The detection of anthelmintic resistance by the faecal egg count reduction test: an examination of some of the factors affecting performance and interpretation

This study examines and compares the possible effects of several procedural variants with those of a currently recommended faecal egg count reduction test for the detection of anthelmintic resistance. The results suggest that the failure of an anthelmintic to reduce the arithmetic mean egg counts of 10-15 animals by at least 90%, from either their pre-treatment levels or from those of an untreated control group 5-10 days later, is likely to be an appropriate procedure. It is recommended that such evaluations of drench performance be regularly conducted on farms. Some suggestions as to how this might be encouraged are made, and the role of larval cultures both in increasing the sensitivity and in enhancing the value of the faecal egg count reduction test are discussed.     

Criteria for diagnosing anthelmintic resistance by the faecal egg count reduction test

This study examines the possible outcome of adopting criteria recommended by the Australian Working Party for defining anthelmintic resistance by the faecal egg count reduction test, namely a mean percentage reduction of less than 95%, together with a lower 95% confidence limit of less than or equal to 90%. Based on an analysis of 313 veterinary diagnostic cases submitted for faecal egg count reduction testing in sheep and goats over a 7-year period, it was found that lower confidence limits of 90% or less were always associated with mean percentage reductions of less than 95%. These results suggest that if anthelmintic resistance is to be defined on the basis of mean faecal egg count reductions of this magnitude, then little practical purpose is likely to be served by further consideration of the lower confidence limits of these estimates.     

Criteria for diagnosing anthelmintic resistance by the faecal egg count reduction test

This study examines the possible outcome of adopting criteria recommended by the Australian Working Party for defining anthelmintic resistance by the faecal egg count reduction test, namely a mean percentage reduction of less than 95%, together with a lower 95% confidence limit of less than or equal to 90%.

Based on an analysis of 313 veterinary diagnostic cases submitted for faecal egg count reduction testing in sheep and goats over a 7-year period, it was found that lower confidence limits of 90% or less were always associated with mean percentage reductions of less than 95%. These results suggest that if anthelmintic resistance is to be defined on the basis of mean faecal egg count reductions of this magnitude, then little practical purpose is likely to be served by further consideration of the lower confidence limits of these estimates.     

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.     

Evaluation of the efficacy of anthelmintics sold on Ethiopian markets against <Emphasis Type="Italic">Haemonchus contortus</Emphasis> in experimentally infected Sheep

A total of 54 lambs, aged between 6–8 months were experimentally infected with Haemonchus contortus to evaluate the efficacy of different anthelmintic brands sold on Ethiopian markets using the faecal egg count reduction test (FECRT) and controlled anthelmintic efficacy trial. Accordingly four different albendazole (Alzole®, Analgon-300®, Albenjung_s® and Ahshialben-300®), two tetramisole (Tetsole® and Ashitetra 600) and two tetramisole-oxyclozanide (Tetraclozan sheep® and Tetraclozash 900®) brands were evaluated at the dosage rates recommended by the manufacturers. Animals were allocated into nine groups of six animals each, and balanced for faecal egg counts (FEC), based on their pre-treatment FEC and treatments were randomized among the groups. One group was kept untreated as a control. Faecal egg count was conducted on day 30 post-infection (day of treatment) and on the 10th day post-treatment. Evaluation of anthelmintics based on FECRT revealed high efficacy (99.55–100% reduction in FEC) for all anthelmintic brands tested against H. contortus. The worm count reduction test using controlled anthelmintic efficacy trial also supported the above finding with 99–100% efficacy of the tested anthelmintics. Therefore, the suspicion on the anthelmintic products as being substandard in quality is not credible, at least, for the brands investigated in this study and it might rather be attributed to under dosing. The need for a good extension system for livestock producers with regard to good anthelmintic usage practices, in light of the inevitable development of anthelminitic resistance, is emphasized. Regular surveillance and laboratory quality evaluation of the anthelmintic products in Ethiopia is indicated.     

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.     

Detection of anthelmintic resistance: a comparison of mathematical techniques

Anthelmintic resistance has become an increasing problem particularly to gastrointestinal tract nematodes and appropriate methods are required to detect this phenomenon so the correct action can be taken. This paper compares a number of mathematical techniques that are used to analyse data. The negative binomial distribution is a mathematical distribution used to model aggregated data and hence is suitable to model the intensity of parasite burden and the magnitude of the faecal egg counts. Maximum likelihood techniques are utilised to exploit this mathematical distribution to analyse the magnitude of the faecal egg count reduction and decline in the worm burden in response to anthelmintic treatment. Data from experimental groups of sheep described in the accompanying paper are used. In addition, simulated data sets of faecal egg counts were created using a random number generator following appropriate negative binomial distributions. The results demonstrate this statistical model can detect evidence of anthelmintic resistance with a faecal egg reduction test that otherwise might require a slaughter trial to demonstrate. In addition, the simulated data sets confirm that there is a significant probability of failure to detect low anthelmintic efficacy with commonly used mathematical techniques. Consequently, the use of maximum likelihood mathematical techniques with a negative binomial statistical model would aid in the early detection of anthelmintic resistance using faecal egg count reductions and result in a lower probability of inappropriately assigning an anthelmintic as effective.     

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.     

Fecundity of anthelmintic resistant adult Haemonchus contortus after exposure to ivermectin or benzimidazoles in vivo

A strain of Haemonchus contortus resistant to the benzimidazoles, ivermectin and salicylanilides was used to study the effect of exposure to ivermectin or oxfendazole on the number of eggs produced by adult female parasites. After anthelmintic administration there was a reduction in the faecal egg count of the infected lambs. Ivermectin caused a reduction in the number of eggs within adult parasites for up to 72 hours but this returned to higher than pre-treatment levels at 168 hours after treatment. After oxfendazole treatment there was an increase in the number of eggs stored in the adult parasites at 24 hours and then a significant reduction in the number of eggs for over 168 hours. This suggested that both ivermectin and oxfendazole have a deleterious effect on the reproductive potential of female H contortus although the parasites are resistant to these drugs. These results confirm the importance of allowing up to 10 days to elapse between the time of administration of an anthelmintic and the collecting of faecal samples when using the faecal egg count reduction test to identify anthelmintic resistance, especially when using benzimidazoles. The more prolonged effect on fecundity produced by oxfendazole may reflect the antimitotic action of the benzimidazole anthelmintics.     

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.     

Multiple Anthelmintic Resistance in Haemonchus Contortus on a Sheep Farm in Kenya

Multiple resistance to albendazole, thiophanate, levamisole and orally administered ivermectin was detected in an isolate of Haemonchus contortus in sheep on a farm where benzimidazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the benzimidazole-resistant isolate were treated with the recommended doses of various anthelmintics. Compared to the control group, the percentage reductions in the faecal egg counts of sheep treated with albendazole, thiophanate, levamisole and ivermectin varied between 38.2% and 79.1% and the residual worm counts between 27.3% and 57.5%. The results indicate the presence of multiple anthelmintic resistance in this isolate of H. contortus. Sheep treated with closantel showed 100% reductions in faecal egg and worm counts, indicating that this drug was very effective against the population of H. contortus on the farm.     

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.     

Efficacy of Albendazole and Levamisole against Gastrointestinal Nematodes of Sheep and Goats in Morogoro,Tanzania

A study was conducted to determine the efficacy of albendazole after it had been withdrawn from use due to the development of resistant strains of nematodes about ten years ago. The study also aimed to determine the present efficacy of levamisole, which had been recommended to replace albendazole. On one farm, the sheep and goats were divided into two groups, one group of each serving as the untreated control, while the other was treated with levamisole. The sheep on the other farm were divided into three groups, one serving as the untreated control group, the second being treated with levamisole and the third being treated with albendazole. Faecal samples were collected one day before treatment, and again 10 days after treatment. Anthelmintic efficacy was determined by the faecal egg count reduction test. Ten days after treatment, the sheep treated with levamisole on the first farm had a 98% reduction in faecal egg count, with a 95% confidence limit of 76%. The goats on the same farm had a 97% reduction in faecal egg count, with a 95% lower confidence limit of 81%. At the second farm, 10 days after treatment, sheep treated with levamisole had a 99.4% reduction in faecal egg count, with a 95% lower confidence limit of 88.9%, whereas the sheep treated with albendazole only had a 59.4% reduction in faecal egg count, with a 95% lower confidence limit of –19.6%. The study indicated that the gastrointestinal nematodes of sheep at the Department of Animal Science and Production farm were still resistant to albendazole about ten years after this anthelmintic had been withdrawn from use. A reduced efficacy of levamisole was suspected.     

Multiple anthelmintic resistance in Haemonchus contortus on a sheep farm in India

Multiple resistance to benzimidazoles (fenbendazole, albendazole and mebendazole) in a strain of Haemonchus contortus in sheep was detected on a farm where fenbendazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the fenbendazole-resistant strain were treated with the recommended doses of various anthelmintics. Compared to the control group, percentage reductions in faecal egg counts of sheep treated with fenbendazole, albendazole, mebendazole, levamisole and morantel varied between 56% and 81% and worm counts between 71% and 86%. The results indicate the presence of multiple anthelmintic resistance in this strain of H. contortus on this farm. Sheep treated with ivermectin and closantel showed 100% reductions in faecal egg and worm counts, suggesting high efficacy of these drugs against the population of H. contortus on this farm.     

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.     

Equine helminth infections: control by selective chemotherapy

A programme of selective anthelmintic therapy was used in a herd of 31 horses. Faecal egg counts were done during the months of September, November, January, March, May and the following September. Horses with greater than or equal to 100 eggs per gram (epg) were treated with ivermectin, and those with less than 100 epg were not treated. The criteria for adequate internal parasite control in the herd was a median herd faecal egg count of less than or equal to 100 epg. Effectiveness of selective therapy was assessed by faecal egg count after nine months of treatment and was determined to be adequate when a median herd egg count of 0 epg was obtained. However, on returning from pasture the following September, median herd egg count had risen to 325 epg. A statistically significant correlation was seen in the paired September faecal egg counts of the horses in that initial September faecal egg count was predictive for the following September. Initial September faecal egg count was related to the number of anthelmintic treatments required during the period of selective therapy, whereas age of horse was not. We propose that faecal egg counts be incorporated into strategic anthelmintic programmes as an economical tool for identifying and targeting herd members predisposed to shedding elevated numbers of helminth eggs.     

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.     

High-level pyrantel resistance in the hookworm Ancylostoma caninum

While anthelmintic resistance is now a widely recognized issue in the livestock industries, its existence within companion animal medicine has been rarely established conclusively. We undertook a placebo-controlled in vivo trial to measure the efficacy of pyrantel embonate against pooled isolates of the hookworm Ancylostoma caninum from Brisbane, Australia. A statistically significant fall in adult worm burden was observed among dogs in the pyrantel treatment group compared to the control dogs (178.0+/-24.5 versus 239.7+/-14.0; p=0.02), equating to an efficacy of just 25.7% (95% CI, 15.0-35.1%), as based upon reduction in mean worm burden. Analysis of faecal egg count trends through the course of the study revealed that egg counts rose in both control and pyrantel-treated dogs, with a greater rise observed in the latter group (11.6+/-8.3% versus 17.3+/-7.6%; p=0.04), despite the decrease in adult worm numbers in this group. Our results indicate that high-level anthelmintic resistance does occur in companion animal medicine, and highlight the need for greater vigilance and more judicious use of anthelmintics in small animal practice. They further indicate that the faecal egg count reduction test needs to be used with caution with this parasite.     

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.     

Anthelmintic treatment and the suppression of egg production in gastro-intestinal nematodes of sheep and cattle: fact or fallacy?

Evidence for a temporary suppression of egg production in gastro-intestinal nematodes of sheep and cattle following anthelmintic treatment is reviewed and the possible relevance of this phenomenon to the appropriate sampling time in the faecal egg count reduction test is discussed. The results of this review suggest that if such suppressive effects do occur in sheep nematodes, then they are unlikely to be of much practical significance and that little benefit would be derived from extending the post-treatment sampling interval beyond 10 days. For cattle nematodes, however, the issues are less readily obvious but indicate, at least when testing for resistance to persistent anthelmintics, that adoption of a longer post-treatment interval might be advisable.