Flukicide effects in the juvenile liver fluke, <i>Fasciola hepatica </i>

This thesis exploits advances in the in vitro culture of juvenile liver fluke to characterise the response to triclabendazole (TCBZ), its active metabolite triclabendazole sulfoxide (TCBZ.SO), and rafoxanide. The results highlight differences in the responses of different fluke isolates and juveniles at different developmental stages. Drug susceptibility was found to be multi-factorial, with in vitro delineation of TCBZ-resistant (TCBZ-R) and susceptible (TCBZ-S) isolates exposing diversity in juvenile fluke motility, growth and survival phenotypic responses to drug treatments. The results also provided evidence that informed drug action, supporting a microtubule-inhibiting mode of action for TCBZ and TCBZ.SO and a metabolism-interfering mode of action for rafoxanide. It was also shown that TCBZ and the active metabolite inhibit the proliferation of neoblast-like cells in the TCBZ-S isolate irrespective of age, exposing neoblasts as a potential source of repair in TCBZ-R isolates, and as such, a potential target in fluke control. Subsequently, it was shown that the TCBZ-susceptibility status of juvenile fluke could be altered through the manipulation of neoblast-like cell proliferation and growth dynamics. Exploiting RNA-interference (RNAi) to silence Fasciola hepatica polo-like kinase 1 (FhPLK1) inhibited neoblast-like cell proliferation to significantly decrease growth and survival of the TCBZ-R isolate, transforming its in vitro phenotype to one that more closely resembled the TCBZ-S isolate. RNAi-induced silencing of neuropeptide F (FhNPF4) resulted in an enhanced growth phenotype and increased survival, transforming the phenotype of the TCBZ-S isolate to one that more closely resembled the TCBZ-R phenotype. Overall, this thesis has contributed to our understanding of the F. hepatica flukicide response, providing evidence in support of drug mode of action and the potential mechanism(s) of fluke resistance to identify novel targets for fluke control.Thesis is embargoed until 31 July 2027.<br/

Improved molecular typing of toxigenic <i>Clostridium difficile</i> strains affecting animal and human health

Clostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.<br/

Neoblast-like stem cells of Fasciola hepatica

The common liver fluke, Fasciola hepatica, causes the disease fasciolosis, which results in considerable losses within the global agri-food industry. There is a shortfall in the drugs that are effective against both the adult and juvenile life stages within the mammalian host, such that new drug targets are needed. Over the last decade the stem cells of parasitic flatworms have emerged as reservoirs of novel putative targets due to their roles in development and homeostasis, including of host-parasite interfaces. Here, we investigate and characterise the proliferating cells that underpin development in F. hepatica. We provide evidence that these cells are capable of self-renewal, are capable of differentiation, and are sensitive to ionising radiation - all attributes of neoblasts in other flatworms. Furthermore, we generated transcriptomes from worms following irradiation-based ablation of neoblasts, identifying 124 significantly downregulated transcripts, including known stem cell markers such as fgfrA and plk1. Sixty-eight of these had homologs associated with neoblast-like cells in S. mansoni. Finally, RNA interference mediated knockdown of histone h2b (a marker of proliferating cells), ablated neoblasts and impaired worm development in vitro. In summary, this work demonstrates that the proliferating cells of F. hepatica are equivalent to the neoblasts of other flatworm species and demonstrate that they may serve as attractive targets for novel anthelmintics.<br/