Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP(Sc), a protease-resistant misfolded isoform of the cellular prion protein PrP(C). Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP(Sc). However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP(Sc) in BSE-affected cattle therefore remains unknown.We report here that PrP(Sc) derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP(Sc) in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP(Sc) in blood was not substantiated in the BSE-affected cattle examined.The distribution of PrP(Sc) is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP(Sc) could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials

Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues

BSE-PrP^Sc detection sensitivity.

<p><b>A</b>. The PrP^Sc seed was diluted to 10⁻⁴ to 10⁻¹¹ with PrP^C substrate, and samples were serially amplified in the presence of 0.5% potassium dextran sulfate (DSP). The duplicate amplified samples were analyzed after each round of amplification (R1–R4) by WB after PK digestion. <b>B</b>. No spontaneous generation of PrP^Sc was observed. Samples labeled “1” to “8” contained only PrP^C substrate and were amplified in the presence of 0.5% DSP.</p

Mean incubation time of TgBoPrP mice following intracerebral inoculation.

<p>Mean incubation time of TgBoPrP mice following intracerebral inoculation.</p

Optimal DSP concentration for BSE PrP^Sc amplification.

<p>The PrP^Sc seed was diluted to 10⁻⁴, and amplification was performed in the presence of the potassium dextran sulfate (DSP). “N” designates the control in which only PrP^C substrate was amplified.</p