Simplified media for spiroplasmas associated with tabanid flies

Traditionally, isolation, maintenance, and testing of Spiroplasma species (Mollicutes: Entomoplasmatales) from horse flies (Tabanus spp.) and deer flies (Chrysops spp.) (Diptera: Tabanidae) have been accomplished in the complex M1D medium. A relatively inexpensive, simplified medium for tabanid spiroplasmas could expedite procedures that require large quantities of growth medium. Nine strains of spiroplasmas, eight from tabanids and one from mosquitoes, were cultured in three simplified broth media, R2, R8-1, and C-3G, and in M1D. There was no significant difference in the rate of spiroplasma growth in M1D and the three simplified media. R2 medium supported the growth of tabanid spiroplasmas more consistently and with better morphology through 10 subcultures than did the other simplified media. Primary isolations were made in R2 medium from tabanids collected (i) in Georgia, U.S.A., with 10 isolations from 10 flies and (ii) in coastal Costa Rica, with isolation rates of 70% (28/40) and 73% (27/37), respectively, for R2 and M1D. Of the seven group VIII field isolates from Costa Rica, four were capable of sustained growth in R2, and three were triply cloned in this simplified medium. These results suggest that the simplified medium R2 is suitable for many procedures with tabanid spiroplasmas.