CRISPR-Cas-powered pen-side diagnostic tests for the early detection of the tick-borne pathogens Theileria parva, Anaplasma marginale and Babesia bigemina

Title : CRISPR-Cas-powered pen-side diagnostic tests for the early detection of the tick-borne pathogens Theileria parva, Anaplasma marginale and Babesia bigemina

Abstract:

The apicomplexan parasite Theileria parva causes East Coast fever (ECF), one of the most important and lethal tick-borne diseases of cattle in sub-Saharan Africa, resulting in annual losses exceeding US $300 million to the livestock industry. ECF diagnosis primarily relies on clinical signs, serology, and microscopic identification of parasites in blood or lymph fluid samples, but these methods may not detect low-level infections or ongoing disease. Molecular tests, such as nested and quantitative PCR assays, offer higher sensitivity, yet they are often impractical for use in resource-limited settings where economic losses are most severe. Therefore, a field-deployable, point-of-care test would be highly valuable in managing and controlling ECF in endemic areas. In response to this need, we have developed a CRISPR-Cas12a-powered pen-side diagnostic tool for the sensitive and specific detection of T. parva, targeting the p104 gene. This tool combines a 20-minute recombinase polymerase amplification (RPA) reaction followed by a 60-minute CRISPR-Cas12a reaction with a FAM/Biotin lateral flow strip readout. We tested two RPA primer pairs and four CRISPR-RNAs (crRNAs), and the p104-based assay demonstrated high sensitivity, detecting as few as one infected lymphocyte per three microliters of blood. It was able to universally detect eight T. parva strains without cross-reactivity to other Theileria species, such as Theileria mutans and Theileria lestoquardi. The assay detected the pathogen as early as day three post-infection and showed a kappa coefficient score of 0.74 (good) when compared to the gold standard qPCR assay. Additionally, building on the development of the T. parva test, we created two more CRISPR-Cas12a assays for detecting Anaplasma marginale and Babesia bigemina, two other important tick-borne pathogens of cattle. These assays target the major surface protein 5 (MSP5) for A. marginale and the rhoptry-associated protein 1a (RAP1a) for B. bigemina. The results from these assays demonstrated high specificity, with no cross-reactivity against other tick-borne pathogens, and a limit of detection of 10² DNA copies/µL for each target marker. This work lays the foundation for the development of sensitive, user-friendly, field-applicable diagnostic tools for detecting T. parva, A. marginale, and B. bigemina infections.

Biography:

Dr. Svitek studied Microbiology and Immunology at the University of Montreal, Canada, and graduated with an MSc in 2004. He then joined Prof. von Messling's research group at the INRS-Centre Armand-Frappier Santé-Biotechnologie (University of Quebec/Institut Pasteur International Network), Canada. He received his PhD degree in Virology and Immunology in 2010 at that institution. After a one-year postdoctoral fellowship in Virology at the Duke-NUS Emerging Infectious Diseases Programme in Singapore, he joined Prof. Vish Nene’s group in 2011 at the International Livestock Research Institute (ILRI), Kenya, as a postdoctoral fellow in Cellular Immunology and Reverse Vaccinology. In 2014, he became a scientist and, in 2019, a senior scientist at that same institution. He has published more than 30 research articles in SCI(E) journals and contributed to more than 35 papers presented at international scientific conferences.