Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

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/μl 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.

FIG 1

Serovar prevalence of 117 isolates that had been serotyped by IHA (blue) in comparison with the in silico serovar predicted for the same isolates based on analysis of the capsule loci (red). The agreement between the IHA and in silico serovar prediction was tested using an unweighted Cohen's kappa test, with a P value of 0.0000128, excluding the NT isolates.

FIG 2

Band patterns for the molecular serotyping PCR for all 15 serovars of H. parasuis. Lane M, Quick-Load 100-bp DNA ladder (NEB); S1 to S15 represent the 15 serovars of H. parasuis. Sp-sp, species-specific marker.

FIG 3

Negative-control panel showing specificity of the primer sets across a range commensal and pathogenic bacteria found in the pig respiratory tract (H. parasuis-positive controls serovar 2 and 5, A. minor, A. porcinus, A. indolicus, S. suis, A. pleuropneumoniae, and B. bronchiseptica). Lane M, Quick-Load 100-bp marker (NEB) and H₂O as the negative control.

FIG 4

Determination of the limit of detection for the serotyping multiplex based on pure genomic DNA for the reference strains of serovars 1, 2, 5, 8, 9, and 13. The unit of genomes per microliter is used. Lane M, Quick-Load 100-bp marker (NEB) and H₂O as the negative control.