Screening Of Antigens For Detection Of Fasciola Hepatica Infection In Sheep And Establishment And Application Of Immunological Detection Methods

Fasciola hepatica is a zoonotic parasite that parasitizes in the liver and bile duct of the host.It is distributed worldwide and is commonly found in ruminants such as cattle and sheep.F.hepatica infection can cause malnutrition in the host,hinder the development of young animals,reduce the degree of fattening and lactation,and cause the death of sheep in severe infection,causing serious economic losses to animal husbandry.Therefore,it is of great significance to detect F.hepatica-infected animals in time and take preventive and control measures.Immunological detection method has become the main detection method for F.hepatica infection because of its high sensitivity,good specificity and easy to operation.Among them,the ELISA can process a large number of samples at the same time,which is suitable for batch detection.And the colloidal gold immunochromatographic test strips has a fast response and visible detection results,making it more suitable for on-site detection.Immunological detection methods mainly include antigen detection and antibody detection.The antigen detection method can confirm the current infection of F.hepatica according to the circulating antigen in the blood of the host,and realize accurate detection,and the existence time of circulating antigen is early,which can realize the early detection of F.hepatica infection.The antibody detection method can realize large-scale screening of F.hepatica infection based on the antibody level in the host,confirm the scope of infection,and avoid the risk of missed detection.Based on different detection scenarios and detection requirements,different detection methods can be used to confirm the infection of F.hepatica.Based on this,this study first used the F.hepatica c DNA expression library to screen and obtain candidate detection antigen genes for F.hepatica infection,then selected specific antigen genes to prepare recombinant proteins and monoclonal and polyclonal antibodies,and immunolocalized the candidate detection antigens.Afterwards,three detection methods for F.hepatica infection,indirect ELISA,double-antibody sandwich ELISA,and colloidal gold immunochromatographic test strips were established.Finally,the established methods were used to detect sheep serum samples to evaluate their clinical application value.It is hoped that this study will provide detection methods suitable for different scenarios for the infection of F.hepatica in sheep,and provide technical support for the prevention and control of Fascioliasis.The main contents are as follows:1.Screening of antigens for detection of F.hepatica infection.Sheep F.hepatica infection serum was used to screen c DNA expression library of F.hepatica,and 17 immune dominant antigen genes were obtained.Among them,2 were identified as F.hepatica detection antigen genes,and 15 were newly discovered as F.hepatica candidate detection antigen genes.Fh D35 and Fh D38 are Fasciola sp.specific antigen genes.The obtained Fh D35 and Fh D38 nucleic acid sequences were used to construct recombinant clone vectors and expression vectors,and the protein was induced to obtain recombinant proteins of Fh D35 and Fhd38 with sizes of 24k Da and 42k Da,respectively.The recombinant proteins were incubated with serum artificially infected with F.hepatica,and the results showed that the specific immune reaction could occur,and the Fh D38 protein had a stronger reaction.The purified recombinant protein Fh D38 was used as antigen to prepare rabbit polyclonal antibody.It was found that Fh D38 protein was localized on the body surface and intestinal cavity of adult F.hepatica,and had the potential to be a detection antigen.2.Establishment and application of an indirect ELISA for detection of F.hepatica infection in sheep.Using the recombinant protein of Fh D38,an indirect ELISA for detection of F.hepatica infection was established,and the optimal reaction conditions were determined through a series of experiments.The optimal coating amount of Fh D38 protein was 0.25μg/well,the optimal dilution of serum sample was1:400,the optimal blocking buffer was 5%skim milk powder,the optimal dilution of secondary antibody was 1:6000,and the optimal reaction time of substrate solution was 15 minutes.The results of performance test showed that the sensitivity of this method was 1:3200,and there was no cross-reaction with the positive sera of Haemonchus contortus（H.contortus）,Dicrocoelium lanceatum（D.lanceatum）and Neospora caninum（N.caninum）,and the reproducibility was good.The positive rate of fecal examination was 31.2%（10/32）,and the positive rate of this method was 34.4%（11/32）.By testing 90 sheep blood samples from a region of Inner Mongolia Autonomous Region,20 samples were found to be positive,and the positive rate was22.2%.3.Establishment and application of a sandwich ELISA for detection of F.hepatica infection in sheep.The purified protein of Fh D38 was used as an antigen for mouse immunity,cell fusion,and screening of hybridoma cell lines.And six hybridoma cell lines were obtained,namely 1E9,3F11,3E5,5H7,7D3,and 10B2.After the identification of antibody subtypes,they were Ig G2a,Ig G2a,Ig G2a,Ig G2b,Ig G2a and Ig G2a.The antibody titers were all above 1:204800,among which 3E5 had the highest affinity.Afterwards,using the anti-Fh D38 protein monoclonal antibody,a double-antibody sandwich ELISA for detection of F.hepatica infection was established,and the optimal reaction conditions were determined through a series of experiments.The optimal molar ratio of N-hydroxysuccinimidobiotin（BNHS）to polyclonal antibody was 40:1,the optimal coating concentration of anti-Fh D38monoclonal antibody was 3.75μg/m L,the optimal dilution of anti-Fh D38 polyclonal antibody was 1:2000,the optimal blocking buffer was 1%BSA,the optimal dilution of serum sample was 1:4,the optimal dilution of enzyme labeled avidin was 1:5000,and the optimal reaction time of substrate solution was 10 minutes.The results of performance test showed that the sensitivity of the method was 1:64,and there was no cross-reaction with the positive sera of H.contortus,D.lanceatum,N.caninum and Schistosoma japonicum（S.japonicum）,and the reproducibility was good.Serum samples from sheep artificially infected with F.hepatica were detected,and it was found that serum circulating antigens could be detected as early as 13 days after infection.The feces and serum samples of 42 sheep were detected by this method and feces examination method,the positive rate of feces examination was 23.82%（10/42）,and the positive rate of this method was 26.2%（11/42）.Forty-five sheep serum samples were tested by this method and a commercial kit,and the positive rates of both methods were 24.4%,with a coincidence rate of 100%,which indicated that this method could be used in clinical detection of F.hepatica infection in sheep.82 sheep sera from Inner Mongolia Autonomous Region,182 sheep sera from Jilin Province and 65 sheep sera from Heilongjiang Province were detected,of which 4 were positive in Inner Mongolia Autonomous Region,and the positive rate was 4.88%;17were positive in Jilin Province,and the positive rate was 9.34%;3 were positive in Heilongjiang Province,the positive rate was 4.62%.4.Establishment and application of a double-antibody sandwich colloidal gold immunochromatographic test strips for detection of F.hepatica infection in sheep.The colloidal gold immunochromatographic test strips for detecting F.hepatica infection in sheep was established by monoclonal and polyclonal antibodies against Fh D38 protein,and the optimal reaction conditions were established through a series of experiments.The optimal p H value was 10μL 0.2mol/L K₂CO₃ per m L colloidal gold solution,the optimal labeling amount of monoclonal antibody was 6μg/m L colloidal gold solution,the optimal coating concentration of detection line（T）was0.125mg/m L polyclonal antibody,the optimal coating concentration of quality control line was 16-fold dilution of HRP goat anti-mouse antibody.The results of performance test showed that the sensitivity of the method was 1:4,and there was no immune cross-reaction with the positive sera of H.contortus,D.lanceatum,N.caninum and S.japonicum,and the reproducibility was good.Serum samples from sheep artificially infected with F.hepatica were detected,and it was found that serum circulating antigens could be detected as early as 13 days after infection.329 serum samples from Inner Mongolia Autonomous Region,Jilin Province and Heilongjiang Province were detected by this method,and the positive rate was 6.99%.The coincidence rate between this method and double-antibody sandwich ELISA of positive samples reached 95.8%.In conclusion,Fasciola sp.specific antigen genes Fh D35 and Fh D38 were screened and obtained in this study.The recombinant proteins Fh D35 and Fh D38were obtained by prokaryotic expression.Fh D38 protein was selected to prepare mouse monoclonal antibody and rabbit polyclonal antibody,and the immunolocalization of Fh D38 protein was performed.Three detection methods,including indirect ELISA,double-antibody sandwich ELISA and colloidal gold immunochromatographic test strips,were established for the detection of F.hepatica infection in sheep,and were applied to the detection of clinical samples.The establishment of the three detection methods provides detection means suitable for different scenarios for the infection of F.hepatica in sheep.The detection results of clinical samples provided epidemiological data and epidemic prevention reference for the infection of F.hepatica in sheep in Heilongjiang Province,Jilin Province and Inner Mongolia Autonomous Region.