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This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L 3 ) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 μg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L 3 . A Log-Logistic model was used to determine the L 3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L 3 (T 50 ), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T 50 values obtained for GF, HA, and LL treatments (T 50 = 7.11 - 7.58 h) were higher in comparison to the T 50 of RL (5.72 h) (≥ 70 % probability of difference). The L 3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 - 48.06 % exsheathment reduction) compared to the RL treatment. The T 50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T 50 = 5.34 - 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L 3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T 50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study adapted the in vitro rumen incubation (IVRI) method to evaluate the biological activity of a Gymnopodium floribundum leaves extract against the exsheathment of Haemonchus contortus infective larvae (L 3 ), and to determine the role of plant polyphenols on the biological activity. The incubation protocol followed the IVRI method, adding polyethylene glycol (PEG) as a polyphenol-blocking agent. The L 3 were incubated in ruminal liquor (RL), ruminal liquor with PEG (RL+PEG), ruminal liquor with G. floribundum extract (RLE), and ruminal liquor with G. floribundum extract and PEG (RLE+PEG). Incubation condition controls included phosphate buffered saline (PBS), PBS with PEG (PBS+PEG), incubation medium (without ruminal liquor) (IM), and incubation medium with PEG (IM+PEG). The L 3 were recovered after incubation times of 0, 1, 3, 6, 9, and 24 h (39 °C). The respective L 3 exsheathment kinetics were estimated for the different treatments (RL, RL+PEG, RLE, and RLE+PEG) using Log-Logistic models. The parameters of the different models were compared to determine the impact of the extract, with or without PEG, on the L 3 exsheathment kinetics. The exsheathment in PBS and PBS+PEG remained < 2.71% at each incubation time. The exsheathment in IM and IM+PEG reached 13.58% and 17.18% at 24 h, respectively. The exsheathment percentages for RLE were lower than those for RL at 3, 6 and 9 h of incubation. The inflection point, indicating the time required to reach 50% of the maximal exsheathment (T 50 ), was the only parameter that differed between the ruminal liquor models. The T 50 in RLE (7.106 h) was higher than the values obtained for RL (5.385 h) and RL+PEG (4.923 h) (99.99% probability of being different). Such delay resulted in a reduction of exsheathment in RLE of 62% at 3 h, 38% at 6 h, and 12% at 9 h, relative to RL values. When PEG was added with the extract (RLE+PEG), the T 50 (5.045 h) was similar to that of RL and RL+PEG. The IVRI method was adapted as an in vitro rumen exsheathment test (IVRET). The IVRET showed that H. contortus L 3 exposed to G. floribundum extract delayed their exsheathment kinetics at different time points. The exsheathment delay was attributed to the polyphenol content of the extract., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To counter the global spread of anthelmintic resistance (AR), considerable efforts have been invested in the development and dissemination of sustainable alternatives to control gastrointestinal nematode (GIN) infections in small ruminants. The degree to which these have been accepted and integrated by farmers, particularly in organic systems, where the drive to reduce chemical inputs is arguably even more pressing, has been little studied. To identify whether more comprehensive actions are needed to support the uptake of alternative GIN controls amongst organic farmers, this study conducted a survey in five European countries on organic dairy goat and meat sheep farmers to gain insight into current GIN control strategies and farmer attitudes towards AR and alternative measures in these countries. The structured survey was disseminated in the five European countries Switzerland, France, Netherlands, Lithuania and United Kingdom, receiving a total of 425 responses, 106 from organic dairy goat farmers and 319 from organic meat sheep farmers. Regression analyses were carried out to identify factors impacting anthelmintic drenching on meat sheep production systems, whereas all data were analysed descriptively. Four key findings emerged: i) The frequency of anthelmintic treatments averaged less than two per animal a year in all production systems; ii) Overall, organic farmers seemed well informed on the availability of alternative GIN control methods, but fewer stated to put them into practice; iii) Targeted selective treatment (TST) strategies of anthelmintics appears to be not commonly incorporated by organic farmers; iv) Despite operating under national and EU organic regulations, each of the organic dairy goat (Switzerland, France and Netherlands) and meat sheep (Switzerland, Lithuania and United Kingdom) production systems developed distinct approaches for GIN control. To increase uptake of alternatives to GIN control and optimise anthelmintic use, initiatives that promote research dissemination, farmer participatory and knowledge transfer activities at national level would be desirable., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Haemonchus contortus is a parasite species that affects the health and production of grazing small ruminants in different parts of the world. Scanning electron microscopy (SEM) is an important tool for the study of parasites' morphology and taxonomy as it generates images that appear 3D and are generally easier to interpret than optical microscopy images. This study used the SEM to describe the vulval types of adult H. contortus from a Mexican isolate. A total of 14 adult H. contortus females were obtained from two artificially infected goats. Females were fixed and processed by critical point drying and observed with SEM. A collection of SEM images was obtained from these parasites and those images were used to identify the structures previously described by optical microscopy studies. Two different types of vulval structures were described in this Mexican H. contortus isolate: Type 1 (vulval flap), Type 2 (epiptygma). An unusual vulval structure was reported in a single individual. The Type 1 included vulval flaps of different sizes and spatial dispositions, as well as one or more knobs in different positions around the vulva. The Type 2 shows differences in the epiptygma. The present study suggests that the Mexican H. contortus isolate used in donor animals possess intraspecific polymorphism in vulval structures., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The present study explored the anthelmintic effects of condensed tannins (CT) in carob (Ceratonia siliqua) pods fed to sheep against gastrointestinal nematodes. Three independent in vivo trials tested whether i) carob pod (CaBP)-containing feed had an anthelmintic effect and if yes, which was the optimal concentration in the diet; ii) whether this effect could be attributed to tannins through the polyethylene glycol (PEG) test and iii) whether there were any synergistic effects when combined with another tannin-containing feed (e.g. sainfoin). In all trials 6-month old nematode-naive lambs, experimentally infected with both Haemonchus contortus and Trichostrongylus colubriformis, were used. Faecal egg counts (FEC) were performed regularly and at the end of each trial adult worm counts (AWC) and female worm fecundity were recorded. In trial 1, 35 lambs (five groups of seven lambs) were fed different CaBP concentrations ranging from 0% to 12 % w/w. FEC declined up to 39.2 % only in the group fed with 12 %CaBP, while a declining trend (P < 0.06) was demonstrated for the AWC of T. colubriformis, which was associated with the increasing concentration of CaBP in feed. Female worm fecundity was reduced in groups fed CaBP for both parasites, however this was only significant for H. contortus (P < 0.001), in a dose dependent manner. In trial 2, four groups of six infected lambs each were used, which received the carob diets CaBP or CaBP + PEG, and the tannin-free diets with or without PEG (C or C + PEG). Results showed that FEC of Groups C, C + PEG, and CaBP + PEG were comparable throughout the trial, while the group receiving only CaBP showed lower FEC from DAY 25 onwards. AWC showed a reduction (67.7 %) only for H. contortus (P < 0.03). Reversal of the anthelmintic effect of CaBP after PEG administration suggested that CT contributed to the anthelmintic action. However, no effect of CaBP was observed on T. colubriformis AWC and on female worm fecundity for both species. Finally, for trial 3 four groups of six lambs each received a diet based on CaBP, sainfoin (S) or a combination (CaBP + S) and were compared to a control (C) diet of lucerne. On DAY 37 FEC values in groups CaBP + S and S tended to be lower compared to the two other groups (C, CaBP), while for AWCs no significant differences were observed for both parasites. The fecundity of H. contortus and T. colubriformis demonstrated significant differences between the treated and control groups, with lower values in the animals receiving CaBP + S. Overall, the results supported the hypothesis that carob had an anthelmintic effect due to its CT, but there was no clear indication of a synergistic effect with sainfoin., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L 3 ) 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 L 3 of different ages (1-7 weeks). Each week, the L 3 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 L 3 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 (EC 50 , EC 90 ) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC 50 and EC 90 (39.4 and 65.6μg/mL) compared to older larvae (P<0.05). The EC 50 and EC 90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC 50 and EC 90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC 50 (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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L 3 ) of ten Haemonchus contortus isolates from different geographical origin. The L 3 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 L 3 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 L 3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L 3 was determined for each concentration at 0, 20, 40 and 60min. Effective concentrations 50% (EC 50 ) 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 EC 50 values for A. pennatula extract ranged from 36 to 501μg/mL (RR: 2.11-13.68). Meanwhile, the EC 50 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 (L 3 ) 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 L 3 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 (EC 50 ) 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The worldwide spread of resistance to anthelmintic (AH) drugs in gastrointestinal nematodes (GINs) imposes to explore alternative solutions. Amongst those, the possible use of tannin-containing nutraceuticals appears as a relevant option to replace (or decrease the frequency of) chemical-based treatments. Our objectives were to test the AH efficacy of sainfoin pellets against a multiresistant strain of Haemonchus contortus in experimentally infected lambs and to examine possible interaction between ivermectin (IVM) and condensed tannins (CT)-rich ressource. In vivo study was performed with twenty-four lambs were inoculated (Day 0) with multiresistant H. contortus infective larvae. On D21 Post-Infection, the lambs were assigned to two dietary treatments (sainfoin vs lucerne control pellets). On D39, half of the animals per group received 0.25ml/kg of an oral ivermectin treatment. On D47, animals were slaughtered to count worms. The consumption of sainfoin was associated with higher packed cell volume (PCV) values (P<0.05) and reduced faecal egg counts (FECs) (P<0.05). For the experimental feeding period, FECs were overall reduced by 50% in the sainfoin group. The diet did not have significant effect on the worm number but sainfoin significantly reduced female fertility. Decrease in plasma IVM concentrations was observed in the sainfoin-fed animals and was associated with a decrease of IVM efficiency when compared with the control group. Incubating tannin in vitro with ivermectin and rumen fluid showed a blocking of ivermectin by the tannins. This suggests that tannins lower the IVM intestinal absorption compromising thereby drug plasma bioavailability and efficacy. Tannin-containing nutraceuticals alter the biology of multiresistant nematodes, thus representing an option for their sustainable control. In vivo and in vitro interactions between nutraceuticals and chemicals impose caution when both tannin-rich diet and drug-based treatments are combined. Further studies are required to clarify the mechanisms that support such interactions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016. Published by Elsevier B.V.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study investigated the separate and combined anthelmintic (AH) effects of different phenolic compounds, including condensed tannins and flavonoids, all of which are known to occur in willow leaves, a potentially valuable dry season feed. A range of contrasting model tannins, which span the whole range of willow tannins, were isolated from tilia flowers, goat willow leaves, black currant leaves and red currant leaves. All together, the tested compounds represented the major tannin types (procyanidins and prodelphinidins) and flavonoid types (flavonols, flavones and flavanones). The larval exsheathment inhibition assay (LEIA) was used to assess their in vitro effects on Haemonchus contortus third stage larvae. Arbutin, vanillic acid, and taxifolin proved to be ineffective whereas naringenin, quercetin and luteolin were highly effective at 250 μM concentrations. Procyanidin (PC) tannins tended to be less active than prodelphinidin tannins (PD). Experiments with combinations of tannins and quercetin or luteolin revealed for the first time the existence of synergistic AH effects between tannins and flavonoid monomers. They also provided evidence that synergistic effects appear to occur at slightly lower concentrations of PC than PD. This suggests that the AH activity of condensed tannins can be significantly enhanced by the addition of quercetin or luteolin. This information may prove useful for plant breeding or selection and for designing optimal feed mixtures.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Anthelmintic resistance (AR) in ovine gastro-intestinal nematodes has been reported to affect the health and productivity of sheep globally. The objective of the present study was to evaluate the efficacy of commonly used oral drenches in sheep in France, Greece and Italy. In each country, 10 farms were selected. On each farm, 50 animals were blocked based on the pre-treatment faecal egg count (FEC). Within each block, animals were randomly allocated to one of 5 treatment groups. In addition to an untreated control group, there were 4 groups treated per oral route: moxidectin (MOX) and ivermectin (IVM), both at 0.2mg/kg bodyweight, levamisole (LEV; at 7.5mg/kg bodyweight) and a benzimidazole (BZ; at 3.75-5mg/kg bodyweight). In France, animals were not treated with LEV, but with netobimin (NET; at 7.5mg/kg bodyweight). The FEC was monitored using a modified McMaster technique. Two weeks after treatment, individual faecal samples were taken from all animals and efficacy was calculated as the difference between arithmetic mean FEC of the control group versus each respective treatment group. The results of the present study indicate the high efficacy of treatment with oral formulations of MOX (99-100%) and IVM (98-100%) on all farms, except on 1 farm in Greece. On this farm, multi drug resistance (MDR) was identified involving 4 anthelmintics (efficacy MOX: 91%; IVM: 0%; BZ: 58% and LEV: 87%). In Greece and Italy, AR against LEV and BZ was observed on some farms, with MDR involving both anthelmintics on 3 farms in Greece and on 2 farms in Italy. In France, AR against BZ and NET was observed on all 10 farms included. In all countries, Teladorsagia sp. was the most common nematode larva identified after treatment, followed by Haemonchus sp. and Trichostrongylus sp., with differences among farms and treatments. The current study confirms the high efficacy of oral treatments with MOX and IVM, even on farms with worm populations resistant to BZ, LEV or NET. This study also reports MDR against 4 anthelmintics on one farm in Greece., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011. Published by Elsevier B.V.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Infections with gastrointestinal nematodes (GIN) remain a major threat for ruminant production, health and welfare associated with outdoor breeding. The control of these helminth parasites has relied on the strategic or tactical use of chemical anthelmintic (AH) drugs. However, the expanding development and diffusion of anthelmintic resistance in nematode populations imposes the need to explore and validate novel solutions (or to re-discover old knowledge) for a more sustainable control of GIN. The different solutions refer to three main principles of action. The first one is to limit the contact between the hosts and the infective larvae in the field through grazing management methods. The latter were described since the 1970s and, at present, they benefit from innovations based on computer models. Several biological control agents have also been studied in the last three decades as potential tools to reduce the infective larvae in the field. The second principle aims at improving the host response against GIN infections relying on the genetic selection between or within breeds of sheep or goats, crossbreeding of resistant and susceptible breeds and/or the manipulation of nutrition. These approaches may benefit from a better understanding of the potential underlying mechanisms, in particular in regard of the host immune response against the worms. The third principle is the control of GIN based on non-conventional AH materials (plant or mineral compounds). Worldwide studies show that non conventional AH materials can eliminate worms and/or negatively affect the parasite's biology. The recent developments and pros and cons concerning these various options are discussed. Last, some results are presented which illustrate how the integration of these different solutions can be efficient and applicable in different systems of production and/or epidemiological conditions. The integration of different control tools seems to be a pre-requisite for the sustainable management of GIN infections. This new era of GIN management requires a new paradigm: to achieve enough control to reduce the negative impact of GIN infections enabling an optimum level of production, health and welfare., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bioactive plants with anthelmintic (AH) properties represent a promising alternative solution to chemical treatments. The AH effect of several Mexican tannin-rich (TR) plants has been screened in vitro. The in vivo AH effect of one TR legume, Lysiloma latisiliquum (Tzalam) on nematode larval establishment was confirmed. The present trial aimed at evaluating the direct and indirect effects of L. latisiliquum fodder consumption on adult Haemonchus contortus. Twenty-two parasite-naïve hair sheep lambs were allocated to an infected group (I) (400H. contortus L(3)/kg BW on D0) and a non-infected group (NI). From D0 to D28 post infection (PI), all the lambs were fed a complete diet. On D28, the two groups were sub-divided into four groups. Two control (C) groups maintained on the original basal diet (CI: 6 infected lambs and CNI: 5 non-infected lambs). The two treatment groups (T) received L. latisiliquum fodder ad libitum up to D36 when lambs were humanely slaughtered (TI: 6 infected lambs and TNI: 5 non-infected lambs). From D28 to D36 PI, individual fodder consumption and nematode egg excretion were measured daily. At necropsy, abomasal contents were recovered to obtain worm burdens and measure the female worm length and fecundity. Histological samples were taken from the respective abomasums and small intestines to count mucosal inflammatory cells. An increased consumption of TR fodder was observed in the TI vs. the TNI group (P<0.01). Before L. latisiliquum distribution, faecal egg excretion was similar in TI and CI groups. From D29 PI the TI group showed lower faecal egg counts compared to CI group (P<0.02). Although no differences in worm burdens were observed, worms of the TI group were smaller and, according to their size, contained fewer eggs in utero than worms from the CI group (P<0.05). Only minor differences in mucosal inflammatory cells were observed between groups, indicating that the indirect effect was not evident. Thus, a short-term consumption of L. latisiliquum can modulate directly the biology of adult H. contortus affecting the worm size and female fecundity while the worm burdens were not affected. Infected animals ate more L. latisiliquum fodder than non-infected animals.

This study was carried out to evaluate the in vitro effect of Cucurbita moschata seed against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and dichloromethane) of C. moschata seed were tested in vitro on four developmental stages of H. contortus using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition (LMI) assay and adult worm motility (AWM) assay. The highly significant (P<0.001) ability to stop larval development (inhibition>90% for each extract) and the negative effect of the dichloromethane and methanolic extracts on adult worm motility (inhibition of motility >59.2% after 24h of incubation) compared to the negative controls, suggest anthelmintic properties of C. moschata seed against H. contortus. The active principles responsible for the activity could be secondary metabolites such as amino acid compounds or terpenoid compounds present in the extracts.

The consumption of tannin-rich (TR) forages has been associated with negative effects against gastrointestinal nematodes and with an improved host resilience. It has been hypothesized that tannins affect the capacity of infective larvae to establish in the mucosae of the host. In this study, we aimed at testing this hypothesis using Lysiloma latisiliquum, a tropical TR tree. The objectives were: (i) to evaluate the effect of the consumption of L. latisiliquum on the establishment of nematode third-stage larvae (L3) in goats; (ii) to define the role of tannins in these effects in vivo by using an inhibitor (polyethylene glycol, PEG); and (iii) to examine a possible indirect effect of tannins on the inflammatory response in the digestive mucosa. Eighteen Criollo goats composed three experimental groups. The control group received fresh leaves of Brosimum alicastrum, a plant with a low level of tannins. Two groups received L. latisiliquum leaves either with (L.L.+PEG) or without (L.L.) daily addition of 25 g PEG. After a 7-day adaptation period, each goat was infected with both Haemonchus contortus and Trichostrongylus colubriformis (3000 L3 per species). The goats were slaughtered 5 days after infection and worm counts and histological analyses were performed. No difference in the voluntary feed intake of foliage was observed between the 3 groups. The consumption of L. latisiliquum significantly reduced the larval establishment of both nematode species compared to the control (P<0.01). For both worm species, the effects were totally alleviated with PEG (L.L.+PEG group), suggesting a major role of tannins in the observed effects. Only minor differences in the mucosal cellular response were observed between the 3 groups. These results confirm that the consumption of TR plants reduces the establishment of nematode larvae in the host and that a direct effect is principally involved.

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.

The aim was to assess the benefits obtained from combining supplementary feeding and copper needles (COWP), compared to the use of both approaches independently, for the control of gastrointestinal nematode (GIN) infections in browsing kids. Forty-four nematode free Criollo kids were exposed to natural parasite infection. The kids were divided into six experimental groups: not treated, supplemented (NT-S), not treated, not supplemented (NT-NS), moxidectin treated, supplemented (M-S), moxidectin treated not supplemented (M-NS), copper treated, supplemented (COWP-S) and copper treated, non-supplemented (COWP-NS). Copper treated groups received Copinox (2 g capsules) on day 0 and on day 60 of the trial. Moxidectin treated groups received Cydectin (0.2 mg/kg of body weight s.c.) every 28 days. Three of the groups received individual supplementation (100 g of feed/day fresh basis; 74% sorghum: 26% soybean meal; NT-S, M-S and COWP-S) and the other three groups were not supplemented (NT-NS, M-NS and COWP-NS). Animals browsed native vegetation (6.5 h/day) during the wet season (154 days). Kids were weighed every 14 days to determine live weight gain (LWG) and blood and faecal samples were obtained to determine packed cell volume (PCV), haemoglobin (Hb), peripheral eosinophil counts (PEC) and faecal egg counts (FEC). At the end of the trial, four kids of each group were euthanatized (six kids in each COWP treated group). Worm burdens, female worm lengths and prolificacy were determined. Liver samples were used to determine copper concentration and were stained with haematoxylin-eosin to determine microscopic lesions. Animals receiving the combination of supplementary feeding and COWP improved their LWG, PCV and Hb to similar levels of animals with suppressive AH treatment. This was not the case when COWP was used without supplementation. Liver copper concentration in COWP treated groups increased significantly especially in the COWP-NS kids but this was not associated with liver lesions or clinical signs. Post-mortem Haemonchus contortus and Trichostrongylus colubriformis worm counts had a tendency to be reduced in the different groups (66-35% reduction) compared to NT-NS group at the end of the trial (P>0.05). Also, COWP treatment and/or supplementation reduced female worm length of T. colubriformis and prolificacy of H. contortus and T. colubriformis. This study, confirmed the value of nutritional supplementation in the control of GIN in growing kids. The use of COWP in addition to supplementation had a limited contribution on the kids' resilience against GIN. This may be due to the reduced infection of H. contortus during this trial.

Haemonchus infection was monitored for 2 years (six kidding periods) in a Creole goat flock grazing under oceanic-tropical climate. Two hundred and thirty individual does were involved from late pregnancy to weaning. Anaemia diagnoses using Famacha method and packed cell volume (PCV) were compared. The best agreement (Kappa=0.33) was found if anaemia was declared when PCV values fell to 16 or below and Famacha score was 4 or 5. Drenching policy according to Famacha method was compared to systematic drenching policy. Using the Famacha method allowed a dramatic decrease in anthelmintic use during the periparturient period (0.57 individual dose instead of three doses for the control). The proportion of the nematode population on the pasture not derived from previously-treated goats (in refugia) was estimated to about 79% (65-90%) of the pasture contamination derived from the Famacha group. On the average, goats which needed to be drenched produced less than the control or undrenched goats (kid average daily gain was decreased by about 15%). The repeatability of the need for drenching individual does was estimated to 0.41. The older goats or the goats in poorer body condition at kidding needed more drenching than the younger animals and the animals in good body condition. Consequently the Famacha method may be used as an additional tool for the culling management.

Seriously escalating global anthelmintic resistance in gastrointestinal nematodes of small ruminants has spawned a variety of alternatives to anthelmintics for worm management, based on the need for sustainable Integrated Parasite Management (sIPM). Pivotal to the sIPM approach is the concept of refugia, the proportion of a given parasite population that escapes exposure to control measures. By balancing drug applications with the maintenance of refugia, the accumulation of anthelmintic resistance alleles in worm populations can be considerably delayed, while still providing good levels of control. The over-dispersed nature of parasitic infections provides an opportunity to achieve this balance, by targeting treatments to the members of a flock or herd that are least tolerant to nematode infection. However, implementation of this strategy has only recently become feasible, with the development of the FAMACHA((c)) system for clinical evaluation of anaemia due to haemonchosis. Subsequently, the use of milk yields has proven an effective indicator in dairy goats infected predominantly with nematodes other than Haemonchus contortus. In addition, short-term weight changes and perhaps also body condition scoring may provide indices of parasitism, permitting the rapid identification of animals likely to benefit from treatment. However, sIPM and refugia-based approaches are more complex than whole-flock treatments in conventional programs, and adoption by farmers is most likely where the theoretical basis is understood. As close communication with informed advisors is generally limited, there is a danger that sIPM will remain a theoretical concept without alternative modes of communication. The development of computer-based decision support programs, which use epidemiological, seasonal and clinical information to provide recommendations for specific situations, should be accorded high priority in the future development of worm management systems.

The purpose of this experiment was to determine if an earlier infection with Oestrus ovis would down regulate an infection with Trichostrongylus colubriformis when the larvae of O. ovis were expelled from the nasal cavities of sheep by a specific treatment. Three groups of five lambs were used: group 1 was artificially infected with O. ovis larvae and later with T. colubriformis, group 2 received O. ovis larvae and later was treated with ivermectin 14 days before being infected with T. colubriformis. Group 3 was infected with T. colubriformis only. The criteria examined were: the effects on nematode egg excretion, worm fecundity, nematode burdens and the kinetics of blood eosinophils. Significant decreases of nematode egg excretion, worm fecundity, nematode burdens were observed in group 1 compared to group 3. However, no changes were observed in either group 2 or 3. In group 2 it was noted that antiparasitic treatment induced a rapid decrease in blood eosinophils to a range close to the non-infected control group and this was associated with the removal of the down regulation effects of nematode burdens. This experiment showed that there is no cross immunity between O. ovis and T. colubriformis and that eosinophils may act against any parasite without specific priming.

The objective was to examine the coincidence in the prevalence of benzimidazole (Bz) resistance in smallholder goat herds, as determined by three average-based and two individually-based faecal egg count reduction (FECR) tests. Nineteen smallholder goat herds with more than 30 animals were selected from 84 herds in Yucatan. Animals shedding 150 eggs/g of faeces (EPG) on day zero were randomly divided into two groups. The control group did not receive treatment and the treated group received fenbendazole (10mg/kg body weight per os). Feed was withdrawn for 16 h before treatment. Ten days after treatment, both groups were sampled to determine their FEC. Faecal cultures and identification of infective larvae were performed for estimating the proportions of genera of gastrointestinal nematodes (GIN) present. Presence of resistant GIN was determined with three different average-based FECR (FECR(1), FECR(2) and FECR(3)) and two individually-based FECR (iFECR(1) and iFECR(2)). The prevalence of herds with Bz resistant nematodes (and 95% confidence interval (95% CI)) was calculated using each formula. Coincidence among formulae was estimated with Kappa values. The prevalence (+/- 95% CI) of Bz resistance calculated with FECR(1) (57.89 +/- 22.20) had a high coincidence with iFECR(1) and iFECR(2) (Kappa values of 0.86 and 0.79, respectively). The prevalence with FECR(2) (31.58 +/- 20.90) and FECR(3) (21.05 +/- 18.33) had a low coincidence with FECR(1) (Kappa < 0.50). Trichostrongylids found on Bz resistant farms were mainly Haemonchus spp., however, some Trichostrongylus spp. and Oesophagostomum spp. were found too. The high coincidence between the standard average-based FECR(1) and the individually based formulae is encouraging and may suggest that either formula could be applied to smallholder farmers. Further laboratory studies are needed to confirm the resistance status in the herds.

Mixed parasitic infection of animals is a common phenomenon in nature. The existence of one species often positively or negatively influences the survival of the other. Our experimental study was started with the objectives to demonstrate the interaction of Haemonchus contortus and Oestrus ovis in relation to cellular and humoral immune responses in sheep. Twenty-two sheep of Tarasconnais breed (France) were divided into four groups (O, OH, H and C) of five or six animals. Group O and OH received 5 weekly consecutive inoculations with O. ovis L1 larvae (total = 82 L1) in the first phase of the experiment between days 0 and 28. On the second phase, groups OH and H received 5000 L3 of H. contortus on day 48 while group C served as our control throughout the experimental period. Parasitological, haematological, serological and histopathological examinations were made according to standard procedures and all animals were slaughtered at day 95. There was no significant variation in the number and degree of development of O. ovis larvae between the two infected groups. Furthermore, in tissues examined in the upper respiratory tract (nasal septum, turbinate, ethmoide and sinus), group O and OH has responded similarly on the basis of cellular inflammatory responses (blood and tissue eosinophils, mast cells and globule leucocytes (GL)) and serum antibody responses against the nasal bots. This may indicate that the presence of H. contortus in the abomasa of group OH had no marked influence over the development of O. ovis larvae in the upper respiratory tract. On the other hand, we have observed a significantly lower H. contortus female worm length, fecal egg count (FEC) and in utero egg count in animals harbouring the nasal bot (OH) than in the mono-infected group (H). This was significantly associated with higher blood eosinophilia, higher packed cell volume (PCV) and increased number of tissue eosinophils and globule leucocytes. We conclude that, the establishment of O. ovis larvae in the upper respiratory tract has initiated higher inflammatory cellular activity in group OH there by influencing the development and fecundity of H. contortus in the abomasum.

Due to the high prevalence of anthelmintic resistance in goats, the need to explore novel approaches to control nematodes and to reduce the exclusive reliance on chemotherapy is strongly demanded in this host species. In sheep, several studies have shown that the consumption of tannin-rich legume forages was associated with positive effects on host resilience and resistance to parasite infection. In goats, studies on such interactions between tanniferous plants and nematode infections remain few. The objectives of the current study were to examine under natural conditions the effects of consumption of sainfoin hay by goats on the parasite populations and on host resilience. Eighteen adult cull goats naturally infected with Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis were used in the study. At the start of the assay, the goats were allocated into two groups, balanced according to weight and the levels of egg excretion. The two groups grazed separate pastures for 3 months with similar stocking rates. Goats from group S received each month indoors, for 7 days, sainfoin hay and control goats (group C) received hay of ryegrass. The diets in both groups were made isoenergetic and isoproteic and the refusals measured. Individual parasitological and pathophysiological measurements were performed fortnightly in order to compare host resistance and resilience. At the end of the study, five goats per group were necropsied. The distribution of sainfoin was associated with: (1) a higher consumption of hay; (2) significant, lower levels of nematode egg excretion which was associated with a decrease in worm fertility but no change in worm population; however, the number of intestinal worms was reduced by 50% in group S; (3) a better host resilience. In particular, after 2 months of grazing, two control goats died and half of the remaining animals needed to be treated whereas this was not the case in group S. These differences were related to significant changes in pepsinogen and phosphate values (PCV) but not in pepsinogen and phosphate concentrations. These results demonstrate that a repeated distribution of sainfoin hay to grazing goats might be beneficial in regard of pasture contamination and host resilience. They suggest that administration of sainfoin hay might represent a valuable alternative and adjunct to reduce nematode infections in dairy goat flock.

Twelve lambs were divided into two groups: Group C control, non-infected, and Group O infected once a week for 5 weeks with OEstrus ovis L1 through the same nostril. The first objective of this experiment was to check whether larvae moving through a given nostril remain in the same side nasal cavity or might to spread in both nasal cavities. It has been observed that larvae invade and spread through the entire nasal cavities. The only possible passage way between both sides is via the choanae and velum palatinum. The second objective was to follow the kinetics of blood eosinophilia. A primary peak in eosinophil numbers was noted 4 days following infection, with a higher peak following the second infection. After that, no major changes were seen. Nevertheless, the numbers of eosinophils were always higher than in control animals until the end of the follow-up. The third objective of the study was an enumeration of reactive cells (mast cells, globule leucocytes, and eosinophils) in the mucosae of the upper and lower respiratory tract after necropsy of the animals of the two groups. As observed in previous experiments, there was a large accumulation of these cells in mucosae of the upper respiratory tract. It was also worth noting a significant accumulation of eosinophils in the tissues of the trachea, bronchae and lungs even though OE. ovis was not present there. This "distant" eosinophilic reaction may have important consequences on patho-physiology of other parasites living in these locations: eosinophils have the potential to kill them even though these cells are not activated by their specific antigens.

Concurrent infections of sheep with Oestrus ovis and trichostrongyles of the digestive tract are common in the field. Previous results have shown that a previous infection with O. ovis adversely affects worm populations of either Trichostrongylus colubriformis or Haemonchus contortus. However, no information was available to determine the influence of the succession of infections on the expression of interactions between these parasites located in remote anatomical sites. In order to investigate the role of these modulating factors, an experimental study was conducted on four groups of naïve sheep, examining the consequences of a delayed infection with O. ovis on a pre-existing population of T. colubriformis. group T was infected four times with 4000 T. colubriformis larvae on days 0, 14, 28 and 42 of experiment; group O received multiple infections with O. ovis first instar larvae on days 42, 49, 56, 70 and 77; sheep from group TO received both infections and animals from group C remained as uninfected controls. Faecal egg counts and eosinophilia were measured weekly throughout the study. At necropsy (day 91), the mucosal cellular responses in the nasal cavities (septum, turbinates, ethmoid and sinus) and in the digestive tract (stomach and small intestine) from all animals were analysed from histological sections. Infection of the digestive tract with nematodes did not modify the biology of Oestrus populations, as measured by the number and weight of larvae. In contrast, infections with O. ovis after T. colubriformis infection was related to significant reductions (P < 0.01) in nematode egg excretion and worm burdens. These changes were associated with significant modifications in populations of mast cells, globule leucocytes and eosinophils in the respiratory and digestive tracts. These results indicate that an antagonistic interaction exists between the populations of O. ovis in the nasal cavities and T. colubriformis in the small intestine but that the order of succession of infections with the two parasites is not a major modulating factor for expression of interactions. They also confirm that parasitic infection in one particular anatomical site induces "at distance" inflammatory reactions of the whole mucosal system.

Although the use of tanniferous plants or condensed tannins as an alternative to anthelmintics to control gastrointestinal nematodes has been largely documented in sheep, studies remain scarce in goats. The objective of this study was therefore to assess the possible impact of condensed tannins in goats infected with adult Haemonchus contortus. Two groups of cull goats were experimentally infected with 10.000 L3 of H. contortus. After 4 weeks, quebracho extracts, representing 5% of the diet DM, were administered for 8 days to one of the two groups. Goats of the second group remained as controls. One week after the end of quebracho administration, the goats were euthanised. Individual egg excretion and pathophysiological parameters were measured weekly during the study. At the end of the study, worm counts were assessed and histological samples from the abomasa were taken to count the numbers of mucosal mast cells, globule leukocytes and eosinophils. The administration of tannins was associated with a significant decrease in egg excretion, which persisted until the end of experiment. This reduction was not associated with any difference in worm number but with a significant decrease in female fecundity. No significant changes in the mucosal density of the three inflammatory cell types were detected between the two groups. These results indicate that the major consequence of tannin consumption in goats is a reduction in worm fecundity and egg output, which does not seem related to significant changes in the local mucosal response.

Infection by Oestrus ovis is common in Lacaune dairy ewes of Roquefort cheese area (Aveyron, France). It is believed by local breeders that there is a close relationship between nasal myiasis and the incidence of enzootic nasal tumour. In order to check these anecdotal reports, a serological survey was done on 658 breeding ewes before turn-out and 897 breeding and primiparous (hoggets) ewes at the end of the grazing season. By the time of sampling, it was clear whether the sheep were infected at the end of the winter or had been re-infected over summer. In April and September, 40.7 and 26.3%, respectively, were free of O. ovis infection, indicating that the autumn treatment was not completely effective and that O. ovis adult flies were circulating during the summer in many flocks. There were no differences in the incidence of adenocarcinoma between the groups indicating that there is no relationship between O. ovis infection and the presence of the cancer. Differences in milk production between the three groups were not statistically significant (Anova test P>0.05). In flocks where 1-5% of the ewes were infected or in non-infected flocks, ewes produced 3.6 and 8.56%, respectively, more milk than ewes from flocks where more than 5% of animals were infected. For primiparous ewes, the differences were of 8.5 and 12.24%.

The targeted application of anthelmintic treatments represents one of the current available solutions to slow down the development of anthelmintic resistance within worm populations. Within an experimental flock of dairy goats, control of gastrointestinal parasitism by such selective treatments, targeting the most receptive animals within a flock, was previously found to be effective and to have no detrimental consequences on milk production. The objectives of the current study were to verify the validity of this method in farm conditions. Eleven dairy goat farms from three main areas of production in France were surveyed for 2 years. In six farms, the survey was prolonged for a third year. During year 1, systematic treatments were applied during the grazing season whereas in year 2 and or year 3, treatments were given exclusively to the goats in first lactation and to the multiparous ones with the highest potential of milk production. The level of nematode infection was measured four times per year through individual coproscopical examinations and the mean annual production of milk was recorded. No significant changes in egg excretion nor in milk production were noticed in any farm during years 2 or 3 after switching from the systematic to the selective mode of treatments. These results confirmed that targeted application of anthelmintics might represent a way to combine the control of trichostrongyles and the prevention of anthelmintic resistance., (Copyright 2002 Elsevier Science B.V.)

Forty-two horses from Normandy (France) were examined post-mortem for small strongyle infections from October to March. In the positive horses, total worm numbers ranged from 234 to 90,247 (mean 11,297). Encysted larvae represented the major part of the total cyathostome burdens with a high percentage (83%) being early third stage larvae. They were mostly recovered from the caecum (48%) and ventral colon (40%) and were less present in the dorsal colon (12%). Adult cyathostomes were mainly located in the ventral colon (64%) and less frequently in the dorsal colon (27%) and caecum (9%). Twenty species of Cyathostominae were identified. The 10 most prevalent species (in sequence of prevalence) were Cyathostomum coronatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, Poteriostomum imparidentatum, Cyathostomum labiatum, Cylicocyclus ultrajectinus, Cylicostephanus calicatus and Cylicostephanus minutus which comprised 84% of the total adult population. Twelve species showed a site preference in the ventral colon, five in the dorsal colon and only one in the caecum while two species were collected in nearly equal numbers from the ventral and dorsal colon. The number of species per horse ranged from 1 to 12 with a median of 5. Infections with singletons occurred in 12.5% of the positive horses while multiple infections were encountered in 87.5%. A positive correlation was found between the intensity of cyathostome infection and its diversity, measured either by the number of occurring species or Shanon indexes., (Copyright 2002 Elsevier Science B.V.)

From December 1998 to March 2000, caecum and ascendant colon of 42 horses naturally infected with cyathostomes were collected during routine necropsy or from a local slaughterhouse. Changes in the numbers of mucosal and submucosal mast cells (MMC and SMMC), intraepithelial, mucosal and submucosal eosinophils (IE, ME and SME) in the large intestine were investigated by histochemical techniques in relation to the worm burdens. The effect of age was examined in three subgroups: 6-24-month-old horses (group 1), 2-10-year-old horses (group 2) and horses more than 10 years of age (group 3). No globule leucocytes were detected in any sections. No significant variations with breed or sex were observed in cell counts. The main variations were higher eosinophil counts in groups 2 and 3 and a marked increase of the MMC counts in the oldest horses (group 3). For each cell type, the infiltration was homogeneous and generalised along the large intestine. In the whole horse sample, the IE numbers were the only parameters that correlated with the MMC and SMMC counts. Very few significant relationships were found between mast cells and eosinophils in groups 1 and 3, whereas numerous positive correlations were recorded in group 2. In the whole horse sample, several correlations were found between different cell counts and cyathostome burdens. The numbers of larvae, adult worms, and the total worm burdens were related to some of the tissular eosinophil counts while the percentage of early third stage larvae (EL3) was linked to mast cell densities. These relations between cells and worm populations showed variations with age. In group 1, most of the significant associations were found between eosinophil counts (IE and SME) and the total numbers of larvae and worms; in group 2, they were noticed between the three eosinophil types and the total cyathostome burdens. In group 3, a MMC hyperplasia was observed and correlations were mostly recorded between these MMC and the total numbers of adult worms or the percentage of EL3. Several associations were also detected between eosinophils (mainly ME and/or IE) and different cyathostome burdens. These variations in the relationship between inflammatory cells and cyathostomes seemed to be consistent with the cellular changes observed among the three age groups. These results suggest that eosinophil and mast cell infiltrations quantified in the large intestine wall might be associated with cyathostome infection.

Resistance to anthelmintics in nematode parasite of the digestive tract is a major concern in small ruminants and particularly in goats. One possible solution to limit the development and spread of resistance is to give treatments on a selective basis, i.e. by targeting the most susceptible animals within a flock rather than treating all the animals. In dairy goats, epidemiological studies have shown that, within a flock, goats in first lactation and the multiparous ones with the highest level of milk production were highly receptive to parasite infection. The objective of the study was to assess whether selective treatments could achieve a level of control of nematodes similar to systematic drenching. A similar experimental design was applied for two successive years. An experimental flock of 120 dairy goats was divided into two groups. All the goats from group SYS (systematic treatment) were drenched at mid-grazing season (July). In contrast, anthelmintics in group SEL (selective treatment) were restricted to the goats in first lactation plus the high producers. Overall, these treated goats represented 48% of the flock in year 1 and 66% in year 2. After treatments, both groups grazed on separate pastures. Parasitological and pathophysiological measurements were performed monthly. The results indicate a similar level of egg excretion in the two groups. The pathophysiological parameters (pepsinogen and phosphate concentrations) were also similar in the two groups, as well as the milk production for the 2 years. These results suggest that a targeted use of anthelmintics may allow efficient control of gastrointestinal nematodes whilst resulting in a predicted reduction in the selection pressure for the development of anthelmintic resistance.

Concurrent infections of sheep with Oestrus ovis and trichostrongyles of the digestive tract are common in the field. In order to examine the possible occurrence of interactions between these two parasites and the consequences on parasite biology and the associated pathophysiological changes, an experimental study was conducted on four groups of naïve sheep. Group O was infected repeatedly with O. ovis larvae from D0 to D42 of experiment; group T received a single infection of Trichostrongylus colubriformis on D49; sheep from group OT received both infections, and animals from group C remained uninfected as controls. Parasitological and pathophysiological parameters were measured regularly. At necropsy (D94), the mucosal cellular responses in the nasal cavities and digestive tract from all animals were analysed from histological sections. Infection of the digestive tract with nematodes did not modify the biology of Oestrus populations. In contrast, the presence of O. ovis was related to significant reductions in nematode egg excretion, worm fecundity and worm burdens. These changes were associated with significant modifications in tissular populations of mast cells, globule leucocytes and eosinophils in the respiratory and digestive tracts. These results indicate that parasitic infection in one particular anatomical site induces "at distance" inflammatory reactions of the whole mucosal system. The relationships between the cellular response and the changes in worm biology are discussed.