In silico–derived <i>Actinobacillus equuli</i> –specific DNA markers and development of associated PCR assays

The ability to accurately and rapidly identify causative agents of infectious diseases facilitates precise treatment, improves clinical outcomes, and augments epidemiology studies. For many veterinary and zoonotic pathogens, however, simple molecular tests for species identification are not available. <i>Actinobacillus equuli</i> causes severe diseases, such as sleepy foal disease, septicemia, and meningitis in horses and pigs. <i>A. equuli</i> can also cause severe diseases in humans bitten by infected animals. Existing <i>A. equuli</i> identification methods are biochemical tests, 16S rRNA gene amplification followed by DNA sequencing, and MALDI-TOF MS. Nonetheless, differentiating among <i>Actinobacillus</i> spp. by these methods is still challenging. We identified novel DNA markers specific to <i>A. equuli</i> by computational genome analysis. We then designed PCR primers specific to <i>A. equuli</i> based on <i>A. equuli</i> marker sequences. We validated 2 <i>A. equuli</i>-specific PCR assays using genomic DNA from 10 strains of <i>A. equuli</i>, 15 strains of other <i>Actinobacillus</i> species, and 5 other bacterial species. Both assays gave the PCR products of expected sizes for genomic DNA of all 10 strains of <i>A. equuli</i> but not for those of other <i>Actinobacillus</i> and other bacterial species. Our novel PCR assays can accelerate <i>A. equuli</i> identification and disease diagnosis, leading to timely and appropriate antimicrobial treatment, and enable high-resolution epidemiologic studies.