WB detection of PrPres in tissues from fetus 1 after saPMCA.

<p>After the 4th round of saPMCA, at least one of the eight aliquots analyzed from each tissue showed PrP^Sc amplification. None of the unseeded negative controls showed any detectable signal. NBH: Normal brain homogenate without pK treatment. RLN: retropharyngeal lymph node. ICV: ileocecal valve.</p

PrPres amplification in fetal tissues after 3–4 rounds of saPMCA.

<p>Number of positive aliquots for each sample analyzed (from a total of 8 aliquots).</p><p>RLN: retropharyngeal lymph node. ICV: ileocecal valve. NT: not tested.</p><p>*Fetuses 2 and 3 shared the same uterine horn.</p

IHC detection of PrPres deposition in scrapie-infected dams.

<p>A wide PrP^Sc distribution was observed in the 3 scrapie-infected dams. A high intensity of immunolabeling in an obex (A), a retropharyngeal lymph node (B) and a placentome at the level of feto-maternal interface (C), is shown. In this image (D), the proximity between trophoblast cells presenting PrP^Sc deposits and a fetal vessel (*) can be observed.</p

Determining the Relative Susceptibility of Four Prion Protein Genotypes to Atypical Scrapie

Atypical scrapie is a sheep prion (PrP^Sc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrP^C). To determine the relative amounts of PrP polymorphisms present in atypical scrapie, total PrP was digested with chymotrypsin to generate characteristic peptides spanning relevant polymorphisms at positions 136, 141, 154, 171, and 172 of sheep PrP^C. A multiple reaction monitoring method (MRM), employing ¹⁵N-labeled internal standards, was used to detect and quantify these polymorphisms present in both the PrP^Sc and PrP^C from heterozygous (ALRRY and ALHQY or ALRQD or AFRQY) atypical scrapie-infected or uninfected control sheep. Both polymorphisms of the full length and truncated (C1) natively expressed PrP^C are produced in equal amounts. The overall amount of PrP^C present in the infected or uninfected animals was similar. PrP^Sc isolated from heterozygotes was composed of significant amounts of both PrP polymorphisms, including the ALRRY polymorphism which is highly resistant to classical scrapie. Thus, an atypical scrapie infection does not result from an overexpression of sheep PrP^C. The replication of all atypical scrapie prions occurs at comparable rates, despite polymorphisms at positions 141, 154, 171, or 172

Additional file 2: of Preliminary studies on isolates of Clostridium difficile from dogs and exotic pets

Clostridium difficile field isolates and Clostridium difficile ATCC 43255 (positive control) toxinotyping in agarose gel. a A1 fragment, 3.1 kb: lane 1 E6 isolate, lanes 2–5 field isolates, lane 6 ATTC strain, lane 7 negative control. b A3 fragment, 3.1 kb: lane 1 ATCC strain, lane 2 E6 strain, lane 3 negative control. c B1 fragment, 3.1 kb: lanes 1 and 3 field isolates, lane 2 E6 strain, lane 4 ATCC strain, lane 5 negative control. M: 1 kb molecular mass (upper arrows point to 3 kb fragment and lower arrows point to 1 kb fragment) (JPEG 1765 kb

Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep

Scrapie is a prion (PrP^Sc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrP^C). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M₁₃₂LGS<b><u>X</u></b>MSRPL₁₄₁ (<b><u>X</u></b> = A or V), Y₁₅₃<b><u>X</u></b>ENMY₁₅₈ (<b><u>X</u></b>,= H or R), and Y₁₆₆RPVD<b><u>X</u></b>Y₁₇₂ (<b><u>X</u></b> = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrP^C or PrP^Sc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrP^Sc isolated from sheep heterozygous for their PrP^C proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrP^Sc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrP^Sc and samples containing only the PK-resistant PrP^Sc. These results show that heterozygous animals contain PrP^Sc that is composed of significant amounts of both PrP polymorphisms

Additional file 1: of Preliminary studies on isolates of Clostridium difficile from dogs and exotic pets

cdu1-cdd1 PCR (700 bp) agarose gel image from Clostridium difficile field isolates. Lane 1 C. difficile E6 strain, lanes 2–6 field isolates, lane 7 positive control, lane 8 negative control. M: 100 bp molecular mass (arrows point 500 bp fragment). (JPEG 1439 kb

Additional file 3: of Preliminary studies on isolates of Clostridium difficile from dogs and exotic pets

Clostridium difficile field isolates and Clostridium difficile ATCC 43255 (positive control) toxinotyping in agarose gel. Lanes 1–3 A2 fragment, 2 kb: ATTC strain, E6 strain and negative control respectively; lanes 4–6 B2 fragment, 2 kb: ATTC strain, E6 strain and negative control respectively; lanes 7–9 B3 fragment, 2 kb: ATTC strain, E6 strain and negative control respectively. M: 1 kb molecular mass (upper arrows point to 3 kb fragment and lower arrows point to 1 kb fragment) (JPEG 839 kb

PrP^res detection in PMCA reactions seeded with tissue samples collected from a Sh-BSE clinically affected pig.

<p>Tissues: brain, sciatic nerve, brachial nerve, mesenteric lymph node, oculomotor muscle, ileum, spleen, and white blood cells (WBC). The animal was culled at 30 months after intracerebral inoculation with Sh-BSE. After 3 rounds of PMCA (48 hours each), PrP^res amplification was detected at dilutions of 10⁻¹ (a) and 10⁻² (b) in all the tissues analysed. No PrP^res was amplified in the mesenteric lymph node, ileum, or spleen at a dilution of 10⁻³ (c). Results obtained for 10⁻¹ dilution after 3 rounds of PMCA in reactions seeded with tissues from a negative control pig (d). Control: PK digested classical scrapie isolate.</p

Detection of PrP^res in peripheral tissue in pigs with clinical disease induced by intracerebral challenge with sheep-passaged bovine spongiform encephalopathy agent

<div><p>Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrP^res accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrP^res positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain.</p></div