Evaluation of broad-spectrum anthelmintic activity in a novel assay against Haemonchus contortus, Trichostrongylus colubriformis and T. sigmodontis in the gerbil Meriones unguiculatus

The gerbil Meriones unguiculatus, infected with three species of nematodes, each located in a separate part of the gastrointestinal tract, provided a reliable laboratory assay for the evaluation of broad-spectrum anthelmintic activity. Gerbils harbouring 6-day-old infections of Haemonchus contortus, Trichostrongylus colubriformis and T. sigmodontis were given selected broad-spectrum anthelmintics by gavage. Three benzimidazoles, thiabendazole, oxfendazole and albendazole, a tetrahydropyrimidine, morantel, an imidazothiazole, levamisole hydrochloride, a macrocyclic lactone, ivermectin and an experimental natural product, paraherquamide, were active against all three nematodes at various dosages. Trichostrongylus colubriformis was most sensitive to levamisole hydrochloride, morantel, thiabendazole and paraherquamide whereas ivermectin, oxfendazole and albendazole were more effective against H. contortus. All compounds were active against the caecal nematode T. sigmodontis although it was less sensitive than T. colubriformis. Haemonchus contortus was more sensitive than T. sigmodontis to all anthelmintics tested except thiabendazole.     

Selection for anthelmintic resistance by macrocyclic lactones in Haemonchus contortus.

Two morphologically marked strains of Haemonchus contortus, CAVRS (smooth-macrocyclic lactone resistant) and McMaster (linguiform-macrocyclic lactone susceptible), were used to investigate the selection for anthelmintic resistance following exposure to ivermectin (IVM), a non-persistent anthelmintic. and a more persistent anthelmintic, oral moxidectin (MOX). Three types of selection were investigated: (1) selection of resident worms at the time of treatment (Head selection); (2) selection of incoming-larvae post-treatment (Tail selection); and (3) selection of both resident population and incoming larvae (Head + Tail selection). The experimental animals were adult sheep and lambs. In the controls where there was no anthelmintic selection, the proportion of CAVRS in the adult worm population was the same as the proportion in larvae given to both adults and lambs indicating that CAVRS and McMaster H. contortus were equally infective. There was a significant effect of anthelmintic on total worm numbers in adult sheep with MOX treated adults having less worms, but selection type was non-significant. Anthelmintic type had a significant effect on numbers of resistant worms in adult sheep with less resistant worms in the MOX treated groups, but selection type had no effect. Analysis of variance of arcsine-transformed proportions of resistant worms found that the type of anthelmintic had a highly significant effect, with MOX treated adults having a higher proportion of resistant worms, while type of selection was not significant. In the lambs, nil treated controls and IVM Head + Tail and Tail selected groups had similar geometric mean total worm burdens while Head selected had less total worms. In the MOX treated lamb groups the worm burdens were similar within selection type but less than the IVM treated groups. In the lambs, the types of selection that resulted in more resistant worms were IVM Tail, MOX Head + Tail and MOX Tail. Resistant worm numbers were similar in both adult and lamb groups with Head selection by either MOX or IVM. Moxidectin selected out higher proportions of resistant worms than did IVM in the lambs, with Tail and Head + Tail being stronger selectors than Head. Computer simulations were used to estimate the rate at which resistance developed in the field using the information generated in the present study. The anthelmintic treatments used in the simulation followed a strategic parasite control program for H. contortus in which all sheep receive three Closantel (CLS) treatments in summer. all sheep receive a broad-spectrum (BS) drench or capsule at weaning and lambs receive an additional two BS drenches insummer or no further treatment in the case of the capsule. Moxidectin, IVM-capsule and IVM were the broad spectrum anthelmintics simulated. All simulations were run four times assuming high or low efficacy against resident resistant worms and in the presence or absence of CLS resistance. The simulations indicated that the presence of CLS resistance hastened selection for macrocyclic lactone (ML) resistance. While the IVM-capsule will select most rapidly for ML resistance, IVM oral is expected to be least selective. Moxidectin treatment is intermediate, except in simulations with no CLS resistance and when MOX is assumed to be highly effective against resident ML-resistant worms, in which case MOX can be expected to select more slowly than IVM oral treatments.     

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.     

Prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern France

The occurrence of benzimidazole (BZ) and levamisole resistance was investigated in 18 randomly selected dairy goat herds located in southwestern France and characterized by extensive management. On each of the 18 farms, 45 adult goats were randomly allocated into three groups of 15 animals each: an untreated control group, a group that was orally administered fenbendazole (10 mg kg(-1) body weight) and a group that received orally a levamisole drench (12 mg kg(-1) body weight). Individual faecal egg counts and pooled larval cultures were done 10 days after anthelmintic treatment. Naive lambs were infected with larvae obtained from control and fenbendazole treated groups and were necropsied 35 days after infection for worm recovery. Faecal egg count reductions (FERC) were calculated for fenbendazole and levamisole and, when less than 95 per 100, were considered as indicative of anthelmintic resistance. An in vitro egg hatch test (EHT) was conducted with thiabendazole on eggs isolated from pooled faeces of fenbendazole treated goats in nine farms. Faecal egg count reductions indicated the occurrence of benzimidazole resistance in 15 out of 18 farms. Among these farms, nine had EHT values above 0.1 microg thiabendazole ml(-1) confirming the benzimidazole resistance status. Levamisole resistance was detected in two farms through FECR. Based on necropsy results, the prevalence of benzimidazole resistance was higher in Trichostrongylus colubriformis, medium in Haemonchus contortus and lower in Teladorsagia circumcincta. In nine farms the benzimidazole resistance was monospecific whereas multispecific resistance was found in the six remaining farms. A negative relationship was found between FECR for fenbendazole and the average number of anthelmintic treatments given per year on the farm. Despite extensive management including a low number of treatments, the prevalence of benzimidazole resistance was very high suggesting that the repeated and sometimes exclusive use of benzimidazole drugs, even at low frequency, is probably the main cause in developing nematode resistance in dairy goat herds. The importance of other factors such as under-dosing or buying animals already carrying resistant nematodes are discussed.     

Synthesis and anthelmintic properties of arylquinolines with activity against drug-resistant nematodes

2,4-Disubstituted quinolines with additional substituents in positions 5-8 have been found to have anthelmintic properties. A number of 2,4-dimethoxy-6- or 8-arylquinolines have potent activity against the sheep nematode Haemonchus contortus, with LD99 values of the same order of magnitude as levamisole. These arylquinolines maintain their activity against levamisole-, ivermectin- and thiabendazole-resistant strains of H. contortus.     

Concurrent infections with the ruminant nematodes Haemonchus contortus and Trichostrongylus colubriformis in jirds, Meriones unguiculatus, and use of this model for anthelmintic studies

Haemonchus contortus- and Trichostrongylus colubriformis-infected jirds (Meriones unguiculatus) are useful for anthelmintic studies. With concurrent infections of these parasites established in the jird, questions of not only anthelmintic activity, but to some extent spectrum, could be assessed in a single model system. This report outlines a model using immunosuppressed (0.02% hydrocortisone in feed) jirds concurrently infected with H. contortus and T. colubriformis. Immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of each species, treated per os on day 10 postinoculation (PI), and killed on day 13 PI. Stomachs and small intestines were removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent examination. Contents of both organs were examined using a stereomicroscope (15-45 x). Various standard anthelmintics were evaluated in the model; modern broad-spectrum ruminant anthelmintics (benzimidazoles, febantel, ivermectin, levamisole hydrochloride, and milbemycin D) are active uniformly and in most cases at doses comparable to those required for efficacy against these parasites in ruminants. This model, using worms of 2 genera living in distinct sites, allows preliminary evaluation of anthelmintic activity and spectrum for experimental compounds in a single cost- and resource-efficient experiment.     

Multiple anthelmintic resistance in gastrointestinal nematodes of small ruminants in Bangladesh

Anthelmintic resistance (AR) against gastrointestinal nematodes (GINs) of sheep and goats is a global concern. To address the problem, this study assessed the status of AR in different government and private sheep and goat farms in Bangladesh. We conducted fecal egg count reduction test (FECRT) and Egg hatch assay (EHA) experiments. For the detection of resistant larvae, pooled fecal samples from treated and non-treated groups were subjected to coproculture. Furthermore, 195 adult Haemonchus parasites were genotyped to ascertain benzimidazole (BZ) resistance allele from seven topographic zones of Bangladesh using allele specific PCR (AS-PCR). In FECRT, the percentage reduction along with 95% confidence intervals indicated that GINs were resistant to albendazole (ABZ), levamisole (LEV) and ivermectin (IVM). Coproculture revealed that Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp. were resistant to anthelmintics. ABZ resistance was also confirmed by in vitro EHA in all the farms except the private goat farm in Mymensingh. The genotype frequencies were 6% for homozygous resistant (rr), 59% for heterozygous (rS) and 35% for homozygous susceptible (SS) among different topographic zones. The allelic frequency of the mutation conferring resistance (r) ranged from 25% to 47% signifying resistance to BZ in nematodes of sheep/goats. The genotype frequencies (rr, rS and SS) and allelic frequencies (r and S) varied significantly (p˂0.05) in different zones in Bangladesh. Overall, the data suggest an alarming condition created by multiple AR in Bangladesh.     

A novel anthelmintic model utilizing jirds, Meriones unguiculatus, infected with Haemonchus contortus

Currently, no in vivo laboratory model is available for evaluating anthelmintics against the important ruminant helminth Haemonchus contortus. This report outlines a novel anthelmintic assay utilizing immunosuppressed (0.02% hydrocortisone in feed) jirds, Meriones unguiculatus, infected with H. contortus. Immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of H. contortus, treated per os on day 10 postinoculation (PI), and necropsied on day 13 PI. Each stomach was removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent examination. Stomach contents were examined using a stereomicroscope (15-45x). A variety of standard anthelmintics has been evaluated in the model; modern broad-spectrum ruminant anthelmintics (benzimidazoles, febantel, ivermectin, levamisole hydrochloride, and milbemycin D) are active uniformly and in most cases at doses (mg/kg) comparable to those required for efficacy against H. contortus in ruminants. This model provides an important new tool to assess preliminarily the activity of experimental drugs against H. contortus in vivo prior to studies in ruminants and also may provide a useful tool for studying host-parasite interactions for H. contortus.     

Selection of different genotype larvae and adult worms for anthelmintic resistance by persistent and short-acting avermectin/milbemycins

To understand the factors that influence selection for anthelmintic resistance, it is necessary to examine the impact of drug treatment, particularly persistent drugs, on all phases of the worm life cycle. The efficacy of various avermectin/milbemycin anthelmintics was determined against resident worms, incoming larvae (L3) and development of eggs in faecal culture. Homozygote-resistant and maternal and paternal F1-heterozygote genotypes of Haemonchus contortus were used to infect sheep before or after treatment with ivermectin (IVM) oral, IVM capsule, moxidectin (MOX) oral or MOX injectable. Total worm count and quantitative larval culture were used to determine efficacy against parasitic and free-living stages, respectively. Selection for resistance by IVM capsules occurred at the adult and L3 stages because of poor efficacy against these stages for all resistant genotypes. However, the selective advantage of these surviving worms was reduced due to the low development of their eggs to L3 in faecal culture. For MOX, selection for resistance predominantly occurred after treatment because of high efficacy against resident adult worms of all resistant genotypes but poor efficacy against resistant L3 ingested after drug administration. The results indicated no evidence of sex-linked inheritance for IVM resistance. Mean IVM efficacies against homozygous and heterozygous resistant adult worms were not different, and IVM capsule efficacy against incoming L3 was approximately 70% for all resistant genotypes, consistent with a dominant trait. MOX was highly effective against adults of all resistant genotypes and approximately 76% effective against incoming L3 regardless of resistance genotype, also consistent with a dominant trait. These results will enable the impact of persistent drugs on worm control and anthelmintic resistance to be estimated. The results indicate that IVM capsules should not be used in populations where avermectin/milbemycin resistance is present.     

Investigation of intestinal nematode responses to naphthalophos and pyrantel using a larval development assay.

Responses of several nematode species to naphthalophos and pyrantel/levamisole were examined using a larval development assay in order to determine the potential of this assay for detection of resistance. Haemonchus contortus and Ostertagia circumcincta showed concentration-dependent responses to naphthalophos, however, the assay was unsuitable for Trichostrongylus colubriformis due to the low toxicity of the drug to the larval stages of this nematode. Measurement of concentration-dependent response to pyrantel in susceptible T. colubriformis was limited by a reduced toxicity against larvae at high drug concentrations, resulting in a parabolic response with a development-inhibition maxima of less than 100%. This limits the usefulness of the assay to detect pyrantel resistance in this species as the presence of a small resistant fraction in a field isolate may be indistinguishable from the parabolic susceptible response. On the other hand, responses of susceptible T. colubriformis to levamisole, and susceptible H. contortus to pyrantel and levamisole showed 100% development inhibition over a range of drug concentrations, indicating that the appearance of a resistant fraction in a field population would be readily discernible from the susceptible response, allowing resistance detection for these drug/parasite combinations. This study has highlighted the varied suitability of the larval development assay technique for resistance detection with different combinations of drugs and parasite species.     

Laboratory selection of a benzimidazole-resistant isolate of Trichostrongylus colubriformis for ivermectin resistance

In vivo ivermectin resistance was selected in an isolate of Trichostrongylus colubriformis (TcR) already known to be benzimidazole resistant. This was accomplished in sheep by using levels of ivermectin calculated to reduce the fecal egg output from each generation of T. colubriformis by congruent to 95%. The first indication of ivermectin resistance was observed with the F10. A dosage-titration trial comparing the parent TcR with the ivermectin-selected F21 demonstrated that the latter was congruent to 20 times more resistant to oral ivermectin therapy in experimentally infected sheep than was the parent isolate. Treatment of the F16 generation with 50 mg/kg of thiabendazole resulted in only 54% egg reduction and confirmed that benzimidazole resistance was stable.     

Toxicity of Bacillus thuringiensis to parasitic and free-living life-stages of nematode parasites of livestock

A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pH相似文献   

Resistance of field isolates of Trichostrongylus colubriformis and Ostertagia circumcincta to ivermectin.

Twelve Romney lambs and 10 Angora goats were infected with 7000 infective third-stage larvae (89% Trichostrongylus, 11% Ostertagia) collected from goats suspected of harbouring ivermectin-resistant nematodes. On 28 days p.i., the lambs and goats were divided into treatment and control groups of six and five animals, respectively. The animals in the treatment groups were treated with ivermectin (0.2 mg/kg) and necropsied 35 days p.i. Faecal egg counts were estimated on days 28 and 35 p.i. and larval development assays (LDAs) were conducted on 22, 24, 26, 28, 30, 32, 34 and 35 days p.i. The ivermectin treatment reduced Trichostronglus colubriformis burdens by 39% and 13% and Ostertagia circumcincta by 33% and 0% in lambs and goats, respectively. When compared with a susceptible strain, the LDAs indicated a resistance factor before treatment in lambs for T. colubriformis of 2.6 and 1.5 with ivermectin and avermectin B2, respectively, which rose to 3.4 and 2.0 after treatment. The LD50 values of the two control groups were relatively constant throughout the experiment. Prior to ivermectin treatment the LD50 values of the treated groups were similar (P > 0.05) to the control groups but following ivermectin treatment their LD50 values increased steadily until the animals were killed on 35 days p.i. The LD50 values for ivermectin and avermectin B2 of sheep were always slightly higher and significantly different (P < 0.01) than those of goats indicating a host effect on this parameter. The greater reduction in worm counts in goats suggests a difference in the efficacy of ivermectin between lambs and goats. This is the first confirmed report of ivermectin resistance in a field strain of T. colubriformis.     

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.     

Development of anthelmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies

Development of anthclmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies. International Journal for Parasitology13: 125–132. In a three year (1978, 1979, 1980) field experiment in south-eastern Australia Merino weaner sheep were exposed to one of four worm control programmes. Sheep were treated either 50–51 times, 9–12 times, 3–6 times or 1–3 times annually with anthelmintic. Thiabendazole (TBZ) was used throughout 1978 and for the initial dose in 1980, levamisole (LEV) throughout 1979 and 1980. Sheep were replaced annually each December.During 1978 in vitro egg hatch tests indicated that populations of Haemonchus contenus with resistance factors to TBZ of 10·7, 10·0, 2.5 and 1·8 respectively were selected by these treatment frequencies. No resistance to LEV was detected in 1979 but during 1980 in vivo egg count reduction tests showed that highly resistant populations of Ostertagia spp. were selected in sheep dosed either 49 or 11 times. This was confirmed by an in vitro larval paralysis test. Partial resistance occurred where sheep were dosed 5 times but not where one dose was given. The only population of H. conlortus still present at the end of 1980 in sufficient numbers to allow eggs to be tested in vitro was from the sheep dosed once. This had retained its resistance to TBZ.     

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.     

Anthelmintic resistance in nematodes: extent, recent understanding and future directions for control and research

Resistance has now been reported to all of the broad spectrum anthelmintic types currently available, namely to the benzimidazoles, levamisole/morantel and to ivermectin. The problem causes most concern for parasite control in sheep, but anthelmintic resistance has also been reported in nematodes of horses, goats, pigs and more recently cattle. Our understanding of the factors which select rapidly for resistance has increased and programmes of worm control which minimize selection for anthelmintic resistance are being developed and tested. One of the greatest problems encountered in attempting to reduce the selection for overt drug resistance is the need for more sensitive tests for developing resistance. In the long term, new approaches to chemotherapy and to overcoming anthelmintic resistance problems will arise from improving our understanding of the modes of action of, and mechanisms of resistance to, anthelmintics at the level of the receptor proteins and their genes.     

Genetic variability following selection of Haemonchus contortus with anthelmintics

Genetic diversity in nematodes leads to variation in response to anthelmintics. Haemonchus contortus shows enormous genetic diversity, allowing anthelmintic resistance alleles to be rapidly selected. Anthelmintic resistance is now a widespread problem, especially in H. contortus. Here, I compare the genes involved in anthelmintic resistance in H. contortus with those that confer susceptibility or resistance on the free living nematode Caenorhabditis elegans. I also discuss the latest knowledge of genes associated with resistance to benzimidazoles, levamisole and the macrocyclic lactones and the need for DNA markers for anthelmintic resistance.     

The metabolism of flubendazole and the activities of selected biotransformation enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics

SUMMARY Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g. sheep and goat). The treatment of haemonchosis is complicated because of recurrent resistance of H. contortus to common anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug flubendazole (FLU) and the activities of selected biotransformation enzymes towards model xenobiotics in 4 different strains of H. contortus: the ISE strain (susceptible to common anthelmintics), ISE-S (resistant to ivermectin), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (resistant to all common anthelmintics). H. contortus adults were collected from the abomasums from experimentally infected lambs. The in vitro as well as ex vivo experiments were performed and analysed using HPLC with spectrofluorimetric and mass-spectrometric detection. In all H. contortus strains, 4 different FLU metabolites were detected: FLU with a reduced carbonyl group (FLU-R), glucose conjugate of FLU-R and 2 glucose conjugates of FLU. In the resistant strains, the ex vivo formation of all FLU metabolites was significantly higher than in the susceptible ISE strain. The multi-resistant WR strain formed approximately 5 times more conjugates of FLU than the susceptible ISE strain. The in vitro data also showed significant differences in FLU metabolism, in the activities of UDP-glucosyltransferase and several carbonyl-reducing enzymes between the susceptible and resistant H. contortus strains. The altered activities of certain detoxifying enzymes might protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.     

Licking behaviour induces partial anthelmintic efficacy of ivermectin pour-on formulation in untreated cattle

Licking behaviour in cattle has been reported to account for the disposition of topically administered macrocyclic lactones. However, its impact on anthelmintic efficacy remains to be established. Therefore, we evaluated the impact of ivermectin exchange between cattle on the reduction in the faecal egg count (FEC) after pour-on administration in a group of 10 heifers experimentally infected with Ostertagia ostertagi and Cooperia oncophora. Four treated (500 μg/kg, pour-on) and six untreated animals were put together after treatment and plasma and faecal exposure to ivermectin as well as the FECs were evaluated before and over 40 days after treatment. Ivermectin was detected in plasma and faeces of the six untreated heifers, with maximal exposures two- to three-fold lower than the minimal exposures in treated animals. The interindividual variability of exposure was very high in untreated animals, with a ten-fold difference between the upper and lower limits compared with treated heifers, where there was only a two-fold difference. Anthelmintic efficacy, expressed as an average reduction of the FECs over the experimental period, was maximal in the treated group. In untreated heifers, anthelmintic efficacies ranged from zero to maximal efficacy, with intermediary values between 30% and 80%. The use of a classical pharmacodynamic model demonstrated a clear relationship between exposure and efficacy and enabled us to define the critical plasma or faecal ivermectin concentrations delimiting an exposure window associated with partial anthelmintic efficacy. This range of ivermectin plasma concentrations (0.1-1 ng/mL) could be considered as a potential selection window for anthelmintic resistance. Finally, our results show that macrocyclic lactone exchange between cattle after pour-on administration, resulting from natural grooming behaviour, can significantly impact on anthelmintic efficacy. This raises several issues such as the design of comparative clinical trials and the occurrence of partial efficacy which is considered a risk factor for the development of anthelmintic resistance.