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Biology of Host-Parasite Relationships

Current Studies/Facilities

The colonies of vector mosquitoes are from the United States (Anopheles quadrimaculatus and An. freeborni) and from different areas of the world (An. stephensi from India, An. gambiae from Africa;An. dirus, An. sawadwongporni, and An. minimus from Southeast Asia/Thailand; An. farauti from the island of New Britain in Melanesia; An. atroparvus from Spain; and An. albimanus from El Salvador). They are used in experiments to determine their potential for transmission of malaria parasites that might be introduced into the United States.

Sixteen species of primate malaria parasites are maintained in laboratory cultures or experimental animals, or stored frozen in liquid nitrogen. These include isolates of all four species of human malaria parasites (Plasmodium falciparum, P. vivax, P. ovale, and P. malariae) collected from different areas of the world at different times. They also include monkey malaria parasites (P. cynomolgi, P. knowlesi, P. inui, P. gonderi, P. fieldi, P. simiovale, P. coatneyi, P. fragile, P. simium, and P. brasilianum) that have strong similarities to human malaria parasites. Studying these parasites during passage through mosquitoes, monkeys, and culture allows the modelling of parasite-host relationships as regards immunity, pathology, and response and susceptibility to old and newly developed antimalarial drugs. New isolates and strains of malaria parasites are collected, adapted to laboratory culture or nonhuman primates, and tested using the latest available treatments.

The CDC Chamblee animal facility houses several New World monkey species such as Aotus nancymai, A. vociferans (owl monkeys) and Saimiri boliviensis (squirrel monkeys) and Old World monkeys such as Macaca mulatta and M. fascicularis which are obtained from regulated feral colonies, commercial breeders or are in-house laboratory-born. The New World primates are used for experimental infections with either human malaria species (except P. ovale) or a number of the monkey malarias, which are normally parasites of the Old World monkeys. The simian malaria parasites in their macaque monkey hosts make excellent dependable models for the biology of the human malarias in human hosts. However, only the New world primates can be infected with the human parasites and as such are the only available animal models to study vaccine efficacy or drug susceptibility of human malaria parasites. These nonhuman primate hosts of human malarias and of the simian malaria parasites also offer faithful models to investigate mechanisms and treatments for severe pathology associated with malaria infections such as anemia, cerebral malaria and malaria in pregnancy. The animal facility and all procedures involving the animals are under the direction of the resident clinical veterinarian. All protocols are reviewed and approved by the institutional Animal Care and Use Committee in accordance with procedures described in the U. S. Public Health Service Policy, 1986. Parasite counts and other results of laboratory tests are recorded on a daily basis and entered into a computerized database. Animals are fed a diet of primate chow, fruits, and vegetables shown to be adequate for the maintenance of monkeys in malarial studies.

Monkeys are infected by the intravenous inoculation of parasitized erythrocytes (either freshly collected from a donor animal or from samples that have been stored frozen), or by the intravenous inoculation of sporozoites dissected from the salivary glands of infected mosquitoes. On some occasions, transmission is accomplished by allowing infected mosquitoes to feed directly on a tranquilized monkey. Beginning 1 day after inoculation of parasitized erythrocytes or 5 to 12 days after the inoculation of sporozoites, thick and thin blood films are made by the method of Earle and Perez (J. Lab. Clin. Med. 17: 1124-1130.1932), stained with Giemsa, and examined microscopically. Parasites are recorded per microliter of blood.

Mosquitoes are reared and maintained in a climate-controlled facility with adequate containment and separation of the individual species. The insectaries are maintained at 25°C and 70% relative humidity. Although the colonies have proven to be hardy, the 1st and 2nd instar larvae are relatively fragile and will not tolerate overfeeding, excessive agitation, or overcrowding. Adult mosquitoes are housed on 1-gallon paper ice-cream-carton cages with mesh tops. Seven days after emergence, adult mosquitoes are allowed to feed on an anesthetized rabbit; 3 days after the blood meal, 8 oz. cups containing 50 mL of distilled water is introduced into the cages. Egg laying occurs overnight; eggs are recovered, washed with 2% bleach solution and collected on filter paper via vacuum filtration. Eggs are then washed into 9" X 12" enamelware pans with 500 mL distilled water and allowed to hatch. Larvae are fed on days 0, 1, 2, and 3 with active instant yeast. On day 2, pellets of fish food are also added. As larvae grow, pans are split to avoid overcrowding and a powdered food consisting of a 1:1:1:1 mixture of dried Brewer's yeast, lactalbumin, milled New World Monkey Chow, and fish food, that has been passed through a 40 mesh screen, is sprinkled on the surface once daily. Larval pans are usually split every other day. Pupation usually occurs over 4 days; pupae are harvested by the ice water method.

Boxes containining Anopheles mosquitoes ready for experimental feeding on malaria-infected blood

Approximately 200 pupae are placed into 8 oz. paper cups containing 100 mL distilled water and placed in 1 gallon paper ice-cream carton cages for emergence. Adults are fed 10% sugar solution daily on a cotton pad. Adult mosquitoes, aged 3 to 6 days, are "starved" with water pads the night before being used for feeding studies. The procedures used for feeding, handling, and dissection of the mosquitoes have been standardized (J. Parasitol. 53: 1130-1134. 1967). In some instances, blood is collected from the femoral vein of the animal, diluted 1:8 in heparinized human blood and fed to mosquitoes through a parafilm membrane. After infection, mosquitoes are held in an incubator at 25°C and fed 10% sugar solution on a cotton pledget.

Oocyst counts are made microscopically from mosquito guts suspended in a 2% aqueous solution of mercurochrome; this allows for a contrasting vital staining of the parasites. For collection of sporozoites, the salivary glands are dissected into 20% fetal bovine serum in saline. The glands are crushed under a cover slip and the sporozoites washed into a vial. Sporozoites are counted in a Neubauer Cell Counting chamber. Sporozoites are then injected into the femoral vein of the monkey using a 27 gauge needle.

Mosquitoes being fed experimentally using a parafilm membrane; the blood meal is pumped on top of the membrane. (Beginning)	Mosquitoes fed experimentally using a parafilm membrane (near completion)

Recent Publications

Sullivan et al., 2003. Adaptation of a strain of Plasmodium falciparum from Ghana to Aotus lemurinus griseimembra, A. nancymai, and A. vociferans monkeys. Am J Trop Med Hyg 69: 593-600.

Collins et al., 2002. Potential of the Panama strain of Plasmodium vivax for the testing of malarial vaccines in Aotus nancymai monkeys. Am J Trop Med Hyg 67: 454-458.

Collins et al., 2002. Experimental infection of Anopheles farauti with different species of Plasmodium. J Parasitol 88: 295-298.

Collins et al., 2001. Plasmodium coatneyi: Observations on periodicity, mosquito infection, and transmission in Macaca mulatta monkeys. Am J Trop Med Hyg 64: 101-110.

Collins et al., 2000. Efficacy of vaccines containing rhoptry-associated proteins RAP1 and RAP2 of Plasmodium falciparum in Saimiri boliviensis monkeys. Am J Trop Med Hyg 62: 466-479.

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