Sulfated dextrans enhance in vitro amplification of bovine spongiform encephalopathy PrP(Sc) and enable ultrasensitive detection of bovine PrP(Sc).
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Japan. ymura@affrc.go.jp
Yuichi
Y
Murayama
Miyako
M
Yoshioka
Kentaro
K
Masujin
Hiroyuki
H
Okada
Yoshifumi
Y
Iwamaru
Morikazu
M
Imamura
Yuichi
Y
Matsuura
Shigeo
S
Fukuda
Sadao
S
Onoe
Takashi
T
Yokoyama
Shirou
S
Mohri
D000818:Animals; D002417:Cattle; D003911:Dextrans / Q000737:chemistry / Q000494:pharmacology*; D016643:Encephalopathy, Bovine Spongiform / Q000378:metabolism*; D066298:In Vitro Techniques; D057230:Limit of Detection; D051379:Mice; D018620:PrPSc Proteins / Q000378:metabolism* / Q000472:pathogenicity; D013431:Sulfates / Q000737:chemistry*; D014774:Virulence
D003911:Dextrans; D018620:PrPSc Proteins; D013431:Sulfates