Japanese Strains Research Articles

Nematode isolation from Creophilus maxillosus (L.) (Staphilinidae) with a description of Rhabditidoides eutrophicola n. sp. (Diplogastridae) and some notes on Diplogastrellus gracilis (Bütschli, 1876) Paramonov, 1952 (Diplogastridae)

Summary Nematodes associated with the carcass beetle Creophilus maxillosus were examined. The beetles were collected from the decomposed body of a dead racoon dog Nyctereutes procyonoides in Kyoto, Japan, and dissected for examination of internal parasites. The dissected beetles were kept on agar plates for isolation of phoretic species. Although no parasites were found, three species of phoretic associates, Caenorhabditis plicata, Diplogastrellus cf. gracilis and Rhabditidoides sp., were recognised, and two species were later successfully cultured as laboratory strains. Diplogastrellus cf. gracilis and Rhabditidoides sp. were compared to an authentic strain of D. gracilis (German strain, SB306) and two presumably conspecific strains (German strain RS5443 and Japanese strain NKZ346), respectively, by mating experiments and molecular sequence comparison. The comparisons revealed that D. cf. gracilis is conspecific to D. gracilis SB306, and three Rhabditidoides strains are new conspecific species. The typological and molecular characters of D. gracilis are described and compared with other populations of the species, and Rhabditidoides sp. is taxonomically described as Rhabditidoides eutrophicola n. sp.

First report of Erwinia pyrifoliae causing flower blight and fruit rot on greenhouse-grown Fragaria × ananassa in the United States.

Erwinia pyrifoliae causes disease of pear (Pyrus spp.), apple (Malus spp.), and strawberry (Fragaria × ananassa) (Wenneker and Bergsma-Vlami 2015), which are economically important commodities in the US. Disease symptoms on pear and apple are indistinguishable from those caused by the non-quarantine fire blight pathogen, E. amylovora (Kim et al. 1999), which also causes disease on strawberries (Atanasova et al. 2005). Samples of greenhouse-grown strawberry 'Albion' from Ohio were submitted to the Purdue Plant and Pest Diagnostic Lab in December 2023. Fruits were stunted with brown lesions, while sepals and pedicels had brown-black water-soaked lesions. Cut fruit exuded bacterial ooze from the main vascular bundle. Bacterial streaming was observed microscopically from symptomatic tissue which tested positive with the E. amylovora ImmunoStrip® (Agdia Inc., Elkhart, IN); reported by the manufacturer to cross-react with E. pyrifoliae. Isolation from symptomatic tissue produced pure cultures on sucrose peptone agar and Kings medium B after incubation at 27°C for 48 hr, and colonies appeared circular and white/opaque. Crude DNA extractions were prepared by boiling colony suspensions in Tris-EDTA buffer. Two independent real-time PCR tests specific for E. pyrifoliae (Lehman et al. 2008; Yasuhara-Bell et al. 2024) produced positive results. Conventional PCR using an E. pyrifoliae-specific primer set targeting a divergent region between pstS and glmS genes (Wensing et al. 2011) also produced positive results. The amplicon was Sanger-sequenced and deposited into NCBI GenBank (Accession PP757383). BLASTn analysis using the Nucleotide collection and Whole-genome shotgun contigs revealed top matches (100% query coverage; 97.5% identity to type strain DSM 12163) with E. pyrifoliae only; next closest match was E. amylovora (53% query coverage). To confirm Koch's postulates, immature fruit of six healthy strawberry 'Albion' plants were wounded with a sterile pipette tip and then submersed in a bacterial suspension in sterile deionized water (DI H2O) (3.1×107 cells/ml). Fruit of six additional plants were mock inoculated using sterile DI H2O. Plants were placed in plastic bags for 48 hr at room temperature with a 12-hr photoperiod. Symptoms were first observed on inoculated plants 1.5 days post-inoculation (DPI). Brown discoloration was observed within fruit and as spreading lesions on fruit pedicels by 4 DPI; mock-inoculated plants remained asymptomatic. Bacterial streaming from symptomatic tissue allowed successful re-isolation of the bacterium. Molecular testing confirmed isolates to be E. pyrifoliae, thus completing Koch's postulates. Following initial confirmation, additional samples of infected strawberry ('Albion' and 'GB96') from the same greenhouse were confirmed positive for E. pyrifoliae by molecular testing and sequencing. To our knowledge this is the first time Erwinia pyrifoliae was detected in the US. There are many known pathways of introduction from Asia and Europe; however, pstS-glmS sequence comparison with strawberry isolates from the Netherlands (sequences provided by M.J.C. Pel) suggests this US strawberry strain is unique, but most closely related to Japanese strains (98.5% identity). Potential origin of this strain is unknown, but comparative genomics studies to investigate relatedness among strains are planned.

Identification and Full-Length Sequence Analysis of a Novel Recombinant Goat Astrovirus Genotype in Guangxi, China.

Astroviruses are single-stranded, positive-sense RNA viruses capable of infecting humans as well as a wide range of mammalian and avian species, with a length of approximately 6.6-7.7 kb. In this study, 139 goat fecal samples collected from the Guangxi province were used for the RT-PCR detection, and two of these were positive for goat astrovirus, with a positivity rate of 1.44% (2/139). The complete genome sequence of an astrovirus strain and the partial genome sequence of a strain astrovirus, named GX WZ 2023 and GX HC 2023, were amplified and sequenced, and their sequence lengths were 6284 nt and 6213 nt, respectively. Among them, the capsid protein of goat astrovirus GX HC 2023 showed the highest amino acid identity of 95.9% with ovine astrovirus GX, which belonged to the MAstV-2 genotype. However, the closest relative of the GX WZ 2023 strain was found to be the caprine astrovirus Sichuan, with a nucleotide sequence identity of 76.8%. The ORF1ab nonstructural protein of this strain showed the highest amino acid identities of 89.2 and 95.8% with the ovine astrovirus S5.1 and caprine astrovirus G5.1 strains, respectively. However, its ORF2 capsid protein has 68.4% amino acid identity with the bovine astrovirus (BAstV) 16 2021 CHN strain and only 21.9-64% amino acid identity with all available strains of goat astrovirus. The GX WZ 2023 strain was recombined with the Chinese (BAstV 16 2021 CHN) and Japanese bovine strains (BAstV JPN 2015) in the ORF2 region. Therefore, the goat astrovirus GX WZ 2023 is proposed as a new member of the family goat astroviridae based on the species classification criteria of the International Committee on Taxonomy of Viruses. These findings enhance our understanding of the prevalence and genetic evolution of goat astrovirus and provide a scientific basis for future studies of these viruses in other animals.

Integrative transcriptomics and metabolomics analysis provide insight into the formation of skin pigmentation diversity in Chinese soft-shelled turtle (Pelodiscus sinensis)

Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater breeding species in East and Southeast Asia. The most external differences among different strains are skin pigmentation and blotches, but the mechanism of their formation is unknown. To investigate the mechanism of skin pigmentation diversity in Chinese soft-shelled turtle, three unique phenotypes (Golden strain, Japanese strain and Qingxi black strain) were selected to observe the chromatophores, and the transcriptome and metabolome were compared. The results showed the presence of melanophores, xanthophores and iridophores in the skin of all three strains. However, there were differences in the composition and organelles of chromatophores. The upregulation of gene asip and elevated accumulation of glutathione and cysteine in Golden strain resulted in the production of pheomelanin, leading to yellow pigmentation in the skin. The upregulation of gene dct and elevated accumulation of indole-5, 6-quinone in Qingxi black strain resulted in the production of eumelanin, which made the skin appear black-brown. Japanese strain produced eumelanin and pheomelanin, however, the presence of melanosomes resulted in skin darkening and appeared dark green pigmentation. The white blotches of Gloden strain and Japanese strains were formed through increased production of iridophores, while the black blotches of Qingxi black strain were formed by inducing migration and apoptosis of melanophores within iridophores. In conclusion, the distinctive characteristics of the skin in three strains resulted from combination of melanin type, abundance of melanosomes and interaction between melanophores and iridophores. These results provided valuable reference for investigating the mechanisms underlying skin pigmentation diversity in Chinese soft-shelled turtle and other vertebrates.

Outbreak of equine herpesvirus 4 (EHV-4) in Denmark: tracing patient zero and viral characterization

BackgroundEquine herpesvirus 4 (EHV-4) causes respiratory disease in horses, and the virus is considered endemic in the global equine population. However, outbreaks can occur when several horses are gathered in relation to shows, competitions, breeding units and at hospitals. In the spring year 2022, an EHV-4 outbreak occurred at the Large Animal Teaching Hospital, University of Copenhagen, Denmark. Nine horses were tested EHV-4 positive during the outbreak, which lasted approx. seven weeks. In addition, a tenth horse “Eq10” tested EHV-4 positive almost three weeks after the last of the outbreak horses tested positive. Detailed clinical registrations were obtained from all ten horses as well as their location and movement during hospitalization. Nasal swabs were obtained throughout the outbreak and tested by qPCR for EHV-4. Additionally, pre- and post-infection sera were tested for the presence of EHV-4 antibodies. Selected samples were characterized by partial and full genome sequencing.ResultsThe most common clinical signs of the EHV-4 infected horses during this outbreak were pyrexia, nasal discharge, mandibular lymphadenopathy and increased lung sounds upon auscultation. Based on the locations of the horses, EHV-4 detection and antibody responses the most likely “patient zero” was identified as being “Eq1”. Partial genome sequencing revealed that Eq10 was infected by another wild type EHV-4 strain, suggesting that the hospital was able to eliminate the outbreak by testing and reinforcing biosecurity measures. The complete genome sequence of the outbreak strain was obtained and revealed a closer relation to Australian and Japanese EHV-4 strains rather than to other European EHV-4 strains, however, very limited sequence data are available from Europe.ConclusionThe study illustrated the transmission of EHV-4 within an equine facility/hospital and provided new insights into the viral shedding, antibody responses and clinical signs related to EHV-4 infections. Finally, sequencing proved a useful tool in understanding the transmission within the hospital, and in characterizing of the outbreak strain.

Genetic Characterization of Palyam Serogroup Viruses Isolated in Japan from 1984 to 2018 and Development of a Real-Time RT-PCR Assay for Broad Detection of Palyam Serogroup Viruses and Specific Detection of Chuzan (Kasba) and D'Aguilar Viruses.

We performed whole genome sequencing (WGS) of 15 Palyam serogroup virus (PALV) strains isolated from cattle or Culicoides biting midges in Japan from 1984 to 2018. We found that the PALV strains consisted of Chuzan (Kasba) virus (CHUV), D'Aguilar virus (DAGV), Bunyip Creek virus, and another PALV, Marrakai virus (MARV). The Japanese MARV strains isolated in 1997 were closely related to Australian PALV strains isolated in 1968-1976 in genome segments 2 and 10, but they were most closely related to other Japanese PALV strains in the other genome segments. Our data suggest that the Japanese MARV strains were reassortant viruses between Asian and Australian PALVs. In addition to the WGS, we developed a real-time reverse-transcription polymerase chain reaction assay that can broadly detect PALV and specifically detect CHUV and DAGV, utilizing the data obtained by the WGS in this study. We detected the DAGV gene in bovine stillborn fetuses and congenitally abnormal calves in 2019 using the newly developed assay. To our knowledge, this is the first report of isolation of MARV outside of Australia and the first report of detection of PALV in bovine fetuses or calves with congenital abnormality outside of Africa.

Complete genome sequence of Japanese RSSC phylotype-I strains infecting different host plants.

Ralstonia solanacearum species complex (RSSC) shows a broad host range and is classified into four phylotypes. To compare type III effectors, we have determined the complete genome sequences of several RSSC strains, especially phylotype-I strains isolated in Japan, with different host specificity.

Long-read genome assembly of the Japanese parasitic wasp Copidosoma floridanum (Hymenoptera: Encyrtidae).

Copidosoma floridanum is a cosmopolitan species and an egg-larval parasitoid of the Plusiine moth. C. floridanum has a unique development mode called polyembryony, in which over two thousand genetically identical embryos are produced from a single egg. Some embryos develop into sterile soldier larvae precociously, and their emergence period and aggressive behavior differ between the US and Japanese C. floridanum strains. Genome sequencing expects to contribute to our understanding of the molecular bases underlying the progression of polyembryony. However, only the genome sequence of the US strain generated by the short-read assembly has been reported. In the present study, we determined the genome sequence of the Japanese strain using Pacific Biosciences high-fidelity reads and generating a highly contiguous assembly (552.7 Mb, N50: 17.9 Mb). Gene prediction and annotation identified 13,886 transcripts derived from 10,786 gene models. We searched the genomic differences between US and Japanese strains. Among gene models predicted in this study, 100 gene loci in the Japanese strain had extremely different gene structures from those in the US strain. This was accomplished through functional annotation (GGSEARCH) and long-read sequencing. Genomic differences between strains were also reflected in amino acid sequences of vasa that play a central role in caste determination in this species. The genome assemblies constructed in this study will facilitate the genomic comparisons between Japanese and US strains, leading to our understanding of detailed genomic regions responsible for the ecological and physiological characteristics of C. floridanum.

The effect of silkworms (Bombyx mori) chitosan on rumen fermentation, methanogenesis, and microbial population in vitro.

Ruminant enteric methane (CH4) is one of the largest sources of greenhouse gases that contribute to global warming. To minimize environmental harm caused by ruminants' CH4 production, natural substances can be used to suppress it. Chitosan from crustacean sources had been known to obstruct CH4 generation in the rumen. About 18% of silkworm pupae is chitin, but little is known about the impact of silkworm pupae chitosan on rumen methanogenesis. This study investigated the efficacy of the silkworm chitosan extraction method and its impact on rumen fermentation, methanogenesis, and microbial growth in vitro. This study employed a randomized complete block design featuring five treatments and four batches for rumen incubation as the blocking factor. In this study, five treatments were implemented: Control (CO) (basal diet with no added chitosan), basal diet with 6% chitosan from the Chinese Silkworm strain 804 (CHI804), basal diet with 6% chitosan from the PS 01 Hybrid Silkworm strain (CHIPS01), basal diet with 6% chitosan from the Hybrid F1 Japanese 102 × Chinese 202 races (CHIJC02), and basal diet with 6% commercial shrimp shell chitosan as the positive control (CHICOMM). The in vitro experiments assessed digestibility, pH, total gas generation, CH4 production, ammonia nitrogen (NH3-N), and short-chain fatty acid levels, along with microbial population. Data were analyzed using a general linear model followed by Duncan's test when applicable. A significant effect on dry matter digestibility (DMD), total gas production, CH4, NH3-N, and rumen microbial populations (Methanogens, Ruminoccocus albus, Ruminoccocus flavefaciens, Selonomonas ruminantium, Butyrivibrio fibrisolvens, Streptoccocus bovis, Prevotella spp., and Bacteroides spp.) was observed (p < 0.05). The extracted chitosan (CHIJC02) used in this study exhibited a similar quality to that of commercial chitosan (CHICOMM). CHI804 treatment could reduce gas production, NH3-N production, and B. fibrisolvens population significantly (p < 0.05), while CHIJC02 could reduce CH4 production, methanogen population, acetate (C2) production, and increase propionate (C3) production significantly (p < 0.05). CHIJC02 and CHICOMM treatments could also increase the population of R. flavefaciens, S. ruminantium, and Bacteroides spp. significantly (p < 0.05). Chitosan addition significantly (p < 0.05) reduced DMD but did not impact organic matter digestibility or pH. The extracted chitosan mimics commercial chitosan in physico-chemical properties. Chitosan derived from Japanese and Chinese F1 hybrid silkworm strains demonstrated superior capacity for inhibiting CH4 generation compared to commercial chitosan. The quality and effects on methanogenesis, rumen fermentation, and rumen microbial populations can differ depending on the origin of chitosan.

Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Prevalence and Evolutionary Characteristics of Bovine Coronavirus in China.

Bovine coronavirus (BCoV), bovine rotavirus, bovine viral diarrhea virus, and bovine astrovirus are the most common intestinal pathogenic viruses causing diarrhea in cattle. We collected 1646 bovine fecal samples from January 2020 to August 2023. BCoV was the major pathogen detected, with a positive rate of 34.02% (560/1646). Of the 670 diarrheal samples and 976 asymptomatic samples, 209 and 351 were BCoV-positive, respectively. Studying the relevance of diarrhea associated with BCoV has shown that the onset of diarrheal symptoms post-infection is strongly correlated with the cattle's age and may also be related to the breed. We amplified and sequenced the hemagglutinin esterase (HE), spike protein, and whole genomes of the partially positive samples and obtained six complete HE sequences, seven complete spike sequences, and six whole genomes. Molecular characterization revealed that six strains were branched Chinese strains, Japanese strains, and partial American strains from the GⅡb subgroup. Strains HBSJZ2202 and JSYZ2209 had four amino acid insertions on HE. We also analyzed ORF1a and found disparities across various regions within GIIb, which were positioned on separate branches within the phylogenetic tree. This work provides data for further investigating the epidemiology of BCoV and for understanding and analyzing BCoV distribution and dynamics.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Genomic properties of a Bartonella quintana strain from Japanese macaque (Macaca fuscata) revealed by genome comparison with human and rhesus macaque strains

Bartonella quintana, the causative agent of trench fever, is an intracellular bacterium that infects human erythrocytes and vascular endothelial cells. For many years, humans were considered the only natural hosts for B. quintana; however, it was recently discovered that wild Japanese macaques (Macaca fuscata) also serve as hosts for B. quintana. To elucidate the genetic characteristics of the B. quintana strain MF1-1 isolated from a Japanese macaque, we determined the complete genome sequence of the strain and compared it with those of strain Toulouse from a human and strain RM-11 from a rhesus macaque. General genomic features and orthologous gene cluster profiles are similar among the three strains, and strain MF1-1 is genetically closer to strain RM-11 than strain Toulouse based on the average nucleotide identity values; however, a significant inversion of approximately 0.68 Mb was detected in the chromosome of strain MF1-1. Moreover, the Japanese macaque strains lacked the bepA gene, which is responsible for anti-apoptotic function, and the trwL2, trwL4, and trwL6 genes, which may be involved in adhesion to erythrocytes of rhesus macaque and human. These features likely represent the genomic traits acquired by Japanese macaque strains in their host-associated evolution.

The relationship between lysozyme gene polymorphism and quality changes during the storage of eggs derived from 2 commercial strains of Japanese quail

During the storage irreversible changes occur in eggs that result in a deterioration of their quality. The most significant changes affect the albumen. One of the major proteins of albumen present in egg white is lysozyme, which protects the embryo from microorganisms. This enzyme also contributes to the qualitative characteristics of albumen. It is possible that its polymorphism also affects the quality and stability of the obtained raw material that is, table eggs. Therefore, the aim of this study was to assess the potential effect of polymorphism in the lysozyme gene and protein on the quality changes during the storage of eggs derived from 2 genetic strains of Japanese quail belonging to various utility types. Eggs from selected females of laying and meat-type breeds were stored for 14 wk. During this period the egg quality traits were evaluated 10 times. DNA was isolated from each female and all exons of the lysozyme gene had been sequenced. In total, fourteen SNPs' and one 4-bp indel mutation were identified in exons and adjacent intronic sequences, among which SNP1 (1:32140723) resulted in a substitution of lysine with glutamine (Q21K). The results showed that SNP1 (strain S22), as well as the SNP2, SNP5, SNP7, SNP8, SNP10, SNP11, SNP12 and SNP13 were significantly associated with breaking strength during egg storage in both investigated Japanese quail strains. Furthermore, a 3 haplotype blocks containing nine SNPs (2, 5, 6, 7, 8, 10, 11, 12 and 13) were identified. These blocks displayed 8 distinct haplotypes that had significant association with breaking strength at all storage time points where egg quality analyses were performed. The study also revealed significant effects of breed and storage time on the egg quality traits. These results provide new insights into the genetic basis of egg quality during storage and could be incorporated into the breeding programs involving these strains.

Isolation and whole-genome sequence analysis of Balagodu virus in Japan.

Whole-genome sequencing of a virus isolated from Culicoides biting midges in southern Japan in 2020 revealed that it is a strain of Balagodu virus (BLGV; genus Orthobunyavirus; family Peribunyaviridae; order Bunyavirales). A solitary instance of BLGV isolation occurred in India in 1963. All assembled segments comprise complete protein-coding sequences that are similar to those of other orthobunyaviruses. The consensus 3'- and 5'-terminal sequences of orthobunyaviruses' genomic RNAs are also conserved in the Japanese BLGV strain. Here, we update the geographic distribution of BLGV and provide its complete sequence, contributing to the clarification of orthobunyavirus phylogeny.

High-quality genome of the zoophytophagous stink bug, Nesidiocoris tenuis, informs their food habit adaptation.

The zoophytophagous stink bug, Nesidiocoris tenuis, is a promising natural enemy of micro-pests such as whiteflies and thrips. This bug possesses both phytophagous and entomophagous food habits, enabling it to obtain nutrition from both plants and insects. This trait allows us to maintain its population density in agricultural fields by introducing insectary plants, even when the pest prey density is extremely low. However, if the bugs' population becomes too dense, they can sometimes damage crop plants. This dual character seems to arise from the food preferences and chemosensation of this predator. To understand the genomic landscape of N. tenuis, we examined the whole genome sequence of a commercially available Japanese strain. We used long-read sequencing and Hi-C analysis to assemble the genome at the chromosomal level. We then conducted a comparative analysis of the genome with previously reported genomes of phytophagous and hematophagous stink bugs to focus on the genetic factors contributing to this species' herbivorous and carnivorous tendencies. Our findings suggest that the gustatory gene set plays a pivotal role in adapting to food habits, making it a promising target for selective breeding. Furthermore, we identified the whole genomes of microorganisms symbiotic with this species through genomic analysis. We believe that our results shed light on the food habit adaptations of N. tenuis and will accelerate breeding efforts based on new breeding techniques for natural enemy insects, including genomics and genome editing.

Analysis of the complete genome characterization of 11 human astrovirus strains in Shandong Province

Objective: To study the complete genome characterization of Human Astrovirus (HAstV) in Shandong Province. Methods: Stool samples from acute flaccid paralysis (AFP) surveillance in Shandong Province from 2020 to 2022 were collected, and HAstV nucleic acid was examined by real-time quantitative PCR (qPCR). Next-generation sequencing (NGS) was conducted for the positive samples to obtain complete genome sequences and identify the genotype. Homology comparison and phylogenetic analysis were performed by using BioEdit and Mega software. Results: A total of 667 samples were examined by qPCR, of which 14 were HAstV-positive (2.1%), including HAstV-1 (n=6), MLB1 (n=6), MLB2 (n=1), and VA2 (n=1). The complete genome sequences were obtained from 11 samples. The six HAstV-1 sequences of this study had 98.2% to 99.9% nt similarities with each other and 87.6% to 98.6% with those from other regions. The four MLB1 sequences of this study had 99.1% to 99.9% nt similarities with each other and 92.2% to 99.4% with those from other regions. The VA2 sequence of this study had 96.0% to 96.3% nt similarities with those from other regions. Phylogenetic analysis based on ORF2 region showed that the local HAstV-1 sequences were most closely related to Japanese strains, and had distinct topology with phylogenies based on ORF1a and ORF1b regions. Conclusion: The complete genome sequences of 11 HAstV strains are obtained, and the VA2 complete genome is found.

Association of toxin-producing Clostridioides difficile with piglet diarrhea and potential transmission to humans.

The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST) 11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.

Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.

Pathogenicity chromosome of Fusarium oxysporum f. sp. cepae

Fusarium oxysporum f. sp. cepae (Foc) is the causative agent of Fusarium basal rot disease in onions, which leads to catastrophic global crop production losses. Therefore, the interaction of Foc with its host has been actively investigated, and the pathogen-specific (PS) regions of the British strain Foc_FUS2 have been identified. However, it has not been experimentally determined whether the identified PS region plays a role in pathogenicity. To identify the pathogenicity chromosome in the Japanese strain Foc_TA, we initially screened effector candidates, defined as small proteins with a signal peptide that contain two or more cysteines, from genome sequence data. Twenty-one candidate effectors were identified, five of which were expressed during infection. Of the expressed effector candidates, four were located on the 4-Mb-sized chromosome in Foc_TA. To clarify the relationship between pathogenicity and the 4-Mb-sized chromosome in Foc_TA, nine putative 4-Mb-sized chromosome loss strains were generated by treatment with benomyl (a mitotic inhibitor drug). A pathogenicity test with putative 4-Mb-sized chromosome loss strains showed that these strains were impaired in their pathogenicity toward onions. Genome analysis of three putative 4-Mb-sized chromosome loss strains revealed that two strains lost a 4-Mb-sized chromosome in common, and another strain maintained a 0.9-Mb region of the 4-Mb-sized chromosome. Our findings show that the 4-Mb-sized chromosome is the pathogenicity chromosome in Foc_TA, and the 3.1-Mb region within the 4-Mb-sized chromosome is required for full pathogenicity toward onion.