High-throughput identification and quantification of Haemonchus contortus in fecal samples

Abstract

Haemonchus contortus are gastrointestinal nematodes of the family Trichostrongylidae that naturally infect small ruminants while grazing, posing a risk to both animal health and farm profitability. Current diagnostics depend on exacting lab techniques, including manual egg counts and larval differentiation, all of which require time, effort, and specialized technicians. The goal of this study was to facilitate and accelerate the identification and quantification of H. contortus in fecal samples through the use of fluorescein-isothiocyanate peanut-agglutinin staining in order to allow automated detection using a 96-well microplate reader. Next, the model was to be validated using samples containing unknown quantities of eggs. Automated analysis of fluorescence emission of known quantities of H. contortus eggs confirmed an almost perfect linear correlation (r = 0.9984, p < 0.0001), indicating that this new approach can satisfactorily be used to quantify H. contortus eggs on a comparative fluorescence scale. As validation, clinical samples containing an unknown quantity of H. contortus eggs were then analyzed by comparing two methods: either Wisconsin Sugar Flotation (WSF) and McMaster counting followed by manual fluorescence microscopy, or WSF coupled with automated microplate reading. Pearson analysis revealed highly significant correlation between manual and automated methods (r = 0.9999, p < 0.0001), while Bland-Altman plots demonstrated excellent agreement between the two (bias = -0.817 ± 9.94 with 95% limits of agreement from -20.31 to 18.67). Overall, these results demonstrate that high-throughput screening fluorescence detection and quantification of H. contortus eggs is both accurate and rapid.

Keywords: Automated fluorescence detection/quantification; Diagnostic methods; Fluorescence microscopy; HTS; Haemonchus contortus; Sheep.