Studies on the Transmission of Simian Malaria V. Infection and Transmission of the B Strain of Plasmodium cynomolgi

Abstract

Five species of mosquitoes, Anopheles freebomi, A. balabacensis balabacensis, A. maculatus, A. stephensi, and A. quadrimaculatus, were examined with regard to their susceptibility to infection with the B strain of Plasmodium cynomolgi. It was found that feedings between the 9th and the 16th days of patent parasitemia in intact Macaca mulatta monkeys resulted in the heaviest mosquito infection. Oocyst counts of greater than 20 per gut resulted in heavy salivary gland infections in 50% or more of the test mosquitoes. Transmission to M. mulatta monkeys was obtained in 95 of 103 attempts by the bites of from 2 to 250 mosquitoes. The prepatent period ranged from 7 to 18 days with a median of 11 days. Plasmodium cynomolgi is one of the most widely studied monkey malarias. Being a malaria with a true relapse mechanism, it serves as a model for studies on chemotherapy (Eyles and Coatney, 1962; Schmidt et al., 1948, 1963, 1966, 1970), immunity (Voller et al., 1966; Voller and Rossan, 1969a, b, c), and physiology (Brewer and Coan, 1969; Eaton and Brewer, 1969; Kuvin et al., 1962). Although readily transmitted by a number of experimental vectors (Warren and Wharton, 1963; Coatney et al., 1971), the infection of mosquitoes upon monkeys infected with this parasite has not been altogether predictable; therefore, an analysis was made of the results of mosquito infection studies which have been conducted during the last 12 years in our laboratory to determine if a dependable schedule could be made for the infection of mosquitoes in the laboratory. The results of these studies are presented here. MATERIALS AND METHODS All the results reported here are of studies which employed the isolate described as P. cynomolgi bastianellii by Garnham (1959). We have elected to refer to this parasite as the B strain of P. cynomolgi (Contacos et al., 1962). The parasite was originally isolated from a Macaca irus (= fascicularis) monkey captured in the State of Pahang, West Malaysia, and obtained from Professor P. C. C. Garnham in April, 1959. Macaca mulatta monkeys from northern India were used throughout the study. Prior blood film Received for publication 17 February 1972. examinations indicated that they were free of malarial infection. The Anopheles balabacensis balabacensis, A. freebori, A. maculatus, A. stephensi, and A. quadrimaculatus mosquitoes have been maintained in our laboratory for several to many years (Collins et al., 1967). The procedures used in the feeding, handling, and dissection of the mosquitoes have been presented previously. The intensity of the gland infections was graded 1+ to 4+ as previously described (Collins et al., 1966). For the transmission attempts, mosquitoes were caged and allowed to bite individually on restrained monkeys. Immediately after biting, mosquitoes were dissected and the salivary glands examined for the presence of sporozoites. In some instances, the salivary glands were dissected into 10% monkey serum-saline solution, crushed under a cover slip, and then introduced either intravenously or, at laparotomy, directly into the liver. The remaining bodies of the mosquitoes were triturated in a motar and pestle with monkey serum-saline solution, centrifuged lightly, and the supernatant injected intravenously into the animal. Peripheral blood smears were made daily beginning 5 days after exposure to infection. Blood films, prepared by the Earle-Perez technique, were stained with Giemsa stain and the parasitemias recorded per mm3 of blood. Gametocyte counts were made from the thin film preparation and recorded per 100 WBC.