Name: phenotypic analysis of rodent malaria parasite asexual and sexual blood stages and mosquito stages
Uploaded: 2019-05-30T12:30:00
Description: Watch this Scientific Journal Video about Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages at JoVE.com

Ahmed Aly is supported by funding to Bezmialem Vakif University from the Turkish Ministry of Development grant 2015BSV036, and by funding provided by the Tulane University School of Public Health and Tropical Medicine, and by funding from NIH-NIAID for R21Grant 1R21AI111058-01A1.

All animal experiments described here were conducted according to the approved protocols of the Institutional Animal Care and Use Committee (IACUC) of Tulane University and the animals ethics committee of Bezmialem Vakif University. All other experimental protocols and the use of recombinant DNA were conducted according to the approved protocols of the Institutional Biosafety Committee (IBC) of Tulane University.
1. Infection of Mice with Blood-stage Parasites for Parasitemia Analysis and Mosqui...

In spite of the similarity in the general biology of their life cycles to that of human malaria parasites, mouse malaria models also have many dissimilarities to human Plasmodium species that would limit their use as reliable in vivo models. For instance, with the exception of live-attenuated parasites as vaccines, all vaccine studies with subunit and DNA and other vaccines gave excellent results in the mouse model, but in humans living in endemic areas, the results were far from satisfactory.
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Malaria parasites cause hundreds of millions of malaria infections in humans worldwide, with more than 600,000 deaths every year1. Human infections are caused by five malaria parasite species, namely P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Most clinical malaria mortalities are caused by P. falciparum in sub-Saharan Africa1.&#160;Another human malaria parasite species that causes extensive worldwide morbidities outside of sub-Saharan Africa is P. vivax2.&#160;The other three species are all more geographically restricted and cause benign malaria infections, except the lethal P. knowlesi3. The unavailability of relevant and practical non-human in vivo models of infections has always been and still is an obstacle to malaria vaccine and drug development. Earlier malaria drug targeting and metabolic studies have relied extensively on avian malaria models like P. gallinaceum and P. lophurae, infecting chickens and ducks, respectively4. Thereafter, rodent malaria species were gradually introduced in various vaccines and drug targeting studies as in vivo models. Over the years, evidence of similarities of the biology and host-parasite interactions of life cycle stages of rodent malaria models to human malaria species have accumulated.
In particular, rodent malaria models were extremely important to explore and characterize the biology of mosquito and pre-erythrocytic stages5. However, there are four rodent malaria species (P. berghei, P. yoelii, P. chabaudi, and P. vinckei) that have different biological features, the most notable of which are in the blood stages6. Rodent malaria species differ in the synchronicity of blood stages, where blood stages of P. chabaudi and P. vinckei strains are mostly synchronous, while the blood stages of P. berghei and P. yoelii are not6,7. Another notable difference is the self-clearance of blood stages that occurs in some strains (e.g., P. yoelii 17X-NL, P. berghei NK65, and P. vinckei lentum), whereas the blood infection of other strains of the same species could be lethal if left untreated (P. yoelii 17X-L, P. berghei ANKA, and P. chabaudi AS). Moreover, P. yoelii 17X-NL strain and P. berghei ANKA strain preferentially invade reticulocytes8,9,10,11, although these features of P. yoelii and P. berghei strains are not a strict growth requirement12,13,14. Therefore, mice are treated with phenylhydrazine prior to an infection with the blood stages of those parasites to increase the parasitemia and gametocytemia needed for a mosquito infection for the P. berghei ANKA strain and for P. yoelii 17X-NL15,16,17,18,19.
Differences in mosquito stages development also exist among different rodent malaria species, the most notable being the temperature and time required for optimal mosquito stages development and the sporozoite length5,6,20.In pre-erythrocytic stages of rodent malaria species, differences include the rodent species and strain that are most susceptible to infectious sporozoite inoculation, the number of sporozoites needed for inoculation in a susceptible rodent strain, the mammalian cell types needed for in vitro liver stage development assays, and the time to complete liver stage development5,21,22,23,24,25,26,27,28,29,30.
Despite these variabilities, rodent malaria parasites were the favorable models early on for the application of reverse genetic approaches, because they were less time- and resource-consuming with a high probability of success31. In fact, rodent malaria models were the best models, and in many instances the only models, available for numerous years to functionally characterize genes expressed in mosquito and liver stages.
In light of the popularity and amenability of reverse genetic approaches in rodent malaria models, a number of different methodologies have been utilized to analyze the phenotypes of transgenic parasite life cycle stages, especially blood stages. However, some of these methodologies are inconsistent; for instance, comparing infections of blood-stage parasites following an IP injection (which are possibly drained to the peritoneal lymph nodes and, from there, can enter the bloodstream; therefore, the injected parasites do not end up equally in the bloodstream), comparing the mosquito transmission of clones with a different number of serial blood-stage transfers or G number (which could affect gametocytogenesis32,33), or comparing transgenic parasites directly to naive wild-type (WT) parasites that were never subjected to electroporation and positive drug selection and the various unstandardized evaluations of male gamete exflagellation. Therefore, it is crucial to standardize protocols that are simple to follow for the phenotypic analysis of any type of transgenic or WT rodent malaria parasites in the blood and in the mosquito to accommodate for the biological variabilities of rodent malaria parasite species.
Herein, we report on a standardized, detailed experimental protocol for the phenotypic analysis of the blood and mosquito life cycle stages of transgenic or wild-type P. yoelii and P. berghei parasites. These protocols are also applicable to P. chabaudi and P. vinckei parasites.

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phenotypic analysis of rodent malaria parasite asexual and sexual blood stages and mosquito stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

The success of applying reverse genetic tools and techniques to malaria parasites has revolutionized the field of malaria research, with the ability to add, delete, or modify specific genomic segments of several Plasmodium species39. Importantly, dispensable genomic loci have been identified and used successfully to introduce fluorescence protein markers in rodent and human malaria parasites by double homologous recombination, to ensure a stable expression...

Watch this Scientific Journal Video about Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages at JoVE.com

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

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