Plasmodium cynomolgi sporozoite production and sporozoite isolation

Here, we are providing the protocol for Plasmodium cynomolgi production as described earlier (Dembélé et al., 2014; Annemarie Voorberg-van der Wel et al., 2013). Our original manuscript in Bio-protocol contains detailed sporozoite isolation protocol and most appropriate references: (Dembélé et al., 2014). In briefly, P. cynomolgi sporozoites were obtained from infected A. dirus mosquitoes. Infected mosquitoes were obtained from the United States army medical directorate; armed forces research institute of medical sciences (AFRIMS) Bangkok 10400, Thailand. Mosquitoes (aged two to five days) were fed on blood from a Macaca fascicularis infected with P. cynomolgi bastianellii (also known as strain B) parasites using a glass feeder system. The mosquitoes were kept in climate chambers at a temperature of 25°C and 80% humidity and they were regularly fed via cotton soaked in a 5% D-glucose solution. Oocysts were counted post one-week infection and an uninfected blood meal was administered to the mosquitoes to facilitate the invasion of sporozoites into the salivary glands. Salivary glands were collected 14–30 days after a blood meal from an infected monkey. Following, sporozoites were isolated from infected salivary glands as described in the protocol section in the current paper.

References:
Dembélé, L., Franetich, J. F., Lorthiois, A., Gego, A., Zeeman, A. M., Kocken, C. H., Le Grand, R., Dereuddre-Bosquet, N., van Gemert, G. J., Sauerwein, R., Vaillant, J. C., Hannoun, L., Fuchter, M. J., Diagana, T. T., Malmquist, N. A., Scherf, A., Snounou, G. and Mazier, D. (2014). Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nat Med 20(3): 307-312.

Annemarie Voorberg-van der Wel, Anne-Marie Zeeman, Sandra M. van Amsterdam, Alexander van den Berg, Els J. Klooster, Shiroh Iwanaga, Chris J. Janse,Geert-Jan van Gemert, Robert Sauerwein, Niels Beenhakker, Gerrit Koopman, Alan W. Thomas, Clemens H. M. Kocken. (2013). Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms. PLoS ONE 8(1): e54888.