Background and Aim: Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most significant pathogens of avian mycoplasmosis. This study aimed to isolate and identify MG and MS from chickens and detect the various virulence genes in the isolates. Moreover, the efficacies of different antibiotics were tested to identify suitable treatment regimens. Materials and Methods: We isolated MG and MS from 487 chicken samples of different ages located in different Governorates in Egypt using conventional isolation methods. The isolates were characterized by polymerase chain reaction (PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method. Results: The prevalence of MG among the isolates was 9.85%, with the highest percentage isolated from air sacs, while the prevalence of MS among the isolates was 1.6%. Moreover, the highest levels of the prevalence of both MG and MS were during the winter and autumn sampling, while the lowest levels were in the summer and spring. Following the 16S rRNA-based detection of Mycoplasma isolates, 14 MG and 5 MS isolates were identified by different PCR-based detection methods for various virulence genes. Nine MG isolates contain the mgc2 gene, six MG isolates contain the gapA gene, and three MS isolates contain the vlhA gene. We validated a duplex PCR method for the simultaneous identification of MG and MS, based on 100% of the MG and MS isolates generating common bands at 55 and 17 kDa, respectively. The MIC method identified tiamulin and spiramycin as the antibiotics of choice for the treatment of MG and MS infections, respectively. Conclusion: For more precise diagnosis of Mycoplasma infections in chicken flocks, conventional isolation methods must be confirmed by PCR. SDS-PAGE analysis helps in epidemiological studies and vaccine preparation. The MIC method can be used to help develop therapies to control avian mycoplasmosis infections.

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