Wir möchten S. Werner für die Bereitstellung der in dieser Studie verwendet und USDA Krähen danken, APHIS, WS, NWRC Tierpfleger für Tierpflege und-überwachung. Erwähnen oder die Verwendung eines Produkts nicht USDA Billigung. Die Finanzierung für diese Studie wurde von USDA APHIS, Veterinary Services zur Verfügung gestellt.

Unser Protokoll wird von einem zuvor veröffentlichten wir 7 angepasst. Alle Verfahren, die Tiere wurden von der Institutional Animal Care und Verwenden Ausschuss des United States Department of Agriculture (USDA), Tier-und Pflanzengesundheit Kontrolle Service (APHIS), Wildlife Services (WS), National Wildlife Research Center (NWRC) zugelassen. 1. Crow Gavaging <ol><li> Schätzen Durchlaufzeit &quot;Pseudo Hirnmaterial&quot; durch den Verdauungstrakt der amerikanischen Krähen. <ol><li> Mix 5 ml gekocht und Rührei ganzes Ei mit blauen Farbstoff und eine Magensonde ein Krähe mit einer 2 in der Schlundsonde Nadel (Abbildung 1). </li><li> Überprüfen Krähe alle 30 min bis blau / grün-gefärbten Kot werden nicht mehr ausgeschieden. </li></ol></li><li> Erhalten infizierten und unheilbar krank RML (Chandler Stamm)-infizierten C57BL / 6 Maus Gehirne. </li><li> Generieren Sie einen 20% wt / vol normal und infizierten Maus-Hirn-Homogenat in einer Kugel Mixer Homogenisator mit 1xPhosphat-gepufferte Salzlösung (PBS) und Glasperlen, dann Zentrifugieren bei 3.000 × g für 1 min, um größere Partikel zu entfernen. Überstand entfernen und verdünnt mit einem gleichen Volumen an 1 × PBS, um eine 10% w / v in steriler PBS zu erzeugen. Einfrieren bei -80 ° C, bis sie benötigt. </li><li> 17 Stunden vor der Magensonde zu entfernen Essen, aber nicht Wasser, von Luftfedern. </li><li> Zufällig zuordnen Krähen Behandlungsgruppen und der Schlundsonde oral jede Krähe mit 5 ml entweder normal oder infizierten Mausgehirnhomogenat mit einer 2 in der Schlundsonde Nadel. </li><li> Über kräht auf einzelne Käfige und sammeln und bündeln alle Kot in jedem Käfig für 4 Stunden nach der Magensonde (Abbildung 2). </li><li> Homogenisieren gepoolten Fäkalien für jede Krähe in einem Kugel-Homogenisator Mixer mit Glasperlen, bis eine gleichmäßige Struktur erzielt wird. * Crow Kot sind meist flüssig und kann leicht gemischt werden. </li><li> Für jede Krähe, verdünnte 500 ul der fäkalen Homogenat in 9,5 ml 1x PBS für ein Gesamtvolumen von 10 ml. </li><li> Zentrifugieren Sie die fäkale homogenate für 15 min bei 1.400 xg und extrahieren Sie den Überstand. </li><li> Um der Gefahr eines sekundären mikrobiellen Infektion zu minimieren, behandeln Stand mit 1 ul von 100 Einheiten / ml Penicillin und 100 ug / ml Streptomycin (Invitrogen, NY) pro 100 ul Homogenat dann unter UV-Licht bei Raumtemperatur für 20 min reduzieren Risiko viraler und bakterieller Kontamination. Nach der UV-Belichtung, beschallen Proben in einem Beschallungsgerät 3000 Mp bei Einstellung 70 für 30 Sekunden, um die Membran der verbleibenden Mikroorganismen stören. </li></ol> 2. Maus-Impfung <ol><li> Zufällig Mäuse zuweisen, um die folgenden Behandlungsgruppen (Tabelle 1): <ol><li> Gruppe 1 - Positive Behandlung Mäusen intraperitoneal geimpft (IP) mit 1 ml der fäkalen Homogenat von Krähen oral mit 5ml infizierten Mäusehirnen zwangsernährt. </li><li> Gruppe 2 - Negative Behandlung Mäuse geimpft IP mit 1 ml der fäkalen Homogenat von Krähen oral mit 5ml normalen Gehirn der Maus zwangsernährt. </li><li> Verdünnen infected und Normal Mausgehirnhomogenat für Gruppen 3 und 4 bis 1:100 w / v in 1x PBS. </li><li> Gruppe 3 - Positive Kontrollmäusen eingeimpft IP mit 1 ml der infizierten Maus-Hirn-Homogenat. </li><li> Gruppe 4 - Negative Kontrollmäusen eingeimpft IP mit 1 ml normalen Maus-Hirn-Homogenat. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Treatment Group </td><td> Anzahl der Tiere </td></tr><tr><td> Gruppe 1 </td><td> Scrapie + Crow Kot </td><td> 100 </td></tr><tr><td> Gruppe 2 </td><td> Scrapie - Krähen-Kot </td><td> 25 </td></tr><tr><td> Gruppe 3 </td><td> Scrapie + Maus Gehirn </td><td> 10 </td></tr><tr><td> Gruppe 4 </td><td> Scrapie - Maus Gehirn </td><td> 5 </td></tr></tbody></table> . Tabelle 1 Scrapie Status des Inokulums (positive + negative -) und Anzahl der verwendeten Tiere 7. <ol start="2"><li> Intraperitoneal impfen Maus: <ol><li> Scruff Maus durch dorsalen Nackenfell mit Daumen und Zeigefinger und sanft drehen, um Bauchseite aus. </li><li> Elevate hinteren Ende der Maus, so Kopf ist etwas niedriger. </li><li> Legen Sie 25 G-Nadel 1 cm durch die Haut, 1 cm lateral der Mittellinie, und 1-2 cm vor dem Iliosakralgelenk mit einer Nadel sichernde Spritze. </li><li> Spritzen 1 ml langsam in Maus impfen Körperhöhle. </li></ol></li></ol> 3. Maus-Überwachung <ol><li> Überwachung der Mäuse täglich, bis sie klinische Zeichen der Maus Scrapie auszudrücken. Klinische Symptome können sein: Kyphose, Ataxie, steifen Schwanz, mangelnde Pflege, Abmagerung und Lethargie. </li><li> Ergebnis Mäuse für jede der sechs klinischen Zeichen, wenn sichtbare Anzeichen sind offensichtlich, wobei 0 = keine sichtbar, 1 = mäßig, und 2 = schwere. </li><li> Euthanize Mäuse, wenn die gesamte tägliche Punktzahl für jedes Zeichen zu erreichen ≥ 8 für 1 Tag, ≥ 6 kontinuierlich für 3 Tage, oder bei 365 Tage nach der Inokulation (dpi). </li><li> Ernte Gehirn unmittelbar nach euthanasien und bei -70 ° C </li><li> Scrapie, eine Diagnose zu bestätigen, Digest Gehirnproben mit 3 ul einer 50 ug / ml Proteinase-K-Lösung (PK) wie folgt verdünnt: 3,1. PK ul, 12,5 ul 500 mM EDTA, pH 8, 109,39 ul 1x PBS für 30 min bei 45 ° C, dann inaktivieren PK durch Zugabe von 8 &amp;mgr; l Ladepuffer und Inkubation Proben bei 95 ° C für 5 min. Last Proben auf ein 12% SDS-PAGE-Gel Elektrophorese und Übertragung auf Immobilon-PVDF-Membran und Block mit 5% fettfreier Milch in 0,2% Tween 20 in PBS für 1 h bei Raumtemperatur. Sonde dann mit BAR224 anti-PrP monoklonaler Antikörper, der mit Meerrettich-Peroxidase, verdünnt in Superblock, für 1 Stunde bei Raumtemperatur konjugiert. Spülen Membran für 1 Stunde mit PBS-Tween 20 0,2%. Um zu visualisieren, Inkubation Western-Blot 5 min mit Chemilumineszenzsubstrat und Bild auf einem G-Feld-Gel-Dokumentationssystem. </li></ol>

<ol>
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Wir zeigen ein Verfahren um den Durchgang von PrP Res Material durch das Verdauungssystem von Krähen zu dokumentieren. Wir verwendeten konventionellen Methoden, um festzustellen, ob Krähen haben die Fähigkeit, PrP Res Material, um die krankheitsfreie geografischen Gebieten transloziert. Andere Widerstand von PrP Res ausgewertet 19-21 und Nagetier 22,23 Verdauungsflüssigkeiten, die beide nicht zu beseitigen Wiederkäuer. Die künftige Anwendung dieser Techniken a...

Übertragbare spongiforme Enzephalopathien (TSE) sind tödliche Infektions neurodegenerative Erkrankungen, die Tierwelt auswirken, Haustieren und Menschen. Der Erreger der TSE-Erkrankungen scheint falsch gefaltete oder pathogenen Isoformen (PrP Res) von Prion-Proteinen 1 sein. Tier TSE-Erkrankungen gehören Chronic Wasting Disease (CWD) in Maultierhirsch (Odocoileus hemionus), Weißwedelhirsche (Odocoileus virginianus), Elch (Cervus elaphus) und Elch (Alces alces), Scrapie bei Schafen und Ziegen, die bovine spongiforme Enzephalopathie ( BSE) in Hausrinder; tragbare Nerz Enzephalopathie bei Zucht Nerz, Feline Spongiforme Enzephalopathie bei Katzen, exotische Huftiere spongiforme Enzephalopathie in exotischen Zoo sinniert der Familie Hornträger und spongiforme Enzephalopathie in nicht-menschlichen Primaten 8. Der einzelne Mensch TSE-Erkrankung, Variante der Creutzfeldt-Jakob-Krankheit ist selten und vermutlich durch den Verzehr von PrP Res-Contamin erworben werdenATED Essen 9. Ebenso BSE Menschen infizieren können, wenn verseuchtes Rindfleisch verbraucht 10. Von all den TSE-Erkrankungen, Scrapie und CWD sind nur zwei mit sich selbst erhaltende Epidemien und Quellen für die Infektion sind vermutlich von Tier zu Tier 2,3,11 sowie aus Umweltquellen 4-6 sein. Forschung schlägt vor, dass die meisten TSE-Erkrankungen erfordern bemerkenswerte längere Inkubationszeiten aus natürlichen Belichtung Ereignisse des PrP Res Material zur Manifestation klinischer Symptome 2-4,6,8 und Scheinspeziesbarrieren zu minimieren, aber nicht das Potenzial für die, Übertragung zwischen 12-14 beseitigen . Identifizieren Mechanismen für die Ausbreitung von Infektions Prionen (PrP Res) Material ist für die Beantwortung von Fragen, wie TSE-Erkrankungen durch die Landschaft bewegen extrem wichtig. Experimentelle Untersuchungen haben ergeben, dass 15,16, Geflügel und Schweine 17, und amerikanische Krähen (Corvus brac Insektenhyrhynchos) 7,18 passive Träger oder Dispergatoren von PrP Res Material. Passage von PrP Res Material durch das Verdauungssystem von Krähen hat kürzlich dokumentiert, was die Rolle, die sie in Ausbreitung von TSE-Erkrankungen 7 zu spielen. Diese Ergebnisse machen es plausibel, dass Krähen, ein Aasfresser, auftreten können, zu konsumieren und zu transportieren infektiösem Material über Kot Abscheidung, um seuchenfreie Gebiete. Die Verfahren, die wir hier zeigen, wurden verwendet, um den Durchgang von PrP Res Material durch das Verdauungssystem von Krähen zu dokumentieren und die Anwendung dieser Methoden auf andere Fänger und Raubtierarten spezifische Modelle in verwandten zukünftige Forschung erheblich erleichtern. In dieser Studie wurden konventionelle Methoden verwendet werden, um eine unkonventionelle Mittel des Menschenhandels PrP Res Material, das für die Verbreitung und der Gesamtbelastung von PrP Res Material beitragen könnten, zu untersuchen.

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

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

Infektiöse Prionen (PrP Res) Material ist wahrscheinlich die Ursache der tödlichen, neurodegenerative übertragbare spongiforme Enzephalopathie (TSE) Krankheiten ein. Übertragung von TSE-Erkrankungen wie Chronic Wasting Disease (CWD), wird vermutet, von Tier zu Tier 2,3 sowie aus Umweltquellen 4-6 sein. Reiniger und Fleischfresser haben Potenzial, PrP Res Material durch Konsum und die Ausscheidung von CWD-kontaminierten Aas transloziert. Neuere Arbeiten haben Gang von PrP Res Material durch das Verdauungssystem der amerikanischen Krähen (Corvus brachyrhynchos), einer gemeinsamen nordamerikanischen Schnitzel 7 dokumentiert. Wir beschreiben Verfahren verwendet werden, um Durchgang von PrP Res Material durch amerikanische Krähen zu dokumentieren. Krähen wurden mit RML-Dehnungs-Maus-adaptierten Scrapie zwangsernährt und ihren Kot gesammelt wurden 4 Stunden nach der Magensonde. Crow Kot wurden dann gesammelt und in intraperitonealC57BL / 6 Mäusen. Die Mäuse wurden täglich überwacht, bis sie ausgedrückt klinischen Zeichen der Maus Scrapie und wurden danach getötet. Asymptomatische Mäuse wurden bis zu 365 Tage nach der Impfung überwacht. Western-Blot-Analyse wurde durchgeführt, um Krankheitsstatus zu bestätigen. Ergebnisse zeigten, dass Prionen infektiös bleiben nach einer Reise durch das Verdauungssystem von Krähen und im Kot vorhanden sind, was zu Krankheit in Versuchsmäusen.

Die angewendeten Verfahren zeigen, dass das Verdauungssystem von Krähen nicht PrP Res Infektiosität 4 Stunden nach orale Gabe von Scrapie Hirnhomogenat 7 zu beseitigen. Alle zwanzig Krähen, die mit PrP Res Material zwangsernährt wurden anschließend PrP Res Material, um Mäuse übertragen über Kot. Kranke Mäuse wurden durch klinische Manifestation der Maus-Scrapie-Krankheit Anzeichen und Bestätigung wurde durch Western-Blot-Analyse abgeschlossen identifiziert. <p cl...

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

Aasfresser haben Potenzial, infektiöse Prionen übertragbaren spongiformen Enzephalopathie in ihren Kot zu krankheitsfreien Gebieten transloziert. Wir detailliert Methoden verwendet werden, um festzustellen, ob der Maus-adaptierten Scrapie-Prionen bleiben nach dem Durchgang aber den Verdauungstrakt der amerikanischen Krähen (Corvus brachyrhynchos), einer gemeinsamen Verbraucher toter Tiere ansteckend.

Verfahren zur Identifizierung von Infektions Prionen Nach Passage durch das Verdauungssystem von eine Vogelart

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 Aufrufe.  USDA.  Aasfresser haben Potenzial, infektiöse Prionen übertragbaren spongiformen Enzephalopathie in ihren Kot zu krankheitsfreien Gebieten transloziert. Wir detailliert Methoden verwendet werden, um festzustellen, ob der Maus-adaptierten Scrapie-Prionen bleiben nach dem Durchgang aber den Verdauungstrakt der amerikanischen Krähen (Corvus brachyrhynchos), einer gemeinsamen Verbraucher toter Tiere ansteckend. 

Serie: 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.

Überwachung Immunzellen Trafficking Fluorescent Prion Rods Stunden nach intraperitonealer Infektion

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

Forschung

Immunologie und Infektion

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.

Schnelle Diagnose der Aviären Influenza Virus bei Wildvögeln: Verwendung eines tragbaren rRT-PCR und gefriergetrocknet Reagenzien in das Feld

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.

Avian Influenza Surveillance mit FTA-Karten: Field Methods, Biosafety-und Transportprobleme gelöst

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.

Proteinfehlfaltungen Cyclic Amplification von Prionen

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.

Klinisches Prüfungsprotokoll zur Erkennung atypischer und klassischer Scrapie bei Schafen

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

Medizin

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.

Transsynaptische Tracing von Peripherie Targets mit Pseudorabies-Virus durch Cholera Toxin und biotinyliertes Dextran Amine Doppelmarkierung Gefolgt

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

Neurowissenschaften

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.

Die Beurteilung Transmissible Spongiforme Enzephalopathie Artgrenzen mit ein<em&gt; In-vitro-</em&gt; Prion Protein-Umwandlungsassay

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.

Echtzeit-Beben-induzierte Umwandlung Test zur Erkennung von CWD Prionen in Fäkalien

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.

Prion Safety Laboratory Wischtest

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.

Verfahren zur Identifizierung von Infektions Prionen Nach Passage durch das Verdauungssystem von eine Vogelart

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