Primary culture of Borrelia burgdorferi from Ixodes ricinus ticks

A total of 971 Ixodes (I.) ricinus ticks field-collected in May and June 1993 and 1994 in two different geographic regions (Middle Hesse and Lower Saxony) were examined for the presence of Lyme borreliosis spirochetes (Borrelia burgdorferi sensu lato) by primary culture using nonselective and selective BSK II medium. Modifications of BSK II medium with varying concentrations of bovine serum albumin and with rabbit serum being replaced by horse serum were examined for their ability to support growth of borreliae from ticks. Spirochetes were isolated from 17.8% each of female and male I. ricinus from Lower Saxony. Isolation rates from female and male I. ricinus originating from Middle Hesse were 13.5% and 19.6%, respectively. In comparison to nonselective BSK II medium the isolation rate of borreliae from the tick midgut was increased significantly (35%) by additional culture in selective BSK II medium. Most spirochetal isolates (85%) were detected within three weeks of incubation. Compared to isolation in standard BSK II medium, reduction of bovine serum albumin by 50% did not diminish the isolation rate of borreliae from ticks. Spirochetal isolates were identified as Borrelia burgdorferi sensu lato by PCR using a primer set based on ribosomal DNA sequences to amplify the variable spacer region between two conserved structures, the 3'end of the 5S rRNA and the 5'end of the 23S rRNA.