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.
Discrepancies between methods were limited to PCR-positive samples and were consistently associated with targets detected at high Ct values, indicating low pathogen abundance. Some targets, specifically IAV and respiratory coronavirus, performed below expectations. For the RNA viruses, this is likely due to lack of adequate primers to detect sequence differences. Future redesign of primers should improve the detection of these pathogens. Notably, no instances were observed in which tNGS detected respiratory pathogens in samples deemed negative by PCR, reinforcing the conservative and clinically appropriate nature of the assay’s detection criteria.
Evaluation of analytical sensitivity using serially diluted clinical samples confirmed that the tNGS assay retains robust detection capability across a wide range of pathogen concentrations, including those commonly encountered in routine diagnostic submissions. Beyond simple presence or absence, the tNGS panel was designed to include genotype and strain, informative genomic regions for selected viral pathogens of interest. This design enabled additional genetic resolution for several viruses, including differentiation of PRRSV North American (Type 2) versus European (Type 1) genotypes, identification of PCV2 genotype D versus non-D genotypes, and determination of influenza A virus subtypes when SIV was detected.
However, full lineage level discrimination was not consistently achieved for all targets, particularly for PRRSV in clinical specimens. PRRSV results were conservatively reported at the genotype level rather than at the lineage level. These findings indicate that, while the current assay reliably detects PRRSV presence, additional optimization or redesign of lineage-targeting primers may be required to consistently achieve lineage-level resolution, which was an intended objective of the panel design.
The sequence level data generated by the tNGS approach supports improved interpretation of circulating viral diversity and may aid outbreak investigations and epidemiologic assessments when sufficient sequence coverage is achieved. Equally important is the assay’s compatibility with herd level sample types, including pooled and population representative specimens. This expands its utility from individual case diagnosis to broader monitoring programs, where understanding pathogen diversity and circulation patterns is often more informative than identifying a single agent in a single animal.
Overall, this project demonstrates that a multiplex tNGS assay can function as a reliable and informative diagnostic tool for swine respiratory disease, with performance closely aligned to established PCR testing while offering substantially broader pathogen coverage and genomic insight. Further, tNGS can help overcome inherent limitations of targeted PCR testing alone. The study supports the use of tNGS as a complementary diagnostic and surveillance tool that can improve outbreak investigations, inform vaccine strategies, strengthen swine health monitoring programs, and expand pathogen surveillance capacity for emerging diseases.
The Swine Health Information Center, launched in 2015 with Pork Checkoff funding, protects and enhances the health of the US swine herd by minimizing the impact of emerging disease threats through preparedness, coordinated communications, global disease monitoring, analysis of swine health data, and targeted research investments. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. For more information, visit http://www.swinehealth.org or contact Dr. Megan Niederwerder at [email protected] or Dr. Lisa Becton at [email protected].
