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.     

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--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.     

The in vitro diagnosis of anthelmintic resistance in cyathostomins

Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resistance in different nematode populations. At present, the faecal egg count reduction test is considered the most suitable method for initial screening for anthelmintic resistance in equine nematode populations. However, in its current state, this test lacks sensitivity. It is also costly and time-consuming to perform. Laboratory-based techniques, such as the egg hatch assay, larval development assay, larval migration inhibition assay and the larval feeding inhibition assay offer alternative options for assessing anthelmintic resistance in nematode populations. All of these tests have been investigated for their utility in measuring drug resistance in sheep nematode populations and some have proven useful. The egg hatch assay, larval development assay and larval migration inhibition assay have been investigated for use in measuring levels of drug resistance in equine nematode populations. However, at best, the results obtained thus far indicate that these tests require further refinement.     

How widespread is resistance to invermectin among gastrointestinal nematodes in sheep in The Netherlands?

In Autumn 2009, a faecal egg count reduction test (FERCT) was carried out on three sheep farms. Groups of 8-11 lambs were treated with ivermectin or moxidectin, with a 14-day interval between treatment and sampling. Ivermectin resistance was present on all three farms. Treatment with ivermectin resulted in a reduction in faecal egg numbers of 94.6%, 63%, and 59%. On two farms, 14 days after treatment pooled faecal samples yielded predominantly larvae of Hamonchus contortus (100% and 98%, respectively). On the third farm, H. contortus and (probably) Teladorsagia circumcincta were resistant to ivermectin (64% and 36% of the larvae, respectively). Treatment with moxidectin resulted in a 100% reduction in egg output in sheep on all three farms. More sensitive culture techniques failed to detect any larvae in samples taken from two farms, but a few Ostertagia-type larvae, probably of T. circumcincta, were detected in samples from the third farm. It can be concluded that gastrointestinal nematodes in sheep from these three farms were resistant to ivermectin, whereas resistance to moxidectin was not detected.     

Benzimidazole resistance of sheep nematodes in Norway confirmed through controlled efficacy test

Background

Resistance against benzimidazoles (BZ) has recently been detected in Norwegian sheep flocks through a large scale prevalence survey based on the faecal egg count reduction test (FECRT). The use of this test in combination with bulk larval culture only gives an indication of which gastrointestinal nematodes genera that are involved and these results have to be confirmed by a controlled efficacy test (CET) to get accurate information about resistant nematodes populations at species level. A CET was therefore performed with larvae from two flocks where BZ resistance was previously detected through FECRT.

Results

The latter test confirmed the previous results in both flocks. In flock A, the BZ resistant nematode population consisted solely of Haemonchus contortus, whereas H. contortus and Teladorsagia circumcincta comprised the resistant worm population in flock B.

Conclusions

Some discrepancies that have been recorded between FECRT and CET results regarding time for post-treatment coproscopical examination and a temporary suppression of faecal egg excretion are discussed.     

A larval development test for the detection of anthelmintic resistance in nematodes of sheep

First stage larvae of a number of species of parasitic nematodes of sheep have been shown to develop to third stage larvae in the presence of a defined medium consisting of Earle's balanced salt solution and yeast extract. A larval development test, based on this culture technique, was used as a screen for detecting the presence of anthelmintic resistance in nematodes of sheep. It was found to be sensitive and simple to use and also appeared capable of detecting resistance to any of the main anthelmintic groups. Available anthelmintic sensitive and resistant strains of Haemonchus contortus and Ostertagia circumcincta showed differences in development when incubated in the presence of either thiabendazole, levamisole and ivermectin. These differences were expressed as the minimum inhibitory concentration required to prevent larval development over the incubation period.     

Resistance of Teladorsagia circumcincta in goats to ivermectin in the Netherlands

Resistance of Teladorsagia circumcincta in goats to ivermectin In 2005, suspected ivermectin resistance of gastrointestinal nematodes was investigated in 17 goats on a farm in the Netherlands, using a faecal egg count reduction test combined with faecal larval cultures. Eight goats were treated orally with 0.2 mg/ kg ivermectin on 1 August 2005 and the other nine goats served as untreated controls. Faeces were collected from all goats on 1 August and on 12 August. Faecal egg counts were reduced by 80.6% in goats receiving ivermectin. On the basis of faecal larval cultures and the egg counts, ivermectin was found to be 100% effective against Haemonchus contortus, 15.0% effective against Teladorsagia circumcincta, and 93.3% effective against Trichostrongylus spp. The effect of levamisole and oxfendazole on gastrointestinal nematodes was also evaluated. Nine goats were treated subcutaneously with 5 mg/kg levamisole and seven goats orally with 4.5 mg/kg oxfendazole on 12 September On the basis of faecal egg counts performed on 12 and 22 September, levamisole reduced the total egg count by 95.4% and oxfendazole reduced the egg count by 85.8%. Differentiation of pooled faecal larval cultures revealed that levamisole was 99.9% effective against H. contortus, 81.9% effective against T. circumcincta, and 99.5% against Trichostrongylus spp. Oxfendazole was 75.5% effective against H. contortus, 33.2% effective against T. circumcincta, and 100% effective against Trichostrongylus spp. On 28 October the nine goats that were still present on the farm were orally treated with 0.4 mg/kg ivermectin. Faecal egg counts and differentiation of pooled larval cultures of faecal samples collected on 28 October and 9 November, respectively, revealed that total egg counts were reduced by 93.1%. Ivermectin was 49.1% effective against T. circumcincta but 100% effective against H. contortus and Trichostrongylus spp. Thus H. contortus appears to be resistant to oxfendazole and T. circumcinta appears to be resistant to oxfendazole and ivermectin. This is the first documented case of ivermectin resistance of a gastrointestinal nematode of small ruminants in the Netherlands.     

An evaluation of techniques used for the detection of anthelmintic resistance in nematode parasites of domestic livestock

In vitro and in vivo techniques used for the detection of anthelmintic resistance are reviewed in terms of their versatility, reliability, accuracy, cost and simplicity. The faecal egg count reduction test is considered the most suitable for field screening of resistance but the larval development assay is likely to prove a valuable adjunct in such investigations. Other in vitro techniques, such as egg hatch tests, larval motility and tubulin binding assays are recommended as suitable only for research investigations. Similarly, in vivo procedures such as the critical and controlled anthelmintic efficacy tests are considered to be appropriate only for specific research purposes because of the costs involved in such studies.     

Drought and flock isolation may enhance the development of anthelmintic resistance in nematodes

A survey of anthelmintic resistant nematodes was conducted in sheep and goat flocks in Greece using in vivo and in vitro tests. Faecal egg count reduction tests in Macedonia were all greater than 99% indicating very high sensitivity of the nematodes to anthelmintics. In vitro tests showed benzimidazole resistant Teladorsagia circumcincta in 17 out of 106 flocks on small islands. On the mainland there were only three cases of benzimidazole resistance out of 310 flocks and animals had recently been introduced to the flocks. Flocks on the islands are isolated and there are higher temperatures than on the more mountainous mainland, where flocks tend to intermingle. It is concluded that drought and isolation are likely to be the major factors accounting for the development of anthelmintic resistance in nematodes in the island flocks.     

Application of in vitro anthelmintic sensitivity assays to canine parasitology: detecting resistance to pyrantel in Ancylostoma caninum

Resistance of the canine hookworm Ancylostoma caninum to anthelmintic therapy with pyrantel is an emerging problem in canine veterinary practice. Detecting anthelmintic resistance in parasites of pets is problematic because traditional resistance-monitoring techniques used with livestock parasites, such as the faecal egg count reduction test, are often impractical for use in small animals. We used two field-collected isolates of A. caninum in an abbreviated critical trial to test their pyrantel resistance status. The strains showed high-level and low-level resistance, with in vivo pyrantel efficacies of 28% and 71%, respectively. We noted a distinct worm density dependence effect on faecal egg count during the critical trial; egg counts in the dogs containing the low-level resistant isolate were 41% higher 6 days after drug treatment, despite the removal of 71% of the adult worms by the drug treatment. We then assessed four candidate in vitro assays for their ability to detect pyrantel resistance in A. caninum larvae, using these two isolates. The assays included a new format termed the larval arrested morphology assay (LAMA), based on observation of the effects of pyrantel on the body shape adopted by infective stage A. caninum larvae in vitro. Our data suggests that three of these assays, the LAMA, the larval motility assay (LMA), and larval feeding inhibition assay (LFIA) show promise with regards to detection of pyrantel resistance in A. caninum, but the complexity of the LFIA would likely limit its suitability for field studies. In vivo pyrantel efficacies of 28% and 71% in the two A. caninum isolates were associated with a 17-fold shift in the in vitro IC(50) values measured using the LAMA. Further testing with isolates of varying degrees of resistance is required to determine which of these assays is suitable as a rapid in vitro laboratory test for pyrantel resistance in A. caninum. The present study also indicates that potential exists for the novel LAMA or the LMA to be of use in detecting pyrantel resistance in the human hookworms, Necator americanus and Ancylostoma duodenale.     

A survey of anthelmintic resistance on ten goat farms in the Manawatu region in 1988

A survey of anthelmintic resistance on ten goat farms was carried out. The criterion for resistance was a faecal egg count reduction (FECR) of <90 percent based on arithmetic means of 11 to 19 goats per group. It was found that nine of the ten farms harboured nematodes resistant to oxfendazole when used at 5mg/kg although on two of these, the FECR's were 89.2 percent and 89.8 percent. Post treatment larval cultures indicated Haemonchus, Ostertagia and Trichostrongylus were the resistant genera involved. On none of the ten farms was resistance to oral ivermectin at 0.2mg/kg detected.     

河北省坝上地区绵羊消化道线虫对丙硫苯咪唑抗药性的检测

目的　为了查明绵羊消化道线虫对丙硫苯咪唑的抗药性。方法　应用粪便虫卵减少试验 (FECRT)对河北省坝上地区绵羊消化道线虫的抗药性进行了检测。结果　应用 5 m g/ kg丙硫苯咪唑对 5组绵羊进行驱虫的虫卵减少率分别为 4 3.9% ,38.4 6 % ,4 9.0 9% ,37.6 3%和 4 3.35 % ,驱净率分别为 2 0 % ,15 % ,15 % ,2 5 %和 10 %。结论　根据 FECRT 95 %置信域小于 90 % ,表明试验羊对该药有抗药性     

Anthelmintic Resistance on an Organized Sheep Farm in India

Monitoring anthelmintic resistance in strongyle nematodes by the faecal egg count reduction test and a commercial larval development assay on an organized sheep farm in the semi-arid area of Rajasthan revealed the emergence of resistance to benzimidazoles and rafoxanide and a potential risk of the development of levamisole/tetramisole resistance. A benzimidazole/levamisole combination, avermectins and closantel were each found to be efficacious.     

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.     

Avermectin-resistance in gastrointestinal nematodes of Boer goats and Dorper sheep in Switzerland

Anthelmintic resistance in gastrointestinal nematodes among small ruminants is widespread in South Africa and Dorper sheep and Boer goats have been imported into Switzerland from this country on a number of occasions. Therefore, this study aimed to investigate the occurrence of avermectin (AVM) resistant gastrointestinal nematodes (GIN) in these breeds in Switzerland. A total of 24 Boer goat farms and 12 Dorper sheep farms participated in the study. According to the faecal egg count reduction test (FECRT) AVM-resistant GIN populations were confirmed in 7 of the 24 Boer goat farms and suspected in a further 8 farms. Likewise AVM-resistance was confirmed in 2 of 12 Dorper sheep farms and suspected in a further 6 farms. Haemonchus contortus and Trichostrongylus spp. were the dominant resistant species according to larval cultures. In the farms with detected AVM-resistance the animals were additionally treated with levamisole after natural reinfection. With the exception of one farm with a 'close-to cutoff-result' the FECRT gave no indication for resistance against levamisole. The results indicate that AVM-resistance is widespread in Swiss small ruminant farms keeping Boer goats and Dorper sheep. The common tradition of grazing animals from different farms on prealpine and alpine pastures could favour the spread of resistant populations within the country.     

A survey of anthelmintic resistance on ten goat farms in the Manawatu region in 1988

A survey of anthelmintic resistance on ten goat farms was carried out. The criterion for resistance was a faecal egg coynt reduction (FECR) of <90 percent based on arithmetic means of 11 to 19 goats per group. It was found that nine of the ten farms harboured nematodes resistant to oxfendazole when used at Smg/kg although on two of these, the FECR's were 89.2 percent and 89.8 percent. Post treatment larval cultures indicated Haemonchus, Ostertagia and Trichostrongylus were the resistant genera involved. On none of the ten farms was resistance to oral ivermectin at 0.2mg/kg detected.     

Repeated treatment faecal egg counts to identify gastrointestinal nematode resistance in a context of low-level infection of sheep on farms in eastern Algeria

The efficacy of benzimidazoles and ivermectin was tested in 14 pilot farms in eastern Algeria. Resistance was detected in strongyle nematodes against benzimidazoles (five farms) and ivermectin (one farm). The main genera in the region were resistant: Teladorsagia, Trichostrongylus, Marshallagia and Nematodirus. The evaluations based on FECRT (faecal egg count reduction tests) were not always consistent due to the low-level of infection, and we developed a simplified, repeated treatment strategy to investigate resistance. The results obtained with repeated treatment faecal egg counts match globally those obtained with conventional faecal egg count reduction tests. This simplified, repeated treatment method should be investigated further since it is quicker, and this could make it possible to test larger farm samplings.     

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.     

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.