Effects Of Salmonella-drived Outer Membrane Vesicles On Production Performance And Prevention Of Salmonella Infection Of Broilers

Outer membrane?vesicles(OMVs)are bilayered nano-vesiclessecreted by bacteria into the extracellular environment during growth.OMVs contain many bioactive compounds,such as bacterial cell membrane protein,lipid and nucleic acid,and they can transmit chemical signals between cells.OMVs have various biological functions,such as mediation of bacteria-host interactions,transportation of bacterial antigens and manipulation of host immune system.Along with the research on the biological characteristics and functions of OMVs,it has been drawn wide concern that OMVs can function as the carrier of bacterial antigen and regulate host immune system.In this study,we investigate the effects of Salmonella typhimurium(ST)-drived OMVs(ST-OMVs)on broiler growth performance,serum immune parameters,andthe prevention of ST infection.This will provide the basis for the prevention and control of Salmonella disease in poultry.Exp.1Isolation,identificationandbiologicalcharacteristicsofSalmonella typhimurium-drived OMVsSalmonella typhimurium was cultured in a sterile environment.The ST-OMVs were enriched from ST culture supernatant by differential centrifugation,then purified by density gradient centrifugation to obtain purified ST-OMVs.(1)By scanning electron microscope,the results showed that the ST-OMVs are different sizes and unadulterated.The visual field was clear and the average diameter of ST-OMVs was about 100 nm.The ST-OMVs particles were consistent with the extracellular vesicles of bacteria.(2)The particle size distribution of the purified ST-OMVs was measured using a laser nanoparticle size analyzer.The results showed that the peak value of the MODE particle size was 76 nm,and the curve was smooth and fluent,indicating well-purified ST-OMVs obtained.The MODE particle size was basically consistent with the theoretical particle size value of the bacterial OMVs,and the small deviation might be related to the degree of adhesion between the particles.(3)The protein content of ST-OMVs was detected by BCA.The results showed that the protein content of ST-OMVs at 1×10~100 particles/mL was 7.3?g/mL.Exp.2 Effect of Salmonella typhimurium-drived OMVs on performance and prevention of Salmonella infection of broilerThree hundred and twenty 1-day-old AA broilers were randomly allocated to five treatments with eight replicates,eight broilers in each replicate.Experimental treatments were the uninfected control group(PBS),ST infection group(ST group)and three OMVs immunization groups with different dosages(injected 5,10 and 20 mu g/d/bird OMVs;OMV5,OMV10 and OMV20).Three doses of OMVs were injected into the peritoneal cavity of chicks at 8-10,15-17,and 22-24 days 3 times,3 days each time,and once daily.The birds of PBS groups and ST groups were injected with equal volumes of PBS solution.When the experimental birds were 30 days old,the ST group and three OMVs immunized chickens were challenged intraperitoneally with 1×10~9 CFU/mL of ST,and the PBS group was injected with a PBS solution of equal volume.The birds were observed for 5 days to monitor the health status changes.When the birds were 50 days old,the optimal immunization dose groups were challenged with a single-injection of 5×10~9 CFU/mL of ST and were observed for 5 days.The results showed as follows:(1)Effects of ST-OMVs on performance and immune function of broilersCompared with the PBS group,the daily feed intake,average daily weight gain and feed conversion ratio of the OMV5 and OMV10 groups had no significant difference(P>0.05),while the average daily feed intake of the OMV20 group was similar to that of the OMV20group.The average daily weight gain was significantly decreased(P<0.05)and the feed conversion rate was significantly increased(P<0.05).The mortality and immune organ indexes of three OMVs immunization groups had no significant difference with the PBS group(P>0.05).The OMVs immunization could significantly increase serum lysozyme activity,albumin content,C3 content and SOD activity in broilers(P<0.05).(2)Immune protection of ST-OMVs against ST infection in broilersCompared with the ST group,OMVs immunization were able to prevent(P<0.05)the weight losses of the experimental chickens caused by ST infection and significantly increased(P<0.05)the survival rate of birds challenged with ST,especially better protective effects observed in the OMV10 and OMV20 groups.In addition,10?g/d/bird OMVs significantly reduced the levels of IL-1?and TNF-?in blood after ST infection(P<0.05),and there was a tendency to decrease IL-6 levels(P=0.074).On day 50,the PBS group and the OMV10 group had been observed for 5 days after the ST challenge.There was no significant difference in the survival rate between the two groups(P>0.05),indicating good persistence of immunity to broilers resulted from OMVs.Conclusions:(1)The well purified ST OMVs can be obtained with a combination of differential centrifugation and density gradient centrifugation.The combination method of scanning electron microscopy and Nanosight particle size analysis was used to identify OMVs morphology accurately.(2)The ST-OMVs can enhance the immune function by improving serum lysozyme,SOD activity,albumin and C3 levels in broiler chicks,and prevent the weight loss of broiler chickens challenged with ST and increase the survival rates of birds challenged with ST.The ST-OMVs have good persistence in the ability of broilers to resist ST infection.(3)The ST-OMVs enhance broiler immunity with a significant dose effect.The higher doses(eg,20?g/d/bird)have a negative effect on broiler production performance or even survival and the moderate doses do not have these effects.In this study,the optimal immunization dosage is 10?g/d/bird.