Development and Validation of Two Diagnostic Real-Time PCR (TaqMan) Assays for the Detection of Bordetella avium from Clinical Samples and Comparison to the Currently Available Real-Time TaqMan PCR Assay

Amro Hashish,Mohamed El-Gazzar,Amr Mekky,Yuko Sato,Nubia Macedo,Avanti Sinha

doi:10.3390/microorganisms9112232

Amro Hashish, Mohamed El-Gazzar + Show 4 more

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https://doi.org/10.3390/microorganisms9112232

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Abstract

Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 103 plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA.

Highlights

The genus Bordetella is comprised of fifteen different species [1,2]
Two new TaqMan probe-based qPCR assays for the detection of Bordetella avium (BA) were developed and validated
Numerous qPCR assays have been developed for the diagnosis of poultry pathogens [51,52,53,54,55], including one assay for the diagnosis of BA

Summary

Introduction

The genus Bordetella is comprised of fifteen different species [1,2]. B. bronchiseptica, B. holmesii, B. parapertussis, and B. pertussis are adapted to mammalian hosts and are phylogenetically closely related, while the more distantly related B. avium and B. hinzii are more associated with avian species [3].Bordetella avium (BA) is a Gram-negative, non-fermentative, motile, aerobic bacilli [4]causing bordetellosis, known as Turkey Coryza, in domesticated turkeys [5] and is an opportunistic pathogen in chickens [6]. The genus Bordetella is comprised of fifteen different species [1,2]. B. bronchiseptica, B. holmesii, B. parapertussis, and B. pertussis are adapted to mammalian hosts and are phylogenetically closely related, while the more distantly related B. avium and B. hinzii are more associated with avian species [3]. Bordetella avium (BA) is a Gram-negative, non-fermentative, motile, aerobic bacilli [4]. Causing bordetellosis, known as Turkey Coryza, in domesticated turkeys [5] and is an opportunistic pathogen in chickens [6]. Mortality associated with uncomplicated bordetellosis in turkey is low, morbidity often approaches 100%, and infected turkeys are susceptible to secondary bacterial infection [5]. BA can infect a variety of wild birds [8,9] and is associated with the Lockjaw Syndrome in Psittacine birds [10]

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