A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes.

A microlarval development test for the detection of anthelmintic resistance in nematodes is described. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis eggs were cultured to third stage larvae in the presence of Earle's balanced salt solution, yeast extract and bacteria in a total volume of 150 microliters. Good dose-response data were obtained with thiabendazole, levamisole, pyrantel tartrate and ivermectin allowing the determination of the 50 per cent lethal concentration and of resistance factors when resistant strains were available. The test was found to be accurate, sensitive, easy to carry out and applicable to the routine detection of resistance.     

Larval development test for detection of anthelmintic resistant nematodes

The growth, using freshly cultured Escherichia coli with ampicillin or heat-treated lyophilised E coli as a food source, of the larvae of the mouse nematode Nematospiroides dubius and the infectivity of resulting third stage larvae were determined. Concentrations of E coli between 0.5 and 1 mg dry weight ml-1 permitted optimal larval development for both N dubius and Trichostrongylus colubriformis. Development of larvae of susceptible and cambendazole-resistant strains of Haemonchus contortus in thiabendazole solutions showed clear differences between the strains and the larval development test was more sensitive than the egg hatch test. The test also detected a levamisole resistant strain of H contortus, although the degree of resistance could not be adequately measured. It is concluded that the test can be run with any anthelmintic to which resistance is suspected.     

Evaluation of a larval development assay (DrenchRite) for the detection of anthelmintic resistance in cyathostomin nematodes of horses

A larval development assay (LDA, DrenchRite) was evaluated to determine the effectiveness of this method in detecting anthelmintic resistance in cyathostomin nematodes of horses. A total of 15 horse farms from Georgia and South Carolina (USA) and Population S ponies from the University of Kentucky (USA) were included in this study. Nematode eggs were extracted from pooled fecal samples and placed into the wells of a DrenchRite plate for testing against thiabendazole (TBZ), levamisole (LEV) and 2 ivermectin (IVM) analogs (IVM-1, IVM-2). After a 7-day incubation larvae in each well were counted and data were analyzed by logistic regression. Resistance status of each farm for different drugs was determined in a separate study using a fecal egg count reduction test. LDA were performed on the 15 farms once, however, the Population S cyathostomins were assayed on 3 separate occasions to estimate the consistency of results between assays. Mean TBZ LC50 for oxibendazole resistant, suspected resistant and sensitive farms were 0.2015, 0.1625, and 0.1355 microM, respectively. For LEV, mean LC50 for PYR resistant, suspected resistant and sensitive farms were 1.590, 1.8018 and 1.4219 microM, respectively. All 15 farms had worms susceptible to IVM; mean LC50 for IVM-1 and for IVM-2 were 7.5727 and 87.9718 nM, respectively. A linear mixed model was fitted to the data to determine the relationship between LC50 and LC95 and resistance status for each farm. No meaningful relations were found. Consistency of assays varied between drugs, being best for TBZ and worst for LEV and IVM-1. All farms in this study had benzimidazole-resistant nematodes; therefore usefulness of DrenchRite for discriminating susceptibility versus resistance to this drug class could not be accurately assessed. Moreover, since all farms tested were sensitive to IVM and resistance to this drug class has not yet been reported in cyathostomins, it is not possible to assess accurately the usefulness of DrenchRite LDA for detecting IVM resistance at this time. Assay results for LEV suggest that LEV in a LDA does not yield data that is useful in estimating PYR efficacy in vivo. Based on results for PYR/LEV, the current high prevalence of benzimidazole resistance, no known cases of IVM resistance, and the sometimes extreme variation in results seen in many of the assays, DrenchRite LDA cannot be considered a useful tool for the diagnosis of resistance in cyathostomins of horses at present.     

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.     

Reliability and reproducibility of the larval paralysis test as an in vitro method for the detection of anthelmintic resistance of nematodes against levamisole and morantel tartrate

In order to study the reliability of the larval paralysis test as an in vitro assay for the detection of resistance of nematodes to levamisole and morantel tartrate, the influence of different parameters was evaluated using resistant and susceptible Ostertagia ostertagi strains. The operator, the sample (10% of the larvae present in the suspension), the incubation time (24, 48 or 72 h), the incubation temperature (20 or 25 degrees C) and the observation period of the larvae (5 or 15 s) had no statistically significant influence on the test results. Statistical differences were obtained only when L3 larvae of different ages (1 or 2 months) were used. Reversibility of the paralysis did not occur when concentrations of levamisole of less than or equal to 200 micrograms ml-1 or morantel tartrate of less than 2000 micrograms ml-1 were used. The reproducibility of the test was fairly good, with a mean standard deviation of 21.3% for the LC50 values. Morantel resistance in a strain of O. ostertagi was not confirmed as such by the larval paralysis test. The resistance factor was less than or equal to 1, in spite of an efficacy of morantel of 77.4% as shown in vivo.     

Potential limitations of the undifferentiated faecal egg count reduction test for the detection of anthelmintic resistance in sheep

Larval cultures were used to determine the identities and occurrences of those parasites (excluding Nematodirus) represented by strongylid eggs at the time of anthehnintic administration in ovine faecal egg count reduction tests submitted to the Batchelar Animal Health Laboratory between 1992 and 1994. The numbers of individual nematode genera recorded in pre-treatment samples from these cases ranged from one to five and included infections of Haemonchus, Ostertagia, Ttichostrongylus, Cooperia and Oesophagostomum/Chabertia. Adequate egg representation for testing purposes by all five genera simultaneously was found to occur in only 17 (10%) of the 163 cases examined, with the majority (71%) of them containing between one and three nematode genera. The greatest representation occurred in those tests conducted during the months of February to May. However, even during this period, worm eggs of all five genera were concurrently present on only 16% of occasions. The importance of knowing what nematode genera are adequately represented at the time of routine faecal egg count reduction testing and the relevance of this information to reducing the likelihood of being misled when under- taking assessments of farm resistance status are discussed.     

Further potential limitations of the undifferentiated faecal egg count reduction test for the detection of anthelmintic resistance in sheep

Fifteen (36%) of 42 mixed gastro-intestinal nematode infections in sheep, identified as drench-susceptible by the undifferentiated faecal egg count reduction test, were found to harbour anthelmintic-resistant worms when analysed on the basis of changes in the egg counts of individual nematode genera. Most of these cases involved resistance in a single nematode genus, with Ostertagia and Trichostrongylus being implicated most frequently. The possible contributory role of larval culture results in helping to reduce the chances of the faecal egg count reduction test producing these and similar types of errors is discussed.     

Update on the prevalence of anthelmintic resistance in gastrointestinal nematodes of sheep in New Zealand

Abstract

Extract

Recently, a comparison was made between the prevalence of anthelmintic resistance, determined using faecal egg count reduction tests (FECRT), on randomly selected sheep farms in a systematic national survey (Waghorn et al. 2006 Waghorn, TS, Leathwick, DM, Rhodes, AP, Lawrence, KE, Jackson, R, Pomroy, WE, West, DM and Moffat, JR. 2006. Prevalence of anthelmintic resistance on sheep farms in New Zealand. New Zealand Veterinary Journal, 54: 271–277. [Taylor & Francis Online], [Web of Science ®] , [Google Scholar]) and that derived from similar case material submitted to a veterinary pathology laboratory on a more ad-hoc basis (McKenna 2008 McKenna, PB. 2008. An examination of the relative reliability of laboratory case submissions in determining the prevalence of anthelmintic resistance in sheep nematodes in New Zealand and the possible influence of test analysis methodology on such data. New Zealand Veterinary Journal, 56: 55–59. [Taylor & Francis Online], [Web of Science ®] , [Google Scholar]). The results of that comparison showed that while there were some differences between them, there were considerable similarities in the prevalence figures obtained from either source. Those similarities, which were particularly evident in terms of the overall pattern of involvement of the various worm genera and the types of anthelmintic concerned, led to the conclusion that FECRT case submissions to veterinary laboratories may offer a useful source of information regarding changes in the prevalence of anthelminticresistant sheep nematodes in New Zealand (McKenna 2008 McKenna, PB. 2008. An examination of the relative reliability of laboratory case submissions in determining the prevalence of anthelmintic resistance in sheep nematodes in New Zealand and the possible influence of test analysis methodology on such data. New Zealand Veterinary Journal, 56: 55–59. [Taylor & Francis Online], [Web of Science ®] , [Google Scholar]). Accordingly, the present study was undertaken to make further use of this material, to try to ascertain what, if any, such changes may have taken place over the last few years.     

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

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

Comparison of two versions of larval development test to detect anthelmintic resistance in Haemonchus contortus

Larval development (LDT) and micro-agar larval development tests (MALDT) were used to compare the reliability and sensitivity of two methods for detecting anthelmintic resistance in Haemonchus contortus. The tests were conducted using three resistant and four susceptible isolates of H. contortus. Both versions of the tests provided comparable results with regard to the characterization of benzimidazole and levamisole susceptibility but neither test was sufficiently sensitive to discrimination between an ivermectin (IVM) susceptible and an IVM resistant isolate. Each test has its own merits with the LDT having the advantage of being less time-consuming.     

The prevalence of anthelmintic resistance in sheep nematodes on the central tablelands of New South Wales

A survey was undertaken to assess the prevalence of anthelmintic resistance in sheep nematode populations on 40 commercial farms distributed throughout the central tablelands of New South Wales. Representatives of the 2 major groups of broad spectrum anthelmintics with different modes of action (thiabendazole and levamisole) were used at the manufacturer's recommended dose rates. Efficacy was assessed on the basis of the reduction in faecal strongyle egg counts 7 days after treatment. An efficacy of less than 90% using both anthelmintics was obtained on 4 farms. Thiabendazole had an efficacy of less than 90% on a further 21 farms and levamisole had an efficacy of less than 90% on an additional 4 farms. There was no evidence of anthelmintic resistance on 8 farms, while the remaining 3 had insignificant parasite burdens. Based on larval cultures from faeces, Ostertagia and Trichostrongylus were the most significant species in resistant populations. Haemonchus burdens were sporadic and levels of resistance relatively low. Nematodirus burdens were widespread but no evidence of resistance was detected.     

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.