このプロジェクトは、化学、生物、放射線及びカナダ食品検査庁の核（CBRN）研究技術イニシアティブ（CRTI）、およびアルバータプリオン研究所からの資金調達を実施した。中国国家自然科学基金は、W. Xuさんへの奨学金（第30620120430）を提供する。著者は彼らの技術支援のためにブラントベイカーやフレッドヴァンHerkだけでなく、ルースバルビエリ、リサKalischuk - Tymensen、アンドリューオルソン、ローナSelinger、ジェフWallinsとホマZahiroddiniに感謝。

重複した堆肥の構造はレスブリッジ、アルバータ州、カナダのレスブリッジ研究センター（LRC）（図1）で建設された。大きな長方形の大麦のわら俵（260 × 120 × 80センチメートル、長さx幅x高さ）は、壁、および床のための小さな俵（100 × 40 × 45 cm）のために使用された。壁は壁の安定性と保温性を最大化され、120 cm厚だったので、大俵が指向された。床を形成する小さな俵は45 cm厚の床をもたらし、より糸がリークのイベントで吸光度を最大にするために水平に実行されていると志向した。長さx幅x高さ）、構造物の全体の寸法は、25 mxの5 mxの2.4メートルだった。建設現場は、約1の傾きを持っていた、および構造体は、我々は実験的な評価のために挿入されたサンプリングポートに向かって浸出水の流れを促進するために指向した。モイスト厩肥は肥料散布機を通して処理と前封じ込め構造物の建設に24時間積んで混合し、曝気した。 堆肥のバンカーは、一般的にサイレージ杭をカバーするために使用される大量の白/黒のビニールシートが並んでいた。十分な余剰がバンカー内に堆肥のベッドの最終的な折り畳み式とシールを有効にするには、各方向に上から残っていた。周囲の風に対抗するために、タイヤは藁がロードされるまでバンカーでプラスチックを重み付けするために使用されたが、これらは前に置かれている死体に削除されました。浸出水のサンプリングポートが（唯一の実験的なサンプリングのために必要）をインストールした後は、緩やかな大麦のわらは、約40cmの厚さ、ベース層を形成する生物封じ込め構造に追加されました。これはフロントエンドローダを持つ単一のラウンドベールを提供し、手動でバンカーの長さに沿って藁を配布することによって達成された。 シックスティーン肥育牛の死亡率は、わらのベッドの上に置かれた。これらは、近隣の商業的フィードロットで以前の48時間以内に死亡した牛だった、大部分は、牛呼吸器疾患に屈しした。死体は、死体の間に約0.5 mの空間で、堆肥のバンカー（図1）内で横方向に整列させた。受動曝気を提供するために、ミシン目フレキシブルなプラスチックの排水チューブ（15cmの直径）の長さがゆるいわらの基地内に埋め込まれ、隣接した死骸の間に敷設された。チューブの端面は、バンカーの上を越えて伸びる側の壁に沿って垂直に向け、および一時的に壁の上面を越えて拡張するタイヤを使ってその場で開催されました。 プレコンディショニング厩肥は、1.6 mの最終的な深さに死体をカバーするためにバンカーにロードされた肥料は、他に向かって構造の一方の端から移動し、バンカーの側壁によってロードされています。後述のサンプル取得のピラミッドは、この段階で肥料層内での所定の深さで配置した。バンカーが満たされた後は、ビニールシートを積み上げた堆肥の上に折り畳まれ、穴あき管の両端は、それらはラップの外側に位置していたようにそれを通過させた。プラスチックはわら、死骸や糞の周り封じ込め障壁を確立するためにテープで密封した。八通気孔（50 × 50 × 15 cm）を受動的通気を促進するため、各構造の上に置かれ、T字型コネクタは、チューブに空気の流れを促進するための穴あき管の長さの各端部に取り付けられた。 このパイロット研究では、堆肥の構造への実験的な改正は、静的な堆肥化の過程で病原体不活化と組織の劣化の調査を可能に組み込まれました。組織や微生物試料組織の重量や微生物の列挙によって事前に定量化は、ナイロンサンプルの袋（5 × 9センチメートル、50μmの孔径）にヒートシールされた、とベイカー検索ピラミッド（BRP）として指定され、専門的なピラミッド型のスチール製ケージに詰め込ま1。同じ肥料に埋め込まれていたこれらのピラミッドは、真剣にBRP、8袋（一eightサンプルタイプの各々）の中で封じ込めを犠牲にするか、堆肥化処理のダイナミクスを変えることなく堆肥化プロセスの間に一定の間隔で堆肥の行列から、その取得を可能にするために設計されたバンカーを埋めるために使用された、各袋は肥料に囲まれていない袋が互いに直接接触していたされなかったように配置。堆肥と袋充填BRPsは、ログチェーンの長さに取り付けられており、堆肥のマトリックス内に80センチメートルと160 cmの深さで懸濁した。チェーンは、1.5 m間隔（図1）でバンカーをスパニング木の棒に固定された。各BRPに配置されたT型熱電対は、外部から堆肥の構造に配置さロガーデータへのチェーンに沿って実行されました。指定された間隔で、BRPsはratchetedケーブルプラー-やってきて、そしてラップが再封印されたを使用して堆肥から垂直に撤回された。アルバータ州南部の自然に乾燥した気候では、ほとんど降雨がtの間に受信した彼の時代が、プラスチック製のラップや堆肥の材料のドーム型の形状は、わらのバンカーの壁によって吸収される横方向の流出を促進するための効果的であった。

<ol>
	<li>Reuter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+temporal+collection+of+tissue+and+microbial+samples+from+static+composting+systems.">A simple method for temporal collection of tissue and microbial samples from static composting systems.</a> Canadian Biosystems Engineering. 50, 6.17-6.20 (2008).</li><li>Xu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biosecure+composting+system+for+disposal+of+cattle+carcasses+and+manure+following+infectious+disease+outbreak.">A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.</a> J. Environ. Qual. 38, 437-450 (2009).</li></ol>

利害の衝突は宣言されません。

静的な堆肥の147日後に、総質量、乾物（DM）、有機物、全炭素および全窒素の損失は、それぞれ2 23.7、35.6、52.9、49.6、、41.4％であった。各構造の中で、材料の量は118から71 m 3に減少し堆肥化。ウシの組織の分解が脳&gt;蹄&gt;骨にランク監視。堆肥の7日後でのみ、脳組織DMの&gt; 90％が分解していた、と蹄DMの80％は堆肥の56 D内で分解していた。 大腸菌 O157の生存能力の完全な損失：H7およびニューカッスル病は、14日以内に達成された。 集約的な家畜生産システムは、自然または人為的な感染症の流行に対して特に脆弱です。狭いエリア内での動物の多数を収容することは人口全体に急速に普及するほとんどの感染性病原体を生じさせる。封じ込めは、任意の伝染病の発生を制御する鍵となる、このように過疎化が大きな家畜の個体に感染性病原体のまん延を防止する手段として頻繁に使用されます。家畜の死体と迅速な処理を必要とする汚染された肥料の大量の過疎化のシナリオの結果。コンポスト化は、迅速な応答処理感染した死体のための方法と同様に感染性病原体を抱くことができる肥料及び土壌として適しています。我々は、ファーム上またはローカルファーム供給店から入手可能な材料を使用して、病気の発生の現場で行うことができる堆肥化の手順の概要を説明します。我々の研究では、肉牛の死骸や糞に関連付けられている感染性病原体が堆肥の14日以内に不活性化された、堆肥の温度は1ヶ月以上、55 ° Cを超えた。浸出水の生産は、緩やかなわらベース層のため、吸収性の性質の可能性が極めて低く、私たちの堆肥化開始時のDMの内容を最適化した。浸出水の総収量は、初期の堆肥の質量（すなわち、構造当たり&lt;300 g）の3未満ppmであった。大腸菌群は14日で、最大で5.8 log10のCFU / mLに浸出水で検出されたが、堆肥の101日後に検出不可能であった。 147日後に、ウシ死体はわずかに長い骨が認識され、ほぼ完全に分解された。骨は、土地の用途に適して最終的な成熟した堆肥をもたらし、biocontained構造が開かれた後に追加のオープンウィンドロウの堆肥化サイクル中にさらに分解された。 結論として、堆肥はほとんどの病原微生物の生存に大きな課題を提示する条件を作成します。無料の細菌、原虫およびウイルスは急速に堆肥の中高温、アルカリ性、高プロテアーゼおよびヌクレアーゼの活動によって不活化されています。成熟した肥育牛の枝肉のこの成功を分解では、この静的な堆肥化処理の手順はすべて共通の家畜に適しているということを示しています。ケアは、その最適な炭素を確保するために、しかし、注意する必要があります：窒素比率と水分量は、達成すべき微生物を殺す条件のために存在しています。このような形の胞子（例えば、炭疽菌）、またはそのようなプリオンのような異常に反抗的であるものが、、まだ堆肥化後に感染に留まることができる細菌のような本質的に、より耐熱性病原体、。堆肥化過程で微生物のこれらのタイプの運命を解明する研究は、当研究室で現在進行中です。 <img src="/files/ftp_upload/1946/1946fig1.jpg" alt="図1" /> 図1。ストローベイルの壁や床、プラスチックシートのエンクロージャ、ゆるいわらのベース、牛の死骸、糞尿、あいたプラスチック製の換気チューブ、および通気口を構成するbiosecure堆肥システムの模式図だけでなく、実験的な改正（浸出液ポート、サンプルの検索ピラミッドと温度センサ（A）横ビュー（断面積）。（B）縦ビュー（サイドウォールが削除）。全ての寸法はcmになります。Xuら（2009）2から

biocontained carcass composting for control of infectious disease outbreak in livestock

集約的な家畜生産システムは、自然または意図的な（バイオテロ）感染症の流行に対して特に脆弱です。狭いエリア内に収容された動物の多数は、群れ全体で最も感染性病原体の急速な普及が可能になります。迅速な封じ込めがあらゆる伝染病の発生を制御する鍵となる、このように過疎化は、しばしば大規模な家畜​​の個体群に病原体のまん延を防ぐために行われている。そのような状況では、家畜の死体や汚染された肥料の多くは、迅速な処理を必要と生成されます。 コンポスト化は、迅速な応答処理感染した死体のための方法と同様に感染性病原体を抱くことができる肥料及び土壌として適しています。我々は、バイオ完結型の死亡率の堆肥化手順を設計し、ウシ組織の劣化と微生物の不活性化のためにその有効性をテストした。我々は、システムが病気の発生の現場で実施されることができるために地元の農産物供給店から、または購入可能な農場で入手可能な材料を使用していました。本研究では、気温は55を超えました° Cを一ヶ月以上と牛の死体や糞尿堆肥の14日以内に不活性化された中に注入された感染病原体のために。 147日後、死体はほぼ完全に分解された。残りの少数の長い骨は、オープンwindrowsで追加の堆肥化のサイクルでさらに分解されたと最終的な成熟した堆肥は、土地のアプリケーションに適していた。 （最終的な寸法25 MX 5 mxの2.4メートル、長さx幅x高さ）堆肥の構造を複製するには大麦のわら俵を使用して構築し、重い黒サイレージのプラスチックシートが並んでいた。それぞれが〜95000キロを合計ゆるいわら、死骸や糞がロードされました。緩い大麦のわらの40センチのベース層は、約0.5メートルの間隔で横方向にアラインされた16肥育牛の死亡率を（平均体重343キロ）に置かれた上に、それぞれのバンカーに入れていた受動曝気の場合は、柔軟性のある、穴あきプラスチック製の排水管（15cmの直径）の長さは両方の内側の壁に沿って垂直に伸びる、と外装にプラスチックも渡される端部と、隣接した死骸の間に配置された。死体はバンカーの上に湿った曝気厩肥（〜1.6メートル深さ）を重層した。プラスチックは、折り返しされたトップと封じ込め障壁と8つの通気孔（50 × 50 × 15 cm）の受動的通気を促進するため、各構造の上に置かれたを確立するためにテープで密封。 147日後に、堆肥材料の体積と質量の損失は、それぞれの構造で、それぞれ、39.8パーセントと23.7％の平均値。

Watch this Scientific Journal Video about Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock at JoVE.com

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

容易に入手可能な材料を使用して、このbiocontained堆肥システムでは、感染症のアウトブレイクが発生した場合に発生する大規模な動物の死体の効果的なオンサイト処理が可能になります。この手順では、死体や汚染された肥料で、ほとんどの感染性病原体を殺します。感染性病原体が確認されれば、非現実的な、成熟した堆肥は肥料として展開することができます。

家畜の感染症アウトブレイクの制御のためのBiocontained枝肉コンポスト

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of ...

biocontained-carcass-composting-for-control-infectious-disease

Research

JoVE Journal

Immunology and Infection

10.8K ビュー.  Lethbridge Research Centre. 容易に入手可能な材料を使用して、このbiocontained堆肥システムでは、感染症のアウトブレイクが発生した場合に発生する大規模な動物の死体の効果的なオンサイト処理が可能になります。この手順では、死体や汚染された肥料で、ほとんどの感染性病原体を殺します。感染性病原体が確認されれば、非現実的な、成熟した堆肥は肥料として展開すること?...

ビデオ: Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

のための負の選択マーカーの開発赤痢アメーバ</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

免疫・感染学

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 ...

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 ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

表面上のヒトノロウイルスの検出のためのスワブサンプリング方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

新世界Zikaウイルス感染クローンからの組換えウイルスの救出とキャラクタリゼーション

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

試金を使用して SIAT1 MDCK 細胞のインフルエンザ a (h3n2) ウイルス中和によるひと血清中の抗体を中和することを測定

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

感染マウス感染モデルにおける髄膜炎の連鎖球菌性バイオ フィルムの役割の調査のための頭蓋内クモ膜下ルート

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

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 ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

ジカ ウイルス特異 Immunoprivileged 臓器感染 Ifnar1-/-マウスの T 細胞応答の評価

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

マウスと次世代シーケンサーによるひと抗体のレパートリーの同定

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...