Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II.

The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture-positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II-negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2%) PCR confirmation assays were performed. Of those, 2,259 (80%) were both ESP and PCR positive; 104 (4%) were ESP positive and PCR negative; 423 (15%) were ESP negative and PCR positive; 28 (1%) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15%) more PCR-positive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5%) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non-M. paratuberculosis isolates.     

Both anti-oxidation and lipid-carbohydrate conversion enhancements are involved in priming-improved emergence of Echinacea purpurea seeds that differ in size

This study evaluated the effects of priming on emergence responses of purple coneflower (Echinacea purpurea L. Moench) seeds. The seeds that differ in seed size were either primed with moistened vermiculite (solid matrix priming) or primed in non-aerated −0.5 MPa polyethylene glycol 6000 solution at 25 °C for 6 days (osmopriming), followed by air-drying to their initial moisture level. The tetrazolium staining tests indicated that both large and small seeds were biochemically viable. No notable difference in germination percentage was found between large and small seeds. However, extensive cavity was visible in portions of small seeds in comparison with large seeds. Large seeds accumulated more antioxidants and had greater activities of anti-oxidative enzymes than small seeds. They also had greater isocitrate lyase and malate synthase activities than small seeds. As a result, large seeds had higher emergence percentage and faster emergence speed as compared to that of small seeds. Both solid matrix priming and osmopriming increased emergence percentage and shortened mean emergence time of purple coneflower seeds by increasing the activities of anti-oxidative enzymes and decreasing the levels of malondialdehyde and total peroxide accumulation. Moreover, priming also enhanced the anti-oxidative activities of treated seeds. The activities of isocitrate lyase and malate synthase were also increased in primed seeds. The enhanced anti-oxidation and lipid-carbohydrate conversion activities might explain in part why primed purple coneflower seeds emerged better than non-primed seeds.     

Differences in lipid peroxidation and Cu,Zn-superoxide dismutase in the hippocampal CA1 region between adult and aged dogs

Reactive oxygen species have been long associated with oxidative stress relevant to many pathological damages. In brain, 4-hydroxy-2E-nonenal (HNE), a major cytotoxic end product of lipid peroxidation, is produced. In contrast, superoxide dismutase (SOD), one of the major antioxidant enzymes, protects neurons from oxidative stress. The aim of this study is to observe differences in the distribution of HNE and Cu,Zn-superoxide dismutase (SOD1) in the hippocampal CA1 region of adult (2-3 years of age) and aged (10-12 years of age) dogs. The HNE immunoreactivity and protein level in the CA1 region were significantly high in the aged dogs compared to those in the adult dogs. SOD1 immunoreactivity and its protein level were also higher in the aged dogs than those in the adult dogs. However, there were not significant differences in NeuN (a neuron-specific soluble nuclear antigen) immunoreactivity in CA1 neurons between the adult and aged dogs. These differences may be associated with oxidative stress in aged dogs compared to that in adult dogs.     

Attractiveness of various protein sources to juvenile rockfish (Sebastes schlegeli,Hilgendorf 1880)

ABSTRACT

The attractiveness of various protein sources of 16 feed ingredients was determined in juvenile rockfish (Sebastes schlegeli) by using reinforced acrylic tank composed of three equally divided rectangular attracting chambers and an acclimatization chamber. Thirty fish were held in the acclimatization chamber at a time and tournament comparison of feed ingredients was applied to evaluate attractiveness. Jack mackerel (JM) (40.0%), sardine (SM) (33.3%), Pollack (PM) (40.0%), shrimp (SHM) (36.7%), mussel meal (40.0%) and oyster (43.3%) meals achieved the highest feeding attractiveness to rockfish in the 1^st through 6^th preliminary test, respectively. JM (40.0%), SHM (36.7%), squid meal (SQM) (33.3%), SM (40.3%), PM (40.0%) and PM (36.7%) achieved the highest feeding attractiveness to fish in the 7^th through 12^th preliminary test, respectively. Among the top five feed ingredients showing high attractiveness to rockfish, JM achieved higher attractiveness than PM and SHM in the 1^st trial. In the 2^nd trial, attractiveness of JM to rockfish was higher than SM and SQM. SM achieved higher attractiveness to rockfish than SQM, but not different from PM throughout the 30-min observation in the 3^rd trial. The strongest feeding attractant response of rockfish was observed in JM, followed by SM, SQM, PM, and SHM, in order among various feed ingredients.     

Substitution effect of Undaria pinnatifida with citrus (Citrus unshiu,Marcovitch) peel by‐product in feed on the growth,body composition and air exposure stressor of juvenile abalone (Haliotis discus,Reeve 1846)

Substitution effect of Undaria pinnatifida with citrus peel by‐product (CPB) on growth, body composition and air exposure stressor of abalone was determined. A total of 1,080 abalone were distributed into 18 net cages. Five formulated diets were prepared in triplicate. The CPB0 diet contained 200 g/kg U. pinnatifida. The 250, 500, 750 and 1,000 g/kg U. pinnatifida were substituted with the equal amount of CPB, referred to as the CPB250, CPB500, CPB750 and CPB1000 diets, respectively. Finally, dry U. pinnatifida was prepared. Abalone were fed for 16 weeks and then subjected to air exposure stressor for 24 hr. The cumulative mortality of abalone was monitored for the following 4 days after 24‐hr air exposure. Survival, weight gain and specific growth rate (SGR) of abalone fed all formulated diets were greater than those of abalone fed the U. pinnatifida. The greatest weight gain and SGR were achieved in abalone fed the CPB500 diet. The chemical composition of the soft body of abalone was not affected by the experimental diets. Higher cumulative mortality was observed in abalone fed the CPB0 and dry U. pinnatifida at 16 hr after 24‐hr air exposure compared to abalone fed all other diets. In conclusion, U. pinnatifida could be completely substituted with CPB in abalone feed.     

Astaxanthin Reduces Stemness Markers in BT20 and T47D Breast Cancer Stem Cells by Inhibiting Expression of Pontin and Mutant p53

Astaxanthin (AST) is a product made from marine organisms that has been used as an anti-cancer supplement. It reduces pontin expression and induces apoptosis in SKBR3, a breast cancer cell line. Using Western blotting and qRT-PCR analyses, this study revealed that in the T47D and BT20 breast cancer cell lines, AST inhibits expression of pontin and mutp53, as well as the Oct4 and Nanog cancer stem cell (CSC) stemness genes. In addition, we explored the mechanism by which AST eradicates breast cancer cells using pontin siRNAs. Pontin knockdown by pontin siRNA reduced proliferation, Oct4 and Nanog expression, colony and spheroid formation, and migration and invasion abilities in breast cancer cells. In addition, reductions in Oct4, Nanog, and mutp53 expression following rottlerin treatment confirmed the role of pontin in these cells. Therefore, pontin may play a central role in the regulation of CSC properties and in cell proliferation following AST treatment. Taken together, these findings demonstrate that AST can repress CSC stemness genes in breast cancer cells, which implies that AST therapy could be used to improve the efficacy of other anti-cancer therapies against breast cancer cells.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD80

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0 kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48 h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.