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Mosquito-borne diseases such as dengue and filariasis are a growing public health concern in endemic countries. Biological approaches, such as the trans-infection of Wolbachia pipientis in mosquitoes, are an alternative vector control strategy, especially for arthropod-borne viruses such as dengue. In the present study, the effect of Wolbachia (wMel strain) on the vectorial capacity of Aedes aegypti for Dirofilaria immitis was studied. Our results showed that Wolbachia does not affect the phenotype of mosquito survival or the prevalence, number, and molting rate of third-stage larvae in both susceptible and resistant strains of Ae. aegypti. RNA-seq analysis of Malpighian tubules at 2 days post-infection with D. immitis showed the differentially expressed genes (DEGs) with and without wMel infection. No characteristic immune-related gene expression patterns were observed among the DEGs. No significant change in the amount of Wolbachia was observed in the Ae. aegypti after D. immitis infection. Our results suggest that infection of D. immitis in Ae. aegypti populations will not interfere with Wolbachia-based vector control strategies in dengue-endemic areas where cases of D. immitis are present. This study demonstrated the veterinary medical validity of a dengue control program using Wolbachia., (© 2024. The Author(s).)

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research., (© 2024. The Author(s).)

Background: Dirofilaria 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., Methods: Six 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., Results: Purified 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., Conclusions: CultureSure 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.