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Production and purification of epsilon prototoxin produced by Clostridium perfringens type D

Produção e purificação de prototoxina epsilon produzida pelo Clostridium perfringens tipo D

Abstract

A prototoxina epsilon foi obtida a partir de cultivo líquido, rico em carboidratos, a partir de amostra de C. perfringens tipo D (INTA - Argentina), incubado em condições de anarobiose à 37ºC por oito horas. O sobrenadante da cultura foi concentrado utilizando-se o sistema Amicon com membrana de "cut off" de 10 kDa. O concentrado foi precipitado com sulfato de amônio (350 g/ml) e dissolvido em tampão fosfato 0,01 M (pH 7,2). A prototoxina purificada por cromatografia de troca iônica (DEAE-Sepharose CL 6B) e isolada no primeiro pico, mostrou uma banda única de aproximadamente 34kDa de peso molecular em SDS-PAGE, além de ser tóxica em camundongos.

Clostridium perfringens; enterotoxemia; purificação; toxina epsilon; cromatografia de troca iônica; SDS-PAGE


Clostridium perfringens; enterotoxemia; purification; epsilon toxin; ion exchange chromatography

Clostridium perfringens; enterotoxemia; purificação; toxina epsilon; cromatografia de troca iônica; SDS-PAGE

COMMUNICATION

[Comunicação]

Production and purification of epsilon prototoxin produced by Clostridium perfringens type D

[Produção e purificação de prototoxina epsilon produzida pelo Clostridiumperfringens tipo D]

P.M. Parreiras1, F.C.F. Lobato1,C.F.L.G.D. Heneine2, R.A. Assis1, G.M. Balsamão3, R.A.P. Nascimento4

1Departamento de Medicina Veterinária Preventiva da Escola de Veterinária da UFMG

Caixa Postal 567

30123-970 – Belo Horizonte, MG

2Departamento de Pesquisa – FUNED

3Laboratório TECSA-Brasília

4Ministério da Agricultura, Pecuária e Abastecimento

Recebido para publicação em 9 de agosto de 2000.

Recebido para publicação, após modificações, em 13 de março de 2002.

E-mail: parreira_2000@yahoo.com.br

Enterotoxemia is a term applied to a group of enteric infections associated to different types of pathogenic Clostridium perfringens, being the type D, the more frequently found in herbivorous. These microorganism is of wide distribution in the world and determines an infectious process of high lethality. Enterotoxemia is produced by an abnormal proliferation of the agent in the intestinal tract, and it is cause of disease only in special circumstances, which are not completely elucidated (Silveira et al., 1995).

Enterotoxemia from C. perfringens occurs mainly in animals between three days and six months of age. However, the disease has been described in adult animals (Sigurdarson & Thorsteinson, 1990). Although the diagnosis of enterotoxemia is usually based on clinical signs and post-mortem examination, the laboratorial analysis is important to confirm the presence of the epsilon toxin produced by C. perfringens type D (Silveira et al., 1995).

The test commonly used for the diagnosis of the enterotoxemia is the toxin neutralization test in mice, which requires long periods of observation and a large number of animals for its accomplishment (Wood, 1991; El Idrissi & Ward, 1992a,b).

Among the in vitro tests used for detection of epsilon toxin, the enzyme linked immunosorbent assays - ELISA has presented the best results regarding sensitivity. In addition, it is a quantitative, fast and specific test (Uzal et al., 1997). Standardization of ELISAs requires the use of prototoxin and specific antibodies; however their production and purification constitute one of the greatest constraints for its implementation.

This paper describes the production, concentration and purification of the epsilon prototoxin from C. perfringens type D for use in the standardization of an ELISA to diagnose enterotoxemia in herbivorous.

Epsilon prototoxin was produced from C. perfringens type D, provided by INTA - National Institute of Agricultural Technology, Argentina, cultured in broth thioglycollate (Diagnolab, Barcelona-Spain) and incubated at 37ºC under anaerobic conditions. After 16 hours of growth, 10ml of culture were transferred to 500ml of medium, as previously described by Lobato et al. (2000), and incubated at 37ºC under anaerobic conditions for eight hours. The culture suspension was centrifuged at 8,000×g, at 4ºC, for 30 minutes (Azevedo et al., 1998).

The culture supernatant was concentrated 10 times using a titration amicon system (Amicon, Bervely, USA) with a 10kDa membrane (Amicon, Bervely, USA) for retention of the epsilon prototoxin. The material was precipitated with saturated ammonium sulphate (350g/l) and ressuspended in 0.01M phosphate buffer, pH 7.2 (Sigma, St. Louis, USA). The prototoxin was purified by ionic change chromatography (DEAE - Sepharose CL 6B) (Pharmacia, Uppsala-Sweeden) (Uzal et al., 1997) and the first pick was analysed by electrophoresis.

The purified prototoxin was activated with a solution of trypsin (Sebald & Petit, 1997) and incubated at 37ºC for 30 minutes and its toxigenicity was tested in mice. After activation, 0.2 ml of toxin was inoculated intravenously in four mice (Swiss, lineage Webster) of both sexes, weighing around 17-20 g. The same filtrate, without trypsin activation, was inoculated as control in four mice. The animals were observed for 72 hours regarding emergence of symptoms and death (Sebald & Petit, 1997).

The electrophoresis showed a pure band of approximate molecular weight of 34 kDa (Fig.1). According to Sakurai et al. (1997), this molecular weight corresponds to the epsilon prototoxin, produced by C. perfringens type D. The animals that received prototoxin treated with trypsin, died within 24 hours, while control animals remained alive.


Uzal et al. (1997) used two columns of DAE - Sepharose CL6B equilibrated with 0,01M Tris, pH 7.5, followed by Sephadex G25 column, equilibrated with 0,01M Tris, pH 7.5. In the present work the methodology used to purify the epsilon prototoxin was similar to that used by Uzal et al. (1997), but a single DEAE – Sepharose CL6B column was satisfactory, providing a simpler and faster manner to purify the prototoxin. The production of purified epsilon prototoxin will allow the production of specific hyperimmune sera enabling the standardization ELISAS for the diagnosis of enterotoxemia.

Keywords: Clostridium perfringens, enterotoxemia, purification, epsilon toxin, ion exchange chromatography

RESUMO

A prototoxina epsilon foi obtida a partir de cultivo líquido, rico em carboidratos, a partir de amostra de C. perfringens tipo D (INTA – Argentina), incubado em condições de anarobiose à 37ºC por oito horas. O sobrenadante da cultura foi concentrado utilizando-se o sistema Amicon com membrana de "cut off" de 10 kDa. O concentrado foi precipitado com sulfato de amônio (350 g/ml) e dissolvido em tampão fosfato 0,01 M (pH 7,2). A prototoxina purificada por cromatografia de troca iônica (DEAE-Sepharose CL 6B) e isolada no primeiro pico, mostrou uma banda única de aproximadamente 34kDa de peso molecular em SDS-PAGE, além de ser tóxica em camundongos.

Palavras-chave: Clostridium perfringens, enterotoxemia, purificação, toxina epsilon, cromatografia de troca iônica, SDS-PAGE

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  • WOOD, K.R. An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi type B and Clostridium perfringens type D epsilon components of multivalent sheep vaccines. Biologicals, v.19, p.281-286, 1991.

Publication Dates

  • Publication in this collection
    16 Dec 2002
  • Date of issue
    June 2002

History

  • Received
    09 Aug 2000
Universidade Federal de Minas Gerais, Escola de Veterinária Caixa Postal 567, 30123-970 Belo Horizonte MG - Brazil, Tel.: (55 31) 3409-2041, Tel.: (55 31) 3409-2042 - Belo Horizonte - MG - Brazil
E-mail: abmvz.artigo@gmail.com