Yuichi Tagawa

Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains.

ABSTRACT Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.

Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrPC) into its pathogenic, amyloidogenic isoform (PrPSc) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron‐binding glycoprotein belonging to the transferrin (TF) family, which 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 have examined the effects of LF on PrPSc formation by using cell culture models. Bovine LF inhibited PrPSc accumulation in scrapie‐infected cells in a time‐ and dose‐dependent manner, whereas TF was not inhibitory. Bioassays of LF‐treated cells demonstrated prolonged incubation periods compared with non‐treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrPC by diminishing its internalization and was capable of interacting with PrPC in addition to PrPSc. 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.

Genetics and infectivity of H5N1 highly pathogenic avian influenza viruses isolated from chickens and wild birds in Japan during 2010-11.

Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds.

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

Western blot assessment of prion inactivation by alkali treatment in the process of horticultural fertilizer production from meat meal

Abstract Western blot detection of the abnormal isoform of the prion protein (PrPSc) was used to assess prion inactivation by heating under alkaline conditions during the manufacturing process used to produce horticultural fertilizer from meat meal. PrPSc was detected in the sample prepared from the horticultural fertilizer spiked with a 0.25 µg equivalent of scrapie-infected mouse brain. In contrast, PrPSc was not detected in a sample containing 0.25 g brain equivalent prepared from a small-scale processed mixture of scrapie-infected mouse brain and meat meal using the same method as that used to produce horticultural fertilizer. Our results indicated that the amount of PrPSc decreased to at least 1/106 by 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.

A Predominant Clonal Thromboembolic Meningoencephalitis Group of Histophilus somni Assigned by Major Outer Membrane Protein Gene Sequencing and Pulsed-Field Gel Electrophoresis

Histophilus somni, a member of the family Pasteurellaceae, causes a variety of diseases, including thromboembolic meningoencephalitis (TEME) and respiratory diseases, which result in considerable economic losses to the cattle and sheep industries. In this study, 132 chronologically diverse isolates from cattle in Japan and 68 isolates from other countries comprising 49 from cattle and 19 from sheep were characterized using major outer membrane protein (MOMP) gene sequence and pulsed-field gel electrophoresis (PFGE) analyses. The H. somni isolates formed nine MOMP genetic clades (clade Ia, Ib, and II–VIII) and 10 PFGE clusters (HS1–HS10). Except for two (1.0%), all isolates fell into one of the nine MOMP genetic clades, while 62 (31.0%) isolates belonged to no PFGE cluster. MOMP genetic clade Ia and PFGE cluster HS1 were the major groups, and all HS1 isolates possessed the clade Ia MOMP gene. Isolates from TEME cases were significantly associated with these major groups (chi-square test, p < 0.0001), as 88.2% of the TEME isolates belonged to MOMP genetic clade Ia and PFGE cluster HS1, which formed the most predominant clonal group. After an inactivated vaccine using an HS1 strain with the clade Ia MOMP gene was introduced in Japan in late 1989, the number of TEME cases and isolates assigned into the clonal group decreased simultaneously. However, the proportions of clade Ia and cluster HS1 isolates from TEME cases remained high after 1990. These results suggest a close association of TEME with PFGE cluster HS1 and MOMP genetic clade Ia, and imply the presence of factors or characteristics commonly possessed by those strains that contribute to the development of TEME.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

The inhibitory effect of the ScFv of an anti-prion protein antibody secreted from N2a58 cells on abnormal prion protein accumulation in scrapie-infected cells, ScN2a

The central event in molecular prion pathogenesis is the conformational change of the cellular prion protein (PrPC) to an abnormal prion protein (PrPSc), and the subsequent accumulation of PrPSc in infected human and animals. Recently, reports have shown that the exposure of scrapie-infected cells (ScN2a) to an anti-prion protein (PrP) antibody inhibited this conformational change and accumulation of PrPSc. This immunological approach has some problems for in vivo applications because of the difficulty of the blood brain barrier and the resulting lack of accessibility of the antibody to brain tissue. We developed an alternative intervention system using gene expression. In this study, we demonstrated that PrPSc accumulation in ScN2a cells could be prevented by this expression system in vitro. We cloned a cDNA of the single chain antibody variable region fragment (ScFv) of an anti-PrP monoclonal antibody T2, and transfected it into a mouse neuroblastoma cell, N2a58 (N2a58-T2ScFv). The T2-ScFv was expressed stably in the N2a58-T2ScFv cells and gave no effects for the PrPC expression. The cells were co-cultivated with ScN2a cells for 4days or 7days, and the quantity of PrPSc was assayed by western blotting and enzyme-linked immunosorbent assay (ELISA). PrPSc in ScN2a cells reduced significantly with co-cultivation. This suggests that insertion of a PrP-specific anti-body gene into a neuronal cell will be a potential therapeutic tool for prion diseases.

A Novel BSE screening kit with simplified preparation method for EIA

Introduction In Japan, all slaughtered and deceased bovine materials are tested for BSE infection. The primary screening test is undertaken by the meat inspection office or live stock hygiene service center in each prefecture. In these circumstances, BSE kits adapted for relatively small number of samples are required. Here we describe the development of a neŵ BSE screening system, which has simpler and safer protocol for sample preparation steps for EIA. This assay system has been named the NippIBL BSE Assay system.

Surveillance of chronic wasting disease (CWD) in Japan

Chronic wasting disease (CWD) in cervids including elk, mule deer, and white-tailed deer, is a member of the transmissible spongiform encephalopathies (TSEs). CWD is a serious problem in North America. The detection of abnormal isoforms of prion protein (PrPSc) is a key factor for the diagnosis of CWD, similar to other TSEs. The surveillance program for TSEs in animals is conducted by the Ministry of Agriculture, Forestry, and Fishery (MAFF) and is targeted to sheep, goats, and deer. In Japan, several different anti-prion protein (PrP) monoclonal antibodies (mAbs) are utilized for bovine spongiform encephalopathy (BSE) confirmation. Since CWD does not occur naturally in Japan, the immunoreactivity of the antibodies against PrPSc found in deer was not known. In this study, we examined the immunoreactivities of these antibodies against PrPSc found in CWD. The protocols that are used in Japan for confirmation of BSE cases are Western blot (WB) and immunohistochemistry (IHC). We used these same protocols to examine CWD positive brain samples which were provided by Dr. A. Davis of the National Veterinary Service Laboratory, USA. Mabs. T1, T2, 44B1, and 72-5, were used successfully to detect PrPSc in CWD affected mule deer brains by WB. In IHC, PrPSc was detected with mAbs T2, 44B1, and polyclonal antibody B103. These results determined that the antibodies used for BSE confirmation are also applicable to CWD, as for scrapie. These same antibodies could detect PrPC from Japanese deer by WB without proteinase digestion. The amino acid sequence of PrP of Japanese deer was found to be the same as sequence as the one reported for mule deer. These antibodies were then used for CWD surveillance in Japan. When 127 of hunter-killed deer from Hokkaido were examined, PrPSc was not detected in any of the animals.