Serological and molecular detection of Theileria equi in horses from Sinop, Mato Grosso state, Brazil / Detecção sorológica e molecular de Theileria equi em equinos oriundos de Sinop, Mato Grosso, Brasil

Equine piroplasmosis is the most important tick-borne disease to affect horses in Brazil. Theileria equi is one of the causative agents of equine piroplasmosis. Chronic cases are expected, in which the animals show no apparent signs of infection and remain asymptomatic but constitute a source of the infectious agent that ticks can spread. This study was conducted across 81 ranches located in the municipality of Sinop, State of Mato Grosso, Brazil. A sample calculation was performed to estimate the apparent prevalence of T. equi among horses. A total of 1,853 animals were included in the sampling analysis based on the information available from the Institute of Agricultural and Livestock Defense of Mato Grosso State. The serological analysis of 367 serum samples using an indirect enzyme-linked immunosorbent assay (ELISA) to detect anti-T. equi antibodies revealed that 337 animals were positive, representing a frequency of 90.70%. The molecular analysis to amplify the EMA-1 gene showed positivity in 20 of 89 tested samples. The fragments of four samples were sequenced and analyzed to determine their similarities to sequences from other species, based on sequences deposited at GenBank. All showed 100% similarity with T. equi. Our study represents the first report of T. equi antibodies among the equids in north-central region of Mato Grosso, revealing the widespread distribution of seropositive animals.

A piroplasmose equina é a doença transmitida por carrapatos mais importante em cavalos no Brasil. Theileria equi é um dos agentes causadores da piroplasmose equina. São esperados casos crônicos, nos quais os animais não apresentam sinais aparentes de infecção e permanecem assintomáticos, mas constituem uma fonte de infecção e disseminação por carrapatos. Este estudo foi realizado em 81 fazendas localizadas no município de Sinop, Estado de Mato Grosso, Brasil. Um cálculo amostral foi realizado para estimar a prevalência aparente de T. equi entre cavalos. No total, 1.853 animais foram incluídos na análise amostral com base nas informações disponíveis no Instituto de Defesa Agropecuária do Estado de Mato Grosso. A análise sorológica de 367 amostras de soro por meio de ensaio imunoenzimático indireto (ELISA) para detecção de anticorpos anti-T. equi revelou que 337 animais eram positivos, representando uma frequência de 90,70%. A análise molecular para o gene EMA-1 mostrou positividade em 20 das 89 amostras testadas. Os fragmentos de quatro amostras foram sequenciados e analisados para determinar suas semelhanças com sequências de outras espécies, a partir das sequências depositadas no GenBank. Todos mostraram 100% de similaridade com T. equi. Nosso estudo representa o primeiro relato de anticorpos contra T. equi entre os equídeos na região centro norte de Mato Grosso, revelando a ampla distribuição de animais soropositivos.

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil / Avaliação longitudinal da resposta immune humoral e diversidade de antígenos de superfície de merozoítos em bezerros naturalmente infectados com Babesia bovis, em São Paulo, Brasil

Abstract Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

Resumo A babesiose é uma doença infecciosa economicamente importante que afeta o gado bovino em todo o mundo. Para avaliar longitudinalmente a resposta imune humoral contra B. bovis e a diversidade genética de antígenos de superfície de merozoítos de B. bovis, entre bezerros naturalmente infectados em Taiaçu, Brasil, amostras de soro e DNA de 15 bezerros, foram obtidos trimestralmente, desde o nascimento até aos 12 meses de idade. Os anticorpos IgG para B. bovis foram detectados pelos testes de Imunofluorescência Indireta e Ensaio de Imunoadsorção Enzimático Indireto. A Reação em Cadeia da Polimerase foi utilizada para investigar a diversidade genética de B. bovis, com base em genes que codificam antígenos de superfície de merozoítos (MSA-1, MSA-2b e MSA-2c). Os resultados da sorologia demonstraram que até seis meses de idade todos os bezerros desenvolveram imunidade ativa contra B. bovis. Entre as 75 amostras de DNA avaliadas, foram obtidas 2, 4 e 5 sequências dos genes msa-1, msa-2b e msa-2c. O presente trabalho demonstrou que as sequências dos genes msa-1 e msa-2b amplificadas do DNA do sangue de amostras positivas a B. bovis de bezerros de Taiaçu foram geneticamente distintas, e msa-2c conservadas. Todos os animais foram soropositivos ao ELISA e ao IFAT, os quais utilizaram o repertório completo de antígenos parasitários, apesar da diversidade genética dos MSAs.

Molecular and antigenic characterisation of ribosomal phosphoprotein P0 from Babesia bovis

Babesia bovis is a tick-borne pathogen that remains an important constraint for the development of cattle industries in tropical and subtropical regions of the world. Effective control can be achieved by vaccination with live attenuated phenotypes of the parasite. However, these phenotypes have a number of drawbacks, which justifies the search for new, more efficient immunogens based mainly on recombinant protein technology. In the present paper, ribosomal phosphoprotein P0 from a Brazilian isolate of B. bovis was produced and evaluated with regard to conservation and antigenicity. The protein sequence displayed high conservation between different Brazilian isolates of B. bovis and several Apicomplexa parasites such as Theileria, Neospora and Toxoplasma. IgG from cattle experimentally and naturally infected with B. bovisas well as IgG1 and IgG2 from naturally infected cattle reacted with the recombinant protein. IgG from cattle experimentally infected with Babesia bigemina cross-reacted with B. bovis recombinant P0. These characteristics suggest that P0 is a potential antigen for recombinant vaccine preparations against bovine babesiosis.

Aislamiento de una cepa de campo de Babesia bigemina (Piroplasma: Babesiidae) y establecimiento del cultivo in vitro para la Producción de antígenos / Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production. Bovine b abesiosis, caused by Babesia bigemina, is a barrier for livestock development; it results in high economic loss to Mexican livestock. Control requires adequate antigens for diagnosis and vaccination programs. However, because of antigenic variation among Babesia strains, it is necessary to use antigens prepared from local strains. The purpose of the present study was to isolate a local field strain and to establish the in vitro culture of B. bigemina by the evaluation of the constituent's concentration of culture media. Thirty engorged female Boophilus microplus were collected from cattle suffering clinical babesiosis (B. bigemina) in Yucatan state, Mexico. These ticks were sent to the laboratory for detection of Babesia sp. vermicules. Eggs were kept at 83-85 % humidity and 27 degrees C until hatching. Larvae were transferred to an esplenectomized calf (B-1). The resulting nymphs were transferred to an esplenectomized calf (B-2). Twelve days later, B. bigemina (local strain) was detected in calf B-2 and its infected blood was frozen in liquid nitrogen to initiate the in vitro culture. The Microaerophilus Stationary Phase (MASP) in vitro culture method was used to reactivate the parasite. Three different concentrations of culture media (70, 60 and 50%), serum (30, 40 and 50%) and uninfected red blood cells (5, 10 and 15 %) were used in order to know the convenient concentrations to obtain the highest percentage of infected red blood cells (PEI). The cultured strain was used to prepare antigens for the Immunofluorescence Antibody Test (IFAT) and several concentrations of serum and conjugate were tested. Strain isolation was successful; 30 days were needed to obtain a PEI of 1.5%. The isolated strain was frozen in liquid nitrogen and the parasites were reactivated with the in vitro culture MASP method...

Pesquisaje de Babesia en trabajadores agropecuarios y donantes en la provincia de Ciego de Avila / Screening of Babesia un agricultural workers and donors in the province of Ciego de Avila

Se realizó un estudio descriptivo de casos, en éste se investigaron 781 sueros con la técnica de inmunofluorescencia indirecta con antígenos de Babesia bovis y Babesia bigemina en el Centro Nacional de Sanidad Agropecuaria. De ellos, 371 provenían de trabajadores agropecuarios y 410 de donantes de la provincia de Ciego de Avila. Se partió de diluciones de 1/64, a cada seropositivo se le confeccionó una encuesta epidemiológica para conocer factores de riesgo. Se empleó la prueba de diferencia de proporciones para comparar los porcentajes de positivos. Se calcularon las medias geométricas en ambos grupos y se procedió a su comparación con la prueba U de Mann Whitney. En los trabajadores agropecuarios se detectó un 7 por ciento con anticuerpos contra el 3,9 por ciento de los donantes (p < 0,05). Se encontró mayor circulación de Babesia bovis que de Babesia bigemina

Análise de testes de conglutinaçäo rápida para detecçäo de anticorpos contra Babesia bovis e Babesia bigemina / Evaluation of rapid conglutination tests for detection of antibodies against Babesia bovis and Babesia bigemina

Testes de conglutinaçäo rápida foram desenvolvidos para detecçäo de anticorpos contra Babesia bovis e B. bigemina. O primeiro (TCR-B. bovis) apresentou resultados idênticos à imunofluorescência indireta (IFI), na detecçäo de anticorpos resultantes da vacinaçäo com cepas atenuadas de B. bovis, enquanto que o segundo teste de conglutinaçäo (TCR-B. bigemina) divergiu em três dos seis animais na fase inicial da soroconversäo. Aos 28 e 56 dias pós-vacinaçäo (PV) houve coincidência total de resultados. A correlaçäo entre os TCR-B. bovis e IFI no exame dos soros de bovinos de sete estados brasileiros foi de 86,2 por cento, enquanto que a correlaçäo entre TCR-B. bigemina e a prova de imunofluorescência foi de 95,6 por cento. Os resultados demonstraram que os testes de conglutinaçäo podem ser empregados em estudos epidemiológicos com eficiência comparável à imunofluorescência indireta

Successful vaccination against Boophilus microplus and Babesia bovis using recombinat antigens

Current methods for the control of the cattle tick Boophils microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials