Journal Article

The Challenge of Developing a Test to Differentiate Actinobacillus pleuropneumoniae Serotypes 9 and 11

Fodor, László, Arnal Bernal, José Luis, Lacouture, Sonia, Bossé, Janine T., Langford, Paul R., Gantelet, Hubert, Solans Bernad, Luis, Gottschalk, Marcelo, Fernández Ros, Ana Belén, Li, Yanwen

2025

Overview

Actinobacillus pleuropneumoniae 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 cps 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 cps and O-Ag loci. Ten isolates had a cpsF 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 cpsF 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 cpsF gene mutations. Our results suggest that, currently, a comprehensive analysis of the cpsF 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.

• Publisher: MDPI AG,MDPI
• Subject: Actinobacillus pleuropneumoniae / Analysis / Antibodies / Antigens / Assaying / Biosynthesis / Capsular polysaccharides / Clinical isolates / Collection / cpsF / Diagnostic software / DNA probes / DNA sequencing / Enzymes / Gene mutations / Gene sequencing / Genes / Genetic diversity / Genomes / Genomics / Laboratories / Mutation / nanopore sequencing / Nucleic acids / Nucleotide sequencing / Nucleotides / Point mutation / Polysaccharides / serotype 11 / serotype 9 / Serotypes / Single nucleotide polymorphisms / Single-nucleotide polymorphism / Strain analysis / Swine / Whole genome sequencing / whole-genome sequencing (WGS)