Development of a pen-side diagnostic test for Fasciola hepatica
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Infection and Global Health
Abstract
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People |
ORCID iD |
| Diana Williams (Principal Investigator) |
Publications
Beesley NJ
(2018)
Fasciola and fasciolosis in ruminants in Europe: Identifying research needs.
in Transboundary and emerging diseases
Graham-Brown J
(2019)
Composite Fasciola hepatica faecal egg sedimentation test for cattle.
in The Veterinary record
Walsh TR
(2021)
Differences in the antibody response to adult Fasciola hepatica excretory/secretory products in experimentally and naturally infected cattle and sheep.
in Veterinary parasitology
Williams D
(2020)
Update on liver fluke in sheep
in In Practice
| Description | The project had two objectives: Establish that a pen-side diagnostic test (the liver fluke lateral flow test) for the parasite, Fasciola hepatica (liver fluke) will work with whole blood Establish that the test does not cross react with a newly introduced trematode, Calicophoron daubneyi (rumen fluke) Lateral flow (LF) antigen was made and sent down to collaborator, Mologic Ltd, Bedfordshire for construction of a new batch of LF kits. Two dimensional gel electrophoresis, Western blotting and dot blots showed that this new batch of antigen was consistent with the previous batch. Prototype kits were redesigned to include a two port format, batches of 100 kits were made and sent to Liverpool. Objective 1: Whole blood was collected from cattle and sheep housed at the University farm and known to be free from liver fluke infection. Blood was collected and added immediately to the LF kits on farm. Serum was also collected and run on the LF kits for comparison. As whole blood contains blood cells and has a higher viscosity, it moves more slowly through the LF test than serum, increasing the chance of non-specific binding (NSB) to the test line. Bovine LF kits performed well using whole blood and there was no binding to the test line using blood from uninfected cows. Whole blood produced higher cube reader units (CRU) compared to serum but were still below the arbitrary cut-off of 20 CRU. This is likely to be due to blood staining the test strip red. The sheep blood gave some non-specific background binding around the test line on this batch of kits. We are currently exploring the reasons for this, it appears to be specific to this particular batch of kits. Production methods are being investigated. Whole blood was also collected from 14 cows from two farms (Farm 1, n = 10; Farm 2, n = 4); each farm has a history of liver fluke infection. All 14 cows tested negative in the LF test with cube reader results below the arbitrary cut off of 20 units. Serum and faecal samples were collected from the same animals, both the serum antibody detection ELISA and liver fluke faecal egg counts were negative for liver fluke but four cows were positive for rumen fluke eggs. This suggests there was no cross reaction between liver fluke and rumen fluke although the number tested was small. The cattle on this farm had tested positive for fluke earlier in the year and had been treated three months before collection of the whole blood samples. None of the cattle tested for this project were fluke infected. This is likely to be because 2018 was an unusually dry year and samples were collected in the summer, a low risk season for fluke. For this reason we spiked negative whole blood with the high positive control serum used in the ELISA. Two µl of serum was mixed with 2µl of blood and tested on the LF kit. A weak band was observed, the CRU increased from 6.9 to 10.6. This was below the arbitrary cut off of 20CRU, but adding larger volumes of serum to the blood affected the sample viscosity so was not a true test of the kit. Objective 2: Serum samples were obtained from calves experimentally infected with Cooperia oncophora (University of Ghent), and Ostertagia ostertagi (Moredun Research Institute), and animals at abattoir with confirmed rumen fluke and/or liver fluke infections (University of Liverpool and University of Edinburgh, The Roslin Institute). All samples were tested using our standard diagnostic antibody detection ELISA, the readout for this test is the percent positivity compared to a positive control serum (PP). The diagnostic cut off value for the bovine ELISA is 15PP and for the sheep ELISA is 23PP. Calves experimentally infected with C. oncophora (n=6) and O. ostertagi (n=8) all had negative PP values by ELISA (mean values of 3.36 and 4.23). Serum was collected from cattle at slaughter. Each animal was examined for the presence of liver fluke/lesions and rumen fluke. The serum was tested for antibodies to liver fluke in the antibody detection ELISA. The proportion of samples negative for liver fluke on post mortem which would be classed as positive (> 15PP) on ELISA does not significantly differ between those samples negative and positive for rumen fluke (estimate and exact binomial confidence interval 0.10 (0.01- 0.30) versus 0.20 (0.09 - 0.34)). Similarly, the proportion of samples positive for liver fluke on post mortem which would be classed as positive (> 15PP) on ELISA does not significantly differ between those samples negative and positive for rumen fluke (estimate and exact binomial confidence interval 0.68 (0.52 - 0.82) versus 0.82 (0.66- 0.93) ). This, coupled with graphical inspection of the results, provides preliminary evidence that there is no cross reaction between antibodies to rumen fluke and liver fluke ES antigens. The data are confounded by the fact the samples were collected from naturally infected animals and it was not possible to confirm that the animals had not recently been exposed to each parasite. Both parasites are transmitted by Galba truncatula and frequently seen as co-infections. Serum from sheep pre and post experimental infection with T. circumcinta (Moredun Research Institute) showed no evidence of cross reaction in the ES ELISA These samples were run on the LF kit and were negative. One hundred and sixty three serum samples were collected from sheep at slaughter from local abattoirs from which liver and rumen fluke infection status was known. Twenty seven samples were collected from sheep from a farm which had a history of rumen and liver fluke infections. Infection status of each sheep was confirmed by faecal egg counts. All samples were tested by ES ELISA. Overall, the data suggested that there is no evidence of cross reaction between F. hepatica, C. daubneyi and other common gastro-intestinal nematodes of sheep. In conclusion the two objectives were met, we showed that whole blood can be used directly in the lateral flow kits and that there is no evidence of cross reaction between antibodies raised to other common helminth parasites, including Calicophoron daubneyi, the rumen fluke of sheep and cattle, and the ES antigen of F. hepatica used as the basis of the lateral flow kit. |
| Exploitation Route | An application to BBSRC Follow on Fund was successful and is now active. |
| Sectors | Agriculture, Food and Drink |
| Description | We have presented results on the development of a penside diagnostic test to a range of farmer organisations including Beef Expo June 2018, AHDB Beef and Lamb webinar and to farmers' groups. We have had significant interest in the penside test from across the industry. |
| First Year Of Impact | 2018 |
| Sector | Agriculture, Food and Drink |
| Impact Types | Economic |
| Description | EU Discontools |
| Geographic Reach | Europe |
| Policy Influence Type | Membership of a guideline committee |
| Description | STAR IDAZ |
| Geographic Reach | Multiple continents/international |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Description | Application of a lateral flow diagnostic test for Fasciola hepatica that can be used on-farm in sheep and cattle |
| Amount | £33,438 (GBP) |
| Funding ID | 91110114 |
| Organisation | Agricultural and Horticulture Development Board |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 09/2021 |
| End | 02/2022 |
| Description | Development of a penside diagnostic test for Fasciola hepatica |
| Amount | £201,773 (GBP) |
| Funding ID | BB/T016981/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 07/2020 |
| End | 09/2021 |
| Description | Collaboration with Mologic |
| Organisation | Mologic |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | collaboration over development of lateral flow test for diagnosing fluke infection in sheep and cattle |
| Collaborator Contribution | Mologic brought their expertise in lateral flow technology |
| Impact | BBSRC pathfinder follow on fund application and award |
| Start Year | 2017 |
| Description | STAR-IDAZ |
| Organisation | European Union |
| Country | European Union (EU) |
| Sector | Public |
| PI Contribution | Collaborated in designing the research needs roadmap for veterinary helminth diseases of livestock for the EU |
| Collaborator Contribution | STAR-IDAZ was charged by EU country states to develop research needs roadmaps for global animal diseases. I led the diagnostics for liver fluke section of the veterinary helminth diseases roadmap and contributed to the overall roadmap. The results will be presented to the DISCONTOOLS meeting in Brussels in April 2020. |
| Impact | DOI: 10.1111/tbed12682. |
| Start Year | 2020 |
| Description | The Farmer Network |
| Organisation | Farmer Network Cumbria |
| Country | United Kingdom |
| Sector | Charity/Non Profit |
| PI Contribution | Working with TFN to validate our lateral flow diagnostic test for liver fluke in sheep and cattle |
| Collaborator Contribution | partnering with us to gather, collect blood and faecal samples from sheep and cattle. These samples then tested using the prototype lateral flow test and compared to standard laboratory tests. |
| Impact | partnership on validating lateral flow test in collaboration with AHDB funded by BBSRC |
| Start Year | 2012 |
| Description | AHDB WEBINAR |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | Webinar for sheep and cattle farmers on improving sustainable and resilient fluke control |
| Year(s) Of Engagement Activity | 2020 |
| Description | COWS technical chapters updated |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | I rewrote the Fasciola hepatica technical chapters for the Control of Parasites Sustainably [https://www.cattleparasites.org.uk/] for the farming industry |
| Year(s) Of Engagement Activity | 2019,2020 |
| URL | https://www.cattleparasites.org.uk/ |
| Description | Penside diagnostic lateral flow test for liver fluke in sheep and cattle |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | summary of progress with the lateral flow test for farmers, vets and SQP/RAMAs. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Presentation to the Research and Development Board of Hybu Cig Cymru |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presented results of the BBSRC IPA research grant to the HCC board - they part funded the project |
| Year(s) Of Engagement Activity | 2020 |
| Description | The Farmer Network webinar |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Industry/Business |
| Results and Impact | Evening discussion and presentation for sheep farmers on improved diagnosis and control of liver fluke. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Webinar on fluke control at housing for Boehringer Ingelheim |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | webinar on best practice around fluke control at housing |
| Year(s) Of Engagement Activity | 2021 |
| Description | five regional farmer and vet workshops |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | five regional workshops organised by AHDB to present findings from BBSRC IPA award: Improving control of liver fluke in cattle in the UK. Attended by over 50 farmers and vets at each meeting. Day long workshops with presentation and then farm walk to assess fluke habitat on farm and consider control programmes. |
| Year(s) Of Engagement Activity | 2018 |
| Description | on line seminar for the agriculture and horticulture development board |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | seminar on fluke control to farmers, vets and SQP/RAMAs |
| Year(s) Of Engagement Activity | 2021 |