Egg Hatch Assay Research Articles

Potential anthelmintic effect of Capparis spinosa (Capparidaceae) as related to its polyphenolic content and antioxidant activity

Capparis spinosa is cultivated for several proprieties and the different parts of the plant (flower buds, fruits, leaf and seeds) have been employed in drugs, foods and cosmetics. This study aimed to explore the antioxidant potential effect and to assess the anthelmintic efficacy of an aqueous extract of Capparis spinosa. In vitro antioxidant activity of aqueous extracts from the fresh leaves and flower buds of C. spinosa was measured by determining free radical-scavenging activity against DPPH and ABTS radical cations. In vitro anthelmintic activities against eggs and adult worms of Haemonchus contortus from sheep were investigated in comparison to albendazole. DPPH and ABTS activities of both aqueous extracts increased in a dose dependent manner. The IC50 values of aqueous extracts from fresh leaves, flowers buds and ascorbic acid in the DPPH radical scavenging assay were 101.40; 70.40 and 57.56 μg/ml, respectively. The IC50 values of aqueous extracts fresh leaves, flowers buds and ascorbic acid in the ABTS radical scavenging assay were 110; 87.89 and 58.17 μg/ml, respectively. Both plant extracts showed ovicidal activity at all tested concentrations. The aqueous extract from flower buds (IC50 = 2.76 mg/ml) showed higher inhibitory effects (P < 0.05) than aqueous extract from leaves (IC50 = 8.54 mg/ml) in the egg hatching assay. The aqueous extract from flower buds inhibited more worms than the aqueous extract from leaves at all tested concentrations. After 6 h of exposure, the highest tested concentrations of aqueous extracts from flower buds and leaves induced 100 and 41.66% inhibition of motility, respectively. These results show for the first time that C. spinosa possesses in vitro anthelmintic properties which may be related to its high content of phenolic compounds such as flavonoids and tannins.

Ovicidal activity of four bioactive plant extracts against the cattle nematode, Cooperia spp. multi-resistant strain

Veterinaria México OA ISSN: 2448-6760Cite this as:von Son de Fernex E, Alonso Díaz MÁ, Mendoza de Gives P, Valles de la Mora B, Zamilpa A, González Cortasar M. Ovicidal activity of extracts from four plant species against the cattle nematode Cooperia punctata. Veterinaria México OA. 2016;3(2). doi: 10.21753/vmoa.3.2.365.Bioactive plants might represent an alternative for Cooperia punctata control in grazing cattle. The objectives of this study were (1) to assess the ovicidal activity of extracts from 4 plant species against C. punctata, (2) to determine the role of the polyphenols in the plants’ anthelmintic (AH) activity, and (3) to evaluate the best plant extraction procedure when searching for ovicidal activity. The egg hatch assay was used with different extraction procedures, aqueous (AQ), acetone:water (AW) and acetonic (AC), to evaluate the ovicidal activity of Leucaena leucocephala, Gliricidia sepium, Guazuma ulmifolia and Cratylia argentea. Eggs of C. punctata were exposed in quadruplicate to 0.6, 1.2, 2.4, 4.8 and 9.6 mg mL-1 of each plant extract. The roles of the polyphenols were assessed using polyethylene glycol (PEG). The 12 plant extracts inhibited egg hatching in a dose-dependent manner. Best-fit LC50 values were 1.03 ± 0.17 and 7.90 ± 1.19 mg mL-1 for G. sepium-AC and L. leucocephala-AQ, respectively. Differences in AH activity were found among the extraction procedures (P &lt; 0.05). At the highest concentration, L. leucocephala-AQ inhibited more than 50% of C. punctata hatching. The G. sepium-AC extract fully inhibited egg hatching. The addition of polyethylene glycol revealed the role of the polyphenols in the bioactivity of most plant extracts; however, for G. sepium-AC, the polyphenols were not the main bioactive compounds. Overall, acetone:water extraction represented the best extraction procedure to obtain both ovicidal activity and higher yield. The inhibition rates suggested that L. leucocephala and G. sepium should be evaluated as a means of reducing larval density in pastures.Table 1. Lethal concentrations required to inhibit 50% of Cooperia punctata egg hatching (LC50), after a 48-h incubation period with bioactive extracts (mg mL-1).

Ovicidal activity of four bioactive plant extracts against the cattle nematode, Cooperia spp. multi-resistant strain

Veterinaria Mexico OA ISSN: 2448-6760 Cite this as: von Son de Fernex E, Alonso Diaz MA, Mendoza de Gives P, Valles de la Mora B, Zamilpa A, Gonzalez Cortasar M. Ovicidal activity of extracts from four plant species against the cattle nematode Cooperia punctata . Veterinaria Mexico OA. 2016;3(2). doi: 10.21753/vmoa.3.2.365 . Bioactive plants might represent an alternative for Cooperia punctata control in grazing cattle. The objectives of this study were (1) to assess the ovicidal activity of extracts from 4 plant species against C. punctata , (2) to determine the role of the polyphenols in the plants’ anthelmintic (AH) activity, and (3) to evaluate the best plant extraction procedure when searching for ovicidal activity. The egg hatch assay was used with different extraction procedures, aqueous (AQ), acetone:water (AW) and acetonic (AC), to evaluate the ovicidal activity of Leucaena leucocephala , Gliricidia sepium , Guazuma ulmifolia and Cratylia argentea . Eggs of C. punctata were exposed in quadruplicate to 0.6, 1.2, 2.4, 4.8 and 9.6 mg mL -1 of each plant extract. The roles of the polyphenols were assessed using polyethylene glycol (PEG). The 12 plant extracts inhibited egg hatching in a dose-dependent manner. Best-fit LC 50 values were 1.03 ± 0.17 and 7.90 ± 1.19 mg mL -1 for G. sepium -AC and L. leucocephala -AQ, respectively. Differences in AH activity were found among the extraction procedures (P < 0.05). At the highest concentration, L. leucocephala -AQ inhibited more than 50% of C. punctata hatching . The G. sepium -AC extract fully inhibited egg hatching. The addition of polyethylene glycol revealed the role of the polyphenols in the bioactivity of most plant extracts; however, for G. sepium -AC, the polyphenols were not the main bioactive compounds. Overall, acetone:water extraction represented the best extraction procedure to obtain both ovicidal activity and higher yield. The inhibition rates suggested that L. leucocephala and G. sepium should be evaluated as a means of reducing larval density in pastures. Table 1. Lethal concentrations required to inhibit 50% of Cooperia punctata egg hatching (LC 50 ), after a 48-h incubation period with bioactive extracts (mg mL -1 ).

In vitro anthelmintic activity of the Zizyphus joazeiro bark against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells

This study examined the in vitro effect of the Zizyphus joazeiro bark against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells. The ovicidal activity of the crude hydroethanolic extract (CE), its partitioned hexane (HE) and aqueous extract (AE) and saponins fraction (SF), including betulinic acid (BA), a biogenetic compound from this plant found in HE, were investigated using the inhibition of egg hatch assay (EHA). Thereafter, the extracts and the SF were evaluated through the larval motility assay (LMA) and larval migration inhibition assay (LMIA). The AE and SF promoted a complete inhibition of the egg hatch, and the effective concentration to inhibit 50% (EC50) values was 1.9 and 1.3mg/mL, respectively. The highest percentages of inhibition in EHA observed after treatments with CE, HE and BA corresponded to 79, 48 and 17%, respectively. The extracts and SF did not show larvicidal activity in LMA and LMIA. The AE and SF demonstrated cytotoxic effects in 3-4,5-dimethylthiazol-2-yl, 2,5diphenyltetrazolium bromide (MTT) and trypan blue tests; however, SF was more toxic (50% inhibitory concentration, IC50=0.20mg/mL). The chemical characterization of the SF was made through Proton Nuclear Magnetic Resonance (1H NMR) and Electrospray Ionization Mass Spectrometry (ESI–MS) analyses, which led to the identification of two saponins known as Joazeiroside B and Lotoside A. The results obtained from the research of this saponin content provide important information about the biological activity, especially the anthelmintic effect present in the plant investigated. That also suggests the types of bioactive compounds that may be responsible for this antiparasitic activity exhibited by the plant extracts.

Anthelmintic activity of acetone extracts from South African plants used on egg hatching of Haemonchus contortus.

The nematode, Haemonchus contortus, is responsible for major economic losses in the livestock industry. The management of parasites such as H. contortus has been through the use of synthetic parasiticides. This has resulted in the presence of residues in meat and milk, which affects food safety. The development of resistance to available anthelmintics coupled with their high cost has further complicated matters. This has led to the investigation of alternative methods to manage nematodes, including the use of plants and plant extracts as a potential source of novel anthelmintics. Acetone extracts were prepared from 15 South African plant species and their anthelmintic activity determined using the egg hatch assay (EHA). The leaf extract of Cleome gynandra had the best inhibitory activity (68% ± 3%) at a concentration of 2.5 mg/mL, followed by the stem extract of Maerua angolensis (65% ± 5%). The extracts had a relatively low toxicity on Vero cells determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cellular assay.

Anthelmintic activity of Sesbania grandiflora against gastrointestinal nematodes of sheep

The study was carried out to validate the anthelmintic efficacy of Sesbania grandiflora and to standardize the effective dose of the plant extract required for worm control in livestock. In vitro and in vivo studies were conducted to determine the direct anthelmintic effect of ethanolic and aqueous extracts of S. grandiflora towards mixed ovine gastrointestinal nematodes. Egg hatch assay for ovicidal and larval migration inhibition and larval development assay for larvicidal properties were used to investigate in vitro effect of extracts on strongyle egg and larvae. Faecal egg count reduction test was conducted in vivo to evaluate the therapeutic efficacy of the extacts administered orally @ 125, 250, 500 mg/kg to sheep naturally infected with mixed gastrointestinal nematodes. Aqueous extract of S. grandiflora demonstrated significant inhibition of egg hatch and larval migration @ 40 and 80 mg/ml. The ED50 value of egg hatch inhibition and LM50 value for larval migration inhibition were 1.489 and 0.683 mg/ml respectively. In faecal egg count reduction test (FECRT), aqueous extract of S. grandiflora at 500 mg/kg caused significant reduction in eggs per gram (98.10 %) higher than the result obtained with albendazole (93.25 %). Although there were slight variations in the haematological parameters (PCV, haemoglobin, RBC and WBC) in all the groups between day 0 and 12, all the parameters were within the normal range reported for sheep. Except for blood urea nitrogen, overall mean of all the serum biochemical profile was within the normal range for sheep. Based on the results obtained by in vitro and in vivo assay, the aqueous extract of S. grandiflora possess anthelmintic activity and could offer an alternative source for the control of gastrointestinal nematodes of sheep.

In vitro anthelmintic activity of aqueous extract of Crassocephalum crepidioides (Benth.) S. Moore on Haemonchus contortus.

Objectives: Gastrointestinal parasites are recognized as major constraint to livestock production throughout the tropics. The use of plants and plant extracts remain the most serious alternative to modern anthelmintic drugs. This study evaluates the in vitro anthelmintic activity of aqueous leaves extract of Crassocephalum crepidioides against Haemonchus contortus. Methods: Aqueous extract at concentrations of 75 to 2400 µg/ml was tested in vitro on three development stages of H. contortus using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worms motility inhibition assay (AMIA). Results: EHA showed significant reduction (P < 0.05) on H. contortus egg hatch. The inhibition of egg hatching was concentration dependent with the greatest inhibition (50.52%) at the highest concentration (2400 µg/ml) of the extract. Thiabendazole used as positive control showed significant inhibition (P < 0.001) with rate of 76.68 % at the highest concentration (500µg/ml). On LMIA the extract significantly (P < 0.05) inhibited larval migration of L3 in a concentration dependent manner compared to Phosphate Buffered Saline, but less than levamisole (P < 0.001). The highest inhibition rate was 35.30 % at the concentration of 1200 µg/ml. The addition of polyvinylpolyrrolidone to the aqueous extract slightly reduces (4.9%) the inhibition of the effect induced by the extract on larval migration. Effect of extract on AMIA was concentration-dependent with significant increase (P < 0.05) in percentage of inhibition after 12 h. Levamisole (500 µg/ml) kills 100% worms after 18 h post-exposure but by this time the plant exhibited only 16.67% inhibition at the highest concentration (2400 µg/ml). Conclusion: These finding indicate that C. crepidioides leaves have anthelmintic properties against H. contortus. This activity may be due to secondary metabolites such as saponins, flavonoids and tannins present in the extract. Further studies are needed to evaluate deeply the anthelmintic potential of this plant.

Influence of Co-Administration of Parbendazole on The Disposition Kinetics and Efficacy of Albendazole in Goats

A study was undertaken to understand the pharmacokinetics and efficacy of albendazole (ABZ) in goats with and without co-administration of ABZ with parbendazole (PBZ) at 7.5 and 4.5 mg/kg body weight, respectively. Plasma albendazole sulphoxide metabolite (active moiety), reached its maximum concentration of 0.322 and 0.325 μg/ml at 18 h of oral administration of ABZ alone and co-administration of ABZ with PBZ. No significant difference (P>0.05) in the values of other pharmacokinetic parameters could be observed except for elimination half-life, where significantly (P<0.05) increased values were observed in ABZ + PBZ treated goats. The nematodes were found resistant against ABZ alone (ED 50 on egg hatch assay was 0.196 μg thiabendazole/ml) while susceptible against the drug combination. The dominant nematode exhibiting resistance was Haemonchus spp. Almost unaltered or slightly reduced drug-parasite contact was observed in goats receivingABZ and PBZ combination.

Anthelmintic activity of Indigofera tinctoria against gastrointestinal nematodes of sheep.

Aim:Gastrointestinal (GI) nematodes are considered as a major constraint for successful sheep production. Control of these parasites heavily relies on the use of chemical anthelmintics. Over the past decades, the development of anthelmintic resistance to various groups of anthelmintics and problem of drug residues in animal products has awakened interest in medicinal plants as an alternative source of anthelmintics. Hence, this study was undertaken to evaluate the anthelmintic efficacy of Indigofera tinctoria by scientifically validated in vitro and in vivo tests approved by the World Association for the Advancement of Veterinary Parasitology.Materials and Methods:In vitro assays such as egg hatch assay for ovicidal and larval migration inhibition and larval development assay for larvicidal properties were used to investigate in vitro effect of extracts on strongyle egg and larvae, respectively. Fecal egg count reduction test was conducted in vivo to evaluate the therapeutic efficacy of the extracts administered orally at dose rates of 125, 250, 500 mg/kg to sheep naturally infected with mixed GI nematodes.Results:Ethanolic extract of I. tinctoria demonstrated significant (p<0.01) inhibition on egg hatching at concentrations of 40 mg/ml and 80 mg/ml. In in vivo assay, the ethanolic extract of I. tinctoria reduced the fecal egg count ranging between 30.82% and 47.78% at various doses (125, 250 and 500 mg/kg). Although there was a slight variation, all the hematological parameters were within the normal range reported for sheep. Except for alanine transaminase, the overall mean of all the serum biochemical profile was within the normal range for sheep.Conclusion:Based on the results obtained by in vitro and in vivo assay, the ethanolic extract of I. tinctoria possesses anthelmintic activity and could replace the chemical anthelmintics used presently.

Fractionation of Heteromorpha arborescens var abyssinica (Apiaceae) leaf extracts based on polarity leads to a marked change in cytotoxicity that may yield a commercially useful product

Parasitic gastroenteritis caused by helminths remains a major cause of mortality and morbidity especially in small ruminants in the tropics. Haemonchus contortus is the major helminth responsible for this disease. Chemical anthelmintics are the most reliable means of treatment. The frequent use of these anthelmintics has led to resistance development. The issue of residues in meat and milk also motivates the use of plants and plant derived products as alternate to chemical anthelmintics. The anthelmintic, antimicrobial activity and cytotoxicity of the fractions of Heteromorpha arborescens var abyssinica were investigated. The egg hatch assay and the larval development test were used to determine the anthelmintic activity. A serial microdilution method with iodo nitrotetrazolium violet as growth indicator was used to determine the antibacterial and antifungal activities. The cellular cytotoxicity of the fractions was determined using the MTT assay. The egg hatch assay led to an EC50 of 0.68, 0.73, 1.58 and 2.88mg/ml for the butanol, ethyl acetate, chloroform and hexane fractions respectively. The larval development test of the butanol, chloroform, ethyl acetate and hexane fractions led to EC50 values of 0.64, 1.05, 1.05 and 3.28mg/ml respectively. Because extracts or fractions can be more useful if they have activity against more than one indication the antimicrobial activity of the fractions was also determined. The best antifungal minimum inhibitory concentrations (MICs) of 0.08mg/ml was against Cryptococcus neoformans and 0.63mg/ml against Aspergillus fumigatus and Candida albicans. Pseudomonas aeruginosa was most susceptible bacterium in this study. The cytotoxicity LC50 values of the fractions of hexane, ethyl acetate, butanol and chloroform were 1.40, 0.91, 2.06 and 2.04mg/ml. Fractionation of the acetone extract of H. arborescens var abyssinica led to a remarkable 35 to 50-fold reduction in cytotoxicity of the fractions compared to the original extract.The anthelmintic activities of the crude leaf extract and fractions were not that high, but due to the decreased cytotoxicity the butanol fraction had a three-fold higher efficacy against H. contortus in both assays than toxicity against the cells. This may lead to a product that can be used to treat helminthiasis in animals if animal safety is in line with cellular safety. The results stress the importance of determining the cytotoxicity of plant extracts with a potential to be used in animal studies. The selectivity index may be more important than the activity of an extract against microorganisms or parasites.

Temperature dependent embryonic development of Trichuris suis eggs in a medicinal raw material

The therapeutic potential of infective pig whipworm eggs, Trichuris suis ova (TSO), is currently tested in several clinical trials on immune-mediated diseases. This paper studied the embryonic development of TSO in a medicinal raw product, where the parasite eggs were suspended in sulphuric acid (pH1). Unembryonated T. suis egg batches were stored at 5, 10, 15, 20, 25, 30, and 40°C (±1°C) and examined at 2, 4, 8, and 14 weeks. Subsequently, sub-batches from each temperature were allowed to embryonate for additional 14 weeks at 25°C, and selected samples were tested for infectivity in Göttingen minipigs.Both male and female pigs were used to evaluate eventual gender specific infectivity. Storage at 30°C up to 14 weeks and subsequent embryonation for 14 weeks at 25°C did not significantly reduce the overall larval establishment in minipigs, as compared to storage at 5°C and subsequent embryonation at 25°C. As marked impairment of egg development was observed during storage at 40°C, a second set of unembryonated egg batches were incubated at 30, 32, 34, 36, 38, and 40°C (±1°C) for 1–8 weeks. The development of the eggs was repeatedly examined by manual light microscopy, multispectral analysis (OvaSpec), and an egg hatching assay prior to the final testing in minipigs (Trial 1). These methods showed that the development started earlier at higher temperatures, but the long-term storage at higher temperature affected the egg development. The present study further documents tolerance of the TSO to storage at temperature 5–15°C, at which temperature development of larvae is not initiated.

The association between antifungal activity and antiparasitic activity of plant extracts against helminths

Haemonchus contortus is globally the most important helminth that causes tremendous losses in small animal production. Treatment cost with anthelmintics to manage infestations was more than $175 million in South Africa, Kenya and India in 2004 [1]. Many poor farmers cannot afford the cost of the anthelmintics. The situation is aggravated by the strong development of resistance by the parasite against currently used anthelmintics. Plants have been used to treat helminth infections by rural farmers with some positive results. The isolation of anthelmintic compounds is challenging due to the demanding assays required for bioassay-guided fractionation. A class of the important anthelmintics the benzimidazoles have good antifungal activity [2]. We separated plant extracts into different fractions based on polarity and determined the activities. When the ethyl acetate fraction was omitted there was an excellent correlation between egg hatch assay and minimum inhibitory concentration (MIC) (R2= 0.9) and a good correlation between larval development and MIC, (R2= 0.6). We determined the anthelmintic activity of 13 tree leaf extracts with known antifungal activity. In many cases there appeared to be a good association between egg hatch assay and the larval development assay of H. contortus. When we analysed the data there was some correlation between the MIC of Cryptococcus neoformans and the egg hatch assay (R2= 0.15) and also between Aspergillus fumigatus and the egg hatch assay (R2= 0.15). The correlations between the MICs and the larval development was much lower (R2= 0.01). By deleting outliers the correlation was improved. We conclude that if the mechanism of antifungal activity is related to the anthelmintic activity it may be worthwhile to isolate antifungal compounds and then determine their anthelmintic activities.

Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

In vivo assessment of closantel ovicidal activity in Fasciola hepatica eggs

Anthelmintic resistance in livestock parasites is currently a worldwide problem.Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment.Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs.As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.

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 8000μg/ml PBS). Additional concentrations were tested for O. viciifolia (75, 100, 200 and 400μg/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, 5000μg/ml, 5000μg/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 14106μg (RR from 2.01 to 6.40). The O. viciifolia extract showed higher variability with EC50 values ranging from 104 to 4783μg (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.

Elucidation of Leucaena leucocephala anthelmintic-like phytochemicals and the ultrastructural damage generated to eggs of Cooperia spp.

Leucaena leucocephala is a tropical forage legume suggested as an alternative method to control gastrointestinal parasitism in ruminants. This study: (1) performed a bio-guided fractionation of an aqueous extract of L. leucocephala using the egg hatch assay (EHA) to identify the anthelmintic (AH)-like phytochemicals present in fresh leaves, and (2) assessed the ultrastructural damage to eggs of Cooperia spp. after incubation with the final fraction. Phytochemicals were isolated using silica gel columns and identified using high performance liquid chromatography and standards for comparison. The final fraction was evaluated using EHA at 0.06, 0.125, 0.250, 0.500 and 1.1mgml−1. The lethal concentration to inhibit 50% of Cooperia spp. egg hatching (LC50) was calculated using a Probit analysis. Scanning and transmission electron microscopy revealed the ultrastructural changes present in Cooperia spp. eggs. Bio-guided isolation procedures led to the recognition of an active fraction (LlC1F3) mainly composed of quercetin (82.21%) and caffeic acid (13.42%) which inhibited 90.49±2.8% of Cooperia spp. egg hatching (P<0.05), and an LC50 of 0.06±0.14mgml−1. Scanning electron microscopy (SEM) showed eggs exposed to the active fraction had an irregular external layer with small projections and ruptures of lateral eggshell walls. Transmission electron microscopy (TEM) showed changes to Cooperia spp. eggs in electro-density, including the thickness of the eggshell layers and fractures after incubation with the final fraction (LlC1F3).Changes in bioactivity after purification suggest synergistic interactions between quercetin and caffeic acid.

Assessment of in vitro and in vivo anthelminthic potential of extracts of Allium sativum bulb against naturally occurring ovine gastrointestinal nematodiosis

Background: The rapid development of anthelminthic resistance has limited the success of traditional control programmes, thereby forcing researchers to search for ethno-veterinary alternatives.Objective: The objective is to assess the anthelminthic potential of various extracts of the bulb of Allium sativum in naturally infected sheep.Animals and methods: In vitro anthelminthic activities of crude aqueous and methanolic extracts of the bulb of A. sativum were investigated against the egg (500 eggs/ml) and larvae of naturally infected sheep. The aqueous extract of A. sativum was also investigated for in vivo anthelminthic activity in three groups (n = 15 each) of naturally infected Chokla sheep with a negative control group receiving no treatment, a positive control group was given a single oral dose of albendazole at 7.5 mg/kg bodyweight, and a group administered a single oral dose of an aqueous extract at 5 g/animal. Data were analysed using the general linear model.Results: Aqueous extract showed better efficacy in egg hatch assay and larval development test. However, in larval paralysis test, reverse trend was seen as methanolic extract was more potent than the aqueous counterpart. A significant amount of 57% faecal egg count reduction was observed in in vivo trail using the aqueous extract on day 21 post-treatment, although in initial stages it showed 30% and 83% effectiveness on days 7 and 14 post-treatment, respectively. No deleterious ill effect was found in any of the haematological and biochemical parameters.Conclusions: Bulb of A. sativum possesses good anthelminthic efficacy and further research is thereby warranted before recommending it for nematode control programme in ovines.

Comparing an in vivo egg reduction test and in vitro egg hatching assay for different anthelmintics against Fasciola species, in cattle

This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A–E). On day zero; groups A–D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P<0.0001), however, praziquantel did not record any reduction of Fasciola egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P<0.01). In albendazole, the hatching varied according to the drug concentration. At the highest two concentrations; 0.2 and 2.0μg/ml, the hatching percentages were 7.4±1.6 and 5.6±1.5 (P<0.01) respectively. On the contrary, there were no significant differences in egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect.

Screening anthelmintic resistance to triclabendazole in Fasciola hepatica isolated from sheep by means of an egg hatch assay.

BackgroundIn the present study, the detection of anthelmintic resistance to triclabendazole (TCBZ) in sheep infected by Fasciola hepatica was studied using an egg hatch assay (EHA). F. hepatica eggs were recovered from bile and faeces of infected animals by isolates with different grade of anthelmintic resistance to TCBZ: i) a resistant isolate (RT); ii) a susceptible isolate (ST); iii) naturally infected sheep by a susceptible field strain (FST). In the EHA the percentage of hatched eggs were calculated according to the following concentrations of TCBZ diluted in dimethyl-sulfoxide (DMSO): 0.05, 0.2, 1, 5, and 25 μg/ml.ResultsIn relation to the EHAs carried out with the eggs from bile of sheep infected by ST, differences were found in the percentage of hatched eggs between the control well, only with DMSO, and the two highest concentrations of TCBZ (5 and 25 μg/m) (p < 0.05). However, when we tested the drug with the eggs from the bile of sheep infected by RT, the percentage of hatched eggs was similar among all concentrations. Since the range of hatching varied between isolates, we calculated the ratio of the results of each concentration to its control value confirming the higher hatching in RT than in ST.We developed an EHA with eggs recovered from faeces in order to avoid the slaughter of sheep. The results of the EHAs with the isolate ST showed differences in the percentage of hatching between the highest concentration (25 μg/ml) and the control well (p < 0.05); however, these differences were not confirmed under field conditions with the strain FST.ConclusionsThe ovicidal effect of TCBZ in F. hepatica eggs from bile was shown using a commercial formulation diluted in DMSO with a minimum concentration of 5 μg/ml. However, in eggs recovered from faeces the results are not conclusive. The cleaning of eggs recovered from faeces is an important issue that should be reviewed and standardized before comparing results between susceptible and resistant isolates in this kind of EHA.

Bacteria-induced egg hatching differs for Trichuris muris and Trichuris suis.

BackgroundEggs of the porcine whipworm Trichuris suis are currently explored in human clinical trials as a treatment of immune-mediated diseases. In this context, only the infective, embryonated eggs, constitute the Active Pharmaceutical Ingredient (API). The rodent whipworm, Trichuris muris is commonly used as a laboratory model to study Trichuris biology. The embryonated eggs (containing a fully developed larva) are biologically active and will invade the large intestinal mucosa of the host. This study aims to assess the in vitro hatching of T. muris and T. suis eggs in various bacterial cultures as a measure for their biological activity.MethodsEggs of T. muris and T. suis were incubated with Escherichia coli strain (BL-21) at three concentrations in a slightly modified in vitro egg hatching assay previously developed for T. muris. Additionally, E. coli strains (M15, SG13009, PMC103, JM109, TUNER, DH5alpha, TOP10) and five Gram-positive bacteria (Enterococcus caccae, Streptococcus hyointestinalis, Lactobacillus amylovorus, L. murinus, and L. reuteri) were tested as a hatching stimulus for T. muris and T. suis eggs.ResultsWhereas T. muris eggs hatched, T. suis did not, even when exposed to different concentrations and strains of E. coli after 4 and 24-hour incubation. When incubated with Gram-positive bacteria, only T. muris eggs showed noticeable hatching after 20 h, although with high variability.ConclusionsThe observed difference in hatching of T. muris and T. suis eggs incubated with selected bacteria, indicate significant biological differences which may reflect specific adaptation to different host-specific gut microbiota.