Occurrence of Clostridium perfringens in Wild Mammals in the Amazon Biome.

The objective of this study was to evaluate the occurrence of Clostridium perfringens in stool samples and swabs collected from wild mammals in the Amazon biome. Sixty-five faecal and swab samples were collected in situ and ex situ from 16 species and three genera of wild mammals, some of which were in good health and some of which had diarrhoea. After pre-enrichment, the samples were plated on selective agar for C. perfringens. Characteristic colonies were subjected to multiplex PCR for the detection of genes encoding the main C. perfringens toxins (alpha, beta, epsilon, and iota toxin and enterotoxin). Among the 65 samples, 40 (61.5%) were positive for the gene encoding the alpha toxin and were classified as type A, 36 of which were asymptomatic animals and four were diarrheal. No other toxinotypes were found. The findings of this study suggest that C. perfringens type A is commonly found in mammal species of the Amazon biome. This seems to be the first study to identify C. perfringens type A in species such as B. variegatus (common ground sloth), C. didactylus (two-toed sloth), P. flavus (Jupará), T. tetradactyla (anteater), S. collinsi (squirrel monkey), S. niger (black marmoset), and S. apella (Guyana capuchin) and in the genus Didelphis sp. (opossum).

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry's production of vaccines against the action of C. perfringens in equine species.

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Necrohemorrhagic enteritis outbreak in a cattle feedlot in Nova Crixás, Goiás, Brazil / Surto de enterite necro-hemorrágica em um confinamento de bovinos no município de Nova Crixás, Goiás, Brasil

This study described an outbreak of necrohemorrhagic enteritis in a beef cattle feedlot in Nova Crixás, State of Goiás, Brazil, with emphasis on epidemiological, lesional, and laboratory aspects. Visits to the property were carried out and a necroscopic examination was performed on the bovine cadavers (N=57), which presented similar macroscopic alterations. Epidemiological data were collected, mainly referring to the feeding management of animals, and tissue samples were submitted to histopathological examination. Samples of feces and intestinal contents were also collected for bacterial isolation and PCR genotyping to detect the etiological agent, being confirmed Clostridium perfringens type A strains in 100% of the samples. Furthermore, 33.3% of strains isolated from intestinal contents and 40% of those isolated from feces were positive for beta-2 encoding gene. Considering the history, macroscopic and microscopic findings, as well as bacterial isolation and PCR, the diagnosis of bovine necrohemorrhagic enteritis was determined.(AU)

Descreve-se um surto de enterite necro-hemorrágica em um confinamento de bovinos de corte no município de Nova Crixás, Estado de Goiás, Brasil, com ênfase nos aspectos epidemiológicos, lesionais e laboratoriais. Foram realizadas visitas à propriedade e todos os cadáveres bovinos (N=57) foram submetidos ao exame necroscópico, os quais apresentaram alterações macroscópicas semelhantes. Foram compilados dados epidemiológicos, sobretudo referentes ao manejo alimentar dos animais e amostras de tecido foram submetidas a exame histopatológico. Foram colhidas, também, amostras de fezes e conteúdo intestinal para isolamento bacteriano e genotipagem por PCR para detecção do agente etiológico, sendo confirmadas estirpes de Clostridium perfringens tipo A em 100% das amostras. Ainda, 33,3% das cepas de Clostridium perfringens isoladas no conteúdo intestinal e 40% daquelas isoladas nas fezes foram positivas para o gene codificador da toxina beta-2. Considerando o histórico, os achados macroscópicos e microscópicos, o isolamento bacteriano e o PCR, foi estabelecido o diagnóstico de enterite necro-hemorrágica por C. perfringens tipo A.(AU)

Necrohemorrhagic enteritis outbreak in a cattle feedlot in Nova Crixás, Goiás, Brazil / Surto de enterite necro-hemorrágica em um confinamento de bovinos no município de Nova Crixás, Goiás, Brasil

This study described an outbreak of necrohemorrhagic enteritis in a beef cattle feedlot in Nova Crixás, State of Goiás, Brazil, with emphasis on epidemiological, lesional, and laboratory aspects. Visits to the property were carried out and a necroscopic examination was performed on the bovine cadavers (N=57), which presented similar macroscopic alterations. Epidemiological data were collected, mainly referring to the feeding management of animals, and tissue samples were submitted to histopathological examination. Samples of feces and intestinal contents were also collected for bacterial isolation and PCR genotyping to detect the etiological agent, being confirmed Clostridium perfringens type A strains in 100% of the samples. Furthermore, 33.3% of strains isolated from intestinal contents and 40% of those isolated from feces were positive for beta-2 encoding gene. Considering the history, macroscopic and microscopic findings, as well as bacterial isolation and PCR, the diagnosis of bovine necrohemorrhagic enteritis was determined.(AU)

Descreve-se um surto de enterite necro-hemorrágica em um confinamento de bovinos de corte no município de Nova Crixás, Estado de Goiás, Brasil, com ênfase nos aspectos epidemiológicos, lesionais e laboratoriais. Foram realizadas visitas à propriedade e todos os cadáveres bovinos (N=57) foram submetidos ao exame necroscópico, os quais apresentaram alterações macroscópicas semelhantes. Foram compilados dados epidemiológicos, sobretudo referentes ao manejo alimentar dos animais e amostras de tecido foram submetidas a exame histopatológico. Foram colhidas, também, amostras de fezes e conteúdo intestinal para isolamento bacteriano e genotipagem por PCR para detecção do agente etiológico, sendo confirmadas estirpes de Clostridium perfringens tipo A em 100% das amostras. Ainda, 33,3% das cepas de Clostridium perfringens isoladas no conteúdo intestinal e 40% daquelas isoladas nas fezes foram positivas para o gene codificador da toxina beta-2. Considerando o histórico, os achados macroscópicos e microscópicos, o isolamento bacteriano e o PCR, foi estabelecido o diagnóstico de enterite necro-hemorrágica por C. perfringens tipo A.(AU)

Necrohemorrhagic enteritis outbreak in a cattle feedlot in Nova Crixás, Goiás, Brazil

ABSTRACT: This study described an outbreak of necrohemorrhagic enteritis in a beef cattle feedlot in Nova Crixás, State of Goiás, Brazil, with emphasis on epidemiological, lesional, and laboratory aspects. Visits to the property were carried out and a necroscopic examination was performed on the bovine cadavers (N=57), which presented similar macroscopic alterations. Epidemiological data were collected, mainly referring to the feeding management of animals, and tissue samples were submitted to histopathological examination. Samples of feces and intestinal contents were also collected for bacterial isolation and PCR genotyping to detect the etiological agent, being confirmed Clostridium perfringens type A strains in 100% of the samples. Furthermore, 33.3% of strains isolated from intestinal contents and 40% of those isolated from feces were positive for beta-2 encoding gene. Considering the history, macroscopic and microscopic findings, as well as bacterial isolation and PCR, the diagnosis of bovine necrohemorrhagic enteritis was determined.

RESUMO: Descreve-se um surto de enterite necro-hemorrágica em um confinamento de bovinos de corte no município de Nova Crixás, Estado de Goiás, Brasil, com ênfase nos aspectos epidemiológicos, lesionais e laboratoriais. Foram realizadas visitas à propriedade e todos os cadáveres bovinos (N=57) foram submetidos ao exame necroscópico, os quais apresentaram alterações macroscópicas semelhantes. Foram compilados dados epidemiológicos, sobretudo referentes ao manejo alimentar dos animais e amostras de tecido foram submetidas a exame histopatológico. Foram colhidas, também, amostras de fezes e conteúdo intestinal para isolamento bacteriano e genotipagem por PCR para detecção do agente etiológico, sendo confirmadas estirpes de Clostridium perfringens tipo A em 100% das amostras. Ainda, 33,3% das cepas de Clostridium perfringens isoladas no conteúdo intestinal e 40% daquelas isoladas nas fezes foram positivas para o gene codificador da toxina beta-2. Considerando o histórico, os achados macroscópicos e microscópicos, o isolamento bacteriano e o PCR, foi estabelecido o diagnóstico de enterite necro-hemorrágica por C. perfringens tipo A.

Quercetina atenúa la virulencia de Staphylococcus aureus al disminuir la secreción de alfa toxina / Quercetin attenuates Staphylococcus aureus virulence by reducing alpha-toxin secretion

Alfa toxina, una proteína formadora de poros con actividad citotóxica, es uno de los principales factores de virulencia secretados por la mayoría de las cepas de Staphylococcus aureus. Se ha establecido la relevancia de esta proteína en la patogenia de la neumonía asociada a infecciones por S. aureus. Por lo tanto, la inhibición de la secreción de alfa toxina puede ser una alternativa en el control de las infecciones causadas por este microorganismo. En este trabajo mostramos que quercetina, un flavonoide de origen natural, inhibe de manera dosis dependiente la actividad hemolítica y disminuye la secreción de alfa toxina en sobrenadantes de cultivos de S. aureus sensible y resistente a meticilina. Además, quercetina previene de manera significativa el daño de células alveolares humanas cuando se co-cultivan con S. aureus. Nuestros datos sugieren que quercetina puede disminuir la virulencia de S. aureus al afectar la secreción de alfa toxina.

Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S. aureus infections has already been esta blished. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S. aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S. aureus. Our results suggest that quercetin can reduce S. aureus virulence by affecting alpha-toxin secretion.

Immunization with a nontoxic naturally occurring Clostridium perfringens alpha toxin induces neutralizing antibodies in rabbits.

Clostridium perfringens alpha toxin, encoded by plc gene, has been implicated in gas gangrene, a life threatening infection. Vaccination is considered one of the best solutions against Clostridium infections. Although studies have identified many low quality clostridial vaccines, the use of recombinant proteins has been considered a promising alternative. Previously, a naturally occurring alpha toxin isoform (αAV1b) was identified with a mutation at residue 11 (His/Tyr), which can affect its enzymatic activity. The aim of the present study was to evaluate whether the mutation in the αAV1b isoform could result in an inactive toxin and was able to induce protection against the native alpha toxin. We used recombinant protein techniques to determine whether this mutation in αAV1b could result in an inactive toxin compared to the active isoform, αZ23. Rabbits were immunized with the recombinant toxins (αAV1b and αZ23) and with native alpha toxin. αAV1b showed no enzymatic and hemolytic activities. ELISA titration assays showed a high titer of both anti-recombinant toxin (anti-rec-αAV1b and anti-rec-αZ23) antibodies against the native alpha toxin. The alpha antitoxin titer detected in the rabbits' serum pool was 24.0 IU/mL for both recombinant toxins. These results demonstrate that the inactive naturally mutated αAV1b is able to induce an immune response, and suggest it can be considered as a target for the development of a commercial vaccine against C. perfringens alpha toxin.

[Quercetin attenuates Staphylococcus aureus virulence by reducing alpha-toxin secretion]. / Quercetina atenúa la virulencia de Staphylococcus aureus al disminuir la secreción de alfa toxina.

Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S.aureus infections has already been established. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S.aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S.aureus. Our results suggest that quercetin can reduce S.aureus virulence by affecting alpha-toxin secretion.

Vaccination with recombinant Clostridium perfringens toxoids α and ß promotes elevated antepartum and passive humoral immunity in swine.

Due to the increasingly restricted use of antimicrobials in animal production systems, the prevention and control of Clostridium perfringens type A- and C-induced diarrhea in piglets should be based on passive immunization via the prepartum vaccination of sows. Given the current obstacles in the production of conventional clostridial vaccines, the use of recombinant proteins has been considered to represent a promising alternative. In the present study, the neutralizing antibody response of immunized sows and their litters to a bivalent vaccine containing the C. perfringens recombinant toxoids alpha (rTA) and beta (rTB) produced in Escherichia coli was assessed. Rabbits (n=8) and pregnant sows (n=7) were immunized with 200µg of each recombinant antigen using Al(OH)3 as adjuvant. The alpha and beta antitoxin titer detected in the rabbits' serum pool was 9.6 and 20.4IU/mL, respectively. The mean alpha and beta antitoxin titers in the sows' sera were 6.0±0.9IU/mL and 14.5±2.2IU/mL, and the corresponding individual coefficients of variation (CV) were 16.04% and 14.91%, respectively. The mean alpha and beta antitoxin titers in the litters' serum pools were 4.2±0.4IU/mL and 10.9±1.7IU/mL, and the CV between litters was 9.23% and 9.85%, respectively. The results showed that the rTA and rTB proteins produced and tested in the present study induced an immune response and can be regarded as candidates for the development of a commercial vaccine against C. perfringens type A- and C-induced diarrhea in pigs.

Avaliação da potência de vacinas contra clostridium septicum comercializadas no brasil

ABSTRACT Clostridium septicum, a ubiquitous organism, is the main pathogen causing malignant edema. Because of its strong cytotoxic alpha toxin, infections are often lethal. Losses in livestock can be prevented through vaccination with alpha-toxoid vaccines. The antibody response to the C. septicum component of 12 commercial vaccines was evaluated by the mouse serum neutralization test using rabbit and bovine sera. The vaccines coded as T1, T2, T4, T10 and T11 induced titers of alpha antitoxin higher than the minimum test level of 2.5 IU/mL. None of the other vaccines induced the formation of antibodies. The majority of the C. septicum vaccines commercialized in Brazil were unable to induce an immune response as recommended for the control of this product.

RESUMO Clostridium septicum, um organismo ubiquitário, é o principal patógeno responsável pelo quadro de edema maligno. Devido à ação citotóxica da toxina alfa as infecções geralmente são fatais. Para o controle da doença é utilizada a vacinação com toxóide alfa. Neste trabalho, avaliouse a eficiência de 12 vacinas comerciais contra clostridioses, que continham em sua composição C. septicum, através da soroneutralização em camundongos com soros de coelhos e bovinos vacinados. As vacinas codificadas como T1, T2, T4, T10 e T11 apresentaram títulos de antitoxina alfa superiores ao nível mínimo exigido pelo teste de 2,5 UI/mL. As demais vacinas não induziram a formação de anticorpos. Em sua maioria, as vacinas comercializadas no Brasil, contra C. septicum, foram ineficientes em estimular resposta imune compatível com os níveis de teste recomendados para controle deste produto.

Detection of bovine Clostridium perfringens by polymerase chain reaction

A polymerase chain reaction (PCR) assay to detect Clostridium perfringens alpha toxin gene (cpa) was used to identify eighty-nine C. perfringens strains obtained from bovine clinical material. The strains were biochemically characterized as C. perfringens. The isolated strains were cultured on plates containing brain heart infusion agar with 5% sheep blood under anaerobic conditions. DNA extraction was performed by boiling. The 324 bp amplification product of cpa was observed in all isolates. C. sordellii, C. botulinum, C. novyi, and C. septicum were also tested but did not produce any alpha toxin gene amplification. These findings suggest that PCR is a useful assay in identifying C. perfringens toxin types.