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

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 would 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.

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

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), closantel, clorsulan (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.