Benzimidazole Resistance In Haemonchus Contortus Research Articles

First identification of benzimidazole resistant Haemonchus contortus in sheep in Greece

The present study investigated for the first time benzimidazole (BZ) resistance in Haemonchus contortus originating from sheep in the area of Thessaloniki, Northern Greece, using an allele-specific PCR. This method detects a polymorphism (Phe/Tyr) that affects the amino acid at position 200 of the β-tubulin protein, and is known to be linked to BZ resistance. Forty adult worms were collected from local abattoir from 8 flocks. From each flock a total of 5 sheep were sampled and from each sheep one worm was collected. It was found that all 40 worms were homozygous benzimidazole resistant, which demonstrates for the first time that resistant strains of H. contortus do exist in sheep farms in Northern Greece. These results have practical implications in sheep farmer consulting in Greece, because sensitive screening of important worms using molecular methods may offer in the future a valid estimation on the correct use of anthelmintics towards a more effective control of gastrointestinal nematode parasites of small ruminants.

Phenotypic and genotypic characterisation of benzimidazole susceptible and resistant isolates of Haemonchus contortus

The study was designed to compare the in vitro egg hatch test (EHT) and molecular allele-specific polymerase chain reaction PCR (AS-PCR) methods as tools for detection of benzimidazole resistance in Haemonchus contortus, a nematode parasite of small ruminants. Comparisons were made during the course of an experimental infection and changes in EHT and AS-PCR were monitored to measure the correlation between in vitro and molecular tests. Both methods were carried out according to World Association for the Advancement of Veterinary Parasitology (WAAVP) recommendations. The molecular test was used to discriminate TAC/TTC polymorphism in the beta-tubulin 200 codon of eight (four resistant and four susceptible) isolates of H. contortus. Using DNA from 100 third-stage larvae, the polymerase chain reaction (PCR)-based analysis revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals in all resistant isolates. Both methods showed comparable and reliable results with regard to detection of benzimidazole resistance. The molecular test has an advantage over the EHT because of its higher sensitivity. On the other hand, EHT is less time-consuming, allows reliable detection of <10% resistance allele frequency and is fairly reliable for the detection of benzimidazole resistance under field conditions.

In vitro detection of benzimidazole resistance in Haemonchus contortus: Egg hatch test versus larval development test

The present study was designed to compare the egg hatch test (EHT) and the larval development test (LDT) as in vitro tools for detection of benzimidazole (BZ) resistance in Haemonchus contortus, a nematode parasite of small ruminants. Comparisons were made during a course of infection and changes in both EHT and LDT were monitored to measure the correlation between resistance and susceptibility in different parasite stages (eggs and larvae). In addition, mixed doses of known numbers of susceptible and BZ-resistant H. contortus eggs were used to assess the sensitivity of LDT for the detection of low levels of resistance. The degree of resistance for each test was expressed as resistance factor (RF). The LDT showed a greater ability to distinguish between four susceptible and four resistant isolates of H. contortus with higher resistance factors compared to the EHT. For the EHT the RF by using ED 50 criterion ranged from 3.2 to 13.3 and from 7.4 to 25.2 by using LC 99. For LDT the resistant isolates were 4.3–63.1 times more tolerant than the susceptible isolates using the ED 50 criterion and 91.1–1411.0 times more tolerant using the LC 99 criterion. The LDT was also able to clearly indicate the presence of low level (4%) of resistant larvae amongst a susceptible background population.

Diagnosis of Benzimidazole Resistance in Haemonchus contortus of Sheep by Allele Specific PCR

The study was conducted on 162 adult male Haemonchus contortus of sheep collected from Avikanagar, Jaipur and Bikaner regions to diagnose the benzimidazole (BZ) resistance in H. contortus. The BZ resistance is primarily linked with the mutation in β-tubulin isotype 1 gene which substitute phenylalanine (Phe) into tyrosine (Tyr) at the 200 codon of the gene. An allele specific polymerase chain reaction (AS-PCR) technique was used for diagnosis of BZ resistance in H. contortus. In AS-PCR, one reverse primer (TGG 312) was used in two separate reactions with each of 2 forward primers (resistant TGG 331 and susceptible CAW 106 primer) that differed only at 3' nucleotide position. Therefore, the amplified products from resistant and susceptible parasites were produced 267 and 266 bp, respectively. A total of 162 parasites were genotyped, of which 130 parasites found homozygous resistant 'rr', 22 heterozygous 'rS' and 10 homozygous susceptible 'SS' type. The prevalence of 'rr' individuals was higher in Jaipur (98%) followed by Avikanagar (93%) and Bikaner (50%) regions. Overall, the prevalence of BZ resistant allele (r) was higher (87%) as compared to 13% of BZ susceptible allele (S).

The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep.

Resistance to the benzimidazole (BDZ) class of anthelmintics in nematodes of sheep has become a common and global phenomenon. The rate at which the selection process and development of resistance occurs is influenced by a number of factors. Of these, the effects of stage-specific exposures to anthelmintic were investigated with a BDZ-resistant strain of Haemonchus contortus (HCR) over five parasite generations. Sheep were infected at each generation with the HCR strain and were treated with thiabendazole (TBZ), either 5 days post-infection (p.i.) (larval line), 21 days p.i. (adult line), or left untreated (no selection line). Additionally eggs from each generation were exposed to TBZ (egg line). Geometric worm burdens were calculated from post-mortem worm counts, both at the start of the study, and after the final selection studies for each of the selection lines. Egg hatch assays (EHAs) were also conducted throughout the study. All data relating to worm burdens and EHAs for each generation were analysed by linear regression to produce dose titration curves and lethal dose(50) (LD(50)) values for each of the selection lines. Over the five generations, LD(50) values on dose-response were increased and worm survival occurred at higher dose rates of TBZ irrespective of the parasite stage exposed to treatment. A similar picture was seen with ED(50) values, which showed a fluctuating but generally upward trend for each of the three selection lines. In contrast, LD(50) and ED(50) values were decreased in the no selection line, indicating some degree of reversion albeit to levels still considered to be BDZ-resistant.

86. Effects of stage-specific selection on the development of benzimidazole-resistance in Haemonchus contortus in sheep

86. Effects of stage-specific selection on the development of benzimidazole-resistance in Haemonchus contortus in sheep

The efficacy of levamisole, and a mixture of oxfendazole and levamisole, against the arrested stages of benzimidazole-resistant Haemonchus contortus and Ostertagia circumcincta in sheep

Sheep were allowed to graze pasture that had been seeded with benzimidazole-resistant Haemonchus contortus and Ostertagia circumcincta in order to acquire a burden of arrested larvae. Following housing, sheep were dosed orally with either oxfendazole at a dose rate of 4.7 mg/kg (to confirm the benzimidazole-resistant status of the species of nematode), levamisole at a dose rate of 7.5 mg/kg, or an oxfendazole/levamisole mixture at a dose rate of 4.6 mg/kg oxfendazole and 8.1 mg/kg levamisole. The efficacies of the treatments were assessed by estimation of the arrested larval burden in the abomasum of each sheep, either at 10 or 11 days (oxfendazole and oxfendazole/levamisole mixture), or 12 or 13 days (levamisole), after treatment. Compared to the untreated controls, the protection afforded by a single dose of either levamisole or the oxfendazole/levamisole mixture was >99% against the arrested stages of both Haemonchus contortus and Ostertagia circumcincta. Treatment with oxfendazole confirmed the benzimidazole-resistance status of the two species.

The effect of reduced feed intake on the efficacy of oxfendazole against benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis in sheep

Within 12 h of reducing the daily intake of a 50:50 lucerne and wheaten hay ration from 800 to 400 g, there was an increase in digesta marker concentration indicating a reduction in the flow rate of digesta fluid and particulates through the abomasum of sheep. The efficacy of the recommended dose of oxfendazole (OFZ) was then tested against benzimidazole resistant Trichostrongylus colubriformis and Haemonchus contortus in sheep whose feed intake was halved 36 h before and after drug treatment. In animals fed the reduced ration there was a 60% reduction of T. colubriformis and 94% reduction of H. contortus compared with a 19% and 60% reduction respectively on the higher feed intake. It is therefore suggested that greater anthelmintic efficacy can be achieved in sheep by temporary feed reduction.

Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in β-tubulin isotype 1

Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in β-tubulin isotype 1

Effect of Selection for Benzimidazole Resistance in Haemonchus contortus on β-Tubulin Isotype 1 and Isotype 2 Genes

In vitro selection for benzimidazole (BZ) resistance in the nematode Haemonchus contortus, a parasite of sheep, suggested the involvement of β-tubulin genes. Two distinct β-tubulin isotype classes were analyzed. Selection with stepwise increased concentrations of BZ showed for the first time that the increasing degrees of resistance correlate to a dual phase adaptation mechanism. At lower degrees of developing resistance, selection for a single β-tubulin isotype 1 variant takes place. Subsequently, at higher degrees of drug resistance, an abrupt elimination of individuals carrying β-tubulin isotype 2 genes in their genome is seen. To evaluate the in vitro experiments we carried out a survey of resistant field populations and found that in the most resistant population (RNO) apparently the same mechanism had happened.

An evaluation of an oral formulation of moxidectin against selected anthelmintic-resistant and -susceptible strains of nematodes in lambs

The efficacy of an oral formulation of the newly developed parasiticide, moxidectin, was tested against benzimidazole-resistant Haemonchus contortus, Trichostrongylus colubriformis, and Nematodirus spathiger, levamisole-resistant Ostertagia circumcincta, and susceptible Cooperia curticei infections in weaned lambs. Thirty-two lambs were experimentally infected with mixed doses of the above strains of nematodes. They were allocated into four treatment groups by stratified randomisation using liveweights and faecal egg counts 28 days later. One group received moxidectin at 0.2 mg/kg liveweight, one group oxfendazole at 4.5 mg/kg liveweight, one group levamisole at 7.5 mg/kg liveweight and the last group remained untreated as the control. Worm burdens in the lambs at slaughter 10 days after oral treatment confirmed the resistance status of the nematode strains used, and showed that moxidectin had a greater than 99.9% efficacy (p<0.01) against all of them. No adverse effects due to treatment with moxidectin were observed in any of the animals.

Specific interaction of benzimidazole anthelmintics with tubulin: high-affinity binding and benzimidazole resistance in Haemonchus contortus

The specific (high-affinity) binding of tritiated benzimidazole ([ 3H]BZ) anthelmintics-mebendazole ([ 3H]MBZ) and oxibendazole ([ 3H]OBZ) to, and the specific displacement (inhibition) of these radioligands by unlabelled BZs (oxibendazole, mebendazole, oxfendazole, albendazole, fenbendazole and thiabendazole) from crude tubulin extracts prepared from thiabendazole (TBZ)-susceptible (S) or TBZ-resistant (R) strains of adult Haemonchus contortus, have been examined. The most striking difference between R and S was that the drug specifically bound at infinite ligand concentration (B max), was markedly reduced for the R strain, with no apparent change in association constant ( K a). Thus, resistance was associated with a loss of high-affinity receptors. TBZ-resistance was not associated with a change in low-affinity binding. There was a greater loss of high affinity receptors for [ 3H]OBZ than for [ 3H]MBZ. Using the displacement data. BZs were ranked according to their K a and IC 50 (concentration of BZ inhibiting 50% of radioligand binding) values. The K a and IC 50 values and the rank order of the BZs were approximately independent of the radioligand displaced or source (S or R) of the tubulin extracts used. The results are consistent with tubulin binding being the primary mechanism of action for all of these BZs.

Comparison of three in vitro techniques to estimate benzimidazole resistance in Haemonchus contortus of sheep

Three in vitro assays to detect benzimidazole resistance, namely, the egg-hatch assay, tubulin-binding assay, and a larval-development assay, were evaluated by estimating the level of benzimidazole resistance in three field isolates of Haemonchus contortus compared with a susceptible reference strain. Comparisons were also made with estimates of benzimidazole resistance of the three field strains obtained from an in vivo controlled anthelmintic efficacy test. All three in vitro tests showed similar, consistent results which also suggested greater sensitivity than the in vivo assay. These results indicate that selection of an in vitro technique to determine benzimidazole resistance should therefore be based on considerations other than precision, such as technical expertise, availability of equipment, cost and speed in which diagnosis is required.

Further investigations of the primary mechanism of benzimidazole resistance in Haemonchus contortus

The binding of the [ 3H] benzimidazole carbamates (BZCs)—albendazole (ABZ), oxibendazole (OBZ), parbendazole (PBZ), mebendazole (MBZ), fenbendazole (FBZ) and oxfendazole (OFZ)—to tubulin from three ecologically-related isolates of adult Haemonchus contortus has been examined. The extent of binding of each BZC was inversely proportional to the known resistance status of the isolate. Biochemically, the change in the formation of the BZC-tubulin complex was due to a reduction in the amount of drug bound to resistant tubulin, with no significant change in the association constant of the complex. The resistance factors derived from the binding data support the hypothesis that the complex is ligand-dependent, with the aryl-substituted BZCs—MBZ, OFZ and FBZ—demonstrating lower resistance factors than those of the alkyl-substituted BZCs—ABZ, OBZ and PBZ. Examination of the slope derived from plots of binding against protein concentration demonstrated that the failure of resistant or partially resistant isolates to bind was due to either a decrease in the number of binding sites or, more likely, to reduced stability of the BZC-tubulin complex rendering it unstable to charcoal extraction.

Efficiency of oxfendazole administered as a single dose or in a controlled release capsule against benzimidazole-resistant Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis

Laboratory strains of Haemonchus contortus and Ostertagia circumcincta selected for thiabendazole resistance were found to have a strong side resistance to a single dose of oxfendazole. The LD50 and LD95 in mg of drug per host liveweight were respectively 4.28 and 18.46 mg/kg for H contortus and 3.61 and 11.20 mg/kg for O circumcincta. A field strain of Trichostrongylus colubriformis that had not been selected with thiabendazole for seven years also had a strong side resistance to oxfendazole with approximately 50 per cent of its population resistant to the recommended dose rate of 5 mg/kg. Prolonged administration of oxfendazole by intraruminal controlled release capsules was found to be effective against both susceptible and resistant strains of the above parasites. The first observed effect of oxfendazole, from controlled release capsules, on resistant worms was a decrease in the percentage of eggs developing to third stage larvae. This was followed by a decrease in egg count and in worm numbers.

Synergistic action of mebendazole and levamisole in the treatment of a benzimidazole-resistant Haemonchus contortus in sheep

One-year old worm free, merino wethers, were each infected with 5000 H. contortus larvae of a strain resistant to mebendazole at a rate of 52 mg/kg body weight of sheep. After 21 days, they were assigned to two trials. The preliminary trial showed that mebendazole and levamisole acted synergistically on the H. contortus infection. In the second trial, sheep were treated with 0.35 mg/kg levamisole (one seventh the minimum effective dose against susceptible worms) or 40 mg/kg mebendazole (40 times the minimum effective dose against susceptible worms). In each case the anthelmintics did not reduce worm burdens, although mebendazole depressed egg production. However, when mebendazole and levamisole, at the above dose rates, were administered simultaneously, total worm counts in sheep were reduced by almost 60%. Similar results were obtained when the levamisole was administered 8 h or 14 h after mebendazole treatment. The implications of these observations for the treatment of benzimidazole-resistant haemonchiasis in sheep are discussed.

THE ANTHELMINTIC EFFICACY OF HALF‐DOSE RATES OF LEVAMISOLE AGAINST BENZIMIDAZOLE RESISTANT HAEMONCHUS CONTORTUS

Australian Veterinary JournalVolume 56, Issue 4 p. 203-204 THE ANTHELMINTIC EFFICACY OF HALF-DOSE RATES OF LEVAMISOLE AGAINST BENZIMIDAZOLE RESISTANT HAEMONCHUS CONTORTUS B. A. FORSYTH, B. A. FORSYTH ICI Australia Operations Pty Ltd, Merrindale Research Station, Dorset Road, Croydon, Victoria, 3136 22 October 1979Search for more papers by this authorA. J. GIBBON, A. J. GIBBON ICI Australia Operations Pty Ltd, Merrindale Research Station, Dorset Road, Croydon, Victoria, 3136 22 October 1979Search for more papers by this author B. A. FORSYTH, B. A. FORSYTH ICI Australia Operations Pty Ltd, Merrindale Research Station, Dorset Road, Croydon, Victoria, 3136 22 October 1979Search for more papers by this authorA. J. GIBBON, A. J. GIBBON ICI Australia Operations Pty Ltd, Merrindale Research Station, Dorset Road, Croydon, Victoria, 3136 22 October 1979Search for more papers by this author First published: April 1980 https://doi.org/10.1111/j.1751-0813.1980.tb05692.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume56, Issue4April 1980Pages 203-204 RelatedInformation

The anthelmintic efficacy of albendazole against Fasciola hepatica and benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep

The anthelmintic efficacy of albendazole (methyl [5-(propylthio) - 1H - benzimidazole -2 -yl] carbamate) against immature and adult Fasciola hepatica and against standardised strains of benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis was evaluated in experimentally infected sheep. A single intrarumenal treatment of dose rates of 3.8 and 7.6 mg/kg was ineffective against immature (six weeks old) F hepatica. Dose rates of 5.7 and 7.6 mg/kg reduced the number of mature (12 weeks old) F hepatica by 70 and 91 per cent respectively. Dose rates of 5.7 and 7.6 mg/kg removed 92 and 99 per cent of four-week-old, benzimidazole resistant H contortus and 89 and 99 per cent of four-week-old, benzimidazole resistant T colubriformis.

Levels of benzimidazole resistance in Haemonchus contortus and Trichostrongylus colubriformis recorded from an egg hatch test procedure

A method of using commercially available benzimidazole anthelmintics in an egg hatch test, to record levels of resistance is described. The test shows that a side-resistance is present to all the benzimidazole anthelmintics tested against both Haemonchus contortus and Trichostrongylus colubriformis after selection with thiabendazole. Additional selection on the field resistant strain of H contortus with thiabendazole showed increased levels of resistance to all the compounds tested while similar selection of T colubriformis failed to change the level of resistance after 20 generations. The level of resistance recorded by the egg hatch test in vitro is greater than that recorded for adults in vivo for the CF strain of H contortus and T colubriformis. Problems associated with reading the test are mentioned as are the different levels for the threshold of activity.

Instability of benzimidazole resistance in nematode eggs

The resistance of the eggs of benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis is not stable and will decrease in the absence of drug challenge. Dosing animals harbouring resistant H contortus with either thiabendazole or mebendazole selects for nematodes producing thiabendazole resistant eggs. The eggs of a thiabendazole selected strain of H contortus were more resistant to thiabendazole, parbendazole and oxibendazole but less resistant to cambendazole and mebendazole.