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.

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.

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.

Asia-wide phylogeography of wild boar (Sus scrofa) based on mitochondrial DNA and Y-chromosome: Revising the migration routes of wild boar in Asia.

Genetics of pigs has been well studied in Europe and Asia, but most of previous studies of molecular phylogeny of Sus scrofa have been based on sequences of both wild and domestic forms. In this study we analysed genetic traits of Sus scrofa from 13 regions in Asia (including previously undisclosed Eastern Caucasus and Trans-Baikal regions) using purely wild boar samples. Mitochondrial control region and Y-chromosome genes (AMELY & USP9Y) were employed to resolve phylogeographic relationships. We discussed spatio-temporal dynamics of wild boar distribution and compared molecular data to morphological and cytogenetic data on wild boar variability and taxonomy. A total of 51 haplotypes were detected in mtDNA control region and five haplotypes were found in combined sequences of Y-chromosome genes. The phylogeography of Asia-wide wild boars supported a hypothesis of migration from South-East Asia to South Asia, followed by migration to East and West Asia. We present a hypothesis about independent dispersal of wild boars into West Asia from South and North-East Asia. Mitochondrial DNA phylogeny generally fits the morphologically based intraspecies taxonomy. Distribution of chromosomal variants of wild boar presently does not show clear correlation with mtDNA clades.

Differential expression of serum amyloid A1 and A3 in bovine epithelia.

Serum amyloid A (SAA) is both an amyloidogenic protein of amyloid A amyloidosis and an acute phase protein in most animal species. Although SAA isoforms, such as SAA1, 2, 3, and 4, have been identified in cattle, their biological functions are not completely understood. Previous studies using mice indicated that SAA3 mRNA expression increased by stimulation with Escherichia coli and lipopolysaccharide (LPS) in colonic epithelial cells, and subsequently the SAA3 protein enhanced the expression of mucin2 (MUC2) mRNA, which is the major component of the colonic mucus layer. These results suggest that SAA3 plays a role in host innate immunity against bacterial infection in the intestine. In this study, a novel anti-bovine SAA3 monoclonal antibody was produced and SAA3 expression levels in bovine epithelia were examined in vitro and in vivo using real-time PCR and immunohistochemistry (IHC). SAA3 mRNA expression, but not that of SAA1, was enhanced by LPS stimulus in bovine small intestinal and mammary glandular epithelial cells in vitro. Moreover, in bovine epithelia (small intestine, mammary gland, lung, and uterus) obtained from four Holstein dairy cows from a slaughterhouse, SAA3 mRNA expression was higher than that of SAA1. Furthermore, using IHC, SAA3 protein expression was observed in bovine epithelia, whereas SAA1 protein was not. These results suggest that in cattle, SAA3 plays an immunological role against bacterial infection in epithelial tissues, including the small intestine, mammary gland, lung, and uterus.

A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity.

A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Interestingly, there was no amino acid sequence similarity among the epitopes on the three proteins. These epitopes had high binding affinities to G2 (KD = ∼10-7 M for monovalent binding and KD = ∼10-9 M for divalent binding), as determined using a SPR biosensor. This is the first report on a three-in-one mAb recognizing completely different epitope sequences with high affinity. Additionally, competitive ELISA indicated that the binding sites on G2, specific for the three different epitopes, overlapped, suggesting that the antigen-binding site may be flexible in the free form and capable of adapting to at least three different conformations to enable interactions with three different antigens.

The N-terminal region of serum amyloid A3 protein activates NF-κB and up-regulates MUC2 mucin mRNA expression in mouse colonic epithelial cells.

Serum amyloid A (SAA) is the major acute-phase protein and a precursor of amyloid A (AA) in AA amyloidosis in humans and animals. SAA isoforms have been identified in a wide variety of animals, such as SAA1, SAA2, SAA3, and SAA4 in mouse. Although the biological functions of SAA isoforms are not completely understood, recent studies have suggested that SAA3 plays a role in host defense. Expression of SAA3 is increased on the mouse colon surface in the presence of microbiota in vivo, and it increases mRNA expression of mucin 2 (MUC2) in murine colonic epithelial cells in vitro, which constitutes a protective mucus barrier in the intestinal tract. In this study, to identify responsible regions in SAA3 for MUC2 expression, recombinant murine SAA1 (rSAA1), rSAA3, and rSAA1/3, a chimera protein constructed with mature SAA1 (amino acids 1-36) and SAA3 (amino acids 37-103), and vice versa for rSAA3/1, were added to murine colonic epithelial CMT-93 cells, and the mRNA expressions of MUC2 and cytokines were measured. Inhibition assays with NF-κB inhibitor or TLR4/MD2 inhibitor were also performed. Up-regulation of MUC2 mRNA expression was strongly stimulated by rSAA3 and rSAA3/1, but not by rSAA1 or rSAA1/3. Moreover, NF-κB and TLR4/MD2 inhibitors suppressed the increase of MUC2 mRNA expression. These results suggest that the major responsible region for MUC2 expression exists in amino acids 1-36 of SAA3, and that up-regulations of MUC2 expression by SAA3 and SAA3/1 are involved with activation of NF-κB via the TLR4/MD2 complex.

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.

Establishment of an on-site diagnostic procedure for detection of orf virus from oral lesions of Japanese serows (Capricornis crispus) by loop-mediated isothermal amplification.

Orf virus infection has been prevalent continuously in the population of wild Japanese serows (Capricornis crispus), goat-like grazing cloven-hoofed mammal species that live mainly in mountainous areas of Japan. Currently, definitive diagnosis of infection requires time-consuming laboratory work. To diagnose rapidly on-site, we developed a field-friendly procedure for the detection of orf virus from oral cavity lesions. DNA was extracted from goat saliva spiked with orf virus as a proxy for Japanese serows by a commercial kit without the use of electricity, and the quality of the extracted DNA was evaluated by conventional polymerase chain reaction (PCR). Extracted DNA was amenable to DNA amplification, the same as when extracted in a laboratory. Next, to find optimal conditions for DNA amplification by loop-mediated isothermal amplification (LAMP), Bst and Csa DNA polymerases and 3 colorimetric indicators for visual diagnosis, hydroxy naphthol blue (HNB), malachite green and D-QUICK, were compared using a portable cordless incubator. The combination of Bst or Csa DNA polymerase with HNB was found to be easiest for visual diagnosis by the naked eye, and viral DNA was successfully amplified from all orf virus strains used. These results suggest that the procedure established here can work completely on-site and can be useful for definitive diagnosis and differentiation of orf virus infection in Japanese serows in remote mountainous areas.

Computed tomography examination and mitochondrial DNA analysis of Japanese wolf skull covered with skin.

A Canis skull, right half of the mandible and part of the left half of the mandible were subjected to three-dimensional (3D) computed tomography (CT) observation and mitochondrial DNA (mtDNA) analysis in order to determine whether the specimens belonged to the extinct Japanese wolf, Canis lupus hodophilax (Temminck, 1839). Osteometric analysis of the skull and right half of the mandible revealed that the material (JW275) was indeed typical of the Japanese wolf. Sequence analysis of a 600-bp mtDNA region revealed that the JW275 belonged to haplotype Group B, which is characterized by an 8-bp deletion in the mtDNA control region. The findings of this study suggest that 3D CT analysis is well suited to examining fragile and valuable biological samples, as it removes the need for destructive sampling.

Experimental transmission of systemic AA amyloidosis in autoimmune disease and type 2 diabetes mellitus model mice.

AA amyloidosis is a protein misfolding disease characterized by extracellular deposition of amyloid A (AA) fibrils. AA amyloidosis has been identified in food animals, and it has been postulated that AA amyloidosis may be transmissible to different animal species. Since the precursor protein of AA fibrils is serum amyloid A (SAA), which is an inflammatory acute phase protein, AA amyloidosis is considered to be associated with inflammatory diseases such as rheumatoid arthritis. Chronic diseases such as autoimmune disease and type 2 diabetes mellitus could be potential factors for AA amyloidosis. In this study, to examine the relationship between the induction of AA amyloidosis and chromic abnormalities such as autoimmune disease or type 2 diabetes mellitus, amyloid fibrils from mice, cattle, or chickens were experimentally injected into disease model mice. Wild-type mice were used as controls. The concentrations of SAA, IL-6, and IL-10 in autoimmune disease model mice were higher than those of control mice. However, induction of AA amyloidosis in autoimmune disease and type 2 diabetes mellitus model mice was lower than that in control mice, and the amount of amyloid deposits in the spleens of both mouse models was lower than that of control mice according to Congo red staining and immunohistochemistry. These results suggest that factors other than SAA levels, such as an inflammatory or anti-inflammatory environment in the immune response, may be involved in amyloid deposition.

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.

[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.

Effect of heating on the stability of amyloid A (AA) fibrils and the intra- and cross-species transmission of AA amyloidosis.

Amyloid A (AA) amyloidosis is a protein misfolding disease characterized by extracellular deposition of AA fibrils. AA fibrils are found in several tissues from food animals with AA amyloidosis. For hygienic purposes, heating is widely used to inactivate microbes in food, but it is uncertain whether heating is sufficient to inactivate AA fibrils and prevent intra- or cross-species transmission. We examined the effect of heating (at 60 °C or 100 °C) and autoclaving (at 121 °C or 135 °C) on murine and bovine AA fibrils using Western blot analysis, transmission electron microscopy (TEM), and mouse model transmission experiments. TEM revealed that a mixture of AA fibrils and amorphous aggregates appeared after heating at 100 °C, whereas autoclaving at 135 °C produced large amorphous aggregates. AA fibrils retained antigen specificity in Western blot analysis when heated at 100 °C or autoclaved at 121 °C, but not when autoclaved at 135 °C. Transmissible pathogenicity of murine and bovine AA fibrils subjected to heating (at 60 °C or 100 °C) was significantly stimulated and resulted in amyloid deposition in mice. Autoclaving of murine AA fibrils at 121 °C or 135 °C significantly decreased amyloid deposition. Moreover, amyloid deposition in mice injected with murine AA fibrils was more severe than that in mice injected with bovine AA fibrils. Bovine AA fibrils autoclaved at 121 °C or 135 °C did not induce amyloid deposition in mice. These results suggest that AA fibrils are relatively heat stable and that similar to prions, autoclaving at 135 °C is required to destroy the pathogenicity of AA fibrils. These findings may contribute to the prevention of AA fibril transmission through food materials to different animals and especially to humans.

Prevalence of amyloid deposition in mature healthy chickens in the flock that previously had outbreaks of vaccine-associated amyloidosis.

Avian amyloid A (AA) amyloidosis is commonly observed in adult birds with chronic inflammation, such as that caused by bacterial infection. We previously described vaccine-associated AA amyloidosis in juvenile chickens. In this study, the prevalence of amyloid deposition was measured in mature healthy chickens that survived a previous outbreak of avian AA amyloidosis while they were juveniles. Herein, we analyzed the amyloid deposition in mature chickens and compared the prevalence of amyloid deposition with juvenile chickens obtained in our previous study (Murakami et al., 2013). We found that: 1) amyloid deposition in the liver was absent in mature chickens, while juvenile chickens had a rate of 24%; 2) amyloid deposition in the spleen was observed in 36% of juvenile chickens and in 40% of mature chickens; 3) amyloid deposition in the pectoral muscle of mature chickens (43.75%) was approximately half that of juvenile chickens (88%). These results suggest that additional amyloid deposition in chickens previously exposed to AA amyloidosis may not worsen with age. Further, amyloid deposition in chickens may tend to regress when causative factors, such as vaccinations and/or chronic inflammation, are absent.

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.

Systemic AA amyloidosis as a prion-like disorder.

Amyloidosis is a collective term for a group of disorders that induce functional impairment of organs and occurs through the accumulation of amyloid, or misfolded protein in beta-sheets. AA amyloidosis is a lethal systemic amyloidosis with SAA as the precursor protein, and is observed in various animal species, including humans. AA amyloidosis can be induced artificially by continuously administering inflammatory stimuli in experimental animal models. In this process of experimental induction, the administration of AA amyloids from either the same or different species is known to markedly expedite AA amyloidosis development, and this is also termed transmission of AA amyloidosis. Similarly to prion disease, AA amyloidosis is considered to be transmitted via a "seeding-nucleation" process. In this manuscript, we reviewed the pathology and transmissibility of AA amyloidosis in animals.

On-site visual diagnosis of parapoxvirus infection using a portable cordless incubator.

A portable cordless incubator was developed for on-site visual diagnosis of parapoxvirus infection on farms and in areas with no electricity or laboratory equipment. The battery-powered thermoregulator can maintain a stable temperature for more than 1 h. The incubator successfully amplified parapoxvirus DNA isolated from sheep and wild Japanese serows (Capricornis crispus) by loop-mediated isothermal amplification (LAMP). Although different absorbance values were obtained for the LAMP reactions performed using the same sample on the incubator and the turbidimeter, visual assessments of whether the results were positive or negative were the same irrespective of the platform used to perform the LAMP reaction. Consequently, using the portable cordless incubator in conjunction with the LAMP assay is considered to be a powerful tool for on-site visual diagnosis of parapoxvirus infection on farms with no electricity.