私たちは心からこのデモの準備の支援のためにパルの研究室のメンバーに感謝。この研究は、NIH / NIAIDからPHS助成AI076684とAI080615によってサポートされていました。

1。若虫マダニ属のマイクロインジェクションscapularisダニ 1。準備針<ol><li>加熱とガラスマイクロピペットプラー装置（成茂）に1mmのガラス毛細管（世界の精密機器を）引いて、いくつかのマイクロインジェクションの針を作製する。慎重に壊れやすいキャピラリーチューブを取り外します。 </li><li>ストアは、ペトリ皿の中で粘着テープで針を（先端を上に向けた状態で）引っ張った。 </li></ol> 2。 B.の準備ブルグドルフェリ <ol><li> B.を育てるmL当たり約10 7の細胞の濃度でまで、BSK文化メディア7のブルグドルフェリ 。スピロヘータはPetroff - Hausser計数室（Hausser科学）を使用して暗視野顕微鏡下でカウントされます。 </li><li>ペレットB.室温で10分間、3,000 xgで遠心分離によってブルグドルフェリ 。 </li><li>上清を除去し、完全に優しくmL当たり10 9細胞の最終濃度に200μL滅菌マイクロチップを通過することによってBSK培地でペレットを再懸濁します。細胞は、高い細胞密度で再懸濁させ、そして、細胞懸濁液は直ちにマイクロインジェクションのために一緒に凝集を使用する必要があります可能性があるため。 </li></ol> 3。準備ティック<ol><li>スライドガラス上に透明な、両面テープを置きます。 </li><li>粘着マット上での作業、慎重に小さなブラシを使用してコンテナから若ダニを除去する。 </li><li>同じ方向を向いて、粘着テープで注入されるダニの必要数、腹側を上に置きます。 </li></ol> 4。注入ティック<ol><li> B.の5μLをロードする20μLmicroloaderピペットチップ（エッペンドルフ）を使用してプルキャピラリー針にブルグドルフェリの文化。閉じ込められた空気の泡のために針を点検し、必要に応じて、細菌培養で針をリロードします。 </li><li>解剖双眼顕微鏡下の目盛りを表示し、ダニの肛門開口部分に焦点を合わせる。ゆっくりと直径がマイクロインジェクションの針を形成し、ダニの肛門開口部のそれよりわずかに小さいチューブを打開するためにキャピラリー針の先端に触れる。非適切な針先でダニを注入しようとするが、注入されたダニの傷害および可能な死の原因として、この手順は重要です。 </li><li>解剖双眼顕微鏡下に固定化したダニを置き、二つの可動肛門のプレートで覆われている肛門絞り、焦点を当てる。細かい鉗子を使用して、軽く触れると肛門開口の近くに任意の領域に非常に穏やかな圧力を適用する。これは、肛門板と腸に接続している肛門細孔の開口部の分離が可能になります。肛門板の強制的な開口部を通って肛門の開口部にわずかに針の先端を慎重に挿入します。ガラスの先端が直腸に接続する半透明の後腸を損傷する可能性があるため、針挿入は最小限に抑える必要があります。自動化されたフットコントロール（エッペンドルフ）を搭載したマイクロインジェクターを使用して、Bを注入する次のパラメータを使用して、 ブルグドルフェリのソリューション：1,000ヘクトパスカル（hPaの）射出圧力、0.2秒噴射時間と8 hPaの補償圧力。それぞれの目盛りは、単回注射を受け取ります。 </li><li>マイクロインジェクションの後、ダニは注射前の状態と同じように動作する必要があります。例えば、ダニは、刺激に応答してクロールする必要があります。 </li><li>我々は通常、ダニは24 °、16時間/ 8時間とC /暗光周期療法、湿度95％に設定した環境室に48時間で回復することができます。チャンバーが使用できない場合は、注入されたダニは、正規のデシケーター室として、密閉容器内で湿った状態で室温で保存することができます。必要に応じてマウスの摂食のために使用される前に、ダニの回復時間も数時間に短縮することができます。 </li></ol> （2）B.共焦点免疫蛍光顕微鏡によるブルグドルフェリのローカライゼーション 1。解剖ティック<ol><li>リン酸の液滴を配置するには、顕微鏡のためのポリ- L -リジンコートしたスライド（シグマ）で解剖し、別のクリーンなガラススライド上に生理食塩水（PBS）を。両面粘着テープで別のスライドガラスを準備する。 </li><li>小さなブラシを使って両面粘着テープで容器と場所からダニを取り除く。 </li><li>解剖顕微鏡でダニを表示し、フォーカス。足の第一および第二の対の間にダニに鋭いカミソリの刃を置きます。しっかりと腹部へのアクセスを許可する2つの部分にダニをカット押し下げます。すぐに、水没PBSの液滴の腹部。 </li><li>非常に微細な鉗子を使用して、カットのサイトの周りに背と腹外骨格をつかむ。慎重に茶色っぽい色の腸憩室を公開するダニから持ち上げ、背側シールドを引き出します。すべての回でPBSの下に解剖ダニを保つように注意してください。 </li><li>半透明の唾液腺のパンdlesは、腸の前方領域の両側に配置され、必要に応じて、この時点で削除することができます。鉗子との場所に残っている外骨格を持って慎重に腹部から腸を引き出します。 </li><li>優しくどんなトラップの組織（例えば、気管を）除去することにより腸をきれいに。 </li><li>微細な鉗子の先端を使用して、すぐにポリ- L -リジンスライドガラス上でPBSの液滴でダニの腸を転送する。微細な刃を使用してより小さな部分に腸を切り離したり、軽く細かい鉗子の先端を押す。慎重に腸組織の周りに余分なPBSを吸引除去する。 </li><li>腸の組織は、室温で風乾する。 </li><li> 10分間アセトンに浸してダニの腸を修正。室温で風​​乾する。スライドは、気密容器内に-20℃で数ヶ月の​​ために、この手順で保存することができます。 </li></ol> 2。染色<ol><li>ティッシュペーパーを使用して、組織の周りに余分な水分を除去し、PAP -ペンなど疎水性のバリアーのライニング装置によって乾燥ダニの腸の周りに円を描く。これにより、その後のインキュベーションステップの間に染色液を保持するのに役立ちます。 </li><li>室温で30分間つまたはブロッキングバッファーの数滴（0.05％PBSのTween - 20、5％ヤギ血清）でダニの腸をカバーしています。ブロッキングのために使用される血清は、抗体の宿主動物のソースによって異なります。スライドが前方にこの点から乾燥させないでください。 </li><li>ゆっくりブロッキングバッファーを吸引除去する。適切なプライマリおよび/または二次抗体溶液と腸をインキュベートします。我々は、フルオレセインイソチオシアネート（FITC）を使用-アンチラベル B室温で1時間ブロッキングバッファーで100倍希釈でブルグドルフェリ抗体（Kirkegaard＆ペリーラボラトリーズ）。光への曝露を制限するためにアルミホイルで容器を覆う。 </li><li>穏やかな吸引によって抗体溶液を取り外します。 4などのラベルティック組織、&#39;、6 - ジアミジノ-2 - フェニルインドール（DAPI）またはヨウ化プロピジウムの蛍光色素で腸をインキュベートする。我々は通常、5分間PBSで20μg/ mLのヨウ化プロピジウム（Sigma）を使用してください。室温で。 </li><li> 0.05％のTween - 20 PBSにて3回洗浄する。 </li><li>このようなSlowfade（Invitrogen社）などの褪色防止試薬を含む緩衝グリセロールでスライドをマウントし、慎重にガラスのカバースリップでカバーしています。スライドは、気密容器内で4℃で数ヶ月ごとにこの手順℃で保存することができます。画像とローカライズB.共焦点顕微鏡下でブルグドルフェリ 。 </li></ol> 3。代表的な結果マイクロインジェクションのための若ダニの位置A と B を表す画像ダニの腸内ブルグドルフェリの局在を図1に提示されます。 <img src="/files/ftp_upload/2544/2544fig1.jpg" alt="図1" /> 図1。マイクロインジェクションとBの局在ダニの腸内にブルグドルフェリ 。 幼虫I.の（A）腹ビューscapularisティックはマイクロインジェクションのために配置されます。挿入したマイクロインジェクションの針（矢印）と肛門絞り（矢じり）がはめ込みに倍率の下に表示されます。細かいforcepは肛門板とマイクロインジェクションのための肛門孔の開口部の分離を許可されて身体に優しい圧力を適用するために使用されます。針が視認性を高めるために、クマシーブリリアントブルー溶液で満たされている。（B）Bの共焦点免疫蛍光イメージングの代表結果ダニの腸内ブルグドルフェリ 。腸憩室の前方領域が表示されます。腸の核とスピロヘータが（矢印）、ヨウ化プロピジウム（赤色）またはFITC結合抗Bと表示されていますそれぞれブルグドルフェリ （緑色）、。バー= 20μmの。

<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>

利害の衝突は宣言されません。

ここでは、細菌性病原体Bと若虫マダニ属のダニの迅速かつ効率的な感染症のためにマイクロインジェクションベースの手順を示すブルグドルフェリ 。我々はまた、Bの検出のための共焦点蛍光抗体法の手順を説明します。 その場でダニの腸内ブルグドルフェリ 。私たちのデモでは、幼虫の腸を含むものの、同様の手順ではまた、幼虫、大人8,9のよう...

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

ライム病はダニ媒介げっ歯類の感染サイクル1で自然に維持されるスピロヘータの病原体ボレリアブルグドルフェリ 、感染によって引き起こされる。ダニ媒介性マウスモデル2は、実験室でライム病を研究するために開発されました。素朴なダニはB.に感染することができますが、 ブルグドルフェリは、感染マウスにそれらを供給することにより、脱皮のプロセスが完了するまでに数ヶ月に数週間かかる。したがって、このようなマイクロインジェクションベースの手続きとして、より迅速かつ効率的なダニの感染のテクニック、、の開発は、ライム病3,4の研究のための重要なツールです。手順は時間が感染したダニを生成するために必要とし、ダニのコホートにおけるスピロヘータの等量の配信を制御できます。これは、B.の世代としては特に重要です。マウスを使って自然な給餌法によるブルグドルフェリ感染したダニは葉ダニの間で病原体の負担の変動で100％の感染率と潜在的に結果を確実に失敗します。さらに、マイクロインジェクションは、B.とダニに感染するために使用することができます。 ブルグドルフェリは、弱毒株は、マウスでは感染を確立できないため、自然にダニ5によって取得できない場合には隔離。この手法はまた、例えば、特異的な抗体または二重のRNA 6座礁、ダニに他の生物学的、さまざまな素材を提供するために使用することができます。この記事では、Bをin vitroで成長にと若ダニのマイクロインジェクションのデモンストレーションを行いますブルグドルフェリ 。我々はまた、共焦点免疫蛍光顕微鏡を用いてダニの腸内のライム病の病原体の局在化のための方法を説明します。

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

ライム病の調査研究は、多くの場合、病原体ボレリアブルグドルフェリ、通常は数週間かかるプロセスに感染したダニの発生を必要とする。ここでは、時間内で達成可能なマイクロインジェクションベースのダニの感染症の手順を示しています。我々はまた、ダニの中でB.ブルグドルフェリのin situ局在のための免疫蛍光法を示しています。

ティック腸内ライム病の病原体の迅速な伝達とローカリゼーションのための方法

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

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Immunology and Infection

11.5K ビュー.  University of Maryland. ライム病の調査研究は、多くの場合、病原体ボレリアブルグドルフェリ、通常は数週間かかるプロセスに感染したダニの発生を必要とする。ここでは、時間内で達成可能なマイクロインジェクションベースのダニの感染症の手順を示しています。我々はまた、ダニの中でB.ブルグドルフェリのin situ局在のための免疫蛍光法を示しています。

ビデオ: 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.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

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

免疫・感染学

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.

呼吸器病原体に対する宿主細胞応答を研究するために偏ヒト気道上皮Calu-3細胞の液体に覆われた文化の確立<em&gt;インビトロ</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.

ライム病研究における送信および外因診断法のために動物にダニの摂食

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.

cDNAからの組換えニューカッスル病ウイルスのレスキュー

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.

で水平遺伝子伝達の研究のための方法論<em&gt;黄色ブドウ球菌</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).

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

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.

ホルマリン固定組織からのリサウイルス抗原検出のための免疫組織化学試験

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

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

腸内調節制御T細胞誘導の生体内増強

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

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.

嚢胞性線維症患者の喀痰中の 緑膿菌 の可視化

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.

ライム病スピロヘータ・ボレリア・ブルグドルフェリのファージ媒介遺伝子操作

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