In silico-derived Actinobacillus equuli-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. Actinobacillus equuli causes severe diseases, such as sleepy foal disease, septicemia, and meningitis in horses and pigs. A. equuli can also cause severe diseases in humans bitten by infected animals. Existing A. equuli identification methods are biochemical tests, 16S rRNA gene amplification followed by DNA sequencing, and MALDI-TOF MS. Nonetheless, differentiating among Actinobacillus spp. by these methods is still challenging. We identified novel DNA markers specific to A. equuli by computational genome analysis. We then designed PCR primers specific to A. equuli based on A. equuli marker sequences. We validated 2 A. equuli-specific PCR assays using genomic DNA from 10 strains of A. equuli, 15 strains of other Actinobacillus species, and 5 other bacterial species. Both assays gave the PCR products of expected sizes for genomic DNA of all 10 strains of A. equuli but not for those of other Actinobacillus and other bacterial species. Our novel PCR assays can accelerate A. equuli identification and disease diagnosis, leading to timely and appropriate antimicrobial treatment, and enable high-resolution epidemiologic studies.