Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

Wolbachiaare intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. AlthoughWolbachiaDNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or notWolbachiacan infect and grow in tick cells, cell lines from three tick species,Ixodes scapularis,Ixodes ricinusandRhipicephalus microplus, were inoculated withWolbachiastrainswStri andwAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into anI. scapulariscell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments ofWolbachiagenes. ThewStri strain infectedIxodesspp. cells and was maintained through 29 passages. ThewAlbB strain successfully infectedIxodesspp. andR. micropluscells and was maintained through 2-5 passages. A novel strain ofWolbachiabelonging to the supergroup F, designatedwCfeF, was isolated inI. scapulariscells from a pool ofCtenocephalidessp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of aWolbachiastrain from a flea and the first isolation of anyWolbachiastrain outside the "pandemic" A and B supergroups. The study demonstrates that tick cells can host multipleWolbachiastrains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains ofWolbachia.