Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8−1.2×10−5 per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.

Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array-surfaceenhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.

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Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies.

Groups of eight chickens were challenged with 10-fold dilutions of one of two strains of Mycoplasma synoviae (MS); each challenge group contained two noninfected sentinels. Both strains were highly efficient in colonizing the respiratory tract with challenge doses as low as 76 and 24 color-changing units/bird. Infection spread rapidly (within 7 days) to sentinels, while uninfected control chickens separated from infected chickens by two empty pens remained uninfected for the 56-day experimental period. Although sentinels and birds challenged with the lowest doses had weaker or slightly slower antibody responses in some cases as measured by serum plate agglutination, enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI), they generally exhibited a typical antibody response. Agglutination reactions tended to be weak, but a high percentage of tests (generally >30% from day 14 postchallenge) were positive. ELISA results were variable, and in some cases reactor rates were low (generally <20%), even though the chickens were colonized in the upper respiratory tract. The HI test was reliable in detecting infected groups; usually >50% were positive from 14 days postchallenge. Mean HI titers were higher when using hemagglutination antigens prepared from the homologous MS strain as compared with antigen prepared from the heterologous strain or with standard antigen prepared from WVU 1853.