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Fasciola hepatica infections cause morbidity and mortality in sheep and have a significant economic impact on farmers. The commonly used diagnostic tests; faecal egg count (FEC), anti-Fasciola antibody ELISA (AbELISA) and the biochemical assays (measuring GLDH and GGT) all have limitations, particularly in detection of pre-patent infections in sheep. A coproantigen ELISA (cELISA) is reported to detect low burdens of infection from 4 weeks post-challenge (wpc) and to only detect current infection. A faecal PCR has been used for early detection of infection, but is limited by inhibitory factors in faecal samples. Loop-mediated isothermal amplification (LAMP) is more resistant to inhibitory factors and has the potential to be a pen-side assay. Triclabendazole (TCBZ) is the drug of choice to treat immature F. hepatica but there have been increasing reports of TCBZ treatment failure in the UK. Treatment outcome is determined using a FEC reduction test (FECRT). A cELISA reduction test (CRT) has recently been proposed. Within this thesis the cELISA, along with FEC, and where feasible the AbELISA and the use of GLDH and GGT concentrations, are evaluated in (1) an experimental challenge model in sheep, (2) individual naturally exposed sheep, in early infection, pre- and post-treatment situations, (3) groups of naturally exposed sheep, including composite samples, in pre- and post-treatment situations and evaluating the FECRT and CRT, lastly a LAMP assay is developed for the detection of F. hepatica, and evaluated against cELISA, FEC and PCR based detection. Two groups of 6 sheep were challenged with F. hepatica metacercarial cysts. In both studies, AbELISA was first to detect infection (3-4 weeks post-challenge (wpc)), followed by cELISA (3-10 wpc) and then FEC (9-10 wpc). Minor fluctuations were seen in both FEC and cELISA levels over both studies and a transient increase in cELISA levels was seen in the first study at 3-8 wpc. All animals were dosed with TCBZ 2 weeks prior to slaughter. The highest FECR was 37% and all sheep had live fluke present in their livers post-mortem. 27 lambs were sampled monthly between June and November with AbELISA, GLDH, GGT, FEC and cELISA tests performed. GLDH and GGT concentrations were above reference ranges from June. AbELISA detected infection in most animals by September and in all but one animal by November. FEC and cELISA both detected some very early positive results, most likely false-positive results, but the majority of animals became positive in November. Twelve lambs were followed to slaughter and all had low burdens of fluke (≤10). A cross-sectional study was conducted including 36 British farms, comprising 812 and 528 sheep pre- and post-treatment, respectively. Low FEC and cELISA results were seen, with better agreement between the two tests pre- than post-treatment. Disagreements between the two tests were more frequently seen where the FEC detected infection but the cELISA did not. This was true both before and after treatment. 80 animals from 2 Scottish farms were confirmed to be infected with liver fluke and given either a TCBZ or closantel treatment and followed for 56 days. A closantel treatment was given to animals that were still infected at 21 days post-treatment (dpt). The highest FECR and CR of the TCBZ-treated groups was 60.3% and 56.4%, respectively, and the lowest FECR and CR of the closantel-treated groups was 83.7% and 94.9%, respectively. A small proportion of closantel-treated animals maintained a low FEC following treatment. Both the FECRT and CRT indicated treatment outcome from 7 dpt. In a postal survey, 41 sample packs were sent to British farmers, of which 25 farmers participated. Samples from 44 and 36 groups were submitted pre- and post-treatment, respectively. Individual and composite faecal samples from each group were tested by FEC and cELISA. Group mean FECs were low and prevalence of infection on farms did not follow a normal distribution. The composite cELISA was more sensitive than the average cELISA, whilst the opposite was true for FEC. The composite cELISA was less sensitive than the composite FEC in low burden situations. A modified version of the composite CRT showed good agreement with the composite FECRT and appears promising in situations where burden was sufficiently high. A faecal LAMP assay, specific to F. hepatica, was developed and evaluated using samples from one of the groups of 6 experimentally challenged animals described above. FEC, cELISA and PCR testing were also performed and compared to the LAMP results. LAMP first detected infection at 3 wpc, followed by cELISA (7 wpc), FEC (10 wpc) and PCR (13 and 14 wpc). The studies within this thesis (1) confirm that cELISA can detect experimental infection of sheep with F. hepatica later than AbELISA but earlier than FEC, and confirm the TCBZ resistant status of a British isolate (Moredun isolate), (2) demonstrate that in animals naturally exposed to F. hepatica, the cELISA does not have an advantage of earlier detection over FEC and is not as sensitive as FEC in established infections (3) show that the modified CRT and composite CRT appear to give a good indication of treatment outcome from 7 dpt, but is of limited use in flocks with a low burden of infection, and (4) demonstrate that a faecal LAMP can detect F. hepatica infection at 3 wpc.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Tereza Kněžíková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Liver fluke - treatment and resistence Fasciola hepatica is a parasite of global importance that we find both in farm animals and in humans. This thesis aims to summarize information on the potential of drugs and treatment alternatives that are suitable for treatment of F. hepatica. Given that for a number of drugs used in the past, as well as the drugs currently administered, resistance developed, this thesis is also focused on this phenomenon, especially the mechanisms of its origin. The drugs used to treat fasciolosis are called antitrematodal drugs. They can be divided into five chemical groups, of which the most important group are currently benzimidazoles and their representative triclabendazole. Also other drugs as albendazole, clorsulon, hexachlorophene, closantel, diamphenitide, bithionol, rafoxanide are important. The rate of resistance development is affected by many factors that may be genetic, biological or functional. F. hepatica actively uses its enzymatic system, especially oxidation enzymes or efflux transporters. The influence on the development of resistance, apart from the parasite itself,...

This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite’s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™ Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Fasciolosis, caused by the trematode parasite Fasciola hepatica, is a multi-host parasitic disease affecting many countries worldwide. It is a well-recognized clinically and economically important disease of food producing animals such as cattle and sheep. In the UK, the incidence and distribution of fasciolosis has been increasing in the last decade while the timing of acute disease is becoming more variable and the season suitable for parasite development outside the mammalian host has been extended. Meanwhile control is proving increasingly difficult due to changing weather conditions, increased animal movements and developing anthelmintic resistance. Forecasting models have been around for a long time to aid health planning related to fasciolosis control, but studies identifying management related risk factors are limited. Moreover, the lack of information on the accuracy of meat inspection and available liver fluke diagnostic tests hinders effective monitoring of disease prevalence and treatment. So far, the evaluation of tests available for the diagnosis of the infection in cattle has mainly been carried out using gold standard approaches or under experimental settings, the limitations of which are well known. In cattle, the infection mainly manifests as a sub-clinical disease, resulting in indirect production losses, which are difficult to estimate. The lack of obvious clinical signs results in these losses commonly being attributed to other causes such as poor weather conditions or bad quality forage. This further undermines establishment of appropriate control strategies, as it is difficult to convince farmers to treat without demonstrating clear economic losses of sub-clinical disease. This project explores the value of slaughterhouse data in understanding the changing epidemiology of fasciolosis, identifying sustainable control measures and estimating the effect of infection on production parameters using data collected at one of the largest cattle and sheep abattoirs in Scotland. Data used in this study include; a) abattoir data routinely collected during 2013 and 2014, b) data collected during 3 periods of abattoir based sampling, c) data collected through administration of a management questionnaire and d) climatic and environmental data from various online sources. A Bayesian extension of the Hui Walter no gold standard model was used to estimate the diagnostic sensitivity and specificity of five diagnostic tests for fasciolosis in cattle, which were applied on 619 samples collected from the abattoir during three sampling periods; summer 2013, winter 2014 and autumn 2014. The results provided novel information on the performance of these tests in a naturally infected cattle population at different times of the year. Meat inspection was estimated to have a sensitivity of 0.68 (95% BCI 0.61-0.75) and a specificity of 0.88 (95% BCI 0.85-0.91). Accurate estimates of sensitivity and specificity will allow for routine abattoir liver inspection to be used as a tool for monitoring the epidemiology of F. hepatica as well as evaluating herd health planning. Linear regression modelling was used to estimate the delay in reaching slaughter weight in beef cattle infected with F. hepatica, accounting for other important factors such as weight, age, sex, breed and farm as a random effect. The model estimated that cattle classified as having fluke based on routine liver inspection had on average 10 (95% CI 9-12) days greater slaughter age, assuming an average carcass weight of 345 kg. Furthermore, estimates from a second model indicated that the increase in age at slaughter was more severe for higher fibrosis scores. More precisely, the increase in slaughter age was 34 (95% CI 11-57) days for fibrosis score of 1, 93 (95% CI 57-128) days for fibrosis score 2 and 78 (95% CI 30-125) days for fibrosis score 3. Similarly, in a third model comparing different burden categories with animals with no fluke burden, there was a 31 (95% CI 7-56) days increase in slaughter age for animals with 1 to 10 parasites and 77 (95% CI 32-124) days increase in animals with more than 10 parasites found in their livers. Lastly, a multi-variable mixed effects logistic regression model was built to estimate the association between climate, environmental, management and animal specific factors and the risk of an animal being infected by F. hepatica. Multiple imputation methodology was employed to deal with missing data arising from skipped questions in the questionnaire. Results of the regression model confirmed the importance of temperature, rainfall and cattle movements in increasing the risk for fasciolosis, while it indicated that the presence of deer can increase the risk of infection and that male cattle have a reduced risk of infection. Overall, this project has used slaughterhouse data to fill important knowledge gaps regarding F. hepatica infection in cattle. It has provided valuable information on the accuracy of routine abattoir meat inspection, as well as other diagnostic tests. It has also provided estimates of the effect of infection on the time cattle take to reach slaughter weight at different levels of infection and identified relevant risk factors related to the infection. In conclusion, knowledge of the effect of infection on slaughter age, as well as regional risk factors for F. hepatica infection, along with an improved use of abattoir inspection results in the evaluation of treatment strategies, can provide farmers and veterinarians with better incentives and tools to improve their herd health strategies and in the longer term help reduce the incidence of liver fluke in cattle.