Adaptation of a strain of Plasmodium falciparum from Ghana to Aotus lemurinus griseimembra, A. nancymai, and A. vociferans monkeys

A strain of Plasmodium falciparum from Ghana was adapted to Aotus lemurinus griseimembra, A. nancymai, and A. vociferans monkeys. Gametocytes in splenectomized A. nancymai were infective to Anopheles freeborni mosquitoes. Sporozoite transmission was accomplished in two splenectomized A. nancymai with prepatent periods of 22 and 25 days. The Ghana III/CDC strain of P. falciparum is susceptible to treatment with chloroquine and mefloquine.     

Plasmodium coatneyi: observations on periodicity, mosquito infection, and transmission to Macaca mulatta monkeys.

Plasmodium coatneyi has adapted well to experimental studies with Macaca mulatta monkeys and Anopheles dirus mosquitoes. Studies were made to determine 1) the course of asexual parasitemia, 2) periods when infective gametocytes were produced, 3) the laboratory-reared mosquitoes susceptible to infection, 4) the mosquito most capable of transmitting the infection to monkeys via bite, 5) the pattern of recrudescence, and 6) the prepatent periods following the bites of infected An. dirus mosquitoes. The period when infective gametocytes are produced is concentrated primarily in the first week when parasitemia exceeds 1,000/microl. Mosquitoes were more heavily infected on days when the asexual parasite counts were highest. Gametocyte counts were generally low. Mature forms of the parasite markedly sequestered giving a pattern of high-low periodicity. Anopheles dirus and An. freeborni mosquitoes were nearly equal in terms of their ability to support oocyst development. Other species (An. stephensi, An. maculatus, and An. gambiae.) were less supportive. High sporozoite densities in the salivary glands were frequently produced in An. dirus and sporozoite transmission was obtained via the bites of these mosquitoes after 12-18 days of extrinsic incubation. Prepatent periods ranged from 10 to 15 days. The presence of frequent parasitic recrudescences suggests mechanisms similar to that seen in human infections with P. falciparum. It is proposed that P. coatneyi in M. mulatta monkeys can be a suitable model for studies on cerebral pathology, vaccine efficacy, and the testing of antimalarial drugs.     

Plasmodium simium and Saimiri boliviensis as a model system for testing candidate vaccines against Plasmodium vivax

Observations on Plasmodium simium infections in Saimiri boliviensis boliviensis monkeys suggest that this host-parasite combination would be a suitable model for the testing of candidate vaccines against Plasmodium vivax. To evaluate the normal course of infections, parasitemia in 52 splenectomized S. boliviensis boliviensis monkeys infected with P. simium were analyzed. The mean maximum parasite count for 31 monkeys after injection with trophozoite-infected erythrocytes was 77,580/microL. Twenty-one monkeys were infected via sporozoites, and prepatent periods ranged from 14 to 24 days with a median of 15 days. The mean maximum parasite count was 29,234/microL. The mean maximum parasite count for monkeys previously infected with Old World P. vivax was 26,337/microL versus 56,362/microL for those previously infected with New World P. vivax, possibly suggesting a closer antigenic relationship between P. simium and the Old World parasites.     

Mosquito infection studies with Aotus monkeys and humans infected with the Chesson strain of Plasmodiun vivax

Oocyst counts were compared between mosquitoes that fed on humans versus mosquitoes that fed on Aotus monkeys, both of which were infected with the Chesson strain of Plasmodium vivax. Oocyst counts obtained from mosquitoes fed on humans were almost 10-fold higher in number. Mosquitoes were more likely to be infected and with a higher rate of infection when they fed on monkeys before the peak in the asexual parasite count. Mosquitoes that fed on humans were more likely to be more heavily infected when fed after the peak in the asexual count. Of several species of owl monkeys, Aotus vociferans was infected at a higher frequency. On the basis of oocyst counts, Anopheles dirus were the most susceptible and An. maculatus were the least susceptible of the mosquito species tested.     

A retrospective examination of sporozoite-induced and trophozoite-induced infections with Plasmodium ovale: development of parasitologic and clinical immunity during primary infection

A retrospective analysis was made of clinical and parasitologic parameters in patients with induced Plasmodium ovale infection to document the initial clinical and parasitologic response and their subsequent development of clinical and parasitologic immunity, and to determine the effect of previous homologous and heterologous malaria on subsequent infection with this parasite. The prepatent periods were relatively uniform. Eight patients injected with sporozoites that had been stored frozen had a median prepatent period of 14 days (range = 14-20 days). Thirty-five patients infected via the bites of infected mosquitoes had a median prepatent period of 15 days (range = 12-18 days). In eight patients previously infected with P. vivax, the median prepatent period was 16 days. High-intensity fever (> or = 104 degrees F) was frequently seen, with instances of fever > or = 106 degrees F recorded on many occasions. Fever > 101 degrees F and > 104 degrees F occurred for much shorter periods of time than had been observed in patients infected with P. falciparum. Parasite counts > 10,000/microL were infrequent; in most patients, such parasite counts rarely lasted more than two or three days. Gametocytemia was generally of low density and lasted only a few days. The overall length of the clinical and parasitologic period was much shorter compared with that seen in patients infected with P. falciparum. Previous infection with P. ovale did not prevent reinfection, but resulted in reduced levels of parasitemia and fever. Previous infection with heterologous species of Plasmodium did not prevent infection; some reduction in the frequency and intensity of fever and parasite counts was evident. Previous infection with homologous or heterologous parasites failed to eliminate the production of infective gametocytes. A total of 462 lots of mosquitoes were fed on 67 patients with no previous history of infection. Of these feedings, 168 (36.4%) resulted in infection as determined by the presence of oocysts on the midguts of dissected mosquitoes. As shown, the infection rate increased with the density of gametocytes even though 48 (23.4%) of 205 lots of mosquitoes fed when no gametocytes were detected were infected.     

Susceptibility of Panamanian Aotus lemurinus lemurinus to sporozoite-induced Plasmodium falciparum (Santa Lucia) infection.

Aotus monkeys are good models for erythrocyte-induced Plasmodium falciparum and P. vivax infections and have been extensively used in malarial drug and vaccine development. Recently, it has been shown that certain species of Aotus can be infected with sporozoites, and that the degree of susceptibility varies among species. We demonstrate here that Panamanian Aotus lemurinus lemurinus are susceptible to a sporozoite-induced infection, opening the possibility that this species of Aotus could be used as models for testing the efficacy of pre-erythrocytic P. falciparum vaccines and drug candidates directed at the pre-erythrocytic stages of P. falciparum and P. vivax malaria. In this species, we compared sporozoite infection rates. Two of four animals splenectomized prior to infection with sporozoites developed patent parasitemias. Seven of eight animals splenectomized either 7 or 35 days after infection became parasitemic. Additionally, we used a P. falciparum-specific polymerase chain reaction (PCR) method to detect the early appearance of parasitized erythrocytes in the blood prior to detection by conventional microscopy, and found that the parasitemia was detected first in five animals by the PCR method, first in three animals by blood film, with one parasitemia detected simultaneously. We also demonstrated the feasibility of infecting monkeys located in Panama with sporozoites isolated at an insectary in Atlanta, thus documenting the feasibility of similar studies where the insectary and monkey colony are not in the same location. A subsequent attempt to infect these monkeys using sporozoites was not successful, suggesting that this model of human malaria is not yet ready for routine use in vaccine or drug efficacy screening. This model merits further study because of the importance of testing pre-erythrocytic P. falciparum malaria vaccines and drugs in animals.     

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Four laboratory-raised colonies of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and C (Chiang Mai and Mae Hong Son strains), were experimentally infected with Plasmodium falciparum and P. vivax using an artificial membrane feeding technique and dissected eight and 12 days after feeding for oocyst and sporozoite rates, respectively. The results revealed that An. aconitus Form B and C were susceptible to P. falciparum and P. vivax, i.e., Form B (Chiang Mai and Phet Buri strains/P. falciparum and P. vivax) and Form C (Chiang Mai and Mae Hong Son strains/P. vivax). Comparative statistical analyses of the oocyst rates, average number of oocysts per infected midgut and sporozoite rates among all strains of An. aconitus Form B and C to the ingroup control vectors, An. minimus A and C, exhibited mostly no significant differences, confirming the high potential vector of the two Plasmodium species. The sporozoite-like crystals found in the median lobe of the salivary glands, which could be a misleading factor in the identification of true sporozoites in salivary glands were found in both An. aconitus Form B and C.     

Infection of chimpanzees with the Uganda I/CDC strain of Plasmodium malariae

Nine splenectomized chimpanzees were infected with the Uganda I/CDC strain of Plasmodium malariae. Two had no history of previous malarial infection, whereas 6 had been infected with P. vivax and 1 with P. vivax and P. ovale. The animals with no previous infection had maximum parasitemias of 8,740 and 10,800/mm3. The other animals had maximum parasite counts of 930-75,700/mm3. Anopheles freeborni, An. stephensi, An. dirus, An. maculatus, An. quadrimaculatus, An. culicifacies, An. arabiensis, and An. gambiae were readily infected by feeding through membranes on heparinized blood from these animals.     

Adaptation of a chloroquine-resistant strain of Plasmodium vivax from Indonesia to New World monkeys

The spread of chloroquine-resistant Plasmodium vivax from Papua New Guinea and Indonesia poses a serious health threat to areas of Southeast Asia where this species of malaria parasite is endemic. A strain of P. vivax from Indonesia was adapted to develop in splenectomized Aotus lemurinus griseimembra, Aotus vociferans, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission to splenectomized Saimiri monkeys was obtained via sporozoites. Chemotherapeutic studies indicated that the strain was resistant to chloroquine and amodiaquine while sensitive to mefloquine. Infections of chloroquine-resistant P.vivax in New World monkeys should be useful for the development of alternative treatments.     

Cryopreservation of the blood stages of Plasmodium falciparum and Plasmodium vivax for in vivo studies

Cryopreservation in Alsevers-glycerol of the blood stages of Plasmodium falciparum (5 strains) and P. vivax (2 strains) indicated that parasite infectivity for Aotus was retained for more than 1,100 days.     

Clinico-pathological observations on the pathogenesis of severe thrombocytopenia and anemia induced by Plasmodium vivax infections during antimalarial drug efficacy trials in Aotus monkeys

During routine antimalarial drug efficacy trials, we observed, for the first time, severe thrombocytopenia developing in Aotus monkeys infected with Plasmodium vivax. Data obtained from 26 Aotus infected with the AMRU-1 strain showed that 77% developed severe thrombocytopenia, whereas only 15% had severe anemia, with hemorrhagic diathesis ensuing in 31%. In general, thrombocytopenic monkeys either failed primary treatment with experimental antimalarial drugs or were found to have higher-density parasitemias, longer patency duration, and lower hematocrits. In these monkeys, severe thrombocytopenia inversely correlated to parasitemia (R = -1.0), and animals that received a blood transfusion had significantly higher platelet counts (P < 0.05) by day 38 after inoculation. In conclusion, the AMRU-1 strain of P. vivax, was considered to be highly pathogenic to Aotus monkeys, and thrombocytopenia rather than anemia should be regarded an early indicator of drug treatment failure with this strain.     

Preliminary observations on the efficacy of a recombinant multistage Plasmodium falciparum vaccine in Aotus nancymai monkeys

A vaccine trial was conducted to determine the efficacy of a multicomponent candidate vaccine, FALVAC-1, against Plasmodium falciparum in Aotus nancymai monkeys. After two immunizations, animals were challenged intravenously with parasites of the Vietnam Oak Knoll (FVO) strain of P. falciparum. The primary outcome was to determine the protective response of the monkeys to immunization with the FALVAC-1 antigen produced in baculovirus when combined with different adjuvants (alum, QS-21, ASO2a, CRL1005/oil, and CRL1005/saline) as compared with FALVAC-1 with FCA/FIA and antigen alone. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other 5 antigen-adjuvant combinations were able to provide discernable levels of protection based on log(parasitemia) and log(cumulative parasitemia) to Day 11.     

Biting behavior and Plasmodium infection rates of Anopheles arabiensis from Sille, Ethiopia

The man-biting behavior and Plasmodium infection rates of anopheline mosquitoes were investigated in Sille, a hyperendemic malarious area in southern Ethiopia. Seven Anopheles species were identified from all night landing collections, conducted from 18:00 to 06:00h between October 2001 and August 2002. The predominant species was Anopheles arabiensis (55.8%), followed by Anopheles coustani (31.5%), Anopheles pharoensis (9.5%), Anopheles funestus (2.2%), Anopheles nili (0.5%), Anopheles marshallii (0.4%) and Anopheles demeilloni (0.2%). Dissection of A. arabiensis showed an average parous rate of 73.2%. A large proportion of the parous mosquitoes were caught biting in the latter part of the night. Malaria sporozoite rates were determined by ELISA for A. arabiensis, with 0.5% (4/796) infective with Plasmodium falciparum and 1.76% (14/796) with Plasmodium vivax; there were no mixed infections. From our small sample of sporozoite positives we found no association between biting behavior and sporozoite infection status.     

Plasmodium falciparum and Plasmodium vivax infections in the owl monkey (Aotus trivirgatus). I. The courses of untreated infections.

This study, the first of three designed to determine the feasibility of using owl monkeys infected with human plasmodia in the search for new, more broadly active antimalarial drugs, dealt with the characteristics of untreated infections with eight strains of Plasmodium falciparum and two strains of P. vivax. Such infections, induced by standardized inocula of these strains in 1,733 monkeys, all Aotus trivirgatus griseimembra, were followed from day of inoculation to death of self-cure. The virulence of the various strains differed strikingly. Incidences of fatal reactions, ranging from 24.4--89.4% and 8.1--45.8%, respectively, in infections with strains of P. falciparum and P. vivax, were closely related to the rate at which parasitemia evolved, the height of parasitemia in the primary attack, and/or the time period over which a high parasite level was sustained. Antemortem symptom complexes and gross tissue and organ reactions in infections with P. falciparum varied with survival time, but within that boundary, were the same for infections with all eight strains of this plasmodium. Morbidity in both fatal and self-limited infections with both plasmodial species was related to height of parasitemia; however, at comparable parasite levels, symptoms exhibited in infections with P. vivax were more severe than in infections with P. falciparum. Overall, the characteristics of infections with these plasmodia in owl monkeys were remarkably similar to those of human infections. With respect to biological features, infections with P. falciparum and P. vivax in this simian host appear to have much to offer in the search for new antimalarial drugs.     

Human malaria transmission studies in the Anopheles punctulatus complex in Papua New Guinea: sporozoite rates, inoculation rates, and sporozoite densities

Malaria sporozoite rates and inoculation rates were measured over periods up to 25 months in the different anopheline species biting humans in 13 villages in Madang Province, Papua New Guinea. Analysis of three members of the Anopheles punctulatus complex, 68,458 An. farauti, 36,779 An. koliensis, and 11,667 An. punctulatus caught in landing catches was made using monoclonal antibody based ELISAs to detect sporozoites of Plasmodium falciparum and P. vivax. Sporozoite rates ranged from 0%-5.5% in An. farauti, 0.2%-3.8% in An. koliensis, and 0%-3.3% in An. punctulatus. In addition, over 3,000 An. longirostris were analyzed and sporozoites were not detected in this species. No significant differences were observed between the three vector species in the densities of P. falciparum sporozoites (geometric mean 2,320). However, the geometric mean P. vivax sporozoite density was significantly higher in An. punctulatus (350) than in either An. koliensis (160) or An. farauti (150). An. koliensis was less susceptible to infections of P. falciparum or P. vivax than either An. farauti or An. punctulatus. Variations in average sporozoite and inoculation rates were found among different villages, despite their close geographic proximity. Sporozoite and inoculation rates varied greatly within a village over time, but malaria transmission was perennial with a higher transmission during the wet season by An. koliensis and An. punctulatus.     

The epidemiology of malaria in an epidemic area of the Peruvian Amazon

A longitudinal study of malariometric indicators and their association with potential risk factors was conducted during August 1997-July 1998 at Padre Cocha, a village of 1,400 residents in the Peruvian Amazon. The incidence of Plasmodium falciparum infections during the study year was 166/1,000 persons; that of P. vivax was 826/1,000 persons. The mean duration of symptoms prior to diagnosis was 2 days; presenting geometric mean parasite densities were 3,976 parasites/microl for P. falciparum infections and 2,282 parasites/microl for P. vivax. There were no malaria-associated deaths. Consistent with the epidemic nature of malaria in the area, the incidence of both parasite species increased with age and there were no age-specific differences in mean parasite densities. No specific occupational risks for malaria were identified. Activities significantly associated with malaria risk reflected local vector behavior and included strolling outdoors after 6:00 PM and arising before 6:00 AM for adults, and attending evening church services for children.     

Estimate of Plasmodium falciparum sporozoite content of Anopheles stephensi used to challenge human volunteers

Plasmodium falciparum infected Anopheles stephensi, taken from a group of mosquitoes which had been used to challenge recipients of (NANP)3-TT vaccine, were tested for P. falciparum sporozoite content by an immunoradiometric assay. Seventy-six percent were infected with mean and median sporozoite equivalents per mosquito of 220,994 and 217,398, respectively (SD = 54,911). This sporozoite density is greater than that usually found in the field. These data suggest that this challenge for evaluating P. falciparum sporozoite vaccines is a demanding test of immunity.     

The number of sporozoites produced by individual malaria oocysts

Mature oocysts of Plasmodium falciparum and P. vivax from western Thailand were separated from the midguts of Anopheles dirus by collagenase digestion, and the number of sporozoites contained in each was counted. For 26 P. vivax oocysts, the mean count was 3, 688 (range 1, 954-5, 577) and for 14 P. falciparum, the mean count was 3, 385 (range 1, 359-4, 554); a single P. cynomolgi oocyst contained 7, 521. Counts were not significantly correlated with oocyst density, oocyst age, or identity of the examiner. There may have been strain differences in fecundity, particularly between P. falciparum lines maintained in vitro. Mosquitoes receiving a second, uninfected blood meal seven days after feeding on P. vivax-infected volunteers developed no additional sporozoites per oocyst, but had salivary glands 3.4 times as infected. By calculation, more than 20% of P. vivax sporozoites released from oocysts subsequently invade the salivary glands.     

Immunogenicity and efficacy trials in Aotus nancymai monkeys with model compounds representing parts of a 75-kD merozoite surface antigen of Plasmodium falciparum.

We tested the ability of a recombinant DNA-encoded fragment (C7Ag) of a Plasmodium falciparum merozoite protein (p75) and of two carrier-free peptide models (28-mer and 76-mer) to stimulate boostable antibody responses in Aotus nancymai monkeys. In addition, we evaluated protection against challenge with the Uganda Palo Alto (FUP) strain of this parasite. The data indicate that C7Ag elicited a strong and boostable IgG antibody response in all the monkeys immunized. However, studies with the peptide models demonstrated that various animals produce antibodies to different portions of this structure. When the post-boost sera from monkeys immunized with C7Ag were analyzed for reactivity against two major portions of C7Ag, most of the antibody response was observed against the disulfide-bonded 76-residue region that forms a conformational immunogenic epitope. In the same sera, antibody levels against the charged helical region modeled with a 28-mer were generally low. Immunization with synthetic peptides revealed that the 76-mer stimulated an antibody response almost as strong as C7Ag, with substantial cross-reactivity against the parasite antigen. The 28-mer evoked a response that was not efficient or uniform, and showed little reactivity with the authentic parasite antigen. Aotus nancymai was shown to be susceptible to infection with the Uganda Palo Alto strain of P. falciparum; however, maximum parasitemia varied markedly in both immunized and control monkeys. Statistical analysis failed to recognize differences in maximum parasitemia between the vaccine and control groups. The variation in maximum parasitemia suggests that the FUP strain in this species of Aotus is a poor model for the detection of differences in efficacy based on maximum parasitemia. This initial study with structures based on parts of the 75-kD merozoite surface antigen of P. falciparum indicated that both the recombinant-produced protein C7 and the 76-mer synthetic peptide, when combined with a Syntex adjuvant formulation, were safe and immunogenic in A. nancymai monkeys. However, the data emphasize the problems of using animal models to evaluate the potential effects of immunogens in humans.     

Strain specificity in the liver-stage development of Plasmodium falciparum in primary cultures of new world monkey hepatocytes

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.