Name: plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding
Uploaded: 2020-07-03T15:30:00
Description: Watch this Scientific Journal Video about Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding at JoVE.com

Forfattere takker Bloomberg Philanthropies for økonomisk støtte til Johns Hopkins Malaria Research Institute (JHMRI). Dette arbeidet ville ikke vært mulig uten ekspertisen fra JHMRI insekt- og parasitologikjerneanlegg.

Blodsamlinger beskrevet nedenfor er godkjent av Institutional Review Board ved Johns Hopkins University. P. falciparum dyrkes i ferske RBCer under sterile forhold i et biosikkerhetsanlegg på nivå 2 (BSL2), og forsiktighet brukes til å håndtere biologiske materialer. Etter hvert trinn som involverer blod eller blodprodukter, skylles hvert plasttøy eller glass med 10% blekemiddel i hetten før riktig avhending.
1. Reagenser og forberedelser
<ol><li>Parasitten isolerer P. falc...

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+production+of+Plasmodium+falciparum+gametocytes+for+malaria+drug+discovery.">Large-scale production of Plasmodium falciparum gametocytes for malaria drug discovery.</a> Nature Protocols. 11 (5), 976-992 (2016).</li><li>Delves, M. 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=Routine+in+vitro+culture+of+P.+Falciparum+gametocytes+to+evaluate+novel+transmission-blocking+interventions.">Routine in vitro culture of P. 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>

Metoder beskrevet her har blitt vellykket brukt ved Johns Hopkins Malaria Research Institute i mer enn 10 år15,16,17,18,19,20,21,22. Gametocytter produsert ved hjelp av denne protokollen har blitt brukt til høy gjennomstrømning gametocytocidal analyser<s...

Malaria er forårsaket av Plasmodium parasitter og overføres til vertebratvertene sine via smittsom bite av kvinnelige Anopheles mygg. Ifølge rapporten fra Verdens helseorganisasjon (WHO) for 2019 var det anslagsvis 405 000 dødsfall, fra totalt 228 millioner tilfeller av malaria1. De fleste malariarelaterte dødsfallene var konsentrert i den afrikanske regionen, spesielt blant barn under fem år. Mens den totale forekomsten av malaria har gått ned globalt fra 2010, har nedgangen de siste årene platået og det haster med ytterligere kontrollstrategier for å eliminere sykdommen.
Sykliske aseksuelle blodstadier av malariaparasitter forårsaker sykdomspatogenese og en liten undergruppe av disse skiller seg ut i kvinnelige og mannlige gametocytter. Plasmodium falciparum gametocytes er unike i naturen som de tar 7-10 dager å utvikle gjennom fem morfologisk distinkte stadier. Umodne gametocytter fra stadium I til IV er beslaglagt i benmargsparenchyma og forblir i stor grad fraværende fra perifersirkulasjon 2,3,4,5. Erytrocytter smittet med modne stadium V gametocytter frigjøres i blodet og sirkulerer fritt for å bli tatt opp av mygg. En gang inne i mygg midgut aktiveres gametocytter, gjennom en temperaturendring og eksponering for midgutmiljøet, forvandles til kvinnelige og mannlige gameter og begynner utviklingen av myggstadiene, som kulminerer med de smittsomnde stadiene av sporozoitter i myggspyttkjertlene6,7.
Siden Trager og Jenson8 beskrev en standardisert metode for kultur P. falciparum, studier på de aseksuelle blodstadiene har sterkt avansert. Mangelen på et pålitelig kultursystem for seksuelle stadier har imidlertid gjort det vanskelig å studere P. falciparum gametocytter, overføringsbiologi og myggstadier. De siste årene har det blitt publisert flere metoder som har hjulpet laboratorier med å etablere gametocyttkulturer9,10,11,12. Dette manuskriptet beskriver standardisert og pålitelig protokoll til kulturen P. falciparum gametocytes som kan representere en verdifull ressurs for malariaforskningssamfunnet. Denne metoden muliggjør robust produksjon av modne og smittsomme gametocytter som sammen med en standardisert myggfôringsprotokoll resulterer i svært pålitelig mygginfeksjonsevne. Disse metodene ble etablert for å opprettholde uavbrutt tilførsel av gametocytter og myggstadiumparasitter. I dette manuskriptet beskriver vi en grundig gametocyttkulturprotokoll (Figur 1), fremstilling av glassmembranmatere og infeksjon av mygg ved hjelp av disse membranmatere (figur 2), disseksjon av midgut (figur 3) og spyttkjertel av mygg (figur 4), og kvantifisering av infeksjon i mygg etter midgut og spyttkjertel disseksjon.

<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&#176;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&#34; 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&#176;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>Plasmodium falciparum 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>
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plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding

Malaria er fortsatt et av de viktigste folkehelseproblemene, noe som forårsaker betydelig sykelighet og dødelighet. Malaria er en myggbåren sykdom som overføres gjennom en smittsom bite fra den kvinnelige Anopheles mygg. Malariakontroll vil til slutt stole på en rekke tilnærminger, som inkluderer måter å blokkere overføring til, gjennom og fra mygg. For å studere myggstadier av malariaparasitter i laboratoriet, har vi optimalisert en protokoll for å dyrke svært smittsomme Plasmodium falciparum gametocytter, et parasittstadium som kreves for overføring fra den menneskelige verten til myggvektoren. P. falciparum gametocytes modnes gjennom fem morfologisk distinkte trinn, som tar ca 1-2 uker. Gametocyttkulturen beskrevet i denne protokollen er fullført på 15 dager og er smittsomme for mygg fra dag 15-18. Disse protokollene ble utviklet for å opprettholde en kontinuerlig syklus av infeksjon kompetente gametocytter og for å opprettholde uavbrutt tilførsel av mygg stadier av parasitten. Her beskriver vi metodikken til gametocyttkulturen og hvordan man infiserer mygg med disse parasittene ved hjelp av glassmembranmatere.

Her presenterer vi resultater fra en rekke membranfôr ved hjelp av P. falciparum NF54 gametocyttkulturer generert ved hjelp av protokollen ovenfor (se figur5). Gametocyttkulturen ble initiert med omtrent 0,5% blandet stadium aseksuell kultur på dag 0, som vokste til en topp parasittmia på omtrent 15% ved dag 4 og dag 5. Som vist i figur 5A på denne høye parasittmien, er parasittene stresset og den aseksuelle scenekulturen krasjer. Imidlertid result...

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

Detaljerte undersøkelser om myggstadier av malariaparasitter er avgjørende for å designe effektive overføringsblokkeringsstrategier. Denne protokollen demonstrerer hvordan du effektivt kan dyrke smittsomme gametocytter og deretter mate disse gametocyttene til mygg for å generere myggstadier av P. falciparum.

Plasmodium falciparum Gametocyttkultur og mygginfeksjon gjennom kunstig membranfôring

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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Biology

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

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