Peggy S. Stanfill

SPOROZOITE-INDUCED INFECTIONS OF THE SALVADOR I STRAIN OF PLASMODIUM VIVAX IN SAIMIRI SCIUREUS BOLIVIENSIS MONKEYS

Twenty Saimiri sciureus boliviensis monkeys from Bolivia were inoculated intravenously with sporozoites of the Salvador I strain of Plasmodium vivax. All animals were splenectomized 7 days after inoculation. Inoculation of 100,000 sporozoites resulted in prepatent periods averaging 16.6 days; all monkeys developed high-level parasitemias with an average maximum of 103,000 per mm3. Inoculation of 10,000 sporozoites resulted in average prepatent periods of 19.4 days; one of the resulting infections produced a transient low-level parasitemia. Of 5 monkeys inoculated with 1,000 sporozoites, 4 had prepatent periods of from 24 to 35 days and 1 failed to demonstrate any parasitemia; 1 monkey supported a low-level transient parasitemia, whereas the other 3 monkeys had high-level parasitemias. It is proposed that by using a minimum inoculum of 10,000 sporozoites, the model system may be useful in the testing of anti-sporozoite vaccines directed against P. vivax.

Community-based malaria control in Saradidi, Kenya: description of the programme and impact on parasitaemia rates and antimalarial antibodies.

A community-based malaria control programme initiated in Saradidi, Kenya in 1982 is described. Antimalarial treatment provided by volunteer community health workers was made available in each village. Malaria was holoendemic. Parasitaemia rates by age were high and did not change after the control programme began. Plasmodium falciparum was the most common species and was present alone or mixed in 98.2% of 8105 infections. Virtually all (98.5%) of 2040 blood samples collected in May 1981 were positive (reciprocal titre greater than or equal to 80) to P. falciparum by the indirect fluorescent antibody (IFA) test. Seropositivity rates to P. falciparum in the IFA test or the enzyme-linked immunosorbent assay (ELISA) were high in all age groups and did not change significantly in longitudinal surveys or in a cohort of children zero to nine years old followed at intervals. While the malaria control programme was successful in bringing treatment to each village, malaria prevalence was not reduced. Parasitologic and serologic studies alone were not adequate to describe the impact of the community-based malaria control programme in Saradidi. Morbidity and mortality rates caused by malaria can decline, significantly improving the health of the population, in the absence of any decrease in parasitaemia rates.

Studies on human malaria in Aotus monkeys. I. Sporozoite transmission of Plasmodium vivax from El Salvador.

Monkey-to-monkey transmission of a strain of Plasmodium vivax from El Salvador was obtained on 12 occasions via the bites or intrahepatic and/or intravenous inoculation of infected salivary glands from Anopheles freeborni, A. albimanus, A. maculatus, and A. balabacensis balabacensis mosquitoes. The prepatent periods ranged from 14 to 48 days. The mean prepatent period was 34 days in intact monkeys and 29 days in splenectomized animals. The transmission rate was approximately the same in intact (57%) and splenectomized (53%) monkeys. Since Porter and Young (1966) reported the successful infection of Aotus trivirgatus monkeys with Plasmodium vivax from Panama, there has been a great deal of interest in the use of these animals as biological models for many different malarial studies. Young et al. (1966) and Porter and Young (1966) reported the successful transmission of P. vivax from the owl monkey to man via the bites of Anopheles albimanus mosquitoes. Subsequently, Baerg et al. (1969) reported transmission of P. vivax from A. trivirgatus and Ateles fusiceps to A. fusiceps, A. trivirgatus, and Saguinus geoffroyi via the bites of A. albimanus mosquitoes. Ward et al. (1969) showed that the Chesson strain of P. vivax could be transmitted with relative ease to A. trivirgatus monkeys via the bites of A. balabacensis balabacensis, A. stephensi, and A. quadrimaculatus mosquitoes. Since 1966, we have studied a number of different isolates of the human malarias in Aotus monkeys. This paper, the first of a series, reports the results of attempts to transmit a strain of P. vivax from El Salvador from monkey to monkey by sporozoite inoculation. MATERIALS AND METHODS The Sal II strain of P. vivax was isolated from a natural infection in the area of Las Guarumas, in the state of La Paz, El Salvador. Blood was inoculated into an A. trivirgatus monkey (AO-219) which was then provided to us by the staff of the Central America Malaria Research Station, CDC, San Salvador. The strain has been maintained by Received for publication 9 March 1973. * Center for Disease Control, Malaria Program, 1600 Clifton Road, Atlanta, Georgia 30333. serial blood or sporozoite passage in A. trivirgatus monkeys. The A. trivirgatus monkeys were obtained commercially, their origin being Colombia, South America. Prior parasitologic and serologic examinations indicated that they were free of natural malarial infection. Some of the animals were splenectomized prior to infection according to the technique of Sodeman et al. (1970). The A. freeborni mosquitoes were originally from Marysville, California, and have been maintained in the laboratory since 1944. The A. maculatts were obtained from the Institute for Medical Research, Kuala Lumpur, Malaysia, in 1964 and have been maintained in our laboratory since then. The A. balabacensis balabacensis colony was originally from Thailand and was obtained from the Walter Reed Army Institute of Research. The A. albimanus were the Apastepeque strain from El Salvador and obtained from CAMRS, San Salvador, in 1971. For the transmission attempts, sporozoites were introduced either by the bites of infected mosquitoes or by the intrahepatic and/or intravenous inoculation of sporozoites from dissected salivary glands. The technique for the intrahepatic inoculation has been reported previously (Held et al., 1967; Sodeman et al., 1969). For the transmission attempts by mosquito bite, mosquitoes were individually caged and allowed to feed on the restrained monkey. After biting, the mosquitoes were dissected and the salivary glands examined for the presence of sporozoites. The gland infections were graded 1+ (1 to 10 sporozoites), 2+ (11 to 100 porozoites), 3+ (101 to 1,000 sporozoites), or 4+ (greater than 1,000 sporozoites). Daily blood smears were made beginning 7 days after sporozoite inoculation to determine the prepatent period. Blood smears were stained with Giemsa.

INFECTION OF AOTUS AZARAE BOLIVIENSIS MONKEYS WITH DIFFERENT STRAINS OF PLASMODIUM FALCIPARUM

Eleven strains of Plasmodium falciparum from Asia, Africa, and Central America were inoculated into a total of 58 splenectomized Aotus azarae boliviensis monkeys. Eight of the strains produced high-level parasitemias, whereas 3 (2 from Honduras and 1 from Zaire) produced only low-level parasitemias. Mosquito infections were only obtained during the first 2 linear passages of the Santa Lucia strain from El Salvador. The results indicate that this species of Aotus monkey is highly susceptible to infection with strains of P. falciparum from different geographic areas, and therefore may be useful for a number of chemotherapeutic or immunologic studies. Its usefulness for mosquito infection studies is very limited.

SUSCEPTIBILITY OF MACACA FASCICULARIS MONKEYS FROM MAURITIUS TO DIFFERENT SPECIES OF PLASMODIUM

Macaca fascicularis monkeys from Mauritius were shown to be susceptible via sporozoite inoculation to 7 species of Plasmodium (P. fragile, P. coatneyi, P. gonderi, P. inui, P. cynomolgi, P. knowlesi, and P. fieldi), indigenous to macaques in southeastern Asia. Four monkeys were sequentially infected with different species of Plasmodium to determine maximum and course of parasitemia. In 2 nonsplenectomized monkeys, P. fragile developed maximum parasite counts of only 134 and 155/microliters. For Plasmodium knowlesi, a parasite that is life-threatening to rhesus monkeys, maximum parasite counts were 4,278 and 7,440/microliters. Plasmodium coatneyi developed to what must be considered as moderate levels. After animals underwent splenectomy, parasite counts of P. coatneyi were 58,280, 89,094, 4,464, and 43,524/microliters. The maximum parasite counts for P. gonderi (13,508 and 21,576/microliters) and P. fieldi (1,767 and 17,836/microliters) were lower than would be expected in M. mulatta. In 2 monkeys that developed patent parasitemia with P. inui, the maximum parasite counts (95,046 and 728,748/microliters) indicated that this parasite may be the best adapted species for development in these animals once infection is established. Finally, the reinfection of 2 monkeys with P. cynomolgi suggested that some animals may be basically more resistant than others, whether splenectomized or not, to the production of high-density parasitemia.

Infection of Peruvian Aotus nancymai Monkeys with Different Strains of Plasmodium falciparum, P. vivax, and P. malariae

Aotus nancymai (karyotype I) monkeys from Peru were studied for their susceptibility to infection with Plasmodium falciparum, P. vivax, and P. malariae. Three strains of P. falciparum (Santa Lucia from El Salvador, Indochina I/CDC from Thailand, and Uganda Palo Alto) were inoculated into 38 monkeys. The results indicated that this species of Aotus monkey is highly susceptible to infection. The Uganda Palo Alto and the Santa Lucia strain parasites appear to be the most useful for immunologic and chemotherapeutic studies. Five strains of P. vivax (Chesson, ONG, Vietnam Palo Alto, Salvador I, and Honduran I/CDC) were inoculated into 28 monkeys. The Vietnam Palo Alto strain produced the highest level parasitemias ranging from 23,800 to 157,000/mm3. Mosquito infections were obtained with the ONG, Chesson, and Salvador I strains. Two out of 6 attempts to transmit P. vivax via sporozoite inoculation to splenectomized monkeys were successful with prepatent periods of 39 and 57 days. Five monkeys were infected with the Uganda I/CDC strain of P. malariae. Maximum parasitemias ranged from 10 to 5,390/mm3.

INFECTION OF AOTUS VOCIFERANS (KARYOTYPE V) MONKEYS WITH DIFFERENT STRAINS OF PLASMODIUM VIVAX

Twenty splenectomized Aotus vociferans (karyotype V) monkeys were infected with strains of Plasmodium vivax from New Guinea, North Korea, Indonesia, El Salvador, and Honduras. Peak parasite densities ranged from 4,840 to 75,500 per mm3. Gametocytes infective to different species of mosquitoes were produced with all strains of P. vivax studied. Two transmissions of the Chesson strain of P. vivax were made by the intravenous inoculation of dissected sporozoites from An. dirus mosquitoes. Prepatent periods were 16 days.

Malaria chemoprophylaxis to pregnant women provided by community health workers in Saradidi, Kenya. III. Serologic studies.

Parasitaemia and antimalarial antibodies were examined from May 1983 to March 1984 in monthly samples taken from 930 pregnant women attending antenatal clinics in Saradidi, Kenya, and 317 of their infants; 104 women were taking chloroquine phosphate 300 mg base weekly for chemoprophylaxis. Seropositivity rates in pregnant women were uniformly high, and mean enzyme-linked immunosorbent assay (ELISA) absorbance values were not related to presence of parasitaemia or history of chemoprophylaxis. Parasitaemia was present in 26.5% of 1677 slides from pregnant women and there was little variation by month of sample. Mean ELISA absorbance values varied by month of sample. Seropositivity rates in infants were high as measured in both the indirect fluorescent antibody (IFA) test (81.6% of 938) and ELISA at 1:100 (83.8% of 1025) and 1:1000 (34.8% of 1025) serum dilutions. Seropositivity rates decreased slightly after birth but by four months of age rates were again high. Parasitaemia was present in 26.5% of 1677 slides from pregnant women. Paired comparisons were made on maternal samples collected less than two months before parturition and samples from the infants collected within two months after birth. The paired antibody response by IFA or ELISA was not dependent on the presence of detectable parasitaemia in the mother. Infants from mothers with a history of antimalarial chemoprophylaxis had significantly (P = 0.04) lower IFA titres than other infants. Measuring the absorbance of a 1:100 serum dilution by ELISA appeared to be an excellent method with which to measure longitudinal serologic changes in a population.

Studies on human malaria in Aotus monkeys. IV. Development of Plasmodium falciparum in two subspecies of Aotus trivirgatus.

Comparative susceptibility studies with a strain of Plasmodium falciparum from Panama indicated that the Aotus trivirgatus griseimembra subspecies of douroucoulis monkeys is more susceptible to infection than is the A. t. trivirgatus subspecies. Serum electrophoretic studies indicated that all of the latter contained a third alpha globulin whereas the former contained only 2. Preinoculation serum electrophoretic studies may be useful as indicators of the level of susceptibility of these animals to this parasite. As reported previously (Collins et al., 1973), a strain of Plasmodium falciparum from Panama (Panama II) has been established in Aotus trivirgatus monkeys. Previous observations had indicated, however, that differences exist in the susceptibility of these monkeys to infection, particularly in the orange-throated variety. All of our previous studies with this strain had been with the grey-legged douroucoulis, A. trivirgatus griseimembra. The availability of a number of the orange-throated animals, identified as A. t. trivirgatus, the three-lined douroucoulis, at the same time made possible a comparison of the susceptibility of these two subspecies to the Panama II strain, the results of which are reported here. MATERIALS AND METHODS The monkeys were procured primarily from Tarpon Zoo, Tarpon Springs, Florida, which imported the animals directly from Colombia, South America. Prior parasitologic and serologic examination indicated that the animals were free of natural malarial infection. Some of the animals were splenectomized prior to infection according to the technique of Sodeman et al. (1970). All but 2 of the A. t. trivirgatus monkeys (AO-419 and AO-426) were infected with microfilariae which were usually abundant on the routine blood films. All animals were infected by the intravenous inoculation of parasitized heparinized blood via the femoral vein. Thick and thin blood films (Earle-Perez) were stained with Giemsa stain and the parasite counts recorded per mm3. During the early and late stages of the infections, blood films were made 3 times a week; during other periods, blood films were made daily. Total proteins in the serum samples were deReceived for publication 15 November 1973. termined by the routine biuret method (Weichselbaum, 1946) and read on a Beckman? DU spectrophotometer at 540 mAu. Serum protein electrophoretic studies were conducted using a Beckman? Model R-100 Microzone? electrophoresis system utilizing cellulose acetate membranes. Membranes were scanned in an R-110 Microzone? densitometer using a 520-m,t interference filter.

Transmission of Plasmodium fragile to Saimiri monkeys

Saimiri monkeys from Bolivia and Guyana were infected with the Nilgiri and Ceylon strains of Plasmodium fragile. Of 20 attempted sporozoite transmissions of the Ceylon strain involving 11 splenectomized Saimiri sciureus boliviensis, only 8 were successful, 2 by mosquito bite and 6 by intravenous injection of sporozoites dissected from salivary glands. Prepatent periods ranged from 18 to 30 days with a mean of 25.8 days.