Fluorescent Protein Labeling Of Pathogenic Bacteria And The Effect Of Probiotics Against Bacterial Infection In Juvenile Zebrafish

Probiotics have been widely used in aquaculture in recent decades and have been shown to be effective in reducing and eliminating bacterial infections.This paper will use probiotics to study the antagonistic protective effect of aquatic pathogenic bacteria infection in model organism zebrafish,in order to effectively use probiotics for disease prevention and control in the aquatic field,achieve the goal of green ecological aquaculture,and provide safe and healthy aquatic products,to meet the growing consumer demand of the people.Aeromonas hydrophila and Vibrio parahaemolyticus are important pathogens of zoonotic diseases from freshwater and seawater,respectively.They are the main causes of bacterial diseases of a variety of fish and animals.They also often cause human food poisoning,diarrhea and even sepsis,resulting in very serious public health problems.Because the hosts of these two kinds of bacteria are diverse and the pathogenic process and mechanism are complex,it is necessary to study them in detail.Green fluorescent protein（GFP）and red fluorescent protein（RFP）are widely used molecular markers to label bacteria or other pathogens.In this study,we aimed to construct GFP labeled A.hydrophila and RFP labeled V.parahaemolyticus,and the antagonistic effect of probiotic Bacillus pumilus on A.hydrophila bacteria in vitro and in vivo（zebrafish（Danio rerio））.The following are the main results of this paper.（1）A.hydrophila was labeled with GFP by chemical method.The plasmid pWSK129containing GFP was chemically labeled with Ah1 strain,and its stability,growth curve and toxicity were tested.The results showed that strain Ah1,named Ah 1^GFP,which was successfully labeled with GFP,showed green fluorescence under the fluorescence microscope,and the correct bands containing GFP plasmid could be obtained by gel electrophoresis.The detection results of plasmid stability showed that when Ah1^GFPwas transmitted to the 4th d,the expression of plasmid was still 100%,and the stability of the 5th to 10th d gradually decreased.Compared with the wild-type strain Ah1,there was no difference in the growth curve and toxicity of the labeled strain Ah 1^GFP.The growth curves of Ah 1^GFPand Ah1 are similar.The logarithmic growth period is 2-8 h and enters the stable period after 8 h.The LD₅₀of Ah1 was 7.52 x 10⁶CFU/mL and that of Ah 1^GFPwas 3.47 x 10⁷CFU/mL.All these results laid the foundation for the tracer experiment with Ah 1^GFPlater.（2）V.parahaemolyticus was labeled with RFP by three parent hybridization.The plasmid p VSV209 containing RFP was labeled with Vp13 strain by three parent hybridization,and the stability,growth curve and toxicity of the plasmid were tested.The experimental results showed that Vp13 strain,named Vp13^RFP,which was successfully labeled with RFP,showed red fluorescence under the fluorescence microscope.The correct band containing RFP plasmid could be obtained by gel electrophoresis,and red colonies visible to the naked eye grew on the LBS plate.The results of plasmid stability test showed that the stability of the first three days of plasmid expression was 100%,and then gradually decreased to the 10th d,and the stability of the 10th d was 5%.The growth curves of Vp13^RFPand Vp13 are similar.The logarithmic growth period is within 1-8 h and enters the stable period after 8 h.The LD₅₀of Vp13 was 3.77 x 10⁷CFU/mL and that of Vp13^RFPwas 3.59 x 10⁷CFU/mL.All these results laid the foundation for the tracer experiment with Vp13^RFP.（3）Toxicity test of probiotic B.pumilus to zebrafish.In order to study the protective effect of probiotics B.pumilus on fluorescent labeled bacteria in zebrafish,the toxicity of probiotics to zebrafish was tested first.The toxicity of B.pumilus at different concentrations(10²,10⁴,10⁶,10⁸,10⁹,10¹⁰CFU/mL)to 3 dpf zebrafish larvae was measured by immersion infection method,and the mortality of each group at each time point in 4 d was recorded continuously.The results showed that under the conditions of10¹⁰CFU/mL,10⁹CFU/mL and 10⁸CFU/mL,the mortality of these three high concentrations of B.pumilus were 100%,85%and 25%,respectively.All zebrafish died on the third day.At low concentrations of 10²CFU/mL,10⁴CFU/mL and 10⁶CFU/mL,the mortality of B.pumilus was 10%,15%and 15%,respectively.More than 50%of zebrafish still survived on the 3rd d.Therefore,we decided to use these three lower concentrations of B.pumilus to test its antagonistic effect in the next step.（4）Protective effect of B.pumilus on zebrafish infected with Ah 1^GFP.The infected objects were 3 dpf zebrafish juveniles,20 in each group.The concentration of A.hydrophila in each group was 1.7×10⁸CFU/mL,short spore rod,three different concentrations（10²,10⁴,10⁶CFU/mL）were used in each group.The first group soaked zebrafish with A.hydrophila and B.pumilus.In the second group,zebrafish were soaked in B.pumilus for 12 h and then infected with A.hydrophila.The third group was infected with A.hydrophila with zebrafish for 6 hours,and then soaked with B.pumilus.The results showed that the highest protection rate of the three groups against B.pumilus was 10⁶CFU/mL,and the mortality was low.In the first group,the mortality rate was 35%in the 10²CFU/mL group,30%in the 10⁴CFU/mL group and5%in the 10⁶CFU/mL group,In the second group,the mortality rate was 15%in the10²CFU/mL group,10%in the 10⁴CFU/mL group and 5%in the 10⁶CFU/mL group.In the third group,after 12 hours,the mortality rate was 45%in the 10²CFU/mL group,30%in the 10⁴CFU/mL group and 10%in the 10⁶CFU/mL group.In conclusion,this paper provides an effective method to label pathogenic A.hydrophila and V.parahaemolyticus by fluorescent protein.A.hydrophila（Ah1 strain）was successfully labeled by chemical transformation with green fluorescent protein plasmid pWSK129.V.parahaemolyticus（Vp13 strain）was successfully labeled by red fluorescent protein particle p VSV209 by using three parent hybridization method.Finally,B.pumilus in the concentration range of 10⁶CFU/mL had a protective effect on zebrafish larvae infected by A.hydrophila.This study provides a theoretical basis for the application of probiotics in the prevention and control of aquatic bacterial diseases.