Effect of three feeding levels on the pathogenesis and establishment of Haemonchus contortus in parasite-naïve Pelibuey hair sheep lambs during their first infection.

This study evaluated the effect of three feeding levels on the pathogenesis and establishment of H. contortus upon the first infection of parasite-naïve Pelibuey hair sheep lambs. Forty-two 6-month-old hair sheep lambs (24 ± 4 kg) raised parasite free from birth were used. The lambs were assigned to 3 groups (n = 14), and each was fed a diet designed for different daily weight gain (DWG): 75 g/d (Diet 1), 125 g/d (Diet 2) and 200 g/d (Diet 3). After four weeks of diet adaptation, 10 lambs/group were infected with 450 L3H. contortus/kg BW (infected), and 4 lambs/group were kept parasite-free (NInf). DWG, hematocrit (Ht), hemoglobin (Hb), peripheral eosinophils (EOS), IgG concentration against H. contortus, and eggs per gram (EPG) of feces were measured in each lamb from day 14 before infection until day 29 postinfection (PI). On day 29 PI, the lambs were slaughtered to determine the total number of adult parasites (TAW), the length of the female worms, and the number of eggs in utero (EIU). Each group reached the expected DWG (P = 0.001), and there was no effect of infection or the diet × infection interaction. Ht was lower in infected lambs than in NInf lambs, and this difference was significant for animals on Diets 1 and 2 (P = 0.044). From day 14 PI onward, Hb was lower in the infected lambs than in the NInf lambs (P = 0.001). Furthermore, compared with NInf lambs, the infected lambs had higher EOS from day 7 PI and higher IgG from day 14 PI. Neither EOS nor IgG were affected by diet. Lambs on Diet 3 had lower EPG during patency than those fed Diets 1 or 2 (days 25 and 28 PI; P = 0.002). Furthermore, lambs fed Diet 3 had lower TAW (Diet 1 vs 3 P = 0.037; Diet 2 vs 3 P = 0.049) and EIU (P = 0.004) than lambs fed Diet 1 or 2. Lambs were resilient to infection regardless of diet. Although EOS and IgG were higher in all infected animals than in Ninf animals, EPG, TAW and EIU decreased only in lambs fed Diet 3. Thus, a diet targeting a DWG of 200 g/d can significantly limit the establishment of H. contortus in Pelibuey lambs infected for the first time.

Impact of Dietary Condensed Tannins and Haemonchus contortus Infection in Growing Sheep: Effects on Nutrient Intake, Digestibility, and the Retention of Energy and Nitrogen.

PURPOSE: A controlled study evaluated the effect of condensed tannins (CT) from Gymnopodium floribundum leaf meal (GF), infection with Haemonchus contortus (I) and their interaction, on feed intake, diet digestibility and retention of N (NR) and energy (ER) in hair sheep lambs. METHODS: Thirty-six, worm-free hair sheep lambs (14.9 ± 1.56 kg body weight) were housed in metabolic cages. Eighteen animals were infected with 6000 H. contortus L3, while other 18 lambs were kept non-infected. On day 28th post-infection (PI), infected lambs were assigned to three diet groups: a diet without GF (I-NONGF), a diet with GF (I + GF) and a diet with GF + polyethylene glycol (PEG) (I + GF + PEG). Non-infected (NI) lambs were assigned to similar diet groups: NI-NONGF, NI + GF and NI + GF + PEG. The packed cell volume (% PCV), ante-mortem faecal egg counts and post-mortem worm burdens were also evaluated. RESULTS: Infection did not affect digestibility, NR and ER. Meanwhile, CT intake from the GF diet reduced the digestibility of dry matter, organic matter and crude protein, as well as NR, compared to lambs consuming the NONGF and GF + PEG diets (P < 0.05). Although, the digestible energy was similar between lambs consuming NONGF and GF + PEG diets, the ER was higher for lambs consuming the control NONGF diet. Diets did not affect the PCV, or the ante-mortem and post-mortem parasitological variables. CONCLUSION: The costs on N and energy metabolism were mainly associated with the CT content of the GF diet, but other features of the diet such as the high lignin content, seemed to affect animals consuming GF meal. Meanwhile, the H. contortus infection had a non-significant impact.

Nitrogen retention in hair sheep lambs with a gradient of Haemonchus contortus infection.

The impact of Haemonchus contortus infections on nitrogen retention (NR) of hair sheep lambs is unknown. This study estimated the feed intake, digestibility, NR, haematocrit (HT), and their relationship with the gradient of H. contortus infection in hair sheep lambs. Twenty-four hair sheep lambs, 3-4 months old, reared parasite-free were used (live weight (LW) 16.7 ± 1.7 kg). Six lambs were kept parasite-free, nine lambs received a dose of 300 H. contortus infective larvae (L3)/kg LW, and the remaining nine lambs received 500 H. contortus L3/kg LW. Infection doses ranged from 3870 to 9,500 L3. Lambs were individually fed for an average daily gain (ADG) of 100 g. On days 1 (P < 0.01) and 7 (P < 0.001) post-infection (PI), the L3 doses were positively associated with HT, possibly due to platelet recruitment. On week 1 PI, the infection (L3) reduced the crude protein (CP) intake (g/kg metabolic LW) (P < 0.05). A reduction of 1% HT corresponded to 1200 eggs per gram of faeces (EPG) for day 21 PI (P < 0.05), 2300 EPG for day 28 PI (P < 0.001), 2400 EPG for day 35 (P < 0.001), and 1300 EPG for day 41 PI (P < 0.001). However, the HT levels were never lower than 21 %. A lower CP digestibility was associated with EPG only on week 5 PI (P < 0.05). There was no significant association between EPG, Total egg in faeces, or worm burden (WB) with intake, dry matter digestibility (DMD), ADG or NR. The NR of lambs was positive along the different weeks of study irrespective of infection level. The WB was negatively associated only with HT on day 41 PI (P < 0.001), where a 1 % HT reduction was estimated for every 257 worms hosted. In conclusion, no relationship was found between H. contortus infection and DMD, ADG or NR in hair sheep lambs. The cost of H. contortus infection resulted only from the HT reduction and depended on the gradient and stage of the infection.

Criollo goats limit their grass intake in the early morning suggesting a prophylactic self-medication behaviour in a heterogeneous vegetation.

The present study compared the feeding behaviour of goats in the early morning (EM = 7:00-8:30 a.m.) and late morning (LM = 9:30-11:00 a.m.) in response to their natural gastrointestinal nematode (GIN) infection status. Twelve female adult goats (37 ± 7.7 kg live weight (LW); 5 ± 1 years) with browsing experience in the tropical deciduous forest were divided into two groups (n = 6): INF group, with natural GIN infection, and non-infected (NI) group, dewormed with moxidectin (0.4 mg/kg LW subcutaneous). Feeding behaviour (dry matter intake (DMI)) of two resource types (grasses vs. shrubs + herbs) was estimated by direct observation for 4 weeks on two grazing moments (EM vs. LM). Environmental temperature and relative humidity at pasture level were measured twice weekly. The GIN egg counts and goats' LW were measured on days 0, 14 and 28. Temperature (mean ± standard deviation) at EM (26.2 ± 1.5 °C) was lower than at LM (38.7 ± 1 °C; P < 0.01). Humidity was higher on the EM (85.1 ± 2.6%) compared to LM (60.4 ± 5.6%; P < 0.01). Irrespective of the infection status, goats consumed similar amounts of grass and shrubs + herbs during EM (P > 0.05). On the other hand, the experimental groups consumed more grass than shrubs + herbs during LM (P < 0.05). The latter suggested prophylactic behaviours strategies to (a) avoid GIN infective larvae, (b) balance the protein:energy ratio of the diet and (c) avoid saturation of detoxification pathways for the secondary compounds consumed from shrubs + herbs. The constant consumption of shrubs + herbs during the study may have reduced the GIN egg count of the INF group.

The worm burden of tracer kids and lambs browsing heterogeneous vegetation is influenced by strata harvested and not total dry matter intake or plant life form.

This study assessed the effect of total dry matter intake (DMI), plant life form and strata harvested on the gastrointestinal nematode (GIN) worm burdens of tracer kids and lambs browsing heterogeneous vegetation during the rainy season (August-November). The rainy season was divided into 6 2-week periods (P1-P6), and environmental conditions (rainy days, rainfall, temperature and humidity) were recorded daily. Five pairs each of tracer kids and tracer lambs raised free of GIN infections were used. Every 15 days, different pairs of kids and lambs were introduced to a 2.2-ha plot and co-grazed with a flock of 30 sheep and 70 goats for a period of 3 weeks. Feeding behaviour of each pair of tracers was measured in weeks 2 and 3. The continuous bite monitoring method was used to estimate total DMI, DMI of plant life forms and DMI from plants of different strata. After each 3-week period, the tracer pair was maintained indoors for 28 days and necropsied on day 29 to recover the worm burden. The feeding behaviour of the tracers was compared between periods (P2-P6) and between kids and lambs. The differences in the worm burdens of the tracers between periods were not associated with total DMI or DMI from plant life forms. Worm burdens were highest during P5 and P6 in tracer kids and lambs (P < 0.05), suggesting a build-up of infective larvae in the tropical deciduous forest (TDF). The lower worm burdens of tracer kids compared with lambs (P < 0.05) seemed to be associated with less low- and more medium-stratum ingestion.

Gymnopodium floribundum fodder as a model for the in vivo evaluation of nutraceutical value against Haemonchus contortus.

This study validated a protocol to identify the nutraceutical value of Gymnopodium floribundum (GF) foliage based on its effects on sheep nutrition, production and health and its impact on adult Haemonchus contortus. Thirty animals (17.8 ± 3.44 kg BW) were distributed into five experimental groups (n = 6). Groups T1-T3 received feed including 20%, 30% and 40% GF content, respectively, and group T4 received feed with 0% GF. Groups T1-T4 were all infected with 6000 H. contortus infective larvae (L3). Group T5 included six worm-free lambs fed a diet without GF. Feed intake, dry matter digestibility (DMD) and organic matter digestibility (OMD) were measured, in addition to the faecal excretion of eggs per gram (EPG) and total faecal egg count (TFEC) of H. contortus. On day 39 post-infection, lambs were humanely slaughtered to recover and count adult parasites and assess the length and fecundity of female worms. Higher levels of GF in the diet reduced DMD and OMD (P < 0.05), with the lowest values in T3 (40% GF) (P < 0.05). Body weight gain was similar for all groups (0.18 to 0.2 kg/day; P > 0.05). EPG and TFEC were lower in T1 and T3 than in T4 (P < 0.05), and the number of adult female worms was lowest in T3 (P < 0.05). In conclusion, GF leaf meal can be considered a nutraceutical when included at 40% (T3) in the diet of lambs, based on its ability to decrease the EPG and TFEC of H. contortus by reducing the female worm burden. A 40% inclusion level had no negative impact on lamb diet intake, production and health, although a reduction of digestibility was observed.

Impact of gastrointestinal parasitism on dry matter intake and live weight gain of lambs: A meta-analysis to estimate the metabolic cost of gastrointestinal nematodes.

This study aimed to estimate the effect of gastrointestinal nematodes (GIN) on the productive performance assessed by the live-weight change (LWC) of lambs, and the metabolic cost associated with parasitism by means of a meta-analysis. Data used in the meta-analyses were obtained from twenty papers selected using the following criteria: (a) lambs with (I) and without (NI) GIN; (b) lambs fed ad libitum; (c) LWC data; (d) feed consumption data; and (e) chemical composition of diets. The effect of diet composition (crude protein [CP] and metabolizable energy [ME]) on dry matter intake (DMI), and the effect of composition and nutrient intake (DMI and CP intake [CPI]) and ME intake (MEI) on LWC was evaluated using respective regression analyses. The metabolic cost of worm burden was determined as the difference in CP and ME requirements between NI and I lambs for each adult parasite. The CP and ME cost were evaluated for lambs at two different levels: maintenance level 0 g LWC and 100 g LWC. The worm burden had an impact on the DMI and LWC of lambs (P < 0.05). The association of CP x infection level was the best predictor of DMI. The association between MEI and infection level were the best predictors of LWC. The metabolic cost of GIN increased to 0.30 mg CP/kg LW0.75 and 0.0056 kJ ME/kg LW0.75 for each adult parasite. The metabolic cost was not modified by diet quality (maintenance level 0 g LW/day or 100 g LW/day). In conclusion, GIN has a negative effect on DMI and LWC. The metabolic cost of GIN infection can be covered by supplying the additional requirements for protein and energy in the diet of infected lambs.

Gastrointestinal nematode infection and feeding behaviour of goats in a heterogeneous vegetation: No evidence of therapeutic self-medication.

The aim of this study was to identify modifications in the feeding behaviour of goats browsing a tropical deciduous forest (TDF) when natural gastrointestinal nematode (GIN) infection was suppressed. Continuous bite monitoring through direct observation was implemented in 12 Criollo goats (adults, non-pregnant) foraging for 4 h per day during the rainy season. In the first Period (P1, one observation point) all goats were maintained with natural GIN infection. In the second Period (P2, three observation points), goats were equally distributed into 2 groups: i) moxidectin treated group (TG) used in a suppressive scheme; and ii) naturally infected group (IG). For each observation point, goats were monitored at three timepoints per day (80 min each), for three consecutive days, to estimate their intake of dry matter (DM), condensed tannins (CT), crude protein, metabolizable energy and digestible DM. Live weight (LW), faecal samples and blood samples were obtained every 28 days to determine LW change, faecal egg counts (FEC) and packed cell volume (PCV). During P1 and P2, the TG and IG had similar LW change and PCV. During both periods, the intake of DM, CT and all macronutrients were similar for TG and IG. The suppression of GIN infection did not modify the feeding behaviour of goats. Therefore, a therapeutic self-medicative behaviour was not identified in Criollo goats browsing a TDF.

Do condensed tannins from Lysiloma latisiliquum have a role in its anthelmintic activity?

We would like to comment on a recent paper by Hernandez-Bolio et al. (2017, Nat. Prod. Res. DOI: 10.1080/14786419.2017.1317774) where an extract free of condensed tannin (CT) was evaluated and concluded that Lysiloma latisiliquum CT does not have a role in its anthelmintic (AH) activity. This letter discusses the AH potential of L. latisiliquum based on previously published evidence of a partial role of CT using different AH assays and Haemonchus contortus isolates. Because the experimental design employed in the above-mentioned paper did not allow a clear-cut conclusion regarding the role of CT, this suggests that a specific experiment must be designed to confirm or reject the involvement of CT fraction(s) on L. latisiliquum AH activity.

An in vitro approach to evaluate the nutraceutical value of plant foliage against Haemonchus contortus.

Nutraceutical plants provide nutrients for the animal as well as secondary compounds that can affect the biology and survival of gastrointestinal nematodes (GIN). Current screening of nutraceutical plants is based on in vitro evidence of anthelmintic (AH) activity against different life stages of GIN, but nutritional information is omitted or scarce. This study proposes an integral in vitro screening protocol to identify the nutraceutical value of the foliage from plant species consumed by small ruminants, using Haemonchus contortus as a biological model. The leaves from Acacia collinsii, A. pennatula, Bunchosia swartziana, Gymnopodium floribundum, Havardia albicans, Leucaena leucocephala, Lysiloma latisiliquum, Mimosa bahamensis, Piscidia piscipula, and Senegalia gaumeri were evaluated for their chemical composition and in vitro digestibility. Acetone:water extracts (70:30) from leaves of each plant were evaluated using the egg hatch assay and larval exsheathment inhibition assay. Respective effective concentrations 50% (EC50) were determined for each assay. The ten plant species showed good nutritional value for ruminants, including crude protein (> 10%), metabolizable energy (> 2.9 MJ/kg DM), and varied CT content (from 1.0 to 37.6%). The best AH activity against H. contortus eggs (EC50 = 401.8 µg/mL) and L3 (EC50 = 83.1 µg/mL) was observed for S. gaumeri extract. Although all the plant species showed in vitro nutraceutical potential, the leaves of S. gaumeri had the best values. The proposed in vitro protocol showed to be useful for the integral assessment of the nutraceutical potential of different plant species as it included the nutritional value and the AH activity against eggs and L3 in the selected plant species.

Simpler intake estimation using direct observation in small ruminants: grouping bites by plant structure and morphology.

OBJECTIVE: To validate the estimation of dry matter intake (DMI) obtained from bite categories (BC) and weight for every plant species (method 1: M1) vs. an alternative method (method 2: M2) grouping plants based on structure and leaf morphology. A dataset containing 80,813 bites and 33 plant species obtained by M1 for sheep and goats browsing a tropical forest was used. Plant species and their respective bite weight were regrouped according to M2. BC weights within each morphological group were compared using ANOVA and post hoc Tukey's honest significant difference comparisons. DMI was estimated for sheep, goats and DMI obtained with both approaches was compared using the t-test, Pearson correlation and orthogonal regression analyses. RESULTS: Dry matter intake estimations were: M1 = 369 ± 153 vs. M2 = 425 ± 161 gDM for sheep and M1 = 567 ± 190 vs. M2 = 681 ± 203 gDM for goats. DMI estimations by M1 and M2 were similar and strongly correlated. Orthogonal regression showed both procedures yielded a similar DMI estimation (P < 0.001). M2 reduces the amount of work required to estimate DMI in heterogeneous vegetation without reducing accuracy. M2 reduced the time required and made simpler to include data from larger number of animals/replicates.

A protocol of human animal interaction to habituate young sheep and goats for behavioural studies.

Animal habituation is key to obtain reliable data on behavioural studies but detailed procedures to achieve it are scarce. This study designed a set of actions to habituate sheep and goats to human observers. Pelibuey sheep (n = 15) and Criollo goats (n = 10) were classified as (a) avoider, flight from human interaction, or (b) follower, seek human interaction. Habituation was measured by the reduction of flight distance by avoiders, or number of followers in the presence of observers. The habituation protocol consisted of a gradually increased series of five manoeuvres, either challenge (for avoiders) or evasion (for seekers), performed first inside a pen and subsequently in a grass paddock. Habituation was considered successful when animals could be observed from a 1-m distance without flight or following the observer. In the pen, habituation took 12 and 13 days for sheep and goats, respectively. Meanwhile, in the grass paddock habituation took 10 days, for both species. The number of challenge and evasion series was negatively correlated with the flight distance in sheep and with the number of followers in goats. This protocol is simple and practical to implement and enables animal habituation for behavioural studies.

Feed resource selection of Criollo goats artificially infected with Haemonchus contortus: nutritional wisdom and prophylactic self-medication.

Previous cafeteria studies suggested that a moderate natural gastrointestinal nematode (GIN) infection did not modify the resource selection of adult Criollo goats towards tannin-rich plants compared with worm-free goats. A higher infection with Haemonchus contortus could trigger a change in the resource selection behaviour towards tannin-rich foliage. Alternatively, goats might select plant species solely to meet their nutritional requirements. A cafeteria study investigated the effect of a high artificial infection with H. contortus on the feed resource selection of goats. Adult Criollo goats (37.5±4.8 kg BW) with browsing experience were distributed in two groups: the infected group (IG) with six animals artificially infected with H. contortus (6000 L3/animal); and the non-infected group (NIG) with six animals maintained worm-free. The experiment included two 5-day periods with additional 5-day adaptation period. In the first period, animals were offered foliage of five plant species with a decreasing gradient of condensed tannins (CT) (Mimosa bahamensis, Gymnopodium floribundum, Havardia albicans, Acacia pennatula, Lysiloma latisiliqum), and three plant species with negligible CT content (Leucaena leucocephala, Piscidia piscipula and Brosimum alicastrum). In the second period the foliage of B. alicastrum was withdrawn. A grain-based concentrate feed was offered daily at 1% BW in DM basis. Dry matter and nutrient intake was determined. Foliage selection of each experimental group was determined using the Chesson selection index. The H. contortus egg count per gram of faeces (EPG) was determined for infected goats twice daily. Chesson index showed a similar pattern of foliage selection on periods 1 and 2. Mean EPG of goats in IG was 2028±259 EPG during period 1 and 1 293±198 EPG during period 2 (P>0.05). During period 1, the selection pattern was highest for B. alicastrum (tannin-free), followed by a tannin-rich plant (M. bahamensis). These two plants remained as highly selected during period 2. The Chesson index showed that both experimental groups (IG and NIG) selected the same plant species in both periods. Thus, a high H. contortus infection did not affect selection of goats fed with CT-rich plants. Apparently, goats balanced their nutrient intake with the plants selected, showing evidence of nutritional wisdom. This balance may have helped to prevent excess protein in the diet and also to maintain a low GIN infection, both considered as examples of prophylactic self-medication.

Is there a negative association between the content of condensed tannins, total phenols, and total tannins of tropical plant extracts and in vitro anthelmintic activity against Haemonchus contortus eggs?

In vitro studies using plant extracts suggest a relationship between their polyphenol contents and their anthelmintic (AH) activity against Haemonchus contortus. High polyphenol content appears to increase the efficacy of plant extracts against H. contortus as assessed by the larval exsheathment inhibition assay (LEIA) while appearing to reduce the AH efficacy measured using the egg hatch assay (EHA). In addition, some plants lack AH activity. Therefore, the present study investigated the relationship between the contents of condensed tannins (CT), total phenols (TP), and total tannins (TT) in methanol:water extracts (70:30) obtained from ten tropical plant species consumed by small ruminants as well as their AH activity against H. contortus evaluated by LEIA and EHA. Extracts of Acacia collinsii, Lysiloma latisiliquum, Havardia albicans, Senegalia gaumeri, Mimosa bahamensis, Piscidia piscipula, Acacia pennatula, Gymnopodium floribundum, Leucaena leucocephala, and Bunchosia swartziana were examined. Positive correlations were found between the effective concentration 50% (EC50) (EHA) of extracts and their CT (r = 0.6809, P < 0.05, n = 10) and TP (r = 0.9152, P < 0.05, n = 10) content, suggesting that their concentration negatively affected AH activity against eggs. Based on the LEIA, there was no significant association between the EC50 and the CT, TP, or TT of all extracts evaluated. Thus, if sheep and goats consume a complex feed mixture with high amounts of CT, TP, and TT, it might be difficult to observe an AH effect against H. contortus egg hatching. However, the AH effect upon L3 establishment might be feasible.

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

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

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

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

Gastrointestinal nematode infection does not affect selection of tropical foliage by goats in a cafeteria trial.

It is important to determine whether gastrointestinal nematodes (GINs) affect foliage choice of goats leading to confirm the expression of a self-medication behavior. This study investigated the effect of GIN infection on tropical foliage selection by goats. During experimental stage 1 (10 days), goats had a natural mixed GIN infection, and at stage 2 (10 days), goats were treated with effective anthelmintics to maintain them free of GIN infection. During stage 1 the twelve adult goats (32 ± 2.3 kg live weight [LW]) were assigned to three groups (n = 4) according to their initial GIN infection status: HI group, with fecal egg count (FEC) between 1450 and 2150 eggs per g/feces (EPG); MI group, medium FEC (592-1167 EPG); and the NI group, free from GIN infection. Fresh foliage of four tropical plants were offered to goats ad libitum for 1 h daily: Gymnopodium floribundum (high condensed tannin [CT] content, 37-40 %), Mimosa bahamensis (medium CT content, 16-17 %), Leucaena leucocephala (low CT content, 3-5 %), and Viguiera dentata (negligible CT content, 0.6-0.9 %). Jacobs' selection indexes (JSIs) were estimated for the experimental foliage based on dry matter (DM), CT, or crude protein (CP) intake. During both study stages, individual fecal egg counts were estimated. The JSI patterns of different plant species, based on DM, CT, or CP, were similar irrespective of infection level during stage 1 (HI, MI, and NI) or no GIN infection (stage 2). Thus, irrespective of GIN infection, goats actively selected M. bahamensis (high CT, low CP content) and V. dentata (negligible CT, high CP content) but avoided G. floribundum (high CT, low CP content) and L. leucocephala (medium CT and high CP content). Thus, natural GIN infection did not influence goats' foliage selection.

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

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

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

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