この研究は国立衛生研究所（NIH）の中小企業イノベーション研究プログラム（NIAID、1R43AI082698 - 01）と食糧農業のUSDA国立研究所（NIFA、2009-33610-20028）によってサポートされていました。

（1）Y.ペストのプレートの接種<ol><li>ストリークY.ペスト A1122（BeiResources＃NR15）ストック培養ルリア-ベルターニ（LB）寒天（ミラー）上に。無菌操作を使用し、すべてのYを行うクラスII型バイオセーフティーキャビネット。Y.におけるペストの操作ペスト A1122はバイオセーフティレベル（BSL）2除外選択エージェントの株である。それは、プラスミド75キロバイトのペア低カルシウム応答（LCR）病原性の両方を欠いている、と病原性に必要なPGMの軌跡。 Yを拡大28℃ ペスト ℃の空気インキュベーターを制御する静的な温度で48時間。 Yの成長率ペスト菌は 1.25 H 9の世代時間と遅いです。 </li></ol> （2）Y.ペスト菌の液体培地接種<ol><li>単一のYを転送する滅菌金属接種ループを使用してLBブロス2 mLを含有する滅菌17 × 100mmの培養チューブにペスト菌のコロニー。サイズの均一なコロニーを選択し、他のコロニーを示している、と明らかにプレート上のコロニーの残りの部分から分離されている。軽くボルテックスチューブと28で文化を育てる° C振盪インキュベーター（225 rpm）で約20時間。 </li></ol> （3）Y.ペスト菌の伸長、レポーターファージ加え、そして生物発光検出<ol><li>一晩Yを薄めるペスト菌の培養50mlファルコンチューブに新鮮なLBブロス（例えば、培地の9.5 mLに細胞を500μL）に1:20 ° C（225 rpm）で振とうしながら28で成長する。 0.2の600 nm（A 600）での光学密度（約5時間）に達するまで成長する。生物発光反応を形質導入するファージの能力は、宿主細菌の生存と適応度に相関しているので、活発に増殖する細胞は、検出のために好ましい。それにもかかわらず、検出システムは、成長のサイクル7のすべての段階で採取された細胞との互換性であることが示されている。 </li><li> 2パーセントのn -デカナール溶液を調製することにより生物発光反応に必要な基質を生成する。 LBブロスの9.8 mLでのn -デカナール200μLを混合。 5秒間激しくボルテックスプライム2％のn -デカナール溶液2mLを持つマイクロプレートルミノメーターインジェクタ。ルミノメーターはよくして、各プレートにデカナールソリューションの67μLをautoinject直ちに10秒間のサンプルを読み込むようにあらかじめ設定さ</li><li> 3文化のチューブにLBブロス1 mLのアリコートを分注する。各チューブにレポーターファージストック溶液（5 × 10 9プラーク形成単位の株式[PFU] / mL）を20μLを追加し、ファージとメディアだけではサンプルは、ネガティブコントロールとして機能します。 Yの不在でペスト菌の細胞は、レポーターファージの追加により、生物発光反応を引き出すしないでください。 </li><li> Y. 1mLのアリコートを分注する6培養管にペスト菌の培養（ステップ3.1から）。文化の3（試験培養）にレポーターファージストックの20μLを追加。残りの3の文化は&quot;単独で細胞のネガティブコントロール（バックグラウンドのautobioluminescence）として機能する。手短にボルテックスで文化をミックスし、各サンプルから収穫200μL（時間0読み取り用）と白色96ウェルマイクロタイタープレートに分注する。 28で、残りの培養液を、Cを（225 rpm）で振盪しながら。 </li><li>マイクロプレートルミノメーターを用いて生物発光の時間0サンプルを（相対発光ユニット、RLU）を測定します。 </li><li> 10以降の各サンプルの収穫200μL、20、30、40、50、および60分後のレポーターファージのほか、生物発光用の試料を測定する。 Y.場合ペスト菌の細胞が存在し、レポーターファージは、特に、細胞に結合し、そのDNAを注入し、luxABレポーター遺伝子（図1）転写と翻訳するの転写と翻訳機構をホストが使用されます。 </li><li>信号の強さと信号の応答時間が存在する細胞の数に比例します。細胞（10 5〜10 8 CFU / mL）を、対照と比較したRLUの大幅な増加が高濃度で20分以内に明らかにする必要があります。低細胞濃度では、（10 2 -10 4 CFU / mL）を、RLUの著しい増加は40〜60分以内に明らかにする必要があります。 </li><li>また、Y.を必要とせずに検出プロセスを迅速化するペスト菌の伸長は、新鮮な成長プレートからコロニーを、レポーターファージを保有するLBブロス200μLのを1.5mlのエッペンドルフチューブに直接（ボルテックス）混合することができる。マイクロタイタープレートのウェルに細胞/ファージ混合液を分注する。 28℃でマイクロタイタープレートをインキュベートし、60分（シングル時のポイントのみ）後の生物発光のためにサンプルをお読みください。 </li></ol> 4。代表的な結果： Yのレポーターファージを介した検出の代表的なタイムコース実験ペスト菌を図2に描かれている。 1の陰性コントロール）ファージ単独で（無細胞）、または2のみ）セル（無ファージ）60分株式会社を通じて約20 RLUの生物発光のベースラインレベルを提供ubation（ベースラインのレベルはルミノメーター固有のもの）。対照的に、テストサンプル（レポーターファージや細胞）のための生物発光の増加は、ファージ添加後15分で明らかである。信号強度は、60分かけて徐々に増やす必要があります。長期期間（80分以​​上）のためのインキュベーションは、宿主細胞の媒介溶解をファージに起因するピーク信号から減少する信号となります。同様の結果が37のインキュベーションの温度で得られる° Cにもかかわらず、Yの最適温度ペスト菌の成長は28℃で9です。 <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="図2"/> 図2。 Yのファージを介した生物発光検出ペスト 。0の時点で、レポーターファージ、細胞を28℃でインキュベートし、混合し、そして生物発光は（RLU）を経時的にモニターした。 RLUの大幅な増加は、（*学生t -検定、p &lt;0.05）が15分以内に明らかである。

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

利害の衝突は宣言されません。

このメソッドは、急速にYを検出するレポーターファージの能力を示していますレポーターファージからペスト菌は、培養Yに生物発光シグナルの応答を伝達することができるファージの添加後20分以内ペスト菌の細胞。レポーターファージはまた、直接Yを検出することができる分離とその後の栽培7の前提条件なしに臨床マトリックスにおけるペス?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> Difco社製LB寒天、ミラー</td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco社製LBブロス、ミラー</td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 × 100 mmの培養管</td><td>米国サイエンティフィック</td><td> 1485-0810 </td></tr><tr><td> N -デカナール</td><td>シグマ</td><td> D7384 </td></tr><tr><td> VERITASマイクロプレートルミノメーター</td><td>ターナーバイオシステムズ</td><td> 9100-001 </td></tr><tr><td>マイクロライトマイクロタイター96ウェルプレート</td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

ペスト菌と炭疽菌は、それぞれ1、ペストや炭疽の原因物質である細菌性病原体カテゴリです。両疾患&quot;の自然発生は、今は比較的まれですが、生物兵器としてこれらの病原体を使用して、テロリスト集団の可能性は本当のです。病気の本質的な伝達性、急速な臨床経過、及び高い死亡率のため、発生を迅速に検出することが重要です。したがって、迅速な検出と診断を提供する方法論は、公衆衛生対策と危機管理の活性化の即時実施を確保するために不可欠です。 組換えレポーターファージは、Yの検出のための迅速かつ特異的なアプローチを提供することがありますペストとB.炭疽菌。疾病管理予防センターは、現在、これらの細菌性病原体2-4の確認同定のために古典的なファージの溶解アッセイを使用してください。これらのアッセイは、自然に彼らの細菌のホストの特定および溶解性であるファージを発生し活用する。特定のファージの存在下で培養細菌の一晩増殖した後、プラーク（細菌の溶解）の形成は、細菌のターゲットの肯定的な識別を提供する。これらのアッセイは堅牢ですが、彼らは3つの欠点に苦しむ：1）彼らは、実験室ベースで、2）彼らが疑われる試料から細菌の分離培養を必要とし、3）彼らは、完了までに24〜36時間かかります。これらの問題に対処するため、組換え&quot;光-タグ&quot;レポーターファージは遺伝的にYのゲノムにビブリオharveyi luxABの遺伝子を統合して設計されたペストとB.炭疽菌の特定のファージ5-8。結果luxABレポーターファージが急速にすることで、特定のターゲットを検出することができた（分以内）と敏感に受容細胞に生物発光表現型を付与する。重要なのは、検出が培ってきたレシピエント細胞にまたはモック感染臨床検体7とどちらが得られた。 デモンストレーションの目的で、ここでは知られているYのファージを介した検出のための方法を説明します。 ペスト菌は CDCペスト診断ファージΦA11226,7（図1）から構成さluxABレポーターファージを用いて分離する。軽微な変更（成長温度やメディアの例：変更）、と同様の方法は、Bの検出に使用されることがあります炭疽菌はB.を使用して隔離炭疽菌のレポーターファージWβ：：luxAB 8。方法は、栽培Yにbiolumescent表現型のファージを介した情報伝達を説明しますその後、マイクロプレートルミノメーターを用いて測定しているペスト菌の細胞。伝統的なファージの溶解アッセイに比べてこの方法の主な利点は、使いやすさ、迅速な結果、そして96ウェルマイクロタイタープレートフォーマットで同時に複数のサンプルをテストする機能です。 <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="図1"/> 図1。検出の回路図。ファージを試料と混合され、ファージが細胞に感染する、luxABが発現し、細胞のbioluminescesされています。サンプルの処理が不要です。ファージや細胞を混合し、続いて光を測定している。

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

優先細菌性病原体の同定のための簡単​​な方法は、遺伝子組み換えレポーターファージを使用することです。その特定の宿主種に固有のこれらのレポーターファージは、、急速に宿主細胞に生物発光信号応答を伝達することが可能です。ここで、我々は、検出のためのレポーターファージの使用について説明します。<em&gt;ペスト菌</em&gt;。

カテゴリの検出細菌性病原体のための&quot;生物発光&quot;レポーターファージ

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Research

JoVE Journal

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

13.3K ビュー.  Guild Associates, Inc.. 優先細菌性病原体の同定のための簡単​​な方法は、遺伝子組み換えレポーターファージを使用することです。その特定の宿主種に固有のこれらのレポーターファージは、、急速に宿主細胞に生物発光信号応答を伝達することが可能です。ここで、我々は、検出のためのレポーターファージの使用について説明します。ペスト菌。

ビデオ: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

細胞内細菌性病原体を持つゼブラフィッシュ胚の感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物発光細菌のイメージング<emインビボで&gt;</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容<em&gt;クリプトコックス·ネオフォルマンス</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

ナノスケールPCRによる動物標本における呼吸器病原体のハイスループット検出

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

病原性細菌に対する免疫学的応答を評価するための Opsonophagocytic 殺害アッセイ

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

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. ...