalis in a gravel and a sandy soil.

    PubMed

    Hijnen, Wim A M; Brouwer-Hanzens, Anke J; Charles, Katrina J; Medema, Gertjan J

    2005-10-15

    To define protection zones around groundwater abstraction wells and safe setback distances for artificial recharge systems in watertreatment, quantitative information is needed about the removal of microorganisms during soil passage. Column experiments were conducted using natural soil and water from an infiltration site with fine sandy soil and a river bank infiltration site with gravel soil. The removal of phages, bacteria, bacterial spores, and protozoan (oo)-cysts was determined at two velocities and compared with field data from the same sites. The microbial elimination rate (MER) in both soils was generally >2 log, but MER in the gravel soil was higher than that in the fine sandy soil. This was attributed to enhanced attachment, related to higher metal-hydroxides content. From the high sticking efficiencies (>1) and the low influence of flow rate on MER it was deduced that straining played a significant role in the removal of Escherichia coli and Cryptosporidium parvum oocysts in the gravel soil. Lower removal of oocysts than the 4-5 times smaller E. coli and spores in the fine sand indicates that the contribution of straining is variable and needs further attention in transport models. Thus, simple extrapolation of grain size and particle size to the extent of microbial transport underground is inappropriate. Finally, the low MER of indigenous E. coli and Clostridium perfringens observed in the soil columns as well as under field conditions and the second breakthrough peak found for Cryptosporidium and spores in the fine sandy soil upon a change in the feedwater pH indicate a significant role of detachment and retardation to microbial transport and the difficulty of extrapolation of quantitative column test results to field conditions. PMID:16295848

  10. Discrimination Efficacy of Fecal Pollution Detection in Different Aquatic Habitats of a High-Altitude Tropical Country, Using Presumptive Coliforms, Escherichia coli, and Clostridium perfringens Spores

    PubMed Central

    Byamukama, Denis; Mach, Robert L.; Kansiime, Frank; Manafi, Mohamad; Farnleitner, Andreas H.

    2005-01-01

    The performance of rapid and practicable techniques that presumptively identify total coliforms (TC), fecal coliforms (FC), Escherichia coli, and Clostridium perfringens spores (CP) by testing them on a pollution gradient in differing aquatic habitats in a high-altitude tropical country was evaluated during a 12-month period. Site selection was based on high and low anthropogenic influence criteria of paired sites including six spring, six stream, and four lakeshore sites spread over central and eastern parts of Uganda. Unlike the chemophysical water quality, which was water source type dependent (i.e., spring, lake, or stream), fecal indicators were associated with the anthropogenic influence status of the respective sites. A total of 79% of the total variability, including all the determined four bacteriological and five chemophysical parameters, could be assigned to either a pollution, a habitat, or a metabolic activity component by principal-component analysis. Bacteriological indicators revealed significant correlations to the pollution component, reflecting that anthropogenic contamination gradients were followed. Discrimination sensitivity analysis revealed high ability of E. coli to differentiate between high and low levels of anthropogenic influence. CP also showed a reasonable level of discrimination, although FC and TC were found to have worse discrimination efficacy. Nonpoint influence by soil erosion could not be detected during the study period by correlation analysis, although a theoretical contamination potential existed, as investigated soils in the immediate surroundings often contained relevant concentrations of fecal indicators. The outcome of this study indicates that rapid techniques for presumptive E. coli and CP determination may be reliable for fecal pollution monitoring in high-altitude tropical developing countries such as those of Eastern Africa. PMID:15640171

  11. Discrimination efficacy of fecal pollution detection in different aquatic habitats of a high-altitude tropical country, using presumptive coliforms, Escherichia coli, and Clostridium perfringens spores.

    PubMed

    Byamukama, Denis; Mach, Robert L; Kansiime, Frank; Manafi, Mohamad; Farnleitner, Andreas H

    2005-01-01

    The performance of rapid and practicable techniques that presumptively identify total coliforms (TC), fecal coliforms (FC), Escherichia coli, and Clostridium perfringens spores (CP) by testing them on a pollution gradient in differing aquatic habitats in a high-altitude tropical country was evaluated during a 12-month period. Site selection was based on high and low anthropogenic influence criteria of paired sites including six spring, six stream, and four lakeshore sites spread over central and eastern parts of Uganda. Unlike the chemophysical water quality, which was water source type dependent (i.e., spring, lake, or stream), fecal indicators were associated with the anthropogenic influence status of the respective sites. A total of 79% of the total variability, including all the determined four bacteriological and five chemophysical parameters, could be assigned to either a pollution, a habitat, or a metabolic activity component by principal-component analysis. Bacteriological indicators revealed significant correlations to the pollution component, reflecting that anthropogenic contamination gradients were followed. Discrimination sensitivity analysis revealed high ability of E. coli to differentiate between high and low levels of anthropogenic influence. CP also showed a reasonable level of discrimination, although FC and TC were found to have worse discrimination efficacy. Nonpoint influence by soil erosion could not be detected during the study period by correlation analysis, although a theoretical contamination potential existed, as investigated soils in the immediate surroundings often contained relevant concentrations of fecal indicators. The outcome of this study indicates that rapid techniques for presumptive E. coli and CP determination may be reliable for fecal pollution monitoring in high-altitude tropical developing countries such as those of Eastern Africa. PMID:15640171

  12. Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

    PubMed

    Cocco, Emiliano; Shapiro, Erik M; Gasparrini, Sara; Lopez, Salvatore; Schwab, Carlton L; Bellone, Stefania; Bortolomai, Ileana; Sumi, Natalia J; Bonazzoli, Elena; Nicoletti, Roberta; Deng, Yang; Saltzman, W Mark; Zeiss, Caroline J; Centritto, Floriana; Black, Jonathan D; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Rutherford, Thomas J; Schwartz, Peter E; Pecorelli, Sergio; Santin, Alessandro D

    2015-12-01

    Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-nave and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment. PMID:26060989

  13. Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer

    PubMed Central

    2014-01-01

    Background Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. Methods In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Results Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Conclusions Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer. PMID:24990559

  14. In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin

    PubMed Central

    Uppalapati, Siva Ramakrishna; Kingston, Joseph Jeyabalaji; Qureshi, Insaf Ahmed; Murali, Harishchandra Sripathy; Batra, Harsh Vardhan

    2013-01-01

    Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture. PMID:24349173

  15. Gene expression profiling within the spleen of Clostridium perfringens-challenged Broilers fed antibiotic-medicated and non-medicated diets

    PubMed Central

    Sarson, Aimie J; Wang, Ying; Kang, Zhumei; Dowd, Scot E; Lu, Yang; Yu, Hai; Han, Yanming; Zhou, Huaijun; Gong, Joshua

    2009-01-01

    Background Clostridium perfringens (Cp) is a Gram-positive anaerobic bacterium that causes necrotic enteritis (NE) in poultry when it overgrows in the small intestine. NE disease has previously been controlled through the use of growth-promoting antibiotics. This practice was recently banned in European countries, leading to significantly increased incidence of NE threatening the poultry industry. Control strategies and technology as substitutes to dietary antibiotics are therefore urgently required. To develop the substitutes, it is important to understand host immune responses to Cp infection. However, the knowledge is still lacking. We therefore investigated gene expression profiles within immunologically-relevant tissue, the spleen, in order to identify factors that are involved in immunity to NE and have potential as therapeutic targets. Results Use of a 44 K Agilent chicken genome microarray revealed significant up-regulation of many immune-associated genes in Cp-challenged chickens, including galectin 3, IFNAR1, IgY-receptor, TCRγ, granzyme A, and mannose-6-P-R, which were subsequently validated by quantitative PCR assays. Functional annotation of differentially expressed genes was conducted using the High Throughput Gene Ontology Functional Annotation database. Medicated and Non-medicated chickens had similar annotation profiles with cell activities and regulation being the most dominant biological processes following Cp infection. Conclusion Broiler chickens demonstrated an intricate and holistic magnitude of host response to Cp challenge and the development of NE. Although the influence of dietary antibiotics appeared to be less significant than the disease process, both had a considerable impact on the host response. Markers previously identified in intestinal inflammatory diseases of other species, including humans, and indicators of enhanced antibody responses, appeared to be involved in the chicken response to Cp challenge. The significance in host immune responses of immune mediators identified from the present study warrants further studies to verify their functions during NE development and to determine their potential application to control NE disease. PMID:19500416

  16. EFFECT OF SPICES AND ORGANIC ACIDS ON THE GROWTH OF CLOSTRIDIUM PERFRINGENS FROM SPORE INOCULA DURING COOLING OF SOUS-VIDE COOKED GROUND BEEF PRODUCTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat treatments given to minimally processed food products are not sufficient to kill C. perfringens spores when present. Thus, the heat resistant spores may survive, germinate, outgrow and multiply into high numbers of vegetative cells if the rate and extent of cooling is inadequate. There is a ...

  17. A Possible Route for Foodborne Transmission of Clostridium difficile?

    PubMed Central

    Peck, Michael W.

    2015-01-01

    Abstract Spores of toxigenic Clostridium difficile and spores of food-poisoning strains of Clostridium perfringens show a similar prevalence in meats. Spores of both species are heat resistant and can survive cooking of foods. C. perfringens is a major cause of foodborne illness; studies are needed to determine whether C. difficile transmission by a similar route is a cause of infection. PMID:25599421

  18. A Randomized Controlled Trial Assessing Infectious Disease Risks from Bathing in Fresh Recreational Waters in Relation to the Concentration of Escherichia coli, Intestinal Enterococci, Clostridium perfringens, and Somatic Coliphages

    PubMed Central

    Wiedenmann, Albrecht; Krger, Petra; Dietz, Klaus; Lpez-Pila, Juan M.; Szewzyk, Regine; Botzenhart, Konrad

    2006-01-01

    We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a better scientific basis for the definition of recreational water quality standards. A total of 2,196 participants were recruited from the local population and randomized into bathers and non-bathers. Bathers were exposed for 10 min and had to immerse their head at least three times. Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentrationresponse effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95% CI, 1.22.6) to 4.6 (95% CI, 2.110.1), depending on the definition of gastroenteritis. The effect of swallowing water provided additional evidence for true doseresponse relationships. Based on the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli, 25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL. Recreational water quality standards are intended to protect the health of those consumers who are not already immune or resistant to pathogens that may be associated with indicator organisms. In contrast to current World Health Organization recommendations, we concluded that standards should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of the cohorts. Although in theory there is no threshold in real concentrationresponse relationships, we demonstrated that a NOAEL approach would be a more robust and practical solution to the complex problem of setting standards. PMID:16451859

  19. epsilon. prime /. epsilon. : Review and recent progress

    SciTech Connect

    Franzini, P.J.

    1991-04-19

    The evolution of the theoretical perspective on {epsilon}{prime}/{epsilon} is reviewed. The introduction of the Z{sup O} penguin and the effects of high M{sub t} are discussed, in particular the possibility for {epsilon}{prime}/{epsilon} to be identically zero. Recent calculations of {epsilon}{prime}/{epsilon} based on current estimates and bounds on the input parameters are presented. 41 refs., 13 figs.

  20. Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Turner, Rebecca; Price, A.; Henden, A.

    2009-05-01

    The IYA 2009 working group on Research Experiences for Students, Teachers, and Citizen-Scientists is planning a multi-year project involving the bright star Eps Aur. The project will go beyond simple observing and also include a major data analysis component. The goal is to introduce the participant to the full scientific process from background research to paper writing for a peer-reviewed journal. It begins with a 10 Star Training Program of several types of binary and transient variable stars that are easy to observe from suburban locations with the naked eye. Participants will be trained both in observing and also in basic data analysis of photometric datasets (light curve and period analysis). Eventually it will lead to a capstone project: monitoring the rare and mysterious 2009-2011 eclipse of Epsilon Aurigae. In the summer of IYA 2009, third-magnitude Eps Aur will experience its next eclipse, which occurs every 27.1 years and lasts 714 days, nearly two years. The star is bright enough to be seen with the naked eye from most urban areas. If fully funded, the project will also involve two public workshops on observing and data analysis in the summers of 2009 and 2010, respectively.

  1. In vitro inhibition of growth of Escherichia coli, Salmonella Typhimurium, and Clostridia perfringens using Probiotics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella Typhimurium, Escherichia coli and Clostridium perfringens are pathogenic organisms found in horses [1] and they cause disease in animals or humans [2]. Due to concern over pathogens such as these, there is increasing interest in antimicrobial alternatives to prevent or reduce the prevalen...

  2. Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences.

    PubMed

    Kuhnert, P; Capaul, S E; Nicolet, J; Frey, J

    1996-10-01

    The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical. PMID:8863454

  3. epsilon-Hexachlorocyclohexane (epsilon-HC)

    Integrated Risk Information System (IRIS)

    epsilon - Hexachlorocyclohexane ( epsilon - HC ) ; CASRN 6108 - 10 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard

  4. Monitoring of anti-C. perfringens bacteriophage CpV1 persistence in gastrointestinal tracts of broilers.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A factor limiting promotion of poultry products to the world market is any contamination of birds with pathogens, including Clostridium perfringens. The latter is often accountable for significant economical losses in production of commercial birds because of a possibility of the development of necr...

  5. How to measure epsilon'/epsilon with lattice QCD

    SciTech Connect

    Sharpe, S.R.

    1987-04-01

    A pedagogical discussion is given of a lattice calculation of epsilon'. The method is outlined, and preliminary results are presented. They suggest that epsilon'/epsilon may be reduced from previous estimates by 60-70%.

  6. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... prescribed in this test. Such solution shall be made by dissolving 1 gram of peptone and 0.25 gram of sodium chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 250 °F. for 25...

  7. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... cause sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice... until injections of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall...

  8. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard... of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for...

  9. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 250 °F. for 25 minutes...) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until...

  10. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 0.25 gram of sodium chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving... Toxin. (v) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour, and hold in ice...

  11. Synthesis and evaluation of a non-radioactive gene probe for the detection of C.perfringens alpha toxin.

    PubMed

    Schlapp, T; Blaha, I; Bauerfeind, R; Wieler, L H; Schoepe, H; Weiss, R; Baljer, G

    1995-04-01

    The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C. septicum and C.histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc. PMID:7603469

  12. Latest experimental information on {epsilon}{prime}/{epsilon}

    SciTech Connect

    Yee B. Hsiung

    2000-08-17

    The authors review the latest experimental results in search for direct CP-violation by measuring the CP-violating parameters Re({epsilon}{prime}/{epsilon}) in neutral kaon decays. The recent result from Fermilab-KTeV Re({epsilon}{prime}/{epsilon}) = (28.0 {+-} 4.1) x 10{sup {minus}4}, and the new preliminary result from CERN-NA48 Re({epsilon}{prime}/{epsilon}) = (14.0 {+-} 4.3) x 10{sup {minus}4}, are presented. Both experiments, though using very different techniques, have now performed very well by collecting millions of events for all four relevant decay modes of K{sub L,S} to {pi}{sup +}{pi}{sup {minus}} and {pi}{sup 0}{pi}{sup 0} simultaneously. The current world average on this important measurement is Re({epsilon}{prime}/{epsilon}) = 19.3 {+-} 10{sup {minus}4} with a {chi}{sup 2}/ndf = 11.1/5, establishing the existence of direct CP-violation. The experimental status of such crucial measurements and the future prospects are also discussed here.

  13. epsilon Expansion for a Defect Space

    NASA Astrophysics Data System (ADS)

    Ideura, K.

    1984-03-01

    The renormalizability of the system in the presence of a defect plane is investigated. The O(epsilon(2) ) calculation of the exponent mu_{||} is performed in epsilon expansion. The consistency between epsilon expansion and 1/n expansion is examined.

  14. Antibacterial activity against Clostridium genus and antiradical activity of the essential oils from different origin.

    PubMed

    Ka?niov, Miroslava; Vukovi?, Nenad; Horsk, Elena; Salamon, Ivan; Bobkov, Alica; Hleba, Luk; Fiskelov, Martina; Vat?k, Alexander; Petrov, Jana; Bobko, Marek

    2014-01-01

    In the present study, the antimicrobial and antiradical activities of 15 essential oils were investigated. The antimicrobial activities were determined by using agar disc diffusion and broth microdilution methods against Clostridium genus and antioxidant properties of essential oils by testing their scavenging effect on DPPH radicals activities. We determined the antibacterial activity of Clostridium butyricum, Clostridium hystoliticum, Clostridium intestinale, Clostridium perfringens and Clostridium ramosum. We obtained the original commercial essential oils samples of Lavandula angustifolia, Carum carvi, Pinus montana, Mentha piperita, Foeniculum vulgare Mill., Pinus sylvestris, Satureia montana, Origanum vulgare L. (2 samples), Pimpinella anisum, Rosmarinus officinalis L., Salvia officinalis L., Abies alba Mill., Chamomilla recutita L. Rausch and Thymus vulgaris L. produced in Slovakia (Calendula a.s., Nova Lubovna, Slovakia). The results of the disk diffusion method showed very high essential oils activity against all tested strains of microorganisms. The best antimicrobial activity against C. butyricum was found at Pimpinella anisum, against C. hystoliticum was found at Pinus sylvestris, against C. intestinale was found at Satureia hortensis L., against C. perfringens was found at Origanum vulgare L. and against C. ramosum was found at Pinus sylvestris. The results of broth microdilution assay showed that none of the essential oils was active against C. hystoliticum. The best antimicrobial activity against C. butyricum was found at Abies alba Mill., against C. intestinale was found at Abies alba Mill., against C. perfringens was found at Satureia montana and against C. ramosum was found at Abius alba and Carum carvi. Antioxidant DPPH radical scavenging activity was determined at several solutions of oil samples (50 ?L.mL(-1)-0.39 ?L.mL(-1)) and the best scavenging effect for the highest concentration (50 ?L.mL(-1)) was observed. The antioxidant properties were different in particular plant species. The highest% of inhibition after 30 min. of reaction was observed at Origanum vulgare (93%), Satureia montana (90.66%) and Lavandula augustifolia (90.22%). PMID:24813985

  15. Apparition of Clostridium sp. and Bacteroides in the intestine of the newborn delivered by cesarian section.

    PubMed

    Bezirtzoglou, E; Romond, C

    1990-01-01

    Anaerobic flora plays a key role in preventing intestinal colonization with potential pathogens. Nowadays, the mechanisms involved in the colonization resistance provided by the anaerobic microflora are to be clarified. Numerous factors seem to intervene in the regulation of the intestinal flora. The purpose of the present study was to correlate the presence or relative absence of Clostridium sp. and Bacteroides with the colonization by C. perfringens, which is involved in lethal infections in an immunologically compromised host. The intestinal bacterial colonization of 20 newborns delivered by cesarian section was assessed sequentially over the first 14 days of life. C. perifringens is a strongly reducing microorganism and undoubtedly causes a decrease in the oxidoreduction potential of the newborn feces favouring the subsequent colonization by other putrefactive bacteria. C. perfringens seems to be the precursor for installation of putrefractive bacteria, as Bacteroides and other Clostridium sp. PMID:2076608

  16. Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

    PubMed Central

    Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

    2014-01-01

    Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

  17. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain.

    PubMed

    Popoff, M R; Rubin, E J; Gill, D M; Boquet, P

    1988-09-01

    By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin. PMID:3137166

  18. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain.

    PubMed Central

    Popoff, M R; Rubin, E J; Gill, D M; Boquet, P

    1988-01-01

    By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin. Images PMID:3137166

  19. Myonecrosis by Clostridium septicum in a dog, diagnosed by a new multiplex-PCR.

    PubMed

    Ribeiro, Márcio Garcia; Silva, Rodrigo Otávio Silveira; Pires, Prhiscylla Sadanã; Martinho, Anna Paula Vitirito; Lucas, Thays Mizuki; Teixeira, Ana Izabel Passarela; Paes, Antonio Carlos; Barros, Claudenice Batista; Lobato, Francisco Carlos Faria

    2012-10-01

    Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog. PMID:22975141

  20. PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Uzal, F A; Hugenholtz, P; Blackall, L L; Petray, S; Moss, S; Assis, R A; Fernandez Miyakawa, M; Carloni, G

    2003-02-01

    The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues. PMID:12458172

  1. Necrotic Enteritis in Chickens Associated with Clostridium sordellii.

    PubMed

    Rimoldi, Guillermo; Uzal, Francisco; Chin, R P; Palombo, Enzo A; Awad, Milena; Lyras, Dena; Shivaprasad, H L

    2015-09-01

    Three outbreaks of necrotic enteritis-like disease associated with Clostridium sordelii were diagnosed in commercial broiler chicken flocks with 18,000 to 31,000 birds between 18 and 26 days old. Clinical signs in the affected flocks included high mortality up to 2% a day, depression, and diarrhea. The main gross changes included segmental dilation of the small intestine with watery contents, gas, mucoid exudate, and roughened and uneven mucosa, occasionally covered with a pseudomembrane. Microscopic lesions in the small intestine were characterized by extensive areas of coagulative necrosis of the villi, fibrinous exudate in the lumen, and high numbers of large, Gram-positive rods, occasionally containing subterminal spores, seen in the necrotic tissue and lumen. These rods were identified as C. sordellii by immunohistochemistry. Clostridium sordellii was isolated in an almost pure culture from the intestine of affected birds. A retrospective study of commercial broiler chicken and turkey submissions to the California Animal Health and Food Safety Laboratory System revealed that C. sordellii had been isolated from intestinal lesions in outbreaks of necrotic enteritis-like disease in 8 of 39 cases, 5 times together with Clostridium perfringens and 3 times alone. The latter three cases are reported here. PMID:26478166

  2. Motility in the epsilon-proteobacteria.

    PubMed

    Beeby, Morgan

    2015-12-01

    The epsilon-proteobacteria are a widespread group of flagellated bacteria frequently associated with either animal digestive tracts or hydrothermal vents, with well-studied examples in the human pathogens of Helicobacter and Campylobacter genera. Flagellated motility is important to both pathogens and hydrothermal vent members, and a number of curious differences between the epsilon-proteobacterial and enteric bacterial motility paradigms make them worthy of further study. The epsilon-proteobacteria have evolved to swim at high speed and through viscous media that immobilize enterics, a phenotype that may be accounted for by the molecular architecture of the unusually large epsilon-proteobacterial flagellar motor. This review summarizes what is known about epsilon-proteobacterial motility and focuses on a number of recent discoveries that rationalize the differences with enteric flagellar motility. PMID:26590774

  3. Clostridium botulinum type E occurs and grows in the alga Cladophora glomerata

    USGS Publications Warehouse

    Byappanahalli, M.N.; Whitman, R.L.

    2009-01-01

    In recent years, massive avian die-offs from Clostridium botulinum type E infection have occurred in the Sleeping Bear Dunes National Lakeshore (SLBE) area of Lake Michigan. These outbreaks have been coincidental with massive blooms of the green algae Cladophora, mostly Cladophora glomerata. We tested the hypothesis that Clostridium botulinum type E can grow under suitable conditions in these algal mats. In a lab mesocosm study, Cladophora from four outbreak-impacted beaches from SLBE were compared with four unimpacted beaches in the Milwaukee–Racine area for bontE gene of Clostridium botulinum. Frequency of the bontE gene was higher after incubation (25 °C for up to 6 weeks) of Cladophora from impacted vs. the unimpacted area. Since no type E gene was detected initially in Cladophora from any of the eight locations, we infer that the increased occurrence of type E gene arose from spore germination or vegetative Clostridium growth within the existing algal mats of SLBE. Moreover, we found that the congener Clostridium perfringens readily grows in mesocosms containing Cladophora.

  4. Purification and characterization of the lethal toxin (alpha-toxin) of Clostridium septicum.

    PubMed

    Ballard, J; Bryant, A; Stevens, D; Tweten, R K

    1992-03-01

    Clostridium septicum lethal (alpha-toxin) was purified and found to be a basic protein (pI 8.4) of approximately 48 kDa that is both lethal and hemolytic. The alpha-toxin had a hemolytic activity of approximately 2 x 10(7) hemolytic units per mg and a 50% lethal dose of approximately 10 micrograms/kg of body weight for mice. The alpha-toxin formed concentration-dependent, sodium dodecyl sulfate-resistant aggregates of approximately 230 kDa. Mice immunized with alpha-toxin showed a significant increase in survival time over mock-immunized mice when challenged with C. septicum. Rabbit polyclonal antibody was generated against the purified toxin and was used to confirm that toxin with the same molecular weight was present in seven different C. septicum isolates. No proteins in the supernatants from cultures of Clostridium perfringens, Clostridium histolyticum, Clostridium chauvoei, or Clostridium difficile were found to react with the C. septicum alpha-toxin-specific antibody. PMID:1541552

  5. Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

    PubMed

    Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R

    2010-08-01

    Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues. PMID:20399850

  6. Purification and characterization of the lethal toxin (alpha-toxin) of Clostridium septicum.

    PubMed Central

    Ballard, J; Bryant, A; Stevens, D; Tweten, R K

    1992-01-01

    Clostridium septicum lethal (alpha-toxin) was purified and found to be a basic protein (pI 8.4) of approximately 48 kDa that is both lethal and hemolytic. The alpha-toxin had a hemolytic activity of approximately 2 x 10(7) hemolytic units per mg and a 50% lethal dose of approximately 10 micrograms/kg of body weight for mice. The alpha-toxin formed concentration-dependent, sodium dodecyl sulfate-resistant aggregates of approximately 230 kDa. Mice immunized with alpha-toxin showed a significant increase in survival time over mock-immunized mice when challenged with C. septicum. Rabbit polyclonal antibody was generated against the purified toxin and was used to confirm that toxin with the same molecular weight was present in seven different C. septicum isolates. No proteins in the supernatants from cultures of Clostridium perfringens, Clostridium histolyticum, Clostridium chauvoei, or Clostridium difficile were found to react with the C. septicum alpha-toxin-specific antibody. Images PMID:1541552

  7. Gas discharge plasmas are effective in inactivating Bacillus and Clostridium spores.

    PubMed

    Tseng, Shawn; Abramzon, Nina; Jackson, James O; Lin, Wei-Jen

    2012-03-01

    Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance. PMID:22075631

  8. Detection of Clostridium septicum hemolysin gene by polymerase chain reaction.

    PubMed

    Takeuchi, S; Hashizume, N; Kinoshita, T; Kaidoh, T; Tamura, Y

    1997-09-01

    A polymerase chain reaction (PCR) was developed for the detection of the hemolysin (alpha toxin) gene of Clostridium septicum. The PCR primers were designed from the sequence of the hemolysin gene and synthesized. A DNA fragment of 270 bp was amplified from 10 strains of C. septicum, but was not from strains of C. chauvoei, C. perfringens, C. novyi, or C. haemolyticum. When the PCR product was digested with Sau3AI, two DNA fragments of the expected 148 bp and 122 bp were recognized. The lowest detectable threshold of PCR for the hemolysin gene was 3.8 x 10(3) cells/ml. The PCR technique may be useful for rapid detection or identification of C. septicum associated with malignant edema. PMID:9342717

  9. Detection and characterization of Clostridium species in soil of Zambia.

    PubMed

    Hang'ombe, B M; Isogai, E; Lungu, J; Mubita, C; Nambota, A; Kirisawa, R; Kimura, K; Isogai, H

    2000-10-01

    In the retrospective study of soil-borne diseases of cattle in Zambia, malignant edema and blackquarter were widespread. One hundred and sixty-five cases with malignant edema and 103 cases with blackquarter were reported between 1985 and 1997. It was found that specific soil-conditions associate the emergence of the soil-borne diseases. Soil samples from five areas in Zambia were examined for the presence of genus Clostridium. Direct immunofluorescent assay (IFA) examination showed that C. septicum, C. novyi and C. chauvoei were detected in the soil of specific areas in Zambia, respectively. Causal organisms such as C. perfringens were isolated from the soil samples. The information of area-specific distribution of Clositridium species may give an efficient program in protecting cattle and man. PMID:11038129

  10. Clostridium Difficile Infections

    MedlinePLUS

    Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Nausea Abdominal pain or tenderness You might get C. difficile disease if you have an illness that ...

  11. Collagenase Clostridium Histolyticum Injection

    MedlinePLUS

    ... treated hand painful and swollen glands in the elbow or underarm area For men receiving collagenase Clostridium histolyticum for Peyronie's disease: tenderness around the injected area (along and above ...

  12. The search for companions to Epsilon Eridani.

    PubMed

    Lawton, A T; Wright, P

    1990-12-01

    The authors review efforts to examine the star Epsilon Eridani and determine the possibility for the existence of an Earth-like planet. Early data indicated that there must be a habitable ecosphere about 82.5 million Km from the primary. Research into the existence of another planetary system determined that Epsilon Eridani was a binary star with an Oort cloud system, indicating the possibility of planet formation. A review of the evidence suggests that the presence of the small red Dwarf companion star precludes the existence of a planetary system surrounding Epsilon Eridani. It is suggested that observations continue to provide further data about the formation of binary systems. PMID:11540498

  13. Structure of a C. perfringens enterotoxin mutant in complex with a modified Claudin-2 extracellular loop 2.

    PubMed

    Yelland, Tamas S; Naylor, Claire E; Bagoban, Tannya; Savva, Christos G; Moss, David S; McClane, Bruce A; Blasig, Ingolf E; Popoff, M; Basak, Ajit K

    2014-09-01

    CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells. PMID:25020226

  14. Systematic effects of the quenched approximation on the strong penguin contribution to epsilon-prime / epsilon

    SciTech Connect

    Aubin, C.; Christ, N.H.; Dawson, C.; Laiho, J.W.; Noaki, J.; Li, S.; Soni, A.; /Brookhaven

    2006-03-01

    We discuss the implementation and properties of the quenched approximation in the calculation of the left-right, strong penguin contributions (i.e. Q{sub 6}) to {epsilon}{prime}/{epsilon}. The coefficient of the new chiral logarithm, discovered by Golterman and Pallante, which appears at leading order in quenched chiral perturbation theory is evaluated using both the method proposed by those authors and by an improved approach which is free of power divergent corrections. The result implies a large quenching artifact in the contribution of Q{sub 6} to {epsilon}{prime}/{epsilon}. This failure of the quenched approximation affects only the strong penguin operators and so does not affect the Q8 contribution to {epsilon}{prime}/{epsilon} nor ReA{sub 0}, ReAP{sub 2} and thus, the {Delta}I = 1/2 rule at tree level in chiral perturbation theory.

  15. Systematic effects of the quenched approximation on the strong penguin contribution to {epsilon}{sup '}/{epsilon}

    SciTech Connect

    Aubin, C.; Christ, N. H.; Li, S.; Dawson, C.; Noaki, J.; Laiho, J. W.; Soni, A.

    2006-08-01

    We discuss the implementation and properties of the quenched approximation in the calculation of the left-right, strong penguin contributions (i.e. Q{sub 6}) to {epsilon}{sup '}/{epsilon}. The coefficient of the new chiral logarithm, discovered by Golterman and Pallante, which appears at leading order in quenched chiral perturbation theory is evaluated using both the method proposed by those authors and by an improved approach which is free of power divergent corrections. The result implies a large quenching artifact in the contribution of Q{sub 6} to {epsilon}{sup '}/{epsilon}. This failure of the quenched approximation affects only the strong penguin operators and so does not affect the Q{sub 8} contribution to {epsilon}{sup '}/{epsilon} nor ReA{sub 0}, ReA{sub 2} and thus, the {delta}I=1/2 rule at tree level in chiral perturbation theory.

  16. Nosocomial diarrhea: evaluation and treatment of causes other than Clostridium difficile.

    PubMed

    Polage, Christopher R; Solnick, Jay V; Cohen, Stuart H

    2012-10-01

    Diarrhea is common among hospitalized patients but the causes are distinct from those of diarrhea in the community. We review existing data about the epidemiology of nosocomial diarrhea and summarize recent progress in understanding the mechanisms of diarrhea. Clinicians should recognize that most cases of nosocomial diarrhea have a noninfectious etiology, including medications, underlying illness, and enteral feeding. Apart from Clostridium difficile, the frequency of infectious causes such as norovirus and toxigenic strains of Clostridium perfringens, Klebsiella oxytoca, Staphylococcus aureus, and Bacteroides fragilis remains largely undefined and test availability is limited. Here we provide a practical approach to the evaluation and management of nosocomial diarrhea when tests for C. difficile are negative. PMID:22700831

  17. Nosocomial Diarrhea: Evaluation and Treatment of Causes Other Than Clostridium difficile

    PubMed Central

    Polage, Christopher R.; Solnick, Jay V.; Cohen, Stuart H.

    2012-01-01

    Diarrhea is common among hospitalized patients but the causes are distinct from those of diarrhea in the community. We review existing data about the epidemiology of nosocomial diarrhea and summarize recent progress in understanding the mechanisms of diarrhea. Clinicians should recognize that most cases of nosocomial diarrhea have a noninfectious etiology, including medications, underlying illness, and enteral feeding. Apart from Clostridium difficile, the frequency of infectious causes such as norovirus and toxigenic strains of Clostridium perfringens, Klebsiella oxytoca, Staphylococcus aureus, and Bacteroides fragilis remains largely undefined and test availability is limited. Here we provide a practical approach to the evaluation and management of nosocomial diarrhea when tests for C. difficile are negative. PMID:22700831

  18. Inhibition of lipases by epsilon-polylysine.

    PubMed

    Tsujita, Takahiro; Sumiyoshi, Maho; Takaku, Takeshi; Momsen, William E; Lowe, Mark E; Brockman, Howard L

    2003-12-01

    Oral administration of epsilon-polylysine to rats reduced the peak plasma triacylglycerol concentration. In vitro, epsilon-polylysine and polylysine strongly inhibited the hydrolysis, by either pancreatic lipase or carboxylester lipase, of trioleoylglycerol (TO) emulsified with phosphatidylcholine (PC) and taurocholate. The epsilon-polylysine concentration required for complete inhibition of pancreatic lipase, 10 microg/ml, is 1,000 times lower than that of BSA required for the same effect. Inhibition requires the presence of bile salt and, unlike inhibition of lipase by other proteins, is not reversed by supramicellar concentrations of bile salt. Inhibition increases with the degree of polylysine polymerization, is independent of lipase concentration, is independent of pH between 5.0 and 9.5, and is accompanied by an inhibition of lipase binding to TO-PC emulsion particles. However, epsilon-polylysine did not inhibit the hydrolysis by pancreatic lipase of TO emulsions prepared using anionic surfactants, TO hydrolysis catalyzed by lingual lipase, or the hydrolysis of a water-soluble substrate. In the presence of taurocholate, epsilon-polylysine becomes surface active and adsorbs to TO-PC monomolecular films. These results are consistent with epsilon-polylysine and taurocholate forming a surface-active complex that binds to emulsion particles, thereby retarding lipase adsorption and triacylglycerol hydrolysis both in vivo and in vitro. PMID:12951365

  19. Clostridium chauvoei in hens.

    PubMed

    Prukner-Radovcic, E; Milakovic-Novak, L; Ivesa-Petricevic, S; Grgic, N

    1995-03-01

    The bacterium Clostridium chauvoei causes disease in certain animals, most frequently in cattle and sheep. It occurs rarely in pigs, while equines and poultry appear to be resistant to infection. Two cases are presented in which C. chauvoei was isolated from disease of complex aetiology in hens. In Case I, 15-week-old light hybrid chickens were affected with chronic respiratory disease, coccidiosis, ascariasis and inflammation of the skin on the head, with necrosis of the comb. Growth was uneven and mortality reached 24%. Clostridium chauvoei was isolated from two of three combs examined. In Case II a flock of broiler breeders aged 11 weeks developed coccidiosis and, owing to disease or death, 60% were excluded from production. Clostridium chauvoei was isolated from all of 10 livers examined. These results demonstrate that C. chauvoei can infect chickens and that its possible role as a pathogen under certain circumstances should be further investigated. PMID:18645777

  20. Tadpole diagram,. delta. /ital I/=1/2 enhancement and EPSILON'/EPSILON

    SciTech Connect

    Zhang Bo; Zhu Zhong-yuan

    1989-04-01

    The contributions of tadpole diagrams including /ital t/ and /ital c/ quark intermediate states to the amplitude of /ital K//r arrow/2..pi.. and EPSILON'/EPSILON are analyzed. The result shows that it is quite possible that their contributions are the most important ones. They may give enough ..delta../ital I/=1/2 enhancement for a reasonable quark relative-momentum cutoff value which depends on the /ital K/ wave function. However, the calculated value of /vert bar/EPSILON'/EPSILON/vert bar/ is too large if we suppose all CP violation effects to come from the Kobayashi-Maskawa phase delta. Perhaps one approach to solve this difficulty is to assume that EPSILON comes mainly from superweak CP violation.

  1. Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens

    PubMed Central

    Smith, Louis Ds.

    1975-01-01

    A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 102 to 3.3 106 per g. The organisms most frequently isolated were Clostridium subterminale, C. sordellii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridum. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content. PMID:238468

  2. PREDICTIVE MODEL FOR GROWTH OF CLOSTRIDIUM PERFRINGENS DURING COOLING OF COOKED GROUND CHICKEN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traditional methodologies for development of microbial growth models under dynamic temperature conditions do not take adequate account for the organisms history. Such models were shown to be inadequate in predicting growth of the organisms under dynamic conditions commonly encountered in the food i...

  3. A probabilistic analysis of Clostridium perfringens growth during food service operations.

    PubMed

    Fazil, Aamir M; Ross, Tom; Paoli, Greg; Vanderlinde, Paul; Desmarchelier, Patricia; Lammerding, Anna M

    2002-03-01

    The purpose of this study was threefold: first, the study was designed to illustrate the use of data and information collected in food safety surveys in a quantitative risk assessment. In this case, the focus was on the food service industry; however, similar data from other parts of the food chain could be similarly incorporated. The second objective was to quantitatively describe and better understand the role that the food service industry plays in the safety of food. The third objective was to illustrate the additional decision-making information that is available when uncertainty and variability are incorporated into the modelling of systems. PMID:11934039

  4. Enteritis associated with Clostridium perfringens type A in 9-month-old calves

    PubMed Central

    Savic, Bozidar; Prodanovic, Radisa; Ivetic, Vojin; Radanovic, Oliver; Bojkovski, Jovan

    2012-01-01

    Four 9-month-old Simmental male calves were presented with a history of sudden death. The necropsy and microscopic findings allowed a diagnosis of enteritis and severe intraluminal hemorrhage with blood clots in the jejunum, suggestive of jejunal hemorrhage syndrome. PMID:22851779

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scrapple is an ethnic food produced/consumed almost exclusively in the Middle Atlantic states of the U.S. It is typically made from ground pork trimmings, seasonings, cornmeal, and flour. This mixture is cooked and then shaped into loaves that are cooled and subsequently stored refrigerated until sl...

  6. Predictive model for growth of Clostridium perfringens during cooling of cooked ground chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traditional methodologies for development of microbial growth models under dynamic temperature conditions do not take into account the organism’s prior history. Such models were shown to be inadequate in predicting growth of the organisms under dynamic conditions commonly encountered in the food ind...

  7. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... inactivated and is nontoxic. Each serial shall meet the applicable requirements in § 113.100 and shall be tested for purity, safety, and potency as prescribed in this section. Any serial found unsatisfactory by... product from each serial and each subserial shall be tested for viable bacteria and fungi as provided...

  8. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... inactivated and is nontoxic. Each serial shall meet the applicable requirements in § 113.100 and shall be tested for purity, safety, and potency as prescribed in this section. Any serial found unsatisfactory by... product from each serial and each subserial shall be tested for viable bacteria and fungi as provided...

  9. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... distilled water; adjusting the pH to 7.2; autoclaving at 250 °F for 25 minutes; and storing at 4 °C until... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  10. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... dissolving 1 gram of peptone and 0.25 grams of sodium chloride in each 100 ml of distilled water; adjusting... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  11. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... dissolving 1 gram of peptone and 0.25 grams of sodium chloride in each 100 ml of distilled water; adjusting... toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of...

  12. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... distilled water; adjusting the pH to 7.2; autoclaving at 250 °F for 25 minutes; and storing at 4 °C until... temperature for 1 hour and hold in ice water until injections of mice can be made. (vi) Five Swiss white...

  13. FERREDOXIN OF CLOSTRIDIUM THERMOSACCHAROLYTICUM

    PubMed Central

    Wilder, Martin; Valentine, R. C.; Akagi, J. M.

    1963-01-01

    Wilder, Martin (University of Kansas, Lawrence), R. C. Valentine, and J. M. Akagi. Ferredoxin of Clostridium thermosaccharolyticum. J. Bacteriol. 86:861865. 1963.An electron-transferring agent has been isolated from Clostridium thermosaccharolyticum. This factor was found to participate as an electron carrier in the phosphoroclastic reaction of pyruvate, with the subsequent formation of acetyl phosphate and molecular hydrogen. It can be employed interchangeably with the ferredoxin of C. pasteurianum in various reactions. Thermal-stability studies indicated that this factor from C. thermosaccharolyticum was comparatively more heat-resistant than the carrier obtained from C. pasteurianum. It was concluded that this carrier was ferredoxin or a ferredoxin-like substance. PMID:14066486

  14. A DIM CANDIDATE COMPANION TO {epsilon} CEPHEI

    SciTech Connect

    Mawet, D.; Mennesson, B.; Serabyn, E.; Stapelfeldt, K.; Absil, O.

    2011-09-01

    Using a vector vortex coronagraph behind the 1.5 m well-corrected subaperture (WCS) at Palomar, we detected a second object very close to {epsilon} Cephei, a {delta} Scuti F0 IV star. The candidate companion, {approx}50 times fainter than {epsilon} Cephei, if physically associated, is a late-type K or early M star, and lies at an angular separation of 330 mas, or 1.1 {lambda}/D for the WCS, making it the smallest angle detection ever realized with a coronagraph in terms of {lambda}/D units. The projected separation of the putative companion is {approx}8.6 AU, most likely on a highly eccentric orbit. The recently detected near-infrared excess is thus likely not due to hot dust. Moreover, we also show that the previously reported IRAS 60 {mu}m excess was due to source confusion on the galactic plane.

  15. Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis.

    PubMed

    Popescu, F; Wyder, M; Gurtner, C; Frey, J; Cooke, R A; Greenhill, A R; Posthaus, H

    2011-11-21

    Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans. PMID:21411248

  16. Threonine requirement of broiler chickens during subclinical intestinal Clostridium infection.

    PubMed

    Star, L; Rovers, M; Corrent, E; van der Klis, J D

    2012-03-01

    The aim of this study was to determine the threonine requirement of broilers during a subclinical Clostridium infection. Three experiments were performed: experiments 1 and 2 to investigate the dose-response of threonine supplementation during infection and experiment 3 to validate the threonine requirement during infection. In each experiment, 1-d-old Ross 308 male broilers were used. An infection model was used with inoculation of Eimeria maxima and Clostridium perfringens at d 9 and 14 of age, respectively. Control birds were inoculated with saline and liver broth at d 9 and 14 of age, respectively. From d 9 of age, infected birds were fed diets differing in the standardized digestible threonine-to-lysine ratio (realized ratios experiment 1: 0.55, 0.58, 0.63, 0.69, and 0.72; realized ratios experiment 2: 0.64, 0.65, 0.67, 0.69, and 0.72; and realized ratios experiment 3: 0.63 and 0.67). Uninfected birds were fed diets with a realized Thr:Lys ratio of 0.63 in experiments 1 and 2 and of 0.63 or 0.67 in experiment 3. The incidence of lesions, lesion severity, and mortality rate of infected birds was not affected by the Thr:Lys ratio. Experiments 1 and 2 showed that the decrease in BW gain and feed intake was less severe in infected birds fed a diet with a Thr:Lys ratio of 0.69 and 0.67, respectively (not significant). Validation of the Thr:Lys ratio in experiment 3 showed that the BW gain and feed intake were higher for infected birds with a Thr:Lys ratio of 0.67 compared with infected birds with a Thr:Lys ratio of 0.63. This resulted in an increased BW gain and feed intake of 129 and 148 g, respectively, with a higher Thr:Lys ratio over a production period of 37 d. This indicates that a higher Thr:Lys ratio in infected birds improved production performance during infection with C. perfringens, although intestinal damage (incidence and lesion severity) was not affected. PMID:22334739

  17. Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

    SciTech Connect

    Banerjee, A; Leang, C; Ueki, T; Nevin, KP; Lovley, DR

    2014-03-25

    The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

  18. The genome sequence of Clostridium tetani, the causative agent of tetanus disease

    PubMed Central

    Brggemann, Holger; Bumer, Sebastian; Fricke, Wolfgang Florian; Wiezer, Arnim; Liesegang, Heiko; Decker, Iwona; Herzberg, Christina; Martnez-Arias, Rosa; Merkl, Rainer; Henne, Anke; Gottschalk, Gerhard

    2003-01-01

    Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of ?1 ng/kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is Clostridium tetani, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic C. tetani E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulence-related factors could be identified, such as an array of surface-layer and adhesion proteins (35 ORFs), some of them unique to C. tetani. Comparative genomics with the genomes of Clostridium perfringens, the causative agent of gas gangrene, and Clostridium acetobutylicum, a nonpathogenic solvent producer, revealed a remarkable capacity of C. tetani: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of C. tetani are presented. PMID:12552129

  19. Diarylacylhydrazones: Clostridium-Selective Antibacterials with Activity Against Stationary-Phase Cells

    PubMed Central

    Casadei, Gabriele; Bremner, John B.; Lewis, Kim; Kelso, Michael J.

    2014-01-01

    Current antibiotics for treating Clostridium difficile infections (CDI), i.e. metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This paper reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and C. perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure-activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI. PMID:24360560

  20. Latest results on the direct CP violation measurements: {Epsilon}{prime}/{epsilon}

    SciTech Connect

    Hsiung, Yee B.; E731 Collaboration

    1992-03-01

    Preliminary results, based on the full data sample from Fermilab-E731 and the combined data sample from CERN-NA31, on the ``direct`` CP-violation measurements {epsilon}{prime}{epsilon} in neutral kaon decay have been reviewed. The E731`s results if Re({epsilon}{prime}{epsilon}) = (6.0 {plus_minus} 5.8(stat) {plus_minus} 3.2(syst) {plus_minus} 1.8(MC)) {times} 10{sup {minus}4}, which provides no evidence for ``direct`` CP-violation, thus supporting the Superweak model; while the NA31`s combined result (1986 + 1988 + 1989 data) is Re({epsilon}{prime}/{epsilon} = (23{plus_minus}3.4(stat) {plus_minus}6.5(syst)){times}10{sup 4}, three standard deviations from zero, which provides evidence for the ``direct`` CP-violation in the Standard Model. Comparisons of the two experiments are made. The Fermilab-E731 group has also fit for the other parameters of the neutral kaon system in their 2{pi} data sample, such as: the K{sub S} life time {tau}{sub S}; the K{sub L}-K{sub S} mass difference {delta}m; the phase difference between {eta}{sub +-}, {delta}{phi} = ({minus}0.6{plus_minus}1.6){degrees}). Which is a test of CPT invariance; the Superweak phase {phi}{sub SW} = (43.37{plus_minus}0.22){degrees}, and the phase of {eta}{sub {plus_minus}}, {phi}{sub {plus_minus}} = (43.2{plus_minus} 1.6){degrees}, which is predicted by CPT invariance to equal {phi}{sub SW}.

  1. Bifidobacterium, Bacteroides, and Clostridium spp. in fecal samples from breast-fed and bottle-fed infants with and without iron supplement.

    PubMed Central

    Mevissen-Verhage, E A; Marcelis, J H; de Vos, M N; Harmsen-van Amerongen, W C; Verhoef, J

    1987-01-01

    Bifidobacterium, Bacteroides, and Clostridium spp. isolated from the feces of 23 neonates during the first 3 months of life were identified. Of the 23 neonates, 10 were breast fed, 6 received an infant formula with iron supplement (5 mg/liter), and 7 received the formula without iron supplement (iron concentration, less than 0.5 mg/liter). The Bifidobacterium spp. most frequently isolated from the three groups of infants were B. longum, B. breve, B. adolescentis, and B. bifidum. The bacteroides spp. most frequently isolated were B. fragilis and B. vulgatus. The most common Clostridium sp. in the three groups of infants was C. perfringens. The type of milk did not select for species of Bifidobacterium, Bacteroides, or Clostridium, except for Clostridium butyricum, which was isolated significantly more often from bottle-fed infants with iron supplement than from the other groups, and Clostridium tertium, which was more often isolated from breast-fed infants. The species of the three anaerobic genera did not persist for a long period of time in the three groups of infants. PMID:3818925

  2. Clostridium difficile infection

    PubMed Central

    Vedantam, Gayatri; Clark, Andrew; Chu, Michele; McQuade, Rebecca; Mallozzi, Michael; Viswanathan, V. K.

    2012-01-01

    Clostridium difficile infection is the leading cause of antibiotic- and healthcare-associated diarrhea, and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics/outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few. Efforts to elucidate C. difficile pathogenic mechanisms have been hampered by a lack of molecular tools, manipulatable animal models, and genetic intractability of clinical C. difficile isolates. However, in the past 5 y, painstaking efforts have resulted in the unraveling of multiple C. difficile virulence-associated pathways and mechanisms. We have recently reviewed the disease, its associated risk factors, transmission and interventions (Viswanathan, Gut Microbes 2010). This article summarizes genetics, non-toxin virulence factors, and host-cell biology associated with C. difficile pathogenesis as of 2011, and highlights those findings/factors that may be of interest as future intervention targets. PMID:22555464

  3. Epsilon Aur monitoring during predicted pulsation phase

    NASA Astrophysics Data System (ADS)

    Waagen, Elizabeth O.; Templeton, Matthew R.

    2014-09-01

    Dr. Robert Stencel (University of Denver Astronomy Program) has requested that AAVSO observers monitor epsilon Aurigae from now through the end of the observing season. "Studies of the long-term, out-of-eclipse photometry of this enigmatic binary suggest that intervals of coherent pulsation occur at roughly 1/3 of the 27.1-year orbital period. Kloppenborg, et al. noted that stable variation patterns develop at 3,200-day intervals' implying that 'the next span of dates when such events might happen are circa JD ~2457000 (2014 December)'. "These out-of-eclipse light variations often have amplitudes of ~0.1 magnitude in U, and ~0.05 in V, with characteristic timescales of 60-100 days. The AAVSO light curve data to the present may indicate that this coherent phenomenon has begun, but we encourage renewed efforts by observers...to help deduce whether these events are internal to the F star, or externally-driven by tidal interaction with the companion star." Nightly observations or one observation every few days (CCD/PEP/DSLR, VUBR (amplitude too small for visual)) are requested. Finder charts with sequence may be created using the AAVSO Variable Star Plotter (http://www.aavso.org/vsp). Observations should be submitted to the AAVSO International Database. Epsilon Aur was the subject of major international campaigns and the AAVSO's Citizen Sky project as it went through its 27.1-year eclipse in 2009-2011. Over 700 observers worldwide submitted over 20,000 multicolor observations to the AAVSO International Database for this project. Much information on eps Aur is available from the AAVSO, including material on the Citizen Sky website (http://www.aavso.org/epsilon-aurigae and http://www.citizensky.org/content/star-our-project). The Journal of the AAVSO, Volume 40, No. 2 (2012) was devoted to discussion of and research results from this event. See full Alert Notice for more details and observations.

  4. Nonlinear models in 2 + epsilon dimensions

    SciTech Connect

    Friedan, D.H.

    1980-08-01

    The general nonlinear scalar model is studied at asymptotically low temperature near two dimensions. The low-temperature expansion is renormalized, and effective algorithms are derived for calculation to all orders in the renormalized expansion. The renormalization group coefficients are calculated in the two-loop approximation, and topological properties of the renormalization group equations are investigated. Special attention is paid to the infrared instabilities of the fixed points, since they provide the continuum limits of the model. The model consists of a scalar field phi on Euclidean 2 + epsilon space whose values phi(x) lie in a finite-dimensional differentiable manifold. 4 figures.

  5. Epsilon Aurigae at the End of Eclipse

    NASA Astrophysics Data System (ADS)

    Hoard, Donald; Stencel, R.; Howell, S.

    2011-05-01

    We request a small investment of 24 minutes of Spitzer time, to obtain four IRAC observations of epsilon Aurigae. A naked eye object located near Capella, epsilon Aurigae is the eclipsing binary star with the longest known orbital period, showing a single long duration (~2 yr) eclipse every 27.1 yr. For much of the last 150 years, the nature of the eclipsing object defied explanation. We recently demonstrated that epsilon Aurigae consists of a high luminosity F0 post-AGB star in orbit with a B5 V star surrounded by a solar system sized (~8 AU diameter) disk of cool, dust-dominated material. The eclipse of epsilon Aurigae is a rare event; moreover, it is a unique astrophysical opportunity, since the backlighting of the disk by the high luminosity eclipsed star reveals details that cannot be detected in similar dusty disks around single stars. The current eclipse started in August 2009 and is expected to reach its photometric conclusion in May 2011 (with the spectroscopic conclusion as late as December 2011). The goals for these observations include: (1) extend our ongoing IRAC monitoring campaign covering the current eclipse to late-phase and post-eclipse visits; (2) provide a consistent, well-calibrated space-based set of IR photometry for comparison with ongoing ground-based work; and (3) use the composite results to constrain the thermal profile of the disk. A key expectation of these particular observations is to reveal the irradiation-heated portion of the disk, which will be visible on its trailing side following eclipse. Observations of this side of the disk will be crucial to test and constrain new models of disk structure. As part of our overall monitoring campaign with Spitzer, Hubble, Herschel, and numerous ground-based facilities, these proposed observations will make an important contribution to the understanding of stellar evolution in binary stars, including mass transfer and evolution studies, along with new insights into astrophysical disks and post-AGB star evolution.

  6. Revealing the Hot Side of Epsilon Aurigae

    NASA Astrophysics Data System (ADS)

    Hoard, Donald; Stencel, Robert; Howell, Steve

    2012-12-01

    We request a small investment of 24 minutes of Spitzer time, to obtain four IRAC observations of epsilon Aurigae. A naked eye object located near Capella, epsilon Aurigae is the eclipsing binary star with the longest known orbital period, showing a single long duration (~2 yr) eclipse every 27.1 yr. For much of the last 200 years, the nature of the eclipsing object defied explanation. We recently demonstrated that epsilon Aurigae consists of a high luminosity F0 post-AGB star in orbit with a B5 V star surrounded by a solar system sized (~8 AU diameter) disk of cool, dust-dominated material. The eclipse of epsilon Aurigae is a rare event; moreover, it is a unique astrophysical opportunity, since the backlighting of the disk by the high luminosity eclipsed star reveals details that cannot be detected in similar dusty disks around single stars. The current eclipse started in August 2009 and ended in July 2011; we are now in the post-eclipse phase, when the irradiation-heated side of the disk will begin rotating into view. The goals for these observations include: (1) extend our ongoing IRAC monitoring campaign covering the current eclipse to post-eclipse visits; (2) provide a consistent, well-calibrated space-based set of IR photometry for comparison with ongoing ground-based work; and (3) use the composite results to constrain the thermal profile of the disk. A key expectation of these particular observations is to reveal the irradiation-heated portion of the disk, which will be visible on its trailing side following eclipse. Observations of this side of the disk will be crucial to test and constrain new models of disk structure. As part of our overall monitoring campaign with Spitzer, Hubble, Herschel, and numerous ground-based facilities, these proposed observations will make an important contribution to the understanding of stellar evolution in binary stars, including mass transfer and evolution studies, along with new insights into astrophysical disks and post-AGB star evolution.

  7. Classical closure theory and Lam's interpretation of epsilon-RNG

    NASA Technical Reports Server (NTRS)

    Zhou, YE

    1995-01-01

    Lam's phenomenological epsilon-renormalization group (RNG) model is quite different from the other members of that group. It does not make use of the correspondence principle and the epsilon-expansion procedure. We demonstrate that Lam's epsilon-RNG model is essentially the physical space version of the classical closure theory in spectral space and consider the corresponding treatment of the eddy viscosity and energy backscatter.

  8. The Final Measurement of Epsilon'/Epsilon from KTeV

    SciTech Connect

    Worcester, E.T.

    2009-09-01

    We present precise measurements of CP and CPT symmetry based on the full dataset of K {yields} {pi}{pi} decays collected by the KTeV experiment at FNAL. We measure the direct CP violation parameter Re({epsilon}{prime}/{epsilon}) = (19.2 {+-} 2.1) x 10{sup -4}. We find the KL-KS mass difference {Delta}m = (5265 {+-} 10) x 10{sup 6} hs{sup -1} and the K{sub S} lifetime {tau}{sub S} = (89.62 {+-} 0.05) x 10{sup -12} s. We test CPT symmetry by finding the phase of the indirect CP violation parameter {epsilon}, {phi}{sub {epsilon}} = (44.09 {+-} 1.00){sup o}, and the difference of the relative phases between the CP violating and CP conserving decay amplitudes for K {yields} {pi}{sup +}{pi}{sup -} ({phi}{sub {+-}}) and for K {yields} {pi}{sup 0}{pi}{sup 0} ({phi}{sub 00}), {Delta}{phi} = (0.29 {+-} 0.31){sup o}. These results are consistent with other experimental results and with CPT symmetry.

  9. The Final Measurement of Epsilon'/Epsilon from KTeV

    SciTech Connect

    Worcester, E.T.

    2009-10-01

    The authors present precise measurements of CP and CPT symmetry based on the full dataset of K {yields} {pi}{pi} decays collected by the KTeV experiment at Fermi National Accelerator Laboratory during 1996, 1997, and 1999. This dataset contains about 15 million K {yields} {pi}{sup 0}{pi}{sup 0} and 70 million K {yields} {pi}{sup +}{pi}{sup -} decays. They measure the direct CP violation parameter Re({epsilon}'/{epsilon}) = (19.2 {+-} 2.1) x 10{sup -4}. they find the K{sub L}-K{sub S} mass difference {Delta}m = (5265 {+-} 10) x 10{sup 6} {bar h}s{sup -1} and the K{sub S} lifetime {tau}{sub S} = (89.62 {+-} 0.05) x 10{sup -12} s. They test CPT symmetry by finding the phase of the indirect CP violation parameter {epsilon}, {phi}{sub {epsilon}} = (44.09 {+-} 1.00){sup o}, and the difference of the relative phases between the CP violating and CP conserving decay amplitudes for K {yields} {pi}{sup +}{pi}{sup -} ({phi}{sub +-}) and for K {yields} {pi}{sup 0}{pi}{sup 0} ({phi}{sub 00}), {Delta}{phi} = (0.29 {+-} 0.31){sup o}. these results are consistent with other experimental results and with CPT symmetry.

  10. CDP-diacylglycerol synthase activity in Clostridium perfingens

    SciTech Connect

    Carmen, G.M.; Zaniewski, R.L.; Cousminer, J.J.

    1982-01-01

    CTP: phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimium between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition on the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C. (Refs. 24).

  11. Epsilon Metal Summary Report FY 2011

    SciTech Connect

    Strachan, Denis M.; Crum, Jarrod V.; Zumhoff, Mac R.; Bovaird, Chase C.; Windisch, Charles F.; Riley, Brian J.

    2011-09-30

    The Epsilon-metal ({var_epsilon}-metal) phase was selected in FY 2009 as a potential waste form to for immobilizing the noble metals found in the undissolved solids + aqueous stream, and the soluble Tc from ion-exchange process, each resulting from proposed aqueous reprocessing. {var_epsilon}-metal phase is observed in used nuclear fuel and the natural reactors of Oklobono in Gabon, where the long-term corrosion behavior was demonstrated. This makes {var_epsilon}-metal a very attractive waste form. Last fiscal year, {var_epsilon}-metal was successfully fabricated by combining the five-metals, Mo, Ru, Rh, Pd and Re (surrogate for Tc), into pellets followed by consolidation with an arc melter. The arc melter produced fully dense samples with the epsilon structure. However, some chemistry differences were observed in the microstructure that resulted in regions rich in Re and Mo, and others rich in Pd, while Ru and Rh remained fairly constant throughout. This year, thermal stability (air), and corrosion testing of the samples fabricated by arc melting were the main focus for experimental work. Thermal stability was measured with a differential scanning calorimeter - thermogravimetric analyzer, by both ramp heating as well as step heating. There is clear evidence during the ramp heating experiment of an exothermic event + a weight loss peak both beginning at {approx}700 C. Step heating showed an oxidation event at {approx}690 C with minimal weight gain that occurs just before the weight loss event at 700 C. The conclusion being that the e-metal begins to oxidize and then become volatile. These findings are useful for considering the effects of voloxidation process. Three different pellets were subjected to electrochemical testing to study the corrosion behavior of the epsilon-metal phase in various conditions, namely acidic, basic, saline, and inert. Test was done according to an interim procedure developed for the alloy metal waste form. First an open circuit potential was measured, followed by linear polarization sweeps. The linear polarization sweep range was the Tafel equation was fit to the linear polarization sweep data to determine the corrosion rate of each pellet in each test solution. The average calculated corrosion rates of the three pellets according to solution conditions were: -1.91 x 10{sup -4} mm/yr (0.001 M NaOH), -1.48 x 10{sup -3} mm/yr (0.01 M NaCl), -8.77 x 10{sup -4} mm/yr (0.001 M H{sub 2}SO{sub 4}), -2.09 x 10{sup -3} mm/yr (0.001 M NaOH + 0.01 M NaCl), and -1.54 x 10{sup -3} mm/yr (0.001 M H{sub 2}SO{sub 4} + 0.01 M NaCl). Three single-pass flow through (SPFT) test were conducted at a flow rate of 10 ml/day, at 90 C, and pH of 2.5, 7.0, and 9.0 for up to 322 days. Results of the tests indicate that dissolution rates were 5 x 10{sup -4} g m{sup 2} d{sup -1} at pH 9.0, 1.2 x 10{sup -4} g m{sup -2} d{sup -1} at pH 7.0, and 2 x 10{sup -4} g m{sup -2} d{sup -1} at pH 2.5. The sample used for the pH 7.0 SPFT test contains extra Re compared to samples used for the other two SPFT test, which came from a single pellet. The corrosion data measured this year indicate that the {var_epsilon}-metal phase is chemically durable. The two chemically different phases, but structurally the same, behave differently during dissolution according to the microstructure changes observed in both the electrochemical and in SPFT test. Characterization of the test specimens after testing suggests that the dissolution is complex and involves oxidative dissolution followed by precipitation of both oxide and metallic phases. These data suggest that the dissolution in the electrochemical and SPFT tests is different; a process that needs further investigation.

  12. Serotyping of Clostridium difficile.

    PubMed Central

    Toma, S; Lesiak, G; Magus, M; Lo, H L; Delme, M

    1988-01-01

    A total of 246 live Clostridium difficile cultures were serotyped by a slide agglutination technique. Fifteen grouping antisera were produced which serotyped 98% of the cultures (241 of 246). Our results indicated that certain serogroups may have specific pathogenicity. Strains of serogroups A, G, H, K, S1, and S4 were cytotoxigenic and were isolated mainly from adult patients with pseudomembranous colitis or antibiotic-associated diarrhea. Nontoxigenic strains of serogroups D and Cd-5 were isolated mainly from asymptomatic neonates and small children. Some cross-reactions occurred among some strains of serogroups A, Cd-5, G, and K. These strains were further examined by analysis of protein profiles and restriction endonuclease patterns to elucidate their serology. Typing of C. difficile by using slide agglutination is a simple technique suitable for routine examination. Serogrouping may be a useful epidemiological marker and could help in elucidating the medical relevance of some C. difficile isolates. PMID:2833528

  13. Biosynthesis of daunorubicin glycosides: role of epsilon-rhodomycinone.

    PubMed Central

    McGuire, J C; Thomas, M C; Stroshane, R M; Hamilton, B K; White, R J

    1980-01-01

    Daunorubicin (daunomycin; NSC 82151) is a fermentation-derived anthracycline antibiotic that is clinically useful in the treatment of human leukemias. Daunorubicin itself is found rarely in microbial fermentations, but is present normally in the form of glycoside derivatives that yield the free drug on simple acid hydrolysis. A major by-product of daunorubicin fermentations is usually the structurally related anthracyclinone epsilon-rhodomycinone. We have used mutants of a daunorubicin-producing Streptomyces species to study the biosynthetic relationship between epsilon-rhodomycinone and daunorubicin. We found that exogenously added epsilon-rhodomycinone can be converted to daunorubicin glycosides by a nonproducing mutant and by a mutant that produces daunorubicin glycosides but not epsilon-rhoeomycinone. Molar conversion efficiences were in the 15 to 30% range. The latter mutant was also shown to convert exogenous 14C-labeled epsilon-rhodomycinone to 14C-labeled daunorubicin glycosides, again at conversion efficiencies of about 25%. The same biotransformation was observed with daunorubicin production strain C5, which normally accumulates both epsilon-rhodomycinone and daunorubicin glycosides. A significant percentage (16 to 37%) of exogenously added epsilon-[14C]rhodomycinone was metabolized by strain C5, and 22 to 32% of the metabolized radioactivity could be recovered as daunorubicin glycosides. A mathematical model of epsilon-rhodomycinone metabolism was constructed based on plausible assumptions concerning the kinetics of epsilon-rhodomycinone accumulation and catabolsim. When analyzed according to this model, our data indicate that most (63 to 73%), but not all, of the daunorubicin glycosides accumulated in the experiments with production strain C5 derived from epsilon-rhodomycinone. A pathway network for the biosynthesis of daunorubicin glycosides is proposed that is in agreement with these data. In this proposed pathway network, epsilon-rhodomycinone is an intermediate in one of at least two pathways which yield daunorubicin glycosides. Images PMID:7425613

  14. Cuticular Architecture of Hassalstrongylus epsilon (Nematoda: Trichostrongyloidea).

    PubMed

    Oliveira-Menezes, A; De Souza, W; Lanfredi, R M

    2003-07-01

    Hassalstrongylus epsilon is a small nematode, whose adult forms are found among the intestinal microvilli of the water rat Nectomys squamipes, Brants 1827 (Rodentia: Muridae). The external appearance of the cuticle, which presents transversal striations and longitudinal ridges, is described using scanning electron microscopy. Transmission electron microscopy of thin sections and replicas of quick-frozen, freeze-fractured, deep-etched and rotatory shadowed samples showed the presence in the cuticle of struts that arise from the fluid median layer, extending outward to the epicuticle. The cuticle showed the presence of five layers: epicuticle, cortical, fibril-rich, fluid median and fibrous. The cuticle layers were made of an assemblage of fibers that create compartments, which were larger in the fluid region than in the fibril-rich median layer. PMID:12884020

  15. Perturbative matching of the staggered four-fermion operators for {epsilon}'/{epsilon}

    SciTech Connect

    Lee, Weonjong

    2001-09-01

    Using staggered fermions, we calculate the perturbative corrections to the bilinear and four-fermion operators that are used in the numerical study of weak matrix elements for {epsilon}'/{epsilon}. We present results for one-loop matching coefficients between continuum operators, calculated in the naive dimensional regularization (NDR) scheme, and gauge invariant staggered fermion operators. In particular, we concentrate on Feynman diagrams of the current-current insertion type. We also present results for the tadpole improved operators. These results, combined with existing results for penguin diagrams, provide a complete one-loop renormalization of the staggered four-fermion operators. Therefore, using our results, it is possible to match a lattice calculation of K{sup 0}-{bar K}{sup 0} mixing and K{yields}{pi}{pi} decays to the continuum NDR results with all corrections of O(g{sup 2}) included.

  16. Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit.

    PubMed Central

    Ito, I; Futai, K; Katagiri, H; Watanabe, M; Sakimura, K; Mishina, M; Sugiyama, H

    1997-01-01

    1. We have explored the effects of targeted disruption of the N-methyl-D-aspartate (NMDA) receptor epsilon 1 or epsilon 2 subunit gene on NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) and long-term potentiations (LTPs) at the two types of synapse in mouse hippocampal CA3 pyramidal neurons: those formed by the commissural/associational (C/A) and fimbrial (Fim) inputs. 2. Electrophysiological experiments were performed in hippocampal slices prepared from both wild-type and epsilon 1- or epsilon 2-disrupted mice using extracellular and whole-cell patch recording techniques. To assess the epsilon 1, epsilon 2 and zeta 1 subunit expression at cellular levels, we performed non-isotopic in situ hybridization with digoxigenin-labelled cRNA probes. 3. We could record EPSCs in response to the stimulations to either of the C/A and Fim afferents from a single CA3 pyramidal neuron. The epsilon 1, epsilon 2 and zeta 1 subunits were expressed together in individual CA3 neurons. 4. The epsilon 1 subunit disruption selectively reduced NMDA EPSCs and LTP in the C/A-CA3 synapse without significantly affecting those in the Fim-CA3 synapse, whereas the epsilon 2 subunit mutation diminished NMDA EPSCs and LTP in the Fim-CA3 synapse with no appreciable functional modifications in the C/A-CA3 synapse. 5. These results suggest that NMDA receptors with different subunit compositions function within a single CA3 pyramidal cell in a synapse-selective manner. Images Figure 2 PMID:9147327

  17. Genomics of Clostridium tetani.

    PubMed

    Brüggemann, Holger; Brzuszkiewicz, Elzbieta; Chapeton-Montes, Diana; Plourde, Lucile; Speck, Denis; Popoff, Michel R

    2015-05-01

    Genomic information about Clostridium tetani, the causative agent of the tetanus disease, is scarce. The genome of strain E88, a strain used in vaccine production, was sequenced about 10 years ago. One additional genome (strain 12124569) has recently been released. Here we report three new genomes of C. tetani and describe major differences among all five C. tetani genomes. They all harbor tetanus-toxin-encoding plasmids that contain highly conserved genes for TeNT (tetanus toxin), TetR (transcriptional regulator of TeNT) and ColT (collagenase), but substantially differ in other plasmid regions. The chromosomes share a large core genome that contains about 85% of all genes of a given chromosome. The non-core chromosome comprises mainly prophage-like genomic regions and genes encoding environmental interaction and defense functions (e.g. surface proteins, restriction-modification systems, toxin-antitoxin systems, CRISPR/Cas systems) and other fitness functions (e.g. transport systems, metabolic activities). This new genome information will help to assess the level of genome plasticity of the species C. tetani and provide the basis for detailed comparative studies. PMID:25638019

  18. Distribution of Clostridium botulinum.

    PubMed Central

    Huss, H H

    1980-01-01

    The distribution of Clostridium botulinum in the natural environments of Denmark, The Faroe Islands, Iceland, Greenland, and Bangladesh was examined. A total of 684 samples were tested. Type E was found in 90% of samples from the aquatic environment of Denmark, including sediments from young artificial lakes, and in 86% of samples from the marine environment of Greenland. Type E was not found in Danish cultivated soil and woodlands, including cultivated soil from reclaimed sea beds, but type B was frequently demonstrated in these environments. C. botulinum types A, B, or E were found in 2.6% of samples from the environments of the Faroe Islands and Iceland, whereas types C or D were demonstrated in 42% of samples from Bangladesh. The incidence of type E in aquatic sediments was not related to general industrial pollution or a high content of rotting vegetation. Fish or a rich aquatic fauna, on the other hand, appeared to contribute to a high incidence of type E. Based on these findings, it is suggested that type E is a true aquatic organism, because this environment offers the best conditions for survival of the spore in nature. It is further suggested that its presence in aquatic bottom deposits is based on sedimentation after proliferation in the carrion of the aquatic fauna and dissemination by water currents and migrating fish. PMID:6990867

  19. Isolation of Clostridium thermocellum auxotrophs

    SciTech Connect

    Mendez, B.S.; Gomez, R.F.

    1982-02-01

    The conversion of biomass of fuels and chemical feedstocks by microbial fermentation offers the potential of solving two of today's important problems: waste accumulation and exhaustion of fossil fuels. Microorganisms with the capabilities of converting biomass components such as cellulos and hemicellulose to chemicals and fuels in a single step are of particular interest. One such microorganism is Clostridium thermocellum, a thermophilic anaerobe which degrades cellulose to ethanol and organic acids. For efficient industrial use, the cellulolytic capacity of this strain must be improved by genetic means. Spontaneous and UV irradiation-induced auxotrophic mutants of Clostridium thermocellum, an anaerobic cellulolytic thermophile, were isolated after penicillin enrichment in a chemically defined medium.

  20. The September epsilon Perseids in 2013

    NASA Astrophysics Data System (ADS)

    Gajdo, tefan; Tth, Juraj; Korno, Leonard; Koukal, Jakub; Piffl, Roman

    2014-04-01

    An unexpected high activity (outburst) of the meteor shower September epsilon Perseids (SPE) was observed on 2013 September 9/10. The similar event occurred in 2008. We analysed SPE meteors observed in a frame of the European stations network (EDMONd) and collected in the video meteor orbits database EDMOND. Also, we compared two AMOS all-sky video observations of SPE meteors, performed at the Astronomical and Geophysical Observatory in Modra (AGO) and Arboretum in Tesarske Mlynany (ARBO) stations of the Slovak Video Meteor Network (SVMN). We obtained activity profiles of the 2013 SPE outburst during four hours around its maximum. Along with SPE activity profiles binned at 10 minutes for single-station meteors, we gained orbital characteristics of SPE meteors observed during the outburst, as well as a mean orbits of the SPE meteor stream in interval 2001-2012. The SPE outburst was confirmed by radio forward-scatter observations as well. The obtained observational results might be the starting point for modeling and explanation of SPE outbursts.