| Mycoplasma synoviae(MS)is a significant pathogen that poses a threat to the health of poultry farming.It can cause inflammation of the air sacs,synovitis,and tenosynovitis,as well as subclinical respiratory symptoms.Infection with MS in laying hens can lead to a decrease in egg production and quality.Broiler chickens,after infection,exhibit degraded carcasses and increased abandonment rates,causing significant economic losses to the poultry industry.Therefore,it is essential to strengthen the monitoring of daily MS infection in chicken flocks.Monitoring the level of MS antibodies is an important means of evaluating the field MS infection.This study aims to optimize the MS serum plate agglutination method and indirect ELISA method for detecting MS antibodies.Additionally,the SPA test,HI test,rMSPBELISA test,and commercial ELISA test were used to monitor MS infection in chicken flocks.The qPCR detection method was also employed to monitor MS pathogens in chicken flocks with choanal cleft swabs.The objective is to explore a stable and reliable monitoring method for MS wild virus infection in chicken flocks.Corresponding MS infection monitoring plans tailored to different farm conditions can achieve rapid diagnosis of MS infection.This is of great significance for preventing and controlling MS infection and blocking its transmission.1.Optimization of MS Plate Agglutination Test MethodThis investigation involved a comparison of various preparation techniques for plate agglutination antigens,with the aim of determining the most effective method for detecting antigens.In the preparation of MS serum plate agglutination antigen,a final concentration of 7%bovine serum albumin was added to minimize the occurrence of non-specific reactions in the SPA test.After diluting the MS positive serum by a factor of 16,the agglutinating particles were still visible to the naked eye when mixed with the antigen in a 1:1 ratio.The results of the shelf life test indicated that the SPA antigen remained viable even after being stored at 4℃ for 12 months.2.Optimization of MS antibody indirect ELISA detection method based on recombinant protein rMSPBIn the initial stages of our laboratory’s research,we successfully produced a recombinant protein rMSPB carrying a His tag through the pET prokaryotic expression system.We then established an indirect ELISA method for detecting MS antibodies using this recombinant protein as the coating antigen.However,we encountered limitations in constructing the regression equation for serum antibody titers,as we relied mainly on commercial kits.To address these limitations and optimize our experimental plan,we utilized the PNT baseline method to determine the endpoint titers of different serum samples.By calculating the correction absorbance of different serum samples at a 1:500 dilution using the subtraction formula,we were able to obtain a regression equation of Log10(ET)=1.155 for the S/P value and endpoint titers at 1:500 dilution × Log10(S/P at 1:500)+3.661,with a high correlation coefficient(R2)of 0.9333.These findings have significant implications for the development of a MS detection kit.The single dilution method for detecting serum samples is highly advantageous as it eliminates errors caused by continuous multiple dilutions,reduces reagent costs,and improves detection efficiency.The rMSPB-ELISA method and a commercially available ELISA method were employed to detect clinical serum samples and generate a trend chart of MS antibody titers.The results indicated that the trend of MS antibody titers measured by both methods was consistent,with a positive coincidence rate of 96.89%and a negative coincidence rate of 90.09%.The sensitivity and specificity of the rMSPB-ELISA method were found to be 91.44%and 98.20%,respectively,which were in agreement with the IDEXX method.3.Application of three serological methods in detecting MS antibodiesThis study utilized various methods,including SPA test,HI test,rMSPB-ELISA method,and commercialized MS ELISA antibody detection method,to monitor the infection of MS in chicken flocks.Additionally,the RT-qPCR method was used to monitor the infection status of MS.By continuously tracking and detecting MS antibodies and pathogens in four groups of chicken flocks,it was observed that the overall trend of MS antibodies detected by the three serological methods was consistent.The titers of MS antibodies in uninfected chickens initially decreased and then increased with MS wildvirus infection.In the early stages of infection,the SPA testing method can be used to detect MS antibodies and conduct preliminary screening for chicken flocks.Furthermore,ELISA and qPCR testing methods should be employed to continuously monitor chicken flocks,and based on clinical test results,further MS prevention,control,and treatment should be carried out on large-scale farms.Therefore,it is recommended to use the qPCR method for regular and systematic MS monitoring of chicken flocks in breeding farms.This method has a fast detection speed,strong sensitivity,and high detection rate.Additionally,the SPA/ELISA method can be used for batch screening of chicken serum,and HI/qPCR can be used to recheck the test results according to different monitoring situations,thereby achieving rapid diagnosis of MS infection.To sum up,the optimized SPA technique and rMSPB-ELISA method utilized in this study exhibit significant clinical relevance.Both methods can leverage their respective monitoring strengths in the detection of chicken MS virus,while also reducing detection expenses.This technical support facilitates the continuous monitoring of breeding farms. |
