Detection and Differentiation of Avian Mycoplasmas by Surface-Enhanced Raman 1 Spectroscopy Based on Silver Nanorod Array

21 Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause 22 annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines 23 recommend regular surveillance and intervention strategies to contain M. gallisepticum 24 infections and ensure mycoplasma-free avian stocks, but several factors make its detection 25 and diagnosis a major challenge. Current techniques are laborious, require special 26 expertise, and are typically plagued by false results. In this study we describe a novel 27 detection strategy which uses silver nanorod array-surface-enhanced Raman spectroscopy 28 (NA-SERS) for direct detection of avian mycoplasmas. As proof-of-concept for use in 29 avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian 30 mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, 31 Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine 32 strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to 33 classify these species rapidly and accurately with >93% sensitivity and specificity. 34 Furthermore, NA-SERS had a lower limit of detection 100-fold greater than standard PCR 35 and comparable to real-time quantitative PCR. Detection of M. gallisepticum in choanal 36 cleft swabs from experimentally infected birds yielded good sensitivity and specificity 37 suggesting that NA-SERS is applicable for clinical detection. 38