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Malignant melanoma is aggressive cancer with a high rate of local invasiveness and metastasis. Currently, the treatment options for patients with advanced-stage and metastatic oral melanoma are limited. A promising treatment option is oncolytic viral therapy. This study aimed to evaluate novel therapies for malignant melanoma using a canine model. Oral melanoma, which frequently occurs in dogs is used as a model for human melanoma, was isolated and cultured and used for the evaluation of the tumor lytic effect induced by viral infection. We constructed a recombinant Newcastle disease virus (rNDV) that promotes the extracellular release of IFNγ from the virus-infected melanoma. The expression of oncolytic and apoptosis-related genes, the immune response by lymphocytes, and IFNγ expression were evaluated in virus-infected melanoma cells. The results showed that the rate of rNDV infection varied according to the isolated melanoma cells and the oncolytic effect differed between melanoma cells owing to the infectivity of the virus. The oncolytic effect tended to be greater for the IFNγ-expressing virus than for the GFP-expressing prototype virus. Additionally, lymphocytes co-cultured with the virus showed induced expression of Th1 cytokines. Therefore, recombinant NDV expressing IFNγ is expected to induce cellular immunity and oncolytic activity. This oncolytic treatment shows promise as a therapeutic approach for melanoma treatment once evaluated using clinical samples from humans.

Borna disease virus (BoDV) is a neurotropic virus that causes several infections in humans and neurological diseases in a wide range of animals worldwide. BoDV-1 has been molecularly and serologically detected in many domestic and wild animals in Japan; however, the genetic diversity of this virus and the origin of its infection are not fully understood. In this study, we investigated BoDV-1 infection and genetic diversity in samples collected from animals in Hokkaido between 2006 and 2020. The analysis was performed by focusing on the P region of BoDV-1 for virus detection. The presence of BoDV-1 RNA was observed in samples of brain tissue and various organs derived from persistently infected cattle. Moreover, after inoculation, BoDV-positive brains were isolated from neonatal rats. The gene sequences of the P region of BoDV obtained from the rat brain were in the same cluster as the P region of the virus isolated from the original bovine. Thus, genetic variation in BoDV-1 was extremely low. The phylogenetic analysis revealed that BoDV-1 isolates obtained in this study were part of the same cluster, which suggested that BoDV-1 of the same cluster was widespread among animals in Hokkaido.

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014–2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan., Here, Kodama et al. describe the discovery, isolation and characterization of a novel tick-borne orthonairovirus, designated Yezo virus (YEZV), from patients with an acute febrile illness in Japan. Serological testing of wildlife and molecular screening of ticks suggest an endemic circulation of YEZV in Japan.

Mosquitoes are an important arthropod vector of arboviruses. Family Phenuiviridae includes several medically important arboviruses, such as Rift Valley fever phlebovirus and Toscana phlebovirus. Recent comprehensive genetic analyses identified many novel mosquito-specific viruses that are phylogenetically related to Phenuiviridae. We collected mosquitoes in Hokkaido, northern Japan, and conducted reverse transcription polymerase chain reactions (RT-PCRs) targeting the RNA-dependent RNA polymerase (RdRp) gene of Phenuiviridae. A total of 285 pools comprising 3,082 mosquitoes from two genera and eight species were collected. Partial RdRp sequences were detected from 97 pools, which allowed us to classify the viruses into three clusters provisionally designated Etutanne virus (ETTV) 1, 2, and 3. The closest related virus was Narangue virus (family Phenuiviridae, genus Mobuvirus), which was detected from Mansonia mosquitoes, and the similarities of its nucleotide and amino acid sequences with those of the ETTVs were 58.4%-66.2% and 64.7%-86.7%, respectively. PCRs and RT-PCRs using DNA and RNase digestion methods showed that the ETTVs were RNA viruses, but they do not form non-retroviral integrated RNA virus sequences in the mosquito genome.

Autoimmune, allergic, and respiratory inflammatory diseases are some of the most important health issues worldwide. Disorders of the gut microbiota have been associated with the induction of allergic and inflammatory diseases, and probiotics are being tested for disease prevention. We examined functional Lactiplantibacillus plantarum RGU (Lp-1) to mice with ovalbumin (OVA)-induced asthma model to elucidate the inhibitory effect on pathological progression in asthma model. Prior to the experiments, the intestinal lactic acid bacteria were reduced by administering multiple antibiotics (MAB) to evaluate the administration effect of lactic acid bacteria. Mice were administered with Lp-1 or comparative control lactic acid bacteria in each group. After that, OVA-induced asthma was induced, and cytokine gene expression and histological findings were compared. Exacerbation of lung lesions was confirmed in the MAB group. The Lp-1 group mice had alleviated lung lesions with a decrease in IL-1β, IL-13, IL-17 and an increase in IL-10 of the splenocytes and bronchial lymph nodes compared with the MAB group, but not in the other groups. In OVA-induced asthma, administration of specific Lactiplantibacillus was confirmed to induce anti-inflammatory cytokines.

We compared the Mx expression and anti-viral function and the 3D structure of Mx protein in four species: chicken (Gallus gallus), whooper swan (Cygnus cygnus), jungle crow (Corvus macrorhynchos), and rock dove (Columba livia). We observed different mortalities associated with highly pathogenic avian influenza virus (HPAIV) infection to understand the relationship between Mx function as an immune response factor and HPAIV proliferation in bird cells. Different levels of Mx were observed among the different bird species after virus infection. Strong Mx expression was confirmed in the rock dove and whooper swan 6 hr after viral infection. The lowest virus copy numbers were observed in rock dove. The virus infectivity was significantly reduced in the BALB/3T3 cells expressing rock dove and jungle crow Mx. These results suggested that high Mx expression and significant Mx-induced anti-viral effects might result in the rock dove primary cells having the lowest virus copy number. Comparison of the expected 3D structure of Mx protein in all four bird species demonstrated that the structure of loop L4 varied among the investigated species. It was reported that differences in amino acid sequence in loop L4 affect antiviral activity in human and mouse cells, and a significant anti-viral effect was observed in the rock dove Mx. Thus, the amino acid sequence of loop L4 in rock dove might represent relatively high anti-viral activity.

Babesia divergens is the major causal agent of zoonotic human babesiosis across Europe. Previously, we reported the detection of a B. divergens Asia lineage in wild sika deer (Cervus nippon) in Japan which was genetically closely related to the European B. divergens. To further elucidate its etiology, we conducted a large epidemiological survey by combining lineage-specific PCR system and blood direct PCR. The infection rate of the Asia lineage was 6.6% (116/1,747) throughout Japan, where Hokkaido (45%), Nagano (17%), Iwate (12%), Gunma (11%), and Yamanashi (11%) were highly enzootic (> 10%) among the 30 prefectures examined. European B. divergens was not detected. A geographical information system (GIS) map revealed dense populations of PCR-positive deer in the mountains including the Japanese Alps in eastern Honshu, and Hokkaido. These areas markedly overlapped with the major habitats of Ixodes persulcatus, a principal tick vector responsible for the transmission of the Asia lineage. Other areas in southern Japan including Miyazaki, Kagoshima, and Shimane Prefectures, where positive sika deer were sporadically detected, may be habitats for other tick species involved in the enzootic cycle as I. persulcatus were scarce. The rise in human babesiosis cases is occasionally attributed to healthy blood donors who were unaware of tick bites and Babesia infection. Therefore, there is an urgent need to investigate whether infections in humans have occurred in Japan.

The thermal stability of relevant viruses in gelatin liquid formulations for medical research and application is poorly understood. Bovine herpesvirus (BHV) was used as a model virus to examine the molecular weight (MW), concentration and gelatin type and to optimize virus stability in liquid formulations at 25 °C and 4 °C. Using the model virus stable liquid formulation, the stability of multiple enveloped and nonenveloped RNA and DNA viruses, including parainfluenza virus (PIV), reovirus (RV), BHV, and adenovirus (AdV), was monitored over up to a 30-week storage period. The BHV model virus was considered stable after 3 weeks in hydrolyzed gelatin (MW: 4000) with a 0.8 LRV (log10 reduction value) at 25 °C or a 0.2 LRV at 4 °C, compared to the stabilities observed in higher MW gelatin (60000 and 160000) with an LRV above 1. Based on the gelatin type, BHV in B-type gelatin samples were unexpectantly more stable than in A-type gelatin sample. All four viruses exhibited stability at 4 °C for at least 8 weeks, BHV or AdV remained stable for over 30 weeks of storage, and at 25 °C, AdV and RV remained stable for 8 weeks. The results demonstrated that 5% hydrolyzed gelatin can act as a relevant stabilizer for the thermal stability of viruses in medical research and application.

The Gobi Desert is a major source of Asian dust events, and the resulting health hazards have increased significantly in recent years. We reported that a variety of live bacteria were distributed in the Gobi Desert in relation to land use. Bacterial distribution was confirmed in the environment and on the land used by animals, however, bacterial saltation due to dust events has not been investigated in detail. In this study, to understand the distribution of surface bacteria in the atmosphere by dust saltation, live bacteria in four dust-generating areas in the Gobi area were monitored using an artificial dust generating device. The live bacteria were detected by experimental saltation at a wind speed of 6.5–8 m/s in all areas. A certain number of live bacteria are constantly saltated by dust events, and these bacteria depend on land use. Moreover, the bacterial saltation strain depended on land use and diversity, indicating that live bacteria are lifted into the environment by dust events. These findings indicate that dust events saltate environmental bacteria on the ground, suggest the risk of animal-derived bacterial saltation affected by land use, and present cross-border public health challenges to be considered in the future.

The Gobi Desert is a major source of dust events, whose frequency of occurrence and damage caused have recently significantly increased. In the present study, we investigated the types of live bacteria present in the surface soil of the Gobi Desert in Mongolia, and determined their genetic identification as well as their geographical distribution. During the survey, four different topographies (dry lake bed, wadi, well, and desert steppe) were selected, and land characteristics were monitored for moisture and temperature. The surface soil was aerobically cultured to isolate bacterial colonies, and their 16s rDNA regions were sequenced. The sequence data were identified through NCBI-BLAST analysis and generated phylogenetic trees. The results revealed two phyla and seven families of isolates from the sample points. Each isolate was characterized by their corresponding sample site. The characteristics of land use and soil surface bacteria were compared. Most of the bacteria originated from the soil, however, animal-derived bacteria were also confirmed in areas used by animals. Our findings confirmed the existence of live bacteria in the dust-generating area, suggesting that their presence could affect animal and human health. Therefore, it is necessary to further investigate dust microbes based on the One Health concept.

Background Hepatitis E virus (HEV) is a zoonotic disease and has been reported around the world. The main objective of this study was to evaluate the sero-prevalence and phylogenetic analysis of HEV in Vietnam. Pig blood and fecal pooled samples were collected to assess the prevalence of HEV. We assessed the true prevalence (TP) of HEV from apparent prevalence (AP) by taking into account the sensitivity and specificity of diagnostic tests using a Bayesian approach. For phylogenetic analysis, the data compared with worldwide HEV reference strains including all eight genotypes (G1-G8) which were identified in previous study. Results A total of 475 sera and 250 fecal pooled samples were collected at slaughterhouses and pig farms from five provinces, in Viet Nam. Overall, the sero-AP of HEV was 58.53% (95% confidence interval: 53.95–62.70) while the sero-TP was slightly higher (65.43, 95% credible interval: 47.19–84.70). In terms of pooled samples, overall, the RNA-AP was 6.80% (95% confidence interval: 4.01–10.66). One strain in Hanoi, two strains in Dak Lak, seven strains in An Giang, four strains in Son La and two strains in Nghe An were isolated. The phylogenetic tree demonstrated that 19 Vietnamese strains were clustered into HEV 3 and 4. Conclusions This study provided evidence that HEV is circulating in domestic pigs in Vietnam. From a public health perspective, it is very important to raise public awareness for high-risk groups (e.g. slaughterhouse workers, pig traders, farmers and market sellers) who have more opportunities to come in contact with pig and contaminated meats.

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.

Additional file 1.

Rabies is a fatal zoonosis, and in Vietnam, it remains problematic despite the availability of dog rabies vaccination. The purpose of this study was to clarify the socio-economic factors associated with voluntary rabies control measures among the general population using a “Knowledge, Attitudes, and Practice” framework to provide health and veterinary authorities in Vietnam with baseline information for better planning of policy supports. A questionnaire survey with interviews was conducted in 495 households (64 mountainous and 431 plain-area households) in Thai Nguyen Province in September 2016. After the survey, uni- and multivariable analyses were performed to detect factors associated with the practices of dog rabies vaccination and tethering dogs. Structural equation modelling (SEM) was performed to understand the structures associated with practice decisions. Contingent valuation was performed to calculate willingness-to-pay for vaccination. Vaccination coverage was 77.4% (724/935 dogs), and was significantly lower in mountainous areas dominated by ethnic minorities (63.8%, 67/105) than in plains (79.2%, 657/830, x2 = 11.7, df = 1, p Our study suggested that rabies education targeted to mountainous areas using local languages for ethnic minority groups as well as the national language, and higher subsidies on dog rabies vaccination for poor households may improve vaccination coverage. Dog management may be improved by promotion campaigns.

The composition of the intestinal microbiota is related to the health and immune function of the host. Administration of antibiotics affects the composition of the intestinal microbiota. However, the effects of immune function on the composition of the intestinal microbiota are still unclear. In this study, we investigated the lymphocyte composition and determined the relationships between lymphocyte function and the intestinal microbiota following antibiotic treatment in mice. To change the composition of the intestinal microbiota, mice were treated with or without antibiotics. Analysis of intestinal microbiota was performed by metagenomic analysis targeting 16S rRNA. Lymphocyte subsets of splenocytes were measured by flow cytometry. For functional analysis of T cells, splenocytes were stimulated with concanavalin (Con A), and cytokine gene expression was measured by real-time polymerase chain reaction. Firmicutes were predominant in the control group, whereas Bacteroidetes predominated in the antibiotic-treated group, as determined by metagenomic analysis. The diversity of the microbiota decreased in the antibiotic-treated group. Analysis of lymphocyte subsets showed that CD3(+) cells decreased, whereas CD19(+) cells increased in the antibiotic-treated group. All cytokine genes in splenocytes treated with Con A were downregulated in the antibiotic-treated group; in particular, genes encoding interferon-gamma, interleukin (IL)-6, and IL-13 significantly decreased. Taken together, these results revealed that changes in the composition of the intestinal microbiota by antibiotic treatment influenced the population of lymphocytes in splenocytes and affected the immune response.

Newcastle Disease Virus (NDV) is an RNA virus, which infects several tumor cells and shows cellular toxicity towards them. The antitumor activity of NDV has been reported in several tumors. In this study, we evaluated the antitumor effect of a NDV-infected melanoma cell vaccine on lung metastasis based on tumor-specific immune responses in a mouse model. B16 mouse melanoma cells were infected with the GFP-expressing recombinant NDV (rNDV) to prepare the vaccine (rNDV-BV). C57BL/6 mice were then immunized twice with rNDV-BV and intravenously inoculated with B16 cells, and the number of metastasis dots in the lungs was evaluated 21 days later. The mice were divided into three groups: pre-inoculation (mice were vaccinated before inoculation with B16 cells), post-inoculation (mice were vaccinated after inoculation with B16 cells), and control (mice were inoculated with DMEM) groups. To evaluate the immune responses, the mouse splenocytes were monitored for lymphocyte subsets and dendritic cells, and IFN-γ and IL-10gene expression after metastasis was measured. The mice receiving rNDV-BV showed prolonged survival and a lower number of metastasis dots. Furthermore, lung metastasis was significantly decreased upon post-metastasis vaccination with rNDV-BV. In pre-inoculation group, cytokine responses against tumor antigens were also significantly affected: IFN-γ levels were increased, but IL-10 levels were decreased. The vaccination also increased the T cell population along with the number of CD8+ dendritic cells during early metastasis. These results indicated that rNDV-BV induced an IFN-γ response against the tumor antigen and suppressed metastasis in the mouse model. Keywords: Autologous-tumor Vaccine; Newcastle Disease Virus; Melanoma Lung Metastasis Mouse Model; Cell-Mediated Immunity; IFN-γ; Abbreviations: NDV: Newcastle Disease Virus; rNDV: Recombinant Newcastle Disease Virus; rNDV-BV: Recombinant Newcastle Disease Virus to Prepare the Vaccine; B16: B16 Mouse Melanoma Cell; IFN: Interferon; IL: Interleukin.

Tick-borne encephalitis (TBE) and severe fever with thrombocytopenia syndrome (SFTS) are both tick-borne zoonotic diseases caused by TBE virus (TBEV) and SFTS phlebovirus (SFTSV). In 2016, a second domestic TBE case was reported in Hokkaido, Japan, after an absence of 23 years. We conducted IgG ELISA for TBEV and SFTSV on 314 deer (Cervus nippon yesoensis) serum samples collected from 3 places in Hokkaido. There were 7 seropositive samples for TBEV but none for SFTSV by ELISA. The specificity of the 7 positive samples was confirmed by neutralization tests against TBEV, and 5 sera showed 320 to 640 of 50% focus reduction endpoint titers. Our results provide information about the infectious status of TBEV in wild deer in Hokkaido, Japan.

Background Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. Methods We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. Results The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. Conclusion This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

Infectious diseases including those acquired through direct or indirect contact with people and livestock threaten the survival of wild great apes. Few studies have reported enterobacterial pathogens in chimpanzees. We used multiplex PCR to screen faeces of chimpanzees sharing a landscape with villagers and livestock in Bulindi, Uganda for Salmonella spp., enterohemorrhagic Escherichia coli (E. coli) and Shigella spp./enteroinvasive E. coli. All three potentially zoonotic pathogens were detected. Individual prevalence ranged between 7 and 20%, with most infections observed in mature male chimpanzees. These preliminary findings suggest detailed investigation of enterobacterial infections in people, primates and livestock in this ecosystem is warranted.

Hiroyuki Onodera,1 Takeru Urayama,2 Kazue Hirota,3 Kazuhiro Maeda,3 Ritsuko Kubota-Koketsu,3,4 Kazuo Takahashi,5 Katsuro Hagiwara,6 Yoshinobu Okuno,3 Kazuyoshi Ikuta,3,4 Mikihiro Yunoki,2,4,6 1Medical Information Department, 2Research and Development Division, Japan Blood Products Organization, Tokyo, 3Research and Development Division, The Research Foundation for Microbial Diseases of Osaka University, Kagawa, 4Former Department of Virology, Research Institute for Microbial Diseases, Osaka University, 5Virology Division, Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka, 6Pathogenic Risk Evaluation, Graduate School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan Abstract: Influenza viruses A/H1N1, A/H3N2, and B are known seasonal viruses that undergo annual mutation. Intravenous immunoglobulin (IVIG) contains anti-seasonal influenza virus globulins. Although the virus-neutralizing (VN) titer is an indicator of protective antibodies, changes in this titer over extended time periods have yet to be examined. In this study, variations in hemagglutination inhibition (HI) and VN titers against seasonal influenza viruses in IVIG lots over extended time periods were examined. In addition, the importance of monitoring the reactivity of IVIG against seasonal influenza viruses with varying antigenicity was evaluated. A/H1N1, A/H3N2, and B influenza virus strains and IVIG lots manufactured from 1999 to 2014 were examined. The HI titer was measured by standard methods. The VN titer was measured using a micro-focus method. IVIG exhibited significant HI and VN titers against all investigated strains. Our results suggest that the donor population maintains both specific and cross-reactive antibodies against seasonal influenza viruses, except in cases of pandemic viruses, despite major antigen changes. The titers against seasonal influenza vaccine strains, including past strains, were stable over short time periods but increased slowly over time. Keywords: IVIG, influenza, seasonal, neutralization, vaccine

The intestinal microbiota of newborn calves changes during the early postnatal period and influences their health and immune function. We studied the compositional changes in the intestinal microbiome of newborn calves during the first week after birth by metagenomic analysis. In feces from newborn calves, we identified 4 bacterial phyla, namely, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The relative abundance of Lactobacillaceae significantly increased from day 1 to day 7. We evaluated Lactobacillus spp. colony numbers using selective agar plates and confirmed that the abundance of Lactobacillus spp. significantly increased during the first 7 days after birth. In conclusion, Lactobacillus spp. colonized the intestinal tract of calves during the first 7 days after birth.

Babesia divergens is the major causal agent of zoonotic human babesiosis across Europe. Previously, we reported the detection of a B. divergens Asia lineage in wild sika deer (Cervus nippon) in Japan which was genetically closely related to the European B. divergens. To further elucidate its etiology, we conducted a large epidemiological survey by combining lineage-specific PCR system and blood direct PCR. The infection rate of the Asia lineage was 6.6% (116/1,747) throughout Japan, where Hokkaido (45%), Nagano (17%), Iwate (12%), Gunma (11%), and Yamanashi (11%) were highly enzootic (10%) among the 30 prefectures examined. European B. divergens was not detected. A geographical information system (GIS) map revealed dense populations of PCR-positive deer in the mountains including the Japanese Alps in eastern Honshu, and Hokkaido. These areas markedly overlapped with the major habitats of Ixodes persulcatus, a principal tick vector responsible for the transmission of the Asia lineage. Other areas in southern Japan including Miyazaki, Kagoshima, and Shimane Prefectures, where positive sika deer were sporadically detected, may be habitats for other tick species involved in the enzootic cycle as I. persulcatus were scarce. The rise in human babesiosis cases is occasionally attributed to healthy blood donors who were unaware of tick bites and Babesia infection. Therefore, there is an urgent need to investigate whether infections in humans have occurred in Japan.

Background Enterocytozoon bieneusi and Cryptosporidium spp. are prevalent zoonotic parasites associated with a high burden among children. To date only limited molecular epidemiological data on E. bieneusi and Cryptosporidium spp. in humans living in Thailand has been published. Methods PCR-based tools were used to detect and characterize E. bieneusi and Cryptosporidium spp. The internal transcribed spacer (ITS) region of the rRNA gene was used to investigate E. bieneusi, and the small subunit (SSU) rRNA gene was used to investigate Cryptosporidium spp., and 697 fecal samples from villagers and school children in rural areas in Thailand were analyzed. Results The infection rates were 2.15% (15/697) for E. bieneusi and 0.14% (1/697) for Cryptosporidium spp. The prevalence of E. bieneusi was significantly high in Loei province. Sequence analysis indicated that the Cryptosporidium isolate was C. parvum. Nine E. bieneusi genotypes were identified, EbpC, Peru12, TMH6, TMH3, TMH7, H, D, and two novel genotypes TMLH1 and TMLH2. E. bieneusi prevalence was significantly higher in male participants than in female participants, and in children aged 3–15 years than in participants aged > 15 years. Conclusions The prevalence, genotypes, and zoonotic potential of E. bieneusi were found to vary significantly high even in one country. Transmission routes and key animal carriers of E. bieneusi may be associated with differences in hygiene, sanitation, and cultural behaviors. Further molecular studies including longitudinal studies will be required to unveil epidemiological characteristics of these opportunistic intestinal protozoa in all over the countries.

The distribution and transport of windblown dust that occurred in Northeast Asia from 28 March to 2 April 2012 was investigated. Data of particulate matter less than 10 micrometers (PM10) near the surface and light detection and ranging (LiDAR) measurements from the ground up to 18 km were used in the study. A severe dust event originated over southern Mongolia and northern China on 28 March 2012, and the widespread dust moved from the source area southeastward toward Japan over several days. Windblown dust reached Japan after two days from the originating area. LiDAR measurements of the vertical distribution of the dust were one to two km thick in the lower layer of the atmosphere, and increased with the increasing distance from the source area.

Monoclonal antibodies (mAbs) that recognize cluster of differentiation (CD) molecules on lymphocytes are useful tools for the study of different lymphocyte subsets in flow cytometry (FCM) analysis. CD4 is a glycoprotein found on the surfaces of helper T cells, monocytes, macrophages, and dendritic cells. In this study, we describe Japanese Black (JB) calves in a farm whose peripheral blood mononuclear cells (PBMCs) did not react with a CD4-specific mAb. To identify calves with PBMCs with low mAb reactivity, PBMCs from 21 JB calves (1–12 months of age) bred at the same farm were examined using two different bovine CD4 mAbs (clones #CC8 and #CACT138A). FCM analysis indicated that the calves fell into two groups based on reactivity against the two mAbs, i.e., double-positive (DP) calves, whose PBMCs were recognized by both mAbs clones, and single-positive (SP) calves, whose PBMCs were only recognized by #CACT138A. PBMCs from seven calves were not recognized by #CC8, although they had normal reactivity with another mAb, #CACT138A. Sequencing analysis of the CD4 gene in these calves revealed four nucleotide substitutions (G918 T, A930C, G970A, and G1074A) in the coding region in the SP group when compared to the DP group. Three of the four mutations were associated with amino acid substitution (Q306H, K310 N, and A324 T). The substitution at A324 T was located in the D4 domain of CD4 gene. Homology modeling based on the amino acid sequences revealed that the surface structure of this part of the molecule was significantly different between the SP and the DP groups. Therefore, the epitope recognized by the #CC8 CD4 mAb was altered in calves with this genetic mutation, and this led to the low reactivity of the PBMCs from calves in the SP group aginst the #CC8 mAb. In conclusion, this is the first study to identify CD4 variants in JB cattle. We confirmed that the variants did not affect lymphocyte functions, such as mitogen stimulation or lipopolysaccharide-induced cytokine gene expression.

Bacterial keratitis of the horse is mainly caused by staphylococci, streptococci, and pseudomonads. Of these bacteria,Pseudomonas aeruginosasometimes causes rapid corneal corruption and, in some cases, blindness. Antimicrobial resistance can make treatment very difficult. Therefore, new strategies to control bacterial infection are required. A bacteriophage (phage) is a virus that specifically infects and kills bacteria. Since phage often can lyse antibiotic-resistant bacteria because the killing mechanism is different, we examined the use of phage to treat horse bacterial keratitis. We isolatedMyoviridaeorPodoviridaephages, which together have a broad host range. They adsorb efficiently to host bacteria; more than 80% of the ΦR18 phage were adsorbed to host cells after 30 s. In our keratitis mouse model, the administration of phage within 3 h also could kill bacteria and suppress keratitis. A phage multiplicity of infection of 100 times the host bacterial number could kill host bacteria effectively. A cocktail of two phages suppressed bacteria in the keratitis model mouse. These data demonstrated that the phages in this study could completely prevent the keratitis caused byP. aeruginosain a keratitis mouse model. Furthermore, these results suggest that phage may be a more effective prophylaxis for horse keratitis than the current preventive use of antibiotics. Such treatment may reduce the use of antibiotics and therefore antibiotic resistance. Further studies are required to assess phage therapy as a candidate for treatment of horse keratitis.IMPORTANCEAntibiotic-resistant bacteria are emerging all over the world. Bacteriophages have great potential for resolution of this problem. A bacteriophage, or phage, is a virus that infects bacteria specifically. As a novel therapeutic strategy against racehorse keratitis caused byPseudomonas aeruginosa, we propose the application of phages for treatment. Phages isolated in this work hadin vitroeffectiveness for a broad range ofP. aeruginosastrains. Indeed, a great reduction of bacterial proliferation was shown in phage therapy for mouse models ofP. aeruginosakeratitis. Therefore, to reduce antibiotic usage, phage therapy should be investigated and developed further.

Background and objectives Human hepatitis E virus (HEV) is prevalent worldwide. Iatrogenic HEV has recently been identified based on the reports of transfusion-transmitted cases. The detection rate of HEV-RNA and seroprevalence of HEV-IgG/IgM have been regionally evaluated in Japan, and donor plasma collected in Hokkaido is currently screened by nucleic acid amplification testing. However, the detection rate of HEV-RNA in blood donors in Japan outside of Hokkaido has not been reported. Materials and Methods A total of 620 140 qualified donor plasma samples from Japanese regions excluding Hokkaido were tested for HEV-RNA (pools of 50 or 500) between 2004 and 2014. HEV-RNA-positive plasma bags were identified, and the HEV viral load, genotype and anti-HEV immunoglobulin (Ig)G/IgM were evaluated. Results The detection rate of HEV-RNA (pools of 50) was 1/15 075 and higher in eastern than in western Japan. All 36 HEV-RNA-positive samples were genotype 3 with viral load ranging from

Borna disease virus (BDV) is a virus that causes a neurological disease in domestic animals, including a variety of animal species in Japan. Few studies have examined the mode of transmission of this virus in cattle, and the exact mechanisms underlying the transmission of the virus need to be elucidated. This study aimed to examine the contribution of vertical transmission of the virus, which occurs when the virus is transmitted from the mother to offspring during gestation or birth. We used an epidemiological approach. The relative risk (RR) was calculated for cattle born to BDV sero-positive cows from farms with a higher within-herd prevalence of BDV (56.8%). We tested the sera of 1,122 dairy cattle from 24 dairy herds in Hokkaido Prefecture, Japan, for BDV infection using the ELISA and western blotting method. The overall level of BDV sero-prevalence was 22.1%. Seroprevalence was significantly higher in closed-breeding herds that do not have buying in cows (39.7%) than in farms that restock cattle by buying in cows (4.4%, P

Newcastle disease virus (NDV) is an oncolytic virus. As immunogenicity of tumor cells is enhanced by NDV infection, recombinant NDV-infected tumor vaccines (rNDV-TV) are effective methods for inducing specific immunity. However, several tumor cells resist NDV infection, and tumor specific immunity is not sufficiently induced by rNDV-TV. Therefore, we clarified the factor contributing to the suppression of NDV infection and attempted to improve rNDV-TV. Initially we investigated the correlation between the NDV infection rate and interferon-related gene expression in six murine tumor cell lines. A significant negative correlation was observed between the constitutive gene expression of Interferon-stimulated genes (ISGs) and NDV infectivity. The NDV infection rate was examined in each tumor cell treated with the Janus kinase (JAK) inhibitor ruxolitinib (Rux). Furthermore, we evaluated the Th1 response induction by Rux-treated rNDV-TV (rNDV-TV-Rux). In Rux-treated tumor cells, Oasl2 gene expression was significantly decreased and viral infectivity was increased. In immunized mice, the number of CD8+ cells, and those expressing the IFN-&gamma, gene, were significantly increased as compared with Rux-untreated rNDV-TV. The infectivity of the virus was dependent on the degree of ISGs expression in tumor cells. To remedy for this problem, rNDV-TV-Rux was expected to have a Th1 immune response.

The objectives of our research on equine piroplasmosis were to clarify the prevalence of the piroplasms Babesia caballi and Theileria equi among reintroduced Przewalski's horses (takhi, Equus ferus przewalskii) in comparison with horses (Equus caballus) of nomads from around Hustai National Park in Mongolia and to determine the mortality among takhi during 2012 to 2015. The prevalence of T. equi was high in 2014 at 80% among nomadic horses and 84% among takhi. We found no evidence of B. caballi infection among takhi. A homology comparison of the 18S rRNA base sequence of T. equi showed the same sequence in both domestic horses and takhi. According to the national park records, piroplasmosis is a cause of high mortality in young takhi (19%). The results suggested that takhi were infected with T. equi in this park, and the infection is an important issue when reintroducing the takhi into the wild and increasing the numbers of the animals in nature. Continuous monitoring and vector control were noted as important issues in future conservation programs.

This study shows the results of field experiments of soil particles saltation and laboratory experiments of imaging of the surface structure of dust particles. In the Gobi area, dust occurs when the wind speed at ground level exceeds 7 m/s. It has been reported that bacteria are attached to dust, but the details of its attachment are unknown. It is also expected that these bacteria will fly at the time of occurrence of dust, and fundamental research is important to clarify the relationship between dust components and bacteria.