Bacillus Subtilis Facilitates Inactivated Transmissible Gastroenteritis Virus To Enhance The Mucosal Immunity Of Pigs

Transmissible gastroenteritis coronavirus(TGEV)is an enteric pathogen and mainly colonized and disseminated through the digestive tract,is the causative agent of severe diarrhea and a high fatality rate in newborn pigs.Oral immunization can efficiently block viral attachment and colonization,which could played an important role in protection and clearance of TGEV infection.Whereas,oral immunization with inactivated whole virus alone is not sufficiently effective to stimulate mucosal immune response.It is therefore necessary to use adjuvants capable of adequately strengthening the immune effect of inactive vaccines.Some suitable mucosal adjuvants could change the efficiency of inactivated virus to mucosal immunity.Previous studies have shown that B.Subtilis and its spores could induce mucosal immune responses in the gut,so they have become an investigativehot spots in recent years.It has been proved that B.Subtilis can be used as an effective mucosal adjuvants in the mice,but very few reports on the pig.Therefor the aim of our study is to investigate whether the effect of oral immunization with inactivated TGEV on the level of local and systemic immune response is improved by co-administration of B.subtilis,and finally provides convenient and effective strategies for the TGE prevention.In order to investigate the immune effect of inactivated TGEV plus B.Subtilis,a total of 24 six-week-old crossbred pigs were randomly divided into four groups for the first immunization,were administered orally to the pigs twice at 7 days intervals.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.Combined with a piglet intestine ligation experiment:twelve ileum segments were injected with inactivated virus and adjuvant and removed after 6 h sustainment to detect the levels of IL-6,IL-12A,IFN-y mRNAs and CD4+,CD8+ T lymphocytes.To investigate the local humoral immunity in digestive tract,the levels of TGEV specific SIgA antibody in feces 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 three parts:1.The effects of inactivated transmissible gastroenteritis virus immunized by oral route on the local cellular immunity in digestive tractApplication of real-time PCR detection was used to detect the expression of IL-6,IL-12A and IFN-y mRNA in intestinal segments which were injected with inactivated virus and adjuvant for 6 h.The results showed expressions of IL-6,IL-12A and IFN-y mRNAs in intestinal segments increased significantly after injected with the ITGEV plus B.Subtilis than injected with the ITGEV alone(P<0.01).Alone use of ITGEV could not make IL-6 and IFN-y mRNA levels increase,but it could increase the amount of mRNA expression of IL-12A(P<0.01).The activation of CD4+ and CD8+ T cell responses were measured in the small intestine cells harvested after the intestinal sterilization experiment.The results showed that the number of CD4+ and CD8+ T lymphocytes increased significantly after injecting with by ITGEV-B.subtilis(P<0.01).Injected with ITGEV plus B.subtilis could increase the number of CD4+ and CD8+ T lymphocytes significantly compared with the small intestine injected with ITGEV alone(P<0.01).SABC immunohistochemical technique was applied to show the distribution and abundance of CD3+T cells in ileum.The results showed that the number of CD34+ T lymphocytes increased significantly after oral immunization by B.subtilis,ITGEV and ITGEV-B.subtilis,and oral immunization with ITGEV plus B.subtilis could increase the number of CD3+ T cell significantly compared with piglets immunized with ITGEV alone(P<0.01).Ileum tissues were fixed,paraffin-embedded and produced into histological sections,HE coloration method was applied to show the distribution and abundance changes of IELs.The results showed that the number of IELs increased significantly after oral immunization by B.subtilis,ITGEV and ITGEV-B.subtilis(P<0.01).Especially,the number of IELs increased four times after oral immunization with ITGEV plus B.subtilis.The results of four essays above prompted that oral immunization with the ITGEV adjuvanted with B.subtilis may have increased significantly the local cellular immunity in intestinal tract of pigs.2.The effects of the inactivated transmissible gastroenteritis vaccine immunized by oral route on the local humoral immunity in digestive tractIndirect 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 the specific anti-TGEV SIgA in the feces washing liquid was measured in the first five weeks.Oral immunization with inactivated TGEV adjuvanted with B.subtilis could increase the specific SIgA significantly in the first three weeks compared with piglets immunized with ITGEV alone(P<0.01).The SIgA level reached to the peak in the second week,then decreased gradually.Indirect ELISA was applied to detect the level of TGEV specific SIgA antibody intissues of ileum.The results showed that oral immunization with ITGEV plus B.subtilis could increase the specific SIgA significantly(P<0.05)compared with piglets immunized with inactivated TGEV alone.The results of two essays above prompted that oral immunization with the ITGEV adjuvanted with B.subtilis may have increased significantly the local humoral immunity in intestinal tract of pigs.3.The effects of the inactivated transmissible gastroenteritis vaccine immunized by oral route on the systemic immunityIndirect ELISA was applied weekly to detect the level of TGEV-specific IgG antibody in serum.The results showed that oral immunization with ITGEV plus B.subtilis could increase the specific IgG significantly in the first four weeks compared with piglets immunized with ITGEV alone(P<0.01).The IgG level also won the highest in the second week,then decreased gradually.The results indicated that intranasal immunization with the ITGEV coupled with B.subtilis could improve the systemic immunity level to some extent.