A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry.

Suresh V Kuchipudi,Michele Yon,Patricia Dunn,Bhushan M Jayarao,Rhiannon M Barry,Jen Williams,Ruth H Nissly,Sherrill Davison,Deepanker Tewari,Traci Pierre,Sean Loughrey,Denise Barnhart,Tammy Mathews,Maurice Byukusenge,Dona J Kelly,Meera Surendran Nair,Eva Walner-Pendleton

doi:10.3389/fvets.2021.609126

Abstract

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.

Highlights

Infectious coryza (IC) is an acute upper respiratory disease of growing broilers and layers, caused by Avibacterium paragallinarum, a gram-negative bacterium previously called Haemophilus paragallinarum [1, 2]
Bacteriological diagnosis of A. paragallinarum is often challenging due to its fastidious nature
It is well-known that real-time PCR assays employing amplicon-specific probes are highly sensitive and precise for the rapid and accurate detection of pathogens from clinical samples [12,13,14]

Summary

Introduction

Infectious coryza (IC) is an acute upper respiratory disease of growing broilers and layers, caused by Avibacterium paragallinarum, a gram-negative bacterium previously called Haemophilus paragallinarum [1, 2]. The most common clinical signs in chickens infected with A. paragallinarum include facial edema, nasal exudates, sneezing, and conjunctivitis [3]. A tentative diagnosis of IC is often made based on history, clinical signs, and characteristic gross lesions. The chronic stage of infection, prior antimicrobial treatments and any delay in sample processing have been shown to interfere with effective recovery of A. paragallinarum from diagnostic samples using conventional bacteriological methods [1]. Despite the worldwide distribution of A. paragallinarum and a major cause of significant economic losses to the poultry industry; the true prevalence, incidence, and overall disease dynamics of IC in poultry flocks is not well-understood.

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