Авторы благодарят Bloomberg Philanthropies за финансовую поддержку Научно-исследовательского института малярии Джона Хопкинса (JHMRI). Эта работа была бы невозможна без экспертных знаний, предоставленных основными учреждениями JHMRI по насекомым и паразитологии.

<ol>
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L., Mlambo, G., Tripathi, A., Dong, Y., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+plasmodium+is+anopheles+species+specific+and+infection+intensity+dependent.">Immune regulation of plasmodium is anopheles species specific and infection intensity dependent.</a> mBio. 8 (5), 01631 (2017).</li><li>Oakley, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+based+detection+of+Plasmodium+falciparum+development+in+Anopheles+stephensi+mosquitoes.">Transcriptome analysis based detection of Plasmodium falciparum development in Anopheles stephensi mosquitoes.</a> Scientific Reports. 8, 11568 (2018).</li><li>Saraiva, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromobacterium+spp.+mediate+their+anti-Plasmodium+activity+through+secretion+of+the+histone+deacetylase+inhibitor+romidepsin.">Chromobacterium spp. mediate their anti-Plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin.</a> Scientific Reports. 8, 6176 (2018).</li><li>Tao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-partitioning+of+the+Plasmodium+falciparum+stage+V+gametocyte+proteome+provides+insight+into+falciparum-specific+cell+biology.">Sex-partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology.</a> Molecular and Cellular Proteomics. 13 (10), 2705-2724 (2014).</li><li>Grabias, B., Zheng, H., Mlambo, G., Tripathi, A. K., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+enhanced+chemiluminescent-ELISA+for+the+detection+of+Plasmodium+falciparum+circumsporozoite+antigen+in+midguts+of+Anopheles+stephensi+mosquitoes.">A sensitive enhanced chemiluminescent-ELISA for the detection of Plasmodium falciparum circumsporozoite antigen in midguts of Anopheles stephensi mosquitoes.</a> Journal of Microbiological Methods. 108, 19-24 (2015).</li><li>Ferrer, P., Vega-Rodriguez, J., Tripathi, A. K., Jacobs-Lorena, M., Sullivan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimalarial+iron+chelator+FBS0701+blocks+transmission+by+Plasmodium+falciparum+gametocyte+activation+inhibition.">Antimalarial iron chelator FBS0701 blocks transmission by Plasmodium falciparum gametocyte activation inhibition.</a> Antimicrobial Agents and Chemotherapy. 59 (3), 1418-1426 (2015).</li><li>Sanders, N. G., Sullivan, D. J., Mlambo, G., Dimopoulos, G., Tripathi, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gametocytocidal+screen+identifies+novel+chemical+classes+with+Plasmodium+falciparum+transmission+blocking+activity.">Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.</a> PLoS One. 9 (8), 105817 (2014).</li><li>Lindner, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomics+and+proteomics+reveal+two+waves+of+translational+repression+during+the+maturation+of+malaria+parasite+sporozoites.">Transcriptomics and proteomics reveal two waves of translational repression during the maturation of malaria parasite sporozoites.</a> Nature Communications. 10, 4964 (2019).</li><li>McLean, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Transmission-Competent+Human+Malaria+Parasites+with+Chromosomally-Integrated+Fluorescent+Reporters.">Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters.</a> Scientific Reports. 9, 13131 (2019).</li><li>Espinosa, D. 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Авторам нечего раскрывать.

Методы, описанные здесь, успешно используются в Научно-исследовательском институте малярии Джона Хопкинса более 10 лет15,16,17,18,19,20,21,22. Гаметоц...

Малярия вызывается паразитами Plasmodium и передается их позвоночным хозяевам через инфекционный укус самок комаров Anopheles. Согласно докладу Всемирной организации здравоохранения (ВОЗ) за 2019 год, было зарегистрировано 405 000 смертей из 228 миллионов случаевмалярии1. Большинство случаев смерти от малярии было сосредоточено в африканском регионе, особенно среди детей в возрасте до пяти лет. В то время как общий уровень заболеваемости малярией снизился во всем мире с 2010 года, в последние годы спад стабилизировался, и для ликвидации этого заболевания срочно необходимы дополнительные стратегии борьбы.
Циклические стадии бесполой крови малярийных паразитов вызывают патогенез заболевания, и небольшое подмножество из них дифференцируется на женские и мужские гаметоциты. Гаметоциты Plasmodium falciparum уникальны по своей природе, так как им занимает 7-10 дней для развития через пять морфологически различных стадий. Незрелые гаметоциты от I до IV стадии секвестрируются в паренхиме костного мозга и в значительной степени остаются отсутствующими в периферическом кровообращении2,3,4,5. Эритроциты, инфицированные зрелыми гаметоцитами V стадии, высвобождаются в кровотоке и свободно циркулируют, чтобы быть захваченными комарами. Попав внутрь москитовой средней кишки, гаметоциты активируются, посредством изменения температуры и воздействия среды средней кишки, трансформируются в женские и мужские гаметы и начинают развитие стадий комара, которое завершается инфекционными стадиями спорозоитов в слюнных железах комара6,7.
С тех пор, как Трагер и Дженсон8 описали стандартизированный метод культивирования P. falciparum,исследования стадий беспологий крови значительно продвинулись. Однако отсутствие надежной системы культивовки половых стадий затруднило изучение гаметоцитов P. falciparum, биологии передачи и стадий комаров. В последние годы было опубликовано несколько методов, которые помогли лабораториям в создании культур гаметоцитов9,10,11,12. Эта рукопись описывает стандартизированный и надежный протокол к культуре гаметоцитов P. falciparum, который может представлять собой ценный ресурс для сообщества исследователей малярии. Этот метод обеспечивает надежную выработку зрелых и инфекционных гаметоцитов, что наряду со стандартизированным протоколом кормления комаров приводит к высоконадежной инфекционности комаров. Эти методы были установлены для поддержания бесперебойного снабжения гаметоцитами и паразитами стадии комаров. В этой рукописи мы описываем тщательный протокол посева гаметоцитов(Рисунок 1),приготовление кормушек со стеклянной мембраной и заражение комаров с помощью этих мембранных кормушек(Рисунок 2),рассечение средней кишки(Рисунок 3)и слюнных желез комаров(Рисунок 4),а также количественную оценку инфекции у комаров после рассечения средней кишки и слюнных желез.

<table><tbody><tr><td>10% Sugar solution</td><td></td><td></td><td></td></tr><tr><td>10ml serological pipet</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>15 ml conical tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>1ml serological pipet</td><td>Falcon</td><td>357521</td><td></td></tr><tr><td>25 ml serological pipet</td><td>Falcon</td><td>357535</td><td></td></tr><tr><td>37&amp;deg;C Incubator</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>5ml serological pipet</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>6 well tissue culture plates</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>9&quot; glass pipet</td><td>Fisherbrand</td><td>13-678-6B</td><td></td></tr><tr><td>Anopheles Mosquitoes</td><td>JHMRI, Insectary core</td><td></td><td>We use A. stephensi or A. gambiae (keele)</td></tr><tr><td>cell counter</td><td></td><td></td><td></td></tr><tr><td>Circulating water bath</td><td></td><td></td><td></td></tr><tr><td>fine tip forceps</td><td>Fisherbrand</td><td>12-000-122</td><td></td></tr><tr><td>Geimsa stain</td><td>Sigma</td><td>GS1L</td><td></td></tr><tr><td>Glass desiccator</td><td></td><td></td><td></td></tr><tr><td>Glass membrane feeder</td><td>Chemglass Life Sciences</td><td>CG183570</td><td></td></tr><tr><td>Glass slides</td><td>Fisherbrand</td><td>12-552-3</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H6648</td><td></td></tr><tr><td>Human Blood O+</td><td>JHU</td><td></td><td>Wash RBCs three times with RPMI and refrigerate at 50% heamatocrit</td></tr><tr><td>Human Serum O+</td><td>Interstate blood bank</td><td></td><td>Pool at-least 6 units of serum from different donors and freeze down aliquots at -20&amp;deg;C.</td></tr><tr><td>Hypoxanthine</td><td>Sigma</td><td>H9337</td><td>Make 500x stock in 1M NaOH</td></tr><tr><td>Mercurochrome</td><td>Sigma</td><td>M7011</td><td>Prepare 1% stock solution in PBS that can be diluted to 0.1% when needed</td></tr><tr><td>Micro Pipette</td><td></td><td></td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td></td><td>Any microscope with 10x, 40x and 100x objective will work.</td></tr><tr><td>Mosquito cups</td><td>Neptune cups</td><td></td><td></td></tr><tr><td>N-acetylglucosamine</td><td>Sigma</td><td>A3286</td><td>Optional and needed only when pure gametocytes are required.</td></tr><tr><td>Netting</td><td></td><td></td><td>Make sure it can contain mosquitoes and allow blood feeding</td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Petri dish</td><td>Thermo Scientific</td><td>249964</td><td></td></tr><tr><td>Pipet tips</td><td></td><td></td><td></td></tr><tr><td>Pipetman</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Plasmodium falciparum&lt;/em&gt; NF54</td><td>BEI Resources</td><td>MRA-1000</td><td>Freeze down large numbers of early passage culture to make sure you have a constant supply</td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>CV-041-CV</td><td>Media contains glutamine and HEPES</td></tr><tr><td>Slide warmer</td><td></td><td></td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6297</td><td>Optional for media, add only when using malaria gas mix during culture incubation</td></tr><tr><td>water bath</td><td></td><td></td><td></td></tr><tr><td>Xanthurenic Acid</td><td>Sigma</td><td>D120804</td><td>For flagellation media</td></tr></tbody></table>

plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding

Малярия остается одной из наиболее важных проблем общественного здравоохранения, вызывающей значительную заболеваемость и смертность. Малярия является заболеванием, передаваемым комарами, передаваемым через инфекционный укус от самки комара Anopheles. Борьба с малярией в конечном итоге будет опираться на множество подходов, которые включают способы блокирования передачи комарам, через них и от них. Для изучения стадий малярийных паразитов комаров в лаборатории мы оптимизировали протокол культивирования высокоинфекционных гаметоцитов Plasmodium falciparum, стадии паразита, необходимой для передачи от человека-хозяина комару-переносчику. Гаметоциты P. falciparum созревают через пять морфологически различных этапов, что занимает примерно 1-2 недели. Культура гаметоцитов, описанная в этом протоколе, завершается за 15 дней и заразна для комаров с 15-18 дней. Эти протоколы были разработаны для поддержания непрерывного цикла заражения компетентными гаметоцитами и для поддержания бесперебойного снабжения комаров стадиями паразита. Здесь мы описываем методологию культивированию гаметоцитов и то, как заразить комаров этими паразитами с помощью стеклянных мембранных кормушек.

Здесь мы представляем результаты серии мембранных кормов с использованием культур гаметоцитов P. falciparum NF54, полученных с использованием протокола выше (см.рисунок 5). Культура гаметоцитов была начата примерно с 0,5% смешанной стадии бесполой культуры на 0-й день, котор?...

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Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Детальные исследования стадий малярийных паразитов комаров имеют решающее значение для разработки эффективных стратегий блокирования передачи. Этот протокол демонстрирует, как эффективно культивировать инфекционные гаметоциты, а затем скармливать эти гаметоциты комарам для генерации стадий комаров P. falciparum.

Плазмодий фальципарум Культура гаметоцитов и инфекция комаров через искусственное мембранное питание

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

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Research

JoVE Journal

Biology

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

7.4K Views.  Johns Hopkins University. Детальные исследования стадий малярийных паразитов комаров имеют решающее значение для разработки эффективных стратегий блокирования передачи. Этот протокол демонстрирует, как эффективно культивировать инфекционные гаметоциты, а затем скармливать эти га...

Video: Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Альфавирус трансдуцирующих Система: Инструмент для визуализации инфекции у комаров-переносчиков

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Immunology and Infection

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.

Протокол по производству Генетические Креста Паразиты грызунов малярией

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 Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Протокол денге инфекций в Комары (A. aegypti) и определение инфекции Фенотип

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

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.

Протокол Plasmodium Инфекции в тропической Определение фенотипа Комары и инфекции

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

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.

Экспериментальная модель для изучения Туберкулез-малярией Коинфекция на естественной передачи Микобактерии туберкулеза И Plasmodium berghei</i

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

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.

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.

Фенотипический анализ грызунов малярии паразита асексуальных и сексуальной крови этапов и комаров этапов

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

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.

In Vivo Отслеживание развития отек и микрососудистая патология в модели экспериментальной церебральной малярии с использованием магнитно-резонансной томографии

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.

Стандартный анализ мембранного питания для выявления инфекции Plasmodium falciparum у комаров-переносчиков Anopheles

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.

Генерация генетически модифицированных спорозоитов Plasmodium berghei

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

<meta charset="utf8"></head><body>Идентификация перестройки киназ у малярийного паразита: химический генетический подход

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

<meta charset="utf8"></head><body>В центре внимания автора: Идентификация компенсаторных путей у малярийных паразитов, содержащих гипоморфный аллель эссенциальных белковкиназ

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

Плазмодий фальципарум Культура гаметоцитов и инфекция комаров через искусственное мембранное питание

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