Rozprawy doktorskie: „Clostridium perfringens”

1

Canard, Bruno. "Organisation genomique de clostridium perfringens". Paris 7, 1991. http://www.theses.fr/1991PA077145.

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Clostridium perfringens est un bacille a gram positif, pathogene pour l'homme et les animaux. Nous avons construit une carte physique et genetique du chromosome de la souche de reference cpn50, de type a, ainsi que de sept autres isolats appartenant aux serotypes a, b, d et e. Nous avons ainsi pu etudier comparativement l'organisation genomique des genes et loci impliques dans la maintenance des fonctions vitales de la bacterie, dans la virulence, et dans la plasticite genomique. Nous avons caracterise respectivement au niveau moleculaire les operons ribosomiques rrn, le gene nagh codant pour une n-acetyl-beta-d-glucosaminidase, et neuf regions genomiques sujettes a des polymorphismes de restriction. Quatre de ces dernieres sont associees a des facteurs de virulence, et une de ces quatre tolere des variations en taille de l'ordre de 0,5 megabase. L'organisation des genes de virulence et la plasticite genomique sont etroitement imbriquees

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2

Stiles, Bradley G. "Purification and characterization of Clostridium perfringens iota toxin". Diss., Virginia Polytechnic Institute and State University, 1987. http://hdl.handle.net/10919/76516.

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Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i_b), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i_a) antigen, had little to no biological activity. When fractions containing i_a and i_b were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i_a or i_b, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i_a and i_b, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i_a and i_b were mixed. Monospecific antisera against purified i_a and i_b neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i_a and i_b proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i_a protein is heat stable (85° C/15 min) while i_b lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i_a, but not i_b, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.
Ph. D.

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3

Pérez, Janampa David Remy. "Caracterización toxigénica de la fosfolipasa C del Clostridium perfringens (Cp-PLC) y su relación con aislados de C. perfrigens de casos de enterotoxemia en alpacas". Master's thesis, Universidad Nacional Mayor de San Marcos, 2010. https://hdl.handle.net/20.500.12672/3262.

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La enterotoxemia, causada por el Clostridium perfringens, es la enfermedad infecciosa más importante que afecta a las alpacas, debido a que ocasiona elevadas tasas de mortalidad neonatal de hasta 70%. Recientes estudios han sugerido la participación de la Cp-PLC (C. perfringens fosfolipasa C) como factor de virulencia responsable del cuadro enterotoxemico en alpacas y otras especies domesticas. El presente estudio evaluó las características toxigénicas de la Cp-PLC y de sobrenadantes de diferentes aislados de C. perfringens obtenidos de casos de enterotoxemia en alpacas relacionándolos con sus niveles de producción de Cp-PLC. El protocolo de purificación de Cp-PLC mostró ser exitoso, mostrando su comportamiento como una enterotoxina incapaz de generar lesiones entéricas. Asimismo, los aislados de C. perfringens analizados evidenciaron distintas características toxigénicas independientes de la presencia de Cp-PLC. Al parecer, la Cp-PLC no seria un factor esencial del C. perfringens en la producción de lesiones entéricas en casos de enterotoxemias en alpacas. Palabras Claves: Cp-PLC, Clostridium perfringens, enterotoxigenico
--- Enterotoxemia caused by Clostridium perfringens, causes a mortality neonatal rate up to 70%, this is why it is considered as the most important infections disease. Recent studies has suggested that Cp-PLC (Clostridium perfringens phospholipase C) is a main virulence factor responsible of the enterotoxemic lesions found in alpacas and other domestic animals. This study evaluated the toxigenic characteristics of Cp-PLC and of supercultures of C. perfringens isolates from enterotoxemia in alpacas associated with their levels of Cp-PLC production. The Cp-PLC purification protocol used was successful, showing that Cp-PLC as an enterotoxin enteric unable to cause injury. Similarly, C. perfringens isolates analyzed showed different toxigenic characteristics independently of the Cp-PLC production. Apparently, Cp-PLC does not be a essential factor from C. perfringens in the production of enteric lesions in cases of enterotoxemia in alpacas. Key Word: Cp-PLC, Clostridium perfringens, enterotoxigenic.
Tesis

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4

Perelle, Sylvie. "Toxine IOTA de "Clostridium perfringens" et toxines apparentées". Paris 11, 1996. http://www.theses.fr/1996PA114811.

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5

Xie, Xinye. "Purification and characterization of a blood group A₂degrading [alpha]-N-acetylgalactosaminidase from clostridium perfringens". free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3012978.

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6

Eaton, Julian Timothy. "Structural studies of Clostridium perfringens alpha toxin". Thesis, Birkbeck (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417896.

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7

Griffiths, Nicola Jane. "Studies on Clostridium perfringens in the horse". Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367091.

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8

Hunter, Sophie Emma Clare. "Molecular genetics of Clostridium perfringens epsilon-toxin". Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316420.

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9

Justin, Neil. "Structural studies of clostridium perfringens alpha toxin". Thesis, Birkbeck (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392355.

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10

Russell, Katherine Margaret. "Intestinal responses to Clostridium perfringens in broilers". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25514.

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Clostridium perfringens is the aetiological agent of Necrotic enteritis (NE); a disease that impacts on the health and welfare of broilers. This disease is a large cost to the industry and presents as lesions in the small intestine hindering productivity. Antibiotics are commonly used to treat NE but as pressure increases to limit their use further information about disease onset and broiler responses to the bacteria and it’s virulence factors during infection is required to implement new preventative measures and treatments. NetB is a secreted toxin from C. perfringens which has an important role in NE onset. Using an in situ intestinal loop model we have been able to characterise: I) temporal broiler responses to NetB positive bacterial culture supernatant (Chapter 2), ii) early host responses to different isolates possessing NetB (virulent) or not (avirulent) in the presence or absence of bacterial cells (Chapter 3) and iii) the responses of two commercial broiler breeds (Chapter 4) four hours post exposure. Samples collected from these experiments have been used for histology, mRNA expression and immunohistology. We have shown differences in mRNA expression in the duodenum of broilers after exposure to C. perfringens cells as well as the culture supernatant from the isolates used after four hours. The presence of bacteria cells resulted in up-regulation of pro-inflammatory cytokine, IFN-γ, mRNA, whereas it resulted in down-regulation of B-LA, mRNA a gene involved in presentation of pathogens to immune cells. IL-6 mRNA expression was also reduced in the presence of virulent isolates. This could indicate a possible evasion strategy for C. perfringens in broilers. Immunohistochemical analysis indicated that slower growing broilers have increased numbers of immune cells (macrophages and γδ T cells) in their duodenum compared with faster growing broilers, although this did not appear to have an effect on mRNA expression levels of pro-inflammatory cytokines, 4h post antigen infusion. Overall we detect greater changes when bacteria are included with culture supernatant and have highlighted possible mechanisms for C. perfringens to avoid the broiler immune system. Induction of NE in the literature requires pre-disposing factors, including co-infection with other intestinal pathogens and dietary manipulation of the host. The final experiment trialled protocols administering a virulent isolate of C. perfringens in-feed and via gavage along with an increased protein source to induce NE (Chapter 5). These models were not considered to be consistent for further investigation of NE in the future.

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11

Zimmer, Markus. "Complete genome sequence and characterization of the lysis system of the temperate Clostridium perfringens bacteriophage f3626 [phi3626]". [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=965243400.

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12

Flores, Díaz Marietta. "A UDP-glucose deficient mutant cell line as a model to study the cytotoxicity of Clostridium perfringens PLC /". Stockholm : Karolinska Univ. Press, 2001. http://diss.kib.ki.se/2001/91-7349-087-3/.

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13

Cole, Ambrose Ralph. "Structural studies of the toxins of Clostridium perfringens". Thesis, Birkbeck (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408277.

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14

Miyashiro, Simone. "Caracterização de isolados de Clostridium perfringens de ruminantes". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-16092014-155341/.

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C. perfringens é uma bactéria anaeróbia presente no intestino delgado do homem e animais em equilíbrio e, sob a ação de alguns fatores predisponentes como mudança brusca de alimentação ou super alimentação, stress no manejo ou alto parasitismo intestinal, há a proliferação do microrganismo com a consequente produção de potentes toxinas que provocam a morte do animal. Dentre as toxinas principais destaca-se a toxina alfa, importante fator de virulência, produzida por todos os tipos de C. perfringens, sendo os pertencentes ao tipo A os maiores produtores. A fim de caracterizar o microrganismo em suspeitas de enterotoxemia em ruminantes, trabalhamos com 61 amostras de intestino delgado de bovinos e 12 de ovinos como grupo estudo e no grupo controle composto de animais hígidos levados ao abate, 73 amostras de intestino delgado de bovinos e 24 de ovinos. Foram realizados procedimentos de isolamento e tipagem molecular de C. perfringens e quantificação celular, detecção molecular da toxina β2, além de avaliações moleculares qualitativa (PCR convencional) e quantitativa (PCR em tempo real) do gene da toxina alfa dos diferentes isolados. Em 29 amostras do grupo estudo bovino (47,54%) e em 4 (33,33%) do grupo estudo ovino isolou-se o microrganismo, em contrapartida no grupo controle bovino não houve isolamento do bacilo e 5 amostras do grupo controle ovino (20,83%) foram positivas. Houve diferença estatisticamente significante somente entre os grupos de bovinos (p<0,05). Todos os isolados (100%) foram classificados como tipo A, e os resultados das quantificações celulares de C. perfringens revelaram que todos os bovinos controle apresentaram <10 UFC/g de conteúdo enquanto que o grupo estudo apresentou mediana de 104 UFC/g com variações de <10 UFC/g até 108 UFC/g. Nos ovinos, a mediana no grupo controle foi 101 UFC/g assim como no grupo estudo, entretanto com clara separação de valores entre os grupos. Tanto na PCR convencional quanto na PCR em tempo real para detecção do RNAm da toxina alfa foi observado limiar de detecção de 102 cópias de cDNA por reação, porém provavelmente devido aos valores das amostras estarem próximos ao limite da sensibilidade analítica da reação, não foi observada boa reprodutibilidade da última. Já na reação molecular convencional, observou-se a presença de detecção de RNAm da toxina alfa em 60,52% dos isolados o que revela alguma diferença da presença do transcrito entre as culturas, já que nas cepas restantes não foi detectada a presença do RNAm em questão. A pesquisa do gene da toxina β2 revelou sua presença em 54,55% dos isolados de C. perfringens corroborando com a afirmativa de que o gene está amplamente distribuído entre os ruminantes. A metodologia aplicada para avaliação da expressão do gene da toxina alfa nos isolados mostrou que há diferenças dos níveis de transcrição porém não permitiu quantificar esses valores. A tipagem molecular corrobora com outros estudos quanto à importância epidemiológica do tipo A nos quadros de enterotoxemia em ruminantes, e os dados da quantificação celular permite-nos concluir que animais sadios possuem um nível basal de C. perfringens <10 UFC/g de conteúdo que não possibilita o seu isolamento.
C. perfringens is an anaerobe present in small intestine of man and animals in equilibrium, and under some predisposing factors such as sudden feeding change or super feeding, rough management or high intestinal parasitism, the microorganism multiplies with the consequent production of potent toxins that can cause animal death. Amongst the main toxins, alpha toxin is an important virulence factor, that is produced by all C. perfringens types, and those belonging to type A are its higher producer. Aiming to characterize the microorganism in ruminants suspect of enterotoxaemia, we evaluated 61 bovine small intestinal samples and 12 sheep small intestines as the study group, and for the control group composed by higid animals led to slaughterhousing, 73 bovine small intestines and 24 ovine samples. We performed microbiological culture and molecular typing of C. perfringens isolates, cellular quantification, molecular detection of 2 toxin, and qualitative and quantitative molecular evaluations of alpha toxin from different isolates by means of conventional PCR and real time PCR, respectively. In 29 samples from the bovine study group (47.54%) and in 4 (33.33%) from ovine study group the microorganism was isolated, however in the bovine control group there was no isolation success and 5 samples from sheep control group (20.83%) were positive. There was statistically significant difference only between bovine groups (p<0,05). All isolates (100%) were classified as type A, and C. perfringens cellular quantification results showed that every control bovine presented <10 CFU/g of intestinal contents while the study group presented a median of 104 CFU/g with results ranging from <10 CFU/g to 108 CFU/g. In sheep, the median value in the control group was 101 CFU/g as in the study group, but with a clear division of values between the groups. We observed the threshold detection of 102 cDNA copies per reaction in both conventional and real time PCR reactions for alpha toxin mRNA detection, however since the samples quantification values were close to the analytical sensitivity of the test, we could not observe the reproducibility in the last technique. In the conventional PCR reaction, alpha toxin mRNA was detected in 60.52% of the isolates. This result reveals some difference in the transcript presence among the cultures, since we could not detect the presence of the described mRNA in the other isolates. Beta2 toxin gene was detected in 54.55% of C. perfringens isolates corroborating with the affirmative that this gene is widely distributed among ruminants. The methodology presented herein for the evaluation of alpha toxin gene expression showed that there are differences in the transcription levels, however it didnt allow to quantify these values. Molecular typing results agree with other studies regarding the epidemiological importance of type A in the enterotoxaemia processes in ruminants, and the cellular quantification data allow us to conclude that healthy animals show a basal level of C. perfringens <10 CFU/g of intestinal content that doesnt allow its isolation.

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15

O'Brien, David Kenneth. "The Interactions of Clostridium Perfringens With Phagocytic Cells". Diss., Virginia Tech, 2003. http://hdl.handle.net/10919/27164.

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Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischemic tissues become contaminated with C. perfringens. C. perfringens quickly multiplies in ischemic tissues and spreads to healthy areas, leading to high levels of morbidity and mortality. As a species, the bacterium can synthesize thirteen different toxins. The alpha toxin (PLC) and perfringolysin O (PFO) are thought to be important virulence factors in gangrene. We wished to understand how C. perfringens is capable of avoiding killing by the host immune system, and determine if PLC and PFO play a role in this avoidance. We found C. perfringens was not killed by J774-33 cells or mouse peritoneal macrophages under aerobic or anaerobic conditions. Using electron microscopy, we showed that C. perfringens could escape the phagosome of J774-33 and mouse peritoneal macrophages. We believe the ability of C. perfringens to survive in the presence of macrophages is due to its ability to escape the phagosome. Using a variety of inhibitors of specific receptors, we identified those used by J774-33 cells to phagocytose C. perfringens. The scavenger receptor, mannose receptor(s), and complement receptor (CR3) were involved in the phagocytosis of C. perfringens. To determine if PFO or PLC were involved in the ability of C. perfringens to survive in the presence of macrophages, we constructed C. perfringens strains lacking these toxins. The ability of C. perfringens to survive in the presence of J774-33 cells is dependent on PFO, while survival in mouse peritoneal macrophages is dependent on PFO and PLC. The ability of C. perfringens to escape the phagosome of J774-33 cells and mouse peritoneal macrophages is mediated by either PFO or PLC. Using a mouse model, we found that PFO and PLC were necessary for C. perfringens to survive in vivo using infectious doses 1000 times lower than those required to initiate a gangrene infection. We propose that PFO and PLC play a critical role in the survival of C. perfringens during the early stages of gangrene infections, when phagocytic cells are present and bacterial numbers are low.
Ph. D.

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16

Netherwood, Trudy. "The association of Clostridium perfringens with foal diarrhoea". Thesis, Open University, 1995. http://oro.open.ac.uk/57556/.

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Several case reports of Clostridium perjringens involvement in equine enteric disease have not identified the prevalence and statistical association of these bacteria with foal diarrhoea. Each of five methods which favoured the recovery of C. perjringens in different physiological states were chosen to improve the sensitivity of isolation in a survey of foal diarrhoea for C. perjringens and other pathogens. C. perjringens was significantly associated with foal diarrhoea (isolated from 57% of 421 scouring anjmals but from only 33% of 222 controls; odds ratio 7.4; p < 0.001 by multivariate analysis); it was also associated with fatal diarrhoea (odds ratio 2.7; p=0.047). Rotavirus, Cryptosporidium sp. and large numbers of Strongyloides westeri were the only other pathogens associated with diarrhoea although they were less prevalent than C. perjringens; Salmonella sp. was the only other pathogen associated with fatal diarrhoea. Enterotoxin production was detected by reverse passive latex agglutination test (RPLA) amongst isolates of C. perjringens from scouring and healthy foals. The enterotoxin gene from an equine strain was cloned and its sequence was essentially identical to that published for a human isolate. Less than 5% of C. perjringen isolated from scouring foals and 0.5% from controls were positive for the enterotoxin gene by polymerase chain reaction (PCR) (odds ratio 19.1; p<0.005). Presence of the enterotoxin gene was confirmed in representative isolates with a gene probe of chromosomal DNA and PCR product as well as neutralisation of cytotoxicity by antitoxin. Enterotoxigenicity of half ofRPLA positive isolates could not be confirmed in this way. Enterotoxigenic C. perfringens were a probable cause of foal diarrhoea. However, a greater proportion of the disease was associated with nonenterotoxigenic . perjringens. There is now a need to identify molecular differences between non-enterotoxigenic C. perjringens strains from scouring and healthy foals which might be associated with pathogenicity.

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17

Eberl, Steven G. "Clostridium Perfringens: An Adjunctive Indicator in Nonpoint Pollution". DigitalCommons@USU, 1986. https://digitalcommons.usu.edu/etd/4397.

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Clostridium perfringens (CP) was evaluated as an additional indicator in assessing impacts and sources of microbial pollution in the Idaho-Utah Cache Valley . Point , nonpoint, river water, and animal fecal samples were analyzed for CP, total coliforms, fecal collforms, and fecal streptococci. Monthly river samples consistently contained <20 CP/100 mL , but concentrations of the other indicators varied significantly by location and date. Two sample stations consistently had CP concentrations greater than 20 / 100 mL . One of these stations was influenced by an upstream wastewater discharge . Chlorinated effluent from this trickling filter plant contained greater than 103 CP / 100 mL, but met a 400 FC/100 mL discharge standard. A consistent decrease in CP concentrations in samples taken downstream from this wastewater source were fo und, despite significant impact from adjacent nonpoint pollution. Lagoon and oxidation ditch wastewater effluents sampled contained <20 CP/100 mL. Nonpoint sources sampled (e.g . , cattle feedlot runoff) contained <20 CP / 100 mL and 102-104/100 mL coliforms and fecal streptococcus. Cattle, horse, and sheep feces analyzed contained 104-107/g coliforms and fecal streptococcus, but less than 102 CP/g. Nonpoint pollution from such animals may contribute significant coliforms and streptococci but not CP. Wastewater treatment effluents may or may not contain elevated levels of CP depending on factors such as wastewater residence time and particular treatment process employed. The occurrence of relatively high, i.e., >102 CP/100 mL, in areas impacted by nonpoint sources may suggest a municipal wastewater input. Coliform and streptococci indicators may not be able to distinguish municipal or domestic microbial loading in the presence of nonpoint source interferences in many circumstances.

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ALBERTIN, FILIPPI ANNE-MARIE. "Les toxi-infections alimentaires collectives a clostridium perfringens". Strasbourg 1, 1990. http://www.theses.fr/1990STR15021.

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Anderson, Michael Anthony. "Porcine Enteric Disease Caused by Clostridium difficile and Clostridium perfringens: Epidemiology, Pathogenesis and Immunity". Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/195681.

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Clostridium difficile and Clostridium perfringens are among the most commonagents of enteric disease in both humans and domestic animals. The former continues to increase in prevalence and diseases caused by the latter persist. Infection with a recently emergedhypervirulent strain (NAP1/027/III) of C. difficile is increasingly common andserious sequelae and fatalities are much more common in these patients. In neonatalpiglets, C. difficile infection (CDI) has become a common occurrence. Historically,isolation of C. perfringens type A from patients with enteric disease has been consideredinconsequential due to its presence in the normal intestine and to the mild nature ofdisease syndromes such as porcine enteritis. However, both CDI and type A diseasecause losses to the swine industry and pigs have been implicated as a possible source ofC. difficile for infection in humans. We investigated the epidemiology and pathogenesisof porcine CDI, and immunity against porcine CDI and type A enteritis. The occurrenceof CDI in integrated swine production facilities was most common in neonatal pigs.Infection in sows was rare, and finisher pigs were culture negative. All C. difficile strainswere ribotype 078. Hypervirulent strain NAP1/027/TTIII was more virulent in neonatalpigs than both a historic human historic human strain and a porcine strain with toxinproducing potential similar to ribotype 027 strains. Inoculation of anti-microbial-treatedadolescent pigs with NAP1/027/III did not cause disease. Ingra-gastric inoculation ofpigs with purified TcdA resulted in severe small intestine damage which isuncharacteristic of natural disease; effects of TcdB were minimal. Passive immunizationof piglets against C. difficile TcdA or C. perfringens type A alpha (CPA) and beta 2(CPB2) toxins did not prevent disease.

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Iwanejko, Lesley Ann. "Cloning the enterotoxin gene from Clostridium perfringens type A". Thesis, University of Nottingham, 1991. http://eprints.nottingham.ac.uk/12195/.

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A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a single plasmid, with an apparently altered copy number, pLWl, that carried the CPE gene, cpe, on a 6.8 kbp insert. A sequence deduced primer strategy for direct plasmid sequencing was initiated using a primer deduced in a similar manner to the 26 bp probe, obviating the need for prior mapping and subcloning of the insert. The amino acid sequence for the conceptual gene product of the single open reading frame differed only slightly from the known CPE sequence but lacked the C terminal residues. The biased cpe codon usage reflected the low %G+C content of the DNA. The %G+C content was even lower in the upstream region and possessed properties characteristic of bent DNA. The region 5' to the ATG translational start codon contained a Shine-Dalgarno sequence and several sequences with significant homology to the putative transcriptional control regions for the tetanus toxin gene. The N-terminal coding region contained a direct repeat of an upstream sequence that shared considerable homologies with the crossover point in site 1 of the Tn3 res region. Southern blot analyses of chromosomal and plasmid DNAs from several isolates indicated that the majority of strains were cpe-. The chromosomal location and architecture of cpe appeared identical in all cpe+ strains. A second copy, pLW2, of the 5' end of cpe, on a 4.5 kbp Pst I/Eco RI restriction fragment, was cloned during one of many unsuccessful attempts to clone the 3' end. A separate re-cloning experiment isolated several different clones that contained the 0.6 kbp Hind III located = 2.5 kbp 5' to the ATG codon of both cloned copies of cpe but none of them carried the CPE gene. The fragment was used as a DNA probe to show that it was present in high copy number in some strains of C. perfringens but completely absent from others. An hypothesis describing the possible involvement of a mobile genetic element in C. perfringens enterotoxin production offers explanations for the cloning of a complex mixture of plasmids, the apparent alteration in plasmid copy number, the identification of putative DNA crossover points, the failure to clone the 3' end of cpe and the isolation of a novel DNA fragment.

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21

Brown, Robert Christopher. "Development of a novel expression system in Clostridium perfringens". Thesis, Open University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321561.

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22

Orsburn, Benjamin. "Factors Affecting the Heat Resistance of Clostridium perfringens Spores". Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/27697.

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The bacterium Clostridium perfringens is a gram-positive anaerobe responsible for many diseases in man and other animals, the most common of which is acute food poisoning (AFP). It is estimated that nearly 240,000 cases of AFP occur each year in the U.S. The C. perfringens spore plays an important role in this infection. The heat resistance of the spore allows the organism to survive the cooking process, grow in the cooling food, and infect the victim. Despite the occurrence of this disease and the importance of the spore to this process, little work has been performed to determine how heat resistance is obtained and maintained by C. perfringens spores. In this work we study the spores and sporulation process of C. perfringens to determine what factors are most important in the formation of a heat resistant spore. We analyzed the spores produced by nine wild-type strains, including five heat-resistant food poisoning isolates and four less heat-resistant environmental isolates. We determined that threshold core density and a high ratio of cortex peptidoglycan relative to germ cell wall were necessary components of a highly heat-resistant spore. In order to test these observations, we constructed two mutant strains. The first could not achieve the necessary level of core dehydration and rapidly lysed in solution. The second mutant had a reduced amount of cortex relative to germ cell wall, and suffered a corresponding decrease in heat resistance as compared to our wild-type strains. The mutant strains supported the observations drawn from our wild-type strains. Dipicolinic acid is a major component of bacterial spores and is necessary for spore heat resistance. The Cluster I clostridia, including C. perfringens, lack the known DPA synthase operon, spoVF. We developed an in vitro assay for detecting DPA synthetase activity and purified the active enzyme from sporulating C. perfringens crude extract and identified the proteins with mass spectrometry. These results identified the electron transfer flavoprotein alpha chain (EtfA) as the DPA synthase of C. perfringens. Inactivating the etfA gene in C. perfringens resulted in a strain that could begin, but not complete, the sporulation process and produced dramatically lower amounts of DPA than the wild-type. The purified enzyme was shown to produce DPA in vitro and utilized FAD as a preferred cofactor. The results of this research may lead to future techniques to decrease the occurrence of the diseases caused by C. perfringens spores and treatments which may carry over to the diseases caused by similar organisms.
Ph. D.

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23

Abraham, Lawrence Joseph. "Molecular genetics of antibiotic resistance determinants from Clostridium perfringens". Thesis, Abraham, Lawrence Joseph (1986) Molecular genetics of antibiotic resistance determinants from Clostridium perfringens. PhD thesis, Murdoch University, 1986. https://researchrepository.murdoch.edu.au/id/eprint/53043/.

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Clostridium perfringens is a sporulating, Gram positive, anaerobic bacterium that is a normal inhabitant of the gastrointestinal tract and is known to cause a number of diseases in both man and animals. Conjugative tetracycline resistance plasmids from C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analysed by comparison with the reference plasmid, pCW3. This conjugative 47 kilobasepair (kb) plasmid was isolated from a human C. perfringens strain and confers inducible tetracycline resistance. Fifteen of the plasmids encoded tetracycline resistance whereas three carried both tetracycline and chloramphenicol resistance. Nine of the tetracycline resistance plasmids had restriction profiles that were identical to those of pCW3. The remaining nine R-plasmids were different to pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contain at least 17 kb of DNA that was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The results showed that a pCW3-like element forms the basis of all transferable R-plasmids found in C. perfringens and that this element was widely disseminated in nature. To compare regions of homology between the conjugative plasmids it was necessary to construct a detailed restriction map of pCW3. The five Cla I fragments that together comprise the entire pCW3 molecule were cloned in Escherichia coli. The recombinant plasmids were mapped and the restriction data were used to construct an unambiguous pCW3 map. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed Eco RI fragments that together encompass a 4 kb region of pCW3. Deletion and transposon mutagenesis experiments showed that the tetracycline resistance gene was contained within a 1. 4 kb segment of the 4 kb region. This gene, designated tetP, was compared with those of other bacteria by Southern hybridization. The results indicated that there was no homology between tetP and any of the other classes of tetracycline resistance determinant. Consequently, tetP represents a new class of tetracycline resistance determinant that is distinct from those of other bacteria. Three of the conjugative tetracycline resistance plasmids that were characterised as being related to pCW3, also coded for chloramphenicol resistance. The homologous 6. 2 kb chloramphenicol resistance regions delete with high frequency upon transfer of these plasmids. The chloramphenicol resistance regions from two of these plasmids, pIP401 and pJIR27, were cloned in E. coli. The recombinant plasmids, pJIR45 and pJIR97 respectively, confer chloramphenicol resistance upon E. coli. However, cultures carrying these plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. Loss of resistance was associated with the loss of the 6. 2 kb segments from both plasmids. Transposition of these segments to different sites on the E. coli chromosome was demonstrated after cloning the 6. 2 kb region onto a temperature sensitive replicon. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences. Comparison of the end sequences of Tn4451 with those of the Tn3 family of transposons indicated some similarity. In contrast to the Tn3- like transposons which contain ca. 30 basepair (bp) inverted repeat sequences at their ends and duplicate 5 bp of the target sequence, TN4451 contained 3 bp inverted repeat sequences at its ends, and appeared to generate 2 bp direct repeats of the target DNA upon insertion. The regions surrounding the site of deletion of TN4451 also were sequenced. Deletion fragments derived from excision of TN4451 in both C. perfringens and E. coli were sequenced and it was shown that the deletion of TN4451was precise. The chloramphenicol resistance transposons TN4451 and TN4452 represent the first transposons to be identified from C. perfringens.

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Meer, Ralph Raymond. "Rapid methods for the detection of toxigenic Clostridium perfringens". Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/290593.

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Clostridium perfringens may be the most widely occurring bacterial pathogen and is responsible for a variety of diseases in both humans and animals. The virulence of this organism is associated with the ability to produce an estimated 17 potential exotoxins. The production of one or more of the five major toxins (α,β,ε, and ι) is the basis for placing isolates into five toxigenic types, A through E. Enterotoxin (CPE), is not used in typing but is considered a major virulence factor. A multiplex PCR genotyping assay was developed, utilizing primers derived from sequences of cpa, cpb, etx, iA, and cpe, yielding products of 324, 196, 655, 446, and 233 bp, respectively. Template for this assay was derived from individual colonies suspended in 200 μl of HPLC-grade water, boiled for 20 min or heated in a microwave oven for 10 min at 700 W. Included in the 50 μl reaction volume was 10 μl of template, 0.15 to 0.7 μM of each primer, 0.1 mM dNTPs, 2 mM MgCl₂, and 2 units of Taq DNA polymerase. The PCR products were examined by electrophoresis in a 1.5% agarose gel stained with EtBr. Correlation of genotype with toxin phenotype in strains examined by mouse inoculation was excellent, and it was possible to provide results rapidly, usually in < 4 h. An ELISA procedure was established for detection of β toxin produced by C. perfringens types B and C. The ELISA was used to differentiate Cpb⁺ from Cpb⁻ isolates grown in overnight broth cultures and to measure β toxin in commercial fermentations of type C organisms. In addition to the above assays, preliminary work was initiated on the development of a PCR procedure for quantitation of C. perfringens in clinical or environmental samples, and involved the construction of a 233 bp homologous, competitive mimic from a restriction digest of a 323 bp PCR product generated from cpa.

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Camiade, Émilie. "Étude de deux autolysines à activité N-acétylglucosaminidase chez Clostridium perfringens et Clostridium difficile". Rouen, 2010. http://www.theses.fr/2010ROUES025.

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L'objectif de ce travail était de caractériser et d'étudier le rôle de deux peptidoglycane hydrolases à activité N-acétylglucosaminidase chez Clostridium perfringens et Clostridium difficile. Les peptidoglycane hydrolases possèdent en effet un rôle essentiel dans la croissance bactérienne, peuvent contribuer à la virulence de certaines espèces pathogènes, et peuvent être impliquées dans l'activité bactéricide des antibiotiques inhibiteurs de la synthèse de la paroi. Le lien phylogénétique des genres Staphylococcus et Clostridium nous a permis de caractériser, par analyse génomique, deux gènes de peptidoglycane hydrolases, acp et acd chez C. Perfringens et C. Difficile. Acp et Acd ont une organisation modulaire comparable comprenant un domaine catalytique à activité N-acétylglucosaminidase en C-terminal et un domaine d'ancrage composé de séquences répétées à motifs SH3_3 en position N-terminale. L'étude des fonctions de ces deux peptidoglycane hydrolases a ensuite été réalisée grâce à la construction de leurs mutants respectifs. Acp se révèle être impliquée (i) dans la séparation des cellules filles de C. Perfringens lors de sa croissance et (ii) dans la réponse à la lyse induite par les sels biliaires et la vancomycine. En ce qui concerne Acd de C. Difficile, son activité est vraisemblablement compensée par d'autres peptidoglycane hydrolases actuellement en cours de caractérisation
The objective of this work was to characterize and study the function of two peptidoglycan hydrolases from Clostridium perfringens and Clostridium difficile. Peptidoglycan hydrolases are implicated into the bacterial growth, may contribute to the virulence of certain pathogenic species and can be implicated in the bactericidal effect of cell wall-targeting antibiotics. The phylogenetic link between Staphylococcus and Clostridium allowed us to characterize, by genomic analysis, two genes of peptidoglycan hydrolases, Acp and Acd in C. Perfringens and C. Difficile. Acp and Acd have a modular organization composed of a C-terminal catalytic domain with N-acetylglucasaminidase activity and an anchoring domain composed of SH3_3 repeated sequences. Acp is involved (i) in the daughter cells separation of C. Perfringens during growth and (ii) in response to bile salts and vancomycin induced-lysis. For Acd of C. Difficile, its activity seems to be compensated by other peptidoglycan hydrolases that are actually in characterization

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Beraldo, Massoli Mariana Casteleti [UNESP]. "Prevalência de Clostridios sulfito redutores e Clostridium perfringens na mucosa intestinal de frangos de corte". Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/115976.

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O gênero Clostridium é amplamente distribuído na natureza, está presente no solo e conteúdo intestinal dos animais, produzem toxinas que são capazes de provocar doenças e intoxicações. O Clostridium perfringens está envolvido com prejuízos na avicultura, pois a doença (Enterite Necrotica) na maioria das vezes é subclínica, e o produtor só observa na hora do abate que a ave não ganhou o peso esperado. O controle dessa e de outras enfermidades e agentes patogênicos na avicultura de corte tem grande importância uma vez que o Brasil está entre os principais países exportadores de carne. O objetivo deste trabalho primeiramente foi pesquisar Clostridium perfringens em frangos de corte sadios, provenientes de dois grandes frigoríficos com Inspeção federal, um no Estado de São Paulo e outro no Estado de Minas Gerais. Foram obtidos 300 raspados intestinais, os quais foram semeados em Agar SPS e incubados em anaerobiose. A identificação dos tipos A, B, C, D e E de C. perfringens foi realizada mediante o emprego da PCR multiplex por amplificação dos genes codificadores das toxinas alfa, beta, épsilon e iota do mesmo. A partir das amostras positivas na PCR (37), procedeu-se o isolamento do agente. Depois de muitas tentativas, verificou-se por meio do sequenciamento da região 16S rDNA, que as colônias sulfito redutoras que estavam sendo isoladas eram Enterococcus spp, microrganismo coexistente na microbiota intestinal de frango de corte, que compete com o C. perfringens pelo crescimento das colônias no mesmo meio de cultura, ainda que este seja seletivo e diferencial para clostrídios sulfito redutores. Dessa forma, notou-se que o crescimento de colônias de enterococos interferia muito na identificação e enumeração do C. perfringens e ainda apresentavam colônias muito semelhantes às de C. perfringens nos oito meios de cultura testados. Visto isto, foram feitos alguns testes para avaliar o comportamento dos ...
Clostridium genus is widely distributed in nature and is present in soil and intestinal contents of animals, produce toxins which are capable of causing diseases and intoxication. Clostridium perfringens is involved with losses in the poultry industry, because the disease is most often subclinical, and producer only observed at the time of slaughter the bird has not gained the expected weight. The control of this and other diseases and pathogens in poultry production is very important since Brazil is among the major meat exporting countries. The aim of this study was to evaluate the presence of Clostridium perfringens in poultry coming from two large slaughter-house, one from São Paulo state and another from Minas Gerais. Were obtained 300 intestinal scraped and cecal contents, which were sown onto SPS agar and incubated anaerobically. The identification of C. perfringens types A, B, C, D and E was performed by the use of multiplex PCR. After identification of the presence of C. perfringens in 37 of 300 samples by amplification of the cpa gene, alpha toxin encoder, the isolation of pure colonies was proceeded. However, through the sequencing of 16S rDNA region, it was also found the presence of Enterococcus spp. that lives in the intestinal microbiota of poultry and disputes in growth on the same culture medium, even been selective and differential for clostridia. Thus, it was observed that colony morphology of enterococci is very similar to C. perfringens in this medium what make isolation of pure colonies difficult. Having on mind the difficulty of C. perfringens isolation under these circumstances, the PCR is an rapid and secure alternative for detection of C. perfringens and its different types, being effective for diagnostics

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Beraldo, Massoli Mariana Casteleti. "Prevalência de Clostridios sulfito redutores e Clostridium perfringens na mucosa intestinal de frangos de corte /". Jaboticabal, 2014. http://hdl.handle.net/11449/115976.

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Orientador: Ruben Pablo Schocken-Iturrino
Banca: Clovis Wesley Oliveira de Souza
Banca: Caroline Petters Pigato de Nardi
Banca: Luiz Augusto do Amaral
Banca: Antonio Carlos Paulillo
Resumo: O gênero Clostridium é amplamente distribuído na natureza, está presente no solo e conteúdo intestinal dos animais, produzem toxinas que são capazes de provocar doenças e intoxicações. O Clostridium perfringens está envolvido com prejuízos na avicultura, pois a doença (Enterite Necrotica) na maioria das vezes é subclínica, e o produtor só observa na hora do abate que a ave não ganhou o peso esperado. O controle dessa e de outras enfermidades e agentes patogênicos na avicultura de corte tem grande importância uma vez que o Brasil está entre os principais países exportadores de carne. O objetivo deste trabalho primeiramente foi pesquisar Clostridium perfringens em frangos de corte sadios, provenientes de dois grandes frigoríficos com Inspeção federal, um no Estado de São Paulo e outro no Estado de Minas Gerais. Foram obtidos 300 raspados intestinais, os quais foram semeados em Agar SPS e incubados em anaerobiose. A identificação dos tipos A, B, C, D e E de C. perfringens foi realizada mediante o emprego da PCR multiplex por amplificação dos genes codificadores das toxinas alfa, beta, épsilon e iota do mesmo. A partir das amostras positivas na PCR (37), procedeu-se o isolamento do agente. Depois de muitas tentativas, verificou-se por meio do sequenciamento da região 16S rDNA, que as colônias sulfito redutoras que estavam sendo isoladas eram Enterococcus spp, microrganismo coexistente na microbiota intestinal de frango de corte, que compete com o C. perfringens pelo crescimento das colônias no mesmo meio de cultura, ainda que este seja seletivo e diferencial para clostrídios sulfito redutores. Dessa forma, notou-se que o crescimento de colônias de enterococos interferia muito na identificação e enumeração do C. perfringens e ainda apresentavam colônias muito semelhantes às de C. perfringens nos oito meios de cultura testados. Visto isto, foram feitos alguns testes para avaliar o comportamento dos...
Abstract: Clostridium genus is widely distributed in nature and is present in soil and intestinal contents of animals, produce toxins which are capable of causing diseases and intoxication. Clostridium perfringens is involved with losses in the poultry industry, because the disease is most often subclinical, and producer only observed at the time of slaughter the bird has not gained the expected weight. The control of this and other diseases and pathogens in poultry production is very important since Brazil is among the major meat exporting countries. The aim of this study was to evaluate the presence of Clostridium perfringens in poultry coming from two large slaughter-house, one from São Paulo state and another from Minas Gerais. Were obtained 300 intestinal scraped and cecal contents, which were sown onto SPS agar and incubated anaerobically. The identification of C. perfringens types A, B, C, D and E was performed by the use of multiplex PCR. After identification of the presence of C. perfringens in 37 of 300 samples by amplification of the cpa gene, alpha toxin encoder, the isolation of pure colonies was proceeded. However, through the sequencing of 16S rDNA region, it was also found the presence of Enterococcus spp. that lives in the intestinal microbiota of poultry and disputes in growth on the same culture medium, even been selective and differential for clostridia. Thus, it was observed that colony morphology of enterococci is very similar to C. perfringens in this medium what make isolation of pure colonies difficult. Having on mind the difficulty of C. perfringens isolation under these circumstances, the PCR is an rapid and secure alternative for detection of C. perfringens and its different types, being effective for diagnostics
Doutor

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Lippke, Ricardo Tesche. "Estudo caso controle avaliando a freqüencia dos principais agentes causadores de diarréia neonatal em suínos". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2008. http://hdl.handle.net/10183/12706.

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A diarréia é o principal evento clínico observado no período neonatal em leitões (como conseqüência das doenças entéricas). Além de contribuir para piora no ganho de peso diário e conversão alimentar do animal causa aumento na mortalidade e gastos com medicamentos. O presente trabalho teve como objetivo determinar a freqüência dos principais agentes virais (rotavírus), bacterianos (E. coli, Clostridium perfringens tipo A e C e Clostridium difficile) e parasitários (coccídeos e Cryptosporidium spp.) envolvidos na diarréia neonatal em leitegadas caso (com diarréia) e controle (sem diarréia). Foram examinadas 276 amostras de fezes provenientes de 147 leitegadas com diarréia e 129 leitegadas sem diarréia, com idade variando entre 1 e 7 dias de vida em 28 unidades produtoras de leitões localizados no Estado do Rio Grande do Sul. Entre as amostras coletadas, 29,34% (81/276) foram positivas para pelo menos um agente pesquisado. Os coccídeos (20/276) e o C. perfringens tipo A (19/276) foram os agentes mais freqüentes. Nenhum dos enteropatógenos pesquisados obteve diferença significativa (p>0,05) entre as leitegadas caso e controle. Apenas o rotavírus (p=0,20) e o C. perfringens tipo A (p=0,16) apresentaram tendência de serem mais freqüentes em leitegadas com diarréia. Uma forte associação foi observada entre a ocorrência das diarréias e leitegadas mais novas (p<0,014). Foi observada pela primeira vez no Brasil a infecção pelo C. difficile, em 13,6% (17/132) das amostras, todavia a presença das toxinas nas fezes não teve relação com a diarréia. Os resultados obtidos indicam a necessidade de cuidados especiais quando da coleta de amostras para diagnósticos de monitoria de diarréias no período neonatal, pois é alta a chance de se obterem resultados falso positivos naqueles casos em que as doenças estiverem ocorrendo numa forma endêmica.
Diarrhea is the main clinical event occurring in pigs in the neonatal period,(as consequence of enteric disease). Besides contributing to losses in daily weight gain and feed conversion, diarrhea causes increased mortality and medication costs. The present work aimed to determine the frequency of the main viral agents (rotavirus), bacterial (E. coli, Clostridium perfringens type A and C and Clostridium difficile) and parasitic (coccidian and Cryptosporidium spp.) involved in neonatal diarrhea in case groups (with diarrhea) and controls (without diarrhea). We examined 276 fecal samples originating from 147 litters with diarrhea and 129 litters without diarrhea, with ages varying between 1 and 7 days, in 28 pig units of the state of Rio Grande do Sul, Brazil. Among the examined samples, 29.34% (81/276) were positive for at least one agent. Coccidia (20/276) and C. perfringens type A (19/276) were the most frequently isolated agents. None of the enteropathogens studied showed significant difference (p>0.05) between case and control litters. Only rotavirus (p=0.20) and C. perfringens A (p=0.16) had a tendency to present higher frequency in piglets with diarrhea. A strong association was observed between occurrence of diarrhea and litters with smaller age (p<0,014). Infection with C. difficile was diagnosed for the first time in Brazil, in 13.6% (17/132) samples; however the presence of the toxin in feces was not related to diarrhea. The present results suggest the need for special attention when sampling for the diagnosis or monitoring diarrhea in the neonatal period, as chances for obtaining false-positive results are high, especially when diseases are occurring endemically.

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Sawires, Youhanna Sobhy. "GENETIC ANALYSIS OF CLOSTRIDIUM PERFRINGENS: INSIGHT INTO EVOLUTION OF VIRULENCE". Diss., Tucson, Arizona : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1231%5F1%5Fm.pdf&type=application/pdf.

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Johansson, Anders. "Clostridium perfringens the causal agent of necrotic enteritis in poultry /". Uppsala : Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/200634.pdf.

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Correa, Alberto Enrique Estrada. "Studies of Clostridium perfringens Type A enteritis in the pig". Thesis, University of Glasgow, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328223.

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Leslie, Dario Lyall. "Genetic analysis of alpha toxin (phospholipase C) from Clostridium perfringens". Thesis, University of Newcastle Upon Tyne, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.346420.

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Bullifent, Helen Lisa. "The regulation of the alpha-toxin gene of Clostridium perfringens". Thesis, University of Sheffield, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296729.

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Nikraftar, Sarah. "Localization of Type IV Pilin Polymerization Proteins in Clostridium perfringens". Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/71742.

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Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS. We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT. In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells.
Master of Science

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Harry, Kathryn Helene. "Sporulation and enterotoxin regulation by sigma factors in Clostridium perfringens". Thesis, Virginia Tech, 2008. http://hdl.handle.net/10919/42517.

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Clostridium perfringens is a leading cause of food poisoning annually in the United States. Ingested C. perfringens vegetative cells respond to the acidic conditions of the stomach by initiating sporulation. The process of sporulation is essential in the formation of an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. During sporulation, the cell must differentiate into the mother cell and the forespore. Studies in 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 coordinate the development of the differentiating cell. These sigma factors are Ï F, Ï E, Ï G, and Ï K. The C. perfringens cpe gene, encoding the enterotoxin CPE, is transcribed from three promoters, P1, P2, and P3. P2 and P3 were previously proposed to be Ï E-dependent, and P1 was proposed to be Ï K-dependent based on consensus recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. In the sigE and sigK mutants, promoter fusion assays indicated that there was no transcription of cpe in either mutant. We also determined through transcriptional analyses that Ï E-associated RNA polymerase and Ï K-associated RNA polymerase co-regulate the transcription of each other. RT-PCR analyses indicated that Ï K is a very early acting sigma factor. The evidence provided here shows that the regulation of sporulation in C. perfringens is not the same as it is in B. subtilis, as previously proposed.
Master of Science

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Hendrick, William Anthony. "Molecular Analysis of Type IV Pilus Assembly in Clostridium perfringens". Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/81696.

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Clostridium perfringens is a Gram-positive anaerobe capable of causing disease in humans and many animals. C. perfringens is able to move across surfaces in a manner that is dependent on growth and type IV pili. Type IV pili are filaments that can be extended away from the cell by rapid polymerization, and retracted by depolymerization. Furthering the understanding of the initial and final energetic states of the pilins will reveal insights into possible mechanisms of type IV pilus assembly. Toward that end, a pilin was purified from the Gram-negative pathogen Pseudomonas aeruginosa and incorporated into an artificial membrane. The pilin was probed by a solid state nuclear magnetic resonance (ssNMR) technique that can determine the angle and depth of insertion of a helical peptide, as well as fluorescent and electron microscopy. All type IV pilus systems involve the action of an assembly ATPase to provide energy to polymerize the pilus. One proposed mechanism involves two primary proteins: an ATPase and an integral membrane core protein (IMCP). Other type IV pilus proteins are thought to play supportive roles in aiding the traversal of the cell envelope. In order to evaluate this model, the assembly ATPase PilB2 and IMCP PilC2 from C. perfringens were purified and examined for interactions. The evidence presented here suggest that PilB2 and PilC2 do not interact directly, and cannot function as a core assembly apparatus. The carbonic anhydrase (Cpb) from C. perfringens strain 13 was characterized both biochemically and physiologically. Cpb belongs to the type I subclass of the β class and is the first β class enzyme investigated from a strictly anaerobic bacteria. Kinetic analyses revealed a two-step, pingpong, zinc-hydroxide mechanism of catalysis. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semi-defined medium and an atmosphere without CO₂. The grew well 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.
Ph. D.

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37

Botlhoko, Tuelo David. "Performance of Clostridium perfringens-challenged broilers inoculated with effective microorganisms". Pretoria : [s.n.], 2010. http://upetd.up.ac.za/thesis/available/etd-02192010-172630.

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Azevedo, Edisio Oliveira de. "Avaliação de vacinas contra Clostridium perfringens tipos C e D". Universidade Federal de Minas Gerais, 1997. http://hdl.handle.net/1843/BUOS-8PQKNM.

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Seven commercial vaccines against clostridiosis. which had in their composition type C and/or D Closmdzum pemingens toxoids. have been evaluated for sterility, residual toxicity and potency. Six vaccines were produced in Brazil and one was imported, a bivalent toxoid standard of C. perjiingens type C and D has been used as a test control. All tested vaccines were sterile when cultivated in bacteria and fungi growth media. and were innocuous when administrated to mice via intraperitoneal route. ln relation to the potency, evaluated by mice semm neutralization tests using immunized rabbits sera pooled the imported vaccine and the standard toxoid have presented higher serum antibodies titres than the required minimum level of 10 and 5 IU/mL for beta and epsilon toxins produced from type C and D C. peyiirigens, respectively. The brazilian made vaccines were ineffective in stimulating serum antibodies levels specetie to beta and epilon toxins, according to the recommended test level for quality control. Four out of six brazilian made toxoids were also tested in goats but did not presefnt detectable antitoxins levels within the test level of L+ or L+/W used for beta and epsilon toxins. respectively. The toxins utilized have been produced cultivating C. pemingens within in dialisys membrane.
Sete vacinas comerciais contra clostridioses, que continham em sua composição toxóides de Clostridium perfringens tipos C e/ou D, foram avaliadas quanto à esterilidade, inocuidade c eficiência. Seis vacinas foram produzidas no Brasil e uma foi importada. Como controle dos testes, empregou-se um toxóide bivalente padrão de C. perfringens C e D. Todas as vacinas testadas foram estéreis quando semeadas em meios para pesquisa dc bactérias e fungos e mostram-se inócuas quando administradas por via intraperitoneal em camundongos. Quanto à eficiência, determinada pelo teste de soro-neutralização em camundongos a partir do `pool" de soros de coelhos imunizados, a vacina importada e o toxóide padrão, apresentaram títulos de anticorpos séricos superiores aos níveis mínimos exigidos de 10 e 5 UI/mL para as toxinas beta e épsilon, produzidas por C. perfringens tipos C e D, respectivamente. As vacinas produzidas no Brasil foram ineficientes em estimular níveis sorológicos de beta e épsilon antitoxinas compatíveis com os níveis de teste recomendados para controle destes produtos. Quatro dos seis toxóides de origem nacional, testados em caprinos, não apresentaram níveis de antitoxinas detectáveis com os níveis de testes de L+ ou L+/10 para beta e épsilon toxinas, respectivamente. As toxinas utilizadas para realização dos testes foram produzidas em membrana de diálise.

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Alnassan, Alaa Aldin. "Kokzidien und Clostridium perfringens: Studien an Koinfektionsmodellen zur Induktion und Bekämpfung der Nekrotischen Enteritis beim Huhn". Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-190110.

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Die nekrotische Enteritis (NE) und Kokzidiose des Huhnes sind häufige Darmerkrankungen weltweit und führen jedes Jahr zu hohen Wirtschaftsverlusten als Folge der Mortalität und Kosten für Behandlung und Bekämpfung. Die beiden Erkrankungen werden meistens bei Mastbroilern zwischen der 3. und 6. Lebenswoche festgestellt. Weil die leistungsfördernden Antibiotika im Geflügelfutter in der EU nicht mehr eingesetzt werden dürfen, ist das Risiko der NE in den letzten Jahren gestiegen. Fischmehl, Stress und Krankheiten sind prädisponierende Faktoren für die Entstehung der NE aber auch die Hühnerkokzidiose spielt in der Hinsicht eine wichtige Rolle. Die Mechanismen der Pathogenese bei der Wechselwirkung zwischen Kokzidiose und NE sind unklar. Bisher wurden verschiedene Infektionsmodelle zur Erforschung der NE unter Beteiligung von Ko-Infektionen zwischen Eimerien und C. perfringens eingesetzt. Dabei steht neben der Krankheitsentstehung auch die effiziente Bekämpfung dieser Erkrankung als großes Problem der Geflügelindustrie im Forschungsmittelpunkt. C. perfringens ist auch bei gesunden Tieren ein häufiger Darmbewohner, wobei bestimmte Stämme verschiedene Toxintypen wie Alpha-, TpeL- und Beta-Toxine produzieren können und damit ursächlich zur Entstehung der NE beitragen, aber ohne andere prädisponierende Auslöser kommt es in der Regel nicht zum Ausbruch. In der vorliegenden Arbeit wurden zwei In-vivo-Modelle zur experimentellen Induktion der NE des Huhnes entwickelt. Ziele waren sowohl die genauere Untersuchung der Pathogenese der NE durch Ko-Infektionen mit Eimerien und Kokzidien als auch die Evaluierung von potentiellen neuartigen Bekämpfungsmöglichkeiten. Außerdem wurde ein In-ovo-Modell etabliert.

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Petit, Laetitia. "Toxine epsilon de clostridium perfringens : mode d'action dans le modele cellulaire mdck et analyse de sa production chez c. perfringens (doctorat : micrbiologie)". Paris 11, 1998. http://www.theses.fr/1998PA114842.

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Farrow, Kylie Ann 1973. "Analysis of clostridial MLS resistance determinants". Monash University, Dept. of Microbiology, 2001. http://arrow.monash.edu.au/hdl/1959.1/8319.

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Marvaud, Jean-Christophe. "Contribution a l'etude des proteines associees aux neurotoxines clostridiennes et vectorisation de proteines dans les cellules (doctorat : microbiologie)". Paris 11, 1998. http://www.theses.fr/1998PA114851.

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Ali, Ali Abdulkareem Ali. "Isolation and characterization of Clostridium perfringens bacteriophages and optimization of electro-transformation parameters for Clostridium difficile". Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42289.

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Clostridium perfringens (C. perfringens) is responsible for a variety of diseases in humans and animals. Clostridium difficile (C. difficile) is the leading cause of antibiotic-associated diarrhoea. The available treatments for infections caused by both pathogens are not effective. Bacteriophage (phage) therapy is a promising treatment strategy to tackle and treat any infections or diseases caused by C. perfringens or C. difficile. The aim of this study was to isolate bacteriophages that infect C. perfringens and characterize them, and to optimize electroporation parameters for C. difficile with the end goal being to be able to genetically modify C. difficile phages. Five phages that infect C. perfringens were isolated and characterized from environmental samples. Two phages were sequenced and annotated, one was found to be a Podovirus and the other a Siphovirus. The Podovirus is a strictly lytic phage that does not possess any undesired genes such as the transduction gene, antibiotic resistance gene, and toxin genes. The endolysin of the Podovirus was cloned, expressed, and purified. The muralytic activity of the enzyme was confirmed by a zymogram. This endolysin has the ability to completely lyse 85.7 % of the tested Clostridium perfringens strains. A series of electroporation experiments were carried out using many experimental settings and varying parameters in order to deliver engineered phage and plasmid DNA genome to C. difficile. The electroporation refractory C. difficile R076 was treated with a cysteine protease inhibitor E- 64 and lysostaphin to facilitate electroporation. E-64 was able to reduce the thickness of the C. difficile surface layer proteins. The treatment with lysostaphin resulted in cell lysis. Unfortunately, all the attempts made to optimize the electroporation protocol for C. difficile were unsuccessful as no cells were transformed. However, the experimental observations provide a strong foundation for further work to develop an effective electro-transformation protocol for C. difficile.

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Pérez, Janampa David Remy. "Genotipificación y subtipificación de Clostridium perfringens aislados de crías de alpacas muertas por enterotoxemia". Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2006. https://hdl.handle.net/20.500.12672/665.

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La enterotoxemia, causada por el Clostridium perfringens, es la enfermedad infecciosa más importante que afecta a las alpacas, debido a que ocasiona elevadas tasas de mortalidad neonatal de hasta 70%. A pesar de esto, existe poca información sobre los factores de virulencia (toxinas) del C. perfringens que participan en la etiopatogénesis de la enfermedad. El presente estudio tuvo como objetivo determinar el genotipo de los C. perfringens aislados de casos de enterotoxemia en base a la presencia de los genes (cpa, cpb, etx e iap) codificantes de las toxinas principales (α, β, ε y ι) así como el subtipo en base a la presencia de los genes cpe y cpb2 codificantes de la enterotoxina (CPE) y la toxina β2, respectivamente. En el estudio se analizaron 47 aislamientos de C. perfringens obtenidos de intestino de crías muertas de alpaca con signos clínicos y lesiones anatomopatológicas e histopatológicas correspondientes a enterotoxemia. El ADN de estos aislados fue extraído y analizado por la técnica de PCR Múltiple conteniendo iniciadores específicos para los genes codificantes de las toxinas mencionadas, encontrándose en 33/47 (70.2%) aislamientos sólo al gen cpa (genotipo A subtipo cpe-cpb2-), en 13/47 (27.7%) a los genes cpa y cpb2 (genotipo A subtipo cpe-cpb2+) y en 1/47 (2.1%) a los genes cpa, cpb y cpe ( genotipo A subtipo cpe+cpb2-). Estos resultados evidencian principalmente a la toxina α, así como a la β2 y β participar en la etiopatogénesis de la enterotoxemia en las alpacas. Palabras Clave: Clostridium perfringens, genotipificación, enterotoxemia, alpacas.
--- Enterotoxemia, caused by the Clostridium perfringens, is the most important infectious disease which affects alpacas, causing up to 70% neonatal mortality. In spite of this, little information exists on the virulence factors (toxins) of C. perfringens which play an important role in the etiopathogenesis of the disease. The objective of the present study was to determine the genotype of C. perfringens isolated from cases of enterotoxemia based on the presence of genes (cpa, cpb, etx and iap) which encode the main toxins (α, β, ε and ι), as well as the subtypes based on the presence of genes cpe and cpb2 which encode the enterotoxin (CPE) and β2-toxin respectively. A total of 47 isolations of C. perfringens were obtained from the small intestine of neonatal alpaca mortalities which had both clinical signs and gross and histological injuries typical of enterotoxemia. The DNA was extracted from these isolates and analyzed by PCR Multiplex using specific primers for the toxin genes. The cpa gene genotype A subtype cpe-cpb2-) was the only gene found in 70.2% (33/47) of the isolations. In 27.7% (13/47) of the cases the cpa and cpb2 (genotype A subtype cpe-cpb2+) genes were found and in 2.1% (1/47) the cpa, cpb and cpe (genotype C subtype cpe+cpb2-) genes were present. These results demonstrate the primary role of α-toxin, as well as the presence of β2 and β-toxins in the etiopathogenesis of enterotoxemia in alpacas. Key Words: Clostridium perfringens, genotypification, enterotoxemia, alpacas.
Tesis

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Jepson, Marie Alice. "The role of the C-Domain of clostridium perfringens Î±-toxin". Thesis, Birkbeck (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247080.

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McGinley, Susan. "Clostridium perfringens: New Ways to Type Strains of a Deadly Bacteria". College of Agriculture and Life Sciences, University of Arizona (Tucson, AZ), 1999. http://hdl.handle.net/10150/622290.

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GUILLOUARD, ISABELLE. "Organisation structurale et fonctionnelle de la toxine alpha de clostridium perfringens". Paris 7, 1997. http://www.theses.fr/1997PA077115.

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La toxine-alpha de clostridium perfringens, principal facteur de virulence produit par les souches pathogenes, joue un role preponderant dans le developpement de la gangrene gazeuse. Phospholipase c, elle peut hydrolyser la phosphatidylcholine (lecithine) et la sphingomyeline, acides gras majeurs des membranes des cellules eucaryotes. Elle est aussi letale, necrosante et hemolytique. Bien caracterisee biochimiquement, les relations existant entre la structure et le mode d'action de la toxine alpha sont encore imprecises. Grace a la remarquable similitude existant entre les 2/3 n-terminaux de la toxine-alpha et la phospholipase c de bacillus cereus (pc-plc) bien decrite structuralement, nous avons determine la position du site catalytique dans la region n-terminale de la toxine alpha. Par mutagenese dirigee, nous avons identife les residus qui composent son site actif et qui sont tous conserves dans la pc-plc : residus histidine impliques dans la coordination avec les atomes de zinc, residus acides (asp et glu) constituant la partie chargee du site actif et residus aliphatiques necessaires pour sa structure. De plus, grace au variant asp56asn completement inactif, nous avons montre que l'activite phospholipasique etait responsable de la letalite due a la toxine-alpha. Pour determiner le role du domaine c-terminal de 120 acides amines completement depourvu d'activite enzymatique in vitro, nous avons examine les residus tyrosine indispensables pour l'interaction avec les membranes cellulaires et la reconnaissance du phospholipide. De plus, un site de fixation a faible affinite pour le calcium, cation necessaire a l'activite de la toxine-alpha, a aussi ete caracterise au niveau de residus aspartate de la partie c-terminale. Le calcium interviendrait dans l'interface entre la proteine et les membranes cellulaires.

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Horton, William Henry Clay. "Characterization of the Components of Carbon Catabolite Repression in Clostridium perfringens". Thesis, Virginia Tech, 2004. http://hdl.handle.net/10919/36119.

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Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens
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Gomes, Alexis de Matos. "Isolamento e tipifícação genotípica de Clostridium Perfringens em frangos de corte". Universidade Federal de Minas Gerais, 2007. http://hdl.handle.net/1843/VETC-7AXLVJ.

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Clostridium perfringens is an anaerobic Gram-positive bacterium which causes gaseous gangrene and enterotoxaemias in humans and domestic animals besides being the primary cause of necrotic enteritis in poultry. Clostridium perfringens strains were isolated 171/250 (68.4%) from the intestinal content of broiler chickens sampled in a slaughterhouse in Pará de Minas, MG. Clostridium perfringens were identified according to Gram staining, lecithinase test on agar TSC-egg yolk, haemolysis in desfibrinated horse blood agar and biochemical tests. Clostridium perfringens strains were classified into five toxigenic types (A -E), which were based on the detection of codifying alfa (cpa), beta (cpb), epsilon (etx) e iota (iA) genes, using multiplex PCR assay for genotyping of the principal and lethal toxins, beta2 toxin (cpb2) and enterotoxin (cpe). Clostridium perfringens strains were isolated in 62/125 (49.60%) from jejunum content and 109/125 (87.20%) strains from ileum. From a total of 62 Clostridium perfringens jejunum isolates obtained 42/62 (67.74%) were type A, 1/62 (1.61%) type A with amplification of products for beta2 toxin gene, 0/62 (0%) type B, 17/62 (27.42%) type C and 1/62 (1.61%) type D. A total of 109 ileum Clostridium perfringens isolates were obtained being 62/109 (56.88%) type A, 3/109 (2.75%) type A with amplification of products for beta2 toxin gene, 1/62 (0.92%) type B, 38/109 (34.86%) type C, 1/109 (0.92%) type D. Clostridium perfringens A (60.82%) and Clostridium perfringens C (32.16%) toxigenic types were the most prevalent types in the analyzed parts of the intestinal content of broiler chickens. Five (2.92%) out of a total of 171 Clostridium perfringens strains were not typified. Clostridium perfringens codifying the iota toxin gen and enterotoxin gene were not detected.
Clostridium perfringens, bactéria anaeróbica Gram positiva, além de causar gangrena gasosa e enterotoxemia em humanos e animais domésticos, constitui a principal causa de enterite necrótica em aves. Amostras de 171/250 (68.4%) Clostridium perfringens foram isolados de conteúdos intestinais de frangos de corte provenientes de um frigorífico da região de Pará de Minas-MG foram identificados pela coloração de Gram, reação de lecitinase em ágar TSC-gema de ovo, colônias com dupla hemólise em ágar sangue desfibrinado de eqüino e provas bioquímicas. As amostras de Clostridium perfringens podem ser classificadas em cinco tipos toxigênicos (A-E), pela detecção dos genes codificadores alfa (cpa), beta (cpb), épsilon (etx) e iota (iA), utilizando a técnica da PCR multiplex para tipificação genotípica das toxinas letais principais, da toxina cpb2 (cpb2) e enterotoxina (cpe). Clostridium perfringens foi isolado em 62/125 (49.60%) amostras de conteúdo do jejuno e em 109/125 (87.20%) de amostras do íleo. Das 62 amostras de Clostridium perfringens isolados do jejuno foram obtidos 42/62 (67.74%) tipo A, 1/62 (1,61%) tipo A com produto de amplificação para o gene da toxina beta2, 0/62 (0%) tipo B, 17/62 (27.42%) tipo C, 1/62 (1.61%) tipo D. Das 109 amostras de Clostridium perfringens isolados do íleo foram obtidos 62/109 (56.88%) tipo A, 3/109 (2.75%) tipo A com produto de amplificação para o gene da toxina beta2, 1/62 (0.92%) tipo B, 38/109 (34.86%) tipo C, 1/109 (0.92%) tipo D. Clostridium perfringens A (60.82%) e Clostridium perfringens C (32.16%) foram os tipos toxigênicos predominantes em conteúdo intestinal de frango de corte. Cinco (2.92%), das 171 amostras de Clostridium perfringens isolados não foram tipificadas. Não foram identificados os genes codificadores das toxinas iota e enterotoxina.

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Fohler, Svenja [Verfasser]. "Occurrence of Clostridium botulinum neurotoxin genes and toxin-genotypes of Clostridium perfringens in dairy cattle / Svenja Fohler". Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2016. http://d-nb.info/1104403897/34.

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