bAbilities to detect heterogeneity of ospC genotypes of the Lyme disease spirochete Borrelia burgdorferi in the tick vector by in vitro culture (IVC) and direct PCR (dPCR) were compared. IVC failed to detect one-third of the ospC genotypes detected by dPCR. Among IVC results, common ospC genotypes were overrepresented while occurrence of rare genotypes was underestimated.A bility to culture Borrelia burgdorferi has been essential for clinical, epidemiological, and pathogenicity studies of B. burgdorferi (1). Various culture medium formulations for Borrelia growth have been widely used to isolate B. burgdorferi from ticks, hosts, and clinical samples (2-4). More recently, culture-independent means of Borrelia detection, usually involving PCR, have become the gold standard (5). One widely used genetic marker for detection of B. burgdorferi is the gene encoding outer surface protein C (ospC). The ospC gene encodes an important major surface protein that has proven useful for discriminating several genotypes (6-9).A comparison of B. burgdorferi strains cultured from ticks with those cultured from clinical samples has led to conclusions that a limited set of ospC genotypes (A, B, I, and K) are invasive and cause systemic disease while the others are limited to ticks or persist as localized infections (8, 10). While several investigators have reported a cultivation effect of B. burgdorferi, all results are based on clinical samples and/or conserved genetic markers. In this study, we compared performances of direct PCR (dPCR) and in vitro cultivation (IVC) to detect 17 distinct Borrelia burgdorferi ospC genotypes from tick specimens.A total of 926 adult deer ticks were surface sterilized in providone-iodine and then rinsed in 70% EtOH. Ticks in one treatment group (n ϭ 420) were bisected by a dorsoventral medial cut, and one half of the ticks were placed directly into BSK-H medium (Sigma-Aldrich, St. Louis, MO) while the other half were used for DNA extraction. Ticks in a second treatment group (n ϭ 506) were dissected, and gut contents were placed in 1.5 ml BSK-H medium; the remainder of the tick body was used in DNA extraction as described above. All cultures were incubated at 34°C and checked biweekly for 4 weeks by dark-field microscopy.After 4 weeks, 25 l of each culture was transferred to a 96-well plate containing 75 l H 2 O, heated to 95°C for 10 min, and centrifuged at 6,000 ϫ g for 10 min, and the resulting supernatant was used as a template for PCR. DNA extraction was performed using the Epicentre Master Complete DNA and RNA purification kits (Epicentre Technologies, Madison, WI). PCR conditions for ospC and the reverse line blotting technique were as described previously (7,11). In addition to the 15 published probes, two new ospC probes were used in this study. We named these type-V (5=-GAG CCG CTT GAG CAG TTA AAC CAT TTG CAC C-3=) and type-W (5=-TCG TTT CGA TTT GCT TCT ACA CCC-3=).dPCR of ticks detected a higher overall infection rate (61.9%) than IVC (34.3%) ( 2 ϭ 140.64; degrees of freedom [df] ϭ 1; P Ͻ...
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