Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276.

Exosomes are small extracellular vesicles (EVs) found in culture supernatants, blood, and breast milk. The size of these nanocomplexes limits the methods of EV analyses. In this study, nitrobenzoxadiazole (NBD), a fluorophore, conjugated endosome-lysosome imager, GIF-2250 and its derivative, GIF-2276, were evaluated for exosome analyses. A correlation was established between GIF-2250 intensity and protein maker levels in bovine milk exosomes. We found that high-temperature sterilization milk may not contain intact exosomes. For precise analysis, we synthesized GIF-2276, which allows for the covalent attachment of NBD to the Lys residue of exosome proteins, and labeled milk exosomes were separated using a gel filtration system. GIF-2276 showed chromatographic peaks of milk exosomes containing >3 ng protein. The area (quantity) and retention time (size) of the exosome peaks were correlated to biological activity (NO synthesis suppression in RAW264.7 murine macrophages). Heat denaturation of purified milk-derived exosomes disrupted these indicators. Proteome analyses revealed GIF-2276-labeled immunomodulators, such as butyrophilin subfamily 1 member A1 and polymeric immunoglobulin receptor. The immunogenicity and quantity of these factors decreased by heat denaturation. When milk exosomes were purified from market-sourced milk we found that raw and low-temperature sterilization milk samples, contained exosomes (none in high-temperature sterilization milk). These results were also supported by transmission electron microscopy analyses. We also found that GIF-2276 could monitor exosome transportation into HEK293 cells. These results suggested that GIF-2250/2276 may be helpful to evaluate milk exosomes.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

First Report of Mermithidae (Enoplea: Mermithida) Parasitizing Adult Stable Flies in Japan.

Mermithidae is a family of nematodes that parasitize a wide range of invertebrates worldwide. Herein, we report nematodes that were unexpectedly found in three of 486 adult stable flies (Stomoxys calcitrans) captured from three farms (F1, F2, and F3) in different regions of Gifu Prefecture, Japan. We aimed to characterize these nematodes both at the morphological and molecular level. Morphological studies revealed that the nematodes were juveniles of Mermithidae. Phylogenetic analysis based on 18S and 28S rDNA indicated that the mermithids from farms F1 and F2 could be categorized into the same cluster as Ovomermis sinensis and Hexamermis sp., whereas the mermithid from farm F3 clustered with Amphimermis sp. Additionally, these mermithids could be categorized within the same clusters as related mermithids detected in Japan that parasitize various arthropod orders. Our findings suggest that these stable flies may have been parasitized by mermithids already present in the region and that genetically distinct species of mermithids occur across Japan. To the best of our knowledge, this is the first report of mermithids parasitizing adult stable flies in Japan.

Comparing microRNA in milk small extracellular vesicles among healthy cattle and cattle at high risk for bovine leukemia virus transmission.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by bovine leukemia virus (BLV) infection. In Japan, cattle diagnosed with EBL are not permitted for human consumption by the law, thereby causing serious economic losses to farmers. The prevalence of BLV is high in Japan (40.9% in dairy cattle and 28.7% in beef cattle, respectively), which makes it difficult to perform the test-and-slaughter of BLV-infected cattle. This necessitates preventing the spread of BLV infection in cattle by early detection, segregation, and the removal of BLV-infected cattle with high proviral load, which are considered high risk for BLV transmission. We aimed to identify cattle that were at high risk for BLV transmission by comparing microRNA (miRNA) profiles in milk small extracellular vesicles (sEV). At first, miRNA profiles in sEV were compared among 4 uninfected cattle and 4 BLV-infected cattle with high proviral load by using a microarray containing mixed probes for miRNA of cattle and humans. Significantly lower amounts of hsa-miR-557 and hsa-miR-19b-1-5p, and insignificantly but higher amounts of hsa-miR-424-5p were observed in milk sEV from BLV-infected cattle than those from uninfected cattle. Next, to evaluate the utility of the aforementioned miRNAs for the identification of cattle that were at high risk for BLV transmission, we performed quantitative real-time PCR using milk sEV newly collected from 5 uninfected cattle and 17 BLV-infected cattle with high proviral load. The cycle threshold value of hsa-miR-424-5p was significantly lower in milk sEV from BLV-infected cattle. The PCR detection was unavailable or a significant difference was not observed for hsa-miR-557 and hsa-miR-19b-1-5p, respectively. These results suggest that the amount of hsa-miR-424-5p was higher in milk sEV from BLV-infected cattle and increasing the hsa-miR-424-5p in milk sEV could be one of the characteristic trends in cattle that are high risk for BLV transmission. Moreover, assessing characteristic miRNA amounts in milk sEV, which can be recovered twice a day by milking, could be useful for the routine monitoring of cattle in dairy herds instead of blood collection.

Characterization of miRNAs in Milk Small Extracellular Vesicles from Enzootic Bovine Leukosis Cattle.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). Most BLV-infected cattle show no clinical signs and only some develop EBL. The pathogenesis of EBL remains unclear and there are no methods for predicting EBL before its onset. Previously, it was reported that miRNA profiles in milk small extracellular vesicles (sEVs) were affected in cattle in the late stage of BLV infection. It raised a possibility that miRNA profile in milk sEVs from EBL cattle could be also affected. To characterize the difference in milk of EBL cattle and healthy cattle, we examined the miRNA profiles in milk sEVs from four EBL and BLV-uninfected cattle each using microarray analysis. Among the detected miRNAs, three miRNAs-bta-miR-1246, hsa-miR-1290, and hsa-miR-424-5p-which were detectable using quantitative real-time PCR (qPCR) and are associated with cancers in humans-were selected as biomarker candidates for EBL. To evaluate the utility of these miRNAs as biomarkers for EBL, their levels were measured using milk that was freshly collected from 13 EBL and seven BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p, but not hsa-miR-1290, were detected using qPCR and their levels in milk sEVs from EBL cattle were significantly higher than those in BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p in sEVs may promote metastasis by targeting tumor suppressor genes, resulting in increased amounts in milk sEVs in EBL cattle. These results suggest that bta-miR-1246 and hsa-miR-424-5p levels in milk sEVs could serve as biomarkers for EBL.

Enzootic nasal tumor virus type 2 envelope of goats acts as a retroviral oncogene in cell transformation.

Enzootic nasal tumor virus type 1 (ENTV-1) (ovine nasal tumor virus) and ENTV-2 (caprine nasal tumor virus) are known to be causative agents of enzootic nasal adenocarcinoma (ENA) in sheep and goats, respectively. Although the nucleotide and amino acid sequences of ENTV-1 and ENTV-2 are quite similar, they are recognized as phylogenetically distinct viruses. The envelope protein of ENTV-1 functions as an oncoprotein in the in vitro transformation of epithelial cells and fibroblasts. Thus, it is the primary determinant of in vivo tumorigenesis in ENA. As per our knowledge, no previous studies have reported in detail the role of ENTV-2 in ENA tumorigenesis. Here, in order to investigate the molecular mechanism of caprine ENA oncogenesis by ENTV-2, we have attempted to identify the transforming potential of ENTV-2 envelope, and investigated the activation of cell signaling pathways in oncogenic transformation. Our findings confirmed that ENTV-2 envelope was capable of inducing oncogenic transformation of rat cell lines in vitro. Further, we found that MAPK, Akt, and p38 were constitutively activated in ENTV-2 envelope-transformed clone cells. In addition, inhibitor experiments revealed that MEK-MAPK and PI3K-Akt signaling pathways are involved in the ENTV-2 envelope-induced cell transformation. These data indicate that ENTV-2 envelope could induce oncogenic transformation by signaling pathways that are also utilized by ENTV-1 envelope.

Food Workers as a Reservoir of Extended-Spectrum-Cephalosporin-Resistant Salmonella Strains in Japan.

Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella, especially extended-spectrum-ß-lactamase (ESBL)-producing Salmonella, is a concern worldwide. Here, we assessed Salmonella carriage by food workers in Japan to clarify the prevalence of ESC-resistant Salmonella harboring blaCTX-M We then characterized the genetic features, such as transposable elements, of blaCTX-M-harboring plasmids using whole-genome sequencing. A total of 145,220 stool samples were collected from food workers, including cooks and servers from several restaurants, as well as food factory workers, from January to October 2017. Isolated salmonellae were subjected to antimicrobial susceptibility testing (disk diffusion method), and whole-genome sequencing was performed for Salmonella strains harboring blaCTX-M Overall, 164 Salmonella isolates (0.113%) were recovered from 164 samples, from which we estimated that at least 0.113% (95% confidence interval [CI]: 0.096 to 0.132%) of food workers may carry Salmonella Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) individuals among the 3,075,330 Japanese food workers are likely to carry Salmonella Of the 158 culturable isolates, seven showed resistance to ESCs: three isolates harbored blaCMY-2 and produced AmpC ß-lactamase, while four ESBL-producing isolates harbored blaCTX-M-14 (n = 1, Salmonella enterica serovar Senftenberg) or blaCTX-M-15 (n = 3, S. enterica serovar Haardt). blaCTX-M-15 was chromosomally located in the S Haardt isolates, which also contained ISEcp1, while the S Senftenberg isolate contained an IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid plasmid carrying blaCTX-M-14 along with ISEcp1 This study indicates that food workers may be a reservoir of ESBL-producing Salmonella and associated genes. Thus, these workers may contribute to the spread of blaCTX-M via plasmids or mobile genetic elements such as ISEcp1IMPORTANCE Antimicrobial-resistant Salmonella bacteria arise in farm environments through imprudent use of antimicrobials. Subsequently, these antimicrobial-resistant strains, such as extended-spectrum-ß-lactamase (ESBL)-producing Salmonella, may be transmitted to humans via food animal-derived products. Here, we examined Salmonella carriage among food handlers in Japan. Overall, 164 of 145,220 fecal samples (0.113%) were positive for Salmonella Among the 158 tested isolates, four were identified as ESBL-producing isolates carrying ESBL determinants blaCTX-M-15 or blaCTX-M-14 In all cases, the genes coexisted with ISEcp1, regardless of whether they were located on the chromosome or on a plasmid. Our findings suggest that food workers may be a reservoir of ESBL-producing strains and could contribute to the spread of resistance genes from farm-derived Salmonella to other bacterial species present in the human gut.

Placental and breastmilk transfer of voriconazole to offspring from pregnant and lactating bottlenose dolphins (Tursiops truncatus).

Fungal pneumonia is a common disease in bottlenose dolphins (Tursiops truncatus), including pregnant and lactating ones. Voriconazole (VRCZ) is commonly used to treat respiratory fungal infections in this species; however, it is unknown whether VRCZ is transferred via the placenta and breastmilk and whether its usage is safe in pregnant and lactating dolphins. We measured VRCZ concentrations in breastmilk and dams', umbilical cord, and calves' plasma samples from four dam-calf dolphin pairs in the Port of Nagoya Public Aquarium, Japan, treated with or without VRCZ. Three pregnant and/or lactating dams were administered VRCZ (loading dose 1.5-2.3 mg/kg, for 3 days; maintenance dose 1.5-3.1 mg/kg, every 5-18 days), twice daily, orally, without side effects in dams or calves. VRCZ was detected in two dams' umbilical cord plasma (0.14 and 2.35 µg/ml) and in one calf's plasma (0.18 µg/ml), collected immediately after birth. Further, VRCZ was detected in breastmilk samples (maximum 13.45 µg/ml) from three VRCZ-administered dams and in plasma from three calves (maximum 7.54 µg/ml) given or nursed from VRCZ-administered dams' breastmilk. The calves' plasma VRCZ concentrations varied, depending on the amount of breastmilk and food consumed. VRCZ concentrations were higher in breastmilk samples than in dams' plasma. To our knowledge, this is the first report on placental and breastmilk VRCZ transfer to offspring in bottlenose dolphins. During VRCZ medication in pregnant and lactating bottlenose dolphins, it is crucial to monitor plasma VRCZ concentrations and any side effects in dams as well as in their calves.

Bovine papular stomatitis virus and pseudocowpox virus coinfection in dairy calves in Japan.

Two cases of coinfection with bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) in dairy calves in Tochigi Prefecture, Japan, are reported. Sequences of BPSV and PCPV were simultaneously detected in the same polymerase chain reaction (PCR) amplicons, which were obtained from the DNA of two dairy calves using a pan-parapoxvirus primer set. PCR amplification using BPSV- and PCPV-specific primer sets were able to distinguish between the two viruses in coinfected clinical samples. Based on these data, further studies on the occurrence BPSV/PCPV coinfections in cattle in Japan are warranted.

In vitro and ex vivo expression of serum amyloid A3 in mouse lung epithelia.

BACKGROUND AND PURPOSE: Serum amyloid A (SAA), an acute-phase protein whose level tracks infection and inflammation, is the precursor protein of amyloid A (AA) fibrils that is thought to cause AA amyloidosis in human and animals. SAA protein has several isoforms based on the difference of amino acid sequence, such as SAA1 to SAA4 in mice. AA fibrils are associated with chronic inflammation and are mainly originated from SAA1 produced in the liver. SAA3 reportedly contributes to the innate immune response in epithelia; however, little is known about its role at the lung epithelia. Therefore, we investigated SAA3 expression in the lung epithelium activated by bacterial antigens. MATERIALS AND METHODS: The expressions of SAA3 and SAA1 mRNA were investigated using quantitative real-time PCR, in vitro using mouse Clara (Club) cells and ex vivo using surgically removed mouse lungs, after their stimulation by using either lipopolysaccharide (LPS), the major outer membranous antigen of gram-negative bacteria, or lipoteichoic acid (LTA), the major outer membranous antigen of gram-positive bacteria. In addition, SAA3 and SAA1/2 proteins in treated lung samples were detected by immunohistochemistry (IHC). RESULTS: SAA3 mRNA expression increased in cells and lungs treated with either LPS or LTA. SAA3 mRNA was more sensitively expressed in LPS than LTA treatment. In contrast, SAA1 mRNA expression did not increase by either LPS or LTA treatment. Furthermore, SAA3 mRNA expression increased in a dose-dependent manner in cells treated with tumor necrosis factor-alpha. By IHC, SAA3 protein was highly expressed in the luminal side of the bronchial epithelium, while SAA1/2 was not expressed. CONCLUSION: These results obtained from in vitro and ex vivo experiments suggest that SAA3 plays an important role in the innate immune response to bacterial infection in the lung epithelia.

Efficient method for isolation of exosomes from raw bovine milk.

OBJECTIVE: This study aimed to establish a rapid and simple method for isolating exosomes from raw bovine milk and to compare the quality of the isolated exosomes with those isolated by a standard method involving ultracentrifugation (UC). METHODS: To remove caseins, which are major milk proteins consisting more than 80% of milk protein (35% in human breast milk) and hamper isolation and purification of exosomes, hydrochloride (HCl) was added to milk for isoelectric precipitation (IP). The effects of acidification on morphological features, particle size distribution, surface charge, and exosome surface proteins were analyzed by electron microscopy, tunable resistive pulse sensing (TRPS), and Western blot (WB) analysis, respectively. RESULTS: Electron microscopy showed that some of the exosomes isolated using IP had rough surfaces; most exosomes were successfully isolated without breakage, and their morphological features were similar to those of exosomes isolated by UC. TRPS showed that their surface charge and peaks (mode) for particle size distribution did not significantly differ between both methods. WB analysis using antibodies against the exosome surface marker proteins - milk fat globule-epidermal growth factor 8 (MFG-E8) and CD63 - revealed that the structures of exosome surface proteins were not affected by adding HCl. CONCLUSIONS: IP can be used to remove caseins to reduce operation time. This method will be useful for efficient isolation and purification of bovine milk exosomes and contribute to progression of research on health management of dairy cattle and drug delivery systems in human medicine, which require large amounts of milk exosomes.

First isolation and genetic characterization of pseudocowpox virus from cattle in Japan.

BACKGROUND: Pseudocowpox virus (PCPV) infects cattle worldwide with zoonotic potential but has not been isolated in Japan. Thus, the epidemiological status of PCPV infection in cattle is undetermined. RESULTS: In May 2016, a cattle in a farm in Yamaguchi Prefecture showed white vesicles and hyperemia in the mucosa under the tongue surface, but not on the teats and coronary cushions. A parapoxvirus was isolated from the oral lesion swab and was genetically characterized based on the full-length sequence of B2L gene encoding viral envelope. Phylogenetic analysis showed that the isolated virus was classified into PCPV. CONCLUSION: This case indicates its potential spread in Japan. This is the first report of isolation of PCPV in Japan.

Normal hematology and serum chemistry of northern fur seals (Callorhinus ursinus) in captivity.

Northern fur seals (Callorhinus ursinus) are endemic to the North Pacific Ocean. They were hunted for their fur and became endangered in the late 1800s, but their populations recovered following the introduction of protection laws. Recently, populations have been decreasing again, although the reasons are unclear. For individuals that are bred and reared in captivity as part of ex situ conservation projects, details of blood characteristics are essential to ensure good health. However, the normal ranges of hematology and serum chemistry of captive northern fur seals have not been defined. This study determined the normal ranges of hematology and serum chemistry of captive fur seals. Blood samples were collected every month for 2 years from four captive northern fur seals in Japan (three born in an aquarium and one kept in the same aquarium following rescue). Fifteen blood characteristics and 29 serum chemistry properties were compared with those previously reported for wild northern fur seals in the USA. Several parameters were not within the normal ranges reported previously in wild northern fur seals. In particular, levels of alkaline phosphatase was outside of the normal ranges previously reported. The hematological and serum chemistry ranges in this study can help provide a guideline for understanding the health of northern fur seals in captivity.

Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin.

This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, ITALIC! Tursiops truncatus mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg(-1)) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body.

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.

Midazolam and butorphanol combination for sedating beluga whales (Delphinapterus leucas).

Safe sedation doses for performing minor procedures such as bronchoscopy, endoscopy, and tooth extraction for beluga whales (Delphinapterus leucas) require elucidation. This study aimed to provide suggestions for determining appropriate midazolam and butorphanol doses to adequately sedate beluga whales to complete procedures and minimize the risk of side effects. We administered midazolam and butorphanol to six captive beluga whales (9-44 years old). Topical lidocaine anesthesia was administered during bronchoscopy. The sedation doses for the beluga whales varied from 0.020 to 0.122 mg/kg for midazolam and from 0.020 to 0.061 mg/kg for butorphanol. In beluga whales, optimal midazolam and butorphanol doses were lowest in old whales. These findings contribute to knowledge regarding appropriate sedation and prevention of overdose accidents during minor procedures in beluga whales.