Identification of Strain-Specific Sequences That Distinguish a Mycoplasma gallisepticum Vaccine Strain from Field Isolates.

Camir Ricketts,Larissa Pickler,John Maurer,Saravanaraj Ayyampalayam,Maricarmen García,Naola M Ferguson-Noel,B W Fenwick

doi:10.1128/jcm.00833-16

Abstract

ABSTRACTDespite attempts to control avian mycoplasmosis through management, vaccination, and surveillance, Mycoplasma gallisepticum continues to cause significant morbidity, mortality, and economic losses in poultry production. Live attenuated vaccines are commonly used in the poultry industry to control avian mycoplasmosis; unfortunately, some vaccines may revert to virulence and vaccine strains are generally difficult to distinguish from natural field isolates. In order to identify genome differences among vaccine revertants, vaccine strains, and field isolates, whole-genome sequencing of the M. gallisepticum vaccine strain ts-11 and several “ts-11-like” strains isolated from commercial flocks was performed using Illumina and 454 pyrosequencing and the sequenced genomes compared to the M. gallisepticum Rlow reference genome. The collective contigs for each strain were annotated using the fully annotated Mycoplasma reference genome. The analysis revealed genetic differences among vlhA alleles, as well as among genes annotated as coding for a cell wall surface anchor protein (mg0377) and a hypothetical protein gene, mg0359, unique to M. gallisepticum ts-11 vaccine strain. PCR protocols were designed to target 5 sequences unique to the M. gallisepticum ts-11 strain: vlhA3.04a, vlhA3.04b, vlhA3.05, mg0377, and mg0359. All ts-11 isolates were positive for the five gene alleles tested by PCR; however, 5 to 36% of field isolates were also positive for at least one of the alleles tested. A combination of PCR tests for vlhA3.04a, vlhA3.05, and mg0359 was able to distinguish the M. gallisepticum ts-11 vaccine strain from field isolates. This method will further supplement current approaches to quickly distinguish M. gallisepticum vaccine strains from field isolates.

Highlights

Despite attempts to control avian mycoplasmosis through management, vaccination, and surveillance, Mycoplasma gallisepticum continues to cause significant morbidity, mortality, and economic losses in poultry production
No major genomic changes were identified among ts-11 avirulent and virulent isolates; there were numerous single-nucleotide polymorphisms (SNPs) that need to be further analyzed and confirmed
Forty of the virulence genes identified in the M. gallisepticum ts-11 genome were annotated as coding for the variable surface protein VlhA, and these vlhA genes mapped to one of 6 loci within the ts-11 genome

Summary

Introduction

Despite attempts to control avian mycoplasmosis through management, vaccination, and surveillance, Mycoplasma gallisepticum continues to cause significant morbidity, mortality, and economic losses in poultry production. M. gallisepticum isolates vary widely in their relative degrees of pathogenicity in animal challenge experiments, depending on the route of infection and the number of in vitro passages [20,21,22] Serial passaging of this organism in vitro has been used to create attenuated strains for use as vaccines [7], but the likelihood of reversion to wild-type virulence is inherent to this attenuation method. Mycoplasma genitalium was the second complete bacterial genome ever published [25], and since over 50 Mycoplasma genomes, including pathogens of humans, animals, and plants, have been reported, including M. gallisepticum [26] Despite these facts, compared to other pathogens, few virulence-related genes have been identified in M. gallisepticum. The expression of MalF, an ABC transporter, has been shown to be essential for persistence [31]

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Results

Conclusion