Larval Migration Inhibition Assay Research Articles

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.

In vitro anthelmintic activity of Zanthoxylum simulans essential oil against Haemonchus contortus

The need for new anthelmintic agents with low impact on the environment is becoming urgent. Phytotherapy is an alternative method to control gastrointestinal nematodes in small ruminants. This study aims to determine the composition of Zanthoxylum simulans essential oil (ZSEO) and evaluate the in vitro ovicidal and larvicidal effects of ZSEO on Haemonchus contortus using egg hatch assay, larval development assay (LDA), and larval migration inhibition assay (LMIA). The chemical composition of ZSEO was determined through gas chromatography and mass spectrometry and 94 compounds were identified from the ZSEO. The major constituents of ZSEO were borneol (18.61%), β-elemene (10.87%). ZSEO and borneol both at 40mg/mL inhibited larval hatching by 100%, with LC50 values of 3.98 and 1.50mg/mL, respectively. The LC50 value of β-elemene was not determined because of its insufficient activity. The results of LDA showed that ZSEO, borneol, and β-elemene all at 40mg/mL inhibited larval development by 99.8%, 100%, and 55.4%, respectively, and exhibited dose-dependent responses with LC50 values of 4.02, 1.99, and 32.17mg/mL, respectively. The results of LMIA showed that ZSEO, borneol, and β-elemene all at 40mg/mL inhibited larval migration by 74.3%, 97.0%, and 53.2%, respectively. ZSEO presented ovicidal and larvicidal activities in vitro. Therefore, Zanthoxylum may be an alternative source of anthelmintic agents to control gastrointestinal nematodes in sheep.

Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.

Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration–response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration–response curves for all isolates to the left, again eliminating differences in EC50 values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.

Effects of tannic acid on Haemonchus contortus larvae viability and immune responses of sheep white blood cells in vitro

Direct inhibitory effects of tannic acid on Haemonchus contortus viability were studied in vitro using the larval migration inhibition (LMI) assay. Sheep white blood cells (WBC) were preincubated with 5 and 50 lg/mL tannic acid or not followed by whole H. contortus antigen (WHA). Cells were harvested at 24 h post-incubation to test host immune responses. Concentrations of 50, 100, 500, 1000, 3000 and 5000 lg/mL tannic acid inhibited larvae migration by 19.8, 42.4, 46.3, 92.0, 93.7 and 100%, respectively, within 96 h post-incubation (P < 0.001). The relative mRNA levels of interferon (IFN)-c, interleukin (IL)-2, IL-4 and IL-10 were increased by WHA stimulation without tannic acid. However, the increased effects on IFN-c and IL-2 were inhibited by tannic acid preincubation (P < 0.001), while the increases in IL-4 and IL-10 were greatly enhanced by tannic acid preincubation (P < 0.001). Changes in protein levels of all cytokines essentially paralleled the changes in their corresponding mRNA levels. In conclusion, tannic acid is directly harmful to larvae in a dose- and time-dependent manner and modulates immune responses of sheep WBC stimulated by H. contortus antigen by inhibiting Th1 cytokines and increasing Th2 cytokine expression in vitro.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100μg/ml of PB or TCZ following incubation in vitro for 1–24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1μg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10μg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10μg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100μg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100μg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100μg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

Effect of molecular weight and concentration of legume condensed tannins on in vitro larval migration inhibition of Haemonchus contortus

The effect of molecular weight of condensed tannins (CT) from a variety of warm-season perennial legumes commonly consumed by sheep and goats on anthelmintic activity has not been previously explored. The objectives of this study were to determine if molecular weight of CT from warm-season perennial legumes could predict the biological activity of CT relative to anthelmintic activity against ivermectin resistant L3 stage Haemonchus contortus (HC) using a larval migration inhibition (LMI) assay. A second objective was to determine if CT from warm-season perennial legumes possess anthelmintic properties against L3 stage (HC). Lespedeza stuevei had the greatest concentration of total condensed tannin (TCT; 11.7%), whereas, with the exception of Arachis glabrata, a CT-free negative control, Leucaena retusa had the least TCT (3.3%). Weight-average molecular weight of CT ranged from 552Da for L. stuevei to 1483Da for Lespedeza cuneata. The treatments demonstrating the greatest percent LMI were L. retusa, L. stuevei and Acacia angustissima var. hirta (65.4%, 63.1% and 42.2%, respectively). The ivermectin treatment had the smallest percent LMI (12.5%) against ivermectin resistant L3 HC. There was a weak correlation (R2=0.34; P=0.05) between CT MW and percent LMI, suggesting that molecular weight of CT is a weak contributing factor to CT biological activity as it relates to LMI of L3 stage HC. L. stuevei, L. retusa and A. angustissima var. hirta STP5 warrant further evaluation of anthelmintic properties in vivo.

The effect of purified condensed tannins of forage plants from Botswana on the free-living stages of gastrointestinal nematode parasites of livestock

The effect of condensed tannins (CT) extracted from forage plants from Botswana on the free-living stages of a number of species of gastrointestinal nematode parasites derived from infected sheep were investigated using in vitro assays. Fresh samples of five different plants (Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius, Grewia flava and Ipomoea sinensis) were collected over two summers (February 2009 and 2010). Fractionation of each crude extract on a Sephadex LH-20 column yielded low molecular weight phenolics and CT-containing fractions. The effect of each purified CT fraction on parasites was evaluated using either egg hatch, larval development or larval migration inhibition assays. Three gastrointestinal nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta) derived from infected sheep were evaluated in the study. CT from V. rotundifolium and I. sinensis fractions from samples collected in 2009 and 2010 did not inhibit larval development. However, CT isolated from V. verrucosum, T. oleifolius and G. flava collected in 2009 completely inhibited the development of all parasite species. These CT fractions were more potent in inhibiting larval development of H. contortus than fractions from the same plant species collected in 2010. However, a slight effect on larval migration was observed with some CT extracts. The results suggest that CT extracts of some forage plants from Botswana have anti-parasitic properties in vitro, and that further research is required to determine any in vivo efficacy from feeding the plants to goats in a field situation.

Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in Dirofilaria immitis

For more than 20years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose–response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose–response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56μM (1.26–16.4μM), greater than that for EPR at 2.02μM (1.68–2.42μM), and this difference was significant (p=0.0428). The R2 value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose–response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.

The in vitro assay profile of macrocyclic lactone resistance in three species of sheep trichostrongyloids

Anthelmintic resistance has emerged as an important problem in animal industries. Understanding resistance mechanisms, especially against macrocyclic lactones (MLs), is the first step in developing better diagnostic tools. Effects of several MLs including ivermectins and milbemycins were tested using two well established in vitro assays: the larval development assay (LDA) and the larval migration inhibition assay (LMIA). These were performed on free-living stages of susceptible and ML-resistant isolates of three trichostrongyloid nematode species of sheep. In general, dose response curves shifted to the right in the resistant isolates. Data showed that resistance was present to ivermectin and its two components suggesting that both components contribute to action and resistance. There were no consistent patterns of potency and resistance of the tested substances for the different isolates in the LDA except that moxidectin (MOX) tended to have lower resistance ratios than ivermectin (IVM). MOX was the most potent inhibitor in the LMIA in susceptible Haemonchus contortus while being less potent in Trichostrongylus colubriformis and particularly in Ostertagia circumcincta. MOX showed high resistance ratios in the LMIA in all three species. Based on these results, resistance to MOX has unique characteristics and the LMIA may perform better in detecting resistance to MOX in these parasite species.

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

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

Effect of using redberry juniper (Juniperus pinchotii) to reduce Haemonchus contortus in vitro motility and increase ivermectin efficacy

A modified larval migration inhibition assay was used to determine if redberry juniper (Juniperus pinchotii Sudw.) can reduce Haemonchus contortus in vitro motility and increase ivermectin (IVM) efficacy. Ruminal fluid was mixed with buffer solution and either no material (CNTL) or Tifton 85 Bermudagrass hay (T85), dried juniper (DRY), fresh juniper (FRE), or distilled juniper terpenoid oil (OIL) to make treatment solutions and anaerobically incubated for 16h. For Trial 1, larvae were incubated in CNTL, T85, DRY, or IVM. During Trial 2, larvae were incubated in CNTL, DRY, FRE, or OIL for 4h. Trials 3 (CNTL or OIL) and 4 (CNTL, DRY or FRE) evaluated larvae after incubation in treatment solution for 2h, then incubated an additional 2h in various IVM doses (0, 0.1, 1, 3, and 6μg/mL IVM) and placed onto a screen. Larvae that passed through the 20-μm screen within a 96-well plate were considered motile. Larvae incubated in CNTL or T85 had similar (P=0.12) motility, but larvae incubated in DRY were less (P<0.02) motile than larvae incubated in CNTL or T85 (Trial 1). During Trial 2, adding DRY, FRE, or OIL reduced (P<0.001) larval motility as compared to CNTL. A treatment×IVM dose interaction (P=0.02) was observed during Trial 3, due to OIL unexpectedly decreasing IMV efficacy at IVM concentrations of 1 (P=0.07), 3, and 6 (P<0.002)μg/mL. No treatment×IVM dose interaction (P=0.57) was observed during Trial 4, but larvae incubated in DRY had less (P<0.004) total motility than larvae incubated in CNTL or FRE. Juniper forage material reduced in vitro H. contortus larval motility, but IVM efficacy was increased only by initially incubating larvae in DRY.

Potential contribution of P-glycoproteins to macrocyclic lactone resistance in the cattle parasitic nematode Cooperia oncophora

Resistance against macrocyclic lactones such as ivermectin is widespread among parasitic gastrointestinal nematodes of small ruminants and is rapidly increasing in cattle parasites. ABC transporters of the subfamily B, the so-called P-glycoproteins (Pgps) have been frequently implicated in ivermectin resistance and are a major cause of multi-drug resistance in protozoa and helminths. The Pgp inhibitor verapamil (VPL) dramatically enhanced susceptibility of the cattle parasitic nematode Cooperia oncophora to ivermectin in vitro as measured in a larval developmental assay and a larval migration inhibition assay using third stage larvae. Moreover, VPL completely restored susceptibility to ivermectin in a resistant isolate resulting in virtually identical dose–response curves of susceptible and resistant isolates in the presence of VPL. Further characterisation of the molecular mechanisms resulting in Pgp-mediated ivermectin resistance is still hampered by the lack of molecular and biochemical information for Pgps of parasitic nematodes. Using PCR with degenerate primers, fragments of four different C. oncophora Pgps could be amplified and the Conpgp-2, previously implicated in macrocyclic lactone resistance in Haemonchus contortus, and Conpgp-3 full-length cDNAs were obtained by RACE PCR. The pgp sequences presented here contribute important data required to systematically screen resistant C. oncophora isolates for up- or down-regulation of Pgps and for the detection of single nucleotide polymorphisms in Pgps to detect selection of specific Pgp alleles by anthelmintics as early as possible.

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

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

Evaluation of the Egg Hatch Assay and the Larval Migration Inhibition Assay to detect anthelmintic resistance in cattle parasitic nematodes on farms

Resistance to anthelmintic drugs, particularly to the widely used benzimidazoles (BZs) and macrocyclic lactones (MLs) is an increasing problem in cattle industries worldwide. Reliable methods for the assessment of anthelmintic efficacy in the field are required in order to react before resistance becomes an obvious problem on individual properties. The ability of the Egg Hatch Assay (EHA) and the Larval Migration Inhibition Assay (LMIA) to detect anthelmintic resistance under field conditions was evaluated on cattle farms in Northern Germany. As published previously Faecal Egg Count Reduction Test (FECRT) was performed using oral albendazole (Valbazen®) or injectable ivermectin (Ivomec®). Herein the FECRT results described earlier were compared with data from EHAs or LMIAs, respectively, performed with eggs from fresh faeces or larvae from faecal cultures of the tested animals before and after treatment. The obtained EC50 values allowed the assessment of efficacy of albendazole and ivermectin on farm level. The results of the FECRTs and the results of both in vitro assays were comparable. In comparison to the FECRT the in vitro assays are less time, labour and cost intensive and are able to assess the susceptibility status of a worm population without treatment. Therefore both are beneficial alternatives for the reliable detection of reduced efficacy of these two drug classes on farms.

In vitro anthelmintic activity of five tropical legumes on the exsheathment and motility of Haemonchus contortus infective larvae

This study investigated the in vitro anthelmintic (AH) activity of five tropical legume plants [Arachis pintoi CIAT 22160 (A.p. 22160), Gliricidia sepium, Cratylia argentea (C.a. Yacapani), C. argentea CIAT 22386 (C.a. 22386), C. argentea Veranera (C.a. Veranera)] against Haemonchus contortus infective larvae and the role of tannins/polyphenolic compounds in the AH effect. Lyophilized leaf extracts of each plant were evaluated using the Larval Exsheathment Inhibition Assay (LEIA) and the larval migration inhibition assay (LMIA). The role of tannins/polyphenolic compounds in the AH effect was evaluated in both assays using polyethylene glycol (PEG) to remove tannins from the solutions. At the highest concentration (1200μg of extract/ml), A. pintoi 22160, C.a. Yacapani, C.a. Veranera and C.a. 22386 completely inhibited the exsheathment process of H. contortus (P<0.01). At the same concentration (1200μg of extract/ml), the inhibition of larval migration for C.a. 22386, C.a. Veranera and G. sepium was 66.0%, 35.9% and 39.2% (relative to the PBS control), respectively. In both bioassays (LEIA and LMIA), the AH effect shown by each plant was blocked after the addition of polyethylene glycol (PEG), corroborating the role of tannins/polyphenolic compounds.

The in vitro diagnosis of anthelmintic resistance in cyathostomins

Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resistance in different nematode populations. At present, the faecal egg count reduction test is considered the most suitable method for initial screening for anthelmintic resistance in equine nematode populations. However, in its current state, this test lacks sensitivity. It is also costly and time-consuming to perform. Laboratory-based techniques, such as the egg hatch assay, larval development assay, larval migration inhibition assay and the larval feeding inhibition assay offer alternative options for assessing anthelmintic resistance in nematode populations. All of these tests have been investigated for their utility in measuring drug resistance in sheep nematode populations and some have proven useful. The egg hatch assay, larval development assay and larval migration inhibition assay have been investigated for use in measuring levels of drug resistance in equine nematode populations. However, at best, the results obtained thus far indicate that these tests require further refinement.

In vitro anthelmintic activity of the essential oils of Zanthoxylum zanthoxyloides and Newbouldia laevis against Strongyloides ratti

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: β-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 μg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50) = 2.5 μg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50) = 46 μg/ml), N. laevis (IC(50) = 51 μg/ml), and the control [levamisole (IC(50) = 36 μg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 μg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.

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

The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200μg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P<0.001) and A. gaumeri (P<0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L3 larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.

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

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