real-time-quaking-induced-conversion-assay-for-detection-cwd-prions

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 ...

Lethbridge Laboratories

An oligonucleotide suspension microarray (Luminex microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV1 and BVDV2), and border disease virus (BDV). Species-specific and pestivirus-common oligonucleotide probes were designed to the 5' UTR region and conjugated to individual color-coded Luminex carboxy beads (probe beads). Target pestivirus sequences were amplified by asymmetric PCR using a biotinylated reverse primer and a forward and reverse primer ratio of 1:5. The biotinylated products were hybridized to eight probe beads in a multiplex assay and analyzed using streptavidin conjugated to a fluorescent reporter molecule. The assay was able to detect and differentiate all 40 strains of CSFV, BVDV1, BVDV2 and BDV tested. The analytical sensitivity was determined to be 0.2-10 TCID50/ml. The major advantages of the DNA-microsphere suspension microarray, as a low density array, are its ease of handling and ability to simultaneously detect and type multiple infectious agents.

A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses.

To gain insight into the disease progression of transmissible spongiform encephalopathies (TSE), we searched for disease-specific patterns in circulating nucleic acids (CNA) in elk and cattle. In a 25-month time-course experiment, CNAs were isolated from blood samples of 24 elk (Cervus elaphus) orally challenged with chronic wasting disease (CWD) infectious material. In a separate experiment, blood-sample CNAs from 29 experimental cattle (Bos taurus) 40 months post-inoculation with clinical bovine spongiform encephalopathy (BSE) were analyzed according to the same protocol. Next-generation sequencing provided broad elucidation of sample CNAs: we detected infection-specific sequences as early as 11 months in elk (i.e. at least 3 months before the appearance of the first clinical signs) and we established CNA patterns related to BSE in cattle at least 4 months prior to clinical signs. In elk, a progression of CNA sequence patterns was found to precede and correlate with macro-observable disease progression, including delayed CWD progression in elk with PrP genotype LM. Some of the patterns identified contain transcription-factor-binding sites linked to endogenous retroviral integration. These patterns suggest that retroviruses may be connected to the manifestation of TSEs. Our results may become useful for the early diagnosis of TSE in live elk and cattle.

Disease-specific motifs can be identified in circulating nucleic acids from live elk and cattle infected with transmissible spongiform encephalopathies.

Scrapie, a transmissible spongiform encephalopathy of sheep and goats, exists in most small ruminant-producing countries of the world. A novel form of this disease was recently recognized and is known by various names, including Nor98, Nor98-like, and atypical scrapie. Differing from classic scrapie in epidemiology, histopathology, and biochemical characteristics, atypical scrapie cases have been identified throughout Europe and in the United States. Enhanced scrapie surveillance efforts recently identified 3 cases of atypical scrapie in Canada.

Identification of atypical scrapie in Canadian sheep.

&#x6D41;&#x884C;&#x75C5;&#x5B66;&#x8C03;&#x67E5;&#x53CA;&#x53EF;&#x80FD;&#x7684;&#x725B;&#x6D77;&#x7EF5;&#x72B6;&#x8111;&#x75C5; (BSE) &#x75C5;&#x56E0;&#x6709;&#x6700;&#x8FD1;&#x88AB;&#x516C;&#x8BA4;&#x662F;&#x5F02;&#x7C7B;&#x3002;&#x5C24;&#x5176;&#x662F;&#xFF0C;&#x4E09;&#x79CD;&#x7C7B;&#x578B; [&#x53E4;&#x5178; (C) &#x548C;&#x4E24;&#x4E2A;&#x975E;&#x5178;&#x578B; (H&#xFF0C;L)] &#x5DF2;&#x7ECF;&#x786E;&#x5B9A;&#xFF0C;&#x5F88;&#x5927;&#x7A0B;&#x5EA6;&#x4E0A;&#x57FA;&#x4E8E;&#x7279;&#x5F81;&#x7684;&#x86CB;&#x767D;&#x9176; K (PK)-&#x8010;&#x6838;&#x5FC3;&#x7684;&#x7EA0;&#x7F20;&#x7684;&#x670A;&#x86CB;&#x767D;&#x75C5; (PrP(res))&#x3002;&#x672C;&#x7814;&#x7A76;&#x8FDB;&#x884C;&#x4E86;&#x5B9A;&#x6027; 17 &#x52A0;&#x62FF;&#x5927;&#x7684;&#x75AF;&#x725B;&#x75C5;&#x53D1;&#x751F;&#x7684;&#x5B97;&#x5728; 2009 &#x5E74; 11 &#x6708;&#x57FA;&#x4E8E;&#x5176; PrP(res)&#xFF0C;&#x5305;&#x62EC;&#x514D;&#x75AB;&#x53CD;&#x5E94;&#x6027;&#x3001; &#x5206;&#x5B50;&#x91CF;&#x3001; glycoform &#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x548C;&#x76F8;&#x5BF9; PK &#x654F;&#x611F;&#x6027;&#x7684;&#x5206;&#x5B50;&#x548C;&#x751F;&#x5316;&#x5C5E;&#x6027;&#x4E4B;&#x524D;&#x3002;&#x4E24;&#x8D77;&#x6848;&#x4EF6;&#xFF0C;&#x5C55;&#x793A;&#x5206;&#x5B50;&#x91CF;&#x548C; glycoform &#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x7C7B;&#x4F3C;&#x4E8E;&#x4EE5;&#x524D;&#x62A5;&#x544A;&#x7684;&#x975E;&#x5178;&#x578B;&#x6848;&#x4EF6;&#xFF0C;&#x4E00;&#x79CD;&#x5BF9;&#x5E94;&#x4E8E;&#x75AF;&#x725B;&#x75C5; H &#x578B; &#xFF08;&#x6848;&#x4F8B; 6&#xFF09; &#x548C;&#x5176;&#x4ED6;&#x5BF9;&#x75AF;&#x725B;&#x75C5; L &#x578B; &#xFF08;&#x6848;&#x4F8B; 11&#xFF09;&#x3002;&#x6240;&#x6709;&#x5176;&#x4ED6;&#x60C5;&#x51B5;&#x4E0B;&#x88AB;&#x5F52;&#x7C7B;&#x4E3A; C &#x578B;&#x3002;PK &#x6D88;&#x5316;&#x6E29;&#x548C;&#x548C;&#x4E25;&#x683C;&#x7684;&#x6761;&#x4EF6;&#x4E0B;&#x53D1;&#x73B0;&#x8FD9;&#x4E24;&#x79CD;&#x60C5;&#x51B5;&#x76F8;&#x6BD4;&#xFF0C;C &#x7C7B;&#x578B;&#x6848;&#x4EF6;&#x51CF;&#x5C11;&#x7684;&#x86CB;&#x767D;&#x9176;&#x62B5;&#x6297;&#x3002;&#x4E0E;&#x897F;&#x65B9;&#x514D;&#x75AB;&#x5370;&#x8FF9;&#x6CD5;&#x3001; N-&#x7EC8;&#x7AEF;&#x7279;&#x5F02;&#x6027;&#x6297;&#x4F53;&#x7ED1;&#x5B9A;&#x5230;&#x4ECE;&#x6848;&#x4EF6; 6 PrP(res) &#x800C;&#x4E0D;&#x662F;&#x4ECE;&#x6848;&#x4F8B; 11 &#x6216; C &#x7C7B;&#x578B;&#x6848;&#x4EF6;&#x3002;C-&#x7EC8;&#x7AEF;&#x7279;&#x5F02;&#x6027;&#x6297;&#x4F53;&#x63ED;&#x793A; glycoform &#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x4E2D;&#x7684;&#x8F6C;&#x53D8;&#xFF0C;&#x5E76;&#x6848; 6&#xFF0C;&#x6307;&#x793A;&#x6027;&#x7684;&#x4E24;&#x4E2A; PrP(res) &#x4E9A;&#x7FA4;&#x4E2D; H &#x578B;&#x75AF;&#x725B;&#x75C5;&#x68C0;&#x6D4B;&#x5230;&#x7B2C;&#x56DB;&#x7684;&#x86CB;&#x767D;&#x7247;&#x6BB5;&#x3002;&#x6697;&#x793A;&#x9057;&#x4F20;&#x75C5;&#x56E0;&#x8005;&#x88AB;&#x6D4B;&#x5E8F;&#x7684;&#x5145;&#x5206;&#x7684; PrP &#x7F16;&#x7801;&#x4E2D;&#x4EFB;&#x4F55; 17 &#x52A8;&#x7269;&#x53D1;&#x73B0;&#x6CA1;&#x6709;&#x7A81;&#x53D8;&#x7684;&#x5E8F;&#x5217;&#x4E2D;&#x7684;&#x670A;&#x75C5;&#x6BD2;&#x57FA;&#x56E0;&#x7B2C; 3 &#x5916;&#x663E;&#x5B50;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6BCF;&#x4E2A;&#x4E09;&#x4E2A;&#x5DF2;&#x77E5;&#x75AF;&#x725B;&#x75C5;&#x7C7B;&#x578B;&#x5728;&#x52A0;&#x62FF;&#x5927;&#x725B;&#x5DF2;&#x7ECF;&#x786E;&#x8BA4;&#xFF0C;&#x5E76;&#x663E;&#x793A;&#x5206;&#x5B50;&#x7279;&#x6027;&#x975E;&#x5E38;&#x7C7B;&#x4F3C;&#x4E8E;&#x53E4;&#x5178;&#x548C;&#x975E;&#x5178;&#x578B;&#x75AF;&#x725B;&#x75C5;&#x53D9;&#x8FF0;&#x7684;&#x6848;&#x4EF6;&#x4ECE;&#x6B27;&#x6D32;&#x3001; &#x65E5;&#x672C;&#x548C;&#x7F8E;&#x56FD;&#x3002;&#x75AF;&#x725B;&#x75C5;&#x75AF;&#x725B;&#x75C5;&#x6D41;&#x884C;&#x4E0E;&#x4F20;&#x64AD;&#x98CE;&#x9669;&#x4F4E;&#x52A0;&#x62FF;&#x5927;&#x7B49;&#x56FD;&#x7684;&#x975E;&#x5178;&#x578B;&#x4E2A;&#x6848;&#x7684;&#x53D1;&#x751F;&#x8FA9;&#x53D1;&#x751F;&#x96F6;&#x661F;&#x5F62;&#x5F0F;&#x7684;&#x75AF;&#x725B;&#x75C5;&#x7684;&#x4E16;&#x754C;&#x5404;&#x5730;&#x3002;

&#x5728;&#x52A0;&#x62FF;&#x5927;&#x7684;&#x75AF;&#x725B;&#x75C5;&#x7684;&#x5206;&#x5B50;&#x3001; &#x751F;&#x5316;&#x548C;&#x9057;&#x4F20;&#x7279;&#x5F81;&#x3002;

The epidemiology and possibly the etiology of bovine spongiform encephalopathy (BSE) have recently been recognized to be heterogeneous. In particular, three types [classical (C) and two atypical (H, L)] have been identified, largely on the basis of characteristics of the proteinase K (PK)-resistant core of the misfolded prion protein associated with the disease (PrP(res)). The present study was conducted to characterize the 17 Canadian BSE cases which occurred prior to November 2009 based on the molecular and biochemical properties of their PrP(res), including immunoreactivity, molecular weight, glycoform profile and relative PK sensitivity. Two cases exhibited molecular weight and glycoform profiles similar to those of previously reported atypical cases, one corresponding to H-type BSE (case 6) and the other to L-type BSE (case 11). All other cases were classified as C-type. PK digestion under mild and stringent conditions revealed a reduced protease resistance in both of these cases compared to the C-type cases. With Western immunoblotting, N-terminal-specific antibodies bound to PrP(res) from case 6 but not to that from case 11 or C-type cases. C-terminal-specific antibodies revealed a shift in the glycoform profile and detected a fourth protein fragment in case 6, indicative of two PrP(res) subpopulations in H-type BSE. No mutations suggesting a genetic etiology were found in any of the 17 animals by sequencing the full PrP-coding sequence in exon 3 of the PRNP gene. Thus, each of the three known BSE types have been confirmed in Canadian cattle and show molecular characteristics highly similar to those of classical and atypical BSE cases described from Europe, Japan and the USA. The occurrence of atypical cases of BSE in countries such as Canada with low BSE prevalence and transmission risk argues for the occurrence of sporadic forms of BSE worldwide.

Molecular, biochemical and genetic characteristics of BSE in Canada.

L&#x26;#39;&#xE9;pid&#xE9;miologie et, &#xE9;ventuellement, l&#x26;#39;&#xE9;tiologie de l&#x26;#39;enc&#xE9;phalopathie spongiforme bovine (ESB) ont &#xE9;t&#xE9; r&#xE9;cemment reconnu comme h&#xE9;t&#xE9;rog&#xE8;ne. En particulier, les trois types [classique (C) et deux atypique (H, L)] ont &#xE9;t&#xE9; identifi&#xE9;s, en grande partie sur la base des caract&#xE9;ristiques de la prot&#xE9;inase (PK) K-noyau r&#xE9;sistant de la prot&#xE9;ine prion mal repli&#xE9;e associ&#xE9;e &#xE0; la maladie (PrP (res)). La pr&#xE9;sente &#xE9;tude a &#xE9;t&#xE9; men&#xE9;e afin de caract&#xE9;riser les 17 cas d&#x26;#39;ESB au Canada qui ont eu lieu avant Novembre 2009 sur la base des propri&#xE9;t&#xE9;s mol&#xE9;culaires et biochimiques de leur PrP (RES), y compris immunor&#xE9;activit&#xE9;, poids mol&#xE9;culaire, le profil et la sensibilit&#xE9; glycoforme PK relative. Deux cas pr&#xE9;sentaient poids mol&#xE9;culaire et des profils de glycoformes semblables &#xE0; ceux de signal&#xE9;s pr&#xE9;c&#xE9;demment, les cas atypiques, l&#x26;#39;une correspondant &#xE0; H de type ESB (cas 6) et l&#x26;#39;autre &#xE0; la L-Type de l&#x26;#39;ESB (cas 11). Tous les autres cas ont &#xE9;t&#xE9; class&#xE9;s comme C-type. La digestion PK dans des conditions douces et rigoureux a r&#xE9;v&#xE9;l&#xE9; une r&#xE9;sistance &#xE0; la prot&#xE9;ase r&#xE9;duite dans ces deux cas par rapport &#xE0; des cas de type C. Avec Western immunoblot, les N-terminale des anticorps sp&#xE9;cifiques li&#xE9;s &#xE0; la PrP (RES) &#xE0; partir de cas n &#xB0; 6, mais pas &#xE0; celle de cas 11 ou C-Type de cas. C-terminale d&#x26;#39;anticorps sp&#xE9;cifiques r&#xE9;v&#xE9;l&#xE9; un changement dans le profil d&#xE9;tect&#xE9; et glycoforme un fragment de prot&#xE9;ine quatri&#xE8;me cas 6, indicatif de deux PrP (RES) dans les sous-populations de type H ESB. Aucune mutation sugg&#xE9;rant une &#xE9;tiologie g&#xE9;n&#xE9;tique ont &#xE9;t&#xE9; trouv&#xE9;s dans l&#x26;#39;un des 17 animaux par s&#xE9;quen&#xE7;age de la pleine PrP-s&#xE9;quence codante de l&#x26;#39;exon 3 du g&#xE8;ne PRNP. Ainsi, chacun des trois types connus d&#x26;#39;ESB ont &#xE9;t&#xE9; confirm&#xE9;s chez des bovins canadiens et pr&#xE9;sentent des caract&#xE9;ristiques mol&#xE9;culaires tr&#xE8;s similaires &#xE0; celles des cas d&#x26;#39;ESB classique et atypique d&#xE9;crits &#xE0; partir de l&#x26;#39;Europe, le Japon et les &#xC9;tats-Unis. L&#x26;#39;apparition de cas atypiques d&#x26;#39;ESB dans les pays comme le Canada avec l&#x26;#39;ESB &#xE0; faible pr&#xE9;valence et le risque de transmission plaide pour la survenue de formes sporadiques d&#x26;#39;ESB dans le monde entier.

Caract&#xE9;ristiques mol&#xE9;culaires, biochimiques et g&#xE9;n&#xE9;tiques de l&#x26;#39;ESB au Canada

Epidemiologie und m&ouml;glicherweise die &Auml;tiologie der bovinen spongiformen Enzephalopathie (BSE) haben vor kurzem erkannt, heterogen sein. Vor allem drei Arten [klassische (C) und zwei atypische (H, L)] wurden, weitgehend nach Merkmalen des Proteinase K (PK)-resistenten Kern von misfolded Prionproteins verbunden mit der Krankheit (PrP(res)). Die vorliegende Studie wurde durchgef&uuml;hrt, um die 17 kanadische BSE-F&auml;lle charakterisieren die vor November 2009 aufgrund der molekularen und biochemischen Eigenschaften von ihrer PrP(res), einschlie&szlig;lich Immunoreactivity, Molekulargewicht, Glycoform Profil und relative Sensitivit&auml;t der PK aufgetreten. Zwei F&auml;llen stellte Molekulargewicht und Glycoform Profilen &auml;hnlich denen von fr&uuml;her berichteten atypischen F&auml;llen eine entsprechende H-Typ BSE (Fall 6) und die andere mit L-Typ BSE (Fall 11). Allen anderen F&auml;llen wurden als C-Typ klassifiziert. PK Verdauung milde und strenge Bedingungen ergab einen reduzierten Protease-Widerstand in beiden F&auml;llen im Vergleich zu den C-Typ-F&auml;llen. Mit westlichen Immunoblotting gebundenen N-Terminal-spezifische Antik&ouml;rper PrP(res) aus Fall 6 aber nicht, dass aus Fall 11 oder C-f&ouml;rmige F&auml;lle. C-Terminal-spezifische Antik&ouml;rper ergab eine Verschiebung im Glycoform Profil und eine vierte Protein-Fragment im Fall 6, bezeichnend f&uuml;r zwei PrP(res) Subpopulationen in H-Typ BSE festgestellt. Keine Mutationen, was auf eine genetische &Auml;tiologie fanden sich in jeder der 17 Tiere durch Sequenzierung der vollst&auml;ndigen PrP-Codierung Sequenz in Exon 3 im PRNP-gen. So jeder der drei bekannten BSE in kanadische Rinder best&auml;tigt worden und Molekulare Eigenschaften sehr &auml;hnlich denen der klassische und atypische BSE-F&auml;lle beschrieben aus Europa, Japan und den USA zu zeigen. Das Vorkommen der atypische F&auml;lle von BSE in L&auml;ndern wie Kanada mit geringem Risiko f&uuml;r die BSE-Pr&auml;valenz und &Uuml;bertragung argumentiert f&uuml;r das Auftreten von BSE sporadische Formen weltweit.

Molekularen, biochemischen und genetischen Merkmale der BSE in Kanada.

Epidemiologia e possibilmente l&#39;eziologia dell&#39;encefalopatia spongiforme bovina (BSE) recentemente sono stati riconosciuti per essere eterogenee. In particolare, sono stati identificati tre tipi [classica (C) e due atipiche (H, L)], in gran parte sulla base delle caratteristiche di proteinasi K (PK)-resistente core della proteina prionica malpiegata associata con la malattia (PrP(res)). Il presente studio &egrave; stato condotto per caratterizzare i 17 casi di BSE canadese che si &egrave; verificato prima del novembre 2009 sulla base delle propriet&agrave; molecolare e biochimiche della loro PrP(res), tra cui immunoreattivit&agrave;, peso molecolare, profilo glycoform e sensibilit&agrave; relativa PK. Due casi esposti glycoform profili simili a quelli precedentemente segnalati casi atipici, uno corrispondente a BSE di tipo H (caso 6) e l&#39;altro di tipo L BSE (11 casi) e peso molecolare. Tutti gli altri casi sono stati classificati come tipo C. Digestione PK in condizioni severe e mite ha rivelato una resistenza ridotta proteasi in entrambi i casi rispetto ai casi di tipo C. Immunoblotting Western, anticorpi specifici per N-terminale legano a PrP(res) dal caso 6 ma non a quello da 11 caso o i casi di tipo C. Anticorpi specifici per C-terminale ha rivelato un cambiamento nel profilo glycoform e rilevato un quarto frammento di proteina in caso 6, indicativo di due sottopopolazioni PrP(res) in H-tipo ESB. Nessuna mutazione suggerendo un&#39;eziologia genetica sono stati trovati in uno qualsiasi degli 17 animali sequenziando il PrP-codificazione completa sequenza nell&#39;esone 3 del gene PRNP. Cos&igrave;, ognuno dei tre tipi noti BSE sono state confermate nel bestiame canadese e mostrano caratteristiche molecolari altamente simili a quelle dei casi di BSE classici e atipici, descritti da Europa, Giappone e Stati Uniti. Il verificarsi di casi atipici di BSE in paesi come il Canada con basso rischio di trasmissione e prevalenza BSE sostiene per la comparsa di forme sporadiche di BSE in tutto il mondo.

Caratteristiche molecolari, biochimiche e genetiche della BSE in Canada.

&#x75AB;&#x5B66;&#x3001;&#x304A;&#x305D;&#x3089;&#x304F;&#x725B;&#x6D77;&#x7DBF;&#x72B6;&#x8133;&#x75C7;&#xFF08;BSE&#xFF09;&#x306E;&#x539F;&#x56E0;&#x306F;&#x6700;&#x8FD1;&#x3001;&#x7570;&#x7A2E;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x8A8D;&#x8B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002;&#x7279;&#x306B;&#x3001;3&#x7A2E;&#x985E;&#x306E;[&#x53E4;&#x5178;&#x7684;&#x306A;&#xFF08;C&#xFF09;&#x3068;&#xFF08;H&#x3001;L&#xFF09;&#x306F;&#x3001;2&#x3064;&#x306E;&#x975E;&#x5B9A;&#x578B;]&#x4E3B;&#x306B;&#x30D7;&#x30ED;&#x30C6;&#x30A4;&#x30CA;&#x30FC;&#x30BC;K&#xFF08;PK&#x200B;&#x200B;&#xFF09;&#x62B5;&#x6297;&#x6027;&#x75BE;&#x60A3;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x7570;&#x5E38;&#x306A;&#x6298;&#x308A;&#x7573;&#x307F;&#x69CB;&#x9020;&#x306E;&#x30D7;&#x30EA;&#x30AA;&#x30F3;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x30B3;&#x30A2;&#xFF08;PrP&#x306E;&#x7279;&#x6027;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x3001;&#x7279;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#xFF08;RES&#xFF09;&#xFF09;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x4E8B;&#x524D;&#x306E;&#x514D;&#x75AB;&#x53CD;&#x5FDC;&#x6027;&#x3001;&#x5206;&#x5B50;&#x91CF;&#x3001;&#x7CD6;&#x578B;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3068;&#x76F8;&#x5BFE;&#x7684;&#x306A;PK&#x306E;&#x611F;&#x5EA6;&#x3092;&#x542B;&#x3081;&#x305F;PrP&#x306E;&#x5206;&#x5B50;&#x304A;&#x3088;&#x3073;&#x751F;&#x5316;&#x5B66;&#x7684;&#x7279;&#x6027;&#xFF08;RES&#xFF09;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x3001;2009&#x5E74;11&#x6708;&#x306B;&#x767A;&#x751F;&#x3057;&#x305F;17&#x30AB;&#x30CA;&#x30C0;&#x306E;BSE&#x75C7;&#x4F8B;&#x3092;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x308B;&#x305F;&#x3081;&#x306B;&#x5B9F;&#x65BD;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x4E8C;&#x3064;&#x306E;&#x30B1;&#x30FC;&#x30B9;&#x306F;&#x3001;&#x4EE5;&#x524D;&#x306B;&#x5831;&#x544A;&#x3055;&#x308C;&#x305F;&#x975E;&#x5B9A;&#x578B;&#x4F8B;&#x3068;&#x985E;&#x4F3C;&#x306E;&#x5206;&#x5B50;&#x91CF;&#x3068;&#x30B0;&#x30EA;&#x30B3;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x3001;H&#x578B;BSE&#xFF08;&#x30B1;&#x30FC;&#x30B9;6&#xFF09;&#x304A;&#x3088;&#x3073;L&#x578B;BSE&#xFF08;&#x30B1;&#x30FC;&#x30B9;11&#xFF09;&#x306B;&#x4ED6;&#x306E;&#x306B;&#x5BFE;&#x5FDC;&#x3059;&#x308B;&#x3082;&#x306E;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x4ED6;&#x306E;&#x3059;&#x3079;&#x3066;&#x306E;&#x5834;&#x5408;&#x306F;C&#x578B;&#x306B;&#x5206;&#x985E;&#x3055;&#x308C;&#x305F;&#x3002;&#x7A4F;&#x3084;&#x304B;&#x306A;&#x3001;&#x53B3;&#x3057;&#x3044;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x306E;PK&#x6D88;&#x5316;&#x306F;&#x3001;C&#x578B;&#x306E;&#x30B1;&#x30FC;&#x30B9;&#x306B;&#x6BD4;&#x3079;&#x3066;&#x3053;&#x308C;&#x3089;&#x306E;&#x4F8B;&#x306E;&#x4E21;&#x65B9;&#x3067;&#x6E1B;&#x5C11;&#x3059;&#x308B;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x62B5;&#x6297;&#x6027;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002;&#x30B1;&#x30FC;&#x30B9;6&#x304B;&#x3089;&#x3067;&#x306F;&#x306A;&#x304F;&#x305D;&#x306E;&#x3088;&#x3046;&#x306A;&#x5834;&#x5408;&#x304B;&#x3089;11&#x307E;&#x305F;&#x306F;C-&#x578B;&#x306E;&#x30B1;&#x30FC;&#x30B9;&#x306B;PRP&#xFF08;RES&#xFF09;&#x306B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3055;&#x308C;&#x305F;&#x897F;&#x6D0B;&#x306E;&#x514D;&#x75AB;&#x30D6;&#x30ED;&#x30C3;&#x30C8;&#x6CD5;&#x3001;N&#x672B;&#x7AEF;&#x7279;&#x7570;&#x6297;&#x4F53;&#x3092;&#x7528;&#x3044;&#x305F;&#x3002; C&#x672B;&#x7AEF;&#x7279;&#x7570;&#x7684;&#x6297;&#x4F53;&#x306F;&#x3001;&#x30B0;&#x30EA;&#x30B3;&#x306E;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306E;&#x5909;&#x5316;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x3001;&#x30B1;&#x30FC;&#x30B9;6&#x3067;4&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x65AD;&#x7247;&#x3092;&#x691C;&#x51FA;&#x3057;&#x3001;H&#x578B;BSE&#x306E;2 PRP&#xFF08;RES&#xFF09;&#x96C6;&#x56E3;&#x3092;&#x793A;&#x3059;&#x3002;&#x907A;&#x4F1D;&#x7684;&#x75C5;&#x56E0;&#x3092;&#x793A;&#x5506;&#x3059;&#x308B;&#x5909;&#x7570;&#x306F;PRNP&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30A8;&#x30AF;&#x30BD;&#x30F3;3&#x306E;&#x30D5;&#x30EB;PRP-&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x914D;&#x5217;&#x3092;&#x914D;&#x5217;&#x6C7A;&#x5B9A;&#x306B;&#x3088;&#x308A;17&#x306E;&#x52D5;&#x7269;&#x306E;&#x3044;&#x305A;&#x308C;&#x306B;&#x3082;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;3&#x3064;&#x306E;&#x65E2;&#x77E5;&#x306E;BSE&#x306E;&#x7A2E;&#x985E;&#x306E;&#x5404;&#x3005;&#x306F;&#x3001;&#x30AB;&#x30CA;&#x30C0;&#x306E;&#x725B;&#x3067;&#x78BA;&#x8A8D;&#x3055;&#x308C;&#x3001;&#x30E8;&#x30FC;&#x30ED;&#x30C3;&#x30D1;&#x3001;&#x65E5;&#x672C;&#x3001;&#x7C73;&#x56FD;&#x304B;&#x3089;&#x8AAC;&#x660E;&#x3057;&#x3001;&#x53E4;&#x5178;&#x7684;&#x304A;&#x3088;&#x3073;&#x975E;&#x5B9A;&#x578B;BSE&#x75C7;&#x4F8B;&#x306E;&#x3082;&#x306E;&#x3068;&#x975E;&#x5E38;&#x306B;&#x985E;&#x4F3C;&#x3057;&#x305F;&#x5206;&#x5B50;&#x306E;&#x7279;&#x6027;&#x3092;&#x793A;&#x3057;&#x3066;&#x304D;&#x305F;&#x3002;&#x4F4E;BSE&#x6709;&#x75C5;&#x7387;&#x3068;&#x4F1D;&#x9001;&#x30EA;&#x30B9;&#x30AF;&#x3068;&#x30AB;&#x30CA;&#x30C0;&#x306A;&#x3069;&#x306E;&#x56FD;&#x3067;BSE&#x306E;&#x975E;&#x5B9A;&#x578B;&#x4F8B;&#x306E;&#x767A;&#x751F;&#x306F;&#x3001;BSE&#x5168;&#x4E16;&#x754C;&#x3067;&#x306E;&#x6563;&#x767A;&#x7684;&#x306A;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x306E;&#x767A;&#x751F;&#x3092;&#x4E3B;&#x5F35;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;

&#x30AB;&#x30CA;&#x30C0;&#x3067;&#x306E;BSE&#x306E;&#x5206;&#x5B50;&#x3001;&#x751F;&#x5316;&#x5B66;&#x7684;&#x304A;&#x3088;&#x3073;&#x907A;&#x4F1D;&#x7684;&#x7279;&#x6027;

&#xC5ED;&#xD559; &#xBC0F; &#xBCD1; &#xC778; &#xC18C; &#xD574; &#xB1CC; &#xC99D; (&#xAD11;&#xC6B0;&#xBCD1;)&#xC758; &#xCD5C;&#xADFC; &#xC774;&#xC885; &#xC218; &#xC778;&#xC815;&#xC744; &#xBC1B;&#xACE0; &#xC788;&#xB2E4;. &#xD2B9;&#xD788;, &#xC138; &#xAC00;&#xC9C0; &#xC720;&#xD615; [&#xD074;&#xB798;&#xC2DD; (C)&#xC640; &#xB450; &#xD754;&#xD558;&#xC9C0; &#xC54A;&#xC740; (H, L)] &#xD655;&#xC778; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;, &#xAC00;&#xC218;&#xBD84;&#xD574; K (PK)&#xC758; &#xD2B9;&#xC131;&#xC5D0; &#xADFC;&#xAC70; &#xD558; &#xC5EC; &#xD06C;&#xAC8C;-&#xB0B4;&#xC131; &#xCF54;&#xC5B4; (PrP(res)) &#xC9C8;&#xBCD1;&#xACFC; &#xAD00;&#xB828; &#xB418;&#xC5C8;&#xB358; misfolded &#xD504;&#xB9AC;&#xC628; &#xB2E8;&#xBC31;&#xC9C8;&#xC758;. &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C;&#xB294; &#xADF8;&#xB4E4;&#xC758; PrP(res), immunoreactivity, &#xBD84;&#xC790;&#xB7C9;, glycoform &#xD504;&#xB85C;&#xD544; &#xC0C1;&#xB300; PK &#xAC10;&#xB3C4; &#xB4F1;&#xC758; &#xBD84;&#xC790; &#xBC0F; &#xC0DD;&#xD654;&#xD559; &#xC801;&#xC778; &#xC18D;&#xC131;&#xC744; &#xAE30;&#xBC18;&#xC73C;&#xB85C; 2009 &#xB144; 11 &#xC6D4; &#xC774;&#xC804;&#xC5D0; &#xBC1C;&#xC0DD; &#xD55C; 17 &#xCE90;&#xB098;&#xB2E4; &#xAD11;&#xC6B0;&#xBCD1; &#xC0AC;&#xB840;&#xB97C; &#xC2E4;&#xC2DC; &#xD588;&#xB2E4;. &#xB450; &#xAC00;&#xC9C0; &#xACBD;&#xC6B0;&#xB294; &#xBD84;&#xC790;&#xB7C9; &#xBC0F; &#xC774;&#xC804;&#xC5D0; &#xBCF4;&#xACE0; &#xB41C; &#xBE44;&#xC815;&#xD615; &#xACBD;&#xC6B0; &#xD574;&#xB2F9; H &#xD615; &#xAD11;&#xC6B0;&#xBCD1; (&#xC0AC;&#xB840; 6)&#xACFC; L &#xD615; &#xAD11;&#xC6B0;&#xBCD1; (&#xC0AC;&#xB840; 11)&#xC5D0; &#xB2E4;&#xB978; &#xD558; &#xD558;&#xB098; &#xADF8;&#xC640; &#xBE44;&#xC2B7;&#xD55C; glycoform &#xD504;&#xB85C;&#xD544; &#xC804;&#xC2DC;. &#xB2E4;&#xB978; &#xBAA8;&#xB4E0; &#xACBD;&#xC6B0;&#xB294; C &#xD615;&#xC73C;&#xB85C; &#xBD84;&#xB958; &#xB410;&#xB2E4;. &#xC628;&#xD654; &#xD558; &#xACE0; &#xC5C4;&#xACA9;&#xD55C; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; PK &#xC18C;&#xD654; C &#xD615;&#xC758; &#xACBD;&#xC6B0;&#xC5D0; &#xBE44;&#xD574; &#xB450;&#xC774; &#xACBD;&#xC6B0; &#xBAA8;&#xB450;&#xC5D0;&#xC11C; &#xAC10;&#xC18C; protease &#xC800;&#xD56D;&#xC744; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xC11C;&#xC591; immunoblotting N &#xD130;&#xBBF8;&#xB110; &#xD2B9;&#xC815; &#xD56D; &#xCCB4; &#xACBD;&#xC6B0; 6&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; Prp(res)&#xB97C; &#xD558;&#xC9C0;&#xB9CC; &#xADF8;&#xC744; &#xACBD;&#xC6B0; 11 &#xB610;&#xB294; C &#xD0C0;&#xC785;&#xC758; &#xACBD;&#xC6B0; &#xBC14;&#xC778;&#xB529;. C &#xD130;&#xBBF8;&#xB110; &#xD2B9;&#xC815; &#xD56D; &#xCCB4; glycoform &#xD504;&#xB85C;&#xD544;&#xC5D0; &#xBCC0;&#xD654;&#xB97C; &#xACF5;&#xAC1C; &#xD558; &#xACE0; H &#xD615; &#xAD11;&#xC6B0;&#xBCD1;&#xC758; &#xACBD;&#xC6B0; 6, &#xB450; PrP(res) &#xBD80;&#xBD84;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xB124; &#xBC88;&#xC9F8; &#xB2E8;&#xBC31;&#xC9C8; &#xC870;&#xAC01;&#xC744; &#xBC1C;&#xACAC;. &#xC544;&#xB2C8; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC720;&#xC804;&#xC790; &#xBCD1; &#xC778; &#xC804;&#xCCB4; PrP &#xCF54;&#xB529; &#xC2DC;&#xD000;&#xC2F1; &#xD558; &#xC5EC; 17 &#xB3D9;&#xBB3C;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; &#xB41C; &#xC81C;&#xC548; PRNP &#xC720;&#xC804;&#xC790;&#xC758; exon 3&#xC758;&#xC5D0;&#xC11C; &#xC2DC;&#xD000;&#xC2A4;&#xC785;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, 3 &#xC54C;&#xB824;&#xC9C4; &#xAD11;&#xC6B0;&#xBCD1; &#xD615;&#xC2DD;&#xC758; &#xAC01; &#xCE90;&#xB098;&#xB2E4; &#xC18C;&#xC5D0; &#xD655;&#xC778; &#xD558; &#xACE0; &#xB192;&#xC740; &#xD074;&#xB798;&#xC2DD;&#xACFC; &#xBE44;&#xC815;&#xD615; &#xAD11;&#xC6B0;&#xBCD1; &#xC0AC;&#xB840; &#xC720;&#xB7FD;, &#xC77C;&#xBCF8; &#xBC0F; &#xBBF8;&#xAD6D;&#xC5D0;&#xC11C; &#xC124;&#xBA85; &#xB41C; &#xAC83;&#xACFC; &#xC720;&#xC0AC;&#xD55C; &#xBD84;&#xC790; &#xD2B9;&#xC131;&#xC744; &#xBCF4;&#xC5EC;. &#xD754;&#xD558;&#xC9C0; &#xC54A;&#xC740; &#xACBD;&#xC6B0; &#xB0AE;&#xC740; &#xAD11;&#xC6B0;&#xBCD1; &#xC815;&#xB3C4;&#xC640; &#xC804;&#xC1A1; &#xC704;&#xD5D8; &#xCE90;&#xB098;&#xB2E4; &#xB4F1;&#xC758; &#xAD6D;&#xAC00;&#xC5D0;&#xC11C; &#xAD11;&#xC6B0;&#xBCD1;&#xC758; &#xBC1C;&#xC0DD;&#xC740; &#xC804;&#xC138;&#xACC4; &#xAD11;&#xC6B0;&#xBCD1; &#xC0B0;&#xBC1C;&#xC801; &#xC778; &#xD615;&#xD0DC;&#xC758; &#xBC1C;&#xC0DD;&#xC5D0; &#xB300; &#xD55C; &#xC8FC;&#xC7A5;.

&#xCE90;&#xB098;&#xB2E4;&#xC5D0;&#xC11C; &#xAD11;&#xC6B0;&#xBCD1;&#xC758; &#xBD84;&#xC790;, &#xC0DD;&#xD654;&#xD559; &#xBC0F; &#xC720;&#xC804; &#xD2B9;&#xC131;.

La epidemiolog&#xED;a y, posiblemente, la etiolog&#xED;a de la encefalopat&#xED;a espongiforme bovina (EEB) ha sido reconocida recientemente a ser heterog&#xE9;neas. En particular, los tres tipos [cl&#xE1;sica (C) y dos at&#xED;pico (H, L)] han sido identificados, en gran medida sobre la base de las caracter&#xED;sticas de la proteinasa K (PK)-n&#xFA;cleo resistente de la prote&#xED;na pri&#xF3;nica mal plegada asociada con la enfermedad (PrP (res)). El presente estudio se realiz&#xF3; para caracterizar a los canadienses 17 casos de EEB que se produjeron con anterioridad a noviembre de 2009 sobre la base de las propiedades moleculares y bioqu&#xED;micos de la PrP (res), incluyendo inmunoreactividad, peso molecular, el perfil de glicoforma y la sensibilidad en relaci&#xF3;n PK. Dos casos presentaron peso molecular y perfiles de glicoforma similares a los de informes anteriores de los casos at&#xED;picos, que corresponde al tipo H EEB (caso 6) y el otro a la L-tipo de la encefalopat&#xED;a espongiforme bovina (caso 11) una. Todos los dem&#xE1;s casos fueron clasificados como tipo C. Digesti&#xF3;n PK bajo condiciones suaves y estrictas revel&#xF3; una resistencia reducida proteasa en ambos casos en comparaci&#xF3;n con los casos de tipo-C. Con inmunotransferencia, N-terminal de anticuerpos espec&#xED;ficos ligados a la PrP (res) de la caja 6, pero no a ese caso de 11 o C de tipo casos. C-terminal de anticuerpos espec&#xED;ficos revel&#xF3; un cambio en el perfil glicoforma y detecta un fragmento de la prote&#xED;na cuarto en el caso 6, indicativo de dos PRP (RES) subpoblaciones de tipo H EEB. No hay mutaciones que sugieren una etiolog&#xED;a gen&#xE9;tica se encuentra en cualquiera de los 17 animales por secuenciaci&#xF3;n completa la secuencia de codificaci&#xF3;n PrP en el ex&#xF3;n 3 del gen PRNP. Por lo tanto, cada uno de los tres tipos conocidos de la EEB se han confirmado en ganado canadiense y mostrar las caracter&#xED;sticas moleculares muy similares a los de casos de EEB cl&#xE1;sicos y at&#xED;picos que se describen de Europa, Jap&#xF3;n y los EE.UU.. La aparici&#xF3;n de casos at&#xED;picos de EEB en pa&#xED;ses como Canad&#xE1; con baja prevalencia de la EEB y riesgo de transmisi&#xF3;n argumenta a favor de la aparici&#xF3;n de formas espor&#xE1;dicas de la encefalopat&#xED;a espongiforme bovina en todo el mundo.

Caracter&#xED;sticas moleculares, bioqu&#xED;micos y gen&#xE9;ticos de la EEB en Canad&#xE1;

Bovine spongiform encephalopathy (BSE) surveillance programs have been employed in numerous countries to monitor BSE prevalence and to protect animal and human health. Since 1999, the European Commission (EC) authorized the evaluation and approval of 20 molecular based tests for the rapid detection of the pathological prion protein (PrP(sc)) in BSE infection. The diagnostic sensitivity, convenience, and speed of these tests have made molecular diagnostics the preferred method for BSE surveillance. The aim of this study was to determine the analytical sensitivity of 4 commercially available BSE rapid-test kits, including the Prionics®-Check WESTERN, the Prionics® Check-PrioSTRIP™, the BioRad® TeSeE™ ELISA, and the IDEXX® HerdChek™ EIA. Performances of these tests were then compared to 2 confirmatory tests, including the BioRad® TeSeE™ Western Blot and the modified Scrapie Associated Fibrils (SAF)/OIE Immunoblot. One 50% w/v homogenate was made from experimentally generated C-type BSE brain tissues in ddH₂O. Homogenates were diluted through a background of BSE-negative brainstem homogenate. Masses of both positive and negative tissues in each dilution were calculated to maintain the appropriate tissue amounts for each test platform. Specific concentrated homogenization buffer was added accordingly to maintain the correct buffer condition for each test. ELISA-based tests were evaluated using their respective software/detection platforms. Blot-protocols were evaluated by manual measurements of blot signal density. Detection limitations were determined by fitted curves intersecting the manufacturers' positive/negative criteria. The confirmatory SAF Immunoblot displayed the highest analytical sensitivity, followed by the IDEXX® HerdChek™ EIA, Bio-Rad® TeSeE™ Western Blot, the Bio-Rad® TeSeE™ ELISA, Prionics®-Check PrioSTRIP™, and Prionics®-Check WESTERN™, respectively. Although the tests performed at different levels of sensitivity, the most sensitive and least sensitive of the rapid tests were separated by 2 logs in analytical sensitivity, meeting European performance requirements. All rapid tests appear suitable for targeted BSE surveillance programs, as implemented in Canada.

A study on the analytical sensitivity of 6 BSE tests used by the Canadian BSE reference laboratory.

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease.

The preferred method to determine the prevalence of bovine spongiform encephalopathy (BSE) in a country is to use immunology-based rapid-tests. Though these tests are validated to detect C-type BSE disease-associated prion (PrP(sc)), test-specific properties may influence their ability to detect H- and/or L-type BSE PrP(sc), where both are atypical from C-type PrP(sc). Molecular characterization shows atypical BSE PrP(sc) to have a different sensitivity to proteinase activity and different affinities for certain prion-specific antibodies. It is important to understand how atypical BSE PrP(sc) may affect the performance of rapid-tests, which are typically dependent on the use of specific proteases and antibodies. The current study used experimentally generated C-, H-, and L-type BSE PrP(sc) to evaluate 3 tests used in various national BSE surveillance programs: an immunochromatographic assay, a standard sandwich enzyme-linked immunosorbent assay (stndELISA), and a PrP(sc)-conformation-specific ELISA (confELISA). Although BSE PrP(sc) type had some effects on rapid-test performance, analytical sensitivity for atypical BSE PrP(sc) on all 3 platforms was not significantly compromised. When testing for atypical BSE PrP(sc), the 3 tests were able to meet the same requirements that the European Food Safety Authority set when evaluating the tests for C-type BSE PrP(sc).

Performance analysis of rapid diagnostic tests on atypical bovine spongiform encephalopathy.

Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM) provided that infectious prion proteins (PrP(TSE)) are inactivated. This study investigated the degradation of SRM and the fate of scrapie prions (PrP(Sc)) over 28 days in laboratory-scale composters, with and without feathers in the compost matrices. Compost was mixed at day 14 to generate a second heating cycle, with temperatures exceeding 65°C in the first cycle and 50°C in the second cycle. Approximately 63% and 77% of SRM was degraded after the first and second cycles, respectively. Inclusion of feathers in the compost matrices did not alter compost properties during composting other than increasing (P < 0.05) total nitrogen and reducing (P < 0.05) the C/N ratio. However, addition of feathers enhanced (P < 0.05) SRM degradation by 10% upon completion of experiment. Scrapie brain homogenates were spiked into manure at the start of composting and extracted using sodium dodecyl sulphate followed by detection using Western blotting (WB). Prior to composting, PrP(Sc) was detectable in manure with 1-2 log(10) sensitivity, but was not observable after 14 or 28 days of composting. This may have been due to either biological degradation of PrP(Sc) or the formation of complexes with compost components that precluded its detection.

Biodegradation of specified risk material and fate of scrapie prions in compost.

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrP(TSE)) for 24 h in three replicate studies with PrP(TSE) in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP(C)) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP(TSE)) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP(TSE) from the roots to the stem to be detectable by the methods employed. Undigested PrP(TSE) did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.

Can plants serve as a vector for prions causing chronic wasting disease?

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

Biodegradation of prions in compost.

Transmissible spongiform encephalopathies (TSE) are progressive, neurodegenerative disorders, of which bovine spongiform encephalopathy (BSE) is of special concern because it is infectious and debilitating to humans. The possibility of using fluorescence spectroscopy to screen for BSE in cattle was explored. Fluorescence spectra from the retinas of experimentally infected BSE-positive cattle with clinical disease were compared with those from both sham-inoculated and non-inoculated BSE-negative cattle. The distinct intensity difference of about 4-10-fold between the spectra of the BSE-positive and the BSE-negative (sham-inoculated and non-inoculated) eyes suggests the basis for a means of developing a rapid, noninvasive examination of BSE in particular and TSEs in general.

Fluorescence Spectroscopy of the Retina for the Screening of Bovine Spongiform Encephalopathy.

Bovine spongiform encephalopathy (BSE) is an invariably fatal prion disease of cattle. The identification of the zoonotic potential of BSE prompted safety officials to initiate surveillance testing for this disease. In Canada, BSE surveillance is primarily focused on high risk cattle including animals which are dead, down and unable to rise, diseased or distressed. This targeted surveillance results in the submission of brain samples with a wide range of tissue autolysis and associated contaminants. These contaminants have the potential to interfere with important steps of surveillance tests resulting in initially positive test results requiring additional testing to confirm the disease status of the animal. The current tests used for BSE screening in Canada utilize the relative protease resistance of the prion protein gained when it misfolds from PrP(C) to PrP(Sc) as part of the disease process. Proteinase K completely digests PrP(C) in normal brains, but leaves most of the PrP(Sc) in BSE positive brains intact which is detected using anti-prion antibodies. These tests are highly reliable but occasionally give rise to initially reactive/false positive results. Test results for these reactive samples were close to the positive/negative cut-off on a sub set of test platforms. This is in contrast to all of the previous Canadian positive samples whose numeric values on these same test platforms were 10 to 100 fold greater than the test positive/negative cut-off. Here we explore the potential reason why a sample is repeatedly positive on a sub-set of rapid surveillance tests, but negative on other test platforms. In order to better understand and identify what might cause these initial reactions, we have conducted a variety of rapid and confirmatory assays as well as bacterial isolation and identification on BSE positive, negative and initially reactive samples. We observed high levels of viable bacterial contamination in initially reactive samples suggesting that the reactivity may be related to bacterial factors. Several bacteria isolated from the initially reactive samples have characteristics of biofilm forming bacteria and this extracellular matrix might play a role in preventing complete digestion of PrP(C) in these samples.

Exploring the cause of initially reactive bovine brains on rapid tests for BSE.

Transmissible spongiform encephalopathies (TSEs) are infectious, fatal neurodegenerative diseases that affect production animal health, and thus human food safety. Enhanced TSE detection methods mimic the conjectured basis for prion replication, in vitro; biological matrices can be tested for prion activity via their ability to convert recombinant cellular prion protein (PrP) into amyloid fibrils; fluorescent spectra changes of amyloid-binding fluorophores in the reaction vessel detect fibril formation. In vitro PrP conversion techniques have high analytical sensitivity for prions, comparable with that of bioassays, yet no such protocol has gained regulatory approval for use in animal TSE surveillance programs. This study describes a timed in vitro PrP conversion protocol with accurate, well-defined analytical criteria based on probability density and mass functions of TSE(+) and TSE(-) associated thioflavin T signal times, a new approach within this field. The prion detection model used is elk chronic wasting disease (CWD) in brain tissues. The protocol and analytical criteria proved as sensitive for elk CWD as two bioassay models, and upward of approximately 1.2 log10 more sensitive than the most sensitive TSE rapid test we assessed. Furthermore, we substantiate that timing in vitro PrP conversion may be used to titrate TSE infectivity, and, as a result, provide a comprehensive extrapolation of analytical sensitivity differences between bioassay, TSE rapid tests, and in vitro PrP conversion for elk CWD.

Defining and Assessing Analytical Performance Criteria for Transmissible Spongiform Encephalopathy-Detecting Amyloid Seeding Assays.

Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrPSc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk's prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion.

Three different types of bovine spongiform encephalopathy (BSE) are known and supposedly caused by distinct prion strains: the classical (C-) BSE type that was typically found during the BSE epidemic, and two relatively rare atypical BSE types, termed H-BSE and L-BSE. The three BSE types differ in the molecular phenotype of the disease associated prion protein, namely the N-terminally truncated proteinase K (PK) resistant prion protein fragment (PrP(res)). In this study, we report and analyze yet another PrP(res) type (PrP(res-2011)), which was found in severely autolytic brain samples of two cows in the framework of disease surveillance in Switzerland in 2011. Analysis of brain tissues from these animals by PK titration and PK inhibitor assays ruled out the process of autolysis as the cause for the aberrant PrP(res) profile. Immunochemical characterization of the PrP fragments present in the 2011 cases by epitope mapping indicated that PrP(res-2011) corresponds in its primary sequence to the physiologically occurring PrP-C1 fragment. However, high speed centrifugation, sucrose gradient assay and NaPTA precipitation revealed biochemical similarities between PrP(res-2011) and the disease-associated prion protein found in BSE affected cattle in terms of detergent insolubility, PK resistance and PrP aggregation. Although it remains to be established whether PrP(res-2011) is associated with a transmissible disease, our results point out the need of further research on the role the PrP-C1 aggregation and misfolding in health and disease.

Sandor Dudas

Canadian Food Inspection Agency

Lethbridge Laboratories

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

Sandor Dudas

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Lethbridge Laboratories

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

Canadian Food Inspection Agency