Isolation and Genetic Characterization of the Human Relapsing Fever Spirochetefrom a Dog with Improved Cultivation Techniques.

Abstract

is the causative agent of tick-borne relapsing fever in Israel and is prevalent in the Eastern Mediterranean basin and parts of Asia. Infection withcauses severe illness and potentially life-threatening complications in humans and companion animals. Isolation andcultivation ofin culture medium is difficult and there are only two previous isolates of this spirochete. Here, we describe the first cultivation offrom an ill dog. Isolation from the dog's blood was performed with the Pettenkofer-LMUmedium and spirochetes with vibrant motility and cell density of 2 × 10/ml were observed in culture by dark-field microscopy 3 days after sampling. The isolate was passaged every 3-4 days with cell densities reaching up to 3 × 10/ml achieved over 30 passages. Cryopreservation was made at -80°C without any cryoprotectant additive, and successful growth from thawed culture samples was observed up to 3 months after freezing with repeated freeze and thaw cycles. Generation time during the exponential growth phase was 14.6 hr. Genetic characterization by polymerase chain reaction amplification and DNA sequencing of the flagellin, glycerophosphodiester phosphodiesterase, andgenes indicated that the isolate is agenotype I strain associated with human relapsing fever. The new canine isolate would be helpful for research on the pathogenesis of relapsing fever and the new modifications in cultivation and preservation methods may assist in future studies of relapsing fever by simplifying previously reportedmethods.