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.     

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.     

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.     

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.     

Rio Meta strain of Plasmodium vivax in New World monkeys and anopheline mosquitoes

An archived strain of Plasmodium vivax, isolated from Rio Meta, northern Colombia, in 1972 was adapted to grow in splenectomized Aotus lemurinus griseimembra and A. nancymai monkeys. Anopheles freeborni, An. maculatus, An. dirus, An. culicifacies, and An. albimanus were shown to be susceptible to infection by feeding on infected monkeys. Infections were more readily obtained by feeding on A. L. griseimembra than on A. nancymai. Transmission through sporozoites was obtained in an A. l. griseimembra monkey after a prepatent period of 24 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.     

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.     

The susceptibility of the Indonesian I/CDC strain of Plasmodium vivax to chloroquine.

A strain of Plasmodium vivax from Indonesia was adapted to splenectomized Aotus and Saimiri monkeys and tested for its susceptibility to chloroquine. Animals were infected by intravenous inoculation of heparinized parasitized blood and subsequently treated with 8 or 15 mg (base) of chloroquine by oral intubation. Recrudescence of infection occurred in 4 of 4 Aotus and 5 of 6 Saimiri monkeys treated with 15 mg base of chloroquine, indicating a level of resistance between that of the standard Chesson strain of P. vivax and the recently reported resistant strains from Papua New Guinea.     

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.     

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.     

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.     

The Uganda I/CDC strain of Plasmodium malariae in Saimiri sciureus boliviensis

The Uganda I/CDC strain of Plasmodium malariae, initially adapted to monkeys of the genus Aotus, was studied in splenectomized Saimiri sciureus boliviensis. Mean maximum parasitemia ranged from 248 to 22,134/mm3. Only 1 mosquito was infected of 2,238 examined. After the parasite was adapted to this host, infections were characterized by periods of detectable parasitemia extending up to 269 days and by sustained periods when parasite counts were greater than 1,000/mm3. After 4 linear passages, the developmental time required before the primary peak parasite count was approximately 2 mo.     

Aotus infulatus monkey is susceptible to Plasmodium falciparum infection and may constitute an alternative experimental model for malaria

Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.     

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.     

Studies on the Indochina I/CDC strain of Plasmodium falciparum in Colombian and Bolivian Aotus monkeys and different anophelines

The Indochina I/CDC strain of Plasmodium falciparum was isolated from a physician returning to the United States after working in the refugee camps along the Thailand-Kampuchean border. The strain was established in splenectomized Aotus monkeys from Colombia after being grown in vitro for 50 days. During the first three passages in Colombian monkeys, the parasites were not infective to Bolivian Aotus monkeys. After six intervening passages in Saimiri sciureus monkeys, the parasites produced high parasitemias in both Colombian and Bolivian Aotus, but gametocytes were no longer produced. Mosquito infections were obtained only during the first three passages in the Colombian monkeys. The most susceptible mosquito was Anopheles freeborni, followed by An. dirus, An. stephensi, An. maculatus, An. culicifacies, and, rarely, An. gambiae. Sporozoites were found in the salivary glands of the An. freeborni, An. dirus, An. stephensi, and An. maculatus.     

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.     

Severe anemia affects both splenectomized and non-splenectomized Plasmodium falciparum-infected Aotus infulatus monkeys

Severe anemia is the earliest and a frequently fatal complication of Plasmodium falciparum infection. Here we describe Aotus infulatus as a primate model suitable to study this malaria complication. Both non-splenectomized and splenectomized monkeys receiving different inocula of P. falciparum FVO strain presented large (> 50%) decreases in hematocrit values during infection. Non-splenectomized animals were able to control parasite growth (parasitemia did not exceed 4%), but they had to be treated because of severe anemia. Three of 4 splenectomized monkeys did not control parasitemia and were treated, but developed severe anemia after treatment when presenting a negative blood film. Destruction of parasitized red blood cells alone cannot account for the degree of anemia. Non-splenectomized monkeys repeatedly infected with homologous parasites became rapidly and progressively resistant to reinfection and to the development of severe anemia. The data presented here point to A. infulatus as a suitable model for studying the pathogenesis of severe malarial infection.     

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.     

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.     

Observations on two strains of plasmodium falciparum from Haiti in Aotus monkeys

Two strains of Plasmodium falciparum originating in Haiti were studied in the Aotus monkey. The Haitian I/CDC strain was first adapted to in vitro cultivation and subsequently inoculated into monkeys. The Haitian III/CDC strain was inoculated directly from a human patient into the Aotus monkey. The strains varied in their levels of pathogenicity to the animals. The Haitian I/CDC strain was highly virulent in six splenectomized animals; in one intact animal, the infection could be controlled but not eliminated with periodic doses of quinine and chloroquine. After subsequent splenectomy, the animal developed high parasitemias and died. No gametocytes developed in any of the Haitian I infections. The Haitian III strain was lethal to five of the 14 splenectomized monkeys inoculated, but some were able to control their infections without drug intervention. Gametocytes developed in all infections that persisted for an adequate length of time, and infections of mosquitoes were obtained both during the primary attack and the first recrudescence of the parasitemia. Of the mosquitoes tested, Anopheles freeborni was most susceptible to infection, followed by An. culicifacies, An. dirus, An. maculatus, and An. albimanus. The Haitian III strain was successfully transmitted to four other splenectomized Aotus monkeys via sporozoite inoculation using An. freeborni.