Cultivation and partial characterization of spiroplasmas in cell cultures.

Abstract

Spiroplasmas were propagated in the Drosophila melanogaster cell line Dm-1. Spiroplasma citri and unidentified strains (corn shunt organism, 277F [tick isolate], powder puff, BNR-1, honey bee, and OBMG) grew to 10(8) to 10(9) colony-forming units per ml and could be passaged. Cytopathic effect (CPE) varied with the infecting spiroplasma. The honey bee isolate killed Dm-1 within 2 to 4 days and produced CPE in four mammalian cells tested. At 25 degrees C, suckling mouse cataract agent produced no CPE in Dm-1 cells. Dm-1 cells did not support growth of the spiroplasmal sex ratio organism. Spiroplasmas could be detected in the cell cultures by agar inoculation, dark-field microscopy, scanning electron microscopy, and DNA fluorescent staining. The uridine phosphorylase test showed significant levels of conversion of [14C]uridine to [14C]uracil for all but some plant isolates: S. citri, corn shunt organism, lettuce, cactus, and powder puff strains, the first mycoplasmas to lack the enzyme. Primary isolations of corn shunt organism from infected corn plants were made in Dm-1 and I-XII cultures. The course of corn stunt organism infection of Dm-1 was monitored for three passages. The use of agarose and Dienes staining of the colonies improved growth and colony counting of corn stunt organism. The number of viable infected DM-1 cells decreased from 1.2 x 10(7) at passage 1 to 7.0 x 10(6) at passage 2 and 3 x 10(5) at passage 3.