Cryopreservation and long-term in vitro maintenance of second-stage larvae of Toxocara canis

Abstract

Second stage larvae of Toxocara canis were isolated from developed eggs, frozen in Eagle's Minimal Essential Medium with 5% dimethyl sulfoxide or 10% glycerol as cryoprotectants according to two cooling schedules and maintained in liquid nitrogen for 1 week. After thawing, the previously frozen larvae (FL) and unfrozen controls (CL) were maintained in a chemically defined medium in vitro for 35 weeks. While CL had motility rates around 95% to 97% throughout the experiment, previously frozen larvae (FL) exhibited rates of 48%–58% at the beginning and of 19%–39% at the end of the 35 week in vitro maintenance period. The surviving FL and CL larvae proved to be infective for mice. Excretory/secretory (ES) antigens isolated from several batches of culture medium in which FL and CL had been maintained reacted in the ELISA with human sera containing antibodies against Toxocara. Antigens from FL and CL separated by SDS-PAGE and silver-stained showed some differences in polypeptide patterns. Western-blot analysis revealed that these differences were not related to antigenic polypeptides but were most likely caused by substances without antigenic properties originating from dead and/or degenerating larvae. It can be concluded that ES antigens produced by previously frozen larvae are essentially the same as those derived from unfrozen controls.

The value of cryopreservation of T. canis larvae for routine production of ES antigens will be further evaluated.