Research Update Mid-Year-1 CHF-02528: Developing a Next Generation Diagnostic Platform for TBD

Research update for improving diagnostics for Tick-Borne Disease.

Diagnostic tests based on the detection of DNA from harmful organisms in clinical samples have revolutionized veterinary medicine in the last decades. Currently, diagnostic panels for several vectorborne diseases (VBDs) are available through universities and private labs in the USA and abroad. However, the vast majority of results from sick dogs are negative, which frustrates veterinarians and dog owners trying to reach a definitive diagnosis. These panels are based on the detection of previously known DNA sequences of each pathogen, which limits their ability to detect novel organisms.

Using an innovative approach, our study proposes the adaptation of high-throughput nextgeneration sequencing (NGS) to the detection of tick-borne bacteria in dog blood to overcome the limitations of the current diagnostics. NGS is capable of generating millions of individual sequencing reads from each sample, allowing for the unbiased identification and characterization of multiple organisms from a single sample.

We are pioneering this strategy in the Veterinary Medicine VBD diagnostics field, and important results were already achieved from our previous AKC-CHF grant (#02292). Since we are dealing with a cutting-edge technology, our work is under continuous and systematic adjustments, aiming enhancements in the platform in order to accurately detect infected dogs and precisely determine which bacteria are responsible for disease.

In this current first report of grant #02528, we describe our bioinformatics efforts on comparing the ‘state-of-the-art’ computational tools using dog blood samples as input, as well as provide a comprehensive standard operating procedure (SOP) for best practices of microbiome analyses applied to VBD diagnostics, as part of the specific aim 3 (SA#3). In parallel, as part of SA#2, we have also started a search for a new marker gene (other than 16S rRNA) through computational screening of whole genomes in order to achieve a better discriminatory power on the taxonomic classification of VBD-causing bacteria, thus increasing the diagnostics capability of detecting species and strains.

Regarding improvements on benchtop microbial DNA isolation techniques (SA#1), we have performed initial tests, which have demonstrated to reduce host DNA concentration in infected dog blood samples confirmed by quantitative PCR assay. Our results are in line with the proposed timeline. We truly believe that our ongoing AKC-CHF research will support the development of better diagnostic tools that will simultaneously advance both canine and human health.

Mid-Year-1 Research Update Dr. Diniz for improved diagnostics for Tick-Borne disease using DNA sequencing.