Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

Effect of iron-quercetin complex on reduction of nitrite in in vitro and in vivo systems

This study investigated whether reducing agents such as quercetin and iron(II) facilitate formation of nitric oxide (NO) gas from orally ingested nitrite in an vivo study. When 3 mg/kg Na (15)NO2 was orally administered to rats with or without iron(II) or quercetin, Hb (15)NO, which is indicative of systemic (15)NO, was detected in the blood, with the maximum blood concentration of Hb (15)NO at 15 min after nitrite or nitrite plus quercetin treatment, whereas after administration of nitrite plus iron(II) or nitrite plus iron(II) and quercetin, the time was shortened to 10 min. Interestingly, iron(II), quercetin, or iron(II) plus quercetin did not affect the total amount of Hb (15)NO generated from orally administered Na (15)NO2. However, the systemic nitrite concentration was significantly decreased in the presence of iron(II) or iron(II) plus quercetin. These results may indicate that iron(II) is critical to the generation of NO gas from nitrite, whereas quercetin contributed little under the in vivo experimental conditions.     

Survival time of juvenile pen shell Atrina pectinata (Bivalvia: Pinnidae) in hyposaline water

International Aquatic Research - Survival time of juvenile pen shell Atrina pectinata (shell length ≈ 34 mm) exposed to hyposaline water without sediments was...     

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non‐stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2‐bis(2‐aminophenoxy) ethane N,N,N’,N’‐tetraacetic acid tetrakis‐acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.     

Establishment rate of direct-seeded rice in the relay-intercropping system in Kanto region of Japan

The relay intercropping system for cultivating direct-seeding rice and winter cereal is a low-cost method particularly for double cropping, because it eliminates the steps of raising seedlings, paddling, and transplanting. However, in this system, the seedling establishment rate (SER) of rice is low and unstable. The objective of this study was to identify the factors affecting SER to highlight the ways to improve SER. Experiments were conducted in experimental fields in Tsukuba City, Ibaraki Prefecture in 2015 and 2016. To determine the time of rice seed death and to calculate seed survival rates, ‘Akidawara’ seeds were embedded in soil (depth: 3 cm) and dug up after defined periods. We then analyzed the correlation between SER and meteorological factors at two sites (Ibaraki Prefecture and Gunma Prefecture) over the same two years. Based on mean air temperature (MT), we divided the period from seeding to June 20 (when irrigation had been initiated in almost all the fields) into four phases. In Phase 1, the number of days with rainfall (≥5 mm) and soil-wetting days (water potential ≥ ?100 kPa) were significantly and negatively correlated with SER. We found that most of the seed deaths occurred after germination, and the germination rates were presumably affected by water absorption during the low temperature phase. Further investigations are needed to understand the occurrences during the seedling emergence period. This study contributes to a better understanding of the factors affecting variations in the SER of direct-seeded rice grown in the relay-intercropping system.

List of Abbreviations: DSR: direct-seeded rice; SER: seedling establishment rate; TT: thiuram treatment; SSR: seed survival rate; DS: dead seeds; AGS: already germinated seeds; DSRI: direct-seeding of rice in the inter-row spaces of winter cereals; VWC: volume water content; WP: water potential     

An epidemic of caprine arthritis encephalitis in Japan: isolation of the virus

A disease characterized by arthritis of carpal joints and occasionally by pneumonia was seen among goats on a farm in Nagano prefecture of Japan in the summer of 2002. Serological investigation was done on 30 goats, that is one group on the farm by agar gel immunodiffusion tests using maedi-visna virus as the antigen, and 19 goats were positive. The caprine arthritis encephalitis virus genome was detected in peripheral blood leukocytes from several antibody-positive goats. Two goats among them were necropsied and used for pathological tests and virus isolation. The isolated virus was serially propagated in fetal lamb lung cell cultures. Goats inoculated with the culture fluid became antibody positive. These results suggested that a CAEV-infected goat had been introduced accidentally to the farm and that subclinical infection occurred among the flock.     

Inhibin secretion in the golden hamster (Mesocricetus auratus) testis during active and inactive states of spermatogenesis induced by the restriction of photoperiod

Gonadal function in the male golden hamster (Mesocricetus auratus) was investigated during exposure to a short photoperiod condition. Within 3 weeks of exposure to the short photoperiod condition, FSH and testosterone in the plasma significantly decreased, and subsequently immunoreactive (ir)-inhibin significantly decreased. Testicular contents of ir-inhibin and testosterone, and pituitary contents of LH and FSH also significantly decreased by 3 weeks with regression of weight of testes, epididymis and seminal vesicles and sperm head count. Circulating LH varied but not significantly. Thereafter, all reproductive parameters and secretion of LH, FSH, ir-inhibin and testosterone gradually recovered after 17 weeks of exposure even though animals continued to be subjected to the short photoperiod condition. Plasma concentrations of inhibin B and inhibin pro-alphaC were detectable and were significantly decreased after 15 weeks of exposure to the short photoperiod, but their levels were still detectable. Immunopositive reaction of inhibin alpha and betaB subunits was found in Sertoli cells and Leydig cells in the regressed testes of animals subjected to short photoperiod as was also seen in animals before exposure to the short photoperiod. Although the spermatogenic cycle was suppressed like prepubertal animals, the present study showed that the testicular recovery, so-called refractoriness, is functionally different from the developing stage of immature animals, especially with regard to inhibin secretion. The present results showed that changes in FSH preceded changes in inhibin during the regression and recovery phases, indicating that FSH is a major regulatory factor of inhibin secretion in male golden hamsters. The present study also demonstrated that regressed testes still secrete a small amount of bioactive inhibin during exposure to a short-photoperiod condition.     

Cloning of porcine interleukin (IL)-12 receptor beta2 (IL-12Rbeta2) gene and its application to a rapid biological assay for human/porcine IL-12

Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.     

Low pathogenicity H5N2 avian influenza outbreak in Japan during the 2005-2006

At the end of May 2005, a low-pathogenicity avian influenza (LPAI) virus of subtype H5N2 was isolated for the first time from chickens in Japan. Through active and epidemiological surveillance, 5.78 million chickens on 41 farms were found to be affected and 16 H5N2 viruses were isolated. Antigenic analysis revealed antigenic similarity of these isolates. Phylogenetic analysis showed that they originated from a common ancestor and clustered with the H5N2 strains prevalent in Central America that have been circulating since 1994. Experimental infection of chickens with the index isolate (A/chicken/Ibaraki/1/05) demonstrated that this virus replicated efficiently in the respiratory tract without clinical signs, and dust-borne and/or droplet-borne transmission was considered as a possible mode of transmission. These results suggested that the H5N2 LPAI viruses isolated in Japan were highly adapted to chickens.