Cultivo de esporocinetos de Babesia bigemina em hem?citos e c?lulas embrion?rias de Rhipicephalus (Boophilus) microplus

• Autor(es): Rezende, Jania de
• Data: 2019
• Origem: Oasisbr
• Assunto(s): C?lula de carrapato; cripreserva??o; Babesia spp; tick cell; cripreservation; Medicina Veterin?ria
• Tipo de Documento: Tese de doutoramento
• Idioma: Português

Descrição

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CNPq - Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico

The hemoparasitos belonging to the genus Babesia are intensively studied because of its importance in the livestock economy worldwide. Cultures of embryonic cells and hemocytes of ticks are excellent substrates for the isolation and cultivation of hemoparasites pathogens, including Babesia spp. This methodology contributes for biological and physiopathology studies, as well as control of the species. The present study had as goals to cultivate in vitro sporokinetes of Babesia bigemina in hemocytes and embryonic cells of Rhipicephalus (Boophilus) microplus. After superficial disinfection of engorged females, the hemolymph was collected and transferred to culture flasks with 25 cm2 and tube 10 cm2 and incubated at 28 ? C. To start the primary embryonic culture, engorged females of R. (B) microplus were incubated at 28 ? C and 13 days after oviposition, the eggs were superficially disinfected, broken, filtered and transferred to medium culture supplemented L15 and at a 28?C. Examinations were daily performed by contrast microscope inverted in phase. Cytospin and thick drop samples, which were stained with Giemsa and observed under light microscopy. It was realized PCR for B. bigemina and Babesia bovis, using two pairs of primers to identify the gene 18SrRNA for both species and were also carried to morphometry of the sporokinetes of confirm the species. Sporokinetes of B. bigemina cryopreserved from the culture of hemocytes were thawed, reactivated in hemocytes free of infection and in cell line CTVM/BME2. It was observed the development of sporokinetes B. bigemina from the first day of cultivation, cells after reactivation. The protozoa showed good motility and capacity to adhere to the cell membrane by the apical extremities. In the cytoplasm of hemocytes was observed round shapes, moving, and with visible nuclei sporokinetes of B. bigemina. In the stained samples of 3rd and 17th days of cultivation of B. bigemina sporokinetes in hemocytes were observed pyriformes integrity forms of sporokinetes immature and mature, with dark red stained nuclei, sometimes centralized or near the apical extremities. In samples from the 17th day of cultivation were observed many small round and oval shapes, compatible with immature sporokinetes. By PCR technique, it was possible to amplify the DNA to 18SrRNA gene of B. bigemina, as well as the comparative study of measurements of sporokinetes. The hemocytes and embryonic cells of R. (B.) microplus consisted in efficient substrates for in vitro B. bigemina sporokinetes cultivation. It was possible to cryopreservation B. bigemina sporokinetes in liquid nitrogen and its reactivation in cultured hemocytes of R. (B) microplus and BME2 cell lines.

Os hemoparasitos pertencentes ao g?nero Babesia s?o intensamente estudados devido sua import?ncia na economia da pecu?ria mundial. Culturas de hem?citos e c?lulas embrion?rias de carrapatos constituem excelentes substratos para o isolamento e cultivo de hemoparasitos patog?nicos, incluindo Babesia spp. Esta metodologia contribui para estudos da biologia, fisiopatologia, bem como controle da esp?cie. O presente estudo teve como objetivos cultivar in vitro, esporocinetos de Babesia bigemina em hem?citos e em c?lulas embrion?rias de Rhipicephalus (Boophilus) microplus. Ap?s desinfec??o superficial de f?meas ingurtitadas, a hemolinfa foi coletada e transferida para frascos de cultura com 25 cm2 e tubo de 10cm? e incubados a 28 ?C. Para iniciar o cultivo prim?rio embrion?rio, f?meas ingurgitadas de R. (B) microlpus foram incubadas ? 28?C e ap?s 13 dias de postura, os ovos foram desinfectados superficialmente, macerados, filtrados e transferidos para meio de cultivo L15 suplementado e em temperatura de 28?C. Observa??es foram realizadas diariamente em microsc?pio de contraste de fase invertido. Realizou-se Citospin e gota espessa das amostras, que foram coradas com Giemsa e observadas em microscopia de luz. Foram realizadas PCR para B. bigemina e Babesia bovis, utilizando dois pares de iniciadores para identificar o gene 18SrRNA para ambas esp?cies e tamb?m foi realizado a morfometria dos esporocinetos para confirma??o da esp?cie. Esporocinetos de B. bigemina criopreservados a partir da cultura de hem?citos, foram descongelados, reativados em hem?citos livres de infec??o e em c?lulas de linhagem CTVM/BME2. Observou-se o desenvolvimento dos esporocinetos de B. bigemina a partir do primeiro dia do cultivo, ap?s reativa??o nas c?lulas. Os protozo?rios apresentaram boa motilidade e capacidade de ader?ncia na membrana celular pela extremidade apical. No citoplasma dos hem?citos observou-se formas redondas, m?veis e com n?cleo v?sivel de esporocinetos de B. bigemina. Nas amostras coradas do 3? e 17? dia do cultivo de esporocinetos de B. bigemina em hem?citos foram observadas formas ?ntegras piriformes de esporocinetos imaturos e maduros, com n?cleo corado de vermelho escuro, as vezes, centralizado ou pr?ximo da extremidade apical. Nas amostras do 17? dia de cultivo foram observados muitas formas pequenas redondas e ovais, compat?veis com esporocinetos imaturos. Pela t?cnica PCR foi poss?vel a amplifica??o do DNA para o gene 18SrRNA de B. bigemina, assim como pelo estudo comparativo das mensura??es dos esporocinetos. Os hem?citos e c?lulas embrion?rias de R. (B.) microplus constitu?ram-se em substratos eficientes para cultivo in vitro de esporocinetos de B. bigemina. Foi poss?vel a criopreserva??o de esporocinetos de B. bigemina em nitrog?nio l?quido e a sua reativa??o em cultura de hem?citos de R. (B.) microplus e em c?lulas da linhagem BME2.