Analysis of the Mitochondrial Genomes of Japanese Wolf Specimens in the Siebold Collection, Leiden.

The taxonomic status of extinct Japanese or Honshu wolves (Canis lupus hodophilax) has been disputed since the name hodophilax was first proposed by Temminck in 1839 on the basis of specimens stored in Leiden, the Netherlands. Points of controversy include whether the type specimen of hodophilax (Jentink c: RMNH.MAM.39181) and the other two specimens from Leiden (Jentink a: RMNH.MAM.39182 and Jentink b: RMNH.MAM.39183) represent different varieties or subspecies of Japanese wolves or not. Two Japanese names, ookami and jamainu, used to describe wild Canis species, further complicate the issue. In this study, the taxonomic status of Japanese wolves was clarified using mitochondrial DNA of the three specimens stored at the Naturalis Biodiversity Center in Leiden, in addition to three Japanese wolf specimens stored at the Museum für Naturkunde in Berlin and five new samples from Japan. The mitochondrial genomes of the type specimen of hodophilax (Jentink c) and another sample from Leiden (Jentink b) as well as Berlin specimens were included in the cluster of Japanese wolves distinct from other grey wolves. However, the other sample from Leiden (Jentink a) was identified as a domestic dog. A mitochondrial genome analysis suggested that Japanese wolves could be categorized into two distinct clusters. Studies of nuclear genomes are needed to further clarify the taxonomic status, divergence time, and population genetic structure of Japanese wolves.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments.

Longitudinal study of experimental induction of AA amyloidosis in mice seeded with homologous and heterologous AA fibrils.

OBJECTIVE: To investigate pathogenesis and kinetics of experimentally induced murine AA amyloidosis seeded with homologous (murine) and heterologous (bovine) AA fibrils. METHODS: Experimental AA amyloidosis was induced by administration of inflammatory stimulus and preformed AA fibrils to a total of 111 female C57/Black mice. In this longitudinal study, heterologous (bovine) as well as homologous (murine) AA fibrils were injected intraperitoneally to mice in various combinations. Re-stimulation was done at 120 or 300 days post first inoculation. To analyze the intensity of amyloid depositions in mice organs, immunohistochemical techniques and image J software were used. Assessment of cytokines level in sera was done using a Mouse Th1/Th2/Th17 Cytokine CBA Kit. RESULTS: Incidence and severity of AA amyloidosis were quite low in mice inoculated with heterologous bovine AA fibrils than homologous murine one. Homologous AA fibrils administration at first and second inoculation caused maximum amount of amyloid depositions and severe systemic form of amyloidosis. Increase in the level of pro-inflammatory cytokine IL-6 was observed after first inoculation, while second inoculation caused a further increase in the level of anti-inflammatory cytokine IL-10. CONCLUSIONS: AA amyloidosis can be induced by heterologous as well as homologous AA fibrils. Severity of AA amyloidosis induced with homologous AA fibrils is higher compared to heterologous AA fibrils.

Japanese Wolves are Genetically Divided into Two Groups Based on an 8-Nucleotide Insertion/Deletion within the mtDNA Control Region.

The mitochondrial DNA (mtDNA) control region (198- to 598-bp) of four ancient Canis specimens (two Canis mandibles, a cranium, and a first phalanx) was examined, and each specimen was genetically identified as Japanese wolf. Two unique nucleotide substitutions, the 78-C insertion and the 482-G deletion, both of which are specific for Japanese wolf, were observed in each sample. Based on the mtDNA sequences analyzed, these four specimens and 10 additional Japanese wolf samples could be classified into two groups- Group A (10 samples) and Group B (4 samples)-which contain or lack an 8-bp insertion/deletion (indel), respectively. Interestingly, three dogs (Akita-b, Kishu 25, and S-husky 102) that each contained Japanese wolf-specific features were also classified into Group A or B based on the 8-bp indel. To determine the origin or ancestor of the Japanese wolf, mtDNA control regions of ancient continental Canis specimens were examined; 84 specimens were from Russia, and 29 were from China. However, none of these 113 specimens contained Japanese wolf-specific sequences. Moreover, none of 426 Japanese modern hunting dogs examined contained these Japanese wolf-specific mtDNA sequences. The mtDNA control region sequences of Groups A and B appeared to be unique to grey wolf and dog populations.

Role of cell death in the propagation of PrP(Sc) in immune cells.

A number of studies have suggested that macrophages, dendritic cells, and follicular dendritic cells play an important role in the propagation of PrP(Sc). Both accumulation and proteolysis of PrP(Sc) have been demonstrated in peripheral macrophages. Macrophages may act as reservoirs for PrP(Sc) particles if the cells die during transient PrP(Sc) propagation. However, whether cell death plays a role in PrP(Sc) propagation in macrophages remains unclear. In this study, we investigated the possibility of propagation and transmission of PrP(Sc) between dead immune cells and living neural cells. We found that under specific conditions, transient PrP(Sc) propagation occurs in dead cells, indicating that interaction between PrP(C) and PrP(Sc) on plasma membrane lipid rafts might be important for PrP(Sc) propagation. Co-culturing of killed donor PrP(Sc)-infected macrophages with recipient N2a-3 neuroblastoma cells accelerated PrP(Sc) transmission. Our results suggest that cell death may play an important role in PrP(Sc) propagation, whereas transient PrP(Sc) propagation in macrophages has little effect on PrP(Sc) transmission.

[Evaluating the Stability of Loop-Mediated Isothermal Amplification Reagents at Irregular Storage Temperatures for On-Site Diagnosis].

Temperature-stability of loop-mediated isothermal amplification (LAMP) reagents was determined for their use in on-site diagnosis, such as in farms/pastures. Bst and Csa DNA polymerases and the reagents that were stored at different temperatures (4 or 25°C) for 1, 2, or 4 days were used for the LAMP assay to detect orf virus DNA as a model. After storage at 4 and 25°C for 2 days, the enzymes and reagents were found to retain sufficient activity to carry out successful DNA amplification. Visual diagnosis was also possible with the reagents (Loopamp Fluorescent Detection Reagent or hydroxy naphthol blue, as well as DNA amplification checker, D-Quick) that were stored for 2 days at different temperatures. Although the time taken to obtain the positive/negative results were delayed, the enzymes and reagents, stored at 25°C for 4 days, were active and had the ability to efficiently amplify DNA in less than 50 min. These results indicate that LAMP assay can be successfully utilized for the diagnosis of infectious diseases under non-clinical settings such as for on-site diagnosis in farms/pastures, owing to the fact that the relevant enzymes and reagents does not require restricted temperature storage.

Reconstructing the colonization history of lost wolf lineages by the analysis of the mitochondrial genome.

The grey wolves (Canis lupus) originally inhabited major parts of the Northern hemisphere, but many local populations became extinct. Two lineages of wolves in Japan, namely, Japanese or Honshu (C. l. hodophilax) and Ezo or Hokkaido (C. l. hattai) wolves, rapidly went extinct between 100 and 120years ago. Here we analyse the complete mitochondrial genome sequences from ancient specimens and reconstruct the colonization history of the two extinct subspecies. We show a unique status of Japanese wolves in wolf phylogeny, suggesting their long time separation from other grey wolf populations. Japanese wolves appeared to have colonized the Japanese archipelago in the Late Pleistocene (ca. 25,000-125,000years ago). By contrast, Ezo wolves, which are clearly separated from Japanese wolves in phylogeny, are likely to have arrived at Japan relatively recently (<14,000years ago). Interestingly, their colonization history to Japan tallies well with the dynamics of wolf populations in Europe and America during the last several millennia. Our analyses suggest that at least several thousands of wolves once inhabited in the Japanese archipelago. Our analyses also show that an enigmatic clade of domestic dogs is likely to have originated from rare admixture events between male dogs and female Japanese wolves.

Genetic structure of wild boar (Sus scrofa) populations from East Asia based on microsatellite loci analyses.

BACKGROUND: Wild boar, Sus scrofa, is an extant wild ancestor of the domestic pig as an agro-economically important mammal. Wild boar has a worldwide distribution with its geographic origin in Southeast Asia, but genetic diversity and genetic structure of wild boar in East Asia are poorly understood. To characterize the pattern and amount of genetic variation and population structure of wild boar in East Asia, we genotyped and analyzed microsatellite loci for a total of 238 wild boar specimens from ten locations across six countries in East and Southeast Asia. RESULTS: Our data indicated that wild boar populations in East Asia are genetically diverse and structured, showing a significant correlation of genetic distance with geographic distance and implying a low level of gene flow at a regional scale. Bayesian-based clustering analysis was indicative of seven inferred genetic clusters in which wild boars in East Asia are geographically structured. The level of genetic diversity was relatively high in wild boars from Southeast Asia, compared with those from Northeast Asia. This gradient pattern of genetic diversity is consistent with an assumed ancestral population of wild boar in Southeast Asia. Genetic evidences from a relationship tree and structure analysis suggest that wild boar in Jeju Island, South Korea have a distinct genetic background from those in mainland Korea. CONCLUSIONS: Our results reveal a diverse pattern of genetic diversity and the existence of genetic differentiation among wild boar populations inhabiting East Asia. This study highlights the potential contribution of genetic variation of wild boar to the high genetic diversity of local domestic pigs during domestication in East Asia.

Failure of heterogeneous amyloid-enhancing factor in geriatric squirrel monkeys (Saimiri boliviensis).

BACKGROUND: Cross-species transmission of AA amyloidosis between primates and other animals has not been previously reported. METHODS: Eight geriatric squirrel monkeys were intravenously administered chimpanzee, bovine, or chicken amyloid fibrils and simultaneously received inflammatory stimulation. RESULTS: AA amyloid deposition was not detected in any of the monkeys histopathologically or immunohistochemically. CONCLUSIONS: These results suggest that heterogeneous AA amyloidosis may not be easily transmitted into primates.

Japanese wolves are most closely related to dogs and share DNA with East Eurasian dogs.

Although the domestic dog's origin is still unclear, this lineage is believed to have been domesticated from an extinct population of gray wolves, which is expected to be more closely related to dogs than to other populations of gray wolves. Here, we sequence the whole genomes of nine Japanese wolves (7.5-100x: Edo to Meiji periods) and 11 modern Japanese dogs and analyze them together with those from other populations of dogs and wolves. A phylogenomic tree shows that, among the gray wolves, Japanese wolves are closest to the dog, suggesting that the ancestor of dogs is closely related to the ancestor of the Japanese wolf. Based on phylogenetic and geographic relationships, the dog lineage has most likely originated in East Asia, where it diverged from a common ancestor with the Japanese wolf. Since East Eurasian dogs possess Japanese wolf ancestry, we estimate an introgression event from the ancestor of the Japanese wolf to the ancestor of the East Eurasian dog that occurred before the dog's arrival in the Japanese archipelago.

Quantitative analysis of the skull in the Japanese wolf (Canis lupus hodophilax) using CT.

In this study using computed tomography (CT), the volumes of the internal cranial cavities, such as the braincase, frontal sinus and tympanic cavity, and the ratio of the volume of each cavity to the skull volume in Japanese wolves were quantified, and CT images of the frontal sinus were observed. The results were then compared with those of other wolf subspecies, including Akita, a dog breed, to clarify the characteristics of the internal cranial cavities in Japanese wolves. The present study revealed that the Japanese wolf had a relatively larger braincase volume and a relatively smaller frontal sinus volume than the wolf ssp. (a group of wild wolf subspecies except the Japanese wolf) and Akita. Moreover, the relative and absolute tympanic cavity volumes of the Japanese wolf and Akita were significantly smaller than those of the wolf ssp. In the CT image or macroscopic observations, the frontal sinuses of the wolf ssp. and Akita were relatively well developed to the caudal and dorsal directions, respectively, compared with that of the Japanese wolf, and the tympanic cavity of the wolf ssp. was more largely swelled ventrally and medially than that of other groups.

Characterization of early transient accumulation of PrP(Sc) in immune cells.

PrP(Sc) is known to elicit no specific immune response and the immune cells are suspected to support its accumulation. In the present study, we investigated the response of some immune cell types to PrP(Sc) to characterize an observed early transient accumulation of PrP(Sc). After cells were treated with PrP(Sc)-brain homogenate, PrP(Sc) was transiently accumulated for the first 8-12h post-exposure then completely cleared by the 5th day of the experiment. The accumulated PrP(Sc) was not a de novo product of the cell PrP(C). Further investigation of this phenomenon revealed some potential factors influencing it. These factors included cholesterol homeostasis, temperature, the degradation power of the cell and the availability of sufficient PrP(C). Our in vitro results suggest that immune cells, especially macrophages are potential risk factors for the accumulation and intercellular spread of PrP(Sc) if the complete clearance of PrP(Sc) were not fulfilled.

Alteration of cell responses to PrP(Sc) in prolonged cell culture and its effect on transmission of PrP(Sc) to neural cells.

The mechanisms and processes of the uptake, intracellular trafficking and intercellular spread of PrP(Sc) and its transfer to neural cells are not clearly defined. The involvement of immune, intestinal, mast or peripheral neural cells in this process also remains unclear. The role of these cell types in the accumulation and transfer of PrP(Sc) to neural cells was investigated following short and prolonged exposure to the Chandler and Obihiro strains of scrapie PrP(Sc) for up to 28 days. Eight cell lines of murine immune, neural, intestinal and fibroblast cell types were tested. After transient degradation phases, certain immune, intestinal and neural cells accumulated PrP(Sc) for up to 28 days postinfection. When co-cultured with N2a-3/EGFP neuroblastoma cells for 4 days followed by several passages, the immune, intestinal and the neural cell lines were able to transfer infection to neural cells. Our results suggest that some of these cell types may have a role in PrP(Sc) accumulation and intercellular spread of PrP(Sc) infection to neural cells in vivo.

Characterization of PrP(Sc) transmission from immune cells to neuronal cells.

We investigated PrP(Sc) transmission in neuronal cells, spleen cells and several immune cells using an in vitro cell-to-cell transmission system. The transmission of PrP(Sc) in the supernatant of PrP(Sc)-infected neuronal cells was also investigated. We found that PrP(Sc) transmission was more efficient in the cell-to-cell transmission system than in the supernatant-mediated system. PrP(Sc) was more efficiently transmitted from adherent spleen cells to neuronal cells than from floating spleen cells. The adherent spleen cells were composed of macrophages (80%), dendritic cells (8%) and follicular dendritic cells (3%), indicating that macrophages play an important role in PrP(Sc) transmission from immune cells to neuronal cells. Although PrP(Sc) in the immune cells used as donor cells was gradually degraded, the PrP(Sc) transmitted to neuronal cells was observed by Western blot analysis. Investigation of the mechanism of PrP(Sc) transmission between cells represents an important step towards understanding the pathogenesis of prion diseases.

Distribution of immune cells and expression of interleukin receptors in ileal Peyer's patches of calves.

Newborn calves lack a mature immune system. The immune system develops with age, but the role of the expression of cytokine receptors in the development of immune cells of Peyer's patches (PPs) in the intestines of calves in the first 2 months has not yet been elucidated. In this study, the distribution of immune cells and the expression of interleukin (IL) receptors (R) in the ileal PPs of newborn and 2-month-old calves were investigated immunohistochemically with monoclonal antibodies against bovine CD4, CD8, IgM, γδTCR, T19, WC3, WC5, and WC6 antigens. The expression of ILRs was examined with antibodies against CD25 (IL-2Rα), IL-2Rγ, IL-4R, IL-6R, IL-10R, and IL-13R antigens. CD4(+), CD8(+), γδTCR(+), T19(+), and WC6(+) cells were found to be more widely distributed in the ileal PPs of 2-month-old calves than in those of newborn calves. Moreover, the expression of CD25 (IL-2Rα), IL-4R, and IL-13R in the ileal PPs of 2-month-old calves was more prominent than that in newborn calves. These data suggest that the immune system of calves at 2 months of age is developed by reactions to foreign antigens and aging.

Molecular analysis of parapoxvirus from a spotted seal Phoca largha in Japan.

A spotted seal Phoca largha with nodular and scab lesions on the whole body was brought to an aquarium in Nagoya, Japan. We extracted DNA from the lesions and used the polymerase chain reaction (PCR) method for detecting orthopoxvirus and parapoxvirus DNA. Parapoxvirus but not orthopoxvirus DNA was detected. The partial nucleotide sequence of the envelope gene was determined from the PCR product, and the sequence was seen to be closely related to 2 parapoxvirus strains from spotted seals in Alaska, showing 100% identity at the amino acid level, with one nucleotide substitution. Virus-neutralizing (VN) antibody against canine distemper virus (CDV) was not detected in the serum, indicating that this individual was not infected with CDV or phocine distemper virus (PDV), which both have a high mortality rate for marine mammals. These results suggest that the lesions were caused by infection with pinniped parapoxvirus, and that the viruses spread and are maintained within the habitat range or populations of spotted seals from the Bering Sea to the Japan Sea. This is the first report of molecular analysis of parapoxvirus in marine mammals in Japan.

Successive deaths of a captive snow leopard (Uncia uncia) and a serval (Leptailurus serval) by infection with feline panleukopenia virus at Sapporo Maruyama Zoo.

Feline parvoviruses were isolated from frozen samples of intestines taken from a snow leopard (Uncia uncia) and a serval (Leptailurus serval) that died successively at Sapporo Maruyama Zoo in Hokkaido, Japan. Isolates possessed an antigenic epitope for both the feline panleukopenia virus (FPLV) and mink enteritis virus, identified with a hemagglutination inhibition test. Sequencing analyses of the VP2 region of the isolates revealed that the two isolates were identical and of the FPLV-type. These results suggested that FPLV was introduced from a feral cat which entered the zoo and transmitted the virus inside the zoo.

Intron 1 mediated regulation of bovine prion protein gene expression: Role of donor splicing sites, sequences with potential enhancer and suppressor activities.

Prion protein plays a key role in the pathogenesis of transmissible spongiform encephalopathies. Because changes in expression of the prion protein gene (PRNP) alter the incubation time and severity of prion diseases. Our previous work revealed a strong association between the promoter (spanning base pairs (bp) -88 to -30) and intron 1 (spanning bp +114 to +892) that leads to optimum expression of the bovine PRNP. Here, we employed two mutation analysis strategies (deletion and insertion) and two reporter assay systems (luciferase and GFP expression) to define the regulatory domains within intron 1 and further elucidate its role in regulating the promoter activity of the bovine prion protein gene. We identified DNA sequences with potential suppressor and enhancer activities within the 5' end of intron 1. Moreover stability analyses for PRNP mRNAs demonstrated that splicing sites and mechanism are critical for bovine PRNP expression.

Anti-PrP antibodies detected at terminal stage of prion-affected mouse.

The causative agent of prion diseases is the pathological isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc has an identical amino acid sequence to PrPC; thus, it has been assumed that an immune response against PrPSc could not be found in prion-affected animals. In this study, we found the anti-prion protein (PrP) antibody at the terminal stage of mouse scrapie. Several sera from mice in the terminal stage of scrapie reacted to the recombinant mouse PrP (rMPrP) molecules and brain homogenates of mouse prion diseases. These results indicate that mouse could recognize PrPC or PrPSc as antigens by the host immune system. Furthermore, immunization with rMPrP generates high titers of anti-PrP antibodies in wild-type mice. Some anti-PrP antibodies immunized with rMPrP prevent PrPSc replication in vitro. The mouse sera from terminal prion disease have several wide epitopes, although mouse sera immunized with rMPrP possess narrow epitopes.

Osteological and genetic analysis of the extinct Ezo wolf (Canis lupus hattai) from Hokkaido Island, Japan.

The Ezo wolf (Canis lupus hattai Kishida, 1931 ) is an extinct subspecies that inhabited Hokkaido in Japan until the middle of the Meiji Period. Because there are very few preserved skeletons, no osteological and/or genetic analyses of the Ezo wolf have been conducted. In this study, 20 cranial and eight mandibular characters were measured on Ezo wolf skeletons, and mitochondrial DNA (mtDNA) was analyzed to assess genetic relationships between the Ezo wolf and other wolf lineages, including the Japanese wolf on Honshu. The morphological study showed that the Ezo wolf is larger than the Japanese wolf and similar in size to the grey wolf of the Asian and American Continents. MtDNA control sequences (751 bp) from two Ezo wolves were identical to those from the Canadian grey wolf. The morphological and genetic characters indicate that the ancestor of the Ezo wolf was genetically related to that of the grey wolf in Canada.