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

Abstract

Background: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type 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-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major.

Results: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the 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 the four different classes of insects ranged from 40.6 up to 1.1 × 10⁸ cell equivalents per host.

Conclusions: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.

Keywords: Crithidia; Diagnostic; Epidemiology; Honeybee; Leishmania; Lotmaria; Prevalence.

Fig. 1

Trypanosomatid α-tubulin amplification in multiple insect hosts. a Illustration of the locus of trypanosomatid alpha/beta (α/β) tubulin tandem repeated arrays. As highlighted in dark blue, the 193-bp amplicon is located at the 3´end of each α-tubulin repeat, being the number of repeats variable across trypanosomatid species. b Clustal analysis of the 3′ end of the α-tubulin open reading frame showing conserved blocks of homology (in blue) that were used for designing primers and TaqMan probes. c Representative 2% agarose gel electrophoresis of the 193 α-tubulin trypanosomatid region amplified in samples of bumblebees, wild bees and grasshoppers. Numbers refer to different samples. The negative control (C-) and the positive control (C+) show negative and positive samples, respectively. F, Forward; FAM, dye label; M, molecular size marker; MGB, minor groove binder; R, reverse

Fig. 2

Trypanosomatid yields in the insect samples analyzed with the α-tubulin TaqMan assay. Data are presented as a violin plot showing the density of cell equivalents per insect. The red circles represent 8 infected samples of the western honey bee Apis mellifera, the yellow squares represent 4 infected samples of the buff-tailed bumblebee Bombus terrestris, the blue cross represents an infected sample of Halictus spp. and the blue triangle represents an infected white-banded digger bee Amegilla quadrifasciata. The blue dotted line represents 1 × 10⁴ cell equivalents per insect [Log4(10)] and 1 × 10⁶ cell equivalents per insect [Log6(10)]