EXPERIMENTAL MODELS FOR LABORATORY MALARIA RESEARCH
The study of malaria parasites may be broadly divided into two major disciplines; those that involve observation of human malaria parasites in their natural hosts in endemic areas, and those that rely on experimental manipulation of living parasites in the laboratory. The latter type of study involves either the manipulation of human malaria parasites, most commonly Plasmodium falciparum, in in vitro cultures of human blood or (uncommonly) in a permissible animal host, or the use of non-human malaria parasites in laboratory animals. Depending on the scope and purpose of the experiments, there will be advantages and disadvantages associated with each particular approach. When the aim of the laboratory scientist is to model most closely the natural situation of the malaria parasite, it is apparent that in vitro culture will often be far from satisfactory, as the physiological conditions in the blood of a living animal cannot be replicated accurately in a culture flask. Indeed, as the intricate interplay and vital association between Plasmodium and its host is fundamental to much of the biology of the malaria parasite, so removing it from the natural host will drastically alter its biology. That is not to say, of course, that in vitro studies are without merit, as they have been hugely important in informing a great deal regarding the biology of malaria parasites. But for certain types of studies, such as those that rely on maintenance of the whole life cycle of the parasites, and particularly those studies concerned with host–parasite interactions, there can be no satisfactory alternative to the use of non-human parasites in experimental animals.
Animal malaria parasite studies are not without their disadvantages and limitations. Some of the laboratory host–parasite combinations, such as the rodent malaria parasites and their Mus musculus hosts do not occur in nature, and so, as in the culture flask, the parasites find themselves in an environment somewhat removed from that in which they evolved. Furthermore, there are serious and profound ethical questions to be taken into consideration with the use of animals in any scientific research. Finally, non-human malaria parasites, although often sharing considerable biological and genetic similarity to their human counterparts, do differ from the human parasites in certain ways, and care must be taken when extrapolating results from one species to another.
The best animal models, from a purely scientific standpoint, are those in which the genetic and phenotypic distance between the parasite itself and whichever of the human parasites one wishes to model is small, along with a similarly close relationship between the experimental host animal and man. Ideally, the experimental host animal should also be the natural host. For studies of malaria parasites, the non-human primate parasites most closely fit this description. In this review, we will discuss solely malaria in non-human primates. We will highlight particular host–parasite pairings, and discuss their suitability for studies on particular aspects of human malaria.
THE PRIMATE MALARIAS
The discovery that man was not the only primate to be infected with malaria parasites occurred early on in the history of modern malariology. In 1899, at around the same time that British and Italian parasitologists were discovering the role of the mosquito in vectoring malaria (Ross, Reference Ross1897; Reference Ross1898; Grassi et al. Reference Grassi, Bignami and Bastianelli1899), Alphonse Laveran described Plasmodium kochi, a parasite of African monkeys (Laveran, Reference Laveran1899). Now known as Hepatocystis kochi, this parasite, which has been found to predominantly infect African green monkeys, Cercopithecus aethiops, is a member of the genus Hepatocystis, a sister group to the Plasmodium spp of mammalian malaria parasites that do not undergo erythrocytic schizogony, maturing to gametocytes following initial invasion of red blood cells (Garnham, Reference Garnham1948).
The first true malaria parasite (i.e. belonging to the genus Plasmodium and undergoing erythrocytic schizogony) of non-human primates to be observed was Plasmodium pitheci, again by Laveran, just after the turn of the 20th Century (Laveran, Reference Laveran1905). This species was found in the blood of an orangutan, and is remarkable for being the first great ape parasite to be described (Halberstadter and Von Prowazek, Reference Halberstadter and Von Prowazek1907). At this time the first reports of malaria parasites infecting Southeast Asian macaques also appeared, with descriptions of Plasmodium inui and Plasmodium cynomolgi infecting Macaca fascicularis. Over the next 60 years, a further nine malaria parasite species infecting non-human primates in Southeast Asia were described, along with two parasites of New World monkeys from South America, one parasite of African green monkeys, and two parasites of lemurs from Madagascar (Table 1).
Table 1. The non-human primate malaria parasites, their geographical origin and dates of first observation
The malaria parasites of the great apes have a more convoluted history than those infecting monkeys. Following the discovery of P. pitheci in 1905, it was not until 1920 that malaria parasites of African great apes were observed, firstly by Reichenow, and then by Blacklock and Adler (Reichenow, Reference Reichenow1920; Blacklock and Adler, Reference Blacklock and Adler1922). Both these reports detailed the observation that there existed in chimpanzees three distinct species of malaria parasite that were broadly comparable to the human malaria parasites P. falciparum, Plasmodium malariae and P. vivax. These were later termed Plasmodium reichenowi, Plasmodium rodhaini and Plasmodium schwetzi, respectively (Table 1).
For the next 90 years, our understanding of the great ape malaria parasites changed very little, with the exception of the addition of a second parasite of the orangutan, Plasmodium silvaticum in 1972 (Garnham et al. Reference Garnham, Rajapaksa, Peters and Killick-Kendrick1972). Then, in 2009 the field once again opened up with a number of papers reporting the discovery of a plethora of new malaria parasite species (Ollomo et al. Reference Ollomo, Durand, Prugnolle, Douzery, Arnathau, Nkoghe, Leroy and Renaud2009; Rich et al. Reference Rich, Leendertz, Xu, LeBreton, Djoko, Aminake, Takang, Diffo, Pike, Rosenthal, Formenty, Boesch, Ayala and Wolfe2009; Duval et al. Reference Duval, Fourment, Nerrienet, Rousset, Sadeuh, Goodman, Andriaholinirina, Randrianarivelojosia, Paul, Robert, Ayala and Ariey2010; Krief et al. Reference Krief, Escalante, Pacheco, Mugisha, Andre, Halbwax, Fischer, Krief, Kasenene, Crandfield, Cornejo, Chavatte, Lin, Letourneur, Gruner, McCutchan, Renia and Snounou2010; Prugnolle et al. Reference Prugnolle, Durand, Neel, Ollomo, Ayala, Arnathau, Etienne, Mpoudi-Ngole, Nkoghe, Leroy, Delaporte, Peeters and Renaud2010) of apes in Africa. Following a brief period of some confusion, a landmark paper that proved that P. falciparum had become a human parasite as a result of a host switch from gorillas consolidated our current understanding of the diversity and host preferences of the malaria parasites of great apes (Liu et al. Reference Liu, Li, Learn, Rudicell, Robertson, Keele, Ndjango, Sanz, Morgan, Locatelli, Gonder, Kranzusch, Walsh, Delaporte, Mpoudi-Ngole, Georgiev, Muller, Shaw, Peeters, Sharp, Rayner and Hahn2010).
These newly described species are closely related to P. falciparum of humans, and belong to the subgenus Laverania. Of these, three species, Plasmodium reichenowi, Plasmodium gaboni (also named Plasmodium blillbrayi) and Plasmodium billcollinsi are found predominantly in chimpanzees, and three, Plasmodium praefalciparum, Plasmodium adleri and Plasmodium blacklocki in gorillas (Table 1). There has also been a ‘rediscovery’ in great apes of parasites closely related to P. vivax and P. malariae (Krief et al. Reference Krief, Escalante, Pacheco, Mugisha, Andre, Halbwax, Fischer, Krief, Kasenene, Crandfield, Cornejo, Chavatte, Lin, Letourneur, Gruner, McCutchan, Renia and Snounou2010; Liu et al. Reference Liu, Li, Learn, Rudicell, Robertson, Keele, Ndjango, Sanz, Morgan, Locatelli, Gonder, Kranzusch, Walsh, Delaporte, Mpoudi-Ngole, Georgiev, Muller, Shaw, Peeters, Sharp, Rayner and Hahn2010, Reference Liu, Li, Shaw, Learn, Plenderleith, Malenke, Sundararaman, Ramirez, Crystal, Smith, Bibollet-Ruche, Ayouba, Locatelli, Esteban, Mouacha, Guichet, Butel, Ahuka-Mundeke, Inogwabini, Ndjango, Speede, Sanz, Morgan, Gonder, Kranzusch, Walsh, Georgiev, Muller, Piel and Stewart2014). The former species has now been formally named Plasmodium carteri (Loy et al. Reference Loy, Liu, Li, Learn, Plenderleith, Sundararaman, Sharp and Hahn2016) in honour of Richard Carter, who has long argued that P. vivax has had a longer association with humans in Africa than it has in Southeast Asia (Carter, Reference Carter2003). A recent genome sequencing project (Sundararaman et al. Reference Sundararaman, Plenderleith, Liu, Loy, Learn, Li, Shaw, Ayouba, Peeters, Speede, Shaw, Bushman, Brisson, Rayner, Sharp and Hahn2016) focused on P. reichenowi and P. gaboni has highlighted their distinct nature as separate species, as well as evolutionary developments that allowed P. falciparum to infect humans.
To date there are 27 Plasmodium malaria parasites of non-human primates described in the literature. It is likely that this number will increase with further surveys of wild primate populations. Below we consider these species in the context of their utility as experimental models for human malaria, and highlight the significant advances in malariology achieved through their use.
NON-HUMAN MALARIA PARASITES IN THE LABORATORY
The following sections focus on the use of non-human primate malaria parasites (NHPMPs) in laboratory research.
Subgenus Laverania
The subgenus Laverania originally consisted of just two species; P. falciparum and P. reichenowi, characterized by their crescent shaped gametocytes. Since 2009, numerous additional members of this subgenus have been described. To date, these have been described and classified based solely on sequencing of DNA, and so remain poorly characterized at the morphological and phenotypical level.
Plasmodium reichenowi
Plasmodium reichenowi was the second malaria parasite species of chimpanzees to be described after P. schwetzi. Morphologically similar to P. falciparum, it shares the same length of erythrocytic cycle as well as several other biological features such as crescent-shaped gametocytes (Bray, Reference Bray1956). Its genome has been fully sequenced and reveals strong synteny with P. falciparum, including conserved organisation of the hypervariable var genes involved in immune evasion (Otto et al. Reference Otto, Rayner, Bohme, Pain, Spottiswoode, Sanders, Quail, Ollomo, Renaud, Thomas, Prugnolle, Conway, Newbold and Berriman2014). It has also provided valuable insights into the recent evolutionary rise of P. falciparum (Sundararaman et al. Reference Sundararaman, Plenderleith, Liu, Loy, Learn, Li, Shaw, Ayouba, Peeters, Speede, Shaw, Bushman, Brisson, Rayner, Sharp and Hahn2016). The high degree of conservation between the two species has also made P. reichenowi a comparative model to study the evolution of P. falciparum and its adaptation to the human host, particularly at the antigenic level (Wanaguru et al. Reference Wanaguru, Liu, Hahn, Rayner and Wright2013; Zilversmit et al. Reference Zilversmit, Chase, Chen, Awadalla, Day and McVean2013; Otto et al. Reference Otto, Rayner, Bohme, Pain, Spottiswoode, Sanders, Quail, Ollomo, Renaud, Thomas, Prugnolle, Conway, Newbold and Berriman2014).
While P. reichenowi can readily infect a wide range of anopheline vectors (Collins et al. Reference Collins, Skinner, Pappaioanou, Broderson and Mehaffey1986a ), it is restricted to chimpanzees as its vertebrate host (Martin et al. Reference Martin, Rayner, Gagneux, Barnwell and Varki2005). Thus, in vitro culturing, using protocols adapted from P. falciparum, plays an important role in the study of this parasite (Kocken et al. Reference Kocken, Narum, Massougbodji, Ayivi, Dubbeld, van der Wel, Conway, Sanni and Thomas2000). Still, the use of a relatively fastidious and ethically sensitive animal model limits the scope of studies possible with P. reichenowi. Furthermore, the fact that chimp-adapted P. falciparum can establish an infection not only in splenectomized chimpanzes, but also in chimpanzees with intact immune systems (Taylor et al. Reference Taylor, Wells, Vernes, Rosenberg, Vogel and Diggs1985) further highlights the rather peripheral role played by P. reichenowi as a human malaria model.
Other species of the Laverania subgenus
Six Laverania gorilla and chimpanzee parasites, P. gaboni (referred to as P. billbrayi by Krief et al. (Reference Krief, Escalante, Pacheco, Mugisha, Andre, Halbwax, Fischer, Krief, Kasenene, Crandfield, Cornejo, Chavatte, Lin, Letourneur, Gruner, McCutchan, Renia and Snounou2010)), P. billcollinsi, P. adleri, P. blacklocki, P. praefalciparum have recently been described in addition to P. reichenowi from the earlier literature (Table 1). These species, whilst of potential use in comparative genomics (Pacheco et al. Reference Pacheco, Cranfield, Cameron and Escalante2013; Boundenga et al. Reference Boundenga, Ollomo, Rougeron, Mouele, Mve-Ondo, Delicat-Loembet, Moukodoum, Okouga, Arnathau, Elguero, Durand, Liegeois, Boue, Motsch, Le Flohic, Ndoungouet, Paupy, Ba, Renaud and Prugnolle2015; Larremore et al. Reference Larremore, Sundararaman, Liu, Proto, Clauset, Loy, Speede, Plenderleith, Sharp, Hahn, Rayner and Buckee2015; Roy, Reference Roy2015; Sundararaman et al. Reference Sundararaman, Plenderleith, Liu, Loy, Learn, Li, Shaw, Ayouba, Peeters, Speede, Shaw, Bushman, Brisson, Rayner, Sharp and Hahn2016), are not (to date) utilized in laboratory studies due to important practical limitations. Firstly, collection of blood samples from Great Apes is difficult as they are protected species. Secondly, maintaining parasites in laboratory conditions would either involve the keeping of Great Apes in captivity, a situation which is no longer deemed ethically acceptable, or the adaptation of the parasites to in vitro culture, preferentially in human blood.
Subgenus Plasmodium
All other malaria parasites of prosimians, except those infecting lemurs (which fall into the subgenus Vinckeia), are classified into the subgenus Plasmodium.
Plasmodium knowlesi
Plasmodium knowlesi is probably the most thoroughly characterized and widely used non-human primate malaria parasite species. It is certainly the species most utilized in experimental laboratory studies of malaria, many of which have contributed enormously to malariology. The contribution of this species to our understanding of malaria parasite biology is immense, and the literature pertaining to it enormous. We will attempt to outline some of the major advancements achieved using P. knowlesi, but advise the reader that this is a selective and in no way exhaustive treatment of the subject.
First described in 1932, laboratory investigations of malaria parasite biology using this species were underway by the close of the decade. Initial in vivo work involved studies on immunity and drug responses (Coggeshall, Reference Coggeshall1940; Coggeshall and Kumm, Reference Coggeshall and Kumm1938; Eaton and Coggeshall, Reference Eaton and Coggeshall1939), and this was shortly followed by investigations of parasite cell biology (McKee et al. Reference McKee, Ormsbee, Anfinsen, Geiman and Ball1946; Shen et al. Reference Shen, Fleming and Castle1946; Morrison and Jeskey, Reference Morrison and Jeskey1947). A workable in vitro culture system was established for P. knowlesi during the mid 1960s (Polet, Reference Polet1966), and this led to its use in studies on malaria parasite cellular biology (Polet and Barr, Reference Polet and Barr1968; Polet and Conrad, Reference Polet and Conrad1969; Skelton et al. Reference Skelton, Lunan, Folkers, Schnell, Siddiqui and Geiman1969). A major breakthrough was achieved with the establishment of continuous in vitro cultivation of malaria parasites by Trager and Jensen in Reference Trager and Jensen1976, was soon adapted to P. knowlesi (Butcher, Reference Butcher1979), and huge advances in the understanding of the ultrastructure of parasite invasion of erythrocytes were subsequently carried out using P. knowlesi in vitro (Aikawa et al. Reference Aikawa, Miller, Johnson and Rabbage1978; Haynes et al. Reference Haynes, Dalton, Klotz, McGinniss, Hadley, Hudson and Miller1988; Johnson et al. Reference Johnson, Epstein, Shiroishi and Miller1980; Miller et al. Reference Miller, Hudson and Haynes1988; Barnwell et al. Reference Barnwell, Nichols and Rubinstein1989).
Antigenic variation, a major immune evasion mechanism employed by malaria parasites, was first described in P. knowlesi. Antigenic variation involves a parasite strain expressing different alleles of genes encoding antigenic proteins over the course of an infection in order to escape antibody mediated immune clearance. This phenomenon was first described in P. knowlesi by Brown and Brown (Reference Brown and Brown1965), using the schizont agglutination test developed in this same species by Eaton (Reference Eaton1938). They showed that during a chronic P. knowlesi infection in a Rhesus macaque, successive waves of parasitaemia were composed of serologically distinct parasites. They went on to further characterize this phenomenon (Brown et al. Reference Brown, Brown and Hills1968), work which eventually led to the identification of the variant antigen proteins themselves (Howard et al. Reference Howard, Barnwell and Kao1983).
Plasmodium knowlesi was has also been fundamental to malaria vaccine studies, in which protection has been elicited against asexual blood forms (Collins et al. Reference Collins, Contacos, Harrison, Stanfill and Skinner1977; Mitchell, Reference Mitchell1977; Mitchell et al. Reference Mitchell, Butcher, Langhorne and Cohen1977), gametocytes (Gwadz and Green, Reference Gwadz and Green1978; Gwadz and Koontz, Reference Gwadz and Koontz1984) and sporozoites (Gwadz et al. Reference Gwadz, Cochrane, Nussenzweig and Nussenzweig1979; Moser et al. Reference Moser, Brohn, Danforth and Nussenzweig1978; Nardin et al. Reference Nardin, Gwadz and Nussenzweig1979).
Another landmark advancement in malariology involving the use of P. knowlesi was the discovery, in the laboratory of Professor Louis Miller at the NIH in the USA, of the Duffy erythrocyte receptor's role in malaria parasite invasion. Miller and colleagues first showed that Duffy negative erythrocytes were refractory to invasion by merozoites of P. knowlesi (Miller et al. Reference Miller, Mason, Dvorak, McGinniss and Rothman1975), an observation which led to experiments that proved the absolute requirement of Duffy for erythrocyte invasion by the related human malaria parasite P. vivax (Miller et al. Reference Miller, Mason, Clyde and McGinniss1976), and explained the absence of this parasite from vast swathes of western and central Africa where P. vivax is almost totally absent (Culleton et al. Reference Culleton, Mita, Ndounga, Unger, Cravo, Paganotti, Takahashi, Kaneko, Eto, Tinto, Karema, D'Alessandro, do Rosario, Kobayakawa, Ntoumi, Carter and Tanabe2008). This work led directly to the identification of malaria parasite invasion ligands (Haynes et al. Reference Haynes, Dalton, Klotz, McGinniss, Hadley, Hudson and Miller1988; Miller et al. Reference Miller, Hudson and Haynes1988; Adams et al. Reference Adams, Hudson, Torii, Ward, Wellems, Aikawa and Miller1990; Fang et al. Reference Fang, Kaslow, Adams and Miller1991), a topic addressed in more detail in a previous review (Culleton and Kaneko, Reference Culleton and Kaneko2010).
Also noteworthy is the landmark paper by Dvorak and colleagues in 1975, who captured the first moving images of the invasion of erythrocytes by merozoites using P. knowlesi (Dvorak et al. Reference Dvorak, Miller, Whitehouse and Shiroishi1975).
Plasmodium knowlesi is not, however, a perfect non-human primate malaria model for experimental studies. It is, for example, difficult to transmit to mosquitoes in the laboratory; sporozoites in the salivary glands of two of the most commonly used laboratory vector mosquitoes, Anopheles stephensi and Anopheles gambiae, were observed too rarely to support effective transmission (Murphy et al. Reference Murphy, Weiss, Fryauff, Dowler, Savransky, Stoyanov, Muratova, Lambert, Orr-Gonzalez, Zeleski, Hinderer, Fay, Joshi, Gwadz, Richie, Villasante, Richardson, Duffy and Chen2014). Sporozoites were also completely unable to colonise the salivary glands of Anopheles freeborni mosquitoes (Rosenberg, Reference Rosenberg1985). Indeed, only the salivary glands of mosquitoes of the Leucosphyrus group of Anopheles appear to be suitable for colonization by P. knowlesi sporozoites. Unfortunately, one of the most effective vectors for transmitting P. knowlesi, Anopheles dirus, can be particularly fastidious to maintain in the laboratory, requiring constant forced mating. However, alternative, less fastidious vectors, such as Anopheles cracens have been proposed (Murphy et al. Reference Murphy, Weiss, Fryauff, Dowler, Savransky, Stoyanov, Muratova, Lambert, Orr-Gonzalez, Zeleski, Hinderer, Fay, Joshi, Gwadz, Richie, Villasante, Richardson, Duffy and Chen2014), and may offer a route to genetic crossing and forward genetics studies.
There are considerable biological differences between P. knowlesi and the other human malaria parasite species. Human malaria is usually classified according of the amount of time required by the parasite to undergo a complete shizogonic replication cycle in the blood. The four major species fall into two categories: tertian (48–50 h cycle, including P. falciparum, P. vivax and P. ovale) and quartan (72 h cycle, P. malariae). Plasmodium knowlesi, however, has a distinct 24 h cycle, which is one factor that leads to its high virulence in man (Mideo et al. Reference Mideo, Reece, Smith and Metcalf2013). Plasmodium knowlesi is an excellent resource for experimental studies on malaria due to the wealth of literature dedicated to it as well as the availability of a high quality genome (Pain et al. Reference Pain, Bohme, Berry, Mungall, Finn, Jackson, Mourier, Mistry, Pasini, Aslett, Balasubrammaniam, Borgwardt, Brooks, Carret, Carver, Cherevach, Chillingworth, Clark, Galinski, Hall, Harper, Harris, Hauser, Ivens, Janssen, Keane, Larke, Lapp, Marti and Moule2008). However, other NHPMPs may be more appropriate when studying particular phenotypes, especially those of relevance to human malarial disease.
In 2004, a large outbreak of P. knowlesi in humans in Borneo elevated this species from a useful model for malaria to a pathogen of direct importance to human health (Singh et al. Reference Singh, Kim Sung, Matusop, Radhakrishnan, Shamsul, Cox-Singh, Thomas and Conway2004).
Quartan malaria parasites
Plasmodium brasilianum
Plasmodium brasilianum is the most closely related sister-species to the human parasite Plasmodium malariae. Like P. malariae it is a quartan parasite and undergoes its full erythrocytic cycle in 72 h (Von Berenberg-Gossler, Reference Von Berenberg-Gossler1909). It is found in tropical areas of South America where its natural hosts include a variety of New World Monkeys (Deane and de Almeida, Reference Deane and de Almeida1967; Marinkelle and Grose, Reference Marinkelle and Grose1968; Baerg, Reference Baerg1971; Collins et al. Reference Collins, Skinner, Huong, Broderson, Sutton and Mehaffey1985; Wedderburn et al. Reference Wedderburn, Mitchell and Davies1985). It can be transmitted by various laboratory mosquito vectors, including both A. stephensi and A. gambiae (Collins et al. Reference Collins, Skinner, Huong, Broderson, Sutton and Mehaffey1985).
Recent evidence has indicated that P. brasilianum can infect humans in the wild (Lalremruata et al. Reference Lalremruata, Magris, Vivas-Martinez, Koehler, Esen, Kempaiah, Jeyaraj, Perkins, Mordmuller and Metzger2015) and this, coupled with the cross-reactivity of P. malariae-specific monoclonal antibodies against P. brasilianum (Cochrane et al. Reference Cochrane, Barnwell, Collins and Nussenzweig1985), suggests a close relationship between the two species. Indeed, there is evidence to suggest that P. brasilianum is a strain of P. malariae that has adapted to infect new world monkeys relatively recently (Ayala et al. Reference Ayala, Escalante and Rich1999; Collins and Jeffery, Reference Collins and Jeffery2007). This fact renders it somewhat obsolete as a model for P. malariae, as the human parasite itself also readily infects new world monkeys (Collins and Jeffery, Reference Collins and Jeffery2007). However, the P. brasilianum genome could offer valuable insights on its origins and adaptations, and would be useful for comparative genomics (Rayner, Reference Rayner2015).
Plasmodium inui
Plasmodium inui is the only major NHPMP other than P. brasilianum with a quartan life cycle (Coatney et al. Reference Coatney, Chin, Contacos and King1966). Initially thought to be closely related to P. malariae and used as a model for it, phylogenetic evidence has revealed its inclusion in the clade of primate malaria parasites that includes P. vivax (Mitsui et al. Reference Mitsui, Arisue, Sakihama, Inagaki, Horii, Hasegawa, Tanabe and Hashimoto2010). Indeed, immunological evidence had previously suggested its separate nature from the P. malariae subgroup (Kamboj and Cochrane, Reference Kamboj and Cochrane1988). Originally isolated from a Javan Macaca fascularis, P. inui can infect a wide range of monkeys, including the Platyrrhini of the New World (Collins et al. Reference Collins, Warren, Sullivan and Barnwell2009b ) and can be transmitted by several species of mosquito commonly kept in the laboratory (Collins et al. Reference Collins, Sullivan, Galland, Nace, Williams, Williams and Barnwell2007). It is also infectious to humans, which marks it as a potential zoonotic disease of medical significance (Coatney et al. Reference Coatney, Chin, Contacos and King1966). Unlike other quartan malaria parasite species, P. inui has been adapted to in vitro culture (Nguyen-Dinh et al. Reference Nguyen-Dinh, Campbell and Collins1980). Despite its closer phylogenetic relatedness to P. vivax than to P. malariae, P. inui has recently been used to model human quartan malaria nephrotic syndrome in monkeys (Nimri and Lanners, Reference Nimri and Lanners2014).
Plasmodium vivax clade malaria parasites
Plasmodium cynomolgi
Plasmodium cynomolgi is, after P. knowlesi, the most well characterized and often used NHPMP in experimental malariology. Like P. knowlesi, this species also has the capacity to infect man in nature (Ta et al. Reference Ta, Hisam, Lanza, Jiram, Ismail and Rubio2014), although there is currently little evidence to suggest that this occurs frequently. It is closely related to the human malaria parasite P. vivax and shares with it some crucial features that make it well suited as a model for vivax malaria. Like P. vivax, P. cynomolgi has a 48 h erythrocytic cell cycle (Wolfson and Winter, Reference Wolfson and Winter1946) and, crucially, it also produces hypnozoites, the dormant liver stages that can cause relapse (Krotoski et al. Reference Krotoski, Garnham, Bray, Krotoski, Killick-Kendrick, Draper, Targett and Guy1982a ). It was, in fact, in P. cynomolgi that hypnozoites were first described, a discovery that took place some 34 years after the same species was used for the first description of exoerythrocytic stages of a mammalian malaria parasite (Shortt and Garnham, Reference Shortt and Garnham1948). It offers an ideal model to study both the biological properties of the hypnozoite stages, and the testing of potential treatments to remove them (Deye et al. Reference Deye, Gettayacamin, Hansukjariya, Im-erbsin, Sattabongkot, Rothstein, Macareo, Fracisco, Bennett, Magill and Ohrt2012; Dembélé et al. Reference Dembélé, Franetich, Lorthiois, Gego, Zeeman, Kocken, Le Grand, Dereuddre-Bosquet, van Gemert, Sauerwein, Vaillant, Hannoun, Fuchter, Diagana, Malmquist, Scherf, Snounou and Mazier2014; Joyner et al. Reference Joyner, Barnwell and Galinski2015).
Originally isolated from a Javan macaque, it can also infect Platyrrhini monkeys (Collins et al. Reference Collins, Skinner, Richardson and Stanfill1975, Reference Collins, Warren and Galland1999), which are more tractable laboratory species. Furthermore, P. cynomolgi blood stages (Nguyen-Dinh et al. Reference Nguyen-Dinh, Gardner, Campbell, Skinner and Collins1981), as well as liver stages (Millet et al. Reference Millet, Jiang, Lun, Bray, Canning and Landau1987, Reference Millet, Fisk, Collins, Broderson and Nguyen-Dinh1988), can be routinely maintained in vitro, making it a more appealing model when ethical and financial considerations regarding the use of monkeys as test animals are considered.
Plasmodium cynomolgi is less restricted than P. knowlesi in terms of mosquito vector transmissibility. Beside the natural vectors A. cracens and A. dirus (Cheong et al. Reference Cheong, Warren, Omar and Mahadevan1965; Vythilingam et al. Reference Vythilingam, Noorazian, Huat, Jiram, Yusri, Azahari, Norparina, Noorrain and Lokmanhakim2008), it can also be transmitted by Anopheles farauti (Nace et al. Reference Nace, Williams, Sullivan, Williams, Galland and Collins2004) and by species commonly maintained under laboratory conditions, such as A. gambiae and A. stephensi (Collins et al. Reference Collins, Sullivan, Nace, Williams, Williams and Barnwell2009a ).
In addition to relapse, P. cynomolgi has been used to study a wide variety of medically relevant phenotypes and general malaria biology, for which there is an extensive body of literature.
Several studies related to malaria immunity have been carried out with P. cynomolgi. The activation of components of the immune system during infections have been studied (Praba-Egge et al. Reference Praba-Egge, Montenegro, Cogswell, Hopper and James2002; Li et al. Reference Li, Ruan, Zhang, Peng, Zhao, Qin and Chen2012), while the parasite has also been used as a model for malaria-HIV co-infections (Koehler et al. Reference Koehler, Bolton, Rollins, Snook, deHaro, Henson, Rogers, Martin, Krogstad, James, Rice, Davison, Veazey, Prabhu, Amedee, Garry and Cogswell2009). The genome of P. cynomolgi contains orthologues of the vir-gene family, which in P. vivax are responsible for immune evasion (Prajapati and Singh, Reference Prajapati and Singh2014). Strain-specific immunity, a trait found both in human and rodent malaria (Ciuca et al. Reference Ciuca, Ballif and Chelarescu-Vieru1934; Jarra and Brown, Reference Jarra and Brown1985), has been observed in P. cynomolgi (Wijayalath et al. Reference Wijayalath, Cheesman, Rajakaruna, Handunnetti, Carter and Pathirana2008, Reference Wijayalath, Cheesman, Tanabe, Handunnetti, Carter and Pathirana2012). Additionally, P. cynomolgi has also been used to test the efficacy of potential vaccination strategies (Millet et al. Reference Millet, Kalish, Collins and Hunter1992, Reference Millet, Grady, Olsen, Galland, Sullivan, Morris, Richardson, Collins and Hunter1995; Barnwell et al. Reference Barnwell, Galinski, DeSimone, Perler and Ingravallo1999; Bhardwaj et al. Reference Bhardwaj, Kushwaha, Puri, Herrera, Singh and Chauhan2003; Dutta et al. Reference Dutta, Kaushal, Ware, Puri, Kaushal, Narula, Upadhyaya and Lanar2005).
The species has been studied to understand the interactions between Plasmodium and its vector. Studies on refractoriness to infections and its genetic basis in mosquitoes have been carried out (Collins et al. Reference Collins, Sakai, Vernick, Parkewitz, Seeley, Miller, Collins, Campbell and Gwadz1986b ; Zheng et al. Reference Zheng, Cornel, Wang, Erfle, Voss, Ansorge, Kafatos and Collins1997, Reference Zheng, Wang, Romans, Zhao, Luna and Benedict2003), as well as experiments aimed at understanding factors determining infectivity to mosquitoes (Naotunne et al. Reference Naotunne, Karunaweera, Rathnayake, Jayasinghe, Carter and Mendis1990, Reference Naotunne, Karunaweera, Del Giudice, Kularatane, Grau, Carter and Mendis1991).
Plasmodium cynomolgi has also been regularly used to test the activity of various novel anti-malarial drugs (Puri and Dutta, Reference Puri and Dutta2003; Deye et al. Reference Deye, Gettayacamin, Hansukjariya, Im-erbsin, Sattabongkot, Rothstein, Macareo, Fracisco, Bennett, Magill and Ohrt2012; McNamara et al. Reference McNamara, Lee, Lim, Lim, Roland, Nagle, Simon, Yeung, Chatterjee, McCormack, Manary, Zeeman, Dechering, Kumar, Henrich, Gagaring, Ibanez, Kato, Kuhen, Fischli, Rottmann, Plouffe, Bursulaya, Meister, Rameh, Trappe, Haasen, Timmerman, Sauerwein and Suwanarusk2013; Ohrt et al. Reference Ohrt, Li, Obaldia, Im-Erbsin, Xie and Berman2014; Zeeman et al. Reference Zeeman, Lakshminarayana, van der Werff, Klooster, Voorberg-van der Wel, Kondreddi, Bodenreider, Simon, Sauerwein, Yeung, Diagana and Kocken2016).
Plasmodium cynomolgi has also been included in several studies on malaria evolution and genetic diversity (Nishimoto et al. Reference Nishimoto, Arisue, Kawai, Escalante, Horii, Tanabe and Hashimoto2008; Cornejo et al. Reference Cornejo, Fisher and Escalante2014; Luo et al. Reference Luo, Sullivan and Carlton2015; Sutton et al. Reference Sutton, Luo, Divis, Friedrich, Conway, Singh, Barnwell, Carlton and Sullivan2016), especially since the publication of three assembled and annotated draft reference genomes (Tachibana et al. Reference Tachibana, Sullivan, Kawai, Nakamura, Kim, Goto, Arisue, Palacpac, Honma, Yagi, Tougan, Katakai, Kaneko, Mita, Kita, Yasutomi, Sutton, Shakhbatyan, Horii, Yasunaga, Barnwell, Escalante, Carlton and Tanabe2012).
The existence of optimized protocols for transfection studies (Kocken et al. Reference Kocken, Dubbeld, Van Der Wel, Pronk, Waters, Langermans and Thomas1999; Voorberg-van der Wel et al. Reference Voorberg-van der Wel, Zeeman, van Amsterdam, van den Berg, Klooster, Iwanaga, Janse, van Gemert, Sauerwein, Beenhakker, Koopman, Thomas and Kocken2013) further facilitates experimental work with the species.
Plasmodium coatneyi
Plasmodium coatneyi is a tertian malaria species closely related to P. knowlesi and found primarily in macaques of SE Asia (Fooden, Reference Fooden1994). It is transmitted by Asian Anopheline mosquitoes such as A. dirus and A. freeborni, although transmission through A. stephensi and A. gambiae, while less efficient, has been demonstrated (Collins et al. Reference Collins, Warren, Sullivan and Galland2001). New world monkeys appear to be susceptible to the liver stages of the parasite, although evidence for successful establishment of the erythrocytic cycle is lacking (Sullivan et al. Reference Sullivan, Bounngaseng, Stewart, Sullivan, Galland, Fleetwood and William2005). The liver stages of P. coatneyi have also been successfully cultured in vitro (Millet et al. Reference Millet, Collins, Aikawa, Cochrane and Nguyen-Dinh1990).
Plasmodium coatneyi displays phenotypes with striking similarities to those associated with malignant falciparum malaria in humans, such as the presence of knob protrusions on the surface of infected erythrocytes, cytoadherence to the vascular endothelium, rosetting and the induction of ‘cerebral malaria’ (Kilejian et al. Reference Kilejian, Abati and Trager1977; Udomsangpetch et al. Reference Udomsangpetch, Brown, Smith and Webster1991; Kawai et al. Reference Kawai, Aikawa, Kano and Suzuki1993, Reference Kawai, Kano and Suzuki1995; Maeno et al. Reference Maeno, Brown, Smith, Tegoshi, Toyoshima, Ockenhouse, Corcoran, Ngampochjana, Kyle, Webster and Aikawa1993; Sein et al. Reference Sein, Brown, Maeno, Smith, Corcoran, Hansukjariya, Webster and Aikawa1993; Smith et al. Reference Smith, Brown, Nakazawa, Fujioka and Aikawa1996). It also provides a valuable model to study the multisystemic dysfunction associated with severe malaria in monkeys (Moreno et al. Reference Moreno, Cabrera-Mora, Garcia, Orkin, Strobert, Barnwell and Galinski2013) and has been used in studies involving co-infections with schistosomiasis (Semenya et al. Reference Semenya, Sullivan, Barnwell and Secor2012).
An non-annotated draft genome project is currently available at the PlasmoDB website (http://www.plasmodb.org/common/downloads/Current_Release/PcoatneyiHackeri/) thus providing some framework for genetic studies with this parasite species.
Plasmodium simium
Plasmodium simium is the most closely related NHPMP to P. vivax. Indeed, there is now strong evidence that it may represent an adaptation of P. vivax to transmission and growth in New World monkeys following a host switch (Ayala et al. Reference Ayala, Escalante and Rich1999; Goldman et al. Reference Goldman, Qari, Millet, Collins and Lal1993; Mu et al. Reference Mu, Joy, Duan, Huang, Carlton, Walker, Barnwell, Beerli, Charleston, Pybus and Su2005; Tazi and Ayala, Reference Tazi and Ayala2011). As expected by its phylogenetic location, P. simium shares many features with its human counterpart, including the presence of an extensive repertoire of vir-genes involved in immune evasion (Prajapati and Singh, Reference Prajapati and Singh2014), the expression of a Duffy binding protein that interacts with a host Duffy antigen receptor for chemokines (DARC) (Camargos Costa et al. Reference Camargos Costa, Pereira de Assis, de Souza Silva, Araujo, de Souza Junior, Braga Hirano, Satiko Kano, Nobrega de Sousa, Carvalho and Ferreira Alves de Brito2015) and a tertian erythrocytic cycle (Deane et al. Reference Deane, Deane and Ferreira Neto1966).
Its natural hosts are the Platyrrhine monkeys of South America (Collins et al. Reference Collins, Contacos, Guinn and Skinner1973, Reference Collins, Skinner, Pappaioanou, Broderson, Ma, Stanfill and Filipski1987; Deane et al. Reference Deane, Deane and Ferreira Neto1966) and it can be transmitted by a variety of mosquito vectors, including the commonly used laboratory species A. stephensi (Collins et al. Reference Collins, Warren, Contacos, Skinner and Richardson1979b ). Occasional cases of naturally acquired human infections have been reported (Deane et al. Reference Deane, Deane and Ferreira Neto1966), although such observations should be viewed with caution due to the difficulty of distinguishing P. simium from P. vivax in the pre molecular biology era.
Due to its close relationship to P. vivax, P. simium has been proposed as a model to study the efficacy of vaccine candidates (Collins et al. Reference Collins, Sullivan, Galland, Williams, Nace, Williams and Barnwell2005). No genome, which could provide valuable information both on its origins and on host adaptations, is currently available.
Plasmodium fragile
Plasmodium fragile is a tertian NHPMP species originally isolated from monkeys of the Indian subcontinent (Ramakrishman and Mohan, Reference Ramakrishman and Mohan1961; Dissanaike et al. Reference Dissanaike, Nelson and Garnham1965a ). Laboratory induced infections have demonstrated its capacity to establish infections in New World Monkeys, but it appears somewhat more restricted in vector infectivity and its development has only been successfully observed in A. dirus (Collins et al. Reference Collins, Skinner, Filipski, Broderson, Stanfill and Morris1990, Reference Collins, Stanfill, Richardson and Smith1974, Reference Collins, Warren, Sullivan, Galland, Strobert, Nace, Williams, Williams and Barnwell2006). However, other species from the Anopheles leucosphyrus group (to which A. dirus belongs) have been observed to transmit P. fragile (Sallum et al. Reference Sallum, Peyton and Wilkerson2005).
Two strains of the parasite are currently available with one having lost the ability to produce infective gametocytes in the host (Collins et al. Reference Collins, Warren, Sullivan, Galland, Strobert, Nace, Williams, Williams and Barnwell2006). Additionally, a protocol to grow the parasite in vitro exists (Chin et al. Reference Chin, Moss and Collins1979).
Like P. falciparum and P. coatneyi, P. fragile infected red blood cells adhere to blood vessels (Fremount and Miller, Reference Fremount and Miller1975) and have been shown to form rosettes; a phenomenon typically associated with P. falciparum infections in humans (David et al. Reference David, Handunnetti, Leech, Gamage and Mendis1988). Indeed, P. fragile infections in monkeys have been used as a model for human cerebral malaria (Fujioka et al. Reference Fujioka, Millet, Maeno, Nakazawa, Ito, Howard, Collins and Aikawa1994).
Plasmodium fragile has been adopted as a model to study malaria vaccines (Collins et al. Reference Collins, Chin and Skinner1979a ; Fujioka et al. Reference Fujioka, Millet, Maeno, Nakazawa, Ito, Howard, Collins and Aikawa1994), as well as major surface antigens (Nguyen-Dinh et al. Reference Nguyen-Dinh, Deloron, Barber and Collins1988; Peterson et al. Reference Peterson, Nguyen-Dinh, Marshall, Elliott, Collins, Anders and Kemp1990). Like major human malaria species, P. fragile also evades the immune system by undergoing antigenic switching (Handunnetti et al. Reference Handunnetti, Mendis and David1987). More recently, various aspects of HIV and malaria co-infections have been studied using P. fragile and the Simian Immunodeficiency Virus as models (Trott et al. Reference Trott, Chau, Hudgens, Fine, Mfalila, Tarara, Collins, Sullivan, Luckhart and Abel2011, Reference Trott, Richardson, Hudgens and Abel2013; Frencher et al. Reference Frencher, Ryan-Pasyeur, Huang, Wang, McMullen, Letvin, Collins, Freitag, Malkovsky, Chen, Shen and Chen2013). Finally, P. fragile has been used to test the activity of antimalarial drugs (Tripathi et al. Reference Tripathi, Vishwakarma and Dutta1997; Puri and Dutta, Reference Puri and Dutta2003).
While less studied than P. knowlesi or P. cynomolgi, P. fragile displays several interesting phenotypes that have made it a relatively popular model for human malaria, despite the reliance on the relatively fastidious A. dirus vector for transmission. Its adaptation to in vitro culture also makes it an ideal parasite for future genome sequencing and genome editing studies.
Other vivax-type NHPMPs
There are several other species of monkey malaria belonging to the P. vivax clade that have been less studied than those described above. These include Plasmodium simiovale, Plasmodium fieldi, Plasmodium hylobati and Plasmodium gonderi. Plasmodium fieldi and P. hylobati were originally detected in SE Asia, whereas P. gonderi is of African origin (Mitsui et al. Reference Mitsui, Arisue, Sakihama, Inagaki, Horii, Hasegawa, Tanabe and Hashimoto2010).
Plasmodium simiovale, like P. vivax and P. cynolomgi, produces hypnozoites (Cogswell et al. Reference Cogswell, Collins, Krotoski and Lowrie1991) and the full-life cycle can be maintained in Old World monkeys using A. dirus and A. maculatus, but not A. stephensi mosquito vectors (Collins and Contacos, Reference Collins and Contacos1979). In vitro cultures of the exo-erythrocytic cycle are also possible (Millet et al. Reference Millet, Anderson and Collins1994), but no evidence of adaptation of the parasite to growth in in vitro blood culture or in Platyrrhine monkeys is available.
Plasmodium gonderi can be transmitted by various Anopheline vectors, including A. stephensi, is infective to Old World monkeys (Collins and Contacos, Reference Collins and Contacos1980) and can be cultivated in vitro (Guo et al. Reference Guo, Chin and Collins1983; Millet et al. Reference Millet, Collins, Aikawa, Cochrane and Nguyen-Dinh1990). Like P. simiovale it induces relapse in infected monkeys (Collins and Contacos, Reference Collins and Contacos1971) and the exoerythrocytic stages have been cultured in vitro (Millet et al. Reference Millet, Anderson and Collins1994). However, no complete life-cycle infections could be maintained in more tractable Platyrrhine monkeys (Sullivan et al. Reference Sullivan, Jennings, Guarner, Noland, Kendall and Collins2002).
Plasmodium hylobati has been generally neglected and attempts at growing the parasite in Platyrrhine monkeys failed (Collins et al. Reference Collins, Contacos, Skinner, Stanfill and Richardson1981).
USE OF NON-HUMAN PRIMATES INFECTED WITH HUMAN MALARIA
Both major human malaria parasite species, P. falciparum and Plasmodium vivax, can grow and replicate in non-human primates (usually splenetcomized) with varying degrees of success (Young et al. Reference Young, Baerg and Rossan1975). Splenectomized chimpanzees have been used to produce genetic crosses between P. falciparum strains for genetic studies. An initial cross between strains 3D7 and Hb3, produced by Walliker and colleagues (Walliker et al. Reference Walliker, Quakyi, Wellems, McCutchan, Szarfman, London, Corcoran, Burkot and Carter1987) led to the discovery of mutations associated with pyrimethamine resistance (Peterson et al. Reference Peterson, Walliker and Wellems1988). A second cross (between strains Dd2 and Hb3) was performed to study the basis of chloroquine resistance (Wellems et al. Reference Wellems, Panton, Gluzman, do Rosario, Gwadz, Walker-Jonah and Krogstad1990), which later led to the discovery of mutations in the chloroquine resistance transporter gene (crt) and their association with drug resistance (Fidock et al. Reference Fidock, Nomura, Talley, Cooper, Dzekunov, Ferdig, Ursos, Sidhu, Naudé, Deitsch, Su, Wootton, Roepe and Wellems2000). The same cross was also used to uncover the genetic basis of sulfadoxine resistance (Wang et al. Reference Wang, Read, Sims and Hyde1997). More recently, what is presumed to be the last ever malaria cross to be produced in chimpanzees, was conducted using an artemisinin-resistant strain of P. falciparum (Miles et al. Reference Miles, Iqbal, Vauterin, Pearson, Campino, Theron, Gould, Mead, Drury, O'Brien, Ruano Rubio, MacInnis, Mwangi, Samarakoon, Ranford-Cartwright, Ferdig, Hayton, Su, Wellems, Rayner, McVean and Kwiatkowski2015).
Human malaria parasite infections in monkeys have been used to discover parasite antigens. One of the most prominent studies involved the discovery of a new family of reticulocyte-specific ligands expressed by P. vivax merozoites infecting splenectomized squirrel monkeys (Galinski et al. Reference Galinski, Medina, Ingravallo and Barnwell1992). Homologues of these genes were found in other malaria parasite species, including P. falciparum (P. falciparum reticulocyte-binding protein homologues, PfRh) and are essential for red blood cell invasion (Cowman and Crabb, Reference Cowman and Crabb2006). A more recently discovered member of this family, PfRh5 (Baum et al. Reference Baum, Chen, Healer, Lopaticki, Boyle, Triglia, Ehlgen, Ralph, Beeson and Cowman2009), has recently been tested in a vaccine trial in Aotus monkeys and showed strong, strain-transcending protective immunity (Douglas et al. Reference Douglas, Baldeviano, Lucas, Lugo-Roman, Crosnier, Bartholdson, Diouf, Miura, Lambert, Ventocilla, Leiva, Milne, Illingworth, Spencer, Hjerrild, Alanine, Turner, Moorhead, Edgel, Wu, Long, Wright, Lescano and Draper2015). Indeed, Aotus monkey models represent a useful model for testing the efficacy of putative malaria vaccines (Herrera et al. Reference Herrera, Perlaza, Bonelo and Arévalo-Herrera2002; Curtidor et al. Reference Curtidor, Patarroyo and Patarroyo2015).
Testing of novel antimalarial compounds also relies heavily on the use of P. vivax- and P. falciparum- infected monkeys (Powers and Jacobs, Reference Powers and Jacobs1972; Rossan et al. Reference Rossan, Young and Baerg1975; Bitonti et al. Reference Bitonti, Sjoerdsma, McCann, Kyle, Oduola, Rossan, Milhous and Davidson1988; Nayar et al. Reference Nayar, Baker, Knight, Sullivan, Morris, Richardson, Galland and Collins1997; Wengelnik et al. Reference Wengelnik, Vidal and Ancelin2002; Ye et al. Reference Ye, Van Dyke and Rossan2013). Indeed, many efforts have been undertaken to adapt strains of human malaria to growth in monkeys for the purpose of drug testing (Herrera et al. Reference Herrera, Perlaza, Bonelo and Arévalo-Herrera2002; Obaldía et al. Reference Obaldía, Milhous and Kyle2009).
Relapse in P. vivax and other malaria parasite species is caused by the hypnozoite stage. This stage was originally discovered in rhesus macaques infected with P. cynomolgi (Krotoski et al. Reference Krotoski, Garnham, Bray, Krotoski, Killick-Kendrick, Draper, Targett and Guy1982a ) and later demonstrated in chimpanzees infected with P. vivax sporozoites (Krotoski et al. Reference Krotoski, Collins, Bray, Garnham, Cogswell, Gwadz, Killick-Kendrick, Wolf, Sinden, Koontz and Stanfill1982b ). Non-human primate hosts are still crucial to understand both hypnozoite biology and genetics and also to develop novel antimalarial treatments (Joyner et al. Reference Joyner, Barnwell and Galinski2015).
Concluding remarks
Plasmodium knowlesi and, to a lesser extent, P. cynomolgi are the two most commonly used NHPMP species used in experimental laboratory research on malaria. This is justified by their zoonotic potential (particularly in the case of P. knowlesi, which may represent a fifth species of human malaria), their adaptation to in vitro culture and to growth in Platyrrhine monkeys, the availability of reference genomes and of established protocols for transfection studies.
However, while P. cynomolgi shares many features with P. vivax, none of the other macaque species share such a close phenotypic similarity to severe P. falciparum, with the possible exception of P. knowlesi in certain hosts (Cox-Singh and Culleton, Reference Cox-Singh and Culleton2015). Unfortunately, the strict host specificity of most Laverania species severely constrains their development as model organisms. However, among the less studied NHPMP species, there are alternative models for severe and cerebral malaria that bear striking similarities with P. falciparum infections, such as P. fragile and P. coatneyi. Both species can be maintained in vitro, while P. fragile can also be maintained in Platyrrhine monkeys and a draft genome exists for P. coatneyi. No genome project is currently available for P. fragile, which is rather surprising given the existence of a strain incapable of producing infective gametocytes, a phenotype of prominent biological interest.
While P. cynomolgi represents the model most suited to study P. vivax, P. simium, due to its close relationship to and possible derivation from P. vivax, could provide valuable insights into the evolution of zoonosis and host adaptation.
Finally, P. brasilianum and P. inui represent potential models for quartan malaria, though the capacity of P. malariae to establish infection in Platyrrhine monkeys makes their development less essential. Nonetheless, P. brasilianum represents a possible adaptation of P. malariae to Platyrrhine monkeys and the assembly and annotation of its genome could provide answers to the questions surrounding its origin.
To conclude, while the use of P. knowlesi as the major malaria model in monkeys is justified by resource availability, ease of use and zoonotic threat, other NHPMPs deserve attention for their capacity to model various and diverse aspects of human malaria. As with all scientific endeavour, picking the right tools for a particular experiment is crucial and, with the diversity of phenotypes provided by the NHPMPs, we are blessed with a well-stocked tool shed.
ACKNOWLEDGEMENTS
We thank Dr Janet Cox-Singh for commissioning this review. We are grateful for comments on an earlier version of this manuscript from Prof. Richard Carter, and during the review process from Prof. Louis Miller and an anonymous reviewer.
FINANCIAL SUPPORT
Richard Culleton was supported by grants from the Japanese Society for the Promotion of Science, numbers 16K21233, 25870525 and 24255009. Axel Martinelli would like to thank the GI-CoRE initiative at Hokkaido University for financial support.
