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A nested PCR (nested flagellin PCR) carrying an internal E. coli DNA control was established and compared with an in-vitro culture method for the detection of Borrelia burgdorferi in urine specimens of dogs. The predicted specific amplicon of the flagellin gene fla was generated from all cultured strains of B. burgdorferi tested (comprising three European genospecies). In contrast, all 13 strains of seven other flagellated bacterial species were negative. The PCR detection limit yielded 20 cells of B. burgdorferi per ml of double-distilled water and approx. 250 bacteria per ml of dog urine. Using the bacterial culture method, urine specimens collected from 216 dogs in Germany were all diagnosed negative for spirochetes by in-vitro culture and dark-field microscopy. In contrast, DNA of B. burgdorferi was detected in 32 specimens (14.8%) by PCR. 31 urine specimens (14.4%) showed inhibitory activity in the PCR assay. However, 94 (44%) were inhibitory in the culture assay. The majority of the PCR-positive dogs exhibited major clinical symptoms which have not been reported in the course of B. burgdorferi infection previously, e.g. cystitis (14/32 dogs) or prostatitis (5/32 dogs). Our results indicate that the analysis of urine specimens by the nested flagellin PCR is a highly valuable procedure for the diagnosis of B. burgdorferi infections in dogs.