BackgroundRobust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community ‘gold standard’ for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification.Methodology/Principal findingsTransmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs.Conclusions/SignificanceThe data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies.

Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host–parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host–parasite interaction within the horse host.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross‐species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well‐recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth‐derived EVs in these processes and highlights EVs as an important participant in cross‐phylum communication. While the mammalian EV field is guided by a community‐agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan‐helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth‐derived EVs to complement the MISEV guidelines. We summarise community‐agreed standards for studying EVs derived from this broad set of non‐model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.