M.K. Nielsen

Anthelmintic resistance in important parasites of horses: does it really matter?

Parascaris equorum and cyathostomins are currently considered the most important parasites of horses and have traditionally been controlled with anthelmintics belonging to three drug classes: benzimidazoles, the tetrahydropyrimidine pyrantel, and macrocyclic lactones. Unfortunately, resistance to benzimidazoles, and to a lesser extent pyrantel, is widespread in cyathostomins around the world. Furthermore, resistance to macrocyclic lactones appears to be in the early stages of development in cyathostomins in multiple locations. In contrast, P. equorum populations have remained susceptible to the three anthelmintic drug classes for a considerably longer period of time. However, over the last 10 years, resistance to macrocyclic lactones has been described in multiple countries. In contrast, resistance to pyrantel has only been described in the USA; resistance to benzimidazoles has yet to be reported. Despite the large number of reports of anthelmintic resistance in both cyathostomins and P. equorum, there are presently no reports that definitively link anthelmintic resistance with clinical problems in horses. However, that generally appears to be due to a publication bias toward well managed horse farms and the lack of appropriate diagnostic methods for rapidly quantifying anthelmintic resistance in these parasites. Management-based, and other, reasons likely responsible for this apparent anomaly are also discussed. Finally, future research priorities in this area, identified from a discussion at the 9th International Conference on Equine Infectious Diseases, are summarized.

Analysis of multiyear studies in horses in Kentucky to ascertain whether counts of eggs and larvae per gram of feces are reliable indicators of numbers of strongyles and ascarids present.

Increasing levels of anthelmintic resistance in equine nematodes have led to recommendations of more sustainable anthelmintic treatment protocols with emphasis on parasite surveillance and diagnosis, rather than prophylactic calendar-based treatments. This requires knowledge of the diagnostic test performance of techniques for counts of eggs per gram of feces (EPG) as well as methods for culturing, counting and identifying third stage (L(3)) strongyle larvae per gram of feces (LPG). For horses, such information does not exist in the published literature. The aim of this study was to examine the relationship between worm count and fecal egg count (FEC) data for strongyle and Parascaris equorum infections as well as larval culture counts for diagnosing Strongylus spp. infections. Necropsy data from 693 horses used for critical or controlled tests, including information on total worm counts, fecal egg counts (FEC) and larval culture results collected at the University of Kentucky over a period of 50 years were analyzed. Sensitivity, specificity, positive and negative predictive values, and receiver operator characteristic (ROC) curves were generated for the larval cultures and ascarid egg counts. For the strongyle egg counts, potential FEC cutoff values for treatment were evaluated statistically by comparing the total strongyle worm counts below and above chosen cutoff values. All tests had high positive predictive values (>0.95), but moderate negative predictive values (<0.70). The negative predictive values of the larval counts were negatively affected by increasing egg count levels. Strongyle FEC cutoff values up to the level of 500 EPG yielded significantly higher strongyle worm counts in the treatment group, whereas no differences were found at higher cutoffs. This supports usage of cutoffs for treatment in the 0-500 EPG range. Altogether, the present study yields unique and useful information of widely used methods for parasite surveillance and diagnosis in equine establishments.

Apparent ivermectin resistance of Parascaris equorum in foals in Denmark.

ANTHELMINTIC resistance has been reported in equine nematodes since the 1960s (Kaplan 2002, 2004). Cyathostomins are currently reported to be widely resistant to benzimidazoles and pyrantel salts, leaving avermectin/milbemycins (macrocyclic lactones) as the only fully effective group of drugs. It is therefore of major concern that two recent reports have identified foals infected with Parascaris equorum apparently resistant to drugs in this group (Boersema and others 2002, Hearn and Peregrine 2003). So far, these have been the only two studies reporting this resistance, and it is not known how widespread the phenomenon is on horse farms in different countries. This short communication describes cases of P equorum apparently resistant to ivermectin in foals in Denmark. In 2003, the attending veterinarian at a standardbred trotter farm in Jutland, Denmark, observed decreased efficacy of an ivermectin oral paste formulation (Virbalan Vet; ChemVet) against P equorum infections in foals. Faecal samples from four foals analysed nine days after treatment revealed P equorum eggs in all of them, with egg counts of up to 1150 eggs per gram of faeces (epg). In 2004, five foals were born on the farm between April 8 and May 4, and a protocol for treatments and faecal analyses was established to investigate the efficacy of ivermectin further. Faecal samples from the foals and their dams were analysed on July 19 and August 23. Horses with detectable ascarid or strongyle eggs in the faeces were treated with an ivermectin oral paste formulation (Noromectin Vet; Biovet ApS) on both occasions. On September 2, faecal analyses were performed again, and foals still positive for P equorum were treated with pyrantel pamoate oral paste (Banminth Vet; Orion Pharma Animal Health). Faecal samples were analysed again on September 13, and any foals with ascarid eggs were retreated with pyrantel pamoate. Follow-up faecal samples were analysed on October 4. The faecal egg counts were performed using a modified McMaster method with a sensitivity of 10 epg (Henriksen and Korsholm 1984). The first author administered all the treatments. Each dam received a full syringe and the foals half a syringe of anthelmintic paste on each occasion; this gave doses above the label dosages of 0·2 mg/kg ivermectin and 19 mg/kg pyrantel pamoate for all the animals. Faecal egg count reductions were calculated by the formula:

Sustainable equine parasite control: Perspectives and research needs

Clinically important equine parasites are ubiquitous in managed horse populations. The traditional approach to parasite control is frequent administration of anthelmintics to all horses on a farm. However, increasing levels of anthelmintic resistance is forcing horse owners and veterinarians to shift this control paradigm. Treatment regimens involving routine deworming of all horses throughout the year are now being replaced by more sustainable approaches, which take in to account the importance of maintaining adequate parasite refugia. The selective therapy principle has been recommended for more than 15 years, but there is limited experience with this approach. The relative magnitude of the faecal egg count for an individual horse is a consistent trait, and this provides a reliable basis for selective therapy. But no studies have evaluated the consequences of selective therapy in the long-term, and such studies are strongly needed to validate this approach. Importantly, it remains unclear how selective therapy may affect the prevalence and intensity of other parasites of significant pathogenic potential (e.g. Strongylus vulgaris), which have become uncommon due to years of intensive chemotherapy. Consequently, a selective approach requires vigilant surveillance of the parasite fauna and intensity. This places a demands for reliable diagnostic tools. Also noteworthy is the fact that the majority of equine nematode parasites are more pathogenic during their larval stages, when they cannot be detected by traditional egg counting techniques. Consequently, parasite-specific diagnostic tools capable of assessing prepatent parasite burdens, and able to differentiate between strongyle species of different pathogenic potentials, would be of great value to the equine clinician. Tools for detecting infections with the tapeworm Anoplocephala perfoliata are laborious, difficult to interpret, and at present there is no established method to evaluate treatment efficacy. Thus, better diagnostic tools are needed for tapeworms as well. Biological control, especially the predacious fungi have demonstrated good potential as an adjunct for strongyle control and such a product could easily have a market in equine establishments. In summary, there is general agreement that the traditional treat-all at frequent interval approach should be abandoned, and that optimal parasite control can be maintained with far fewer anthelmintic treatments. But better diagnostic techniques and more evidence documenting the long-term consequences of selective therapy programs are needed to develop and validate systems for sustainable equine parasite control.

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 degrees C), refrigerator (4 degrees C), room (18-24 degrees C), or incubator (37-38 degrees C) temperatures. Egg counts were performed after 0, 6, 12, 24, 48, and 120h of storage. In experiment B, feces were collected rectally and stored airtight or in the open air in the horse barn for up to 24h. Egg counts were performed after 0, 3, 6, 12, and 24h 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 24h. 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 12h. 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 12h of passage.

Strongylus vulgaris associated with usage of selective therapy on Danish horse farms—Is it reemerging?

Nematodes belonging to the order Strongylida are ubiquitous in grazing horses, and the large strongyle Strongylus vulgaris is considered the most pathogenic. This parasite was originally described widely prevalent in equine establishments, but decades of frequent anthelmintic treatment appears to have reduced the prevalence dramatically. Increasing levels of anthelmintic resistance in cyathostomin parasites have led to implementation of selective therapy to reduce further development of resistance. It has been hypothesized that S. vulgaris could reoccur under these less intensive treatment circumstances. The aim with the present study was to evaluate the occurrence of S. vulgaris and the possible association with usage of selective therapy. A total of 42 horse farms in Denmark were evaluated for the presence of S. vulgaris using individual larval cultures. Farms were either using a selective therapy principle based on regular fecal egg counts from all horses, or they treated strategically without using fecal egg counts. A total of 662 horses were included in the study. Covariate information at the farm and horse level was collected using a questionnaire. The overall prevalence of S. vulgaris was 12.2% at the individual level and 64.3% at the farm level. Farms using selective therapy had horse and farm prevalences of 15.4% and 83.3%, respectively, while the corresponding results for farms not using selective therapy were 7.7% and 38.9%. These findings were found statistically significant at both the horse and the farm level. Stud farms using selective therapy were especially at risk, and occurrence of S. vulgaris was significantly associated with the most recent deworming occurring more than six months prior. The results suggest that a strict interpretation of the selective therapy regimen can be associated with an increased prevalence of S. vulgaris. This suggests that modifications of the parasite control programs could be considered on the studied farms, but it remains unknown to which extent this can be associated with increased health risks for infected horses.

Selective anthelmintic therapy of horses in the Federal states of Bavaria (Germany) and Salzburg (Austria): an investigation into strongyle egg shedding consistency.

For 9 consecutive months (March-November 2008), faecal samples were collected monthly from 129 horses residing within 40 km of Salzburg, Austria. Samples were analysed quantitatively using a modified McMaster egg counting technique. Whenever a faecal egg count (FEC) result exceeded 250 eggs per gram (EPG), the horse was treated with pyrantel, ivermectin or moxidectin. In 52 of 129 horses (40.3%), no strongyle eggs were ever detected over the course of 9 months. In 39 horses (30.2%), strongyle eggs were detected in at least 1 sample, but the egg count never exceeded 250 EPG. The remaining 38 (29.5%) horses were treated at least once in response to a FEC that exceeded 250 EPG. As a result of this selective anthelmintic scheme, the total number of anthelmintic treatments was reduced to 54% of the number of treatments administered to the same horses in the previous year. Both the maximum and mean FEC dropped significantly after initiation of the study. A statistically significant, negative correlation was demonstrated between the maximum and mean FEC of a horse and its age. Pasture hygiene appeared to reduce FECs, but the effect was not statistically significant. The magnitude of the initial FEC was significantly correlated with the maximum FECs in the subsequent 8 months (p<0.01). The same relationship was observed for the maximum FEC of the first 2 samples. Furthermore, horses which required several anthelmintic treatments had a higher initial FEC and a greater maximum FEC in the first 2 samples than horses which received only one or no treatment. These results suggest that selective anthelmintic treatment accomplished a reduced pasture contamination with strongyle eggs, while simultaneously decreasing the number of anthelmintic treatments. Sustained implementation of a selective treatment strategy has the potential to reduce selection pressure for anthelmintic resistance. These results reported herein will assist equine practitioners in designing and monitoring sustainable anthelmintic treatment programs.

Determination of ivermectin efficacy against cyathostomins and Parascaris equorum on horse farms using selective therapy.

Ivermectin resistance has recently been described in Parascaris equorum and there have been reports from several countries of a shortened egg reappearance period (ERP) following ivermectin treatment for cyathostomins. This study was aimed at determining the efficacy of ivermectin in treating cyathostomins and P. equorum in Danish horses. A total of 196 animals were selected from 52 farms, all of which were using a selective anthelmintic treatment strategy. ERP was investigated with weekly samples from 96 horses from nine farms. Horses were treated with ivermectin oral paste by their owners at an estimated dose rate of 0.2mg/kg. Overall, faecal egg counts were reduced 10-14 days after treatment by 96.9% and 100% for P. equorum and cyathostomins, respectively. Mean faecal egg count reductions at 4 and 6 weeks post treatment were 99.5% and 96.9%, respectively. No signs of developing ivermectin resistance were found in either cyathostomins or P. equorum in the studied horses.

Selective therapy in equine parasite control—Application and limitations

Since the 1960s equine parasite control has relied heavily on frequent anthelmintic treatments often applied with frequent intervals year-round. However, increasing levels of anthelmintic resistance in cyathostomins and Parascaris equorum are now forcing the equine industry to change to a more surveillance-based treatment approach to facilitate a reduction in treatment intensity. The principle of selective therapy has been implemented with success in small ruminant parasite control, and has also found use in horse populations. Typically, egg counts are performed from all individuals in the population, and those exceeding a predetermined cutoff threshold are treated. Several studies document the applicability of this method in populations of adult horses, where the overall cyathostomin egg shedding can be controlled by only treating about half the horses. However, selective therapy has not been evaluated in foals and young horses, and it remains unknown whether the principle is adequate to also provide control over other important parasites such as tapeworms, ascarids, and large strongyles. One recent study associated selective therapy with increased occurrence of Strongylus vulgaris. Studies are needed to evaluate potential health risks associated with selective therapy, and to assess to which extent development of anthelmintic resistance can be delayed with this approach. The choice of strongyle egg count cutoff value for anthelmintic treatment is currently based more on tradition than science, and a recent publication illustrated that apparently healthy horses with egg counts below 100 eggs per gram (EPG) can harbor cyathostomin burdens in the range of 100,000 luminal worms. It remains unknown whether leaving such horses untreated constitutes a potential threat to equine health. The concept of selective therapy has merit for equine strongyle control, but several questions remain as it has not been fully scientifically evaluated. There is a great need for new and improved methods for diagnosis and surveillance to supplement or replace the fecal egg counts, and equine health parameters need to be included in studies evaluating any parasite control program.

Resistance to avermectin/milbemycin anthelmintics in equine cyathostomins – Current situation

Avermectins and milbemycins (AM) are potent compounds against all major nematode parasites, but their continuous usage has led to the development of widespread resistance in many of the important species of ruminant and equine parasites. The exception to this has been the cyathostomins, where AM resistance was recently first reported only after decades of drug exposure. Data from a Brazilian study suggests that AM resistance has developed in cyathostomins and reports of shortened egg reappearance periods after ivermectin treatment have been published recently from USA and Germany. Thus, AM resistance in cyathostomins is an emerging worldwide concern, but there is only limited amount data on the extent of this problem. To limit the development and spread of AM-resistant cyathostomins the equine industry must implement new strategies for worm control, and the veterinary parasitology community must develop and validate improved protocols for detecting anthelmintic resistance in the field.