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Plant-derived condensed tannins (CT) show promise as a complementary option to treat gastrointestinal helminth infections, thus reducing reliance on synthetic anthelmintic drugs. Most studies on the anthelmintic effects of CT have been conducted on parasites of ruminant livestock. Oesophagostomum dentatum is an economically important parasite of pigs, as well as serving as a useful laboratory model of helminth parasites due to the ability to culture it in vitro for long periods through several life-cycle stages. Here, we investigated the anthelmintic effects of CT on multiple life cycle stages of O. dentatum.

The use of natural plant extracts as de-wormers for humans and livestock has long been practiced, however scientific validation of these practices and identification of active compounds has been lacking [6]-[8]. Anthelmintic effects of plants are normally ascribed to secondary metabolites such as alkaloids, terpenoids or polyphenols such as proanthocyanidins [9], also known as condensed tannins (CT). Proanthocyanidins are a diverse and widely-occurring group of compounds, and consist of polymers of either catechin and/or epicatechin (termed procyanidins - PC), or of gallocatechin and/or epigallocatechin (termed prodelphinidins - PD), with hetero-polymers being common [10]. They are found in plant material from both tropical and temperate areas, and have been widely investigated for their antioxidant and anti-inflammatory properties [11],[12]. It is also apparent that CT can have anthelmintic effects; reduced worm burdens have been reported in rats administered CT in the diet, or in livestock grazing forages containing CT [13],[14]. Moreover, direct anthelmintic effects of purified CT have been confirmed in in vitro assays against, amongst others, Haemonchus contortus[15], Ostertagia ostertagi[16] and Ascaris suum[17]. However, much work remains to be done to establish the spectra of activity of CT, i.e. the range of helminth species that are susceptible, and what stages of the life cycle are targeted by these molecules.

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Acetone/water extracts were first tested in the widely-used assay of free-living larval development, which measures the ability of newly-hatched larvae from eggs to develop to infective L3 in the presence of a putative anthelmintic agent. In negative control wells, 80% of larvae developed to L3 after 7 days. The addition of 50 μg/mL levamisole resulted in 100% inhibition of egg hatching and therefore subsequent larval development. Egg hatching was not inhibited by the plant extracts, but all five plant extracts strongly inhibited the development of L1 to infective L3, with most larvae dying at the L1 or L2 stage (Figure 1). A dose-dependent relationship was evident for all extracts except for hazelnut skin, where all four tested concentrations (125 - 1000 μg/mL) inhibited development by more than 90% (Figure 1). Overall, these results clearly show that CT-containing plant extracts have anthelmintic activity against the free-living stages of O. dentatum.

Inhibition of development of Oesophagostomum dentatum larvae by plant extracts. Percentage development of O. dentatum free living stages, from eggs through L1-L2 to L3, in the presence of water, levamisole or different concentrations from five plant extracts.

Having established that all five extracts had activity against free-living larvae, anthelmintic effects were next assessed against the L3 larval stage that is infective to pigs and develops within the GI tract. First, the migratory ability of exsheathed L3 was quantified after incubation in the extracts. Migration of L3 was reduced (P

Activity of plant extracts and purified condensed tannin fractions against Oesophagostomum dentatum third-stage larvae. A - Percentage O. dentatum L3 migration after overnight incubation in media only, ivermectin (50 μg/mL) or plant extracts (1 mg/mL). Results are the mean of two independent experiments, each performed in triplicate. *P P P B - Percentage of O. dentatum L3 migration after overnight incubation in media only or in plant extracts pre-incubated with PVPP (see materials and methods). Results are mean of two independent experiments, each performed in triplicate. C - Percentage of O. dentatum L3 migration after overnight incubation in derived F2 ractions from hazelnut skin, willow bark or Tilia flowers. Results are mean of two independent experiments, each performed in triplicate. Concentration refers to the amount of CT in each fraction. The dashed line represents the number of migrated larvae after incubation in medium only (negative control). D - Percentage development of O. dentatum from L3 to L4 after 14 days incubation in medium only, plant extracts (1 mg/mL) or diethylcarbamazine (25 mM). Results are from a single experiment performed in triplicate. **P P

Effects of purified condensed tannin fractions on motility of Oesophagostomum dentatum fourth-stage larvae. Inhibition of O. dentatum L4 motility by derived F2 fractions from A) Hazelnut skin, B) white clover flowers, C) blackcurrant leaves, D) willow bark and E) Tilia flowers. Results are mean of two independent experiments, each performed in triplicate. Concentrations refer to the amount of CT in each fraction. IVM - 50 μg/mL ivermectin.

We found that the development of free-living larvae was potently inhibited by all five extracts from CT-containing plants. This larval development assay is commonly used to screen potential anthelmintic compounds [26], however it has the drawback of focusing on stages of the parasite life cycle which are not exposed to the compound in vivo. Therefore, it cannot be automatically assumed that results observed with these free-living stages can be extrapolated to the parasitic stages found within the GI tract. Indeed, we found that a concentration (1 mg/mL) that completely inhibited larval development with all five extracts had only modest inhibitory effects against migration of exsheathed L3 parasites. Hazelnut skin, blackcurrant leaf and white clover flower extracts had no significant effects on larval migration, whilst extracts from willow bark and Tilia flowers did significantly inhibit migration but only by 20-30% - an effect considerably less than the 95% inhibition observed after incubation in ivermectin. Therefore, it is apparent that whilst some CT-containing extracts can inhibit migration of infective larvae, overall the anthelmintic effect is less pronounced against this early parasitic stage than against free-living larvae. Therefore, our results highlight the importance of assessing putative anthelmintic compounds against not only free-living parasites but also parasitic stages. The active compounds within Tilia flowers and willow bark were confirmed as CT by both PVPP-incubation and fractionation experiments, which demonstrated that anthelmintic activity was retained in CT fractions of 80-90% purity. It is interesting to contrast the results obtained here with our previous work with another pig nematode, A. suum, where incubation of L3 in comparable concentrations of CT led to a substantially higher inhibition of larval migration [17]. This suggests that not all helminth species are equally susceptible to the anthelmintic effects of CT, and careful assessment of activity is necessary before the suitability of these natural anti-parasitic compounds can be considered as viable control options in varying host-parasite systems.

Given the overall lower efficacy of the isolated molecules against O. dentatum than comparable studies with A. suum[17], it may be that the mechanism of action of CT against O. dentatum is subtly different than to other helminths. At present, the anthelmintic mode-of-action of CT is not known, but is proposed to involve biochemical interactions between CT and proline-rich proteins on the nematode sheath or cuticle that interfere with both worm motility and feeding, and also key metabolic processes such as exsheathment (and perhaps also moulting, as suggested by our current data). This is supported by electron microscopy studies of worms exposed to CT that demonstrate direct structural damage to the cuticle [17],[35], consistent with the ultrastructural changes we observed in the present study with adult O. dentatum. Such a mechanism would appear to be fairly non-specific and broad-spectrum in nature, hence it is interesting to note the differences in susceptibility between different nematodes. O. dentatum falls within the Strongyloididea superfamily of nematodes, a distinct family from the Ascaraidoidea (e.g. A. suum) and Trichostrongylidae (e.g. T. colubriformis and O. ostertagi) superfamilies [36], and this divergence may represent biological differences that determine susceptibility to CT. Comparative studies of these two nematodes, including transcriptomic and proteomic analyses of worms exposed to equivalent amounts of CT, may shed some light on the more precise mechanisms of the anthelmintic effect, and such studies are on-going in our laboratory. The marked differences in the response of O. dentatum during free-living development and adults to CT, compared to L3 stages, is also deserving of further studies to determine the mechanisms responsible.

We have for the first time shown anthelmintic effects of CT-containing plant extracts and purified CT fractions against O. dentatum, and demonstrated that free-living/non-infective stages and adults appear to be highly susceptible to the effects of CT, whereas L4 are less susceptible and L3 are only modestly affected. Moreover, the moulting of L3 to L4 can be inhibited by CT, suggesting that specific, key processes in the parasite life cycle can be disrupted by CT. These data encourage further investigations to determine in vivo efficacy in pigs. In addition, further mechanistic studies, such as the relationship between the fine structure of CT molecules and anthelmintic activity, are also a high priority. 2ff7e9595c