Clonagem, expressão, caracterizacao molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicacao em urn teste de ELISA por competicao

Abstract

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PRl-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. Te sequencing of the msp5 gene of PR1 isolate showed 98% of identity with the Florida and Saint Maries isolates, 97% with Brazil (Pernambuco) and Havana isolates; and 91% with A centrale. Te cELISA-PRl test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PRl, 380 bovine sera were used, whereas 245 truly positives and 135 truly negatives sera tested by the cELISA-USA. In the standardization of the cELISA-PRl 135 negative and 148 positive bovine sera were used. Te cELISA-PRl and IFI tests showed 100 and 99.3% specifcity, 100 and 98%, sensibility, and a kappa coefcient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PRl and IFI, which showed 96.7 and 69.1% specifcity, 98.9 e 96.3% sensibility and kappa coefcient of 0.956 and 0.699, respectively. Tese results indicate that the cELISA-PRl, likewise the cELISA-USA, could sensitively and specifcally detect cattle naturally infected with A. marginale and would be recommended for epidemiological studies, eradications program, and regulation of international cattle movement, while IFI, which presented lower specifcity should not be used in situations that demand more specifc diagnosis.