Congenital abnormalities in calves associated with Peaton virus infection in Japan.

Peaton virus (PEAV; family Peribunyaviridae, genus Orthobunyavirus) appears to be capable of producing congenital malformations in ruminants; however, its pathogenicity remains unknown given its relatively low incidence. We evaluated the relationship between congenital abnormalities of calves and PEAV infection by serologic, epidemiologic, pathologic, and virologic investigations using specimens from 31 malformed calves in the years 1996-2016 in Japan. Antibody testing was carried out for known teratogenic viruses, including Akabane, Aino, Chuzan, and bovine viral diarrhea viruses, in the precolostral sera of these abnormal calves, but all results were negative. However, all 31 malformed calves were positive for antibodies against PEAV. A PEAV-specific gene was amplified from central nervous system tissues from a stillborn calf delivered in April 2007, and its nucleotide sequence was identical with that of PEAV isolated from healthy sentinel cattle in September 2006. These findings indicate that PEAV can cause bovine congenital anomalies.

Whole genome analysis of a novel neurotropic bovine astrovirus detected in a Japanese black steer with non-suppurative encephalomyelitis in Japan.

While neurotropic bovine astroviruses (BoAstVs) have been identified in North America and Europe, their presence has never been reported in Asia. In this study, we detected BoAstV in the brain of a steer showing neurological signs. Phylogenetic analysis revealed that the identified virus belongs to the Virginia/Human-Mink-Ovine clade, which contains most of the neurotropic astroviruses including the neurotropic BoAstVs. Similarity plot analysis showed that the virus was closely related to the American BoAstV NeuroS1 strain with respect to the ORF regions and to the European BoAstV CH13 strain in the 3' untranslated region, suggesting the occurrence of intra-genotypic recombination events.

Pathogenic characteristics of a novel intranuclear coccidia in Japanese black calves and its genetic identification as Eimeria subspherica.

Bovine intranuclear coccidiosis is caused by the protozoans Eimeria alabamensis and Cyclospora spp. Here, we characterized the disease and genetically identified the causative species in Japanese black calves with chronic and refractory watery diarrhea. Histologic examinations revealed atrophy of the jejunal villi and numerous parasites in the nucleus of epithelial cells in the jejunum. Based on molecular analyses using 18S ribosomal RNA gene-specific primers that we designed, the parasites were found to be formed in the same cluster as Eimeria subspherica in the phylogenetic tree, which was separated from those of other related Eimeria spp. These results constitute the first report of E. subspherica as a cause of bovine intranuclear coccidiosis.

Development of molecular diagnostic protocols for detecting three types of Entamoeba from diarrheal and asymptomatic pigs and environmental moist soils.

Entamoeba suis and Entamoeba polecki subtypes (ST) 1 and 3 have recently been implicated in disease outbreaks in pigs. However, the distributions of these parasites in Japan and the potential sources of infection on farms still remain unclear. Here, we examined a farm of fattening/growing pigs with abnormal feces in Kagoshima Prefecture, Japan, and found the presence of parasites in the farm environment. Examination of intestinal tissues from pigs presenting with ulcerative colitis revealed a large number of trophozoites that had invaded the lesions. We identified single and mixed infections of E. suis and E. polecki ST1 and ST3 in paraffin sections or fecal samples from affected pigs. Two subtypes of Entamoeba were identified using four primer sets by PCR and sequencing. The parasites were detected in moist soil samples obtained around the drinking water source or puddles, implicating transmission of cysts via contaminated soils. Additionally, we found evidence of Entamoeba spp. and coinfections in surveyed pigs without any diarrhea at two neighboring farms. Our results establish methods for successfully identification of parasites, including cases in which multiple infections are present.

Congenital Malformations of Calves Infected with Shamonda Virus, Southern Japan.

In 2015 and 2016, we observed 15 malformed calves that were exposed to intrauterine infection with Shamonda virus, a Simbu serogroup orthobunyavirus, in Japan. Characteristic manifestations were arthrogryposis and gross lesions in the central nervous system. Our results indicate that this arbovirus should be considered a teratogenic virus in ruminants.

Resurgence of bovine ephemeral fever in mainland Japan in 2015 after a 23-year absence.

In September and October 2015, suspected cases of bovine ephemeral fever (BEF) were reported in the mainland region of Kagoshima Prefecture and on Tanegashima Island. The genome of the BEF virus (BEFV) was detected in the diseased cows and the cows that had recovered. The serum obtained from the affected cows contained high titers of BEFV-neutralizing antibody. In total, 18 affected cows were demonstrated to be infected with BEFV during the outbreak. Our findings showed evidence that BEF occurred in mainland Japan after a 23-year absence. Phylogenetic analysis based on the surface glycoprotein (G) gene revealed that BEFVs detected in the affected cows were genetically distinct from previous Japanese BEFVs, but were close to BEFVs circulating in Taiwan and mainland China in recent years. Amino acid substitution in the neutralizing epitope domains of the G protein was limited between the detected viruses and the vaccine strain (YHL isolate), and high titers of the neutralizing antibody against the YHL isolate were induced in the infected cattle during the disease occurrences. Therefore, current BEF vaccines probably elicit protective immunity against the BEFVs detected in 2015, although their effectiveness should be assessed. Since the BEFV vaccination rates are estimated to be low, a BEF outbreak should be considered a possibility in mainland Japan.

Reemergence of Ibaraki disease in southern Japan in 2013.

In Japan in 2013, two cattle in the northwestern part of Kagoshima Prefecture developed fever and swallowing difficulty and were suspected of having Ibaraki disease. The epizootic hemorrhagic virus (EHDV) genome was detected from diseased and asymptomatic cattle by reverse transcription-polymerase chain reaction (RT-PCR). High neutralization antibody titers to Ibaraki virus (IBAV) ranging from 1:128 to 1:1,024 were observed in the RT-PCR-positive cattle, and the virus was isolated in one of the IBAV-positive farms. A pairwise alignment and phylogenetic analysis based on the major outer coat protein VP2 encoded in segment 2 revealed a close relationship between the isolated viruses and previous IBAV isolates. The phylogeny of VP2 also suggested that an IBAV variant isolated in 1997 was distinct from IBAV and sorted into a heterogeneous serotype, EHDV serotype 7. The findings revealed the reemergence of Ibaraki disease in Japan after a 26-year absence. Interestingly, the co-circulation of EHDV serotype 1 with IBAV was observed in the affected region, suggesting the potential reassortment between two heterogeneous serotypes in the field. Sentinel surveillance in Kagoshima Prefecture indicated that the incursion of IBAV occurred in October 2013 and that its spread was limited within the small area. Inadequate environmental temperatures for vector transmission in late autumn might have limited the virus spread to a wider region. The reemergence of Ibaraki disease showed us the importance of continuous vaccination to prevent economic losses.

Homocysteine induced SH-SY5Y apoptosis through activation of NADPH oxidase in U251MG cells.

Epidemiological studies have indicated a correlation between homocysteinemia and dementia, including Alzheimer's disease. However, the mechanism by which homocysteine (Hcy) induces neuronal cell death remains unknown. We found that micromolar concentrations of Hcy induced neuroblastoma SH-SY5Y cell death only when co-cultured with glioblastoma U251MG cells. In this culture system, cysteine had no effect on SH-SY5Y cell death. There was an increase in TUNEL-positive cells and loss of mitochondrial membrane potential following treatment with 100 µM Hcy. Addition of conditioned medium prepared from U251MG cells in the presence of 100 µM Hcy also reduced SH-SY5Y cell viability, while this effect was prevented when using conditioned medium from U251MG cells exposed to 100 µM Hcy+apocynin, a specific NADPH oxidase inhibitor. Following exposure to 100 µM Hcy in U251MG cells, expression of Rac1, a compartment of NADPH oxidase, was translocated to the plasma membrane, and the active form of Rac1 was increased. There was no change in peroxide concentration in the medium of U251MG cells after addition of Hcy. Overall, these data suggest that Hcy stimulates Rac1 activation and NADPH oxidase, resulting in superoxide anion production that may induce SH-SY5Y cell apoptosis.

Homocysteine and copper induce cellular apoptosis via caspase activation and nuclear translocation of apoptosis-inducing factor in neuronal cell line SH-SY5Y.

Hyperhomocysteinemia has been implicated in dementia and neurodegenerative disease. Physiological homocysteine concentrations did not result in apoptosis in SH-SY5Y cells in the present study. The apoptosis was recognized in millimolar level of homocysteine. However, SH-SY5Y cell death was observed following exposure to micromolar level of homocysteine in combination with copper. Exposure to 250microM homocysteine and 10microM CuCl(2) for one day decreased cell viability by 40%. Homocysteine and copper caused apoptosis, because hallmarks of apoptosis were recognized, such as loss of mitochondrial membrane potential, TUNEL-positive cells, release of cytochrome c from mitochondria, and caspase-3 activation, but not nucleosomal DNA fragmentation. Homocysteine and copper generated the intracellular reactive oxygen species, and homocysteine and copper-induced apoptosis was due to an accumulation of intracellular reactive oxygen species, which was inhibited by catalase. Pan-caspase inhibitor, z-VAD-fmk, could not completely inhibited homocysteine and copper-induced cell death. Homocysteine and copper also caused the nuclear translocation of apoptosis-inducing factor. These results suggested that homocysteine and copper induced not only caspase-dependent apoptosis but also caspase-independent apoptosis-inducing factor related apoptosis.