Validation of a real-time PCR assay for high-throughput detection of<i>Avibacterium paragallinarum</i>in chicken respiratory sites

Clothier, Kristin A.; Stoute, Simone; Torain, Andrea; Crossley, Beate

doi:10.1177/1040638719866484

Cited by 6 publications

(3 citation statements)

References 15 publications

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“…In 2008, Corney et al developed another assay for the detection of A. paragallinarum DNA based on real-time PCR with primers for the pyrG gene [ 27 ]. Clothier et al successfully validated this assay as a diagnostic one in 2019 [ 28 ]. In 2021, Kuchipudi et al [ 29 ] developed an assay based on real-time PCR with primers for a region of the A. paragallinarum recN gene encoding a repair protein.…”

Section: Discussionmentioning

confidence: 99%

Development and Validation of PCR Diagnostic Assays for Detection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale

Krylova,

Bogomazova,

Kirsanova

et al. 2023

Veterinary Sciences

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PCR is the most effective method for detecting difficult-to-cultivate pathogens and pathogens that are part of mixed infections in animals, such as Ornithobacterium rhinotracheale, which causes bird ornithobacteriosis, or Avibacterium paragallinarum, which causes infectious coryza. In this work, we developed and validated two efficient and sensitive diagnostic assays for the rapid and accurate detection of A. paragallinarum and O. rhinotracheale DNA in bacterial isolates and clinical samples using real-time PCR with TaqMan-like probes. When designing the PCR assays, we performed in silico analysis, optimized DNA isolation methods and PCR conditions, and assessed the analytical and diagnostic performance of PCR. We designed primers and probes that have no mismatches with published whole-genome sequences of bacteria. The optimization of conditions showed that the PCR assays are sufficiently robust to changes in temperature and oligonucleotide concentration. The validation showed that the developed assays have high analytical and diagnostic sensitivity and specificity. These assays are expected to improve the differential diagnosis of respiratory diseases in chickens and turkeys.

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Section: Discussionmentioning

confidence: 99%

Development and Validation of PCR Diagnostic Assays for Detection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale

Krylova,

Bogomazova,

Kirsanova

et al. 2023

Veterinary Sciences

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“…Previously, a gel-based PCR assay published in 1996 has been extensively used for the detection of A. paragallinarum from diagnostic samples (10). Based on this HPG gel based PCR assay, a 5 ′ Taq nuclease assay targeting a smaller region of the gene called HPG-2 of A. paragallinarum was developed in 2008 (15) and the workflow was recently validated in 2019 (11). However, these assays are designed based on the study published in 1996, when the whole genome sequence information of Avibacterium paragallinarum was not available.…”

Section: Discussionmentioning

confidence: 99%

“…The efficiency of recN based PCR was compared to a previously validated assay targeting the HPG-2 (Haemophilus paragallinarum, the historical name for A. paragallinarum) region of the bacterium (11)…”

Section: Comparative Efficacy Of the New Recn Qpcr Vs Previous Hpg-2 Qpcrmentioning

confidence: 99%

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

et al. 2021

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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%.

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Avibacterium

Blackall¹

2020

Bergey's Manual of Systematics of Archaea and Bacteria

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A.vi.bac.te'ri,um. L. fem. n. avis a bird; N.L. neut. n. bacterium rod; N.L. neut. n. Avibacterium bacterium of birds. Proteobacteria / Gammaproteobacteria / Pasteurellales / Pasteurellaceae / Avibacterium Avibacterium is a genus within the family Pasteurellaceae . Members of this genus are Gram‐negative, nonmotile, rod‐shaped, or pleomorphic cells typically occurring singly. Species in this genus vary in the requirement for external sources of nicotinamide adenine dinucleotide (NAD) (often termed V factor) when grown in agar or broth. Two species, Avibacterium gallinarum and Avibacterium endocarditidis , do not require NAD, while the other species ( Avibacterium avium, Avibacterium paragallinarum, and Avibacterium volantium ) typically require NAD. Notably, some isolates of A. paragallinarum do not require NAD. The requirement for NAD results in the occurrence of satellitic growth on sheep blood agar when a nurse colony is present. Growth is mesophilic, and most isolates, particularly on initial isolation, require an atmosphere of 5% CO 2 . Nitrate is reduced without gas production. The reaction in Hugh–Leifson medium with (+)‐ d ‐glucose is fermentative without gas production. All members of the genus are catalase‐ and oxidase‐positive except that A. paragallinarum is catalase‐negative. Disease conditions associated with this genus are all linked to chickens and include infectious coryza ( A. paragallinarum) , valvular endocarditis and septicemia ( A. endocarditidis ), and a chronic fowl cholera‐like condition ( A. gallinarum) . DNA G + C content (mol%) : 44.2–47 ( T m ). Type species: Avibacterium gallinarum Blackall et al. 2005 VP .

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Validation of a real-time PCR assay for high-throughput detection ofAvibacterium paragallinarumin chicken respiratory sites

Cited by 6 publications

References 15 publications

Development and Validation of PCR Diagnostic Assays for Detection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale

Development and Validation of PCR Diagnostic Assays for Detection of Avibacterium paragallinarum and Ornithobacterium rhinotracheale

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

Avibacterium

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