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

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.