Research And Application Of Induction Of Nasal Immunity By Whole Inactive O Type Foot-and-mouth Disease Virus With Bacillus Subtilis In Cattle

Foot-and-mouth disease virus(FMDV)is the causative agent of contagious and economically devastating diseases that affects cloven-hoofed livestock worldwide.It is highly contagious and the incidence is 100%.An effective defense barrier against FMD would be established if the respiratory tract transmission for virus was being cut off due to FMDV is mainly transmitted by the respiratory tract.At present,it is mainly subcutaneous or intramuscular injection of inactive FMDV vaccine for prevent FMD in China,but those immunization only induce systemic immunity and fail to produce immune protection against FMDV at invasive mucosa sites.Recent studies have found that Bacillus subtilis(B.subtilis)is a good immune enhancer,which can improve immunity and promote growth.Therefore,we selected B.subtilis combined with inactive FMDV use for cattle by intranasal immune.Although research of nasal vaccine for FMD has made some progress,but the effect of the nasal vaccine is very different among the different species.It is necessary to understand the distribution of the important components of mucosal immune response in cattle respiratory tract,which include the distribution of mucosal immunoreactive cells including CD3+lymphocyte and IgA(immunoglobulin A)secretory cells and the Toll like receptors(TLRs).The distribution of immunoreactive cells in the submucosal lymphoid tissues are the key component to play in mucosal immune response.CD3+lymphocytes are the basis of mucosal immune response to against infection.SIgA(Secreory immunoglobulin A,SlgA)is the main effect factor of mucosal immune and plays a key role in preventing virus adhesion and invasion.The number of IgA secretory cells can react to the level of local mucosal SIgA antibody.TLR7 is one of the members of TLRs that recognizes single strand RNA viruses including FMDV and plays an important role in bridging the innate immune responses and adaptive immune responses.However,the distribution of mucosal immunoreactive cells and TLR7 in cattle respiratory tract have not been reported yet,therefore we first explained their distribution in cattle respiratory tract in detail.At the same time,we investigated whether the tongue tonsils that are the first lymphoid tissues to contact with the outside world except for NALTs(Nasal-associated lymphoid tissues)are also an effective mucosal immune inducing site.Then,we confirmed the level of mucosal immune response and innate immune response after inactive FMDV with B.subtilis administered through intranasal spray.Finally,we applied the intranasal immunization to half-year-old calves in practice,and it is found that it had good immune effect and increased the body weight.The paper is divided into the following three parts:1.Distribution of mucosal immunoreactive cells and TLR7 in cattle respiratory tractNasal mucosa,trachea,pulmonary bronchial,lung and tongue tonsil from five health cattle aged seven were collected and the distribution of mucosal immunoreactive cells including CD3+lymphocyte and IgA secretory cells and TLR7 were tested by immunohistochemistry and histological methods.The results showed that the mucosal immunoreactive cells and TLR7 were distributed in cattle nose,trachea,pulmonary bronchial and lung.CD3+lymphocytes were distributed mucosal epithelium,underneath lamina propria and around gland in nasal mucosa,trachea and pulmonary bronchial and lung.IgA secreting cells were distributed mainly underneath the mucosal epithelium and around the glands in nasal mucosa,trachea of cattle.The number of the CD3+ lymphocytes and IgA secreting cells in the respiratory tract was counted by image system.The numbers of the CD3+ lymphocytes are the largest in nasal mucosa and lung,followed by trachea and pulmonary bronchial.The numbers of the IgA secreting cells decreasingly reduce from nasal mucosa to lung.There were TLR7 in the nasal mucosa,trachea,pulmonary bronchus and lung tissue and underneath epithelium of the mucosa which exist mucosal epithelium,lymphocytes and IgA secreting cells in lamina propria and around glands.Distribution of TLR7 is the most trachea,followed by the nasal mucosa and pulmonaiy bronchial and at least lung.A lot of lymphoid tissues existed underneath epithelium of the nasal mucosa and the pulmonary bronchial of lung.Moreover,a lot of lymphoid tissues existed underneath epithelium of the nasal mucosa and the pulmonary bronchial of lung.The distribution of a large number of CD3+ lymphocytes and a small amount of IgA secreting cells in cattle tongue tonsil.Although tongue tonsil participated in local mucosal immune response,but not act as effective mucosal immunity inducing site.Those results illustrated the distribution of mucosal immunoreactive cells including CD3+ lymphocyte,IgA secretory cells and TLR7 in cattle respiratory tract and indicated that the cattle nasal mucosa is a good mucosal immune inducing site.2.Effects of intranasal immunization with inactive FMDV combined with Bacillus subtilis on mucosal immune response in respiratory tract and systemic immune response in cattleIn the current study,health seven-year-old cattle were immunized by 1×107 LD50/mL inactive FMDV along with 1×109/mL B.subtilis in 1:1 volume to administered through intranasal spray to explore the impact on mucosal immune response(respiratory tract)and systemic immunity.The results showed that the CD3+lymphocytes,IgA secretory cells and TLR7 significantly increased in cattle nasal mucosae and pulmonary bronchial(P<0.01)after intranasal immunization with inactive FMDV along with B.subtilis,but the conventional inactive FMDV vaccine had no significant effect on above immune composition(P>0.05).The level of IL-12 and TNF-? significantly increased in nasal,tracheal,pulmonary and pulmonary bronchial(p<0.01,p<0.05),especially in the trachea and pulmonary bronchus,but IL-4,IL-6 and IL-10 level have no significantly difference(P>0.05).The effect of routine inactive FMDV vaccine on IL-4,IL-6,IL-10,IL-12and TNF-? in cattle respiratory tissues were not significant(P>0.05).O type FMDV specific SIgA fr-om nasal,saliva and O type FMDV specific antibodies in serum significantly increased at 3 days after intranasal immunization,and oaintained for 3 months(P<0.01,P<0.05).These results indicated that both cellular immunity and local humoral immunity of the cattle respiratory tract were enhanced by intranasal immunization with inactive FMDV along with B.subtilis,as well as the systemic immune response.3.By intranasal immunization with inactive FMDV and Bacillus subtilis clinically applied to calves186 healthy calves were intranasal immunized with inactive FMDV and Bacillus subtilis,who were half year old in 10 cattle farms from Mangshi and Longchuan city in Dehong Prefecture Yunnan Province.5 test groups were set up in application for calves at the above trial dose,(A)inactive FMDV combined with B.subtilis by intranasal immune group with 146 calves,(B)alone B.subtilis group,(C)routine vaccine group,(D)alone inactive FMDV group and(E)negative control group.There were 10 calves in each group for B,C,D and E.The specific antibody of O type FMDV in serum was detected by ELISA,which come from random 100 caves in group A and 10 calves in each group for B,C,D and E.At the same time,the weight of the selected 50 calves in group A and 10 calves in each group for B,C,D and E was weighed.The results showed that there were 72 serums with 0 type FMDV specific antibodies from 100 calves in group A and the positive rate of population antibody was 72%,which reached the standard of more than 70%population immunity antibody passing rate of the FMDV compulsive immunization in China,and fail to happen immune stress reaction.There was no significant difference between group A and C(P>0.05).Group A and C are extremely significant different from B,D and E(P<0.01).The group B,D and E did not detect specific antibodies.Body weight measurements showed that the monthly weight gain of calves in A and B groups increased significantly compared with the other three groups(P<0.05),while the other three groups had no significant difference(P>0.05).These results indicated that inactivate FMDV intmasal immune was feasible and safe in practical production with certain economic and social benefits.