A Swine Health Information Center-funded study aimed to improve detection of common and emerging swine pathogens using a next generation sequencing panel. Led by Dr. Rebecca Wilkes at Purdue University, the study sought to develop and validate a targeted next-generation sequencing (tNGS) respiratory panel capable of detecting multiple viral and bacterial swine respiratory pathogens in a single test. Swine respiratory disease is often caused by multiple pathogen infections occurring simultaneously, making diagnosis and control difficult when relying on single-pathogen tests. Results from this study showed strong agreement between tNGS and PCR, high specificity, and robust sensitivity across clinically relevant pathogen loads. Moreover, the panel successfully identified mixed infections and detected pathogens that may be missed by targeted PCR testing alone.
Find the industry summary for SHIC project #24-007 here.
Infectious disease syndromes in swine are frequently multifactorial and can be challenging to determine the causative pathogen(s) by clinical signs alone. Co-infections with viral and bacterial pathogens often occur, making accurate diagnoses challenging. Many diagnostic tests utilized in swine are single pathogen assays. Consequently, there is a critical need within the swine industry for comprehensive diagnostic approaches capable of simultaneously detecting co-infections and identifying emerging or unexpected pathogens. This comprehensive approach can help support more informed treatment decisions, guide targeted interventions, and reduce reliance on sequential or repeated testing.
Objectives of the study described herein include 1) develop and optimize primers targeting swine respiratory pathogens, 2) validate specificity and sequencing performance with characterized strains, 3) determine analytical sensitivity in comparison with qPCR and 4) validate diagnostic performance using clinical samples. An array of primers targeting multiple regions across the genomes of common and emerging swine respiratory pathogens was designed in collaboration with the AgriSeq Bioinformatics team (Thermo Fisher Scientific, USA). The panel was developed to enable simultaneous detection of viral, bacterial, and selected virulence-associated targets relevant to swine disease syndromes. Primer design focused on highly conserved genomic regions to ensure broad detection, while selected targets included regions enabling genotyping or lineage assignment for specific pathogens, such as PRRSV and PCV2.
Selected clinical samples were retrospectively obtained from swine cases submitted to the Purdue University Animal Disease Diagnostic Laboratory (ADDL) based on results from the laboratory’s validated real-time PCR assays. Pathogens included PRRSV, Influenza A virus, PCV2, PCV3, M. hyopneumoniae(MHP), Streptococcus suis, Glaesserella parasuis, Actinobacillus pleuropneumoniae (APP), and M. hyorhinis (MHR). Clinical isolates of Streptococcus suis were also included. Sample selection was performed to ensure representation of both viral and bacterial respiratory pathogens as well as mixed infections commonly associated with porcine respiratory disease complex.
A total of 70 clinical samples were included in this study, including 20 positive samples to establish relative limits of detection. An additional 25 PCR-positive respiratory cases and 25 PCR-negative respiratory case controls were used to determine diagnostic sensitivity, specificity and accuracy of the developed test. Feasibility of the targeted sequencing assay was evaluated using a combination of qPCR-positive clinical specimens, and synthetic DNA controls (gBlocks, IDT, Coralville, IA) representing targeted genomic regions.
A comprehensive multiplex primer pool targeting approximately 25 swine respiratory pathogens was successfully developed in collaboration with Thermo Fisher Scientific’s AgriSeq bioinformatics team. Successful amplification and sequencing were achieved for the respiratory pathogens included in the assay, except Actinobacillus suis, for which no positive clinical sample was available to confirm the panel’s ability to detect this pathogen. Assessment of analytical sensitivity across multiple dilutions showed the tNGS assay consistently detected major respiratory pathogens, including PRRSV (multiple lineages), PCV2, PCV3, and M. hyopneumoniae, demonstrating robust performance in complex clinical matrices.
Across the clinical validation cohort, the tNGS panel showed strong concordance with PCR testing, reflected by an overall agreement of 92% and a Cohen’s kappa value of 0.84, indicating near-perfect categorical agreement beyond chance. Importantly, all PCR-negative samples remained negative by tNGS, underscoring the assay’s high specificity and low risk of false-positive reporting. This is a critical consideration for diagnostic laboratories, where false positives can lead to unnecessary interventions, economic loss, and erosion of producer confidence.
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