Development of a duplex qPCR for the differentiation of a live attenuated Escherichia coli aroA mutant vaccine strain from field isolates in chickens
Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carc...
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| creator | Leurs, Kirsten Goossens, Evy Christensen, Henrik Mainil, Jacques G Vancraeynest, Dieter Ducatelle, Richard Van Immerseel, Filip |
| description | Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain. |
| doi_str_mv | 10.1371/journal.pone.0278949 |
| format | Article |
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Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0278949</identifier><identifier>PMID: 36534672</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Amino acids ; Animals ; Attenuated vaccines ; Biology and Life Sciences ; Birds ; Chickens ; Colibacillosis ; Colonization ; Disease ; Diseases ; E coli ; Economic impact ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli Infections ; Escherichia coli Infections - veterinary ; Escherichia coli Vaccines ; Feces ; Gene mutations ; Genetic aspects ; Identification and classification ; Immunization ; International organizations ; Life sciences ; Medicine and Health Sciences ; Multidisciplinary ; Mutants ; Mutation ; Médecine vétérinaire & santé animale ; Peritonitis ; Polymerase chain reaction ; Poultry ; Poultry Diseases ; Poultry Diseases - prevention & control ; Prevention ; Research and Analysis Methods ; Salmonella ; Sciences du vivant ; Sensitivity ; Strains (organisms) ; Vaccination ; Vaccines ; Vaccines, Attenuated ; Veterinary medicine & animal health ; Virulence</subject><ispartof>PloS one, 2022-12, Vol.17 (12), p.e0278949-e0278949</ispartof><rights>Copyright: © 2022 Leurs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</rights><rights>COPYRIGHT 2022 Public Library of Science</rights><rights>2022 Leurs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. 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prevention & control</subject><subject>Prevention</subject><subject>Research and Analysis Methods</subject><subject>Salmonella</subject><subject>Sciences du vivant</subject><subject>Sensitivity</subject><subject>Strains (organisms)</subject><subject>Vaccination</subject><subject>Vaccines</subject><subject>Vaccines, Attenuated</subject><subject>Veterinary medicine & animal health</subject><subject>Virulence</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk89u1DAQxiMEoqXwBggsISE47BLbcZxcKq1KgUqVisqfq-U4410XJ97azqo8Cdc-S58MZ3dbdVEPKIdY49_3jWfsybKXOJ9iyvGHCzf4Xtrp0vUwzQmv6qJ-lO3jmpJJSXL6-N56L3sWwkWeM1qV5dNsj5aMFiUn-9mfj7AC65Yd9BE5jSRqh6WFK3T59egcaedRXABqjdbgE2JkNK7fgNasAMkYoR9khPbm-jioBXijFkYi5ay5uZbezVA3RJnMV1Ip0wMK0UvTI-1dh7QB2yITnE0OAaVwEqtf0Ifn2RMtbYAX2_9B9uPT8fejL5PTs88nR7PTieK0jBNW4ZYUDS8ok4UuqwJXdcUbxZminHGqoOE5aAk8T9u64JSVRDa8wkpXjAI9yF5vfJfWBbHtaRCEM1bltMAsEScbonXyQiy96aT_LZw0Yh1wfi6kj0ZZEJBrrFitQRd1gVXbtBWWBMqaqkYTWiavw222oemgVamjXtod092d3izE3K1EzUvC2GhANwbWwBxS8saIFVkL1-vBptMo0YAgpKwEqSu6Vr3bpvXucoAQRWeCAmtlD264rTbneETf_IM-3JMtNZepbNNrl06rRlMx4zQv81Ts6DV9gEpfC51R6dlqk-I7gvc7gsREuIpzOYQgTr6d_z979nOXfXuPXYC0cZHe3DC-5bALFhtQeReCB313NTgX49TddkOMUye2U5dkr-5f653odszoX5P6Km8</recordid><startdate>20221219</startdate><enddate>20221219</enddate><creator>Leurs, Kirsten</creator><creator>Goossens, Evy</creator><creator>Christensen, Henrik</creator><creator>Mainil, Jacques G</creator><creator>Vancraeynest, Dieter</creator><creator>Ducatelle, Richard</creator><creator>Van Immerseel, Filip</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>Q33</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0003-4238-4658</orcidid><orcidid>https://orcid.org/0000-0001-8520-0834</orcidid><orcidid>https://orcid.org/0000-0001-6476-5932</orcidid></search><sort><creationdate>20221219</creationdate><title>Development of a duplex qPCR for the differentiation of a live attenuated Escherichia coli aroA mutant vaccine strain from field isolates in chickens</title><author>Leurs, Kirsten ; Goossens, Evy ; Christensen, Henrik ; Mainil, Jacques G ; Vancraeynest, Dieter ; Ducatelle, Richard ; Van Immerseel, Filip</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c736t-581d24b7435a4f68418987bc75c37573ceb70efae704f6f473562ab781cf853e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Amino acids</topic><topic>Animals</topic><topic>Attenuated vaccines</topic><topic>Biology and Life Sciences</topic><topic>Birds</topic><topic>Chickens</topic><topic>Colibacillosis</topic><topic>Colonization</topic><topic>Disease</topic><topic>Diseases</topic><topic>E coli</topic><topic>Economic impact</topic><topic>Escherichia coli</topic><topic>Escherichia coli - 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Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>36534672</pmid><doi>10.1371/journal.pone.0278949</doi><tpages>e0278949</tpages><orcidid>https://orcid.org/0000-0003-4238-4658</orcidid><orcidid>https://orcid.org/0000-0001-8520-0834</orcidid><orcidid>https://orcid.org/0000-0001-6476-5932</orcidid><oa>free_for_read</oa></addata></record> |
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| language | eng |
| recordid | cdi_plos_journals_2755803415 |
| source | PLoS; MEDLINE; DOAJ Directory of Open Access Journals; PubMed Central; Free Full-Text Journals in Chemistry; EZB Electronic Journals Library |
| subjects | Amino acids Animals Attenuated vaccines Biology and Life Sciences Birds Chickens Colibacillosis Colonization Disease Diseases E coli Economic impact Escherichia coli Escherichia coli - genetics Escherichia coli Infections Escherichia coli Infections - veterinary Escherichia coli Vaccines Feces Gene mutations Genetic aspects Identification and classification Immunization International organizations Life sciences Medicine and Health Sciences Multidisciplinary Mutants Mutation Médecine vétérinaire & santé animale Peritonitis Polymerase chain reaction Poultry Poultry Diseases Poultry Diseases - prevention & control Prevention Research and Analysis Methods Salmonella Sciences du vivant Sensitivity Strains (organisms) Vaccination Vaccines Vaccines, Attenuated Veterinary medicine & animal health Virulence |
| title | Development of a duplex qPCR for the differentiation of a live attenuated Escherichia coli aroA mutant vaccine strain from field isolates in chickens |
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