Research Update CHF 03137 EY1: Targeted Next Generation Sequencing Panel for Comprehensive Testing for Reproductive and Neurologic Pathogens of Dogs

The goal of this project is to validate respiratory and neurologic disease testing using a targeted next-generation sequencing (tNGS) panel we previously developed. We expect this assay, which includes 34 different pathogens known to be associated with reproductive/neurologic disease in dogs, will perform as well as individual real-time PCR assays designed to detect each of these pathogens individually. This type of comprehensive testing takes the guesswork out of choosing which individual tests or small panel of tests to run to diagnose disease.

We have now completed testing approximately 70% (23/34) of the pathogens that are detectable with this assay, and all but 1 could be detected. The pathogen missed was Campylobacter jejuni. This one was included for its possible role in reproductive disease, but it is more likely to cause diarrhea in dogs. Therefore, we will not redesign the assay at this point. We also began testing the sensitivity of the tNGS assay, compared to real-time PCR testing.

Based on the results from 13 pathogens tested, the approximate limit of detection, based on real-time PCR Ct values is in the mid-30s. One of the pathogens, bluetongue virus, had a detection limit of approximately 25. This suggests additional primers for this pathogen should be added to the assay to increase the sensitivity for this organism. We started testing the diagnostic sensitivity and specificity of the assay by evaluating 22 CSF samples submitted from dogs with neurologic disease for canine distemper virus (CDV) real-time PCR. Only one of these samples was positive for CDV by real-time PCR. We were also able to detect this virus in this sample by the tNGS method. Additionally, we were able to identify the variant as an America-5 genotype with the new assay. All the other real-time PCR negative samples were also negative by the tNGS assay for CDV, and for all other neurologic pathogens in the tNGS assay as well.

We have also begun testing the blinded samples for Aim 4 of the study. We are currently evaluating the data and will share the samples and the primer pools with our collaborator at the University of Pennsylvania. We are currently ahead of schedule, based on the timeline submitted for the project.

End-Year-1 03137 Research Update Targeted Next Generation Sequencing Panel for Comprehensive Testing for Reproductive and Neurologic Pathogens of Dogs