Cyathostomins In Horses Research Articles

Searching for ivermectin resistance in Dutch horses

A study was conducted to evaluate the occurrence of resistance against, in particular, ivermectin in cyathostomins in the Netherlands. Seventy horse farms were visited between October 2007 and November 2009. In initial screening, faecal samples were collected 2weeks after deworming with either ivermectin, moxidectin or doramectin. Pooled faecal samples from a maximum of 10 horses were examined for worm eggs using a modified McMaster technique and for worm larvae after faecal larval cultures. In total 931 horses were involved. On 15 of 70 farms eggs and/or larvae were found. On 8 of these 15 farms a FECRT with ivermectin was performed on 43 horses. Efficacy of ivermectin against cyathostomins of 93% was found in one animal on one farm. Additionally, the strategies and efforts of the horse owners to control cyathostomins, as well as risk factors for the development of macrocyclic lactone resistance were evaluated with a questionnaire. Strikingly, many responders indicated that the control of cyathostomins in horses is achieved through very frequent deworming. Fourteen percent of these owners deworm seven times per year or more. On 34% of the 70 farms treatment was repeated within the Egg Reappearance Period of a product.

Isolation of Potentially Useful Antigens from Cyathostomin Third-Stage Larvae by Using a Fast Protein Liquid Chromatography One-Step Method

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes.

Biological control of cyathostomin (Nematoda: Cyathostominae) with nematophagous fungus Monacrosporium thaumasium in tropical southeastern Brazil

Horses are hosts to a wide variety of helminthes; the most important are the cyathostomin, or small strongyles. The viability of a fungal formulation (pellets) using the nematode-trapping fungus Monacrosporium thaumasium was assessed in biological control of horse cyathostomin. Two groups (fungus-treated and control) consisted of six mares in each group, crossbred (ages of 2.5 and 3.5 years), were placed in pastures of Cynodon sp. naturally infected with horse cyathostomin larvae. In the treated group, each animal received 1g/10kg body weight (0.2g/10kg live weight of fungus) of pellets of sodium alginate matrix containing the fungus M. thaumasium orally, twice a week for 6 months. In the control group, animals received (1g/10kg body weight) of pellets without fungus. The egg count per gram of feces showed difference (p<0.01) in the animals treated with the fungus in relation to the control animals during all months of the experiment. The EPG percentage decrease were 87.5%, 89.7%, 68.3%, 58.7%, 52.5% and 35.2% during June, July, August, September, October and November, respectively. In faecal cultures, there was difference (p<0.05) among animals treated with fungus was found in relation to the control animals during all the experiment month, with percentage reduction of 67.5%, 61.4% and 31.8% in September, October and November, respectively. Difference (p<0.01) was observed in the recovery of infective larvae from pastures that were collected up to 20cm from the dung pats in pastures in the group treated with the fungus in relation to the control group with a reduction of 60.9% and between 0–20 and 0–40cm from the faecal pat reduction (p<0.01) was about 56% in the group treated with the fungus M. thaumasium in relation to the control group pasture. There was no difference (p>0.05) between the average weight gains in both animal groups. The treatment of horses with pellets containing the nematophagous fungus M. thaumasium can be effective in controlling cyathostomin in the tropical region of southeastern Brazil.

Optimizing protease production from an isolate of the nematophagous fungusDuddingtonia flagransusing response surface methodology and its larvicidal activity on horse cyathostomins

Protease production from Duddingtonia flagrans (isolate AC001) was optimized and the larvicidal activity of the enzymatic extract was evaluated on infective horse cyathostomin larvae (L3). Duddingtonia flagrans was grown in liquid medium with eight different variables: glucose, casein, bibasic potassium phosphate (K2HPO4), magnesium sulphate (MgSO4), zinc sulphate (ZnSO4), ferrous sulphate (FeSO4), copper sulphate (CuSO4) and temperature. The Plackett-Burman analysis showed a significant influence of MgSO4, CuSO4 and casein (P < 0.05) on protease production by D. flagrans in liquid medium. Central composite design indicated that the highest proteolytic activity was 39.56 U/ml as a function of the concentrations of casein (18.409 g/l), MgSO4 (0.10 g/l) and CuSO4 (0.50 mg/l). A significant difference (P < 0.01) was found for the larval number between the treated and control groups at the end of the experiment. A reduction of 95.46% in the number of free-living larvae was found in the treated group compared with the control. The results of this study suggest that protease production by D. flagrans (AC001) in liquid medium was optimized by MgSO4, CuSO4 and casein, showing that the optimized enzymatic extract exerted larvicidal activity on cyathostomins and therefore may contribute to large-scale industrial production.

Predatory activity of the nematophagous fungus Duddingtonia flagrans on horse cyathostomin infective larvae

This work was performed to determine the predatory capacity in vitro of the nematophagous fungus Duddingtonia flagrans (isolate AC001) on cyathostomin infective larvae of horse (L(3)). The experimental assay was carried out on plates with 2% water-agar (2% WA). In the treated group, each plate contained 1.000 L(3) and 1.000 conidia of the fungus. The control group without fungus only contained 1.000 L(3) in the plates. Ten random fields (4 mm diameter) were examined per plate of treated and control groups, every 24 h for seven days under an optical microscope (10x and 40x objective lens) for non-predated L(3) counts. After 7 days, the non-predated L(3) were recovered from the Petri dishes using the Baermann method. The interaction there was a significant reduction (p < 0.01) of 93.64% in the cyathostomin L(3) recovered. The results showed that the D. flagrans is a potential candidate to the biological control of horse cyathostomin L(3).

Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil

The viability of a fungal formulation using the nematode-trapping fungus Duddingtonia flagrans was assessed for the biological control of horse cyathostomin. Two groups (fungus-treated and control without fungus treatment), consisting of eight crossbred mares (3–18 years of age) were fed on Cynodon sp. pasture naturally infected with equine cyathostome larvae. Each animal of the treated group received oral doses of sodium alginate mycelial pellets (1 g/(10 kg live weight week)), during 6 months. Significant reduction ( p < 0.01) in the number of eggs per gram of feces and coprocultures was found for animals of the fungus-treated group compared with the control group. There was difference ( p < 0.01) of 78.5% reduction in herbage samples collected up to (0–20 cm) between the fungus-treated group and the control group, during the experimental period (May–October). Difference of 82.5% ( p < 0.01) was found between the fungus-treated group and the control group in the sampling distance (20–40 cm) from fecal pats. During the last 3 months of the experimental period (August, September and October), fungus-treated mares had significant weight gain ( p < 0.01) compared with the control group, an increment of 38 kg. The treatment with sodium alginate pellets containing the nematode-trapping fungus D. flagrans reduced cyathostomin in tropical southeastern Brazil and could be an effective tool for biological control of this parasitic nematode in horses.

Comparison of two fecal egg recovery techniques and larval culture for cyathostomins in horses

To compare the McMaster and centrifugal flotation techniques and larval culture for recovery of cyathostomin (small strongyle) eggs from the feces of horses. Fecal samples from 101 horses. In experiment I, homogenized fresh feces from a single horse were randomly subsampled by use of each technique for 10 replicates. In experiment II, samples from 43 horses that had no anthelmintic treatment were analyzed by use of McMaster, centrifugal flotation, and larval culture techniques. In experiment III, 57 horses were treated with an anthelmintic by owners, and fecal samples were analyzed as for experiment II. In experiment I, use of the McMaster technique recovered 72% of the eggs obtained by use of centrifugal flotation from paired subsamples. In experiment II, use of the McMaster technique recovered 81% of the eggs obtained by use of centrifugal flotation. Only cyathostomins resulted from individual larval cultures. In experiment III, 24 samples had negative results for all 3 tests, 18 samples had positive results only with larval cultures, and 15 samples had positive results of centrifugal flotation (only 5 of which had positive results via the McMaster technique). Centrifugal flotation consistently was superior to the McMaster technique, especially at low fecal egg numbers. The combination of centrifugal flotation and larval culture may provide the best accuracy for evaluation of anthelmintic efficacy.

Comparison of two fecal egg recovery techniques and larval culture for cyathostomins in horses

Comparison of two fecal egg recovery techniques and larval culture for cyathostomins in horses

Equine parasites: diagnosis and control - a current perspective

Equine parasites: diagnosis and control - a current perspective

Faecal Cyathostomin Egg Count distribution and efficacy of anthelmintics against cyathostomins in Italy: a matter of geography?

BackgroundIn the framework of a trial carried out in 2008 in Europe to evaluate the efficacy of major parasiticides against horse cyathostomins, pre- and/or post-treatment Faecal Egg Counts (FEC) were evaluated in a total of 84 yards and 2105 horses from nine different regions from the South, the Center, the North-Center and North-East of Italy. Specifically, on the basis of FECs of the horses present in each property, 60 out of the 84 yards were enrolled for a Faecal Egg Count Reduction Test (FECRT) using fenbendazole, pyrantel, ivermectin and moxidectin.ResultsOf the 1646 horses bred in the 60 recruited yards, 416 animals had a FEC between 50 and 150 Eggs Per Gram (EPG) of faeces and 694 a FEC >150 EPG (i.e. with total of 1110 positive animals). Of the 1110 positive animals, those with the highest FECs (i.e. 988) were included in the FECRT. The FECRT for four anthelmintic compounds showed remarkable differences in terms of prevalence of reduced and equivocal efficacy against cyathostomins in the different areas of Italy. Administration of fenbendazole and pyrantel resulted in resistance present or suspected in about half of the yards examined while resistance to ivermectin was found in one yard from central Italy and suspected resistance was detected in three more yards, one in each the North, the Center and the South. Treatment with moxidectin was 100% effective in all yards examined.ConclusionCyathostomin populations in the South and the Center of Italy were more susceptible to fenbendazole and pyrantel than the populations present in the Center-North and North-Eastern areas of Italy. Fenbendazole and/or pyrantel were ineffective in almost all properties from the North of Italy. The reasons for such a difference among the Italian regions in terms of FECs and efficacy of antiparasitic drugs are discussed, together with the role that veterinarians, and horse owners and managers should have for effective worm control programs in this country.

Anthelmintic resistance in cyathostomins of brood horses in Ukraine and influence of anthelmintic treatments on strongylid community structure

In 2004–2006, 322 brood horses from 11 horse farms were examined using the faecal egg count reduction test (FECRT) to determine the presence and distribution of anthelmintic resistance in strongylids in Ukraine. The anthelmintic drugs “Albendazole-7.5” (7.5 mg of albenazole, Ukraine) at a dose of 5 mg per kg body weight and “Univerm” (0.2% aversectin C, Russia) at a dose of 0.5 mg per kg body weight were used. Seventy-one horses from six farms were examined in vivo to investigate the influence of anthelmintic treatment on the gastrointestinal strongylid community structure. Horses were treated with anthelmintics; faecal sampling (200 g in each sample) for strongylid expulsion was performed 24, 36, 48 and 60 h after treatment; and all strongylids expelled (25,292 specimens) were collected and identified. Fourteen horses from the Dubrovsky horse farm were also examined to determine the benzimidazole-resistant cyathostomin species; 5208 specimens of benzimidazole-resistant cyathostomins were collected and identified. According to the FECRT data, benzimidazole resistance in strongylids was observed only at the Dubrovsky horse farm (FECRT = 68.7%). No resistance to macrocyclic lactones in strongylids or in Parascaris equorum was observed. Twenty-nine strongylid species were found in horses from six horse farms. The number of species per horse ranged from 4–9 (5.8 ± 1.5) to 10–20 (14.4 ± 2.9) and depended on horse anthelmintic treatment strategies. From 4 to 13 strongylid species predominated (prevalence > 66.7%) in the strongylid community. Eleven cyathostomin species ( Cylicocyclus nassatus, C. ashworthi, C. leptostomum, Cyathostomum catinatum, C. pateratum, Cylicostephanus calicatus, C. longibursatus, C. goldi, C. minutus, Coronocyclus coronatus and C. labiatus) were found to be resistant to benzimidazoles at the Dubrovsky horse farm. Ten of these were the dominant species in the strongylid community; only C. labiatus was a rare species (prevalence 29.4%). Species richness and species diversity were significantly higher in horses from farms without treatment or with occasional treatments than from farms with regular treatments. The shape of the prevalence frequency distribution of strongylid species from farms with regular treatments was bimodal (“core” and “satellite” mode). This distribution was multimodal at farms without treatment or with occasional anthelmintic treatments. The results of the current study indicated the possibility of the further spread of anthelmintic resistance on horse farms in Ukraine and the necessity of monitoring the development of resistance in horse parasitic nematodes.

The ecology of horse cyathostomin infective larvae (Nematoda-Cyathostominae) in tropical southeast Brazil

Experimental studies about the recovery, survival and migration to pasture of cyathostomin infective larvae (L 3) from fresh feces depositions were conducted from February 2005 to March 2007 in a tropical region of southeast Brazil. Grass and feces were collected weekly at 8 a.m., 1 and 5 p.m. and processed by the Baermann technique. Multivariate analysis (principal components method) showed the influence of time and environmental variables on the number of infective larvae recovered from the feces and pasture. In the rainy period (October–March), more infective larvae were recovered on the feces and grass apex. In contrast, in the dry period (April–September), the recovery was higher only on the grass base, as well as the L 3 survival on feces and grass. More larvae were recovered at 8 a.m., except from the grass apex, where the highest recovery was at 1 p.m. Few studies investigating the seasonal transmission of equine cyathostomin have been conducted in South American tropical climates. These results demonstrate that in tropical conditions L 3 are available on feces and pasture throughout the year. Knowledge of climatic influences on the development and survival of L 3 is crucial to designing integrated parasite control programs that provide effective protection while slowing the development of anthelmintic resistance.

A Field Study on the Effect of Some Anthelmintics on Cyathostomins of Horses in Sweden

The objective of the study was to investigate different aspects on the efficacy of three anthelmintics on cyathostomin nematodes of Swedish horses. A faecal egg count reduction (FECR) test was performed on 26 farms. Horses were treated orally with recommended doses of ivermectin, pyrantel pamoate or fenbendazole. Faecal samples were collected on the day of deworming and 7, 14 and 21 days later. No resistance was shown against ivermectin; the FECR was constantly >99%. The effect of pyrantel was assessed as equivocal in 6 farms 14 days after treatment; the mean FECR was 99%. As many as 72% of the fenbendazole-treated groups met the criteria for resistance; the mean FECR was 86%, ranging from 56% to 100%. A re-investigation of two farms where pyrantel resistance had been suspected clearly revealed unsatisfactory efficacy of pyrantel on one of these farms; the FECR varied from 72% to 89%. Twenty-six of the horses previously dosed with pyrantel or fenbendazole, and which still excreted >/=150 eggs per gram of faeces 14 days after treatment, were dewormed with ivermectin and fenbendazole or pyrantel in order to eliminate the remaining cyathostomins. A total of 13 cyathostomin species were identified from horses that initially received fenbendazole and seven species were identified from pyrantel-treated individuals. The egg reappearance period (ERP) following treatment with ivermectin and pyrantel was investigated on two farms. The shortest ERP after ivermectin treatment was 8 weeks and after pyrantel was 5 weeks. We conclude that no substantial reversion to benzimidazole susceptibility had taken place, although these drugs have scarcely been used (<5%) in horses for the last 10 years. Pyrantel-resistant populations of cyathostomins are present on Swedish horse farms, but the overall efficacy of pyrantel is still acceptable.

Drug resistance and drug tolerance in parasites

In their recent Trends in Parasitology article on tolerance and antimalarial drug resistance, Hastings and Watkins [1xTolerance is the key to understanding antimalarial drug resistance. Hastings, I.M. and Watkins, W.M. Trends Parasitol. 2006; 22: 71–77Abstract | Full Text | Full Text PDF | PubMed | Scopus (50)See all References][1] made the extremely important point that resistance is not an all-or-nothing effect, and that several mutations can occur before the malarial parasite fails to respond to the full therapeutic treatment in the patient. Clearly, this must be taken into account in any modelling on the development of resistance. The authors use the word ‘tolerance’ to describe the intermediate state between susceptibility and complete drug failure. We made the same mistake in work carried out in Kenya in the 1980s, which showed the ‘resistance’ of Schistosoma mansoni to oxamniquine in an article entitled ‘Tolerance of Kenyan Schistosoma mansoni to oxamniquine’ [2xTolerance of Kenyan Schistosoma mansoni to oxamniquine. Coles, G.C. et al. Trans. R. Soc. Trop. Med. Hyg. 1987; 81: 782–785PubMed | Scopus (33)See all References][2]. It should have been entitled ‘resistance’.To try and correct this misunderstanding, we defined resistance in Schistosoma as the following [3xDefining resistance in Schistosoma. Coles, G.C. and Kinoti, G.K. Parasitol. Today. 1997; 13: 157–158Abstract | Full Text PDF | PubMedSee all References][3]: ‘A population of Schistosoma is resistant when either a susceptible population shows a significant decrease in response to a schistosomicide or is significantly less responsive than a fully susceptible population…It is complete when the maximum dose of drug tolerated by the host has no effect on the parasite population.’ As used by Fallon et al. [4xSchistosome resistance to praziquantel: fact or artifact?. Fallon, P.G. et al. Parasitol. Today. 1996; 12: 316–320Abstract | Full Text PDF | PubMed | Scopus (60)See all References][4] and discussed in our previous letter [3xDefining resistance in Schistosoma. Coles, G.C. and Kinoti, G.K. Parasitol. Today. 1997; 13: 157–158Abstract | Full Text PDF | PubMedSee all References][3], ‘tolerance is an innate insusceptibility of a parasite to a drug, even before the parasite has been exposed to the drug.’ We cited the example of oxamniquine, which does not work against Schistosoma haematobium. Tolerance can be total or it can be stage-specific; for example, ivermectin does not kill inhibited cyathostomins in horses but is extremely effective against the lumenal stages [5xThe effect of ivermectin treatment against inhibited early third stage, late third stage and fourth stage larvae and adult stages of the cyathostomes in Shetland ponies and spontaneous expulsion of these helminths. Eysker, M. et al. Vet. Parasitol. 1992; 42: 295–302Crossref | PubMed | Scopus (43)See all References][5]. There is a similar confusion by farmers when they think anthelmintics are working adequately but low levels of resistance are detected on the farm. This low-level resistance will not necessarily show up as lost productivity but serves as a warning that future problems will develop if changes are not made in the use of that anthelmintic. This slightly lowered efficacy is not tolerance, but resistance.I believe it is important to use the same terminology when discussing drug resistance, whether it is in malaria, nematodes or any other parasites. I would, therefore, like to suggest that the definition we proposed in 1997 [3xDefining resistance in Schistosoma. Coles, G.C. and Kinoti, G.K. Parasitol. Today. 1997; 13: 157–158Abstract | Full Text PDF | PubMedSee all References][3] is adopted by all parasitologists, and that ‘tolerance’ is not used to describe intermediate levels of resistance but is confined to the innate inactivity of a drug towards a stage or a particular species of parasite, in which activity might be expected as a result of drug efficacy against a closely related species.

Larval development assay for detection of anthelmintic resistance in cyathostomins of Swedish horses

The aim of this study was to investigate the suitability of a larval development assay (LDA) for the determination of anthelmintic resistance in cyathostomin nematode populations of the horse. In addition, comparison of results between geographic regions, types of horse establishment, and the use of anthelmintics in Sweden, was established. Seventy horse herds from different parts of Sweden were sampled, and strongyle eggs from the faeces of 54 of those were investigated by an LDA (DrenchRite ™). The following anthelmintics were tested: thiabendazole (TBZ), levamisole (LEV), ivermectin monosaccharide (IVM-MS), ivermectin aglycone (IVM-AG) and pyrantel (PYR). The LC50 values for TBZ and LEV were generally lower than those previously reported in other LDA studies on horse nematodes. This could be related to the infrequent use of these compounds for the past 20 years in Sweden. In this study, there was a great variation within and between assay plates that could not be explained. Still the LC50 values differed significantly between the regions for all anthelmintics, except for pyrantel. The highest LC50s were observed in parasite populations from the south of Sweden. There were no significant differences between riding schools and studs. Limitations of this technique exist, namely the lack of established cut-off values for susceptible and resistant populations and interpretation problems related to multi-species infections. Although there are advantages with LDA such as the possibility of testing several compounds simultaneously without interference with the deworming programmes on the farms, we conclude that LDA currently is not a reliable alternative to the faecal egg count reduction test (FECRT).

EVIDENCE OF P-GLYCOPROTEIN SEQUENCE DIVERSITY IN CYATHOSTOMINS

P-glycoproteins (Pgps) are adenosine triphosphate-binding transporter proteins thought to be associated with multi-drug resistance in mammals and protozoans and have been suggested to be involved in the mechanism of ivermectin (IVM) resistance in Haemonchus contortus. Until now, resistance to IVM has not been reported in cyathostomins in horses in spite of its widespread and frequent use. Reasons for this might be differences in the molecular mechanism of the development of resistance. Based on this hypothesis, the present study was carried out to find homologues of Pgp in cyathostomins. A 416-bp polymerase chain reaction (PCR) product was generated using complementary DNA (cDNA) of Cylicocyclus elongatus and Cylicocyclus insigne and degenerate primers, located in the conserved Pgp nucleotide-binding domains. Resulting PCR products showed interspecific nucleotide and amino acid sequence identities of 73.3 and 76.8%, respectively. Specific primers were designed based on the Cc. elongatus sequence, and a PCR product of 268-bp was amplified from cDNA of single adults of Cylicocyclus radiatus, Cc. insigne, Cylicocyclus nassatus, Cc. elongatus, Cylicostephanus hybridus (2 individuals), Cylicostephanus goldi, Cyathostomum pateratum, Cyathostomum coronatum, and Cyathostomum catinatum. Two clusters of sequences were found representing 2 different internucleotide-binding domains (IBDs). A further distinct IBD is represented by the 416-bp PCR product of Cc. insigne. Therefore, a total of 3 clearly different sequences of the IBD were cloned and sequenced, suggesting that at least 2 Pgp genes exist in cyathostomins.

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 LC 50 for oxibendazole resistant, suspected resistant and sensitive farms were 0.2015, 0.1625, and 0.1355 μM, respectively. For LEV, mean LC 50 for PYR resistant, suspected resistant and sensitive farms were 1.590, 1.8018 and 1.4219 μM, respectively. All 15 farms had worms susceptible to IVM; mean LC 50 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 LC 50 and LC 95 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.