Development and application of loop-mediated isothermal amplification methods targeting the seM gene for detection of Streptococcus equi subsp. equi

Loop-mediated isothermal amplification (LAMP) constitutes a potentially valuable diagnostic tool for rapid diagnosis of contagious diseases. In this study, we developed a novel LAMP method (seM-LAMP) to detect the seM gene of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles in equids. The seM-LAMP successfully amplified the target sequence of the seM gene at 63°C within 60 min. The sensitivity of the seM-LAMP was slightly lower than the 2nd reaction of the seM semi-nested PCR. To evaluate the species specificity of the seM-LAMP, we tested 100 S. equi and 189 non-S. equi strains. Significant amplification of the DNA originating from S. equi was observed within 60 min incubation, but no amplification of non-S. equi DNA occurred. The results were identical to those of seM semi-nested PCR. To investigate the clinical usefulness of the methods, the seM-LAMP and the seM semi-nested PCR were used to screen 590 nasal swabs obtained during an outbreak of strangles. Both methods showed that 79 and 511 swabs were S. equi positive and negative, respectively, and the results were identical to those of the culture examination. These results indicate that the seM-LAMP is potentially useful for the reliable routine diagnosis of Streptococcus equi subsp. equi infections.     

Amputation for histiocytic sarcoma in a cat

A 9-year-old spayed female domestic shorthair cat presented with a skin lesion of the left tarsus. The lesion was biopsied and, based on the microscopic appearance and immunohistochemical characteristics, histiocytic sarcoma was diagnosed. Amputation was performed with improved demeanor seen postoperatively. However, between 44 and 60 days following the surgery, relapse of skin lesions appeared in multiple locations, including at the previous amputation site, and euthanasia was elected. This is the first report of a histiocytic sarcoma treated with amputation in a cat.     

Expression and possible role of 20alpha-hydroxysteroid dehydrogenase in the placenta of the goat

20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone (20alpha-OHP). 20Alpha-HSD is expressed in the murine placenta, suggesting a role, yet unidentified, played by this enzyme during the course of pregnancy. To elucidate the possible roles of 20alpha-HSD during pregnancy, 20alpha-HSD gene expression in the placenta was examined by Northern blot analysis, and progestin (progesterone and 20alpha-OHP) concentrations in the maternal and fetal sera and the amniotic fluid were measured by radioimmunoassay in pregnant Shiba goats. The expression of 20alpha-HSD mRNA was observed in the placenta and the intercaruncular part of the uterus during mid to late pregnancy. Analysis by in situ hybridization revealed that 20alpha-HSD mRNA was mainly localized in the endometrial epithelium of the caruncle side of the placenta. Considerable enzyme activity of 20alpha-HSD was also detected in the cytosolic fraction of the placenta and intercaruncular part of the uterus. Although concentrations of progesterone and 20alpha-OHP in the maternal serum showed similar profiles, progesterone levels in the fetal serum stayed extremely low throughout the pregnancy. The 20alpha-OHP concentration in the fetal serum was always higher than that in the maternal serum. In the amniotic fluid, the concentrations of both progesterone and 20alpha-OHP remained at very low levels throughout the pregnancy. These results support the notion that 20alpha-HSD protects the fetus from the cytotoxic effects of progesterone, and thereby maintains the normal development of the fetus.     

Antigen-specific histamine release in dogs with food hypersensitivity

An in vitro evidence of IgE-mediated hypersensitivity to food allergens was detected by positive results of antigen-specific histamine release in dogs with food hypersensitivity. Eight dogs were diagnosed to have food hypersensitivity based on identification of offending food allergens with food elimination followed by oral food provocation. The percentages of histamine release against the stimulation of offending food allergens in the cases ranged from 2.1% to 70.9%. Six of the 8 cases showed histamine release higher than those of healthy control dogs. Four dogs showed relatively high histamine release at the percentage beyond 10% that was compatible with a positive value of histamine release in humans with food hypersensitivity. These findings would suggest that IgE-mediated hypersensitivity against food allergens could be involved in canine food hypersensitivity.     

Development of novel clubroot resistant rapeseed lines (Brassica napus L.) effective against Japanese field isolates by marker assisted selection

Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from rapeseed fields and found they were classified as pathotype groups 2 and 4 using Japanese F₁ Chinese cabbage cultivars. We produced the resynthesized clubroot resistant Brassica napus harboring two resistant loci, Crr1 and Crr2, by interspecific crossing and developed resistant rapeseed lines for southern and northern regions by marker-assisted selection and backcrossing. We improved the DNA marker for erucic acid content to remove linkage drag between Crr1 and high erucic acid content and successfully selected lines with clubroot resistance and zero erucic acid for northern regions. A novel line, ‘Tohoku No. 106’, suitable for southern regions showed stable resistance against all six isolates and high performance in infested fields. We conclude that Crr1 and Crr2 are important genes for CR rapeseed breeding and marker-assisted selection is effective in improving clubroot resistance.     

Preparation and characterization of fermented seaweed sauce manufactured from low-quality nori (dried and fresh fronds of <Emphasis Type="Italic">Pyropia yezoensis</Emphasis>)

The present study tested processes to manufacture fermented sauce from low-quality nori (dried and fresh fronds of Pyropia yezoensis). The nori sauce was prepared using three tanks with fresh or dried nori cultured in different conditions. In the present study enzymes were not added for the promotion of the degradation of nori, while in a previous study they were. The supernatants of culture mashes obtained from the three tanks were combined, and this low-quality nori sauce (LNS) was characterized and compared with sauces manufactured from high-quality nori, soy, and fish. The LNS showed low concentrations of total nitrogen compounds (0.20 g/100 ml) and free amino acids, and its taste showed a high sourness score as evaluated by a taste-sensing system. On the other hand, the LNS was rich in polysaccharides, which were observed to be readily degraded to lower molecular weight size sugars by heat treatment. The LNS showed little risk for heavy metal or allergen contamination. The obtained sauce product is expected to be commercially utilized as a component of low allergen-risk sauce products after blending with other seasonings without wheat or soy elements.     

Spectral sensitivity of juvenile chub mackerel (<Emphasis Type="Italic">Scomber japonicus</Emphasis>) in visible and ultraviolet light

Although chub mackerel (Scomber japonicus) is widely distributed all over the world, the relevance of its visual sensitivity to its ecology is not yet fully understood. We investigated spectral sensitivity in juvenile chub mackerel in the range of ultraviolet (UV) to visible light (369–652 nm) by electroretinogram (ERG) using light-emitting diodes (LEDs). Sensitivity peaked at a wavelength of approximately 482 nm in dark-adapted fish and 525 nm in light-adapted fish. A secondary sensitivity peak in the UV range at approximately 382 nm was found in both dark- and light-adapted fish. The UV sensitivity of chub mackerel may be attributable to UV transmissibility of the optical media and to the presence of a beta-band of visible light-sensitive visual pigments, and not to an alpha-band of UV visual pigments. This UV sensitivity may be useful for feeding or communication with other fishes.     

Transverse shape analysis of xylem ground tissues by Fourier transform image analysis I: trial for statistical expression of cell arrangements with fluctuation

Periodic cell arrangements of tracheids in Cryptomeria japonica and Agathis sp., and that of wood fibers in Magnolia obovata were examined by Fourier transform image analysis (FTIA). The angular distribution functions on power spectral patterns (PSPs) transformed from the dot maps were traced. As well as this conventional method, we developed the line convolution method and domain transformation for more correct analysis of radial and tangential arrangements. Cell arrangements became clear in Cryptomeria and Agathis. Fluctuations were expressed by the standard deviation (σ) or the relative standard deviation (σ_r) so that we could compare the characteristics of both woods. In Cryptomeria, the most provable distance between tracheids along radial files was 36.7 µm with 17% fluctuation, whereas the tangential interval was 28.3 µm with σ_r of 24%. In Agathis, the radial diameter was 35.1 µm with σ_r of 23% and the tangential interval was 41.5 µm with σ_r of 23%. In Magnolia, the maximal periodicity of wood fibers was 15.8 µm and 20.5 µm along and between radial files, respectively, although the fluctuations could not be estimated because of the remarkable interruptions by numerous vessels.