| Summary: | <i>Wolbachia</i> are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although <i>Wolbachia</i> DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not <i>Wolbachia</i> can infect and grow in tick cells, cell lines from three tick species, <i>Ixodes scapularis</i>, <i>Ixodes ricinus</i> and <i>Rhipicephalus microplus</i>, were inoculated with <i>Wolbachia</i> strains <i>w</i>Stri and <i>w</i>AlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an <i>I. scapularis</i> cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of <i>Wolbachia</i> genes. The <i>w</i>Stri strain infected <i>Ixodes</i> spp. cells and was maintained through 29 passages. The <i>w</i>AlbB strain successfully infected <i>Ixodes</i> spp. and <i>R. microplus</i> cells and was maintained through 2–5 passages. A novel strain of <i>Wolbachia</i> belonging to the supergroup F, designated <i>w</i>CfeF, was isolated in <i>I. scapularis</i> cells from a pool of <i>Ctenocephalides</i> sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a <i>Wolbachia</i> strain from a flea and the first isolation of any <i>Wolbachia</i> strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple <i>Wolbachia</i> strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of <i>Wolbachia</i>.
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