Abstract
BackgroundDirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals. The parasite is endemic worldwide. Vector stage research requires a reliable supply of D. immitis microfilariae (mf). It is believed that cryopreserved mf would retain viability and provide a powerful tool for vector stage research. However, reports on cryopreservation of D. immitis mf are limited. Therefore, this study aimed to validate commercial cryopreservation media to establish a practical, convenient and reproducible storage procedure for D. immitis mf.MethodsSix different commercially available cryopreservation media were compared with the traditional polyvinylpyrrolidone-dimethyl sulfoxide (PVP-DMSO) preservation solution. In vitro viability of purified D. immitis mf and mf-infected total blood was analyzed using a motility assay and propidium iodide staining. In vivo infectivity of Aedes aegypti mosquitoes with cryopreserved mf was assessed using a mosquito survival test and quantifying the number of third-stage larvae (L3) after 13 days post-infection.ResultsPurified mf cryopreserved in CultureSure showed the best viability when compared to mf cryopreserved in the remaining five commercially available media and PVP-DMSO. Viability of mf in mf-infected total blood cryopreserved in CultureSure varied with the ratio of infected blood to CultureSure. Optimum results were obtained with 200 µl mf-infected blood:800 µl CultureSure. CultureSure was also the optimum medium for cryopreserving mf prior to infectivity of A. aegypti. The number of L3 was approximately the same for CultureSure cryopreserved mf (3× concentrated solution) and non-cryopreserved fresh mf.ConclusionsCultureSure is an optimal commercial cryopreservation solution for the storage of D. immitis purified mf, mf-infected total blood, and mf used for in vivo mosquito experiments. Furthermore, this study describes an easy preservation method for clinical D. immitis-infected blood samples facilitating vector stage studies, as well as the study of macrocyclic lactone resistance in heartworms and the education of veterinarians.
Highlights
Dirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals
In vitro viability of purified microfilariae The viability of cryopreserved mf was compared between traditional PVP-DMSO and several commercially available cryopreservation media
Microscopic observation revealed that mf cryopreserved with CultureSure exhibited the highest motility (χ2 = 721.2, df = 7, P < 0.0001, adjusted residuals (AR) = 8.5 on 0 dpt; χ2 = 648.5, df = 7, P < 0.0001, AR = 10.51 on 1 dpt and χ2 = 333.3, df = 7, P < 0.0001, AR = 12.19 on 3 dpt) (Fig. 1a)
Summary
Dirofilaria immitis is a parasitic nematode transmitted by mosquitoes and the cause of heartworm disease in dogs and dirofilariasis in humans and other mammals. Vector stage research requires a reliable supply of D. immitis microfilariae (mf ). It is believed that cryopreserved mf would retain viability and provide a powerful tool for vector stage research. The parasitic nematode Dirofilaria immitis is transmitted by mosquitoes causing potentially lethal heartworm disease in dogs, pulmonary dirofilariasis in humans, and dirofilariasis in various mammals [1]. Despite the availability of effective preventive medicines, D. immitis is endemic worldwide. A better understanding of host-parasite interactions between mosquito vectors and D. immitis is needed to establish a truly effective control strategy. Vector stage experiments require live D. immitis microfilariae (mf ) obtained from infected animals [2]. In the most recent study, published in 2001, Bartholomay et al [6] prepared and tested a 16% polyvinylpyrrolidone (PVP)-6% DMSO solution (PVP-DMSO) as a cryoprotective medium
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call