Fasciolosis, a global parasitic disease of agricultural livestock, is caused by the liver fluke Fasciola hepatica. Management and strategic control of fasciolosis on farms depends on early assessment of the extent of disease so that control measures can be implemented quickly. Traditionally, this has relied on the detection of eggs in the faeces of animals, a laborious method that lacks sensitivity, especially for sub-clinical infections, and identifies chronic infections only. Enzyme linked immunosorbent assays (ELISA) offer a quicker and more sensitive serological means of diagnosis that could detect early acute infection before significant liver damage occurs. The performance of three functionally-active recombinant forms of the major F. hepatica secreted cathepsins L, rFhCL1, rFhCL2, rFhCL3, and a cathepsin B, rFhCB3, were evaluated as antigens in an indirect ELISA to serologically diagnose liver fluke infection in experimentally and naturally infected sheep. rFhCL1 and rFhCL3 were the most effective of the four antigens detecting fasciolosis in sheep as early as three weeks after experimental infection, at least five weeks earlier than both coproantigen and faecal egg tests. In addition, the rFhCL1 and rFhCL3 ELISAs had a very low detection limit for liver fluke in lambs exposed to natural infection on pastures and thus could play a major role in the surveillance of farms and a 'test and treat' approach to disease management. Finally, antibodies to all three cathepsin L proteases remain high throughout chronic infection but decline rapidly after drug treatment with the flukicide, triclabendazole, implying that the test may be adapted to trace the effectiveness of drug treatment.

Fasciola hepatica, or liver fluke, causes fasciolosis in humans and livestock. Following ingestion of vegetation contaminated with encysted parasites, metacercariae, newly excysted juveniles (NEJ) excyst in the small intestine and cross the intestinal wall. After penetrating the liver, the parasite begins an intra-parenchymal migratory and feeding phase that not only drives their rapid growth and development but also causes extensive haemorrhaging and immune pathology. Studies on infection are hindered by the difficulty in accessing these microscopic juvenile parasites in vivo. Thus, a simple and scalable in vitro culture system for parasite development is needed. Here, we find that two-dimensional (2D) culture systems using cell monolayers support NEJ growth to a limited extent. By contrast, co-culture of F. hepatica NEJ with HepG2-derived 3D spheroids, or "mini-livers," that more closely mimic the physiology and microenvironment of in vivo liver tissue, promoted NEJ survival, growth, and development. NEJ grazed on the peripheral cells of the spheroids, and they released temporally regulated digestive cysteine proteases, FhCL3, and FhCL1/2, similar to in vivo parasites. The 3D co-culture induced development of the NEJ gut and body musculature, and stimulated the tegument to elaborate spines and a variety of surface sensory/tango/chemoreceptor papillae (termed S1, S2, and S3); these were especially pronounced around the oral and ventral suckers that sense host chemical cues and secure the parasite in tissue. HepG2 3D spheroid/parasite co-culture methodologies should accelerate investigations into the understanding of F. hepatica NEJ developmental biology and studies on host-parasite interactions, and streamline the search for new anti-parasite interventions.

Immunomodulatory nutraceuticals have garnered special attention due to their therapeutic potential for the amelioration of many chronic inflammatory conditions. Macrophages are key players in the induction, propagation and resolution of inflammation, actively contributing to the pathogenesis and resolution of inflammatory disorders. As such, this study aimed to investigate the possible therapeutic effects bovine casein derived nutraceuticals exert on macrophage immunological function. Initial studies demonstrated that sodium caseinate induced a M2-like macrophage phenotype that was attributed to the kappa-casein subunit. Kappa-casein primed macrophages acquired a M2-like phenotype that expressed CD206, CD54, OX40L, CD40 on the cell surface and gene expression of Arg-1, RELM-α and YM1, archetypical M2 markers. Macrophages stimulated with kappa-casein secreted significantly reduced TNF-α and IL-10 in response to TLR stimulation through a mechanism that targeted the nuclear factor-κB signal transduction pathway. Macrophage proteolytic processing of kappa-casein was required to elicit these suppressive effects, indicating that a fragment other than C-terminal fragment, glycomacropeptide, induced these modulatory effects. Kappa-casein treated macrophages also impaired T-cell responses. Given the powerful immuno-modulatory effects exhibited by kappa-casein and our understanding of immunopathology associated with inflammatory diseases, this fragment has the potential as an oral nutraceutical and therefore warrants further investigation.

The helminth defense molecules (HDM) are a family of immune regulatory peptides exclusively expressed by trematode worms. We have previously demonstrated that in vivo FhHDM‐1, the archetypal member of the HDMs, regulated macrophage responses to inflammatory ligands, thereby ameliorating the progression of immune‐mediated tissue damage in several murine models of inflammatory disease. Accordingly, we postulated that an understanding of the structure–function relationship of the HDMs would facilitate the identification of the minimal bioactive peptide, which would represent a more synthesizable, cost‐effective, potent biotherapeutic. Thus, using a combination of bioinformatics, structural analyses, and cellular assays we discovered a 40 amino acid peptide derivative termed FhHDM‐1.C2. This peptide contains a 12 amino acid motif at its N‐terminus, which facilitates cellular interaction and uptake, and an amphipathic α‐helix within the C‐terminus, which is necessary for lysosomal vATPase inhibitory activity, with both regions linked by a short unstructured segment. The FhHDM‐1.C2 peptide exhibits enhanced regulation of macrophage function, compared with the full‐length FhHDM‐1, and potent prevention of the progression of relapsing–remitting‐experimental autoimmune encephalomyelitis (EAE) when administered prophylactically or therapeutically. The protective effect of FhHDM‐1.C2 is not associated with global immune suppression, which places the HDMs peptides as an improved class of biotherapeutics for the treatment of inflammatory diseases. Comparing the HDMs from several zoonotic trematodes revealed a similar capacity for immune regulation. These important new advances into the structure–function relationship of the lead HDM peptide, FhHDM‐1, encourage further prospecting and screening of the broader trematode family of peptides for the discovery of novel and potent immune‐biotherapeutics.