Development of a Taq-Man qPCR assay for trypanosomatid multi-species detection and quantification in insects

Abstract

Abstract Background: Trypanosomatid parasites are widely distributed in nature, evolving monoxenous and dixenous cycles. These parasites thrive in a wide number of Insect Orders, some of them with an important economic and environmental value, such as bees. The objective of this work was to develop a robust and sensitive qPCR assay for detecting trypanosomatid parasites in any kind of parasitized insect sample. Methods: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity, and versatility of the α-tub TaqMan assay have been tested and validated using field samples from honeybee workers, wild bees, bumblebees, and grasshoppers as well as in the human infective trypanosomatid Leishmania major. Results: This assay showed a detection limit of 1 parasite equivalent/µL and successfully detected trypanosomatids in 10 different hosts belonging to the Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from 4 apiaries (n= 224 worker honeybees) located in Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra colony prevalence of 0 to 13%. Parasite loads in 4 different classes of insects ranged from 40.6 to up to 1.1 x 108 cell equivalents per host. Conclusions: These results showed that α-tubulin TaqMan qPCR assay is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatid parasites in a wide range of environmental settings.