Research Update End Year 1 – Grant 02292: Broad-Range Detection of Canine Tick-Borne Disease and Improved Diagnostics Using Next-Generation Sequencing

Dr. Pedro Paul Diniz, DVM, PhD provides an update at the end of his first year looking for improved diagnostics for tick-borne disease using genetic sequencing.

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. In fact, dogs infected with these microbes have been reported at every US state including Hawaii, and more recently, Alaska. 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. CHF has implemented a new funding mechanism to address such limitation, which supports this study. 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 500 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 billions of DNA sequences from a large number of dogs suspected of infection; and (4) comprehensive qualitycontrol measures in order to support and validate the impact of our results.

In the last six months, expanded the sample size to 537, to account for missing samples. We have completed the preparation of the samples from all canine samples, and have submitted them for deep DNA sequencing. We also design novel family-level primers for the Anaplasmataceae, Bartonellaceae and Mycoplasmataceae
families using a unique computational approach and confirmed their sensitivity and specificity based on rigorous benchtop experiments. We sought further training to update and expand our computational protocols to remain at the cutting-edge workflow of analysis of large-scale DNA sequences in metagenomics. Our bioinformatics pipeline is ready to quickly analyze the raw data coming from our large-scale DNA sequencing effort.

Finally, we already identified key elements to mprove 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 initiatives, breed clubs and other sponsors will have a positive impact in animal health at a global level.

End-Year-1 Research Update Dr. Diniz to improve diagnostics for tick-borne disease.