Mosquito Infection Studies with Aotus Monkeys and Humans Infected with the Chesson Strain of Plasmodiun vivax

doi:10.4269/ajtmh.2012.11-0264

Am J Trop Med Hyg. 2012 March 1; 86(3): 398–402.

doi: 10.4269/ajtmh.2012.11-0264

PMCID: PMC3284352

Mosquito Infection Studies with Aotus Monkeys and Humans Infected with the Chesson Strain of Plasmodiun vivax

William E. Collins,^* JoAnn S. Sullivan, Geoffrey M. Jeffery, Douglas Nace, Tyrone Williams, G. Gale Galland, Allison Williams, and John W. Barnwell

Division of Parasitic Diseases and Malaria, Center for Global Health and Animal Resources Branch, National Center for Preparedness, Detection and Control of Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Atlanta Research and Education Foundation, Decatur, Georgia

*Address correspondence to William E. Collins, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Diseases Control and Prevention, 4770 Buford Highway, Chamblee, GA 30341. E-mail: wec1/at/cdc.gov

Received April 26, 2011; Accepted December 9, 2011.

Abstract

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.

Introduction

In 2009,¹ we described a comparison of parasite levels that can occur between four different species of South American Aotus monkeys (A. lemurinus griseimembra, A. nancymaae, A. vociferans, and A. azarae boliviensis) after infection with the Chesson strain of Plasmodium vivax, and compared these levels with those that occurred in humans as obtained from archived records. The number of humans and monkeys that supported the infection of mosquitoes, the different species of mosquitoes that were fed, and the species of mosquitoes that were used for transmission were presented. It was suggested that splenectomized Aotus monkeys, particularly, A. lemurinus griseimembra, could mimic the course of the Chesson strain of P. vivax malaria in humans regarding parasitemia and infectivity to mosquitoes.

Subsequently, studies were made to determine when during infection in monkeys and humans infectivity to mosquitoes was more likely to occur, and to compare the results of the monkey and the human studies. In addition, further observations were made on the comparative receptivity of the available laboratory host mosquitoes for the Chesson strain of P. vivax. We present the results of these studies.

Materials and Methods

Mosquitoes

Anopheles freeborni (F-1 strain originally from California), An. dirus (from Thailand), An. stephensi (from Delhi, India), An. maculatus (from Malaysia), An. culicifacies (from India), An. gambiae (from The Gambia), and An. quadrimaculatus (from the southeastern United States) were laboratory-reared and maintained at the insectaries at the Division of Parasitic Diseases, Centers for Disease Control and Prevention in Chamblee, Georgia.

During periods of highest asexual parasite count, mosquitoes of different species were allowed to feed on tranquilized monkeys. Mosquitoes were contained in pint-sized ice cream carton cages and allowed to feed directly through netting upon the shaved belly of the animal until engorged to repletion. After feeding, mosquitoes were held in an incubator at 25°C until examined one week later for oocysts on their midguts. If oocysts were present, mosquitoes were kept until sporozoites were present in the salivary glands, usually after 14 or more days of extrinsic incubation. The results presented are based on the oocyst examinations.

All feedings for the human studies were made with laboratory-reared An. quadrimaculatus mosquitoes either in the U.S. Public Health Laboratories in Columbia, South Carolina, or in Milledgeville, Georgia, during 1946–1963. After feeding on the patients, the mosquitoes were held under similar condition during the extrinsic incubation period.

Monkeys

Aotus lemurinus griseimembra and A. azarae boliviensis were wild caught and imported from Colombia and Bolivia, respectively, during periods when export was allowed from those countries. After the ban on exportation by these two countries, animals were vivarium born. Aotus nancymaae and A. vociferans monkeys were largely imported from Peru, and a limited number of these animals were vivarium 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 with the Chesson strain, although some of the animals had been previously infected with other species and strains of Plasmodium. All monkeys were splenectomized before or during exposure. All surgery was 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 1986 U.S. Public Health Policy.

Monkeys generally were pair-housed. In some cases, monkeys were single-housed to avoid injuries caused by fighting with cage mates. Space recommendations for laboratory animals were followed as set forth in the NIH Guide for the Care and Use of Laboratory Animals. All animals were fed a diet that has been proven to provide adequate nutrition and calories to captive Aotus monkeys used in malaria-related research. Feed was free of contaminants and freshly prepared. Daily observations of the animals' behavior, appetite, feces, and condition were recorded. Any medical conditions were treated by an attending veterinarian as needed.

Infections were initiated either through the inoculation of sporozoites or of cryopreserved or fresh infected blood intravenously via the femoral vein. Blood-stage parasitemia was monitored and quantified by the daily examination of Giemsa-stained thick and thin blood films by the method of Earle and Perez.²

Human patient management

Consent for any necessary patient treatment was granted by the families of the patients or by the courts when patients were admitted to the hospitals. The decision to infect a neurosyphilitic patient with malaria was made as part of standard patient care by the medical staff of the hospital. Patient care and evaluation of clinical endpoints (e.g., fever) were the responsibility of the medical staff. As reported,³ during infection, hospital personnel routinely monitored the temperature, pulse, and respiration every four hours and during paroxysms (fevers), every hour. During paroxysms, patients were treated symptomatically.

Patients were infected with the Chesson strain of P. vivax either by sporozoites or the inoculation of infectious blood from a donor patient.¹ The U.S. Public Health Service personnel provided the parasites for inoculation, monitored the daily parasite counts to determine the course of infection, provided the mosquitoes to be fed on the patients, and performed mosquito dissections and examinations. All patients undergoing malaria therapy were confined in screened wards of the hospital to prevent possible infection of local anopheline mosquitoes.

Treatment

Infections were terminated at the direction of the attending physician. Infections were terminated by treatment with various drugs such as chloroquine, pyrimethamine, quinine, chlorguanide, and primaquine, depending on the route of inoculation.

Results

Owl monkeys

Gametocytes were rarely seen in these studies and feedings were generally performed over many days blindly to increase the prospects of getting infection. For all species of Aotus monkeys, during the period between days −12 and +12 of the peak asexual parasite count, no male gametocytes were observed on 65.0% of the 726 days on which gametocyte counts were made. Of the 254 male gametocyte positive days, 138 (54.3%) occurred on day 0 or before and 116 (45.7%) occurred after day 0. The median male gametocyte count before day 0 varied from 30 to 300/μL; afterwards, it varied from 60 to 240/μL.

Aotus lemurinus griseimembra

Six different species of mosquitoes were fed on 27 A. l. griseimembra monkeys infected with Chesson strain of P. vivax (Table 1 ). The median maximum asexual parasite count was 27,100/μL. Of the 1,787 lots of mosquitoes fed, 766 (42.9%) were positive for oocysts. For all species of Anopheles, 4,157 (8.5%) of the 48,941 mosquitoes dissected and examined were positive.

Table 1

Infection of different species of Anopheles mosquitoes on monkeys and humans infected with the Chesson strains of Plasmodium vivax

Aotus azarae boliviensis

Four species of mosquitoes were fed on 15 A. a. boliviensis monkeys (Table 1). The median maximum asexual parasite count was 9,299/μL. Of the 469 lots of mosquitoes fed, 82 (17.5%) were positive for oocysts. For all species, 205 (1.9%) of the 10,527 mosquitoes dissected and examined were positive. The percentages infected were considerably lower than seen for feedings with A. l. griseimembra. In addition, fewer mosquitoes were fed on these animals because, as indicated in the previous report,¹ lower parasite counts occurred in this host and there were fewer indications for mosquito infection.

Aotus nancymaae

Five species of mosquitoes were fed on 18 A. nancymaae monkeys (Table 1). The median maximum asexual parasite count was 12,462/μL. Of the 374 lots of mosquitoes fed, 125 (33.4%) were positive. For all species of Anopheles, 496 (7.0%) of the 7,120 dissected and examined were positive. This value was almost as high as was obtained with A. l. griseimembra monkeys with regard to individual mosquito dissection. However, asexual parasite counts were generally lower and mosquitoes were fed on fewer occasions (similar to that of A. a. boliviensis).

Aotus vociferans

Four species of mosquitoes were fed on 8 A. vociferans monkeys (Table 1). The median asexual parasite count was 21,118/μL. Of the 41 lots of mosquitoes fed, 24 (58.5%) were infected. For all species of Anopheles, 168 (19.6%) of the 857 dissected and examined were positive. Although this finding is based on limited numbers, of the four species of monkeys infected with the Chesson strain of P. vivax, infections in A. l. griseimembra and A. vociferans appeared to be the best at infecting mosquitoes. The median asexual parasite counts were also higher than in the other monkeys.

An examination of mosquito infection patterns showed that they were associated with peaks in the asexual parasite count. Of the 473 infections in An. freeborni fed on Aotus monkeys, 384 (81.2%) were obtained during the 12-day period preceding or over the 12-day period after the peak asexual parasite counts. These 384 infections were plotted in relation to the peak in the asexual parasite count (Figure 1). There were 358 negative feedings during this 25-day period; 130 of these negative lots were fed during the 12 days before the peak during which there were also 212 infected lots (infection rate = 62.0%). For the12-day period after the peak, there were 214 negative lots and 144 infected lots (infection rate = 40.2%) for that 12-day period. The average number of oocysts per positive gut during the period before the peak day was 3.61. On the day of maximum asexual parasite count, the average number of oocysts per positive gut was 2.96. The average number of oocysts per positive gut for the 12-day period after the peak was 2.33. More infectivity, as judged by a higher infection rate, and a greater intensity of infection (oocysts per gut) occurred before the observed peak in the asexual parasite count.

Figure 1.

Lots of Anopheles freeborni mosquitoes infected and the average number of oocysts per positive gut before and after the peak in the asexual parasite count when fed on Aotus monkeys infected with the Chesson strain of Plasmodium vivax. Pos. Lots = positive (more ...)

An examination was made of the comparative intensity in infection of the four most frequently fed mosquitoes (An. freeborni, An. maculates, An. stephensi, and An. dirus) based on the average number of oocysts per positive gut. Based on 115 comparative feedings, An. freeborni had an average of 3.76 oocysts per positive gut versus 2.11 oocysts per positive gut for An. maculatus (ratio = 100:56.1). Based on 49 comparative feedings between An. freeborni and An. stephensi, the mean values were 4.71 versus 4.51 oocysts per positive gut (ratio = 100:96.0). Based on 68 comparative feedings between An. freeborni and An. dirus, the mean numbers of oocysts per positive gut were 4.01 and 5.51 (ratio = 100:137.4). It was apparent that in general, An. dirus were most heavily infected and the An. maculatus the least heavily infected when compared with the standard An. freeborni. A pooled t-test was used to compare the oocyst means for each of the species pairs. There was no statistical difference in any of the paired oocysts counts except for An. freeborni versus An. maculatus (P < 0.0001).

Only nine comparative feedings were conducted between An. freeborni and An. quadrimaculatus. The mean numbers were 3.66 versus 3.03 (ratio = 100:82.7). Thus, the high oocyst counts obtained in the human studies may have been even higher if An. freeborni had been available. Anopheles culicifacies and An. gambiae were not fed comparatively with An. freeborni.

Humans

Anopheles quadrimaculatus mosquitoes were fed on 47 patients infected with the Chesson strain (Table 1). The median asexual parasite count was 22,065/μL. Of the 244 lots examined, 211 (86.5%) were infected. Of the 3,764 mosquitoes dissected and examined, 1,865 were shown to have oocysts; average of 70.11 oocysts per positive gut. Of the 211 positive lots, 166 occurred during the period of 8 days before (n = 37) and 12 days after (n = 110) the peak in the asexual parasite count (Figure 2). Forty-seven (22.3%) positive lots occurred during the 8 days before the peak parasite count, and 140 lots were positive (66.3%) after the day of peak parasite count. Thus, in contrast to the findings with the monkey feedings, the highest number of lots infected and the greater number of oocysts per positive gut were obtained after the peak asexual parasite count. The overall intensity of mosquito infection was markedly higher with humans than seen in any of the monkey species fed on by any of the Anopheles species.

Figure 2.

Lots of Anopheles quadrimaculatus mosquitoes infected and the average number of oocysts per positive gut before and after the peak in the asexual parasite count when fed on humans infected with the Chesson strain of Plasmodium vivax. Infect. = infection. (more ...)

An examination showed that male gametocyte counts recorded for humans were much higher than those recorded for Aotus monkeys. Between day −8 and day +12, there were 387 recordings of male gametocytes and no males were observed on 63 (16.3%) of the days; 32 (25%) of 128 in the period from day −8 through day 0, and 31 of 259 in the period from day +1 through day +12. The mean male gametocyte counts ranged from 13.1 to 111.3/100 leukocytes.

Discussion

The Chesson strain is one of the classic parasites that has been studied for many years in humans and during the past several decades in monkeys and chimpanzees.¹^,⁴^–¹⁵ During recent times, many isolates of this parasite have been stored frozen and subsequently passed through humans, monkeys, and chimpanzees. Whether it is now the same parasite as was studied in humans during the 1940s through 1960s is questionable. A molecular examination of preserved human material from that early era would indicate whether the present day parasites are similar to or the same as the one used in the early human studies. At the present time, we assume that we are working with Chesson stain of P. vivax based only on its genealogy.

It was apparent from an examination of the data from feeding on infected Aotus monkeys that mosquito infection most often occurred before peaks in the asexual blood parasite count. Gametocytes were rarely observed on the blood films and could not be used as predictors of mosquito infection or the intensity of infection. In the monkeys, higher levels of infection occurred in the period preceding the peak in the asexual parasite count than occurred after the peak (Figure 1). This finding contrasts with observations from archival data from human studies (Figure 2), in which a higher percentage of mosquito infection occurred after the peak in the asexual blood parasite count.

In the human studies, the intensity of mosquito infection, (based on oocysts count) was at least 10-fold higher than that obtained by feeding on monkeys. In humans, gametocytes were often observed, although the number of gametocytes present was not always a predictor of the number of oocysts that would be found in the mosquitoes. This high level of infection had also been observed in previous studies with P. vivax in chimpanzees.⁴ It is apparent that splenectomized Aotus monkeys do not support the production of infective gametocytes at the same high density levels as do humans and splenectomized chimpanzees. In addition, it appears that in Aotus monkeys, mosquito infection decreases once the peak in the asexual parasite count is reached, whereas in humans, mosquito infection appears to continue or increase beyond the peak in the asexual parasite count.

The Aotus monkeys, particularly splenectomized A. lemurinus griseimembra, mimic the human host for studies with the Chesson strain of P. vivax with regard to asexual parasitemia, susceptibility to infection by sporozoite inoculation, and recrudescences. However, these animals are apparently unable to produce infective gametocytes at levels approaching that of humans, which results in a much reduced level in the intensity of mosquito infection (oocysts per positive gut). In particular, microgametocytes are rarely seen during P. vivax infections in Aotus monkeys. Therefore, experimental feeding must be scheduled in relation to the anticipated peak in the asexual parasite count. The frequency of infection is reduced or aborted soon after the peak in the asexual count. This finding contrasts with archival data from human studies in which mosquito infection continues after the peak in the asexual parasite count.

This reduced intensity of mosquito infection and the limitation in periods of infectivity define the usefulness of the Aotus monkeys for studies with the Chesson strain where mosquito infection is a vital component. Currently, only A. nancymaae and A. vociferans are available in sufficient numbers for extensive studies. The higher median asexual parasite counts that were obtained in the A. vociferans and the higher level mosquito infection over that of A. nancymaae indicates that A. vociferans would be the preferred host for studies with this parasite. Of the laboratory mosquitoes tested for susceptibility to infection with Chesson strain, An. dirus was the most heavily infected. Thus, at the present time, studies with Chesson strain could best be conducted using A. vociferans monkeys and An. dirus as the vector mosquito.

ACKNOWLEDGMENTS

We thank the staff of the Animal Resources Branch, the National Center for Infectious Diseases, for the care of the animals.

Notes

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.

Footnotes

Authors' addresses: William E. Collins, JoAnn S. Sullivan, Douglas Nace, and John W. Barnwell, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Diseases Control and Prevention, Atlanta, GA, E-mails: wec1/at/cdc.gov, JSSO/at/cdc.gov, DDN4/at/cdc.gov, and WZB3/at/cdc.gov. Geoffrey M. Jeffery, Decatur, GA, E-mail: GJefferey2/at/comcast.net. Tyrone Williams, Atlanta Research and Education Foundation, Decatur, GA, E-mail: TDW1/at/cdc.gov. G. Gale Galland, Division of Global Migration and Quarantine, National Center for Preparedness, Detection and Control of Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, E-mail: GGGO/at/cdc.gov. Allison Williams, Animal Resources Branch, National Center for Preparedness, Detection and Control of Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, E-mail: ZHN7/at/cdc.gov.

References

1. Collins WE, Sullivan JS, Jeffery GM, Williams A, Galland GG, Nace D, Williams T, Barnwell JW. The Chesson strain of Plasmodium vivax in humans and different species of Aotus monkeys. Am J Trop Med Hyg. 2009;80:152–159. [PubMed]

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

3. Collins WE, Jeffery GM. A retrospective examination of sporozoite- and trophozoite-induced infections with Plasmodium falciparum.

Am J Trop Med Hyg (Suppl): 1999:4–48. [PubMed]

4. Ward RA, Rutledge LC, Hickman RL. Cyclical transmission of Chesson strain malaria to subhuman primates. Nature. 1969;224:1126–1127. [PubMed]

5. Schmidt LH. Plasmodium falciparum and Plasmodium vivax infections in the owl monkey (Aotus trivirgatus) I. The course of untreated infections. Am J Trop Med Hyg. 1978;27:671–702. [PubMed]

6. Schmidt LH. Plasmodium falciparum and Plasmodium vivax infections in the owl monkey (Aotus trivirgatus) II. Responses to chloroquine, quinine, and pryrimethamine. Am J Trop Med Hyg. 1978;27:703–717. [PubMed]

7. Schmidt LH. Plasmodium falciparum and Plasmodium vivax infections in the owl monkey (Aotus trivirgatus) III. Methods employed in the search for new blood schizonticidal drugs. Am J Trop Med Hyg. 1978;27:718–737. [PubMed]

8. Collins WE, McClure HM, Swenson RB, Mehaffey PC, Skinner JC. Infection of mosquitoes with Plasmodium vivax from chimpanzees using membrane feeding. Am J Trop Med Hyg. 1986;35:56–60. [PubMed]

9. Collins WE, Warren McW, Contacos PG, Skinner JC, Richardson BB, Kearse TS. The Chesson strain of Plasmodium vivax in Aotus monkeys and anopheline mosquitoes. J Parasitol. 1980;66:488–497. [PubMed]

10. Collins WE, Skinner JC, Broderson JR, Mehaffey P, Sutton BB. Infection of Aotus azarae boliviensis monkeys with different strains of Plasmodium vivax. J Parasitol. 1985;71:239–243. [PubMed]

11. Collins WE, Warren McW, Huong AY, Skinner JC, Sutton BB, Stanfill PS. Studies on the comparative infectivity of fifteen strains of Plasmodium vivax to laboratory-reared anophelines, with special reference to Anopheles cuclicifacies. J Parasitol. 1986;72:521–524. [PubMed]

12. Krotoski WA, Garnham PC, Cogswell FB, Collins WE, Bray RS, Gwadz RW, Killick-Kendrick R, Wolf R, Sinden R, Hollingdale M, Lowrie RC, Jr, Koontz LC, Stanfill PS. Observations on early and late post-sporozoite tissue stages in primate malaria. IV. Pre-erythrocytic schizonts and/or hypnozoites of Chesson and North Korean strains of Plasmodium vivax in the chimpanzee. Am J Trop Med Hyg. 1986;35:263–274. [PubMed]

13. Collins WE, Skinner JC, Pappaioanou M, Broderson JR, McClure HM, Strobert E, Sutton BB, Stanfill PS, Filipski V, Campbell CC. Chesson strain Plasmodium vivax in Saimiri sciureus boliviensis monkeys. J Parasitol. 1987;73:929–934. [PubMed]

14. Collins WE, Skinner JC, Pappaioanou M, Ma NF-S, Broderson JR, Sutton BB, Stanfill PS. Infection of Aotus vociferans (karyotype V) monkeys with different strains of Plasmodium vivax. J Parasitol. 1987;73:536–540. [PubMed]

15. Collins WE, Skinner JC, Pappaioanou M, Broderson JR, Ma NS-F, Filipski V, Stanfill PS, Rogers L. Infection of Peruvian Aotus nancymai monkeys with different strains of Plasmodium falciparum, P. vivax, and P. malariae. J Parasitol. 1988;74:392–398. [PubMed]

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