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Year 2007 - Volume 27, Number 07
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ELISA com MSP5 recombinante truncada para detecção de anticorpos contra Anaplasma marginale em bovinos, p.301-306
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ABSTRACT.- Melo E.S.P., Araújo F.R., Ramos C.A.N., Soares C.O., Rosinha G.M.S., Elisei C. & Madruga C.R. 2007. [ELISA based on recombinant truncated MSP5 for detection of antibodies against Anaplasma marginale in cattle.] ELISA com MSP5 recombinante truncada para detecção de anticorpos contra Anaplasma marginale em bovinos. Pesquisa Veterinária Brasileira 27(7):301-306. Embrapa Gado de Corte, Cx. Postal 154, Campo Grande, MS 79002-970, Brazil. E-mail: flabio@cnpgc.embrapa.br
The objective of this study was the production and solubilization of recombinant truncated MSP5 of Anaplasma marginale and the evaluation of its performance in an enzyme-linked immunosorbent assay (ELISA), to detect antibodies against the rickettsia in cattle. The fragment of msp5 gene, except the hydrophobic N-terminal region, was amplified by PCR, cloned in pTrcHis-TOPO plasmid and expressed in Escherichia coli. Solubilization of the recombinant protein was evaluated in different pHs and concentrations of urea. The sensibility and specificity of the assay were evaluated with 66 sera from cattle experimentally-infected and 96 sera from cattle free of A. marginale defined by polymerase chain reaction for msp5 gene. Serum samples from 1,666 cattle from Brazil - states of Rio Grande do Sul (73), Mato Grosso do Sul (91), Pernambuco (86), Bahia (314) and Minas Gerais (267), Uruguay (32) and Costa Rica (803), were tested by ELISAs with recombinant truncated MSP5 and with recombinant MSP1a, and the agreement between both ELISAs was calculated. ELISA with recombinant truncated MSP5 protein detected infected animals with sensibility of 96.97% and specificity of 100%. In cattle experimentally-infected, the ELISA detected antibodies from the 12th day post-infection (DPI) to the end of the experiment, at the 37th DPI. The agreement between the ELISAs with truncated MSP5 and MSP1a antigens was 95.67%, with a kappa index of 0.81. Disagreement results showed significative difference (p <0.001). Antibodies for A. marginale were detected in animals of the all the region analyzed. The ELISA with recombinant truncated MSP5 showed a good performance in ELISA for detention of antibodies against A. marginale, with high sensitivity and specificity, representing an important tool for the diagnosis of anaplasmose bovine in epidemiological studies. |
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