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 detection of anthelmintic resistance in nematodes of veterinary importance

Before revised World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines on the detection of anthelmintic resistance can be produced, validation of modified and new methods is required in laboratories in different parts of the world. There is a great need for improved methods of detection of anthelmintic resistance particularly for the detection of macrocyclic lactone resistance and for the detection of resistant nematodes in cattle. Therefore, revised and new methods are provided here for the detection of anthelmintic resistance in nematodes of ruminants, horses and pigs as a basis for discussion and with the purpose that they are evaluated internationally to establish whether they could in the future be recommended by the WAAVP. The interpretation of the faecal egg count reduction test has been modified and suggestions given on its use with persistent anthelmintics and continuous release devices. An egg hatch test for benzimidazole (BZ) resistance is described. A microagar larval development test for the detection of benzimidazole and levamisole resistance provides third stage larvae for the identification of resistant worms. The sensitivity of these two tests can be increased by using discriminating doses rather than LD(50) values. Details are given of a PCR based test for the analysis of benzimidazole resistance in strongyles of sheep and goats, horses and cattle. Although promising for ruminant trichostrongyles, quantitative determination of gene frequency using real time PCR requires further development before PCR tests will be used in the field. Apart from faecal egg count reduction tests there are currently no satisfactory tests for macrocylic lactone resistance despite the great importance of this subject. Except for treatment and slaughter trials there are no validated tests for fasciolicide resistance or for the detection of resistance in cestodes.     

Benzimidazole resistance in Trichostrongylus axei in sheep: long-term monitoring of affected sheep and genotypic evaluation of the parasite

This is the first report of benzimidazole (BZ) resistance in the nematode Trichostrongylus axei in sheep. Trichostrongylus axei infects several species of herbivores including sheep, cattle and horses, and the emergence of anthelmintic resistance could lead to significant problems in its control. Benzimidazole resistance in two sheep flocks in central France was detected by post-treatment worm counts. The sequencing of a central region of the isotype 1 beta-tubulin gene from adult T. axei recovered post-mortem revealed only one, non-synonymous single nucleotide polymorphism at position 200 (Phe200Tyr), which had already been reported for other nematodes. Seven years after BZ treatment ceased, T. axei helminths present were still resistant to BZ suggesting these parasites do not revert to susceptibility to this anthelmintic, even when the selection pressure had been removed for many years. The findings also highlight major changes in the make-up of the nematode burden in sheep flocks that accompanies the emergence of BZ resistance.     

Anthelmintic resistance--looking to the future: a UK perspective

The full extent of anthelmintic resistance in nematodes of farm animals is not known. Resistance can be detected with a faecal egg count reduction test and two in vitro tests, the egg hatch and larval development tests. The sensitivity of these two in vitro tests can be increased by using discriminating doses rather than calculating LD50 values. Only benzimidazole resistance can be detected with PCR based tests because the molecular mechanisms of resistance to levamisole and the macrocyclic lactones remain unknown. Resistance detection is important because it enables the appropriate management strategies to be put in place. The development of resistance is delayed by keeping sufficient parasites in refugia (not exposed to anthelmintic), but the necessary management details have not yet been validated in the field. It is probably too late to use combination products to delay the development of resistance, except in cattle but quarantining animals to prevent introduction of resistant helminths onto a farm is important. Dilution of resistant worms with susceptible ones is only at the preliminary research stage and the application of non-chemical methods of control to delay resistance is not yet a practical option. Extensive research is required to manage resistance, especially in the control of resistance in Fasciola hepatica.     

Use of two in vitro methods for the detection of anthelmintic resistant nematode parasites on Slovak sheep farms

A survey of the prevalence of anthelmintic resistant nematode populations was conducted on 32 sheep farms in the Slovak Republic. In vitro egg hatch test and larval development tests were used for the detection of resistance to benzimidazole anthelmintics and they were compared with in vivo faecal egg count reduction tests. There was agreement in the declaration of resistance between the faecal egg count reduction test and both in vitro tests. The presence of resistant populations was determined on two farms using egg hatch test. In both farms, the LD(50) values were higher than 0.1 microg TBZ/ml, indicating resistance. By using LD(99) values it might be possible to reveal relatively small proportion of resistant larvae in the population. The prevalence of benzimidazole resistance has not change on Slovak sheep farms during last decade.     

Anthelmintic resistance in nematode parasites of sheep in Denmark with special emphasis on levamisole resistance in Ostertagia circumcincta.

This study was undertaken to elucidate the presence of anthelmintic resistance in nematode parasites of sheep in Denmark. Twenty two flocks of sheep were selected for Faecal Egg Count Reduction (FECR) tests, based on a prior history of either the same anthelmintic, or anthelmintic class having been used 3 times or more over the previous 5 years. Evidence of anthelmintic resistance was detected in 7 flocks. Two flocks showed FECR of 88% and 94% after treatment with thiabendazole, FECR of another 2 were 90% and 94% following treatment with fenbendazole. Three flocks showed FECR of 73%, 89% and 94%, respectively following the use of levamisole. Ostertagia circumcincta was isolated from 1 of the latter flocks and subjected to an in vivo controlled slaughter assay. Following treatment with levamisole at the recommended dose rate of 7.5 mg/kg, FECR was 44.5% and worm counts were reduced by 67.7%. These results were further substantiated by an in vitro egg hatch paralysis assay and by measuring pepsinogen levels in treated and non-treated lambs. This is the first instance of anthelmintic resistance in sheep nematodes in Scandinavia.     

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.     

Observations on the emergence of multiple anthelmintic resistance in sheep flocks in the south-east of Scotland

Multiple resistance to benzimidazole, imidazothiazole and macrocyclic lactone anthelmintics is an emerging problem in the south-east of Scotland. The general management and nematode control strategies employed in four affected flocks (flocks 1-4) were investigated in an attempt to identify the risk factors which might have led to the appearance of production limiting disease associated with anthelmintic resistance. The important risk factors for multiple anthelmintic resistance could not be confirmed and it proved easier to criticise nematode control practices on theoretical grounds, than to propose practical solutions. It seems likely that different risk factors were involved in the four flocks. Lambs in flocks 1 and 2 had been treated with an anthelmintic at 3-4 weekly intervals with the aim of achieving suppressive nematode control, while sheep in flock 1 had been treated with an anthelmintic after they were moved onto clean grazing. Recently lambed ewes had been treated with moxidectin in three of the four flocks, with the aim of controlling their periparturient rise in faecal nematode egg output. All of these factors might have contributed to the emergence of multiple anthelmintic resistance, because they could have led to anthelmintic treatments at times when the nematode population in refugia was small, compared to that in the sheep. Annual rotation of the anthelmintic group was compromised by the emergence of benzimidazole resistance and did not prevent the emergence of multiple resistance in any of the flocks described, although the practice may have slowed the development of resistance. Underdosing may have selected for benzimidazole and imidazothiazole resistance in flock 2, associated with inaccurate estimation of the weights of terminal sire lambs. These investigations also highlighted problems associated with the diagnosis of anthelmintic resistance, in particular the confounding effects of the onset of host immunity to nematode parasites, the possible influence of the age of the adult nematode population, and the insensitivity of the undifferentiated faecal egg count reduction test in situations where resistance is emerging.     

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance.

Methods have been described to assist in the detection of anthelmintic resistance in strongylid nematodes of ruminants, horses and pigs. Two tests are recommended, an in vivo test, the faecal egg count reduction test for use in infected animals, and an in vitro test, the egg hatch test for detection of benzimidazole resistance in nematodes that hatch shortly after embryonation. Anaerobic storage for submission of faecal samples from the field for use in the in vitro test is of value and the procedure is described. The tests should enable comparable data to be obtained in surveys in all parts of the world.     

Economic evaluation of three anthelmintic strategies for lamb flocks affected by benzimidazole-resistant nematodes

OBJECTIVE: To compare the profitability of three anthelmintic strategies in growing lambs in flocks with nematodes resistant to benzimidazole anthelmintics. METHOD: A partial-budgeting analysis was carried out by means of a stochastic simulation model, which allows inputs to be described as distributions rather than as fixed values, and hence permits variation between farms to be considered in the analysis. RESULTS: The results show that control of nematode parasites by use of an effective anthelmintic provides the highest net returns, yielding a margin over ineffectively treated lambs of A$114 per 100 lambs on average. Suppressive treatment based on the administration of two controlled-release capsules and monthly with moxidectin resulted in an average loss of A$131 per 100 lambs in comparison with animals treated with an ineffective anthelmintic. Analysis of the results from capsule-treated lambs did not take into account the unmeasured benefits associated with less contamination of pastures. Sensitivity analysis using a stochastic model indicates that apart from the effect of treatment on weight gain variation in carcase price greatly influences the profitability of all the parasite control programs examined. CONCLUSION: The results suggest that it is economically important for farmers to adjust their strategy in the presence of anthelmintic resistance. But as a result of uncertainty in the factors influencing economic return, the expected economic benefit is likely to vary substantially.     

Detecting benzimidazole resistance with faecal egg count reduction tests and in vitro assays

Composite strains of Trichostrongylus colubriformis and Ostertagia spp consisting of 0, 1, 10, 25, 50, 75, 90, and 100% of known resistant strains were prepared and tested for benzimidazole resistance using faecal egg count reduction tests, in vitro egg hatch assays and tubulin binding assays. All tests detected resistance where the proportion of the resistant strain in the composite was 50% or more, whereas none of the tests unequivocally detected resistance below 25%. Egg count reduction tests were no less sensitive than the in vitro tests in detecting low levels of resistance but the egg hatch and tubulin binding assays provided a better quantitative estimate of moderate to high levels of resistance. Faecal egg count reduction therefore, provides a suitable means of detecting resistance in the field but tests, more sensitive to low levels of resistance are required. Results indicate that the use of post-treatment counts alone provides an adequate indication of anthelmintic efficiency.     

Multiple anthelmintic resistance and the possible contributory factors in Beetal goats in an irrigated area (Pakistan)

This paper presents the first report of multiple anthelmintic resistance in the gastrointestinal nematodes of goats and its possible contributory factors in an irrigated area (Pakistan). A total of 18 privately owned Beetal goat flocks were selected in order to determine the anthelmintic resistance against commonly used anthelmintics. Forty to 48 animals from each flock were selected according to their weight and egg count. The three anthelmintics viz., oxfendazole, levamisole and ivermectin, were given to three groups at manufacturer’s recommended dose while one group was kept as untreated control. Anthelmintic resistance was determined through faecal egg count reduction and egg hatch tests while assessment of the contributory factors of anthelmintic resistance was measured through the rural participatory approach. Faecal egg count reduction test revealed high prevalence of anthelmintic resistance (83.3%) and it was either single (levamisole) or multiple (oxfendazole and levamisole). Egg hatch test confirmed the resistance against oxfendazole as detected with faecal egg count reduction test. None of the goat flocks was resistant to ivermectin. Copro-cultures revealed that Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta were the most common species exhibiting resistance to levamisole and oxfendazole. Step-wise logistic regression of the data on worm control practices revealed significant role of under-dosing, low-protein diets, healthcare supervision by the traditional healers and mass treatments.     

Cattle nematodes resistant to anthelmintics: why so few cases?

The apparent lack of anthelmintic resistant nematodes in cattle is due to the management systems used with most cattle and the lack of surveys for resistance. With extensive beef grazing or with beef suckler herds a large percentage of worms are in refugia (not exposed to anthelmintic) and few anthelmintic treatments are used. With dairy replacement heifers resistance can become a problem unless only first year animals are treated and different fields are used for grazing the animals each year. Intensive beefproduction on grass is unsustainable and in New Zealand resistance is already becoming a serious problem. Individual farms require monitoring for resistance and steps taken to avoid the introduction of resistance. New validated tests and surveys for resistance are required as the extent of anthelmintic resistant bovine nematodes is not known.     

Teladorsagia circumcincta resistance to moxidectin and multiple anthelmintic groups in ewes following use of the persistent drug before lambing

This paper describes the nematode control strategy adopted by a Scottish hill sheep farmer. It provides an example of the limited use of anthelmintic drugs, targeted towards control of the periparturient rise in faecal nematode egg output in ewes and exploitation of grazing management, resulting in limited exposure of naive sheep to infective larvae on pasture. Resistance to benzimidazole, imidazothiazole and macrocyclic lactone anthelmintics, and to a combination of a macrocyclic lactone and imidazothiazole anthelmintic drugs was diagnosed. The targeted use of a persistent anthelmintic drug to control a periparturient rise in faecal nematode egg output in ewes may be unnecessary and selects strongly for resistance when the reservoir of anthelmintic-susceptible nematodes in refugia is small at the time of treatment. However, the use of a persistent anthelmintic drug in a selective proportion of ewes can be important and probably does not select strongly for resistance when the reservoir of anthelmintic-susceptible nematodes in refugia is large at the time of treatment. The former circumstances might arise on many Scottish hill sheep farms, whereas the latter may occur on upland and lowground farms, depending on previous grazing management, anthelmintic use and winter weather conditions. These factors must be taken into account when preparing sustainable health plans for nematode parasite control in individual sheep flocks.     

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.     

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.     

The present status of anthelmintic resistance in gastrointestinal nematode infections of sheep in the northwest of Spain by in vivo and in vitro techniques

The aim of this study was to update the anthelmintic resistance (AR) status in sheep flocks infected by gastrointestinal nematodes (GIN) by means of in vivo and in vitro methods in the northwest of Spain. With this objective, we studied the efficacy of benzimidazoles (BZs), imidazothiazoles (IMs) and macrocyclic lactones (MLs), between 2006 and 2011. The sampling area was the Autonomous Community of Castilla y León but the majority of the flocks were located in the province of León. When the mean of GIN eggs per gram (epg) in faeces in a flock was higher than 150, the in vivo Faecal Egg Count Reduction Test (FECRT) was carried out. According to this test, AR was present in 63.6% of flocks, independently of the anthelmintic used. Flocks were mainly resistant to levamisole (LEV) (59.0%), followed by ivermectin (IVM) (27.3%) and albendazole (13.6%). Multidrug-resistance was also observed in 27.2% of the flocks, one of them being resistant to all anthelmintic families, including long-acting moxidectin. Comparing the evolution of AR in the last decade, between 1999 and 2011, the level of resistance to BZs and MLs was fairly constant throughout the time by means of the FECRT. However, the resistance to LEV increased significantly in only one decade since during the period 1999–2003 the percentage was 38.5%. The AR status was also measured by in vitro techniques in those flocks with an egg output lower than 150 epg. The prevalence of AR to BZs reached the 35.3% by Egg Hatch Assay. However, the level of resistance reported for LEV and IVM was 61.5% and 23.5%, respectively, by using the Larval Feeding Inhibition Assay, percentages very similar to those reported with the FECRT.     

Slowing the spread of anthelmintic resistant nematodes of sheep and goats in the United Kingdom.

Anthelmintic resistant nematodes pose an increasing threat to animal welfare and lamb production on lowland sheep farms in the United Kingdom. Based on published information on anthelmintic resistance in nematodes and insecticide resistance in arthropods, seven recommendations are made for reducing the development and spread of anthelmintic resistant nematodes. The two most important are probably to prevent the introduction of resistant worms through the purchase of stock and to reduce the reliance on frequent anthelmintic treatments by using epidemiological principles of nematode control.     

RESISTANCE TO BENZIMIDAZOLE ANTHELMINTICS IN EQUINE STRONGYLES

A survey was conducted to determine whether benzimidazole resistant populations of equine strongyles are present in New South Wales and north central Victoria; what is their frequency and geographical distribution; which species are involved; and whether different methods of parasite control could be related to the occurrence and frequency of anthelmintic resistant populations. Resistant populations of strongyles were found over wide areas of New South Wales and in north central Victoria. There was no relationship between geographical location and the occurrence of benzimidazole resistance. The species involved were small strongyles of the sub-family Cyathostominae. There was a direct correlation between the occurrence of resistance (including the level at which it is present) and the frequency of use of benzimidazole anthelmintics. Examination of management practices showed that resistance is not an important problem on farms where different chemical classes of anthelminitcs were used in a slow rotation programme; combination anthelmintic therapy (for example, benzimidazole/piperazine/organophosphates) was used and anthelmintic treatment was given at intervals of not less than 16 weeks. Tentative suggestions are made for the control of small strongyles in the light of an emerging resistance problem.     

The role of combination anthelmintic formulations in the sustainable control of sheep nematodes

Combinations of anthelmintics with a similar spectrum of activity and different mechanisms of action and resistance are widely available in several regions of the world for the control of sheep nematodes. There are two main justifications for the use of such combinations: (1) to enable the effective control of nematodes in the presence of single or multiple drug resistance, and (2) to slow the development of resistance to the component anthelmintic classes. Computer model simulations of sheep nematode populations indicate that the ability of combinations to slow the development of resistance is maximised if certain prerequisite criteria are met, the most important of which appear to concern the opportunity for survival of susceptible nematodes in refugia and the pre-existing levels of resistance to each of the anthelmintics in the combination. Combinations slow the development of a resistant parasite population by reducing the number of resistant genotypes which survive treatment, because multiple alleles conferring resistance to all the component anthelmintic classes must be present in the same parasite for survival. Individuals carrying multiple resistance alleles are rarer than those carrying single resistance alleles. This enhanced efficacy leads to greater dilution of resistant genotypes by the unselected parasites in refugia, thus reducing the proportion of resistant parasites available to reproduce with other resistant adults that have survived treatment. Concerns over the use of anthelmintic combinations include the potential to select for resistance to multiple anthelmintic classes concurrently if there are insufficient parasites in refugia; the potential for shared mechanisms of resistance between chemical classes; and the pre-existing frequency of resistance alleles may be too high on some farms to warrant the introduction of certain combinations. In conclusion, anthelmintic combinations can play an important role in resistance management. However, they are not a panacea and should always be used in accordance with contemporary principles for sustainable anthelmintic use.