私たちは彼の研究室で撮影を行うことを可能にするためシトリンの魚のラインとビル·ジャックマン：著者は、寛大にαカテニンを提供するための博士ルトリン博士とトービンに感謝。著者は、資金源国立衛生研究所（助成5P20RR016463、8P20GM103423とR15AI094406）とUSDAを認める（プロジェクト＃ME0-H-1-00517-13）。この原稿は、メイン農林試験場公開番号3371として公開されている。

注：すべてのゼブラフィッシュケアプロトコルおよび実験は施設内動物管理使用委員会（IACUC）プロトコルA2012-11-03下のNIHガイドラインに従って行った。 4日後に受精1.ゼブラフィッシュ飼育<ol><li>別の映像34に示すように、最初の3時間後に受精内、ABゼブラフィッシュ、または任意の他のトランスジェニック系統を収集する。 </li><li> （; 0.17ミリモルのKCl、0.33のCaCl 2、0.33のMgCl 2、2mMのHEPES、pH6.8の5 mMのNaCl）をメチレンブルー（0.3 / mlの最終E3培地150 mlを含む15センチメートルペトリ皿に120の卵をインキュベート12/12の明/暗光周期で33℃のインキュベーター中の濃度）。 </li><li> E3 + PTU（1-フェニル-2-チオ尿素、10μg/ mlの最終濃度）と6時間後にメディアを交換して、死んだ卵を取り除く。 </li><li> 2日後に新鮮なE3 + PTUと交換媒体、33℃で4日間インキュベートする。 </li></ol> 2.でjectionマイクロニードルの準備<ol><li>次の設定で針プラーを使用したマイクロニードルに（110時間550ヒート; 0プル; 130ベロシティ）;（ID 0.69ミリメートル外径1.2ミリメートル）ホウケイ酸キャピラリーを引き出します。 NOTE：設定は、同様の形状のマイクロニードルを得るために、フィラメントからフィラメントに変化するであろう（ 図1参照）、新たなフィラメントがインストールされるたびに再調整する必要がある。 </li><li> 0.4μmのフィルタリン酸緩衝生理食塩水の3μlのマイクロニードルを埋める（; 137のNaCl、2.7のKCl; 10のNa 2 HPO 4; PBS 1.8 mMのKH 2 PO 4; pH7.4）を20μlのmicroloaderピペットを用いて、先端。 </li><li>高圧噴射システムに接続されているマイクロマニピュレータにマイクロピペットホルダー上のマイクロニードルを設定します。 30 PSIへの射出圧力と30ミリ秒のパルス持続時間を設定します。 </li><li>針の1/5が水になるまで蒸留水でペトリ皿を使用して、マイクロニードルを下げる。マイクロニードルのWiクリップちょうど水位以下に目細かいピンセット。水の中からマイクロニードルを持参し、ボーラスが表示されるまで、ペダルを数回押してください。 </li><li>タイプの一滴浸漬油でレイヤード血球計数器上のマイクロニードルを下げてボーラスの直径を測定します。所望の容積を得るために、パルス持続時間を調整する（ 表1参照）。 </li><li>油で安定した状態を保つ（ボーラスは体積が増加した場合、圧力を下げ、ボーラスボリュームが減少した場合背圧を増やす）するボーラスのための背圧を設定します。各マイクロニードルのためのパルス持続時間を記録します。 </li><li> microloaderピペットチップを使用してそれを吸引することによって、微小針からPBSを削除し、インジェクタに戻ってそれを配置し、連続パルススイッチを切り替えることで、左オーバーPBSを追放。各マイクロニードルを予約。 </li></ol> 3. カンジダ準備<ol><li>コドン最適化dTomato、GFP、BFP、EOSや極東で形質転換ストリークカンジダ·アルビカンス酵母peptineデキストロース（YPD）寒天プレート（10g / Lの酵母エキスが20g / Lペプトン、20グラム/ Lのデキストロースに滅菌木製のダボを用いて凍結ストック（25％グリセロール中で-80℃のストック）から赤、が20g / L寒天;オートクレーブし、ペトリ皿中を25ml注ぎ）、30℃で一晩インキュベートする。 </li><li>滅菌木製のダボを用いてつのコロニーを選択し、YPD液体（10g / Lの酵母エキスが20g / Lペプトン、20グラム/ Lのデキストロース、オートクレーブし、無菌的に16×150mmのガラス培養において5mlのアリコートを5 mlに植菌管）。 </li><li> 60rpmでローラードラムで30℃で一晩インキュベートする。 </li><li> Cの 1ミリリットルを収集1.7ミリリットル滅菌遠心管にアルビカンス文化と転送。 </li><li>秒のカップルのための5000×gで遠心分離する。上清を空にし、徹底的に滅菌PBS、渦の1ミリリットルを追加します。これを2回繰り返します。 </li><li>滅菌PBS 1,000：1ミリリットル株式1に希釈することにより、血球計数器を使用してコロニーを数える。 </li></ol> 4にゼブラフィッシュを注入する浮袋<ol><li> 33℃のインキュベーター中で30分間注入皿（2％アガロース）を温める。 </li><li>所望の時間に、すべてのゼブラフィッシュを吸引することなく、25ミリリットルピペットを用いて注入するために、魚を含む15センチメートルペトリ皿からE3 + PTUの150ミリリットルの100を削除します。 </li><li> （最終濃度は200μg/ ml）を、残りの50ml培地に4 mg / mlの緩衝化トリカインメタンスルホン酸塩ストック（TR）2 mlを加えることにより、ゼブラフィッシュのdpf 4を麻酔し、15分間待つ。 </li><li>解剖顕微鏡下では、膨張した浮袋で魚を選択します。魚はまた、MPOにおける好中球分布として、均質な表現型のために、その時点でスクリーニングすることができる：解剖顕微鏡落射蛍光を用いて、GFPライン 。 </li><li> C.を希釈PBS中1ml当たり形成単位1.5×10 7コロニー（CFU / ml）を基準にした元濃度（ ステップ3.8）からアルビカンス株式を。 </li><li>プラスチック製の移送管を用いて射出皿30ゼブラフィッシュを転送するTTEとは余分なメディアを取り出します。 </li><li>釣りワイヤツールを使用すると、（0.012インチ径釣り糸ホウケイ酸キャピラリー内にスーパー接着）、垂直方向に注入ディッシュを保持し、垂直方向に魚を方向付けることで、10匹の魚の3行を作る。 </li><li>余分なメディアを取り出します。 </li><li>徹底的に1.5×10 7 CFU / mlのCでチューブをボルテックスアルビカンス 。 </li><li> Cの 3μlの1のマイクロニードルを埋めるアルビカンスが新しいmicroloaderピペットチップを使用して、適切な設定にパルス持続時間を設定する（ ステップ2.9参照 ）。 </li><li>浮袋（ 図3A）の背面に向かって目指して、マイクロニードルと魚の頭の間に20°の角度を作るために、注入の皿を回します。 </li><li>針の穴がルーメン内にあることを確認しながら、浮袋にマイクロニードルを押し、一度ペダルを押してください。 </li><li>各魚を繰り返し、E3で料理をあふれさせることで回復皿（25ミリリットルE3 + PTU）に移転Dリカバリー皿にそれを空に。 </li><li>注射皿に別の30魚を転送します。 </li><li>徹底的にC.ボルテックスで満たされた新しいマイクロ針で繰り返し注射アルビカンス 。 </li><li>必要に応じて、汚染を避けるため、別の回復皿（ ステップ4.10）に転送するために異なる注入の皿を使用して、PBS対照を注入して終了。 </li></ol> 5.目的の接種のために魚のスクリーニング<ol><li>溶解するまでマイクロ波処理し、37℃に冷却し、沸騰、E3 40mlのアガロース低溶融物160mgを加えることにより、0.4％低融点アガロース溶液（LMA）を40mlを準備する。一旦冷却TR（最終濃度200μg/ ml）を400μlのを追加します。 </li><li> （25 mLのE3 + PTU、最終濃度を200μg/ mlのにTRの唯一の1ミリリットルを使用してステップ4.3）に記載されているようで30分の回復後、魚を麻酔し。 </li><li>体重ボートでLMAの2ミリリットルに転送（できるだけメディアとの）30魚。 </li><li>トランスファーピペットで吸引し、個々の魚とのみ10×6中央井戸を使用して、96ウェルイメージングプレートにLMAの1滴の魚プレート。 </li><li>画面にすべての魚は、イメージングプレートにロードされるまで繰り返します。 </li><li>媒体を5分間室温で固化しましょう​​。 </li><li>彼らはガラスの底に接触していることを確認しながら、彼らの側に魚を置きます。 </li><li>倒立落射蛍光/共焦点顕微鏡および適切なフィルターで20X対物レンズを用い、C.数を記録する各魚の浮袋でアルビカンス酵母細胞。 </li><li> 、浮袋で15-25酵母細胞魚（または所望の接種材料）を選択して安楽死させる非選択魚（E3におけるTRの800μgの/ mlで）によってE3 + PTU 50mlの深いペトリ皿に選択された魚を戻すイメージングプレート選択したウェルにE3の3滴を追加し、プラ​​スチック製のトランスファーピペットを用いて魚を吸引する。 </li><li>所望の期間のために、33℃でインキュベートする。 </li></ol> 6.魚ポストスクリーニングイメージング<ol><li>所望の時間に、イメージに魚を含む深いペトリ皿からE3 + PTUメディアの50ミリリットルの25を削除します。 </li><li> （25ミリリットルE3 + PTU残り、最終濃度を200μg/ mlのにTRの唯一の1ミリリットルを使用してステップ4.3）に記載されているような魚を麻酔し。 </li><li>適切96ウェルイメージングプレート及び位置に魚プレート（ - 5.7 5.3ステップ ）。 </li><li>適切なレーザーおよび発光フィルターを使用して共焦点顕微鏡を走査して画像。微分干渉コントラスト（DIC）の下で4倍対物レンズを用いて魚に第1焦点と第2マイクロ秒/ピクセル速度および1024×600ピクセルのアスペクト比を有する走査モードで共焦点レーザーで20X対物レンズを用いて関心領域の画像を取得する。 3フレームのカルマン·フィルタモードを適用し、1μmのステップサイズでのZスタックを取得する。 </li><li> 50ミリリットルE3 + PTUまたはindividuaに深くシャーレに戻るリットルウェル（24ウェル培養皿を3ml E3 + PTU）と33℃でインキュベートする。 </li></ol> 7.浮袋を解剖<ol><li>高真空グリースで10ミリリットル注射器を埋める。 </li><li> 2％のLMA（アガロースE3の8ミリリットル中の低融点の160 mgのと歩調5.3）を準備します。 </li><li>所望の時間に、魚（ ステップ5.2）を麻酔し。 </li><li>転送10 E3 1mlの1.7 mlの遠心管に魚や遠心管に4 mg / mlのトリカイン（最終濃度800μg/ ml）を200μlを添加し、室温で15分間インキュベートすることによって安楽死させる。 </li><li>マイクロスライド（75×25ミリメートル、 図2）に1センチメートル離れて、広場に真空グリースの4滴を配置することにより、画像化スライドガラスを準備します。 </li><li>広場の真ん中に2％のLMAの2滴を置きます。 </li><li>解剖顕微鏡下で独立したスライドガラス上に1魚を置き、心臓が鼓動を停止していることを確認して、余分な水分を除去する。 </li><l細かいピンセットを使用してI&gt;は左ピンセットで固定された位置に頭を維持し、右のピンセット（ 図4A）で、前腸を下に引っ張って浮袋を分析。 </li><li>消化管からそれを分離し、左ピンセットで空気圧ダクトによる浮袋をピックアップし、それが固化する前に、2％のLMAの中心に配置します。 </li><li>イメージングスライドガラスの上にカバースリップ（18×18 mm）を置き、真空グリースなどLMA（ 図2）に触れるまで押し込みます。 </li><li>切開の10分以内に画像がステップ6.4で説明したように。 手順を繰り返し7.7から7.11他の魚と。 </li></ol>

The authors have nothing to disclose.

浮袋のマイクロインジェクション疾患モデルの進歩と限界ここで紹介するモデルは、Gratacap らに記載粘膜カンジダ症浸漬モデルの拡張である（2013）。それは、制御された感染時間、再現性の高い感染量、したがって、改善された効率の利点が追加されます。ここでは、非侵襲非常に詳細に感染動態の時間的なドキュメントだけでなく、より高解像度浮袋のe...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

粘膜病原体に対する初期の防衛は上皮バリアと自然免疫細胞の両方で構成されています。両方の免疫適格、と彼らの相互通信は、感染症に対する保護のための最も重要である。病原体上皮および自然免疫細胞の相互作用は最高の複雑な挙動は、時間と空間に繰り広げられる、生体内 、 で調査されている。しかし、既存のモデルは、粘膜レベルでの病原体との戦いの容易な時空間的イメージングを可能にしない。 ここで開発されたモデルは、少年ゼブラフィッシュの浮袋に、真菌病原体、 カンジダ·アルビカンスの直接注射により粘膜感染が作成されます。得られた感染は、上皮および粘膜疾患の発生を通じて自然免疫細胞の挙動の高分解能イメージングを可能にする。この方法の汎用性は、pHをもたらす免疫イベントの詳細なシーケンスをプローブするホストの尋問が可能にagocyte動員および保護において、特定の細胞タイプおよび分子経路の役割を検討する。また、免疫攻撃の関数としての病原体の挙動は、蛍光タンパク質を発現するC.で同時に撮像することができるアルビカンス 。宿主 - 病原体相互作用の増大した空間分解能について迅速浮袋切開技術を用いても可能である。 ここで説明粘膜感染モデルは、粘膜カンジダ症の研究のための貴重なツール作り、簡単な再現性の高いです。このシステムはまた、マイコバクテリア、正常上皮表面を介して感染する細菌またはウイルス、微生物などの他の粘膜病原体に広く翻訳可能であってもよい。

後部浮袋でマイクロインジェクションここに提示された実験方法はC.一貫した用量の注射を記載ゼブラフィッシュのDPF 4の浮袋でアルビカンス酵母細胞。イマージョンモデルと以前の研究は、Cに浮袋免疫応答を示唆しているアルビカンスは、哺乳類の粘膜カンジダ症32と同様である。ここでは、より多くの、簡単な再現性があり、迅速であ...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

浮袋注入による幼虫ゼブラフィッシュにおける粘膜カンジダ症のモデル化

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K ビュー.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

ビデオ: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

ゼブラフィッシュ幼生における播種性カンジダ症の非侵襲的イメージング

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

免疫・感染学

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

インビトロ 病原体誘導性好中球トランス上皮移動を評価するコーカルチャーアッセイ

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

腸管病原体の胆汁酸塩によるバイオ フィルム形成: 同定及び定量化の手法

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

結核抗酸菌 marinum感染成人ゼブラフィッシュをモデル化

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

播種性カンジダ症のハチミツガWaxworm 感染症モデル

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

ブレオマイシンの気管内注射によるマウス肺損傷の誘導

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

薬物を含むリポソームを注射してラーバルゼブラフィッシュマクロファージに薬物を標的にする

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

ヒト粘膜リンパ組織におけるEx Vivo自然免疫応答を研究する扁桃単核細胞の単核細胞の単離

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...