A Universal Approach to Molecular Identification of Rumen Fluke Species Across Hosts, Continents, and Sample Types

Abstract

Rumen fluke are parasitic trematodes that affect domestic and wild ruminants across a wide range of countries and habitats. There are 6 major genera of rumen fluke and over 70 recognized species. Accurate species identification is important to investigate the epidemiology, pathophysiology and economic impact of rumen fluke species but paramphistomes are morphologically plastic, which has resulted in numerous instances of misclassification. Here, we present a universal approach to molecular identification of rumen fluke species, including different life-cycle stages (eggs, juvenile and mature fluke) and sample preservation methods (fresh, ethanol- or formalin-fixed, and paraffin wax-embedded). Among 387 specimens from 173 animals belonging to 10 host species and originating from 14 countries on 5 continents, 10 rumen fluke species were identified based on ITS-2 intergenic spacer sequencing, including members of the genera Calicophoron, Cotylophoron, Fischeroedius, Gastrothylax, Orthocoelium, and Paramphistomum. Pairwise comparison of ITS-2 sequences from this study and GenBank showed >98.5% homology for 80% of intra-species comparisons and <98.5% homology for 97% of inter-species comparisons, suggesting that some sequence data may have been entered into public repositories with incorrect species attribution based on morphological analysis. We propose that ITS-2 sequencing could be used as a universal tool for rumen fluke identification across host and parasite species from diverse technical and geographical origins and form the basis of an international reference database for accurate species identification.

The winner takes it all: dominance ofCalicophoron daubneyi(Digenea: Paramphistomidae) among flukes in Central European beef cattle

“…Trematodes of the family Paramphistomidae, known under the common name rumen fluke, are small pink pear-shaped concave amphistome flukes with two suckers, that infect a broad range of mammalian hosts including cattle, zebu, water buffalo, sheep, goats, deer and also llama and alpaca, and might have a significant impact on livestock productivity (Eduardo, 1983; Deplazes et al ., 2016; Mitchell et al ., 2021). Even though adult rumen flukes are usually well tolerated (Taylor et al ., 2007), heavy infestation with the immature flukes can result in illness associated with the intestinal phase of the infection, usually termed acute larval paramphistomosis.…”

“…Paramphistomids have a complex life cycle involving aquatic or mud snails as intermediate hosts producing cercariae (Tilling, 2013; Horák et al ., 2019; Mitchell et al ., 2021). Ruminants become infected by ingestion of metacercariae attached to grass while grazing or the floating metacercariae may be swallowed while drinking (Rondelaud et al ., 2004).…”

“…However, DNA sequences are available only from a few countries (green). In the UK and Netherlands, the parasite's identity was confirmed by DNA (Ploeger et al ., 2017; Mitchell et al ., 2021); however, the sequences are not available. The map was created based on sequences published in GenBank and following literature: Dinnik (1962), Sey (1974, 1978), Sey and Vishnyakov (1976), Sey and Arru (1977), Graber et al .…”

“…Identification of paramphistomid flukes by traditional morphology and morphometry is quite complicated and a thorough examination of histological sections is required (Eduardo, 1982). On the contrary, DNA barcoding of the internal transcribed spacer 2 (ITS2) region represents a tool allowing identification to the species level also in the case of eggs in fecal samples as the source of the DNA (Mitchell et al ., 2021). In this study, we aimed to investigate the prevalence and diversity of paramphistomid flukes in beef cattle herds across Czech Republic.…”

DNA barcoding of rumen flukes (Paramphistomidae) from bovines in Germany and Austria

“…Thus, species identity and taxonomy of the paramphistomes encountered in the infection of ruminants, intermediate Section Editor: Georg von Samson-Himmelstjerna host specificity and life cycle, have been a matter of intense debate in the 1970s and early 1980s and resulted in several instances in confusion, misidentification, and generation of synonyms (Odening and Gräfner 1979;Sey 1980Sey , 1982Odening 1983). However, work over the past 15 years has shown that these problems can be addressed using molecular identification methods (Mitchell et al 2021).…”