Storage of pathogenic leptospires in liquid nitrogen
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Abstract:
The virulence and viability of various serovars of Leptospira interrogans were successfully preserved by storage in liquid nitrogen. Dimethyl sulphoxide at a final concentration of 2.5% (v/v) was added as cryoprotectant to a culture of leptospires grown in Ellinghausen-McCullough-Johnson-Harris medium. Ampoules were cooled at a controlled rate of 1 degree-3 degrees C/min to -70 degrees C, then transferred to the liquid phase of a liquid nitrogen storage unit. Glycerol was discounted as a cryoprotectant as it was found to be approximately 10 times more toxic than dimethyl sulphoxide to four of five serovars used in this study. The viability of nine strains has so far been observed over a period of 8-22 months storage in liquid nitrogen and full viability of all strains has been preserved over this period. Virulence of strains of serovars pomona and hardjo was well preserved, as demonstrated by challenge tests in guinea pigs and domestic pigs.
Insights
Liquid nitrogen successfully preserves Leptospira interrogans viability and virulence. Dimethyl sulfoxide is an effective cryoprotectant, outperforming glycerol for Leptospira storage.
Area of Science:
- Microbiology
- Veterinary Science
Background:
- Leptospira interrogans serovars are significant pathogens.
- Effective long-term preservation methods are crucial for Leptospira research and diagnostics.
Purpose of the Study:
- To evaluate the efficacy of liquid nitrogen for preserving the virulence and viability of Leptospira interrogans.
- To compare cryoprotective agents for Leptospira storage.
Main Methods:
- Leptospira interrogans cultures were preserved in liquid nitrogen using dimethyl sulfoxide (2.5% v/v) as a cryoprotectant.
- Controlled cooling rates were employed before transfer to liquid nitrogen.
- Cryoprotectant toxicity and strain viability were assessed over 8-22 months.
- Virulence was tested via challenge studies in guinea pigs and domestic pigs.
Main Results:
- Liquid nitrogen storage successfully preserved the viability of nine Leptospira interrogans strains for up to 22 months.
- Dimethyl sulfoxide demonstrated significantly lower toxicity compared to glycerol.
- Virulence was maintained in preserved strains of serovars pomona and hardjo.
Conclusions:
- Liquid nitrogen is a suitable method for long-term preservation of Leptospira interrogans.
- Dimethyl sulfoxide is a preferred cryoprotectant for Leptospira storage.
- Preserved strains retain both viability and virulence for experimental use.