Aeromonas hydrophila is an opportunistic pathogen responsible for a number of diseases in freshwater farming. Moreover, the bacterium has been identified as a zoonotic pathogen that threatens human health. Antibiotics are widely used for treatments of infectious diseases in aquaculture. However, the abuse of antibiotics has led to the emergence of antimicrobial resistant strains. Thus, novel strategies are required against resistant A. hydrophila strains. The quorum sensing (QS) system, involved in virulence factor production and biofilm formation, is a promising target in identifying novel drugs against A. hydrophila infections. In this study, we found that thymol, at sub-inhibitory concentrations, could significantly reduce the production of aerolysin and biofilm formation by inhibiting the transcription of genes aerA, ahyI, and ahyR. These results indicate that thymol inhibits the quorum sensing system. The protective effects of thymol against A. hydrophila mediated cell injury were determined by live/dead assay and lactate dehydrogenase (LDH) release assay. Moreover, the in vivo study showed that thymol could significantly decrease the mortality of channel catfish infected with A. hydrophila. Taken together, these findings demonstrate that thymol could be chosen as a phytotherapeutic candidate for inhibiting quorum sensing system-mediated aerolysin production and biofilm formation in A. hydrophila.

Aeromonas hydrophila is a pathogenic bacterium that can cause serious infections both in humans and aquatic animals. Antibiotics are the main approach for fighting against the pathogen. However, the emergence of antibiotic resistance has resulted in treatment failure. Therefore, drugs with novel strategies need to be developed. Quorum sensing has been recognized as a promising method for identifying anti-virulence drugs against bacterial infections. The aim of this study was to identify novel drugs targeting quorum sensing of A. hydrophila as alternatives of antibiotics in aquaculture. Thus, hemolytic activity, biofilm formation, qPCR and experimental therapeutics assays were conducted. The results showed that sanguinarine inhibited the growth of A. hydrophila at concentrations higher than 16 μg/mL, but the production of aerolysin and biofilm formation was significantly inhibited at sub-inhibitory concentrations by disrupting the quorum sensing system. Cell viability results showed that sanguinarine could provide protection for A549 cells from aerolysin-induced cell injury. In addition, the mortality of channel catfish administered with sanguinarine at a dosage of 20 mg/kg decreased to 40%, which showed a significant decrease compared with fish in positive group. Taken together, these findings demonstrated that anti-virulence strategies can be a powerful weapon for fighting against bacterial pathogens and sanguinarine appears to be a promising candidate in the treatment of A. hydrophila infections.

Niclosamide is a mollusicide used for aquaculture on a large scale in China. However, it has high toxicity to aquatic animals and may cause damage to DNA. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction procedure with high-performance liquid chromatography–heated electrospray ionization-tandem mass spectrometry method was developed for its determination. Samples were extracted with ammoniated acetonitrile, and anhydrous magnesium sulfate was added to remove moisture and precipitate proteins. The extracts were further purified with octadecylsilane. The recoveries of niclosamide in fortified muscle tissue at concentrations of 0.5 to 20 µg/kg were between 76.40% and 95.60% with relative standard deviations less than 9.54%. The limits of detection and quantification were 0.2 µg/kg and 0.5 µg/kg, respectively. The developed method was used to study the adsorption of niclosamide in channel catfish after administration.

The aim of this study was to investigate the effect of different water temperatures (19, 25, and 30°C) on tissue residue depletion of tiamulin in Nile tilapia (Oreochromis niloticus) after five consecutive days of oral administration at the dose of 20 mg/kg body weight and to calculate the corresponding elimination half-life (T_1/2) and withdrawal times (WTs). After oral administration at scheduled 11 time points (1, 2, 3, 5, 7, 9, 12, 15, 20, 25, and 30 days), samples of plasma and tissues (muscle plus skin, liver, kidney, and gill) were collected. Tiamulin concentration in samples were determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).T_1/2was calculated by the equation:T_1/2= ln2/k. WT 1.4 software was used to calculate WT. The results showed that tiamulin was widely distributed in all tissue samples with the highest concentration in liver. At three different water temperatures, theT_1/2were calculated as 2.76, 2.13, and 1.64 days in plasma, 2.71, 1.85, and 1.31 days in muscle plus skin, 2.27, 1.70, and 1.50 days in liver, 2.84, 2.32, and 1.94 day in kidney, and 3.16, 2.42, and 1.74 days in gill, respectively. At 19°C, the order of WT is kidney (11.88 days) > liver (10.41 days) > gill (10.77 days) > plasma (8.83 days) > muscle plus skin (7.14 days). The WT for tiamulin at 25°C was in the following order: kidney (8.40 days) > liver (8.21 days) > gill (8.07 days) > plasma (7.24 days) > muscle plus skin (4.05 days). At 30°C, the WT dropped and shown as follows: gill (6.99 days) > kidney (6.51 days) > liver (6.29 days) > plasma (3.27 days) > muscle plus skin (2.92 days). The present investigations indicated that increasing the temperature from 19 to 30°C shortenedT_1/2and WT of tiamulin in tilapia. To ensure the safety of fish consumption, the longest WT of tissues is suggested for tiamulin in Nile tilapia at the corresponding water temperature; i.e., WTs were 12 days at 19°C, 9 days at 25°C, and 7 days at 30°C, respectively. Overall, we intended to provide a theoretical basis for tissue residue depletion kinetics of tiamulin in fish and improve our understanding of the influence of the temperature on tissue residue depletion kinetics of tiamulin in fish.

IL-10 and IL-6 play important roles in protecting against inflammation and clearing pathogens from the body. In this study, homologous compounds of IL-10 and IL-6 were identified in channel catfish, and their immune responses were analyzed. The CDS sequences of IL-10 and IL-6 were 549 bp and 642 bp, respectively, and showed the highest homology with Ameiurus melas. In addition, the expression of the IL-10 and IL-6 genes was ubiquitous in 10 tissues examined. IL-10 is highly expressed in the liver and slightly expressed in the gill. The high expression of the IL-6 gene was observed in the spleen, heart, and gonad, with the lowest levels in the liver. LPS, Poly(I:C), PHA, and PMA showed a highly significant increase in IL-10 and IL-6 expression 48 h after CCK stimulation (p < 0.01). Otherwise, Yersinia ruckeri, Streptococcus iniae, channel catfish virus, and deltamethrin induced IL-10 and IL-6 expression, varying in intensity between different organs. Our results suggest that IL-10 and IL-6 are involved in the immune response of the host against the pathogen.

We developed a method for determination of imidacloprid and its metabolites 5-hydroxy imidacloprid, olefin imidacloprid, imidacloprid urea and 6-chloronicotinic acid in Procambarus clarkii (crayfish) tissues using quick, easy, cheap, effective, rugged, and safe (QuEChERS) and high-performance liquid chromatography-triple quadrupole mass spectrometry. Samples (plasma, cephalothorax, hepatopancrea, gill, intestine, and muscle) were extracted with acetonitrile containing 0.1% acetic acid and cleaned up using a neutral alumina column containing a primary secondary amine. The prepared samples were separated using reverse phase chromatography and scanned in the positive and negative ion multiple reaction-monitoring modes. Under the optimum experimental conditions, spiked recoveries for these compounds in P. clarkii samples ranged from 80.6 to 112.7% with relative standard deviations of 4.2 to 12.6%. The limits of detection were 0.02–0.5 μg·L⁻¹, the limits of quantification were 0.05–2.0 μg·L⁻¹ and the method of quantification was 0.05–2.0 μg·kg⁻¹. The method is rapid, simple, sensitive and suitable for rapid determination and analysis of imidacloprid and its metabolites in P. clarkii tissues.