Larval Migration Inhibition Assay Research Articles

Phytochemical analysis and anthelmintic activity of Combretum mucronatum leaf extract against infective larvae of soil-transmitted helminths including ruminant gastrointestinal nematodes

BackgroundSoil-transmitted helminths (STH) infect more than a quarter of the world’s human population. In the absence of vaccines for most animal and human gastrointestinal nematodes (GIN), treatment of infections primarily relies on anthelmintic drugs, while resistance is a growing threat. Therefore, there is a need to find alternatives to current anthelmintic drugs, especially those with novel modes of action. The present work aimed to study the composition and anthelmintic activity of Combretum mucronatum leaf extract (CMLE) by phytochemical analysis and larval migration inhibition assays, respectively.MethodsCombretum mucronatum leaves were defatted with petroleum ether and the residue was extracted by ethanol/water (1/1) followed by freeze-drying. The proanthocyanidins and flavonoids were characterized by thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UPLC). To evaluate the inhibitory activity of this extract, larval migration assays with STH and GIN were performed. For this purpose, infective larvae of the helminths were, if necessary, exsheathed (Ancylostoma caninum, GIN) and incubated with different concentrations of CMLE.ResultsCMLE was found to be rich in flavonoids and proanthocyanidins; catechin and epicatechin were therefore quantified for standardization of the extract. Data indicate that CMLE had a significant effect on larval migration. The effect was dose-dependent and higher concentrations (1000 µg/mL) exerted significantly higher larvicidal effect (P < 0.001) compared with the negative control (1% dimethyl sulfoxide, DMSO) and lower concentrations (≤ 100 µg/ml). Infective larvae of Ascaris suum [half-maximal inhibitory concentration (IC50) = 5.5 µg/mL], Trichuris suis (IC50 = 7.4 µg/mL), and A. caninum (IC50 = 18.9 µg/mL) were more sensitive to CMLE than that of Toxocara canis (IC50 = 310.0 µg/mL), while infective larvae of Toxocara cati were largely unaffected (IC50 > 1000 µg/mL). Likewise, CMLE was active against most infective larvae of soil-transmitted ruminant GIN, except for Cooperia punctata. Trichostrongylus colubriformis was most sensitive to CMLE (IC50 = 2.1 µg/mL) followed by Cooperia oncophora (IC50 = 27.6 µg/mL), Ostertagia ostertagi (IC50 = 48.5 µg/mL), Trichostrongylus axei (IC50 = 54.7 µg/mL), Haemonchus contortus (IC50 = 145.6 µg/mL), and Cooperia curticei (IC50 = 156.6 µg/mL).ConclusionsThese results indicate that CMLE exhibits promising anthelmintic properties against infective larvae of a large variety of soil-transmitted nematodes.Graphical

Bacteria Associated with the Parasitic Nematode Haemonchus contortus and Its Control Using Antibiotics

Internal parasitism, a significant cause of production losses in sheep, is routinely controlled by anthelmintic drenches. A better understanding and alternative control strategies are needed to combat the increasing resistance. This study investigated the presence of resident bacteria in the field strain of Haemonchus contortus. Adult female worms were collected from the abomasa of sheep. DNA was extracted from adult female worms and parasite eggs laid in vitro using long or shorter enzymatic incubation methods. Polymerase Chain Reaction (PCR) was performed using universal bacterial and phylum Firmicutes-specific primers; PCR products were cloned and sequenced. The analysis of the sequences shows a majority of the sequences belong to rumen bacteria, mainly Ruminococcus. Streptococcus was detected in four eggs, and adult worm samples and the sequences had a very high homology to the Streptococcus sequences in the database. Clostridium was detected only in the adult samples, whereas Nevskia and Pseudomonas were detected only in the egg samples. Three antibiotics, Ampicillin (Amp), Gentamycin (Gen) and Tetracycline (Tet), individually or combination, were tested to establish proof of concept that abomasal nematode parasites can be controlled by killing the resident bacteria. A larval migration inhibition assay was used to test the hypothesis. Tet (10 and 20 mM) resulted in around 30% mortality in larvae. Amp and Gen did not result in significant levels of larval mortality but, when given in combination, resulted in significant mortality of the larvae, suggesting the role of antibiotics in controlling the parasites by targeting the resident bacteria.

Evaluation of in vitro methods of anthelmintic efficacy testing against Ascaridia galli.

To investigate methods for in vitro assessment of anthelmintic efficacy against the chicken nematode Ascaridia galli this study firstly evaluated sample preparation methods including recovery of eggs from excreta using different flotation fluids and induced larval hatching by the deshelling-centrifugation method and the glass-bead method with or without bile. It then evaluated two in vitro assays, the in-ovo larval development assay (LDA) and larval migration inhibition assay (LMIA), for anthelmintic efficacy testing against A. galli using fresh eggs and artificially hatched larvae, respectively. Four anthelmintics, thiabendazole (TBZ), fenbendazole (FBZ), levamisole (LEV) and piperazine (PIP) were employed using an A. galli isolate of known susceptibility. The results suggested that the LDA and LMIA could successfully be used to generate concentration response curves for the tested drugs. The LDA provided EC50 values for inhibition of egg embryonation of 0.084 and 0.071 μg/ml for TBZ and FBZ, respectively. In the LMIA, the values of effective concentration (EC50) of TBZ, FBZ, LEV and PIP were 105.9, 6.32, 349.9 and 6.78 × 107 nM, respectively. For such in vitro studies, a saturated sugar solution showed high egg recovery efficiency (67.8%) and yielded eggs of the highest morphological quality (98.1%) and subsequent developmental ability (93.3%). The larval hatching assays evaluated did not differ in hatching efficiency but the deshelling-centrifugation method yielded larvae that had slightly better survival rates. For final standardization of these tests and establishment of EC50 reference values, tests using isolates of A. galli of defined resistance status need to be performed.

Anthelminthic effects of extracts of indigenous browses from mid rift valley of Ethiopia

This study was conducted to evaluate the potential anthelminthic properties of extracts of leaves of indigenous browses (Acacia seyal, Acacia senegal, Acacia tortilis, Millettia ferruginea, and Vernonia amygadalina) based on three in vitro assays. Acetone extracts of browses at different concentrations (75 to 1200 μg/ml, for egg and larvae and 100mg/ml for an adult) were tested on three developmental stages of Haemonchus contortus (eggs, infective larvae, and adult worms) using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worm motility inhibition assay (AMIA). Significant effects were obtained with all five browses but differences were observed depending on the parasitic stages. The effects of five browse extracts on egg hatching were concentration-dependent, the highest (P&lt;0.05) egg hatch inhibition rate was observed at 1200 μg/ml concentration for all browses. All extracts had a higher effect (P&lt;0.01) than that of the negative control, phosphate buffer saline (PBS). In contrast, no concentration-response relationship was found for infective larvae and adult worms, although more potent effects were observed with the highest concentrations. The LMI rate (70%) induced by Vernonia amygadalina extract, at a concentration of 300 μg/ml, was the highest (P&lt;0.05) of all other browses, even at higher concentrations. The highest LMI rate (62%) induced by Acacia senegal extract at higher concentration, was lower than that of LMI rate (70%) induced by Vernonia amygadalina, at 300 μg/ml concentration. Vernonia amygadalina was found to be highly and rapidly effective against adult worms inducing the highest mortality rate (90%) as soon as 4 hrs after incubation. Overall, the in vitro results suggest that these five browses do possess anti-parasitic properties and Vernonia amygadalina showed the most effective anti-parasitic property. These effects remain to be confirmed through in vivo study.

Susceptibility of entomopathogenic nematodes to ivermectin and thiabendazole

The objective of the present study was to determine the susceptibility of entomopathogenic nematodes to ivermectin and thiabendazole. Soil samples collected from the municipalities of Irapuato and León, Guanajuato, Mexico, were obtained, from which the entomopathogenic nematodes (EPNs) of the Steinernematidae and Heterorhabditidae families were isolated. The samples were classified from livestock and nonlivestock soils, and the susceptibility of EPNs to anthelmintics was determined with the larval motility assay (LMA, 24 h) and the larval migration inhibition assay (LMI assay, 48 h). Sterile distilled water (T1) and treatments with 1% ivermectin diluted in 5% DMSO (dimethyl sulfoxide) (T2) and 5% thiabendazole diluted in 5% DMSO (T3) were applied to infective juvenile larvae. Analysis of variance was performed with a factorial design and Tukey’s test at 0.05 probability. In addition, different concentrations of ivermectin (0.1, 0.5, 1, 1.2, 1.5, and 2 μg) and thiabendazole (1, 5, 10, 12, 15, and 20 mg) were evaluated to perform a Probit analysis to determine their LC50. All strains of EPNs were susceptible to ivermectin in both the LMA and LMI assay. The results show that EPNs are susceptible to ivermectin and thiabendazole, and the degree depends on the type of test performed, the chemical product used, and the origin of the strain of EPN.

In vitro anthelmintic activity of Psidium guajava hydroalcoholic extract against gastro-intestinal sheep nematodes.

Tanniferous plants have been used for ruminants verminosis control and represent a possibility to minimize the pharmacological resistance against conventional antiparasitics. This study aimed to evaluate the antihelminthic activity of the hydroalcoholic extract of stem bark of guava tree (PgHA). It was performed the hatchability and larval migration inhibition assays to evaluate PgHA at the following concentrations 0.62, 1.25, 2.5 and 5.0 mg mL-1 and the control treatments. The total polyphenol, flavonoid and tannin contents were determined by phytochemical analysis, high performance liquid chromatography coupled to mass spectrometry. The antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl, ferric reducing antioxidant power and thiobarbituric acid reactive substances tests. It was also determinated total protein, intracellular H2O2 and antioxidant activity of enzimes: glutathione S-transferase and superoxide dismutase. PgHA was able to inhibit both hatchability and larval migration, but only hatchability inhibition presented dose-dependent pattern. The antioxidant activity was demonstrated by linear regression with IC50 corresponding to 534.02 μg mL-1. The antiparasitic mechanism occurred through pro-oxidative activity by the increase of total proteins, intracellular H2O2 and the lipid peroxidation products, as well as the increase of the enzymes above related. Thus, the PgHA showed antiparasitic activity in vitro.

In vitro anthelmintic activity of grape pomace extract against gastrointestinal nematodes of naturally infected sheep

Grape pomace obtained as a byproduct of industrial processing of grapes retains nutrients and substances with anthelmintic potential such as saponins, tannins, and flavonoids. Therefore, this study evaluated the in vitro ovicidal and larvicidal activity of hydroalcoholic grape pomace extract against gastrointestinal nematodes of sheep. The anthelmintic evaluation was performed by in vitro assays with eggs and larvae of nematodes obtained from naturally infected donor sheep. The grape pomace extract showed high in vitro ovicidal and larvicidal activity with LD50 values of 0.30 mg/mL for egg hatching inhibition, 1.01 mg/mL for larval development inhibition and 100% efficacy in larval migration inhibition assays at all concentrations evaluated. The effect of tannins was evaluated by the addition of 50 mg/mL polyvinyl polypyrrolidone to grape pomace extract at the concentration of 12.5 mg/mL. The in vitro ovicidal activity of grape pomace was reduced by only 15% after polyvinyl polypyrrolidone addition, indicating that other phytochemicals also contribute to the ovicidal activity displayed by the extract. Our results demonstrate that grape pomace exhibits in vitro anthelmintic activity, suggesting that, beyond its nutritional potential, this pomace can also be an ally for gastrointestinal nematode control in sheep.

In vitro effects of aqueous extract from Maytenus senegalensis (Lam.) Exell stem bark on egg hatching, larval migration and adult worms of Haemonchus contortus

BackgroundMaytenus senegalensis is a common shrub which is scattered in tropical Africa. Different parts of this plant have been reported to be useful in traditional medicine against gastrointestinal disorders and intestinal worms. This study evaluated the anthelmintic activity of the aqueous stem bark extract of M. senegalensis using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worms’ motility inhibition assay (AMIA).ResultsOn EHA, the extract concentrations tested resulted in a significant (p < 0.01) inhibition of egg hatching in concentration-dependent manner and ranged between 31.86% at 75 μg.mL− 1 to 54.92% at 2400 μg.mL− 1 after a 48 h post-exposure with eggs. For the LMI assays, the aqueous extract of M. senegalensis showed a significant (p < 0.05) inhibition of larval migration in a concentration-dependent manner. The highest concentration used (2400 μg.mL− 1) showed a 37.77% inhibition. The use of polyvinyl polypyrrolidone (PVPP) indicated that tannins and flavonoids were partly involved in the effect since the larval migration was inhibited by 15.5%, but other biochemical compounds were also implicated. On AMIA, M. senegalensis was associated with a reduced worm motility after a 24 h post exposure compared to phosphate buffered saline as control (p < 0.05). By this time 66.66% of the worms’ were found immotile or dead in the wells containing plant extract at 2400 μg.mL− 1. The Phytochemical analysis of aqueous extract of M. senegalensis by HPLC-ESI-MSn detected the presence of proanthocyanidins (20%) and flavonoids (> 50%).ConclusionsThese in vitro results suggest the presence of some anthelmintic properties in M. senegalensis extract, which is traditionally used by small farmers in west and central Africa. These effects may be due to the flavonoids and proanthocyanidins present in the extract and need to be studied under in vivo conditions.

Motility in the L3 stage is a poor phenotype for detecting and measuring resistance to avermectin/milbemycin drugs in gastrointestinal nematodes of livestock

Motility is a commonly used in vitro phenotype for assessing anthelmintic activity of candidate compounds, and for detecting anthelmintic resistance in nematodes. Third-stage larvae (L3) of parasitic nematodes are commonly used in motility-based assays because L3 are simple to obtain and can remain viable in storage for extended periods. To improve the measurement of motility of microscopic stages of nematodes, our laboratory developed the Worminator, which quantitatively measures motility of parasites. Using the Worminator, we compared the dose-response characteristics of several avermectin/milbemycin (AM) compounds using L3 from both AM-susceptible and AM-resistant Cooperia spp. (abamectin, doramectin, eprinomectin, ivermectin, moxidectin) and Haemonchus contortus (eprinomectin, ivermectin, moxidectin). Concentrations tested with the Worminator ranged from 0.156 to 40 μM. Differences in EC50 between AM-susceptible and AM-resistant isolates of Cooperia spp. and Haemonchus contortus were small, with resistance ratios ranging from 1.00 to 1.34 for Cooperia spp., 0.99 to 1.65 for Haemonchus contortus. Larval migration inhibition assays were conducted using the same isolates and were equally ineffective for detection of resistance with resistance ratios less than 2.0. These results contrast with those of the Larval Development Assay where we obtained a resistance ratio of 16.48 using the same isolates of Haemonchus contortus. Moreover, even at the highest concentration tested (40 μM), 100% inhibition of motility was never achieved and EC50 for Worminator assays were more than 100× higher than peak plasma levels achieved in vivo following treatment. These data demonstrate that dose-response characteristics for inhibition of motility in L3 of gastrointestinal nematodes of livestock do not significantly differ for AM-susceptible and AM-resistant isolates. These data challenge the suitability of motility as a phenotype for detecting and measuring resistance to AM drugs in gastrointestinal nematodes of livestock.

In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0). The extract was fractionated using ammonium sulfate (0–90%) and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction), SPF (stem protein fraction) and RPF (root protein fraction), and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA), larval exsheathment inhibition (LEIA) and larval migration inhibition (LMIA) assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC) chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1). During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1). According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H. contortus.

Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56μM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.

Anthelmintic evaluation of Cucurbita pepo againstgastrointestinal nematodes of sheep

The study was aimed at to investigate the anthelmintic efficacy of aqueous and ethanolic extracts of Cucurbita pepo against gastrointestinal nematodes of sheep by scientifically validated in vitro and in vivo tests. In vitro anthelmintic activity was determined by egg hatch assay, larval migration inhibition assay and larval development assay against eggs and larvae of mixed gastrointestinal nematodes of sheep. The extracts which were found to be effective in in vitro assays were further evaluated by in vivo faecal egg count reduction test against mixed species of sheep gastrointestinal nematodes with 3 different doses (125, 250 and 500 mg kg-1). Haematological and serum parameters were estimated before and after treatment to determine the impact of plant extracts on health of the animal. Aqueous extract of C. pepo demonstrated significant inhibition of egg hatch at 40 and 80 mg/ml. The ED50 value of egg hatch inhibition was 3.51mg/ml. In larval migration inhibition assay, both aqueous and ethanolic extracts of C. pepo demonstrated inhibition of larval migration and the LM50 was 1.75 and 0.32 mg/ml respectively. However, in larval development assay, both aqueous and ethanolic extracts showed only marginal inhibition of larval development at all concentrations tested. In faecal egg count reduction test (FECRT), aqueous extracts of C. pepo at 500 mg/kg caused significant reduction in eggs per gram (96.61 per cent) the reduction being higher than the result obtained with albendazole, the positive control (93.25 per cent). There was a significant difference in EPG reduction between sheep treated with albendazole and C. pepo. Haematological and serum parameters were not affected in both the treated and untreated sheep indicating that the extracts did not cause toxic effects. Based on the results obtained in this study, the aqueous extract of C. pepo possesses anthelmintic activity and could offer an alternative source for the control of gastrointestinal nematodes of sheep.

Prosopis juliflora Pods Alkaloid-rich Fraction: In vitro Anthelmintic Activity on Goat Gastrointestinal Parasites and Its Cytotoxicity on Vero Cells.

Background:This study was designed to assess the in vitro anthelmintic activity of the fraction containing alkaloid from Prosopis juliflora pods on goat gastrointestinal nematodes using the egg hatch assay (EHA), larval migration inhibition assay (LMIA), and larval motility assay (LMA).Materials and Methods:The alkaloid-rich fraction (AF) – content juliprosopine as major alkaloid – was obtained from ethyl acetate extract after fractionation in Sephadex LH-20 chromatography column and its characterization were made by nuclear magnetic resonance analysis together with literature data comparison. The concentrations tested were 4.0, 2.67, 1.78, 1.19, and 0.79 mg/mL (EHA) and 4 mg/mL (LMIA and LMA). The in vitro cytotoxicity on Vero cell cultures was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue tests.Results:High ovicidal activity was observed with IC50 and IC90 values at 1.1 and 1.43 mg/mL for AF. On the other hand, this fraction showed low larvicidal activity and high toxic effect.Conclusion:Thus, P. juliflora pod alkaloid rich-fraction has ovicidal activity in vitro against goat gastrointestinal nematodes and cytotoxic in Vero cell cultures.SUMMARY Prosopis juliflora alkaloid-rich fraction (AF) showed in vitro anthelmintic effect against gastrointestinal nematodes of goatsThe AF was more effective against eggs than third larval stage (L3) of gastrointestinal nematodesThe AF showed cytotoxicity activity on Vero cell lineThe juliprosopine was the main alkaloid found in the AF from P. juliflora pods. Abbreviations used: AF: Alkaloid-rich fraction; DMSO: Dimethyl sulfoxide; EE: Ethyl acetate extract; EHA: Egg hatch assay; IC50: Inhibitory concentration 50%; IC90: Inhibitory concentration 90%; L3: Infective larvae; LMA: Larval motility assay; LMIA: Larval migration inhibition assay; MTT: Bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR: Nuclear magnetic resonance; PBS: Phosphate buffered saline; RPMI: Roswell Park Memorial Institute médium; TLC: Thin Layer Chromatography.

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 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.

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.

Characterisation of ivermectin and multi-drug resistance in two field isolates of Teladorsagia circumcincta from Irish sheep flocks

Ivermectin resistant Teladorsagia circumcincta were isolated from two Irish sheep farms. Parasite naïve lambs were artificially challenged with the isolates in order to test their sensitivity to benzimidazole (BZ), levamisole (LEV) and ivermectin (IVM) by means of a faecal egg count reduction test (FECRT). Both isolates were found to be resistant to all three anthelmintics with reductions of 47% (C.I. 10–68), 92% (C.I. 74–97) and 50% (C.I. 24–68) for the Farm A isolate and of 85% (C.I. 59–94), 89% (C.I. 69–96) and 73% (C.I. 49–86) for the Farm B isolate for BZ, LEV and IVM, respectively. A controlled efficacy test (CET) was undertaken to confirm resistance to IVM and to determine the efficacy of moxidectin (MOX). In agreement with the FECRT, IVM resistance was confirmed for both isolates, with reductions in worm burden of 78% (C.I. 67–85) and 80% (C.I. 45–93) for the Farm A and B isolates respectively. Both isolates were found to be MOX susceptible, with reductions in worm burden of 97% (C.I. 91–99) and 100% for the Farm A and B isolates respectively. Additionally, in vitro tests confirmed the results of the FECRT; results from the larval development assay (LDA) confirmed that both isolates were resistant to BZ and LEV with development observed at the discriminating dose for each drug, while the larval migration inhibition assay (LMIA) confirmed IVM resistance and indicated MOX susceptibility in both isolates. These results represent the first confirmed cases of IVM resistance on Irish sheep farms as well as the first report of multiple anthelmintic resistant gastrointestinal nematodes in Ireland.

Potent anthelmintic activity of galloylated proanthocyanidins from shea meal (Vitellaria paradoxa)

Proanthocyanindins (PAs) from shea meal (SM), a by-product obtained after lipid extraction of the nuts, contained B-type linkages, had a high ratio of prodelphinidins (73%) and were galloylated (42%). The average polymer size was 8 flavan-3-ol subunits (≈2384 Daltons) and epigallocatechin gallate was the major subunit. Purified PA fractions from SM were tested in vitro for anthelmintic properties against gastrointestinal nematodes from ruminants (H. contortus and T. colubriformis) [1] by the larval exsheathment inhibition assay and from pigs (A. suum) by the larval migration inhibition assay. Results showed that PAs from SM have a potent anthelmintic activity against those parasites similar to white clover (Trifolium repens) flowers (WCF) [1, 2] (EC50 µg/mL; SM: 55.1, 16.5, 75.9; WCF: 37.4, 14.5, 110.1 for A. suum, H. contortus and T. colubriformis respectively). WCF PAs are constituted almost exclusively of prodelphinidin (PD) compared to SM (98% vs. 73%) but do not contained galloylated PAs. Studies [1, 2] have shown that anthelmintic activity of PAs was mainly associated with their PD ratio but our current results suggest that galloylation can be a major factor to anthelmintic activity and SM as a potential nutraceutical anthelmintic feed for controlling parasitic nematodes.

Uso del ensayo de inhibición de migración larval para la evaluación de resistencia antihelmíntica de novillas lecheras en Puerto Rico

La resistencia antihelmíntica de ivermectina (IVM) en Cooperia spp. de novillas lecheras de Puerto Rico se evaluó mediante el ensayo de inhibición de la migración de larvas (LMIA, por sus siglas en inglés). La tercera etapa larval (L3) se recuperó a partir de cultivos fecales pre tratamiento en ocho fincas donde se evaluó previamente la resistencia antihemíntica con el uso de una prueba de reducción del conteo de huevos fecales (FECRT, por sus siglas en inglés). La concentración efectiva donde se inhibe el 50% de la migración de las larvas (EC50) se obtuvo a partir de cepas susceptibles y resistentes de Cooperia spp. aisladas en una de las fincas. Las larvas susceptibles se caracterizaron por un EC50 de 0.7224 uM y las resistentes por un EC50 de 7.0778 uM. Este resultado indica que en promedio las larvas del aislado resistente son 10X más resistentes que las del aislado susceptible. Sin embargo, el LMIA no fue capaz de discriminar consistentemente entre las larvas susceptibles y resistentes obtenidas de las fincas evaluadas. En uno de los casos, un valor de EC50 de 0.7263 uM, cercano al valor obtenido con el aislado susceptible, no coincidía con el resultado de FECR de -136.19% documentado en esa finca. En dos ocasiones más la prueba no pudo discriminar entre los aislados resistentes y susceptibles. De hecho, los valores de EC50 de LMIA obtenidos de algunos aislados resistentes eran menores que los valores de EC50 de los aislados susceptibles, aunque sus resultados de FECRT indicaran que había resistencia a IVM. Posiblemente, el FECRT dio resultados falso positivos debido a la presencia de resistencia de lactonas macrocíclicas en esas dos fincas. Por lo tanto, se requieren más estudios para validar la efectividad de LMIA para detectar la resistencia antihelmíntica a lactonas macrocíclicas en larvas de Cooperia spp. aisladas de muestras fecales de bovinos.