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Previous issue date: 2010-02-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq, Brasil. / Cell cultures provide a simplified system of observation that can be particularly useful for
studies of intracellular and epicellular microorganisms. The aim of this study was to establish
in vitro embryonic cells primary culture of the tick Rhipicephalus sanguineus to cultivate the
spirochete Borrelia burgdorferi american strain G39/40. The culture was established from
embryonated eggs of engorged females of R. sanguineus to 12 days after the beginning of the
oviposition, using the culture medium Leibovitz's L-15B supplemented with 20% of
inactivated fetal calf serum, 10% of tryptose phosphate broth, 0.1% fraction V bovine
albumin, 1% of glutamine and 0.1% of gentamicin antibiotic, pH 6.8. After the formation of a
monolayer, the initial culture medium L-15B was removed from the tubes and replaced by
Barbour-Stoenner-Kelly medium (BSK) or BSK with L-15B without antibiotics. Spirochetes
previously grown in BSK were counted and inoculated into tubes, with final concentration of
approximately 6.2 x 105 spirochetes/mL. B. burgdorferi from the inoculated tubes were
countered when the means showed yellow color, indicative of high acidity due to the
multiplication of spirochetes. On the third day after the start of primary culture of R.
sanguineus embryonic cells, we observed the fixation of cell aggregates on the surface of the
bottles. From these clusters, there were several cell types, such as large fibroblast-type cells
and structures like vesicles and tubes. In the second week, we observed the appearance of
round or flattened epithelial-type cells, and after 21 days of culture, we realized the formation
of a monolayer due to the appearance of confluent cells. The L-15B medium proved to be
efficient for the development of primary culture of R. sanguineus embryonic cells. There was
a great multiplication of spirochetes cultivated with cultured embryonic cells when compared
to the initial concentration, as well as the spirochetes grown in the absence of the tick cells,
observing an increase of 100 times the number of B. burgdorferi. Seven days after
inoculation, the tubes in which we used only the BSK medium, higher concentrations of B.
burgdorferi were recovered when compared to the tubes where the medium BSK and
Leibovitz's L-15B were used. Regardless of the culture media tested, the final concentration
of B. burgdorferi of the tubes with embryonic tick cells was lower than that of seamless
embryonic cells. In observation of the culture tubes on microscopy phase contrast, spirochetes
were presented adhered to epithelial-type and fibroblast-type tick cells in an epicelular way
and with great motility. R. sanguineus embryonic cells grown in BSK medium, with or
without B. burgdorferi inoculation, stopped its propagation, showed membrane degeneration
and many of them broke away from the surface of the bottle. The cells grown in BSK and L-
15B continued to multiply, many were still intact and attached to the bottle, with the presence
of tissues in development, with fewer degenerated and floating cells than those cultivated in
BSK. The spirochete B. burgdorferi strain G39/40, adhered, grew, multiplied and showed
great motility in cultures of embryonic cells of R. sanguineus tick, using BSK and Leibovitz?s
L-15B media. / As culturas celulares oferecem um simplificado sistema de observa??o que pode ser
particularmente ?til para estudos de microrganismos intracelulares e epicelulares. O objetivo
deste estudo foi estabelecer cultura prim?ria in vitro de c?lulas embrion?rias do carrapato
Rhipicephalus sanguineus para cultivo da espiroqueta Borrelia burgdorferi, cepa americana
G39/40. A cultura foi estabelecida a partir de ovos embrionados de f?meas ingurgitadas de R.
sanguineus com 12 dias ap?s o ?nicio da postura, utilizando o meio de cultivo Leibovitz?s L-
15B, suplementado com 20% de soro fetal bovino inativado, 10% de caldo triptose fosfato,
0,1% fra??o V de albumina bovina, 1% de glutamina e 0,1% de antibi?tico gentamicina, pH
6,8. Com a forma??o de uma monocamada celular, o meio de cultura inicial L-15B foi
retirado dos tubos e trocado por meio Barbour-Stoenner-Kelly (BSK) ou BSK com L-15B
sem antibi?tico. As espiroquetas previamente cultivadas em BSK foram contadas e inoculadas
nos tubos, apresentando concentra??o final de aproximadamente 6,2 x 105 espiroquetas/mL. A
contagem de B. burgdorferi dos tubos inoculados foi realizada quando o meio apresentou
colora??o amarela, indicativa de elevada acidez devido ? multiplica??o das espiroquetas. No
terceiro dia ap?s o in?cio da cultura prim?ria de c?lulas embrion?rias de R. sanguineus, foi
poss?vel observar a fixa??o de agregados celulares na superf?cie dos frascos. A partir destes
agregados, surgiram diversos tipos celulares, como grandes c?lulas fibroblast?ides e
estruturas semelhantes a ves?culas e tubos. Na segunda semana, foi observado o aparecimento
das c?lulas epiteli?ides ou redondas e, com 21 dias de cultivo, visualizou-se a forma??o de
uma monocamada celular devido ao aspecto confluente das c?lulas. O meio de cultivo L-15B
demonstrou ser eficiente para o desenvolvimento da cultura prim?ria de c?lulas embrion?rias
de R. sanguineus. Houve grande multiplica??o das espiroquetas cultivadas com c?lulas
embrion?rias quando comparada ? concentra??o inicial, assim como das espiroquetas
cultivadas na aus?ncia das c?lulas de carrapato, observando-se um aumento em 100 vezes do
n?mero de B. burgdorferi. Sete dias ap?s a inocula??o, foram recuperadas maiores
concentra??es de B. burgdorferi nos tubos onde se utilizou somente o meio BSK, do que nos
tubos onde foi utilizado BSK juntamente com Leibovitz?s L-15B. Independente dos meios de
cultivo testados, a concentra??o final de B. burgdorferi dos tubos com c?lulas embrion?rias de
carrapato foi menor do que a dos tubos sem c?lulas embrion?rias. Na observa??o dos tubos de
cultivo ? microscopia de contraste de fase, as espiroquetas apresentaram-se aderidas ?s c?lulas
de carrapato epiteli?ides e fibroblast?ides de maneira epicelular e com grande motilidade. As
c?lulas embrion?rias de R. sanguineus cultivadas em meio BSK, com ou sem in?culo de B.
burgdorferi, pararam sua multiplica??o, apresentaram degenera??o na membrana e muitas
desprenderam-se da superf?cie do frasco. As c?lulas cultivadas em meio BSK e L-15B
continuaram a se multiplicar, muitas ainda estavam ?ntegras e aderidas ao frasco, com
presen?a de tecidos em desenvolvimento, com menos c?lulas degeneradas e flutuantes que as
cultivadas somente em BSK. A espiroqueta B. burgdorferi, cepa G39/40, aderiu, cresceu,
multiplicou e apresentou grande motilidade nos cultivos com c?lulas embrion?rias do
carrapato R. sanguineus, utilizando meios BSK e Leibovitz?s L-15B.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:jspui/1896 |
| Date | 25 February 2010 |
| Creators | C?mara, Teixeira, Rafaella |
| Contributors | Fonseca, Adivaldo Henrique da, Ribeiro, M?cio Fl?vio Barbosa, Oliveira, Angela de, Massard, Carlos Luiz Massard |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Medicina Veterin?ria (Patologia e Ci?ncias Cl?nicas), UFRRJ, Brasil, Instituto de Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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