私たちは、動物のケアとモニタリングのために、S.、本研究と米国農務省で使用されるカラスを提供するためのヴェルナー、APHIS、WS、NWRC動物管理スタッフに感謝したいと思います。言及または製品を使用するには、米国農務省の承認を意味するものではありません。この研究のための資金は、米国農務省、APHIS、獣医サービスによって提供された。

我々のプロトコルは、我々が以前に7を発表した1から採用されている。動物に関わるすべての手続きは、米国農務省（USDA）、動植物衛生検査局（APHIS）、野生動物サービス（WS）、国立野生生物研究センター（NWRC）の施設内動物管理使用委員会によって承認された。 1。カラスGavaging <ol><li>アメリカのカラスの消化管を通して「疑似脳物質 &#39;の通過時間を見積もります。 <ol><li>調理の5ミリリットルを混合し、強制経口投与針2（ 図1）を用いた青色色素および強制経口投与1カラスと全卵をスクランブル。 </li><li>青/緑に染色された糞はもはや排泄されるまで、30分ごとにカラスないことを確認してください。 </li></ol></li><li>感染していないと末期RML（チャンドラー株）に感染したC57BL / 6マウスの脳を入手します。 </li><li> 1Xと弾丸ブレンダーホモジナイザーの通常20％重量/容量、感染したマウス脳乳剤を生成リン酸緩衝生理食塩水（PBS）とガラスビーズを、次いで、より大きな粒子状物質を除去するために1分間3,000×gで遠心する。上清を除去し、無菌のPBS中の10％重量/体積を生成するために、1×PBS等容量の希釈した。必要になるまで-80℃で凍結する。 </li><li>前胃管栄養への17時間は、カラスのペンから、水食料を削除ではなく。 </li><li>無作為に処置群にカラスを割り当て、強制経口強制栄養針2を使用して正常または感染したいずれかのマウス脳ホモジネート5mlでそれぞれカラス。 </li><li>転送は、個々のケージにカラスや4時間後、胃管栄養法（ 図2）のために、各ケージ内のすべての糞を集め、プール。 </li><li>均一な組織が得られるまでガラスビーズと弾丸ブレンダーホモジナイザーの各カラスのためにプールされた糞を均質化する。 *カラスの糞は主に液体であり、容易に混合することができる。 </li><li>各カラスのために、10ミリリットルの全容量のために9.5ミリリットルの1X PBSに糞便ホモジネート500μlの希釈する。 </li><li>糞便homogenaを遠心分離1400×gで15分間のTE、上清を抽出します。 </li><li>二微生物感染の脅威を最小限ホモジネート、100μlの1〜100単位の液/ mlペニシリンおよび100μg/ mlのストレプトマイシン（Invitrogen社、NY）を用いて上清を治療するためにその後減少させるために20分間室温でUV光下に置くウイルスおよび細菌汚染のリスク。 UV露光した後、残りの微生物の膜を破壊するために30秒間70を設定することで、3000 Mpは超音波処理における超音波処理のサンプル。 </li></ol> 2。マウス接種<ol><li>無作為に以下の処置群（ 表1）にマウスを割り当てる。 <ol><li>グループ1 - 陽性の処置マウスは経口5ミリリットル感染マウスの脳を強制飼養カラスからの糞便ホモジネート1mlで腹腔内（IP）接種した。 </li><li>グループ2 - 負の処置マウスは、経口5ミリリットル、通常のマウスの脳を強制飼養カラスからの糞便ホモジネート1mlのIPアドレスを接種した。 </li><li> infecteを希釈1×PBSで1:100重量/容量のグループ3および4のdおよび正常マウス脳ホモジネート。 </li><li>第3群 - 陽性対照マウスは、感染マウス脳ホモジネート1mlのIPを接種した。 </li><li>グループ4 - 陰性対照マウスは正常マウス脳ホモジネート1mlのIPを接種した。 </li></ol></li></ol><table border="1"><tbody><tr><td></td><td>治療グループ</td><td>動物の数</td></tr><tr><td>グループ1 </td><td>スクレイピー+クロウ糞</td><td> 100 </td></tr><tr><td>グループ2 </td><td>スクレイピー - クロウ糞</td><td> 25 </td></tr><tr><td>グループ3 </td><td>スクレイピー+マウス脳</td><td> 10 </td></tr><tr><td>グループ4 </td><td>スクレイピー - マウス脳</td><td> 5 </td></tr></tbody></table> 表1スクレイピー接種の状況（負正+、 - ）と動物の数が7を使用していました。 <ol start="2"><li>腹腔内にマウスに接種： <ol><li>優しく首筋の親指と人差し指で背面の首の毛皮によるマウスとは、腹側を露出させ回転させます。 </li><li>ヘッドがやや低いので、マウスの後端部を上昇させる。 </li><li>針ロック注射器を用いて仙腸関節にCM前部皮膚を通して25 G針1センチ、正中線の左右1cmの1-2を挿入します。 </li><li> 1mlを、マウスの体腔内に徐々に接種注入する。 </li></ol></li></ol> 3。マウスの監視<ol><li>彼らはマウススクレイピーの臨床徴候を表現まで毎日マウスを監視します。臨床症状は含まれる：脊柱後、運動失調、硬い尾、グルーミング、衰弱、および嗜眠の欠如を。 </li><li> 0目に見える=なし、1 =中等度、および2 =が激しいところ目に見える兆候は、明らかであるとき6臨床徴候のそれぞれにマウスを獲得。 </li><li> 3日間、または365日後に接種（DPI）で連続6それぞれの記号の1日の総得点は1日の≥8に到達したときに、マウスを安楽死させる≥。 </li><li>収穫脳直後のeuthan-70℃でのアジアと店舗</li><li> 、スクレイピー診断を確認、次のように希釈されたプロテイナーゼK溶液（PK）の50μg/ mlのの3μlの脳サンプルを消化するために：3.1。 μlのPK、500mMのEDTA12.5μlの、pH8で、45℃で30分間、1×PBSの109.39μlを、次いで、8μlのローディング緩衝液を添加し、95℃で5分間、サンプルをインキュベートすることによってPKを不活性化する。 12％SDS-PAGEゲル電気泳動させると、室温で1時間、PBS中の0.2％のTween 20でイモビロンPVDF膜を5％脱脂乳でブロックへの転送上に負荷サンプル。次いで、室温で1時間のSuperblock中で希釈西洋ワサビペルオキシダーゼ、Bar224にコンジュゲート抗プリオンモノクローナル抗体でプローブする。 PBS-0.2％Tween 20で1時間、膜を洗浄します。可視化するために、G-ボックスゲルドキュメンテーションシステムに化学発光基質およびイメージを使用してウエスタンブロット5分間インキュベートする。 </li></ol>

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利害の対立が宣言されていません。

私たちは、カラスの消化器系を通じてプリオンのRES物質の通過を文書化するための手順を示しています。私たちは、カラスが無病の地域へのプリオン解像度素材を転位する能力を持っているかどうかを判断するために、従来の方法を使用していました。他はそれを排除することができなかった、どちらも19〜21と齧歯類22,23消化液を反芻するプリオン解像度...

伝達性海綿状脳症（TSE）は、野生生物に影響を与える致命的な感染性の神経変性疾患、家畜、そしてヒトである。 TSE疾患の感染性病原体はプリオンタンパク質1のミスフォールドまたは病原性アイソフォーム（PRP RES）であるように思われる。ヒツジおよびヤギにおけるスクレイピー;、牛海綿状脳症（動物TSE疾患はラバ鹿（ ミュールジカ ）、オジロジカ（ オジロジカ ）、エルク（ アカシカ ）、およびムース（ ヘラジカ ）の慢性消耗病（CWD）が含まれ国内の牛のBSE）;養殖ミンクにおける伝染性ミンク脳症、ネコのネコ海綿状脳症、ヒト以外の霊長類8にし、海綿状脳症、エキゾチックな動物園のエキゾチックな有蹄動物海綿状脳症は、家族ウシ科の反芻。単一のヒトTSE病、変異型クロイツフェルト·ヤコブ病は、稀で、 解像度のPrP-contaminを消費することによって取得されると考えられているated食品9。汚染された牛肉が10消費された場合も、BSEはヒトに感染することができます。すべてのTSE疾患のうち、スクレイピーおよびCWDは、動物から動物2,3,11にだけでなく、環境源4-6からのものであると推定される自律的な流行と感染のソースでのみ2である。研究では、ほとんどのTSE疾患は臨床徴候が発現する前にプリオン解像度素材の自然曝露イベントからの注目すべき拡張潜伏期間が必要2-4,6,8、見かけ種の壁を最小限に、しかし、種間伝播12月14日の可能性を排除しないことを示唆している。 感染性プリオン（PRP RES）材料の普及のためのメカニズムを識別することはTSE疾患が風景を越えて移動する方法についての質問に答えるために非常に重要です。実験的研究は、昆虫15,16、家禽および豚17、およびアメリカのカラス（ カラスのブラチことが示唆されているhyrhynchos）7,18は、PrP解像度材料の受動的なキャリアまたは分散機である。カラスの消化器系を通じてプリオンのRES物質の通過は最近、TSE疾患7の分散でプレイしてもよい役割を示す、文書化されています。これらの結果は、カラス、スカベンジャーは、遭遇する可能性があること、それがもっともらしい作る消費し、無病領域に、糞の堆積により感染性物質を輸送する。 我々はここに証明する手続きは、カラスの消化システムを介してプリオンのRES物質の通過を記録するために使用されたと大いに関連する今後の研究の他のスカベンジャーと肉食種特異的なモデルに、これらの方法の適用を容易にします。本研究では、従来の方法は、プリオン解像度素材の広がりと全体的な重荷に貢献できるプリオン解像度素材を、人身売買の型破りな手段を調査するために使用された。

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

感染性プリオン（PRP RES）材料は、おそらく致命的な神経変性伝達性 ​​海綿状脳症（TSE）の病気1の原因である。例えば、慢性消耗病（CWD）などのTSE疾患の送信は、動物から動物への2,3並びに環境源4-6からであると推定される。スカベンジャーと肉食動物は、CWDに汚染された死肉の摂取と排泄を通してプリオン解像度材料を移動させる可能性を持っている。最近の研究はアメリカのカラスの消化器系（ アメリカガラス ）、一般的な北アメリカのスカベンジャー7を通してプリオン解像度素材の通過を文書化しました。 我々はアメリカのカラスを通じてプリオン解像度素材の通過を記録するために使用される手順について説明します。カラスはRML - ひずみマウス適応スクレイピーで強制経口投与し、それらの糞は4時間後に強制経口投与を収集した。カラスの糞をし、その後プールし、腹腔内に注射したC57BL / 6マウス。彼らはマウススクレイピーの臨床徴候を表明し、その後、安楽死させたまではマウスを毎日モニターした。無症候性マウスは365日後に接種するまで監視した。ウェスタンブロット解析は、疾患状態を確認するために行った。結果は、プリオンがカラスの消化器系を通って移動した後に感染性のままであり、試験マウスにおいて疾患を引き起こす、糞便中に存在することが明らかになった。

使用手順は、カラスの消化器系は、スクレイピー脳ホモジネート7の強制経口投与した後にPrPの解像度感染性4時間を排除しないことを示している。プリオン解像度材料で強制経口投与されたすべての20カラスはその後、マウスに糞を経由したPrP 解像度素材を送信した。ウェスタンブロット分析によって完了した罹患したマウスは、臨床マウススクレイピー徴候...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

スカベンジャーは無病領域に糞中の感染伝達性海綿状脳症プリオンを移動させる可能性を持っている。私たちはマウス適応スクレイピープリオンはアメリカのカラスの消化管（ アメリカガラス ）、死んだ動物の共通の消費者も通過した後、感染のままかどうかを判断するために使用される詳細な方法。

鳥類の消化器系を通過した後の感染プリオンを識別するための手順

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.0K ビュー.  USDA. スカベンジャーは無病領域に糞中の感染伝達性海綿状脳症プリオンを移動させる可能性を持っている。私たちはマウス適応スクレイピープリオンはアメリカのカラスの消化管（ アメリカガラス ）、死んだ動物の共通の消費者も通過した後、感染のままかどうかを判断するために使用される詳細な方法。 

ビデオ: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

免疫・感染学

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

野鳥の鳥インフルエンザウイルスの迅速な診断：フィールドのポータブルRRT - PCR及び凍結乾燥試薬の使用

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

フィールドメソッド、バイオセーフティ、および交通の問題解決：FTAカードによる鳥インフルエンザのサーベイランス

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

プリオンのタンパク質のミスフォールディングサイクリック増幅

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

羊の非定型および古典スククレープを検出する臨床検査プロトコル

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

医学

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

コレラ毒素とビオチン化デキストランアミン二重標識が続く仮性狂犬病ウイルスとペリフェラルターゲットからトレース経シナプス

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

神経科学

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

と伝達性海綿状脳症種の障壁を評価する<em&gt;インビトロ</em&gt;プリオンタンパク質変換アッセイ

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

リアルタイム揺れる誘起変換用糞便材料の CWD プリオンの検出

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

プリオン安全研究所 スワイプ試験

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

鳥類の消化器系を通過した後の感染プリオンを識別するための手順

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この動画の章

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

野鳥の鳥インフルエンザウイルスの迅速な診断：フィールドのポータブルRRT - PCR及び凍結乾燥試薬の使用

フィールドメソッド、バイオセーフティ、および交通の問題解決：FTAカードによる鳥インフルエンザのサーベイランス

プリオンのタンパク質のミスフォールディングサイクリック増幅

羊の非定型および古典スククレープを検出する臨床検査プロトコル

コレラ毒素とビオチン化デキストランアミン二重標識が続く仮性狂犬病ウイルスとペリフェラルターゲットからトレース経シナプス

と伝達性海綿状脳症種の障壁を評価する

リアルタイム揺れる誘起変換用糞便材料の CWD プリオンの検出

プリオン安全研究所スワイプ試験

免疫組織化学による異常プリオンタンパク質の検出

鳥類の消化器系を通過した後の感染プリオンを識別するための手順

要約

さらに動画を探す

この動画の章

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

野鳥の鳥インフルエンザウイルスの迅速な診断：フィールドのポータブルRRT - PCR及び凍結乾燥試薬の使用

フィールドメソッド、バイオセーフティ、および交通の問題解決：FTAカードによる鳥インフルエンザのサーベイランス

プリオンのタンパク質のミスフォールディングサイクリック増幅

羊の非定型および古典スククレープを検出する臨床検査プロトコル

コレラ毒素とビオチン化デキストランアミン二重標識が続く仮性狂犬病ウイルスとペリフェラルターゲットからトレース経シナプス

と伝達性海綿状脳症種の障壁を評価する

リアルタイム揺れる誘起変換用糞便材料の CWD プリオンの検出

プリオン安全研究所 スワイプ試験

免疫組織化学による異常プリオンタンパク質の検出

プリオン安全研究所スワイプ試験