The use of high resolution melting analysis of ITS-1 for rapid differentiation of parasitic nematodes Haemonchus contortus and Ashworthius sidemi

Abstract

Among gastrointestinal nematodes, haematophagous strongylids Haemonchus contortus and Ashworthius sidemi belong to the most pathogenic parasites of both domestic and wild ruminants. Correct identification of parasitic taxa is of crucial importance in many areas of parasite research, including monitoring of occurrence, epidemiological studies, or testing of effectiveness of therapy. In this study, we identified H. contortus and A. sidemi in a broad range of ruminant hosts that occur in the Czech Republic using morphological/morphometric and molecular approaches. As an advanced molecular method, we employed qPCR followed by High Resolution Melting analysis, specifically targeting the internal transcribed spacer 1 (ITS-1) sequence to distinguish the two nematode species. We demonstrate that High Resolution Melting curves allow for taxonomic affiliation, making it a convenient, rapid, and reliable identification tool.

The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

“…Ashworthius detection was included in the presented multiplex assay for the following reasons. This hematophagous abomasal nematode is phylogenetically related and morphologically/morphometrically almost indistinguishable in immature stages from H. contortus , and poses a threat of becoming one of the most widespread pathogenic GINs of autochthonous European ruminants [ 29 ]. This invasive parasite, originally endemic in Asian deer, was probably introduced to Europe by sika deer in the late nineteenth and early twentieth century, and since then it has successfully spread among new hosts (such as red deer, roe deer, fallow deer, or moose) and is highly pathogenic in some, such as European bison [ 38 ].…”

“…The gDNA of adult nematodes, which served as reference template DNA during the optimization experiments, was extracted from a single individual of each given nematode species using previously published protocols [ 28 , 29 ] and based on overnight incubation at 55 °C in 50 μl of extraction buffer (100 mM Tris–HCl, 10 mM EDTA, 100 mM NaCl, 1% SDS, 1.5 mM dithiothreitol) containing 0.06 mg proteinase K, followed by alcohol precipitation. Extracted gDNA was stored at −20 °C until processed.…”

The Evaluation of Gastrointestinal Nematodes in Faecal Samples Using Multiplex Real-Time PCR Assays

“…Ashworthius detection was included in the presented multiplex assay for the following reasons. This hematophagous abomasal nematode is phylogenetically related and morphologically/morphometrically almost indistinguishable in immature stages from H. contortus, and poses a threat to become one of the most widespread pathogenic GINs of autochthonous European ruminants [29]. This invasive parasite originally endemic in Asiatic deer was probably introduced to Europe by sika deer in the late 19th and early 20th century and since then it has successfully spread among new hosts (such as red deer, roe deer, fallow deer, or moose) and is highly pathogenic in some e.g.…”

“…DNA extraction. The gDNA of adult nematodes, which served as reference template DNA during the optimization experiments, was extracted from a single individual of each given nematode species using previously published protocols [28,29] and based on overnight incubation at 55 o C in 50 µl of extraction buffer (100 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 1% SDS, 1.5 mM dithiothreitol) containing 0.06 mg proteinase K, followed by alcohol precipitation. Concentration, yield, and purity of extracted gDNA were measured by NanoDrop 8000 Spectrophotometer (Thermo Fisher Scienti c).…”

Soil-Borne Nematodes: Impact in Agriculture and Livestock and Sustainable Strategies of Prevention and Control with Special Reference to the Use of Nematode Natural Enemies

“…The use of traditional specialised taxonomic identification keys is an invaluable tool for morphological identification of adult nematodes at necropsy [77][78][79] and L3 from faecal cultures [79,80], for epidemiological studies, for research proposes, and for establishing control strategies. However, advanced molecular methods, e.g., qPCR followed by a high resolution melting analysis of ITS-1, open other convenient, rapid, and reliable alternative methods for taxonomic affiliation [81]. Similarly, new molecular tools, e.g., DNA metabarcoding using only faecal samples, have been claimed to provide a non-invasive method for assessing parasitic nematode populations [82].…”