Takahiro Shirozu

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.

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.

The Iriomote cat (Prionailurus bengalensis iriomotensis) is an endangered leopard cat that inhabits only Iriomotejima Island, Japan. The number of Iriomote cats is estimated to be approximately 100-110 and has been almost stable but slightly decreased in the coastal lowland. Infectious diseases are one of the major concerns in the conservation of Iriomote cats. Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis. Toxoplasmosis is fatal in immunocompromised hosts, especially kittens and cat fetuses. We investigated the seroprevalence of T. gondii in Iriomote cats. Based on seroprevalence data, we simulated the effect of T. gondii infection on the population dynamics of Iriomote cats. The seroprevalence of T. gondii in Iriomote cats was significantly higher than that in domestic cats and dogs. Furthermore, the seroprevalence of T. gondii in domestic cats was significantly higher on Iriomotejima Island than on Okinawajima Island. Our simulation demonstrated that T. gondii eradication may help increase the population of Iriomote cats by an average of 11.5 (23.1%) cats in 2120. Therefore, controlling T. gondii may be an effective strategy for the conservation of Iriomote cats.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

Vector control is crucial for mitigation of mosquito-borne diseases such as malaria. The emergence of insecticide resistance has begun to hamper vector control in recent years. Novel vector control strategies that do not depend on chemical insecticides are needed. We evaluated the potential of venoms, the poisonous secretion of the animals, as sources of novel mosquitocidal molecules. We screened for mosquitocidal effects of seven venoms, using Melittin (Mel) as a positive control by microinjection using the malaria vector Anopheles stephensi mosquitoes. Tf2, Hm3a (Hm3), Chlorotoxin (Ctx) and Mel caused significantly high mortality among these mosquitoes. To identify the most effective venom, we assayed for the lowest effective dose of Tf2, Hm3, Ctx, and Mel. Tf2 displayed the highest mosquitocidal potency. We next determined the LD₅₀ of Tf2 against An. stephensi mosquitoes. The LD₅₀ of Tf2 against An. stephensi was 5.9×10⁻¹³ mol/mosquito. Our results indicated that Tf2 was the most effective candidate venom molecule for a novel vector control agent. Further research regarding Tf2 is expected to contribute to the control of mosquito-borne diseases such as malaria.