Research Update Mid-Year-2 CHF-02292: Broad-Range Detection of Canine Tick-Borne Disease and Improved Diagnostics Using Next-Generation Sequencing

Research update for expansion and improvement of diagnostics for definitive diagnosis of tick-borne disease in dogs.

Dogs from any breed, age or gender can be infected with microbes transmitted by ticks or fleas. These diseases can cause devastating effects and even death not only to dogs but also to humans. Ticks are present everywhere in the US, and they bring the risk of transmitting the microbes to dogs and humans. It is still very difficult to diagnose these conditions, and approximately 95% of suspected dog cases are negative when we use current diagnostic technology. One of the biggest limiting factors for the development of better diagnostic tools is the insufficient funding opportunities for large-scale projects. The long-term goal of our research team is to expand the current diagnostic tools to include a larger spectrum of potentially hazardous microorganisms.

Our innovative approach is based on four pillars: (1) large-scale DNA sequencing to identify known and potentially new organisms present in blood of dogs naturally exposed to vector-borne diseases; (2) increase in sensitivity and specificity of large-scale sequencing by targeting major families of potentially hazardous organisms, (3) advanced bioinformatic analysis of millions of DNA sequences from a large number of dogs suspected of infection; and (4) comprehensive quality-control measures in order to support and validate the impact of our results.

At this point, we have completed the Specific Aim #1 by performing Universal 16S-NGS on >500 canine samples previously tested by a conventional PCR panel and comparing the results from both techniques. We found high sensitivity rates on the comparison between Universal 16S-NGS and PCR panel for some pathogens. We were also able to detect a known pathogen in approximately one of every five samples previously reported as negative by the conventional PCR panel. We also genetically characterized potential novel strains or species of pathogens. We have now advanced our progress towards Specific Aim #2, which is based on the improvement of the NGS platform for better detection of pathogens. We have expanded the sensitivity of the NGS platform by designing and validating family-level primers for the Anaplasmataceae, Bartonellaceae and Mycoplasmataceae families, as well as for the 18S rRNA gene for the detection of protozoans such as Babesia spp. Our bioinformatics pipeline has proven to be ready to quickly analyze the raw data coming from our largescale DNA sequencing effort.

Finally, we believe we already identified key elements to improve the use of this new technology in veterinary diagnostics. Our results will probably become a paradigm shift in the detection and identification of the cause of vector-borne diseases in dogs, by unveiling thousands of novel potentially hazardous microorganisms. Our data will serve as the new foundation for the implementation and expansion of novel diagnostic methods, and will ultimately support early diagnosis and better medical care to dogs worldwide. By sponsoring this CHF research initiative, breed clubs and other sponsors will have a positive impact on animal health at a global level.

Mid-Year-2 Research Update Dr. Diniz for improved diagnostics for Tick-Borne disease using DNA sequencing.