Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax

Abstract

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Introduction

Continuous in vitro culture of Plasmodium parasites has only been achieved for a few species, of which only one, P. falciparum specifically infects humans. This has greatly advanced knowledge on this parasite's biology, biochemistry, genetics, etc. To date, in vitro cultivation of P. vivax, a parasite responsible for increasing proportions of global malaria infection [1] has yet to be achieved [2]. Laboratory-based investigations on this important species have thus been restricted to brief ex-vivo manipulations of samples collected from patients, often harbouring genetically diverse strains, making comparative studies challenging. Traditionally, P. cynomolgi, a parasite species that naturally infects macaques in southeast Asia, has served as the animal model for P. vivax, because it recapitulates the morphological and biological characteristics of P. vivax, and it has now been confirmed to be phylogenetically highly close to P. vivax [3]. Previous demonstration that some strains of P. cynomolgi can be maintained in culture have recently spurred the development of a robust routine cultivation protocol for the P. cynomolgi Berok strain, the usefulness of which has been validated through extended length drug assays, isolation of a cloned line, etc. [4]. Although this surrogate model has made it possible to address long-standing issues on P. vivax, researchers have faced challenges specific to the in vitro maintenance of P. cynomolgi.

It was early realised that many of the standard conditions used for P. falciparum cultivation were not suitable for P. cynomolgi. For instance, P. cynomolgi unlike P. falciparum, does not grow under standard 5% CO₂, and serum replacements such as Albumax result in abnormal growth. Furthermore, the standard use of the antibiotic gentamycin to minimise risk of bacterial contamination, a particularly important consideration when cultures need to be maintained for extended periods, is not compatible with P. cynomolgi cultivation because this species is susceptible to gentamycin. Finally, the need for non-human primate blood and serum can impose substantial costs both monetary and bureaucratic if these are to be imported.

Here we present the results of our investigations aimed at optimizing P. cynomolgi culture conditions, which included testing serum substitutes, diverse antibiotic combinations, and synchronization methodologies, and we provide thereby optimised cultivation conditions that facilitate the maintenance and use of this parasite.