Characterization and Differentiation of Chicken Mycoplasma Isolates Using 16S-23S Intergenic Spacer Region Sequencing

Ff Santos,Mdm Brandão,J Li,Vla Pereira,Mi Khan,Nm Soares,Er Nascimento,Ls Machado

doi:10.1590/1806-9061-2018-0834

Abstract

ABSTRACT The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: Sao Paulo, Minas Gerais, Rio de Janeiro and Espirito Santo. The isolates were recovered from the Sao Paulo, Rio de Janeiro and Espirito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

Highlights

Mycoplasma infections are highly prevalent among poultry flocks, and three species are considered to be pathogenic: Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Mycoplasma meleagridis (MM)
Samples were obtained from commercial layer flocks in four Brazilian states
The isolates were recovered from seven different flocks in São Paulo, Rio de Janeiro and Espírito Santo (Table 1)

Summary

INTRODUCTION

Mycoplasma infections are highly prevalent among poultry flocks, and three species are considered to be pathogenic: Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Mycoplasma meleagridis (MM). The economic losses attributed to mycoplasmosis are due to decreases in egg production and quality, poor hatchability, poor feed efficiency, high mortality rates and high carcass condemnation rates on slaughter (Kleven, 2008; Nascimento et al, 2005; Nascimento; Pereira, 2009). These microorganisms prefer the mucous and serous membranes of birds, causing respiratory, joint and urogenital pathologies. In addition to the notably pathogenic mycoplasma, another 20 species are known to infect birds (Kleven, 2008). The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using a 16S–23S rDNA IGSR sequencing method. Phylogenetic analyses were conducted using the MEGA version 6 software program (Tamura et al, 2013)

RESULTS AND DISCUSSION

99 USA-R-CK60