Anthelmintic resistance in strongylids (Nematoda: Strongylidae) parasitizing wild and domestic equids in the Askania Nova Biosphere Reserve, Ukraine

Resistance of strongylids in domestic horses to benzimidazole anthelmintics (BZ) has been detected worldwide; however, information on the presence of BZ-resistance in wild equids has not been published to date. The purpose of this study was to analyze the manifestations of the BZ resistance in strongylids in domestic and wild equids kept in the Askania Nova Biosphere Reserve, Ukraine. Four species of equids: domestic horses and Shetland ponies (Equus caballus), donkeys (E. asinus), plains zebras (E. burchelli) and Grévy's zebras (E. grevyi) kept under semi-free conditions were examined using the Fecal Egg Count Reduction Test (FECRT) in order to detect the presence of resistance to the BZ anthelmintics. Analysis of long-term data (2009­2019) revealed a decrease in the efficacy of BZ drugs against strongylids in these four species of equids from 97.6% in 2009 to <75% in 2019. The efficacy of anthelmintic treatments was low in all species of equids: in plains zebras ­ 69.4%, Grévy's zebras ­ 72.7%, horses ­ 66.4%, ponies ­ 61.1% and donkeys ­ 45.2%. Ten species of cyathostomins (Cyathostomum catinatum, Cylicocyclus nassatus, C. ashworthi, C. leptostomus, Cylicostephanus calicatus, C. goldi, C. longibursatus, C. minutus, Coronocyclus labiatus, C. labratus) were found after horse deworming with albendazole. Our results are the first detection of BZ-resistance in strongylids of wild equids kept under semi-free conditions in the Reserve.

Can an entomopathogenic nematode serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds?

As gastro-intestinal nematodes (GINs) become increasingly resistant to chemical anthelmintics, and because consumers scrutinize chemical residues in animal products, the use of herbal anthelmintics and in particular, phenolic compounds, has become attractive. Most life stages of GINs cannot be grown in the lab as they are obligatory parasites, which limits our understanding of the effects of phenolic compounds on their parasitic stages of life. We hypothesized that a species phylogenetically close to GINs and grown in vitro, the insect-parasitic nematode Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade), when fed with Photorhabdus luminescens exposed to plant phenolics, can serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds. We compared the development of H. bacteriophora infective juveniles (IJ) and the exsheathment rate of L3 larvae of the strongyle Teladorsagia circumcincta and Trichostrongylus colubriformis when exposed to catechin, rutin, chlorogenic and gallic acids, and myricetin. Gallic acid had the highest impact in terms of IJ mortality but the highest impairment of IJ development to adulthood was imposed by myricetin. The studied compounds were not lethal to GINs stricto sensu but we consider that the practical implications of total exsheathment inhibition and mortality on GIN populations are similar. Catechin and rutin had similar effects on rhabditid and strongyles: they imposed ca. 90% lethality of IJs at concentrations higher than 1200 ppm and the remaining live IJs did not develop further, and they also totally inhibited strongyle L3 exsheathment in a dose-response fashion. Gallic acid was 100% lethal to IJs exposed above 300 ppm and chlorogenic acid caused 87% mortality above 1200 ppm, with no development for the surviving IJs but for all lower concentrations, all the IJs developed to adult stages. Likewise, gallic and chlorogenic acids did not affect the exsheatment of GIN L3 larvae. Therefore, a discrepancy between the effects of gallic and chlorogenic acids on the development of rhabditid IJs and exsheathment of GIN L3 larvae was found only when they were exposed to high concentrations. A dose-response of IJ lethality to myricetin was found, with no IJ development between 150 and 2400 ppm; but contrary to the other compounds, myricetin also impaired IJ development of IJs above 10 ppm in a dose-response manner and showed dose-responses in the L3 exsheathment. Apart for the high rates of lethality imposed on IJs by gallic and chlorogenic acids at high concentration, these results suggest that H. bacteriophora fed P. luminescens exposed to phenolics shows potential to serve as model in studies of the anthelmintic effects of phenolics in GIN.

Modelling the development of anthelmintic resistance in cyathostomin parasites: The importance of genetic and fitness parameters.

Previously described models for the free-living and parasitic phases of the cyathostomin life-cycle were combined into a single model for the complete life-cycle. The model simulates a single free-living population on pasture utilising parasite egg output from the horses and localised temperature and rainfall data to estimate infective larval density on herbage. Multiple horses of different ages are possible, each with an individualised anthelmintic treatment programme. Genotypes for anthelmintic resistance are included allowing for up to three resistance genes with 2 alleles each. Because little is known of the genetics of resistance to anthelmintics in cyathostomins, the first use of this model was to compare the effect of different assumptions regarding the inheritance of resistance on model outputs. Comparisons were made between single and two-gene inheritance, where the heterozygote survival was dominant, intermediate or recessive under treatment, and with or without a fitness disadvantage associated with the resistance mechanism. Resistance developed fastest when the heterozygotes survived anthelmintic treatment (i.e., were dominant) and slowest when they did not (i.e., were recessive). Resistance was slower to develop when inheritance was poly-genic compared to a single gene, and when there was a fitness cost associated with the resistance mechanism, although the latter variable was the least influential. Importantly, while these genetic factors sometimes had a large influence on the rate at which resistant genotypes built up in the model populations, their order of ranking was always the same, when different anthelmintic use strategies were compared. Therefore, the described model is a useful tool for evaluating different treatment and management strategies on their potential to select for resistance.

The effect of climate, season, and treatment intensity on anthelmintic resistance in cyathostomins: A modelling exercise.

Anthelmintic resistance is widespread in equine cyathostomin populations across the world, and with no new anthelmintic drug classes in the pharmaceutical pipeline, the equine industry is forced to abandon traditional parasite control regimens. Current recommendations aim at reducing treatment intensity and identifying high strongylid egg shedders in a targeted treatment approach. But, virtually nothing is known about the effectiveness of these recommendations, nor their applicability to different climatic regions, making it challenging to tailor sustainable recommendations for equine parasite control. This study made use of a computer model of the entire cyathostomin life-cycle to evaluate the influence of climate and seasonality on the development of anthelmintic resistance in cyathostomin parasites. Furthermore, the study evaluated the impact of recommended programs involving selective anthelmintic therapy on delaying anthelmintic resistance development. All simulations evaluated the use of a single anthelmintic (i.e., ivermectin) over the course of 40 model years. The study made use of weather station data representing four different climatic zones: a cold humid continental climate, a temperate oceanic climate, a cold semi-arid climate, and a humid subtropical climate. Initially, the impact of time of the year was evaluated when a single anthelmintic treatment was administered once a year in any of the twelve months. The next simulations evaluated the impact of treatment intensities varying between 2 and 6 treatments per year. And finally, we evaluated treatment schedules consisting of a combination of strategic treatments administered to all horses and additional treatments administered to horses exceeding a predetermined fecal egg count threshold. Month of treatment had a large effect on resistance development in colder climates, but little or no impact in subtropical and tropical climates. Resistance development was affected by treatment intensity, but was also strongly affected by climate. Selective therapy delayed resistance development in all modelled scenarios, but, again, this effect was climate dependent with the largest delays observed in the colder climates. This study is the first to demonstrate the value of cyathostomin parasite refugia in managing anthelmintic resistance, and also that climate and seasonality are important. This modelling exercise has allowed an illustration of concepts believed to play important roles in anthelmintic resistance in equine cyathostomins, but has also identified knowledge gaps and new questions to address in future studies.

A model for the dynamics of the parasitic stages of equine cyathostomins.

A model was developed to reproduce the dynamics of the parasitic stages of equine cyathostomins. Based on a detailed review of published literature, a deterministic simulation model was constructed using the escalator boxcar-train approach, which allows for fully-overlapping cohorts of worms and approximately normally distributed variations in age/size classes. Key biological features include a declining establishment of ingested infective stage larvae as horses age. Development rates are constant for all the parasitic stages except the encysted early third stage larvae, for which development rates are variable to reflect the sometimes extended arrestment of this stage. For these, development is slowed in the presence of adult worms in the intestinal lumen, and when ingestion of infective larvae on herbage is high or extended. In the absence of anthelmintic treatments, the life span of adult worms is approximately 12 months, and the presence of an established adult worm burden largely blocks the transition of luminal fourth stage larvae to the adult stage, resulting in mortality of the larvae. This inhibition is removed by effective anthelmintic treatment allowing the rapid replacement of adult worms from the pool of mucosal stages. Within the model, the rate and seasonality at which infective stage larvae are ingested strongly influences the dynamics of the pre-adult stages. While the adult worm burden remains relatively stable within a year, due to the negative feedback they have on developing stages, the numbers and proportions of larval stages relative to the total worm burden increase with the numbers of infective larvae ingested. Further, within the model, the seasonal rise and fall of encysted stages is largely driven by the seasonal pattern of infective larvae on pasture. Because of this, the model reproduces the contrasting seasonal patterns of mucosal larvae, typical of temperate and tropical environments, using only the appropriate seasonality of larvae on pasture. Thus, the model reproduces output typical of different climatic regions and suggests that observed patterns of arrested development may simply reflect the numbers and seasonality of free-living stages on pasture as determined by different management practices and weather patterns.

Proteolytic and nematicidal potential of the compost colonized by Hypsizygus marmoreus.

Spent mushroom compost (SMC) is a residue generated in edible mushrooms production, such as Hypsizygus marmoreus. Its genome was recently sequenced, demonstrating cuticle-degrading protease genes. The present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae. The extraction of the crude extract directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ±â€¯18.38 U g-1 of compound. Moreover, the zymogram and SDS-PAGE indicated the presence of two proteases with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, there was a significant reduction in the number of Panagrellus redivivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively. A0A151VWY3 mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.

Anthelmintic efficacy against equine strongyles in the United States.

Equine strongyle parasites are ubiquitous in grazing equids across the world. Anthelmintic resistance is widely developed in cyathostomin populations, but very few surveys have evaluated anthelmintic efficacy in equine populations in the United States, and most of these are over 15 years old. The present study was carried out as part of the National Animal Health Monitoring Systems (NAHMS) Equine 2015-2016 study. The aims were to investigate anthelmintic treatment efficacy by means of the fecal egg count reduction test (FECRT) and identify parameters associated with decreased efficacy. Data were collected from equine operations in 28 states via questionnaires and fecal samples submitted for fecal egg count analysis. Participants were instructed to collect samples from six equids at the day of anthelmintic treatments and 14 days later, and they were asked to include an empty syringe with a legible label of the anthelmintic product used in the shipment. Overall, dewormer treatment was effective for 76.3% of operations (84.6% of animals). Macrocyclic lactone use was effective for 88.7% of operations (95.0% of animals) while pyrimidine/benzimidazole use was effective for 21.4% of operations (43.5% of animals). Univariate analysis revealed that overall, macrocyclic lactones exhibited significantly higher efficacy than the pyrimidine and benzimidazole drug classes (p < 0.0001). There were no statistically significant differences observed between geographic regions (West, South Central, North East, and Southeast). Body weight (p = 0.0355), amount of anthelmintic administered (p = 0.0119), and operation size (p = 0.0162) were statistically associated with anthelmintic efficacy, while anthelmintic treatment frequency in the previous 12 months was not (p = 0.7081). Multiple, mixed-effect logistic regression revealed that anthelmintic drug class (p < 0.0001) was the most impactful factor in predicting anthelmintic efficacy, after accounting for operation size, region and clustering of equids at the operation level. Pasture rotation (p = 0.0129) also demonstrated a significant effect using this model. These data document widespread occurrence of reduced anthelmintic efficacy of benzimidazole and pyrimidine products against strongyle infections in equids in the United States. Anthelmintic efficacy patterns were relatively uniform between the four studied regions, and some epidemiological factors were identified to be associated with anthelmintic efficacy against strongyle infections. This information can be useful in devising sustainable parasite control strategies in the future.

Risk factors associated with strongylid egg count prevalence and abundance in the United States equine population.

Equine strongyle parasites are considered ubiquitous in grazing equids across the world, and cyathostomin parasites are known pathogens causing well-described disease complexes in horses. Decades of intensive anthelmintic treatments have led to anthelmintic resistance in cyathostomins, and current recommendations are to lower treatment intensity and base control strategies on fecal egg count surveillance. Little is known about risk factors associated with strongyle parasite egg shedding patterns in the United States equine population, as the most recent national survey was conducted 20 years ago. The present study was carried out as part of the National Animal Health Monitoring Systems (NAHMS) Equine 2015-2016 study. The aims were to describe strongyle parasite egg shedding patterns in the United States equine population and identify risk factors associated with prevalence and egg count magnitude. Data were collected from equine operations in 28 states via questionnaires and fecal samples submitted to a parasitology research laboratory for fecal egg count analysis and the data gathered underwent comprehensive statistical analyses. Though region and season were related, overall, the summer months and the fall in the southeast tended to have the greatest odds of presence of strongyles eggs on a FEC. Generally, equids resident in the Western region (Arizona, California, Colorado, Montana, Oregon, and Wyoming) had significantly lower strongyle prevalence, no matter the season, as well as a markedly different distribution between strongyle egg shedding levels (p = 0.0005). Overall, egg counts were over-dispersed with about 27% of equids (95% Confidence Interval (CI): 20-34%) contributing 80% of the egg output. Pasture history was significantly associated with strongyle egg prevalence (p = 0.0003) and egg shedding levels (p = 0.0063) with daily access in the previous 30 days being associated with higher odds of presence and greater median egg count levels. Equid gender was significantly associated with strongylid presence (p = 0.0081) and egg count level (p = 0.0008), with male equids having significantly lower odds and median egg counts than female equids, and age was significantly negatively associated with strongylid prevalence (p < 0.0001). Time since last deworming was significantly positively associated with prevalence of strongyle eggs, and this was dependent on the class of dewormer used (p = 0.0086), with equids treated with macrocyclic lactone class of drugs having lower odds of strongyle egg presence at 120 days since the last deworming. These data provide useful insights into strongylid egg shedding patterns in the United States equine population, and they can help refine parasite control recommendations depending on region, pasture access, and age distribution.

The behaviour of the nematode, Steinernema feltiae (Nematoda: Steinernematidae) in sand contaminated with the industrial pollutant chromium VI.

This study set out to determine the suitability of the nematode Steinernema feltiae as a bioindicator for heavy metal pollution, specifically chromium VI. Nematodes were introduced into sand contaminated with concentrations of Cr VI+, in a range between 10 and 100 ppm, in increments of 10. Reproductive potential, development times and infectivity vs exposure times to Cr VI were employed as endpoints. It was observed that infective juveniles (IJ) from this nematode can survive and successfully infect host insects in the presence of Cr VI for as much as 13 days, and that the nematode increases its reproductive potential at concentrations up to 100 ppm Cr VI+. Conversely, development times (time in days taken for progeny to emerge after larval host death) and IJ infectivity rates were observed to reduce with increasing concentrations of Cr VI. The ability of this nematode to survive in the presence of high concentrations of Cr VI, and its ability to increase progeny numbers at the early stages of Cr VI exposure may provide a survival advantage for this nematode at contaminated sites. It may also demonstrate potential for development as a model species for toxicological assessment in in-situ field sampling.

Evaluation of the mucosal inflammatory responses to equine cyathostomins in response to anthelmintic treatment.

Members of Cyathostominae are pervasive parasites of equids that can cause larval cyathostominosis, a potentially life-threatening disease that occurs when a multitude of encysted larvae synchronously excyst from the wall of the large intestine. Moxidectin and fenbendazole are the two current labeled drugs that target the encysted larval stages; however, there is limited knowledge of the local inflammatory response to the larvae and to the two treatments in clinically healthy horses. This study is the first to evaluate the local inflammatory response to cyathostomin larvae and to larvicidal treatment at 2 and 5 weeks post treatment. Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into 3 groups: Group 1, fenbendazole at 10 mg/kg for 5 days, Group 2, a single dose of moxidectin at 0.4 mg/kg, and Group 3, untreated controls. Tissue samples from the cecum and dorsal and ventral colons were used for histopathological and immunohistochemical evaluation. Tissues were stained with routine hematoxylin and eosin (H&E) for light microscopy and immunohistochemically for MAC387, CD20, and CD3 for differentiation of activated macrophages, B cells, and T cells, respectively. Semiquantitative scores were assigned for all inflammatory cell types and fibrous connective tissue. Larvae observed by light microscopy were enumerated and classified by stage. Mucosal ulcerations and submucosal granulomas were also enumerated. Mean macrophage scores were higher in the moxidectin group than the fenbendazole group (p = 0.0185) and the control group had a higher activated macrophage score than both treatment groups (p = 0.0104, p = 0.0004). T lymphocyte scores were higher in the moxidectin group when compared to the control group (p = 0.0069). Goblet cell hyperplasia scores were elevated at 5 weeks post treatment compared to 2 weeks post treatment (p = 0.0047) and were elevated in the ventral colon compared to the dorsal colon (p = 0.0301). Eosinophil scores were elevated surrounding degenerative larvae when compared to intact larvae (p = 0.0001). Mucosal ulcerations were found only in the control group at 2 weeks post treatment. This study found subtle inflammatory differences between treatment groups but provided new information about goblet cells and eosinophils in relation to encysted cyathostomin larvae.

In-vitro Anthelmintic Effects of Aqueous and Ethanolic Extracts of Marrubium vulgare Leaves Against Bovine Digestive Strongyles.

OBJECTIVE: The aim of the present study was to evaluate in vitro the anthelmintic activity of Marrubium vulgare L. growing in Algeria against digestive strongyles in naturally infected bovine. METHODS: The anthelmintic activities of the extracts were evaluated using the egg hatch assay and larval mortality assay. Leaves powder of M. vulgare as extracted by maceration. Ethanolic (EE) and aqueous extracts (AE) were tested at 0.78, 1.55, 3.1, 6.2, 12.5, 25, and 50 mg/ml. Albendazole and dimethyl sulfoxide were used as positive and negative controls at concentrations20 mg/ml and 3%, respectively. RESULTS: The mean embryonation rate was maximum in AE and EE (48.4±3.47% and 54.2±2.87%, respectively) of M. vulgare leaves. The extracts of M. vulgare leaves high effects were observed with 50 mg/ml, but the lowest reduction on parasite eggs hatchability was observed in cultures exposed to 0.78 mg/ml to both extracts. The larval mortality rate of both AE and EE from M. vulgare showed that the extracts at 50 mg/ml exhibited 45.8±1.99% and 51±2.53%, respectively, at 24h. CONCLUSION: The findings of the present study showed that AE and EE of M. vulgare leaves have a potential anthelmintic activity on eggs and larvae of bovine strongyles parasites in vitro.

Procyanidin A2 in the Australian plant Alectryon oleifolius has anthelmintic activity against equine cyathostomins in vitro.

There is a need to investigate new methods of controlling cyathostomins in horses due to increasing anthelmintic resistance amongst these parasites. In a previous study we identified the Australian plant Alectryon oleifolius as having anthelmintic activity towards cyathostomins. This study aimed to isolate and identify the bioactive compound(s) responsible for all or part of this anthelmintic activity and quantify its activity in vitro. The condensed tannin procyanidin A2 was isolated from the plant through a process of bioassay guided fractionation and identified using 1D and 2D nuclear magnetic resonance spectroscopy and high performance liquid chromatography with mass spectrometry. Procyanidin A2 demonstrated significant anthelmintic activity in larval development assays, completely inhibiting development from egg to third larval stage at concentrations as low as 50µg/mL and having an IC50 value of 12.6µg/mL. Procyanidin A2 also significantly inhibited larval migration at concentrations of 25µg/mL. This study indicates that procyanidin A2 is the principal anthelmintic compound in extracts from A. oleifolius, and further highlights the potential for the use of this plant as a component of cyathostomin control programs in the future.

Anthelmintic therapy of equine cyathostomin nematodes - larvicidal efficacy, egg reappearance period, and drug resistance.

Cyathostomins are ubiquitous in grazing horses across the world, and anthelmintic resistance has been reported with increasing levels over past decades. The aims of the present study were (i) to investigate the efficacy against encysted larval stages of moxidectin (0.4 mg/kg) and fenbendazole (10 mg/kg daily for five consecutive days) and compare these regimens at 2 and 5 weeks post-treatment, (ii) to investigate individual cyathostomin species associated with shortened egg reappearance periods, and (iii) to document species exhibiting decreased susceptibility to the evaluated compounds. Thirty-six ponies were allocated to treatment groups with half euthanatized 2 weeks post-treatment, and the remainder necropsied after 5 weeks. Luminal and mucosal worm counts were conducted and strongyle egg counts were determined at weekly intervals. At 2 weeks, mean reductions of early L3s were 50.4% and 73.8% for fenbendazole and moxidectin, respectively. At 5 weeks, the respective efficacies were 51.3% and 71.8%. Two week efficacies against late L3s and L4s (LL3s/L4s) were 70.8% and 74.6% for fenbendazole and moxidectin, respectively, whereas very low numbers were found in all three groups at 5 weeks. None of the mucosal counts were significantly different between treatment groups. Fenbendazole and moxidectin reduced luminal worm counts by 93.2% and 98.3% at 2 weeks following administration, with moxidectin group adult counts being significantly lower than the other two groups (P < 0.0001). Both treatment groups had increased counts 3 weeks later (P = 0.0415). A moxidectin ERP of 4 weeks was associated with surviving luminal L4s, and adult species contributing to this were Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus ashworthi and Cylicocyclus nassatus. This study documented (i) larvicidal efficacy of fenbendazole much lower than historical standards, (ii) survival of luminal immatures (L4) following moxidectin administration, and (iii) new information about cyathostomin species associated with these phenomena.

Are sainfoin or protein supplements alternatives to control small strongyle infection in horses?

The spread of anthelmintic resistance in equine strongyle nematodes has become a major problem, advocating for the development of alternative control for strongyles. Our study consisted of both in vivo and in vitro experiments. We investigate for the first time the efficacy of a short-term consumption of tannin-rich sainfoin (Onobrychis viciifolia) or extra proteins in naturally infected horses. We used 30 horses allocated into three groups of 10 individuals that received for 18 days either (i) a tannin-rich diet with 70% DM sainfoin pellets (Sd), (ii) a protein-rich diet with 52% DM Italian rye-grass pellets and 18% DM grinded linseed expeller (Pd), or (iii) a control diet with 45% DM barley and 25% DM cereal-based pellets (Cd). The three diets were isoenergetic, covering 94% of animal energy requirements on average, and the Sd and Pd diets were isoproteic and provided extra proteins (227% of protein requirements v. 93% for the Cd diet). Pd and Cd were compared to test for benefits of receiving extra proteins, while Sd and Pd were compared to account for the effect of sainfoin secondary metabolites. There were no between-diet differences in faecal egg counts (FEC) or in worm burden evaluated from worm counts in faeces of drenched horses at the end of the experiment. However, coprocultures from the faeces collected in each group at the beginning and at the end of the experiment suggested a lower rate of strongyle larval development in the Sd group at the end of the experiment (Sd=8.1%, Pd=30.5%, Cd=22.6%). In vitro tests using sainfoin solutions evidenced the influence of sainfoin on strongyle larval development: adding 29% of sainfoin pellets to faeces reduced the strongyle egg development into infective larvae by 82% (P<0.001) and using solutions with sainfoin concentrations higher than 7.5 mg/ml reduced egg hatching by 37% (P<0.05). The short-term use of tannin-rich plants in horse diet could thus constitute a promising strategy to reduce the risk of infection by strongyles at pasture.

First report of anthelmintic resistance of equine cyathostomins in Cuba.

Anthelmintic resistance in equine cyathostomins has been described worldwide, with resistance to the benzimidazole class being particularly widespread. The status of anthelmintic efficacy in Cuba has been virtually unknown due to the lack of equine labelled products. One recent report documented suboptimal efficacy levels of extra-label albendazole products against cyathostomins, but it remains unknown to which extent benzimidazole resistance exists in the population. The aim of the present study was to evaluate the anthelmintic efficacy of two benzimidazole products labelled for equines, fenbendazole and oxibendazole. A fecal egg count reduction test (FECRT) was carried out on 132 horses aged 4 months to 18 years in 14 herds, belonging to six provinces. Ten herds exhibited signs of resistance to at least one of the benzimidazoles (mean FECRT<90%). Overall, oxibendazole exhibited higher efficacy than fenbendazole (p = 0.0062), and higher efficacy levels were found in horses never dewormed before compared to those treated within 3-12 months prior to the study (p = 0.0015). Pre-treatment larval cultures revealed the presence of large strongyles and cyathostomin larvae in all herds, while only cyathostomin larvae were detected post treatment. The present work is the first report of anthelmintic resistance in equine cyathostomins in Cuba, and suggests pre-selection for resistant strains by extra-label use of albendazole on the studied farms.

Anthelminthic activity of methanol extracts of Diospyros anisandra and Petiveria alliacea on cyathostomin (Nematoda: Cyathostominae) larval development and egg hatching.

Methanol extracts of plant structures are promising alternatives to traditional pharmaceutical anthelminthic treatments. An in vitro evaluation was done of how methanol extracts of Diospyros anisandra bark and leaves, and Petiveria alliacea stems and leaves, collected during the rainy and dry seasons, effected cyathostomin larval development and egg hatching. Seven concentrations (600, 300, 150, 75, 37.5, 18.7 and 9.3µg/ml) were tested using the egg hatch assay. An ANOVA was applied to identify differences between the concentrations and the controls. Fifty percent lethal concentration (LC50) and the 95% confidence interval were calculated with a probit analysis. At and above 37.5µg/ml, the D. anisandra bark extracts from both seasons exhibited ≥95% egg hatch inhibition (EHI), while the D. anisandra leaf extracts had >90% EHI at and above 75µg/ml. For P. alliacea, the extracts from leaves and stems from either season exhibited >97% EHI at and above 300µg/ml, although similar efficacy was also observed at lower concentrations with the rainy season stems (75µg/ml) and leaves (150µg/ml). Values for LC50 were lowest for the rainy season D. anisandra bark (10.2µg/ml) and leaf extracts (18.4µg/ml), followed by the rainy season P. alliacea stems extract (28.2µg/ml). In the D. anisandra extracts, EHI was largely due to its ovicidal activity (≥96% beginning at 37.5µg/ml), whereas in the P. alliacea extracts it was due to L1 larval hatch failure (≥90% beginning at 75µg/ml). Overall, the rainy season D. anisandra bark extracts had a strong in vitro anthelminthic effect against cyathostomins by inhibiting larval development, and the rainy season P. alliacea stem extracts had a strong effect by preventing egg hatching. Both are possible control alternatives for these nematodes.

P-glycoproteins play a role in ivermectin resistance in cyathostomins.

Anthelmintic resistance is a global problem that threatens sustainable control of the equine gastrointestinal cyathostomins (Phylum Nematoda; Superfamily Strongyloidea). Of the three novel anthelmintic classes that have reached the veterinary market in the last decade, none are currently licenced in horses, hence current control regimens focus on prolonging the useful lifespan of licenced anthelmintics. This approach would be facilitated by knowledge of the resistance mechanisms to the most widely used anthelmintics, the macrocyclic lactones (ML). There are no data regarding resistance mechanisms to MLs in cyathostomins, although in other parasitic nematodes, the ABC transporters, P-glycoproteins (P-gps), have been implicated in playing an important role. Here, we tested the hypothesis that P-gps are, at least in part, responsible for reduced sensitivity to the ML ivermectin (IVM) in cyathostomins; first, by measuring transcript levels of pgp-9 in IVM resistant versus IVM sensitive third stage larvae (L3) pre-and post-IVM exposure in vitro. We then tested the effect of a range of P-gp inhibitors on the effect of IVM against the same populations of L3 using the in vitro larval development test (LDT) and larval migration inhibition test (LMIT). We demonstrated that, not only was pgp-9 transcription significantly increased in IVM resistant compared to IVM sensitive L3 after anthelmintic exposure (p < 0.001), but inhibition of P-gp activity significantly increased sensitivity of the larvae to IVM in vitro, an effect only observed in the IVM resistant larvae in the LMIT. These data strongly implicate a role for P-gps in IVM resistance in cyathostomins. Importantly, this raises the possibility that P-gp inhibitor-IVM combination treatments might be used in vivo to increase the effectiveness of IVM against cyathostomins in Equidae.

Risk factor analysis of equine strongyle resistance to anthelmintics.

Intestinal strongyles are the most problematic endoparasites of equids as a result of their wide distribution and the spread of resistant isolates throughout the world. While abundant literature can be found on the extent of anthelmintic resistance across continents, empirical knowledge about associated risk factors is missing. This study brought together results from anthelmintic efficacy testing and risk factor analysis to provide evidence-based guidelines in the field. It involved 688 horses from 39 French horse farms and riding schools to both estimate Faecal Egg Count Reduction (FECR) after anthelmintic treatment and to interview farm and riding school managers about their practices. Risk factors associated with reduced anthelmintic efficacy in equine strongyles were estimated across drugs using a marginal modelling approach. Results demonstrated ivermectin efficacy (96.3% ± 14.5% FECR), the inefficacy of fenbendazole (42.8% ± 33.4% FECR) and an intermediate profile for pyrantel (90.3% ± 19.6% FECR). Risk factor analysis provided support to advocate for FEC-based treatment regimens combined with individual anthelmintic dosage and the enforcement of tighter biosecurity around horse introduction. The combination of these measures resulted in a decreased risk of drug resistance (relative risk of 0.57, p = 0.02). Premises falling under this typology also relied more on their veterinarians suggesting practitionners play an important role in the sustainability of anthelmintic usage. Similarly, drug resistance risk was halved in premises with frequent pasture rotation and with stocking rate below five horses/ha (relative risk of 0.53, p < 0.01). This is the first empirical risk factor analysis for anthelmintic resistance in equids. Our findings should guide the implementation of more sustained strongyle management in the field.

The route of administration drastically affects ivermectin activity against small strongyles in horses.

The goal of the current study was to evaluate the comparative efficacy of ivermectin (IVM) against small strongyles (cyathostomins) following its oral and intramuscular (IM) administration, in naturally parasitized horses. The parasitological data were complemented with the assessment of the plasma disposition kinetics of IVM. The trial included two different experiments. In experiment I, 40 horses naturally infected with small strongyles were randomly allocated into four experimental groups (n=10) and treated with IVM (0.2mg/kg) as follows: IVM oral paste, animals were orally treated with Eqvalan® (IVM 1.87% paste, as the reference formulation) by the oral route; IVM oral solution, animals were orally treated with Remonta® (IVM 2% solution, as a test formulation); IVM IM solution, animals were IM treated with the test product (Remonta® IVM 2% solution); and control, animals were kept without treatment as untreated controls. In experiment II, 24 horses naturally parasitized with small strongyles were randomly allocated into the same four experimental groups (n=6) described for experiment I. Faecal samples were individually collected directly from the rectum of each horse prior (day -1) and at 7 and 15 (Experiment I) or 7, 15 and 21 (Experiment II) days after-treatment, to assess the eggs per gram (epg) counts and estimate the efficacy of the treatments. Additionally, the comparative plasma disposition kinetics of IVM in treated animals was assessed in experiment II. In both experiments, an excellent (100%) IVM efficacy was observed after its oral administration (test and reference formulations). However, the IM administration of IVM resulted in a low efficacy (36-64%). Similar IVM plasma concentration was observed after its oral administration as a paste or as a solution. The higher IVM plasma profiles observed after the IM administration accounted for an enhanced systemic availability. The improved IVM efficacy observed against adult cyathostomins after its oral administration can be explained by an enhanced drug exposure of the worms located at the lumen of the large intestine. These findings may have a direct impact on the practical use of macrocyclic lactones in horses.

Shortened egg reappearance after ivermectin or moxidectin use in horses in the UK.

This study reports ivermectin and moxidectin egg reappearance periods (ERP) from UK horses with persistently positive faecal egg counts (FEC), defined as positive FEC within the ERP of an anthelmintic post-treatment, or with FECs that remained positive after the normal ERP post-anthelmintic treatment. A selected population of UK pleasure horses deemed at high risk of strongyle infection was studied. The earliest ERP recorded after ivermectin or moxidectin, using first positive FEC, was 5 weeks. From 16 premises where moxidectin was used, five had ERP ≥12 weeks using two further metrics. For premises where moxidectin was administered to only one animal (present or tested), and evaluated as one group (n = 61), ERP was ≥10 weeks. For premises where ivermectin was used, the ERP was ≥5 weeks. Premises with only one horse (present or tested), dosed with ivermectin (n = 31), analysed as one group, demonstrated egg reappearance ≥6 weeks. These field data suggest shortened ERPs following macrocyclic lactone treatment compared to previously published values (8-10 and >13 weeks respectively) when these drugs were first marketed.