Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

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.

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.

Supplementation with dry Mimosa caesalpiniifolia leaves can reduce the Haemonchus contortus worm burden of goats.

Gastrointestinal nematodes (GINs) cause considerable economic losses in grazing goat herds. At present, GIN control cannot rely on conventional anthelmintic (AH) drugs because parasites have developed resistance against such drugs. Thus, alternative control methods are being sought to reduce the dependence on AH. Many tannin-rich plants exhibit AH activity and may be used as alternatives for GIN control. Mimosa caesalpiniifolia is a tannin-rich shrub consumed by small ruminants in Brazil. This study evaluated the in vivo AH effect of M. caesalpiniifolia leaf powder supplementation on GIN egg fecal excretion and worm burden in goats. Plant leaves were harvested, dried and ground to obtain a powder. Twenty-four castrated male goats, aged six to eight months, with a mean body weight of 15.0 ±â€¯2.5 kg were used in the experiment. Animals were infected orally with 16,000 larvae comprising 50% Haemonchus spp., 41% Trichostrongylus spp. and 9% Oesophagostomum spp. Once the infection was patent, the goats were distributed into four groups of six animals. The control group received concentrate without condensed tannins (CTs) and did not receive any drench against GINs. The monepantel group received concentrate without CTs and were drenched once with monepantel. The other two groups received the M. caesalpiniifolia leaf powder in two periods of seven consecutive days (days 1-7 and 14-21), with one of the groups also receiving 10 g of polyethyleneglycol (PEG)/day. The animals were weighed weekly, and individual fecal eggs counts (FECs) were performed daily. After 28 days, the animals were humanly slaughtered, and the worm burden was estimated. Although live weight gain and FECs did not differ among the groups (P > 0.05), post-mortem worm counts showed a reduction in Haemonchus contortus adult worm burden (57.7%) in goats of the CT group compared to control goats (P < 0.05). The addition of PEG did not diminish AH activity in the CT + PEG group (66.9% reduction compared to the control). No AH effect against other GIN species was found. The result for the addition of PEG suggested that the observed AH activity was associated with plant secondary compounds, as opposed to CTs. As expected, no AH effect against Oesophagostomum columbianum was found for the monepantel group showed. Thus, feeding dry leaves of M. caesalpiniifolia represent a promising alternative for the control of GIN infections in goats.

In vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla (Oxalidaceae) hydroalcoholic extract against Haemonchus contortus in lambs.

The in vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla hydroalcoholic extract (HE), against Haemonchus contortus in experimentally infected lambs, were assessed. We used a microtitration plate method, comprising the following two stages. Stage 1: 20 µl of water containing 200 sheathed H. contortus infective larvae (ShHcl) were deposited in every well of three series; then, the series 2 and 3 wells were treated with 80 µl 1% ivermectin and O. tetraphylla HE at 20 mg/ml, respectively. Stage 2: the same procedure was performed replacing the ShHcl with exsheathed larvae (ExShHcl). Evaluations were performed after 24 and 48 h. The total numbers of dead and live larvae were counted. A second experiment evaluated the reduction in nematode egg populations in the faeces of lambs treated orally with the O. tetraphylla HE. The 27 lambs used were divided into Groups 1, 2 and 3 (n = 9), which were administered water (positive control), levamisole 1 m (7.5 mg/kg body weight (BW), as a unique dose) and O. tetraphylla HE (20 mg/kg BW), respectively. The plant HE was administered daily for 8 days. The in vitro assay showed 80.9% and 86.5% larval mortality of ShHcl after 24 and 48 h, respectively, while the corresponding mortality values for ExShHcl were 97 and 99%, respectively. The in vivo assay showed variability in the eggs/gram of faeces (epg) values; however, at the end of the trial, the average reduction in the epg values of the O. tetraphylla HE group was 45.6% (P < 0.05). Oxalis tetraphylla HE contains compounds that belong to the flavonol group with anthelmintic activity.

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.

Plant products and secondary metabolites with acaricide activity against ticks.

The present review documents the results of studies evaluating the acaricidal activity of different plant products and secondary metabolites against ticks that are resistant and susceptible to conventional acaricides. Studies published from 1998 to 2016 were included. The acaricidal activity of plant extracts, essential oils and secondary compounds from plants have been evaluated using bioassays with ticks in the larval and adult stages. There is variable effectiveness according to the species of plant and the concentrations used, with observed mortalities ranging from 5 to 100% against the Rhipicephalus (Boophilus), Amblyomma, Dermacentor, Hyalomma, and Argas genera. A number of plants have been reported to cause high mortalities and/or affect the reproductive capacity of ticks in the adult phase. In the majority of these trials, the main species of plants evaluated correspond to the families Lamiaceae, Fabaceae, Asteraceae, Piperaceae, Verbenaceae, and Poaceae. Different secondary metabolites such as thymol, carvacrol, 1,8-cineol and n-hexanal, have been found to be primarily responsible for the acaricidal activity of different essential oils against different species of ticks, while nicotine, dibenzyldisulfide and dibenzyltrisulfide have been evaluated for plant extracts. Only thymol, carvacrol and 1,8-cineol have been evaluated for acaricidal activity under in vivo conditions. The information in the present review allows the conclusion that the secondary metabolites contained in plant products could be used as an alternative for the control of ticks that are susceptible or resistant to commercial acaricides.

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

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

In vitro 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.

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

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

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

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

In vivo anthelmintic activity of Phytolacca icosandra against Haemonchus contortus in goats.

The in vivo anthelmintic (AH) activity of the ethanolic extract from leaves of Phytolacca icosandra was evaluated in goats artificially infected with Haemonchus contortus. Parasite naïve goats were artificially infected with 3000 H. contortus infective larvae per animal. Once the infection was patent (day 28 post-infection) all the animals were sampled to determine the faecal egg counts (FEC) for five consecutive days. Two groups of animals were formed balanced for their FEC and body-weight (BW) (n=6/group): the non-treated control group and the treated group in which goats were individually administered with the ethanolic extract of P. icosandra. The extract was administered orally using gelatin capsules (250 mg/kg BW) which were dosed on two consecutive days using a pill-dispenser. Faecal samples were collected from each animal from the day of dosage (Day 0) on a daily basis to determine the number of eggs per gram of feces (EPG) for 15 days post-treatment (PT). The FEC of the two groups were compared using the repeated measures analyses of variance using the log transformed data Ln (FEC+1). The presence of saponins, coumarins, flavonoids, steroids and terpenoids were detected by standard methodologies in the extract. The P. icosandra ethanolic extract was further analyzed by gas chromatography (GC) coupled to a mass spectrometry (GC-MS). A significant reduction in FEC was observed in the treated group compared to the control from day 7 until day 15 PT (P<0.05). The highest percentage reduction (72%) was found on day 11 PT. No adverse reactions were observed in all treated animals for the entire trial. The GC-MS analysis of the organic extracts revealed the presence of three fatty acids as compounds with highest abundance. The three compounds that were identified by their mass fragmentation patterns were: 2-Pentadecanone, 6, 10, 14-trimethyl (RT 10.3 min), Pentadecanoic acid, 14-methyl-, methyl ester (RT 10.8 min) and Hexadecanoic acid, ethyl ester (RT 11.2 min). It is concluded that the P. icosandra ethanolic extract obtained from leaves showed in vivo anthelmintic activity against H. contortus when administered orally to goats at a dose of 250 mg/kg BW on two consecutive days. The dose used did not cause any negative effects on the health of goats.

Maize supplementation of Pelibuey sheep in a silvopastoral system: fodder selection, nutrient intake and resilience against gastrointestinal nematodes.

This trial evaluated the effect of maize supplementation on the ingestive behavior, nutrient intake and the resilience against gastrointestinal nematode (GIN) infection of hair sheep in a silvopastoral system containing tropical grasses and legume trees. In addition, it attempted to determine the metabolic cost of the natural GIN infection in supplemented and non-supplemented animals. Twenty-nine 3-month-old lambs (male and female), raised nematode free, were allocated to four groups: I-NS (infected, not supplemented, n = 8), I-S (infected, supplemented with maize at 1.5% live weight (LW), n = 7), T-NS (treated with moxidectin 0.2 mg/kg LW every 28 days, and not supplemented, n = 7) and T-S (treated with moxidectin and supplemented with maize at 1.5% LW, n = 7). During the 70-day trial, fodder intake, fodder selection, LW change (LWC), red blood cell counts (RBC), hemoglobin (Hb), hematocrit (Ht) and eggs per gram of feces (EPG) were measured every 14 days. Supplement consumption was recorded daily. Metabolizable energy (ME) and protein (MP) consumption from the feeds were estimated. Maize supplementation helped to improve the resilience of hair sheep lambs against GIN infections. The I-S and T-NS groups showed similar LWC, RBC, Hb and Ht (P > 0.05) and both were higher than those in the I-NS group (P < 0.05). No difference was found in EPG between the I-NS and the I-S groups (P > 0.05). No effect of sex was observed in the different variables. Although all groups showed low dry matter intake (DMI) (< 2% LW), supplemented groups (T-S and I-S) showed higher total DMI (fodder + maize; P < 0.05), hence higher ME and MP intakes than the non-supplemented groups (T-NS and I-NS). All groups showed similar fodder selection patterns. The estimated metabolic cost of parasitism was ME = 0.70 MJ/day and MP = 9.2 g/day in the I-S animals. Meanwhile, the cost in the I-NS animals was ME = 1.46 MJ/day and MP = 12.71 g/day. Maize supplementation was an economically viable strategy to control GIN compared with no intervention.

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

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

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

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

Ovicidal and larvicidal activity of the crude extracts from Phytolacca icosandra against Haemonchus contortus.

The development of anthelmintic resistance has impacted on the success of conventional anthelmintics (AH) for the control of gastrointestinal nematodes in grazing/browsing sheep and goats. Medicinal plants from the traditional herbolary in Mexico may provide new candidates that can be explored as alternative sources of AHs for ruminants. This study evaluated the leaf extracts derived from Phytolacca icosandra against infective L(3) larvae and eggs from Haemonchus contortus collected from sheep. Three extracts of different polarities were obtained from the leaf plants using ethanol, n-hexane and dichloromethane as the solvents. The effectiveness of the in vitro AH activity of the plant extracts was evaluated using larval migration inhibition (LMI) and egg hatch (EHA) assays. For the LMI assays, the ethanolic extract of P. icosandra showed 55.4% inhibition of larval migration at 2mg/mL (p<0.05). The dichloromethane extract of P. icosandra showed 67.1% inhibition of migration at 3mg/mL (p<0.05) and a dose-dependent response with an LD(50) of 0.90 mg/mL. The n-hexane extract failed to show inhibition of larval migration at any concentration explored. In the EHA for the ethanol extract, the lowest concentration tested (0.15 mg/mL) resulted in inhibition of egg hatching greater than 72.6%. Therefore, the LD(50) could not be calculated for this extract. The LD(50) of the dichloromethane extract of P. icosandra was 0. 28 mg/mL. An egg hatch inhibition greater than 90% was observed with both the ethanolic and dichloromethane extracts when using a concentration of 0.90 mg/mL or higher. The n-hexane extract failed to show egg hatch inhibition at any concentration tested. The AH activity reported for P. icosandra could be attributable to the flavonoids, steroids, terpenoids, coumarins and/or saponins that were present in the ethanolic and dichloromethane extracts. A combination of more than one component may also explain the observed AH activity against the H. contortus life stages that were evaluated. In conclusion, the ethanolic and dichloromethane extracts of P. icosandra showed clear in vitro AH activity against the H. contortus eggs and the L(3) larvae. However, the hexanic extract of the plant leaves failed to show any in vitro AH activity.

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 µg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 µg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.