Topics in Animal Health Research. 8-9 (2009)

  A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 197.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion-inoculated mice showed no clinical signs of BSE for up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrP^Sc were pathologically observed in the brain of BSE/JP24 prion-affected TgBoPrP mice. The molecular weight and glycoform ratio of PrP^Sc in BSE/JP24 mice were different from those in C-BSE mice, and PrP^Sc in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties compared to those of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 ± 3.1 days). This result implies that the BSE/JP24 prion has newly emerged and shows that the L-type BSE prion might be classified into multiple strains.

Published papers and other works:

• Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle.
  Masujin K., Shu Y., Yamakawa Y., Hagiwara K., Sata T., Matsuura Y., Iwamaru Y., Imamura M., Okada H., Mohri S., Yokoyama T.
  Prion. 2, p.123-128 (2008).

Topics in Animal Health Research. 8-18 (2009)

  In the interspecies transmission of prions, the species barrier influences the susceptibility of the host. Bovine spongiform encephalopathy (BSE) prions affect a wide range of host species but do not affect hamsters. To study this species barrier, we analyzed the transmissibility of BSE prions to several lines of transgenic (Tg) mice, including those expressing mouse and hamster chimeric prion proteins (PrP^Cs) (MH2M and MHM2 mice). BSE prions were transmitted to ‘susceptible mice (MHM2, ICR)’, which harbored the mouse sequence at sub-region PrP131-188. However, BSE prions were not transmitted to ‘resistant mice (MH2M, TgHaNSE)’, which harbored the hamster sequence at that subregion. After the BSE prions were passaged once in wild-type mice, they could be transmitted to resistant mice. A BSE-like glycoform pattern of proteinase K-resistant prion protein (PrPcore) was detected in all of the susceptible mice. In contrast, in addition to PrPcore, further truncated PrP fragments existed in the resistant mice. These mixed PrP fragments might have resulted from the adaptation of resistant mice to BSE prions.

Published papers and other works:

• Both host prion protein 131-188 subregion and prion strain characteristics regulate glycoform of PrP^Sc.
  Yokoyama T., Shimada K., Masujin K., Iwamaru Y., Imamura M., Ushiki K.Y., Kimura K.M., Itohara S., Shinagawa M.
  Archives of Virology. 152, p.603-609 (2007).
• Alteration of the biological and biochemical characteristics of bovine spongiform encephalopathy prions during interspecies transmission in transgenic mice models.
  Yokoyama T., Masujin K., Iwamaru Y., Imamura M., Mohri S.
  Journal of General Virology. 90, p.261-268 (2009).

Topics in Animal Health Research. 8-19 (2009)

  Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP^C) into its pathogenic, amyloidogenic isoform (PrP^Sc) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin family that accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study we examined the effects of LF on PrP^Sc formation by using cell culture models. We demonstrated that LF can mediate the clearance of PrP^Sc and the reduction of prion infectivity in cells persistently infected with prion. The mechanism of action of LF potentially involves the cell-surface retention of PrP and/or binding to PrP. Furthermore, LF partially inhibited the formation of protease-resistant PrP, as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities. Elucidation of the antiprion mechanisms of LF may contribute not only to the establishment of a new class of therapeutic agent but also to comprehension of the prion replication mechanisms.

Published papers and other works:

• Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation.
  Iwamaru Y., Shimizu Y., Imamura M., Murayama Y., Endo R., Tagawa Y., Ushiki-Kaku Y., Takenouchi T., Kitani H., Mohri S., Yokoyama T., Okada H.
  Journal of Neurochemistry. 107, p.636-646 (2008).

Topics in Animal Health Research. 7-14 (2008)

  Prions, infectious agents causing transmissible spongiform encephalopathy (TSE), are composed primarily of the pathogenic form (PrP^Sc) of the host-encoded prion protein. Although very low levels of infectivity have been detected in urine from scrapie-infected rodents, no reports of urinary PrP^Sc have been substantiated. Studies on the dynamics of urinary PrP^Sc during infection are needed to ensure the safety of urine-derived biopharmaceuticals and to assess the possible horizontal transmission of prion diseases. Using the protein misfolding cyclic amplification technique, a time-course study of urinary excretion and blood levels of PrP^Sc was performed in Sc237-infected hamsters and a high rate of PrP^Sc excretion was found during the terminal stage of the disease. Following oral administration, PrP^Sc was present in all buffy coat samples examined; it was also present in most of the plasma samples obtained from hamsters in the symptomatic stage. PrP^Sc was excreted in urine for a few days after oral administration; subsequently, urinary PrP^Sc was not detected until the terminal disease stage. These results represent the first biochemical detection of PrP^Sc in urine from TSE-infected animals.

Published papers and other works:

• Urinary excretion and blood level of prions in scrapie-infected hamsters.
  Murayama Y., Yoshioka M., Okada H., Takata M., Yokoyama T., Mohri S.
  Journal of General Virology. 88, p.2890-2898 (2007).

Topics in Animal Health Research. 7-23 (2008)

  Chronic wasting disease (CWD) in cervids is a transmissible spongiform encephalopathy; however, its risk to humans has been obscure. The increasing number of diseased deer in North America has raised concerns regarding the risk CWD poses to humans. In this study, we demonstrated the applicability of the available commercial diagnostic kits and confirmatory procedures for bovine spongiform encephalopathy (BSE) in Japan for the diagnosis of CWD. No CWD was confirmed in the surveillance of 558 Japanese deer that were examined between 2003 and 2006. We concluded that the present BSE diagnostic procedure in Japan will be a powerful tool for CWD control.

Published papers and other works:

• Applicability of current bovine spongiform encephalopathy (BSE) diagnostic procedures for chronic wasting disease (CWD).
  Masujin K., Shimada K., Kimura K.M., Imamura M., Yoshida A., Iwamaru Y., Mohri S., Yokoyama T.
  Microbiology and Immunology. 51, p.1039-1043 (2007).

Topics in Animal Health Research. No.6-8 (2007)

  Recently, an abnormal isoform of prion protein (PrP^Sc) was detected in the peripheral nervous system (PNS) of bovine spongiform encephalopathy (BSE)-affected cattle. The data showed that the infectivity was not limited to the central nervous system (CNS) of BSE-affected cattle. It has been reported that prions spread to the CNS via the PNS in sheep scrapie, but the pathogenesis of BSE in cattle is less well understood. To determine whether parts of the PNS become positive before or after the CNS is affected, we investigated PrP^Sc distribution by a highly sensitive western blotting (WB) in the PNS, adrenal gland and CNS of cattle that were orally inoculated with BSE-affected brain and sequentially culled. In experimental BSE-affected cattle, PrP^Sc was first detected in the CNS and dorsal root ganglia, and subsequently PrP^Sc accumulation was detected in the peripheral nerve trunks. PrP^Sc was also detected in the adrenal glands of cattle that showed clinical signs. This study shows that PrP^Sc is detected in the PNS during the disease course at the same time as, or after, it accumulates in the CNS.

Published papers and other works:

• Prions in the peripheral nerves of bovine spongiform encephalopathy-affected cattle.
  Masujin K., Matthews D., Wells G.A.H., Mohri S., Yokoyama T.
  Journal of General Virology. 88, p.1850-1858 (2007).

Topics in Animal Health Research. No.6-13 (2007)

  Abnormal isoforms of prion proteins (PrP^Sc), which are infectious agents associated with prion diseases, retain infectivity after undergoing routine sterilization processes. A sensitive method for detecting the infectivity is a bioassay, and it has been used for assessing prion inactivation. However, the result is obtained after several hundred days. Here, protein misfolding cyclic amplification (PMCA), in which PrP^Sc can be amplified in vitro, was applied to assess prion inactivation by dry heating and autoclaving. Scrapie-infected hamster brains were inactivated under various conditions, and residual infectivity and PrP^Sc were detected by bioassay and PMCA, respectively. The PMCA results were in good agreement with those of the bioassay. In samples autoclaved at temperatures below 150°C, while infected mice died in the bioassay, protease-resistant PrP (PrP^res) signals were detected in the second or third round of PMCA. Three rounds of PMCA require only 6 days, which means that the PMCA method is much faster than the bioassay.

Published papers and other works:

• Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation.
  Murayama Y., Yoshioka M., Horii H., Takata M., Yokoyama T., Sudo T., Sato K., Shinagawa M., Mohri S.
  Biochemical and Biophysical Research Communications. 348, p.758-762 (2006).

Topics in Animal Health Research. No.6-27 (2007)

  In prion diseases, microglia have been implicated in the pathogenic events involving the loss of neurons. To elucidate the roles of microglia in the pathogenesis of prion diseases, physiological and biochemical characteristics of prion-infected microglial cells should be clarified. Here, we establish a novel microglial cell line (MG20 cell) from the brains of neonatal tga 20 mice that overexpressed murine prion protein (PrP) and show that this cell line can be infected with murine prion strains. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of PrP in MG20 cell[s] up to the 15th passage and showed that Chandler scrapie maintained biological properties in this cell culture. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie and bovine spongiform encephalopathy agents. Thus, MG20 cell lines infected with various murine prion strains can be a useful model for the study of [the] pathogenesis of prion diseases and prion strain determinants.

Published papers and other works:

• Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains.
  Iwamaru Y., Takenouchi T., Ogihara K., Hoshino M., Takata M., Imamura M., Tagawa Y., Hayashi-Kato H., Ushiki-Kaku Y., Shimizu Y., Okada H., Shinagawa M., Kitani H., Yokoyama T.
  Journal of Virology. 81, p.1524-1527 (2007).

Topics in Animal Health Research. No.5-4 (2006)

  Rapid western blot (WB) procedure to detect an abnormal isoform of prion protein (PrP^Sc) in lymphoid tissues has been established and applied to the surveillance of fallen stock. Using this method, we examined brain and palatal tonsil tissues with WB and detected three sheep scrapie. While one clinically scrapie-infected sheep harbored PrP^Sc in the brain and palatal tonsil, two sheep in the pre-clinical stage harbored PrP^Sc in the brain but not in the palatal tonsil. This study shows that PrP^Sc accumulation in the palatal tonsil is variable in natural scrapie, even among genetically-susceptible sheep.

Published papers and other works:

• Rapid PrP^Sc detection in lymphoid tissue and application to scrapie surveillance of fallen stock in Japan: variable PrP^Sc accumulation in palatal tonsil in natural scrapie.
  Shimada K., Hayashi H.K., Ookubo Y., Iwamaru Y., Imamura M., Takata M., Schmerr M.J., Shinagawa M., Yokoyama T.
  Microbiology and Immunology. 49(8), p.801-804 (2005).

Topics in Animal Health Research. No.5-7 (2006)

  We developed an effective method for detecting prion (PrP) antigenic determinants remaining in bovine meat and bone meal (MBM) using pressurized fluid extraction (PSE) equipment and the flow microbead immunoassay (FMI). With the FMI, bovine recombinant PrP could be determined quantitatively in the 7 pmol-7 nmol range using anti-PrP peptide polyclonal antibody-coupled microbeads and anti-PrP monoclonal antibody (SAF61) as detection antibody. PSE extraction at 120°C for 5 min under high pressure was the most effective way to elute PrP determinants from bovine MBMs. The FMI was capable of detecting PrP determinants in bovine MBM extracts with high specificity and indicated that the MBMs contained high levels of PrP determinants. This assay was also applied to the detection of PrP^Sc determinants in bovine MBM spiked with scrapie-infected brain at a weight ratio of 50:1. These data indicate that this assay was effective for the specific detection of PrP determinants contained in bovine MBM extracts.

Published papers and other works:

• Specific detection of prion antigenic determinants retained in bovine meat and bone meal by flow microbeads immunoassay.
  Murayama Y., Yoshioka M., Horii H., Takata M., Miura K., Shinagawa M.
  Journal of Applied Microbiology. 101(2), p369-376 (2006).

Topics in Animal Health Research. No.5-22 (2006)

  Western blot detection of the abnormal isoform of the prion protein (PrP^Sc) was used to assess prion inactivation by heating under alkaline conditions during the manufacturing process that is used to produce horticultural fertilizer from meat meal. PrP^Sc was detected in the sample prepared from the horticultural fertilizer spiked with a 0.25 μg equivalent of scrapie-infected mouse brain. By contrast, PrP^Sc was not detected in a sample containing a 0.25 g brain equivalent prepared from a small-scale processed mixture of scrapie-infected mouse brain and the meat meal made by the same method as that used to produce the horticultural fertilizer. Our results indicated that the amount of PrP^Sc decreased to at least 1/10⁶ through processing with heating under alkaline conditions. Although the bioassay suggests that prion infectivity was reduced under these conditions, this procedure did not completely remove high-titer prion infectivity.

Published papers and other works:

• Western blot assessment of prion inactivation by alkali treatment in the process of horticultural fertilizer production from meat meal.
  Yokoyama T., Shimada K., Tagawa Y., Ushiki Y.K., Iwamaru Y., Hayashi H.K., Shinagawa M.
  Soil Science and Plant Nutrition. 52(1), p.71-76 (2006).

Topics in Animal Health Research. No.4-22 (2005)

  Heterogeneity in transmissible spongiform encephalopathy is thought to have derived from conformational variations in an abnormal isoform of the prion protein (PrP^Sc). To characterize PrP^Sc in bovine spongiform encephalopathy (BSE) and scrapie, we analyzed the newly generated N-terminus of PrP^Sc isoforms by digestion with proteinase K (PK). With a lower concentration of PK, the terminal amino acid of BSE PrP^Sc converged at N₉₆. Under the same conditions, however, the terminal amino acid of scrapie PrP^Sc was G₈₁ or G₈₅. Furthermore, with an increase of PK concentration, the N-terminal amino acid was shifted and converged at G₈₉. The results suggest that the PK cleavage site of BSE PrP^Sc is uniform and is different from the cleavage site of scrapie PrP^Sc.

Published papers and other works:

• The N-terminal cleavage site of PrP^Sc from BSE differs from that of PrP^Sc from scrapie.
  Hayashi H.K., Takata M., Iwamaru Y., Imamura M., Ushiki Y.K., Shinagawa M., Yokoyama T.
  Biochemical and Biophysical Research Communications. 328, p.1024-1027 (2005).

• Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein
• Two different scrapie prions isolated in Japanese sheep flocks
• A novel proteolytic enzyme that digests PrP^Sc
• Neuropathological and immunohistochemical evaluation of the first case of bovine spongiform encephalopathy