| Aeromonas hydrophila belongs to the Aeromonadaceae family and the genus Aeromonas,and is a zoonotic pathogen.When humans are infected with Aeromonas hydrophila,symptoms such as severe diarrhea and sepsis can occur.At present,the main way to treat aquatic animal diseases caused by Aeromonas hydrophila in the aquaculture industry is to use antibiotics,and the massive use of antibiotics has also brought about increasingly serious problems.Therefore,research and development of green and healthy drugs to replace antibiotics is an urgent task for the aquaculture industry.Traditional Chinese medicine has attracted much attention because of its many advantages,such as no residue,no environmental pollution and obvious antibacterial effect.In view of this,this study selected the three Chinese medicines of Astragalus,Sanguis,and Chinese gall.Through the in vitro antibacterial test,the in vitro inhibitory effect of the three Chinese medicines decoction and their combined use on Aeromonas hydrophila was investigated,and the effects on Aeromonas hydrophila were analyzed.The influence of bacteria-related virulence gene expression.Feed the crucian carp with three kinds of single Chinese medicinal water decoctions and a combination of coated fish food by oral immunization,and detect its growth indicators,related immune indicators,cytokine expression levels,and after the challenge to Aeromonas hydrophila To explore the protective effect of the three-flavor traditional Chinese medicine decoction and its combination on the immune protection of fish.This test is mainly divided into two parts:Test1:In Vitro Inhibition Test of Three Kinds of Traditional Chinese Medicine Decoction and Combination on Aeromonas hydrophilaDetermine the minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),growth curve,biofilm,and inhibitory virulence of three single Chinese medicine decoctions of Astragalus,Sanyu,and Chinese gallnut and their combined use against Aeromonas hydrophila Gene expression.To explore the in vitro antibacterial effect of single Chinese medicine decoction and its combination.The two-fold dilution method was used to determine the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of each Chinese medicine against Aeromonas hydrophila.The results showed that the MIC and MBC of single-flavored Radix Astragali and Sanguis sylvestris were 2.5g/ml and5g/ml,respectively;the MIC and MBC of single-flavored Galla Chinensis were 1.25g/ml and2.5g/ml,respectively;the MIC and MBC after combined use The lowest concentration is0.625g/ml and 2.5g/ml respectively.Select the minimum inhibitory concentration of each traditional Chinese medicine decoction to act on Aeromonas hydrophila,measure the OD600 of the bacterial liquid every 1h within 10 h,and draw the curve,and find that the three single-flavored Chinese medicines reacted to the hydrophile before 4h.The bacterium has obvious inhibitory effect,and after the combined use,it has an inhibitory effect on Aeromonas hydrophila within 7 hours.Detecting its effect on the biofilm of Aeromonas hydrophila found that: single-flavored Astragalus,Sanguis,and Chinese gall can inhibit the biofilm of Aeromonas hydrophila at a concentration of 25%,and after combined use at a concentration of 15% When it can inhibit the biofilm of Aeromonas hydrophila.Each in vitro inhibition test showed that the combined use of Chinese herbal medicine water decoction has stronger inhibitory effect on Aeromonas hydrophila than single Chinese herbal medicine water decoction.The minimum inhibitory concentration measured by each traditional Chinese medicine acted on Aeromonas hydrophila,and its influence on the expression level of related virulence genes of Aeromonas hydrophila was analyzed.The results showed that the expression level of the hemolysin hly gene of Aeromonas hydrophila was not significantly different from that of the blank control group(P<0.05),and the expression of the omp gene of Aeromonas hydrophila in the astragalus group and the combination group The amount was significantly different from that of the blank control group(P<0.01),and the expression of the luxs gene of Aeromonas hydrophila in the Sanguisorba group and the combination group was significantly different from that of the blank control group(P<0.001).It shows that each Chinese medicine can inhibit the expression of omp gene and luxs gene of Aeromonas hydrophila.Test2:Study on the immunoprotective effects of three kinds of single Chinese medicine decoctions and their combined use.Crucian carp was used as a test animal.After the feed was coated with traditional Chinese medicine,the crucian carp was fed regularly and quantitatively.The tissues and blood of each group of crucian carp were collected before immunization and during each immunization cycle to detect immune related indicators.After the end of the immunization,a challenge with Aeromonas hydrophila was performed to detect the immune protection rate.ELISA method was used to detect serum Ig M,l ZM,SOD,CAT,C4,C3,GSH-PX indicators.The results showed that the various indicators of crucian carp fed with traditional Chinese medicine have been improved.The serum of crucian carp after oral immunization can detect a higher level of Ig M,and the serum contains LZM,SOD,CAT,C3,C4,and GSH-PX.All indicators showed upward adjustment trends to varying degrees.It is preliminarily shown that various Chinese medicines can enhance the humoral immunity of fish.QRT-PCR was used to detect the transcription levels of NF-KB,MYD88,IL-1β,TNF-α and IFN-γ cytokines in various tissues of crucian carp.The results showed that the transfer rate of IL-1β,TNF-α,IFN-γ,NF-KB,MYD88 in the kidney and spleen of the crucian carp in each test group showed an upward-regulated trend.In particular,the expression of IL-10,IL-1β,IFN-γ,TNF-α,NF-KB and MYD88 genes in the intestine of crucian carp is significantly up-regulated,indicating that traditional Chinese medicine may stimulate the relevant cytokines in the intestine of fish to achieve the improvement of crucian carp.The role of immune level.After being challenged by Aeromonas hydrophila,it was observed for 14 days.It was found that Chinese medicine can relieve the infection of Aeromonas hydrophila on crucian carp,improve the survival rate of crucian carp,and play a protective role to a certain extent.In summary,the traditional Chinese medicine selected in this study can inhibit Aeromonas hydrophila both in vivo and in vitro,and feeding it as a feed additive to fish schools can provide medication basis for the prevention and control of fish diseases caused by the bacteria and provide references for fish Immunity played a role in promoting. |
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