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 stunt organism, 277F [tick isolate], powder puff, BNR-1, honey bee, and OBMG) grew to 108 to 109 colonyforming 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°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

Insect cell cultures in the study of attachment and pathogenicity of spiroplasmas and mycoplasmas

“…Fluorescent DNA staining was performed by using the fluorochrome Hoechst 33258. The procedure was as previously described [13] and included the following steps: fixation with 2% glutaraldehyde in serum-free M1A (Dm-1) or BSR (AS-2 and AC-20) medium, 3 washings followed by air-drying, staining and mounting.…”

“…After cell seeding and spiroplasma inoculation, the Leighton tubes were incubated at 25° C for a spiroplasma-specific period of 2 to 7 days. The cultures were then fixed in 2% glutaraldehyde and the coverslips processed for SEM as described [12,13]. For TEM, the cells were seeded in T25 tissue culture flasks.…”

“…For our studies of interactions between insect cells and mycoplasmas, we chose 3 cell cultures: the Drosophila melanogaster Dm-1 line, and the 2 Agallian leafhopper lines AS-2 and AC-20, derived from the species Aceratagallia sanguinolenta and Agallia constricta. Some of the results have been published [13,14] and show interesting aspects of such interactions which will undoubtedly help to better understand the host-parasite relationship.…”

Infection of Three Insect Cell Cultures by Spiroplasma Citri and Other Spiroplasmas

“…Spiroplasmas as well as mammalian mycoplasmas and Acholeplasma laidlawii multiplied to high titers in Dm-1 cultures (9,10). Spiroplasma dependent cytopathogenic effects (CPE) and culture death was observed.…”