Coccidiostatic effects of tannin-rich diets in rabbit production.

The potential anti-eimerial effect of tannin containing resources such as sainfoin and carob in rabbits was tested on does at pre-weaning and to growing rabbits in their feed. The trial began at parturition (D0), when 24 does and their litters were assigned into three groups. They were fed either with a control (Group CO), a carob (containing 10% carob pods meal) (Group CP) or a sainfoin diet (containing 34% dehydrated sainfoin pellets) (Group SA). All diets were made isoproteic and isoenergetic and also balanced for crude fibre but differed by their tannin content. Weaning occurred at D37, and growing rabbits remained in the same cage until D51. Then, they were transferred to fattening cages until the end of the trial (D104) and slaughtering. Weight gain of young rabbits among the three groups (mean = 31.2 g/day) did not differ statistically. The mortality rates were 10% (SA), 15% (CP) and 20% (CO), respectively, but the differences were not statistically significant. Post-weaning economical feed conversion ratio (FCR) was reduced between rabbits of group SA compared to CO and CP groups. Faecal oocyst count (FOC) in group SA was 60% lower than in CO and CP groups. Areas under the curve (AUCs) calculated between sampling days and FOC, after transfer to fattening cages, was 62% lower in group SA than in CO and CP groups. The main Eimeria species identified (from D59 to D83) was Eimeria magna (53% of oocysts). AUCs for E. magna did not differ according to diet. In conclusion, the diet containing sainfoin reduced oocyst excretion of Eimeria spp. by 60%, and improved the economical FCR.

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40µg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6µg/mL) compared to older larvae (P<0.05). The EC50 and EC90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC50 (r≥-0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.

Susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of polyphenol-rich plants. Part 2: Infective L3 larvae.

This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L3) of ten Haemonchus contortus isolates from different geographical origin. The L3 of 10 different isolates were exposed either to the acetone:water extract of a temperate plant (Onobrychis viciifolia) or a tropical plant (Acacia pennatula) and were evaluated with the larval exsheathment inhibition assay (LEIA). The L3 of each isolate were incubated with different concentrations of each extract (0, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200µg/mL of phosphate buffered saline (PBS)). After incubation, the exsheathment process of L3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L3 was determined for each concentration at 0, 20, 40 and 60min. Effective concentrations 50% (EC50) and the corresponding 95% confidence intervals (95% CI) were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated for each extract to compare isolates, using the most susceptible isolate as the respective reference for each extract. To determine the role of polyphenols on the reported effect, a second set of incubations was made for each isolate and each extract, using the extracts at a concentration of 1200µg/mL PBS with or without polyvinylpolypyrrolidone (PVPP), a polyphenol blocking agent, and controls without extract. The ten different H. contortus isolates showed variation in susceptibility for each of the 2 extracts tested (P<0.05). The EC50 values for A. pennatula extract ranged from 36 to 501µg/mL (RR: 2.11-13.68). Meanwhile, the EC50 values for O. viciifolia extract ranged from 128 to 1003µg/mL (RR: 1.25-7.82). The use of PVPP revealed that polyphenols were responsible for the anthelmintic activity recorded for both extracts. However, tested H. contortus isolates suggested that susceptibility to one polyphenol-rich extract did not determine the susceptibility to the other polyphenol rich extract. The latter result indicated that the different H. contortus isolates varied in their susceptibility to the polyphenols present in each extract evaluated.

The in vitro anthelmintic properties of browse plant species against Haemonchus contortus is determined by the polyphenol content and composition.

The aims of the present study were to (a) evaluate the anthelmintic activity of 10 East African browse plant extracts, (b) examine their role in inhibition of Haemonchus contortus larval exsheathment, (c) establish relationship between inhibition of larval exsheathment and browse plant extract polyphenol composition. Acetone/water (70/30%) extracts of air dried leaves of Acacia etbaica, Cadaba farinosa, Capparis tomentosa, Dichrostachys cinerea, Dodonaea angustifolia, Euclea racemosa, Maerua angolensis, Maytenus senegalensis, Rhus natalensis and Senna singueana were used. The larval exsheathment inhibition assay (LEIA) was applied using H. contortus third stage larvae (L3) and browse plant extract concentrations of 0, 150, 300, 600, 1200µg/ml in phosphate buffered saline (PBS). Data were analysed using the PROC MIXED procedure of SAS. Polyvinylpolypyrrolidone (PVPP) was used to evaluate whether polyphenols were involved in L3 exsheathment inhibition. All browse plant extracts significantly (P≤0.001) inhibited larval exsheathment in a dose dependent manner. The dose required to inhibit 50% of the larvae (EC50) was highest in C. farinosa and lowest in E. racemosa and M. senegalensis. Significant differences (P<0.001) between the control and PVPP treated A. etbaica, C. tomentosa, M. angolensis, R. natalensis and D. cinerea indicates that larval inhibition was largely due to non-phenol compounds. For E. racemosa, M. senegalensis, D. angustifolia and S. singueana, PVPP treatment reversed inhibition activity and in these extracts, inhibition was mostly attributable to tannin and other polyphenols (kaempferol, quercetin and myricetin based glycosides). Overall, the browse plant extracts have anthelmintic property against H. contortus and larval inhibition resulting from the presence of phenolic and non-phenolic compounds.

Nutritive value and anthelmintic effect of sainfoin pellets fed to experimentally infected growing rabbits.

Alternative strategies to synthetic chemical drugs are needed in livestock and are a key issue in organic farming today. This study aimed at examining the potentialities of sainfoin, a legume rich in condensed tannins, as a nutraceutical that combines nutritive and antiparasitic effects in rabbits. To test the effect of infection with a helminth (I: infected groups; NI: not infected groups) and the effect of substituting 40% of the alfalfa in a control diet (C) with sainfoin (diet S), four groups of 16 weaned rabbits were arranged according to a 2×2 bifactorial design. The sainfoin diet differed from the control by its tannin concentration (1.8% v. 1.0% tannic acid equivalent) and its ADL concentration (84 v. 43 g/kg). For each diet, 16 rabbits were infected with 2125 third-stage larvae of Trichostrongylus colubriformis. Growth, feed intake, feed conversion ratio and nematode faecal egg counts (FECs) were controlled for 6 weeks. A digestibility trial was performed. After necropsy, adult worms and eggs in utero per female were counted and egg-hatching rate calculated. Growth tended to be lower for S groups than for C groups (38.2 v. 39.5 g/day; P=0.06). Feed intake was higher for S groups compared with C groups (+5.2 g dry matter/day; P<0.01), as was the feed conversion ratio (3.2 v. 2.9; P<0.001), probably in relation to the dietary ADL level. Protein digestibility was reduced in S groups compared with C groups (-6.0 points; P<0.001), probably associated with the effect of the tannin concentration. Digestibility of hemicelluloses was reduced in infected rabbits compared with non-infected ones (-5 points; P=0.01). Using the substitution method, the digestible energy of dehydrated sainfoin pellets used as raw material was calculated at 11.12 MJ/kg and digestible proteins at 110 g/kg. The infection did not produce any clinical signs of digestive disorders. No differences were observed according to the diet, neither in the number of adult worms (972; P=0.50), the number of eggs in utero per female (14; P=0.95), nor FEC (400 eggs/g; P=0.57). In contrast, the rate of faecal egg hatching in the S group tended to be lower than in the control (58.3% v. 85.2%; P=0.08). In conclusion, sainfoin seems to fit nutritive requirements for rabbits, supplies a large quantity of fibre and particularly lignins, and limits the development of nematode eggs in faeces.

Interactions Between Nutrition and Infections With Haemonchus contortus and Related Gastrointestinal Nematodes in Small Ruminants.

Interactions between host nutrition and feeding behaviour are central to understanding the pathophysiological consequences of infections of the digestive tract with parasitic nematodes. The manipulation of host nutrition provides useful options to control gastrointestinal nematodes as a component of an integrated strategy. Focussed mainly on the Haemonchus contortus infection model in small ruminants, this chapter (1) illustrates the relationship between quantitative (macro- and micro-nutrients) and qualitative (plant secondary metabolites) aspects of host nutrition and nematode infection, and (2) shows how basic studies aimed at addressing some generic questions can help to provide solutions, despite the considerable diversity of epidemiological situations and breeding systems.

In vitro assessment of the anthelmintic activity of Hedysarum carnosum Desf. at different phenological stages and from six locations in Tunisia.

Gastrointestinal nematodes are compromising productivity of grazing sheep and goats. Therefore, scientists have been looking for cost-effective alternative options. Forage legumes (Fabacea Family) contain tannins that could improve livestock performance and their health as well. The present study aimed to (i) determine the in vitro anthelmintic (AH) activity of 19 acetonic extracts of Hedysarum carnosum Desf on Haemonchus contortus by a larval exsheathment assay (LEA); (ii) test the anthelmintic activity of condensed tannins using a deactivating reagent, polyvinylpolypyrrolidone (PVPP); (iii) study the effect of location and the phenological stage on the percentage of exsheathment. The LEA was used at different concentrations (150, 300, 600, 1200 µg mL-1 of acetonic extract/mL of purified buffer solution (PBS)). The larval exsheathment is concentration, location, phenological stage dependent. All extracts, caused a delay of the percentage of exsheathment over 50% so the AH activity of H. carnosum was confirmed. After addition of PVPP, the % exsheathment was similar to the 150 µg mL-1 concentration. The biplot showed that Loc1(S), Loc4(B), Loc 5(PF), Loc 6(BM) and Loc 6(PF) were isolated from other plant extract sample. Our in vitro study showed that H. carnosum seems to be a promising alternative to AH drugs.

In vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of two tannin rich plants.

The aim of the study was to examine the variation in the in vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins using respective egg hatch assays (EHA) with acetone:water extracts of two tannin containing plants, chimay (Acacia pennatula) and sainfoin (Onobrychis viciifolia). Fresh eggs were incubated in PBS with different concentrations of each extract (0, 600, 1200, 2400, 3600, 5000 and 8000 µg/ml PBS). Additional concentrations were tested for O. viciifolia (75, 100, 200 and 400 µg/ml PBS). Effective concentrations 50% (EC50), with the corresponding 95% confidence interval (95% CI), were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated to compare the isolates, using the most susceptible isolate for each extract as the respective reference. A second set of incubations were made using polyvinylpolypyrrolidone (PVPP) (0, 5000 µg/ml, 5000 µg/ml+PVPP) to determine the influence of polyphenols on the AH effect. The proportion of morulated eggs, eggs with L1 larvae failing eclosion (%LFE), and emerged larvae were estimated at different extract concentrations. Data of each isolate was used to calculate the effective concentration 50% (EC50) for each extract. The EC50 of each isolate was used to determine resistance ratio (RR) for the different isolates. For the 2 extracts, a susceptibility variation in egg hatching was observed for the different H. contortus isolates. The EC50 values for A. pennatula ranged from 2203 to 14106 µg (RR from 2.01 to 6.40). The O. viciifolia extract showed higher variability with EC50 values ranging from 104 to 4783 µg (RR from 3.66 to 45.74). The main AH effects of the two extracts tested on the ten isolates consisted in blocking the emergence of L1 larvae (higher% LFE). Additional observations on emerged larvae showed that extract exposure caused alterations in the internal structure, separating the cuticle from the pharynx, bulb and intestinal cells. The use of PVPP revealed that (a) condensed tannins were not the sole plant secondary metabolites responsible for the AH effects, and (b) different H. contortus isolates showed variability in the role of tannins either on the ovicidal effect or the %LFE.

Chemical Structures of Plant Hydrolyzable Tannins Reveal Their in Vitro Activity against Egg Hatching and Motility of Haemonchus contortus Nematodes.

The use of synthetic drugs against gastrointestinal nematodes of ruminants has led to a situation where resistance to anthelmintics is widespread, and there is an urgent need for alternative solutions for parasite control. One promising approach is to use polyphenol-rich bioactive plants in animal feeds as natural anthelmintics. In the present work, the in vitro activity of a series of 33 hydrolyzable tannins (HTs) and their hydrolysis product, gallic acid, against egg hatching and motility of L1 and L2 stage Haemonchus contortus larvae was studied. The effect of the selected compounds on egg and larval structure was further studied by scanning electron microscopy. The results indicated clear relationships between HT structure and anthelmintic activity. While HT size, overall flexibility, the types and numbers of functional groups, together with the linkage types between monomeric HTs affected the activity differently, the optimal structure was found with pentagalloylglucose.

Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock.

Parasitic infections with gastrointestinal nematodes (GINs) still represent a worldwide major pathological threat associated with the outdoor production of various livestock species. Because of the widespread resistance to synthetic chemical anthelmintics, there is a strong impetus to explore novel approaches for a more integrated management of these infections. The use of nutraceuticals in the control of GINs is one of the alternatives which has been widely studied for 20 years. The objectives of this review are: (i) to define and illustrate the concept of 'nutraceutical' in the context of veterinary parasitology based on data obtained on the most studied models to control GINs in small ruminants, the tannin-containing legumes (Fabaceae); (ii) to illustrate how the 'nutraceutical concept' could be expanded to other plants, other livestock production systems and other GI parasitic diseases, and (iii) to explain how this concept is opening up new research fields for better understanding the interactions between the host, the digestive parasites and the environment.

Anthelmintic activity of acetone-water extracts against Haemonchus contortus eggs: interactions between tannins and other plant secondary compounds.

This study aimed at (i) describing the effects of acetone-water extracts obtained from a range of different plant materials, on the hatching process of Haemonchus contortus eggs under in vitro conditions and (ii) identifying the role of tannins and other plant secondary compounds (PSC), on these AH effects by using polyvinylpolypyrrolidone (PVPP), an inhibitor of tannins and other polyphenols. An egg hatch assay (EHA) was used to determine the AH effect. Acetone-water (70:30) extracts from different foliages (Lysiloma latisiliquum, Laguncularia racemosa, Rizophora mangle, Avicennia germinans) and plant by-products (Theobroma cacao seed husk and pulp, and percolated Coffea arabica) were obtained. Fresh H. contortus eggs were incubated in PBS with increasing concentrations of each extract (0, 600, 1200, 2400 and 3600 µg/ml PBS). A general linear model was used to determine the dose effect of each extract. A mild ovicidal activity was only recorded for T. cacao extracts (seed husk and pulp). The main anthelmintic (AH) effect for all the extracts, except for C. arabica, was to block the eclosion of larvated eggs. The use of PVPP at 3600 µg/ml PBS showed that tannins of the L. racemosa extract were responsible for blocking eclosion of larvated eggs. Extracts of L. latisiliquum, A. germinans, T. cacao seed husk and pulp also blocked eclosion of larvated eggs but the addition of PVPP indicated that tannins were not responsible for that activity. In contrast, it suggested unfavorable interactions between polyphenols and other PSC contained in those extracts, limiting the AH effect on the egg hatching process. The present results suggest that the interactions between tannins and other PSC are complex and may reduce the AH effects against H. contortus eggs.

In vitro effect of heather extracts on Trichostrongylus colubriformis eggs, larvae and adults.

This study was carried out to evaluate the in vitro effects of different heather species on Trichostrongylus colubriformis eggs, larvae and adult worms, and obtain scientific evidence to attribute these effects to the action of their phenolic compounds and/or tannins. Total phenolic extracts of three heather species (Calluna vulgaris, Erica cinerea, and Erica umbellata) and an equal mixture of these three extracts were tested in vitro in the three development stages of T. colubriformis using an egg hatching assay (EHA), larval exsheathment inhibition assay (LEIA), and adult motility inhibition assay (AMIA). The egg hatching rate was measured after incubation with heather extracts for 48 h at 25°C. Infective third-stage larvae (L3) were incubated for 3h at 25°C with heather extracts. The evolution of artificial exsheathment over time was measured with repeated observations at 20-min intervals for 60 min. Adult worms were obtained from one donor goat and incubated with the extracts at 37°C for 5 days in 48-multiwell plates. Worm motility was measured at 0, 19, 24, 43, 48, 67, 72, 96 and 115 h after the beginning of the experiment. The extracts were tested at concentrations of 75, 150, 300, 600 and 1,200 µg/ml. All extracts significantly (P<0.001) inhibited egg hatching and the effect was dose dependent. All extracts inhibited or delayed the exsheathment of T. colubriformis L3, and the effect was dose dependent for C. vulgaris. Incubation with heather extracts induced a reduction in adult worm motility compared to control, although significant (P<0.05) differences were only found at the highest concentrations. Additional studies showed that purified tannins of the same heather species disturbed T. colubriformis larval exsheathment. All these results confirm the anthelmintic properties of heather against T. colubriformis, and suggest that not only tannins but also some other phenolic compounds might be involved.

In vitro effect of heather (Ericaceae) extracts on different development stages of Teladorsagia circumcincta and Haemonchus contortus.

The aim of the present study was to evaluate the in vitro effects of heather (Ericaceae) phenolic extracts on the abomasal nematodes Teladorsagia circumcincta and Haemonchus contortus. Extracts of three heather species (Calluna vulgaris, Erica cinerea, Erica umbellata and a balanced mixture of all three) were tested in vitro on different development stages of T. circumcincta (eggs, infective larvae and adult worms) and H. contortus (eggs and infective larvae) using an egg hatching assay (EHA), a larval exsheathment inhibition assay (LEIA) and an adult motility inhibition assay (AMIA). The egg hatching rate was measured after incubation with heather extracts for 48 h at 25 °C. Ensheathed infective larvae were incubated for 3h at 20 °C with heather extracts. Artificial exsheathment was induced in vitro by adding hypochloride solution to the larval suspension. The progress of exsheathment over time was measured by repeated observations at 10-min (T. circumcincta) and 20-min (H. contortus) intervals for 60 min. Adult T. circumcincta worms were obtained from two donor goats and incubated with the extracts at 37 °C for 3 days in 48-well multiwell plates. Worm motility was measured at 0, 19, 24, 43, 48, 67 and 72 h. The extracts were tested at concentrations of 75, 150, 300, 600 and 1200 µg/mL. Incubation with E. cinerea, E. umbellata and mixed heather extracts had a significant (P<0.01) dose-dependent effect on T. circumcincta egg hatching. H. contortus egg hatching was significantly (P<0.01) inhibited only by the E. cinerea extract. All extracts had a significant (P<0.01) dose-dependent effect on the exsheathment of T. circumcincta and H. contortus infective larvae. The incubation with all heather extracts induced a reduction in adult T. circumcincta motility compared to the control, although significant (P<0.05) differences were only found at the highest concentration (1200 µg/mL). The effect of the mixed extract was significant at all concentrations and significant effects were also observed for C. vulgaris and E. umbellata at 600 µg/mL. These results show anthelmintic properties of heather phenolic extracts against T. circumcincta and H. contort, thus confirming observations from previous in vivo studies.

Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions.

The structural changes induced in adult Haemonchus contortus after in vitro and in vivo contact with a tannin-rich (TR) plant, either tzalam (Lysiloma latisiliquum) or sainfoin (Onobrychis viciifolia), were assessed using scanning electron microscopy (SEM). All the worms used in the study were adult females. The Haemonchus adult worms were obtained from the abomasum of infected donor goats. Adult H. contortus were kept in contact with the extracts of TR plants for 24h for the in vitro assays and were compared to worms maintained in PBS (control). For the in vivo studies, the adult H. contortus parasites were obtained from artificially infected goats. All the goats were fed a tannin-free diet until D27 post-infection when infection was patent. On D28 some goats were fed fodder of one of the TR plants for seven consecutive days. Thus, their H. contortus were in contact with TR fodder within the gastrointestinal tract. The control worms were obtained from goats fed only a tannin-free diet. In the in vitro assays and in vivo studies, the SEM observations revealed structural alterations in the worms after contact with TR plants when compared to the control worms (i.e.: longitudinal and transversal folds and thicker cuticular ridges, material aggregates around the buccal capsule and/or vulva or anus). The main changes concerned the cuticle and the buccal area. The structural changes found in the worms exposed to TR plants might affect their motility and nutrition with possible consequences on their reproduction, as suggested by previous in vivo trials.

In vitro anthelmintic activity of the essential oils of Zanthoxylum zanthoxyloides and Newbouldia laevis against Strongyloides ratti.

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: ß-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 µg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50) = 2.5 µg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50) = 46 µg/ml), N. laevis (IC(50) = 51 µg/ml), and the control [levamisole (IC(50) = 36 µg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 µg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.

Direct and indirect effects of bioactive tannin-rich tropical and temperate legumes against nematode infections.

Parasitic infections with gastrointestinal nematodes (GINs) represent a major pathological threat associated with the outdoor production of various livestock species. Up to now, the control of these parasitic diseases essentially relied on the use of commercial anthelmintic drugs. However, resistance to anthelmintics is nowadays widespread in worm populations. Recent results indicate that bioactive tanniniferous plants represent a valuable option as an alternative to commercial drugs for the control of GINs. The pertinent use of tannin-containing fodders as nutraceuticals supposes a clear understanding of the mode of action against the worms. The objectives of this paper are: (1) to discuss the nature and quantity of the active molecules involved in the anthelmintic activity; and (2) to review and analyze the changes provoked to the various parasitic stages. The possible involvement of some main polyphenols to explain the bioactivity of some tannin-rich plants will be discussed as well as the possible effects on the various nematode stages, relying on data obtained either with the temperate forage, sainfoin (Onobrychis viciifoliae) or with the tropical legume tree (Lysiloma latisiliquum). The information on the mode of action will be related to the potential consequences for better field applications under entirely different environmental and ecological conditions of productions.

Ultrastructural changes in the third-stage, infective larvae of ruminant nematodes treated with sainfoin (Onobrychis viciifolia) extract.

Plants rich in condensed tannins are an alternative to chemical anthelmintics to control gastrointestinal nematodes (GINs) in ruminants. Previous functional studies have shown that sainfoin extracts affect the two forms of infective larvae (L3), ensheathed and exsheathed. However, the mechanisms of action remain unknown. The aim of this study was thus to compare ultrastructural changes in ensheathed and exsheathed L3 of two GIN species after in vitro contact with sainfoin extracts using transmission electron microscopy. The main changes identified were an alteration of the hypodermis, the presence of numerous vesicles in the cytoplasm and degeneration and/or death of muscular and intestinal cells. The changes suggested similar and nonspecies-specific lesions in the two nematode species. Comparison of the modifications found in the ensheathed vs. exsheathed L3s revealed different locations of the main cellular changes depending on the larval form. It is hypothesized that these spatial differences in lesions are mainly influenced by the presence of the sheath which favors contact between the active compounds and either the cuticle or the digestive tract. Overall, our observations suggest that the functional changes observed in the biology of GIN L3s after contact with sainfoin extracts are mediated through a direct mode of action, i.e. different interactions between the bioactive plant metabolites and the nematode structure depending on the route of contact.

Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus.

The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 µg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P<0.001) and A. gaumeri (P<0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L(3) larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.

Involvement of tannins and flavonoids in the in vitro effects of Newbouldia laevis and Zanthoxylum zanthoxyloïdes extracts on the exsheathment of third-stage infective larvae of gastrointestinal nematodes.

The present study aimed at examining the possible role of tannins and flavonoids on the in vitro anthelmintic properties of the extracts of two plants from the southern area of Western Africa, i.e. Newbouldia laevis and Zanthoxylum zanthoxyloïdes. Extracts of the two plants were prepared by use of acetone/water (70/30) and their anthelmintic activity was measured by use of the larval exsheathment inhibition assay (LEIA) applied on the abomasal species, Haemonchus contortus and the intestinal species Trichostrongylus colubriformis. Three concentrations of extracts were evaluated to examine the possible dose effect. In addition, the possible involvement of tannins and flavonoids was examined by comparing the levels of inhibition of larval exsheathment obtained with the same extracts, after of not addition of PVPP which forms complexes with these compounds. The results indicate significant effects with both plants and both nematode species. In the range of concentrations examined, the results were dose-dependent for N. laevis extracts but not for Z. zanthoxyloïdes because the three doses applied provoked a similar highly significant inhibition whatever the tested dose. The use of PVPP indicated for both plant and nematode species, that tannins and flavonoids are involved partly in the effect but that some other biochemical compounds were also involved in both plants.

Anthelmintic activity of some Mediterranean browse plants against parasitic nematodes.

The anthelmintic properties of tannin-rich plants are being explored as an alternative to chemical drugs. Most data have been acquired on legume forages, but only few on browse plants. The present study aimed to (i) screen the in vitro effects of extracts from 7 Mediterranean plants on Haemonchus contortus, (ii) verify the role of tannins using an inhibitor, polyvinyl polypyrrolidone (PVPP) and (iii) verify the in vivo effects of extracts from 4 plants. Significant inhibition was shown in vitro using a larval migration inhibition (LMI) assay for all extracts except that from Olea europaea var. koroneiki. After adding PVPP, the LMI values were restored to control levels for all plants except Pistacia lentiscus and Ceratonia siliqua, confirming a role for tannins in the activity. In the in vivo experiment, 48 lambs composed 6 groups, depending on diet. On Day 0, groups G1-G5 received H. contortus and Trichostrongylus colubriformis larvae and G6 remained uninfected. The various diets were distributed from Days 14 to 45: P. lentiscus (G1), Quercus coccifera (G2), C. siliqua (G3), Onobrychis viciifolia (G4), or Medicago sativa for the 2 control groups (G5, G6). Egg excretion, packed cell volumes (PCVs) and inorganic phosphate were measured weekly throughout the entire experimental period. At slaughter, the worms were enumerated and their fecundity assessed. Consumption of the 4 browser plants did not provoke differences in pathophysiological measurements but there were significant decreases in egg excretion, mainly explained by significant decreases in worm fecundity for both species, without any statistical difference in worm numbers.