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.     

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.     

Further studies on anthelmintic resistance in sheep at an organised farm in arid region of Rajasthan

Faecal egg count reduction test (FECRT) and in vitro egg hatch assay (EHA) was performed on Indian Karakul (3/4 crosses of Karakul with Malpura, Marwari and Sonadi) transported from arid region campus, Bikaner, where resistance was reported in 1996. FECRT revealed that fenbendazole and levamisole were 100% effective in reducing the egg counts. LC50 value on EHA was 0.074 +/- 0.015 microg thiabendazole ml(-1). The faecal culture examination revealed the presence of Haemonchus contortus only. It was concluded that H. contortus was fully susceptible to both benzimidazole and levamisole.     

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.     

Characterisation of two triple resistant field isolates of Teladorsagia from Scottish lowland sheep farms

The anthelmintic resistance status of two field isolates derived from farms (farm A and B) located near Edinburgh were examined using both controlled efficacy tests (CET) and faecal egg count reduction tests (FECRT). Efficacies against fenbendazole (FBZ), levamisole (LEV) and ivermectin (IVM) and, for one isolate, against combinations of these anthelmintics and moxidectin were determined in na?ve lambs, artificially infected with the isolates and treated with the compounds at the manufacturers recommended dose rates. (FBZ, 5mg/kg bodyweight (BW); LEV, 7.5mg/kg BW; IVM, 0.2mg/kg BW; Moxidectin (MOX) 0.2mg/kg BW). In both field isolates, the predominant species found pre-treatment and the only species found post-treatment was Teladorsagia circumcincta. Resistance to FBZ, LEV and IVM was confirmed in CET and FECRT on farm A and to the latter two compounds on farm B, which had a history of benzimidazole resistance and where TBZ resistance was also demonstrated using an egg hatch assay (EHA). For the farm A isolate CET efficacies against FBZ; IVM; LEV; FBZ + IVM; FBZ + LEV; FBZ, LEV + IVM and MOX were 59, 60, 88, 94,93, 92 and 98%, respectively. The CET efficacies for the farm B isolate were 51% and 72% for LEV and IVM, respectively.     

Impaired efficacy of ivermectin against Parascaris equorum, and both ivermectin and pyrantel against strongyle infections in trotter foals in Finland

In order to assess the resistance situation against macrocyclic lactones in Parascaris equorum and against tetrahydropyrimidine derivatives in strongyles in Finnish trotter horses, 112 foals on 18 farms, mostly 1 year old, were examined for these parasites with a modified McMaster faecal flotation method. P. equorum positive foals (n=24) were given ivermectin orally at a dose of 200 μg/kg b.w., while strongyle positive but P. equorum negative foals (n=38) received pyrantel embonate orally at a dose of 19 mg/kg. Sixteen P. equorum infected foals, treated with ivermectin, also harboured strongyles. During the anthelmintic treatment visit to the farm, Faecal Egg Count Reduction Test (FECRT) reference (first) samples were collected. Fourteen days later, the second sampling (reduction samples) was done. The FECR was calculated for each foal/parasite combination. The reduction efficacies of ivermectin against P. equorum (mean 52%, calculated from the individual egg count reductions) and pyrantel against strongyles (43%) were strongly indicative of widespread resistance. Also indication of ivermectin resistance among strongyles was seen. The widespread use of anthelmintics for Finnish horses obviously has resulted in resistance, as has happened elsewhere, too.     

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.     

Resistance to macrocyclic lactone anthelmintics and associated risk factors on sheep farms in the lower North Island of New Zealand

AIM: To estimate the prevalence of resistance to macrocyclic lactone (ML) anthelmintics over a 3-year period on sheep farms in the Taihape area, and to determine associations between the presence of ML resistance and farm management practices. METHODS: All farmers with >1,000 lambing ewes served by one veterinary practice in the Taihape area of the central North Island of New Zealand were invited to take part in the study (n=157); respondents were selected on their willingness to participate. Resistance to ML was measured during 2004-2006, using a standard faecal egg count reduction test (FECRT) and a half dose of oral ivermectin (IVM; 0.1 mg/kg; n=84). Additional FECRTs were conducted separately on some farms, using full doses of IVM (n=47), levamisole (LEV; n=49), a benzimidazole (BZ; n=76), and a BZ/LEV combination (n=42). Resistance was defined as <95% faecal nematode egg count reduction (FECR) 7-10 days post-treatment. Larval cultures were performed on pooled faecal samples from all pre- and post-treatment groups for which any positive faecal nematode egg counts (FEC) were recorded. To ascertain whether particular management or drenching strategies were associated with the presence of resistance, a questionnaire comprising 50 questions was developed, and each farmer interviewed individually. RESULTS: Of all farmers invited, 84/157 (54%) participated in this study. Based on undifferentiated FEC, ML resistance to the half-dose IVM was evident on 46/84 (55%) participating farms. The majority of these cases (71%) involved Ostertagia (Teladorsagia) circumcincta. Resistance was also evident to full doses of IVM on 22/47 (47%), BZ on 41/76 (54%), LEV on 18/49 (37%), and BZ/LEV on 2/42 (5%) farms. Positive associations (p<0.05) were evident between the presence of ML resistance and management practices, such as: weaning over half of the lambs onto paddocks not grazed by lambing ewes since June; not always returning lambs to the same paddock after drenching; and the use of visual signs to assess 'worminess'. Measuring drench efficacy in January to March rather than later in the year, and presence of resistance to BZ or LEV were associated with a higher apparent prevalence of ML resistance (p<0.05). CONCLUSIONS: Anthelmintic resistance, and particularly ML resistance, was widespread on sheep farms in the Taihape area, and mostly involved O. circumcincta. The prevalence of resistance was lower on farms on which management practices, particularly those involving lambs, maintained refugia of unselected nematodes. Further investigation is required to develop control strategies which minimise selection for resistant worms, and how these may vary with climate.     

Equine strongyles II. Occurrence of anthelmintic resistance in Switzerland

The occurrence of anthelmintic resistance in strongyles was investigated in 440 horses on 90 farms in Switzerland. The egg hatch assay suggested that benzimidazole (BZ)-resistance was present in 40 of 82 farms (49%). Faecal egg count reduction after pyrantel-treatment was above 96% in 14 of 15 farms. In the remaining farm the efficacy was only 80%. Ivermectin efficacy was investigated on 5 farms and the efficacy was recorded at 98-100%. Faecal cultures undertaken after treatment revealed almost exclusively larvae of the family Cyathostominae. Data about management practices, pasture hygiene and anthelmintic usage were obtained with a questionnaire. Horses were treated on average 3.5 times per year. In 75% of the farms BZ were a component of the seasonal treatment schedule. Only the use of BZ had a significant correlation with the presence of BZ-resistance (P < 0.01). Recommendations for the control of equine strongyles should include measures that minimize the risk of resistance developing against remaining effective anthelmintics.     

A small scale survey of ivermectin resistance in sheep nematodes using the faecal egg count reduction test on samples collected from Scottish sheep

Thirty-eight sheep flocks, predominantly from the south/central Scotland, were examined using a faecal egg count reduction test (FECRT) for the presence of ivermectin (IVM) resistant nematodes. Efficacies of less than 95%, 14-17 days post-treatment, were identified in 6 of 17 naturally grazing flocks where pre-treatment faecal egg counts were in excess of 150 eggs per gram. Efficacies on these IVM resistant farms ranged from 66 to 92%. One other suspected cases of IVM resistance was also identified in returned material. The larvae detected in post-treatment coprocultures from resistant flocks were from the genera Teladorsagia (4 from 6) and Trichostrongylus (2 from 6).     

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.     

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.     

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.     

Further comparison of faecal egg count reduction test procedures: sensitivity and specificity

In a recent communication (McKenna 2006), a comparison was made between four different methods for calculating results from faecal egg count reduction (FECR) tests (FECRTs). The first and most complex of these, referred to as FECRT1, involved the use of the formula: FECR = 100 x (1-[T2/T1][C1/C2]), where T1 and T2 represented the mean pre- and post-treatment faecal nematode egg counts (FECs) of a treated group, and C1 and C2 represented the mean pre- and post-treatment FECs of an untreated control group, respectively. The other three formulae consisted of more simplified versions of this procedure. In one of them (FECRT2), only post-treatment samples were considered, whereas the other two were based on comparisons between the FECs of groups of animals sampled at the time of anthelmintic treatment (pre-treatment) with those sampled several days later (post-treatment). Thus, FECRT2 was determined according to the formula: FECR = 100 x (1-[T2/C2]), while FECRT3 was calculated from FECR = 100 x (1-[T2/T1]). The fourth procedure (FECRT4) was based on a further simplification of FECRT3, where pre-treatment FECs from only one treatment group were used for comparison with all post-treatment results. This base-line pre-treatment group thus effectively functioned as an untreated control group and hence the formula for FECRT4 was FECR = 100 x (1-[T2/C1]). The study was based on an analysis of 210 previously published FECRTs performed in sheep or goats. In each case, FECRs were calculated using all four of these FECRT formulae, and their results compared. The results of these comparisons indicated that the use of any one of them was likely to result in similar estimates of anthelmintic efficacy and the detection of comparable numbers of cases of anthelmintic resistance continued.     

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

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

Efficacy of moxidectin and other anthelmintics against small strongyles in horses

Objective To compare the efficacy of moxidectin to ivermectin, oxibendazole and morantel against some gastrointestinal nematodes in horses.
Design Faecal egg count reduction after treatment.
Procedure A farm was selected where the population of small strongyles in horses was known to be resistant to oxibendazole. Horses were allocated to treatment groups based on faecal egg counts. After treatment, faecal samples were taken up to 109 days after treatment and faecal egg counts estimated. Faecal cultures were used to estimate the contribution of small and large strongyles to the faecal egg counts at each sampling.
Results Moxidectin (0.4 mg/kg) suppressed faecal egg counts for 109 days after treatment in most horses compared to 40 days with ivermectin (0.2 mg/kg), 13 days with morantel (9.4 mg/kg) and less than 13 days with oxibendazole (10 mg/kg). Most of the faecal egg count was attributable to small strongyles based on faecal culture, although Strongylus vulgaris was present in some samples in low numbers. Oxibendazole resistance in small strongyles was confirmed and a less than expected efficacy of morantel was also seen.
Conclusion Moxidectin was highly effective in reducing faecal egg counts after treatment for at least 12 weeks and up to 16 weeks in most horses. These horses were infected with a population of small strongyles known to be resistant to oxibendazole and possibly morantel. The duration of the reduction in faecal egg counts after treatment with moxidectin (0.4 mg/kg) was at least twice that of ivermectin (0.2 mg/kg) and greater than four times that for morantel and oxibendazole.     

A survey on anthelmintic resistance in nematode parasites of sheep in the Slovak Republic

The prevalence of anthelmintic resistance on 27 sheep farms in Slovakia was investigated in 2003 and 2004 using the faecal egg count reduction test (FECRT) according to the WAAVP guidelines. Resistance to albendazole was detected on one farm (3.7%) and suspected on two farms (7.4%) out of 27 sheep flocks. Resistance to ivermectin was tested on 26 farms. On six (23.1%) farms, results indicated the presence of ivermectin resistance. Resistance to ivermectin was suspected on eight farms (30.8%). However, it is also possible that generic ivermectin anthelmintics used in survey have a lower efficacy against sheep nematodes.     

Efficacy of benzimidazole anthelmintics in goats and sheep in the Philippines using a larval development assay

The negative effects of nematodes in small ruminants can be reduced by use of dewormers but their effectiveness is increasingly limited by the emergence of anthelmintic resistance. The efficacy of benzimidazole (BZ) anthelmintics in the Philippines was estimated by an in vitro larval development assay using worm eggs recovered from faeces collected from goats and sheep. Two hundred and eighteen farms were selected to represent areas of the country with high goat and sheep populations and the full range of farm sizes, from smallholders with just a few animals to commercial and institutional farms with several hundred. Initial surveys of worm control advisers indicated that BZs have been in continuous widespread use for up to 20 years with little use of other chemical groups. A larval development assay (LDA: DrenchRite) was modified for use with BZs alone to allow up to five samples to be analysed on a single microtitre plate. The assay was validated by comparison with the faecal egg count reduction test (FECRT). The dominant nematode genera were Haemonchus and Trichostrongylus with small numbers of Oesophagostomum. The range of BZ efficacy estimated from the LDA results was 0-100% and the distribution of efficacy levels was continuous, with mean efficacy of 82 and 64% for goats and sheep, respectively. There were significant associations between efficacy and parameters measured to characterize the sampled farms: size of animal management group, FEC of sample, recent importation of stock and no access to common grazing were all correlated with decreased efficacy. Likewise, low efficacy was associated with reported frequency and number of years that BZ drenches had been used. The LDA was found to be highly suited to estimate efficacy in nematode populations from small farms where performance of a FECRT for even one chemical would be impractical. Using a larval development assay, we have demonstrated a wide efficacy range for BZs against nematodes from all sizes of goat and sheep farms in the tropics.     

The bias, accuracy and precision of faecal egg count reduction test results in cattle using McMaster, Cornell-Wisconsin and FLOTAC egg counting methods

The faecal egg count reduction test (FECRT) is the recommended method to monitor anthelmintic drug efficacy in cattle. There is a large variation in faecal egg count (FEC) methods applied to determine FECRT. However, it remains unclear whether FEC methods with an equal analytic sensitivity, but with different methodologies, result in equal FECRT results. We therefore, compared the bias, accuracy and precision of FECRT results for Cornell-Wisconsin (analytic sensitivity = 1 egg per gram faeces (EPG)), FLOTAC (analytic sensitivity = 1 EPG) and McMaster method (analytic sensitivity = 10 EPG) across four levels of egg excretion (1-49 EPG; 50-149 EPG; 150-299 EPG; 300-600 EPG). Finally, we assessed the sensitivity of the FEC methods to detect a truly reduced efficacy. To this end, two different criteria were used to define reduced efficacy based on FECR, including those described in the WAAVP guidelines (FECRT <95% and lower limit of 95%CI <90%) (Coles et al., 1992) and those proposed by El-Abdellati et al. (2010) (upper limit of 95%CI <95%). There was no significant difference in bias and accuracy of FECRT results across the three methods. FLOTAC provided the most precise FECRT results. Cornell-Wisconsin and McMaster gave similar imprecise results. FECRT were significantly underestimated when baseline FEC were low and drugs were more efficacious. For all FEC methods, precision and accuracy of the FECRT improved as egg excretion increased, this effect was greatest for McMaster and least for Cornell-Wisconsin. The sensitivity of the three methods to detect a truly reduced efficacy was high (>90%). Yet, the sensitivity of McMaster and Cornell-Wisconsin may drop when drugs only show sub-optimal efficacy. Overall, the study indicates that the precision of FECRT is affected by the methodology of FEC, and that the level of egg excretion should be considered in the final interpretation of the FECRT. However, more comprehensive studies are required to provide more insights into the complex interplay of factors inherent to study design (sample size and FEC method) and host-parasite interactions (level of egg excretion and aggregation across the host population).