Yaling Wang

A sensitive and validated immunomagnetic-bead based enzyme-linked immunosorbent assay for analyzing total T-2 (free and modified) toxins in shrimp tissues

Accurate analyses of total T-2 (free and modified) in aquatic organisms including shrimp are important as the hazard caused by T-2 has been caught increasing attention. Therefore, acurate analysis of free T-2 especially of modified T-2 in shrimp tissues is important. A rapid, sensitive, and validated method for quantitative determination of free T-2 and modified T-2 toxin was developed using immunomagnetic-bead based enzyme-linked immunosorbent assay (IMB-ELISA). Super paramagnetic particles with a carboxyl group activated by an ester method coupled with envelope antigen 3- acetylneosolaniol- hemisuccinate - ovalbumin (3-Ac-NEOS-HS-OVA) was used to form immunomagnetic beads which could bind to T-2 skeletal structure antibodies. The conditions for magnetic bead coating of T-2 skeletal structure antibodies, and the concentrations of the polyclonal antibody and HRP-labeled goat anti-rabbit antibody were optimized. A good linear relationship with T-2 concentrations ranging from 5-75ng/mL (R2 =0.9965) was observed. The detection limit of different shrimp tissues of the IMB-ELISA ranged from 2.53 to 3.20ng/mL. And the IC50 was 63ng/mL. The recovery varied from 86% to 99% with a standard deviation of 2.8-5.8%. The application of this method to study the distribution in tissues showed that the total T-2 concentration in hepatopancreas was 26.7µg/kg > blood > head > muscle in the highest dose group of 12.2mg/kg. Our research showed a combination of ELISA and immunomagnetic bead technology provide a new, convenient approach to significantly improve the accuracy and sensitivity of total T-2 measurement in shrimp tissues.

Pathogenicity of Vibrio parahaemolyticus in Different Food Matrices.

The pathogenicity and virulence factors of Vibrio parahaemolyticus in four food matrices--shrimp, freshwater fish, pork, and egg-fried rice--were compared by measuring the thermostable direct hemolysin activity and total hemolytic titer. Significantly high thermostable direct hemolysin and also hemolytic titers (P < 0.05) were produced by V. parahaemolyticus in egg-fried rice > shrimp > freshwater fish > pork. Filtrates of V. parahaemolyticus in shrimp given intraperitoneally induced marked liver and kidney damage and were highly lethal to adult mice compared with filtrates of V. parahaemolyticus in freshwater fish > egg-fried rice > pork. From in vitro and in vivo pathogenicity tests, it seems the type of food matrix has a significant impact on the virulence of V. parahaemolyticus. These results suggest that hemolysin may not necessarily be the only virulence factor for pathogenicity of V. parahaemolyticus. This is the first report that shows that virulence factors produced by V. parahaemolyticus in seafood such as shrimp are more toxic in vivo than in nonseafood.

Modeling antimicrobial activity of lipopeptides from Bacillus amyloliquefaciens ES-2 against Shewanella putrefaciens in shrimp meat using a response surface method.

Bacillus amyloliquefaciens ES-2 can produce antimicrobial lipopeptides, including surfactin and fengycin. In this study, the model of antimicrobial activity against Shewanella putrefaciens in shrimp meat by antimicrobial lipopeptides from B. amyloliquefaciens ES-2 was researched by response surface methodology. The results showed that S. putrefaciens had high sensitivity to antimicrobial lipopeptides, which had a MIC of 0.6 mg/ml. A quadratic mathematical model representative of the action of antimicrobial lipopeptides on S. putrefaciens in shrimp meat was developed as a function of concentration, time, and temperature. A reduction of S. putrefaciens cells of over 2 log cycles could be realized when the temperature was below 5.4°C, the time was over 6 h, and the concentration of the lipopeptides was over 0.3 mg/g.

Effect of T-2 toxin-injected shrimp muscle extracts on mouse macrophage cells (RAW264.7)

Abstract Following intramuscular injections of 0.1 mL, 3 mg kg−1 BW−1(1/10 LD50) T-2 toxin (T-2), the tissue concentration of T-2 in shrimp was quantitatively detected using LC-MS/MS. The biological half-time (t1/2) of T-2 in blood was 40.47 ± 0.24 min. The highest number of intramuscular T-2 shrimp could tolerate when given at blood t1/2 intervals was 4. The shrimps which were injected 5 T-2 died. The T-2 toxin highest accumulation was 0.471 ± 0.012 ng g−1 BW−1. The effect of toxic shrimp muscle subjected to different processing conditions (high pressure, trifluoroacetic acid, acid and alkali digestions, artificial digestive juice [to simulate exposure to gastric and intestinal juices]) on mouse macrophage cells (RAW267.4) were evaluated by the MTT assay. The inhibition ratio of 2% muscle extract on RAW267.4 was 85.70 ± 2.63%. The immunocytotoxicity of muscle extracts to RAW264.7 was highest in muscle extracts subjected to physical and chemical digestion (high pressure > NaOH > trifluoroacetic acid > 0.02 M HCl > 0.2 M HCl > controls), and also artificial digestion (artificial intestinal juice > artificial gastric juice > N type intestinal juice > N type gastric liquid > controls). Results showed that high-pressure and artificial intestinal juice were most effective in the release of modified T-2 to free T-2 thus enhancing toxicity. These results can be interpreted as measurement of T-2 in food being of little value because of enhanced toxicity of T-2-contaminated food as they pass through the gastrointestinal tract.

Analysis of T-2 Toxin Removal Factors in a Lactococcus Fermentation System

The objective of this work was to determine the bacterial strains and factors that most efficiently degrade T-2 toxin in foods or animal feed. To determine the most efficient strain and optimal incubation times for degradation of T-2, the rate of T-2 removal by three lactic acid bacteria strains was quantified by liquid chromatography plus tandem mass spectrometry after incubation in de Man Rogosa Sharpe broth with 50 ng mL-1 T-2 at 37°C for 96 h. Various components of the most efficient degradation strain fermentation systems were extracted, and the ability to remove T-2 was assayed. Lactococcus lactis CAMT22361 was the most efficient degradation strain for removing T-2. Yeast extract powder interfered with L. lactis CAMT22361 in the degradation process. T-2 toxin was removed by various components of the L. lactis CAMT22361 cells in the following order: nonprotein material of the extracellular fraction > protein in the extracellular fraction > whole cell ≈ cell wall > cell intracellular matrix fluid. T-2 removal rates were 54.08% ± 0.79%, 43.65% ± 0.84%, 43.09% ± 0.87%, 41.98% ± 0.8%, and 23.45% ± 0.66%, respectively. The nonprotein fraction in the extracellular fluid was most likely the key component in L. lactis CAMT22361 and hence would be the most desirable cellular component to be used to remove T-2 from food or feed.

Effects of T-2 Toxin on Pacific White Shrimp Litopenaeus vannamei: Growth, and Antioxidant Defenses and Capacity and Histopathology in the Hepatopancreas

Modified-masked T-2 toxin (mT-2) formed during metabolism in edible aquatic animals may go undetected by traditional analytical methods, thereby underestimating T-2 toxicity. The effects of T-2 on growth and antioxidant capacity and histopathological changes in the hepatopancreas were studied in Pacific white shrimp Litopenaeus vannamei exposed for 20 d to 0, 0.5, 1.2, 2.4, 4.8, and 12.2 mg/kg of T-2 in their feed. The concentration of mT-2 in the hepatopancreas was detected by liquid chromatography-tandem mass spectrophotometry before and after trifluoroacetic acid (TFA) treatment that converted mT-2 to free T-2. A dose-dependent increase in mT-2 concentration was observed in the hepatopancreas. Dietary exposure to T-2 significantly decreased (P < 0.05) shrimp growth and survival rate compared with the controls. The malondialdehyde (MDA) concentration was significantly increased in shrimp exposed to feed with ≥2.4 mg/kg T-2 (P < 0.05). The antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), total antioxidant capacity (T-AOC), and also glutathione (GSH) content increased in shrimp dosed with 2.4-4.8 mg/kg T-2 but declined at the highest dose (12.2 mg/kg), probably indicating an inability to cope with high concentrations of reactive oxygen species (ROS) as evident from a marked increase in MDA (P < 0.05) culminating in cellular toxicity. Histopathological changes in the hepatopancreas were dose dependent, with cell autophagy evident at the highest exposure dose. This is the first report in shrimp of a dose-dependent increase in ROS, SOD enzyme activity, and T-AOC at low T-2 exposures, and associated histopathological changes in the hepatopancreas, in response to dietary T-2. Received January 26, 2016; accepted October 9, 2016.

Modeling the effects of different conditions on the inhibitory activity of antimicrobial lipopeptide (AMPNT-6) against Staphylococcus aureus growth and enterotoxin production in shrimp meat

Several bacteria, including Staphylococcus aureus (S. aureus), have been identified as food-borne pathogenic bacteria that cause many cases of food poisoning. This has prompted researchers to identify novel antimicrobial compounds. NT-6 antimicrobial lipopeptide (AMPNT-6) secreted by Bacillus subtilis can inhibit S. aureus growth and enterotoxin B production. The effects of temperature, sodium chloride, pH and sodium metabisulfite on the activity of AMPNT-6 applied to control S. aureus growth and enterotoxin B production in shrimp were investigated by using the response surface method. Design-Expert software was chosen to conduct data analysis and develop the models to demonstrate whether AMPNT-6 is able to control the growth of S. aureus and toxin production in shrimp exposed to different environmental conditions. The results showed that temperature, sodium chloride concentration, pH and sodium metabisulfite concentration had no significant effects on the antimicrobial activity of AMPNT-6 and there were no significant interactions between them. Quadratic polynomial mathematical models were established, and the quadratic regression equations of predicted value LgN (logarithm of bacterial colony number) and Y (enterotoxin B) to the independent variables x1 (temperature), x2 (sodium chloride concentration), x3 (pH), x4 (sodium metabisulfite concentration) and x5 (AMPNT-6 concentration) were constructed as:

Regulatory effects of Shewanella putrefaciens isolated from shrimp Penaeus orientalis on the virulence factors of Vibrio parahaemolyticus and evaluation of the role of quorum sensing in virulence factors regulation

{\text{LgN}} = 6.93 + 0.29x_{1} - 0.11x_{3} - 0.46x_{5} - 0.35x_{1}^{2} - 0.30x_{3} x_{4} \left( {R^{2 } = 0.9049} \right);