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Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines
摘要： 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.
关键词： Fluorescent membrane marker,Tick cell lines,Borrelia burgdorferi,Phagocytosis
更新于2025-11-21 11:24:58
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A luminescence-based assay for evaluating bactericidal antibody to <i>Borrelia Burgdorferi</i> in vaccinated horses’ serum
摘要： Background: Current serological tests cannot discriminate between bactericidal B. burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. Objective: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. Study design: Prospective validation study and method comparison. Methods: Serum samples were obtained either from archives of Animal Health Diagnostic Center at Cornell University (N = 7) or a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and Borrelia burgdorferi ML23 pBBE22luc, which is able to process luciferin with luciferase and produce luminescence in viable Borrelia. After incubation, a light signal can be detected by using a luminometer and calculate the borreliacidal antibody titer. Results: Components of reaction mixture including spirochetes and complement from various sources and concentrations were tested to identify a reliable recipe for our complement mediated L-SBA. We also applied this L-SBA on measuring bactericidal antibody activities and calculated IC50 of serum samples from clinical collections. Furthermore, we analysed the L-SBA titers and anti-OspA antibody levels from vaccinated horses using the multiplex assays and found that there is a relationship between results generated using these two different assays. The increases of L-SBA titers correlated with increases of anti-OspA antibody titer in sera (r = 0.423). Main limitations: Immunoreactivity of commercial complement may differ from different batches. Clinical protection of borreliacidal antibody levels has not been determined. Conclusions: The L-SBA provided a sensitive and easy-operating platform for evaluation of bactericidal antibody to Borrelia burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.
关键词： luciferase,horse,Borrelia burgdorferi,bactericidal,luminescence
更新于2025-09-19 17:15:36