The Challenge of Developing a Test to Differentiate Actinobacillus pleuropneumoniae Serotypes 9 and 11
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.011 | 0.014 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.003 | 0.002 |
| Insufficient payload (model declined to judge) | 0.001 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".