ABSTRACT
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.
Uma amostra brasileira de Anaplasma marginale com apêndice foi estabelecida e mantida in vitro em uma linhagem de células de carrapatos (IDE8). A infecção foi confirmada através de microscopia ótica e eletrônica de transmissão. Além disso, os primers MSP1aNF2 e MSP1aNR2 amplificaram produtos do DNA extraído das células infectadas. Comparações de sequências parciais do gene msp1α e do genoma completo de A. marginale confirmaram que as sequências dos fragmentos amplificados pertenciam ao genoma de A. marginale. Este é o primeiro estabelecimento in vitro de uma amostra brasileira de A. marginale em células de carrapatos, representando um novo sistema para estudos biológicos e moleculares, além de ser uma nova fonte de material para o desenvolvimento de testes diagnósticos e de vacinas.
Subject(s)
Animals , Anaplasma marginale/genetics , In Vitro Techniques , Tick Infestations/genetics , Ixodes/cytology , Diagnostic Techniques and Procedures , Base Sequence , Methods , MethodsABSTRACT
This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.
Subject(s)
Babesia/growth & development , Ixodes/parasitology , Animals , Babesia/genetics , Babesia/isolation & purification , Cattle , Cattle Diseases/parasitology , Cell Line , DNA, Protozoan/analysis , Female , Hemolymph/parasitology , Ixodes/cytology , Ixodes/embryology , Polymerase Chain Reaction , Rhipicephalus/parasitologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Cell Line , Ixodidae/cytology , Preservation, Biological , Animals , Cell Survival , Cryopreservation , Ixodidae/embryology , RefrigerationABSTRACT
A atividade lipásica por grama de forma farmacêutica de apresentaçäo (comprimido, drágea ou cápsula) e o custo em cruzeiros de uma dose correspondente a 20.000 UI de lipase foram determinados em nove medicamentos indicados no tratamento da insuficiência pancreática exócrina e comercializados no Brasil. Os seguintes resultados foram alcançados: Viokase, 5.451 UI e 0,054 ORTN; Pankreoflat, 8.212 UI e 0,039 ORTN; Cotazym B, 8.940 UI e 0,020 ORTN; Car-Nutrizim, 11.334 UI e 0,020 ORTN; Pankreon Composto, 11.184 UI e 0,012 ORTN; Pancreatina CEME, 6.104 UIe, os três últimos, Digeplus. Éssen e Frutenzima com 0 UI de atividade. Os resultados demosntram grande variaçäo na atividade lipásica e custo financeiro dos preparados à base de enzimas pancreáticas, indicando a necessidade de maior controle dos órgäos oficiais sobre a qualidade e custo destes medicamentos e de análise cuidadosa na prescriçäo dos mesmos, no sentido de escolher aqueles que contenham maior atividade lipásica