Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans.

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the "HitFinder" library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

Effect of nutraceuticals on acanthocephalan Neoechinorhynchus buttnerae and its toxicity to the host tambaqui Colossoma macropomum.

The production of tambaqui Colossoma macropomum has been undergoing financial losses due to parasitic infection by the acanthocephalan Neoechinorhynchus buttnerae, raising an alert for aquaculture in South America. The lack of adequate treatment and use of unlicensed chemicals encourages research for alternative solutions with minimal side effects. The objectives of this study were to evaluate the in vitro antiparasitic potential of commercial nutraceutical products (Natumix® and BioFish®) against N. buttnerae and to assess the respective in vivo toxic effects on the host tambaqui. For in vitro assays, parasitized fish were necropsied for acanthocephalans sampling. The parasites were exposed to three concentrations (0.078, 0.313 and 1.25 mg/ml) of each product, as well as controls (one without product and another with a solubilizer). For the in vivo acute toxicity test, juvenile fish (<0.1 g) were exposed to five increasing concentrations of each product. Mortality of tambaqui was recorded at 24, 48, 72 and 96 h. The estimated lethal concentration (LC) for 10, 50, 90 and 99% of fish was determined to classify the toxicity of the products on the target species. After in vitro efficacy tests, the highest concentrations (1.25 mg/ml) caused 100% mortality of the parasites in both products, but only Natumix® caused 100% mortality using the intermediate concentration (0.313 mg/ml) after 24 h. According to the acute toxicity result, the LC50 classified the nutraceutical products as slightly toxic for tambaqui. The tested products had a parasiticidal effect on N. buttnerae, and the toxicity test showed that both products have therapeutic potential when added to the diet.

Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes.

Soil-transmitted nematodes (STNs), namely hookworms, whipworms, and ascarids, are extremely common parasites, infecting 1-2 billion of the poorest people worldwide. Two benzimidazoles, albendazole and mebendazole, are currently used in STN mass drug administration, with many instances of low/reduced activity reported. New drugs against STNs are urgently needed. We tested various models for STN drug screening with the aim of identifying the most effective tactics for the discovery of potent, safe and broad-spectrum agents. We screened a 1280-compound library of approved drugs to completion against late larval/adult stages and egg/larval stages of both the human hookworm parasite Ancylostoma ceylanicum and the free-living nematode Caenorhabditis elegans, which is often used as a surrogate for STNs in screens. The quality of positives was further evaluated based on cheminformatics/data mining analyses and activity against evolutionarily distant Trichuris muris whipworm adults. From these data, two pairs of positives, sulconazole/econazole and pararosaniline/cetylpyridinium, predicted to target nematode CYP-450 and HSP-90 respectively, were prioritized for in vivo evaluation against A. ceylanicum infections in hamsters. One of these positives, pararosaniline, showed a significant impact on hookworm fecundity in vivo. Taken together, our results suggest that anthelmintic screening with A. ceylanicum larval stages is superior to C. elegans based on both reduced false negative rate and superior overall quality of actives. Our results also highlight two potentially important targets for the discovery of broad-spectrum human STN drugs.

Araticum (Annona crassiflora Mart.) as a source of nutrients and bioactive compounds for food and non-food purposes: A comprehensive review.

Araticum (Annona crassiflora Mart.) is a fruitful tree native to the Brazilian Cerrado biome that holds high nutritional, functional and economic potential. This plant has been used since ancient times by folk medicine for the treatment of several pathological conditions. There has been increasing interest in the development of pulp-based food products as well as the by-products utilization to obtain value-added ingredients. Understanding the chemical composition and biological activities of different botanical parts of Annona crassiflora Mart. provides a basis to support future researches and applications. In this context, this paper carries out an exhaustive review of the scientific literature, on the main phytochemicals of different botanical parts of Annona crassiflora Mart. (fruit, leaves, stem and root) and their biological activities, assessing their potential uses for several industrial segments. Annona crassiflora Mart. fruits and especially their by-products (peel and seeds) and leaves have been shown a wide range of bioactive compounds such as phenolic compounds, alkaloids, annonaceous acetogenins, tocols, carotenoids, phytosterols, dietary fiber, vitamins, minerals and essential oils. These compounds contribute to various biological activities, including antioxidant, hepatoprotective, anti-inflammatory, antitumoral, analgesic, antidiabetic, skin healing, antidiarrhoeic, antimicrobial, antiparasitic, insecticide and herbicide activities of Annona crassiflora Mart. extracts. Therefore, these findings demonstrate that Annona crassiflora Mart. fruit, by-products and leaves can be excellent candidates to be used as functional foods and/or sources for obtaining bioactive compounds for the food, cosmetics and pharmaceutical applications.

The Anthelmintic Effects of Medicinal Plant Extracts Against Paramphistome Parasites, Carmyerius spatiosus.

INTRODUCTION: Paramphistomosis is a disease caused by the rumen flukes which cause an acute gastroenteritis and anemia with high mortality particularly in young ruminants. MATERIALS AND METHODS: In this study, we have investigated the anthelmintic effect of medicinal plant extracts from leaves and heartwoods of Cassia siamea L., roots of Plumbago zeylanica L. and Plumbago indica L., and leaves of Terminalia catappa L. against Carmyerius spatiosus. RESULTS: The highest anthelminthic effect on the flukes after 24 h of exposure was found in heartwood ethyl acetate extract of C. siamea (LC50 = 374.30; LC90 = 749.03 ppm), root n-butanol extract of P. zeylanica (LC50 = 1005.12; LC90 = 2411.55 ppm), root hexane, ethyl acetate, and n-butanol extract of P. indica (LC50 = 34.38, 211.34, 506.92; LC90 = 64.09, 496.05, 934.86 ppm), and leaf n-butanol and water extract of T. catappa (LC50 = 487.17, 470.28; LC90 = 913.27, 848.23 ppm). When observed by scanning electron microscopy, the tegument showed similar sequence of morphological changes after treatments with all plant extracts, comprising of swelling of ridges and folds, blebbing, rupturing of the blebs, erosion, lesion and disruption of the tegument. CONCLUSION: This study is the first report on the anthelmintic activity of plant extracts to C. spatiosus; therefore, these plant extracts are highly effective in the elimination of adult rumen flukes.

Virtual screening of natural anti-filarial compounds against glutathione-S-transferase of Brugia malayi and Wuchereria bancrofti.

Glutathione-S-transferase also referred as GST is one of the major detoxification enzymes in parasitic helminths. The crucial role played by GST in various chronic infections has been well reported. The dependence of nematodes on detoxification enzymes to maintain their survival within the host established the crucial role of GST in filariasis and other related diseases. Hence, this well-established role of GST in filariasis along with its greater nonhomology with its human counterpart makes it an important therapeutic drug target. Here in this study, we have tried to explore the inhibitory potential of some of the well-reported natural ant-filarial compounds against the GST from Wuchereria bancrofti (W.bancrofti) and Brugia malayi (B.malayi). In silico virtual screening, approach was used to screen the selected natural compounds against GST from W.bancrofti and B.malayi. On the basis of our results, here we are reporting some of the natural compounds which were found to be very effective against GSTs. Along with we have also revealed the characteristic of the active site of BmGST and WbGST and the role of important active site residues involve in the binding of natural compounds within the active site of GSTs. This information will oped doors for using natural compounds as anti-filarial therapy and will also be helpful for future drug discovery.

Evaluation of Antioxidant Properties, Phenolic Compounds, Anthelmintic, and Cytotoxic Activities of Various Extracts Isolated from Nepeta cadmea: An Endemic Plant for Turkey.

Nepeta cadmea Boiss. is a species endemic to Turkey that belongs to the Nepeta genus. Several species of this genus are used in folk medicine. This study was designed to investigate the phenolic compounds, antioxidant, anthelmintic, and cytotoxic activities of various extracts (ethanol, methanol, acetone, and water) of N. cadmea. The antioxidant activities of these extracts were analyzed using scavenging methods (DPPH, ABTS, and H2 O2 scavenging activity), the ß-carotene/linoleic acid test system, the phosphomolybdenum method, and metal chelating activity. Among the 4 different extracts of N. cadmea that were evaluated, the water extract showed the highest amount of radical scavenging (DPPH, 25.54 µg/mL and ABTS, 14.51 µg/mL) and antioxidant activities (ß-carotene, 86.91%). In the metal chelating and H2 O2 scavenging activities, the acetone extract was statistically different from the other extracts. For the phosphomolybdenum method, the antioxidant capacity of the extracts was in the range of 8.15 to 80.40 µg/mg. The phenolic content of the ethanol extract was examined using HPLC and determined some phenolics: epicatechin, chlorogenic, and caffeic acids. With regard to the anthelmintic properties, dose-dependent activity was observed in each of the extracts of N. cadmea. All the extracts exhibited high cytotoxic activities. The results will provide additional information for further studies on the biological activities of N. cadmea, while also helping us to understand the importance of this species. Furthermore, based on the results obtained, N. cadmea may be considered as a potentially useful supplement for the human diet, as well as a natural antioxidant for medicinal applications. PRACTICAL APPLICATION: The plants of the Nepeta genus have been extensively used as traditional herbal medicines. Nepeta cadmea Boiss., one of the species belonging to the Nepeta genus, is a species endemic to Turkey. In our study, we demonstrated the antioxidant capacities, total phenolic, flavonoid, tannin content, anthelmintic, and cytotoxic activities of various extracts of Nepeta cadmea. The present study could well supply valuable data for future investigations and further information on the potential use of this endemic plant for humans, in both dietary and pharmacological applications.

Anthelmintic efficacy of hydro-methanolic extracts of Larrea tridentata against larvae of Haemonchus contortus.

An in vitro study was conducted to determine the anthelminthic activity of hydro-methanolic extracts of Larrea tridentata on sheathed and exsheathed larvae of Haemonchus contortus. Larvae of the parasite were incubated at 20-25 °C in hydro-methanolic extracts at concentrations of 12.5, 25, 50, 100, and 200 mg/mL for 24, 48, or 72 h. Ivermectin and water were the positive and negative controls, respectively. Total phenolic compounds of leaves of L. tridentata were 97.88 ± 10.45 mg/g of dry matter. Other compounds detected in this shrub by HPLC-mass spectrometry were sesamin, galocatechin, peonidin 3-O rutinoside, methyl galangin, epigallocatechin 7-O-glucuronide, and epigalocatechin. Mortality rate of sheathed and exsheathed H. contortus was low (16-34%) with doses ≤ 100 mg/mL of the extracts. At 200 mg/ml, the hydro-methanolic extracts of L. tridentata killed 32.1 and 68.4% of sheathed and exsheathed larvae, respectively, regardless of incubation time. The effective concentration of the L. tridentata extract for 50% larvae mortality (EC50) after 24 h of incubation was 36 mg/mL (CI = 6-94). Microscopic observations revealed damage to the cuticle of this parasite exposed to extracts of L. tridentata. These in vitro results provided evidence that L. tridentata extracts possess anti-Haemonchus contortus properties, particularly during the exsheathed stage of this nematode. It would be necessary to assess the safety of this shrub in vivo and also to carry out in vivo efficacy studies.

Anthelmintic effects of indigenous multipurpose fodder tree extracts against Haemonchus contortus.

Condensed tannins (CT) extracted from Balanites aegyptiaca, Tamarindus indica, and Celtis toka browses were used to evaluate their anthelmintic effect on different developmental stages of Haemonchus contortus. To achieve this objective, various serial concentrations of each CT extract of the foliages were used to test adult motility, inhibition of egg hatchability, and larval development. The fodders were selected based on their multipurpose advantage and accessibility to use as fodder for livestock in the low lands of the Gambella region. The fastest and slowest adult motility rate was observed in 2-ml (4 min) and 0.125-ml dose of C. toka, respectively, which is better than that in ivermectin. Egg hatchability inhibition was observed with dose difference within species, but there is no difference between B. aegyptiaca and T. indica. The foliage extracts of the studied browses were observed to inhibit the larvae by 100% at 2 ml, which is similar to ivermectin. There is no significant difference observed in larvae development inhibition between the species and ivermectin (p > 0.05). CT extracts of studied plants have found to own significant anthelmintic activity in a dose-dependent manner. They could serve as anthelmintic economically and eco-friendly after further and series of in vivo experiments.

Risk-based approach to developing a national residue sampling plan for testing under European Union regulation for veterinary medicinal products and coccidiostat feed additives in domestic animal production.

A ranking system for veterinary medicinal products and coccidiostat feed additives has been developed as a tool to be applied in a risk-based approach to the residue testing programme for foods of animal origin in the Irish National Residue Control Plan (NRCP). Three characteristics of substances that may occur as residues in food are included in the developed risk ranking system: Potency, as measured by the acceptable daily intake assigned by the European Medicines Agency Committee for Medicinal Products for Veterinary Use, to each substance; Usage, as measured by the three factors of Number of Doses, use on Individual animals or for Group treatment, and Withdrawal Period; and Residue Occurrence, as measured by the number of Non-Compliant Samples in the NRCP. For both Number of Doses and Non-Compliant Samples, data for the 5-year period 2008-12 have been used. The risk ranking system for substances was developed for beef cattle, sheep and goats, pigs, chickens and dairy cattle using a scoring system applied to the various parameters described above to give an overall score based on the following equation: Potency × Usage (Number of Doses + Individual/Group Use + Withdrawal Period) × Residue Occurrence. Applying this risk ranking system, the following substances are ranked very highly: antimicrobials such as amoxicillin (for all species except pigs), marbofloxacillin (for beef cattle), oxytetracycline (for all species except chickens), sulfadiazine with trimethoprim (for pigs and chickens) and tilmicosin (for chickens); antiparasitic drugs, such as the benzimidazoles triclabendazole (for beef and dairy cattle), fenbendazole/oxfendazole (for sheep/goats and dairy cattle) and albendazole (for dairy cattle), the avermectin ivermectin (for beef cattle), and anti-fluke drugs closantel and rafoxanide (for sheep/goats); the anticoccidials monensin, narasin, nicarbazin and toltrazuril (for chickens). The risk ranking system described is a relatively simple system designed to provide a reliable basis for selecting the veterinary medicinal products and coccidiostat feed additives that might be prioritised for residue testing.

Las plantas medicinales en el control de nemátodos gastrointestinales en cabras: potencial de las plantas que crecen en la región de Coquimbo, Chile / Medicinal plants in the control of gastrointestinal nematodes in goats: the potential of the plants which grow at the Coquimbo region, Chile

RESUMO O objetivo deste estudo foi determinar o potencial de plantas que crecem na região de Coquimbo, no controle de nematódeos gastrintestinais em caprinos. Nós usamos uma abordagem não-experimental para validar a eficácia potencial das plantas medicinais como anti-helmínticos. Esta validação foi realizada com base em uma revisão da extensa literatura consultada a respeito de plantas medicinais. Determinou-se o um potencial de eficácia das plantas, da consistência das informações sobre o uso tradicional, os compostos presentes e farmacologia. Analisamos também os dados dos potenciais efeitos adversos que impedem seu uso em animais. Antecedentes apoiam a um alto potencial da eficácia como anti-helmíntico para se três espécies de plantas que crescem na região de Coquimbo: Allium sativum L., Artemisia absinthium L. e Chenopodium ambrosioides L. Os possíveis efeitos secundários do consumo de plantas medicinais ou seus derivados devem ser avaliadas em testes de campo antes de serem utilizados nas em escala produtiva.

ABSTRACT The aim of this study was to determine the potential of the plants which grow at the Coquimbo region in the control of gastrointestinal nematodes in goats. We used a non-experimental approach to evaluate the potential efficacy of medicinal plants as anthelmintics. This research was conducted with exhaustive bibliography referred to the medicinal use of plants in the international literature. We determined the potential of effectiveness of the plants, the consistency of the information about the traditional use, the present compounds and the pharmacology. We also performed an analysis about the potential adverse effects of the plants" application in animals. The collected data support the efficacy of these three species of plants growing at the Coquimbo region as efficient anthelmintic ones: the Allium sativum L., the Artemisia absinthium L. and the Chenopodium ambrosioides L. The possible side effects of the consumption of medicinal plants or their derivatives must be evaluated in field trials before they start being used in a productive scale.

Heavy metals analysis, phytochemical, phytotoxic and anthelmintic investigations of crude methanolic extract, subsequent fractions and crude saponins from Polygonum hydropiper L.

BACKGROUND: Polygonum hydropiper L decoctions are traditionally used in the treatment of various ailments including inflammation, dyspepsia, diarrhea, menorrhagia, hemorrhoids, helminthiasis and CNS disorders. Present study was undertaken to investigate P. hydropiper L. for heavy metals content, phytoconstituents, Phytotoxic and anthelmintic activities to explore its toxicological and pharmacological potentials and rationalize its ethnomedicinal uses. METHODS: Plant crude powder, methanolic extract, fractions and soil samples were analyzed for heavy metals using atomic absorption spectrophotometer. Qualitative phytochemical analysis of the plant extracts was carried out for the existence of alkaloids, flavonoids, glycosides, anthraquinones, saponins, terpenoids, sterols and tannins. Radish seeds phytotoxicity assay was used to study phytotoxic action of plant extracts. Pheretima posthuma and Ascaridia galli were used to study anthelmintic potential of the plant using albendazole and levamisole HCl as standard drugs. RESULTS: Plant crude powder, methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) and soil samples were found to contain copper (Cu), iron (Fe), chromium (Cr), zinc (Zn), lead (Pb), nickel (Ni), cadmium (Cd) and lead (Pb) in different concentrations. In crude powder of the plant, heavy metals concentrations were within WHO specified limits, whereas different fractions and soil samples exhibited high metals content. Ph.Cr was tested positive for the presence of alkaloids, flavonoids, saponins, tannins, triterpenoids and anthraquinone glycosides. Among different fractions Ph.EtAc, Ph.Sp, Ph.Chf and Ph.Bt were most effective causing 89.32, 89.25, 86.68 and 85.32% inhibition of seeds in phytotoxicity assay, with IC50 values of 50, 60, 35 and 100 µg/ml respectively. In anthelmintic study, Ph.Sp, Ph.Chf, Ph.EtAc and Ph.Cr were most effective against P. posthuma at 10 mg/ml concentration with an average death time of 50, 64.67, 68.67 and 71 minutes respectively. Ph.EtAc, Ph.Chf and Ph.Aq were most effective against A. galli with average death time of 7, 9 and 10 min respectively at 1 mg/ml concentration. CONCLUSIONS: Our findings indicate that P. hydropiper contains different heavy metals and secondary metabolites. Different fractions exhibited phytotoxic and anthelmintic activites comparable to control drugs, thus provide pharmacological basis for ethnomedicinal uses of this plant.

Ruminant self-medication against gastrointestinal nematodes: evidence, mechanism, and origins.

Gastrointestinal helminths challenge ruminants in ways that reduce their fitness. In turn, ruminants have evolved physiological and behavioral adaptations that counteract this challenge. Ruminants display anorexia and avoidance behaviors, which tend to reduce the incidence of parasitism. In addition, ruminants appear to learn to self-medicate against gastrointestinal parasites by increasing consumption of plant secondary compounds with antiparasitic actions. This selective feeding improves health and fitness. Here, we review the evidence for self-medication in ruminants, propose a hypothesis to explain self-medicative behaviors (based on post-ingestive consequences), and discuss mechanisms (e.g., enhanced neophilia, social transmission) that may underlie the ontogeny and spread of self-medicative behaviors in social groups. A better understanding of the mechanisms that underlie and trigger self-medication in parasitized animals will help scientists devise innovative and more sustainable management strategies for improving ruminant health and well-being.

Phytochemical, anti-oxidant and Anthelmintic activities of various leaf extracts of Flacourtia sepiaria Roxb.

OBJECTIVE: The present study was carried out to investigate the phytochemical constituents, In vitro antioxidant potential and anthelmintic activities of Flacourtia sepiaria Roxb leaves. METHODS: The dried powdered leaves of Flacourtia sepiaria were extracted using petroleum ether, chloroform, ethyl acetate and methanol by a soxhlet extractor and preliminary phytochemical screening was performed using standard protocols. All the extract was evaluated for their potential antioxidant activities using test such as DPPH, superoxide anion radical, hydroxyl radical, nitric oxide radical scavenging abilities, ferrous chelating ability and total phenolic and flavanoid content. Anthelmintic activity of extract was screened in adult Indian earthworm model. RESULTS: Preliminary screening revealed the presence of bioactive compounds especially phenolics, tannins and terpenoids in all extracts. The phenolic and flavanoid content was highest in methanolic extract and lowest in petroleum ether extract. The paralytic (9.46±0.212) and death time (31.43±0.148) of methanolic extract was found to be significant (P<0.05) when compared with paralytic (7.33±0.206) and death time (18.60±0.229) of standard piperazine citrate at 100 mg/mL concentration. CONCLUSIONS: The results of the present study indicate that the leaf extracts of Flacourtia sepiaria exhibited strong antioxidant activity and possess significant anthelmintic activity and thus it is a good source of antioxidant and anthelmintic constituents.

Persistence of the efficacy of copper oxide wire particles against Haemonchus contortus in grazing South African goats.

A study was conducted to examine the duration of anthelmintic effect of copper oxide wire particles (COWP) in grazing goats, as data for the persistence of efficacy of COWP in this host species is limited. Forty-eight indigenous male goats were infected naturally by grazing them on Haemonchus contortus-infected pasture. When the faecal egg count (FEC) in the goats was 3179 ± 540 eggs per gram of faeces (mean ± standard error), half the animals were treated with 4 g COWP (day 0; mean live weight=25.5 ± 0.8 kg). Eight treated (COWP) and eight non-treated (CONTROL) goats were removed from the pasture on each of days 7, 28 and 56, maintained for 27 or 29 days in concrete pens and then humanely slaughtered for nematode recovery. Mean liver copper levels were in the high range in the goats removed from pasture at day 7 (treated: 191 ± 19.7 ppm; untreated: 120 ± 19.7 ppm; P=0.022), but had dropped to normal levels at days 28 and 56. The mean H. contortus burdens of the treated versus the non-treated goats were, respectively, 184 ± 48 and 645 ± 152 for the goats removed from pasture at day 7 (71% reduction; P=0.004), 207 ± 42 and 331 ± 156 at day 28 (37% reduction; P=0.945) and 336 ± 89 and 225 ± 53 at day 56 (-49% reduction; P=0.665). Weekly monitoring of FECs after treatment until slaughter indicated that the COWP-treated goats had lower FECs than the controls, the treatment main effect being significant at days 7, 28 and 56 (P<0.01). The day main effect and the treatment × day interaction were only significant for the goats removed from pasture at day 28 (P ≤ 0.001). Packed cell volumes increased during the course of the experiment (day, P<0.001), but the treatment main effect was significant only for the goats removed from pasture at day 28 (CONTROL 28 d, 28.65 ± 0.52%

Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs.

Anthelmintic drugs are used in clinical and veterinary practice for the treatment of infections caused by parasitic worms. Their extensive use in food-producing animals can cause the presence of residues in food. For consumer protection it is necessary to monitor the levels of anthelmintic residues to ensure that they remain within the legally permitted maximum acceptable concentrations. For this purpose, the use of multiplex screening methods is advantageous. Biochip array technology allows the simultaneous determination of multiple analytes from a single sample at a single point in time. This study reports the development of an Evidence biochip array for the multiplex screening of anthelmintic drugs. Simultaneous competitive chemiluminescent immunoassays are employed. The solid support and vessel is the biochip, which contains an array of discrete test sites. The assays were applied to the semiautomated bench-top analyser Evidence Investigator. The aminobenzimidazoles assay detected aminomebendazole, albendazole 2-aminosulphone and aminoflubendazole, the avermectins assay detected emamectin benzoate, eprinomectin, abamectin, ivermectin and doramectin, the benzimidazoles assay detected albendazole sulphone, albendazole, albendazole sulphoxide, oxibendazole, oxfendazole and flubendazole, the thiabendazole assay detected cambendazole, thiabendazole and 5-hydroxythiabendazole and the triclabendazole assay detected ketotriclabendazole, triclabendazole and triclabendazole sulphoxide. The limits of detection ranged from 0.3 ppb (aminobenzimidazoles) to 2.0 ppb (levamisole) in milk and from 0.15 ppb (aminobenzimidazoles) to 6.5 ppb (levamisole) in tissue. The average recovery range was 71-135 %. This multianalytical approach on a biochip platform is applicable to the screening of more than 20 anthelmintic drugs in different food matrices, leading to consolidation of tests and enhancement of the test result output.

Synthesis and biological evaluation of some novel 2-phenyl benzimidazole-1-acetamide derivatives as potential anthelmintic agents.

The present study describes synthesis of a series of 2-phenyl benzimidazole-1-acetamide derivatives and their evaluation for anthelmintic activity using Indian adult earthworms, Pheretima posthuma. The structure of the title compounds was elucidated by elemental analysis and spectral data. The compounds 4-({[2-(4-nitrophenyl)-1H-benzimidazol-1-yl]acetyl}amino) benzoic acid (3a), N-ethyl-2-[2-(4-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3c), N-benzyl-2-[2-(4-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3d), N-(4-hydroxyphenyl)-2-[2-(4-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3f), 2-[2-(4-nitrophenyl)-1H-benzimidazol-1-yl]-N-phenyl acetamide (3h), 2-[2-(4-chlorophenyl)-1H-benzimidazol-1-yl]-N'-phenylacetohydrazide (3k), 2-[2-(4-chlorophenyl)-1H-benzimidazol-1-yl]-N-(4-nitrophenyl) acetamide (3n) and 2-[2-(4-chlorophenyl)-1H-benzimidazol-1-yl]-N-phenyl acetamide (3q) were found better to paralyze worms whereas N-ethyl-2-[2-(4-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3c), N-(4-nitrophenyl)-2-[2-(4-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3e), 4-({[2-(4-chlorophenyl)-1H-benzimidazol-1-yl] acetyl}amino) benzoic acid (3j), 2-[2-(4-chlorophenyl)-1H-benzimidazol-1-yl]-N-ethyl acetamide (31) and 2-[2-(4-chlorophenyl)-1H-benzimidazol-1-yl]-N-phenyl acetamide (3q) were better to cause death of worms compared to the anthelmintic drug albendazole.

Animal grazing selectivity and plant chemistry issues impact on the potential of Rhagodia preissii as an anthelmintic shrub.

Rhagodia preissii had shown significant in vitro anthelmintic activity in a previous study, we examined the effect of including this shrub in the diet of sheep infected with Trichostrongylus colubriformis. Worm-infected merino wethers were grazed for 7 weeks on either R. preissii or annual pasture, and faecal egg counts (FECs) were conducted weekly. Plant material was collected weekly from eaten and uneaten plants, and analysed for levels of plant secondary metabolites (tannins, oxalates, saponins) and in vitro anthelmintic activity. While mean FECs were consistently lower in sheep grazing R. preissii compared to pasture (reductions of 20-74%), the differences were not significant. There was no relationship between grazing preference (eaten or uneaten) and in vitro anthelmintic activity of plant extracts. The levels of saponins and oxalates did not correlate with grazing preference or in vitro anthelmintic activity, while tannins were not responsible for the anthelmintic activity. While the identity of the grazing deterrent and in vitro anthelmintic compounds remain unknown, the presence of plants which were both highly preferred by the sheep and showed in vitro anthelmintic activity indicates a potential to develop the species as an anthelmintic shrub through selection of shrub populations dominated by such plants.

A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility.

BACKGROUND: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50) values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.