Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Ovine haemonchosis: a review.

Sheep farming is the backbone of a rural economy in developing countries, and haemonchosis is a major impediment in the way of its progress. Haemonchus contortus (H. contortus) infection persists all over the world particularly in the tropical and sub-tropical regions. Various review articles have been published to substantially cover one or more aspects of its morphology, prevalence, pathogenesis, symptoms, diagnosis, immune response, drug resistance, treatment, and control measure. The objective of this paper is to briefly review past and present information available in the aforementioned areas in one place to enable the readers to fully understand the problem from a broader perspective. H. contortus parasite harbours in abomasum of affected animal and feeds on its blood, producing mild to severe symptoms and even death in acute form. The parasite thus inflicts heavy production losses and is of economic importance. H. contortus has developed diverse characters over the years leading to limited success in the production of vaccines. Indiscriminate use of the anthelmintics has produced drug resistance against almost all conventional products. Efficacy of medicinal plants and non-conventional chemicals has been reported under controlled experiments; however, research on their adverse effects on growth and fertility is yet to be studied. Research on molecular tools for identification and introduction of resistant genes into the flock is also underway but still a long journey to find its field application. Crossbreeding may compromise the production traits of the existing flock. In given circumstances, a targeted selective treatment approach along with selective breeding, culling of more susceptible animals, and maintaining a good body condition score through the provision of a balanced diet remains a workable strategy to control haemonchosis in sheep.

Can the foregut nematode Haemonchus contortus and medicinal plants influence the fecal microbial community of the experimentally infected lambs?

The abomasal parasitic nematode Haemonchus contortus can influence the abomasal microbiome of the host. On the other hand, no information occurs on the influence of the parasite on the hindgut microbiome of the host. We evaluated the impact of Haemonchus contortus on the fecal microbial community of the experimentally infected lambs treated with a mixture of medicinal herbs to ameliorate the haemonchosis. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with a blend of medicinal herbs (Inf+Herb), uninfected control animals (Control), and uninfected animals supplemented with medicinal herbs (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herb blend (Herbmix) consisted of dry medicinal plants of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis, Melisa officinalis, Foeniculum vulgare, and Artemisia absinthium. Each animal was fed meadow hay and a commercial concentrate (600 + 350 g DM/d). Inf+Herb and C+Herb lambs were fed Herbmix (100 g DM/d and animal). Treatment lasted for 50 days. The fecal microbial fermentation parameters (short-chain fatty acids, ammonia, and pH) were evaluated at intervals of 0, 20, 32, and 50 days. The fecal eubacterial populations were evaluated by denaturing gradient gel electrophoresis (DGGE) at day 32 when H. contortus infection was the highest. No substantial effects of the H. contortus infection and the herbal treatment on fecal microbial fermentation parameters and fecal eubacterial populations were observed. Evaluation of DGGE patterns by Principal component analysis pointed to the tendency to branch the C+Herb group from the other experimental groups on Day 32. The results indicate that hindgut microbial activity was not disturbed by H. contortus infection and herbal treatment.

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.

Evaluation of encapsulated anethole and carvone in lambs artificially- and naturally-infected with Haemonchus contortus.

Molecules from natural sources, such as essential oils, have shown activity against parasites in vitro, but have not yet been explored extensively in vivo. Anethole and carvone (10% each), encapsulated with 80% of a solid matrix, referred to as EO (encapsulated oils), were tested in vivo in 2 experiments. In Experiment 1: Lambs were artificially infected with multidrug resistant Haemonchus contortus, or left uninfected, and treated (or not) with 50 mg/kg bw (body weight) of EO in a controlled environment. Thirty-two male lambs were kept in individual cages for a period of 45 days, after which animals were evaluated for parasitological, hematological, toxicological, and nutritional parameters. After 45 days of treatment, EO at 50 mg/kg bw provided a significant (P ≤ 0.05) reduction in fecal egg count (FEC). Although FEC was reduced, animals from both treatments had similar counts of total adult worms. The low FEC was caused probably by a significant reduction (P ≤ 0.05) in both male worm size and female fecundity. Dry matter intake of uninfected controls was significantly (P ≤ 0.05) reduced, although no toxicity was observed in treated animals. Thus, in Experiment 2, conducted for five months we used an EO dose of 20 mg/kg bw. Thirty-four weaned lambs, free of parasites, were divided in two groups and kept in collective pens. One group received EO at 20 mg/kg bw mixed with concentrate for 5 months and the other was kept as a control group (CTL). Parasitological and hematological parameters as well as body weight were evaluated. In the first 2.5 months, CTL and EO groups were confined, and both presented similar clinical parameters. Then, animals were allotted to graze on contaminated pastures to acquire natural infection for the next 2.5 months. The infection was patent after 25 days and both groups had similar decreases in weight gain, increases in FEC, and decreases in blood parameters. Coprocultures from CTL and EO groups established that parasite population was 90% Haemonchus sp. We concluded that the technology of encapsulation is safe and practical to deliver to lambs at the farm level and anethole and carvone at 50 mg/kg bw caused a significant decrease in FEC and, consequently, in pasture contamination by free living stages of H. contortus. However, EO at 20 mg/kg bw was not effective to prevent or treat sheep naturally-infected with gastrointestinal nematodes.

Arylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus contortus in vitro.

Due to widespread drug resistance in parasitic nematodes, there is a need to develop new anthelmintics. Given the cost and time involved in developing a new drug, the repurposing of known chemicals can be a promising, alternative approach. In this context, we tested a library (n = 600) of natural product-inspired pesticide analogues against exsheathed third stage-larvae (xL3s) of Haemonchus contortus (barber's pole worm) using a whole-organism, phenotypic screening technique that measures the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from chemical scaffolds of arylpyrrole or fipronil. The seven most promising compounds, selected based on their anthelmintic activity and/or limited cytotoxicity, are arylpyrroles that reduced the motility of fourth-stage larvae (L4s) with significant potency (IC50 values ranged from 0.04 ±â€¯0.01 µM to 4.25 ±â€¯0.82 µM, and selectivity indices ranged from 10.6 to 412.5). Since the parent structures of the active compounds are uncouplers of oxidative phosphorylation, we tested the effect of selected analogues on oxygen consumption in xL3s using the Seahorse XF24 flux analyser. Larvae treated with the test compounds showed a significant increase in oxygen consumption compared with the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study have identified natural product-derived molecules that are worth considering for chemical optimisation as anthelmintic drug leads.

Essential oils of Citrus aurantifolia, Anthemis nobile and Lavandula officinalis: in vitro anthelmintic activities against Haemonchus contortus.

BACKGROUND: Infections of sheep with gastrointestinal parasites, especially Haemonchus contortus, have caused serious losses in livestock production, particularly after the emergence of resistance to conventional anthelmintics. The search for new anthelmintic agents, especially those of botanical origin, has grown substantially due to the perspective of less contamination of meat and milk, as well as other advantages related to their cost and accessibility in less developed countries. The aim of this study was to evaluate the in vitro anthelmintic activity of essential oils of the plant species Citrus aurantifolia, Anthemis nobile and Lavandula officinalis against the main developmental stages of the parasite H. contortus. RESULTS: Plant species were selected based on substantial ethnopharmacological information. Analysis of the composition of each oil by gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of limonene (56.37%), isobutyl angelate (29.26%) and linalool acetate (35.97%) as the major constituents in C. aurantifolia, A. nobile and L. officinalis, respectively. Different concentrations of each oil were tested in vitro for their capacity to inhibit egg hatching (EHT), larval development (LDT) and adult worm motility (AWMT) using a multidrug-resistant strain of H. contortus (Embrapa 2010). The IC50 values obtained for the oils of C. aurantifolia, A. nobile and L. officinalis were 0.694, 0.842 and 0.316 mg/ml in the EHT and 0.044, 0.117 and 0.280 mg/ml in the LDT, respectively. The three oils were able to inhibit adult worm motility completely within the first 8-12 h of observation in the AWMT. CONCLUSIONS: The present results demonstrate significant anthelmintic activity of the three oils against the different developmental stages of H. contortus. Furthermore, this study is of ethnopharmacological importance by validating the anthelmintic activity of the oils studied. Although new experiments are necessary, these data contribute to the development of pharmaceutical-veterinary products for sheep farming by opening up new therapeutic possibilities against gastrointestinal infections caused by H. contortus.

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.

ANTHELMINTIC EFFECTS OF DRIED GROUND BANANA PLANT LEAVES (MUSA SPP.) FED TO SHEEP ARTIFICIALLY INFECTED WITH HAEMONCHUS CONTORTUS AND TRICHOSTRONGYLUS COLUBRIFORMIS.

BACKGROUND: Helminths is a endoparasites that cause the major losses for profitable sheep production in Brazil. The increased development of resistant strains of endoparasites have enforced the search for sustainable alternatives. The aim of this paper was to provide information about endoparasites control with banana leaves in infected sheep as alternative control strategies and see its viability. MATERIALS AND METHODS: In this study, we performed two trials to investigate the anthelmintic properties of banana leaves on endoparasites in sheep. In Trial 1, twelve sheep were artificially infected with Trichostrongylus colubriformis; in Trial 2, eleven sheep were artificially infected with Haemonchus contortus. Clinical examinations, packed cell volume, total protein, faecal egg counts (FECs) and egg hatchability tests (EHTs) were performed. At the end of the trials, the sheep were humanely slaughtered, and total worm counts were performed. RESULTS: In Trial 1 and 2, no significant FEC decreases were note but significant diference in EHTs were observed. Total worm counts, clinical and haematological parameters did not reveal significant changes between the treatment and control groups. These results suggest that feeding dried ground banana plant leaves to sheep may reduce the viability of Trichostrongylus colubriformis eggs, and this anthelmintic activity is potentially exploitable as part of an integrated parasite management programme. CONCLUSION: However, further investigation is needed to establish the optimal dosage, develop a convenient delivery form and confirm the economic feasibility of using banana plantation byproducts as feed for ruminant species. Abbreviations: Coproculture test (CT)., Faecal egg count (FEC)., Egg hatchability test (EHT).

Effect of changes in the nutritional status on the performances of growing Creole kids during an established nematode parasite infection.

In this study, we evaluated the effect of changes in the nutritional status on the performances of growing Creole kids during an established experimental gastrointestinal nematode (GIN) infection. Eighteen 6-month-old Creole kids were distributed in two main groups infected (I) and non-infected (NI) and were placed for a period of 4 weeks on each of three diets differing in their nutritional values: (1) fresh grass (FG, 6.7 MJ/kg dry matter (DM) and 7.9% crude protein (CP)) non-supplemented, (2) FG supplemented with a commercial concentrate (CC, 12.2 MJ/kg DM and 20.6% of CP), and (3) FG supplemented with dried banana (Ban, 11.1 MJ/kg DM and 4.3% CP). The experiment was designed as a split-plot with experimental infection (I and NI) as the main plot and the diets (FG, CC, and Ban) as the subplots with three replicates. We showed a significant effect of the diet changes on the fecal egg counts. A higher dry matter intake, digestibility, and growth rate were observed with the CC diet but together with a slight but significant increase of the intensity of the GIN infection. These data suggest that the improvement of the protein nutritional status during an establish GIN infection would improve the animal performance at the expense of the mechanism involved in the control of the infection.

Supplementation with copper edetate in control of Haemonchus contortus of sheep, and its effect on cholinesterase's and superoxide dismutase activities.

The aim of this study was to evaluate the effect of copper edetate on biochemical parameters, oxidative profile, cholinesterase's activities, as well as its capacity to control gastrointestinal parasites in infected sheep. Thus, Lacaune sheep (n = 18) infected by Haemonchus contortus were used and divided into three groups of six animal each: the group A was composed of untreated animals (the control group), the group B was formed by animals treated with 0.3 mg/kg of copper edetate, and the group C was composed of animals treated with 0.5 mg/kg of copper edetate. Blood collection was performed on days 0, 10, 20 and 30 after mineral supplementation and different variables were measured. Cholinergic system was evaluated to determine the acetylcholinesterase (AChE) activity in total blood and butyrylcholinesterase (BChE) activity in serum. Eggs per gram of feces (EPG) were evaluated. There were no significant differences (P > 0.05) between groups regarding total protein, albumin, globulin and urea levels, GGT activity, as well as the hematocrit, and EPG. ALT activity decreased (P < 0.05) on groups B and C on day 30 compared to the control group (the group A). AChE activity decreased (P < 0.05) in the group C on days 10 and 30 compared to the control group, such decrease (P < 0.05) was also observed for BChE activity in the group C on day 10. ROS levels increased in the group C compared to groups A and B on day 10, while the SOD activity increased in the group C on days 20 and 30 compared to the control group (P < 0.05). CAT activity did not differ between groups (P > 0.05). In summary, the copper edetate was not efficient to control gastrointestinal parasites, but efficiently activated SOD, an important antioxidant enzyme. In addition, copper edetate was able to partially inhibit cholinesterase's activities when supplementated at its highest dose.

Larvicidal activity of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus.

Effective control of gastrointestinal parasites is necessary in sheep production. The development of anthelmintics resistance is causing the available chemically based anthelmintics to become less effective. Biological control strategies present an alternative to this problem. In the current study, we tested the larvicidal effects of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus larvae. Bacterial suspensions [2 × 108 colony-forming units (CFU) g-1 of the feces] of B. thuringiensis var. israelensis and recombinant Escherichia coli expressing Cry11Aa toxin were added to naturally H. contortus egg-contaminated feces. The larvae were quantified, and significant reductions of 62 and 81% (P < 0·001) were, respectively observed, compared with the control group. A 30 mL bacterial suspension (1 × 108 CFU mL-1) of B. thuringiensis var. israelensis and recombinant E. coli expressing Cry11Aa toxin were then orally administered to lambs naturally infected with H. contortus. Twelve hours after administration, feces were collected and submitted to coprocultures. Significant larvae reductions (P < 0·001) of 79 and 90% were observed respectively compared with the control group. The results suggest that the Cry11Aa toxin of B. thuringiensis var. israelensis is a promising new class of biological anthelmintics for treating sheep against H. contortus.

Screening of the 'Pathogen Box' identifies an approved pesticide with major anthelmintic activity against the barber's pole worm.

There is a substantial need to develop new medicines against parasitic diseases via public-private partnerships. Based on high throughput phenotypic screens of largely protozoal pathogens and bacteria, the Medicines for Malaria Venture (MMV) has recently assembled an open-access 'Pathogen Box' containing 400 well-curated chemical compounds. In the present study, we tested these compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility, growth and development of H. contortus. We also studied the effect of the 'hit' compound on mitochondrial function by measuring oxygen consumption. Among the 400 Pathogen Box compounds, we identified one chemical, called tolfenpyrad (compound identification code: MMV688934) that reproducibly inhibits xL3 motility as well as L4 motility, growth and development, with IC50 values ranging between 0.02 and 3 µM. An assessment of mitochondrial function showed that xL3s treated with tolfenpyrad consumed significantly less oxygen than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. Given that tolfenpyrad was developed as a pesticide and has already been tested for absorption, distribution, excretion, biotransformation, toxicity and metabolism, it shows considerable promise for hit-to-lead optimisation and/or repurposing for use against H. contortus and other parasitic nematodes. Future work should assess its activity against hookworms and other pathogens that cause neglected tropical diseases.

Chemical composition and in vitro activity of Calotropis procera (Ait.) latex on Haemonchus contortus.

Calotropis procera is among the species of medicinal plants that have traditionally been used for the treatment of parasites in small ruminants, stimulating the scientific validation of anthelmintic effects. This study aimed to investigate the chemical composition of ethyl acetate extract of Calotropis procera latex (EAECPL), assess the in vitro effect against Haemonchus contortus and the structural changes caused in the adult worm. The latex was collected, lyophilized and subjected to washing with the ethyl acetate solvent to obtain EAECPL. The constituents of the extract were isolated by column chromatography and identified by (13)C and (1)H nuclear magnetic resonance spectroscopy. The egg hatching test (EHT), larval development test (LDT) and adult worms motility test (WMT) were conducted to evaluate the effectiveness of EAECPL on eggs, larvae and adult of H. contortus, respectively. The worms obtained from the WMT, after 24h exposure to EAECPL or controls were observed on a scanning electron microscope (SEM). The results were analysed by variance analysis and compared with Tukey's test (P<0.05). Three compounds were isolated from EAECPL and identified as urs-19(29)-en-3-yl acetate, (3ß)-Urs-19(29)-en-3-ol, and 1-(2',5'-dimethoxyphenyl)-glycerol. In the EHT, EAECPL inhibited larval hatching by 91.8% at dose of 4mg/ml. In the LDT 1mg/ml inhibited 99.8% larval development. In the WMT, EAECPL in the concentration of 100µg/ml inhibited 100% motility of worms, 12h post-exposition. In the SEM, obvious differences were not detected between the negative control worms and the worms treated with EAECPL. In this study, EAECPL showed an effect on inhibition egg hatching, larval development and motility of the adult worms of H. contortus. This should be related both to the identified compounds, as well as the other compounds present in the EAECPL, acting alone or synergistically.

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.

Anthelmintic activity of selected ethno-medicinal plant extracts on parasitic stages of Haemonchus contortus.

BACKGROUND: Parasitic roundworms (nematodes) cause substantial morbidity and mortality in livestock animals globally, and considerable productivity losses to farmers. The control of these nematodes has relied largely on the use of a limited number of anthelmintics. However, resistance to many of these these anthelmintics is now widespread, and, therefore, there is a need to find new drugs to ensure sustained and effective treatment and control into the future. METHODS: Recently, we developed a screening assay to test natural, plant extracts with known inhibitory effects against the free-living worm Caenorhabditis elegans. Using this assay, we assessed here the effects of the extracts on motility and development of parasitic larval stages of Haemonchus contortus, one of the most important nematodes of small ruminants worldwide. RESULTS: The study showed that two of five extracts from Picria fel-terrae Lour. have a significant inhibitory effect (at concentrations of 3-5 mg/ml) on the motility and development of H. contortus larvae. Although the two extracts originated from the same plant, they displayed different levels of inhibition on motility and development, which might relate to the presence of various active constituents in these extracts, or the same constituents at different concentrations in distinct parts of the plant. CONCLUSIONS: These results suggest that extracts from P. fel-terrae Lour. have promising anthelmintic activity and that more broadly, plant extracts are a potential rich source of anthelmintics to combat helminthic diseases.

Chemical Structures of Plant Hydrolyzable Tannins Reveal Their in Vitro Activity against Egg Hatching and Motility of Haemonchus contortus Nematodes.

The use of synthetic drugs against gastrointestinal nematodes of ruminants has led to a situation where resistance to anthelmintics is widespread, and there is an urgent need for alternative solutions for parasite control. One promising approach is to use polyphenol-rich bioactive plants in animal feeds as natural anthelmintics. In the present work, the in vitro activity of a series of 33 hydrolyzable tannins (HTs) and their hydrolysis product, gallic acid, against egg hatching and motility of L1 and L2 stage Haemonchus contortus larvae was studied. The effect of the selected compounds on egg and larval structure was further studied by scanning electron microscopy. The results indicated clear relationships between HT structure and anthelmintic activity. While HT size, overall flexibility, the types and numbers of functional groups, together with the linkage types between monomeric HTs affected the activity differently, the optimal structure was found with pentagalloylglucose.

In vitro anthelmintic activity of aqueous leaf extract of Annona muricata L. (Annonaceae) against Haemonchus contortus from sheep.

Despite the overall progress of sheep farming in Brazil, infections with the gastrointestinal parasite Haemonchus contortus represent one the most important problems in sheep production, aggravated by the increasing resistance of nematodes to traditional anthelmintic drugs caused by inadequate sheep flock management by breeders. Ethnopharmacological data indicate Annona muricata as a promising alternative for the control of gastrointestinal nematodes because of its general anthelmintic properties. The aim of this work was to evaluate the in vitro anthelmintic effects of A. muricata aqueous leaf extract against eggs, infective larvae and adult forms of parasitic nematode H. contortus. At higher doses, A. muricata extract showed 84.91% and 89.08% of efficacy in egg hatch test (EHT) and larval motility test (LMT), respectively. In the adult worm motility test, worms were completely immobilized within the first 6-8h of nematode exposition to different dilutions of extract. Phytochemical analysis indicated the presence of phenolic compounds in A. muricata aqueous leaf extract that may be responsible for the anthelmintic effects observed. Moreover those results validate the traditional use of A. muricata as a natural anthelmintic and then the pharmacological potential of its compounds for future in vivo investigations.

Anthelmintic activity of Arisaema franchetianum and Arisaema lobatum essential oils against Haemonchus contortus.

ETHNOPHARMACOLOGICAL RELEVANCE: Arisaema franchetianum and Arisaema lobatum are two perennial plants native to China. Arisaema franchetianum is universally used to promote the subsidence of induration and swelling, quicken blood and relieve pains, and kill intestinal parasites in humans and animals. Arisaema lobatum is used to treat malaria, intestinal parasites, and snake and insect bites in humans and animals. The aim of this study was to determine the composition of the essential oils from Arisaema franchetianum and Arisaema lobatum and evaluate the anthelmintic effect against Haemonchus contortus. MATERIALS AND METHODS: Two oils were investigated by GC and GC-MS. The anthelmintic bioassay tests of Arisaema franchetianum and Arisaema lobatum essential oil, linalool and carvacrol were performed using egg hatch assay (EHA), larval development assay (LDA) and larval migration inhibition assay (LMIA). RESULTS: Fifty six components representing 96.88% of the Arisaema franchetianum oil and 64 components representing 96.88% of the Arisaema lobatum oil were identified. Carvacrol and linalool were found to be the major constituents of two oils. In the EHA, greater than 99% inhibition were observed with Arisaema franchetianum oil at 10mg/mL (CE50 1.63mg/mL) and Arisaema lobatum oil at 5 and 10mg/mL (CE50 0.48mg/mL). In the LDA, both oils induced complete inhibition at 10mg/mL, with the CE50 being 1.10mg/mL for Arisaema franchetianum oil and 0.73mg/mL for Arisaema lobatum oil. In the LMIA, the Arisaema franchetianum oil and Arisaema lobatum oil at best inhibited 74.1% and 95.6% of larval migration at 10mg/mL, respectively. Carvacrol exhibited similar activity to Arisaema lobatum essential oil and linalool did not show high activity in every assay. CONCLUSIONS: These data show for the first time that the essential oils obtained from Arisaema franchetianum or Arisaema lobatum had promising anthelmintic activity against Haemonchus contortus. Arisaema plant may offer an alternative source for the control of gastrointestinal nematodes of sheep and goats.

In vitro ovicidal and larvicidal activity of the essential oil of Artemisia lancea against Haemonchus contortus (Strongylida).

Prolonged use of chemical anthelmintics has been found to result in anthelmintic resistance and environmental issues, thereby limiting the application of these drugs in domestic animals and prompting interest in the study of plant extracts as alternative sources thereof. The aim of this study was to evaluate the in vitro effect of the essential oil (EO) of Artemisia lancea against the parasitic nematode Haemonchus contortus using egg hatch assay, larval development assay, and larval migration inhibition assay. The EO yield of extraction was 0.63% (w/w), and the major constituents were 1,8-cineole (34.56%) and camphor (16.65%). In the egg hatch assay, an inhibition greater than 99% was observed with the EO at 10 mg mL(-1) and the LC50 was 1.82 mg mL(-1). 1,8-Cineole demonstrated moderate ovicidal activity with a LC50 of 4.64 mg mL(-1), whereas camphor did not show enough activity to have its LC50 determined. In the larval development assay, the EO, 1,8-cineole, and camphor inhibited 93.6%, 65.2%, and 57% of larval development at 10 mg mL(-1) and exhibited dose-dependent responses with LC50 values of 1.66, 5.07, and 7.80 mg mL(-1), respectively. In the migration inhibition assay, the EO and 1,8-cineole at best inhibited 77% and 60.3% of larval migration at 10 mg mL(-1), respectively. Camphor showed low inhibition capacity, and its efficacy was not dose dependent. The results indicate that the in vitro anthelmintic activity of the EO of A. lancea may be associated with the additive action of the two major constituents, as well as other more minor terpenoid components.