T. Yokoyama:Prion Disease Research Center, National Institute of Animal Health
In 2001 September, the first bovine spongiform encephalopathy (BSE) case in Japan was confirmed. For the first Prionics WESTERN (PW) test which performed at August 15, this case could not confirm as BSE. From two aspects, the test was performed with a different protocol form manufacture's one; 1) obex region was not subjected to the test and 2) semi-dry blotting apparatus was used. Here, I reported the results of PW retest for the first BSE case, and related samples.
All obex regions were subjected to pathological examination and the remainder was kept as frozen sample. Brain homogenate was generated newly from this remaining frozen tissue. Thus, obex was also unavailable in this experiment. The caudal portion from obex might be subjected to the test (the accuracy portion of used samples could not be identified). I also used the four BSE positive samples from UK, as positive control. Samples were prepared by both PW kit and conventional method for western blot (WB), and tank-blotting apparatus were used. Though, the signal of abnormal isoform of prion protein (PrPSc) is slightly weaker than lane 3, and 4 (UK samples), the Japanese BSE was positive in both tests (Fig.1, lane 2). One UK BSE sample (lane 1) showed weaker signal than others.
Next, I examined the PrPSc distribution in BSE cattle with PW kit. Four BSE samples from UK were sectioned and detection of PrPSc was carried out. As shown in Fig.2, PrPSc does not accumulated evenly in BSE cattle, even at the brain stem. Especially in caudal region from obex harbor lesser PrPSc than cranial region. This result showed the importance of collecting tissue portion for homogenization.
From these experiences, surveillance procedure has revised as; 1) obex region is longitudinally divided into two and subjected one for biochemical and the other for pathological tests. 2) Tank blotting system is applied for WB of BSE test. 3) Diluted mice positive PrPSc has examined simultaneously to give guarantee for sensitivity of the test.
Acknowledgments: I thank to Dr. Danny Matthews at veterinary laboratory agency, Weybridge for providing BSE materials, and also thank to Dr. Hiroko HAYASHI for her technical help, and Dr. Morikazu SHINAGAWA for his advisement.
