저자는 존스 홉킨스 말라리아 연구소 (JHMRI)에 재정 지원을 블룸버그 자선 사업가에게 감사드립니다. 이 작품은 JHMRI 곤충 및 기생충학 핵심 시설에서 제공하는 전문 지식없이는 가능하지 않았을 것입니다.

<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>

저자는 공개 할 것이 없습니다.

여기에 설명된 방법은 10년 이상 존스 홉킨스 말라리아 연구소에서 10년 이상15년,16년,17년,18년,19,20,21,22이상에성공적으로 사용되어 왔다. 이 프로토콜을 사용하여 생산된 게임토세포는 높은 처리...

말라리아는 플라스모듐 기생충에 기인하고 여성 아노페Les 모기의 전염성 바이트를 통해 그들의 척추 동물 호스트에게 전달됩니다. 2019년 세계보건기구(WHO) 보고서에 따르면 총 2억 2,800만 건의 말라리아1건에서약 405,000명의 사망자가 발생했다. 말라리아 관련 죽음의 대부분은 아프리카 지역에 집중되었습니다, 특히 5 세 이하의 아이들 중. 말라리아의 전반적인 부각 비율은 2010년부터 전 세계적으로 감소하는 동안, 최근 몇 년 동안 쇠퇴는 고원하고 추가 통제 전략은 질병을 제거하기 위하여 긴급하게 필요합니다.
말라리아 기생충의 순환 무성 혈액 단계는 질병 병인을 일으키는 원인이 되고 이들의 작은 부분 집합은 여성과 남성 게임토세포로 분화합니다. 플라스모듐 falciparum 게임 토세포는 5 개의 형태학적으로 뚜렷한 단계를 통해 개발하는 데 7-10 일이 걸리기 때문에 자연에서 독특합니다. 단계 I에서 IV까지미성숙한 게임토세포는 골수 parenchyma에서 격리되고 주로 말초 순환2,3,4,5에서결석남아 있습니다. 성숙한 단계 V 게임토세포에 감염된 적혈구는 혈류량에 방출되고 모기에 의해 채택되기 위하여 자유롭게 순환합니다. 일단 모기 미드구트 안에, 게임토세포는 미드구트 환경에 온도 및 노출의 변화를 통해 활성화되고, 여성과 남성 게임으로 변환하고 모기 단계의 발달을 시작, 이는 모기 타액선지6에서 산발성 의 감염 단계로절정6,7.
트레이거와 젠슨8이 P. falciparum배양에 표준화된 방법을 설명했기 때문에 무성혈액 단계에 대한 연구가 크게 진행되었습니다. 그러나, 성적 단계에 대한 신뢰할 수있는 문화 시스템의 부족은 P. falciparum 게임 토세포, 전염 생물학 및 모기 단계를 연구하기 어렵게 만들었습니다. 최근에는 게임토사이클 문화 9,10,11,12를설정하는실험실을도왔던 여러 가지 방법이 발표되었습니다. 이 원고는 말라리아 연구 커뮤니티에 귀중한 자원을 나타낼 수있는 문화 P. falciparum 게임 토세포에 표준화되고 신뢰할 수있는 프로토콜을 설명합니다. 이 방법은 표준화 된 모기 먹이 프로토콜과 함께 매우 신뢰할 수있는 모기 감염성을 초래하는 성숙하고 전염성 있는 게임토세포의 견고한 생산을 가능하게합니다. 이러한 방법은 게임토세포와 모기 단계 기생충의 끊임없는 공급을 유지하기 위해 설립되었다. 본 원고에서는 철저한 게임토세포 배양프로토콜(도 1),유리막 피더의 준비 및 모기의 감염(도2),미드구트의해부(도 3)및 모기의 타액선(도 4)및 중구 및 타액 선해부 후 모기감염의 정량화를 설명한다.

<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

말라리아는 중요한 이환율과 사망을 일으키는 원인이 되는 가장 중요한 공중 위생 문제 의 한개 남아 있습니다. 말라리아는 여성 Anopheles 모기에서 전염되는 바이트를 통해 전염되는 모기 매개 질병입니다. 말라리아 통제는 결국 모기를 통해 그리고 투과를 막는 쪽을 포함하는 접근의 다수에 의지할 것입니다. 실험실에서 말라리아 기생충의 모기 단계를 연구하기 위하여는, 우리는 모기 벡터에 인간 호스트에서 전송을 위해 요구되는 기생충 단계인 전염성이 높은 Plasmodium falciparum 게임토사이클을 배양하기 위하여 프로토콜을 최적화했습니다. P. falciparum 게임토세포는 약 1-2주가 걸리는 5개의 형태학적으로 뚜렷한 단계를 통해 성숙합니다. 이 프로토콜에 기재된 게임토세포 배양은 15일 이내에 완료되며 15-18일부터 모기에 감염된다. 이러한 프로토콜은 감염 유능한 게임토세포의 지속적인 주기를 유지하고 기생충의 모기 단계의 중단없는 공급을 유지하기 위해 개발되었다. 여기에서는 게임토세포 배양의 방법론과 유리 막 피더를 사용하여 이러한 기생충으로 모기를 감염시키는 방법을 설명합니다.

여기서 우리는 위의 프로토콜을 사용하여 생성된 P. falciparum NF54 게임토세포 배양체 배양체를 사용하여 일련의 멤브레인 피드의 결과를 제시한다(그림5참조). 게임토세포 문화는 0일째에 약 0.5%의 혼합단계 무성문화로 시작되었으며, 4일째와 5일째에는 약 15%의 충수 기생충증으로 성장했습니다. 이 높은 기생충에서 그림 5A에 나타난 바와 같이, 기...

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

말라리아 기생충의 모기 단계에 대한 상세한 조사는 효과적인 전송 차단 전략을 설계하는 데 중요합니다. 이 프로토콜은 전염성 있는 게임토세포를 효과적으로 배양한 다음 이러한 게임토세포에 먹이를 주어 P. falciparum의모기 단계를 생성하는 방법을 보여줍니다.

플라스모듐 팔시파룸 인공 막 수유를 통한 게임토세포 배양 및 모기 감염

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

plasmodium-falciparum-gametocyte-culture-mosquito-infection-through

Research

JoVE Journal

Biology

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

7.4K 조회수.  Johns Hopkins University. 말라리아 기생충의 모기 단계에 대한 상세한 조사는 효과적인 전송 차단 전략을 설계하는 데 중요합니다. 이 프로토콜은 전염성 있는 게임토세포를 효과적으로 배양한 다음 이러한 게임토세포에 먹이를 주어 P. falciparum의모기 단계를 생성하는 방법을 보여줍니다.

비디오: 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.

Alphavirus Transducing 시스템 : 모기 벡터의 감염을 떠올리을위한 도구

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

연구

면역학 및 감염병학

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.

쥐 말라리아 기생충의 유전자 크로스의 생산을위한 프로토콜

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.

모기 (A. aegypti)와 감염 표현형 결정에 뎅그열 감염 프로토콜

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 ...

생물학

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.

모기와 감염 표현형 결정에서 Plasmodium falciparum 감염에 대한 프로토콜

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.

자연의 전송에 결핵 말라리아 동시 감염을 연구하는 실험 모델 결핵균와 변형체 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.

설치류 말라리아 기생충 무성 및 성적 혈액 단계 및 모기 단계의 현상형 분석

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.

자기 공명 영상을 이용한 실험용 뇌 말라리아 모델에서의 부종 발달 및 미세 혈관 병리학 적 추적

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 ...

의학

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.

Anopheles 모기 벡터에서 플라스모듐 팔시파룸 감염 검출을 위한 표준 막 공급 분석

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.

유전자 변형 Plasmodium berghei Sporozoites 생성

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>말라리아 기생충에서 키나아제 재배선 식별: 화학 유전학 접근법

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>저자 스포트라이트: Identifying comcomcomatory pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases

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.

플라스모듐 팔시파룸 인공 막 수유를 통한 게임토세포 배양 및 모기 감염

요약

더 많은 비디오 탐색

PLAYLIST