Isolation, identification, genotyping, antibiotic resistance, and pathogenicity of Mycoplasma galliscepticum from a chicken farm in Shandong

Abstract:

[Background] Mycoplasma galliscepticum (MG) is the main cause of chronic respiratory diseases in poultry. The different susceptibility to antibiotics and pathogenicity of MG strains from different regions increases the difficulty in the prevention and control of MG infection. [Objective] To clarify the genotypes, antibiotic resistance, resistance gene mutations, and pathogenicity of MG isolates from a chicken farm in Yantai, Shandong, so as to provide a scientific basis for the prevention and control of MG in this area. [Methods] The MG isolates were obtained from the cleft palate swabs of chicken tested positive for MG. Seven housekeeping genes (atpG, plsC, mraW, ugpA, DUF3196, lgT, and dppC) were used for multilocus sequence typing (MLST), and a phylogenetic tree was constructed. The minimum inhibitory concentrations (MICs) of eight antibiotics including chlortetracycline, spectinomycin, lincomycin, tylosin, tylvalosin, tiamulin, valnemulin, and doxycycline against the isolates were determined. The 23S rRNA V domain and L4 and L22 protein genes were sequenced. SPF-grade chickens of 28 days old were infected by eye challenge and trachea challenge, respectively. [Results] Nine MG isolates were obtained in this study, including seven isolates (YTX2, YTX5.6, YTX11, YTX12, YTX13, YTX14, and YTX15) of sequence type 97 (ST97) and two isolates (YTX9 and YTX10) of ST16. New alleles of plsC and mraW were identified in the seven strains of ST97, with the ID of 33 and 31, respectively. All the MG isolates were sensitive to valnemulin and tiamulin (MIC≤0.25 μg/mL), moderately sensitive to tylvalosin and doxycycline (MIC≤4 μg/mL), and resistant to chlortetracycline and lincomycin (MIC≥16 μg/mL). The seven MG isolates of ST97 were moderately resistant to tylosin (MIC: 4-8 μg/mL). The mutation A2069G was identified in the V domain of the 23S rRNA gene in strains YTX5.6 and YTX2. The mutation G568T occurred in the L4 protein gene of strains YTX5.6 and YTX2, and the mutations T102C, T129C, T252C, C336T, and G404A occurred in the L22 protein gene. The strain YTX10 sensitive to tylosin, tylvalosin, tiamulin, valnemulin, and doxycycline showed the same sequence compared with the control strain MG R_low, with no mutation detected. The morbidity of air sacculitis was 80%, 60%, and 80%, respectively, 10, 20, and 30 days after tracheal challenge with the strain YTX5.6. Air sacculitis occurred 20 and 30 days after tracheal challenge with YTX10, with the morbidity of 40% and 20%, respectively. The tracheal mucosal thickening was severer in the tracheal challenge group than in the eye challenge group (P<0.05). Severe tracheal thickening was observed 20 days after tracheal challenge with strain YTX5.6, with an average thickness of 300.6 μm. Tracheal challenge with strain YTX5.6 or YTX10 resulted in significantly higher MG loads than eye challenge with same strain 10, 20, and 30 days post-infection. [Conclusion] In this study, nine MG strains of ST97 and ST16 were isolated from a chicken farm in Yantai, Shandong. The nine isolates demonstrated varied resistance to antibiotics and different pathogenicity to SPF-grade chickens.