The Immune Protection Of Porcine Enterotoxigenic Escherichia Coli Total RNA

Enterotoxigenic Escherichia coli(ETEC)induced piglets diarrhea is a common disease which is widespread in the world.It could cause many diseases such as jaundice,white diarrhea,edema disease and post-weaning diarrhea in piglets,resulting in serious economic loss in pig industry.Piglets diarrhea is mainly controlled by the treatment of antibiotic or ZnO,however,the use of antibiotics will lead to increased E.coli drug resistance,and ZnO excreted by feces will cause heavy metal contamination in soil.Therefore,it is urgent to find an efficient and more environment-friendly method for immunotherapy and prevention.Some recent studies have shown that pathogen RNA is an important pathogen-associated molecular patterns(PAMPs),such as Listeria RNA,reovirus RNA,Sendai virus RNA,and non-pathogenic E.coli RNA can induce innate immune responses in human or mouse cells.However,study of ETEC PAMPs has been limited to protein level such as adhesins and enterotoxin,whether ETEC total RNA can be one of the PAMPs to induce host immune responses is still unknown.This study was designed to explore the function of ETEC total RNA in innate and adaptive immunity at the cellular and animal level,in order to provide theoretical foundation and new ideas for RNA-based vaccine adjuvants or immunostimulants.Firstly,at the cellular level,we used pig-derived ETEC strains or isolated ETEC total RNA to infect or stimulate porcine intestinal epithelial cells(IPEC).The ELISA results showed that both ETEC and ETEC total RNA could induce the secretion of IL-1β,producing innate immune responses.Secondly,at the animal level,piglet E.coli trivalent inactivated vaccine,ETEC total RNA and PBS were intramuscularly injected in the mouse models,subsequently serum IL-1β,IgG1,IgG2 b,IgA and IgM level were tested using ELISA on the day before immunization,10 days and 20 days post immunization.The results showed that the serum IL-1β level of ETEC total RNA immunized group was significantly higher than that of PBS injected group(P<0.05),and the serum IgG1,IgG2 b,IgA and IgM level of ETEC total RNA immunized group were significantly higher than those of the PBS injected group(P<0.05).The serum IgM concentration of the RNA immunized group was extremely significantly higher than vaccine immunized group(P<0.01),and was 2-3 times higher than that of the vaccine immunized group and the PBS injected group.It was demonstrated that the ETEC total RNA can induce high level adaptive immunity in mouse models.Finally,piglet E.coli trivalent inactivated vaccine,ETEC total RNA,vaccine/RNA mixture and PBS were intramuscularly injected in mouse models,and the ETEC challenge experiments were conducted 23 days later.Mice was intraperitoneally injected with absolute lethal dose of ETEC,and continuous observed for 5 days.The results showed that the survival rate of the ETEC RNA immunized group was 75%,that of the vaccine immunized group was 12.5%,that of the vaccine/RNA mixture immunized group was 75%,and that of the PBS injected group was 0%.The survival rate of the ETEC RNA immunized group and the vaccine/RNA mixture immunized group were higher than that of the PBS group and the vaccine only immunized group.It indicated that vaccination with ETEC total RNA has a high protective effect on ETEC infected mouse model,and adding ETEC could significantly improve the protection level of vaccine.This study also investigated the effects of different dosages of ETEC total RNA on the adaptive immunity of mouse model,the results showed that the serum IgG1,IgG2 b,IgM and IgA level of the high-dose immunized group were significantly higher than that of the low-dose immunized group(P<0.05).In the ETEC challenge experiment,the survival rate of high-dose immunized group was higher than that of the low-dose immunized group.Therefore,it suggested that the immune effect of ETEC total RNA on mouse model is positively correlated with the RNA doses.In summary,we conclude that the ETEC total RNA can stimulate IPEC to induce the innate immunity at the cellular level,and induce adaptive immunity at the animal level.ETEC total RNA has immunoprotective effects against ETEC infection and the effect is dose-dependent.