Survey of anthelmintic resistance in Western Australian sheep flocks 1. Prevalence

Between July 1981 and December 1983, 116 randomly selected sheep farms in the south west of Western Australian were surveyed for resistance to anthelmintics. A faecal worm egg count reduction test was carried out on each farm. Anthelmintics tested were thiabendazole (44 mg/kg) and levamisole (7.5 mg/kg) given by intra-ruminal injection and comparisons were made with an untreated group on each farm. Successful tests were carried out on 84 farms and 68% of these had resistant worms present. The prevalence of thiabendazole resistant populations was for H. contortus 18%; Teladorsagia, 41% and Trichostrongylus, 48% and for levamisole resistant populations H. contortus, 10%; Teladorsagia, 41%; Trichostrongylus, 24%, and Nematodirus, 10%. Multiple resistant populations were found on 17% of farms. Although the distribution of nematode genera varied between the 400 to 750 mm and the greater than 750 mm rainfall zones there was no significant difference in the prevalence of resistance between zones. About one third of resistant populations were severely resistant (less than 60% reduction). It is likely that resistant worms were present on many farms without causing clinical disease and continued anthelmintic selection pressure will result in further development of resistance.     

Anthelmintic resistance survey in New Zealand

The prevalence and level of thiabendazole resistance of sheep nematodes in the North Island of New Zealand was investigated by means of an in vitro egg-hatch technique; samples from sheep on 52 properties in six country areas were examined. Resistance of Haemonchus contortus and Trichostrongylus spp. was identified on the basis of egg hatch in 0.1 ppm thiabendazole. Evidence of resistance was obtained on 11(21.2%) of the properties. LD 50's and resistance ratios were calculated for the resistant nematode populations found.     

Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes

In order to monitor and eventually control the spread of drug-resistant Haemonchus contortus, knowledge of the molecular mechanisms underlying resistance is essential. Here we phenotypically and genotypically characterize three multidrug-resistant H. contortus field isolates from Australia and South Africa. All were significantly less susceptible to thiabendazole than a sensitive reference strain in an in vitro egg-hatch assay. The sensitivity was further reduced in a surviving population after treatment of infected sheep with albendazole. The beta-tubulin genes were amplified from genomic DNA of the H. contortus isolates, cloned, and sequenced. There was a high degree of sequence variation. The known mutation phenylalanine-200 to tyrosine (F200Y) occurred in 60% of the sequences from resistant isolates, but not in the sensitive reference. Interestingly, 90% of the beta-tubulin sequences from resistant isolates lacking tyrosine-200 carried another mutation nearby, glutamate-198 to alanine (E198A). This mutation was not found in the sensitive isolate, nor in sequences from resistant isolates carrying the mutation F200Y. However, the mutation E198A is known from benomyl-resistant isolates of phytopathogenic fungi such as Monilinia fructicola. The finding that alanine-198 correlates with thiabendazole resistance in H. contortus isolates from South Africa and Australia suggests that also in nematodes, the mutation E198A plays a role in benzimidazole resistance. Alanine-198 alleles of beta-tubulin can be detected by PCR-RFLP and we suggest to include this test in future surveys of H. contortus field populations.     

Changes in resistance in mixed infections of susceptible and benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis passaged in sheep

Benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis were each diluted with equal numbers of their respective susceptible genotypes and passaged in separate worm-free sheep. The progeny of the mixed susceptible and resistant infections were diluted with equal numbers of susceptible genotypes before passaging on two further consecutive occasions in worm-free sheep. In H contortus the amount of thiabendazole required to prevent a 50 per cent egg hatching (LC50) was reduced at each generation, but the reduction was significant (P less than 0.05) only at the third passage. In T colubriformis the LC50 for thiabendazole was reduced only at the first passage and thereafter remained constant. The reduction was not significant. In another experiment, the dilution with susceptible genotypes was made only at the first passage. Thereafter for two further generations the progeny produced from the mixed infection were passaged in worm-free sheep. In H contortus a reduced amount of thiabendazole was required to prevent a 50 per cent egg hatching at each generation, but did not reach a level of significance. The result for T colubriformis was identical to the continuous dilution experiment.     

Prevalence of anthelmintic resistant nematodes in sheep in south-east England

The prevalence of anthelmintic resistant nematodes among 52 commercial flocks in south-east England was investigated by comparing the faecal egg counts of groups of lambs before, and seven days after, treatment with thiabendazole and levamisole. Evidence of thiabendazole resistance was found on seven farms. in each case Haemonchus contortus was the only species of nematode involved. In vitro egg hatch assays carried out for isolates from these farms gave ED50 estimates of 0.065 to 0.332 micrograms thiabendazole/ml compared with estimates of 0.027 to 0.031 micrograms thiabendazole/ml for a known susceptible strain of H contortus assayed at the same time. In a series of slaughter trials, there was a 17 to 85 per cent reduction, as compared with controls, in the mean worm burdens of groups of lambs infected with these isolates and killed seven days after treatment with thiabendazole, confirming their resistance to this anthelmintic.     

THE INCIDENCE OF THIABENDAZOLE RESISTANCE IN FIELD POPULATIONS OF HAEMONCHUS CONTORTUS ON THE NORTHERN TABLELANDS OF NEW SOUTH WALES

A survey was undertaken to assess the incidence of thiabendazole resistance in field populations of H. contortus in sheep flocks on 40 farms distributed throughout the sheep grazing area of the northern tablelands of New South Wales. Treatment with thiabendazole at 66 mg/kg reduced H. contortus ova output by 96-100% in 18 flocks while in the remaining 22 there was a range of responses down to levels where the anthelmintic was without apparent effect. Factors which may contribute to the high incidence of anthelmintic resistance are discussed and the possibility of widespread resistance occurring in other sheep raising areas is considered.     

Prevalence of benzimidazole resistance of nematodes in sheep in The Netherlands

A survey was carried out in The Netherlands to determine the prevalence of benzimidazole resistance in sheep nematodes. It was shown that 47 per cent of the 59 investigated flocks harboured trichostrongylid populations with resistance to benzimidazoles, as demonstrated by an LD50 value of 0.12 micrograms ml-1 thiabendazole or higher in an in vitro egg hatch assay. On these farms Haemonchus contortus was found to be the dominant species. In two flocks resistant Ostertagia and, or, Trichostrongylus species populations were found. Farms with resistance were revisited to determine the anthelmintic efficacy in vivo by means of treatment and egg count. In only four of the 19 flocks which showed resistance in vitro was an efficacy of less than 95 per cent in vivo achieved. This means that there is a discrepancy between the in vivo and in vitro results. Resistance was found more frequently on pure sheep farms than on mixed farms. It is suggested that mixed farms offer more refuge for susceptible H contortus populations.     

The dose response of several benzimidazole anthelmintics against resistant strains of Haemonchus contortus and Trichostrongylus colubriformis selected with thiabendazole

Dose response lines for eight benzimidazole anthelmintics and thiophanate were determined, using standardised strains of thiabendazole selected and resistant Haemonchus contortus and Trichostrongylus colubriformis. Against H contortus, thiophanate, thiabendazole, parbendazole and oxibendazole were inactive. Mebendazole was inactive at dose rates of 6.26 and 12.5 mg/kg, although significant activity occurred at 25 mg/kg. Fenbendazole, cambendazole, oxfendazole and albendazole demonstrated significant activity at dose rates equal to or greater than the recommended therapeutic level. Thiophanate was inactive against resistant T colubriformis. The remaining compounds only showed significant activity when used at dose rates in excess of the recommended therapeutic level. These results show that a side resistance exists among the benzimidazole anthelmintics and suggests that changes in dose response lines could be expected to occur if resistant strains are selected with benzimidazoles other than thiabendazole.     

Controlled tests on activity of contemporary parasiticides on natural infections of helminths in lambs, with emphasis on strains of Haemonchus contortus isolated in 1955.

Ten controlled tests were done between 1972 and 1989, in lambs on pasture, evaluating activity of fenbendazole (FBZ; 5 mg/kg of body weight), oxfendazole (OFZ; 3.5 and 10 mg/kg), oxibendazole (OBZ; 10 mg/kg), pyrantel pamoate (PRT; 25 mg of base/kg), and thiabendazole (TBZ; 44 and 50 mg/kg) against natural infections of helminths, with emphasis on 2 strains (A and B) of Haemonchus contortus. Strain A was phenothiazine-susceptible and strain B was phenothiazine-resistant when isolated in 1955. For approximately 10 years prior to these tests, sheep infected with both strains had been treated periodically each year with several compounds, including thiabendazole, which was used many more times than the other drugs. For this study, 4 (FBZ, OFZ, OBZ, and PRT) of the 5 compounds were evaluated in either 1 or 2 controlled tests. The fifth compound, TBZ, was used for 5 tests. Strain A H contortus was resistant to TBZ for all years tested, but more susceptible to FBZ, OFZ, OBZ, and PRT. Overall, strain B was susceptible to TBZ (with a few exceptions), and also to FBZ, OFZ, OBZ (activity less on immature forms), and PRT. Other abomasal parasites (2 species of Ostertagia and 3 of Trichostrongylus) were found in low numbers, but removal overall was good for the compounds tested. Trichostrongylus axei, found in higher numbers than species of Ostertagia and other species of Trichostrongylus, were effectively removed by all compounds in most cases. Activities of TBZ and PRT were also evaluated against several species of intestinal helminths, most of which were found in low numbers. Cooperia curticei were inconsistently removed by TBZ, but activity of PRT was effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE ANTHELMINTIC EFFICACY OF NON-BENZIMIDAZOLE ANTHELMINTICS AGAINST BENZIMIDAZOLE RESISTANT STRAINS OF HAEMONCHUS CONTORTUS AND TRICHOSTRONGYLUS COLUBRIFORMIS IN SHEEP

The anthelmintic efficacy of 6 non-benzimidazole compounds and thiabendazole against standardised, benzimidazole resistant strains of H. contortus and T. colubriformis in sheep was determined using a controlled test. All compounds were administered intraruminally at their recommended therapeutic dose rates and 4 of the compounds were also assessed at half their recommended therapeutic dose rates. Levimisole at a dose of 6.4 mg/kg body weight, morantel at 4.4 mg/kg and 8.8 mg/kg, naphthalophos at 6.25 mg/kg and 12.5 mg/kg, rafoxanide at 7.5 mg/kg and phenothiazine at 530 mg/kg were 98% or more effective in removing adult infections of benzimidazole resistant H. contortus. Rafoxanide at 3.75 mg/kg, phenothiazine at 265 mg/kg and carbon tetrachloride at 0.05ml/kg were less effective, removing 80%, 73% and 72% respectively of the benzimidazole resistant H. contortus worm burdens. Against the benzimidazole resistant T. colubriformis levamisole at 6.4 mg/kg, and morantel at 4.4 mg/kg and 8.8 mg/kg removed 99%, 68% and 86% respectively of the adult infections. All other anthelmintics had little or no efficacy at the dose rates tested. Thiabendazole at 44 mg/kg had no significant effect against these strains of H. contortus and T. colubriformis. Several non-benzimidazole anthelmintics have shown high efficacy in this experiment and should be suitable for treating infections with benzimidazole resistant H. contortus. Of the anthelmintics tested, only levamisole and morantel show high efficacy against benzimidazole resistant T. colubriformis.     

High-level ivermectin resistance in a field isolate of Haemonchus contortus associated with a low level of resistance in the larval stage: implications for resistance detection

The IVPro isolate of Haemonchus contortus was isolated in 1999 after significant numbers of the parasite survived an ivermectin capsule treatment of grazing sheep acquiring a natural infection at Prospect, NSW, Australia. The isolate shows high-level resistance to ivermectin (faecal egg count is unaffected by ivermectin oral treatment at 0.2mg kg(-1)). The larval LC(50), as assessed by larval development assays (LDAs), is only approximately two-fold higher than several susceptible isolates, making it difficult to detect the resistance using larval LC(50) as an indicator. However, the isolate shows the presence of a small proportion of the population (<20%) able to develop at significantly higher drug concentrations than the susceptible isolates. Hence, if the IVPro and susceptible isolates are compared at the LC(99) level, the IVPro isolate is readily identifiable as resistant. This degree of distinction at the LC(99) allows the IVPro isolate to be identified as resistant by simply observing the highest drug concentration in the development assay at which some larvae develop relative to the susceptible isolates. Assessing the development assay using this criterion allows a distinction between IVPro and the susceptible isolates equivalent to 10-fold differences in drug concentration, greatly increasing the likelihood of detecting the resistant isolate in routine resistance tests. This study highlights the need to examine this aspect of LDAs in order to detect the type of resistance displayed by IVPro H. contortus.     

Effect of cool storage of faecal samples containing Haemonchus contortus eggs on the results of an in vitro egg development assay to test anthelmintic resistance

An in vitro egg development assay was used to test the resistance of eggs from a benzimidazole susceptible and a resistant strain of Haemonchus contortus to four benzimidazoles. Storage of eggs contained in faecal samples at 4 degrees C for four, 24 or 72 hours did not appear to impair the results from the in vitro egg development assay or alter the LD50. Storage for one week, however, proved to be lethal for the eggs. The average LD50 values for thiabendazole, albendazole, mebendazole and cambendazole were 0.06, 0.07, 0.96 and 2.47 ppm for the susceptible strain. For the resistant strain the values averaged 0.26, 0.24, 1.07 and 5.11 ppm.     

Efficacy of moxidectin, nemadectin and ivermectin against an ivermectin-resistant strain of Haemonchus contortus in sheep.

The efficacies of ivermectin, nemadectin and moxidectin were evaluated when administered orally to lambs infected with either a susceptible laboratory strain of Haemonchus contortus or a strain reported to be resistant to ivermectin. Groups of 24 Dorset cross Cheviot cross Suffolk lambs were infected with either the susceptible or resistant strain of H contortus and allocated to treatment groups according to their faecal egg counts 27 days after infection. One day later the lambs were dosed orally with one of the three anthelmintics at 0.2 mg/kg bodyweight, and they were killed and surviving worms were recovered 13 or 14 days after treatment. Against the ivermectin resistant strain, ivermectin did not significantly reduce the egg count or the numbers of adult H contortus; however, both nemadectin and moxidectin reduced the nematode egg counts and the numbers of H contortus by 99 and 100 per cent, respectively. Against the susceptible strain, all the anthelmintics reduced the egg counts by 100 per cent as early as four days after treatment and reduced the numbers of susceptible H contortus by 100 per cent. No adverse reactions to any of the drugs were observed.     

The effect of route of administration on the anthelmintic efficacy of benzimidazole anthelmintics in sheep infected with strains of Haemonchus contortus and Trichostrongylus colubriformis resistant or susceptible to thiabendazole

Observations of erratic anthelmintic activity of fenbendazole against known standardised thiabendazole-resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep were investigated. Fenbendazole at a dose rate of 10 mg/kg body weight was administered by oral, intra-ruminal or intra-abomasal routes, and was most effective against both resistant strains following intra-ruminal administration. In addition thiabendazole, oxibendazole, fenbendazole, parbendazole and mebendazole plus two unrelated compounds, levamisole and morantel tartrate, were used at one and a half times their suggested or recommended therapeutic dose rate against thiabendazole-resistant strains of H contortus and T colubriformis in sheep; each drug being administered by the intra-ruminal or intra-abomasal routes. Fenbendazole was more effective against both strains following intra-ruminal administration. Parbendazole was more effective against the resistant strain of T colubriformis following intra-ruminal administration. At the dose rate chosen for the other benzimidazoles used against these resistant strains, there was no difference in anthelmintic efficacy due to route of administration. Levamisole was highly effective against both resistant strains, irrespective of the route of administration. In the groups treated with morantel tartrate, the results obtained were difficult to interpret due to mortalities and a highly variable response in the surviving sheep. Fenbendazole, thiabendazole and mebendazole when used at their suggested or recommended therapeutic dose rate in sheep, were highly effective against known thiabendazole-susceptible strains of H contortus and T colubriformis following both intra-ruminal or intra-abomasal administration.     

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.     

Further studies on anthelmintic resistance in sheep at an organised farm in arid region of Rajasthan

Faecal egg count reduction test (FECRT) and in vitro egg hatch assay (EHA) was performed on Indian Karakul (3/4 crosses of Karakul with Malpura, Marwari and Sonadi) transported from arid region campus, Bikaner, where resistance was reported in 1996. FECRT revealed that fenbendazole and levamisole were 100% effective in reducing the egg counts. LC50 value on EHA was 0.074 +/- 0.015 microg thiabendazole ml(-1). The faecal culture examination revealed the presence of Haemonchus contortus only. It was concluded that H. contortus was fully susceptible to both benzimidazole and levamisole.     

Decreased ivermectin and moxidectin sensitivity in Haemonchus contortus selected with moxidectin over 14 generations.

Ivermectin resistance in the nematode Haemonchus contortus has been reported in many parts of the world and many ivermectin resistant isolates have been found to have reduced sensitivity to moxidectin. However, it is unclear whether parasites that are selected with moxidectin would demonstrate reduced sensitivity to ivermectin. In this study, the effects of moxidectin and ivermectin on an unselected strain and a strain of H. contortus derived from the unselected strain but selected over 14 generations with moxidectin, were compared in jirds. The recovery of adult worms and fourth stage (L4) larvae following treatment were compared between strains and anthelmintics. Moxidectin-selected H. contortus showed reduced sensitivity to ivermectin as well as to moxidectin. Doses of 0.1 mg/kg of moxidectin and 0.4 mg/kg of ivermectin were necessary to obtain an efficacy of 95% or above against the moxidectin-selected strain of H. contortus compared with 0.025 mg/kg for moxidectin and 0.1 mg/kg for ivermectin required for a similar efficacy in the unselected strain.     

Selection for resistance to macrocyclic lactones by Haemonchus contortus in sheep.

An anthelmintic-sensitive Haemonchus contortus strain was selected for moxidectin and ivermectin resistance concurrently for 22 generations. Treatment with 0.002 mg moxidectin/kg BW or 0.02 mg ivermectin/kg BW produced >99% efficacy against the susceptible parent strain passaged for 22 generations without any anthelmintic exposure. However, to obtain similar efficacy the moxidectin-selected and the ivermectin-selected strains of H. contortus required 0.05 mg moxidectin/kg BW or 0.4 mg ivermectin/kg BW. These results indicate that development of resistance to one macrocyclic lactone, simultaneously results in resistance to another macrocyclic lactone. However, rates of resistance development differ between compounds and occurs more slowly with moxidectin than with ivermectin.     

Critical controlled test to evaluate resistance of field strains of Haemonchus contortus to thiophanate

Following reports of suspected gastrointestinal strongylid nematode resistance to thiophanate in sheep and goats at Sokoine University of Agriculture (SUA), Tanzania, field strains of Haemonchus contortus, one from SUA and another from Kimamba, Tanzania, were selected for a critical controlled test to evaluate the efficacy of thiophanate. The SUA H. contortus strain in goats showed a marked resistance to thiophanate at an oral dose of 50 mg kg-1 liveweight (efficacy = 24.1%), while the Kimamba H. contortus strain showed very marked susceptibility (efficacy = 99.7%) to thiophanate at the same oral dose. This is the first report from East Africa of a field strain of H. contortus resistant to thiophanate. The incidence of resistant strains of strongyloid parasites to benzimidazole anthelmintics in sheep and goats in East Africa is on the increase. The magnitude of this problem should be evaluated and remedial solutions sought.     

EFFECTIVENESS OF BROAD SPECTRUM ANTHELMINTICS AGAINST SELECTED STRAINS OF TRICHOSTRONGYLUS COLUBRIFORMIS

SUMMARY A field population of Trichostrongylus colubriformis was divided into 4 lines for exposure to selection in the laboratory. The first line was selected with 50 mg/kg thiabendazole, the second with 4 mg/kg morantel tartrate, the third with 50 mg/kg thiabendazole followed by 4 mg/kg morantel tartrate and the fourth line was not selected for drug resistance. Following at least 9 generations of selection there was no difference in LD₅₀ or LD₉₅ between the unselected and single selected strains of worms. The strain selected by⁵⁰both thiabendazole and morantel tartrate had a significantly higher LD₅₀ against thiabendazole, morantel tartrate and levamisole than did the other three strains. The single selected strains had LD₉₅'s of 172, 21.5 and 2.3 mg/kg for thiabendazole, morantel tartrate and levamisole respectively, compared with corresponding values of 111, 17.3 and 2.4 in the unselected strain and 124, 15.5 and 3.0 in the double selected strain. The estimated efficiency of the recommended dose of each anthelmintic against the unselected field strain was:—thiabendazole (44 mg/kg) 50% efficient, morantel tartrate (8.8 mg/kg) 76% efficient and levamisole (7.0 mg/kg) 99.9% efficient.