Biz bu çalışmada ve USDA kullanılan kargalar sağlamak için S. Werner teşekkür etmek istiyorum, APHIS, hayvan bakımı ve izlenmesi için WS, NWRC hayvan bakım personeli. Mansiyon veya bir ürünün kullanımı USDA onaylandığı anlamına gelmez. Bu çalışma için fon USDA, APHIS, Veteriner Hizmetleri tarafından sağlanmıştır.

Bizim protokol daha önce 7 yayınlanmış bir uyarlanmıştır. Hayvanları içeren tüm işlemler Amerika Birleşik Devletleri Tarım Bakanlığı (USDA), Hayvan ve Bitki Sağlığı Denetim Servisi (Aphis), Yaban Hayatı Hizmetleri (WS), Ulusal Yaban Hayatı Araştırma Merkezi (NWRC) Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1.. Crow Gavaging <ol><li> Amerikan karga sindirim sistemi yoluyla &#39;sahte beyin malzemenin&#39; geçişi zamanı tahmin. <ol><li> Pişmiş ve (Şekil 1) gavaj iğne bir 2 kullanarak mavi boya ve sonda 1 karga ile tam pişmiş yumurta 5 ml karıştırın. </li><li> Mavi / yeşil-lekeli dışkı atılır ediliyor artık kadar Crow her 30 dakikada bir kontrol edin. </li></ol></li><li> Bulaşmamış ve ölümcül hasta RML (Chandler suşu) enfekte C57BL / 6 fare beyinleri edinin. </li><li> 1x bir kurşun blender homojenleştiricide normal bir% 20 ağırlık / hacim ve enfekte fare beyin Homojenat oluşturmakfosfat tamponlu tuzlu su (PBS) ve cam boncuklar, daha sonra daha büyük bir parçacık maddenin ayrılması için 1 dakika için 3000 x g &#39;de santrifüj. Süpernatantı ve steril PBS içinde bir% 10 ağırlık / hacim üretmek için 1x PBS eşit hacmi ile seyreltilir. Ihtiyaç kadar -80 ° C&#39;de dondurun. </li><li> Önce sonda 17 saat karga kalemler dışında, su değil gıda kaldırmak, ancak. </li><li> , Tedavi gruplarına rastgele tahsis karga ve oral gavaj iğnesi bir 2 kullanılarak, normal ya da enfekte ya da fare beyin homojenatının, 5 ml her karga sonda. </li><li> Transferi bireysel kafeslere kargalar ve 4 saat sonrası gavaj (Şekil 2) için her bir kafes içinde tüm dışkısını toplamak ve havuz. </li><li> Homojen bir doku elde edene kadar cam boncuk ile bir mermi blender homojenleştiricisinde her karga için toplandı dışkı homojenize. * Crow dışkı daha çok sıvı ve kolayca karıştırılabilir. </li><li> Her karga için, 10 ml&#39;lik toplam hacim 9.5 ml 1 x PBS içine dışkı homojenat 500 ul seyreltik. </li><li> Fekal homogena santrifüj1,400 x g&#39;de 15 dakika boyunca te ve süpernatant ekstrakte edin. </li><li> , Ikinci bir mikrobiyal enfeksiyon tehlikesini en aza indirmek homojenat 100 ul başına 100 birim 1 ul / ml penisilin ve 100 ug / ml streptomisin (Invitrogen, NY) ile tedavi edilmesi için daha sonra süpernatant azaltmak için 20 dakika boyunca oda sıcaklığında, UV ışığı altında yer viral ve bakteriyel kontaminasyon riski. UV maruz kaldıktan sonra, herhangi bir geri kalan mikropların membran bozulması için 30 saniye boyunca 70 ayar bir 3000 Mp sonikatör sonikasyon örnekleri. </li></ol> 2. Fare aşılama <ol><li> Şu tedavi gruplarında (Tablo 1) için fareler tahsis: <ol><li> Grup 1 - Tedavi ile farenin ağızdan 5 ml enfekte Fare beyinleri ile zorla ağız yolundan verilmiştir karga dışkı homojenat, 1 ml (IP) intraperitonal olarak aşılandı. </li><li> Grup 2 - negatif bir tedavi farenin ağızdan 5 ml normal fare beyinleri ile zorla ağız yolundan verilmiştir karga dışkı Homojenat 1 ml IP aşılandı. </li><li> Enfeksiyonlandı sulandırmak1:100 w / v 1x PBS için Grup 3 ve 4 için d ve normal fare beyni homojenatı. </li><li> Grup 3 - pozitif kontrol fareleri enfekte fare beyni homojenatından 1 ml IP olarak aşılanmıştır. </li><li> Grup 4 - Negatif kontrol fareleri, normal fare beyni homojenatından 1 ml IP olarak aşılanmıştır. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> Tedavi Grubu </td><td> Hayvanların sayısı </td></tr><tr><td> Grup 1 </td><td> Scrapie + Crow Dışkı </td><td> 100 </td></tr><tr><td> Grup 2 </td><td> Scrapie - Crow Dışkı </td><td> 25 </td></tr><tr><td> Grup 3 </td><td> Scrapie + Fare Beyin </td><td> 10 </td></tr><tr><td> Grup 4 </td><td> Scrapie - Fare Beyin </td><td> 5 </td></tr></tbody></table> . Tablo 1 Scrapie inokülumunun durumu (negatif pozitif +, -) ve hayvan sayısı 7 kullanılır. <ol start="2"><li> Periton fareyi aşılamak: <ol><li> Nazikçe Scruff başparmak ve işaret parmağı ile sırt boyun kürk fare ve ventral yüzü ortaya çıkarmak için döndürün. </li><li> Kafası biraz daha düşük, bu nedenle fare arka ucunu yükseltin. </li><li> 1 cm deri, orta hatta 1 cm lateraline aracılığıyla 25 G iğne yerleştirin ve 1-2 cm anterior sakroiliak eklem iğne kilitleme şırınga kullanarak. </li><li> 1 ml fare vücut boşluğuna yavaş inoküle enjekte edilir. </li></ol></li></ol> 3. Fare İzleme <ol><li> Onlar fare skrapinin klinik belirtilerini ifade kadar günlük fareler izleyin. Klinik bulgular içerebilir: kifoz, ataksi, sert kuyruk, bakım eksikliği, zayıflatma, ve uyuşukluk. </li><li> 0 görünür = yok, 1 = orta, 2 = şiddetli nerede görünür işaretler, belirgin olduğunda 6 klinik bulguların her biri için fareler skor. </li><li> Her oturum için günlük toplam puanları için 1 gün ≥ 8 ulaştığınızda fareler Euthanize, ≥ 6 sürekli olarak 3 gün boyunca, ya da 365 gün aşılama (dpi) göndermek. </li><li> Hasat beyinleri hemen aşağıdaki euthan-70 ° C&#39;de asya ve mağaza </li><li> , Bir scrapie teşhisi doğrulamak aşağıdaki gibi seyreltilmiş proteinaz-K çözeltisi (PK) içindeki bir 50 ug / ml &#39;lik 3 ul beyin örnekleri sindirimi için: 3.1. PK ul, 500 mM EDTA, 12.5 ul, pH 8, 45 ° C&#39;de 30 dakika boyunca 1x PBS 109,39 ul, daha sonra 8 ul yükleme tamponu ilave edildikten ve 5 dakika boyunca 95 ° C&#39;de inkübe edilerek örnekleri PK etkisiz hale getirirler. Bir% 12 SDS-PAGE jeli, Electrophorese ve oda sıcaklığında 1 saat boyunca PBS içerisinde% 0.2 Tween-20 içerisinde Immobilon PVDF membran ve% 5 yağsız süt ile bloğuna transfer üzerine yük örnekleri. Daha sonra, oda sıcaklığında 1 saat boyunca Superblock içerisinde seyreltilmiş Horseradish peroksidaz, konjuge edilmiş anti-PrP Bar224 monoklonal antikoru ile prob. PBS-% 0.2 Tween 20 ile, 1 saat boyunca membran durulayın. , Görselleştirmek bir G-box jel dokümantasyon sistemi üzerinde kemolüminesans ve görüntü ile Western Blot 5 dakika inkübe. </li></ol>

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Çıkar çatışması ilan etti.

Biz karga sindirim sistemi yoluyla PrP Res malzemenin geçişini belgelemek için bir prosedür ortaya koymaktadır. Biz kargalar hastalıksız coğrafi bölgelere PrP Res malzeme translokasyonunda yeteneğine sahip olup olmadığını belirlemek için geleneksel yöntemleri kullanılır. Diğerleri bunu ortadan kaldırmak için başarısız ikisi de 19-21 ve kemirgen 22,23 sindirim sıvıları, geviş PrP Res direncini değerlendirdik. Onlar da potansiyel leş PrP ...

Bulaşıcı Süngerimsi Ensefalopati (TSE) yaban hayatı etkileyen ölümcül bulaşıcı nörodejeneratif hastalıklar, evcil hayvanlar ve insanlar vardır. TSE hastalıkları, bulaşıcı madde prion proteinlerinin 1 yanlış katlanmış veya patojenik izoformu (PrP S) olduğu görülmektedir. Koyun ve keçilerde scrapie;; sığır süngersi ensefalopati (TSE Hayvan hastalıkları karaca (Odocoileus hemionus), beyaz kuyruklu geyik (Odocoileus virginianus), geyik (Cervus elaphus) ve geyik (Alces alces) kronik israf hastalığı (CWD) içerir yerli sığırlarda BSE); Yetiştirilmiş vizon transmissible vizon ensefalopati; kedilerde feline süngerimsi ensefalopati; primatlarla 8 ve süngerimsi ensefalopati; egzotik hayvanat bahçesinde egzotik toynaklı süngerimsi ensefalopati aile Bovidae ruminates. Tek bir insan TSE hastalığı, Creutzfeldt-Jakob hastalığı, nadir ve PrP Res-Kirlenmiş alıcı tarafından satın alınması için düşünülmüşated gıda 9. Kontamine sığır 10 tüketilen Benzer şekilde, eğer BSE insanları enfekte edebilir. Tüm TSE hastalıkları, scrapie ve CWD hayvandan hayvana 2,3,11 yanı sıra çevresel kaynaklardan 4-6 olduğu tahmin edilmektedir kendini idame ettiren ve salgın enfeksiyon kaynakları ile sadece iki tanesi. Araştırma çoğu TSE hastalıkları klinik bulguların tezahürü PrP Res malzemenin doğal maruz olaylardan önemli uzun kuluçka dönemleri gerektiren 2-4,6,8 ve belirgin tür engelleri en aza indirmek, ancak, türler arası iletim 12-14 potansiyelini ortadan kaldırmak olmadığını göstermektedir . Bulaşıcı prion (PrP Res) malzeme yayılması için mekanizmalar belirlenmesi TSE hastalıkları manzara karşısında nasıl hareket hakkında soruları yanıtlayan için son derece önemlidir. Deneysel araştırmalar 15,16, kümes hayvanları ve domuz 17, ve Amerikan kargaları (Corvus eşyaları böcekler düşündürmektedirhyrhynchos) 7,18 PrP Res malzemenin pasif taşıyıcılar veya dağıtıcılar bulunmaktadır. Karga sindirim sistemi yoluyla PrP Res malzemenin geçişi geçenlerde TSE hastalıkları 7 dağılması oynayabileceği rolü göstererek, belgelenmiştir. Bu sonuçlar, kargalar, bir çöpçü, karşılaşma olabilir o akla yatkın hale tüketmek, ve hastalıksız bölgelere, dışkı biriktirme yoluyla bulaşıcı malzeme taşımak. Burada göstermek prosedürler kargalar sindirim sistemi yoluyla PrP Res malzemenin geçişini belgelemek için kullanılan ve büyük ölçüde gelecekteki ilgili araştırmalarda diğer çöpçü ve etobur türe özgü modeller için bu yöntemlerin uygulanmasını kolaylaştıracaktır. Bu çalışmada, geleneksel yöntemler PrP Res malzemenin yayılması ve genel yüküne katkıda bulunabilecek PrP ​​Res malzeme kaçakçılığı bir alışılmamış araçlar araştırmak için kullanılmıştır.

<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

Bulaşıcı prion (PrP Res) malzeme olasılıkla ölümcül, nörodejeneratif Bulaşıcı süngerimsi ensefalopati (TSE) hastalıklar 1 nedenidir. Bu tür kronik halsizlik hastalığı (CWD) ve TSE hastalıkları, Transmission, hayvan 2,3 hayvana hem de çevresel kaynaklarından 4-6 olduğu tahmin edilmektedir. Süpürücüleri ve etobur tüketim ve CWD kirlenmiş leş boşaltım yoluyla PrP Res malzemeyi translokasyonunda potansiyele sahiptir. Son çalışma Amerikan kargalar sindirim sistemi (Corvus brachyrhynchos), ortak bir Kuzey Amerika çöpçü 7 ile PrP Res malzemenin geçişini belgelemiştir. Biz Amerikan kargalar aracılığıyla PrP Res malzemenin geçişini belgelemek için kullanılan prosedürleri açıklamaktadır. Kargalar RML-deformasyon fare adapte skrapili zorla ağız yolundan ve onların dışkı 4 saat sonrası sonda toplandı. Crow dışkı içine daha sonra bir araya getirilmiş ve periton içine enjekte edildiC57BL / 6 fareleri. Bu fare skrapinin klinik belirtileri ifade edilmiş ve daha sonra kurban edilmiştir kadar Fareler, günlük olarak izlendi. Asemptomatik fareler 365 gün Aşıdan kadar takip edildi. Western Blot analizi hastalık durumunu teyit etmek için yapılmıştır. Sonuçlar prionların testi farelerde hastalığa neden kargalar sindirim sistemi yoluyla seyahat sonra bulaşıcı kalır ve dışkı mevcut olduğunu ortaya çıkarmıştır.

Kullanılan prosedürler karga sindirim sistemi scrapie beyin homojenatı 7 arasında oral gavaj sonrası PrP Res enfekte 4 saat ortadan etmediğini göstermektedir. PrP Res malzeme ile zorla ağız yolundan verilmiştir olan tüm yirmi karga, daha sonra farelere dışkı yoluyla PrP Res malzeme aktarılır. Hastalıklı farelerin fare klinik işaretler ve scrapie hastalık onay Western blot analizi ile tamamlandı tezahürü ile belirlenmiştir. K...

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

Süpürücüleri hastalıksız bölgelere kendi dışkılarıyla bulaşıcı bulaşıcı süngerimsi ensefalopati prionları translokasyonunda potansiyele sahiptir. Biz fare adapte skrapi prıonlar Amerikan kargalar sindirim sisteminde (Corvus brachyrhynchos), ölü hayvanların ortak bir tüketici olsa geçtikten sonra bulaşıcı kalırsa belirlemek için kullanılan ayrıntı yöntemleri.

Bir Kuş Türlerinin Sindirim Sistemi geçtikten sonra Bulaşıcı prions belirlenmesi için prosedürler

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 Görüntüleme.  USDA.  Süpürücüleri hastalıksız bölgelere kendi dışkılarıyla bulaşıcı bulaşıcı süngerimsi ensefalopati prionları translokasyonunda potansiyele sahiptir. Biz fare adapte skrapi prıonlar Amerikan kargalar sindirim sisteminde (Corvus brachyrhynchos), ölü hayvanların ortak bir tüketici olsa geçtikten sonra bulaşıcı kalırsa belirlemek için kullanılan ayrıntı yöntemleri. 

Video: 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.

İntraperitoneal Enfeksiyon sonra Bağışıklık Hücreleri Ticareti Floresan Prion Çubuklar Saat İzlenmesi

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

İmmünoloji ve Enfeksiyon

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.

Yabani Kuşlar Kuş Gribi Virüsü Hızlı Teşhis: Alanında Taşınabilir RRT-PCR ve Dondurulmuş kuru Reaktifler Kullanımı

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.

Kuş FTA Kartları İnfluenza Sürveyans: Alan Yöntemleri, Biyogüvenlik ve Ulaşım Sorunları çözüldü

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.

Prionlar protein misfolding Döngüsel Amplifikasyon

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.

Koyunlarda Atipik ve Klasik Sıyrık Tespit Etmek için Klinik Muayene Protokolü

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

Tıp

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.

Transsinaptik Kolera Toksin ve Biotinlenmiş Dekstran Aminler Çift Etiketleme Ardından Pseudorabies&#39;e Virüs ile Periferik Hedefler gelen İzleme

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

Nörobilim

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.

Bir ile Bulaşıcı Süngerimsi Ensefalopati Türler Engelleri değerlendirilmesi<em&gt; İn Vitro</em&gt; Prion Proteini Dönüştürme Deneyi

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.

Gerçek zamanlı depremin dönüşüm tahlil CWD Prionlar fekal madde olarak algılanması için kaynaklı

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 Güvenlik Laboratuvarı Kaydırma Testi

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.

Bir Kuş Türlerinin Sindirim Sistemi geçtikten sonra Bulaşıcı prions belirlenmesi için prosedürler

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