Multiplex PCR method for differentiating highly pathogenic Yersinia enterocolitica and low pathogenic Yersinia enterocolitica,and Yersinia pseudotuberculosis

A multiplex PCR method for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as the highly pathogenic Y. enterocolitica, including serotype O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis, was developed. Four primer pairs were chosen to detect the genes fyuA, ail, inv, and virF, responsible for the virulence in pathogenic Yersinia species. Under the multiplex PCR conditions, the unique band patterns for the highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis were generated from Yersinia strains. The detection limit of this method was 10¹–10³ CFU per reaction tube. This multiplex PCR method could detect highly pathogenic Y. enterocolitica O8 from the wild rodent fecal samples that were culture-positive. Therefore, the new multiplex PCR method developed in this study is a useful tool for rapid and sensitive diagnosis, distinguishing three pathogenic Yersinia groups.     

Inosine supplementation effectively provokes the growth,immune response,oxidative stress resistance and intestinal morphology of juvenile red sea bream,Pagrus major

A feeding trial was conducted to determine effects of dietary inosine on growth, immune responses, oxidative stress resistance and intestinal morphology of juvenile red sea bream. A semi‐purified basal diet supplemented with 0 (D1, control), 2 g kg^?1 (D2), 4 g kg^?1 (D3), 6 g kg^?1 (D4) and 8 g kg^?1 (D5) dietary inosine, respectively, to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (initial weight: 8 g). After 50 days of feeding trial, fish fed diets with 4 g kg^?1 inosine had the highest (p < .05) final weight, weight gain and specific growth rate. Similarly, improved feed intake, feed conversion ratio and protein efficiency ratio were also found at 4 g kg^?1 inosine supplemented group. Some non‐specific immune parameters such as total serum protein, lysozyme activity and bactericidal activity tended to be higher for fish fed diets supplemented with inosine. Peroxidase and catalase activity also influenced (p < .05) by dietary inosine supplementation. Fish fed 2 g kg^?1 and 4 g kg^?1 inosine supplemented diets showed the least oxidative stress condition. Inosine supplementation significantly increased (p < .05) anterior enterocyte height and posterior fold height, enterocyte height and microvillus height compared with control. We concluded that 4 g kg^?1 dietary inosine supplementation effectively provokes growth and health performance of red sea bream by increasing growth, immune response, oxidative stress resistance and intestinal health condition.     

Effects of dietary astaxanthin and vitamin E and their interactions on the growth performance,pigmentation, digestive enzyme activity of kuruma shrimp (Marsupenaeus japonicus)

A 56‐day feeding trial was done to investigate the interactive effects of astaxanthin (Ax) and vitamin E (α‐Toc) on the performance of kuruma shrimp (Marsupenaeus japonicus). A 2 × 3 factorial experiment was conducted with six experimental diets containing two levels of Ax (0 and 0.6 g/kg diet [Ax₀ and Ax_0.6]) and three levels of α‐Toc (0, 0.2 and 1 g/kg diet [α‐Toc₀, α‐Toc_0.2 and α‐Toc₁]). The results indicated that growth performance was significantly (p < 0.05) increased in shrimp fed with the Ax_0.6 × α‐Toc_0.2 diet. Interactive effects between Ax and α‐Toc on the growth parameters were observed. Furthermore, pigmentation performance was significantly (p < 0.05) better in the Ax_0.6 groups. Interaction between Ax and α‐Toc was also found in the Ax content of shrimp body parts. Interestingly, dietary α‐Toc helped to reduce the Ax consumption rate, promote the absorption and increase the deposition of Ax in the muscle. Shrimps from the Ax_0.6 groups showed significantly (p < 0.05) improved hepatopancreatic digestive enzyme activities compared with those of the Ax₀ groups. Although no interactive (p > 0.05) effects were found between dietary α‐Toc and Ax on total haemocyte count and tolerance against freshwater, dietary Ax and α‐Toc supplementation showed better performance on these two parameters. It was concluded that dietary Ax and α‐Toc functioned synergistically, and the shrimp fed with the diet containing 0.6 g Ax/kg diet Ax and 0.2 g α‐Toc/kg diet showed improved growth and pigmentation performance compared with the other groups in the current study.     

Effects of water-soluble and fat-soluble choline sources on the performances of juvenile kuruma shrimp, Marsupenaeus japonicus Bate

The present study was undertaken to clarify the effect and interaction of choline chloride (CC) and phosphatidylcholine (PC), the water‐ and fat‐soluble sources of choline, on the performances of kuruma shrimp, Marsupenaeus japonicus juveniles using as response variables such as percent weight gain (%WG), specific growth rate (SGR), survival (SR%), feed efficiency ratio (FER), protein efficiency ratio (PER), proximate composition, fatty acids profile and free choline and PC contents in the whole body of kuruma shrimp. A 40‐day feeding trial was conducted as a 2 × 3 factorial experiment with six test diets containing three levels of CC (0.3, 0.6 and 1.2 g kg^?1) and two levels of soybean PC (SPC; 2 and 4 g kg^?1). Both of the choline sources (CC and SPC) showed a significant interaction (P<0.05) regarding all the evaluated response variables except for the proximate and fatty acid contents. Interestingly, there was a positive effect (P<0.05) of CC observed with increasing its level at the low level of SPC and vice versa. However, the magnitudes of the beneficial effect of CC in terms of the evaluated parameters were changed with the different dietary levels of SPC. Also, the inclusion of higher levels of SPC (4 g kg^?1) or CC (0.6 and 1.2 g kg^?1) diminished the effect of the other source.     

Genetic homogeneity between adult and juvenile populations of <Emphasis Type="Italic">Scombrops gilberti</Emphasis> (Percoid,Scombropidae) in the Pacific Ocean off the Japanese Islands

Scombrops gilberti is a member of the percoid family Scombropidae, which includes a single genus and three to four species worldwide. Little is known about the ecology of this species. Juvenile S. gilberti have been found in the waters off northern Japan (Iwate Prefecture), whereas adults are found in the waters around the southern counterpart (Izu Islands), approximately 700 km from the northern waters. In the present study, we captured immature S. gilberti (106–248 mm standard length, SL) in the northern waters by set net at 8–80 m depth, whereas larger individuals (150–328 mm SL) were captured by trawling at 150–500 m depth. By contrast, only adult S. gilberti (422–590 mm SL) were captured in the southern waters. The genetic composition of the adult population of S. gilberti from the southern waters and of the juvenile population from the northern waters was compared using the nucleotide sequence of the mitochondrial DNA (mtDNA) cytochrome b gene. No significant differences in genetic parameters such as fixation index, neutrality test or mismatch distribution analysis were found between these geographically distinct populations of S. gilberti, showing that these populations are genetically homogeneous.     

Growth, stress tolerance and non-specific immune response of Japanese flounder Paralichthys olivaceus to probiotics in a closed recirculating system

ABSTRACT:   Effects of probiotics on growth, stress tolerance and non-specific immune response in Japanese flounder Paralichthys olivaceus were evaluated in a closed recirculating system. Survival and growth of flounder treated by supplying commercial probiotics either in the diet (the probiotic diet group), or into the rearing water (the water supply group), were higher compared to the untreated group (the control group). Water quality parameters, pH, NH₄-N, NO₂-N and PO₄-P showed lower concentration in the probiotic diet group compared with the control group and the supply group. Plasma lysozyme activity in the probiotic diet group and the water supply group was significantly higher ( P  < 0.05) than that in the control group. In heat shock stress tests, flounder in the probiotics-treated groups showed greater heat tolerance (measured by 50% lethal time, LT50) than the control group. Pathogen challenge tests with Vibrio anguillarum (2 × 10⁷ c.f.u./mL) resulted in significantly higher survival in the probiotics-treated groups than the control group. Results indicated that probiotics supplied in the rearing water and the diet of fish enhanced the stress tolerance and the non-specific immune system of Japanese flounder, providing them a higher resistance against stress conditions and pathogens.     

Dystrophin-deficient muscular dystrophy in a Toy Poodle with a single base pair insertion in exon 45 of the Duchenne muscular dystrophy gene

A 10-month-old, intact male Toy Poodle was referred for a postural abnormality. Blood biochemical tests revealed a marked increase in plasma creatine phosphokinase (CPK) concentration. The isoenzyme test showed that 99% of serum CPK consisted of CPK-MM. Histopathological evaluation of muscle biopsy samples confirmed scattered degeneration and necrosis of myofibers. Immunohistochemistry for dystrophin showed an absence of staining in muscle cells. Based on these findings, the dog was diagnosed with dystrophin-deficient muscular dystrophy. Whole genome sequencing using genomic DNA extracted from blood revealed a single base pair insertion in exon 45 of the Duchenne muscular dystrophy (DMD) gene. This is the first report on muscular dystrophy in Toy Poodles and identified a novel mutation in the DMD gene.     

Oolong tea theasinensins attenuate cyclooxygenase-2 expression in lipopolysaccharide (LPS)-activated mouse macrophages: structure-activity relationship and molecular mechanisms

Oolong tea theasinensins are a group of tea polyphenols different from green tea catechins and black tea theaflavins. The present study reports the inhibitory effects of oolong tea theasinensins on the expression of cyclooxygenase-2 (COX-2) and underlying molecular mechanisms in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. The structure-activity data revealed that the galloyl moiety of theasinensins played an important role in the inhibitory actions. Theasinensin A, a more potent inhibitor, caused a dose-dependent inhibition of mRNA, protein, and promoter activity of COX-2. An electrophoretic mobility shift assay (EMSA) revealed that theasinensin A reduced the complex of NF-κB- and AP-1-DNA in the promoter of COX-2. Signaling analysis demonstrated that theasinensin A attenuated IκB-α degradation, nuclear p65 accumulation, and c-Jun phosphorylation. Furthermore, theasinensin A suppressed the phosphorylation of MAPKs, IκB kinase α/β (IKKα/β), and TGF-β activated kinase (TAK1). These data demonstrated that the down-regulation of TAK1-mediated MAPKs and NF-κB signaling pathways might be involved in the inhibition of COX-2 expression by theasinensin A. These findings provide the first molecular basis for the anti-inflammatory properties of oolong tea theasinensins.     

Methionine requirement of juvenile Japanese flounder Paralichthys olivaceus estimated by the oxidation of radioactive methionine

Growth and amino acid oxidation studies were conducted to estimate methionine requirement of juvenile Japanese flounder, Paralichthys olivaceus , by using the purified diets containing 500 g kg^–1 crude protein from casein, gelatine and crystalline amino acids (CAA). Diets with six graded levels of methionine (5.3, 8.3, 11.3, 14.3, 17.3 and 20.3 g kg^–1 diet) were fed to triplicate groups of the juvenile (initial weight 2.8 ± 0.05 g) twice a day for 40 days. To prevent leaching losses, CAA were precoated using carboxymethylcellulose (CMC), and further diets were bound by CMC and κ-carrageenan. Based on broken-line analysis of percentage weight gain and feed conversion efficiency, the methionine requirements of Japanese flounder in the presence of 0.6 g kg^–1 of cystine were 14.9 and 14.4 g kg^–1 dry diet, respectively. After the growth study was finished, a direct estimate of methionine requirement was made by examining the influence of dietary methionine level on ¹⁴C-methionine oxidation by determining radioactive carbon dioxide, protein and nonprotein fractions of the whole body. The dose–response curve between expired radioactive CO₂ and dietary methionine levels showed that the optimum methionine level for the flounder was estimated to be within the range of 14.3–17.3 g kg^–1 of diet in high agreement with values obtained from the growth study.     

Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 μg g⁻¹ body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL⁻¹ for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.