Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

First report of molecular identification of Cystoisospora suis in piglets with lethal diarrhea in Japan.

Cystoisospora suis is a pathogen that causes diarrhea in pigs and can lead to serious disease. Species identification, especially by histopathological examination, is often difficult because of morphologically similar parasites such as Eimeria species. In this study, we used histopathological, bacteriological, virological, and parasitological methods to identify the cause of the disease in two piglets with severe diarrhea. Villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of examined piglets, and coccidian parasites were found in the cytoplasm of the epithelial cells. In some merozoites in the meronts, the presence of two nuclei indicated type 1 merozoites, characteristic of C. suis. According to Cystoisospora-specific PCR targeting the rRNA internal transcribed spacer 1 (ITS1) gene, the sequences of the products were 98.5% similar to those of C. suis. Escherichia coli (O149 serogroup) exhibiting a virulence factor profile (LT, STb, and EAST1 as toxins and F4 as a colonization factor) was detected in one piglet. No other bacteria or significant enteric viruses were found. Co-infection with C. suis and E. coli could imply aggravation of the disease, although further study is needed to assess the pathogenicity of this interaction. This study is the first to clarify by molecular analysis the sequences of C. suis detected in piglets in Japan.

Bovine epizootic encephalomyelitis caused by Akabane virus in southern Japan.

BACKGROUND: Akabane virus is a member of the genus Orthobunyavirus in the family Bunyaviridae. It is transmitted by hematophagous arthropod vectors such as Culicoides biting midges and is widely distributed in temperate to tropical regions of the world. The virus is well known as a teratogenic pathogen which causes abortions, stillbirths, premature births and congenital abnormalities with arthrogryposis-hydranencephaly syndrome in cattle, sheep and goats. On the other hand, it is reported that the virus rarely induces encephalomyelitis in cattle by postnatal infection. A first large-scale epidemic of Akabane viral encephalomyelitis in cattle occurred in the southern part of Japan from summer to autumn in 2006. The aim of this study is to define the epidemiological, pathological and virological properties of the disease. RESULTS: Nonsuppurative encephalomyelitis was observed in cattle that showed neurological symptoms such as astasia, ataxia, opisthotonus and hypersensitivity in beef and dairy farms by histopathological analysis. Akabane viral antigen and genome were consistently detected from the central nervous system of these animals, and the virus was isolated not only from them but also from the blood samples of clinically healthy calves in the epidemic area. The isolates were classified into genogroup I a containing the Iriki strain, which caused encephalitis of calves almost twenty years ago in Japan. Most of the affected cattle possessed the neutralizing antibody against Akabane virus. Seroconversion of the cohabitated and sentinel cattle in the epidemic area was also confirmed during an outbreak of the disease. CONCLUSION: The ecological and epidemiological data we have obtained so far demonstrated that the Akabane virus is not endemic in Japan. No evidence of Akabane virus circulation was observed in 2005 through nation-wide serological surveillance, suggesting that a new strain belonging to genogroup I a invaded southern Japan from overseas in the summer of 2006 and caused an unprecedented epizootic of encephalomyelitis mainly in susceptible calves. It will be necessary to reconsider the vaccine strategy to control the disease effectually.