Abstract
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.
Highlights
Lyme borreliosis (LB) is the most prevalent vectorborne bacterial disease of humans in the western world [1]
The sequence amplified from the flgE flagellin gene of B. burgdorferi s.s. aligned with several species of the genus Borrelia; it had the greatest degree of sequence identity with B. burgdorferi, and it shared 100% identity with the sequences BORFLAA (Genbank M67456.1), BOR1FLA (Genbank L42876.1) and BORFLAA (Genbank M67456.1), among others
Flow cytometry PKH stained B. burgdorferi efficiently without affecting bacterial viability or motility when observed in dark field after 24 h, retaining approximately 95% motility
Summary
Lyme borreliosis (LB) is the most prevalent vectorborne bacterial disease of humans in the western world [1]. LB has risen from relative obscurity to become a prototypal emerging infectious disease [1]. Mammalian cell cultures have provided insights into the pathogenesis of LB in the vertebrate host. They have supported the identification of cellular receptors for spirochete adherence in addition to various strategies for inducing an adaptive immune response against spirochetes in vitro [5]. Similar studies using tick cells have elucidated the phenomenon of spirochete tropism within tick tissues and cells, as well as spirochete transmission mechanisms [6,7,8]
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