Exploring cryopreservation alternatives for Dirofilaria immitis microfilariae

Area of Science:

• Veterinary Parasitology
• Cryobiology

Background:

• Canine heartworm disease, caused by Dirofilaria immitis, impacts canids and felids.
• Previous cryopreservation studies used -70°C with suboptimal microfilariae (mf) survival and morphology, or expensive liquid nitrogen.
• A need exists for cost-effective, accessible cryopreservation methods for D. immitis mf.

Purpose of the Study:

• To develop and validate a cryopreservation protocol for D. immitis mf at -80°C.
• To create a method suitable for laboratories with limited specialized equipment.

Main Methods:

• Developed a cryoprotectant medium: 5% DMSO, 20% newborn calf serum, 75% saline.
• Diluted infected dog blood 1:1 with medium and stored at -80°C using a controlled-rate freezing container.
• Assessed survival, motility, length, and morphology of mf monthly for up to 150 days.

Main Results:

• Achieved 99% survival of D. immitis mf at 1 day post-cryopreservation.
• Survival decreased to 63% by 120 days, with observed shifts in motility patterns.
• Over 50% survival was maintained at 150 days, with no detectable morphological alterations but reduced length.

Conclusions:

• This study establishes the first protocol for cryopreserving D. immitis mf at -80°C.
• The protocol integrates cryoprotectants and gradual temperature transition, offering an accessible method for long-term preservation.
• This method supports research and diagnostics for canine heartworm disease in resource-limited settings.