| Summary: | Sheep are at risk from the disease fasciolosis, caused by Fasciola hepatica. For sheep, triclabendazole (TCBZ) is the drug of choice as it kills the early, immature stages of the parasite. However, TCBZ is becoming increasingly ineffective due to emergence of resistance. The aim of this thesis was to identify the most appropriate diagnostic test to detect infection with F. hepatica in sheep, the optimum faecal egg count reduction method to detect TCBZ resistance, and thirdly to assess the prevalence of TCBZ resistance in Britain. In Chapter 2, three different techniques that have been described in the literature to diagnose F. hepatica infection: faecal egg counts (FEC), coproantigen ELISA and a PCR-based assay, were compared. The objectives were to compare the sensitivity of these techniques to detect early infection in experimentally infected sheep and measure the efficacy of TCBZ against a susceptible isolate. For this, a total of 12 sheep were infected with 200-215 TCBZ susceptible metacercariae and infection was tracked on a weekly basis from 0 to 14 weeks post-infection (wpi) at which point they were divided into two groups of six. Group 1 was left untreated and Group 2 animals were treated with TCBZ (Fasinex®; 10 mg/kg) per os. Faecal samples were collected on the day of treatment and daily until 10 days post treatment (dpt). At 10 dpt, all sheep were killed for liver fluke recovery and enumeration. Results showed that the coproantigen ELISA could detect infection from 5 wpi (2/12 sheep), and all sheep were positive by 8 wpi. FEC was less sensitive at detecting early infection, 1/12 animal became positive at 7 wpi and all sheep were positive at 11 wpi. The PCR failed to detect infection at any time point. The efficacy of TCBZ against F. hepatica was 97% with p < 0.001. There was no correlation between the coproantigen levels and FEC for all sheep, R2 = 0.031 (Spearman’s test p = 0.21) at 14 wpi. The study confirmed that the coproantigen ELISA is more sensitive in detecting early fluke infection compared to FEC. All six sheep in Group 2 were positive by FEC and 4 of these were also positive by coproantigen ELISA at 10 days post treatment with TCBZ; at post-mortem, between one and 13 flukes were recovered in all but one animal. Chapter 3 describes a pilot study conducted to improve the design of a previously described composite faecal egg count reduction test (cFECRT). Firstly we determined if it was necessary to sample the same 20 sheep before and post treatment, or if two random groups of 20 sheep could be sampled for the two counts. Samples from 44 sheep from Farm 1 and 105 sheep from Farm 2 were collected and individual egg counts determined. Bootstrap analysis showed that the same 20 sheep had to be sampled pre-treatment and at 21 dpt. The coproantigen ELISA and FEC were compared on individual samples and composite samples from 20 sheep on five farms. Faecal samples were collected prior to treatment, 7 and 21 dpt to determine the optimum time to collect the post-treatment sample. A comparison revealed that the results of the individual coproantigen ELISA were more informative compared to composite ELISA values for detection of infected sheep. The results also showed that sample testing at 21 dpt can help avoid false positive results. Chapter 4 describes two TCBZ resistance prevalence studies. Firstly a total of 20 farms in the county of Cumbria were approached. Sixteen farms submitted samples and 13 farms had pre-treatment counts sufficiently high to conduct the FECRT, and evidence of TCBZ failure was detected on all 13 farms. Secondly a survey of TCBZ resistance was conducted in three regions of Britain, North East England, South West England and South Wales. Two hundred and fifty farms were contacted, 42 farms took part in the study. Of the 42 farms who submitted pre-treatment samples, seven had pre-treatment counts of 100eggs per gram or higher. Evidence of TCBZ failure was observed in 4 of those 7 farms, all from North East England. Using questionnaire data, descriptive statistics showed that there was no significant difference (T-test p = 0.82) between resistance status and total number of ewes on a farm. Evidence from the present study suggests that TCBZ resistance is common on sheep farms in some parts the UK but further work is needed to establish a national prevalence. Overall, this study demonstrated that the coproantigen ELISA test is able to detect pre-patent fluke infections when compared to FEC. However, this work highlights that the coproantigen ELISA performs differently in experimentally infected sheep compared with naturally infected sheep. Further evaluation of the coproantigen ELISA is needed if it is to be used with confidence for detecting TCBZ resistance in the field. Whilst this study identified TCBZ to be a problem on some sheep farms it was not possible to determine the prevalence of TCBZ resistance in England and Wales, This highlights the need to monitor TCBZ efficacy on a farm by farm basis in England and Wales.
|
|---|
