Arthropod cell lines in the isolation and propagation of tickborne spiroplasmas

The ability of several continuous tick cell culture lines to support growth of tickborne spiroplasmas (helical, wall-less prokaryotes in the classMollicutes) was assessed. Seven triturates, prepared from pools ofIxodes pacificus ticks naturally infected with theSpiroplasma sp. (group VI) organism, were retrieved from frozen (–70°C) storage and passaged in three distinct tick cell lines, in antibiotic-free tick cell culture medium alone, or in spiroplasma culture medium (SP-4 formulation). Six spiroplasma strains were recovered in the RML-19 cell line fromDermacentor variabilis, and five isolations were made in another cell line (RML-15) from this tick species. None was recovered in aRhipicephalus sanguineus cell line (RML-23), in tick cell culture medium, or in SP-4 broth medium. One of the spiroplasma isolates (Y43) was maintained through four consecutive weekly refeedings of theD. variabilis cell line and for three feedings ofR. sanguineus cells, where numbers of spiroplasmas in cell supernatants reached levels comparable to those obtained in the SP-4 medium.A laboratory-adapted strain (SMCA) ofSpiroplasma mirum, a second helical mollicute of tick origin (the suckling mouse cataract agent), grew in three tick cell lines (RML-15, RML-23, and RML-16 cells fromD. parumapertus), in three mosquito cell lines (fromAedes albopictus, Ae. aegypti, andCulex quinquefasciatus), and in both cell culture medium alone and in SP-4 medium. The organisms survived for 1–2 weeks, but failed to multiply, in cell lines fromC. tritaeniorhynchus, Antheraea eucalypti, orXenopus laevis. Some evidence of cytopathic effect ofS. mirum on tick cell lines was seen, although growth of the organism in mosquito cell cultures was not associated with cell toxicity. The use of arthropod cell lines appears to have value in the primary isolation of arthropod- or insect-derived mollicutes and for the study of cytopathogenicity of these wall-less prokaryotes.