The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae.

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) 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 L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 - 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 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 T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 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 L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 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.

Adapting the in vitro rumen incubation method to evaluate the effect of a plant extract on the exsheathment inhibition of Haemonchus contortus infective larvae.

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 (L3), 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 L3 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 L3 were recovered after incubation times of 0, 1, 3, 6, 9, and 24 h (39 °C). The respective L3 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 L3 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 (T50), was the only parameter that differed between the ruminal liquor models. The T50 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 T50 (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 L3 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.

Two in vitro anthelmintic assays of four Paraguayan medicinal plants for proof of concept of the role of polyphenols in their biological activities and LC-HRMS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Haemonchus spp., and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. THE AIMS OF OUR STUDY: Four medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occidentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. MATERIALS AND METHODS: To evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without polyvinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). RESULTS: C. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42-41.80 µg/mL) and A. absinthium on egg hatching processes (EC50 = 121.70-137.34 µg/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus, and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20-67.00% of larvae exsheathment, (p < 0.05) and 40.51-24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. CONCLUSIONS: The present study demonstrated that C. occidentalis, A. absinthium, and K. odorata, which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.

Reducing anthelmintic inputs in organic farming: Are small ruminant farmers integrating alternative strategies to control gastrointestinal nematodes?

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.

An in vitro rumen incubation method to study exsheathment kinetics of Haemonchus contortus third-stage infective larvae.

This study developed and evaluated an in vitro rumen incubation (IVRI) method to describe the exsheathment kinetics of Haemonchus contortus third-stage infective larvae (L3) in ruminal liquor (RL). The specific objectives were (i) to standardize the IVRI method to facilitate the contact between L3 and RL as well as the larval recovery, and (ii) to apply the IVRI method to describe the exsheathment kinetics of H. contortus and to select the best fitting nonlinear model. Incubation devices containing H. contortus larvae were incubated according to the IVRI technique in cattle RL or PBS. The incubation conditions included RL mixed with a nitrogen-rich media, maintained at 39 °C, with pH = 7.0, vented with CO2 and manual agitation. The larvae were recovered after 0, 1, 3, 6, 9, 12, and 24 h. The exsheathed and ensheathed larvae were counted to estimate the exsheathment (%) in RL or PBS. Exsheathment in RL was analyzed with nonlinear regression models: Exponential, Gompertz, Logistic, Log-Logistic, and Weibull. The models' fit was compared to select the one that best described the exsheathment kinetics. The exsheathment in RL reached 6.52%, 20.65%, 58.22%, 69.24%, 73.08%, and 77.20% in 1, 3, 6, 9, 12, and 24 h, respectively. Although the Gompertz, Weibull, and Logistic models were adequate to describe the observed exsheathment, the Log-Logistic model had the best fit. The IVRI method using bovine RL represents a suitable tool for the study of the in vitro exsheathment kinetics of H. contortus L3.

Impact of Seasonal and Organ-Related Fluctuations on the Anthelmintic Properties and Chemical Profile of Cladium mariscus (L.) Pohl Extracts.

The use of plants and their metabolites stands as a promising option to tackle parasitic infections by gastrointestinal nematodes (GIN) in integrated control strategies. Still, the influence of environmental and phenological factors, and their interactions, in the wild on the metabolomics and biological properties of target plant species, is often disregarded. In this work, we hypothesized that variations in the anthelmintic (AH) properties and chemical composition of extracts from the salt tolerant species Cladium mariscus L. Pohl (sawgrass) may be influenced by seasonal factors and organ-parts. To test this hypothesis, acetone/water extracts were prepared from dried biomass obtained from aerial organs collected from sawgrass in consecutive seasons and tested against Haemonchus contortus and Trichostrongylus colubriformis by the larval exsheathment inhibition assay (LEIA) and egg hatching inhibition assay (EHIA). To ascertain the role of plant organ, the activity of leaves and inflorescences extracts from summer samples was compared. The role of polyphenols in the anthelmintic activity depending on GINs and fluctuations across seasons and plant organs was assessed using polyvinylpolypyrrolidone (PVPP), coupled with an in-depth chemical profiling analysis using high-performance liquid chromatography completed with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn). Main differences in anthelmintic activities were observed for summer and autumn samples, for both assays. Moreover, inflorescences' extracts were significantly more active than those from leaves against both parasite species on EHIA and against H. contortus on LEIA. Application of PVPP totally inhibit the AH effects based on EHIA and only partly for LEIA. Non-treated PVPP extracts were predominantly composed of flavan-3-ols, proanthocyanidins, luteolin and glycosylated flavonoids, while two flavonoid glycosides were quantified in all PVPP-treated samples. Thus, the activity of such compounds should be further explored, although some unknown metabolites remain to be identified. This study reinforces the hypothesis of the AH potential of sawgrass and of its polyphenolic metabolites uses as nutraceutical and/or phytotherapeutic drugs.

Use of agro-industrial by-products containing tannins for the integrated control of gastrointestinal nematodes in ruminants.

Previous studies have illustrated that different bioactive legume fodders containing condensed tannins might represent one of the options for integrated sustainable control of gastrointestinal nematodes (GIN) in ruminants, which may help address the worldwide development of resistance to synthetic anthelmintics. More recently, impetus has been given to assess the potential antiparasitic activity of less conventional resources, represented by different agro-industrial by-products (AIBPs). This review presents in vitro and in vivo results obtained with a range of tannin-containing AIBPs of various geographical and botanical origins, namely AIBP of nuts, temperate and tropical barks, carob, coffee and cocoa. They tend to confirm the "proof of concept" for their antiparasitic effects and also for other aspects of ruminant production in an agro-ecological context. Socio-economic aspects of the exploitation of such non-conventional resources are also discussed as potential models of the circular economy, by using waste. The different modes of use of these resources are presented in this review, as well as strengths, weaknesses, opportunities, and threats (SWOT) analyses to illustrate the advantages and limitations of on-farm use.

TITLE: Utilisation de sous-produits agro-industriels contenant des tanins pour le contrôle intégré des nématodes gastro-intestinaux chez les ruminants. ABSTRACT: Plusieurs études antérieures ont illustré le fait que des légumineuses bioactives contenant des tannins condensés peuvent représenter une des alternatives à intégrer avec d'autres options pour une maitrise durable des nématodes gastro-intestinaux en réponse au développement constant et à l'expansion continue à l'échelle mondiale des résistances aux anthelminthiques de synthèse. Des recherches plus récentes se sont intéressées au potentiel d'application de ressources moins conventionnelles que représentent des coproduits agroindustriels (CPAI). Cette revue vise à présenter des résultats in vitro et in vivo obtenus avec une gamme de CPAI d'origines géographiques et botaniques diversifiées (coproduits de l'industrie des noix, du bois (en régions tempérées et tropicales), du caroubier, du café et du cacao). Ces résultats ont confirmé la preuve de concept pour les effets antiparasitaires, et aussi pour d'autres volets de la production des ruminants dans un contexte agro écologique de l'élevage. Par ailleurs, les aspects socio-économiques d'exploitation de ces ressources, considérées jusqu'à présent comme des déchets, dans un modèle de circuits courts sont aussi évoqués. Les avantages et inconvénients des différentes modalités d'exploitation des CPAI sont aussi discutés dans le cadre d'une analyse SWOT.

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

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

Scanning electron microscopy of different vulval structures in a Mexican Haemonchus contortus isolate.

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.

In vitro Evaluation of the Nutraceutical Potential of Theobroma cacao pod Husk and Leaf Extracts for Small Ruminants.

PURPOSE: Some agroindustrial by-products could be used as nutraceutical materials for small ruminants helping with their nutrition while controlling their gastrointestinal nematodes. This study evaluated the potential in vitro nutraceutical value of pod husks and leaves of three varieties of Theobroma cacao using two Haemonchus contortus isolates with different polyphenol susceptibility. METHODS: Leaves and husks from three T. cacao varieties (AZT, CAL and CEY) were evaluated for their bromatological composition, in vitro dry matter digestibility and polyphenol content. Acetone:water extracts (70:30) of each plant variety were evaluated using the egg hatch and larval exsheathment inhibition tests, using two isolates (FESC and PARAISO) of H. contortus. Effective concentrations 50% (EC50) were determined for both tests. The role of polyphenols was confirmed using polyvinylpolypyrrolidone. L3 exposed to CAL leaf extract were submitted to transmission electron microscopy. RESULTS: Both plant materials showed a good nutritional value to complement protein-rich diets for small ruminants. Extracts inhibited exsheathment of H. contortus L3 more effectively than the egg hatching, and the leaf extracts were more active than husk extracts in the L3 exsheathment inhibition. The FESC isolate was more sensitive to extracts. Polyphenols blocked exsheathment inhibition of leaf extracts. Structural damage was observed in the sheath and muscles of L3 exposed to CAL leaf extracts. CONCLUSION: The two T. cacao materials tested showed their potential to be used as ruminant feeds. Extracts affected H. contortus by blocking L3 exsheathment, particularly with the leaf extracts. The in vivo nutraceutical value should be confirmed in small ruminants.

A systematic review on the ethnoveterinary uses of mediterranean salt-tolerant plants: Exploring its potential use as fodder, nutraceuticals or phytotherapeutics in ruminant production.

ETHNOPHARMACOLOGICAL RELEVANCE: Salt-tolerant plants are well adapted to the harsh conditions of the Mediterranean region, where have been used traditionally as food and medicines for human and animals. In addition, various species are currently recognized as sources of metabolites with pharmacological, cosmetical and nutraceutical interest. Nevertheless, ethnoveterinary data on salt-tolerant plants are dispersed in the literature and there are few discussions on its veterinary potential. Having in mind the rising interest on organic farming, alternatives to chemical substances in livestock production and concern for animal health and welfare practices, these plants may represent an untapped resource for animal management and veterinary purposes. In this sense, the purpose of this work is to summarize the ethnoveterinary knowledge on salt-tolerant plants described in the Mediterranean region, raising awareness to the potential of this group of plants to be used in veterinary science, targeting especially ruminants. MATERIAL AND METHODS: Literature search (2000-2020) was conducted using Web of Science and Science Direct databases. Ethnoveterinary reports (EVR) concerning salt-tolerant plants were summarized and filtered for ruminants. From the final 29 publications, EVR concerning therapeutic uses were categorized according to its ATCvet code and results analyzed. RESULTS: A total of 221 EVR were identified from 39 plants, belonging to 21 plant families, targetting ruminants. Ten EVR (4.5%) concerned uses of salt-tolerant species as animal feed, while around 75% of therapeutic uses was represented by three categories: alimentary tract and metabolism (QA; n = 75), dermatologicals (QD; n = 53) and genitourinary system and sex hormones (QG; n = 41). Pistacia lentiscus L., Foeniculum vulgare Mill., Dittrichia viscosa (L.) Greuter, Plantago major L. and Hordeum vulgare L. were the most cited species in the latter categories. CONCLUSIONS: The ethnoveterinary knowledge on salt-tolerant species hints some plants of veterinary pharmacological potential, but other species deserve further notice. This information should serve as a basis and, coupled with the currently available scientific data on bioactive properties and chemical composition of salt-tolerant species, inspire additional research on the exploitation of this botanical group, as sources of novel products for ruminant nutrition, health and quality of its products.

Feeding of carob (Ceratonia siliqua) to sheep infected with gastrointestinal nematodes reduces faecal egg counts and worm fecundity.

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.

Ellagitannins Inhibit the Exsheathment of Haemonchus contortus and Trichostrongylus colubriformis Larvae: The Efficiency Increases Together with the Molecular Size.

Worldwide, parasitic gastrointestinal nematodes continue to threaten animal health, welfare, and production in outdoor breeding systems of small ruminants. For more than 50 years, the control of these parasitic worms has relied on the use of commercial synthetic anthelmintics. However, anthelmintic resistance in worm populations is nowadays widespread and requires novel solutions. The use of tannin-rich plants has been suggested as an alternative to synthetic anthelmintics to control gastrointestinal nematodes. The majority of previous studies have focused on the activity of proanthocyanidins (syn condensed tannins), and less is known about ellagitannins. In this study, the effects of 30 structurally unique ellagitannins on the exsheathment of third-stage infective larvae were examined on Haemonchus contortus and Trichostrongylus colubriformis by the in vitro larval exsheathment inhibition assay. Ellagitannins were found to be promising natural anthelmintics as they showed direct inhibition on larval exsheathment for both nematode species. In general, ellagitannins were more efficient at inhibiting the exsheathment of H. contortus larvae than those of T. colubriformis. The efficiency of inhibition increased as the degree of oligomerization or the molecular weight of the ellagitannin increased. Otherwise, we found no other structural features of ellagitannins that significantly affected the anthelmintic activity on the third-stage infective larvae. The effective concentrations were physiologically relevant and should be achievable in the gastrointestinal tract also in in vivo conditions.

Tannin supplementation modulates the composition and function of ruminal microbiome in lambs infected with gastrointestinal nematodes.

This study was carried out to evaluate the effects of tannin supplementation on ruminal microbiota of sixteen lambs infected and non-infected with Haemonchus contortus and Trichostrongylus colubriformis. Animals were fed with hay, concentrate and supplemented with Acacia mearnsii (A. mearnsii). The animals were divided into four treatments: two control groups without infection, either receiving A. mearnsii (C+) or not (C-), and two infected groups, one with A. mearnsii (I+) and another without A. mearnsii (I-). Ruminal short-chain fatty acids (SCFA) and metagenome sequencing of ruminal microbiota were used to evaluate the effect of tannin and infection on ruminal microbiome. For SCFA, differences were observed only with A. mearnsii. Total SCFA and acetate molar percentage were decreased in C+ and I+ (P<0.05). Butyrate, valerate and isovalerate were higher in lambs that received A. mearnsii in the diet (P<0.05). The infection changed the microbiome structure and decreased the abundance of butyrate-producing microorganisms. In addition, A. mearnsii supplementation also affected the structure the microbial community, increasing the diversity and abundance of the butyrate-producing and probiotics bacteria, amino acid metabolic pathways, purine, pyrimidine and sphingolipid metabolism. Together, our findings indicate that A. mearnsii supplementation modulates important groups related to nitrogen, amino acid, purine and pyrimidine metabolism, in rumen microbiome, affected by gastrointestinal nematodes infection in lambs.

Ultrastructural study of adult Haemonchus contortus exposed to polyphenol-rich materials under in vivo conditions in goats.

This study assessed the ultrastructural changes caused in adult Haemonchus contortus obtained from goats fed fodder based on polyphenol-rich plants Lysiloma latisiliquum or Onobrychis viciifolia or from goats drenched with quebracho extract, Schinopsis spp. The H. contortus were obtained from artificially infected goats used as models to investigate the anthelmintic effect of feeding or drenching with the polyphenol-rich materials. Nematode populations were exposed to polyphenol-rich plant materials by feeding host goats for 8 consecutive days (D28 to D35 post-infection) with (a) L. latisiliquum fodder at 800 g fresh basis/day, (b) O. viciifolia fodder offered ad libitum, and (c) drenched with a solution containing quebracho extract (90 g/day). Meanwhile, control H. contortus were obtained from goats fed polyphenol-free diets. The H. contortus specimens were recovered from the goats on D36 post-infection, and transmission electron microscopy (TEM) was used to identify ultrastructural changes. In vivo exposure to different polyphenol-rich plant materials caused vacuolization of the nematodes' intestinal, muscular and hypodermal cells. These alterations represent the first evidence of cell damage caused in H. contortus when hosts were fed or drenched with polyphenol-rich materials. Ultrastructural changes affecting several types of cells could explain modifications in worm motility and nutrition, eventually affecting H. contortus reproductive success. This study contributes to our understanding of the mechanisms of action of polyphenol-rich plants against H. contortus when given as nutraceuticals to goats.

TITLE: Étude ultrastructurale d'Haemonchus contortus adultes exposés in vivo à des matériaux riches en polyphénols chez les chèvres. ABSTRACT: Cette étude a examiné les lésions ultrastructurales provoquées chez les adultes d'Haemonchus contortus obtenus chez des chèvres alimentées par des fourrages riches en polyphénols, Lysiloma latisiliquum ou Onobrychis viciifolia, ou des chèvres recevant des extraits de quebracho, Schinopsis spp. Les H. contortus ont été obtenus à partir de chèvres artificiellement infestées pour vérifier les effets anthelminthiques de l'alimentation ou l'administration de ressources riches en polyphénols. Les populations de nématodes ont été exposées à des ressources végétales contenant des polyphénols chez des hôtes infestés recevant pendant 8 jours (J28 à J35 après infestation) : (a) un fourrage à base de L. latisiliquum (800 g/jour), (b) un fourrage à base de sainfoin (O. viciifolia) offert ad libitum, et (c) une solution d'extrait de quebracho (90 g/jour). Pendant ce temps, une population témoin d'H. contortus a été obtenue à partir de chèvres alimentées avec un régime à base de fourrages sans polyphénols. Des échantillons d'H. contortus ont été obtenus à partir des divers groupes de chèvres à J36 après infestation, et la microscopie électronique à transmission a été utilisée pour identifier les modifications ultrastructurales. L'exposition in vivo à différents matériaux végétaux riches en polyphénols chez l'hôte a provoqué chez les nématodes une vacuolisation des cellules intestinales, musculaires et hypodermiques. Ces altérations représentent la première preuve de lésions cellulaire provoquées chez H. contortus lorsque l'hôte reçoit des ressources riches en polyphénols. Les changements ultrastructuraux affectant divers types de cellules pourraient expliquer des modifications fonctionnelles de motilité et de nutrition des vers, et finalement modifier le succès reproducteur d'H. contortus. Cette étude contribue à la compréhension des mécanismes d'action des plantes contenant des polyphénols utilisées comme alicament contre H. contortus.

Effects of Acacia mearnsii supplementation on nutrition, parasitological, blood parameters and methane emissions in Santa Inês sheep infected with Trichostrongylus colubriformis and Haemonchus contortus.

Gastrointestinal nematodes are a main problem for ruminant production, reducing animal performance and increasing environmental impact per unit of animal product generated. Tannin supplementation may lead to positive results regarding aspects such as parasitic infections and methane (CH4) emissions. Therefore, the objective of this experiment was to evaluate the effects of the condensed tannins (CT) extract made of powdered Acacia mearnsii bark (PAB) on nutrition, parasitic status and CH4 emissions in sheep artificially infected with Trichostrongylus colubriformis and Haemonchus contortus. Twenty 10-month old Santa Inês lambs (24.7 ± 3.14 kg of initial body weight) were used in a 50-day trial. Animals were divided in four treatment groups according to parasitic infection and PAB supplementation: two control groups without infections, one without PAB (C-) (n = 4) and one with PAB (C+) (n = 4); two infected groups, one without PAB (I-) (n = 6) and another receiving PAB (I+) (n = 6). Initially, animals were kept in individual pens where they were fed ad libitum chopped tifton 85 hay (Cynodon spp.) and 210 g/animal/day of concentrate. On the first day of experiment, animals of I- and I+ groups were artificially infected with infective larvae (L3) of T. colubriformis and H. contortus. Lambs were weighed fortnightly to calculate average daily body weight gain (ADG). Blood and faeces samples were also collected in the same moment of weighing for the evaluation of blood parameters and faecal egg count (FEC) respectively. After 40 days of experiment, measurements of CH4 emissions in small chamber system started and following that, apparent total tract digestibility (ATTD) assay was carried out in metabolic cages. In the end of experimental period (50 days), lambs were slaughtered and samples of abomasum and small intestine content were collected for worm count, identification, and eggs/female count. No significant (p > 0.05) treatment effects were verified for ADG, ATTD and worm count. Blood parameters were affected in both infected groups (p < 0.05) from the 28th experimental day onwards, when these animals started to show reduced red blood cells, haemoglobin and packed cell volume when compared to C- and C+. Decreased FEC was verified in I+ when compared to I- and also, H. contortus eggs/female worm for I+ was lower than for I- (p < 0.05). Both infected groups showed higher CH4 emissions than the control groups (p < 0.05). Results highlighted the anthelmintic potential of PAB and indicated methanogenic effect of parasitic nematode infections.

100 Questions in Livestock Helminthology Research.

An elicitation exercise was conducted to collect and identify pressing questions concerning the study of helminths in livestock, to help guide research priorities. Questions were invited from the research community in an inclusive way. Of 385 questions submitted, 100 were chosen by online vote, with priority given to open questions in important areas that are specific enough to permit investigation within a focused project or programme of research. The final list of questions was divided into ten themes. We present the questions and set them briefly in the context of the current state of knowledge. Although subjective, the results provide a snapshot of current concerns and perceived priorities in the field of livestock helminthology, and we hope that they will stimulate ongoing or new research efforts.

Condensed Tannin Changes along the Digestive Tract in Lambs Fed with Sainfoin Pellets or Hazelnut Skins.

The variable anthelmintic efficacy of condensed tannins (CT) against gastrointestinal nematodes may depend on CT concentration, composition, or fate along the digestive tract. We analyzed CT concentration and composition by acetone-HCl-butanol and thiolysis coupled to HPLC-MS in digesta and feces of lambs. Lambs had been infected with Haemonchus contortus and Trichostrongylus colubriformis and received sainfoin pellets and hazelnut skins of contrasting prodelphinidin/procyanidin ratios. The digesta and feces had lower CT concentrations than the original feeds but similar concentration patterns across the digestive compartments. The changes in assayable CT concentrations between rumen, abomasum, and small intestine may be due to complex formation between CT and other dietary components. However, the large CT disappearance (61-85%) from feed to feces could also indicate that CT may have been structurally modified, degraded, or absorbed during digestion. Interestingly, there were no changes in the structural features of assayable CT in the digesta.

Comparison of the in vitro anthelmintic effects of Acacia nilotica and Acacia raddiana.

Gastrointestinal nematodes are a major threat to small ruminant rearing in the Sahel area, where farmers traditionally use bioactive plants to control these worms, including Acacia nilotica and Acacia raddiana. The main aim of this study was to screen the potential anthelmintic properties of aqueous and acetone extracts of leaves of these two plants based on three in vitro assays: (1) the egg hatch inhibition assay (EHA); (2) the larvae exsheathment inhibition assay (LEIA) using Haemonchus contortus as a model; and (3) an adult mortality test (AMT) applied on Caenorhabditis elegans. For the EHA, only A. raddiana was effective with IC50 = 1.58 mg/mL for aqueous extract, and IC50 = 0.58 mg/mL for acetonic extract. For the LEIA, all extracts inhibited the exsheathment of larvae compared to the controls, and the aqueous extract of A. nilotica was more larvicidal with IC50 = 0.195 mg/mL. In general, all responses to the substances were dose-dependent and were significantly different from the control group (p < 0.05). For the AMT, the extracts of the two Acacia species were effective but A. raddiana showed greater efficacy with 100% mortality at 2.5 mg/mL and LC50 = 0.84 mg/mL (acetonic extract). The addition of polyvinyl polypyrrolidone (PVPP) to the extracts suggested that tannins were responsible for blocking egg eclosion and inducing adult mortality but were not responsible for exsheathment inhibition. These results suggest that the leaves of these Acacia species possess ovicidal and larvicidal activities in vitro against H. contortus, and adulticidal effects against C. elegans.