Mycoplasmosis in Poultry: An Evaluation of Diagnostic Schemes and Molecular Analysis of Egyptian Mycoplasma gallisepticum Strains

Abstract

Infections with Mycoplasma gallisepticum (MG) in poultry are associated with a wide range of disease conditions, including those affecting the respiratory and reproductive systems. The purpose of this study was to endorse the more sensitive diagnostic scheme for MG infection and identify the best molecular marker for MG phylogenetic analysis using six housekeeping genes: mgc2, mraW, atpG, ugpA, DUF31196, and lgT. For these purposes, 55 poultry flocks of different species were screened using either qRT-PCR or PCR techniques analogous to conventional culturing from non-cultured and cultured swabs on PPLO broth. The rate of MG positivity was the highest when using qRT-PCR from cultured broth (89.0%) and the lowest when using conventional culturing (34.5%). Compared to qRT-PCR from broth, statistical analysis using the Roc curve in MedCalc statistical software showed that the PCR schemes (qRT-PCR from swabs and PCR from swabs and broth) performed better than conventional culturing in terms of sensitivity, accuracy, and area under the curve (AUC), suggesting that they may be more reliable schemes. Further support was added by Cohen's kappa test, showing moderate agreement between the molecular approaches. Among the six screened genes, mgc2 and mraW had the highest detection rates (69% and 65.4%, respectively). The comparative phylogenetic analysis revealed that mgc2 or atpG gene sequences distinguished MG isolates into different clades with high discriminatory power.

Keywords: DUF31196; Mycoplasma gallisepticum; atpG; lgT; mgc2; mraW; qRT-PCR; ugpA.

Figure 1

Flowchart of the study design and numbers of flocks analyzed.

Figure 2

Postmortem lesions of the investigated poultry flocks: (A) a Hubbard broiler chicken, 29 days of age (flock 8-B) with Fibrinous perihepatitis (black arrow), (B) a layer chicken (Lohmann) of 27 weeks (flock 2-L) showing slight turbidity on the air sac (red circle), (C) a breeder quail (Baladi breed), 77 days of age (Flock 3-Q-BR), showing fibrinous air sacculitis (black arrow), (D) a 37-day-old turkey Poults (B6 breed (Flock 4-TR) showing caseous exudate in opened infraorbital sinus (blue circle).

Figure 3

Sensitivity and specificity of four diagnostic schemes (qRT PCR from swabs, PCR from swabs, PCR from broth, and conventional culturing) for MG based on qRT PCR from broth as the gold standard test.