The equine tapeworm, Anoplocephala perfoliata, is the principal tapeworm species commonly infecting horses worldwide. Clusters of A. perfoliata are found primarily attached to the caecal wall, close to the ileocaecal valve. A light infection is often asymptomatic, whereas a severe infection from large clusters of A. perfoliata is likely to cause significant damage and dysfunction at the site of attachment and has been linked to colic. Given a status as a neglected helminth, many aspects of the fundamental biology, such as understanding of A. perfoliata infection and its interaction with the digestive physiology and health of its host, have yet to be thoroughly researched. In the current thesis, I have generated molecular tools using a polyomic approach, transcriptomics and proteomics, supported with bioinformatics to explore A. perfoliata. In doing so, this work has successfully generated the first de novo assembled transcriptome for adult A. perfoliata providing an in–depth insight into the parasites fundamental biology. Transcriptomic analysis revealed various key sequences likely expressed as proteins potentially involved in host–parasite interactions including glutathione transferases (both Sigma and Omega classes), Heat shock protein 90 alpha and alpha–Enolase. During infection, helminths secrete excretory–secretory products (ESPs), designated as the secretome, into the host environment. Of note are the extracellular vesicles (EVs) which are secreted as part of this secretome and are likely involved in cell–cell communication and host–parasite interactions. Given this, I first confirmed that EVs are secreted within A. perfoliata secretome during in vitro maintenance;these EVs were purified using size exclusion chromatography (SEC). Furthermore, I present the first analysis into the A. perfoliata secretome using GeLC (for whole EVs and EV depleted ESPs>10 kDa) and Gel free (for EV surface shaves) proteomic approaches supported by the newly developed A. perfoliata transcriptome. EV proteomic profiles resolved contained known EV biomarkers (i.e. annexins and tetraspanins) alongside putative immune modulators highlighting EVs secreted into the host environment as likely capable of interacting directly or indirectly with the host. I further investigated A. perfoliata EV interaction with mammalian host cells, including EV uptake (confocal and flow cytometry analyses), cell viability (trypan blue, MTT assays and flow cytometry) and cytokine secretion (Milliplex) after THP–1 cells were exposed to EVs. This study demonstrated that A. perfoliata EVs are taken up by mammalian cells and have immune modulatory activities and functions, primarily stimulating a Th1–driven immune response. The current thesis also includes a praziquantel (PZQ)–equine hindgut microbiome interaction study, examining the effect of PZQ on the equine gut microbiome in in vitro hindgut microbial fermentation model over 72 hours. Results demonstrated PZQ at any dosage seems unlikely to have widespread changes on the overall equine hindgut microbiome. However, PZQ at a higher than recommended dose may alter the fermentation pathways in the equine hindgut, which could impact on the nutritional function of the caecum. All discoveries from the current research have uncovered many aspects of A. perfoliata fundamental biology including the dataset of A. perfoliata transcriptome and proteomics of the secretome, key secretory proteins and potential immune modulators. In conjunction with the establishment of an interaction between EVs and mammalian immune cells, this thesis provides a basis for the further study into the host–parasite interaction. Furthermore, my investigation into the interaction between PZQ and the equine hindgut microbiome gives further insight into the impact of parasite treatment methods on the host hindgut microbiome and nutritional functioning. The comprehensive data generated from my research serve as a foundation for future studies, mainly aiming to advance my understanding of this infection. Furthermore, this knowledge holds the potential to drive the creation of efficacious strategies for further diagnosis, treatment, and prevention of A. perfoliata infections.
Horsing around with Anoplocephala perfoliata: Polyomic Investigation of the Host–Parasite Interface
Wititkornkul, B. (Author). 2023
Student thesis: Doctoral Thesis › Doctor of Philosophy