Co-cultivo de Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) em cultura primária de células embrionárias de Dermacentor nitens (Acari: Ixodidae)

Abstract

The aims of this study were to establish the primary culture of embryonic cells from Dermacentor nitens and evaluate the influence of the Barbour-Stoenner-Kelly (BSK) and Leibovitz's L-15B media in co-culture with Borrelia burgdorferi (American strain G39/40). The culture was established from embryonated eggs of ingurgitated females of D. nitens with 13 days of laying, using the Leibovitz's L-15B medium supplemented. After the formation of monolayer in flasks of 10cm2, the L-15B medium was removed from the flasks. Three groups with different media were formed. A group consisting only of BSK medium (group I), a group with L-15B medium with 40% BSK (group II) and a third group with L-15B medium with 10% BSK (group III). For each group three repetitions were done, which received the spirochetes inoculum of B. burgdorferi, with final concentration of approximately 1.1 x 106 spirochetes/mL. It was prepared another flask without tick cells with 5 mL of BSK, in order to assess the development of spirochetes in the absence of embryonic cells. The count of B. burgdorferi was performed three days after inoculation of spirochetes. Since the second day of culture it was observed fixation of most cells on the surface of the flask, with numerous cell aggregates. After fixation, it was observed a wide variety of cell types that began to differentiate into fibroblastoid cells and posteriorly they start to differentiate into epithelioid and rounded cells. There was extensive proliferation of spirochetes cultured with embryonic cells in comparison to the initial concentration. Most spirochetes was epicellular and was attached to the cells longitudinally or by their edges, with few free spirochetes. Group II showed the best results, since the medium caused less damage to tick cells in comparison to Group I with good multiplication of spirochetes in comparison to groups III and control. Although D. nitens is not species-specific vector of B. burgdorferi, the spirochete culture in this study was successful, proving to be a useful tool in the isolation of strains or species of Borrelia spp.