Development of a quantum dots based immunochromatographic strip for rapid and on-site detection of African swine fever virus.

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.

Impact of Corn Starch Molecular Structures on Texture, Water Dynamics, Microstructure, and Protein Structure in Silver Carp (Hypophthalmichthys molitrix) Surimi Gel.

This study systematically investigates the impact of corn starch molecular structures on the quality attributes of surimi gel products. Employing molecular analyses to characterize corn starch, three amylopectin fractions (A, B1, and B2), categorized by the degree of polymerization ranges (6 < X ≤ 12, 12 < X ≤ 24, and 24 < X ≤ 36, respectively) were specifically focused on. The surimi gel quality was comprehensively assessed through texture profile analysis, nuclear magnetic resonance, scanning electron microscopy, stained section analysis, and Fourier transform infrared spectroscopy. Results indicated the substantial volume expansion of corn amylopectin upon water absorption, effectively occupying the surimi gel matrix and fostering the development of a more densely packed protein network. Starch gels with higher proportions of A, B1, and B2 exhibited improved hardness, chewiness, and bound water content in the resultant surimi gels. The weight-average molecular weight and peak molecular weight of corn starch showed a strong positive correlation with surimi gel hardness and chewiness. Notably, the secondary structure of proteins within the surimi gel was found to be independent of corn starch's molecular structure. This study provides valuable insights for optimizing formulations in surimi gel products, emphasizing the significance of elevated A, B1, and B2 content in corn starch as an optimal choice for crafting dense, chewy, water-retaining surimi gels.

Grass carp Il-2 promotes neutrophil extracellular traps formation via inducing ROS production and autophagy in vitro.

Interleukin (IL)-2 has been reported to regulate neutrophil functions in humans, mice, pigs and chicken although it is a key regulator of T cells. Consistently, we found that grass carp (Ctenopharyngodon idellus) interleukin-2 (gcIl-2) is capable of modulating the antimicrobial activities of neutrophils via regulating granzyme B- and perforin-like gene expression in our previous study. In the present study, stimulation of gcIl-2 on neutrophil extracellular traps (NETs) formation in grass carp neutrophils was demonstrated by detecting free DNA release, histone H3 citrullination and morphological changes of the cells. Further investigation revealed that reactive oxygen species (ROS) production from NADPH oxidase but not mitochondria was involved in NETosis induced by gcIl-2. Aside from ROS, autophagy was disclosed to be indispensable for NETosis induced by gcIl-2. These converging lines of evidence suggested that fish Il-2 could induce NETs formation via NADPH oxidase-derived ROS- and autophagy-dependent pathways in fish species which is evolutionarily conserved with that in mammals. It is noteworthy that these two pathways did not interplay with each other in Il-2-stimulated NETosis. The mechanisms governing autophagy induced by Il-2 were also explored in the present study, showing that Il-2 modulated the action of high mobility group box 1 (HMGB1) protein to stimulate autophagy, leading to NETs formation in fish neutrophils. These results provided a new insight to the function of Il-2 in fish neutrophils, and a clue about the regulation of NETosis in the lower vertebrates.

Characterization and in vitro antibacterial activity of grass carp (Ctenopharyngodon idella) serum amyloid A.

Serum amyloid A (SAA) predominantly synthesized by hepatocytes is a classical acute phase protein and has been extensively studied in mammals. However, the studies on the structure and properties of fish SAA are limited although SAA genes have been cloned and identified from various fishes. In the present study, a cDNA of grass carp (Ctenopharyngodon idella) SAA (gcSAA) was cloned and characterized, displaying a high homology with its counterparts in vertebrates. gcSAA mRNA was expressed with highest abundance in the liver and its levels were increased by a 24-hour infection of Aeromonas hydrophila (A. hydrophila) for more than 5 folds in the intestine, 15 folds in the spleen, 75 folds in the head kidney and 100 folds in the liver, implying that it is an acute phase protein in grass carp. Subsequently, recombinant gcSAA protein (rgcSAA) was prepared from a prokaryotic expression system after codon optimization of its coding sequence. The direct antibacterial activity assay and the plate count assay disclosed that gcSAA inhibited the growth and survival of A. hydrophila but not Edwardsiella piscicida (E. piscicida) which both are common bacterial pathogens in aquaculture. The propidium iodide (PI) uptake assay confirmed the bactericidal property of gcSAA, showing that it is able to enhance the uptake of PI in A. hydrophila but not E. piscicida. These findings revealed the molecular features of gcSAA and its roles in host defense against bacterial infection.

A monoclonal antibody for identifying capsaicin congeners in illegal cooking oil and its applications.

In this work, we studied the preparation of a high-affinity antibody and its immunochromatographic applications to simultaneously identify capsaicin(LJJ), dihydrocapsaicin(HLJ), nordihydrocapsaicin, homodihydrocapsaicin, and other congeners in illegal cooking oil. We used dihydrocapsaicin hapten-conjugated carrier protein BSA as the immunogen according to the formaldehyde method, and conjugated capsaicin and OVA as the coated detection antigen according to the formaldehyde method. We subsequently screened and cloned a hybridoma cell line 2B3 with the highest affinity, which could stably secrete monoclonal antibodies against compounds in the capsaicin family. We then established a capsaicin indirect ELISA standard curve, which was fitted using the linear regression equation R = 0.9987, curve y = -2.3x + 0.2, and IC50 = 0.2 ng/mL. The cross-reaction rate for capsaicin was 100%, 116% for dihydrocapsaicin, 88% for homodihydrocapsaicin, and 94% for nordihydrocapsaicin. In the second application, we established a simple and accurate sample pretreatment method and a quantum dot-labeled test strip to quickly and quantitatively detect capsaicin family compounds in illegal cooking oil in 8 min. The average recovery rates for each spiked concentration were between 75% and 107.8%, and the coefficient of variation values for each spiked concentration were less than 15%. The high-affinity antibody we identified could simultaneously identify capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin, and other congeners in illegal cooking oil, and the antibody could be quickly and accurately applied for the qualitative and quantitative detection of capsaicin family residues in illegal cooking oil.

A Calibration Curve Implanted Enzyme-Linked Immunosorbent Assay for Simultaneously Quantitative Determination of Multiplex Mycotoxins in Cereal Samples, Soybean and Peanut.

In this study, a rapid and sensitive immunoassay method has been established based on calibration curve implanted enzyme-linked immunosorbent assay (C-ELISA) for the simultaneously quantitative determination of aflatoxin B1, deoxynivalenol and zearalenone in cereal samples, soybean and peanut. The C-ELISA avoids using the standard substances during the detection. The principle of the C-ELISA is to implant the optimized standard curve data into the matched analysis software which can make data processing more convenient and faster. The implanted calibration curve software was programmed with C plus plus. In the new immunoassay system for aflatoxin B1, deoxynivalenol and zearalenone, their linear detection ranges were from 0.03~0.81, 1.00~27.00 and 5.00~135.00 ng/g, respectively. Recovery rates from spiked samples ranged from 85% to 110% with the intra-assay coefficients of variation under 5%. Compared with HPLC method, the new method showed consistence in all the observed contents of the three mycotoxins in real samples. The new method can rapidly and reliably high throughput simultaneously screen for multiplex mycotoxins.

Efficient removal of zinc by multi-stress-tolerant yeast Pichia kudriavzevii A16.

Heavy metal bioaccumulation by growing microorganisms is a potential technique for treating the heavy metal pollution in food materials, e.g. fishery processing wastes. In this study, a multi-stress-tolerant yeast with high Zn tolerance and efficient Zn removal ability was screened and renamed as Pichia kudriavzevii A16 after identification. High salinity and low pH obviously increased the Zn bioaccumulation capacity of P. kudriavzevii A16, contributing to the increasing Zn removal rate of P. kudriavzevii A16 at 0.5 mmol/L Zn from 67.69% to 77.03% and 96.09%, respectively. P. kudriavzevii A16 displayed high specificity of Zn removal at high concentrations of Cu, while high concentrations of Cd significantly inhibited the Zn removal by restraining the yeast growth. P. kudriavzevii A16 possessed more powerful Zn removal ability than Saccharomyces cerevisiae CICC1211 under various environmental stresses. The multi-stress-tolerant P. kudriavzevii A16 can be developed into a potential Zn removal agent using in complex food environments.

Modulation of cadmium bioaccumulation and enhancing cadmium tolerance in Pichia kudriavzevii by sodium chloride preincubation.

Application of growing microorganisms for cadmium removal is limited by the sensitivity of living cells to cadmium. The effects of sodium chloride (NaCl) preincubation on the cadmium bioaccumulation and tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation significantly reduced the intracellular and cell-surface cadmium bioaccumulation of P. kudriavzevii at both 6 and 20 mg L(-1) cadmium, while no obvious effect was observed in S. cerevisiae except that the intracellular cadmium bioaccumulation at 20 mg L(-1) cadmium was reduced obviously by 20-60 g L(-1) NaCl. For both yeasts, the improved contents of protein and proline after NaCl preincubation contributed to the cadmium tolerance. The thiol contents in P. kudriavzevii under cadmium stress were alleviated by NaCl preincubation, which might be due to the decrease of intracellular cadmium bioaccumulation. NaCl preincubation enhanced the contents of glycerol and trehalose in P. kudriavzevii under cadmium stress, while no acceleration was observed in S. cerevisiae. The results suggested that NaCl preincubation could be applied in cadmium removal by growing P. kudriavzevii to increase the cadmium tolerance of the yeast.

Halomonas shantousis sp. nov., a novel biogenic amines degrading bacterium isolated from Chinese fermented fish sauce.

A Gram-negative, aerobic, short rod-shaped and non-motile bacterium, designated SWA25(T), was isolated from Chinese fermented fish sauce in Shantou, Guangdong Province, China. Strain SWA25(T) was moderately halophilic, formed colourless colonies and grew at 10-45 °C (optimum, 37 °C) and pH 4-9 (optimum, 6-7) in the presence of 0.5-22.5 % (w/v) NaCl (optimum, 3 %). The major cellular fatty acids (>10 %) were identified as C18:1 ω7C, C16:0, C16:1 ω7c, and C19:0 cyclo ω8c, and the predominant respiratory ubiquinone was Q-9. The genomic DNA G+C content was 61.3 ± 2.1 mol %. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SWA25(T) belonged to the genus Halomonas in the family Halomonadaceae. The closest relatives were Halomonas xianhensis A-1(T) (96.5 % 16S rRNA gene sequence similarity), H. lutea DSM 23508(T) (96.5 %) and H. muralis LMG 20969(T) (96.1 %). DNA-DNA hybridization assays showed 30.7 ± 2.6 % relatedness between strain SWA25(T) and H. xianhensis A-1(T), and 39.4 ± 4.1 % between strain SWA25(T) and H. lutea DSM 23508(T). On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain SWA25(T) should be placed in the genus Halomonas as a representative of a novel species. The name Halomonas shantousis sp. nov. is proposed, with SWA25(T)(=CCTCC AB 2013151(T) = JCM 19368(T)) as the type strain.

The phosphoenolpyruvate carboxykinase of Mycobacterium tuberculosis induces strong cell-mediated immune responses in mice.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes guanosine or adenosine mononucleotide-dependent reversible conversion of oxaloacetate (OAA) and phosphoenolpyruvate (PEP). Mycobacterium (M) tuberculosis possesses a putative GTP-dependent PEPCK. To analyze the immune responses caused by PEPCK, the effects of PEPCK on the induction of CD4(+) T cells and cytokines such as IFN-gamma, IL-12 and TNF-alpha were evaluated in mice. It was found that the number of CD4(+) T cells was increased in the PEPCK immunized mice although the change of the number of CD8(+) T cells was not significant. The cytokines IFN-gamma, IL-12 and TNF-alpha were increased significantly in the mice immunized with PEPCK than those of incomplete adjuvant. These characteristics were further demonstrated in the mice infected by pckA mutated BCG strain. The results indicate that PEPCK can effectively induce cell-mediated immune response by increasing activity of cytokines and PEPCK may be a promising new subunit vaccine candidate for tuberculosis.

Biological role of surface Toxoplasma gondii antigen in development of vaccine.

AIM: To analyze the biological role of the surface antigen of Toxoplasma gondii (T gondii) in development of vaccine. METHODS: The surface antigen of T gondii (SAG1) was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of T gondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on T gondii tachyzoites under electromicroscope. RESULTS: The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-gamma, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-alpha were undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION: SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against T gondii may be regulated by both hormone- and cell-mediated immune response.

pckA-deficient Mycobacterium bovis BCG shows attenuated virulence in mice and in macrophages.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation and phosphorylation of oxaloacetate (OAA) to form phosphoenolpyruvate (PEP). In this study, the regulation of the PEPCK-encoding gene pckA was examined through the evaluation of green fluorescent protein expression driven by the pckA promoter. The results showed that pckA was upregulated by acetate or palmitate but downregulated by glucose. Deletion of the pckA gene of Mycobacterium bovis BCG led to a reduction in the capacity of the bacteria to infect and survive in macrophages. Moreover, mice infected with DeltapckA BCG were able to reduce the bacterial load much more effectively than mice infected with the parental wild-type bacteria. This attenuated virulence was reflected in the degree of pathology, where granuloma formation was diminished both in numbers and degree. The data indicate that PEPCK activity is important during establishment of infection. Whether its role is in the gluconeogenic pathway for carbohydrate formation or in the conversion of PEP to OAA to maintain the TCA cycle remains to be determined.