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

Background

Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods.

Methodology/Principal Findings

Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC₅₀ values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus.

Conclusions/Significance

Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus

Martin P. J., Le Jambre L. F. and Claxton J. H. 1981. The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus. International Journal for Parasitology11: 35–41. Beginning with a mixture of 5% thiabendazole-resistant and 95% non thiabendazole-resistant Haemonchus contortus larvae, worm free sheep were artificially infected with either 10,000, 9000, 7000, 2500 or zero larvae. Four weeks later, the infected sheep were treated with 44 mg/kg of thiabendazole. Seven days after anthelmintic treatment the same sheep were infected with zero, 1000, 3000, 7500 or 10,000 larvae respectively so that each sheep was given a total of 10,000 larvae. The larvae given after thiabendazole treatment are referred to as being in refugia (defined as an area where individual members of a population can escape exposure to a drug). This was repeated for six generations of parasites, maintaining the same proportion of larvae in refugia in each generation. The egg hatch assay for resistance indicated that refugia delayed the development of resistance in Haemonchus contortus. Where none or a small proportion of larvae were in refugia, a rapid increase in resistance occurred. With an increased proportion of larvae in refugia, resistance was slower to develop. Population size, as estimated by faecal egg counts done after anthelmintic treatment and corrected for the number of larvae given in each treatment, was lower at the higher levels of refugia. This was due to a lower level of resistance in the higher refugia lines and consequently a greater effect of the drug. However, the estimate of population size, from egg counts done after the larvae introduced from refugia had matured, were similar in all lines.     

Haemonchus contortus: in vitro drug screening assays with the adult life stage

Motility and feeding assays were assessed as in vitro systems for screening of novel compounds for anthelmintic activity against adult Haemonchus contortus. The study aimed to develop an assay with the parasitic adult stage of this species that could be used in conjunction with, or as an alternative to, the free-living larval stage screens commonly used for drug discovery with many parasitic nematode species. The feeding assay showed limitations due to the apparent continuation of a significant degree of feeding in worms showing greatly reduced motility in the presence of some drugs. Hence, it appeared most likely that the feeding assay would underestimate the toxicity of these drugs. The motility assay was able to detect toxicity of known anthelmintics, including the 'slow-acting' benzimidazoles. A small-scale screening exercise used the motility assay to detect toxicity towards adult parasites in 10 compounds out of a group of 200 chemicals (selected due to known toxic effects in larval development assays). The motility assay appeared suitable for drug screening against adult H. contortus. The use of the adult stage for drug screening in this way ensures that the drug is toxic towards the parasite life stage to be targeted in vivo. A lack of activity in subsequent in vivo trials could, therefore, be most likely attributable to host pharmacokinetic factors rather than an intrinsic lack of activity of the drug towards the adult parasite.     

The effect of egg embryonation on field-use of a hookworm benzimidazole-sensitivity egg hatch assay in Yunnan Province, People's Republic of China

Background

Current efforts to control human soil-transmitted helminths (STHs) involve the periodic mass administration of benzimidazole drugs to school aged children and other at- risk groups. Given that high levels of resistance to these drugs have developed in roundworms of livestock, there is a need to monitor drug efficacy in human STHs. The current study aimed to evaluate an in vitro egg hatch assay for measuring the sensitivity of human hookworms to benzimidazole drugs in an isolated field setting in southern Yunnan province, People''s Republic of China.

Methodology/Principal Findings

Egg hatch assays were performed with hookworm (Necator americanus) eggs extracted from 37 stool samples received from local school-aged children. The mean IC₅₀ was 0.10 ug/ml thiabendazole (95% CIs: 0.09–0.12 ug/ml). Observation of the eggs immediately prior to assay set-up revealed that a small percentage had embryonated in some samples. Scoring of % embryonation of eggs prior to the assay allowed for corrections to be made to IC₅₀, IC₉₅ and IC₉₉ values. Examination of the data with and without this correction revealed that the embryonation of a small number of eggs did not affect IC₅₀ values, but did increase IC₉₅ and IC₉₉ values for some samples.

Conclusions/Significance

This study has highlighted the impact of egg embryonation on the use of benzimidazole drug sensitivity assays for human hookworms in field settings. Given the greater flexibility required in human stool collection procedures compared to livestock studies, we suggest that embryonation of some eggs may be an unavoidable issue in some human studies. Hence, it needs to be measured and accounted for when analysing dose response data, particularly for generation of IC₉₅ and IC₉₉ values. The protocols used in this study and our suggested measures for accounting for egg embryonation should have widespread application in monitoring benzimidazole sensitivity at field sites worldwide.     

Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep

The cyclotides are a novel family of backbone-cyclized cystine-knot containing peptides from plants that have been shown to possess insecticidal activity against Helicoverpa larvae, an important pest of corn and cotton. In the current study, we investigated the in vitro effects of the cyclotides on the viability of egg, larval, and adult life stages of two species of economically important gastrointestinal nematode parasites of livestock, Hemonchus contortus and Trichostrongylus colubriformis. The cyclotides showed significant activity in inhibiting development of nematode larvae and motility of adult worms. Activities were comparable to some currently used anthelmintic compounds in these in vitro assay systems. A series of alanine mutants of the prototypic cyclotide kalata B1 were assayed against larvae to determine regions of the peptide responsible for activity. It was observed that anthelmintic activity was dramatically reduced as a consequence of the mutation of a large number of residues that are found clustered on one surface. Activities toward larvae were equivalent in the naturally occurring L-isomer of kalata B1 and a synthetic all-D-isomer, indicating that there is no chiral requirement for anthelmintic activity. The clustering of important residues and the lack of chiral selectivity further support the proposed mode of action of the cyclotides, which involves a membrane-based interaction rather than an interaction at a specific receptor. The cyclotide-induced leakage of a fluorescent dye from vesicles used as a model membrane mimetic further confirms the membrane lytic ability of cyclotides. The relative potency of kalata B1 and kalata B2 in causing membrane leakage is consistent with the order of their anthelmintic activity. These results demonstrate that the cyclotides show potential for use in the control of gastrointestinal nematode parasites.     

In vitro effects of four tropical plants on the activity and development of the parasitic nematode, Trichostrongylus colubriformis

The in vitro effects of extracts of four tropical plants (Zanthoxylum zanthoxyloides, Newbouldia laevis, Morinda lucida and Carica papaya) on the egg, infective larvae and adult worms of Trichostrongylus colubriformis were screened for potential anthelmintic properties. Significant effects were observed with the four plants on T. colubriformis but they differed depending on the stage of the parasite. Extracts of each plant induced a dose-dependent inhibition of egg hatching. Using a larval inhibition migration test, the effects on the infective larvae were also detected with the four plant extracts. In contrast, for adult worms, the effects were statistically significant only for N. laevis and C. papaya. No significant activity was shown for M. lucida and Z. zanthoxyloides. These in vitro results suggest the presence of some anthelmintic properties associated with these four plants, which are traditionally used by small farmers in western Africa. These effects need to be studied under in vivo conditions.     

Efficacy of the cyclooctadepsipeptide PF1022A against Heligmosomoides bakeri in vitro and in vivo

The cyclooctadepsipeptide PF1022A derived from the fungus, Mycelia sterilia, is characterized by a broad spectrum of activity against different parasitic gastrointestinal nematodes of livestock. In the present work the anthelmintic activity of PF1022A against Heligmosomoides bakeri, a widely used laboratory model was studied. Albendazole, ivermectin and levamisole served as reference. In vitro, PF1022A showed low activity on embryonation but significantly inhibited egg hatch (10 and 100 μg/ml), whereas albendazole (10 and 100 μg/ml) revealed statistically significant inhibitions of both embryonation and egg hatch. PF1022A (1-100 μg/ml) completely inhibited larval movement at most examination points. Comparable significant anthelmintic activity on the larval stages of H. bakeri was observed with levamisole (48-100%), while slightly lower activities were observed with ivermectin (20-92%) and albendazole (0-87%) at 1-100 μg/ml. PF1022A and levamisole significantly inhibited motility and egg release of adult worms, while albendazole and ivermectin failed to demonstrate activity. Significant worm burden reductions were achieved with PF1022A, levamisole and ivermectin in vivo. For example, at 0·125 mg/kg PF1022A a worm burden reduction of 91·8% was observed. The use of drug combinations did not further enhance the in vitro and in vivo activity of PF1022A. In conclusion, further investigations are warranted with PF1022A, as the drug is characterized by significant larvicidal and nematocidal activity in vitro and in vivo.     

To hatch or not to hatch? Egg hatch response to larval density and to larval contact in a treehole mosquito

Abstract.

• 1 We investigated the effect on egg hatch of exposure to: (1) varying larval density, and (2) larval contact in Aedes triseriatus Say (Diptera: Culicidae). For 2 days in the laboratory we submerged eggs into a treehole water medium containing 0 (control), 4, 12 or 24 larvae that could either contact the eggs directly or were separated from them by a screen. Following treatment, abundance of microorganisms on the egg surfaces, a food source for newly hatched larvae and a proposed hatching stimulus, was assessed by counts made from serial dilutions of samples.
• 2 We discovered a complex hatching response to larval contact and to larval density, and an interaction between these two factors in their effect on microbial growth. Hatching was inhibited in the 0-larva control, even though microorganisms grew abundantly on the eggs. Hatch rate, as well as microbial counts, were high for eggs in direct contact with 4 larvae. As density increased in the larval contact treatment, microorganisms disappeared from the egg surfaces and hatch rate declined.
• 3 When protected from larval grazing, eggs supported numerous microbial colonies irrespective of larval density. In contrast to the contact treatment group, egg hatch increased with increasing larval density. These observations suggest that the combination of microbial growth and a larval factor stimulates hatch. This hatching response may have evolved because both abundant microorganisms and numerous larvae reflect a habitat of good quality.

     

The community ecology of Aedes egg hatching: implications for a mosquito invasion

Abstract. 1. A recently introduced treehole mosquito from Asia, Aedes albopictus , is spreading throughout eastern North America, especially in tyre-refuse piles. Previous studies have identified inhibitory effects of larvae on egg hatch as a potential population regulatory mechanism within Aedes. Larva-egg interactions may also occur between species. This experiment assesses the ability of larvae of A. albopictus and two possible competitors in North America, A.triseriatus and A.aegypti , to suppress hatching of conspecific and congeneric eggs.
2. We exposed eggs of each species to varying combinations of larval species and density for 24h and assessed subsequent hatch rates. Aedes albopictus eggs exhibited the lowest level of inhibition when exposed to high larval densities; moreover, at the lowest larval density they imposed the most intense interspecific hatch inhibition.
3. Discretionary hatching in response to larval density may influence community composition by promoting the spread of A.albopictus , perhaps even leading to its dominance within North American Aedes communities.     

Hatching Response of Meloidogyne incognita acrita to Electric Shock

The influence of electric shock on hatch of Meloidogyne incognita acrita from egg masses taken from roots of ''Acala SJ-I'' cotton (Gossypium hirsutum L.) was studied. Egg masses in tap water were individually placed between the tips of needle electrodes 1 mm apart and exposed to potentials of l, 10, 20, and 60 vdc/mm at 1, 1, 1, and 86 milliamperes dc, respectively, for periods of 2 and 60 seconds. Hatched larvae were counted at five-day intervals for 60 days. Of the eight treatment combinations used, six gave a greater egg hatch than the control. The largest hatch, 520 percent greater than the control, resulted from exposure to 1 vdc/mm for 60 seconds; 60 vdc/mm for 2 and 60 sec decreased egg hatch 11 and 94 percent of the untreated control. Hatched larvae from all treatments except the 60 vdc/mm, 60-second exposure were infective and reproduced on young cotton plants in a glasshouse.     

THE INFLUENCE OF SOIL MOISTURE ON THE EMERGENCE OF LARVAE FROM CYSTS OF THE BEET EELWORM, HETERODERA SCHACHTII SCHMIDT

The significance of soil moisture in relation to aeration and larval emergence from cysts of Heterodera schachtii Schmidt is discussed. The rate of larval emergence increased as oxygen concentration increased. A comparison of the moisture characteristics of a mass of cysts and of sand of about the same particle size showed that water was removed from between both the cysts and the sand particles at 12–16 cm. of water-pressure deficiency. There was an indication that water was removed from between the eggs within the cysts at 100–135 cm - of water-pressure deficiency. The rate of larval emergence showed a gradual decline is suction was increased beyond 20 cm. of water-pressure deficiency, approaching zero at 175 cm. of water-pressure deficiency. Results suggest that egg hatch is not directly dependent on pressure deficiency and that the presence of free larvae within the cyst inhibits further egg hatch. A technique is described for measuring larval motility in sand. There is a decline in motility when most of the water has been removed from the sand pore-spaces. It is suggested that the relatively low rates of larval emergence at high-pressure deficiencies are due to inhibition of larval migration from the cyst by the surface forces of the water film.     

Wheat germ oil and its effects on a liquid larval rearing diet for oriental fruit flies (Diptera: Tephritidae)

Wheat germ oil was added to a larval liquid diet for rearing Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) to optimize fruit fly quality. Effects of various concentrations of wheat germ oil at 0.04, 0.07, 0.15, 0.30, and 0.66% and their possible mode of action were evaluated. Results suggest that addition of wheat germ oil does not affect pupal weight, larval developmental period, adult emergence, mating ability, or peak time for egg production. But there was a significant increase in pupal recovery, percentage of adult fliers, egg production, or egg hatch for larvae fed the diet with wheat germ oil compared with those reared on the liquid diet without wheat germ oil. The increase in egg hatch and fliers was dose dependent. Therefore, addition of wheat germ oil to fruit fly rearing diet is a novel way to improve fruit fly quality, especially in egg hatch, fliers, egg production, and pupal recovery.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Speckle interferometry applied to pharmacodynamic studies: evaluation of parasite motility

The work reported here describes the application of the optical technique known as dynamic speckle interferometry to evaluate the motility of nematode parasites exposed to different anthelmintic drugs. This technique, a well proven tool for assessing the time evolution of different phenomena, is here successfully used to quantify parasite motility in pharmacodynamic assays. The characterization of the pharmacological properties of anthelmintic drugs is critical to optimize their use in parasite control. Besides, the evaluation of nematode motility is a relevant indicator of the pharmacodynamic effect of anthelmintic drugs. The application of this approach to study the motility of Haemonchus contortus (used as a model of nematode parasites) larvae exposed to different drugs is presented, showing its usefulness.Abbreviations ABZ albendazole - BZD benzimidazole - IVM ivermectin - LVM levamisole     

Tebufenozide targeted against codling moth (Lepidoptera: Tortricidae) adults, eggs, and larvae

The effectiveness of tebufenozide applied against the adult, egg, and larvae of codling moth, Cydia pomonella (L.), was evaluated. Significant reductions in fecundity and egg hatch occurred after 1-h and 24-h exposures of females and 24-h exposures of males-only to residues in plastic plates. A significant reduction in egg hatch was also found after a 1-h exposure of males. The ovicidal effects of tebufenozide in field trials did not significantly differ for eggs laid on residues or treated topically. Corrected egg mortality exceeded 95% for cohorts laid <130 degree-days after sprays were applied. Fecundity and egg hatch were measured after either a 24-h exposure of moths or a 10-d exposure of moths and eggs on apple trees. Significant reductions in both fecundity and fertility occurred compared with an untreated control up to 7 d in the 24-h assays and accounted for 60-70% fewer larvae produced per mated female. The mean numbers of larvae produced per mated female after the 10-d exposure were reduced 100-75% in assays started on day 0-21 after the spray application compared with the untreated control. Fecundity was significantly reduced for 7 d and egg hatch was reduced for the entire 21-d test period in these trials. Residues had a 14-d half-life on apple foliage. Residues applied to foliage or to foliage and fruit did not significantly increase the proportion of uninjured fruit compared with the untreated control in bioassays where neonates were placed on foliage 10 cm from fruit. However, the proportion of injured fruits with shallow stings versus deep entries was higher on the treated versus the untreated plants. Field applications of tebufenozide reduced fruit injury >90% when applied early during emergence. Plots treated after the beginning of egg hatch had a larger proportion of injured fruits with shallow larval feeding.     

Population dynamics of Trichostrongylus colubriformis in sheep: computer model to simulate grazing systems and the evolution of anthelmintic resistance

A computer model was developed to simulate Trichostrongylus colubriformis populations, their level of resistance to the common anthelmintics, host mortalities and acquired immunity. Predictions were based on sheep management practices such as lambing, weaning, sheep/paddock rotation, anthelmintic treatment, the use of controlled release devices (capsules) for anthelmintic delivery and daily meteorological records to determine the development and survival of infective larvae (L3) on pasture. Evolution of drug resistance was determined by a simple genetic system which allowed for up to three genes, each with two alleles, to give a maximum of 27 genotypes associated with one drug or three genotypes for each of three drugs. The model was validated against egg counts, L3 counts on pasture and host mortalities observed in a grazing trial, however, aspects of the model such as the development of drug resistance and use of the model in a variety of climatic zones have yet to be tested against field observations. The model was used to examine the impact of grazing management and capsule use on anthelmintic resistance and sheep production over 20 years using historical weather data. Predictions indicated that grazing management can play a dominant role in parasite control and that capsule use will reduce sheep mortalities and production losses, and in some circumstances will not cause a substantial increase in anthelmintic resistance for up to 5 years.     

The role of epidemiological knowledge and grazing management for helminth control in small ruminants

There is no single requirement more crucial to the rational and sustainable control of helminth parasites in grazing animals than a comprehensive knowledge of the epidemiology of the parasite as it interacts with the host in a specific climatic, management and production environment. In its absence, anthelmintic treatment is either given suppressively, which provokes resistance, or therapeutically, which risks clinical disease and production losses. Sustainable parasite-control programmes require knowledge of seasonal larval availability, origin of larvae contributing to any peaks and climatic requirements for worm egg hatching, larval development and survival. Control measures based on this knowledge include strategic anthelmintic treatments and various forms of grazing management. While these measures can reduce the frequency of anthelmintic treatment required, their effect on selection for drench resistance is more problematical, unless they can be combined with other forms of control to reduce our current dependence on anthelmintics.     

The influence of water and humidity on the hatching of Nematodirus battus eggs

This paper examines the influence of water on the ecology of the eggs of Nematodirus battus, with a view to estimating the importance of including rainfall in mathematical models of parasite abundance. The literature suggests that, under pasture conditions, the availability of moisture is unlikely to be limiting for egg development, while eggs and infective larvae are highly resistant to desiccation. In the presented experiment, eggs that had been kept in salt sludges at 95% and 70% RH and were subsequently put at 15°C produced only a mildly accelerated, but not a mass, hatch, in the first few days after return to water. Eggs kept at higher osmotic pressures died. Mass hatching of infective larvae, described at pasture when spells of rain follow periods of drought, is unlikely to occur as the result of a sudden water influx into eggs. Since water is not necessary for migration of infective larvae from the soil on to grass, such peaks in larval abundance are more likely to arise from the effects of temperature on hatching of eggs.     

The consequences of short-term grazing of bioactive forages on established adult and incoming larvae populations of Teladorsagia circumcincta in lambs

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.