Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

Özgür Çelebi ,Mitat Şahin ,Fatih Büyük ,A Ü Baca ,Salih Otlu ,Aliye Gülmez Sağlam ,Elif Çelik ,Umit Sevimli ,Eray Büyük ,Mustafa Reha Çoşkun ,S.h Erpek ,Murat Karahan ,Doğan Akça

doi:10.1590/1806-9061-2021-1522

Özgür Çelebi , Mitat Şahin + Show 11 more

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https://doi.org/10.1590/1806-9061-2021-1522

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Abstract

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Frey’s Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.

Highlights

Mycoplasma species have been defined as the microorganisms that lead to several clinical infections, primarily reproductive system infections, peritonitis, airsacculitis, pneumonia and sudden death in poultry
Molecular analysis findings Species-specific PCR findings Molecular confirmation of the phenotypically identified Mycoplasma species was performed by species-specific PCR that amplified the dnaX, pcrA, rpoB gene regions, and the identification of 32 M. anatis, 9 M. anseris and 5 M. cloacale species was confirmed by the PCR (Figure 1)
Three (5.88%) of the 5 isolates that were defined as Mycoplasma spp. via phenotypic methods but could not be identified at the species level were later identified as M. anserisalpingitis by speciesspecific PCR

Summary

Introduction

Mycoplasma species have been defined as the microorganisms that lead to several clinical infections, primarily reproductive system infections, peritonitis, airsacculitis, pneumonia and sudden death in poultry. Mycoplasma gallisepticum and Mycoplasma synoviae are responsible for respiratory system infections, arthritis and embryonal deaths in chicken and turkeys, the latter of which results from the widespread in ovo transmission property of the bacteria. In waterfowls such as geese and ducks, Mycoplasma anatis, Mycoplasma anseris, Mycoplasma cloacale and Mycoplasma anserisalpingitis 1220) are common pathogenic Mycoplasma species (Stipkovits & Szathmary, 2012; Otlu, 2016; Grózner et al, 2019b; Gyuranecz et al, 2020) These Mycoplasma species, together with other infectious microorganisms and environmental factors, play an important role in the immune system of geese. Respiratory system infections caused by Mycoplasma species progress with nasal discharge, wheezing, coughing and have a moderate mortality rate (5-9%) in geese eRBCA-2021-1522

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