The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11
<i>Actinobacillus pleuropneumoniae</i> is a major swine pathogen, classified into 19 serotypes based on capsular polysaccharide (CPS) loci. This study aimed to improve the diagnostic method to differentiate between serotypes 9 and 11, which are challenging to distinguish using convention...
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2025-01-01
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| author | José Luis Arnal Bernal Ana Belén Fernández Ros Sonia Lacouture Janine T. Bossé László Fodor Hubert Gantelet Luis Solans Bernad Yanwen Li Paul R. Langford Marcelo Gottschalk |
| author_facet | José Luis Arnal Bernal Ana Belén Fernández Ros Sonia Lacouture Janine T. Bossé László Fodor Hubert Gantelet Luis Solans Bernad Yanwen Li Paul R. Langford Marcelo Gottschalk |
| author_sort | José Luis Arnal Bernal |
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| description | <i>Actinobacillus pleuropneumoniae</i> is a major swine pathogen, classified into 19 serotypes based on capsular polysaccharide (CPS) loci. This study aimed to improve the diagnostic method to differentiate between serotypes 9 and 11, which are challenging to distinguish using conventional serological and molecular methods. A novel qPCR assay based on locked nucleic acid (LNA) probes was developed and validated using a collection of reference strains representing all known 19 serotypes. The assay demonstrated specificity in detecting the nucleotide variation characteristic of the serotype 9 reference strain. However, the analysis of a clinical isolate collection identified discrepancies between LNA-qPCR and serological results, prompting further investigation of the <i>cps</i> and O-Ag loci. Subsequent nanopore sequencing and whole-genome sequencing of a collection of 31 European clinical isolates, previously identified as serotype 9, 11, or undifferentiated 9/11, revealed significant genetic variations in the <i>cps</i> and O-Ag loci. Ten isolates had a <i>cpsF</i> sequence identical to that of the serotype 11 reference strain, while six isolates had single-nucleotide polymorphisms that were unlikely to cause significant coding changes. In contrast, 15 isolates had interruptions in the <i>cpsF</i> gene, distinct from that found in the serotype 9 reference strain, potentially leading to a serotype 9 CPS structure. In the O-Ag loci, differences between serotypes 9 and 11 were minimal, although some isolates had mutations potentially affecting O-Ag expression. Overall, these findings suggest that multiple genetic events can lead to the formation of a serotype 9 CPS structure, hindering the development of a single qPCR assay capable of detecting all <i>cpsF</i> gene mutations. Our results suggest that, currently, a comprehensive analysis of the <i>cpsF</i> gene is necessary to accurately determine whether the capsule of an isolate corresponds to serotype 9 or 11. Although such analyses are feasible with the advent of third-generation sequencing technologies, their accessibility, cost, and time to result limit their use in routine diagnostic applications. Under these circumstances, the designation of the hybrid serovar 9/11 remains a valid approach. |
| format | Article |
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| institution | DOAJ |
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| series | Microorganisms |
| spelling | doaj-art-5312cd0bc835463d813a71ef99775e1b2025-08-20T03:12:19ZengMDPI AGMicroorganisms2076-26072025-01-0113228010.3390/microorganisms13020280The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11José Luis Arnal Bernal0Ana Belén Fernández Ros1Sonia Lacouture2Janine T. Bossé3László Fodor4Hubert Gantelet5Luis Solans Bernad6Yanwen Li7Paul R. Langford8Marcelo Gottschalk9Exopol. Polígono, río Gállego D14, 50840 San Mateo de Gállego, SpainExopol. Polígono, río Gállego D14, 50840 San Mateo de Gállego, SpainResearch Group on Infectious Diseases in Production Animals and Swine and Poultry Infectious Diseases Research Centre, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, Saint-Hyacinthe, QC J2S 2M2, CanadaSection of Paediatric Infectious Disease, Imperial College London, St Mary’s Campus, London W2 1PG, UKMicrobiology and Infectious Diseases Department, University of Veterinary Medicine Budapest, Szent Istvan, No. 2, H-1078 Budapest, HungaryResearch and Development Department, Ceva Biovac Campus, 49070 Beaucouzé, FranceExopol. Polígono, río Gállego D14, 50840 San Mateo de Gállego, SpainSection of Paediatric Infectious Disease, Imperial College London, St Mary’s Campus, London W2 1PG, UKSection of Paediatric Infectious Disease, Imperial College London, St Mary’s Campus, London W2 1PG, UKResearch Group on Infectious Diseases in Production Animals and Swine and Poultry Infectious Diseases Research Centre, Faculty of Veterinary Medicine, University of Montreal, 3200 Sicotte, Saint-Hyacinthe, QC J2S 2M2, Canada<i>Actinobacillus pleuropneumoniae</i> is a major swine pathogen, classified into 19 serotypes based on capsular polysaccharide (CPS) loci. This study aimed to improve the diagnostic method to differentiate between serotypes 9 and 11, which are challenging to distinguish using conventional serological and molecular methods. A novel qPCR assay based on locked nucleic acid (LNA) probes was developed and validated using a collection of reference strains representing all known 19 serotypes. The assay demonstrated specificity in detecting the nucleotide variation characteristic of the serotype 9 reference strain. However, the analysis of a clinical isolate collection identified discrepancies between LNA-qPCR and serological results, prompting further investigation of the <i>cps</i> and O-Ag loci. Subsequent nanopore sequencing and whole-genome sequencing of a collection of 31 European clinical isolates, previously identified as serotype 9, 11, or undifferentiated 9/11, revealed significant genetic variations in the <i>cps</i> and O-Ag loci. Ten isolates had a <i>cpsF</i> sequence identical to that of the serotype 11 reference strain, while six isolates had single-nucleotide polymorphisms that were unlikely to cause significant coding changes. In contrast, 15 isolates had interruptions in the <i>cpsF</i> gene, distinct from that found in the serotype 9 reference strain, potentially leading to a serotype 9 CPS structure. In the O-Ag loci, differences between serotypes 9 and 11 were minimal, although some isolates had mutations potentially affecting O-Ag expression. Overall, these findings suggest that multiple genetic events can lead to the formation of a serotype 9 CPS structure, hindering the development of a single qPCR assay capable of detecting all <i>cpsF</i> gene mutations. Our results suggest that, currently, a comprehensive analysis of the <i>cpsF</i> gene is necessary to accurately determine whether the capsule of an isolate corresponds to serotype 9 or 11. Although such analyses are feasible with the advent of third-generation sequencing technologies, their accessibility, cost, and time to result limit their use in routine diagnostic applications. Under these circumstances, the designation of the hybrid serovar 9/11 remains a valid approach.https://www.mdpi.com/2076-2607/13/2/280<i>Actinobacillus pleuropneumoniae</i>serotype 9serotype 11<i>cpsF</i>whole-genome sequencing (WGS)nanopore sequencing |
| spellingShingle | José Luis Arnal Bernal Ana Belén Fernández Ros Sonia Lacouture Janine T. Bossé László Fodor Hubert Gantelet Luis Solans Bernad Yanwen Li Paul R. Langford Marcelo Gottschalk The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11 Microorganisms <i>Actinobacillus pleuropneumoniae</i> serotype 9 serotype 11 <i>cpsF</i> whole-genome sequencing (WGS) nanopore sequencing |
| title | The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11 |
| title_full | The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11 |
| title_fullStr | The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11 |
| title_full_unstemmed | The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11 |
| title_short | The Challenge of Developing a Test to Differentiate <i>Actinobacillus pleuropneumoniae</i> Serotypes 9 and 11 |
| title_sort | challenge of developing a test to differentiate i actinobacillus pleuropneumoniae i serotypes 9 and 11 |
| topic | <i>Actinobacillus pleuropneumoniae</i> serotype 9 serotype 11 <i>cpsF</i> whole-genome sequencing (WGS) nanopore sequencing |
| url | https://www.mdpi.com/2076-2607/13/2/280 |
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