Development and Validation of PCR Assays for Improved Diagnosis of Infectious Coryza by Differentiating Pathogenic and Nonpathogenic Avibacterium paragallinarum.

Abstract

Avibacterium paragallinarum (AP) is a primary bacterial pathogen of chickens that leads to infectious coryza (IC) disease. Recently, multiple commercial layer flocks in several U.S. states reported positive real-time quantitative PCR (qPCR) results without any history of clinical signs. Owing to the proven specificity of the current IC qPCR assays, these results suggested the existence of AP strains that do not lead to clinical disease in layers, i.e., nonpathogenic AP (npAP) strains. This was further proven by isolating and characterizing npAP strains from these normal layer flocks. Although these strains are clinically nonpathogenic in layers, current IC qPCR assays fail to distinguish them from the pathogenic AP, leading to qPCR-positive flocks with no apparent disease. Therefore, the purpose of this study was to develop and validate TaqMan real-time PCR assays that can differentiate between pathogenic and the newly discovered npAP strains. Whole-genome sequences of six npAP isolates were generated, and genomic comparison was conducted against 43 pathogenic AP strains. Analysis revealed two consistent features. First, the capsular polysaccharide transporter gene hctA was exclusively present in the pathogenic AP strains but absent in npAP strains. Second, unique lengthy insertions within the HMTp210 gene were observed only in the npAP strains. The HMTp210 insertions were chosen as the qPCR target to identify the newly discovered npAP strains (np-HMTp210 assay). On the other hand, hctA was selected to identify the pathogenic AP strains. During the validation process, 28 isolates and 10 oropharyngeal (OP) swab pools representing the pathogenic AP strains as well as six isolates and 86 OP pools of npAP strains were tested. A wide panel of respiratory, bacterial and viral, pathogens were included in the validation. Both assays demonstrated high performance in terms of analytical specificity in relation to each other and when tested against various bacterial and viral pathogens. Moreover, the hctA and np-HMTp210 assays displayed high sensitivity, with a limit of detection of 1 copy/µl and 2.5 copies/µl, respectively, and PCR efficiencies of 94.62% and 92.99%, respectively. Both assays showed 100% diagnostic specificity and sensitivity. However, after the validation process, an ongoing surveillance effort in clinically normal layer flocks uncovered a new population of npAP strains. This new npAP population refutes our original qPCR design goals to distinguish AP strains from npAP strains because the latest finding renders the differential capacity of this newly developed PCR incomplete. However, the newly developed qPCR in its current status is still useful in differentiating the great majority of cases and is still useful for diagnostic laboratories to provide much needed IC diagnostic answers to the poultry industry. Meanwhile, we will continue to investigate new targets that could either complement or replace the current targets to achieve our goal of the complete differentiation between these two AP populations.