Development and Validation of a LC-MS/MS Method for the Determination of Nitrofuran Metabolites in Soft-Shell Turtle Powder Health Food Supplement.

Soft-shell turtle (SST; freshwater terrapin or tortoise) is a popular and important health functional food (HFF) product in many Asian countries. HFFs containing SST must be safe, but several HFFs have been found to be contaminated with dangerous substances, such as nitrofuran metabolites (NFMs). This finding suggests that the consumption of HFFs results in the regular exposure of vulnerable individuals to hazardous substances. Importantly, nitrofuran antibiotics have been banned for use in food-producing animals since the 1990s by the European Union. Thus, in this study, we propose a reliable and quick method to reduce the time required for the detection of four NFMs in SST powder that conventional methods are unable to quantify. Our method involves the derivatization and hydrolysis of SST powder and was validated in accordance with the requirements of European Commission Decision 2002/657/EC. The method achieves an apparent mean recovery of 82.2-108.1%, repeatability of 1.5-3.8%, and reproducibility of 2.2-4.8% for 0.5-10.0 µg kg-1 of 1-aminohydantoin, semicarbazide, 3-amino-2-oxazolidinone, and 3-amino-5-morpholinomethyl-2-oxazolidinone. In addition, linearity was achieved with correlation coefficients of 0.999, and the detection capability (CCß) and decision limit (CCα) were found to be reliable, indicating that this is a fast and accurate method for the analysis of SST powder. The validated method was successfully applied to detect NFMs in SST powder in commercial HHFs.

Effectiveness of the guava leaf extracts against lipopolysaccharide-induced oxidative stress and immune responses in Cyprinus carpio.

The anti-inflammatory activity of the guava leaf extracts (GLE) against LPS-induced inflammatory responses in fish macrophage cell lines is well documented. Here, we evaluated the effects of dietary GLE on LPS-induced oxidative stress, immune responses, and glucocorticoid receptor-related gene expression in Cyprinus carpio. Basal diet was supplemented with 0 (control), 100, 150, 200, or 250 mg kg-1 GLE for eight weeks. Highest (p < 0.05) weight gain rate was obtained in fish group supplemented with 200 mg kg-1 of GLE. The results showed that superoxide dismutase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, lysozyme, and complement C3 decreased, while malondialdehyde level increased in the liver and spleen upon LPS-challenge. Dietary GLE supplementation (especially 200 or 250 mg kg-1) alleviated LPS-induced changes. Similarly, GLE (150-250 mg kg-1) reversed LPS-induced alteration of serum biochemical parameters such as alkaline phosphatase, aspartate transaminase, alanine transaminase, and myeloperoxidase. LPS treatment markedly induced increased the mRNA levels of TNF-α, IL-1ß, and NF-κB p65 in both the liver and kidney tissues; however, GLE pre-treatment attenuated LPS-induced elicitation of TNF-α, IL-ß, and NF-κB p65. Moreover, dietary GLE supplementation significantly increased the expression of HSP70 and HSP90, and glucocorticoid receptor in the liver and kidney after LPS challenge. Thus, GLE attenuated LPS-induced inflammation response by up-regulating glucocorticoid receptor-related gene expression in carp. Finally, GLE supplementation reduced carp mortality after LPS-challenge. These results suggest that dietary supplementation with 200 mg kg-1 GLE is adequate for effectively attenuating LPS-induced oxidative stress and immune-suppressive effects in C. carpio.

Vaccination of fish against Aeromonas hydrophila infections using the novel approach of transcutaneous immunization with dissolving microneedle patches in aquaculture.

The aim of this study was to develop and evaluate a novel route of administration for vaccinating fish against Aeromonas hydrophila infection using a dissolving microneedles (MNs) patch. The A. hydrophila JUNAH strain was inactivated with formalin and used as a vaccine antigen. It was mixed with dissolvable carboxymethyl cellulose (CMC) as the matrix material to produce the MNs patches. When examined with a scanning electron microscope, each patch has 282 uniformly distributed, pyramid-shaped needles on a circular base. In the skin insertion experiment, the MNs patches were confirmed to be capable of penetrating the skin of the fish. Through agglutination assay and analysis of non-specific parameters like lysozyme and superoxide dismutase, it was verified that the antigen embedded into the patch induced adaptive and innate immune responses in the fish. In the challenge experiment, the group inoculated with the MNs patch and the group injected with formalin killed cells (FKC) showed a similar survival rate. Our results suggest that the FKC-loaded MNs patch is a wholly viable method alternative to injection for the vaccination of fish.

Enhanced bath immersion vaccination through microbubble treatment in the cyprinid loach.

Immunization by bath immersion is likely the simplest method of fish vaccination. Although the route of immunogenicity has not been fully identified, immersion vaccination is clearly a useful labor-saving technique. In this study, microbubble (MB) treatment was assessed for its ability to improve the efficacy of bath immersion vaccination in the cyprinid loach. MBs are commonly defined as minute particles of gas with a diameter of less than 100 µm, which generated free radicals. Here, the efficacy of MB treatment for vaccination enhancement in the cyprinid loach was assessed in direct challenge experiments using the virulent Aeromonas hydrophila JUNAH strain; assessments comprised agglutination titer assay and non-specific parameter analysis. Agglutination titers were high in loaches that were immunized via injection with inactivated cells (FKC group); however, non-specific immune activation parameters (e.g., lysozyme, superoxide dismutase, and phagocytic activity) were more increased in loaches that were immunized via bath immersion with MB treatment. Moreover, MB-treated loaches showed comparable survival rates, relative to loaches immunized via injection with formalin inactivated cells. Thus, higher levels of non-specific immune parameters suggest increased efficacy of this vaccine approach. Improving the effectiveness of bath immersion vaccine will increase its affordability and ease of application in aquaculture.