Enhanced protection against Heligmosomoides polygyrus in IL-2 receptor β-chain overexpressed transgenic mice with intestinal mastocytosis

IL-2 receptor β-chain overexpressed transgenic (Tg2Rβ) mice lack NK cells, but the development of other lymphocyte subsets and macrophages remained apparently intact. These mice also exhibit intestinal mastocytosis. Helminth infection induces various immune responses, such as mast cells, goblet cells, eosinophils and IgE, mediated by Th2 cytokines. IL-4 is also important in the regulation of resistance and susceptibility to Heligmosomoides polygyrus infection. However, there are contradictory results about the relation between resistance to H. polygyrus and intestinal mastocytosis. The present study showed that Tg2Rβ mice suppressed worm fecundity with mastocytosis without an increase of the levels of goblet cells, eosinophils and IgE compared with control mice. These results clearly indicated that mast cells have the ability for to protect against H. polygyrus infection. However, additional studies are required to evaluate protective effector mechanisms against H. polygyrus.     

Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli

In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.     

Establishment of a quantitative ELISA for the measurement of allergen-specific IgE in dogs using anti-IgE antibody cross-reactive to mouse and dog IgE

As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable to measure absolute quantity of serum antigen-specific IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantification of antigen-specific IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize canine IgG. The reactivity of CRE-DM to canine IgE was also confirmed by an inhibition ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In order to know whether canine IgE specific to an allergen could be quantitatively measured with an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, a standard curve was created by using concentration-predetermined Cry j 1-specific monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-specific IgE in dogs that were experimentally sensitized to Japanese cedar pollen could be calculated and determined in ng/ml. The specificity of the Cry j 1-specific IgE ELISA using CRE-DM was also confirmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate was 97.0%. Reproducibility of the ELISA in three independent assays was determined using groups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml. Intra- and inter-assay reproducibility was determined with coefficient of variation ranging between 3.1-5.2% and 2.2-8.0%, respectively. These results demonstrated that the ELISA utilizing CRE-DM was a specific, reliable and robust new laboratory test that could quantify absolute amount of antigen-specific IgE in canine serum. The ELISA will serve as a useful tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments as well as during seasonal fluctuation of allergen exposure.     

Inferring phenotypic causal structure among farrowing and weaning traits in pigs

Direct selection for litter size or weight at weaning in pigs is often hindered by external interventions such as cross‐fostering. The objective of this study was to infer the causal structure among phenotypes of reproductive traits in pigs to enable subsequent direct selection for these traits. Examined traits included: number born alive (NBA), litter size on day 21 (LS21), and litter weight on day 21 (LW21). The study included 6,240 litters from 1,673 Landrace dams and 5,393 litters from 1,484 Large White dams. The inductive causation (IC) algorithm was used to infer the causal structure, which was then fitted to a structural equation model (SEM) to estimate causal coefficients and genetic parameters. Based on the IC algorithm and temporal and biological information, the causal structure among traits was identified as: NBA → LS21 → LW21 and NBA → LW21. Owing to the causal effect of NBA on LS21 and LW21, the genetic, permanent environmental, and residual variances of LS21 and LW21were much lower in the SEM than in the multiple‐trait model for both breeds. Given the strong effect of NBA on LS21 and LW21, the SEM and causal information might assist with selective breeding for LS21 and LW21 when cross‐fostering occurs.     

Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off‐target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off‐target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non‐specific cleavage of off‐target sites.     

The estimation of duration of maternally-derived antibodies against Akabane,Aino, and Chuzan virus in calves by the receiver operating characteristic analysis

The duration of maternally-derived antibodies against three arboviruses was investigated in calves, using the results of arbovirus serosurveillance performed in Kagoshima Prefecture during 2002–2016. The duration of maternally-derived antibodies against Akabane virus (AKAV), Aino virus (AINOV), and Chuzan virus (CHUV) was estimated to be 178 (sensitivity: 0.769, specificity: 0.730), 156 (sensitivity: 0.806, specificity: 0.791), and 156 days of age (sensitivity: 0.845, specificity: 0.814), by receiver operating characteristic analysis. The duration of maternally-derived antibodies against AKAV, AINOV, and CHUV differed 7–14, 22–28, and 20–31 days in the same calf types between the regions far from each other although it was similar between the adjacent regions. The dairy calves showed 6–29 days longer duration than the beef calves rearing in a similar region.     

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass

Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collected tissue was conducted for culture, and the growing epithelial-like cells were passaged. The cells showed continuous proliferation for over 6 months. After 40 passages, the cells were named CMG-1. Lipid droplets in the cytoplasm of CMG-1 cells were confirmed by Oil Red O staining. As reported in studies with human sebaceous epithelial cell lines, lipogenesis in CMG-1 cells was promoted by linoleic acid, whereas transforming growth factor-β (TGF-β) suppressed it. Additionally, real-time PCR revealed that the expression levels of chemokines and cytokines, including CC chemokine ligand (CCL)-2, CCL-20, CXCL-10, Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1α, IL-1β, and IL-8, were significantly increased in CMG-1 cells following treatment with lipopolysaccharide. In conclusion, we successfully established a new canine sebaceous epithelial cell line. Our data indicated that lipogenesis and inflammatory responses were quantitatively evaluable in this cell line. CMG-1 cells could be useful for the pathological analysis of sebaceous gland diseases in dogs.     

Effect of nitrogen application at the flowering stage on the quality of soybean seeds

Application of nitrogen (N) fertilizer at the flowering stage changed the contents of storage compounds in seeds of Soybean (Glycine max L. cvs Enrei and Tamahomare). The effects of the N application on the maturation of soybean seeds were examined by comparing changes in the contents of amino acids, sugars, water, protein, and oil in seeds from N‐dressed plants (NDS) with those from undressed plants (UDS) during maturation. The application resulted in a decrease of contents of total and some amino acids (glutamine and asparagine) in developing seeds except for at the early maturation stage and in a decreased protein content of mature seeds. On the other hand, the N application led to faster accumulation of oil in developing seeds and to an increased oil content of mature seeds. Based on these results, it was concluded that the N application at the flowering stage changed the composition of solutes imported by developing seeds and resulted in variations in the contents of storage compounds. The results indicate that it is possible to improve seed quality by fine control of N application.     

Investigations on Cambodian soils

Basic information on the nature of soils, their potentialities, and their limitations is necessary to evaluate Cambodian agriculture properly, and to develop a program for increasing food and other crops. Suitable information has, however, been limited, especially as there is not to be found in any report much data relating to soils of different origins over the whole country. There is, needless to say, some information on confined areas of Cambodia.     

Psychoacoustical evaluation of floor-impact sounds from wood-framed structures

Floor-impact sounds may introduce noise problems in dwelling-type environments. In particular, floor-impact sounds from lightweight structures like wood-framed buildings should be paid significant attention. In this study, such floor-impact sounds were investigated from the viewpoint of residents. The floor specifications of wood-framed structures were improved by using high-density underlay, and heavy floor-impact sounds were evaluated by using both psychoacoustical and conventional methods. The results showed that the floor-impact sound insulation grades specified in the Japanese Industrial Standards (JIS) did not correspond to the floor specifications of wood-framed structures. The nonstationary loudness, which is a psychoacoustical index for nonstationary sounds, changed with the specifications of wood-framed structures in a wider range of floor-impact sound levels in comparison with the maximum A-weighted sound pressure level, which is a single-number index of the JIS.