Screening Of Essential Oils For Inhibiting Aeromonas Schubertii And Its Effect On Diease Resistance Of Hybrid Snakehead

Aeromonas schubertii（A.schubertii）is the pathogen of visceral sarcoidosis in hybrid snakehead[Channa maculate♀×Channa argus♂],which brought serious harm to hybrid snakehead.But there has had no effective prevention and control measures aiming to A.schubertii except antibiotics by far.In order to reduce the use of antibiotics in aquaculture,essential oils were studied as antibacterial substance to cure the fish diseases.Hybrid snakehead with typical symptoms of A.schubertii was selected for the isolation and identification of pathogenic bacteria,and the bacteria was used as an indicator to screen essential oils.Moreover,the selected essential oils were further analyzed for their antibacterial mechanism,effects on hybrid snakehead about their disease resistance and relative expression of MHCⅠ.The main study contents and results are presented as follows:Isolation and identification of A.schubertiiA dominant strain was isolated from the kidney of the diseased hybrid snakehead and named 1907056.The results of artificial challenge showed that 1907056 had strong pathogenicity against hybrid snakehead with an LD₅₀ value of 2.6×10⁶CFU/m L.The1907056 strain was identified as A.schubertii based on morphological and biochemical identification,together with 16S r DNA and gyr B gene sequencing.The growth curve of the bacterium was described by measuring the OD₆₀₀,and the results showed that the bacterium grows extremely rapidly,and its logarithmic phase is between 2 h and 6 h,and entered to plateau phase after 10 h.Screening of plant essential oils for inhibiting A.schubertiiThe agar diffusion method was used to screen essential oils with bacteriostatic activity,and the minimum inhibitory concentration（MIC）and the minimum bactericidal concentration（MBC）of the selected essential oils against strain 1907056were determined by the double dilution method and the plate coating method.The test results show that cinnamaldehyde,oregano essential oil and tannin all have an inhibitory effect on the test bacteria.Among them,cinnamaldehyde has the strongest inhibitory effect.The diameter of the inhibition zone is（42.83±1.25）mm,and the MIC and MBC are 0.1μL/m L and 0.2μL/m L,indicating that cinnamaldehyde has a very strong inhibitory effect on the test bacteria 1907056.Inhibitory mechanism of cinnamaldehyde on A.schubertiiThe effects of cinnamaldehyde on the cell wall,cell membrane and DNA of A.schubertii were studied by measuring alkaline phosphatase（AKP）activity,intracellular metabolic enzyme activity,protein content,and genomic DNA electrophoresis patterns in bacterial culture medium.It was found that cinnamaldehyde can change the permeability of cell wall and cell membrane of the bacteria,and cause the DNA molecules to break,eventually leading to the death of the bacteria.The effects of cinnamaldehyde on disease resistance and MHCⅠgene expression in hybrid snakeheadThe feeding experiment was lasted for 35 days.After breeding,the fish were challenged with A.schubertii,and the results showed that the addition of 0.04%and0.08%cinnamaldehyde in the feed could significantly improve the resistance of the hybrid snakehead to A.schubertii.And the survival rate after challenged were（48.61±11.95）%and（55.56±7.86）%.In addition,high dose of cinnamaldehyde can significantly promote the expression of MHC I in the liver and kidney,but inhibit its expression in the spleen.In a word,cinnamaldehyde has a strong inhibitory effect on A.schubertii,and has a positive effect on the disease resistance of hybrid snakehead and the expression of MHC I,which can be used as a new direction for the development of new aquatic drugs.