Am. J. Trop. Med. Hyg., 80(1), 2009, pp. 160-164
Copyright © 2009 by The American Society of Tropical Medicine and Hygiene
Plasmodium inui shortii: Studies in Old World and New World Monkeys
William E. Collins*,
McWilson Warren,
Joann S. Sullivan, AND
John W. Barnwell
Division of Parasitic Diseases, National Center for Vector Borne, Zoonotic, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
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ABSTRACT
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Plasmodium inui shortti was studied in monkeys (66 Macaca mulatta, 2 M. fascicularis, 12 Saimiri boliviensis, 4 Aotus lemurinus griseimembra, and 1 A. nancymaae). Prepatent periods for 30 sporozoite transmissions by Anopheles stephensi, An. dirus, and An. maculatus mosquitoes ranged from 10 to 48 days with a median of 15.5 days. In rhesus monkeys, mean maximum parasite counts for intact animals were 181,970/µL; for splenectomized animals, the mean maximum parasite count was 1,167,890/µL.
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INTRODUCTION
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Plasmodium inui shortti was isolated in southern India and first described as a new species by H. E. Shortt in 1961.1 The parasite was kindly provided to the National Institutes of Health, Far East Research Unit of the U.S. Public Health Service as blood from animal 266 (see genealogy in Figure 1
) in 1962. The parasite has been maintained since then by either blood or sporozoite passage by the U.S. Public Health Service Laboratories at Chamblee, Georgia. The parasite was compared with an older strain of P. inui (Mulligan strain) by Donald Eyles, who in 1963 proposed that the parasite was most likely a subspecies rather than a distinct species.2 He proposed the recognition of two subspecies, P. inui inui and P. inui shortti. Subsequently, numerous isolates of P. inui were established in the laboratory for study in monkeys.3 No attempts have been made to further separate this complex. Since the report by Coatney and others in 1966 on the experimental infection of human volunteers with the OS strain of Plasmodium inui, (P. inui shortti), this parasite has been of particular interest.4
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FIGURE 1. Genealogy of the OS strain of Plasmodium inui shortti. Solid line = trophozoite passage; dotted line = sporozoite passage.
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After three passages in the Far East Research Unit, monkey R-0430 was infected and then shipped to Chamblee, where additional studies with P. inui shortti were initiated. Vector susceptibility and transmission studies using rhesus monkeys indicated that Anopheles stephensi, An. maculatus, and An. dirus were capable vectors after mosquito feedings on rhesus monkeys. 5,6 With the establishment of suitable vectors, An. maculatus and An. stephensi were used to transmit infections to human volunteers that established that the parasite would readily infect humans by sporozoite and erythrocytic inoculation.4 Serologic studies using the indirect immunofluorescence test were conducted to determine the relationships between the different strains that were available. Although this is a subjective test, it suggested that there were immunologic differences between the different strains based on serologic responses in rhesus monkeys.7
It was important for the continued study of this parasite that it be adapted to in vitro culture. This was accomplished by Nguyen-Dinh and others in 1980.8 One of the interesting similarities between the human and monkey malarias has been that those parasites that will develop in humans often will also develop in erythrocytes of New World monkeys and that those that do not will likewise not develop in human erythrocytes. Thus, attempts were made to adapt P. inui shortti to different species of Aotus and Saimiri monkeys and to transmit the parasite with different species of anopheline mosquitoes.9–11 Sullivan and others described the exoerythrocytic stages of P. inui shortti in New world monkeys. 12 Thus, ready development of the parasite in New World monkeys was not unexpected because of its previous ready passage to humans.
We report a summary of sporozoite-induced and erythrocytic-induced studies in Old World and New World monkeys with P. inui shortti after its isolation in 1961.
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MATERIALS AND METHODS
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All monkeys were obtained commercially. The Macaca mulatta originated from northern India where malarial infections are not present in these animals; some animals were laboratory born. The two M. fascicularis were imported from Mauritius where malaria is not endemic in monkeys, and the M. radiata was from southern India where malaria occurs in this host. Aotus lemurinus griseimembra originated in Colombia, A. nancymaae originated in Peru, and Saimiri boliviensis monkeys were colony born. Upon arrival at the 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 inoculation. Many monkeys were splenectomized before exposure to infection. All surgeries were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care, International, Inc. 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.
New World monkeys generally were housed doubly or in some cases singly. 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, Bethesda, MD). 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 animals were treated as medical conditions arose by an attending veterinarian. Animals were fed a diet of animal chow, fruits, and vegetables considered suitable for their maintenance in captivity. Splenectomy was performed under sterile conditions by a qualified veterinarian using standard procedures. Early studies at Institute for Medical Research in Malaysia were conducted using animal care and housing practices appropriate and approved by the Institution.
Monkeys were infected by intravenous inoculation of infected erythrocytes either freshly collected from a donor animal or that had been stored frozen over liquid nitrogen. At times, infected mosquitoes were allowed to feed directly on a tranquilized animal for transmission of the parasite. Alternatively, sporozoites were dissected from the salivary glands of infected mosquitoes and then injected intravenously or intrahepatically into the monkeys. Beginning 1 day after injection of parasitized erythrocytes or 7 days after sporozoite inoculation, thick and thin blood films were prepared by the method of Earle and Perez, 13 stained with Giemsa stain, and examined microscopically. Parasite counts were recorded per microliter of blood.
In the Division of Parasitic Diseases, Centers for Disease Control and Prevention insectary in Chamblee, Georgia, Anopheles freeborni originally from California, An. quadrimaculatus from the southeastern United States, An. stephensi from India, An. dirus from Thailand, An. maculatus from Malaysia, and An. gambiae from The Gambia were maintained continuously. The procedures for feeding, handling, and dissection of the mosquitoes have been reported previously.5
Oocyst counts were made microscopically from mosquito guts suspended in a 2% solution of mercurochrome; this allowed for a contrasting vital staining of the parasites. After dissection, salivary glands of mosquitoes were examined and the density of sporozoites present was scored as 1+ (1–10 sporozoites), 2+ (11–100 sporozoites), 3+ (101–1,000 sporozoites), or 4+ (> 1,000 sporozoites). For transmission by sporozoite injection, infected salivary glands were harvested in 20% fetal bovine serum/saline, the number of sporozoites present was determined using a Neubauer cell counting chamber, and the sporozoite suspension was injected intravenous into the femoral vein of the recipient monkey. On five occasions, sporozoites from the dissected salivary glands were injected directly into the liver. 14
Infections were cured by treatment with chloroquine by oral intubation. Rhesus monkeys were given a total of 300 mg (base) over 3 days, and New World monkeys were given 30 mg (base) over 3 days. To modify parasitemias, small doses of quinine (150 mg) were given by oral intubation. Sporozoite-induced infections were also treated with primaquine; rhesus monkeys were given 7.5 mg a day for 7 consecutive days, and New World monkeys were given 2.5 mg a day for 7 days by oral intubation.
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RESULTS
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Sporozoite-Induced Infections
There were 15 sporozoite-induced infections to M. mulatta monkeys, 10 by bites and 5 by intra-hepatic injection (Table 1). Prepatent periods ranged from 10 to 31 days with a median of 14 days. The mean number of bites for the 10 monkeys was 5.6. None of the 11 animals receiving sporozoites (that were not treated) were splenectomized before the peak in their primary parasite count. Thus, the maximum parasite counts for the 11 sporozoite-induced infections in intact rhesus monkeys ranged from 15,220 to 840,000/µL with a mean of 129,941/µL. These counts occurred between day 9 and day 27 of patent parasitemia (mean = day 16.6). After splenectomy of 6 of the animals, maximum parasite counts ranged from 7% to 43.0%. These counts occurred between 6 and 12 days after splenectomy. Two Macaca fascicularis were exposed to infection by the intravenous injection of sporozoites of P. inui shortti. The prepatent periods were 19 and 21 days.
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TABLE 1 Prepatent periods and maximum parasite counts for 30 monkeys infected with sporozoites of Plasmodium inui shortti
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Twelve S. boliviensis monkeys were infected with P. inui shortti, 4 by intravenous injection of sporozoites dissected from the salivary glands and 8 by bites of An. dirus mosquitoes (Table 1
). Prepatent periods ranged from 12 to 17 days (median = 15 days). All monkeys had been splenectomized. The maximum parasite counts ranged from 43,400 to 508,000/µL with a median of 324,000/µL. The peak parasite count occurred, on average, on the 18th day of patent parasitemia. One transmission was obtained to splenectomized Aotus nancymaae monkey AI-0280 by bites of An. dirus mosquitoes. The prepatent period was 28 days and the maximum parasite count was 384,000/µL.
Trophozoite-Induced Infections
Sixteen intact and 13 splenectomized M. mulatta were injected with parasitized erythrocytes of P. inui shortti (Table 2). Maximum parasite counts for the intact animals ranged from 8,385 to 800,000/µL with a median count of 204,215/µL. For the 13 splenectomized monkeys, the maximum parasite counts ranged from 760,000 to 2,680,000/µL with a median of 1,120,000/µL. The day of maximum parasite count was on day 14.5 for the intact animals and day 17.2 for the splenectomized monkeys. The prepatent periods for splenectomized animals T-0175 through T-0189 (Table 2) were extended because the parasites were all from human volunteers who had been infected with this parasite. Apparently adaptation of the parasite back to the monkeys required longer than direct passage with infected rhesus erythrocytes.
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TABLE 2 Maximum parasite counts in intact and splenectomized Macaca mulatta monkeys infected with Plasmodium inui shortti by inoculation of parasitized erythrocytes
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Twelve additional rhesus monkeys were infected by the intravenous inoculation of parasitized erythrocytes. Nine of these splenectomized animals were treated with chloroquine either before they reached the maximum parasite count or near the maximum parasite count (Table 3). Eight of these animals had parasite counts 
600,000/µL at the time of treatment between 8 and 19 days of patent parasitemia. The other 4 monkeys were treated between days 3 and 15. The other 3 animals were treated when the counts were less than 100,000/µL.
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TABLE 3 Maximum parasite counts in 22 Macaca mulatta monkeys infected with Plasmodium inui shortti by trophozoite inoculation*
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Four monkeys (Table 3) that had been previously infected with other strains of P. inui were infected with P. inui shortti. In the 3 monkeys that had been splenectomized, the maximum parasite counts were 240,000, 640,000, and 720,000/µL, counts that were below that of their primary infections. The maximum count for the animal that was intact (840,000/µL) was only slightly higher.
Six additional M. mulatta monkeys with prior history of infection with various species of monkey malaria were infected with P. inui shortti. Four had been splenectomized and two were intact. Parasite counts in the splenectomized animals ranged from 16,554 to 380,000/µL. The parasite counts in the two intact animals were 25,414 µL and 1,172,000/µL.
Four splenectomized A. lemurinus griseimembra monkeys with extensive previous history of infections were injected with parasitized erythrocytes of P. inui shortti. Maximum parasite counts ranged from 97,092 µL to 212,040/µL.
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DISCUSSION
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Plasmodium inui shortti is a parasite that was isolated from a bonnet macaque and transferred into a rhesus monkey (M. mulatta). From there it was readily transmitted to humans by bite of mosquitoes. Subsequently, it was readily transmitted to New World monkeys. In Old and New World monkeys, this parasite produced high density parasite counts and readily infected laboratory maintained mosquito hosts, thus making it a suitable parasite for a variety of studies.
Plasmodium inui has many biologic characteristics similar to those of P. malariae, which makes this parasite useful and attractive in studies of growth cycle and similar pathology associated with the kidneys. 15–18 Both parasites also persist in their primate host for many years and decades, often for the life of the host. This finding contrasts with that of other infections, such as P. vivax or P. falciparum, which usually persist only several years. The mechanisms for such a long-term relationship giving rise to chronic parasitemia from a single infection are yet to be understood in either P. malariae in humans or P. inui in macaque monkeys.
Interestingly, a previous study had suggested that the spleen played a role in the longevity of P. inui infections in the host M. mulatta.19 In spleen-intact animals, parasites persisted for up to 13 years, whereas in splenectomized monkeys, paraasitemia persisted for less than 1 year.
There are areas in Southeast Asia where susceptible humans and competent vectors co-exit. The similarity of P. inui blood stages to those of P. malariae on thick blood films would make diagnostic separation extremely difficult such as was experienced with P. knowlesi.20 What are the characteristics of this parasite that permit it to infect humans and both New World and Old World monkey erythrocytes? Most primate malaria parasites do not infect all three types of hosts or support such high-density parasite counts for extended periods. No doubt, there is much to be learned by studies with this parasite in nonhuman primates that may provide clues or answers to questions concerning the long-term relationships between the human parasite P. malariae and its human host.
Aliquots of P. inui shortti have been deposited with the American Type Culture Collection.
Received September 26, 2008.
Accepted for publication September 30, 2008.
Acknowledgments: We 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.
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.
Financial support: This study was supported in part by an Interagency Agreement 936-3100-AA6-P-00-0006-07 between the United States Agency for International Development and the Centers for Disease Control and Prevention.
* Address correspondence to William E. Collins, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mailstop F-36, 4770 Buford Highway Chamblee, GA 30341. E-mail: wec1{at}cdc.gov 
Authors’ addresses: William E. Collins, Joann S. Sullivan, and John W. Barnwell, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Mailstop F-36, 4770 Buford Highway, Chamblee, GA 30341. McWilson Warren, Millbrook Road, Box 417, Grafton, NH 03240.
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REFERENCES
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ABSTRACT
INTRODUCTION
