Anthelmintic resistance in ruminants: challenges and solutions.

Anthelmintic resistance (AR) is a growing concern for effective parasite control in farmed ruminants globally. Combatting AR will require intensified and integrated research efforts in the development of innovative diagnostic tests to detect helminth infections and AR, sustainable anthelmintic treatment strategies and the development of complementary control approaches such as vaccination and plant-based control. It will also require a better understanding of socio-economic drivers of anthelmintic treatment decisions, in order to support a behavioural shift and develop targeted communication strategies that promote the uptake of evidence-based sustainable solutions. Here, we review the state-of-the-art in these different fields of research activity related to AR in helminths of livestock ruminants in Europe and beyond. We conclude that in the advent of new challenges and solutions emerging from continuing spread of AR and intensified research efforts, respectively, there is a strong need for transnational multi-actor initiatives. These should involve all key stakeholders to develop indicators of infection and sustainable control, set targets and promote good practices to achieve them.

Relationship between the excretion of eggs of parasitic helminths in roe deer and local livestock density.

Because of their continuing expansion, wildlife ruminant species that prosper in rural landscapes may be increasingly affected by and/or contribute to the circulation of certain generalist pathogens also infecting domestic ruminants, when they share common spaces or resources. In this study, we aimed to test the hypothesis that parasitism with gastrointestinal nematodes (GINs) of wild roe deer inhabiting different rural landscapes is correlated with livestock density. We used faecal egg counts of GINs and spatial data of 74 GPS-collared roe deer, inhabiting various landscapes from closed forests to open fields, together with weekly records of livestock abundances on pasture. We tested whether the excretion of GIN eggs in roe deer was influenced by the density of livestock in their home range over the grazing season. Our results showed that all of the roe deer home ranges, except four, contained pastures occupied by livestock. Excretion of GIN eggs occurred in 77% of the roe deer. The excretion of GIN eggs in roe deer tended to increase with livestock density in their home range. This result suggests, but does not prove, a higher risk of ingesting GIN larvae originating from livestock dung. In the context of increasing overlap between roe deer and livestock ranges, the exchange of pathogens between both hosts is plausible, although species identity of the parasites present was not determined. Assessing which GIN species are shared between wild and domestic ruminants, and how this may affect the health of both hosts, is a central question for future research in the context of interspecific pathogen circulation.

Relative abilities of young sheep and goats to self-medicate with tannin-rich sainfoin when infected with gastrointestinal nematodes.

Plant secondary metabolites (PSM) are one of the promising options to control gastrointestinal nematodes in sheep and goats. The objective of this study was to assess the abilities of sheep and goats to self-medicate with tannin-rich sainfoin (Onobrychis viciifolia) (SF) when infected with gastrointestinal nematodes, using a cafeteria and an operant conditioning trial. Hypotheses were that parasitized (P) lambs and goat kids would show greater intake and preference for SF than their non-parasitized (NP) counterparts, that kids would eat more SF than lambs (due to their lower resistance against parasites and their greater ability to consume PSM), and that SF intake would increase over time for P animals. We used 20 female kids and 20 ewe lambs aged 3 months. Half of the animals per species (n = 10) were experimentally infected with 170 L3 larvae of Haemonchus contortus/kg of BW (P). The other half were free from parasites throughout the study (NP). Five weeks after infection, animals were exposed to a 24-day cafeteria trial (three 8-day periods) offering a free choice between two legume pellets: SF (3.8% condensed tannins) and alfalfa (ALF, Medicago sativa; no tannin). Subsequently, animals were involved in an operant conditioning trial of two 4-day long sessions, to assess in short-term tests their motivation to walk for a SF reward when offered in choice with freely available ALF. In the cafeteria trial, SF preference was greater in kids than in lambs, particularly in the first two periods. We did not observe a greater preference for SF in P animals, which was even greater in NP animals for periods 1 and 2. Sainfoin intake increased through periods for P animals, which led to similar SF preferences for all groups during period 3. In the operant-conditioning trial, motivation to get the SF reward was similar between P and NP animals. These results support the hypotheses that goats are more willing to consume tanniferous feeds than sheep, and that P animals increased SF intake through time. However, the emergence of a curative self-medicative behaviour was not supported, as P individuals did not show greater SF intake, preference, nor a greater motivation to get SF than NP animals, regardless of animal species. These findings are discussed with previous results and some explanations are presented.

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.

Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs.

Gastrointestinal (GI) nematode control has an important role to play in increasing livestock production from a limited natural resource base and to improve animal health and welfare. In this synthetic review, we identify key research priorities for GI nematode control in farmed ruminants and pigs, to support the development of roadmaps and strategic research agendas by governments, industry and policymakers. These priorities were derived from the DISCONTOOLS gap analysis for nematodes and follow-up discussions within the recently formed Livestock Helminth Research Alliance (LiHRA). In the face of ongoing spread of anthelmintic resistance (AR), we are increasingly faced with a failure of existing control methods against GI nematodes. Effective vaccines against GI nematodes are generally not available, and anthelmintic treatment will therefore remain a cornerstone for their effective control. At the same time, consumers and producers are increasingly concerned with environmental issues associated with chemical parasite control. To address current challenges in GI nematode control, it is crucial to deepen our insights into diverse aspects of epidemiology, AR, host immune mechanisms and the socio-psychological aspects of nematode control. This will enhance the development, and subsequent uptake, of the new diagnostics, vaccines, pharma-/nutraceuticals, control methods and decision support tools required to respond to the spread of AR and the shifting epidemiology of GI nematodes in response to climatic, land-use and farm husbandry changes. More emphasis needs to be placed on the upfront evaluation of the economic value of these innovations as well as the socio-psychological aspects to prioritize research and facilitate uptake of innovations in practice. Finally, targeted regulatory guidance is needed to create an innovation-supportive environment for industries and to accelerate the access to market of new control tools.

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.

Tropical tannin-rich fodder intake modifies saliva-binding capacity in growing sheep.

We evaluated the effect of feeding dietary tannins from Lysiloma latisiliquum fresh forage on the saliva tannin-binding capacity of hair sheep lambs without previous exposure to tannin-rich (TR) fodder. Twenty-four hair sheep lambs (13.6±3.04 kg LW) were fed a tannin-free diet at the beginning of the experimental period (from day 10 to 13). On day 14, lambs were distributed into three groups (n=8): control group (CG), fed with the tannin-free diet (from D10 to D112); tannin short-term group (TST), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D55); tannin long-term group (TLT), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D112). Saliva samples were collected from the mouth of each lamb in the morning before feeding time on D10 and D14 (baseline period), on D49 and D56 (period 1) and on D97 and D112 (period 2). The tannin binding response of salivary protein (∆% turbidity) was determined with the haze development test (HDT) using either tannic acid or L. latisiliquum forage acetone extract. A turbidity protein index (TPI) was calculated as (∆% turbidity/[salivary protein (mg)]). Differences in HDT and TPI in the different groups were compared by repeated measures ANOVA using Proc Mixed. All groups had similar ∆% turbidity throughout the experiment (P>0.05). At baseline and period 1, the TPI of the different groups was similar (P>0.05). On period 2 the TLT group showed higher TPI compared with CG (P<0.05). Meanwhile, CG and TST showed similar salivary TPI. The saliva of hair sheep lambs consuming TR L. latisiliquum fresh fodder (TLT group) increased their TPI compared with control lambs not exposed to tannins.

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.