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.     

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.     

Recent advances in laboratory models for evaluation of helminth chemotherapy

Extensive research on nematodes of veterinary importance has resulted in new tests based on incubation of parasitic nematodes in culture media, with a variety of detection systems including visual observation, inhibition of aggregation, reduced secretion of acetylcholinesterase and electronic motility measurements. There has been a proliferation of tests to detect benzimidazole resistant nematodes including egg embryonation, egg hatch, larval motility, differences in esterases, and tubulin binding by labelled benzimidazoles. A simple larval development test, which detects resistance to all types of anthelmintics, is likely to become the preferred test for evaluation of resistance in nematodes of grazing animals. By contrast the relative unimportance of trematodes and cestodes from a commercial point of view has resulted in few recent developments in models for evaluating chemotherapy. But the emergence of drug resistant strains of both schistosomes and liver fluke indicates the need for simple tests to detect 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.     

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.     

A survey of anthelmintic resistant nematode parasites in Scottish sheep flocks

The state of anthelmintic resistance in Scottish sheep flocks was evaluated during April-August 2000 using in vitro bioassays, egg hatch assays for detecting thiabendazole (TBZ) resistance and larval development assays for levamisole, ivermectin and TBZ resistance. Anaerobic sampling kits and detailed questionnaires outlining farm demographics and current management practices were mailed to 227 Moredun Foundation member farms, replies were received from 98 farms. Kits received from 90 farms contained sufficient material to conduct one or more of the assays. A majority of the farms examined (64%) exhibited TBZ resistance but there were both farm locality and regional variations in the percentage of resistant farms. Teladorsagia was the predominant genera detected from farms. No resistance to levamisole or ivermectin was detected in any of the samples. There was no strong evidence from this survey that any of the management practices examined greatly affected TBZ resistance.     

Studies on mutlispecific resistance of gastrointestinal nematodes to benzimidazoles on dairy-goat farms

Multispecific resistance to benzimidazoles was studied in three selected farms. These farms had bred dairy goats for more than 15 years. The helminths were introduced with the goats at the establishment of the farms which afterwards remained isolated. Nematode resistance could then be related to their own management practices. Faecal egg count tests and egg hatch assays were performed to assess intensity of resistance. The generic (infective larvae in faecal cultures) and specific richness (adult worms) were assessed. The resistant species were Trichostrongylus colubriformis, Teladorsagia circumcincta, Haemonchus contortus and Oesophagostomum venulosum. Faecal egg count reduction tests and egg-hatch assays did not match exactly. Faecal larval counts after treatments gave a distorted picture of multispecific resistance: Haemonchus and Oesophagostomum were very largely over represented. The number of species found in the three farms was relatively low compared with other reports in goat farms of the area. This reduction of diversity might also be due in part to characteristics of breeding management and history (use of permanent pasture and introduction of goats at the establishment of farm).     

Field evaluation of the efficacy of fenbendazole in captive wild ruminants

The efficacy of in-feed fenbendazole at a dose rate of 7.5 mg/kg bodyweight for three consecutive days was assessed in five Arabian oryx (Oryx leucoryx), six scimitar-horned oryx (Oryx dammah), 14 slender-horned gazelles (Gazella leptoceros), eight Soay sheep (Ovis aries aries soay), 13 alpine ibex (Capra ibex ibex), six red deer (Cervus elaphus hippelaphus) and 11 Nelson's elk (Cervus elaphus nelsoni) kept in five herds in a zoo. The efficacy was assessed by means of repeated faecal egg count reduction (fecr) tests and in vitro egg hatch assays. Fenbendazole was highly effective against nematodes in five of the seven species, consistently reducing egg shedding by more than 90 per cent. In the egg hatch assays of the five herds, 50 per cent inhibition of hatching (ld50) was observed at a concentration of thiabendazole below 0.1 microg/ml. In the Arabian oryx and alpine ibex the efficacy of fenbendazole was less than 90 per cent, and the ld50 in the egg hatch assays was between 0.1 and 0.2 microg/ml thiabendazole.     

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.     

Studies on an Ostertagia ostertagi strain suspected to be resistant to benzimidazoles.

Four calves were infected with a susceptible (laboratory) strain of Ostertagia ostertagi and four with a field strain suspected to be resistant to benzimidazoles. After 25 days two calves from each group were treated with 3.5 mg kg-1 fenbendazole. Egg output fell to zero in all treated calves. Treated calves did not harbour worms at slaughter 35 days after infection. Significant differences between the strains were shown for ED50 values for thiabendazole in the egg hatch assay, but not in the tubulin binding assay. It is concluded that benzimidazole resistance in the suspected strain cannot be confirmed.     

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.     

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.     

Isolation of benzimidazole resistant strains of Ostertagia circumcincta from British sheep

Two strains of Ostertagia circumcincta were isolated from sheep in Great Britain; one (CVL strain) from a breeding flock maintained at the Central Veterinary Laboratory, the other (H2 strain) from a commercial flock in southern England. Their resistance to benzimidazole anthelmintics was assessed by means of in vitro egg hatch assays and slaughter trials. In vitro egg hatch assays gave calculated ED50 estimates of 0.799 micrograms thiabendazole/ml for the CVL strain and 0.794 micrograms thiabendazole/ml for the H2 strain, compared with ED50 estimates of 0.038 micrograms thiabendazole/m and 0.036 micrograms thiabendazole/ml for two known susceptible strains of O circumcincta. There was a 40.7, 28.4 and 66.9 per cent reduction in the group mean worm burdens of lambs infected with the CVL strain following treatment with thiabendazole, fenbendazole and oxfendazole, respectively, and 23.8, 0.0, 79.6, 52.7, 99.9 and 100 per cent reduction in the group mean worm burdens of lambs infected with the H2 strain following treatment with thiabendazole, fenbendazole, oxfendazole, albendazole, levamisole and ivermectin, respectively. Detailed field histories for both strains are given.     

A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles.

This study reports a comparison between faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) for detecting anthelmintic resistance in equine strongyles. Resistance to benzimidazoles was demonstrated in 33 of 42 (79%) farms tested by FECRT and in 32 (62%) of the 52 farms tested by EHA. As the reference strain used was not fully susceptible to benzimidazoles it was not possible to determine the level of resistance by LDA. Pyrantel resistance was indicated on three of 15 farms by faecal egg count reduction. Resistance was also indicated by LDA for one of these farms. In addition resistance was indicated by LDA on two more farms that were not tested by FECRT. Further testing is needed to confirm if these findings are truly indicative of resistance. Generally, correlations between the tests were poor and it was not possible to use the outcome of one test to predict the outcome of another.     

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.     

Prevalence of anthelmintic-resistant nematodes in fibre-producing goats in Scotland.

Six cashmere goat herds in Scotland were surveyed to assess the prevalence of anthelmintic resistance and the species of nematode involved. Resistance was determined in vivo by a faecal egg count reduction test and in vitro using an egg hatch assay and specific faecal egg identification techniques. Benzimidazole resistance was present in five of the herds, insufficient eggs were obtained from the other herd to draw firm conclusions. Teladorsagia (Ostertagia) species predominated in four of the five herds in which resistance was detected and Haemonchus contorus was the dominant species in the other.     

Isolation of oxfendazole resistant Cooperia oncophora in cattle

A field strain of Cooperia oncophora resistant to oxfendazole was isolated from a commercial cattle rearing property in Waikato, New Zealand. Resistance to oxfendazole was assessed by means of a faecal egg count depression test and an in vitro egg hatch test. This is the first documented case of anthelmintic resistance in Cooperia spp. and the first report of anthelmintic resistance in cattle in New Zealand.     

Comparison of in vitro methods and faecal egg count reduction test for the detection of benzimidazole resistance in small strongyles of horses

The objective of the study was to compare the in vitro egg hatch test (EHT), larval development test (LDT) and in vivo faecal egg count reduction test (FECR test) for the detection of benzimidazole resistance in equine strongyles. The presence of resistant or susceptible strongyle populations was determined in 25 stud farms using the in vivo FECR test and in vitro EHT. On the basis of the FECR values, resistance to fenbendazole was detected on 15 of the 25 farms (60%). The ED₅₀ value (anthelmintic concentration producing 50% inhibition of hatching) for suspected resistant populations varied from 0.110 to 0.222 g/ml thiabendazole (TBZ). Final LD₅₀ values (anthelmintic concentration inhibiting development of 50% of eggs into L₃ infective larvae) above 0.029 g/ml TBZ in the in vitro larval development test on samples from 11 stud farms revealed the presence of populations of small strongyles suspected of being benzimidazole-resistant.     

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.     

虫卵孵化试验对某羊场羊线虫抗药性的检测

为了解家畜寄生线虫对使用较久的驱虫药丙硫咪唑的抗药性情况, 用虫卵孵化试验对江苏某羊场进行抗药性检测, 测得丙硫咪唑虫卵孵化半数抑制量ＩＤ５０ ＝ ０－０６０ ２ μｇ／ｍＬ, 约是敏感株（０－０２０ ４ μｇ／ｍＬ） 的３ 倍, 但较世界兽医寄生虫促进协会提出的线虫抗药性临界值（０－１ μｇ／ｍＬ） 小。虫卵减少试验也证实了该羊场不存在抗药性。因此该场羊寄生线虫尚无抗药性。