PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Ramos C.A.N., Araújo F.R., Souza I.I.F., Guedes Jr D.S., Oliveira R.H.M., Farias T.A., Oliveira J.B., Alves L.C. & Faustino M.A.G. 2010. [Comparison between several antigens for diagnosis of Anaplasma marginale by ELISA.] Comparação entre diversos antígenos para o diagnóstico de Anaplasma marginale por ELISA. Pesquisa Veterinária Brasileira 30(1):37-41. Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE 52171-900, Brazil. E-mail: carlosanramos@yahoo.com.br Bovine anaplasmosis is a major disease in tropical and subtropical regions of the world by determine economical loss due mortality and productive reduction. The disease is caused by Anaplasma marginale, an intraerythrocytic rickettsia whose control requires, besides an efficient vaccine, the accurate identification of chronically infected cattle. Although the existence of diverse methods of diagnosis of this rickettsia, the serological methods, in particular the enzyme immunosorbent assays (ELISAs), are the most used due to its versatility and practice. However, due to the high number of antigens currently available, an evaluation becomes necessary to define which antigens present the better performance in the diagnosis of anaplasmosis. Sera from cattle positive or negative to A. marginale by PCR, and sera from cattle proceeding from Brazil and Costa Rica, were tested by ELISAs based in recombinant MSP1a, MSP2, and MSP5, a pool of the three recombinant proteins, and initial body lisate antigen (CI). Using sera from A. marginale positive cattle by PCR, the highest sensitivity was shown by CI ELISA. Nevertheless, the highest specificity, with sera from negative cattle by PCR, was shown by recombinants ELISAs. The percentiles of positive cattle from Brazil and Costa Rica were higher with CI ELISA. Reasons for such differences were discussed.

• Anaplasma marginale

Abstract in Portuguese:

RESUMO.- Ramos C.A.N., Araújo F.R., Souza I.I.F., Guedes Jr D.S., Oliveira R.H.M., Farias T.A., Oliveira J.B., Alves L.C. & Faustino M.A.G. 2010. [Comparison between several antigens for diagnosis of Anaplasma marginale by ELISA.] Comparação entre diversos antígenos para o diagnóstico de Anaplasma marginale por ELISA. Pesquisa Veterinária Brasileira 30(1):37-41. Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE 52171-900, Brazil. E-mail: carlosanramos@yahoo.com.br Anaplasmose bovina é uma doença com grande importância nas regiões tropicais e subtropicais do mundo por determinar perdas econômicas devido à mortalidade e redução da produtividade. É causada por Anaplasma marginale, uma riquétsia intraeritrocítica obrigatória cujo controle requer, além de uma vacina eficiente, uma acurada identificação de bovinos cronicamente infectados. Apesar de existirem atualmente diversos métodos de diagnóstico dessa riquétsia, os métodos sorológicos, em particular o ensaio de imunoadsorção enzimática–ELISAs, são os mais utilizados devido à sua versatilidade e praticidade. No entanto, devido ao grande número de antígenos disponíveis, atualmente torna-se necessária uma avaliação para definir quais antígenos apresentam um melhor desempenho no diagnóstico da anaplasmose. Soros de bovinos positivos e negativos para A. marginale por PCR, e soros de animais provenientes do Brasil e Costa Rica, foram testados em ELISAs baseados em MSP1a, MSP2 e MSP5 recombinantes, um pool das três proteínas recombinantes, e antígeno de lisado de corpúsculos iniciais da riquétsia (CI). Utilizando soro de bovinos positivos para A. marginale por PCR, uma maior sensibilidade foi observada no ELISA CI. No entanto, uma maior especificidade, com soro de bovinos negativos a PCR, foi observada com os ELISAs recombinantes. O porcentual de bovinos positivos do Brasil e Costa Rica foi maior com ELISA CI. Razões para essas diferenças são discutidas.

Abstract in English:

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.

• Anaplasma marginale ELISA MSP5 solubilization recombinant protein

Abstract in Portuguese:

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.