Name: plasmodium falciparum gametocyte culture and mosquito infection through artificial membrane feeding
Uploaded: 2020-07-03T15:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding at JoVE.com

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>
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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. 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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 처리량 게임토시토시달분석서(22)에사용되어 왔으며, 프로테오믹15뿐만아니라 전사23연구에도 사용되었습니다. 그러나, 이러한 방법을 개발하는 주된 이유는 성숙한 게임토세포를 사용하여 모기 단계및스포로조이트(23,24,25,26)에대한 연구를 위해 모기를 감염시키는 것이다. 이 원고는 유리 막 피더를 사용하여 성숙한 P. falciparum 게임 토세포 및 모기의 감염을 생성하는 상세한 프로토콜을 설명합니다. 이 방법은 전송 차단 전략, sporozoite 단계 및 P. falciparum의 전 적혈구 간 단계에 일하는 모든 실험실에 대 한 중요 한.
1981년 Ifediba와 Vanderberg는 P. falciparum의장기 문화를 설명했으며, 50 μg/mL 저산소산틴이 존재하여 전염성이 높은 성숙한 게임토세포27을생산했습니다. 그 이후로 다른 응용 프로그램에 대한 게임 토세포를 생산하는 방법을설명하는 수많은 간행물이 있었다9,10,11,12. 이러한 간행물의 대부분은 이전에 설명 된 게임 세포 발생 유도 조건을 활용하여 수율을 증가시합니다. 조건부 미디어를 사용하여, 갑작스런 기생충증 증가에 의해 문화를 강조하고, 빈혈을 모방하기 위해 혈통에 있는 강하, 적혈구 용해 및 로그 상 억압을 대량으로, 게임 세포 발생을 유도하는 데 사용될 수 있다. 여기에 설명된 방법은 간단하고 테스트된 시간입니다. 0.3~1% 무성단계 기생충혈을 통해 게임토세포 문화를 개시하여 4%의 혈종염으로 매일 미디어를 변경하여 15-18일까지 미디어를 변경합니다. 일관된 결과를 얻으려면 낮은 통로 무성기 기생충으로 게임토세포 문화를 시작하고, 신선한 RBC(<1주)를 사용하고, 미디어 변화 시 배양 온도가 변동하지 않도록 하는 것이 중요합니다. falciparum 게임토세포 개발 과정은 골수4,5에서사치공간의 정적 조건에서 격리되기 때문에 문화 기간 동안 정착된 RBC 층을 방해하지 않는 것이 중요합니다.
P. falciparum 게임토세포를 일관성있게 배양하는 것은 까다롭지만 모기를 감염시키는 것은 또 다른 수준의 복잡성을 제시합니다. 멤브레인 수유는 모기의 나이 및 적합성, midgut microbiota 및 먹이 행동28,29와같은 게임토세포 이외의 여러 변수의 대상이 됩니다. 일반적으로 SMFA 데이터는 높은 수준의 가변성을 나타내며 다른 실험조건(11,28)의효과를 확인하기 위해 많은 수의 모기가 필요합니다. 게임토세포에 대한 낮은 통로 문화를 사용하여 건강한 3 - 6 일 된 모기와 최적화 된 멤브레인 공급 프로토콜은 난모 세포 수의 변화에 도움이 될 수 있습니다.
여기에 설명된 프로토콜은 게임토세포 재배와 멤브레인 수유를 위해 수년 동안 최적화되어 왔습니다. 이러한 방법은 성숙한 전송 유능한 게임토세포, 표준 멤브레인 수유 분석, 모기 미드구트 해부 및 난소제 정량화뿐만 아니라 타액 선 해부 및 스포로조이트 정량화를 얻기위한 자세한 설명을 제공합니다. 이러한 프로토콜은 신뢰할 수 있는 모기 감염및 강력한 난모세포 수와 스포로조이트 수율을 제공하는 데 필요한 게임토세포 수측면에서 일관되습니다.

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, 기생충은 스트레스와 무성 단계 문화 충돌. 그러나, 이 스트레스는 수반되는 게임 세포 발생의 유도결과. 6일과 7일 이후 조기 게임토세포가 나타났고, 무성 기생충은 서서히 감소했지만 낮은 수준에 머물렀다. 무성 단계 기생충의 존재는 모기 먹이 실험에 영향을 미치지 않았다. 그러나, 게임토세포가 약물 감수성 및 프로테오믹 또는 전사연구와 같은 순수한 배양을 필요로 하는 실험에서 사용될 경우, 잔류 무성단계는 50mM N-아세틸글루코사민으로 치료하여 제거될 수 있다. 대부분의 게임토세포는 15일까지 V단계로 성숙하여 모기에 감염되어 먹이를 받을 준비가 되었습니다. 게임토세포 배양 개시 후 다른 시점에서 지름사 염색 혈액 얼룩의 대표적인 이미지가 도 5B에표시됩니다.
혈액 공급 후에 8에서 10 일 사이에서, 모기는 감염의 보급 그리고 강렬을 결정하기 위하여 해부되었습니다. 보급은 강렬이 각 모기에서 찾아낸 난모의 수 인 동안 난모가 있는 먹이 모기의 백분율입니다. 둘 다 피드의 성공에 대한 중요한 지표입니다. 5개의 독립적인 모기 사료로부터의 데이터는 도 6에도시된다. 사료는 P. falciparum NF54의 16 일 문화에서 0.3 %의 성숙한 게임 토세포로 공급 한 후 감염성 수준의 정상적인 변화를 보여주기 위해 선택되었습니다. 난소증은 게임토세포와 유병률의 모기 감염률을 결정하기 위해 정량적 데이터를 제공하여 감염된 먹이 모기의 비율을 보여줍니다. 이러한 데이터는 전송 차단제를 평가하고 전염 차단 백신 및 약물에 대한 표적을 식별하고 특성화하는 데 사용할 수 있습니다. 난모세포의 수는 실험 내와 사이에 다양하고 다양한 실험 조건의 효과를 결정하기 위해 코호트 당 25-50 모기가 필요했습니다.
난소성 강도는 대부분의 전송 차단 assays 및 전략의 끝점으로 간주됩니다, 그러나 스포로조이트의 수는 일반적으로 산포로조이트 생물학과 간 단계 연구를 위해 중요합니다. 표 1은 12개의 독립적인 혈액 공급에서 모기 당 얻은 평균 수 sporozoites를 보여줍니다. 표에 나타난 바와 같이, 스포로조이트의 평균 수는 일관적이었다, 그러나, 우리가 제로 스포로조이트를 얻은 하나의 실험이 있었다, 분석의 가끔 실패를 나타내는.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61426/61426fig1v2.jpg"> 그림 1: P. falciparum 게임토세포 배양 및 멤브레인 공급 프로토콜의 워크플로우. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61426/61426fig1v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61426/61426fig02.jpg"> 그림 2: 모기 혈액 공급 설정. (A)유리 피더와 파라핀필름(B) 파라필름 막 부착전과후에 두 개의 유리 피더가 모기컵 위에 유리 피더를 표시하고 순환 수조와 연결한다. (D,E) 유리 피더(F) 혈액 공급의 균일분포를 보여주는 유리 피더의 바닥뷰(F) 파라필름 멤브레인을 통해 먹이를 주는 여러 모기를 배관한다. (H)모유 수유 후 모기컵의 최고 보기, 컵 의 바닥에 먹이 모기에 의해 배설 된 혈액 방울을 보여주는. 스케일 바 = 10mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61426/61426fig02large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61426/61426fig03.jpg"> 그림 3: 모기 미드구트 해부의 단계를 보여주는 그래픽 표현. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61426/61426fig03large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61426/61426fig04.jpg"> 그림 4: 모기 침샘 해부의 단계를 보여주는 그래픽 표현. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61426/61426fig04large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61426/61426fig05.jpg"> 그림 5: P. falciparum 게임토세포 문화와 감염된 모기 미드구트에 난모성 시각화. (A)15일 게임토세포 문화의 시간 과정, 처음 4일 이내에 기생충을 피크로 하는 무성단계의 가파른 곱셈을 보여주고, 시간이 지남에 따라 게임세포 발생과 성숙이 뒤따릅니다. (B)게임세포 문화의 다양한 단계를 보여주는 건사 염색 얇은 혈액 얼룩, 1일 초 무성단계, 4일째 성무성 기생충, 6일째 는 기생충 부하, 9일과 12일 조기 게임토세포, 성숙한 남녀 게임토세포로 인한 문화가 강조된다. 형태학적으로 초기 단계 의 게임토세포는 무성 단계와 구별할 수 없었지만, 후기 단계 II는 뾰족한 끝으로 초승달 모양을 보였고 기생충이 단계 III 및 IV로 발전함에 따라 길어졌다. 성숙한 단계 V 게임토사이클은, 그러나, 둥근 끝과 최소한의 숙주 셀가시성14와고전적인 초승달 모양이 특징입니다. 스케일 바 = <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61426/61426fig05large.jpg" target="_blank">10mm이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61426/61426fig06.jpg"> 그림 6: P. falciparum 난모세포는 모기 midgut당 계산됩니다. (A)그래프는 수년에 걸쳐 난모성 수를 나타내며, 각 실험마다 모기가 0.3%의 게임토세포로 공급되었고, 8일째에는 혈류 후 사료가 해부되었다. 각 점은 개별 미드구트의 난모성 수를 나타내며 가로 선은 중앙값입니다. (B)표는 감염한 모기의 평균 난모세포 수, 보급 및 감염 범위를 나타낸다. (C)다른 배율에서 두 개의 별도 혈액 공급 실험에서 모기 미드구트에 난모를 보여주는 사진. 스케일 바 = 150 μm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61426/61426fig06large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>실험</td>
			<td>해부의 날 (후 혈액 공급)</td>
			<td>평균 스포로조이트/모기</td>
		</tr>
<tr>
<td>1</td>
			<td>14</td>
			<td>42, 000</td>
		</tr>
<tr>
<td>2</td>
			<td>14</td>
			<td>40, 000</td>
		</tr>
<tr>
<td>3</td>
			<td>14</td>
			<td>42, 750</td>
		</tr>
<tr>
<td>4</td>
			<td>15</td>
			<td>25, 411</td>
		</tr>
<tr>
<td>5</td>
			<td>15</td>
			<td>33, 750</td>
		</tr>
<tr>
<td>6</td>
			<td>14</td>
			<td>15, 750</td>
		</tr>
<tr>
<td>7</td>
			<td>16</td>
			<td>0</td>
		</tr>
<tr>
<td>8</td>
			<td>15</td>
			<td>33, 200</td>
		</tr>
<tr>
<td>9</td>
			<td>14</td>
			<td>56, 333</td>
		</tr>
<tr>
<td>10</td>
			<td>14</td>
			<td>45, 333</td>
		</tr>
<tr>
<td>11</td>
			<td>17</td>
			<td>43, 750</td>
		</tr>
<tr>
<td>12</td>
			<td>16</td>
			<td>56, 000</td>
		</tr>
</tbody></table>표 1: 2년 동안 12개의 독립적인 주기동안 모기당 타액샘 스포로조이트의 평균 수. A. stephensi 모기는 0.3% P. falciparum 게임토세포와 15-20 모기가 14-17일 사이에 해부되었다. 모기 당 평균 스포로조이트 수가 표시됩니다.

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

Nanyang Technological University

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

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