Polyomic tools for an emerging livestock parasite, the rumen fluke Calicophoron daubneyi; identifying shifts in rumen functionality

Abstract

Background: Diseases caused by parasitic flatworms of rumen tissues (paramphistomosis) are a significant threat to global food security as a cause of morbidity and mortality in ruminant livestock in subtropical and tropical climates. Calicophoron daubneyi is currently the only paramphistome species commonly infecting ruminant livestock in temperate European climates. However, recorded incidences of C. daubneyi infection in European livestock have been increasing over the last decade. Whilst clinical paramphistomosis caused by adult worms has not been confirmed in Europe, fatalities have been attributed to severe haemorrhagic enteritis of the small intestine resulting from the migration of immature paramphistomes. Large numbers of mature adults can reside in the rumen, yet to date, the impact on rumen fermentation, and consequently on productivity and economic management of infected livestock, have not been resolved. Limited publicly available nucleotide and protein sequences for C. daubneyi underpin this lack of biological and economic understanding. Here we present for the first time a de novo assembled transcriptome, with functional annotations, for adult C. daubneyi, which provides a reference database for protein and nucleotide sequence identification to facilitate fundamental biology, anthelmintic, vaccine and diagnostics discoveries.

Results: This dataset identifies a number of genes potentially unique to C. daubneyi and, by comparison to an existing transcriptome for the related Paramphistomum cervi, identifies novel genes which may be unique to the paramphistome group of platyhelminthes. Additionally, we present the first coverage of the excretory/secretory and soluble somatic proteome profiles for adult C. daubneyi and identify the release of extracellular vesicles from adult C. daubneyi parasites during in vitro, ex-host culture. Finally, we have performed the first analysis of rumen fluke impacting upon rumen fermentation parameters using an in vitro gas production study resulting in a significant increase in propionate production.

Conclusions: The resulting data provide a discovery platform (transcriptome, proteomes, EV isolation pipeline and in vitro fermentation system) to further study C. daubneyi-host interaction. In addition, the acetate: propionate ratio has been demonstrated to decrease with rumen fluke infection suggesting that acidotic conditions in the rumen may occur.

Keywords: Calicophoron daubneyi; In vitro; Paramphistome; Proteomics; Rumen fermentation; Transcriptome.

Fig. 1

Annotation of mapped C. daubneyi sequences to Gene Ontology (GO) terms at GO level 3. Numbers of sequences assigned to each term are shown in parentheses with a total of 17,149 sequences returned at least 1 (range 1–15) GO term annotation. a Biological process. b Molecular function. c Cellular component

Fig. 2

Representative 2DE protein array of adult C. daubneyi excretory-secretory (ES) products. 17 cm 2DE protein array of excretory-secretory (ES) products from in vitro culture, annotated to highlight the 50 most abundant spots identified during Progenesis analysis. Proteins were separated across a non-linear pH range of 3–10 using IEF in the first dimension and 14% SDS-PAGE in the second dimension and Coomassie Blue stained. ES products were obtained from 6 h in vitro culture in supplemented DME medium. Numbered protein spots correspond to the order of relative abundance detected by Progenesis analysis and show the spots excised for MS identification

Fig. 3

Representative 2DE protein array of adult C. daubneyi soluble somatic proteins. 17 cm 2DE protein array of soluble somatic proteins annotated to highlight the 50 most abundant spots identified during Progenesis analysis. Proteins were separated across a linear pH range of 3–10 using IEF in the first dimension and 14% SDS-PAGE in the second dimension and Coomassie Blue stained. Numbered protein spots correspond to the order of relative abundance detected by Progenesis analysis and show the spots excised for MS identification

Fig. 4

Extracellular (including exosome-like) vesicles (EVs) isolated from adult C. daubneyi excretory/secretory (ES). ES products were produced during in vitro culture and EVs identified using transmission electron microscopy. Preliminary vesicle characterisation (exosome-like and apoptotic bodies) are based on approximate size only

Fig. 5

KEGG pathway map for propionate metabolism. EC codes identified in C. daubneyi transcript data based on those known in the helminth species C. elegans and S. mansoni available in the KEGG organism database are highlighted in red