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Published on: March 4, 2018
Studies on cryopreservation of Cryptosporidium parvum
1Livestock and Poultry Sciences Institute, U.S. Department of Agriculture, Beltsville, Maryland 20705.
Abstract:
Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.
Insights
Cryopreservation protocols significantly impact Cryptosporidium parvum infectivity. Only unfrozen oocysts and sporozoites, with or without dimethyl sulfoxide (DMSO), successfully established infections in mice.
Area of Science:
- Veterinary Parasitology
- Microbiology
- Cryobiology
Background:
- Cryptosporidium parvum is a significant enteric pathogen.
- Effective cryopreservation of Cryptosporidium parvum is crucial for research and diagnostics.
- Previous studies have explored various cryopreservation methods with limited success.
Purpose of the Study:
- To evaluate the impact of different cryopreservation protocols on the infectivity of Cryptosporidium parvum oocysts and sporozoites.
- To identify cryopreservation conditions that preserve the viability and infectivity of Cryptosporidium parvum.
Main Methods:
- Neonatal BALB/c mice were infected with oocysts (oral) or sporozoites (rectal) pretreated with various cryopreservation methods.
- Cryopreservation involved different cryoprotective agents (e.g., DMSO, glycerol, propylene glycol) at varying concentrations and cooling rates.
- Control groups received fresh, non-cryopreserved oocysts or sporozoites.
Main Results:
- Only unfrozen oocysts and sporozoites, both those without cryoprotectant and those treated with 10% DMSO, successfully established infections.
- Most cryopreservation protocols, including those with cryoprotectants and controlled cooling rates, rendered the organisms non-infectious.
- Freezing without cryoprotectant or with cryoprotectants but without controlled cooling resulted in loss of infectivity.
Conclusions:
- Standard cryopreservation methods, including the use of cryoprotective agents and controlled cooling, are detrimental to Cryptosporidium parvum infectivity.
- Minimal cryopreservation, such as short exposure to 10% DMSO without freezing, may preserve some infectivity.
- Further research is needed to develop effective cryopreservation techniques for Cryptosporidium parvum.
