| Corynebacterium pseudotuberculosis is an intracellular Gram-positive bacteria that can cause chronic infectious diseases in a variety of animals and humans.It is the leading cause of Caseous lymphadenitis(CLA),CLA is a serious infectious disease of sheep and goats worldwide.Clinically,the infected ruminantia are mainly adult goats and sheep.Lambs are not susceptible to CLA.Once the disease occurs in sheep,it is difficult to eradicate,often causing sheep to appear superficial lymph node abscess,caseous lesions,internal organs purulent nodules and so on.Furthermore,C.pseudotuberculosis can infect and cause lymphadenitis and endocarditis,which seriously affects public health and safety.It is difficult to diagnose the disease in the early stage,and the effect of antibiotics is not obvious,and it can not be prevented by adding drugs in the feed,but the operation is complicated and easy to pollute the environment.Therefore,vaccination is the most effective measure to prevent and control the disease.At present,there is no commercial inactivated vaccine to prevent the pathogen in our country,so it is urgent to develop a suitable and effective vaccine to reduce the infection rate and prevalence rate of the pathogen and reduce the economic loss to the sheep industry.PLD(thrombin D)is one of the most important virulence proteins of C.pseudotuberculosis,which is closely related to its spread and pathogenicity in vivo after infected animals.Therefore,PLD has become an important antigen component in the preparation of CLA vaccine,based on the candidate vaccine strain of C.pseudotuberculosis——Wanzhou strain(WZ),which was screened in our laboratory earlier,we plan to further prepare different components of vaccine,firstly,the mice were immunized and the humoral immunity,cellular immunity and immune protection rate induced by the inactivated vaccine were evaluated to determine the best vaccine preparation plan,and secondly,the effects of the inactivated vaccine prepared by different adjuvants were compared,finally,to prepare WZ strain two-component inactivated vaccine to immunize goats,and to evaluate the humoral immunity,cellular immunity,physiological indexes and the number of abscesses induced by the vaccine,to provide research data and basis for CLA vaccine prevention and control.1.Evaluation of immunization effect of different components of inactivated C.pseudotuberculosis vaccine in miceWith ISA 206 VG as adjuvant,to prepare inactivated C.pseudotuberculosis vaccine with different components:WZ bacteria culture solution group(WZ+culture solution,G2),WZ bacteria Group(G3),r PLD Group(G4)and r PLD+WZ bacteria group(G5),the mice were immunized subcutaneously at the back of neck with normal saline(group G1).The interval between the first and second immunization was 21 days.The mice were challenged with the bacteria in abdominal cavity after on the 21th day and 42th day of the second immunization,respectively,the infection and morbidity of mice were observed after 30 days.The levels of total Ig G,IG1 and Ig G2a subtypes in serum were determined by indirect Elisa.The m RNA expression levels of TNF-α,IFN-γ,IL-4 and IL-6 in mouse splenocytes were detected by real-time quantitative PCR.The immune protective effect of inactivated C.pseudotuberculosis vaccine with different components was evaluated by the infection and challenge test of C.pseudotuberculosis in the immunized mice.The results showed that the levels of Ig G,Ig G1 and Ig G2a in Group G2 and G3were significantly higher than those in group G1(bacterial coating),the m RNA expressions of TNF-α,IFN-γand IL-4 in the splenocytes of group G2 were significantly higher than those of group G1,suggesting that the inactivated vaccine of group G 2could induce the expression of Th1 and Th2 cytokines in splenocytes.When the mice were challenged with 7.2×10~6 CFU,the protective rate was 100%in G2,G3 and G5groups,and no C.pseudotuberculosis was found in the livers of G2 and G3 groups,the results indicated that WZ inactivated vaccine(G2)or WZ inactivated vaccine(G3)had good protective effect in mice.However,the titers of Ig G,IG1 and Ig G2a in group G4were not high,and the protective rate was only 25%,although the titers of anti PLD antibody were significantly increased.The inactivated vaccine of WZ strain with good immune effect was screened out by comprehensive evaluation.2.Effects of different adjuvants on mice immunized with inactivated C.pseudotuberculosis vaccineIn order to screen adjuvant with better immune promotion effect for subsequent goat immune test,totally inactivated WZ bacterial suspension(WZ+culture solution)was emulsified with ISA 201 VG and ISA 206 VG adjuvant respectively to obtain two-component bacterial suspension inactivated vaccine with different adjuvant,afterimmunization,the levels of serum Ig G,Ig G1 and Ig G2a antibodies were detected by ELISA to select the best adjuvant.The results showed that the total Ig G,IG1 and Ig G2a of mice immunized with 201adjuvant vaccine were higher than those of mice immunized with 206 adjuvant vaccine,and the total Ig G of mice immunized with 201 adjuvant vaccine was higher than that of mice immunized with 206 adjuvant vaccine,there was no significant difference between Ig G1 and Ig G2a.In addition,two weeks after the first and second immunization,the continuous mean weight gain of mice immunized with 201 adjuvant WZ inactivated vaccine was significantly higher than that of mice immunized with 206 adjuvant WZ inactivated vaccine.Therefore,the subsequent use of 201 adjuvant to prepare WZ strain two-component inactivated vaccine in goats for comprehensive evaluation of the immune effect.3.Evaluation of immunization effect on goats with inactivated C.pseudotuberculosis vaccineThe two-component inactivated WZ vaccine with 201 adjuvant was prepared and injected subcutaneously into the neck and back of the immunized goats.The interval between the first immunization and the second immunization was 28 days,and the second immunization was 30 days,then challenged with 4×10~6CFU C.pseudotuberculosis for 65 days.The immune protective effect of WZ inactivated vaccine was evaluated by the titers of serum antibody,cellular immune response and abscess of immunized goats.The results showed that the serum Ig G antibody of goats immunized with the inactivated WZ strain vaccine prepared with 201 adjuvant was significantly higher than that of the control group,and the serum Ig G antibody of goats immunized with the second adjuvant was significantly higher than that of the first adjuvant,the results showed that the prepared WZ inactivated vaccine could produce good humoral immune response in goats.In addition,the expression levels of Th1 and Th2 cytokines,such as TNF-α,IFN-γand Il-10,were higher in goat mononuclear cells stimulated by WZ inactivated vaccine than those in unstimulated cells.The levels of IFN-γand IL-4 in the serum of the immunized goats were higher than those of the control goats,which indicated that the WZ inactivated vaccine could produce a good cellular immune response in the immunized goats.The protective effect of WZ inactivated vaccine on body weight and body temperature of goats infected with C.pseudotuberculosis was studied,the results showed that the inactivated vaccine of 201 adjuvant WZ had a good protective effect on the immune goats.In conclusion,the inactivated WZ vaccine with ISA 201 adjuvant can induce good humoral and cellular immunity and reduce the number of abscesses in goats infected with C.pseudotuberculosis,the elimination of the infection of the pathogen to lymph nodes can be used for the immunoprophylaxis and treatment of goat CLA. |
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