Survival and Viability of Dirofilaria immitis Microfilariae in Defined and Undefined Culture Media

Abstract

In vitro cultivation of Dirofilaria immitis microfilariae has been utilized to observe worm development (Devaney, 1981, Acta Tropica 38: 251-260) as well as to perform immunological (El-Sadr et al., 1983, Journal of Immunology 130: 428-434; Rzepczyk and Bishop, 1984, Parasite Immunology 6: 443-457), pharmacological (Devaney and Howells, 1984, Annals of Tropical Medicine and Parasitology 73: 139-144), and physiological (Jaffe and Doremus, 1970, Journal of Parasitology 56: 254-260; Ando et al., 1980, American Journal of Tropical Medicine and Hygiene 29: 213-216) studies. The objective of the present study was to test various culture conditions for long-term maintenance of D. immitis microfilariae. Culture conditions that permitted extended microfilarial survival were further evaluated by testing the ability of cultured microfilariae to develop to infective larvae in mosquitoes. Microfilariae, recovered from infected canine blood by a saponin lysis technique (Grieve et al., 1984, Acta Tropica 41: 271-278), were placed in culture in 1 of the following 4 media: L-15, RPMI1640, F12 and F12K (Gibco, Grand Island, New York). Gentamicin (100 Ag/ml) was added to the various media, and culture fluid consisted of media alone or with heat-inactivated fetal calf serum (FCS) (Flow Laboratories, McLean, Virginia) at concentrations of 40%, 20%, 10%, or 5%. Microfilariae were cultured in 2 ml of media in 110 x 16-mm flat-sided tubes (Nunc, Kamstrup, Denmark) at a concentration of 15,000 microfilariae per ml. Cultures were maintained at 37 C in 5% CO2 in air, and microfilarial motility was assessed weekly. All of the media selected for evaluation in this study were equally proficient at maintaining microfilariae if they were supplemented with 40% FCS. At lower FCS concentrations, microfilariae survived for abbreviated time periods in L-15, RPMI and F12. Reducing the concentration of FCS in F12K to as low as 5% did not increase microfilarial mortality. Over 50% of microfilariae cultured in F12K without FCS survived for 4 wk; addition of 5% FCS allowed 50% survival for 6 wk (Fig. 1). Changing media every 8 days did not enhance microfilarial survival. Microfilariae, recovered after 2 wk in culture in F-12K or F-12K with 5% FCS, were inoculated into female Aedes aegypti mosquitoes using the fluid enema technique described by Grieve et al. (1984, Acta Tropica 41: 271-278) based on Klowden (1981, Transactions of the Royal Society of Tropical Medicine and Hygiene 75: 354358). Mosquitoes were also inoculated with microfilariae recovered directly from canine blood. A dose of 30 microfilariae was administered to each mosquito. Nineteen days after inoculation, the mosquitoes were dissected, and numbers and stages of larval D. immitis were recorded. It was determined that after 2 wk in culture in either F12K or F12K with 5% FCS, microfilariae were still capable of normal development in mosquitoes (Table I).