Les auteurs remercient Bloomberg Philanthropies pour son soutien financier au Johns Hopkins Malaria Research Institute (JHMRI). Ce travail n’aurait pas été possible sans l’expertise fournie par les installations de base d’insectes et de parasitologie de JHMRI.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Malaria+Report.">World Malaria Report.</a> World Health Organization. , (2018).</li><li>Sinden, R. E., Smalley, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gametocytogenesis+of+Plasmodium+falciparum+in+vitro:+The+cell-cycle.">Gametocytogenesis of Plasmodium falciparum in vitro: The cell-cycle.</a> Parasitology. 79 (2), 277-296 (1979).</li><li>Sinden, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+Development+of+Malarial+Parasites.">Sexual Development of Malarial Parasites.</a> Advances in Parasitology. 22, 153-216 (1983).</li><li>Joice, R., 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=Plasmodium+falciparum+transmission+stages+accumulate+in+the+human+bone+marrow.">Plasmodium falciparum transmission stages accumulate in the human bone marrow.</a> Science Translational Medicine. 6 (244), 5 (2014).</li><li>Abdulsalam, A. H., Sabeeh, N., Bain, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+Plasmodium+falciparum+gametocytes+in+bone+marrow.">Immature Plasmodium falciparum gametocytes in bone marrow.</a> American Journal of Hematology. 85 (12), 943 (2010).</li><li>Ghosh, A. K., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+sporozoite+invasion+of+the+mosquito+salivary+gland.">Plasmodium sporozoite invasion of the mosquito salivary gland.</a> Current Opinion in Microbiology. 12 (4), 394-400 (2009).</li><li>Bennink, S., Kiesow, M. J., Pradel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+malaria+parasites+in+the+mosquito+midgut.">The development of malaria parasites in the mosquito midgut.</a> Cellular Microbiology. 18 (7), 905-918 (2016).</li><li>Trager, W., Jenson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+malarial+parasites.">Cultivation of malarial parasites.</a> Nature. 273 (5664), 621-622 (1978).</li><li>Duffy, S., Loganathan, S., Holleran, J. P., Avery, V. 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Falciparum gametocytes to evaluate novel transmission-blocking interventions.</a> Nature Protocols. 11 (9), 1668-1680 (2016).</li><li>Habtewold, T., 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=Streamlined+SMFA+and+mosquito+dark-feeding+regime+significantly+improve+malaria+transmission-blocking+assay+robustness+and+sensitivity.">Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity.</a> Malaria Journal. 18 (1), 24 (2019).</li><li>Demanga, C. 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=The+development+of+sexual+stage+malaria+gametocytes+in+a+Wave+Bioreactor.">The development of sexual stage malaria gametocytes in a Wave Bioreactor.</a> Parasites and Vectors. 10 (1), 216 (2017).</li><li>Brockelman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+favoring+gametocytogenesis+in+the+continuous+culture+of+Plasmodium+falciparum.">Conditions favoring gametocytogenesis in the continuous culture of Plasmodium falciparum.</a> Journal of Eukaryotic Microbiology. 29, 454-458 (1982).</li><li>Meibalan, E., Marti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+malaria+transmission.">Biology of malaria transmission.</a> Cold Spring Harbor Perspectives in Medicine. 7, (2017).</li><li>Essuman, 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=A+novel+gametocyte+biomarker+for+superior+molecular+detection+of+the+plasmodium+falciparum+infectious+reservoirs.">A novel gametocyte biomarker for superior molecular detection of the plasmodium falciparum infectious reservoirs.</a> Journal of Infectious Diseases. 216 (10), 1264-1272 (2017).</li><li>Sim&#245;es, M. 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. A., 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=Proteolytic+Cleavage+of+the+Plasmodium+falciparum+Circumsporozoite+Protein+Is+a+Target+of+Protective+Antibodies.">Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.</a> Journal of Infectious Diseases. 212 (7), 1111-1119 (2015).</li><li>Swearingen, K. 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=Interrogating+the+Plasmodium+Sporozoite+Surface:+Identification+of+Surface-Exposed+Proteins+and+Demonstration+of+Glycosylation+on+CSP+and+TRAP+by+Mass+Spectrometry-Based+Proteomics.">Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics.</a> PLoS Pathogens. 12 (4), 1005606 (2016).</li><li>Ifediba, T., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vitro+maturation+of+Plasmodium+falciparum+gametocytes.">Complete in vitro maturation of Plasmodium falciparum gametocytes.</a> Nature. 294 (5839), 364-366 (1981).</li><li>Miura, K., 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=An+inter-laboratory+comparison+of+standard+membrane-feeding+assays+for+evaluation+of+malaria+transmission-blocking+vaccines.">An inter-laboratory comparison of standard membrane-feeding assays for evaluation of malaria transmission-blocking vaccines.</a> Malaria Journal. 15, 463 (2016).</li><li>Miura, K., 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=Qualification+of+Standard+Membrane-Feeding+Assay+with+Plasmodium+falciparum+Malaria+and+Potential+Improvements+for+Future+Assays.">Qualification of Standard Membrane-Feeding Assay with Plasmodium falciparum Malaria and Potential Improvements for Future Assays.</a> PLoS One. 8 (3), 57909 (2013).</li></ol>

Les auteurs n’ont rien à divulguer.

Les méthodes décrites ici ont été utilisées avec succès à l’Institut de recherche sur le paludisme Johns Hopkins depuis plus de10ans15,16,17,18,19,20,21,22. Les gamétocytes produits à l’aide de ce protocole ont été utilisés pour des tests ga...

Le paludisme est causé par les parasites Plasmodium et est transmis à leurs hôtes vertébrés par la piqûre infectieuse de moustiques anophèles femelles. Selon le rapport 2019 de l’Organisation mondiale de la santé (OMS), on estime à 405 000 le nombre de décès, sur un total de 228 millions de cas de paludisme1. La plupart des décès liés au paludisme étaient concentrés dans la région africaine, en particulier chez les enfants de moins de cinq ans. Alors que le taux d’incidence global du paludisme a diminué à l’échelle mondiale à partir de 2010, le déclin a plafonné ces dernières années et des stratégies de lutte supplémentaires sont nécessaires de toute urgence pour éliminer la maladie.
Les stades sanguins asexués cycliques des parasites du paludisme provoquent une pathogenèse de la maladie et un petit sous-ensemble de ceux-ci se différencient en gamétocytes femelles et mâles. Les gamétocytes de Plasmodium falciparum sont uniques par nature car ils prennent de 7 à 10 jours à se développer à travers cinq stades morphologiquement distincts. Les gamétocytes immatures du stade I à IV sont séquestrés dans le parenchyme de la moelle osseuse et restent largement absents de la circulation périphérique2,3,4,5. Les érythrocytes infectés par des gamétocytes matures de stade V sont libérés dans la circulation sanguine et circulent librement pour être pris par les moustiques. Une fois à l’intérieur de l’intestin moyen du moustique, les gamétocytes sont activés, par un changement de température et une exposition à l’environnement de l’intestin moyen, se transforment en gamètes femelles et mâles et commencent le développement des stades de moustique, qui culmine avec les stades infectieux des sporozoïtes dans les glandes salivaires des moustiques6,7.
Depuis que Trager et Jenson8 ont décrit une méthode standardisée de culture de P. falciparum,les études sur les stades sanguins asexués ont considérablement progressé. Cependant, l’absence d’un système de culture fiable pour les stades sexuels a rendu difficile l’étude des gamétocytes de P. falciparum, de la biologie de la transmission et des stades des moustiques. Ces dernières années, plusieurs méthodes ont été publiées qui ont aidé les laboratoires à établir des cultures de gamétocytes9,10,11,12. Ce manuscrit décrit un protocole normalisé et fiable pour la culture des gamétocytes de P. falciparum qui peut représenter une ressource précieuse pour la communauté de recherche sur le paludisme. Cette méthode permet la production robuste de gamétocytes matures et infectieux qui, avec un protocole d’alimentation normalisé des moustiques, se traduit par une infectiosité très fiable des moustiques. Ces méthodes ont été établies pour maintenir un approvisionnement ininterrompu en gamétocytes et en parasites au stade des moustiques. Dans ce manuscrit, nous décrivons un protocole complet de culture de gamétocytes (Figure 1), la préparation de mangeoires à membrane de verre et l’infection des moustiques à l’aide de ces mangeoires membranaires (Figure 2), la dissection de l’intestin moyen (Figure 3) et de la glande salivaire des moustiques (Figure 4), et la quantification de l’infection chez les moustiques après la dissection de l’intestin moyen et des glandes salivaires.

<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

Le paludisme reste l’un des problèmes de santé publique les plus importants, causant une morbidité et une mortalité importantes. Le paludisme est une maladie transmise par les moustiques transmise par une piqûre infectieuse du moustique anophèle femelle. La lutte contre le paludisme reposera éventuellement sur une multitude d’approches, notamment des moyens de bloquer la transmission vers, par et depuis les moustiques. Pour étudier les stades des moustiques des parasites du paludisme en laboratoire, nous avons optimisé un protocole de culture de gamétocytes hautement infectieux de Plasmodium falciparum, un stade parasitaire nécessaire à la transmission de l’hôte humain au moustique vecteur. Les gamétocytes de P. falciparum mûrissent à travers cinq étapes morphologiquement distinctes, ce qui prend environ 1 à 2 semaines. La culture de gamétocytes décrite dans ce protocole est terminée en 15 jours et est infectieuse pour les moustiques des jours 15 à 18. Ces protocoles ont été développés pour maintenir un cycle continu de gamétocytes compétents en matière d’infection et pour maintenir un approvisionnement ininterrompu des stades de moustiques du parasite. Ici, nous décrivons la méthodologie de culture de gamétocytes et comment infecter les moustiques avec ces parasites en utilisant des mangeoires à membrane de verre.

Nous présentons ici les résultats d’une série d’alimentations membranaires utilisant des cultures de gamétocytes P. falciparum NF54 générées à l’aide du protocole ci-dessus (voir (Figure 5). La culture de gamétocytes a été initiée avec environ 0,5% de culture asexuée au stade mixte le jour 0, qui a atteint un pic de parasitémie d’environ 15% au jour 4 et au jour 5. Comme le montre la figure 5A à cette parasitémie élevée, les pa...

Watch this Scientific Journal Video about Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding at JoVE.com

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Des études détaillées sur les stades des moustiques des parasites du paludisme sont essentielles pour concevoir des stratégies efficaces de blocage de la transmission. Ce protocole montre comment mettre en culture efficacement des gamétocytes infectieux, puis nourrir ces gamétocytes avec des moustiques pour générer des stades de moustiques de P. falciparum.

Plasmodium falciparum Culture de gamétocytes et infection par les moustiques par l’alimentation par membrane artificielle

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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Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

7.4K vues.  Johns Hopkins University. Des études détaillées sur les stades des moustiques des parasites du paludisme sont essentielles pour concevoir des stratégies efficaces de blocage de la transmission. Ce protocole montre comment mettre en culture efficacement des gamétocytes infectieux, puis nourrir ces gamétocytes avec des moustiques pour générer des stades de moustiques de P. falciparum.

Vidéo: 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.

Système Alphavirus Transducing: des outils de visualisation des infections dans les moustiques vecteurs

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

Recherche

Immunologie et 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.

Protocole pour la production d&#39;un croisement génétique des parasites du paludisme des rongeurs

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.

Protocole pour la dengue dans les moustiques (Aedes aegypti) et détermination de phénotype d&#39;infection

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 ...

Biologie

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.

Protocole pour les infections à Plasmodium falciparum dans la détermination Phénotype moustiques et les infections

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.

Un modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et 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.

Analyse phénotypique des stades du sang asexué et sexuel du parasite du paludisme des rongeurs et des stades des moustiques

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.

Le suivi in vivo du développement de l&#39;œdème et de la pathologie microvasculaire dans un modèle de paludisme cérébral expérimental utilisant l&#39;imagerie par résonance magnétique

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 ...

Médecine

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.

Dosage standard d’alimentation par membrane pour la détection de l’infection à Plasmodium falciparum chez les moustiques vecteurs anophèles

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.

Génération de sporozoïtes génétiquement modifiés à 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>Identification du recâblage des kinases chez le parasite du paludisme : une approche de génétique chimique

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>Pleins feux sur l’auteur : Identification des voies compensatoires chez les parasites du paludisme contenant un allèle hypomorphe de protéines kinases essentielles

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.

Comprendre le développement de voies compensatoires chez un parasite mutant du paludisme hébergeant un allèle hypomorphe de kinases ressemblant à des plantes

Plasmodium falciparum Culture de gamétocytes et infection par les moustiques par l’alimentation par membrane artificielle

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