Molecular Characterization of Mycoplasma gallisepticum Isolates from Turkeys

Abstract

Mycoplasma gallisepticum was isolated from several turkey flocks at different locations in the United States that were clinically affected with respiratory disease. Five of these isolates from four series of outbreaks had patterns similar to the 6/85 vaccine strain of M. gallisepticum by random amplified polymorphic DNA (RAPD) analysis using three different primer sets, whereas with a fourth primer set (OPA13 and OPA14), only two of the isolates were similar to 6/85. Results obtained by sequencing portions of the pvpA, gapA, and mgc2 genes and an uncharacterized surface lipoprotein gene indicated that the field isolates had DNA sequences that ranged from 97.6% to 100%, similar to the 6/85 results. In some of the outbreaks there was an indirect association with the presence of commercial layers in the area that had been vaccinated with this vaccine strain, but there was no known close association with vaccinated birds in any of the outbreaks. Turkeys were challenged with two of the field isolates and with 6/85 vaccine strain. Turkeys challenged with the field isolates developed respiratory disease with airsacculitis and a typical M. gallisepticum antibody response, whereas birds challenged with 6/85 developed no respiratory signs or lesions and developed only a weak antibody response. Although these isolates were very similar to the 6/85 vaccine strain, it was not possible to prove that they originated from the vaccine strain-it is possible that they could be naturally occurring field isolates.

Development of aMycoplasma gallisepticuminfection model in turkeys

“…Further experiments will be needed to examine strain differences in tissue tropism and the initial sites of colonization. Previous studies have detected thickening only of the upper tracheal mucosa in turkeys exposed to M. gallisepticum by aerosol, possibly because this was the only section of the trachea examined Kleven et al, 2004;Zaki et al, 2004). However, we demonstrated that thickening of the mucosa could be detected along the length of the trachea.…”

“…However, none of these studies used the same criteria to assess infection and disease, so it is difficult to compare the different routes of inoculation that were used. Lin & Kleven (1982) assessed gross air sac lesions and serology, Dingfelder et al (1991) examined reisolation and serological responses, Kleven et al (2004) reported re-isolation, serology, air sac and upper tracheal lesion scores and measurement of upper tracheal mucosal thicknesses, and Sanei et al (2007) assessed serology, reisolation, air sac lesions, clinical signs, tracheal mucosal lesions and thicknesses at three levels of the trachea. There have also been studies that demonstrated the safety of the K5054, ts-11 and 6/85 strains in turkeys and their capacity to protect against air sac lesions and increases in tracheal mucosal thickness caused by exposure to a coarse spray of the R strain Zaki et al, 2004).…”

Differentiation of Mycoplasma gallisepticum strains using molecular methods

“…Because of the use of live MG vaccines, it is important to be able to differentiate the vaccine strain from MG wild strains. The most widely used methods to differentiate MG strains are the immunoblot polyacrylamide gel electrophoresis of mycoplasma proteins (Bencina et al, 1994), the random amplified polymorphic DNA analysis (Geary et al, 1994;Fan et al, 1995;Charlton et al, 1999), restriction enzyme analysis (Kleven et al, 1988;Liu et al, 2001) and the sequence analysis of MG genes (Liu et al, 2001;Kleven et al, 2004).…”

“…This research was complemented with the examination of MG strains using primers specific for different genes with published sequences such as gapA and pvpA (Liu et al, 2001;Kleven et al, 2004). In addition, in our study PCR tests were designed to amplify for different fragments of crmA, crmB, crmC, gapA and mgc2 genes, respectively.…”

“…All own primers were designed using the Primer Designer 2.0 software. The primers specific for gapA and pvpA genes were described by Liu et al (2001) and Kleven et al (2004). The first RAPD PCR was performed with M16SPCR5', M13F and S1OLIGO3' primers (Fan et al, 1995).…”