Researchers have developed a new toolkit for detecting trypanosome parasites in livestock, aiding efforts to monitor and control the disease.
The research, published today as a Reviewed Preprint in eLife, is described by editors as an important study advancing the diagnosis of animal African trypanosomiasis (AAT), also known as Nagana, which adapts a CRISPR-based diagnostic tool (SHERLOCK4AAT) to detect different trypanosome species responsible for AAT. The evidence supporting the conclusions is deemed convincing and in line with the current state-of-the-art diagnostics. The study will be of interest to the fields of epidemiology, public health, and veterinary medicine.
62.7% of free-ranging and farm pigs across Côte d’Ivoire and Guinea were found to be infected with at least one species of trypanosome. Image credit: Sandy Millar via Unsplash
AAT, caused by parasites of the genus Trypanosoma, puts over a million livestock at risk across 37 countries in Africa, causing considerable economic impact. While elimination of human African trypanosomiasis (HAT) – also known as sleeping sickness – is within reach across the continent, AAT remains a significant burden, with an estimated annual loss of $4.75bn (USD) in agricultural GDP in endemic regions. Moreover, these domestic animals provide potential reservoirs for human-infectious trypanosomes, making it essential to map and monitor disease transmission.
“Active and accurate surveillance of the infection status of domestic animals using reliable diagnostic tools will be key to reaching and sustaining the World Health Organization’s goal of eliminating HAT. However, current methods for diagnosing AAT are limited by a lack of sensitivity and reliability,” says co-lead author Roger-Junior Eloiflin, Research Assistant at INTERTRYP, University of Montpellier, CIRAD/IRD, France. Eloiflin served as co-lead author of the study alongside Elena Pérez-Antón, Research Scientist in the Trypanosome Molecular Biology Unit, Institut Pasteur, University of Paris, INSERM, France, and Aïssata Camara, a researcher in the Parasitology Unit, Institut Pasteur of Guinea, Conakry, Guinea.
Among the current methods, molecular diagnostic methods stand out as an exception and offer potentially increased sensitivity by detecting specific regions of the parasites’ nucleic acids. To capitalise on this capability, the team adapted a detection method called Specific High-Sensitivity Enzymatic Reporter unLOCKing (SHERLOCK), that has previously shown success in detecting HAT, to develop a range of assays suitable for detecting animal trypanosomes – called SHERLOCK4AAT, from a single dry blood spot.
They started by identifying DNA targets within highly conserved and species-specific regions of trypanosomes of veterinary importance. The tests were designed using the sequences for 18S ribosomal RNA and GAPDH genes that are highly conserved across species, but that also contain species-specific regions to allow for species discrimination.
For the pan-trypanosome test, the team initially chose a single target previously shown to have a specificity of 93% for detecting HAT in blood. Although this target performed well, they found the sensitivity increased when they added in a second guide RNA to create a so-called multiplexed assay.
For the species-specific tests, although unable to find target genes to differentiate between all species, they found that SHERLOCK4AAT could discriminate between closely related AAT-causing species with a limit of detection between 10 and 1,000 parasites per ml, making them comparable with other existing molecular tests. The one exception was the species-specific test for Trypanosoma vivax, which – based on a single copy of the IFX gene – performed less well, suggesting a different gene target would be required.
Having established the sensitivity of the tests in the lab, the team applied the SHERLOCK4AAT toolbox to profiling trypanosome species in free-ranging and farm pigs across Côte d’Ivoire and Guinea. Overall, 62.7% of pigs were found to be infected with at least one species of trypanosome. T. brucei gambiense – a human-infectious trypanosome – was found in one animal at both sites, suggesting that these animals may act as reservoirs of infection.
“Overall, our results are consistent with the few available studies on the prevalence of trypanosome parasites in domestic pigs, confirming the effectiveness of the SHERLOCK4AAT toolbox in analysing the distribution of trypanosomes in areas where HAT remains at a low level ” concludes co-corresponding author Brice Rotureau, Research Director at the Institut Pasteur, University of Paris, INSERM, and the Parasitology Unit, Institut Pasteur of Guinea. “Because of their proximity to humans and their easy access for frequent sampling, pigs could be used as sentinels to monitor the circulation of human-infectious trypanosomes using the SHERLOCK4AAT toolbox.”
Brice Rotureau served as co-corresponding author of the study alongside Lucy Glover, a group leader in the Trypanosome Molecular Biology Unit, Institut Pasteur, University of Paris, INSERM.
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