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.

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Heparin</td><td>Sigma</td><td>375095-100KU</td><td></td></tr><tr><td>Xanthurenic acid</td><td>Sigma</td><td>D120804-5G</td><td></td></tr><tr><td>Hypoxanthine</td><td>Sigma</td><td>H9377-25G</td><td></td></tr><tr><td>Alsever&#039;s solution</td><td>Sigma</td><td>A3551-500ML</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>Phenylhydrazine</td><td>Sigma</td><td>P26252-5G</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-500ML</td><td></td></tr><tr><td>Giemsa</td><td>Sigma</td><td>GS1L-1L</td><td></td></tr><tr><td>26 G x 3/8 Precision Glide Needle,&amp;nbsp;</td><td>Becton Dickinson</td><td>305110</td><td></td></tr><tr><td>1 mL TB Syringe, 26 G x 3/8</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>1 cc Insulin Syringe, U-100 27 G</td><td>Becton Dickinson</td><td>329412</td><td></td></tr><tr><td>Isoflurane, USB</td><td>Piramal</td><td>2667- 46- 7</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>22400105</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995065</td><td></td></tr><tr><td>Pencillin/Streptomycin</td><td>Gibco</td><td>10378016</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>Fiber Glass Wool</td><td>Corning</td><td>3950</td><td></td></tr></tbody></table>

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. ...

phenotypic-analysis-rodent-malaria-parasite-asexual-sexual-blood

Research

JoVE Journal

Immunology and Infection

11.5K Views.  Bezmialem Vakif University, Istanbul, Turkey. Door de opvallende gelijkenissen van de levenscyclus en biologie van knaagdiermalariaparasieten, de menselijke malariaparasieten, zijn knaagdiermalariamodellen onmisbaar geworden voor malariaonderzoek. Deze standaardmethoden kunnen helpen bij het beantwoorden van belangrijke vragen op het gebied van malariaonderzoek over hoe u een fenotypische analyse van wilde type en transgene knaagdiermalariasoorten uitvoeren. Het aantonen van de procedures zal worden afgestudeerde studenten Gozde Deveci e...

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

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Biology

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicine

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

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

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