*2.9. Killing of F. hepatica NEJ with Superoxide and Protection with SOD and Catalase*

To determine the susceptibility of *F. hepatica* NEJ to superoxide in vitro, the SOD activity assay described in Section 2.5 was repurposed to examine the effect of enzymatically generated superoxide on live NEJ. In this application, assay conditions and enzyme concentrations, except for catalase (CAT, recombinant from Serratia; Abcam, Cambridge, UK) and BS, were as described above in Section 2.5. Tetrazolium salt was omitted from the assay buffer (AB) to avoid any potential toxicity to the NEJ. *F. hepatica* metacercariae were excysted as described above, washed in 1 × PBS and incubated as described below.

At total of 10 NEJ per replicate were incubated in (i) PBS; (ii) AB with XOD; (iii) AB with XOD and BS (0.16 U/mL); (iv) AB with XOD and CAT (43 U/mL); (v) AB with XOD, BS and CAT (43 U/mL); (vi) AB with XOD and CAT (4.3 U/mL); and (vii) AB with XOD, BS and CAT (4.3 U/mL). NEJ were incubated at 37 ◦C and 5% CO2 immediately after addition of XOD. After 24 h incubation, the number of dead NEJ (characterised by a lack of movement, including the absence of gut activity or a complete breakdown of the tegument and internal structure, after a one-minute observation) were counted. All treatments were conducted in duplicate over several days, giving a total of six biological replicates per treatment. To test the capacity of our recombinant enzymes to counteract the impacts of ROS on NEJ, we repeated the assay with the inclusion of rFhSOD1 and rFhSOD3 at concentrations of equal enzyme activity to BS, with and without the addition of catalase.
