#18-040

Complete

Date Full Report Received

07/01/2020

Date Abstract Report Received

07/01/2020

Investigation

Institution:
Primary Investigator:

Funded By

Foreign Animal Diseases (FAD) are black swan events – they come as a surprise, have a major effect, and are often inappropriately rationalized as foreseeable after the fact with the benefit of hindsight. Porcine epidemic diarrhea virus (PEDV) was a wake-up call, but pales in comparison to the consequences of a regulated FAD introduction, such as foot-and-mouth disease virus (FMDV). The challenges addressed in this study were to develop a rapid method for detection of a surprise FAD introduction so that ruinous spread, as occurred with PEDV, and freezing of all animal movement can be avoided. FMDV is particularly challenging since any vesicular disease case must be addressed and FMDV ruled out so that animal transport is permitted. The threat of an undiagnosed FAD introduction, combined with the essential need for rapid rule-outs due to endemic vesicular diseases like Senecavirus A (SVA), requires a robust diagnostic capability. We proposed to develop and benchmark methods and protocols for pen-side sequencing for vesicular disease diagnostics that can provide producers with new options for real-time, robust and rapid molecular detection of foreign and endemic pathogens.

Using SVA as a model for FMDV and PRRSV as a model for endemic pathogens, we evaluated the portable, hand-held MinION sequencing device. The protocols that we developed were compared to the gold standard of detection, diagnostic PCR detection. Our main results showed that MinION sequencing can be used for detection of a pathogen at the species level from a clinical sample without the need for prior knowledge. This can be used for unexpected pathogen detection and co-infection investigations. In addition, our method can improve the detection resolution down to the strain level, which is essential for epidemiological analysis and investigation of other challenging clinical cases, such as a biosecurity breach or vaccine failure. The analytical sensitivity of MinION sequencing as a diagnostic tool was evaluated and compared with real-time PCR. While sequencing at the current state is less sensitive than qPCR diagnostics, it is a great support tool for PCR and provides valuable genomic information.

Pen-side and slaughterhouse nucleic acid sequencing addresses a long-sought goal of moving diagnostic testing into the field for rapid and accurate pathogen identification. It is particularly important for FAD surveillance in suspect cases. We concluded that rapid, direct sequencing can identify pathogens causing clinical disease and in the case of vesicular disease, it was able to identify SVA (even the strain of SVA) as a cause of clinical signs. In addition, this research resulted in a diagnostic method that is immediately adaptable to any pathogen of concern, since it does not require specific primers or specific knowledge of genetic sequence. Thus, it is valuable for a broad range of applications in which timely differential disease diagnosis is needed for case management allowing for improvement in animal care, reduction of costs, and an increase in productivity. Our results have been communicated to the swine community through the SDEC newsletter, the SHMP, the Leman Conference, and AASV.

For more information please contact Dr. Cheryl Dvorak at the Veterinary and Biomedical Sciences Department at the University of Minnesota, email: dvora013@umn.edu.

Key Findings:
• MinION sequencing can be used for robust, unbiased pathogen detection at the species level
• Using additional bioinformatics analysis, the strain of a pathogen can be identified from MinION sequence data
• This study created a disease diagnostic model for FAD outbreaks
• This study accelerated the development of rapid, on-site, real-time disease diagnosis