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Am. J. Trop. Med. Hyg., 80(2), 2009, pp. 228-235
Copyright © 2009 by The American Society of Tropical Medicine and Hygiene

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Studies on the Salvador I Strain of Plasmodium vivax in Non-human Primates and Anopheline Mosquitoes

William E. Collins*, Joann S. Sullivan, Elizabeth Strobert, G. Gale Galland, Allison Williams, Douglas Nace, Tyrone Williams, AND John W. Barnwell
Division of Parasitic Diseases, National Center for Zoonotic, Vector Borne and Enteric Diseases, Geographic Medicine and Health Promotion and Animal Resources Branches, National Center for Preparedness, Detection and Control of Infectious Diseases, Centers for Disease Control and Prevention, US Public Health Service, Atlanta, Georgia; Yerkes Regional Primate Center, Emory University, Atlanta, Georgia; Atlanta Research and Education Foundation, Decatur, Georgia


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A review is presented on studies conducted in New World monkeys and chimpanzees with the Salvador I strain of Plasmodium vivax. This isolate has been adapted to Aotus and Saimiri (squirrel) monkeys and developed as a model for the testing of antimalarial vaccines. After the injection of 10,000 sporozoites, the median prepatent period in S. boliviensis monkeys was 21.5 days. In 103 sporozoite-induced infections in splenectomized monkeys, the median maximum parasite count ranged from 2,139 to 202,368/µL, with a median maximum parasite count of 48,174/µL. Median maximum parasite counts in Aotus lemurinus griseimembra, A. nancymaae, A. azarae boliviensis, and A. vociferans monkeys were 19,902, 18,390, 21,420, and 18,210/µL, respectively and ranged from 124 to 156,000/µL. Mosquito infections were readily obtained in different species of Anopheles mosquitoes. The S. boliviensis monkey and Salvador I strain seems suitable for the testing of sporozoite and liver stage vaccines but not for blood-stage vaccines against P. vivax unless adapted further in spleen-intact Saimiri boliviensis monkeys.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major milestone in malaria research has been the sequencing of the complete genome of Plasmodium vivax.1 Specific for this development was the availability of the Salvador I strain of the parasite from non-human primates. This was an appropriate choice because this particular strain of the parasite has been so widely used in many different experimental studies after its isolation > 30 years ago. Because the genome of this parasite will, no doubt, be probed for continuing study, a review of the biologic activity of the Salvador I strain in non-human primates and different anopheline mosquitoes should provide us with useful information that will assist in understanding the complexities and relationships between the different parts of the genome. Here, we review in some detail the various studies that have been conducted with the Salvador I strain in New World primates, chimpanzees, and vector mosquitoes.

The strain was isolated from a patient in the area of Congrejera in the La Paz Department of El Salvador by staff of Central American Research Station of the Centers for Disease Control in 1969.2 Blood was passaged into an Aotus lemurinus griseimembra monkey and subsequently transferred to the Centers for Disease Control (CDC) laboratories in Chamblee, Georgia. The parasite has subsequently been passaged through New World monkeys, chimpanzees, and to human volunteers.

Because of its ready infectivity to mosquitoes and frequent transmission by sporozoites to different non-human primate hosts, it has served as a model for a number of candidate vaccine trials and for numerous biologic studies.2–34 Over the last 35 years, infections were induced in our laboratory in 393 Saimiri and 125 Aotus monkeys and in 53 chimpanzees.

The first reported studies were those of Contacos and others3 and Collins and others,2 who described the transmission of the Salvador I strain from Aotus monkeys to human volunteers through the bites of An. freeborni mosquitoes. After five linear passages by I&nfected erythrocytes through A. lemurinus griseimembra monkeys, infected mosquitoes were fed on an infected monkey and allowed to feed on and infect volunteers. Prepatent periods ranged from 12 to 16 days. Five of the infections relapsed after periods of 192 to 229 days; a second relapse occurred in one volunteer at Day 229. The other four volunteers had received radical curative therapy before the second relapse occurred. In comparative feeding studies, An. freeborni were shown to be ~10 times more susceptible to infection than An. maculatus mosquitoes.

This parasite strain remained stored frozen until 1983 when Campbell and others4 initiated studies on its development in squirrel monkeys from different regions of South America. Splenectomized animals from Guyana and Peru failed to consistently support the development of high-density parasite counts. However, monkeys from Bolivia (Saimiri boliviensis boliviensis) seemed to much more readily support such development. S. boliviensis monkeys were shown to support the development of gametocytes infective to An. freeborni, An. gambiae, An. culicifacies, An. albimanus, An. stephensi, An. quadrimaculatus, An. dirus, and An. maculatus mosquitoes. Transmission was obtained after injection of sporozoites into two Guyanan, one Bolivian, and two Peruvian squirrel monkeys. This study established that Bolivian Saimiri monkeys had the potential for development as models in malaria research.

At approximately this same time, Colombia ceased exporting A. lemurinus griseimembra monkeys, the most susceptible Aotus host for human malaria parasites, and alternative species’ needed to be developed for P. vivax studies. A. azarae boliviensis were available and were shown to be susceptible to infection with the Salvador I strain and would also support mosquito infection.5

In comparative feedings on Aotus monkeys infected with the Salvador I strain between the North American mosquito An. freeborni and the Indian vector An. culicifacies, it was shown that the former was more heavily infected at a ratio of 100 to 76, based on oocysts numbers per gut.7 In other studies, comparative feedings were made between An. freeborni, An. dirus, An. stephensi, and two strains of An. gambiae with seven different strains of P. vivax, including Salvador I. The results indicated that there were statistically significant differences between each combination of vector and parasite.8 Later studies also showed the comparative infectivity of Salvador I to different strains of An. farauti.9,10 Porter and Collins 11 determined that the mosquito An. hermsi was a potential vector for P. vivax in California using Salvador I as the experimental strain.

Bolivia, by the mid-1980s, had stopped the export of Aotus and Saimiri monkeys, and new hosts for malaria research again needed to be developed. Peruvian A. vociferans and A. nancymaae were possible alternatives. Studies indicated that the Salvador I strain would develop well in A. vociferans and that these monkeys strongly supported gametocyte production. 12 Salvador I also developed well in A. nancymaae; however, mosquito infection was more difficult to obtain by feeding on infections in this host. 13

It was therefore decided to reinvestigate the model developed by Campbell and others4 of S. boliviensis monkeys, even though they could no longer be imported from Bolivia. These animals breed well in captivity, and a breeding colony had been established. Thus, efforts were made to further develop this model for vaccine trials. In the initial study, mosquitoes were infected by feeding on chimpanzees that had been infected with the Salvador I strain by injection of infected parasitized erythrocytes. 14 In this 20 monkey study, 10 monkeys were injected intravenously with 100,000 sporozoites, and the resulting prepatent periods averaged 16.6 days, with all animals developing high-level parasite counts that averaged a maximum parasite count of 103,000 parasites/µL. Five monkeys were injected intravenously with 10,000 sporozoites, and the resulting pre-patent periods averaged 19.4 days. One of the resulting infections produced a transient low-level parasitemia, whereas the other four developed the normal high-density parasite counts. Finally, five monkeys were injected intravenously with 1,000 sporozoites; four had extended pre-patent periods of from 24 to 35 days and one failed to develop detectable parasitemia. One monkey developed a low-level transient parasitemia. Based on these results, it was proposed that, by using 10,000 sporozoites injected intravenously, a predictable model system could be used to test anti-sporozoite vaccines against P. vivax.

Research efforts by 1994 had established S. boliviensis as a suitable model for the testing of sporozoite-induced infections with the Salvador I strain. 15 Of 58 animals inoculated, 45 had developed parasitemia; prepatent periods ranged from 10 to 63 days. Of 41 Aotus monkeys of various species that had been inoculated, only 10 had developed parasitemia. Sporozoites from An. freeborni, An. dirus, and An. gambiae had been used to induce these infections.

Observation of S. boliviensis monkeys after injection of sporozoites of the Salvador I strain of P. vivax resulted in 12 infections where the maximum parasite count was < 625/µL, and the days of patent parasitemia were < 17 days. 16 This suggested that early developing exoerythrocytic schizonts apparently release parasites with different levels of virulence, ranging from those producing high-level parasitemia to a more abundant avirulent form. It suggested that most of the parasites released from the developing liver-stage schizont may be incapable of producing high-density parasitemia in the host and that only a small percentage are capable of inducing high-density parasite counts. This occurred in ~10% of the sporozoite-induced infections in S. boliviensis monkeys, most of which were injected with 10,000 or more sporozoites.

The production of large numbers of infected mosquitoes and large volumes of infected blood for molecular studies and diagnostic studies required the use of a larger host. Thus, chimpanzees, Pan troglodytes, were infected to provide material and mosquitoes to support vaccine trials and other biologic studies. 17 Over the following 20+ years, 25 splenectomized chimpanzees were infected with the Salvador I strain. Median maximum parasite counts for primary infections were 28,400/µL. Mosquitoes were infected by feeding through membranes on blood collected intravenously from the chimpanzees. Sporozoites from An. stephensi, An. dirus, and An. freeborni were used to infect 193 Saimiri and 6 Aotus monkeys and 1 chimpanzee.

In 1985, McCutchan and others6 reported the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax, based on genomic DNA of the Salvador I strain. The initial vaccine trial using Salvador I involved the testing of two recombinant vaccines based on the circumsporozoite protein.18 The first (NS181V20) produced in Escherichia coli contained the repeat region of the CS protein plus 81 N-terminal amino acids derived from a nonstructural (NS1) of the influenza A virus. The other, VIVAX-1 vaccine, contained the entire repeat region plus 15 amino acids of the amino terminal and 48 amino acids of the carboxy-terminal sequence flanking the repeat region of the CS protein. Forty-eight S. boliviensis monkeys were included in the trial, but the results unfortunately failed to show protection. Challenge infections were initiated with 10,000 sporozoites given intravenously to each animal that had been dissected from the salivary glands of mosquitoes that had been infected by feeding on a chimpanzee. The animals developed decent serologic responses to the immunizations, but this did not result in any reduction in pre-patent periods.

A second vaccine trial was conducted using CS protein vaccines rPvCS-2 and rPvCS-3. 19 These were yeast-derive recombinant products combined with alum. Thirty-six S. boliviensis monkeys were used in the trial. Challenge was again with 10,000 sporozoites to each animal from mosquitoes that had been infected by feeding on a chimpanzee. Although there were very high levels of antibody response, the levels of protection were again inconclusive. An examination of pre- and post-immunization sera from these monkeys in regard to the ability to inhibit sporozoite invasion of liver cell hepatocytes in vitro indicated a strong correlation with serologic response. 20 However, no relationship between the peripheral blood mononuclear cell (PBMC) responses and protection was found. 21 Additionally, there were no differences in renal pathology between vaccinated and unvaccinated controls. 22

A passive protection study was conducted in which six S. boliviensis monkeys were injected with 2 mg of monoclonal antibody directed against the sporozoites of the Belem strain of P. vivax and 1 hour later challenged with 10,000 sporozoites of the Salvador I strain of P. vivax.23 Four of the monkeys were protected and two others had delayed pre-patent periods compared with nine control monkeys receiving no monoclonal antibody and six monkeys injected with an anti-trypanosomal antibody. Analysis indicated that protection was directed against the AGDR section of the repeat of the circumsporozoite protein.

A study was also made to determine the duration of immunity after immunization with irradiated sporozoites. Six S. boliviensis were immunized and challenged multiple times with sporozoites over a period of almost 4 years. 24 Complete protection from repeated challenge with infective sporozoites was shown in one monkey; protection in two monkeys was obtained on eight of nine occasions, in one monkey on seven of nine occasions, in one monkey on six of nine occasions, and in one monkey on four of eight occasions. Five of six monkeys were protected against infection during the last six challenges. Inoculation with blood-stage parasites at the end of the trial indicated that all animals were susceptible to infection. These results suggested that this host–parasite model could serve as a model for attenuated sporozoite vaccines and that protection against sporozoite challenge may be reinforced by subsequent exposure to viable sporozoites.

An anti-sporozoite vaccine trial was conducted using a multiple antigen construct [(PvCS)2]2(P2)2 directed against the circumsporozoite protein of P. vivax combined with a nonionic copolymer, with muramyl tripeptide Mf-75, or with alum. Animals were challenged with 10,000 sporozoites of the Salvador I strain. 25,26 Animals were subsequently rechallenged with 30,000 sporozoites. Eleven of the 26 immunized monkeys were protected, and 10 additional animals had delays in their pre-patent periods.

Gibson and others 27 had published the structure of Pv200, better known as major merozoite surface protein-1, from the Salvador I strain, a protein of the C-terminal end of this antigen was expressed in the yeast Saccharomyces cerevisiae. However, it was not until 6 years later that a trial was conducted with this antigen. The efficacy of the recombinant merozoite surface protein (MSP-119) of P. vivax was tested in S. boliviensis monkeys by immunization with yeast expressed yP2P30Pv20019,28,29 followed by monkeys being challenged with 100,000 parasitized erythrocytes of the Salvador I strain. These challenged animals were splenectomized 1 week later to increase parasite densities and improve the course of parasitemia. After 7 weeks of infection, animals were treated. Eighteen weeks later, animals were rechallenged. Only 5 of 14 immunized animals were protected after the first challenge. Identification of a monkey model system other than Salvador I and S. boliviensis that supports higher-density parasitemia without splenectomy seems to be needed for the testing of blood stage vaccines against P. vivax. In this respect, further serial blood passage of the Salvador I strain in non-splenectomized squirrel monkeys may provide a suitable challenge strain for blood stage vaccine trials.

The Salvador I strain of P. vivax was used for the development and testing of transmission-blocking vaccine candidates Pvs25 and Pvs28 with sporozoites produced in chimpanzees. 30–33 In these studies, chimpanzees were infected with the Salvador I strain and gametocytes mixed with sera from animals that had been immunized with various constructs of transmission-blocking vaccine candidates. Percentage inhibition of oocyst numbers was used as an indication of vaccine efficacy. It was shown that alum and Montanide adjuvants when combined Pvs25 antigen directed against the ookinete surface protein could in some cases totally inhibit oocyst development in An. freeborni, An. stephensi, and An. gambiae mosquitoes.

Qari and others 34 described sporozoite stage-specific and blood stage–specific small subunit ribosomal RNA (SSUrRNA)-encoding genes (SSUrDNA) of the Salvador I strain of P. vivax. The comparison with other human malaria parasites showed the presence of seven conserved regions (≥ 90% similarity), four highly variable regions (< 60% similarity), and three semiconserved regions.

As a consequence of the extensive studies that have been made with the Salvador I strain of P. vivax and its ability to consistently produce infective gametocytes, it was selected in 2002 for complete genome sequencing by The Institute for Genomic Research (TIGR), Rockville, Maryland. Parasites to be used for this project were produced from S. boliviensis monkeys infected at the CDC, and the highly purified positive DNA free of host contamination was provided to TIGR for sequencing.

Here we summarize the results of infection of Aotus and Saimiri monkeys and chimpanzees P. troglodytes with the Salvador I strain of P. vivax and the results of comparative infection and transmission of the strain by four of the main laboratory vectors: An. freeborni, An. stephensi, An. gambiae, and An. dirus.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Different species of non-human primates were infected over the course of these studies depending on their availability. These included A. lemurinus griseimembra (originally from Columbia), A. azarae boliviensis (from Bolivia), A. nancymaae (from Peru), A. vociferans (from Peru), and S. boliviensis (from Bolivia). On arrival at the facility, all animals were quarantined for a 2-month conditioning period, weighed, and tested for tuberculosis. Parasitologic and serologic examination indicated that the animals were free of infection with malaria parasites before inoculation. Almost all of the monkeys were splenectomized before exposure to infection. All surgeries were performed in an AAALAC (Association for the Assessment and Accreditation of Laboratory Animal Care, International)-approved surgical suite appropriate for aseptic surgery. Studies were conducted after review and approval by the institutional Animal Care and Use Committees of Emory University and CDC in accordance with Public Health Policy on Humane Care and Use of Laboratory Animals, 1986. P. troglodytes (laboratory-born chimpanzees) were housed at the Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia.

Monkeys generally were housed doubly or in some cases singly 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, NIH. All animals were fed a diet that has been proven to provide adequate nutrition and calories in captive 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. All were treated as medical conditions arose by an attending veterinarian.

The mosquitoes used for comparative studies were An. freeborni from California, An. dirus from Thailand, An. stephensi from India, and An. gambiae from The Gambia. Other mosquitoes shown to be infected or used in feedings were An. maculatus from Malaysia, An. albimanus from El Salvador, An. culicifacies from India, An. farauti from the Southwest Pacific, An. quadrimaculatus from the southeastern United States, An. arabiensis from Kenya, An. minimus from Thailand, An. hermsi from California, and An. atroparvus from Spain.

During periods when gametocytes were present in the blood, mosquitoes were allowed to feed on tranquilized monkeys as previously described. 35 At other times, mosquitoes were allowed to feed through parafilm membranes on blood collected in heparin from monkeys or chimpanzees. After feeding, mosquitoes were held in an incubator at 25°C until examined 1 week later for the presence of oocysts on their midguts. Blood-stage parasitemia was monitored and quantified by the daily examination of thick- and thin-blood films by the method of Earle and Perez. 36 Infections were terminated by treatment with chloroquine (30 mg over 3 days in monkeys; 1,500 mg over 3 days in chimpanzees). Drugs were administered by oral intubation to monkeys and intramuscularly to chimpanzees.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Infection of Saimiri (squirrel) monkeys Saimiri boliviensis monkeys became the animals most often used in vaccine trials for studies with the Salvador I strain of P. vivax. An examination of the pre-patent periods after the injection of 10,000 sporozoites into 88 monkeys resulted in the median pre-patent period of 21.5 days (Figure 1Go). These animals were splenectomized 6 or 7 days after sporozoite injection to allow the parasite count to rise to higher levels. The pre-patent periods ranged from 14 to 46 days. In an additional 37 splenectomized animals that were injected with 15,000–75,000 sporozoites, the pre-patent periods ranged from 14 to 53 days with a median of 19 days. Of the 125 sporozoite-induced infections, 76 were with sporozoites dissected from An. stephensi, 35 from An. dirus, 7 from An. freeborni, and 7 were from An. gambiae mosquitoes.


Figure 1
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  		FIGURE 1. Prepatent periods for 88 splenectomized S. boliviensis monkeys injected with 10,000 sporozoites of the Salvador I strain of P. vivax. The median prepatent period is indicated by the arrow.

 
The course of parasitemia in 103 sporozoite-induced infections in S. boliviensis were examined, and the maximum parasite count ranged from 2,139 to 202,368/µL, with a median maximum count of 48,174/µL. The day of maximum parasite count ranged from Day 12 to Day 54, with the median day being Day 21.5 (Figure 2Go); 44.7% of the maximum parasite counts were ≥ 50,000/µL. The course of parasitemia in 51 trophozoite-induced infections in S. boliviensis had maximum parasite counts ranging from 1,000 to 296,000/µL (Figure 3Go), with a median maximum count of 36,538/µL. The day of maximum parasite count ranged from Day 8 to Day 46, with the median day being Day 23; 43.1% of the maximum parasite counts exceeded 50,000/µL.


Figure 2
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  		FIGURE 2. Day of maximum parasite count for 103 splenectomized S. boliviensis monkeys infected by sporozoites with the Salvador I strain of P. vivax. The median day of maximum parasite count is indicated by the arrow.

 

Figure 3
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  		FIGURE 3. Day of maximum parasite count for 51 splenectomized S. boliviensis monkeys infected by trophozoites with the Salvador I strain of P. vivax. The median day of maximum parasite count is indicated by the arrow.

 
Only 12 intact (non-splenectomized) S. boliviensis were allowed to have a full course of infection with the Salvador I strain. Maximum parasite counts ranged from 93 to 93,000/µL (median, 4,700/µL). The median day of maximum parasite count was Day 14.

Infection of Aotus (owl) monkeys Twenty Aotus monkeys were infected by sporozoites with the Salvador I strain. Most of these animals had been previously infected with P. falciparum, and some had in addition been infected with P. malariae. Three A. lemurinus griseimembra were infected, two by the bites of infected An. freeborni and An. stephensi mosquitoes and one by the injection of 65,000 sporozoites, with pre-patent periods of 17, 39, and 57 days, respectively. Maximum parasite counts were 11,904, 24,738, and 4,650/µL after 13, 16, and 14 days of patent parasitemia, respectively. One A. azarae boliviensis was injected with 50,000 sporozoites; the prepatent period was 24 days, the maximum parasite count was 7,817/µL, and the maximum parasite count day occurred on Day 11.

Nine A. nancymaae were infected with between 50,000 and 450,000 sporozoites of the Salvador I strain. Pre-patent periods ranged from 16 to 47 days, with a median of 23 days. Three A. nancymaae were infected by bites of infected An. gambiae and An. stephensi mosquitoes; pre-patent periods were 21, 27, and 48 days. Maximum parasite counts for these 12 splenectomized monkeys ranged from 1,581 to 78,480/µL and occurred after 7–22 days of patent parasitemia (median, 13 days). Four A. vociferans were infected: three by the injection of 120,000, 190,000, and 220,000 sporozoites and one by the bites of infected An. stephensi mosquitoes. Pre-patent periods were 23, 23, 23, and 19 days, respectively. Maximum parasite counts for these splenectomized monkeys were 2,909, 24,543, 28,800, and 25,000/µL on Days 8, 14, 9, and 18, respectively.

Twenty splenectomized A. lemurinus griseimembra, 12 of which had previously been infected P. falciparum and 3 with P. falciparum and P. malariae, were infected with trophozoites of Salvador I. Maximum parasite counts ranged from 2,330 to 68,634/µL, with a median parasite count of 19,902/µL.

Thirty-four splenectomized A. nancymaae, 18 of which had previously been infected with P. falciparum, were infected with trophozoites of Salvador I. Maximum parasite counts ranged from 155 to 156,000/µL, with a median parasite count of 18,390/µL.

Thirteen A. azarae boliviensis, four of which had previously been infected with P. falciparum and one with P. knowlesi, were infected with trophozoites of Salvador I. Maximum parasite counts ranged from 1,302 to 92,000/µL, with a median parasite count of 21,420/µL.

Eight splenectomized A. vociferans, five of which had previously been infected with P. falciparum, were infected with trophozoites of Salvador I. Maximum parasite counts ranged from 5,310 to 60,000/µL, with a median maximum parasite count of 18,810/µL.

The median maximum parasite counts were remarkably close for each of the four species of Aotus infected with trophozoite stages of Salvador I considering the wide range in maximum parasite counts observed. In 13 monkeys that had experienced infection with heterologous strains of P. vivax or infection with the related parasite P. simium previous to infection with Salvador I, the maximum parasite counts ranged from 124 to 64,000/µL, with a median maximum parasite count of 27,166/µL.

Infection of chimpanzees Twenty-four splenectomized chimpanzees (Table 1Go) with no previous history of infection with P. vivax were infected with Salvador I. Their maximum parasite counts during their primary infections ranged from 527 to 112,000/µL. The median parasite count was 30,876/µL. Mosquitoes were fed on blood collected in heparin through parafilm membranes. Very-high-density infections were routinely obtained. 17 After an interval that ranged from 448 to 2,073 days later, the animals were again infected with P. vivax. During the intervening period, most of them had also been infected with heterologous species of Plasmodium (except for C-591, C-676, and C-682). An additional animal, C-510, had initially been infected with the North Korean strain of P. vivax.


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TABLE 1
Maximum parasite counts in 24 chimpanzees infected and reinfected with the Salvador I strain of P. vivax

 
For the 25 chimpanzees reinfected with the Salvador I strain, the maximum parasite counts ranged from 31 to 14,136/µL, with a median parasite count of 900/µL. No mosquitoes were fed on chimpanzees C-451, C-516, C-472, and C-514 because the parasite counts were only 31, 250, 60, and 60/µL, respectively, and it was thought that infection would be unlikely. No infection was obtained by feeding on two different days on C-510. The maximum count for this animal was 150/µL. No infection was obtained by feeding on C-494 on 3 different days, although the maximum parasite count was 1,457/µL. The remaining 21 chimpanzees were able to support mosquito infection to some level, even though maximum parasite counts were relatively low.

Mosquito infection As indicated in the introductory review and unpublished transmission studies, the Salvador I strain readily infected different species of Anopheles mosquitoes and produced infective sporozoites in mosquitoes for transmission studies. Of 5,706 lots of the various mosquito species fed, dissected, and examined for the presence of infection, 3,449 were shown to contain parasites (60.45%). All of the laboratory species we examined (An. freeborni, An. stephensi, An. gambiae, An. dirus, An. farauti, An. maculatus, An. culicifacies, An. quadrimaculatus, An. albimanus, An. hermsi, An. culicifacies, An. minimus, An. atroparvus, and An. arabiensis) were susceptible to infection; however, some were more heavily infected than were others in side-by-side comparisons. Of the more commonly fed mosquitoes, in such comparisons, based on oocysts per positive gut, An. freeborni had over twice as many oocysts as did An. gambiae. An. gambiae consistently had greater numbers of oocysts per positive gut than did An. stephensi and An. dirus mosquitoes (Figure 4Go).


Figure 4
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  		FIGURE 4. Comparative number of oocysts per positive gut in An. gambiae, An. freeborni, An. stephensi, and An. dirus mosquitoes when fed simultaneously on monkeys or chimpanzees infected with the Salvador I strain of P. vivax.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Salvador I strain of P. vivax was selected for whole genome sequencing because so much was already known about the biology of the strain based on laboratory studies in New World monkeys. These hosts provide models for the examination of vector relationships, potential vaccine candidates, and potential antimalarial drugs. Of even greater importance, the adaptation of the human malarial parasites to these New World primates has allowed for the study of the immunologic and biologic relationships between primates and human pathogens and allows us to relate them to the human experience with these infections. By understanding the limitations and the applications of the models, we can better apply the results of vaccine trials in non-human primates to what might be expected from human studies.

As this review has indicated, the Salvador I strain of P. vivax is one of the parasites that has been studied most extensively in New World monkeys and has been applied in several vaccine trials with Saimiri boliviensis with challenge using sporozoites. Lessons have been learned along the way; for example, it is probably not a suitable model for the testing of blood-stage vaccines at this stage of adaptation. This is because of the fact that parasite counts are too low and unpredictable in non-splenectomized animals. However, to test anti-sporozoite or anti-liver stage vaccines, it seems to be an excellent model. Here, a delayed splenectomy is timed to occur after the development of the liver stage is completed (7 or more days after sporozoite injection) and only used to induce high density blood-stage parasite count. This does not affect the outcome of the anti-sporozoite or liver-stage vaccine assessment. As shown here, sporozoite challenge using S. boliviensis monkeys provides a good model for testing liver-stage or anti-sporozoite vaccines. However, using Salvador I strain to infect various species of Aotus monkeys does not seem to be the model of choice for testing pre-erythrocytic vaccines. Other strains of P. vivax may be more suitable for blood-stage trails in Aotus monkeys, such as Vietnam Palo Alto, 37 although further passages of the Salvador I through intact (non-splenectomized) owl and squirrel monkeys may allow it to adapt to these conditions and be suitable as a model for blood stage vaccine trials.

This examination of Salvador I was meant to review the model and where it now stands in the vaccine testing hierarchy. The model of choice for testing anti-sporozoite and liver-stage vaccines against P. vivax is S. boliviensis and the Salvador I strain. Immunization and challenge is always done in intact animals to allow the host to mount the greatest immune response to immunization. Challenge is with a number of sporozoites that has been shown to uniformly result in all animals developing infection in a predictable time period. In this model system, 10,000 sporozoites seems to be the suitable number. It seems that most of the parasites that are released from the liver stage schizont produce an avirulent infection. Thus, a large number of sporozoites (≥ 10,000) must to be injected into the S. boliviensis monkey to induce a virulent infection. Injections of numbers less than this may result in low-density or subpatent infections. With other combinations of parasite and monkey, such as Plasmodium knowlesi and the Macaca mulatta, 100–200 sporozoites would be the challenge dose. In the P. vivax model system, the animal is splenectomized 7–10 days after sporozoite injection. At that time, any effect of the immunization on the sporozoite or the primary liver stages is complete. Splenectomy now allows for an increase in asexual parasite development. Without this, parasite counts would sometimes remain at very low or even sub-patent levels. The Salvador I parasite readily infects most of the available laboratory-reared vector mosquitoes. Mosquito infection is obtained by feeding on splenectomized monkeys and is especially dense when fed on blood from splenectomized chimpanzees.

Received September 22, 2008. Accepted for publication October 27, 2008.

Acknowledgments: The authors thank the staff of the Animal Resources Branch, the National Center for Preparedness, Detection and Control of Infectious Diseases, for the care of the animals.

Financial support: This study was supported in part by an Interagency Agreement 936-3100-AA6-P-00-0006-07 between the US Agency for International Development and the Centers for Disease Control and Prevention.

Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

* Address correspondence to William E. Collins, Mail Stop F-36, Division of Parasitic Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, Chamblee, GA 30341. E-mail: wec1{at}cdc.gov Back

Authors’ addresses: William E. Collins, JoAnn S. Sullivan, Douglas Nace, and John Barnwell, Mail Stop F-36, Division of Parasitic Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, Chamblee, GA 30341. Allison Williams, Animal Resources Branch, and G. Gale Galland, Geographic Medicine and Health Promotion Branch, National Center for Preparedness, Detection and Control of Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30333. Elizabeth Strobert, Yerkes Regional Primate Research Primate Research Center, Emory University, 954 Gatewood Road, Atlanta, GA 30322. Tyrone Williams, Atlanta Research and Education Foundation, 1670 Clairmont Road, Decatur, GA 30033.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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