Involvement of tannins and flavonoids in the in vitro effects of Newbouldia laevis and Zanthoxylum zanthoxyloïdes extracts on the exsheathment of third-stage infective larvae of gastrointestinal nematodes

The present study aimed at examining the possible role of tannins and flavonoids on the in vitro anthelmintic properties of the extracts of two plants from the southern area of Western Africa, i.e. Newbouldia laevis and Zanthoxylum zanthoxylo?des. Extracts of the two plants were prepared by use of acetone/water (70/30) and their anthelmintic activity was measured by use of the larval exsheathment inhibition assay (LEIA) applied on the abomasal species, Haemonchus contortus and the intestinal species Trichostrongylus colubriformis. Three concentrations of extracts were evaluated to examine the possible dose effect. In addition, the possible involvement of tannins and flavonoids was examined by comparing the levels of inhibition of larval exsheathment obtained with the same extracts, after of not addition of PVPP which forms complexes with these compounds. The results indicate significant effects with both plants and both nematode species. In the range of concentrations examined, the results were dose-dependent for N. laevis extracts but not for Z. zanthoxylo?des because the three doses applied provoked a similar highly significant inhibition whatever the tested dose. The use of PVPP indicated for both plant and nematode species, that tannins and flavonoids are involved partly in the effect but that some other biochemical compounds were also involved in both plants.     

Effects of four tropical tanniniferous plant extracts on the inhibition of larval migration and the exsheathment process of Trichostrongylus colubriformis infective stage

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.     

Polyphenols from Pistacia lentiscus and Phillyrea latifolia impair the exsheathment of gastro-intestinal nematode larvae

The infection of grazing ruminants with gastro-intestinal nematodes (GINs) is a severe problem in the Middle East. However, goats that graze the south-western slopes of the Carmel Heights in Israel have very low faecal egg counts, despite high grazing density. We hypothesized that polyphenols from Pistacia lentiscus L. and/or Phillyrea latifolia L. – both prevalent woody species of the region that are consumed by goats – have anthelmintic bioactivity. We tested this hypothesis by using the larval exsheathment inhibition assay (LEIA). Extracts were prepared from leaves of either plant species using 70% ethanol (E70), 100% ethanol (E100), or boiling water (W). Larvae were incubated in a phosphate-buffered saline solution with or without plant extract (1200 μg/ml) and then exposed to an exsheathment solution expected to elicit 100% exsheathment after one hour. All extraction methods of P. lentiscus were highly effective at inhibiting larval exsheathment, but higher potency was found for the E70 than for E100 extraction method, while W was intermediate. Only the E70 extract of P. latifolia was highly effective relative to the control. The E70 extract of P. lentiscus had more than 7 times the potency of the E70 extract of P. latifolia. Irrespective of solvent and tannin-equivalent used, P. lentiscus contained more than double the quantity of total polyphenols than P. latifolia. The polyphenols of P. lentiscus consisted mainly of galloyl derivatives (63.6%), flavonol glucosides (28.6%), and catechin (7.8%). In P. latifolia, oleuropein and its derivative tyrosol accounted for 49.3 and 23.1% of phenolics, respectively, the remainder being flavones (luteolin and quercetin) and their glucoside derivatives. Results of the LEIA test suggest that extracts of tannin-rich plants interfere with the very early stage of host invasion and that high concentration of galloylated derivatives may explain anthelmintic activity.     

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, 1200 μg/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 L(3) 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.     

In vitro larval migration and kinetics of exsheathment of Haemonchus contortus larvae exposed to four tropical tanniniferous plant extracts

As for some temperate forage, some tropical tanniniferous plants (TTP) from browsing might represent an alternative to chemical anthelmintic. The anthelmintic effect of four TTP (Acacia pennatula, Lysiloma latisiliquum, Piscidia piscipula, Leucaena leucocephala) on Haemonchus contortus was measured using two in vitro assays. First, the effects of increasing concentrations of lyophilized extracts (150, 300, 600, 1200 microg/ml PBS) were tested on H. contortus larvae (L(3)) using the larval migration inhibition (LMI) test. An inhibitor of tannin, polyvinyl polypyrrolidone (PVPP), was used to verify whether tannins were responsible for the AH effect. Secondly, the effects of extracts on larval exsheathment were examined. Larvae (L(3)) were in contact with extracts (1200 microg/ml) for 3h, and then were exposed to an artificial exsheathment procedure with observations of the process at 10 min intervals. A general lineal model (GLM) test was used to determine the dose effect in the LMI test and the difference of the percentage of exsheathed larvae between the control and the treatment groups. A Kruskal Wallis test was used to determine the effect of PVPP on LMI results. The LMI test showed a dose-dependent anthelmintic effect for A. pennatula, L. latisiliquum and L. leucocephala (P<0.01), which disappeared after PVPP addition, confirming the role of tannins. No effect was found for P. piscipula on H. contortus in the LMI test. However, all four plant extracts interfered with the process of L(3) exsheathment which might be involved as a mechanism of action of tannins on H. contortus larvae. A. pennatula, L. latisiliquum and L. leucocephala could be used as an anthelmintic for the control of H. contortus after confirmation based on in vivo studies.     

Effects of artemisinin and Artemisia extracts on Haemonchus contortus in gerbils (Meriones unguiculatus)

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC(50) value of 0.045 mg/ml in EHA, 0.063 mg/ml in LDA, 0.009 mg/ml in LFIA, and 24.66 mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil.     

Direct and indirect anthelmintic effects of condensed tannins in sheep

Anthelmintic activity of condensed tannins (CT) was evaluated both in vitro and in vivo. In vitro tests included egg hatch test and paralysis/mortality assay on adult Haemonchus contortus. In vivo anthelmintic effect was determined by faecal egg count reduction test in lambs. To this end, 18 lambs were divided into three groups (low tannin, high tannin and control). The lambs of low and high tannin groups were fed diets containing 2 and 3% CT while the control group was fed on diets without CT. In vitro trials showed a dose-dependent inhibition of nematode egg hatching; whereas, there was no effect of CT on adult H. contortus. In vivo trials indicated reduction in faecal egg counts in lambs fed diets containing CT. Feed intake and nutrient digestibility of CT-fed sheep was lower and nitrogen balance was higher as compared to control. Maximum weight gain was observed in animals fed diets containing 3% CT. The direct anthelmintic effect of CT, therefore, was evidenced by inhibited egg hatching; whereas, faecal egg counts reduction in sheep was through improved nutrient utilization.     

Condensed tannins act against cattle nematodes

The use of natural plant anthelmintics was suggested as a possible alternative control of gastrointestinal nematodes (GIN) in ruminants. Direct anthelmintic effects of tannin-containing plants have already been shown in sheep and goat GIN. These anthelmintic properties are mainly associated with condensed tannins. In the present study, we evaluated possible in vitro effects of three tannin-containing plants against bovine GIN. Effects of Onobrychis viciifolia, Lotus pedunculatus and Lotus corniculatus condensed tannin (CT) extracts on Cooperia oncophora and Ostertagia ostertagi were determined by a larval feeding inhibition assay (LFIA) and a larval exsheathment assay (LEA). In the LFIA, all three plant extracts significantly inhibited larval feeding behaviour of both C. oncophora and O. ostertagi first stage larvae in a dose-dependent manner. The L. pedunculatus extract, based on EC(50) (effective concentration for 50% inhibition), was the most effective against both nematodes, followed by O. viciifolia and L. corniculatus. The effect of CT extracts upon larval feeding behaviour correlates with CT content and procyanidin/prodelphidin ratio. Larval exsheathment of C. oncophora and O. ostertagi L3 larvae (third stage larvae) was also affected by CT extracts from all three plants. In both in vitro assays, extracts with added polyvinylpolypyrrolidone, an inhibitor of tannins, generated almost the same values as the negative control; this confirms the role of CT in the anthelmintic effect of these plant extracts. Our results, therefore, indicated that tannin-containing plants could act against cattle nematodes.     

In-vitro anthelminthic activity of crude aqueous extracts of Aloe ferox, Leonotis leonurus and Elephantorrhiza elephantina against Haemonchus contortus

Aloe ferox (Mill), Leonotis leonurus (L) R. BR; and Elephantorrhiza elephantina (Burch.) Skeels are plants frequently used by resource-limited farmers in the Eastern Cape Province to control gastrointestinal parasites in goats. A study was conducted to validate their anthelminthic activities in-vitro on the egg and larvae of the nematode parasite Haemonchus contortus. The crude aqueous extracts of leaves of A. ferox and L. leonurus; and roots of E. elephantina were used. Eggs and larvae of the parasite were incubated at 25°C in aqueous extracts at concentrations of 0.625–20 mg/ml for 48 h and 7 days for the egg hatch and larval development assays respectively. Albendazole and water were the positive and negative controls respectively. Inhibition of egg hatching and larval development increased significantly (P?<?0.05) with increasing concentrations of the extracts. E. elephantina and L. leonurus extracts had 100% egg hatch inhibition at concentration as low as 2.5 mg/ml and 1.25 mg/ml respectively, whereas A. ferox extracts had 100% inhibition at concentrations of 20 mg/ml. At the lowest concentration tested (0.625 mg/ml), E. elephantina inhibited egg hatching >96% and this was comparable to albendazole at the same concentration. E. elephantina and L. leonurus also totally inhibited larval development at concentrations of 1.25 mg/ml. The study provided evidence that A. ferox, E. elephantina and L. leonurus extracts possess anthelminthic activity, thus justifying their use in the treatment of GI helminthosis. There is however need to assess the safety of these plants in vivo and also to undertake in vivo efficacy studies.     

Effect of condensed tannins on egg hatching and larval development of Trichostrongylus colubriformis in vitro

The effects of condensed tannins extracted from seven forages on the viability of the eggs and first stage (L1) larvae of the sheep nematode Trichostrongylus colubriformis were evaluated in in vitro assays. The extracts of condensed tannins were obtained from Lotus pedunculatus (LP), Lotus corniculatus (LC), sulla (Hedysarum coronarium), sainfoin (Onobrychus viciifolia), Dorycnium pentaphylum (DP), Dorycnium rectum (DR) and dock (Rumex obtusifolius). Extracts containing 200 to 500 microg/ml reduced the proportion of eggs that hatched. The larval development assay was used to evaluate the effect of the extracts on the development of either eggs or L1 larvae to L3 infective larvae. Development was allowed to proceed for seven days by which time the larvae in control incubations had reached the infective L3 stage. Extracts containing 200 microg/ml from LP, DP, DR or dock prevented egg development, and only 11, 8 and 2 per cent of the eggs developed to L3 larvae with extracts from LC, sulla and sainfoin, respectively. When the concentration was 400 microg/ml no eggs developed to L3 larvae. The addition of the extracts after hatching also inhibited the development of L1 to L3 larvae; 200 microg/ml extracted from LP, LC, sulla, sainfoin, DP, DR and dock resulted in only 14, 18, 17, 15, 14, 16 and 4 per cent of L1 larvae developing to the L3 stage compared with 85 per cent for controls, and 400 microg/ml further reduced the development of L1 larvae. Statistical analyses showed that when the extracts were added before hatching they were significantly (P<0.001) more effective at inhibiting the larval development than when they were added after hatching. The condensed tannins from dock had the greatest inhibitory effect on egg development followed by the tannins from DR, sainfoin, DP, LP, sulla and LC.     

Susceptibility assay of Haemonchus contortus to commonly used anthelmintics in Jimma, southwest Ethiopia

An experimental study to investigate the susceptibility pattern of Haemonchus contortus to commonly used anthelmintics (albendazole, tetramisole, ivermectin, and triclobendazole) was conducted between June and September 2009. Adult H. contortus parasites were collected from a total of 30 sheep slaughtered at Jimma municipal abattoir. The anthelmintics were assessed for egg hatch inhibition ability against H. contortus eggs. The eggs (500 eggs/ml) were incubated with different concentrations (0.25, 0.125, 0.0625, 0.03125, and 0.015625?μg/ml) at 26°C for 48?h. Egg hatching inhibition of different anthelmintics at different concentrations was recorded. The overall mean percent inhibition showed that there was a significant difference (p?相似文献   

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 μg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 μg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.     

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 2mg/mL (p<0.05). The dichloromethane extract of P. icosandra showed 67.1% inhibition of migration at 3mg/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.     

Resistance to thiabendazole, fenbendazole and levamisole in Haemonchus and Trichostrongylus species in sheep on a Kenyan farm

Resistance to thiabendazole (TBZ), fenbendazole (FBZ) and levamisole (LVM) in naturally acquired gastrointestinal nematode parasites in sheep was investigated on a farm where anthelmintic resistance was suspected. This was measured by both the in vitro egg hatch assay, and reductions in faecal egg and worm counts in treated animals. In the egg hatch assay, nematode eggs were incubated in various concentrations of either TBZ or LVM. The level of resistance was expressed as the drug concentration inhibiting 50% of the eggs from hatching (LC50). The nematode population had LC50 values of 0.26 microgram ml-1 TBZ and 3.12 micrograms ml-1 LVM. In the faecal egg and worm count reduction test, naturally infected sheep were treated with either TBZ (88 mg kg-1), FBZ (10 mg kg-1) or LVM (15 mg kg-1). Faecal egg and total worm counts from these sheep were then compared with counts from untreated sheep. TBZ, FBZ and LVM failed to reduce the faecal egg counts and total worm counts by more than 90%. Based on the identification of larvae from faecal cultures, the most predominant nematode species in the resistant population were Haemonchus (62%) and Trichostrongylus (28%). TBZ reduced faecal egg counts for both species by less than 90%. FBZ and LVM also reduced Haemonchus spp. eggs by less than 90%. Other nematode species numbers did not satisfy criteria for the determination of efficacy.     

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.     

Efficacy of anthelmintic properties of medicinal plant extracts against Haemonchus contortus

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This present work evaluated the efficacy of ethyl acetate, acetone, and methanol dried leaf and seed extracts of five medicinal plants were tested in vitro ovicidal and larvicidal activities on Haemonchus contortus. The in vitro assay was based on egg hatch assay (EHA) and larval development assay (LDA), all plant extracts were evaluated at five concentrations 50, 25, 12.5, 6.25 and 3.13 mg/ml. The leaf and seed ethyl acetate, acetone and methanol of Annona squamosa, Eclipta prostrata, Solanum torvum, Terminalia chebula, and Catharanthus roseus extracts were showed complete inhibition (100%) at the maximum concentration tested (50 mg/ml). The overall findings of the present study have shown that our experimental plant extracts contain possible anthelmintic compounds.     

Studies on the development and survival periods of the non-parasitic stages of Boophilus microplus (Canestrini), in the climatic conditions of Ranchi (India).

Engorged female ticks of Boophilus microplus were exposed in wire-gauze cylinders and glass tubes in an experimental grass plot at monthly intervals during 1989, and egg laying, egg development and larval survival periods observed and recorded. Rainfall and atmospheric relative humidity had an important influence on tick activity. Egg production was maximum, hatching percentage was high, incubation and prehatch periods were short, and larval survival and total longevity periods were long for ticks exposed during the warm and humid rainy season from June to September. Dry atmospheric conditions severely affected egg development, egg hatch and larval survival. Eggs failed to hatch in the dry months from December to April and only 29-38% hatched after a long incubation period of 41 days in November and May. On grass, the larvae of ticks exposed in November survived for the shortest period of 28 days and the larvae of ticks exposed in June survived for the longest period of 133 days. Low winter temperatures reduced egg production and prolonged the pre-oviposition, oviposition and incubation periods. It is suggested that the results of this study might be helpful in the development of measures to control tick infestation by planned dipping and restricted grazing during the period from late June to January when the pasture has a substantial load of larval ticks.     

Comparison of in vitro methods and faecal egg count reduction test for the detection of benzimidazole resistance in small strongyles of horses

The objective of the study was to compare the in vitro egg hatch test (EHT), larval development test (LDT) and in vivo faecal egg count reduction test (FECR test) for the detection of benzimidazole resistance in equine strongyles. The presence of resistant or susceptible strongyle populations was determined in 25 stud farms using the in vivo FECR test and in vitro EHT. On the basis of the FECR values, resistance to fenbendazole was detected on 15 of the 25 farms (60%). The ED₅₀ value (anthelmintic concentration producing 50% inhibition of hatching) for suspected resistant populations varied from 0.110 to 0.222 g/ml thiabendazole (TBZ). Final LD₅₀ values (anthelmintic concentration inhibiting development of 50% of eggs into L₃ infective larvae) above 0.029 g/ml TBZ in the in vitro larval development test on samples from 11 stud farms revealed the presence of populations of small strongyles suspected of being benzimidazole-resistant.     

Field evaluation of the efficacy of fenbendazole in captive wild ruminants

The efficacy of in-feed fenbendazole at a dose rate of 7.5 mg/kg bodyweight for three consecutive days was assessed in five Arabian oryx (Oryx leucoryx), six scimitar-horned oryx (Oryx dammah), 14 slender-horned gazelles (Gazella leptoceros), eight Soay sheep (Ovis aries aries soay), 13 alpine ibex (Capra ibex ibex), six red deer (Cervus elaphus hippelaphus) and 11 Nelson's elk (Cervus elaphus nelsoni) kept in five herds in a zoo. The efficacy was assessed by means of repeated faecal egg count reduction (fecr) tests and in vitro egg hatch assays. Fenbendazole was highly effective against nematodes in five of the seven species, consistently reducing egg shedding by more than 90 per cent. In the egg hatch assays of the five herds, 50 per cent inhibition of hatching (ld50) was observed at a concentration of thiabendazole below 0.1 microg/ml. In the Arabian oryx and alpine ibex the efficacy of fenbendazole was less than 90 per cent, and the ld50 in the egg hatch assays was between 0.1 and 0.2 microg/ml thiabendazole.     

The requirement for early exposure of Haemonchus contortus larvae to Bacillus thuringiensis for effective inhibition of larval development

The potential for a nematocidal Bacillus thuringiensis (Bt) to target the free-living larval stages of Haemonchus contortus was examined using in vitro larval development and migration assays. Bt toxicity in larval development assays decreased as the time period between egg hatch and initial exposure to the Bt was increased; a time lag of 48 h resulted in a 350-fold increase in the IC(50) (from 2.6 ng/ml to 910 ng/ml). The effects on larval migration largely paralleled the effects on larval development, indicating that the larvae which reached the infective stage after exposure to Bt were generally as 'fit' as control worms in terms of migration ability. However, a comparison of the two assays also showed the presence of a level of Bt exposure which showed significantly more toxicity in migration assays than development assays, indicating that, in some cases, fully developed Bt-exposed larvae were less able to migrate than controls, and hence may be compromised in their ability to infect sheep. The rapid decrease in toxicity when exposure to the Bt is delayed highlights a significant issue concerning the use of Bt for control of the free-living larval stages of animal-parasitic nematodes. Targeting the larvae by delivering bacterial spores to the faeces through the host animal's digestive tract would require the spores to germinate upon defecation, grow through a vegetative phase, to then produce crystal toxin protein upon subsequent sporulation. This period of bacterial development will introduce a time lag between worm egg hatching and initial exposure of the larvae to the Bt, which, as demonstrated in the present study, may allow the worm larvae to develop to late larval stages which are relatively insensitive to the toxin.