Induced immune response of Escherichia coli BL21 expressing recombinant MSP1a and MSP1b proteins of Anaplasma marginale

This work aims to evaluate the potential of immunization with E. coli BL21 expressing the recombinant rMSP1a and rMSP1b proteins of Anaplasma marginale. E. coli BL21 was transformed with recombinant plasmids pET102/msp1α and pET101/msp1β, and rMSP1a and rMSP1b were expressed after induction by IPTG. BALB/c mice were vaccinated with formolized BL21/rMSP1a and BL21/rMSP1b, and the production in mice sera of whole IgG was determined by ELISA. The mice immunized with BL21/rMSP1a showed a better humoral response for whole IgG when compared to the mice immunized with BL21/rMSP1b; these mice exhibited a small response after the second vaccination. Sera of mice immunized with BL21/rMSP1a reacted via western blot with BL21 and rMSP1a, with molecular masses varying from 70 to 105 kDa. Sera of mice immunized with BL21/rMSP1b reacted with BL21 and rMSP1b with a molecular mass of 100 kDa. These results demonstrate that BL21 containing rMSP1a and rMSP1b in the outer membrane were able to produce an immune response in mice, reinforcing its use in vaccine models against bovine anaplasmosis.

Esse trabalho avaliou o potencial de imunização de Escherichia coli BL21 expressando as proteínas recombinantes rMSP1a e rMSP1b de Anaplasma marginale. A E. coli BL21 foi transformada com os plasmídios recombinantes pET102/msp1α e pET101/msp1β e as proteínas rMSP1a e rMSP1b foram expressas após indução com IPTG. Camundongos BALB/c foram vacinados com BL21/rMSP1a e BL21/rMSP1b formolisadas, e a produção de IgG total foi determinada pelo teste de ELISA nos soros dos camundongos imunizados. Os camundongos imunizados com a BL21/rMSP1a mostraram uma melhor resposta humoral para IgG total, comparada à resposta apresentada pelos camundongos imunizados com BL21/rMSP1b; estes camundongos exibiram uma menor resposta após a segunda vacinação. Soros de camundongos imunizados BL21/rMSP1a reagiram pelo western blot com BL21 e rMSP1a, com massa molecular variando de 70 a 105 kDa. Soro de camundongos imunizados com BL21/rMSP1b reagiram com BL21 e rMSP1b com massa molecular de 100 kDa. Esses resultados demonstram que BL21 contendo rMSP1a e rMSP1b na membrana externa foram capazes de produzir resposta imune em camundongos, reforçando o seu uso em modelos de vacina contra a anaplasmose bovina.

[Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its application in a competitive enzyme-linked immunosorbent test]. / Clonagem, expressão, caracterização molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicação em um teste de ELISA por competição.

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The 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. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 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-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3% specificity, 100 and 98%, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1% specificity, 98.9 e 96.3% sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically 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 specificity should not be used in situations that demand more specific diagnosis.

Clonagem, expressão, caracterização molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicação em um teste de ELISA por competição / Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its application in a competitive enzyme-linked immunosorbent test

No presente trabalho, um teste imunoenzimático por competição (cELISA) foi padronizado com a proteína recombinante de superfície rMSP5, clonada a partir do gene msp5 do isolado PR1 de A. marginale. O sequenciamento mostrou que o gene que codifica a rMSP5/PR1 tem 98 por cento de identidade com os isolados Flórida e Santa Maria, 97 por cento com isolados de Pernambuco, Brasil e Havana, Cuba e 91 por cento com A. centrale. O teste de cELISA-PR1 foi comparado com os testes de IFI e cELISA-USA. Para a padronização e validação do cELISA-PR1, foram utilizados 380 soros bovinos comprovadamente positivos ou negativos pelo cELISA-USA. Desse total, 245 soros positivos eram de animais de área endêmica e 135 soros eram negativos, de área livre de anaplasmose. Na padronização, foram utilizados 283 soros de bovinos, dos quais 135 eram negativos e 148 positivos. Os testes de cELISA-PR1 e IFI apresentaram especificidade de 100 e 99,3 por cento, sensibilidade de 100 e 98 por cento, com coeficiente kappa de 0,993 e 0,978, respectivamente. Na validação do teste, foram utilizados 245 soros de bovinos de áreas endêmicas para anaplasmose, testados pelo cELISA-PR1 e IFI e apresentaram especificidade de 96,7 e 69,1 por cento, sensibilidade de 98,9 e 96,3 por cento e coeficiente kappa de 0,956 e 0,699, respectivamente. Esses resultados permitem afirmar que o teste de cELISA-PR1 apresentou performance equivalente ao cELISA-USA, podendo também ser utilizado em estudos epidemiológicos, programas de controle e movimentação internacional de animais, enquanto a IFI, com os resultados menos precisos apresentados, não deve ser utilizada em situações que requerem maior rigor no diagnóstico.

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The sequencing of the msp5 gene of PR1 isolate showed 98 percent of identity with the Florida and Saint Maries isolates, 97 percent with Brazil (Pernambuco) and Havana isolates; and 91 percent with A. centrale. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 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-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3 percent specificity, 100 and 98 percent, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1 percent specificity, 98.9 e 96.3 percent sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically 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 specificity should not be used in situations that demand more specific diagnosis.

[Occurrence and molecular characterization of Babesia species in a canine hospital population in the Londrina Region, Parana State, Brazil]. / Ocorrência e caracterização molecular de espécies de Babesia em cães de uma população hospitalar da Região de Londrina, PR.

Canine babesiosis is a worldwide disease caused by the protozoan of Babesia genus. Babesia canis and B. gibsoni are both species that naturally infect dogs. The objective of this study was to evaluate the infection of Babesia species in dogs attended at the Londrina State University Veterinary Teaching Hospital (HV-UEL). It was selected 282 dogs seen at the Londrina State University Veterinary Teaching Hospital (HV-UEL) between April of 2005 and May of 2006. They presented anemia (Packed Cell Volume<25%), thrombocytopenia (Platelet count <150000/mm3), leukopenia (White blood cell count<5000/mm3) or a combination of two or three of these alterations at the moment of the consultation. The presence of Babesia sp was determined by the amplification of a specific fragment of DNA of the Babesia genus by PCR. Microscopic examination of Giemsa-stained blood smears detected 38 (13.5%) positive samples against 105 identified by PCR from 282 dogs. The positive samples were submitted to PCR-RFLP by Hinf I that allows distinguishing the species of B. canis vogeli and B. gibsoni. From 282 dogs, Babesia sp infection was identified in 105 (37.2%). From these 105 positive samples, the PCR-RFLP identified 66 (23.4%) samples with a profile compatible to B. canis vogeli and 39 (13, 8%) to B. gibsoni. As conclusions, the results obtained allow to affirm that the babesiose is an important differential for dogs that present anemia, leukopenia and thrombocytopenia and, B. canis vogeli is the subspecies that is present in the most of the cases of babesiose in the population of dogs studied and, that B. gibsoni is also present causing babesiosis in dogs of the Londrina region, Parana State, Brazil.