A serological test based on mutated recombinant Fasciola hepatica cathepsin L protease for the diagnosis of equine fasciolosis

Fasciola hepatica is a common trematode parasite of livestock in many regions, causing significant economic losses and affecting animal welfare. Horses rarely develop patent liver fluke infection. However, liver damage can affect animal health and welfare. Therefore, F. hepatica infection in horses may be underreported. Recently, a serological test for the antibody detection has been reported based on recombinant parasite cathepsin L1 (FhCL1) protease. Here, we optimized this enzyme-linked immunosorbent assay (ELISA) for antibody-detection in horses with equine fasciolosis based on recombinant forms of FhCL proteases with a bioengineered diagnostic epitope mutation. Epitopes from different homologues of FhCL-proteases were modified, and canonical and mutated versions were recombinantly produced. The best performing candidate was evaluated with 175 serum samples from horses in Ireland. Seven horses suspected of having liver fluke infections were monitored for one year following treatment with triclabendazole. Additionally, a cohort of 368 samples from Swiss horses was tested to gather prevalence data. The final diagnostic test based on an epitope-mutated antigen showed a sensitivity and specificity of 65 % and 97.4 %, respectively. Follow-up of horses with suspected F. hepatica infection showed not only improved biochemical liver values but also a decrease in antibody titres, which fell below the test threshold after approximately 6-9 months. This group allowed for the definition of thresholds to distinguish between ambiguous/borderline results and clear positives. The prevalence of equine fasciolosis in Swiss horses was between 3.5 % and 5.7 %, depending on the applied diagnostic threshold. These results suggest that F. hepatica may be a neglected infection in Swiss horses. However, reliable diagnostic tests remain important, particularly for detecting cases in horses with liver disease of unknown origin or those co-grazed with ruminants in endemic regions.