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In silico-derived Actinobacillus equuli-specific DNA markers and development of associated PCR assays.
Abstract: 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. causes severe diseases, such as sleepy foal disease, septicemia, and meningitis in horses and pigs. can also cause severe diseases in humans bitten by infected animals. Existing identification methods are biochemical tests, 16S rRNA gene amplification followed by DNA sequencing, and MALDI-TOF MS. Nonetheless, differentiating among spp. by these methods is still challenging. We identified novel DNA markers specific to by computational genome analysis. We then designed PCR primers specific to based on marker sequences. We validated 2 -specific PCR assays using genomic DNA from 10 strains of , 15 strains of other species, and 5 other bacterial species. Both assays gave the PCR products of expected sizes for genomic DNA of all 10 strains of but not for those of other and other bacterial species. Our novel PCR assays can accelerate identification and disease diagnosis, leading to timely and appropriate antimicrobial treatment, and enable high-resolution epidemiologic studies.
Publication Date: 2025-10-08 PubMed ID: 41063450PubMed Central: PMC12511006DOI: 10.1177/10406387251382186Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study identified new DNA markers unique to Actinobacillus equuli using computational methods and developed PCR assays for rapid, accurate detection of this bacterial species.
- The PCR tests were validated and shown to specifically detect A. equuli, enabling faster diagnosis and better management of related diseases in animals and humans.
Background
- Actinobacillus equuli is a bacterial pathogen causing serious diseases such as sleepy foal disease, septicemia, and meningitis in horses and pigs.
- This bacterium can also cause severe infections in humans when bitten by infected animals, making accurate identification clinically important.
- Current identification methods for A. equuli include biochemical tests, 16S rRNA gene sequencing, and MALDI-TOF mass spectrometry.
- However, these methods face challenges in reliably differentiating A. equuli from closely related Actinobacillus species.
Research Objectives
- To identify novel DNA markers that are specific to A. equuli through in silico (computational) genome analysis.
- To develop PCR assays based on these DNA markers that can specifically detect A. equuli from other species.
- To validate these PCR assays across multiple bacterial strains to ensure sensitivity and specificity.
Methods
- Computational genome analysis was performed to scan available genomic data for sequences unique to A. equuli.
- From these unique sequences, specific PCR primers were designed to target the A. equuli-specific DNA markers.
- The PCR assays were tested using genomic DNA from:
- 10 different strains of A. equuli
- 15 strains from other Actinobacillus species
- 5 other bacterial species as negative controls
- PCR products were analyzed to confirm that the correct DNA fragments were amplified only from A. equuli strains.
Results
- Two PCR assays targeting distinct A. equuli-specific markers were successfully developed.
- Both PCR assays produced expected amplicon sizes consistently from all 10 A. equuli strains tested.
- No PCR products were detected from any other Actinobacillus species or unrelated bacterial species, demonstrating high specificity.
Significance and Applications
- The newly developed PCR assays provide a rapid and reliable method for identifying A. equuli infections compared to conventional testing.
- This enables timely diagnosis and improves the precision of antimicrobial treatment for affected animals and potentially humans.
- The availability of species-specific DNA markers also allows for more detailed epidemiologic studies, aiding in tracking disease outbreaks.
- These molecular tools fill an important gap where existing biochemical and molecular identification methods fall short.
Conclusion
- Through in silico genomic analysis and experimental validation, this study has established novel DNA markers and associated PCR assays specific to Actinobacillus equuli.
- These assays improve diagnostic capabilities for veterinary and zoonotic diseases caused by this pathogen.
- They have potential utility in clinical microbiology labs and epidemiology research focused on this bacterial species.
Cite This Article
APA
Songsungthong W, Pornthanakasem W, Leartsakulpanich U, Srijuntongsiri G.
(2025).
In silico-derived Actinobacillus equuli-specific DNA markers and development of associated PCR assays.
J Vet Diagn Invest, 38(1), 59-65.
https://doi.org/10.1177/10406387251382186 Publication
Researcher Affiliations
- National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Sirindhorn International Institute of Technology, Thammasat University, Pathum Thani, Thailand.
- National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Sirindhorn International Institute of Technology, Thammasat University, Pathum Thani, Thailand.
- National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Sirindhorn International Institute of Technology, Thammasat University, Pathum Thani, Thailand.
- School of Information, Computer, and Communication Technology (ICT), Sirindhorn International Institute of Technology, Thammasat University, Pathum Thani, Thailand.
MeSH Terms
- Animals
- Polymerase Chain Reaction / veterinary
- Polymerase Chain Reaction / methods
- Actinobacillus / genetics
- Actinobacillus / isolation & purification
- Actinobacillus Infections / veterinary
- Actinobacillus Infections / microbiology
- Actinobacillus Infections / diagnosis
- Horse Diseases / microbiology
- Horse Diseases / diagnosis
- Horses
- DNA, Bacterial / genetics
- Genetic Markers
- RNA, Ribosomal, 16S / genetics
- Computer Simulation
Conflict of Interest Statement
Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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