**1. Introduction**

Fasciolosis, a zoonotic disease of humans and livestock, is caused by infection with the digenean trematodes, *Fasciola hepatica* and *Fasciola gigantica*. The economic impact of fasciolosis on livestock production is expected to exceed USD 3 billion/year [1]. The parasite also infects an estimated 17 million people globally, and 180 million people live in endemic regions where they are at risk of infection [2].

Infection occurs when the mammalian host ingests encysted parasites, metacercariae, carried on plant material or water. The metacercariae excyst in the host's intestine and

the newly excysted juveniles (NEJ) penetrate the intestinal wall and migrate through the abdominal cavity to the liver. Upon reaching the liver, the immature parasites spend 8–12 weeks burrowing through the parenchymal tissue, where they rapidly increase in size and mature. After taking up residence in the bile ducts and gall bladder, the parasites complete their development into egg-laying adults [3]. During this period of migration and growth, the infected host mounts an acute proliferative cellular and humoral immune response to block the parasites and mitigate damage and haemorrhaging caused by their migration. This acute response is characterised by marked eosinophilia, along with the infiltration of macrophages and lymphocytes to the parasite tracks, resulting in the local production of damaging reactive oxygen species (ROS) [4].

Genomic, transcriptomic, and proteomic analyses of several life stages of *F. hepatica* (infective metacercariae, NEJ at 1, 3, and 24 h post-excystment, immature juvenile flukes from 21 days post-infection, and mature adults) have identified many developmentally regulated proteins, thereby shedding light on the complex interactions these parasites have with their hosts throughout the infection process [5–7]. The major proteins secreted during these early infection stages include cathepsin-like proteases, protease inhibitors, and a slew of antioxidant enzymes, including superoxide dismutase (FhSOD), peroxiredoxin (FhPrx), thioredoxin (FhTrx), glutathione peroxidase (FhGPx), and glutathione-S-transferase (FhGST) [8]. Together, these proteins are assumed to help tissue invasion, macromolecule digestion, and defend against the onslaught of host-generated ROS [7].

SODs are a class of metalloenzyme antioxidants that, in pathogens, play a role in defence against exogenous ROS produced by the host by catalysing the two-step disproportionation of superoxide anions (O2 •- ) into hydrogen peroxide (H2O2) [9]. Three isoforms of SOD have been described in helminths (worms) and their mammalian hosts based on their localisation and metal co-factors: (1) a cytosolic Cu/Zn SOD, (2) a mitochondrial Mn-SOD, and (3) an extracellular Cu/Zn SOD characterised by the presence of a hydrophobic N-terminal signal peptide [10–14]. To date, only a cytosolic form of Cu/Zn SOD has been described in *F. hepatica* and *F. gigantica* [15–17]. However, our interrogation of the existing *Fasciola* spp. genomes revealed the presence of multiple SOD sequences in these parasites, including the cytosolic form, a mitochondrial form, and a novel extracellular SOD possessing a characteristic N-terminal signal peptide sequence. This extracellular SOD is observed at greater protein abundance in the secretome of *F. hepatica* NEJ relative to that of the mature adults, suggesting that it acts as a specialised enzyme protecting the invading parasites against the oxygen-mediated killing mechanisms of their hosts.

Here, we characterised the role of SODs in the defence of *F. hepatica* against ROS via the production of functional recombinant cytosolic (rFhSOD1) and extracellular (rFhSOD3) forms excreted/secreted by the parasite during early invasion. We show that these antioxidants are highly homologous to their mammalian host counterparts, which accounts for their lack of immunogenicity in infected sheep. Specific anti-rFhSOD1 and anti-rFhSOD3 antibodies were produced and used in immuno-localisation experiments to identify the site of enzyme production and secretion in *F. hepatica* NEJ and adults. Finally, we developed an in vitro biological assay that enzymatically produces ROS and demonstrates the susceptibility of NEJ to both superoxide and hydrogen peroxide. This killing of NEJ was obviated via the addition of SOD and catalase, revealing the power of this cascade in defence against ROS during invasion.
