Effects of sub-zero storage temperatures on endoparasites in canine and equine feces

Fecal samples from wild and domestic carnivores are routinely frozen for three days at -80°C to kill eggs of Echinococcus spp., following recommendations from the World Health Organization (WHO) and World Organization for Animal Health (OIE). This is done to decrease the risk of zoonotic infection with these pathogenic cestodes. In addition, it is often necessary to freeze fecal samples collected for research prior to batch processing by a limited number of personnel, especially large numbers of samples or those collected in remote locations. The effect of freezing on the recovery of endoparasite eggs, cysts and oocysts from fecal samples is not well documented, even in hosts for which veterinary diagnostic submissions are relatively common. In this study, fecal samples from naturally infected dogs and horses were split into four treatment groups: fresh; -80°C for 3 days; -20°C for 30 days; and -80°C for 3 days followed by -20°C for an additional 30 days. Temperatures and times were chosen to simulate diagnostic and research protocols currently in place. Helminth eggs and sporocysts of Sarcocystis spp. were counted using a quantitative double centrifugation sucrose fecal flotation (modified Stoll egg count). Repeated measures ANOVA was used to detect differences in egg/sporocyst counts between the treatment groups for Sarcocystis spp. sporocysts, taeniid eggs (Taenia and/or Echinococcus spp.), ascarid eggs (Parascaris equorum, Toxocara canis, Toxascaris leonina), and strongylid type eggs (Uncinaria stenocephala, and equine strongylids, most likely cyathostomins). Counts for P. equorum and strongylid type eggs (both equine and canine) decreased significantly following freezing. Post-freezing, some samples that had been positive on fresh examination became negative for Parascaris and strongylid eggs. This study suggests that protocols requiring freezing artificially lowers recovery of eggs of Parascaris and strongylid nematodes in fecal surveys; however, recovery rates for eggs of other helminth species as well as sporocysts of Sarcocystis spp. were relatively robust compared to the freshly tested fecal samples. This is reassuring for large scale fecal surveys needed for fecal egg count reduction analyses following treatment, and for surveillance in wildlife and remote human and animal populations.