Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

In vitro cultivation of Anaplasma marginale and A. phagocytophilum in tick cell lines: a review / Cultivo in vitro de Anaplasma marginale e A. phagocytophilum em células de carrapatos: uma revisão

Continuous cell lines have been established from several ixodid and argasid tick species, representing an excellent tool suitable for the isolation of pathogens and their subsequent propagation, which in turn allows the production of antigenic material for diagnostic tests, antibody and vaccine production, and also for studies on host-vector-pathogen relationships. This paper reviews the use of tick cells for culture initiation and maintenance of two obligate intracellular bacterial pathogens, Anaplasma marginale and Anaplasma phagocytophilum. These in vitro cultivation systems have been used in a wide range of studies, covering morphological ultrastructural analysis, genetics, proteomics and biological differences between strains, including genome transcriptional and protein expression approaches, enabling comparisons between host and vector cells. Thus, such systems open a new window for a better understanding of interactions between pathogens and tick cells. Last but not least, such systems contribute to the reduction in usage of animals for experimental research, as antigenic material can be produced in reasonably large quantities without the use of in vivo species-specific systems.

Linhagens contínuas de células já foram estabelecidas a partir de várias espécies de carrapatos ixodídeos e argasídeos e representam uma ferramenta excelente para o isolamento e propagação de patógenos, permitindo a produção de material antigênico para testes diagnósticos, produção de anticorpos e vacinas, e também para estudos das relações entre hospedeiro-vetor-patógenos. Este artigo revisa o uso de células de carrapatos para estabelecimento e manutenção in vitro de dois patógenos intracelulares, Anaplasma marginale e Anaplasma phagocytophilum. Estes sistemas de cultivo in vitro, têm sido utilizados em vários estudos, tais como análises morfológicas, genéticas, proteômicas e estudos diferenciais entre isolados, incluindo genômica transcricional e expressões proteicas, permitindo comparações entre células dos hospedeiros e dos vetores. Tais sistemas constituem, portanto, uma nova abordagem para melhor entendimento das relações entre patógenos e células de carrapatos. Além disso, tais sistemas contribuem para a redução do uso de animais de experimentação, uma vez que permitem a produção de grandes quantidades de material antigênico sem o uso de sistemas espécie-específicos in vivo.

Manutenção in vitro de células IDE8 em dois tipos de soro bovino / In vitro maintenance of IDE8 cells using two types of bovine serum

The present study had the objective of defining the culture conditions, optimizing the maintenance and expansion of an IDE-8 cell line in Brazil, with the aim to propose its use as a model for in vitro infection and multiplication of Brazilian strains of rickettsia and other hemoparasites. The supplementation of IDE-8 cells with two distinct fetal bovine sera (a Brazilian and an imported) was evaluated. Culture media were changed weekly and subcultures were carried out every 15 days. The development of cultures and subcultures was evaluated by the percentage of viability and cellular morphology. The results indicate that the imported SFB can be replaced by the Brazilian SFB one, as no significant differences (P<0.05) were seen among culture viabilities.