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Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis
Kate J Howell1, Sarah E Peters2, Jinhong Wang2
1Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom kjh52@cam.ac.uk.
Abstract:
Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/microl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.
Insights
A new multiplex PCR method offers rapid molecular serotyping for Haemophilus parasuis, differentiating 14 of 15 serovars. This technique is faster, more sensitive, and specific than traditional indirect hemagglutination for diagnosing Glässer's disease and pneumonia in pigs.
Area of Science:
- Veterinary Microbiology
- Bacterial Pathogenesis
- Molecular Diagnostics
Background:
- Haemophilus parasuis is a significant swine pathogen causing Glässer's disease and pneumonia.
- Current serotyping relies on indirect hemagglutination (IHA), which is time-consuming and has limitations in distinguishing all serovars.
- Genetic variation in capsule loci presents an opportunity for molecular-based serotyping.
Purpose of the Study:
- To develop and validate a rapid multiplex PCR (mPCR) assay for molecular serotyping of H. parasuis.
- To improve upon the speed, sensitivity, and specificity of existing serotyping methods.
Main Methods:
- Design of a multiplex PCR assay targeting variable regions within the capsule loci of 15 H. parasuis serovars.
- Validation of the mPCR using a collection of >200 H. parasuis isolates with known in silico serovar data.
- Comparison of mPCR results with traditional IHA serotyping for a subset of isolates.
Main Results:
- The mPCR successfully differentiated 14 of the 15 H. parasuis serovars, with serovars 5 and 12 detected together.
- The assay demonstrated high specificity, with no cross-reactivity against related bacteria.
- Validation showed 100% concordance with in silico results and 90% concordance with IHA for typed isolates.
- The mPCR identified two previously in silico-nontypeable isolates.
Conclusions:
- The developed multiplex PCR provides a rapid, sensitive, and specific method for molecular serotyping of H. parasuis.
- This assay can aid in the accurate diagnosis and epidemiological surveillance of Glässer's disease and pneumonia in swine.
- The mPCR offers a significant advancement over conventional IHA serotyping.
