New Zealand white rabbits 4–6 weeks old were immunized subcutaneously with 30 µg of rIxofin3D-PF or ovalbumin in complete Freund's adjuvant and boosted twice with 30 µg of rIxofin3D-PF or ovalbumin once every 3 weeks in incomplete Freund's adjuvant. Test bleeds were obtained from ear veins 2 weeks after the final boost and reactivity to recombinant rIxofin3D-PF and ovalbumin assessed by western blot. Rabbits were euthanized and serum was obtained by cardiac puncture. Polyclonal IgG was purified from the sera using the Melon Gel IgG purification kit (Thermoscientific, IL). For passive immunization, mice were passively immunized 24 h prior to tick placement by intraperitoneal inoculation with 100 µg of purified rabbit IgG against rIxofin3D-PF or ovalbumin. For active immunization, mice were immunized with 10 µg of rIxofin3D-PF or ovalbumin as described for rabbits. To address the role of rIxofin3D-PF in B. burgdorferi transmission, four B. burgdorferi N40 infected nymphs were placed on each immunized mouse. Nymphs were allowed to feed to repletion. Salivary glands and guts were dissected and combined in pools of 2–3 ticks for quantitative RT-PCR as described above. DNA was isolated from skin punch-biopsies at 7, 14 and 21 days and from heart and joints 21 days post tick-detachment and Borrelia burden assessed by quantitative PCR as described [35] (link).
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