Exploring cryopreservation alternatives for Dirofilaria immitis microfilariae.

Canine Heartworm Disease, caused by Dirofilaria immitis, primarily affects canids and felids. The earliest studies on cryopreservation were carried out at -70°C, achieving acceptable survival rates, however microfilariae (mf) showed alterations both in morphology and motility. Thereafter, liquid nitrogen was used representing an excellent tool for long-term preservation, albeit it is expensive and requires trained personnel. Accordingly, the aim of this study was to develop a protocol for cryopreservation of D. immitis mf at -80°C feasible to laboratories with limited specialized equipment. The cryoprotectant medium was composed by 5 % dimethyl sulfoxide, 20 % of newborn calf serum and 75 % of saline solution. At Study Day (SD) 0 whole blood from a D. immitis naturally infected dog was diluted with the medium at a ratio of 1:1 and stored at -80°C using a freezing container (Nalgene® Mr. Frosty® Cryo 1°C). On the SD1 and then once a month, one cryovial was thawed and examined for survival, motility, length and morphology of mf. On SD 1, the mf showed a survival rate of 99 %. By SD 120 the survival rate gradually decreased (up to 63 %) and a shift in motility patterns between the "medium" and "slow" classes, was observed. On SD 150, the survival rate exceeded 50 % and mf did not exhibit detectable morphological alterations; however, a reduction in length was observed. This study marks the first protocol where the -80°C freezer has been employed for cryopreservation of D. immitis, integrating the application of cryoprotectants and novel techniques for gradual temperature transition.