A Brazilian isolate of Anaplasma marginale with appendage was successfully established and maintained in vitro in a tick cell line (IDE8). Infection was confirmed by optical and transmission electron microscopy. In addition, primers MSP1aNF2 and MSP1aNR2 amplified products from DNA extracted from infected IDE8 cells. Comparisons with partial sequences of the msp1α gene and the complete genome of A. marginale confirmed that the sequences of amplified fragments were from the A. marginale genome. This is the first establishment of a Brazilian A. marginale isolate in tick cells, representing a new system for biological and molecular studies and also a new source of material for diagnosis and development of vaccines.
In vitro cultivation of the IDE8 cell line, derived from embryonic Ixodes scapularis ticks, constitutes an important system for the study of tick-borne pathogens, as these cells support growth of rickettsial species which are not normally transmitted by this tick. However, since cryopreservation of IDE8 cells is not always successful, there is a need to develop alternative ways to preserve these cells. In the present study, a suspension of IDE8 cells in culture medium was kept under refrigeration at 4 degrees C for up to 60 days. Every 15 days, the suspension was mixed and aliquots were re-cultured in 2-ml tubes, under standardized conditions. In addition, three techniques for cryopreservation, using two different cryoprotectants (DMSO and glycerol), were evaluated. Medium changes were carried out every week and subculturing every 2 weeks. The development of cultures and their respective subcultures, after returning to standard culture temperature, was evaluated by percentage viability and by cellular morphology evaluated in Giemsa-stained cytocentrifuge smears. All cultures and subcultures appeared healthy, showing growth rates comparable to cultures that had not been kept under refrigeration. The results demonstrated that storage under refrigeration at 4 degrees C is an efficient method for preservation of IDE8 cells for up to 60 days and that refrigeration may be preferable to cryopreservation for short-term preservation of IDE8 cells.
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