Analysis of the strongylid nematodes (Nematoda: Strongylidae) community after deworming of brood horses in Ukraine

Communities of intestinal helminths in horses are commonly studied post mortem. The study objectives were here to examine the species composition of the strongylid community in brood horses in Ukraine after deworming with an aversectin drug Univerm. The site distribution of the strongylid species was analysed according to dynamics of their expulsion in faeces. Forty-four horses of different ages from Poltavska oblast (22 horses), Kyivska oblast (17 horses) and Sumska oblast (5 horses) of Ukraine were included in the study. Horses were treated with Univerm anthelmintic (0.2% aversectin) at a dose rate of 0.5mg aversectin preparation per kg body weight. Faecal sampling (200 g each) was performed at 24, 36, 48 and 60 h post treatment, and all nematodes expelled were collected and identified. The largest numbers of strongylids were expelled at 24--36 h after treatment. Twenty-five nematode species from the subfamilies Strongylinae and Cyathostominae were identified. The number of strongylid species found per horse ranged from 7 to 20, on an average 11+/-3.6 (S.D.). The number of cyathostomin species found per horse ranged from 7 to 16, on an average 10+/-2.3 (S.D.). Cylicocyclus nassatus and Cyathostomum catinatum were the most dominant species were found in 100% of horses, amounting to 36.3% and 17.6% of the total number of strongylids collected, respectively. C. longibursatus, C. ashworthi, Cylicostephanus calicatus, C. leptostomus and C. minutus were identified in more than 80% horses and represented 39.9% of the total number of strongylids collected. The dynamics of the different strongylid species expelled was irregular. Correlation between the time of cyathostomin species expulsion in faeces and their predicted localisation inside the horse intestine was found. Species mainly localised in the caecum were found in faeces later than those species localised in the dorsal and ventral colons. Larvae and adult Parascaris equorum, Oxyuris equi and botfly larvae from the genus Gasterophilus were also found in horse faeces. This investigation shows that is possible to study the horse strongylid community after deworming with aid of an aversectin drug. The results obtained here correspond to those recorded in previous autopsy surveys in other countries.     

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).     

The in vitro diagnosis of anthelmintic resistance in cyathostomins

Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resistance in different nematode populations. At present, the faecal egg count reduction test is considered the most suitable method for initial screening for anthelmintic resistance in equine nematode populations. However, in its current state, this test lacks sensitivity. It is also costly and time-consuming to perform. Laboratory-based techniques, such as the egg hatch assay, larval development assay, larval migration inhibition assay and the larval feeding inhibition assay offer alternative options for assessing anthelmintic resistance in nematode populations. All of these tests have been investigated for their utility in measuring drug resistance in sheep nematode populations and some have proven useful. The egg hatch assay, larval development assay and larval migration inhibition assay have been investigated for use in measuring levels of drug resistance in equine nematode populations. However, at best, the results obtained thus far indicate that these tests require further refinement.     

An update on cyathostomins: Anthelmintic resistance and worm control

Intestinal nematodes are an important cause of equine disease. Of these parasites, the Cyathostominae are the most important group, both in terms of their prevalence and their pathogenicity. Cyathostomin infections are complex and control is further complicated by ever‐increasing levels of resistance to some of the commonly used anthelmintics. There are no new equine anthelmintics under development, so it is imperative that the efficacy of any currently‐effective drug classes be maintained for as long as possible. It is believed that the proportion of refugia (i.e. the percentage of parasites not exposed to a drug at each treatment) is one of the most crucial factors in determining the rate at which anthelmintic resistance develops. It is important, therefore, that levels of refugia be taken into account when designing nematode control programmes for horses. This can be assisted by knowledge of the local epidemiology of the infection, supplemented by faecal egg count analysis to identify those animals that are making the major contribution to pasture contamination. This type of rational nematode control requires equine veterinary surgeons to get involved in designing and implementing deworming programmes. The advice given must be based on a combination of knowledge of cyathostomin biology and epidemiology as well as an awareness of the parasite population's current drug sensitivity and a sound history of husbandry at the establishment. As anthelmintic resistance will be the major constraint on the future control of cyathostomins, researchers are now actively investigating this area. Studies are underway to develop tests that will enable earlier detection of anthelmintic resistance and an assay that will help identify those horses that require anthelmintic treatments targeted at intestinal wall larvae.     

Anthelmintic resistance in nematodes of horses

Suppressive anthelmintic treatment strategies originally designed to control Strongylus vulgaris in horses were extremely successful in reducing morbidity and mortality from parasitic disease. Unfortunately, this strategy has inadvertently resulted in the selection of drug-resistant cyathostomes (Cyathostominea), which are now considered the principal parasitic pathogens of horses. Resistance in the cyathostomes to benzimidazole drugs is highly prevalent throughout the world, and resistance to pyrantel appears to be increasingly common. However, there are still no reports of ivermectin resistance in nematode parasites of horses despite 20 years of use. It is unknown why resistance to ivermectin has not yet emerged, but considering that ivermectin is the single most commonly used anthelmintic in horses most parasitologists agree that resistance is inevitable. The fecal egg count reduction test is considered the gold standard for clinical diagnosis of anthelmintic resistance in horses, but diagnosis is complicated by lack of an accepted standard for the performance of this test or for the analysis and interpretation of data. Presently there is very little data available on the molecular mechanisms of anthelmintic resistance in cyathostomes; beta-tubulin gene is the only anthelmintic-resistance associated gene that has been cloned. The increasingly high prevalence of anthelmintic-resistant cyathostomes must be taken into account when designing worm control programs for horses. Strategies to decelerate further selection for drug resistance thereby extending the lifetime of currently effective anthelmintics should be implemented whenever possible. Considering the nature of the equine industry in which horses often graze shared pastures with horses from diverse locations, transmission and widespread dispersal of resistant parasites is virtually assured. A proactive approach to this problem centered on understanding the molecular basis of anthelmintic resistance in cyathostomes is required if we are to expect chemical control of nematodes in horses to remain a viable element of parasite control in the future.     

Anthelmintic resistance in non-strongylid parasites of horses

Since 2002, selected populations of Parascaris equorum in several countries have been reported to survive treatment with macrocyclic lactone (M/L) anthelmintics. Clinical treatment failures are characterized by negligible fecal egg count reduction, but M/L resistance has been confirmed in ascarids by controlled efficacy testing. Resistance was selected by current parasite control practices for foals, which often include exclusive and excessively frequent use of M/L dewormers, thereby minimizing refugia within the host and in the environment. Chemical control of M/L-resistant isolates can be accomplished with pyrimidine and/or benzimidazole anthelmintics, but a few M/L-resistant populations have recently exhibited resistance to pyrantel pamoate as well. Some specimens of Oxyuris equi regularly survive treatment with macrocyclic lactones, but it is uncertain whether this constitutes resistance or merely confirms the incomplete oxyuricidal efficacy of virtually all broad spectrum equine anthelmintics. Variations in other biological parameters of Oxyuris and Parascaris, specifically atypical infection of older hosts and shorter prepatent periods, have been reported anecdotally. These changes may represent genetic modifications that have evolved in parallel with resistance as a result of anthelmintic selection pressure.     

Determination of anthelmintic efficacy against equine cyathostomins and Parascaris equorum in France

This article reports the results of a faecal egg count reduction test on 4 farms in France, as an integrated part of the routine deworming strategy against horse cyathostomins and Parascaris equorum. Treatment with fenbendazole (FBZ) or ivermectin (IVM) was evaluated in yearlings on Farms 1 and 2 and treatment with pyrantel embonate (PYR) was tested on Farms 3 and 4. Calculation of the arithmetic mean faecal egg count reduction and the 95% confidence intervals (95% CI) around the mean was performed using bootstrap analysis. For equine cyathostomins, resistance to FBZ was found with an arithmetic mean reduction of 48.8% (95% CI: 1.9–69.3%). On Farms 1 and 2, horses with reduced efficacy were identified. PYR was found to be effective against cyathostomins, with an arithmetic mean reduction of 95.3% (95% CI: 84.6–99.8%), as well as IVM (100%). For P. equorum, both FBZ and PYR were effective (100% reduction). The efficacy of IVM, however, was low (45.5%; 95% CI: 0–96.3%). These results confirm that FBZ resistance in equine cyathostomins is present in France and that anthelmintic resistance to IVM is present in P. equorum. This study underlines the necessity to evaluate the efficacy of horse deworming strategies on a regular basis under field conditions.     

Prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses.

OBJECTIVE: To determine the prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. DESIGN: Prospective study. ANIMALS: 80 horses on 10 farms in a 5-county region of northeast Georgia. PROCEDURE: On each farm, horses were stratified in descending order according to pretreatment fecal egg count (FEC), blocked into groups of 4, and then randomly assigned to 1 of 4 treatment groups: no treatment (controls), and treatment with pyrantel pamoate, fenbendazole, or ivermectin. Fecal samples were collected 24 hours prior to treatment and 2, 4, and 6 weeks after treatment for determination of FEC. Mean percentage of reduction in FEC was then calculated for each treatment group. For horses from each farm, the efficacy of each anthelmintic was categorized on the basis of mean percentage of reduction in FEC at 2 weeks after treatment (< 80% reduction = ineffective; 80 to 90% reduction = equivocal; and > 90% reduction = effective). RESULTS: Pyrantel pamoate was effective at reducing FEC in horses from 7 farms, ineffective in horses from 2 farms, and equivocal in horses from 1 farm. Fenbendazole was ineffective at reducing FEC in horses from 9 farms and equivocal in horses from 1 farm. Ivermectin was effective at reducing FEC in horses from all 10 farms. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cyathostome resistance to fenbendazole is highly prevalent, and resistance to pyrantel pamoate is high enough to warrant concern. Resistance to ivermectin was not detected. On the basis of these data, it appears that ivermectin continues to be fully effective in horses. However, too few farms were used in this study to determine the prevalence of cyathostome resistance to ivermectin. Therefore, the efficacy of ivermectin should continue to be monitored closely.     

Anthelmintic resistance in the field: Changes in resistance status of parasitic populations in response to anthelmintic treatment

Changes in anthelmintic resistance in nematode parasites were monitored in sheep grazing on 2 separate farms, but with the same anthelmintic treatment program, over 16 years. High levels of benzimidazole resistance emerged in Ostertagia and Trichostrongylus spp populations on both farms following 9 years of continuous use of this class of drug. Subsequently, variations in the levels of resistance occurred for the same species between farms and between species on the same farm. A change to levamisole for 2 years resulted in a significant reversion towards benzimidazole susceptibility, but a concomitant rise in levamisole resistance, in Ostertagia on one farm. However, benzimidazole resistance increased rapidly following the re-introduction of oxfendazole into the anthelmintic treatment program. Results from both farms illustrate the pitfalls of using one anthelmintic class for an extended period and provide indirect support for the alternation of anthelmintic classes at approximately yearly intervals.     

In vitro field screening for anthelmintic resistance in strongyles of sheep and horses

Simplified in vitro field methods are described for the detection and assay of benzimidazole-resistance in sheep trichostrongylids and horse strongyles. Worm eggs are recovered from fresh faeces, within 1 hour of collection, by flotation in sugar solution. The separated eggs are then incubated for 20–24 hours at 27–30° C in solutions of thiabendazole in distilled water ranging from 0.1 to 1.1 p.p.m. for sheep trichostrongylids and from 0.05 to 0.5 p.p.m. for horse strongyles. Under controlled conditions, eggs from thiabendazole-susceptible individuals of both sheep and horse nematodes rarely hatch at thiabendazole concentrations of 0.1 p.p.m. Eggs from resistant individuals will hatch at 0.1 p.p.m. and above. Semi-quantitative estimates of the level of resistance can be determined by measuring the % egg-hatch at varying concentrations of thiabendazole. A field method for selecting test animals with low egg-counts, and an in vitro method for the culture of eggs or first-stage larvae to third stage for identification are described.     

Effects of fecal collection and storage factors on strongylid egg counts in horses

Fecal analyses are becoming increasingly important for equine establishments as a means of parasite surveillance and detection of anthelmintic resistance. Although several studies have evaluated various egg counting techniques, little is known about the quantitative effects of pre-analytic factors such as collection and storage of fecal samples. This study evaluated the effects of storage temperature, storage time and airtight versus open-air storage on fecal egg counts. The experimental protocols were replicated in two study locations: Copenhagen, Denmark and Athens, Georgia, USA. In both locations, the experiment was repeated three times, and five repeated egg counts were performed at each time point of analysis. In experiment A, feces were collected rectally and stored airtight at freezer (−10 to −18 °C), refrigerator (4 °C), room (18–24 °C), or incubator (37–38 °C) temperatures. Egg counts were performed after 0, 6, 12, 24, 48, and 120 h of storage. In experiment B, feces were collected rectally and stored airtight or in the open air in the horse barn for up to 24 h. Egg counts were performed after 0, 3, 6, 12, and 24 h of storage. In experiment A at both locations, samples kept in the refrigerator showed no decline in egg counts, whereas storage in the freezer and incubator led to significantly declining egg numbers during the study. In contrast, storage at room temperature yielded marked differences between the two study locations: egg counts remained stable in the U.S. study, whereas the Danish study revealed a significant decline after 24 h. In experiment B, the Danish study showed no differences between airtight and open-air storage and no changes over time, while the U.S. study found a significant decline for open-air storage after 12 h. This difference was attributed to the different barn temperatures in the two studies. To our knowledge, this is the first study to evaluate the pre-analytic factors affecting egg counts in horses using an experimental protocol replicated in two contrasting geographic and climatic locations. Our results demonstrate that refrigeration is the best method for storage of fecal samples intended for egg count analysis, but that accurate results can be derived from fecal samples collected from the ground within 12 h of passage.