| Piglet diarrhea is a common disease in intensive farming,resulting in significant economic losses,of which enterotoxigenic E.coli(enterotoxigenic E.coli,ETEC)is one of the main pathogens of piglets diarrhea.As a substitute for antibiotics,the Egg yolk antibody used to control piglets diarrhea has a certain basis for research.However,the antibody titer and low yield of yolk antibody are the bottlenecks in the application of IgY,in which immunoadjuvant is one of the influencing factors.The commonly used Freund's adjuvant has some side effects,weak immunogenicity,and poor safety.Therefore,it is necessary to study safe and efficient immune adjuvant to obtain a large amount of high titer egg yolk antibody for prevention and treatment of piglet diarrhea caused by ETEC.CpG-ODN(Oligodeoxynucleotide DNA,ODN)is an efficient and safe vaccine adjuvant,which can effectively improve the body's immune response level.As a medicament immune adjuvant,astragalus polysaccharides and chitosan have the advantages of extensive resources and low cost,and the application prospect is good.In this study,four different sequences of CpG-ODN were designed with Cp G-ODN 2135 as the template,according to the relationship between the immunological activity of CpG-ODN and its structural characteristics.The effects of different sequences of CpG-ODN adjuvant,chitosan,Freund's adjuvant and astragalus polysaccharide adjuvant on the cellular and humoral immune responses of laying hens were investigated by in vitro and in vivo experiments.The effect of different adjuvants on chicken spleen lymphocytes was detected by CCK-8 method.The results showed that the immunostimulatory activity of CpG-ODN S2 was the best,and the stimulus index was 3.64.The effects of different adjuvant and ETEC inactivated vaccine on egg laying performance,cellular immunity and humoral immunity were investigated by in vivo experiments.A total of 120 chickens(120 days)were randomly divided into 10 groups and three infants were immunized three times.Group 1 and 2 were treated with PBS and ETEC K88 inactivated whole vaccine respectively.Group 3 to 7 were treated with different sequences of CpG-ODN S1~S5(30 ?g/each)with ETEC K88,respectively.Group 8~10 were treated with ETEC K88 were treated with Freund's adjuvant,chitosan adjuvant(200 mg/m L)and astragalus polysaccharide adjuvant(25 mg/m L),respectively.The results showed that the average egg production rate(71.81%)in the Freund's adjuvant group was significantly lower than that in the other groups during the whole experiment,which seriously affected the laying performance of the laying hens.The levels of IL-2,IFN-?,serum antibody and IgY in serum were detected by ELISA.The results showed that IL-2(229.64 ng/L),IFN-?(P<0.05).The secretion of IL-2 and IFN-? was higher in CpG-ODN S2 group than that in Freund's adjuvant(P<0.05).CpG-ODN S2 group serum antibody,IgY titer up to 95 million,80 million,and the strength and durability of the best,significantly better than Freund's adjuvant(P<0.05);S1 and S4 group IgY titer Respectively,75 million,65 million,compared with Freund's adjuvant was not significant(P>0.05).In summary,the self-designed CpG-ODN S2 sequence as an immunoadjuvant promotes cell and humoral immune responses in laying hens significantly better than Freund's adjuvant,and is immunogenic and can be used to prepare ETEC inactivated vaccines to produce large amounts of The high titer of yolk antibodies against piglets diarrhea. |
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