13TSB_ENDANI Development of a pen-side test for liver fluke in cattle and sheep

Lead Research Organisation: University of Nottingham
Department Name: School of Veterinary Medicine and Sci

Abstract

Fasciolosis is a major disease of cattle and sheep and is a key problem within the farming industry. It is caused by the parasite, Fasciola hepatica (a liver fluke), and is acquired by the ingestion of water or vegetation contaminated by the infectious stage of the parasite. The parasite goes on to colonise the liver of cattle and sheep, following migration across the gut wall and peritoneal cavity, where they feed on liver cells and blood. The presence of these parasites causes reduced anaemia, poor liver function, yields, poor fertility and high perinatal loses, and chronic weight loss in cattle. In addition to these effects seen in cattle, acute disease can cause sudden death from haemorrhage and liver damage in sheep. These effects in turn cause economic losses, estimated to be £1312 million worldwide. Currently diagnosis of fasciolosis is based upon epidemiological data and blood samples (for raised liver enzymes) in the acute/sub-acute disease, and the demonstration of eggs in faeces in chronic disease. A number of enzyme-linked immunosorbent assay (ELISA) techniques have also been developed, however, both these and egg counts have to be carried out in central laboratories with the expertise to do so and can take several days to perform, thus there is a clear need for a simple rapid decentralised test. Additionally, current serology based methods of diagnosis fail to distinguish between previous and current infection, while the faecal egg count is both time and labour intensive. Control of infection following diagnosis is
through the use of flukicides, the efficacy of these drugs is currently under question as drug resistance is now a significant problem for many farmers. Furthermore, the use of classes of these drugs is also now prohibited in dairy animals producing milk for the human food chain. Ultimately this means a more evidence and rational approach to drug usage is needed to prevent further drug resistance and concerns for human food safety. To do woud require a test capable of distinguishing not only current from previous infection but also staging the infection in terms of mature versus immature fluke being present. This project will aim to develop a pen-side lateral flow device (LFD) for the diagnosis of fasciolosis in cattle and sheep from faecal samples, with the aim of producing a simple to use, rapid test that will allow farmers and veterinarians to make informed decisions on whether treatment is required.
We will achieve this through the combined used of the project partners expertise to complete the following aims; 1) Preparation of F. hepatica antigens (proteins) for immunising mice to generate antibody producing cells; 2) Creation of immortal hybridomas in vitro producing antibody to the F. hepatica antigens; 3) Optimise ELISA immunoassays using antibodies produced in WP3 to F. hepatica antigens capable of distinguishing mature from immature flukes; 4) Develop and produce prototype penside lateral flow devices; 5) Evaluate prototype lateral flow devices on farms with a current fluke problem or farms with no history of fluke infection . The resulting lateral flow device will be used for simple, rapid diagnosis
of fasciolosis at the pen-side and will provide evidence of the life-stage of F. hepatica, this is of paramount importance as it influences the choice and efficacy of drug treatments. In diagnosing the parasite and the most efficacious treatment option the novel test will provide a clear economic benefit to the livestock producer. The test will also allow compliance with government policy on the best practice for F. hepatica treatment being on an individual animal basis. In doing so overall drug use will be reduced again reducing costs to the farmer and contributing to the reduction of anthelmintic-resistance.

Technical Summary

Fasciolosis is a major disease of cattle and sheep and is a key problem within the farming industry. It is caused by the parasite, Fasciola hepatica (a liver fluke), and is acquired by the ingestion of water or vegetation contaminated by the infectious stage of the parasite. The parasite goes on to colonise the liver of cattle and sheep, following migration across the gut wall and peritoneal cavity, where they feed on liver cells and blood. The presence of these parasites causes reduced anaemia, poor liver function, yields, poor fertility and high perinatal loses, and chronic weight loss in cattle. In addition to these effects seen in cattle, acute disease can cause sudden death from haemorrhage and liver damage in
sheep. These effects in turn cause economic losses, estimated to be £1312 million worldwide. Currently diagnosis of fasciolosis is based upon epidemiological data and blood samples (for raised liver enzymes) in the acute/sub-acute disease, and the demonstration of eggs in faeces in chronic disease. A number of enzyme-linked immunosorbent assay (ELISA) techniques have also been developed, however, both these and egg counts have to be carried out in central laboratories with the expertise to do so and can take several days to perform, thus there is a clear need for a simple rapid decentralised test. Additionally, current serology based methods of diagnosis fail to distinguish between previous and current infection, while the faecal egg count is both time and labour intensive. Control of infection following diagnosis is through the use of flukicides, the efficacy of these drugs is currently under question as drug resistance is now a significant
problem for many farmers. Furthermore, the use of classes of these drugs is also now prohibited in dairy animals producing milk for the human food chain. Ultimately this means a more evidence and rational approach to drug usage is needed to prevent further drug resistance and concerns for human food safety

Planned Impact

The impact of this project will be felt primarily by the farming stakeholders directly affected by the present of liver fluke infection in their herds/flocks, wider or secondary impacts will be upon society and consumers through the provision of healthier and safer products. The economic benefit to farmers will arise from the ability to target treatment of animals quickly and to only those that require it, current treatment costs are around £3.60/300kg for cattle and £1.50/100kg for sheep. With average UK dairy herd sizes of 117 cows (based on 1.8 million dairy cattle in 15,300 holdings) and flock sizes of 422 sheep (based on 31 million sheep and 73,400 holdings) the cost per treatment could be around £638 to £842 per herd and £442 per flock, with at least two treatments being recommended bringing the total cost to between £1276-£1684 per herd, and £884 per flock, per annum, thus significant savings could be made if the whole herd/flock does not need to be treated. Additonally, optimum use of anthelmintics in terms of agent as well as animal selection and dose will provide added long term production gains for the farmer in terms of reductions in fasciolosis incidence and the incidence of drugresistance. The economic benefit to the food industry will be reduced liver condemnation and there will be better carcass weights for those animals that have been selectively treated.
Environmental/Social Benefits: The anthelmintic chemicals used to treat liver fluke leave residues in the meat and milk of treated animals; they have lengthy withdrawal periods - 60-66 days for beef and a no usage ban now in place in dairy cattle - which prevents the sale of products from treated animals for human consumption during this time. Decreased use of anthelmintics would thus relieve the potential for drug residues to enter the food chain. This project will contribute to
reducing the use of anthelmintic treatments for liver fluke, many of which e.g. TCB, nitroxynil (NXY), closantellan (CLO) and oxyclozane have been withdrawn from the market in dairy cattle due to the presence of anthelmintics in milk destined for the human food chain. Resistance to the anthelmintic tricalbendazole, has been reported in several countries, thus, the reduced overall use of anthelmintics due to more targeted use to only infected animals, as well as the use of optimised doses and optimised agent application (according to the life cycle of the parasite) will slow the spread of drug resistance. To rapidly facilitate the dissemination and impact of our project we will interact with various stakeholders in a number of ways. Veterinary surgeons will be informed by means of publication in practice focused journals, e.g. Veterinary Record. Presentation at conferences such as British Society for the Advancement of Veterinary Parasitology and British Veterinary Association is envisioned. To inform the farming community existing links with the various levy boards will be exploited, all partners in the project have active links with EBLEX and DairyCo, both of which represent the primary target market for this assay. Furthermore, the University of Nottingham School of Veterinary Medicine and Science has an
extremely active outreach programme such that we have a presence at major national agricultural and country shows, e.g. Chatsworth Show, Beef Expo etc... The presence of project partners at these events will help to showcase the benefits that out project can bring to the farming industry. Finally, by improving the rational selection of drug usage and the consequent
production of safer products we will impact upon society and the consumer. These groups will be reached by the University of Nottingham and their extremely active press office which ensures scientific gains made by the University receive widespread mainstream media coverage. A number of partners in the project have already been involved with local and national radio and press media outlets in this fashion.

Publications

10 25 50
 
Description We have developed a panel of antibody, both IgM and IgG isotypes, reagents against a series of Fasciola hepatica specific proteins. Using these we were able to develop three potential ELISA assays which were able to detect liver fluke homogenate spiked into a variety of buffers. These include a competition ELISA using antibody (MBDC) and liver fluke homogenate on the plate; a sandwich ELISA using two anti-liver fluke homogenate antibodies, one of which was biotinylated; and finally an ELISA involving superblock, liver fluke homogenate, and the antibody (MBDC) to exploit an unusual effect we noted of the superblock being able to bind liver fluke homogenate but not Ostertagia.

The sandwich assay was ruled out due to the finding that the biotinylated antibody was binding to the capture antibody. The assay exploiting the capacity of superblock to bind liver fluke homogenate was shown to be promising, but it lacked the sensitivity we would require for such an assay as it was only able to detect relatively large amounts of liver fluke homogenate i.e. limit of detection of ~2.5 µg/ml after 2 hours or ~0.3 µg/ml if left overnight which is not ideal.

The most promising of the three assays, the competition ELISA using antibody (MBDC) and liver fluke homogenate was taken forward for testing with spiked negative/healthy faecal extracts. There were issues with the matrix effects from the faecal samples being too great to be able to detect a liver fluke homogenate therefore a filtration of the faecal samples prior to spiking with liver fluke homogenate was used to try to improve matters. We did not see competition in neat extracts but upon dilution we were able to see competition. Additional healthy faecal samples were collected from the University of Nottingham Dairy Unit to test the reproducibility of this within different sample matrices. These samples only illustrated competition at the higher levels of liver fluke homogenate used for spiking and illustrated sample matrix effects at the lower levels of spike used.

We were unable to detect a liver fluke homogenate spike when it was added to the healthy faecal sample prior to its extraction. It was possible that the spike was binding to the filter in the filtration unit and a test showed this to be the case, therefore different filtration units with low protein binding were tested and this resolved the binding issue.

In terms of LFD production we had hoped to be able to develop a sandwich assay for this. Ideally the sandwich assay uses a secondary or anti-species antibody to complete the T-line however, given that the LFD was required to detect levels of liver fluke in multiple species the assay was developed using two antibodies specific to liver fluke. This form of LFD design most commonly results in a weak T-line due to available binding sites and the accumulation of conjugate-Target molecules passing over the T-line, accumulating at the release pad nitrocellulose membrane overlap and reduced proximity to the T-line. The eventual outcome of the assay identified Ab-Ab Interaction between conjugate and T-line. Modifications aimed at improving the conjugation process: pH buffers, reduced salt solutions, blockers and buffer exchanging material in order to reduce the Ab-Ab interaction were tested, however were unable to reduce the interaction with interfering substances such as Ostertagia and Foetal Bovine Serum.

Unfortunately the outcome of this project was not in line with our expectations as no working lateral flow test or ELISA test has been developed.
Exploitation Route The antibodies are available for use by other researchers. Raised antibodies demonstrated cross-reactivity and thus were unsuitable for multiplex use in an ELISA.
Sectors Agriculture, Food and Drink

 
Description As part of the planning process for our field trail we have been in extensive discussions with farming stakeholders. We have been instrumental in directing stakeholders to sources of best practice and in potential ways in which to implement this best practice. This has involved providing free faecal egg counts for a variety of farmers with varying flock sizes and providing this information to clinicians and SQPs to best advice on the appropriate treatment and management strategies.
First Year Of Impact 2014
Sector Agriculture, Food and Drink
Impact Types Economic,Policy & public services

 
Description I contributed to an updated expert review of Fasciola as an entire disease area for the EU Discontools Project.
Geographic Reach Europe 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
URL http://www.discontools.eu/database.html
 
Description Providing training to junior clinicians and influencing population-wide treatment decisions
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact As part of our plans to validate the diagnostic tool we have acquired access to large naturally infected cohorts of animals through clinical associate practices. We offer a service to these clinicians/herds in providing a diagnostic service but also we are able to retain samples for later use in validation. During the course of this service we have been able to offer training in diagnostic parasitology but also in the how this data is used and its true meaning, i.e. no correlation of egg count data with serology results etc.. This has proved beneficial for interns/residents involved in these practices as they have had the opportunity to spend some time in a laboratory setting.
 
Description Agri-Tech Scheme
Amount £125,692 (GBP)
Funding ID BB/M018369/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2015 
End 08/2016
 
Title Biobank of infected samples 
Description We have developed a collection of matched blood/faecal samples from naturally infected sheep and cattle from a wide geographic range within the UK. This is being used to validate our diagnostic device but will be made available to other researchers as necessary. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact This biobank has facilitated the ongoing collaboration between us and other research groups working in related fields. 
 
Description Phenotyping of Fasciola hepatica knockdowns 
Organisation University of Phayao
PI Contribution Under the auspicious of a Newton Fund/British Council award we are hosting a research from the UNiversity of Phayao to develop knock-down phenotypes for Fasciola hepatica. We are providing molecular biology and genomic experience alongside parasitology support.
Collaborator Contribution Molecular biology and RNAi
Impact Knowledge exchange to date.
Start Year 2018
 
Description Association of Veterinary Teaching and Research Workers Meeting 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact PG presentation to a veterinary focused research meeting.
Year(s) Of Engagement Activity 2017
 
Description Edinburgh Invited Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited seminar at the University of Edinburgh Immunology Group to speak on the role of anergy in developing protective immunity/vaccination against Fasciola hepatica.
Year(s) Of Engagement Activity 2019
 
Description Invited Seminar University of Liverpool regional Immunology group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Invited seminar for the British Society of Immunology Liverpool/Merseyside regional group. The topics of liver fluke disease and host immunity were discussed. As a result of the talk there has already been a collaboration established.
Year(s) Of Engagement Activity 2016
 
Description Invited seminar at the Edinburgh Immunology Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I was recently an invited speaker at the Edinburgh Immunology group seminar series. Giving me the opportunity to share some of the progress we have made in our diagnostic work with like-minded audiences not only in the veterinary field but also in the human/medical field.
There was much stimulating discussion afterwards.

As a result of this we have acquired further access to banked samples which will prove useful when validating our device. Furthermore, in collaboration with a colleague we are developing plans to implement our diagnostic tool as part of a control strategy/treatment intervention strategy.
Year(s) Of Engagement Activity 2014
 
Description Liver Fluke Control 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An overview of the collaborative work being conducted between University of Nottingham School of Veterinary Medicine & Science and ADAS UK. As a result of this publication/newsletter we have had contact and engagement with both farming and veterinary stakeholders globally regarding the use of our control methods.
Year(s) Of Engagement Activity 2013
 
Description Molecular and Cellular Biology of Helminths XI 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact PG presentation at International Parasitology meeting
Year(s) Of Engagement Activity 2017
 
Description Parasitology/Immunology CPD 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Was asked to speak at the British Cattle Veterinary Association annual meeting as a sponsored session by Boehringer Ingelheim. Presented on mucosal immunology in the context of infection.
Main audience was practising vets and some industry members.
Year(s) Of Engagement Activity 2018
 
Description Public Involvement Panel - Speaking Engagement at University of Liverpool 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A 20 min overview of research in the fasciola area was presented and potential problems and future directions were explained. There was discussion afterwards specific to the topic and more generally around public perception of disease control, government involvement and animal experimentation.
Year(s) Of Engagement Activity 2017
 
Description School Visit (Sutton Bonington) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This was an informative visit as part of an active outreach program to local schools. This involved brief talks about the work in our school and some specific examples including the work being conducted as part of our grant.
In addition there was an opportunity to give the visitors a tour of our building.

I have had a request to host two school children (GSCE-level) as "interns" for work experience in my laboratory over the coming Christmas period.
Year(s) Of Engagement Activity 2014