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Abstract
Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are economically relevant respiratory pathogens of poultry which may have analogous clinical presentations to respiratory diseases of several different etiologies. The overarching goal of this research was to facilitate rapid, sensitive, and specific diagnostic testing for poultry respiratory pathogens. There are several hurdles to overcome in achieving that goal as there are different standards and recommendations for different types of pathogens – from collection of samples in the field to processing in the diagnostic lab. In this research, we investigated several of the practical limitations to simplification and unification of molecular testing for multiplex testing of avian respiratory pathogens. The effect of transportation/preparation media on MS and MG quantitative PCR of tracheal swabs was evaluated and brain heart infusion (BHI) broth (already recommended for transport of several agents of poultry respiratory disease) was determined to be the ideal choice. The effect of swab location on MS, MG, and infectious laryngotracheitis virus (ILTv) detection by qPCR was evaluated and choanal cleft swabs were determined to be superior to tracheal and oropharyngeal swabs for detection of the pathogen DNA. Next, two highly sensitive duplex qPCRs for detection of ILTv and either MS or MG were developed in progress toward developing more complicated multiplex assays. And, finally, a novel multiplex targeted nanopore sequencing panel using MS and MG strain typing genes was developed for use on the accessible MinION nanopore sequencer along with a user friendly bioinformatic workflow. The outcomes of this research allow the poultry industry to maximize the benefits of costly diagnostic assays, set scientifically based standards in different laboratories for sample collection and handling, and meet demanding turnaround times for conclusive diagnosis of respiratory diseases.