A luminescence-based assay for evaluating bactericidal antibody to Borrelia burgdorferi in vaccinated horses' serum.

BACKGROUND: Current serological tests cannot discriminate between bactericidal Borrelia 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 the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and B. 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 to calculate the borreliacidal antibody titre. RESULTS: Components of the 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 the half inhibitory concentration (IC50 ) of serum samples from clinical collections. Furthermore, we analysed the L-SBA titres and anti-outer surface protein A (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 titres correlated with increases of anti-OspA antibody titre 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 the evaluation of bactericidal antibody to B. burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.

Antibody-mediated bacterial killing of Ichthyobacterium seriolicida in Japanese amberjack.

Ichthyobacterium seriolicida is the causative agent of bacterial hemolytic jaundice (BHJ) in Japanese amberjack, Seriola quinqueradiata. Fish recovering from BHJ acquire protective immunity against reinfection. In this study, fish were passively immunized to determine whether serum antibody is involved in protection against BHJ. The susceptibility of I. seriolicida to the bactericidal activity of Japanese amberjack serum was also investigated. In passive immunization tests, significantly lower mortality was noted in fish that received convalescent serum. Bacteria were killed when exposed to convalescent serum but not serum from naïve fish. Electron microscopic analyses showed that I. seriolicida cells were morphologically altered by reaction with convalescent serum. Naïve fish serum became bactericidal upon addition of purified IgM from convalescent serum. Involvement of the classical complement pathway in the bactericidal mechanism was confirmed because bactericidal activity was lost upon heating convalescent serum or chelation treatment using EDTA. Convalescent fish serum thus protects against reinfection by I. seriolicida via humoral immunity mediated by activation of the classical complement pathway.