Use this URL to cite or link to this record in EThOS:
Title: Development of a pen-side diagnostic test for liver fluke infections in cattle and sheep
Author: Walsh, T. R.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
Availability of Full Text:
Access from EThOS:
Access from Institution:
Fasciola hepatica is a common trematode parasite of cattle and sheep worldwide. Undiagnosed infections can lead to significant production losses in ruminant livestock, or in worst cases, sudden death from extensive damage to the liver. Over recent years, prevalence of fluke has increased within the UK. This in turn has led to an increased reliance on flukicide drugs for control. Resistant fluke populations have now been reported in many areas. Diagnostic tests are crucial for control of disease. Current diagnostics include faecal egg counts, antibody ELISAs using serum or milk and copro-antigen ELISAs. These tests each have their own limitations, but for all, samples must be sent to laboratories for testing, adding time and costs to the diagnosis. A pen-side diagnostic test would be invaluable for farmers, allowing them to diagnose infections on farm and target individual treatments, avoiding the use of herd blanket treatments which can contribute to spread of resistance. In this thesis, we aim to produce a prototype penside diagnostic test. In Chapter 2, we produced a recombinant CL1 (rCL1) in the yeast Pichia pastoris intended for use in the pen-side test. Using Western blotting we showed that host antibodies from experimentally infected cattle and sheep specifically recognised fluke cathepsin enzymes, L1 (CL1) and L2. In Chapter 3, we compared the rCL1 antigen against native fluke excretory-secretory (ES) products for its ability to detect host antibodies by ELISA in naturally and experimentally infected cattle and sheep. Serum antibodies from experimentally infected animals showed strong recognition of rCL1, whilst recognition by naturally infected animals was inconsistent. This was confirmed by peptide array. In Chapter 4, we investigated the possibility that the poor antibody recognition by naturally infected animals could be due to variation in the rCL1 protein sequence compared to native CL1. We sequenced the CL1 gene from seven parasites and showed that there was variation within the CL1 gene which translated to differences in the amino acid sequence. This variation did not explain the poor host antibody recognition of our rCL1 by naturally infected animals as these changes were outside of the immunogenic areas identified in Chapter 3. The observed variation also did not result in any conformational changes to the rCL1 as determined by modelling the 3D structure of the protein. In Chapter 5 we used 2D Western blotting to show that host antibodies from naturally infected animals recognised multiple antigens within fluke ES products, and these antigens were identified through mass spectrometry. The results suggested that further considerations would be needed for preparation of the pen-side test before it could be made a commercial test. Overall, these results suggest there are differences in the host antibody response to fluke ES products between naturally and experimentally infected cattle and sheep. Whilst there is still much validation needed before the pen-side test can be made commercially available, this test has the potential for diagnosis of fluke infections on farm.
Supervisor: Williams, Diana ; Hodgkinson, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral