Cryopreservation of Trichinella spiralis muscle stage larvae

A cryopreservation protocol for Trichinella spiralis muscle stage larvae is described. Larvae are pretreated in 10% bile at 37 degrees C for 1 hr to induce an increase in surface permeability, than incubated in 20% v/v ethanediol at 37 degrees C for 10 min, transferred to 0 degrees C for a second incubation step of 15 min in a v/v mixture of 33% ethanediol: 33% methanol: 34% saline at 0 degrees C followed by rapid cooling (approximately 5,100 degrees C min-1) of aliquots distributed onto glass coverslips. The larvae are thawed by dropping the coverslips into 2 ml saline at room temperature (20 degrees C) and immediately agitating, which simultaneously dilutes (1:100) the cryoprotectants. Groups of 6 mice were infected with muscle stage larvae by gastric intubation. Five days post-infection 14.5 +/- 3.2% of control untreated unfrozen MSL and 16.5 +/- 4.1% of unfrozen bile and cryoprotectant treated controls were recovered as adult worms from the small intestine. Of the cryopreserved larvae, 1.1 +/- 0.4% were recovered as adults, which represents 7.6% compared to the untreated unfrozen controls. When the bile pretreatment step was omitted fewer adult worms (0.09 +/- 0.04%) were recovered and no second generation muscle stage larvae were produced. Modifying this technique by omitting the first incubation step in 20% ethanediol and extending the second incubation step to 25 min yielded 72.3% recovery of T. nativa adult worms 5 days post-infection compared to unfrozen controls. The reproductive capacity index of bile treated cryopreserved T. spiralis was 2.5 +/- 0.6 compared to 51.8 +/- 18.8 for normal muscle stage controls.