Detecting in vitro anthelmintic effects with a micromotility meter

An in vitro target parasite anthelmintic assay utilizing a micromotility meter has been developed and validated. Haemonchus contortus, an economically important ruminant helminth with worldwide distribution, was the parasite used in the model. Four commercially available ruminant anthelmintics (albendazole, ivermectin, levamisole hydrochloride and coumaphos) were initially evaluated at concentrations of 200, 150, 100 and 50 micrograms ml-1. All four significantly affected helminth motor activity and were active at 200 and 150 micrograms ml-1, and three of the four were active at 100 and 50 micrograms ml-1. An Upjohn compound (p-toluoyl chloride phenylhydrazone) was also assayed and was significantly active at all four levels. In a subsequent titration study, albendazole, levamisole hydrochloride, ivermectin and the hydrazone were significantly active at 100 and 10 micrograms ml-1; only levamisole hydrochloride and the hydrazone were active at 1.0 microgram ml-1. None of the drugs were active at 0.1 microgram ml-1. The data indicate that the in vitro H. contortus assay utilizing the micromotility meter is sensitive, accurate, rapid, repeatable, and inexpensive. With additional effort, this model can be extended to incorporate other target helminth parasites and stages of development. This in vitro assay system should be a valuable addition to the battery of tests used to identify anthelmintic candidates, monitor drug resistance, and define the kinetics and mode of action of drugs.     

THE ISOLATION OF A FIELD STRAIN OF HAEMONCHUS CONTORTUS IN QUEENSLAND SHOWING MULTIPLE ANTHELMINTIC RESISTANCE

SUMMARY Following the apparent failure of levamisole to control infections of Haemonchus contortus in sheep at Lawes in south eastern Queensland, a strain of this parasite was isolated at the Animal Research Institute, Yeerongpilly. This strain was used to infect sheep at Yeerongpilly and the Merrindale Research Station, Victoria where four experiments to classify the resistance pattern of the parasite were carried out. Resistance to thiabendazole was first suspected in 1969, and these experiments confirmed that resistance to this drug was still present. They also showed that a strong degree of resistance had been developed to both levamisole and morantel tartrate. Other benzimidazole anthelmintics and also the organophosphorus compound naphthalophos were only moderately effective against the original isolate but rafoxanide, nitroxynil and phenothiazine were almost 100% effective. Other highly effective chemicals were disophenol and closantel. After passaging the strain for four generations with both levamisole and albendazole, resistance to both naphthalophos and the newer benzimidazole anthelmintics increased dramatically. This is the first report of a field strain of H. contortus exhibiting resistance to benzimidazole, non-benzimidazole and organophosphorus anthelmintics.     

Multiple Anthelmintic Resistance in Haemonchus Contortus on a Sheep Farm in Kenya

Multiple resistance to albendazole, thiophanate, levamisole and orally administered ivermectin was detected in an isolate of Haemonchus contortus in sheep on a farm where benzimidazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the benzimidazole-resistant isolate were treated with the recommended doses of various anthelmintics. Compared to the control group, the percentage reductions in the faecal egg counts of sheep treated with albendazole, thiophanate, levamisole and ivermectin varied between 38.2% and 79.1% and the residual worm counts between 27.3% and 57.5%. The results indicate the presence of multiple anthelmintic resistance in this isolate of H. contortus. Sheep treated with closantel showed 100% reductions in faecal egg and worm counts, indicating that this drug was very effective against the population of H. contortus on the farm.     

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.     

Anthelmintic resistance in Australian sheep nematode populations

SUMMARY The resistance status of gastro–intestinal nematodes to anthelmintics was evaluated on 881 sheep farms throughout Australia during 1991–92. Resistance was shown to be widespread. Overall, 85% of farms had sheep infected with nematodes resistant to benzimidazole, 65% to levamisole and 34% to combination (benzimidazole + levamisole) products. Resistance to ivermectin was not detcted. On only 9% of farms did all anthelmintic groups reduce egg counts by greater than or equal to 95%. The culture of faeces from untreated sheep showed Telodorsagia circumcincta, Trichostrongylus spp, Chabertia ovina and Haemonchus contortus to be the principal species. The nature and prevalence of resistance was not significantly correlated with stocking rate. However, resistance to combination products was almost twice as prevalent on farms in areas with an average annual rainfall of greater than 500 mm.     

Anthelmintic resistance in sheep and goat farms on Peninsular Malaysia.

The faecal egg count reduction test (FECRT) was conducted on 39 sheep farms and 9 goat farms located in Peninsular Malaysia. The anthelmintic groups used in these tests were the benzimidazoles, levamisole, the benzimidazole/levamisole combination, macrocyclic lactones and closantel. Results indicated that the prevalence of resistance to the benzimidazole group was high, with approximately 50% of the sheep farms and 75% of the goat farms having resistant nematode parasite populations present. Resistance to levamisole, closantel and ivermectin was also detected. Differentiation of the infective larvae derived from faecal cultures indicated that by far the most predominant parasite species was Haemonchus contortus.     

Anthelmintic resistance in nematode parasites of goats

Cases of anthelmintic resistance on 3 goat farms in Gippsland were investigated. On the first farm Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia (Teladorsagia) circumcincta were found to be resistant to fenbendazole, but the first 2 species were fully susceptible to levamisole. On the second farm a population of T. colubriformis, resistant to concurrent full doses of levamisole and a benzimidazole, was found to retain this resistance when transferred to sheep. On the third farm, heavy mortality due to Ostertagia and Trichostrongylus spp burdens was arrested only by the use of concurrent full doses of levamisole and oxfendazole; no single anthelmintic was found to be effective. A disturbing rise in the incidence of clinical helminthiosis, often accompanied by anthelmintic resistance, occurred on goat farms in Gippsland early in 1984.     

Efficacy of repeated doses of levamisole, morantel, fenbendazole, and ivermectin against gastrointestinal nematodes in ewes

Levamisole, morantel, fenbendazole, or ivermectin was administered at 2-week intervals from May 1 through Sept 14, 1985, to breeding ewes (20 ewes/drug) infected with various gastrointestinal nematodes. All ewes had fewer gastrointestinal nematode eggs per gram of feces (epg) after 2 treatments, compared with pretreatment epg counts. Ewes administered ivermectin continued to have a low mean epg (0 to 3) throughout the study. The mean epg counts of ewes treated with levamisole increased from 3 to 483 during the study. This increase was similar to that of ewes treated with morantel (7 to 485 epg). The mean epg count of fenbendazole-treated ewes increased from 4 to 192 during the study. By the end of the study, the mean epg counts when expressed as a percentage of the pretreatment epg counts were 4% (ivermectin), 249% (fenbendazole), 627% (levamisole), and 630% (morantel). With the exception of the ivermectin-treated ewes, the epg count increased almost linearly in the ewes after the 2nd anthelmintic treatment. These data indicate that the gastrointestinal nematodes (including Haemonchus contortus) may have developed more resistance to levamisole and morantel than to fenbendazole. On the basis of the epg counts, resistance to ivermectin did not develop during the 4.5-month treatment period. The percentage of ewes shedding eggs after 2, 4, and 6 anthelmintic treatments was lowest for ewes treated with ivermectin (20%) and was similar (40%) for ewes treated with 1 of the other 3 anthelmintics. At the conclusion of the study, most of the ewes (90%) were shedding at least a small number of eggs, regardless of the anthelmintic treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Multiple anthelmintic resistance in a goat herd

Anthelmintic resistance was monitored over a 30 month period within a goat herd in eastern Virginia, USA. Resistance to ivermectin, levamisole and benzimidazole drugs was detected in Haemonchus contortus using the fecal egg count reduction test (FECRT). When levamisole use was discontinued for 1 year, susceptibility to levamisole appeared to return. Although a single treatment with fenbendazole was able to reduce fecal egg counts by only 50%, two doses administered in a 12 h interval increased efficacy to 92%, however, confidence intervals indicated that resistance was still present. When fecal egg counts were determined the following year after several treatment using this protocol, the efficacy of fenbendazole had fallen again to 57% reduction in fecal egg counts. The predominant genus present in cultured composite fecal samples was Haemonchus. Trichostrongylus, Cooperia and Teladorsagia were also present in smaller numbers.     

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.     

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.     

Efficacy of monepantel and anthelmintic combinations against multiple-resistant Haemonchus contortus in sheep, including characterisation of the nematode isolate

Three experiments defined the resistance profile of a population of Haemonchus contortus, which was shown to express multiple resistances to the benzimidazole, levamisole, macrocyclic lactone and salicylanilide anthelmintic classes when given as a registered combination. Study 1 was a faecal egg count reduction (FECR) test and the efficacies for the anthelmintics were monepantel, 100%; abamectin+levamisole+oxfendazole, 40.0%; and abamectin+levamisole+oxfendazole+naphthalophos, 100%. No larvae were recovered from the post-treatment cultures for monepantel or the 4-way treatment, and for the 3-way treatment the culture was 100% Haemonchus spp. Efficacies in Study 2 were calculated from mean post-mortem nematode burdens of H. contortus and were levamisole+oxfendazole, 3.1%; abamectin+levamisole+oxfendazole, 5.0%; ivermectin, 0.4%; moxidectin, 28.4% and closantel, 70.2%. Study 3 was also a FECR test that resulted in efficacies of 100% for monepantel and 83.0% for a formulated 4-way combination of abamectin+levamisole+albendazole+closantel. Larvae recovered from the post-treatment culture for the combination-treated sheep were all Haemonchus spp. Multi-resistant parasites such as examined in these studies are a continuing challenge to be managed by farmers and their advisors. Control programs must be planned and well-managed, and should include on-farm testing for anthelmintic resistance, monitoring of nematode burdens (by FEC and larval culture) to determine appropriate treatment times and the management of pastures to reduce the overall parasite challenge. This should be in balance with the generation, use and maintenance of drug-susceptible nematode populations in refugia.     

Larval development test for detection of anthelmintic resistant nematodes

The growth, using freshly cultured Escherichia coli with ampicillin or heat-treated lyophilised E coli as a food source, of the larvae of the mouse nematode Nematospiroides dubius and the infectivity of resulting third stage larvae were determined. Concentrations of E coli between 0.5 and 1 mg dry weight ml-1 permitted optimal larval development for both N dubius and Trichostrongylus colubriformis. Development of larvae of susceptible and cambendazole-resistant strains of Haemonchus contortus in thiabendazole solutions showed clear differences between the strains and the larval development test was more sensitive than the egg hatch test. The test also detected a levamisole resistant strain of H contortus, although the degree of resistance could not be adequately measured. It is concluded that the test can be run with any anthelmintic to which resistance is suspected.     

Anthelmintic resistance in the field: Changes in resistance status of parasitic populations in response to anthelmintic treatment

Changes in anthelmintic resistance in nematode parasites were monitored in sheep grazing on 2 separate farms, but with the same anthelmintic treatment program, over 16 years. High levels of benzimidazole resistance emerged in Ostertagia and Trichostrongylus spp populations on both farms following 9 years of continuous use of this class of drug. Subsequently, variations in the levels of resistance occurred for the same species between farms and between species on the same farm. A change to levamisole for 2 years resulted in a significant reversion towards benzimidazole susceptibility, but a concomitant rise in levamisole resistance, in Ostertagia on one farm. However, benzimidazole resistance increased rapidly following the re-introduction of oxfendazole into the anthelmintic treatment program. Results from both farms illustrate the pitfalls of using one anthelmintic class for an extended period and provide indirect support for the alternation of anthelmintic classes at approximately yearly intervals.     

Effectiveness of strategic anthelmintic dosing in controlling Haemonchus contortus infections in sheep in the United Kingdom

The strategic use of anthelmintics in the control of infections of Haemonchus contortus in ewes and lambs, was investigated in a series of paddock trials. The levels of infection and clinical signs associated with the presence of either benzimidazole-resistant or benzimidazole-susceptible strains of H contortus in lambs were controlled by regular drenching with levamisole or mebendazole, respectively, or by the strategic use of closantel in combination with mebendazole. In the latter case, control was achieved by dosing either the ewes in the early part of the grazing season, or the lambs from June onwards. It was concluded that worm control strategies based on closantel could provide effective control of both benzimidazole-susceptible and benzimidazole-resistant strains of H contortus on farms in the United Kingdom.     

Multiple species of nematodes resistant to ivermectin and a benzimidazole-levamisole combination on a sheep farm in New Zealand

AIM: To confirm the presence of multiple anthelmintic resistance on a sheep farm in New Zealand. METHODS: Three groups of 10 weaned Romney-cross lambs were treated either with an oral dose of ivermectin (0.2 mg/kg), or a benzimidazole/levamisole (BZ/LEV) combination (4.75 albendazole and 7.5 mg/kg levamisole), or were left untreated. Ten days later, animals were necropsied, and adult worms recovered and identified from the abomasa and small intestines. Pre- and post-treatment faecal nematode egg counts (FEC) were recorded, and larval cultures were performed. RESULTS: In a faecal egg count reduction test (FECRT), adjusted to reflect pre- and post-treatment larval culture results, ivermectin resistance was detected in Teladorsagia (Ostertagia), Trichostrongylus and Haemonchus spp, while BZ/LEV combination- resistant Teladorsagia and Trichostrongylus spp were also present. Adult worm counts confirmed these results, and identified the species involved as Teladorsagia circumcincta, Trichostrongylus colubriformis and H. contortus. CONCLUSION: Multiple, multi-generic anthelmintic resistance was confirmed on a sheep property in New Zealand. This included the first confirmed case of ivermectin resistance in T. colubriformis from sheep in New Zealand.     

The efficacy of netobimin against a benzimidazole susceptible and a resistant strain of Haemonchus contortus in sheep in The Netherlands

Netobimin was tested for efficacy against Haemonchus contortus using 7 groups of 5 parasite-free lambs of six months age. The lambs in group 1 and 2 were infected with 10,000 larvae of a benzimidazole susceptible strain and those in groups 3-7 with the same dose of a resistant strain. The following treatment scheme was applied 21 days after infection: lambs in groups 2 and 4-7.5 mg kg-1 netobimin, in group 5-20 mg kg-1 netobimin, in group 6-5 mg kg-1 oxfendazole and in group 7--3.8 mg kg-1 albendazole. The lambs in groups 1 and 3 remained untreated. All lambs were slaughtered 28 days after infection. Egg counts decreased in all lambs after treatment, but increased again in lambs in groups 4, 6 and 7. There was a slight increase in lambs in group 5, while those in group 2 remained negative. Post-mortem worm counts showed a reduction of 99.8 per cent in lambs in group 2 compared to those in group 1. In lambs in group 4-7 the reduction of worm counts was respectively 40.9, 89.5, 24.7 and 40.7 per cent compared to those in group 3. Egg development assays carried out 20 days after infection showed an average LD50 of 0.46 mg ml-1 thiabendazole for the resistant strain. After treatment (day 27) the LD50 was 0.53, 0.48, 0.58, 0.56 and 0.47 in lambs in the groups 3-7. It is concluded that netobimin and other (pro)-benzimidazoles should not be used in cases of benzimidazole resistance and that levamisole, pyrantel tartrate or ivermectin are preferable.     

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.