Introduction
Fasciola hepatica (the liver fluke) is a common parasite of cattle and sheep that affects animals worldwide and is the cause of the disease, fasciolosis. Recently, it has been shown that prevalence of liver fluke has been increasing significantly within the UK. This has been linked to many factors including climate change, changing farming practices (e.g. extended grazing periods), increased movement of animals and stewardship schemes. In UK dairy herds, infections with liver fluke are estimated to cause decreases in milk yield of approximately 8–15% per cow per year, or 1100 litres per cow. In beef cattle, infections can result in reduced weight gain, leading to individual animals taking an extra 80 days to reach market weight at a cost of £30–200 per animal. Estimates in sheep also show that fluke infection increase costs by £3–5 per ewe, through reduced daily live weight gain.
The liver fluke has a complex life cycle involving an intermediate host, the mud snail Galba truncatula. This means that typical ‘flukey’ areas are wet and muddy pastures which are the main habitat of the snail. The lifecycle begins with an infected host passing eggs onto pasture within its dung. These eggs then develop and hatch to an aquatic larval stage, which is able to infect the snail. Warm (above 10°C) and wet conditions favour an increased rate of development for the stages of the parasite in the environment.
Following a period of development within the snail, infective cysts called metacerariae are released from the snail; they attach onto the grassand are eaten by the cattle or sheep as they graze. Once ingested the juvenile fluke emerge and burrow through the gut wall to the liver. Once there, the juvenile fluke spend about 6 weeks migrating and feeding on the liver tissue and blood, before finally reaching the bile ducts as mature adults which are able to produce eggs which are passed out onto pasture within the animal’s dung. It takes approximately 8–14 weeks from ingestion of the cysts before eggs are detectable.
There are two types of fasciolosis, acute disease and chronic disease, determined by the number of cysts ingested by the animal over time. Acute infections are usually associated with the ingestion of large numbers of infective cysts over a short period of time. This leads to severe abdominal pain, weight loss and anaemia as a result of the extensive liver damage caused by the sudden migration of large numbers of juvenile fluke through the liver tissue. The acute form of the disease results in sudden death in infected sheep, and farmers can lose up to 10% of their flock in a matter of days with little warning. This form of the disease however, is rarely seen in cattle. Chronic infections result from ingestion of smaller numbers of cysts over a longer time period, allowing the adult fluke to establish themselves in the bile ducts of the liver before clinical signs appear.
Anaemia and weight loss are typical of chronic disease and it can occur on both sheep and cattle. Many fluke infections, characterised by low burden are sub-clinical, so rarely diagnosed but they with infection cause reduced weight loss and lower milk yields.
The rising prevalence of liver fluke over recent years has led to an increased dependence on the use of anthelmintics. The drug of choice, triclabendazole (TCBZ) (for example Fasinex, Combinex, Tribex, and Endofluke) is used mainly for its unique ability to target both adult and juvenile fluke within the host and is therefore able to combat the large numbers of migrating juvenile fluke during acute infections. Advice on the treatment of liver fluke can be found on the COWS (Control of Worms Sustainably) website: http://www. cattleparasites.org.uk/fluke.html. The emergence of resistance to TBCZ is therefore an urgent concern to UK farming. These results highlight the need for reliable and sensitive diagnostic tests.
Current diagnostics
The diagnosis of liver fluke infection is traditionally based on the detection of eggs in dung, using faecal egg counts (FEC) and presence of eggs is indicative of current infection. However, over recent years a number of highly sensitive and specific diagnostic tests have been developed to replace these traditional methods. These tests are largely based on the detection of specific molecules within samples.
One such test is the Copro-antigen test, which has been developed to detect specific fluke molecules within the dung of infected animals. Another test is able to detect host antibodies against fluke using individual serum samples or milk samples. Milk samples are a great advantage in dairy farming as they are much easier to collect and less invasive. Bulk milk tank samples can also be tested to estimate how much infection is present within the herd. These tests hold a slight advantage over FEC in being able to detect infection earlier, before the adult fluke are present.
Why we need improved diagnostics
All of these tests have significant limitations. For instance, FEC are only able to confirm infection once the adult fluke have matured and producing eggs at approximately 10–14 weeks after infection. This test does not detect the presence of the migrating juvenile flukes, which can cause clinical disease as early as 3 weeks after exposure. The sensitivity of FEC can also be very low and variable, particularly in cattle; this test may only detect fewer than 7 out of 10 infected cattle. To add to this, studies have also shown that liver fluke eggs can become trapped in the gall bladder leading to intermittent egg detection.The use of composite FEC (pooling dung samples from several individuals within the herd) is able to give an idea whether a herd or group of animals is infected; however it does not give information about the level of infection within the individual. This therefore does not allow for targeted treatment to the individual.
Whilst the Copro-antigen test is able to detect infection slightly earlier that the FEC (at approximately 8 weeks post infection), we have found that the Copro-antigen test is no more sensitive than the FEC. The detection of host antibodies is complicated by the fact antibodies have been found to remain circulating for several weeks following treatment.
All of the above tests require samples to be sent to the laboratory for testing. This can add significant time and cost to the diagnosis, especially in cases of acute disease.
What is the aim of this project?
The aim of this project is produce a pen-side diagnostic test which is able to provide the farmer with accurate and immediate results for detecting liver fluke infection in individual animals. The test itself would be one that farmers could use themselves through for example blood taken from an ear prick, or even through saliva or milk. This would remove the need for samples to be sent to the laboratory for testing and therefore reduce costs for farmers, but also speed up the time taken to get results. By testing individual animals treatment could be targeted at the individual, reducing costs and slowing the spread of resistance to flukicide drugs.
The test could be used to detect liver fluke infection in dairy cattle at drying off as there is a very limited time window for treatment. It would also be valuable in detecting infection in individual beef cattle during housing, and ideally provide a much earlier detection system for liver fluke infections in sheep and help in the treatment of acute fasciolosis.
Designing the test
This design of this test will be based around antibody detection because antibodies are produced early in infection. The first aim of this project was to identify which fluke molecule would be the best to use in the test. The picture in Figure 1 is the results of analysis of the major secretions of the fluke on a gel. The gel shows that a specific molecule, Cathepsin L1 is easily detectable within these samples.
CL1 is a protease enzyme which is secreted in large amounts by the fluke and is thought to permit the parasite to infect a wide range of mammalian hosts and play a key role in its pathogenicity. This enzyme is responsible for digestion of nutrients, facilitating the migration of the parasite through the liver and also has been implicated in the inactivation of host immune defence molecules. This has resulted in this protein being recognised as an important target for vaccine studies.
CL1 is a highly immunodominant molecule, which means that is one of the main targets of the host immune response. The host immune response involves the natural production of antibodies to help combat infection, and it is these antibodies against CL1 that we aim to detect with our diagnostic test. This test will therefore work in a similar way to laboratory antibody detection tests; however this test will aim to provide results much faster. Figure 2a shows the increase in the antibody response to liver fluke infection in an experimentally infected sheep over a 16 week period. It also shows that CL1 is one of the main targets for this host response. Figure 2a also shows that host antibodies against fluke infection can be detectable as early as 4 weeks post infection. Figure 2b shows that the same response can be detected in calves naturally infected over the course of their first year grazing season.
Lateral flow immunoassays (LIFAs) (Figure 3) are used in a wide variety of settings and are rapidly gaining popularity for use as a diagnostic technology. The first tests commercially produced were for detection of human pregnancy and now kits are commercially available for the monitoring of ovulation, detecting infectious disease, such as viruses, bacteria and parasitic infections, analysing drug abuse etc. LIFAs can have several different designs, and some can be made to qualitative (presence or absence of disease), semi quantitative or occasionally fully quantitative (intensity of infection). Currently, no such test exists for the monitoring of liver fluke infection in livestock.
In order to design the LIFA, we require large amounts of the target molecule, CL1. The second aim of the project this year has to isolate or clone the gene for the CL1 enzyme and then put it into yeast. The yeast cells can be stimulated to produce the fluke CL1 enzyme, which is then known as a recombinant protein (Figure 4). The recombinant protein will be identical every time it is produced and therefore will provide good uniformity in the penside test.
Conclusions
A LIFA will provide a much quicker and simpler method for detecting individual animals infected with liver fluke on farm. This will aid in the target treatment of animals reducing the reliance on blanket drug treatments of whole herds or flocks and therefore not only improve health and welfare of infected individuals but also aid in slowing the rapid increase in resistance seen in liver fluke populations. The next steps for the test will involve designing the remaining molecular components of the test, and then the actual test itself. For this we will probably contact a local biotechnology company to help, and once we have the final product, we will validate our LIFA against other currently available diagnostic tests for fluke infections.