Study On Quantity Controlling Of Pathogenic Bacterial In Shrimp Culture Using Chinese Herbal Compounds (HD-4) By Oral Administration

Since 2009,acute hepatopancreatic necrosis disease（AHPND）has occurred in Chinese cultured Litopenaeus vannamei,causing great losses to the whole shrimp industry.The study showed that the outbreak of the disease was related to excessive levels of AHPND causing Vibrio parahaemolyticus(VP_AHPND)in Litopenaeus vannamei.In this study,Sanguisorba officinalis L.,the main component of compound Chinese herbal medicine HD-4,was used to explore the in vitro antimicrobial effect and mechanism of VP_AHPND.Effects of structure of intestinal flora in Litopenaeus vannamei was studied through a small scale feeding experiment.Through the production test,the quantitative control of Vibrio in Litopenaeus vannamei after the intake of HD-4 was checked.The results of this study provided theoretical support for the application of DH-4 in the actual production and the establishment of AHPND prevention and control technology.1、Effects of Sanguisorba officinalic L.on the growth of VP_AHPNDTo explore the inhibition mechanism of HD-4 on VP_AHPND,we select the principal component of HD-4（Sanguisorba officinalic L）as the research object,and conducted some experiment such as the inhibition effect of different extraction methods on VP_AHPND and the growth curve of VP_AHPND with different concentration of Sanguisorba officinalic L.alcoholic extracts.Results show that the inhibitory zone diameters of Sanguisorba officinalic L.aqueous extracts（Ⅰ）,high temperature treated Sanguisorba officinalic L.aqueous extracts（Ⅱ）,Sanguisorba officinalic L.alcoholic extracts（Ⅲ）and Sanguisorba officinalic L.alcoholic and aqueous extracts（Ⅳ）on VP_AHPND are 2.00cm,1.97 cm,2.60 cm and 2.55 cm respectively.The minimal inhibitory concentration（MIC）of drug（Ⅰ）and（Ⅱ）on VP_AHPND is 3.2 mg/mL,while the minimal bactericidal concentration（MBC）is 6.4 mg/mL.The MIC of drug（Ⅲ）and（Ⅳ）is 1.6mg/mL while MBC is 6.4 mg/mL.In the co-culture experiment of drugs and bacteria,the growth curves of the blank control group,0.4mg/mL group and 1.6mg/mL group reached the plateau at 12h,18h and 18h,respectively.The maximum concentration of bacteria at the plateau was 4.60×10¹⁰CFU/mL,9.00×10⁶CFU/mL and 4.40×10⁴CFU/mL respectively,while the bacteria cannot survive in 6.4mg/mL group.In the stress-culture experiment,the growth curve of VP_AHPND in each group showed the same trend before（0-10h）while it was firstly inhibited and then gradually recovered after adding drugs（10-22h）.The maximum value was 4.14×10¹⁰CFU/mL at 20h.It was greatly inhibited in1.6mg/mL group and the maximum value was 6.46×10⁸CFU/mL at 20h.6.4mg/mL group was inhibited greatly after adding drugs,while there was no surviving bacteria at20h.2、Stability evaluation of six reference genes of VP_AHPND under different concentrations of Sanguisorba officinalis L.alcoholic extractsReference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays.The current study was designed to select the optimal reference genes suitable for evaluating the inhibitory effect of Chinese herbal medicine on Vibrio parahaemolyticus.The expression of 6 common candidate reference genes（rec A,pvs A,pvu A,gapdh,16S rRNA and rpo S）of Vibrio parahaemolyticus under the stress of Sanguisorba officinalis L.alcoholic extracts were detected by qRT-PCR,data analysis was then assessed by GeNorm,Norm Finder,Best Keeper,Delta CT and Ref Finder software packages.The results showed that the primers efficiency E（%）were within 1.00±0.08and the amplicons of these six genes had good specificity under the stress of different concentrations of Sanguisorba officinalis L.extraction.16S rRNA got the lowest variation in Ct value（CV=3.88）,while pvs A got the highest variation value（CV=12.53）.The stability of the six reference genes judged by the five methods is listed as follows:The stability order given by GeNorm was rpo S=16SrRNA>gapdh>rec A>pvu A>pvs A.The result of Norm Finder is gapdh>rpo S>pvu A>16S rRNA>rec A>pvs A,Best Keeper is 16SrRNA>rpo S>gapdh>rec A>pvu A>pvs A and Delta Ct is gapdh>rpo S>pvu A>16S rRNA>rec A>pvs A.The comprehensive ranking results made by Ref Finder is rpo S>gapdh>16S rRNA>pvu A>pvs A>rec A.Taking the results of pairwise variations into consideration,it is recommended using both rpo S and gapdh as reference genes in this condition.The results will provide foundation supporting for studying the inhibitory mechanism of Sanguisorba officinalis L.on VP_AHPND from the perspective of gene expression.It is of great significance for the establishment of AHPND-prevention technology using Sanguisorba officinalis L.as the core drug.3、Effect of Sanguisorba officinalic L.alcoholic extracts on VP_AHPNDHPND T6SSVP_AHPND is one of the main pathogens of acute hepatopancreatic necrosis disease（AHPND）in Litopenaeus vannamei.Its pathogenic factor T6SS is closely related to the occurrence of the disease.T6SS provides the adhesion and environmental adaptability of VP_AHPND,which can dominate the inter-species competition of bacteria and play an important role in the process of infecting host.Inhibiting the growth and expression of T6SS can reduce the virulence of VP_AHPND to the host and provide an effective strategy for the prevention of AHPND.In this study,qRT-PCR technology was used to investigate the effects of Sanguisorba officinalic L.alcoholic extracts on growth and expression of related genes（VP1388,VP1393,VP1400,VP1409,VPA1027,VPA1043and VPA104）in T6SS of VP_AHPND to explore the mechanism of Sanguisorba officinalic L.on VP_AHPND.The results showed that the alcoholic extracts of Sanguisorba officinalic L.could significantly inhibit the transcriptional activities of VPA1027,VPA1043 and VPA1044,while it could activate VP1388,VP1400 and VP1409 in a short time,but had little effect on the expression of VP1393.It was concluded that the Sanguisorba officinalic L.alcoholic extracts could weaken the pathogenicity of VP_AHPND to the host by inhibiting the growth and T6SS function,which provided theoretical support for the feasibility of using Sanguisorba officinalic L to prevent and control shrimp AHPND.4.Effects of structure of intestinal flora in Litopenaeus vannamei by oral HD-4To explore the effect of HD-4 on the structure of intestinal flora in Litopenaeus vannamei,we conductd the experiment through feeding medicated feeds.And measured the structure of intestinal flora in Litopenaeus vannamei by Illumina HiSeq high-throughput sequencing technology.The results showed that 650 different types of OTUs were detected in all samples,among which 238 types of OTU were shared by five groups of samples.Before the experiment,Proteobacteria,Bacteroidetes,Firmicutes and Tenericutes are the main bacteriophyta in tested shrimp gut on the classification of phylum.Flavobacteriales,Vibrionales,Rhodobacterales and Mycoplasmatales are the main bacteria in tested shrimp gut on the classification of order.Vibrio and Spongiimonas are the main bacterium in tested shrimp gut on the classification of genus.At the end of the experiment,the relative content of bacteria at each classification level changed.Among them,Vibrio and Spongiimonas showed the most significant difference at the level of genus classification which relative contents decreased to 6.94%and 0.54%,respectively.During the experiment,ACE and Chao1index were significantly increased,indicating that the bacterial abundance in shrimp gut increased.The decrease of Simpson index and the increase of Shannon index indicated that the diversity of intestinal flora of Litopenaeus vannamei increased significantly.Through KEGG function prediction,it was found that the metabolic capacity of xenobiotics biodegradation and energy metabolism in group E was significantly improved compared with group A.In this experiment,HD-4 can significantly improve the intestinal flora structure of Litopenaeus vannamei and promote nutrient metabolism.The results provide data support for the application of HD-4 in shrimp culture.5.Quantitative control and prevention demonstration of pathogenic bacterium in Litopenaeus vannamei by oral HD-4To explore the inhibitory effect of Artemia sp.and HD-4 on Vibrio sp.in Litopenaeus vannamei,this chapter took the number of colonies on solid TSB and TCBS as the measurement index,and carried out the experiment under four period:post larvae cultivation,shrimp cultivation,brood stock cultivation and disease treatment.Results showed that during post larvae cultivation experiment,the quantity of Vibrio sp.in post larvae decreased by 17.2 times throughout the experiment in the test group I.The quantity of Vibrio sp.in control group I and test group II is relatively stable.The quantity of Vibrio sp.in post larvae increased by 34.2 times throughout the experiment in the control group II.During shrimp cultivation period,the quantity of Vibrio sp.in test group fluctuated significantly with the dosing rhythm with the minimum value of 0CFU/g（3d）and the maximum value of 1.89×10⁴CFU/g（13d）.In control group,the quantity of Vibrio sp.increased continuously,with the minimum value of 1.62×10²CFU/g and the maximum value of 9.93×10⁴CFU/g.In brood stock strengthening rearing experiment,the quantity of Vibrio sp.in brood stock in test group showed a fluctuating downward trend with the minimum value of 5.30×10³CFU/g（10d）,which was 12.3 times lower than that of the beginning of the test.The quantity of Vibrio sp.in control group showed a fluctuating trend,with the minimum value of 5.50×10⁴CFU/g（0d）and the maximum value of 9.32×10⁵CFU/g（7d）.During AHPND treatment period,the number of Vibrio sp.decreased continuously,with the minimum value of 8.22×10⁶CFU/g（11d）,which was 78.6 times lower than that of the beginning of the trial.The quantity of Vibrio sp.in control group showed a fluctuating trend,with the value of 1.15×10⁸CFU/g at the end of the experiment.The results of Vibrio sp.in Artemia sp.in the Bohai Sea rimland showed that the model of culturing Artemia sp.in water fertilized by chicken droppings existing safety risks.The quantity of Vibrio sp.in water with chicken droppings was 122 times higher than those without chicken droppings.And in Artemia sp.,it is 42 times higher than those without chicken droppings.The data of weight gain rate showed that there is no significant effect on body weight gain after feeding HD-4.The results of this experiment confirmed the effect of HD-4 in actual production and application,and provided data supporting for the establishment of the AHPND prevention and control technology through HD-4 as the core.