우리는 진심으로이 예제의 준비와 지원을 팔 실험실의 구성원 감사합니다. 이 연구는 NIH / NIAID에서 PHS 부여 AI076684 및 AI080615 지원했다.

1. Nymphal Ixodes scapularis 진드기의 Microinjection 1. 준비 바늘 <ol><li> 난방 및 유리 micropipette 끌어당기는 장치 (Narishige)에 1mm의 유리 모세관 튜브 (세계 정밀 계측기)를 당기는하여 여러 microinjection의 바늘을 조작. 조심스럽게 연약한 모세관 튜브를 제거합니다. </li><li> 저장소는 배양 접시에 접착 테이프에 바늘 (끝 위를 향하게)를 뽑아. </li></ol> 2. B. 준비 burgdorferi <ol><li> B. 그로 ML 당 7 10 세포의 농도에까지 BSK 문화 미디어 7 burgdorferi. Spirochetes은 Petroff - Hausser 계산 실 (Hausser 과학)를 사용하여 어두운 현장 현미경으로 계산됩니다. </li><li> 펠렛 B. 실온에서 10 분 3,000 XG에서 원심 분리하여 burgdorferi. </li><li> 뜨는 제거하고 완전히 부드럽게 ML 당 10 9 세포의 최종 농도 200 μL 살균 microtip 통과하여 BSK 미디어 펠렛을 resuspend. 전지은 높은 세포 밀도에서 resuspended 있으며, 세포 현탁액은 즉시 microinjection 함께 클럼프 사용해야합니다 수 있기 때문에. </li></ol> 3. 준비 거려 <ol><li> 유리 슬라이드에 대한 명확하고 양면 접착 테이프를 넣으십시오. </li><li> 끈적끈적한 매트 작업을 신중하게 작은 브러시를 사용하여 컨테이너에서 nymphal 진드기를 제거합니다. </li><li> 같은 방향, 최대 접착 테이프, 복부 측면에 주입하는 진드기의 필요한 숫자를 놓습니다. </li></ol> 4. 주입 거려 <ol><li> B. 5 μL를 로드할 20 μL microloader 피펫 팁 (Eppendorf)를 사용하여 가져온 모세관 바늘로 burgdorferi 문화. 갇혀있는 공기 방울을 위해 바늘을 검사하고 필요한 경우, 세균성 문화와 바늘을 다시로드합니다. </li><li> 해부 쌍안 현미경 진드기를 확인하고 체크의 항문 조리개 영역에 중점을두고 있습니다. 부드럽게 직경이 microinjection 바늘을 형성 틱의 항문 구멍보다 약간 작은 튜브를 휴식하기 위하여 모세관 바늘의 팁을 터치. 아닌 적절한 바늘 팁과 진드기를 삽입하는 모든 시도가 주입 틱의 부상 가능한 사망 원인으로이 단계는 중요합니다. </li><li> 해부 쌍안 현미경 고정 진드기를 놓고 두 이동식 항문 플레이트에 의해 덮여있다 항문 구멍에 중점을두고 있습니다. 고급 집게를 사용하여 부드럽게 터치와 항문 구멍 근처에있는 지역으로 매우 가벼운 압력을 적용합니다. 이것은 항문 접시와 용기를 연결 항문 구멍의 오프닝의 분리를 허용합니다. 항문 접시의 강제 개방을 통해 항문 조리개에 약간 바늘의 팁을 조심스럽게를 삽입합니다. 유리 끝에서 항문에 연결 반투명 hindgut가 손상될 수로 바늘을 삽입은 최소로 보관해야합니다. 자동 발 제어 (Eppendorf)이 장착된 microinjector 사용하여 B를 주입 1000 hectopascals (고전력 증폭기) 사출 압력, 0.2 초 분사 시간과 8 고전력 증폭기 보상 압력 : 다음 매개 변수를 사용하여 burgdorferi 솔루션입니다. 각 틱은 단일 주사를 받게됩니다. </li><li> microinjection에 따라, 진드기는 미리 분사 상태로 유사한 행동해야합니다. 예를 들어, 틱은 자극에 대한 응답으로 크롤 링해야합니다. </li><li> 우리는 일반적으로 진드기가 24으로 설정 환경 챔버에서 48 시간 동안 복구할 수 있도록 ° 16시간 / 8시간 진한 / 라이트 photoperiod 처방과 95 %의 습도와 C. 챔버 사용할 수없는 경우, 주입 진드기는 이러한 일반 건조기 챔버로 공기 꽉 컨테이너 내의 습한 조건에서 상온에서 보관 수 있습니다. 필요한 경우 생쥐 먹이로 사용하기 전에, 진드기의 복구 시간도 몇 시간이 단축 될 수 있습니다. </li></ol> 2. B. 공촛점 Immunofluorescence 현미경으로 burgdorferi 현지화 1. 해부 거려 <ol><li> 인산염의 비말을 장소 현미경을위한 폴리 - L - 라이신 코팅 슬라이드 (시그마)에 절개하여 다른을위한 깨끗한 유리 슬라이드에 호수 (PBS)를 버퍼. 양면 접착 테이프와 다른 유리 슬라이드를 준비합니다. </li><li> 작은 브러시를 사용하여 양면 접착 테이프에 컨테이너와 장소에서 체크를 제거합니다. </li><li> 해부 현미경으로 체크보기 및 중점을두고 있습니다. 다리의 첫 번째와 두 번째 쌍 사이의 체크에 날카로운 면도날을 넣으십시오. 단단히 복부에 액세스할 수 있도록 두 가지의 체크를 절단 아래 버튼을 누르십시오. 즉시 잠수함 PBS의 비말에서 복부합니다. </li><li> 아주 좋은 집게를 사용하여, 상처의 위치 주변의 지느러미와 복부 외골격를 잡아. 조심스럽게 갈색 색깔 굿 diverticula를 노출 체크에서 지느러미 방패를 당겨 멀리. 항상 PBS에서 해부 진드기를 유지하기 위해주의하십시오. </li><li> 세미 반투명 침샘 롤빵dles은 창자의 앞쪽에 지역의 양쪽에 위치하고 있으며, 원하는 경우이 시점에서 제거할 수 있습니다. 포셉과 장소에 남아있는 외골격을 잡고 조심스럽게 복부의 창자를 끄집어. </li><li> (예를 들어, 기관)에 갇혀있는 조직을 제거하여 직감을 부드럽게 닦아주십시오. </li><li> 좋은 포셉의 팁을 사용하면 신속하게 폴리 - L - 라이신 유리 슬라이드에 PBS의 비말에 체크 굿를 전송할 수 있습니다. 좋은 블레이드를 사용하여 작은 조각으로 창자를 분리하거나 부드럽게 잘 포셉의 끝과 함께 눌러. 조심스럽게 굿 조직 주위에 여분의 PBS를 대기음. </li><li> 굿 조직은 상온에서 건조 공기 수 있습니다. </li><li> 10 분 아세톤에 잠수함이 잠수하여 체크 굿을 수정. 상온에서 건조 공기 수 있습니다. 슬라이드는 공기 꽉 컨테이너에서 -20 ° C에서 몇 달 동안이 단계에서 저장할 수 있습니다. </li></ol> 2. 더럽히는 것 <ol><li> 티슈를 사용하여 조직 주위에 여분의 수분을 제거하고 자궁 - 펜 또는 소수성 장벽 라이닝 장치에 의해 건조 틱 굿 주위에 원을 그립니다. 이것은 이후 부화 단계 동안 얼룩 솔루션을 유지하는 데 도움이 될 것입니다. </li><li> 실온에서 30 분 하나 또는 차단 버퍼의 몇 방울 (0.05 % PBS에서 트윈 - 20, 5 % 염소 혈청)와 체크 굿 표지. 차단에 사용되는 혈청은 항체에 대한 호스트 동물의 소스에 따라 다릅니다. 슬라이드가이 시점에서 건조하는 것을 허용하지 않습니다. </li><li> 천천히 차단 버퍼를 대기음. 적절한 기본 및 / 또는 보조 항체 솔루션의 용기를 품어. 안티 B. 라벨 - 우리는 플루오레신 isothiocyanate (FITC)를 사용 실온에서 1 시간 버퍼를 차단에서 1:100 희석에 burgdorferi 항체 (Kirkegaard &amp; 페리 연구소). 조명에 노출을 제한하기 위해 알루미늄 호일로 용기를 커버. </li><li> 부드러운 흡인하여 항체 솔루션을 제거합니다. 예 : 4로 레이블 틱 조직에 형광 염료 &#39;, 6 diamidino - 2 - phenylindole (DAPI) 또는 propidium 요오드화물의와 창자를 품어. 우리는 일반적으로 5 분 PBS에 20 μg / ML의 propidium 요오드화물의 (시그마)를 사용합니다. 상온에서. </li><li> 0.05 % PBS에서 트윈 - 20 3 번 씻으십시오. </li><li> 이러한 Slowfade (Invitrogen)로 antifade 시약을 포함하는 버퍼 글리세롤에 슬라이드를 장착하고 신중하게 유리 coverslip로 커버. 슬라이드는 공기 꽉 컨테이너 내에 네에서 몇 달 동안이 단계 ° C에 저장할 수 있습니다. 이미지와 B를 집중 공촛점 현미경 burgdorferi. </li></ol> 3. 대표 결과 microinjection에 nymphal 진드기의 위치와 B를 대표 이미지 틱 용기 burgdorferi 로컬 라이제이션은 그림 1에 표시됩니다. <img src="/files/ftp_upload/2544/2544fig1.jpg" alt="그림 1" /> 그림 1. Microinjection와 B의 현지화 틱 굿에 burgdorferi. nymphal I.의 (A) 복부보기 scapularis 진드기는 microinjection을 위해 위치. 삽입 microinjection 니들 (화살표)로 아날 조리개 (화살촉)이 삽입된 페이지의 배율에 따라 표시됩니다. 좋은 forcep은 항문 접시와 microinjection을 위해 항문 구멍의 오프닝의 분리를 허용 신체에 부드러운 압력을 적용하는 데 사용됩니다. 바늘은 가시성을 향상시키기 위해 쿠매시 빛나는 파란색의 솔루션으로 가득 차 있습니다. (B) B.의 공촛점 immunofluorescence 이미징 대표 결과 틱 굿 이내 burgdorferi. 굿 diverticulum의 앞쪽에 지역은 표시됩니다. 굿 핵 및 spirochetes는 (화살표) propidium 요오드화물의 (붉은 색) 또는 FITC - 복합 방지 B.으로 표시 아르 각각 burgdorferi (녹색 색깔). 바 = 20 μm의.

<ol>
	<li>Steere, A. C., Coburn, J., Glickstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+Lyme+disease.">The emergence of Lyme disease.</a> J Clin Invest. 113, 1093-1101 (2004).</li><li>Barthold, S. W., Diego, C., Philipp, M. T., Samuels, D. S., Radolf, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Borrelia, Molecular Biology, Host Interaction and Pathogenesis. , 353-405 (2010).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OspC+facilitates+Borrelia+burgdorferi+invasion+of+Ixodes+scapularis+salivary+glands.">OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands.</a> J Clin Invest. 113, 220-230 (2004).</li><li>Yang, X. F., Pal, U., Alani, S. M., Fikrig, E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+OspA/B+in+the+life+cycle+of+the+Lyme+disease+spirochete.">Essential role for OspA/B in the life cycle of the Lyme disease spirochete.</a> J Exp Med. 199, 641-648 (2004).</li><li>Zhang, X., Yang, X., Kumar, M., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BB0323+function+is+essential+for+Borrelia+burgdorferi+virulence+and+persistence+through+tick-rodent+transmission+cycle.">BB0323 function is essential for Borrelia burgdorferi virulence and persistence through tick-rodent transmission cycle.</a> J Infect Dis. 200, 1318-1330 (2009).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TROSPA,+an+Ixodes+scapularis+receptor+for+Borrelia+burgdorferi.">TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.</a> Cell. 119, 457-468 (2004).</li><li>Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+Lyme+disease+spirochetes.">Isolation and cultivation of Lyme disease spirochetes.</a> Yale J Biol Med. 57, 521-525 (1984).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc Natl Acad Sci U S A. 101, 1141-1146 (2004).</li><li>Narasimhan, 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+tick+antioxidant+facilitates+the+Lyme+disease+agent's+successful+migration+from+the+mammalian+host+to+the+arthropod+vector.">A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.</a> Cell Host Microbe. 2, 7-18 (2007).</li><li>Broadwater, A. H., Sonenshine, D. E., Hynes, W. L., Ceraul, S., DeSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+capillary+tube+feeding:+a+method+for+infecting+nymphal+Ixodes+scapularis+(Acari:+Ixodidae)+with+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme disease spirochete Borrelia burgdorferi.</a> J Med Entomol. 39, 285-292 (2002).</li><li>Policastro, P. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+Ixodes+scapularis+larvae+(Acari:+Ixodidae)+by+immersion+in+low+passage+cultures+of+Borrelia+burgdorferi.">Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi.</a> J Med Entomol. 40, 364-370 (2003).</li><li>Fuente, d. e. l. a., Kocan, J., M, K., Almazan, C., Blouin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기서 우리는 세균성 병원체 B.와 nymphal Ixodes의 진드기의 신속하고 효율적인 감염 microinjection 기반의 절차를 입증 burgdorferi. 우리는 또한 B.의 검색을위한 공촛점 immunofluorescence 절차를 설명 현장에서 틱 용기 burgdorferi. 우리의 시위는 nymphal 직감을 포함하지만, 이와 유사한 절차 또한 유충이나 성인 8,9 등 진드기의 다른 발달 단계에 대해 적용됩니다. 그러?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Glass capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Vertical glass puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC-10</td>
<td>&nbsp;</td>
<tr>
<td>Petroff-Hausser counting chamber</td>
<td>&nbsp;</td>
<td>Hausser scientific</td>
<td>3900</td>
<td>&nbsp;</td>
<tr>
<td>Microloader pipette tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
<td>&nbsp;</td>
<tr>
<td>Femtojet microinjector</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920010504</td>
<td>&nbsp;</td>
<tr>
<td>Foot control FemtJet</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920005098</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP665-1</td>
<td>Filter-sterilized</td>
</tr>		</tbody>
		</table>
		</div>

methods for rapid transfer and localization of lyme disease pathogens within the tick gut

라임 병은 진드기 - 설치류 감염 사이클 1 자연 유지 spirochete 병원체 Borrelia burgdorferi와 감염에 의해 발생합니다. 틱 - 부담 murine 모델 2 실험실에서 라임 병을 연구하기 위해 개발되었습니다. 나이브 진드기는 B.에 감염 될 수 있지만 burgdorferi 감염 생쥐에서 그들을 먹이로, 털갈이 과정을 완료하는 데 개월 몇 주간 소요됩니다. 따라서, 이러한 microinjection 기반의 프로 시저로보다 신속하고 효율적인 틱 감염 기법의 개발 라임 질병 3,4의 연구를위한 중요한 도구입니다. 절차는 시간이 감염된 진드기를 생성하기 위해 필요하며 진드기의 코호트에서 spirochetes의 동일한 수량의 배달 제어할 수 있습니다. 이것은 B.의 세대로서 특히 중요합니다 마우스를 사용하여 자연적인 먹이 프로세스 burgdorferi 감염 진드기는 먹이 거려 사이에 병원균 부담의 변화 100 %의 감염 속도와 잠재적 결과를 보장하기 위해 실패합니다. 또한, microinjection은 B.와 진드기를 감염하는 데 사용할 수 있습니다 burgdorferi는 감쇠 변형 생쥐에 감염을 설정할 수 없습니다이며, 따라서 자연 진드기 5 인수 수없는 경우에는 분리합니다. 이 방법은 또한 진드기에 다른 생물 학적 재료의 다양한 제공하는 데 사용할 수있는 예를 들어, 특정 항체 또는 두 RNA 6 좌초. 이 문서에서는, 우리는 B를 체외에서 재배한에서 함께 nymphal 진드기의 microinjection을 설명합니다 burgdorferi. 우리는 또한 공촛점 immunofluorescence 현미경을 사용하여 체크 굿에서 라임 병의 병원균의 국산화에 대한 방법을 설명합니다.

Watch this Scientific Journal Video about Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut at JoVE.com

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

라임 질병 연구 조사는 종종 병원체 Borrelia burgdorferi, 일반적으로 몇 주 정도 소요 프로세스에 감염된 진드기의 생성을 필요로합니다. 여기 시간 이내에 수행할 수 있습니다 microinjection 기반 틱 감염 절차를 보여줍니다. 우리는 또한 진드기 이내 B. burgdorferi의 현장 지방화에 대한 immunofluorescence 방법을 보여줍니다.

틱 굿 내에서 빠른 전송과 라임 질병 병원균의 현지화을위한 방법

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

methods-for-rapid-transfer-localization-lyme-disease-pathogens-within

Research

JoVE Journal

Immunology and Infection

11.5K 조회수.  University of Maryland. 라임 질병 연구 조사는 종종 병원체 Borrelia burgdorferi, 일반적으로 몇 주 정도 소요 프로세스에 감염된 진드기의 생성을 필요로합니다. 여기 시간 이내에 수행할 수 있습니다 microinjection 기반 틱 감염 절차를 보여줍니다. 우리는 또한 진드기 이내 B. burgdorferi의 현장 지방화에 대한 immunofluorescence 방법을 보여줍니다.

비디오: Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

연구

면역학 및 감염병학

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지<em&gt; 체외에서</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

라임 병 연구의 변속기 및 Xenodiagnosis에 대한 동물에 진드기의 먹이

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

의 cDNA에서 재조합 뉴캐슬 질병 바이러스의 구조

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

수평 유전자 전달의 연구에 대한 방법론<em&gt; 황색 포도상 구균</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

포르말린 고정 조직에서 리사바이러스 항원 검출을 위한 면역조직화학 시험

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

낭포성 섬유증 환자의 객담 내 녹농균의 시각화

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

라임병 스피로체테 보렐리아 부르그도르페리의 파지 매개 유전자 조작

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

SARS-CoV-2 변이체에서 백신 유도 중화항체 정량화

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

저자 스포트라이트: SARS-CoV-2 연구에서 오미크론 하위 변이체 평가 및 중화 항체를 위한 유사형 바이러스 시스템

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

SARS-CoV-2 변이체 및 중화 항체의 신속한 정량화를 위한 Ha-CoV-2 슈도바이러스 적용

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...