Parasitology Today, vol. 6. no I, I 990 3

Fig. I. Merozoites per schizont for various malaria species infecting lizards. (Token from Ref: 7.)

4 10 16 22 28 34 40 46 52 58 >90

Merozoites per schizont

host fitness. As predicted by the ‘para- site’ theory of sex, Burt and Bell showed that the presence of ‘B’ chromosomes is associated with elevated rates of recom- bination in the host genome, lending support for the possible role of more ‘conventional’ parasites in the evol- utionary maintenance of sexual repro- duction.

R.M. May (Oxford and London Universities, UK) and R.M. Anderson (London University, UK) concluded the day by cautioning against isolation of

population genetics from population dynamics. As an example, they illus- trated that the contrast between the long doubling time characteristic of the early existence of HIV infection and the much more rapid spread of infection today need not necessarily be the result of evolutionary changes in the parasite. Instead, heterogeneous transmission within and between the villages, charac- teristic of the time and place of the proposed origin of the virus may have been critical in limiting the epidemic in its

early phase. The much shorter doubling time now in evidence could be a simple consequence of altered social organiz- ation of the host and accompanying changes in parasite population dynamics. As this example illustrates, powerful insights have been gained from the recent integration of population biology with parasitology. We look forward to the results of similar integration with evolutionary biology.

References Harvey, P. ( I989)Nature 342,230 Freeland, W.J. (I 976)Brotrop1co 8, 12-24 Jaenike, J.( I978)Evol. Theory 3, I9 l-l 94 Hamilton, W.D. and Zuk, M. (1982) Scrence 218,384-387 Thompson, G. (1989) B~of. J. Lynn. Sot. 32, 385-393 May, R.M. and Anderson, R.M. ( 1983) Proc. R. Sot. London Ser. B 2 I 9,28 I-3 I 3 Schall, 1.1. In The Evolutionary 61ology of Poro- srtism (Keymer, A.E. and Read, A.F., eds). Cambridge Universtty Press(In press)

Anne Keymer and Andrew Read are at the Department of Zoology, Univers,ty of Oxford, South Parks Rood, Oxford OX I 3PS, UK.

Laboratory Models for Research in vivo and in vitro on Malaria Parasites of

Mammals: Current Status B. Mons and R. E. Sinden

In research aimed at developing strategies for the eradi- cation of human malaria, various species of rodent, avian and non-human primate plasmodia are used as laboratory models. Here Barend Mons and Robert Sin- den attempt to summarize the most common laboratory models for mammalian malaria, and to shed some light on their applicability to different aspects of malaria research.

Despite extensive research efforts, malaria continues to be the major tropical disease of humans. The parasite responsible, Plasmodium, has resisted most effectively all the strategies employed to control it, including a variety of drugs, and initial vaccination trials. The mosquito vector has proved to be equally

Barend Mons IS at the Laboratory of Parasitology, Medical Faculty, University of Leiden, PO Box 9605, Wassenaarsweg 62, 2300 RG Leiden. The Netherlands. Robert Slnden is at the Molecular and Cellular Parasitology Research Group, The Department of Pure and Applied Biology, Imperial College, London SW7 2BB, UK.

0 ,990, Elsewr Sc~enre PubIshers Ltd. (UK) 0 / 69-4707/9@$02 00

resilient to control measures, developing high levels of resistance to the major insecticides, and with- standing environmental measures such as mosquito nets and the eradication of breeding sites for the anopheline vectors.

Our failure to predict the parasite’s capacity to circumvent our attacks often stems from an inad- equate understanding of the basic biology of these organisms, their relationships to both the vertebrate host and the vector, and to their environment. This information is vital to future strategies for the eradi- cation of malaria.

Previously, the opportunities for research on the fundamental biology of the life cycle of human ma- laria parasites (P. falciparum, P. vivax, P. malariae and P. ovale) were limited by the inaccessibility of the parasite within its vertebrate and invertebrate hosts, and today, research involving the human host is sanctioned only in the final stages of vaccine or drug trials. Although several species of non-human primates are partly susceptible to infection with

4 Porositology Today, vol. 6, no. I, I990

Table I. The possibilities for in vivo laboratory maintenance of the various life-cycle stages of the more important malaria parasites of mammals

Species EE AES SES FERT OOK ooc SPOR SYN Human P. falciporum ++ ++ + + + + ++ exp.

P. vivox ++(h) ++ + + + + ++ -

P. ovale + + + + + + + -

P. malorioe + +’ + + + + + -

Non-human primate P. fragile

+ z(h) ++ ++ + + ++ ++ -

P. cynomolgi ++ ++ ++ ++ ++ ++ -

P. brasilianum ++ ++ + + + ++ ++ -

Rodent P. berghei +++ +++ +++ +++ +++ +++ +++ exp.

P. chobaodi ++ +++ + + + + + nat.

Abbreviations: EE. exoerythrocytic (liver) stages; AES, asexual erythrocytic stages; SES, sexual erythrocytic stages; FERT. fertilization; OOK, ookinetes; OOC, oocysts; SPOR, sporozoites; SYN, synchronous infection; h, hypnozoites described; exp./nat., synchronicity induced experimentally or naturally. Symbols: + + +. easy to maintain in readily available host, possibility for harvesting of relatively large numbers of parasitic stages; + +, easy to maintain but host not readily available or harvesting of pure parasitic stage not easy; +, can be maintained but host difficult to obtain and maintain in the laboratory, relatively low numbers of parasites available from in viva sources.

human plasmodia, the ethical and methodological problems involved in using these animals are mani- fold. The more so, because almost all susceptible non-human primates are rare, or even endangered species. On occasion, however, parasites of primates in their natural host can be a valuable alternative, but the use of such models is difficult to justify on a regular basis.

The difficulties of working with primate malaria parasites have meant that numerous species of rodent and avian malaria parasites have been widely used as laboratory models to study the biology of plasmodia. The major drawback of these model organisms is their uncertain phylogenetic relation- ship with human plasmodia, which calls into ques- tion their relevance as biochemical or molecular models. Despite certain similarities between P. ful- ciparum and avian plasmodia (eg. P. relicturn and P. gallinaceum) 1,2 differences in their life cycles, vec- tors, and in the immune systems of their vertebrate hosts limit the usefulness of avian malaria as a model, hence stressing the importance of rodent models in fundamental studies.

A reproducible, in vitro culture method for the pathogenic asexual bloodstages has only been re- alized for P. falciparum and for a limited number of non-human primate and rodent plasmodia (see Table 1). When studying the relationship between the parasite and its vector, laboratory infection of mosquitoes and transmission to new vertebrate hosts are prerequisites for any suitable model system. Such transmission is routine for only a limited num- ber of plasmodial species in a relatively small number of malaria laboratories.

Criteria for a good laboratory model In general, a good laboratory model should meet

two major criteria: (1) it should be relevant to human malaria, and (2) it should offer the ability to study the biology of the parasite at the cellular and molecular levels in different hosts (see Box 1).

The pathogenic phase of the life cycle, the intra- erythrocytic asexual parasite, has been the most intensely studied. However, all stages are of great interest and many research groups believe that the other stages might prove equally important for ma- laria control. This shift of interest has resulted in the development of a variety of techniques for the main- tenance of a wide range of stages of the parasite’s life cycle in viva and in vitro3. The mosquito vectors for the different species of plasmodia may be maintained by near routine techniques (but may require critical and technically fastidious mating methods), and therefore most Plasmodium species are transmissible through the vector in the laboratory. The major limitations to research thus relate to the availability of the relevant vertebrate host and/or a reproducible culture system for the parasite. Tables 1 and 2 give a summary of current possibilities for in viva and in vitro maintenance of some important species of mam- malian malaria parasites.

In vivo models The earlier studies on primate malarias have been

extensively reviewed in the classic works of Garn- ham4 and Coatney et al .5. Much recent work, mainly by Collins and his colleagues, has examined the suitability of models for vaccine studies in monkeys. Plasmodium fulciparum and P. vivax have been adap- ted to a variety of New World monkeys, and trans- mitted by mosquitoes to them, but beyond the description of new and more susceptible (sub)- species of Aotus and Saimiri, no new alternative hosts have been identified6. The remaining malaria para- sites of humans, P. ovaZe7 and P. maluriae, can only be studied in the chimpanzee and in a very limited number of the New World monkeys (Aotus and Saimiri)8. A parasite that is able to infect intact common marmosets (CaZZithrix jacchus) has recently been described; it initially resembled P. vivax’, but further studies suggest that this parasite may in fact be most like P. malariae (G.H. Mitchell, pers. com-

Parasitology Today, vol. 6, no I, 1990 5

mun.). Either host-parasite combination would be a major step forward for the study of human malaria in commonly available primates.

Recent fundamental research using primate hosts has included the description of the dormant liver forms, or hypnozoites, of P. cytu)m~Zgj~~~~~ and P. vi~ax’~. The hypnozoite is widely accepted as the origin of true relapses13. However, more research is needed to prove beyond doubt that this parasite is not a re-invaded merozoite, but a dormant, sporozoite-derived parasite. The assumed close phy- logenetic relationships between certain non-human primate and human malaria parasites (P. fulciparum with P. reichenovi, P. vivax with P. cynomolgi and P. malariae with P. brasiliunum) make the species that infect non-human primates preferred models for the study of the biology of Plasmodium. However, at present, in vivo research using non-human primate plasmodia should be undertaken with extreme dis- cretion by the few institutes that have the necessary resources.

In marked contrast, maintenance of the rodent malarias in vivo is not difficult and techniques for the induction of synchronized asexual infections, and enriched gametocyte infections have been described14’15. For most laboratories, rodent malaria parasites will therefore continue to be the logical first choice for studies on the basic biology of Plasmodium in the vertebrate host.

In vitro culture of malaria parasites A combination of in vivo and in vitro studies is of

the utmost importance for the understanding of the basic cell biology of the parasite and for the study of host responses. In this section we review current possibilities for in vitro propagation of each stage of

the life cycle. A potential danger in the use of parasites maintained in vitro is that they may change in both phenotype and genotype from the original isolate and the possible impact of these changes on individual studies (eg. cytoadherence or chromo- some structure) could be significant.

Asexual erythrocytic stages (AES). The falciparum- type parasites, P. fulciparum, P. fragile and (tenu- ously) P. chabaudi were among the first parasites for which in vitro culture was reported’“” for the pathogenic asexual blood phase. The vivax-type parasites, P. vivax, P. ovale, P. cynomolgi and P. berghei appeared to be much more difficult to culture and although in vitro techniques for the erythrocytic stages of P. cynomolgi”, P. berghei2’y2’, P. yoeZii22, P. vivax23y24, P. inui and P. gonderi have now been described, they remain somewhat problematic24 and only P. berghei is widely cultured on a routine basis. One problem is the ability of the erythrocytic mero- zoites to invade only a limited range of red blood cell types (notably reticulocytes)23P25, and this is still a major obstacle for their mass cultivation. Full syn- chronization in culture has been realized for P. faZciparum26 and P. berghei14 but only partly achieved for P. vivax25.

Sexual erythrocytic stages (SES). In general, ga- metocytes are produced in highly variable numbers in culture27, but this innate property is modulated by environmental factors28y29. For P. falciparum, pure sexual cultures of infective gametocytes can be pro- duced by selective killing of asexual parasites26’30, enabling biochemical and immunological studies of this stage. Employing combined in vivo and in vitro techniques, we have been able to produce pure sexual stages of P. berghei in sufficient amounts for biochemical analysis (C. Janse, unpublished), but

Box I. Summary of Criteria for ‘Ideal’ Laboratory Models in Malaria (I) In vitro culture ofasexual bloodforms possible. (6) Variety of research tools, mAbs, probes, etc., available. (2) Possibility for highly synchronized infections and cultures. (7) Purification of major relevant life-cycle stages in large (3) Cyclical passage in the laboratory possible. numbers possible. (4) Immunological interaction with host (experimental or (8) Combined in vivo and in vitro research possible in normal

natural) studied. laboratory. (5) Varietyofwell-defined clones and lines available that exhibit (9) Complete in vitro development of all vertebrate stages.

natural phenotypes. ( IO) Culture of the early/late sporogonic stages practicable.

I 2 3 4 5 6 7 a 9 IO Human P. falciporum + + - + + + k - - k P. vivax + - - + + - - - - -

P. ova/e - - - - - _ - -

P. molariae - - - - - - _

Non-human primate P. fragile + + - + + - - -

P. cynomolgi + - - + + - - _ -

P. brasilianum + - + + - - - _ _ -

Rodent P. berghei + + + + + + + + + + P. chabaudi + + + + + + - - - -

6 Parasitology Today, vol. 6, no. I, I990

Table 2. Present state-of-the-art of in vitro cultivation techniques for more important species of malaria parasites

Species EE AES SES FERT OOK ooc MOSQ SYN Human P. falciporum + +++(m) f++(m) +++ + -

yes exp (SES. AES)

P. vivax ++ ++ ++ - - -

P. ovale ++ + _ - - -

P. malariae + + - - -

Non-human primate P. fragile _ ++ _ - - - -

P. cynomolgi ++ +++ - - - -

P. brasilianom - + _ _ - - -

P. inui + + _ _ - - -

P. coatneyi + - - - - - -

P. knowlesi + + _ - - -

P. gonderi + + - - - -

Rodent P. berghei +++ +++ +++ +++(m) +++(m) - yes exp.

(SES, AES, OOK) P. chabaudi - + - - -

yes nat.

Abbreviations: EE, exoerythrocytic (liver) stages; AES. asexual erythrocytic stages; SES. sexual erythrocytic stages; FERT. fertilization; OOK, ookinetes; OOC, oocysts; MOSQ, mosquito infection from culture; SYN, synchronous culture; m. mass cultivation possible. Symbols: + ++, optimal culture possibilities. up to infective forms for the next parasitic stage, continuous culture of EA possible; + +, maturation of the complete parasitic stage, infectivity of next parasitic stage not achieved with full reproducibility, continuous culture of EA difficult; +, maturation of parasitic stage not complete; -, culture not realized.

for all the other malaria parasites that have been the human or non-human primate malaria parasites cultured in vitro, the production of infective gameto- would be a great stimulus to malaria research, since it cytes is at present neither predictable nor repro- would enable cyclical transmission without any ducible. involvement of a vertebrate host.

Ookinetes (OOK) and sporogonic stages. Fertiliz- ation under in vitro conditions is readily achieved for most malaria parasites, but the subsequent produc- tion of viable ookinetes has proved more difficult. Reproducible culture of ookinetes from I’. berghei has been described31-33, and several groups have tried to culture the ookinete stage of P. falciparum but with only a single published success34. Notwith- standing infection of mosquitoes with cultured ookinetes, the in vitro propagation of later sporo- gonic stages has met with very limited success3’ and remains one of the major technical challenges to malaria research.

Exo-etythrocytic stages (EE). The recent avail- ability of these stages in culture has provided broad opportunities for studies on possible drug therapy, vaccination and the cell biology of the tissue stages. Sporozoites from many species of plasmodia readily invade hepatocytes or hepatoma cells from a variety of vertebrates,. and a bizarre range of heterologous sources3G38. In contrast to the erythrocytic stages, the EE forms of the vivax-type malaria parasites are more readily cultured in vitro than those of P. ful- ciparum (see Table 2). Complete (morphological) maturation of EE schizonts has been described for P. vivax39, P. berghei 36, P. yoelaa “‘40, P. ovale41, P. ma- lariae42, P. knowlesi, P. coatneyi, P. inui43 and P. cynomolgi42. However, functional maturation, first shown by the inoculation of EE culture supernatants into mice36, has only been obtained reproducibly in vitro with P. berghei44. In that study, up to 20% of the liver merozoites gave rise to gametocytes in the first erythrocytic cycle. To repeat this success with any of

Clearly there is no single PZasmodium species, or method of maintenance in the laboratory that can satisfy all the needs for malaria research, and there is no laboratory model that reliably emulates malaria in humans in the endemic environment. Institutions with large numbers of non-human primates may still choose to carry out basic research using these valu- able hosts. However, we feel that the fundamental biology of the malaria parasite can, in most cases, be studied equally well, and with greater convenience, in rodent models because they offer the unique combination of in vivo and in vitro methods of main- tenance, and the possibility of distinguishing innate properties of the parasite from host-induced phenomena. Results obtained in rodent systems have proved to be of significant, predictive value for monkey trials with the human malarias, notably in the development of vaccination candidates and methods, in genetics and cytology, enzyme analysis, drug evaluation, EM studies, and detailed examin- ation of the mechanisms of antigenic variation; P. chabaudi is claimed to be a useful model for the synchronicity of infection, and capillary seques- tration.

It is in the evaluation of the complex dynamic interchange between the parasite and the host’s immune system that the rodent malarias will continue to play a major role. The availability of numerous immunogenetically defined mouse strains, the knowledge of the normal route and intensity of parasite growth of various cloned para- site lines, and the ability to culture the parasite in vitro will all play significant roles in evaluating the

Paraatology Today, vol. 6, no. I, I990

relative impact of humoral, or cell-mediated responses in, for example, the analysis of the immunogenicity of biochemically purified, or recombinant parasite proteins and synthetic pep- tides. This particular combination of benefits has already been recognized in that the basic biology of a major vaccine candidate (the circumsporozoite pro- tein) is being extensively re-evaluated in rodent hosts (eg. Refs 45, 46). More emphasis on the rodent models would reduce the use of non-human primates for purely fundamental research and thus make these precious animals more widely available when finally needed for applied research.

A significant advance in culture techniques would be the development of continuous, and mass, culti- vation of the blood stages of P. vivax and P. ma- hiue, to reduce the need to use susceptible primates (notably apes) for the simple propagation of the vertebrate stages.

relevant in basic studies, particularly on the mech-

Despite substantial advances in the ability to ma- nipulate malaria parasites in the laboratory, the major objectives for the future development of new in vitro culture methods have remained substantially unchanged for the past five to ten years, namely, the culture of the later sporogonic stages from ookinete to sporozoite for any species, the continuous and mass cultivation of the blood stages of P. vivax, and the complete and routine development of the EE stages of P. falciparum. The completion of either of the two latter objectives might allow us to repeat the success with P. berghei in the total replacement of the vertebrate host by culture in con&w from sporozoite to infective gametocyte. Such cultures would be

anisms of genetic exchange and variation in human 38 Sinden, R.E. et al. Am.3. Trop. Med. Hyg. (in press)

plasmodia. 39 Mazier, D. et al. (1984) Nature 307,367-369

4 Garnham, P.C.C.G. (1966) Malaria Parasites and O&r Haemo- sporidia Blackwell Scientific Publications

5 Coatney, G.R. et al. (1971) The Primate Malatis US Department of Health, Education and Welfare

6 World Health Organization (1988)BulZ. WHO 66,719-728 7 Bray,R.S. (1957)Am.J. Trop. Med. Hyg. 6,638-645 8 Collins, W.E. etal. (1984)J. Parasitol. 70,677-681 9 Mitchell, G.H. etal. (1988)ParasitoZogV96,241-251

10 Krotoski, W.A. etal. (1986)Am.J. Trop. Med. Hyg. 35,263-274 11 Krotoski, W.A. etal. (1980)Br. Med.?. 280,153-154 12 Atkinson,C.T. etal. (1989)Am.J. Trop. Med. Hyg. 40,131-140 13 Gamham, P.C.C. (1988) in Malaria. Principles and Practice ofMala-

riology (Wernsdorfer, W. and McGregor, I., eds), pp 61-69, Chur- chill Livingstone

14 Mons,B. (1986)AccaZxiden. 54,1-83 15 Dearsly, L., Nicholas, J. and Sinden, R.E. (1986) Int. ‘3. Parasitol.

17,1307-1312 16 Trager, W. and Jensen, J.B. (1976)Science 193,673-675 17 Chin. W.. Moss. D. and Collins. W.E. (1979) Am. 7. Trob. Med.

Hyg. i8, i91-59i ~ I ., .

18 Geiman, Q.M., Siddiqui, W.A. and Schnell, J.V. (1966) Milk Med. 131,1015-1025

19 Nguyen-Dinh,P.etal.(1981)Science212,1146-1148 20 Mons, B., Janse, C.J. and Boorsma, E.G. (1985) Parasitology 91,

423-430 21 Janse,C.J. etal. (1984)Znt.J.ParasitoZ. 14,317-320 22 Lewis-Hughes, P.H. and Howell, M. J. (1984) Int. J. Parasitol. 14,

447-451 23 Mons, B. eral. (1988)Inr.J. Parasitol. 18,307-311 24 Mons, B. etal. (1988)&p. Parasitol. 66,183-188 25 Janse,C.J. etal. (1989)Zk.J. Parasitol. 19,509-514 26 Ponnudurai. T. etal. (1986)Parasitolow 93.263-274 27 Sinden, R.i. (1983)Adv. iarasitol. 22,15<215 28 Carter, R. and Miller, L.H. (1979)BuZ1. WHO 57,37-52 29 Mons, B. (1985) Parasitology Today 1,87-89 30 Sinden, R.E. et al. (1984)Parasitology 88,23%247 31 Weiss, M.M. and Vanderberg, J.P. (1977)J. Parasitol. 63,932-934 32 Janse,C. J. etal. (1985)Parasitology91,19-29 33 Sinden, R.E., Hartley, R.H. and Winger, L. (1985)Parasitology91,

227-244 34 Carter,E.H.etal.(1987)ParasitoZogy95,25-30 35 Ball, G.H. (1972) Inverrebrate Tissue Culture (Vol. 2), pp

32 l-350, Academic Press

37 Mazier, D. etal. (1988)Biol. ce1161,165-172 36 Hollingdale, M.R. (1985) Heparology 5,327-335

40 Mazier, D. etal. (1982)CRAcad. Sci. Paris294,963 41 Mazier, D. etal. (1987)Exp. Parasitol. 64,39-00

References 42 Millet, P. etal. (1988)km.j. Trop. Med. Hyg. 38,470-473 1 Peters, W. et al. (1976) Philos. Trans. R. Sot. London Ser. B 275, 43 Millet, P. etal. (1988)Am.J. Trop. Med. Hyg. 39,529-534

439-482 44 Suhrbier, A. etal. (1987) Trans. R. Sot. Trop. Med. Hyg. 82,907-910 2 Sinden, R.E. etal. (1978)Proc. R. Sot. London&r. B 201,375-399 45 Good, M.F. and Miller, L.H. (1989) Vaccine7,3-10 3 Sinden, R.E. (1987)Parasitology Today 3,292 46 Kumar, S. etal. (1988) Nature 334,258-260

UNDPIWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases

Announcement of FIELDLINCS Coordination and Technical Support Grants Field Links for Intervention and Control Studies (FIELDLINCS) is a recently created programme designed to promote high-quality field research on the TDR target diseases (malaria, schistosomiasis, fila- riasis. trypanosomiasis, leishmaniasis and leprosy).

In order to encourage and strengthen multi-disciplinary field research in Latin America, TDR wishes to award up to three FIELDLINCS Coordination and Technical Support Grants on a competitive basis to institutions in Latin America. The focus of these grants should be on networking young investigators interested in or already conducting field research on one of the TDR target diseases.

Contracts will be for a three-year period, renewable for another two years, and can be up to US$lOO 000 for the first year. After evaluating the experience of the first year and depending on the availability of funds, the budget for subsequent years may be increased.

In order to discuss further the form these proposals should take, TDR in conjunction with PAHO will hold a meeting of

representatives from interested institutions from 24 to 26 May I990 in conjunction with and following the 3rd Congreso Latin0 American0 de Medicino Tropical and the 9th Congreso National de Parasitologia in Mexico City. Review of the final proposals will occur at a special meetingofaTDRsub-group in January 1991.

Institutions wishing to send a representative to the May meeting should send letters of application to Dr F.J. Lopez-Anturiano, World Health Organization. Regional Office for the Americas/Pan Ameri- can Sanitary Bureau, 525 23rd Street NW, Washington DC 20037, USA, with a copy to the Coordinator of the FIELDLINCS Pro- gramme at the address below.

For information about the preparation of letters of application or about the Project Development Grants, please address correspon- dence to: jacqueline Cattani, PhD, Coordinator of field Links for Intervention and Control Studies Programme, JDR, World Health Organization, I2 I I Geneva 27, Switzerland.