Molecular Identification and Sequencing of <I>Mycoplasma gallisepticum</I> Recovered from Broilers in Egypt

Abstract

Background and objectives: Avian mycoplasmosis, particularly Mycoplasma gallisepticum (MG) is one of the infectious diseases associated with economic losses in Egyptian poultry industry. Thus, this study was aimed to determine the prevalence, serological identification, molecular characterization, sequencing and minimum inhibitory concentration of M. gallisepticum isolated from diseased broilers in Egypt.

Materials and methods: A total of 351 samples (227 tissue samples "tracheas and air sacs" and 124 tracheal swabs) and 71 sera were collected from diseased broilers. The conventional (isolation and biochemical) and molecular methods (PCR) were performed for detection of M. gallisepticum and virulence-associated gene (mgc2). The serum plate agglutination (SPA) test and enzyme-linked immunosorbent assay (ELISA) were applied on sera for determination of the presence of antibodies against M. gallisepticum. The minimal inhibitory concentration test (MIC) was used to determine the sensitivity of two sequenced M. gallisepticum strains to anti-mycoplasma agents.

Results: The total recovery rate of Mycoplasma from 351 samples from broilers was 45.29% (159) in which M. gallisepticum showed a prevalence of 62.89% (100/159). Serological identification of M. gallisepticum in 71 collected sera using SPA and ELISA were 54.9 and 40.8% with the highest geometric mean titer of ELISA for M. gallisepticum (699.08 and 495.92). Molecular characterization of Mycoplasma using PCR showed that 50% (3/6) of tested isolates were identified as M. gallisepticum based on 16SrRNA. Also, the mgc2 gene was detected in 50% (3/6) M. gallisepticum isolates. Two positive PCR mgc2 specific genes of M. gallisepticum isolates were subjected to gene target sequencing (GTS) to verify that these two isolates were M. gallisepticum. The minimal inhibitory concentration test (MIC) was applied to determine the sensitivity of these two sequenced M. gallisepticum strains to anti-mycoplasma agents. The first M. gallisepticum isolate was sensitive to tilmicosin, tiamulin and spiramycin. The second M. gallisepticum isolate showed sensitivity to tiamulin, spiramycin and tilmicosin.

Conclusion: These results summarized the necessity of monitoring the Egyptian poultry farms for avian mycoplasmosis. Also, further studies are required for controlling of mycoplasma in all stages of the poultry industry production chain to avoid different losses in Egypt.

Keywords: ELISA; Egyptian poultry farms; M. gallisepticum; PCR; avian mycoplasmosis; diseased broiler; mgc2 gene; minimal inhibitory concentration.