Evaluation Of Immune Effect Of Corynebacterium Pseudotuberculosis Pld Deletion Strain On Mice And Goats

Corynebacterium pseudotuberculosis is a Gram-positive facultative intracellular parasite that is pleomorphic and non-sporulating that mainly causes Caseous lymphadenitis（CLA）,which is characterized by the formation of abscesses on the skin surfaces or in the viscera and lymph nodes of small ruminants.Once infection,s necrotic lesions were formed on the skin surface and necrotic lesions were formed in superficial lymph nodes and lungs.It’s infection resulting in wool production decrease,dairy production and reproductive capacity decrease.Although the in vitro search showed that the bacterium was sensitive to some antibiotics,thick and dense fibrogranulomas were formed in the injury area after animal infection with caseous lymphadenitis,in which drugs could not penetrate and effective treatment could not be carried out.In recent years,some places have achieved a bit success in the treatment of caseous lymphadenitis by using the method of abscess laceration and aspiration.However,this method cannot remove the internal lesions,neither to say this peocessing method may cause environmental pollution and accelerate the spread speed of Corynebacterium pseudotuberculosis.At present,there is no effective measure for the treatment of Corynebacterium pseudotuberculosis.Therefore,immunization of sheep is important to prevent the spread of the disease.Our previous research found that after deleted the pld gene,the pathogenicity of Corynebacterium pseudotuberculosis was extremely significantly reduced.In this research,we use Corynebacterium pseudotuberculosis Xuanhan pld gene knock out strain（Xh02Δpld）as an live attenuated vaccine strain to immune mice and host animal-goats,which would provide reference for the development of vaccines for the immune prevention and control of C.pseudotuberculosis in goats.1.Mice immunized with C.pseudotuberculosis XH02△pld protective researchTo investigate whether the pld gene deletion strain XH02△pld has the potential used as a vaccine,.Use Kunming mice PBS-injected as the control（Group G1）.The other mice（1×10⁴ CFU,Group G2;1×10⁵ CFU,group G3;1×10⁶ CFU,G4 group）were subcutaneously immunized with XH02△pld.All of the mice received secondary immunization on 70 days,then use XH02（8×10⁶CFU）and WZ（5×10⁵CFU）were used for challenging mice on 98 days and 114 days after the first immunization.The protective effect of XH02△pld-immunized mice was evaluated by the state of immunized mice,body weight,bacterial load of organ,serum antibody（Ig G,Ig G1,and Ig G2a）levels and cytokine content（TNF-αand IL-4）.m RNA expression of cytokines（TNF-α,IFN-γand IL-4）in splenocyte stimulated by XH02 bacterial protein,and attack protection rate.The results showed that the immunity of XH02△pld had no significant effect on the mice.No Corynebacterium pseudotuberculosis was founded in the ascites,liver,and kidney of the mice at 15,30,and 45 days after the first immunization.After the first immunization,G3 and G4 groups’serum Ig G,Ig G1 and Ig G2a antibody levels were obviously increased.After the second immunization,each immune group’s serum Ig G,Ig G1 and Ig G2a antibody levels in were obviously increased,indicating that the XH02△pld immunization of mice could induce an increase in humoral immune level.Through calculation,there were more cases where the immunized mice Ig G1/Ig G2a was less than 1,suggesting that after XH02△pld immunization,the body mainly produced Th1 immune response.The expression levels of TNF-α,IFN-γand IL-4 after stimulation of mice spleen lymphocytes,G2 group were obviously increased,and the expression levels of TNF-αand IL-4 were significantly higher than those in the G1group,suggesting that XH02△pld might induce Th1 and Th2 responses to a certain extent after immunization.After inoculation with XH02 and WZ strains,the protection rates were 16.7–50%and 16.7–33.3%,indicating that the mice of each immune group had protection against Corynebacterium pseudotuberculosis.2.Evaluation of immune protective ability of goats immunized with Corynebacterium pseudotuberculosis XH02△pldIn order to further explore whether Corynebacterium pseudotuberculosis XH02△pld has the potential as base of the vaccines.In this experiment,goats were immunized subcutaneously with 2×10⁶ CFU XH02△pld bacteria after the first immunization for seven weeks,they were treated with the same dose of adjuvant for the second immunization.During this period,body temperature and weight were recorded every week,and lood separation serum was collected to detect the content of serum antibody Ig G,IFN-γand IL-4.Peripheral blood was collected for blood cell analysis 2 and 11weeks after the first immunization.The peripheral blood mononuclear cells（PBMCs）were collected at the second and tenth weeks after the first immunization.The mononuclear cells（PBMCs）were isolated at the 10th week after the first immunization,and the expressions of TNF-α,IFN-γand IL-10 m RNA were detected after stimulation with XH02 mycoprotein.The mononuclear cells（PBMCs）isolated at the second and tenth weeks after the first immunization were used to detect the proliferation of peripheral mononuclear cells by using the MTT method.Eleven weeks after the first immunization,XH02 was used for bilateral detoxification of goats.After the challenge,the changes of body temperature and weight were recorded,and the abscess at the injection site was observed and scored semi-quantitatively.Eight weeks after the virus attack（that is,19 weeks after the first immunization）,the goat organs and lymph node lesions were observed,and the lymph node changes were scored semi-quantitatively.The organs and lymph nodes were collected for the detection of bacterial load to evaluate the effect of XH02△pld on the immunization of goats.The results showed that XH02△pld had no obvious effect on goats after immunization.After the first immunization,the serum Ig G level of goats increased gradually,and after the second immunization,the serum antibody level increased obviously.The serum Ig G level of goats in the immunization group was significantly higher than that in the control group at the third and fourth weeks after the second immunization（that is,the tenth and eleventh weeks after the first immunization）.The content of IFN-γin goats serum was 470.22%and 419.76%higher than that in control group at 2 weeks and 10 weeks after first immunization,and the content of IL-4 in serum was 211.6%and 237.9%higher than that in control group at 2 weeks and 10weeks after first immunization.The results showed that the weight gain of goats in XH02△pld immunization group was not significantly affected,and their body temperature returned to normal earlier.The overall degree of abscess formation in XH02△pld immune group was lighter than that in control group.After autopsy observation and detection of bacterial load in goats infected with XH02,it was found that the bacterial load in goats lymph nodes in immune group was lower than that in control group.3.Expression of recombinant pld deletion fragment protein and preliminary attempt to establish an ELISA method as a coating antigenIn order to establish a method for distinguishing the antibodies produced by wild virus infection of XH02△pld-immunized goat and Corynebacterium pseudotuberculosis,in this experiment,the Overlap PCR technology was firstly used to carry out plasmid ring opening and r PLD gene sequence amplification,followed by ligation transformation BL21（DE3）,and screening and identification to obtain E.coil/pet-28a（+）-△pld strain.Subsequently,IPTG was used for induction,and expression was induced simultaneously using empty E.coil/pet-28a（+）as the control.After fragmentation by centrifugation,the fragmentation supernatant and precipitate were collected,and the expression of r PLD protein was detected by SDS-PAGE and purified.Finally,the protein was quantified and used as an antigen to coat a 96-well plate,and the combination of r PLD protein and serum antibody was tested by negative and positive serum.It was initially found that there was no significant difference between the negative and positive serum OD₄₅₀ of Corynebacterium pseudotuberculosis infection detected by coating with this protein,and the specific reason required further investigation.