Screening Of Lactobacillus From Swine Intestines And Their Antiviral Activities Against PEDV

Lactic acid bacteria play an important role in medicine,agriculture and food,their safety has been recognized.Porcine epidemic diarrhea virus?PEDV?has a high mortality rate for piglets and has great economic losses to the pig industry.The prevention and treatment of PEDV has become a major problem for the pig industry.To isolate Lactobacillus from Porcine epidemic diarrhea virus?PEDV?from piglet feces,this study firstly used MRS medium to separate and purify Lactobacillus,and then the anti-viral effects of lactic acid bacteria were evaluated on the cell model in vitro using MTT colorimetric assay,the TCID₅₀ method,indirect immunofluorescence assay,and three probiotic strains that inhibited the virus were obtained.This experiment laid the foundation for further research on the antiviral effect of lactic acid bacteria.In this experiment,36 strains with probiotic characteristics were first isolated from swine feces using MRS medium.Then 10 strains of lactic acid bacteria with antiviral effect were initially screened by MTT colorimetry.The isolated 10 strains of lactic acid bacteria?LAB?were subjected to an gallbladder resistance test in vitro acid,and 6 strains were selected as spare strains.According to Biochemical tests and 16S rDNA identification of 6strains,4 strains of Lactobacillus plantarum,1 strain of Facalis and 1 strain of Pediococcus pentosaceus were confirmed.Three strains of bacteria were selected for further antiviral testing.Isolated and identified strains were subjected to vitro for antiviral tests.First,the MTT method was used to calculate the maximum non-toxic doses of the three strains.The starting point was to dilute them to different concentrations for testing.The assay was performed on Vero cells in vitro.LAB or their metabolites are assessed in three ways?first adding LAB or metabolites followed by viruses;LAB or metabolites and viruses added simultaneously;first adding viruses and then adding LAB or metabolites?to assess PEDV's inhibition of cells effect.The results showed that there were different inhibition effects in the three modes.In the live bacteria test,the inhibition rate of the pretreatment group was better.In the metabolite test,the simultaneous access to the metabolites and the virus group had a greater inhibitory effect on PEDV than the other two groups.The effect is good.Co-cultivation of PEDV with lactic acid bacteria and detection of the TCID50 of the virus showed that the TCID₅₀/100?L after treatment with probiotics were 10^-3.76,10^-3.82,and 10^-3.15,respectively,and the TCID50/100?L of the untreated group compared with the control group(10^-4.16)for comparison,all have different degrees of reduction,indicating that lactic acid bacteria have a significant inhibitory effect on the virulence of the virus.The indirect immunofluorescence assay was used to test the effect of the LAB treatment of the cells and the infection of the virus,and the virus was established with a blank control.The inhibition of LAB was determined by observing the amount of fluorescence.The results showed that the amount of fluorescence in the treated group with LAB and their metabolites was lower than that in the virus control group,indicating that LAB and metabolites had a certain blocking effect on PEDV-adsorbed cells.In summary,this experiment successfully isolated and screened out three strains of lactic acid bacteria with anti-PEDV effect from pig feces.Their bacterial and metabolites can reduce the effect of virus on cell viability and block PEDV's infection of cells.