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.
Highlights
The generic ITS-2 Trem primers were successful in amplifying DNA extracted from whole adult rumen fluke (Calicophoron calicophorum, Calicophoron microbothrium, Cotylophoron cotylophorum, C. daubneyi, Fischeroedius elongatus, Gastrothylax crumenifer, Orthocoelium streptocoelium, P. cervi, and P. leydeni); pools of juvenile rumen fluke (C. daubneyi); pools of rumen fluke eggs (C. daubneyi, Calicophoron microbothrioides, and P. leydeni); adult liver fluke (F. hepatica) and lancet fluke (D. dendriticum) and from crude liver fluke lysates (F. gigantica)
DNA extracts from paraffin wax-embedded samples and those stored in formalin for over a year did not yield sufficient quality polymerase chain reaction (PCR) product for sequencing when using the generic ITS-2Trem primers
Accurate identification of rumen fluke species is important for understanding of disease epidemiology and control options
Summary
Paramphistomes, are parasitic trematodes that reside in the digestive tracts of ruminants, including livestock such as cattle [1, 2], sheep [3, 4], goats [5, 6], and farmed deer [7]; camelids [8] and wildlife, such as antelope [9,10,11], buffalo [12], and various species of wild deer [12, 13].The first published information on rumen fluke emerged in 1790, describing adult fluke within the rumen of red deer in Europe [14]. Molecular Identification of Rumen Fluke agreement or consistency around nomenclature in the literature, with the names rumen fluke and stomach fluke or paramphistome and amphistome largely used interchangeably [15]. They mainly belong to six different families: Paramphistomidae, Gastrodiscidae, Gastrothylacidae, Olveriidae, Balanorchiidae, and Stephanopharyngidae; all in the Superfamily Paramphistomoidea, Stiles and Goldberger, 1910 [16]; under which, more than 70 species have been described [17]. Some species are found across multiple host species in geographically distant areas, e.g., Paramphistomum leydeni has been found in goats in China [6], cattle in the Netherlands [23] and Argentina [3], sheep in Ireland [24], and reindeer in Finland [25]
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