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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.
Insights
This study shows the Drosophila melanogaster cell line Dm-1 can propagate various Spiroplasma species. Some Spiroplasma isolates caused cytopathic effects in Dm-1 cells, impacting cell viability.
Area of Science:
- Microbiology
- Cell Biology
- Insect Pathology
Background:
- Spiroplasmas are wall-less bacteria that can infect plants, insects, and mammals.
- The Drosophila melanogaster cell line (Dm-1) is a valuable tool for studying insect-borne pathogens.
Purpose of the Study:
- To evaluate the susceptibility of the Dm-1 cell line to various Spiroplasma isolates.
- To characterize the cytopathic effects (CPE) induced by Spiroplasma infection in Dm-1 cells.
- To assess the utility of Dm-1 cells for primary isolation and propagation of Spiroplasmas.
Main Methods:
- Propagation of Spiroplasma species (S. citri, honey bee isolate, corn stunt organism, etc.) in Dm-1 cells.
- Monitoring of cell viability and observation of cytopathic effects using microscopy and colony counting.
- Detection of Spiroplasmas via agar inoculation, dark-field microscopy, scanning electron microscopy, and DNA fluorescent staining.
- Uridine phosphorylase activity assay to differentiate Spiroplasma strains.
Main Results:
- Dm-1 cells supported the growth of several Spiroplasma strains to high titers (10^8-10^9 CFU/ml).
- The honey bee Spiroplasma isolate caused rapid cell death and CPE in Dm-1 cells and mammalian cells.
- Spiroplasma citri and other plant isolates lacked uridine phosphorylase activity.
- Corn stunt organism infection led to a significant decrease in Dm-1 cell viability over three passages.
Conclusions:
- The Dm-1 cell line is a suitable host for propagating diverse Spiroplasma species and studying their interactions.
- Spiroplasma-induced CPE in Dm-1 cells varies depending on the isolate, with some being highly virulent.
- The Dm-1 system facilitates primary isolation and characterization of Spiroplasmas, including those from plant pathogens.