| Objective:To infect goat tracheal epithelial cells with Mycoplasma capricolum subsp.capripneumoniae(Mccp),establish an infection method that can be used in the laboratory to identify potential protective antigens of Mccp and provide technical means for the identification of potential protective antigen targets.Lay the foundation for the study of the pathogenic mechanism of Mccp;establish a method for Mccp to infect SPF chicken embryos,evaluate the virulence of different strains of Mccp,and be a reliable evaluation method for the screening of vaccine strains.Methods:(1)Mccp-infected goat tracheal epithelial cells(GTC)indirect immunofluorescence(IFA)test:Infect GTC cells with Mccp,the serum after Mccp-infected goats recovered as the primary antibody,and FITC-labeled rabbit anti-goat Ig G as the secondary antibody to confirm Mccp infection Concentration,time and working concentration of primary and secondary antibodies;establish indirect immunofluorescence detection method(IFA).(2)The preliminary application of indirect immunofluorescence method for Mccp-infected GTC:The positive and negative serum prepared in the laboratory,as well as the antiserum and mixed antiserum against Mccp adhesion-related proteins P161,P87,P42,P200R1 and P200R2 screened by reverse vaccinology and immunoproteomics in the previous laboratory were co-incubated with Mccp bacterial solution,inoculated with GTC cells,and the changes of specific fluorescence intensity were observed.The copy number of adhesion cells after co-incubation of each serum with Mccp bacterial solution was detected by real-time PCR,and the adhesion rate was calculated.The blocking/adhesion effect of these proteins on Mccp adhesion to GTC cells was identified,providing a potential protective target for vaccine development.(3)Establishment and preliminary application of the method for Mccp to infect SPF chicken embryos:use different concentrations(dose:0.2 m L/egg)of Mccp bacteria liquid to infect SPF chicken embryos of different ages through the yolk sac and allantoic cavity of chicken embryos.Record the death of chicken embryos.Collect chicken embryo allantoic fluid and yolk sac fluid samples for Mccp real-time PCR identification and isolation and culture identification to determine the optimal inoculation concentration(dose:0.2 m L/egg),inoculation route and inoculation age,and real real-time PCR detects and isolates and cultivates the best samples,and establishes a method for Mccp to infect chicken embryos.Using this method,the Mccp M1801 isolate,M1601 isolate,F38 standard strain and ZLY 1309F isolated from Tibetan antelope were inoculated with SPF chicken embryos at the same age,and the mortality of chicken embryos,the positive rate of real-time PCR test samples and The positive rate of sample isolation and culture identification was compared to compare the four Mccp strains’pathogenicity to chicken embryos.Results:(1)The indirect immunofluorescence method conditions of Mccp infected GTC cells were as follows:the adhesion titer was 1.5～7.0×10~7copies/μL(1×10~6CCU/m L,dose:0.2m L/egg),the adhesion time was 2 h,the optimal working concentration of primary antibody was1:100,and the optimal working concentration of secondary antibody was 1:100.Inhibition of Mccp adhesion by positive serum resulted in decreased fluorescence intensity and adhesion rate.(2)The preliminary application of indirect immunofluorescence(IFA)method for Mccp-infected goat tracheal epithelial cells(GTC):there was almost no specific fluorescence after Mccp bacterial solution was treated with positive serum,and the adhesion rates(0.23%and 0.27%)were also significantly lower than those of positive serum(7.96%and 8.58%).P161 antiserum,P87 antiserum,P42 antiserum,P200R1 antiserum,P200R2 antiserum and mixed antiserum decreased the specific fluorescence intensity of blocking hole,especially P161.After blocking the adhesion of each protein,the adhesion rate decreased from strong to weak as P161 antiserum(adhesion rate was 1.64%and 1.76%),mixed antiserum(adhesion rate was 1.07%and1.47%),P200R2 antiserum(2.03%and 2.48%),P200R1 antiserum(adhesion rate was 2.06%and 2.65%),P87 antiserum(adhesion rate was 2.28%and 2.96%),P42 antiserum(3.44%and4.13%).From the observation of specific fluorescence reduction and adhesion rate,it was speculated that protein P161 was closely related to Mccp adhesion.(3)The optimal conditions for the establishment of Mccp infection SPF chicken embryo test method were as follows:yolk sac route inoculation,inoculation concentration of 1×10~7CCU/m L(dose:0.2 m L/egg),inoculation age of 7～8 days old,and urine sac fluid as the optimal real-time PCR detection and isolation and identification sample.The results showed that the mortality rate of M1801 strain,the positive rate of real-time PCR detection and the positive rate of sample separation and identification were 100%,which were virulent strains in 4 Mccp strains.M1601(mortality rate60%,detection positive rate 100%,isolation and identification positive rate 50%)took the second place,followed by F38(mortality rate 30%,detection positive rate 90%,isolation and identification positive rate 70%)and ZLY 1309F(mortality rate 30%,detection positive rate100%,isolation and identification positive rate 70%).Conclusion:(1)The IFA method of Mccp adhesion to GTC cells was established and the positive serum blocked the adhesion.(2)It is preliminarily identified that Mccp P161 protein has an adhesion effect.(3)The Mccp infection method for chicken embryos has been established,which can be used to evaluate the virulence of Mccp strains. |
