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

Abstract

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. 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.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates

“…According to the method of Ricketts et al (21), out of the 12 examined M. gallisepticum samples, all six Australian samples and one from Italy (IZSVE/2013/3185-5f) were characterized as ts-11 isolates, the results of which reveal poor agreement with the MLST and MAMA-ts11-plpA, MAMA-ts11-glpK, and MAMA-ts11-potC assays (range of adjusted Rand coefficients, 0.198 to 0.327) (Data Set S1).…”

“…These techniques are usually tested on a very limited number of samples from a limited geographical region, need special equipment, or are highly expensive. Recently, PCR tests have been developed for the differentiation of ts-11 from field isolates (21), but these are not suitable in situations when multiple strains are present in a sample. For the genotyping of M. gallisepticum isolates, a core-genome multilocus sequence typing (MLST) system with improved discriminatory power has been established (22), but this method needs the previous isolation and whole-genome sequence of the bacteria.…”

“…The current study describes the development and characterization of rapid and cost-effective PCR-based assays for the simultaneous discrimination of 6/85, ts-11, and F vaccine strains from field isolates. To better evaluate the system, the results were compared with the data from MLST analysis (23) and PCR assays described by Ricketts et al (21), and a total of 239 M. gallisepticum strains and clinical samples originating from 16 countries were examined. Portugal, n ϭ 2; Austria, n ϭ 1; France, n ϭ 1; Jordan, n ϭ 1; and Slovenia, n ϭ 1) and 36 DNA samples from clinical samples (Spain, n ϭ 17; Israel, n ϭ 8; Italy, n ϭ 6; Iraq, n ϭ 3; Albania, n ϭ 1; and Jordan, n ϭ 1) were provided for a blind test from the sample collections (Data Set S1).…”

“…Samples which appeared to be ts-11 reisolates by the developed MAMA tests and/or those originating from Australia were further tested according to Ricketts et al (21). In brief, the presence of three additional genes (vlhA3.04a, vlhA3.05, and mg03659) was investigated for the discrimination of field isolates from ts-11 vaccine strains.…”

When Micro Drives the Macro: A Fresh Look at Disease and its Massive Contributions in the Hindu Kush-Himalaya

“…Chronic respiratory disease (CRD) in chicken, turkeys, game birds, pigeons, and wild birds is caused by Mycoplasma gallisepticum that is evolutionarily related to the low % Guanine and Cytosine Gram-positive Clostridiales (Weisburg et al 1989;Ricketts et al 2017). Serology examination shows about 9.5% prevalence in the chicken in the Nepal (DoAH 2015).…”