Molecular characterization and antimicrobial susceptible profiles of Thai mycoplasma synoviae and immune response chickens received live mycoplasma synoviae vaccine

Abstract

Mycoplasma synoviae infection in chickens which has been increasing worldwide is mostly controlled by pathogen-free flocks’ maintenance, medication in infected flocks and vaccination in high-risk flocks. The effective control strategy requires the information of diagnostic assays for detecting and differentiating M. synoviae strains, the understanding of immune response mechanisms in vaccinated chickens, and the reliable drug susceptible evidents for making decision in antimicrobial usage. This study aimed to develop a convenient assay for evaluation of quality or uniformity of vaccination with live MS-H vaccine, to characterize the immune response mechanisms in chickens receiving 3 different vaccination programs immunizing single dose at either 9 or 12 weeks of age or two doses at both 9 and 12 weeks of age, and to determine the antimicrobial susceptible profiles of current Thai M. synoviae isolates. Based on sequence analysis of partial vlhA gene in this study, Thai M. synoviae isolates collected from articular joint and respiratory tract of chickens during 2020, consisted of types E and L with 19 and 35 amino acid length, respectively, differing from MS-H vaccine strain classified as type C with 32 amino acid length. Consequently, the developed PCR-RFLP assay using restriction enzyme TasI to digest vlhA gene-targeted PCR amplicons, was verified to detect MS-H vaccine strain in vaccinated chickens and differentiate it from non-vaccine strains; WVU1853 reference strain (ATCC 25204) and Thai M. synoviae field strains. Besides, this study also demonstrated that vaccination with live MS-H, whether single or two doses, could similarly stimulate immune response mechanisms including both humoral and cellular immune responses. Antibody response in vaccinated chickens were initially detected at 2 weeks post first vaccination, and continuously increased to the highest level at 3 and 5 weeks post first vaccination examined by RPA assay and ELISA, respectively. Cellular immune response against both homologous and heterologous antigens, examined by MTS Tetrazolium assay, were similar at early period post immunization while the responses against homologous antigen was better at late period post immunization. Furthermore, antimicrobial susceptible profiles of current Thai M. synoviae isolates was performed strong susceptible to tylvalosin, tylosin, and tiamulin at MIC50 value of 0.0098, 0.0391 and 0.0781 µg/ml, respectively; moderate susceptible to doxycycline, oxytetracycline and lincomycin-spectinomycin at MIC50 value of 0.1563, 0.1563 and 0.625 µg/ml, respectively; and resistance to enrofloxacin at MIC50 value of 10 µg/ml. Interestingly, at least three subpopulations of Thai M. synoviae isolates were presented with different MIC values; ranging 0.0195-0.625 µg/ml; from strong susceptibility to resistance to tilmicosin.