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A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry
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...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071849/ https://www.ncbi.nlm.nih.gov/pubmed/33912603 http://dx.doi.org/10.3389/fvets.2021.609126 |
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author | Kuchipudi, Suresh V. Yon, Michele Surendran Nair, Meera Byukusenge, Maurice Barry, Rhiannon M. Nissly, Ruth H. Williams, Jen Pierre, Traci Mathews, Tammy Walner-Pendleton, Eva Dunn, Patricia Barnhart, Denise Loughrey, Sean Davison, Sherrill Kelly, Dona J. Tewari, Deepanker Jayarao, Bhushan M. |
author_facet | Kuchipudi, Suresh V. Yon, Michele Surendran Nair, Meera Byukusenge, Maurice Barry, Rhiannon M. Nissly, Ruth H. Williams, Jen Pierre, Traci Mathews, Tammy Walner-Pendleton, Eva Dunn, Patricia Barnhart, Denise Loughrey, Sean Davison, Sherrill Kelly, Dona J. Tewari, Deepanker Jayarao, Bhushan M. |
author_sort | Kuchipudi, Suresh V. |
collection | PubMed |
description | 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%. |
format | Online Article Text |
id | pubmed-8071849 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-80718492021-04-27 A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry Kuchipudi, Suresh V. Yon, Michele Surendran Nair, Meera Byukusenge, Maurice Barry, Rhiannon M. Nissly, Ruth H. Williams, Jen Pierre, Traci Mathews, Tammy Walner-Pendleton, Eva Dunn, Patricia Barnhart, Denise Loughrey, Sean Davison, Sherrill Kelly, Dona J. Tewari, Deepanker Jayarao, Bhushan M. Front Vet Sci Veterinary Science 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%. Frontiers Media S.A. 2021-04-12 /pmc/articles/PMC8071849/ /pubmed/33912603 http://dx.doi.org/10.3389/fvets.2021.609126 Text en Copyright © 2021 Kuchipudi, Yon, Surendran Nair, Byukusenge, Barry, Nissly, Williams, Pierre, Mathews, Walner-Pendleton, Dunn, Barnhart, Loughrey, Davison, Kelly, Tewari and Jayarao. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Veterinary Science Kuchipudi, Suresh V. Yon, Michele Surendran Nair, Meera Byukusenge, Maurice Barry, Rhiannon M. Nissly, Ruth H. Williams, Jen Pierre, Traci Mathews, Tammy Walner-Pendleton, Eva Dunn, Patricia Barnhart, Denise Loughrey, Sean Davison, Sherrill Kelly, Dona J. Tewari, Deepanker Jayarao, Bhushan M. A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry |
title | A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry |
title_full | A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry |
title_fullStr | A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry |
title_full_unstemmed | A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry |
title_short | A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry |
title_sort | highly sensitive and specific probe-based real-time pcr for the detection of avibacterium paragallinarum in clinical samples from poultry |
topic | Veterinary Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071849/ https://www.ncbi.nlm.nih.gov/pubmed/33912603 http://dx.doi.org/10.3389/fvets.2021.609126 |
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