Studies on two strains of Plasmodium cynomolgi in New World and Old World monkeys and mosquitoes

Infections that cause the Gombak and Smithsonian strains of Plasmodium cynomolgi were induced in Macaca mulatta, Aotus lemurinus griseimembra, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission of the Gombak strain to Aotus spp. monkeys was obtained by the injection of sporozoites dissected from the salivary glands of experimentally infected Anopheles dirus and by the bites of infected An. dirus and Anopheles farauti mosquitoes. Two S. boliviensis monkeys were infected via the injection of sporozoites dissected from An. dirus. Prepatent periods in New World monkeys ranged from 14 to 44 days, with a median of 18 days. The Smithsonian strain was transmitted via sporozoites to 1 A. lemurinus griseimembra and 9 A. nancymai monkeys. Prepatent periods ranged from 12 to 31 days.     

Adaptation of the AMRU-1 strain of Plasmodium vivax to Aotus and Saimiri monkeys and to four species of anopheline mosquitoes.

A chloroquine-resistant strain of Plasmodium vivax (AMRU-1) from Papua New Guinea has been adapted to grow in 4 species of Aotus monkeys (Aotus lemurinus griseimembra, Aotus vaciferans, Aotus nancymai, and Aotus azarae boliviensis), hybrid Aotus monkeys, and Saimiri boliviensis monkeys. Whereas it was possible to infect Saimiri monkeys with this parasite by inoculation of parasitized erythrocytes, only 42% of Saimiri monkeys became infected, compared to 92% of Aotus monkeys attempted. Comparative mosquito feedings showed that only A. vociferans, A. l. griseimembra, and Saimiri boliviensis monkeys produced infections in mosquitoes. Oocysts were observed on the guts of the 4 species of mosquitoes used (Anopheles gambiae, Anopheles stephensi, Anopheles freeborni, and Anopheles dirus), but sporozoite transmission was effected only with the intravenous inoculation of sporozoites from An. dirus into an A. l. griseimembra monkey.     

Observations on the Vietnam Palo Alto strain of Plasmodium vivax in two species of Aotus monkeys

Thirty-three splenectomized Aotus lemurinus griseimembra monkeys with no previous experience with malaria were infected with the Vietnam Palo Alto strain of Plasmodium vivax. The median maximum parasite count was 280,000/microl. Nine splenectomized monkeys with previous infection with Plasmodium falciparum had median maximum parasite counts of 120,000/microl. Splenectomized Aotus nancymai monkeys supported infections at a lower level. Transmission via the bites of Anopheles dirus mosquitoes was obtained in a splenectomized A. lemurinus griseimembra, with a prepatent period of 31 days. It is estimated that between 1.5 x 10(8) and 1.6 x 10(9) parasites can be removed from an infected animal for molecular or diagnostic antigenic studies.     

Adaptation of Plasmodium vivax to growth in owl monkeys (Aotus nancymai)

The purpose of this study was reactivation and adaptation of a strain of Plasmodium vivax to Aotus nancymai monkeys. A need arose for malarial parasites for use in serologic and molecular studies and for teaching slides. This particular strain of parasite had been characterized previously as producing high-density parasitemia in splenectomized New World monkeys and therefore represented a good candidate for reactivation. P. vivax (Vietnam II), isolated in 1970, was reactivated after adaptation in Aotus lemurinus griseimembra monkeys nearly 33 years earlier and adapted to A. nancymai monkeys. Passage was achieved by intravenous inoculation of parasite blood stages into splenectomized A. nancymai monkeys. Parasitemia was determined by analyzing daily blood smears stained with Giemsa. Maximum parasite counts ranged from 10,630 to 94,000 parasites/microl; the mean maximum parasite count for the four animals was 39,565 parasites/microl. Parasite counts of > 10,000/microl were maintained for 2 to 64 days. After only three passages of the parasite, attempts to reactive were successful. A. nancymai proved a suitable animal model for the recovery of this parasite. In conclusion, successful reactivation and adaptation of this parasite offers the capability to perform a series of diagnostic, immunologic, and molecular studies as well as to provide otherwise potentially unavailable teaching materials to healthcare professionals.     

Adaptation of a strain of Plasmodium vivax from India to New World monkeys, chimpanzees, and anopheline mosquitoes.

A strain of Plasmodium vivax from India was adapted to develop in splenectomized Saimiri boliviensis, Aotus lemurinus griseimembra, A vociferans, A. nancymai, A. azarae boliviensis, hybrid Aotus monkeys, and splenectomized chimpanzees. Infections were induced via the inoculation of sporozoites dissected from the salivary glands of Anopheles stephensi and An. dirus mosquitoes to 12 Aotus and 8 Saimiri monkeys; transmission via the bites of infected An. stephensi was made to 1 Aotus monkey and 1 chimpanzee. The intravenous passage of infected erythrocytes was made to 9 Aotus monkeys and 4 chimpanzees. Gametocytes in 13 Aotus monkeys and 4 chimpanzees were infectious to mosquitoes. Infection rates were markedly higher in mosquitoes fed on chimpanzees. PCR studies on 10 monkeys injected with sporozoites revealed the presence of parasites before their detection by microscopic examination. The India VII strain of P. vivax develops in Aotus and Saimiri monkeys and chimpanzees following the injection of parasitized erythrocytes, or sporozoites, or both. The transmission rate via sporozoites to New World monkeys of approximately 50% may be too low for the testing of sporozoite vaccines or drugs directed against the exoerythrocytic stages. However, the strain is highly infectious to commonly available laboratory-maintained anopheline mosquitoes. Mosquito infection is especially high when feedings are made with gametocytes from splenectomized chimpanzees.     

The Uganda I/CDC strain of Plasmodium malariae in Aotus lemurinus griseimembra monkeys

Two lines of the Uganda I/CDC strain of Plasmodium malariae were studied in splenectomized Aotus lemurinus griseimembra monkeys. A line initially adapted to these monkeys from an infected chimpanzee failed to produce high-level parasite counts or mosquito infection in 13 of this type of monkey during 16 linear passages. Another line, originally adapted from the chimpanzee to Aotus azarae boliviensis, after 7 linear passages in 3 different types of Aotus was then passaged to 14 splenectomized A. lemurinus griseimembra. Geometric mean maximum parasitemia in these monkeys was 18,400/mm3. Mosquito infections were readily obtained during the period just after the parasite count rose above 1,000/mm3. Anopheles freeborni, An. stephensi, An. dirus, and 2 strains of An. gambiae supported the development of the parasite to the presence of sporozoites in the salivary glands. Two attempts to transmit the strain to other splenectomized A. lemurinus griseimembra by sporozoite inoculation were unsuccessful.     

Studies on the Cambodian I strain of Plasmodium falciparum in Aotus monkeys

The Cambodian I strain of Plasmodium falciparum, originally from Kampuchea was adapted for development in three different types of Aotus monkeys. High-level parasitemias were readily produced in splenectomized Colombian A. trivirgatus griseimembra monkeys. Initially, only minimal parasitemias developed in A. t. trivirgatus monkeys from Colombia. However, in one animal, adaptation occurred and high-level parasitemias were obtained during the second recrudescence of the infection. Passage to other A. t. trivirgatus monkeys indicated that the parasite was well adapted for development in splenectomized animals; low to moderate parasitemias were still produced in intact animals. This line of the parasite produced high level parasitemias when inoculated into splenectomized Aotus monkeys from Peru. Infections in Anopheles freeborni mosquitoes were obtained as late as the 7th passage in A. t. griseimembra monkeys and as late as the 7th recrudescence of the infection in an individual monkey (348 days after inoculation). The sporogonic cycle was completed in An. freeborni mosquitoes, and one transmission to an A. t. griseimembra monkey via the bites of infected mosquitoes was obtained.     

Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys.

BACKGROUND: The carboxy-terminus of the merozoite surface protein-1 (MSP1) of Plasmodium falciparum has been implicated as a target of protective immunity. MATERIALS AND METHODS: Two recombinant proteins from the carboxy-terminus of MSP1, the 42 kD fused to GST (bMSP1(42)) and the 19 kD (yMSP1(19)), were expressed in Escherichia coli and secreted from Saccharomyces cerevisiae, respectively. To determine if vaccination with these recombinant proteins induces protective immunity, we conducted a randomized, blinded vaccine trial in two species of Aotus monkeys, A. nancymai and A. vociferans. After three injections using Freund's adjuvant, the monkeys were challenged with the virulent Vietnam Oak Knoll (FVO) strain of P. falciparum. RESULTS: All three control monkeys required treatment by Day 19. Two of three monkeys vaccinated with bMSP1(42) required treatment by Day 17, whereas the third monkey controlled parasitemia for 28 days before requiring treatment. In contrast, both of the A. nancymai vaccinated with yMSP1(19) self-resolved an otherwise lethal infection. One of the two yMSP1(19)-vaccinated A. vociferans had a prolonged prepatent period of > 28 days before requiring treatment. No evidence of mutations were evident in the parasites recovered after the prolonged prepatent period. Sera from the two A. nancymai that self-cured had no detectable effect on in vitro invasion. CONCLUSIONS: Vaccination of A. nancymai with yMSP1(19) induced protective immune responses. The course of recrudescing parasitemias in protected monkeys suggested that immunity is not mediated by antibodies that block invasion. Our data indicate that vaccine trials with the highly adapted FVO strain of P. falciparum can be tested in A. nancymai and that MSP1(19) is a promising anti-blood-stage vaccine for human trials.     

Infection of Aotus and Saimiri monkeys with Plasmodium gonderi

Attempts were made to infect 4 species of New World monkeys (Saimiri boliviensis, Aotus nancymai, A. vociferans, A. azarae boliviensis) with Plasmodium gonderi, a malaria parasite of African monkeys. Sporozoites were obtained from Anopheles dirus or A. stephensi mosquitoes that fed on an infected rhesus monkey (Macaca mulatta). Inoculation of sporozoites was by injection of dissected sporozoites by either the intravenous or intrahepatic routes, or by mosquito bite. Liver biopsies done 7 or 8 days after sporozoite inoculation showed that hepatocytes of all 4 species of these New World monkeys supported exoerythrocytic stages of P. gonderi, but daily blood film examination during a 60-day observation period failed to detect blood stages of the parasite.     

Studies on the North Korean strain of Plasmodium vivax in Aotus monkeys and different anophelines

Twenty-two Aotus monkeys of different karyotypes were infected with the North Korean strain of Plasmodium vivax. Aotus lemurinus griseimembra animals from Colombia produced higher maximum parasitemias and more readily infected mosquitoes than did Aotus monkeys from Bolivia (K-VI) or Peru (K-V and K-X). Comparative feedings indicated that the most susceptible mosquito species was Anopheles stephensi, followed by An. gambiae, An. dirus, An. freeborni, An. quadrimaculatus, An. culicifacies, and An. maculatus.     

Infectivity of Plasmodium simium to Aotus trivirgatus monkeys and different anophelines.

Infections of Plasmodium simium were induced in splenectomized and intact Aotus trivirgatus griseimembra monkeys by parasitized blood and by sporozoites from Anopheles freeborni mosquitoes. Eleven of 13 monkeys developed infection after sporozoite inoculation; prepatent periods ranged from 11 to 25 days (mean 15.8 days). Comparative infectivity studies indicated that An, freeborni mosquitoes were the most susceptible followed by An. stephensi, An. Balabacensis balabacensis, An. maculatus, An. quadrimaculatus, An. culicifacies, and An. albimanus. Studies with 3 pupal phenotypes of An. freeborni indicated that lines containing the green and nonstriped pupal phenotypes were more susceptible than the base colony; the striped phenotype was slightly less susceptible.     

Studies on infections with the Berok strain of Plasmodium cynomolgi in monkeys and mosquitoes

Infections with the Berok strain of Plasmodium cynomolgi were induced in Macaca mulatta, Macaca fascicularis, Macaca nemestrina, Aotus lemurinus griseimembra, Aotus azarae boliviensis, and Saimiri boliviensis monkeys. Transmission was obtained with sporozoites developing in Anopheles peditaeniatus, Anopheles maculatus, Anopheles quadrimaculatus, Anopheles culicifacies, and Anopheles dirus mosquitoes. This strain of P. cynomolgi offers significant potential for a number of experimental studies. The parasite induces high-density parasite counts in both Old World and New World monkeys; rhesus monkeys readily support the development of gametocytes infectious to different anopheline mosquitoes routinely maintained in the laboratory; the gametocytes are infective to laboratory-maintained Anopheles albimanus, a vector rarely susceptible to plasmodia of Old World monkeys; encapsulated oocysts are produced in An. culicifacies as well as in Anopheles gambiae; and the parasite has been adapted to long-term in vitro culture.     

Development of a polymorphic strain of Plasmodium vivax in monkeys.

A strain of Plasmodium vivax from Thailand with a polymorphic repeat unit of the circumsporozoite protein was established in Saimiri sciureus boliviensis and 3 species of Aotus monkeys. All 11 attempts to transmit infection via sporozoite inoculation, 4 times to splenectomized S. sciureus boliviensis, 2 times to splenectomized Aotus nancymai, and 5 times to intact Saimiri monkeys, were successful. Anopheles freeborni, Anopheles stephensi, Anopheles dirus, and Anopheles gambiae mosquitoes were infected by feeding on parasitemic blood from a chimpanzee and an Aotus azarae boliviensis monkey. Our results indicate that this strain may be useful in antisporozoite vaccine trials.     

Studies on a newly isolated strain of Plasmodium brasilianum in Aotus and Saimiri monkeys and different anophelines

A strain of Plasmodium brasilianum was isolated from an Aotus vociferans monkey from Peru. The parasite readily infected Aotus monkeys from Bolivia and Columbia and Saimiri sciureus monkeys from Peru and Bolivia. Highest level mosquito infections were obtained by feeding on the Saimiri monkeys. The most susceptible mosquito was Anopheles freeborni, followed by Anopheles dirus, Anopheles stephensi, Anopheles gambiae, Anopheles culicifacies, Anopheles maculatus and Anopheles albimanus. Anopheles quadrimaculatus were also susceptible to infection. Degenerating oocysts were observed in An. dirus mosquitoes infected with this parasite. Transmission via the bites of infected An. maculatus mosquitoes was obtained to 3 Bolivian Saimiri monkeys; prepatent periods were 27, 27, and 29 days.     

Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation

Aotus lemurinus griseimembra is considered one of the best nonhuman primate species for malarial studies because of its susceptibility to infection by Plasmodium falciparum asexual blood stages. However, reproducible transmission of infective P. falciparum sporozoites by mosquito inoculation has been difficult to achieve even in splenectomized monkeys. Characterization of an Aotus-P. falciparum cyclical transmission model has become a top priority as a result of the significant progress toward the development of preerythrocytic malaria vaccines. Herein, we describe a reproducible model developed using intact A. lemurinus griseimembra monkeys intravenously inoculated with sporozoites from a monkey-adapted P. falciparum (Santa Lucia) strain and a wild Falciparum-Cali-Colombia-4 (FCC-4) strain. Sporozoites were obtained by salivary gland dissection of laboratory-reared Anopheles albimanus mosquitoes. Parasitemia was monitored by thick-smear microscopy, parasite lactate dehydrogenase (pLDH) determination, and mosquito xenodiagnosis. The last method proved to be the most sensitive method for monitoring parasitemias. Infection with the Santa Lucia strain showed a mean prepatent period of 16 days (range 6-21 days), whereas infection with the wild FCC-4 strain resulted in a 24-day prepatent period. Mean peak parasite density was approximately 900 parasites/microliter for both parasite strains. The prepatent period, the peak of parasitemia, and the duration of patency were independent of the size of the sporozoite inoculum and the presence of spleen in the host. This model is being successfully used to test the protective efficacy of P. falciparum preerythrocytic vaccine candidates.     

Plasmodium vivax: in vitro growth and reinvasion in red blood cells of Aotus nancymai

Plasmodium vivax was maintained in experimentally infected Aotus nancymai. Positive monkeys were used as donors for culture material. After leucocyte removal with two different methods, including the classic CF11 method and a commercially available filter, parasites were grown under continuous shaking conditions in standard RPMI 1640, containing 20% human AB + serum. When mature schizonts were present, artificially induced reticulocytes from monkeys pretreated with the hemolytic drug phenylhydrazine HCl were added. Addition of reticulocytes and shaking were both necessary to realize a significant reinvasion under in vitro conditions. A strong positive correlation between the percentage of reticulocytes and in vitro invasion was demonstrated, and a preferential invasion into reticulocytes was demonstrated in vivo and in vitro using blood films stained with brilliant cresyl blue and counterstained with Giemsa.     

Mortality and body weight changes in Aotus nancymai shipped from Iquitos, Peru to Richland, Washington

Four hundred and fifty wild-caught owl monkeys (Aotus nancymai) were shipped from Iquitos, Peru to Richland, Washington. Mortality and body weight changes from the time of shipment to 30 days after arrival were evaluated using age, sex, and transit time as comparison criteria. No association between mortality and sex, age, or transit time was observed. The mortality percentage from shipment to 30 days after arrival was 2.44%. Both age and transit time had a significant effect on weight changes, but sex did not. Shipping procedures are described.     

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.     

Erythrocytes and erythrocyte morphologies of healthy and colony-born owl monkeys (Aotus lemurinus griseimembra)

Inadequate availability of hematological reference data seriously restricts optimal utilization of the owl monkey (Aotus lemurinus griseimembra) as an experimental model. The current study investigated erythrocytic morphology in peripheral blood of healthy, colony-born owl monkeys. The blood of the subjects contained discoid erythrocytes, poikilocytes, and showed considerable anisocytosis. Also observed were nucleated erythrocytes, erythrocytes with Howell-Jolly bodies, and reticulocyte types I, II, and III. Heinz bodies were not detected.     

Genus Aotus Q- and G-band karyotypes and and natural hybrids.

Q- and G-banding studies of Aotus trivigatus trivirgatus and Aotus trivirgatus griseimembra are presented. After the analysis of 8 specimens, 2 A.t. trivirgatus and 6 A.t. griseimembra and the review of the literature on the field we state the existence of two different karyotypes, 2n = 54 and 2n = 50, for A.t. trivirgatus and three different karyotypes, 2n = 54, 2n = 52 and 2n = 50, for A.t. griseimembra. It was found that Robertsonian translocations were the mechanism involved in the numerical reduction of the chromosomes. The data presented include the statement of two natural hybrids and the cross-breedings which originated them.