Characterization and arbovirus susceptibility of cultured CERNI cells derived from sika deer (Cervus nippon).

Cervus nippon (sika deer) are widely distributed throughout eastern Asia. Deer possess a variety of antibodies against several zoonotic pathogens, indicating that they act as reservoir of zoonoses. In this study, we reported the characterization of cultured cells derived from sika deer and evaluated their susceptibility to arthropod-borne viruses to clarify their usefulness in virological studies. Cells derived from testicular tissue in Dulbecco's modified eagle medium with 16% fetal bovine serum started growing as primary cultured cells. The diploid cells consisted of 68 chromosomes, consistent with those of Japanese sika deer previously reported. The phylogenetic analysis showed the cells formed a robust clade with Japanese population of C. nippon, indicating that the cultured cells established in this study were originated from the Japanese sika deer. The cells immortalized by the simian virus 40 T-antigen were predominantly spindle-shaped cells exhibiting adhesive properties, and cultivated at 37°C and 5% CO2, which are common culture conditions for many mammalian cell lines. Western blotting analysis indicated that the cultured cells were multiple types of cells that coexist, including at least epithelial, fibroblast, and also Leydig cells. We confirmed that the cells have susceptibility to several arboviruses distributed in Japan: Getah virus, Japanese encephalitis virus, Oz virus, and severe fever with thrombocytopenia syndrome virus, but not to Tarumiz tick virus. From these results, the cells contribute to clarify the role of sika deer as a reservoir of zoonoses in nature and deer-associated experimental research at the cellular and molecular levels.

Seroconversion of anti-Getah virus antibody among Japanese native Noma horses around 2012.

Getah virus (GETV), an arthropod-borne virus transmitted by mosquitoes, has been isolated from several animals. GETV infection in horses shows clinical signs such as fever, rash, and edema in the leg. Noma horses are one of the eight Japanese native horses. The present study aimed to clarify the occurrence of GETV infection in Noma horses. Serum samples collected from Noma horses were analyzed using a virus neutralization test and enzyme-linked immunosorbent assay and showed that the anti-GETV antibody titers in the samples collected in 2017 were significantly higher than those collected in 2012. We concluded that a seroconversion of anti-GETV antibodies was occurred in the Noma horse population around 2012, providing evidence of the GETV epidemic in Japan circa 2012.

[THE CORRELATION OF ALLERGY SPECIFIC IGE MEASUREMENT REAGENT IMMNOCAP, ALASTAT, AND ORITON IGE].

BACKGROUND: The reagent which is available for single allergenic tests is Oriton IgE, ImmnoCAP, Alastat in Japan. No study has investigated the correlations of Oriton IgE and ImmnoCAP or Alastat, and, used for specific IgE antibody testing. METHOD: Six frequently tested allergens (dust mite, cedar pollen, dog dander, egg white, milk, and candida) were measured by three methods, and Spearman rank correlation coefficient and class-judged agreement were evaluated. Furthermore, we did the evaluation like other 2 methods when we made small short sample volumes of Oriton IgE. RESULT: As for the examination result of Oriton IgE and ImmnoCAP or Alastat, constant correlation was confirmed. However, the tendency was a different result by assay method and an allergenic item. No significant differences were observed in the results of the Oriton IgE test when standard sample volumes and small short sample volumes were used. CONCLUSION: These comparison results help us to understand each characteristic and select an optimal test method. In addition, it can be inferred that it is beneficial to choose tests requiring small sample volumes in pediatric patients.

Surveillance of antimicrobial-resistant Escherichia coli in Sheltered dogs in the Kanto Region of Japan.

There is a lack of an established antimicrobial resistance (AMR) surveillance system in animal welfare centers. Therefore, the AMR prevalence in shelter dogs is rarely known. Herein, we conducted a survey in animal shelters in Chiba and Kanagawa prefectures, in the Kanto Region, Japan, to ascertain the AMR status of Escherichia coli  (E. coli) prevalent in shelter dogs. E. coli was detected in the fecal samples of all 61 and 77 shelter dogs tested in Chiba and Kanagawa, respectively. The AMR was tested against 20 antibiotics. E. coli isolates derived from 16.4% and 26.0% of samples from Chiba and Kanagawa exhibited resistance to at least one antibiotic, respectively. E. coli in samples from Chiba and Kanagawa prefectures were commonly resistant to ampicillin, piperacillin, streptomycin, kanamycin, tetracycline, and nalidixic acid; that from the Kanagawa Prefecture to cefazolin, cefotaxime, aztreonam, ciprofloxacin, and levofloxacin and that from Chiba Prefecture to chloramphenicol and imipenem. Multidrug-resistant bacteria were detected in 18 dogs from both regions; ß-lactamase genes (blaTEM, blaDHA-1, blaCTX-M-9 group CTX-M-14), quinolone-resistance protein genes (qnrB and qnrS), and mutations in quinolone-resistance-determining regions (gyrA and parC) were detected. These results could partially represent the AMR data in shelter dogs in the Kanto Region of Japan.

Japanese Orthopaedic Association (JOA) clinical practice guidelines on the management of lateral epicondylitis of the humerus - Secondary publication.

BACKGROUND: The guidelines presented herein provide recommendations for the management of patients with lateral epicondylitis of the humerus. These recommendations are endorsed by the Japanese Orthopaedic Association (JOA) and Japan Elbow Society. METHODS: The JOA lateral epicondylitis guideline committee revised the previous guidelines on the basis of the "Medical Information Network Distribution Service Handbook for Clinical Practice Guideline Development 2014", which emphasized the importance of the balance between benefit and harm, and proposed a desirable method for preparing clinical guidelines in Japan. These guidelines consist of 11 clinical questions (CQs), 9 background questions (BQs), and 3 future research questions (FRQs). For each CQ, outcomes from the literature were collected and evaluated systematically according to the adopted study design. RESULTS: The committee proposed recommendations for each CQ by determining the level of evidence and assessing the consensus rate. Physical therapy was the best recommendation with the best evidence. The BQs and FRQs were answered by collecting evidence based on the literature. CONCLUSIONS: The guidelines presented herein were reviewed systematically, and recommendations were proposed for each CQ. These guidelines are expected to be widely used not only by surgeons or physicians but also by other healthcare providers, such as nurses, therapists, and athletic trainers.

Inactivation of Scrapie Prions by the Electrically Charged Disinfectant CAC-717.

Previous studies have revealed that the electrically charged disinfectant CAC-717 has strong virucidal and bactericidal effects but is safe for humans and animals. In this study, CAC-717 was further evaluated for its potential effects as a disinfectant against scrapie prions. Western blotting showed that CAC-717 reduced the amount of the abnormal isoform of prion protein (PrPSc) in prion-infected cell (ScN2a) lysates. Furthermore, the reduction of prion transmissibility was confirmed by a mouse bioassay, in which mice injected with scrapie prions pre-treated with CAC-717 survived longer than those injected with untreated scrapie prions. Lastly, to evaluate the seeding activity of ScN2a cell lysates treated with CAC-717, quantitative protein misfolding cyclic amplification (PMCA) was performed directly on ScN2a cell lysates treated with CAC-717, which showed that the median dose of PMCA (PMCA50) dropped from log9.95 to log5.20 after CAC-717 treatment, indicating more than a 4 log reduction. This suggests that the seeding activity of PrPSc is decreased by CAC-717. Collectively, these results suggest that CAC-717 has anti-prion activity, reducing both PrPSc conversion activity and prion transmissibility; thus, CAC-717 will be useful as a novel disinfectant in prion diseases.

Acute blocking of forearm supination secondary to tearing of the triangular fibrocartilage complex.

We studied the characteristics of acute blocking of supination of the distal radioulnar joint caused by triangular fibrocartilage complex injuries. Twenty-four patients who were treated for acute blocking of supination were retrospectively assessed. Supination was suddenly blocked after minor trauma to the wrist. Active and passive supination was severely restricted with a mean preoperative range of motion (11°), whereas pronation was almost normal. The cause was identified arthroscopically or at open operation. It was found to be a result of avulsion of the dorsal or palmar portion of the radioulnar ligament, which blocked movement of the ulnar head. Blocking was reduced manually in four cases, by arthroscopic surgery in eight cases and by open surgery in 12 cases. After treatment, forearm supination improved to 84° of the mean range of motion. Distal radioulnar joint blocking from a ruptured triangular fibrocartilage complex should be considered in the differential diagnosis of loss of forearm supination.Level of evidence: IV.

Author Correction: Dakrông virus, a novel mobatvirus (Hantaviridae) harbored by the Stoliczka's Asian trident bat (Aselliscus stoliczkanus) in Vietnam.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dakrông virus, a novel mobatvirus (Hantaviridae) harbored by the Stoliczka's Asian trident bat (Aselliscus stoliczkanus) in Vietnam.

The recent discovery of genetically distinct shrew- and mole-borne viruses belonging to the newly defined family Hantaviridae (order Bunyavirales) has spurred an extended search for hantaviruses in RNAlater®-preserved lung tissues from 215 bats (order Chiroptera) representing five families (Hipposideridae, Megadermatidae, Pteropodidae, Rhinolophidae and Vespertilionidae), collected in Vietnam during 2012 to 2014. A newly identified hantavirus, designated Dakrông virus (DKGV), was detected in one of two Stoliczka's Asian trident bats (Aselliscus stoliczkanus), from Dakrông Nature Reserve in Quang Tri Province. Using maximum-likelihood and Bayesian methods, phylogenetic trees based on the full-length S, M and L segments showed that DKGV occupied a basal position with other mobatviruses, suggesting that primordial hantaviruses may have been hosted by ancestral bats.

Differential Distribution of Salmonella Serovars and Campylobacter spp. Isolates in Free-Living Crows and Broiler Chickens in Aomori, Japan.

Salmonella and Campylobacter cause foodborne enteritis mainly via the consumption of raw/undercooked contaminated poultry meat and products. Broiler flocks are primarily colonized with these bacteria; however, the underlying etiology remains unclear. The present study was conducted in order to obtain further information on the prevalence and genotypic distribution of Salmonella and Campylobacter in free-living crows and broiler flocks in a region for 2 years, thereby facilitating estimations of the potential risk of transmission of C. jejuni from crows to broiler flocks. Salmonella serovars Bredeney and Derby were isolated from 8 and 3 out of 123 captured crows, respectively, both of which are not common in broiler chickens. Campylobacter were isolated from all 89 crows tested and C. jejuni was prevalent (85 crows). Pulsed field gel electrophoresis showed broad diversity in the crow isolates of C. jejuni. However, 3 crow isolates and 2 broiler isolates showing similar banding patterns were assigned to different sequence types in multi-locus sequence typing. These results indicate that crows do not share Salmonella serovars with broilers, and harbor various genotypes of C. jejuni that differ from those of broiler flocks. Thus, our results indicate that crows are not a potential vector of these bacteria to broiler flocks in this region.

Influenza A virus infection in Japanese wild boars (Sus scrofa leucomystax).

Serum samples were collected from 385 wild boars between 2010 and 2013 to examine the seroprevalence of influenza A virus (IAV) in Japan. Antibodies against IAV were identified using a commercial kit in 13 wild boars (3.4%). To identify the serotypes, positive sera were examined by virus-neutralization test using representative serotypes and strains. Three wild boars in Yamaguchi and four in Tochigi showed the highest antibody titers against the pandemic H1N1 2009 virus and classical swine H1N1 virus strains, respectively. These data indicate that wild boars may have close contact with humans and domestic pigs and therefore that there is potential for IAVs to reassort in wild boars as they have been shown to do in pigs.

First isolation and characterization of pteropine orthoreoviruses in fruit bats in the Philippines.

Pteropine orthoreovirus (PRV) causes respiratory tract illness (RTI) in humans. PRVs were isolated from throat swabs collected from 9 of 91 wild bats captured on the Mindanao Islands, The Philippines, in 2013. The nucleic acid sequence of the whole genome of each of these isolates was determined. Phylogenetic analysis based on predicted amino acid sequences indicated that the isolated PRVs were novel strains in which re-assortment events had occurred in the viral genome. Serum specimens collected from 76 of 84 bats were positive for PRV-neutralizing antibodies suggesting a high prevalence of PRV in wild bats in the Philippines. The bat-borne PRVs isolated in the Philippines were characterized in comparison to an Indonesian PRV isolate, Miyazaki-Bali/2007 strain, recovered from a human patient, revealing that the Philippine bat-borne PRVs had similar characteristics in terms of antigenicity to those of the Miyazaki-Bali/2007 strain, but with a slight difference (e.g., growth capacity in vitro). The impact of the Philippine bat-borne PRVs should be studied in human RTI cases in the Philippines.

Molecular phylogeny of a genetically divergent hantavirus harbored by the Geoffroy's rousette (Rousettus amplexicaudatus), a frugivorous bat species in the Philippines.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles (order Eulipotyphla, families Soricidae and Talpidae) prompted a further exploration of their host diversification and geographic distribution by analyzing lung tissues from 376 fruit bats representing six genera (order Chiroptera, suborder Yinpterochiroptera, family Pteropodidae), collected in the Republic of the Philippines during 2008 to 2013. Hantavirus RNA was detected by RT-PCR in one of 15 Geoffroy's rousettes (Rousettus amplexicaudatus), captured in Quezon Memorial National Park on Luzon Island in 2009. Phylogenetic analyses of the S, M and L segments, using maximum-likelihood and Bayesian methods, showed that the newfound hantavirus, designated Quezon virus (QZNV), shared a common ancestry with hantaviruses hosted by insectivorous bats, in keeping with their evolutionary relationships and suggests that ancestral bats may have served as the early or original mammalian hosts of primordial hantaviruses. As the first hantavirus detected in a megabat or flying fox species, QZNV extends our knowledge about the reservoir host range.

Detection of Campylobacter jejuni in rectal swab samples from Rousettus amplexicaudatus in the Philippines.

Bats are the second diversity species of mammals and widely distributed in the world. They are thought to be reservoir and vectors of zoonotic pathogens. However, there is scarce report of the evidence of pathogenic bacteria kept in bats. The precise knowledge of the pathogenic bacteria in bat microbiota is important for zoonosis control. Thus, metagenomic analysis targeting the V3-V4 region of the 16S rRNA of the rectal microbiota in Rousettus amplexicaudatus was performed using high throughput sequencing. The results revealed that 103 genera of bacteria including Camplyobacter were detected. Campylobacter was second predominant genus, and Campylobacter coli and Campylobacter jejuni were identified in microbiome of R. amplexicaudatus. Campylobacteriosis is one of the serious bacterial diarrhea in human, and the most often implicated species as the causative agent of campylobacteriosis is C. jejuni. Therefore, we investigated the prevalence of C. jejuni in 91 wild bats with PCR. As a result of PCR assay targeted on 16S-23S intergenic spacer, partial genome of C. jejuni was detected only in five R. amplexicaudatus. This is the first report that C. jejuni was detected in bat rectal swab samples. C. jejuni is the most common cause of campylobacteriosis in humans, transmitted through water and contact with livestock animals. This result indicated that R. amplexicaudatus may be a carrier of C. jejuni.

Detection of a novel herpesvirus from bats in the Philippines.

Bats are natural hosts of many zoonotic viruses. Monitoring bat viruses is important to detect novel bat-borne infectious diseases. In this study, next generation sequencing techniques and conventional PCR were used to analyze intestine, lung, and blood clot samples collected from wild bats captured at three locations in Davao region, in the Philippines in 2012. Different viral genes belonging to the Retroviridae and Herpesviridae families were identified using next generation sequencing. The existence of herpesvirus in the samples was confirmed by PCR using herpesvirus consensus primers. The nucleotide sequences of the resulting PCR amplicons were 166-bp. Further phylogenetic analysis identified that the virus from which this nucleotide sequence was obtained belonged to the Gammaherpesvirinae subfamily. PCR using primers specific to the nucleotide sequence obtained revealed that the infection rate among the captured bats was 30 %. In this study, we present the partial genome of a novel gammaherpesvirus detected from wild bats. Our observations also indicate that this herpesvirus may be widely distributed in bat populations in Davao region.

Altered social interactions in male juvenile cynomolgus monkeys prenatally exposed to bisphenol A.

Bisphenol A (BPA) is a widespread environmental contaminant, and humans are routinely exposed to BPA. We investigated whether prenatal exposure to BPA influences behavioral development in juvenile cynomolgus monkeys (Macaca fascicularis). Pregnant cynomolgus monkeys were implanted with subcutaneous pumps and exposed to 10µg/kg/day BPA or vehicle (control) from gestational day 20 to 132. Both BPA-exposed and control juvenile monkeys (aged 1-2years) were assessed using the peer-encounter test that was conducted to evaluate behaviors in social interaction with a same-sex, same-treatment peer. In the encounter test, prenatal BPA exposure significantly reduced environmental exploration and presenting, a gesture related to sexual reproduction, and increased visual exploration, but only in males; furthermore, it significantly reduced the typical sexual dimorphism of the aforementioned behaviors normally observed between male and female juvenile cynomolgus monkeys. This study demonstrates that prenatal BPA exposure affects behavioral development during adolescence and results in the demasculinization of key sexually dimorphic behaviors in male juvenile monkeys.

Ubiquitin C-terminal hydrolase l1 is expressed in mouse pituitary gonadotropes in vivo and gonadotrope cell lines in vitro.

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.

Molecular phylogenetic analysis of Orientia tsutsugamushi based on the groES and groEL genes.

DNA sequences encoding the GroES and GroEL proteins of Orientia tsutsugamushi were amplified by the PCR and sequenced. Pairwise alignment of full-length groES and groEL gene sequences indicated high sequence similarity (90.4-100% and 90.3-100%) in O. tsutsugamushi, suggesting that these genes are good candidates for the molecular diagnosis and phylogenetic analysis of scrub typhus. Comparisons of the 56-kD type-specific antigen (TSA) protein gene and the groES and groEL genes showed that genotypes based on the 56-kD TSA gene were not related to a cluster containing the groES and groEL genes in a dendrogram, suggesting that a gene rearrangement may be associated with homologous recombination in mites.

Bartonella jaculi sp. nov., Bartonella callosciuri sp. nov., Bartonella pachyuromydis sp. nov. and Bartonella acomydis sp. nov., isolated from wild Rodentia.

Four novel strains of members of the genus Bartonella, OY2-1(T), BR11-1(T), FN15-2(T) and KS2-1(T), were isolated from the blood of wild-captured greater Egyptian jerboa (Jaculus orientalis), plantain squirrel (Callosciurus notatus), fat-tailed gerbil (Pachyuromys duprasi) and golden spiny mouse (Acomys russatus). All the animals were imported to Japan as pets from Egypt, Thailand and the Netherlands. The phenotypic characterization (growth conditions, incubation periods, biochemical properties and cell morphologies), DNA G+C contents (37.4 mol% for strain OY2-1(T), 35.5 mol% for strain BR11-1(T), 35.7 mol% for strain FN15-2(T) and 37.2 mol% for strain KS2-1(T)), and sequence analyses of the 16S rRNA genes indicated that those strains belong to the genus Bartonella. Sequence comparisons of gltA and rpoB genes suggested that all of the strains should be classified as novel species of the genus Bartonella. In phylogenetic trees based on the concatenated sequences of five loci, including the 16S rRNA, ftsZ, gltA and rpoB genes and the ITS region, and on the concatenated deduced amino acid sequences of three housekeeping genes (ftsZ, gltA and rpoB), all strains formed distinct clades and had unique mammalian hosts that could be discriminated from other known species of the genus Bartonella. These data strongly support the hypothesis that strains OY2-1(T), BR11-1(T), FN15-2(T) and KS2-1(T) should be classified as representing novel species of the genus Bartonella. The names Bartonella jaculi sp. nov., Bartonella callosciuri sp. nov., Bartonella pachyuromydis sp. nov. and Bartonella acomydis sp. nov. are proposed for these novel species. Type strains of Bartonella jaculi sp. nov., Bartonella callosciuri sp. nov., Bartonella pachyuromydis sp. nov. and Bartonella acomydis sp. nov. are OY2-1(T) ( = JCM 17712(T) = KCTC 23655(T)), BR11-1(T) ( = JCM 17709(T) = KCTC 23909(T)), FN15-2(T) ( = JCM 17714(T) = KCTC 23657(T)) and KS2-1(T) ( = JCM 17706(T) = KCTC 23907(T)), respectively.