Effect Of Oral Immunization With The Inactivated Transmissible Gastroenteritis Virus On The Local Intestinal Tract And Systemic Immune Responses In Pigs

Transmissible gastroenteritis coronavirus(TGEV)mainly colonized and disseminated through the intestinal tract,is the causative agent of severe,often fatal,diarrhoea in newborn pigs.Oral immunization can efficiently block viral attachment and colonization,which could played an important role in protecting pigs against TGEV infection.Inactivated virus alone is insufficient to induce effective immune protection for the degradation in the gastrointestinal tract.Therefore,immunopotentiators were used to improve the immune effect of inactive vaccines.CpG-DNA,an effective immunopotentiator,was famous known as an excellent adjuvant in mucosal immunization.The aim of our study is to investigate whether the effect of oral immunization with inactivated TGEV(ITGEV)on the level of local and systemic immune response is improved by administration of CpG-DNA,and finally provides theoretical basis for the prevention of TGE.In vitro test,the immunostimulatory activity ofadjuvant CpG and ITGEV on swine spleenlymphocytes were investigated.In vivo intestine ligation test,15 ileum segments were injected with inactivated virus and adjuvant and removed after 6 h sustainment to detect the levels of IL-6,IL-12 and IFN-y mRNAs.Then 20 six-week-old crossbred pigs were randomly divided into five groups for the first immunization,were immunized again a week later.These pigs were normal feeding,slaughtered 42 days after the first immunization.To investigate the local cellular immunity in digestive tract,the numbers of intraepithelial lymphocytes(IELs)and CD3+ T lymphocytes in ileum were detected.To investigate the local humoral immunity in digestive tract,the areas of IgA secreting cells in ileum,the levels of TGEV specific SIgA antibody in feces samples and in the tissues of ileum were detected.To investigate the systemic immunity,the level of TGEV specific IgG antibody in serum was detected.This study content is divided into the following four parts:1.The immunostimulatory activity of CpG on swine spleenlymphocytesTo test the ability of porcine spleen lymphocytes to respond to CpG byproliferating,we use co-incubation of naive spleen lymphocytes with bacterial CpG-DNA,CpG-ODN(ODN D19),ITGEV and CpG-DNA for 72 h.Proliferation data are shown that co-incubation of naive spleen lymphocytes with bacterial CpG-DNA,CpG-ODN,CpG-DNA plus ITGEV led to a significant level of cellular proliferation,bacterial CpG-DNA could be as effective as CpG-ODN.The results showed that CpG had good immunostimulatory effects on swine spleenlymphocytes.2.The effects of oral immunization with inactivated transmissible gastroenteritis virus on the local cellular immunity in intestinal tractApplication of real-time PCR detection was used to detect the expression of IL-6,IL-12 and IFN-y mRNA in intestinal segments which were injected with inactivated virus and adjuvant for 6 h.The results showed that the levels of IL-6,IL-12 and IFN-y mRNAs in intestinal segments increased significantly after injection with the ITGEV coupled with adjuvant CpG.Injection with CpG alone could increase the levels of IL-6,IL-12 and IFN-γmRNAs as well.Alone use of ITGEV could not make IL-6 and IFN-γ mRNA levels increase,but it could increase the amount of mRNA expression of IL-12.Ileum tissues were fixed,paraffin-embedded and produced into histological sections,SABC immunohistochemical technique was applied to show the distribution and abundance of CD3+T cells in ileum.The results showed that the numbers of CD3+T cells in ileum increased significantly after oral immunization with the ITGEV coupled with adjuvant CpGHE coloration method was applied to show the distribution and abundance changes of IELs.The results showed that the numbers of IELs in ileum increased significantly after oral immunization with the ITGEV coupled with adjuvant CpG.The results of three essays above prompted that oral immunization with the ITGEV adjuvanted with CpG could improve the local cellular immunity in intestinal tract of pigs.3.The effects of oral immunization with inactivated transmissible gastroenteritis virus on the local humoral immunity in intestinal tractSABC immunohistochemical technique was applied to show IgA secreting cells in ileum.The results showed that the areas of IgA secreting cells increased significantly after oral immunization with ITGEV coupled with adjuvant CpG.Indirect enzyme-linked immuno-sorbent assay(ELISA)was applied weekly to detect the level of TGEV specific SIgA antibody in feces samples.The results showed that in the first three weeks after the first immunization the level of TGEV-specific SIgA antibody in feces samples was increased significantly after oral immunization with the ITGEV coupled with adjuvant CpG.Oral use of ITGEV alone could significantly increase the level of specific SIgA antibody in feces samples as well.Four weeks later,TGEV-specific SIgA antibody levels decreased,no significant differences were found between the groups.Indirect ELISA was applied to detect the level of TGEV specific SIgA antibody in tissues of ileum.The results showed that 45 d after oral immunization,the level of TGEV-specific SIgA antibody in ileum still existed.The results of three essays above prompted that oral immunization with the ITGEV adjuvanted with CpG could improve the local humoral immunity in intestinal tract of pigs.4.The effects of oral immunization with inactivated transmissible gastroenteritis virus on the systemic immunityIndirect ELISA was applied weekly to detect the level of TGEV-specific IgG antibody in serum.The results showed that in the early days after immunization,the high levels of IgG exhibited to significantly increase and reached peak at 2 wk after oral immunization with ITGEV plus CpG or ITGEV alone,but the effect would not exhibit the same strengths as ITGEV hypodermic immunization.After the initial growth,the TGEV-specific IgG decreased as time goes on,but there was still significant difference between the immunization groups and the control groups.The results indicated that oral immunization with the ITGEV coupled with CpG could improve the systemic immunity level to some extent.