| Chicken coccidiosis is a protozoan disease caused by one or more coccidia of Eimeria and is distributed worldwide.The disease is highly harmful and has a high infection rate,which is one of the causes of serious economic losses in the poultry industry worldwide.At present,the prevention and treatment of the disease is still dominated by chemical drugs and live vaccines,but the problems caused by drug residues,drug resistance and development of new insecticides are becoming more and more serious.The DNA vaccine has the advantages of being safe and stable,which generally uses cytokines as adjuvants to achieve better effects.As a probiotic,Lactobacillus can be an ideal living carrier for DNA vaccines since it has the characteristics of strong colonization,easy cultivation,and immunoregulation.It can be delivered to the cells through oral administration to induce local or systemic immune responses.In order to improve the delivery efficiency of Lactobacillus,S.aureus fibronectin A(FnBPA)with invasive ability was expressed on the surface of Lactobacillus,so that it has the ability to invade cells,thereby improving the delivery efficiency of DNA vaccine.Using the novel eukaryotic plasmid pValac as the expression vector,Eimeria tenella(E.tenella)microsomal protein 2(EtMIC2)as a protective antigen and chicken interleukin 18(ChIL18)as an adjuvant,we constructed a series of recombinant plasmids which were delivered to chickens by invading Lactobacillus expressing the FnBPA protein.The immune effect and the protective effect were evaluated.The specific research contents and results are as follows:(1)Construction and in vitro validation of DNA vaccine expressing EtMIC2 protein and ChIL18.Referring to the EtMIC2 and IL18 sequences registered by GenBank,the EtMIC2-IL18gene was optimized and routinely synthesized.The EtMIC2 and EtMIC2-IL18 gene fragments were amplified by PCR and ligated to the pValac.pValac-EtMIC2 and pValac-EtMIC2-IL18were successfully constructed after double enzyme digestion identification and sequencing.They were then transfected into 293T cells,Western blot and indirect fluorescent immunoassay to detect the expression of EtMIC2 and EtMIC2-IL18 protein in the cytoplasm.(2)Construction of invasive lactobacillus with DNA vaccine and its ability to invade cells in vitroTwo eukaryotic plasmids,pValac-EtMIC2 and pValac-EtMIC2-IL18,were transformed into NC8-pSIP409 and NC8-pSIP409-FnBPA recombinant Lactobacillus to construct pValac-EtMIC2/pSIP409,pValac-EtMIC2/pSIP409-FnBPA,pValac-EtMIC2-IL18/pSIP409,pVala c-EtMIC2-IL18/pSIP409-FnBPA four strains,after induction by SPPIP inducer,the expression of FnBPA protein was detected by Western blot,and the results showed that the invading Lactobacillus were transferred into eukaryotic plasmid.The FnBPA protein was successfully expressed.The CEF cells were co-cultured with the isolated CEF cells for 2 hours,washed,and lysed at room temperature for 10min.Finally,colony counting was conducted and the cell invasion rate was calculated according to the counting results.The results showed that the cell invasion rate of the invasive Lactobacillus expressing FnBPA protein was significantly higher than that of the non-invasive lactic acid bacteria(P<0.001).The invasion rate of pValac-EtMIC2/pSIP409-FnBPA on CEF cells was up to 0.25%,while that of pValac-EtMIC2/pSIP409 as the control strain was only 0.09%.Similarly,the invasion rate of pValac-EtMIC2-IL18/pSIP409-FnBPA strain on CEF cells was 0.15%,and that of pValac-EtMIC2-IL18/pSIP409 was 0.08%.The cell invasion rate of the invasive Lactobacillus expressing FnBPA protein was significantly higher than that of the non-invasive Lactobacillus(P<0.001).(3)Effect of invasive Lactobacillus on immunity level of chicks and evaluation of protective effect against E.tenellaHealthy 1-day-old broiler chicks were randomly divided into 6 groups Physiological saline,GroupI;challenge,GroupII,pValac/pSIP409,GroupIII;pValac-EtMIC2/pSIP409,GroupIV,pValac-EtMIC2/pSIP409-FnBPA,GroupV;pValac-EtMIC2-IL18/pSIP409-FnBPA,GroupVI).Oral immunization was performed on day 1,3,5,12,14,and 16 at 1×10~9CFU/200?L per chick and at 30d(except GroupI)challenge at 5×10~4.The content of CD3~+CD4~+and CD3~+CD8~+T in blood of each group,lymphocyte proliferation ability in blood,SIgA in intestinal,serum specific antibody and cytokine were detected.The relative weight gain rate,oocyst output,cecal lesion score and anticoccidial index(ACI)of chicks after one week of infection with coccidia were counted.The results of animal experiments showed that the content of CD3~+CD4~+T in blood of GroupIV,GroupV and GroupVI chicks was significantly higher than that of GroupI at 30d(before the challenge)(P<0.05).At 38d(after the challenge),the content of CD3~+CD4~+T lymphocytes in the peripheral blood of GroupV and GroupVI chicks was significantly higher than that of GroupIII(P<0.01).Similarly,the CD3~+CD8~+T content in the blood of GroupVI chicks was up to about 35%at 30d,which was significantly higher than that of GroupIII(P<0.05).On 38d,GroupIV,GroupV and GroupVI were significantly higher than GroupIII(P<0.001),and the highest GroupVI content was about 43%.The results showed that the GroupVI had the strongest proliferation ability before and after the challenge,and the GroupVI stimulation index was about 1.3 at 30d,which was significantly higher than GroupIII(P<0.001).At 38d,the GroupVI stimulation index was about 1.5,and its proliferation level was also significantly higher than that of GroupIII(P<0.001).In this study,the levels of cytokines IL-4,IFN-?and IL-18 in the serum of the chickens in each group were detected before the challenge.The levels of IFN-?in the GroupVI were higher than those in other groups,which was significantly higher than that of GroupIII(P<0.01).The content of IL-18 in GroupVI was significantly higher than that in each group(P<0.001).The secretion of SIgA in the intestinal of GroupVI was the highest and significantly higher than that of GroupV(P<0.05).After the GroupV and GroupVI were challenge,the average weight gain of GroupV and GroupVI was higher,and the relative weight gain rate of GroupI was 79.66%and 84.75%,respectively.The amount of oocyst excretion in GroupIV,GroupV and GroupVI feces was significantly reduced,and the reduction rate of oocyst was 67.73%,69.75%and 76.16%,respectively,compared with that of GroupII.The cecal lesions of GroupIV,GroupV and GroupVI chicks were significantly alleviated after one week of challenge,with an average score of 1.3,1.1 and 0.9 respectively.Finally,ACI values of each group were obtained,and GroupIV,GroupV and GroupVI were162.27,167.66 and 174.75 respectively,all of which could achieve good anti-coccidiasis effect,and GroupVI had the best effect.In this study,the invasive Lactobacillus expressing FnBPA was used as the carrier to deliver the eukaryotic plasmids pValac-EtMIC2-IL18 into the host,which could improve the humoral and cellular immunity and enhance the resistance to E.tenella.The DNA vaccine delivered by the invasive lactobacillus provides a new idea and method for the prevention of E.tenella. |
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