Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach

Abstract

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform–based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites’ ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.Electronic supplementary materialThe online version of this article (10.1007/s00436-019-06414-7) contains supplementary material, which is available to authorized users.

Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus

“…Therefore, we believe this inconsistency between the copy numbers for two amplicons to be a direct consequence of the reference amplicon being situated in a more conserved exonic region, whereas the “sensitive” allele amplicon was in an inherently more variable intron. Nevertheless, unlike in any of our previous approaches for the detection of SNPs ( Baltrušis et al, 2018 ) or genetically variable regions for species differentiation ( Baltrušis et al, 2019 ), this study employs a more robust design, utilizing the simultaneous absolute quantification of two distant regions within the hco-acr-8 for the indirect determination of the frequency of the “resistant” allele.…”

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

“…The whole process of species differentiation using morphological characteristics is time-consuming and laborious, requires an experienced helminthologist, and is not always reliable. Therefore, molecular confirmation [28][29][30] . Until now, multiplex PCR techniques for the detection of A. sidemi and H. contortus tended to target the nuclear ribosomal region and mitochondrial NADH dehydrogenase subunit 4 (ND4) 40,41 .…”

“…Therefore, molecular confirmation is appropriate if not downright necessary. For this reason, GIN diagnostics based on molecular methods is fast becoming a preferred method, because it allows for a rapid , safe , and sensitive species identification 28 – 30 .…”

“…Taxonomic identification based on morphological/morphometric features is possible, especially in adult male specimens, but in adult females and immature nematodes, these methods are unreliable and in the larval stages and eggs they are not applicable at all. Moreover, because morphological examination is time-consuming and requires a helminthologist of considerable experience, we witness a growing use of molecular tools for species identification and demand for novel approaches 28 – 30 .…”

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples

“…Droplet digital PCR (ddPCR) is a versatile and robust platform designed for selective amplification and probe-driven detection of short (typically 50–250 bp long) amplicons. We have previously successfully implemented ddPCR to quantify the presence of strongyle nematodes in larval cultures, derived from livestock samples, in order to distinguish between the major parasite genera with high precision [ 16 , 17 ]. Furthermore, assays, based on ddPCR have been found to exhibit superior sensitivity [ 18 ] as well as increased tolerance to inhibitors in comparison to more conventional PCR approaches [ 19 ].…”