Fasciola hepatica infection in sheep: current and novel diagnostic tests

Abstract

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.