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Hasegawa K.,Advancesoft Corporation | Mohri S.,Japanese National Institute of Animal Health | Yokoyama T.,Japanese National Institute of Animal Health
Prion | Year: 2010

The e200K mutation of the human prion protein (PrP) is known to cause familial creutzfeldt-Jakob disease. In order to elucidate the effects of the mutation on the local structural stability of PrP, we performed ab initio fragment molecular orbital calculations for the wild-type human PrP and the e200K variant modeled under neutral and mild acidic conditions. The calculations revealed that this substitution markedly altered the intramolecular interactions in the PrP, suggesting that the local structural instabilities induced by the e200K mutation might cause initial denaturation of the PrP and its subsequent conversion to a pathogenic form. This work presents a new approach for quantitatively elucidating structural instabilities in proteins that cause misfolding diseases.

Suto J.-I.,Japan National Institute of Agrobiological Science | Satou K.,Japanese National Institute of Animal Health
BMC Research Notes | Year: 2014

Background: Plasma high-density lipoprotein (HDL)-cholesterol level is a clinically important quantitative phenotype that widely varies among inbred mouse strains. Several genes or loci associated with plasma HDL-cholesterol levels have been identified on autosomes and the X chromosome. In contrast, genes or loci on the Y chromosome have not attracted significant attention hitherto. Therefore, we investigated the effects of the Y chromosome on plasma HDL-cholesterol levels in Y- chromosome-consomic (Y-consomic) mouse strains. Findings. Plasma HDL-cholesterol level data from 16 Y-consomic strains demonstrated that the Y chromosome substitutions significantly altered plasma HDL-cholesterol levels, i.e., variations in the plasma HDL-cholesterol level could be partially explained by Y chromosome genes. We obtained the following results from the genotype data on 30 single nucleotide polymorphisms (SNPs), including nonsynonymous and synonymous SNPs and 9 polymorphisms in Sry: (1) Variation in rs46947134 of Uty was significantly associated with plasma HDL-cholesterol levels. (2) A CAG repeat number polymorphism in Sry was significantly associated with plasma HDL-cholesterol levels. (3) Strains with a certain haplotype of the Mus musculus domesticus-type Y chromosome had significantly lower plasma HDL-cholesterol levels than strains with a certain haplotype of the M. m. musculus-type Y chromosome. Conclusions: The effect of the Y chromosome on plasma HDL-cholesterol levels was confirmed in the Y-consomic strains. We identified several variants associated with plasma HDL-cholesterol levels. Because the physiological significance of various Y-linked genes remains unclear, the results of this study will provide further insights into the functions of Y-linked genes in lipid metabolism. © 2014 Suto and Satou; licensee BioMed Central Ltd.

Akiba M.,Japanese National Institute of Animal Health | Kusumoto M.,Japanese National Institute of Animal Health | Iwata T.,Japanese National Institute of Animal Health
Journal of Microbiological Methods | Year: 2011

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non- Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods. © 2011 Elsevier B.V.

Yoshioka K.,Japanese National Institute of Animal Health | Noguchi M.,Japanese National Institute of Animal Health | Suzuki C.,Japanese National Institute of Animal Health
Animal Reproduction Science | Year: 2012

The objective of this study was to enhance procedures for producing piglets derived from in vitro-produced (IVP) pig embryos by non-surgical embryo transfer (ET). The effects of insertion length for the catheter, asynchrony between the age of donor IVP blastocysts and the recipient estrous cycle, and volume of transfer medium were investigated. The IVP blastocysts at 5 days after in vitro fertilization were placed into porcine zygote medium (PZM)-5 supplemented with 10% (v/v) fetal bovine serum (PZM+FBS) in a 0.25mL plastic straw (21-40 blastocysts per straw) and then transferred into one uterine horn of recipients using the Takumi ® catheter for deep intrauterine insertion. Successful production of piglets derived from IVP embryos was achieved following non-surgical ET when the catheter was inserted at more than 30cm anterior to the spiral guide spirette. The efficiency of piglet production (percentage number of piglet(s) born based on the number of embryos transferred) was greater (P<0.05) in recipients whose estrous cycle was asynchronous to that of donors with a 1-day delay (8.3%) than in those with a 2-day (1.5%) or 3-day (0.9%) delay, while pregnancy and farrowing rates (10-40%) did not differ among treatments. When blastocysts were transferred into recipients with 1.0 or 2.5mL PZM+FBS, there were no significant differences in farrowing rate (30-40%) or average litter size (4.5-6.7) between treatments. The results of the present study indicate that the insertion length of the deep intrauterine catheter and the degree of asynchrony between donor embryos and recipient estrous cycle influenced on pregnancy and birth outcome following non-surgical transfer of IVP blastocysts. © 2012 Elsevier B.V..

Tagawa Y.,Japanese National Institute of Animal Health
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2014

Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length. © 2014, Springer International Publishing Switzerland.

Ito T.,Japanese National Institute of Animal Health | Maeno Y.,Japan National Research Institute of Fisheries And Environment of Inland Sea
Diseases of Aquatic Organisms | Year: 2014

In this study, we examined the influence of water temperature on the development of herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus after experimentally induced infection with cyprinid herpesvirus 2 (CyHV-2). In Expt 1, Ryukin goldfish were infected with CyHV-2 by intraperitoneal injection and maintained at 4 different water temperatures. Cumulative mortalities of the 15, 20, 25 and 30°C groups were 10, 90, 90 and 60%, respectively. Therefore, the temperature range of 20-25°C is considered highly permissive for HVHN. One of 6 surviving fish of the 15°C group died after a rapid temperature increase to 25°C at 30 d post infection. All 3 Edonishiki goldfish, co-reared with the surviving Ryukin in tanks where the water temperature was increased from 15 to 25°C, died. In Expt 2, Edonishiki goldfish were exposed to CyHV-2 by bath immersion at 13 or 24°C, resulting in cumulative mortalities of 0 and 87%, respectively, at 28 d post-exposure. No mortality of the surviving Edonishiki in the 13°C treatment was observed when the water temperature was increased to 24°C. In addition, in Expt 2, no mortality was observed in any Ranchu co-reared with CyHV-2-immersed Edonishiki in the group where water temperature was increased from 13 to 24°C, even after re-immersion challenge with CyHV-2. It is interesting to note that CyHV-2 DNA was detected in the kidneys of 4 of the 5 surviving Ranchu co-reared with the CyHV-2-immersed Edonishiki group where the water temperature was increased from 13 to 24°C. Therefore, it is likely that the surviving Edonishiki of the 13°C group were virus carriers. This study indicates that most fish infected with CyHV-2 at 13-15°C acquire resistance to HVHN, but as carriers they are able to infect naïve fish. © Inter-Research 2014 · www.int-res.com.

Yoshioka K.,Japanese National Institute of Animal Health
Journal of Reproduction and Development | Year: 2011

Recent advances in systems for in vitro production (IVP) of porcine embryos, including in vitro oocyte maturation, fertilization and embryo culture, have enabled us to generate viable embryos that can develop to full term after transfer into recipients. This technology is being applied now to developments in gamete/embryo biology and agriculture, as well as in producing cloned and genetically modified pigs. Chemically defined media for IVP of embryos are useful for a precise analysis of the physical action of substances on gametogenesis and early embryogenesis, because they eliminate undefined factors present in biological materials, such as serum or serum albumin. Use of a chemically defined medium also improves the reliability of media formulations, yields a higher reproducibility of results and ensures biosafety of culture media by eliminating protein preparations, which may be contaminated with pathogens. Therefore, it has certain advantages for research and for commercial purposes. We have recently developed a defined IVP system for porcine embryos using a single basic medium based on the composition of porcine oviductal fluid. This paper discusses the developmental ability and normality of porcine IVP embryos, and limitations and advancements in this system. © 2011 by the Society for Reproduction and Development.

Suzuki T.,Japanese National Institute of Animal Health | Hasebe A.,Gifu Prefectural Central Livestock Health and Sanitation Office | Miyazaki A.,Japanese National Institute of Animal Health | Tsunemitsu H.,Japanese National Institute of Animal Health
Virus Research | Year: 2015

Porcine rotavirus C (RVC) has been often detected in sporadic cases or outbreaks of diarrhoea in suckling and weaned pigs. Surveillance studies of RVCs have demonstrated high prevalence in the United States, and Japan, and some other countries. To date, the zoonotic impact and pathogenicity of RVCs are not well understood, and only a few complete sequences of RVCs are available. The aim of this study was to perform sequence and phylogenetic analyses for the VP4 and VP7 genes of the 22 porcine RVCs identified in Japan from 2002 to 2010. The genetic classification of the VP4 genes of the 22 porcine RVCs revealed the presence of six clusters including one cluster each from human and bovine RVCs with a cut-off value of 80%. In addition, VP7 genes of the 22 porcine RVCs were grouped into four of the seven known clusters on the basis of cut-off values of 85% at the nucleotide level reported previously. The data presented here demonstrate that multiple porcine RVC strains with distinctive genotypes based on a combination of the VP4 and VP7 genes are widely distributed and circulated among farms throughout Japan. According to establishment of dual genetic classification for VP4 and VP7 genotypes of porcine RVCs, furthermore, we discovered a possible event of gene reassortment between different rotavirus strains from the same farm. Our findings should advance the understanding of the evolution and pathogenicity of RVCs. © 2014 Elsevier B.V.

BACKGROUND: Salmonella enterica serovar Enteritidis is a zoonotic pathogen. Human infections are associated with contaminated eggs and egg products. In Japan, since 1989, the incidence of food-borne disease caused by S. Enteritidis has increased and a pandemic has occurred; however, little is known about changes that occurred before and after this pandemic event in the dominant lineage of isolates from food-producing animals. This study aimed to determine the S. Enteritidis lineages in Japan over the last few decades by using multilocus variable-number tandem repeat analysis (MLVA).FINDINGS: MLVA was used to analyse 79 S. Enteritidis isolates collected from chickens (n = 63), cattle (n = 12), pigs (n = 2), and goats (n = 2) during 1975-2009. The S. Enteritidis isolates showed 14 different MLVA allele combinations, which were classified into two major clusters (A and C) and a minor cluster (B). All the 62 isolates in cluster A were isolated after 1988, whereas 13 of the 17 isolates belonging to cluster B and C were isolated before 1989.CONCLUSIONS: The MLVA results showed that cluster C was predominant before 1989, and isolates in cluster A disseminated since 1989 and replaced the previous dominant clone, suggesting that isolates of cluster A originated from imported S. Enteritidis infection.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

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