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Characterization of Spiroplasma mirum (suckling mouse cataract agent) in a rabbit lens cell culture
Abstract:
Spiroplasma mirum (suckling mouse cataract agent) was studied in an epithelial cell line AG-4676, derived from rabbit eye lens. Rabbit eye lens is a natural target tissue of S. mirum infection. The organism grew rapidly in this cell line, reaching titers of 10(7) to 10(9) color change units per ml at 7 days after infection. This is the same level as that achieved in SP-4 medium designed specifically for S. mirum. No lag period was apparent in growth in AG-4676. S. mirum did not grow in Dulbecco minimal essential medium-10% fetal bovine serum, the medium for AG-4676, indicating the need for cells or a cellular product. S. mirum-infected AG-4676 cells exhibited vacuolization and granulation and an increase in polynucleation compared with uninfected controls (36/100 versus 14/100, P less than 0.001). Infection significantly decreased the growth rate of AG-4676, especially late in the growth cycle. In a representative experiment, growth of AG-4676 at 11 days was reduced from 9 X 10(5) to 2 X 10(4) cells by S. mirum infection. S. mirum grew to high titers in conditioned medium of AG-4676, obtained from cell-free supernatants of 1- to 5-day-old AG-4676 cultures. This growth promotion was not due to osmotic conditioning of the medium. Preliminary characterization of this growth promotion substance showed it to be active after 0.22-micron filtration, heating at 56 degrees C for 30 min, freezing and thawing, and dilution at 10(-1) but not 10(-2). AG-4676-propagated S. mirum produced death or cataracts in suckling Wistar rats at the same frequency (55/60, 91.7%) as SP-4-propagated organisms (60/65, 92.3%).
Insights
Spiroplasma mirum replicates efficiently in rabbit eye lens epithelial cells (AG-4676), mirroring growth in specialized media. This infection impacts cell morphology and growth, but the resulting Spiroplasma mirum causes cataracts in rats similarly to standard culture methods.
Area of Science:
- Microbiology
- Cell Biology
- Ophthalmology
Background:
- Spiroplasma mirum, the suckling mouse cataract agent, naturally infects rabbit eye lenses.
- Understanding its in vitro growth requirements is crucial for studying its pathogenesis.
Purpose of the Study:
- To investigate the in vitro growth of Spiroplasma mirum in a rabbit eye lens epithelial cell line (AG-4676).
- To characterize the effects of S. mirum infection on AG-4676 cells.
- To assess the infectivity of cell-cultured S. mirum in a susceptible animal model.
Main Methods:
- Culturing Spiroplasma mirum in AG-4676 cells and SP-4 medium.
- Comparing growth kinetics and cell morphology between infected and uninfected AG-4676 cells.
- Inoculating suckling Wistar rats with S. mirum propagated in AG-4676 cells or SP-4 medium.
Main Results:
- S. mirum achieved high titers (10^7-10^9 CCU/ml) in AG-4676 cells, comparable to SP-4 medium, with no lag phase.
- Infected AG-4676 cells showed vacuolization, granulation, increased polynucleation, and significantly reduced growth rates.
- Conditioned medium from AG-4676 cells supported S. mirum growth, suggesting the presence of essential cellular factors.
- AG-4676-propagated S. mirum induced cataracts and death in rats at a high frequency (91.7%), similar to SP-4 propagated organisms (92.3%).
Conclusions:
- Rabbit eye lens epithelial cells (AG-4676) provide a suitable in vitro environment for Spiroplasma mirum replication.
- S. mirum infection adversely affects AG-4676 cell viability and growth.
- Cell-free factors from AG-4676 cultures promote S. mirum growth.
- In vitro propagated S. mirum retains its pathogenic potential for inducing cataracts and mortality in vivo.