• View in gallery

    Parasitemia in Aotus lemurinus griseimembra monkeys AI-331 and AI-4052 infected with the Ghana III/CDC strain of Plasmodium falciparum.

  • View in gallery

    Genealogy of the Ghana III/CDC strain of Plasmodium falciparum in Aotus monkeys. Solid line = blood passage; dotted line = sporozoite passage; * = second inoculation; # = third inoculation with the Ghana III strain.

  • View in gallery

    Parasitemia in Aotus nancymai monkey AI-1767 and A. vociferans monkey AI-2653 following infection and reinfection with the Ghana III/CDC strain of Plasmodium falciparum. Anopheles freeborni mosquitoes were infected.

  • View in gallery

    Parasitemia in Aotus nancymai monkeys AI-2713, AI-2791, and AI-1771 following infection and reinfection with the Ghana III/CDC strain of Plasmodium falciparum.

  • View in gallery

    Parasitemia in Aotus nancymai monkeys AI-1774, AI-1778, AI-1772, and AI-1777 following infection and reinfection with the Ghana III/CDC strain of Plasmodium falciparum.

  • View in gallery

    Restriction fragment length polymorphism analysis of merozoite surface protein-1 (MSP-1) and MSP-2 polymerase chain reaction (PCR) DNA fragments amplified from Ghana III/CDC parasites of Plasmodium falciparum taken at different points during in vitro and in vivo adaptation. A, PCR-amplified fragments of block 2 of MSP-1. B, PCR-amplified fragments of MSP-2. C, Alu I restriction enzyme digests of MSP-1 DNA fragments. D, Hinf I restriction enzyme digests of MSP-2 DNA fragments. Lanes 1 through 8 represent amplification of DNA from 1) Ghana III patient blood, 2) in vitro culture, 3) AI-1767 (25 X 00), 4) AI-331 (22 XI 00), 5) AI-1778 (2 II 01), 6) AI-1778 (27 VII 01), 7) the P. falciparum 7G8 strain, and 8) the P. falciparum Viet Nam-Oak Knoll (FVO) strain. Lanes S = DNA size standards (1.0, 0.85, 0.65, 0.5, 0.4, 0.3, 0.2, and 0.1 kilobases from top to bottom).

  • View in gallery

    Parasitemia in Aotus vociferans monkeys AI-3000, and AI-2990 following infection with the Ghana III/CDC strain of Plasmodium falciparum.

  • View in gallery

    Parasitemia in Aotus nancymai monkey AI-2787 following infection with sporozoites of the Ghana III/CDC strain of Plasmodium falciparum dissected from Anopheles freeborni mosquitoes.

  • 1

    Siddiqui WA, Schnell JV, Geiman QM, 1972. A model in vitro system to test susceptibility of human malaria parasites to antimalarial drugs. Am J Trop Med Hyg 21 :392–399.

    • Search Google Scholar
    • Export Citation
  • 2

    Geiman QM, Meagher MJ, 1967. Susceptibility of a New World monkey to Plasmodium falciparum.Nature 215 :437–439.

  • 3

    Collins WE, Anders RF, Ruebush TK II, Kemp DJ, Woodrow GC, Campbell GH,Brown GV, Irving DO, Gross N, Filipski VK, Coppel RL, Broderson JR, Thomas LM, Pye D, Skinner JC, Wilson C, Stanfill PS, Procell PM, 1991. Immunization of owl monkeys with ring-infected erythrocyte surface antigen of Plasmodium falciparum.Am J Trop Med Hyg 44 :34–41.

    • Search Google Scholar
    • Export Citation
  • 4

    Ruebush TK II, Campbell GH, Moreno AO, Patarroyo ME, Collins WE, 1990. Immunization of owl monkeys with a combination of Plasmodium falciparum asexual blood-stage synthetic peptide antigens. Am J Trop Med Hyg 43 :355–366.

    • Search Google Scholar
    • Export Citation
  • 5

    Sharma P, Ruebush TK II, Campbell GH, Richman SJ, Wilkins PM, Broderson JR, Ardeshir F, Gross M, Silverman C, Skinner JC, Filipski V, Wilson C, Roberts JM, Ma NF-S, Stanfill PS, Reese RT, Collins WE, 1992. Immunogenicity and efficacy trials in Aotus nancymai monkeys with model compounds representing parts of a 75-kD merozoite surface antigen of Plasmodium falciparum.Am J Trop Med Hyg 46 :691–707.

    • Search Google Scholar
    • Export Citation
  • 6

    Berzins K, Adams S, Broderson JR, Chizzolini C, Hansson M, Lovgren K, Millet P, Morris CL, Perlmann H, Perlmann P, Sjolander A, Stahl S, Sullivan JS, Troye-Blomberg M, Collins WE, 1995. Immunogenicity in Aotus monkeys of ISCOM formulated repeat sequences from the Plasmodium falciparum asexual blood stage antigen Pf155/RESA. Vaccine Res 4 :121–133.

    • Search Google Scholar
    • Export Citation
  • 7

    Collins WE, Walduck A, Sullivan JS, Andrews K, Stowers A, Morris CL, Jennings V, Yang C, Kendall J, Lin Q, MArtin LB, Diggs C, Saul A, 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.

    • Search Google Scholar
    • Export Citation
  • 8

    Kumar S, Collins WE, Egan A, Yadava A, Girrard O, Blackman M, Guevara Patino JA, Diggs C, Kaslow D, 2000. Immunogenicity and efficacy in Aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kD C-terminus of the merozoite surface protein 1 (MSP-1). Infect Immun 68 :2215–2223.

    • Search Google Scholar
    • Export Citation
  • 9

    Collins WE, Galland GG, Sullivan JS, Morris CL, 1994. Selection of different strains of Plasmodium falciparum for testing blood-stage vaccines in Aotus nancymai monkeys. Am J Trop Med Hyg 51 :224–232.

    • Search Google Scholar
    • Export Citation
  • 10

    Earle WC, Perez M, 1932. Enumeration of parasites in blood of malarial patients. J Lab Clin Med 17 :1124–1130.

  • 11

    Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN, 1993. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 58 :283–292.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

ADAPTATION OF A STRAIN OF PLASMODIUM FALCIPARUM FROM GHANA TO AOTUS LEMURINUS GRISEIMEMBRA, A. NANCYMAI, AND A. VOCIFERANS MONKEYS

View More View Less
  • 1 Division of Parasitic Diseases and Animal Resources Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Atlanta Research and Education Foundation, Atlanta, Georgia

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.

INTRODUCTION

The adaptation of different strains of Plasmodium falciparum to New World monkeys has made possible the use of these infections for vaccine trials, as well as a number of basic biologic studies. Two of these, Vietnam Oak Knoll (FVO)1 and Uganda Palo Alto2, have become standards for a number of different vaccine trials.3–9 New strains are being adapted to broaden the molecular diversity that is needed for heterologous vaccine challenges. Of particular interest has been the adaptation of strains from Africa to Aotus nancymai and Aotus vociferans, the species of animals currently available for these trials. Our goal has been 1) to obtain parasites representative of parasites currently circulating in Africa, and 2) to passage the parasites in as few animals as possible to determine the predictability of parasitemia and maintenance of the production of gametocytes.

Reported here is an examination of the adaptation process for a strain of P. falciparum originating in Ghana and the selection of parasite populations with the characteristics of high-density asexual parasitemia and maintenance of gametocytemia. Emphasis was directed toward adaptation to the currently available monkeys following initial adaptation to Aotus lemurinus griseimembra. The isolate is identified as Ghana III/CDC.

MATERIALS AND METHODS

Aotus nancymai and A. vociferans monkeys were wild-caught animals imported from Peru. The A. lemurinus griseimembra were laboratory-born animals. Upon arrival at our facility, all animals were quarantined for a two-month conditioning period, weighed, and tested for tuberculosis. Parasitologic and serologic examination indicated that the animals were free of infection with malaria parasites before primary inoculation. All monkeys were splenectomized before or after exposure to infection with the Ghana III parasite. All surgeries were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care International, Inc. (Rockville, MD)-approved surgical suite appropriate for aseptic surgery. Protocols were reviewed and approved by the Centers for Disease Control and Prevention Institutional Animal Care and Use Committee, in accordance with procedures described in the U. S. Public Health Policy, 1986.

Animals were housed singly or doubly to avoid injuries caused by fighting with cage mates. Space recommendations for laboratory animals were followed as set forth in the Guide for the Care and Use of Laboratory Animals, National Institutes of Health. All animals were fed a diet that has been proven to provide adequate nutrition and calories in captive Aotus monkeys used in malaria-related research. Feed was free of contaminants and freshly prepared. Daily observations of the animals’ behavior, appetite, stool, and condition were recorded. An attending veterinarian treated all animals as medical conditions arose.

Anopheles freeborni (F-1 strain originally from California) were laboratory-reared and maintained at the Division of Parasitic Diseases/Centers for Disease Control and Prevention insectaries. Mosquito infection was obtained by allowing the caged anophelines to feed directly on the tranquilized monkey. For sporozoite challenge, sporozoites were dissected from the salivary glands of infected mosquitoes into 20% fetal bovine serum in phosphate-buffered saline (pH 7.2). The glands were crushed under a coverslip; the released sporozoites were washed from the slide into a vial, and an aliquot was transferred to a Neubauer Cell Counting Chamber for quantification. The sporozoites were then injected intravenously into the femoral vein of the monkey.

Blood-stage parasitemia was monitored by the daily examination of thick and thin blood films by the method of Earle and Perez.10 Infections were terminated by treatment with chloroquine (30 mg base over a three-day period) or mefloquine (20 mg). All drugs were administered by oral intubation. Aliquots of the Ghana III/CDC strain of P. falciparum have been deposited with the Malaria Research and Reference Reagent Resource Center (MR4) at the American Type Culture Collection (Manassas, VA).

Genotyping of parasite populations was performed by polymerase chain reaction (PCR) amplification of polymorphic regions of the merozoite surface protein-1 (MSP-1) and MSP-2 genes of P. falciparum, followed by restriction fragment length polymorphism analysis of the PCR product using Alu I for MSP-1 products and Hinf I for MSP-2 products combined with gel electrophoresis. Primer pairs M1-OF2 (CTA GAA GCT TTA GAA GAT GC) and M1-OR2 (ATT CTA ATT CAA GTG GAT CAG) were used to amplify the regions surrounding the block 2 polymorphic domain of MSP-1 and primer pairs M2-F1 (GAA GGT AAT TAA AAC ATT GTC) and M2-R1 (GAT GTT GCT GCT CCA CAG) were used to amplify the central polymorphic repetitive domain of MSP-2. Detection of subpatent malaria infections was accomplished by using a species-specific diagnostic nested PCR amplification method that detects the rRNA genes of Plasmodium.11

RESULTS

Parasites were obtained from a patient (hospitalized locally in Atlanta) who had acquired the infection in Ghana. A heparinized blood sample was introduced into in vitro culture. After 13 days of development in culture, parasitized erythrocytes were injected into A. lemurinus griseimembra monkey AI-331 (Figure 1). The parasitemia was of short duration, with a maximum parasite count of 17,640/μL. The animal was splenectomized on day 54, and subsequently reinoculated with blood from monkey AI-1767. The resulting maximum parasite count was 88,200/μL; the infection was terminated with 30 mg of chloroquine given over a three-day period. The genealogy of the strain to date is shown in Figure 2.

Parasitized erythrocytes from the initial peak in monkey AI-331 were passaged to A. lemurinus griseimembra monkey AI-4052 (Figure 1). However, in spite of splenectomy at day 19, the parasite count remained at a low density. The infection was terminated with chloroquine. Passage to A. nancymai monkey AI-1767 (splenectomized seven days after inoculation) resulted in a maximum parasite count of 95,040/μL; parasitemia > 1,000/μL occurred for 23 days (Figure 3). Gametocytes were detectable at 44 days and mosquito infection was obtained on nine occasions. The infection was terminated with chloroquine. Following reinoculation (passage 8), a maximum parasite count of 1,620/μL was obtained. This infection was cured with 20 mg of mefloquine.

Aotus vociferans monkey AI-2653 had been splenectomized and previously infected with P. vivax. Three recrudescences of infection occurred following passage of Ghana III-infected erythrocytes from monkey AI-331 (Figure 3); maximum parasite counts were 4,410/μL, 8,820/μL, and 29,160/μL, respectively. Thus, it was demonstrated that splenectomized A. nancymai and A. vociferans animals would support asexual infection and the maintenance of gametocytemia. Infections were cured by treatment with 30 mg of chloroquine or 20 mg of mefloquine. The parasitemia in monkey AI-2653 suggested the appearance of different parasite populations with increasing adaptability to grow in its erythrocytes. Upon reinoculation (passage 6), a peak parasite density of 800,000/μL occurred in this animal, accompanied by another extended period of gametocytemia. The infection was treated with 20 of mg mefloquine.

The next passages (monkeys AI-2713 and AI-2791) were to splenectomized A. nancymai monkeys. Monkey AI-2713 (Figure 4) developed an initial maximum parasite count of 140,000/μL. Asexual parasites disappeared for 36 days, at which time the animal was reinoculated (passage 6) and had a maximum parasite count of 440,000/μL that was controlled with treatments of 4 and 2 mg of chlorguanide given on successive days. This was followed by a recrudescence with a peak parasite count of 34,000/μL. The animal was again reinoculated (passage 8); the maximum parasite count was 20,250/μL. The animal was treated with 20 mg of mefloquine. Following another reinoculation (passage 9), a maximum parasite count of 14,580/μL occurred.

Aotus nancymai monkey AI-2791 (Figure 4) had a similar initial peak parasite count of 104,000/μL. The animal was reinoculated (passage 8) on day 55. A subsequent maximum parasite count of 47,160/μL occurred on day 65. The infection was eventually terminated with chloroquine.

Blood was passaged from monkey AI-2791 to A. nancymai monkey AI-1771 (Figure 4). The animal was splenectomized two days after inoculation. A maximum parasite count of 332,000/μL occurred on day 11; the animal was treated with 2 mg of chlorguanide to control, but not cure the infection. Mosquito infection occurred on days 23 and 24. The animal was reinoculated (passage 6) on day 30; this was followed by three recrudescent peak parasite counts of 13,500/μL, 51,840/ μL, and 136,800/μL, respectively. Again, each successive recrudescence peak was higher than the previous one. The animal was again reinoculated (passage 8); this was followed by peak parasite counts of 16,630/μL and 50,400/μL on days 128 and 186.

Blood from monkey AI-1771 (passage 4) was injected into intact A. nancymai monkey AI-1774 (Figure 5). A maximum parasite count of 236,000/μL occurred on day 8. Unfortunately, this animal died; however, during the initial parasitemic period, blood was passaged from monkey AI-1774 to previously infected monkeys AI-1771, AI-1775, AI-2653, AI-2713, and AI-2791 (passage 6) (Figures 3 and 4).

From the courses of parasitemia in many of the animals, it was suggestive that different populations of parasites failed to provide substantial immunity to subsequent infection with the Ghana III strain. The adaptation process for this strain could involve selection from multiple lines of differing genotype and thus potentially different antigenic phenotypes. To determine if selection of genotypes was occurring during adaptation we amplified polymorphic regions of two genetic loci, MSP-1 and MSP-2, from samples of the Ghana III parasite populations collected from the original patient isolate (11 VIII 00), during adaptation to in vitro culture and from monkey AI-331 (22 XI 00), AI-1767 (25 X 00), AI-1778 (2 II 01), and AI-1778 (27 VII 01) during its third infection. Surprisingly, the genotypic profile did not change during either in vitro or in vivo adaptation processes compared with that seen in the Ghana III parasite population present in the blood of the patient (Figure 6).

The salivary glands of An. freeborni mosquitoes infected by feeding on monkey AI-1771 were dissected and 17,000 sporozoites were injected intravenously into A. nancymai monkey AI-1778 (Figure 5). The animal was splenectomized two days after inoculation. The prepatent period was 22 days and the peak parasite count was 120,000/μL. The animal was reinoculated (passage 8); a maximum parasite count of 84,000/μL occurred, and the infection was treated with 20 mg of mefloquine. A PCR-based examination of a blood sample two months later was positive for P. falciparum, suggesting that the animal had not received or retained the entire amount of drug. The animal was reinoculated (passage 9); a maximum parasite count of 108,000/μL occurred and the infection was again treated and cured with 20 mg of mefloquine. Gametocyte production was meager.

Blood was passaged from monkey AI-1778 to A. nancymai monkey AI-1772 (Figure 5). A maximum parasite count of 304,000/μL occurred. This demonstrated that high-density parasitemia could occur in an intact animal, although the period of detectable parasitemia was only 16 days. The animal was splenectomized and then reinoculated (passage 8). A maximum parasite count of 560,000/μL occurred. The gametocytemia in this animal was transient; the infection was treated with 20 mg of mefloquine.

Because of the limited number of animals available for such adaptation, reinfection of animals was considered appropriate to select the more virulent line(s), yet maintaining gametocyte production. However, the selection of a line with high-density parasitemia and continuing gametocytemia was still uncertain, so blood from monkey AI-1771 (Figure 4) was passaged to splenectomized A. nancymai monkey AI-1777 (passage 7). Monkey AI-1777 had a maximum parasite count of 176,000/μL (Figure 5). A peak parasite count during recrudescence was only 1,440/μL. From monkey AI-1777, blood was passaged to nine A. nancymai monkeys for rechallenge (AI-1772, AI-2713, AI-1767, AI-1775, AI-1775, AI-1771, AI-1778, AI-2791, and AI-4052 (Table 1 and Figure 2). Aotus nancymai monkey AI-1792 (passage 8) was inoculated with blood from late in the infection in monkey AI-1777 (day 104). An initial maximum parasite count of only 1,350/μL occurred and the animal was splenectomized 20 days after inoculation. The subsequent maximum parasite count was 68,400/μL, but gametocyte counts were of low density. Blood was used to reinoculate monkeys AI-1778 and AI-2713 and the animal was treated with 20 mg of mefloquine.

It was apparent that relatively high parasite densities could be induced in both intact and splenectomized An. nancymai. However, there were indications that passage had resulted in decreasing gametocyte production. It was therefore decided to reactivate parasites frozen from two points in the passage history to further investigate A. vociferans as a potential host for studies with this parasite.

Splenectomized A. vociferans monkeys AI-3000 and AI-2990 were infected to hopefully select parasites that would produce high density asexual parasite counts and large numbers of infective gametocytes (Figure 7). Monkey AI-3000 was inoculated with blood that had been stored frozen from monkey AI-1767 (passage 2, Table 1 and Figure 2). A maximum parasite count of 68,940/μL occurred on day 25, and gametocytes of relatively low density were produced. Monkey AI-2990 was inoculated with blood that had been stored frozen from monkey AI-1778, the sporozoite-induced infection (passage 5, Table 1 and Figure 2). High-density asexual parasitemia was obtained that was modified by two treatments with 2 mg of chlorguanide; gametocyte densities > 1,000/μL were obtained on eight days during the primary attack. There was a recrudescence of parasitemia that resulted in a maximum parasite count of 128,000/μL, at which time the animal was treated with 4 mg of chlorguanide. Gametocyte densities > 1,000/μL occurred at 13 days. Anopheles freeborni mosquitoes were infected by feeding through membranes on erythrocytes from which the plasma had been removed and diluted 1:8 in heparinized human blood.

Mosquitoes that had been thus infected were dissected and the sporozoites were injected into A. nancymai monkey AI-2787 (Figure 8). The prepatent period was 25 days. A maximum parasite count of 316,000/μL occurred, and the animal was treated with 4 mg of chlorguanide that eliminated the asexual parasitemia; the animal was subsequently cured by treatment with 20 mg of mefloquine. From monkey AI-2787, passage was made to A. nancymai monkeys AI-1739 and AI-1772. Monkey AI-1739 had been infected previously with the Vietnam Oak Knoll strain of P. falciparum and with P. vivax. A maximum parasite count of 9,000/μL was obtained. Monkey AI-1772 had been infected previously with the Ghana III strain of P. falciparum and with P. vivax. A maximum parasite count of 23,940/μL was obtained.

DISCUSSION

Despite many attempts, few isolates of P. falciparum from Africa have been adapted for vaccine trials in A. nancymai monkeys. The Uganda Palo Alto (Hawaii) and Uganda Palo Alto (Cayenne) strains produce high-density asexual parasitemia, but fail to produce infective gametocytes, even when passaged to splenectomized animals.9 Before subjecting a strain to sequential passage in intact Aotus monkeys, we decided to first establish a line or lines that produced infective gametocytes as a model for anti-sporozoite and transmission-blocking studies; our experience has been that mosquito infection is rare unless the monkey is splenectomized. A line or lines could then be selected in intact A. nancymai or A. vociferans monkeys for the testing of blood-stage vaccine candidates.

Aotus lemurinus griseimembra appear to be much more susceptible to primary adaptation of a new isolate than are A. nancymai and A. vociferans. Because the only animals currently available in numbers large enough for vaccine trials are A. nancymai and A. vociferans, our procedure has been to initially produce parasites in vitro, passage to A. l. griseimembra, and, once a parasite develops in this host, transfer infection to the other hosts.

In the current adaptation, the movement from A. lemurinus griseimembra to A. nancymai was successful, but there were some unexpected courses of parasitemia. In most instances, the primary attack in a monkey is the most intense, followed by peak parasite counts of decreasing intensity. However, during the adaptation process with the Ghana III strain, recrudescent peak parasite counts were frequently higher than during the primary attack. In general, previous work with other parasite strains has indicated that once a monkey has been infected with a particular strain of P. falciparum, reinfection has frequently been difficult, resulting in maximum parasite counts of very low intensity. Reinfection of these splenectomized animals suggested that the Ghana III parasite was either 1) being selected for ever-increasing intensity of parasitemia, or 2) that the initial inocula contained a mixture of heterologous parasites. Analysis of the genotypic profile of the Ghana III parasite population throughout the adaptation processes undertaken here indicates that the latter possibility was not occurring during adaptation. The primary goal was to select parasites from a particular recrudescent population that produced high-density parasitemia coupled with the production of large numbers of infective gametocytes.

The demonstration of mosquito infection and subsequent sporozoite transmission to A. nancymai indicates that an anti-sporozoite vaccine trial model could be developed using the Ghana III strain and this host. For vaccine trials against blood-stage antigens of P. falciparum, the Ghana III strain would need to be further selected to produce predictable high-density parasite counts in intact animals. The infection in monkey AI-1772, in which a maximum parasite count of 304,000/μL was obtained in an intact animal, demonstrated that this host-parasite model has significant potential for blood-stage trials.

Table 1

Peak parasite counts in Aotus lemurinus griseimembra, A. nancymai, and A. vociferans monkeys infected with the Ghana III/CDC strain of Plasmodium falciparum*

Monkey no.Species of AotusPassage no.Donor monkeyInoculumSplenectomyPeak parasitemia
* CDC = Centers for Disease Control and Prevention.
† Inoculated with blood that had been stored frozen.
‡ Sporozoites dissected from Anopheles freeborni mosquitoes.
AO-331l. griseimembra1Culture2.1 × 107Day 5417,640
3AI-17675.6 × 106Pre-88,200
AI-1767nancymai2AO-3311.1 × 106Day 795,040
8AI-17775.8 × 106Pre-1,620
AI-4052l. griseimembra2AO-3316.0 × 104Day 192,430
AI-2653vociferans2AO-3316.0 × 104Pre-4,410; 8,820; 29,160
6AI-17741.4 × 107Pre-800,000
AI-2791nancymai3AI-17678.9 × 106Pre-104,000
6AI-17741.4 × 107Pre-47,160
AI-2713nancymai3AI-17678.9 × 106Pre-140,000
6AI-17741.4 × 107Pre-440,000; 34,000
8AI-17775.8 × 106Pre-20,250
9AI-17922.8 × 106Pre-14,580
AI-3000†vociferans3AI-17672.4 × 107Pre-68,940; 2,280
AI-1771nancymai4AI-27911.4 × 106Day 2332,000
6AI-17741.4 × 107Pre-13,500; 51,840; 136,800
8AI-17775.8 × 106Pre-16,630; 50,400
AI-1774nancymai5AI-17711.7 × 108236,000
AI-1778nancymai5AI-17711.7 × 104Day 2120,000
8AI-17775.8 × 106Pre-84,000
9AI-17922.8 × 106Pre-108,000
AI-1772nancymai6AI-17782.1 × 107Day 24304,000
8AI-17775.8 × 106Pre-560,000
8AI-27787.9 × 107Pre-23,940
AI-2990†vociferans6AI-17781.9 × 107Pre-100,000; 184,000; 100,000; 128,000
AI-1777nancymai7AI-17713.6 × 107Pre-176,000
AI-2778nancymai7AI-29903.3 × 104Pre-316,000
AI-1792nancymai8AI-17776.0 × 104Day 201,350; 68,400
AI-1739nancymai8AI-27787.9 × 107Pre-9,000
Figure 1.
Figure 1.

Parasitemia in Aotus lemurinus griseimembra monkeys AI-331 and AI-4052 infected with the Ghana III/CDC strain of Plasmodium falciparum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 2.
Figure 2.

Genealogy of the Ghana III/CDC strain of Plasmodium falciparum in Aotus monkeys. Solid line = blood passage; dotted line = sporozoite passage; * = second inoculation; # = third inoculation with the Ghana III strain.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 3.
Figure 3.

Parasitemia in Aotus nancymai monkey AI-1767 and A. vociferans monkey AI-2653 following infection and reinfection with the Ghana III/CDC strain of Plasmodium falciparum. Anopheles freeborni mosquitoes were infected.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 4.
Figure 4.

Parasitemia in Aotus nancymai monkeys AI-2713, AI-2791, and AI-1771 following infection and reinfection with the Ghana III/CDC strain of Plasmodium falciparum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 5.
Figure 5.

Parasitemia in Aotus nancymai monkeys AI-1774, AI-1778, AI-1772, and AI-1777 following infection and reinfection with the Ghana III/CDC strain of Plasmodium falciparum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 6.
Figure 6.

Restriction fragment length polymorphism analysis of merozoite surface protein-1 (MSP-1) and MSP-2 polymerase chain reaction (PCR) DNA fragments amplified from Ghana III/CDC parasites of Plasmodium falciparum taken at different points during in vitro and in vivo adaptation. A, PCR-amplified fragments of block 2 of MSP-1. B, PCR-amplified fragments of MSP-2. C, Alu I restriction enzyme digests of MSP-1 DNA fragments. D, Hinf I restriction enzyme digests of MSP-2 DNA fragments. Lanes 1 through 8 represent amplification of DNA from 1) Ghana III patient blood, 2) in vitro culture, 3) AI-1767 (25 X 00), 4) AI-331 (22 XI 00), 5) AI-1778 (2 II 01), 6) AI-1778 (27 VII 01), 7) the P. falciparum 7G8 strain, and 8) the P. falciparum Viet Nam-Oak Knoll (FVO) strain. Lanes S = DNA size standards (1.0, 0.85, 0.65, 0.5, 0.4, 0.3, 0.2, and 0.1 kilobases from top to bottom).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 7.
Figure 7.

Parasitemia in Aotus vociferans monkeys AI-3000, and AI-2990 following infection with the Ghana III/CDC strain of Plasmodium falciparum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Figure 8.
Figure 8.

Parasitemia in Aotus nancymai monkey AI-2787 following infection with sporozoites of the Ghana III/CDC strain of Plasmodium falciparum dissected from Anopheles freeborni mosquitoes.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 6; 10.4269/ajtmh.2003.69.593

Authors’ addresses: JoAnn S. Sullivan, James J. Sullivan, Katharine K. Grady, Curtis S. Huber, Douglas Nace, John W. Barnwell, and William E. Collins, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mailstop F-36, 4770 Buford Highway, Atlanta, GA 30341, E-mail: wec1@cdc.gov. Allison Williams and G. Gale Galland, Animal Resources Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30333; Amy Bounngaseng and Tyrone Williams, Atlanta Research and Education Foundation, Veterans Affairs Medical Center of Atlanta, Decatur, GA 30033.

Financial support: This work was supported in part by an interagency agreement between the United States Agency for International Development, Malaria Vaccine Development Program, and the Centers for Disease Control and Prevention, Project # 936-6001, and the Atlanta Research and Education Foundation of the Veterans Affairs Medical Center of Atlanta.

REFERENCES

  • 1

    Siddiqui WA, Schnell JV, Geiman QM, 1972. A model in vitro system to test susceptibility of human malaria parasites to antimalarial drugs. Am J Trop Med Hyg 21 :392–399.

    • Search Google Scholar
    • Export Citation
  • 2

    Geiman QM, Meagher MJ, 1967. Susceptibility of a New World monkey to Plasmodium falciparum.Nature 215 :437–439.

  • 3

    Collins WE, Anders RF, Ruebush TK II, Kemp DJ, Woodrow GC, Campbell GH,Brown GV, Irving DO, Gross N, Filipski VK, Coppel RL, Broderson JR, Thomas LM, Pye D, Skinner JC, Wilson C, Stanfill PS, Procell PM, 1991. Immunization of owl monkeys with ring-infected erythrocyte surface antigen of Plasmodium falciparum.Am J Trop Med Hyg 44 :34–41.

    • Search Google Scholar
    • Export Citation
  • 4

    Ruebush TK II, Campbell GH, Moreno AO, Patarroyo ME, Collins WE, 1990. Immunization of owl monkeys with a combination of Plasmodium falciparum asexual blood-stage synthetic peptide antigens. Am J Trop Med Hyg 43 :355–366.

    • Search Google Scholar
    • Export Citation
  • 5

    Sharma P, Ruebush TK II, Campbell GH, Richman SJ, Wilkins PM, Broderson JR, Ardeshir F, Gross M, Silverman C, Skinner JC, Filipski V, Wilson C, Roberts JM, Ma NF-S, Stanfill PS, Reese RT, Collins WE, 1992. Immunogenicity and efficacy trials in Aotus nancymai monkeys with model compounds representing parts of a 75-kD merozoite surface antigen of Plasmodium falciparum.Am J Trop Med Hyg 46 :691–707.

    • Search Google Scholar
    • Export Citation
  • 6

    Berzins K, Adams S, Broderson JR, Chizzolini C, Hansson M, Lovgren K, Millet P, Morris CL, Perlmann H, Perlmann P, Sjolander A, Stahl S, Sullivan JS, Troye-Blomberg M, Collins WE, 1995. Immunogenicity in Aotus monkeys of ISCOM formulated repeat sequences from the Plasmodium falciparum asexual blood stage antigen Pf155/RESA. Vaccine Res 4 :121–133.

    • Search Google Scholar
    • Export Citation
  • 7

    Collins WE, Walduck A, Sullivan JS, Andrews K, Stowers A, Morris CL, Jennings V, Yang C, Kendall J, Lin Q, MArtin LB, Diggs C, Saul A, 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.

    • Search Google Scholar
    • Export Citation
  • 8

    Kumar S, Collins WE, Egan A, Yadava A, Girrard O, Blackman M, Guevara Patino JA, Diggs C, Kaslow D, 2000. Immunogenicity and efficacy in Aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kD C-terminus of the merozoite surface protein 1 (MSP-1). Infect Immun 68 :2215–2223.

    • Search Google Scholar
    • Export Citation
  • 9

    Collins WE, Galland GG, Sullivan JS, Morris CL, 1994. Selection of different strains of Plasmodium falciparum for testing blood-stage vaccines in Aotus nancymai monkeys. Am J Trop Med Hyg 51 :224–232.

    • Search Google Scholar
    • Export Citation
  • 10

    Earle WC, Perez M, 1932. Enumeration of parasites in blood of malarial patients. J Lab Clin Med 17 :1124–1130.

  • 11

    Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN, 1993. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 58 :283–292.

    • Search Google Scholar
    • Export Citation
Save