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Production and evaluation of a recombinant SeM protein for Strangles?? control

By Carina Martins de MORAES

Abstract

A Adenite Eq??ina ?? uma enfermidade contagiosa do trato respirat??rio superior dos eq????deos causada por Streptococcus equi subesp. equi. Animais portadores assintom??ticos respons??veis pela perman??ncia da infec????o nos rebanhos s?? podem ser detectados por m??todos microbiol??gicos ou sorol??gicos e as vacinas utilizadas no controle da doen??a induzem n??veis de prote????o geralmente n??o superiores a 50 %. Considerando que a prote??na SeM de S. equi ?? o ant??geno mais promissor na prote????o contra a doen??a, este trabalho objetivou produzir a prote??na SeM recombinante de S. equi, visando sua utiliza????o como ant??geno em vacinas e em ELISA. Prote??na SeM recombinante (rSeM) foi produzida mediante a clonagem e express??o em Escherichia coli e purificada por cromatografia de afinidade. Para testar sua capacidade imunog??nica, vacinas elaboradas com rSeM foram aplicadas a camundongos. F??meas Balb/c isog??nicas com 4-6 semanas foram divididas aleatoriamente e inoculadas por via SC com 1/20 da dose vacinal estimada para cavalos, nos dias 0 e 21 do experimento. Um grupo foi vacinado com 250 mL (12 mg mL-1) de prote??na recombinante sem adjuvante, outro com 300 mL de vacina contendo 12 mg mL-1 rSeM adicionada de 20% de hidr??xido de alum??nio, outros dois grupos com duas bacterinas comerciais contra Adenite Eq??ina; dois grupos com as vacinas comerciais, acrescidas de 12 mg mL-1 de rSeM e o grupo restante com uma bacterina contendo cepas de campo. O grupo controle foi inoculado com o mesmo volume de solu????o salina est??ril. Coletou-se sangue por pun????o do plexo venoso retro-ocular nos dias 0, 21 e 42. Os anticorpos foram titulados por ELISA utilizando a prote??na rSeM como ant??geno. A rSeM foi imunog??nica em camundongos com ??ndices de prote????o de 100%. Para a padroniza????o de um ELISA, utilizaram-se grupos de 20 soros equinos de animais negativos, vacinados e positivos. Utilizando um ponto de corte de m??dia das densidades ??pticas dos soros negativos acrescidos de dois desvios padr??o, o teste teve 100% de sensibilidade e especificidade.Strangles is a contagious disease of the upper respiratory tract of horses caused by Streptococcus equi subsp. equi. Asymptomatic carriers responsible for maintaining the infection in the herd can only be detected by serological or microbiological methods and vaccines used for the control of the disease induce levels of protection generally not exceeding 50%. Considering that S. equi SeM protein is considered the most promising antigen to protect against the disease, this research aimed to produce and evaluate as antigen for vaccines and for ELISA, a recombinant S. equi SeM protein (rSeM). rSeM was produced by cloning and expression in Escherichia coli and purified by affinity chromatography. To test its immunogenicity isogenic female Balb-c mice 4-6 weeks-old were randomly divided and inoculated with 1 / 20th of the estimated dose of the vaccine for horses by the SC route, on days 0 and 21 of the experiment. One group was vaccinated with 250mL (12 mg mL-1) of rSeM without adjuvant, another with 300mL of vaccine containing 12 mg mL-1 of rSeM plus 20% of aluminiun hydroxide, two other groups were vaccinated with two commercial bacterins against Strangles, other two groups were vaccinated with the same commercial vaccines containing 12 mg mL-1 of rSeM and the remaining group was inoculated with a bacterin produced with a field strain. The control group was inoculated the same dose of sterile saline. Blood samples were collected from the retro-orbital venous plexus on days 0, 21, 42. The antibodies were titrated by ELISA using rSeM as antigen. rSeM was immunogenic for mice with a protection index of 100%. For the standardization of an ELISA, groups of 20 negative, vaccinated and positive animals were used. Using as Cut-off the mean plus two SD of the Optical Densities of the negatives, the test showed 100% sensitivity and specificity

Topics: adenite equina, proteina recombinante, streptococcus equi, rSeM, vacina, ELISA, strangles, streptococcus equi, rSeM, ELISA, vaccines, CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOGENETICA
Publisher: Universidade Federal de Pelotas
Year: 2008
OAI identifier: oai:agregador.ibict.br.RI_UFPEL:oai:repositorio.ufpel.edu.br:123456789/1268
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