Disclosing the bioactive metabolites involved in the in vitro anthelmintic effects of salt-tolerant plants through a combined approach using PVPP and HPLC-ESI-MSn.

Strategies to reduce dependence on synthetic drugs for the treatment of gastrointestinal nematodes (GIN) infections in ruminants include the search for novel anthelmintic scaffolds on plants, yet salt-tolerant plants remain overlooked. This study aims to evaluate the in vitro anthelmintic properties of selected salt-tolerant plants against GIN, and identify the potential bioactive secondary metabolites involved. For that purpose, 80% acetone/water extracts were prepared from dried biomass of aerial organs of nine salt-tolerant plant species and tested against Haemonchus contortus and Trichostrongylus colubriformis by the Larval Exsheathment Inhibition Assay (LEIA) and Egg Hatching Inhibition Assay (EHIA). Pistacia lentiscus, Limoniatrum monopetalum, Cladium mariscus and Helychrisum italicum picardi were the most active in both GIN and life stages. To investigate the role of polyphenols in the anthelmintic activity, four selected extracts were treated with polyvinylpolypyrrolidone (PVPP), and non-treated and treated samples were further characterized by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn). While polyphenols seem responsible for the EHIA properties, they are partially accountable to LEIA results. Several phenolics involved in the anthelmintic effects were identified and discussed. In sum, these species are rich sources of anthelmintic compounds and, therefore, are of major interest for nutraceutical and/or phytotherapeutic applications against GIN in ruminants.

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design.

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials.

Cyclotide interactions with the nematode external surface.

Cyclotides are a large family of cyclic cystine knot-containing plant peptides that have anthelminthic activities against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes of sheep. In this study, we investigated the interaction of the prototypic cyclotide kalata B1 with the external surface of H. contortus larvae and adult worms. We show that cyclotides do not need to be ingested by the worms to exert their toxic effects but that an interaction with the external surface alone is toxic. Evidence for this was the toxicity toward adult worms in the presence of a chemically induced pharyngeal ligature and toxicity of cyclotides toward nonfeeding larval life stages. Uptake of tritiated inulin in ligated adult worms was increased in the presence of cyclotide, suggesting that cyclotides increase the permeability of the external membranes of adult nematodes. Polyethylene glycols of various sizes showed protective effects on the nonfeeding larval life stage, as well as in hemolytic activity assays, suggesting that discrete pores are formed in the membrane surfaces by cyclotides and that these can be blocked by polyethylene glycols of appropriate size. This increased permeability is consistent with recently reported effects of cyclotides on membranes in which kalata B1 was demonstrated to form pores and cause leakage of vesicle/cellular contents. Our data, together with known size constraints on the movement of permeants across nematode cuticle layers, suggest that one action of the cyclotides involves an interaction with the lipid-rich epicuticle layer at the surface of the worm.

The anthelmintic activity of the cyclotides: natural variants with enhanced activity.

The cyclotides are a family of backbone-cyclised cystine-knot-containing peptides from plants that possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematode parasites of sheep. In the current study, we investigated the in vitro effects of newly discovered natural cyclotides on the viability of larval and adult life stages of these pests. The natural variants cycloviolacin O2, cycloviolacin O3, cycloviolacin O8, cycloviolacin O13, cycloviolacin O14, cycloviolacin O15, and cycloviolacin O16 extracted from Viola odorata showed up to 18-fold greater potency than the prototypic cyclotide kalata B1 in nematode larval development assays. Cycloviolacin O2 and cycloviolacin O14 were significantly more potent than kalata B1 in adult H. contortus motility assays. The lysine and glutamic acid residues of cycloviolacin O2, the most potent anthelmintic cyclotide, were chemically modified to investigate the role of these charged residues in modulating the biological activity. The single glutamic acid residue, which is conserved across all known cyclotides, was shown to be essential for activity, with a sixfold decrease in potency of cycloviolacin O2 following methylation. The three lysine residues present in cycloviolacin O2 were acetylated to effectively mask the positive charge, resulting in a 18-fold decrease in anthelmintic activity. The relative anthelmintic activities of the natural variants assayed against nematode larvae correlated with the number of charged residues present in their sequence.

Immunomodulation of lambs following treatment with a proteasome preparation from infective larvae of Trichostrongylus colubriformis.

Proteinases are known to be capable of prolonging the survival of endoparasites in a host. We were therefore interested in knowing whether immunization of lambs against a proteasome (multisubunit proteinases) preparation obtained from Trichostrongylus colubriformis infective third-stage larvae (L3) would have any effect on the immune response to a single challenge infection with the same organism. A total of 21 penned lambs aged 8 months were divided into 3 equal groups. Group 1 was immunized on three occasions with increasing amounts of a proteasome-enriched fraction obtained from infective L3. Group 2 was given a similar amount of protein from the initial supernatant of homogenized larvae. Group 3 (controls) received adjuvant plus saline solution only. All groups were challenged with 60,000 infective T. colubriformis larvae at 28 days after the last immunization. Significant protection was obtained only when the initial supernatant extract was used to immunize lambs. The proteasome preparation seemed to have immunosuppressive effects through the stimulation of nonspecific IgE production. Significantly lower levels of specific IgE were observed in lambs immunized with the proteasome-enriched fraction, and levels of specific IgG antibodies were increased. We suggest that proteasome fractions of T. colubriformis may serve as useful preparations for the study of mechanisms of IgE production in parasitized sheep.