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.     

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.     

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.     

Anthelmintic resistance and sheep management practices in south western Victoria

Twenty-eight farms in 7 shires in south western Victoria were selected and tested for presence of benzimidazole-resistant nematodes between November 1979 and June 1981. Mean faecal egg counts of sheep were less than 100 strongyloid eggs/g on 11 farms. Faecal egg count reduction tests were conducted on the remaining 17 farms and thiabendazole was less than 90% efficient in reducing egg counts in sheep from 5 (29%) of these farms. Thiabendazole-resistant Teladorsagia circumcincta were identified at necropsy of experimentally infected treated sheep. In further studies a survey of 104 farms was conducted in the Mount Rouse and Dundas shires of western Victoria in 1981 and 1982 respectively to determine the prevalence of anthelmintic resistance in these shires. Mean faecal egg counts among weaner sheep in the winter-spring of both years were less than 100 eggs/g which indicated low levels of parasitic nematode populations. A faecal egg count reduction test was conducted on 10 farms and thiabendazole was less than 90% efficient on 3; levamisole was greater than 90% efficient in all 10 tests. Most of the surveyed farms carried Merino or Merino crossbred sheep at 10 to 15 dry sheep equivalents per ha and weaners were treated with anthelmintics 3 to 6 times per year. Management procedures based mainly on anthelmintic therapy were effective in controlling nematode populations in weaner sheep, although many producers alternated between different groups of anthelmintics within the same year contrary to current recommendations for long-term preservation of anthelmintic efficacy. It was concluded that anthelmintic resistance was not of practical importance to the majority of sheep producers in the region.     

Naphthalophos combinations with benzimidazoles or levamisole as effective anthelmintics for sheep

Objective: To investigate the relative efficacy and safety of the anthelmintic naphthalophos in sheep, either given alone or in combination with benzimidazole (fenbendazole and albendazole) or levamisole anthelmintics.
Design: A parasitological study using faecal egg count reduction tests, a validating slaughter trial and field safety trials.
Procedure: Faecal egg count reduction tests were carried out on 13 farms. Naphthalophos and combinations of naphthalophos with levamisole and fenbendazole were included in the drench tests. On one property a controlled efficacy study was carried out to validate faecal egg count reduction test findings. In this trial, sheep were slaughtered 10 days after treatment and the remaining parasites recovered from the gastro-intestinal tract. Safety trials were carried out on eight farms where approximately 50 000 sheep were treated with naphthalophos and albendazole that were tank mixed in the backpack.
Results: The efficacy of naphthalophos alone in faecal egg count reduction tests ranged from 59 to 98% with one test showing 95% reduction. The efficacy of naphthalophos and levamisole ranged from 74 to 100%, with 5 farms showing 95% reduction. The efficacy of naphthalophos and fenbendazole ranged between 88 and 100% with 95% reduction achieved on 10 farms. The controlled efficacy study showed a good correlation between the faecal egg count reduction tests and numbers of parasites recovered, except for Nematodirus where the faecal egg count reduction tests overestimated efficacy. The mortality rate in the safety trials was 0.05%, with most fatalities occurring on one farm.
Conclusion: The combination of naphthalophos and fenbendazole was more effective than a combination of naphthalophos and levamisole, and will provide a sufficiently safe drench rotation option.     

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.     

Anthelmintic resistance amongst sheep and goats in Kenya

Faecal egg count reduction tests (FECRT) and larval cultures were used to assess the prevalence of anthelmintic resistance amongst sheep and goats on farms served by Kenya's five regional Veterinary Investigation Laboratories. Twenty-four out of 42 farms tested (57%) showed resistance to at least one anthelmintic group. Resistance to levamisole was found in nine out of 35 sheep farms tested (26%) and 12 out of 24 goat farms (50%). Resistance to benzimidazole drugs was found in 10 out of 28 sheep farms (36%) and six out of 20 goat farms (30%). Larval cultures of post-treatment faecal samples showed resistance to be predominantly due to Haemonchus contortus. Twelve farms were routinely using substandard products which appeared to have no anthelmintic activity.     

Comparative use of faecal egg count reduction test,egg hatch assay and beta-tubulin codon 200 genotyping in small strongyles (cyathostominae) before and after benzimidazole treatment

A survey on benzimidazole (BZ) resistance in small strongyles was performed on three farms in the tenth region in Chile. Samples from a total of 100 horses were tested using the faecal egg count reduction test (FECRT), the egg hatch assay (EHA) and an allele-specific PCR for the detection of beta-tubulin isotype 1 genes coding for phenylalanine (phe) or tyrosine (tyr) at codon 200. In the past, BZ-type drugs have been used within anthelmintic campaigns on all the three farms. This has predictably led to a high degree of BZ resistance at the Valdivia and Ri?ihue farms and to a lesser degree at the Frutillar farm, as demonstrated by all the three tests. The FECRT indicated resistance in every farm by faecal egg count reductions (FECR) of 27% (S.D. +/- 33), 26.5% (S.D. +/- 26.9) and 83.9% (S.D. +/- 22.8) for the Valdivia, Ri?ihue and Frutillar farms, respectively. With the EHA, the following mean LD(50) values were found before and after treatment with fenbendazole (FBZ): 0.093, 0.141 and 0.066 microg TBZ/ml and 0.149, 0.158 and 0.091 microg TBZ/ml, respectively, for the Valdivia, Ri?ihue and Frutillar samples. The corresponding LD(96) values were 0.222, 0.263 and 0.188 microg TBZ/ml before treatment and 0.316, 0.322 and 0.221 microg TBZ/ml after treatment, indicating BZ resistance in all the cases. Genotyping was performed on more than 1700 single larvae, at least 10 per faecal sample, for 98 pre- and 66 post-treatment samples. Despite a general trend toward higher percentages of phe/tyr and tyr/tyr individuals following treatment, no statistically significant difference was found between these two and the phe/phe genotype percentages. However, a significantly negative correlation was detected between the LD(50) values and the phe/phe percentages and there was a positive correlation between the FECRT results and the phe/phe percentages. Thus, there seems to be a difference in the significance of the codon 200 polymorphism in the mechanisms of BZ resistance in small strongyles of the horse and sheep trichostrongyles.     

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.     

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

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

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.     

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.     

Effect of repeated benzimidazole treatments with increasing dosages on the phenotype of resistance and the beta-tubulin codon 200 genotype distribution in a benzimidazole-resistant cyathostomin population

This study was designed to investigate the effect of repeated treatments with increasingly high fenbendazole (FBZ) dosages on the phenotype and genotype of a benzimidazole (BZ)-resistant cyathostomin population. An experimentally infected horse was treated repeatedly with FBZ dose rates between 7.5 and 30.0 mg/kg body weight (bw) over approximately 2 years. Faecal egg counts (FECs) and larval cultures were performed weekly. A total of 45 faecal egg count reduction tests (FECRTs) were analysed, revealing a high variability during the course of experiment with a mean value in faecal egg count reduction (FECR) of -17% (S.D. +/- 78). The FECR was always < 90%, providing the evidence of BZ resistance. Nine egg hatch tests were performed during the course of the experiment and revealed LD(50) values between 0.20 and 0.31 microg/ml thiabendazole (TBZ) and LD(96) values of > 0.36 microg/ml TBZ, confirming the phenotype of resistance. The LD(99) varied between 0.40 and 0.63 microg/ml TBZ. Despite consecutive treatments, no noticeable increase of the LD(50), LD(96) and LD(99) values was detected for the duration of the experiment. The molecular analysis of the codon 200 of 106 third stage larvae (L3) was carried out following repeated treatments with 30 mg FBZ/kg bw. Out of these larvae 32% were homozygous TTC/TTC, 60% showed the heterozygous TTC/TAC genotype, and 8% were homozygous TAC/TAC. The resulting allele frequencies were 62% for TTC and 38% for TAC. These findings suggest that repeated BZ treatments with increasing dosages do not alter significantly the FECRT and EHT characteristics of a BZ-resistant cyathostomin population. Furthermore, it may also be concluded that, in contrast to sheep trichostrongyles, such a selection regime does not result in beta-tubulin codon 200 TAC allele autocracy.     

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.     

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 in equine cyathostomes in Slovakia

The present study included 19 stud farms, including 243 horses, that were investigated for the occurrence of anthelmintic resistant cyathostomes. The number of horses on the farms varied from nine to more than 100, and horses of all ages were included. A minimum of seven horses were used for faecal egg count reduction (FECR) tests. The anthelmintics included were: fenbendazole (paste formulation), ivermectin (paste formulation) and pyrantel (powder). Resistance to benzimidazoles was detected on 14 farms, with FECR values ranging from 65.1 to 86.3%. Larval cultures after fenbendazole treatment revealed exclusively cyathostome larvae. Ivermectin was tested on eight farms and proved to be effective on all. Pyrantel was tested on two farms and FECR test indicated high efficacy (92-97%). Egg hatch assay (EHA) results showed that mean concentrations of thiabendazole that inhibited hatching in 50% of the eggs (ED(50)) in resistant populations were over 0.1 microg ml(-1). The results of our study suggest widespread resistance to fenbendazole in equine cyathostomes in Slovakia, and possible strategies to delay anthelmintic resistance are discussed briefly.     

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.     

Anthelmintic resistance survey in New Zealand

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

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.