Gastrointestinal parasite community structure in horses after the introduction of selective anthelmintic treatment strategies.

A relatively new method to study the species richness and diversity of nematode parasites in grazing animals is to perform deep sequencing on composite samples containing a mixture of parasites. In this work, we compared species composition of strongyles in two groups of horses as a function of egg count and age, based on a DNA barcoding approach. Faecal egg counts and larval cultures were obtained from nearly 300 horses, i.e., domestic horses (n = 167) and trotters (n = 130) sampled nationwide. The second internal transcribed spacer region (ITS2) of strongyle nematodes in the larval cultures was first amplified using barcoded universal primers and then sequenced on the PacBio platform. Subsequently, bioinformatic sequence analysis was performed using SCATA to assign operational taxonomic units (OTU). Finally, species occurrence and composition were assessed using R. ITS2 sequences were found in the majority (89%) of larval samples. Sequencing yielded an average of 140 (26 to 503) reads per sample. The OTUs were assigned to 28 different taxa, of which all but three could be identified as species. The average relative abundance of the seven most abundant species (all Cyathostominae) accounted for 87% of the combined data set. The three species with the highest prevalence in both horse groups were Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus calicatus, and they were frequently found in different combinations with other species regardless of horse group. Interestingly, this result is largely consistent with a previous Swedish study based on morphological analysis of adult worms. In addition, two migratory strongylids (Strongylus vulgaris and S. edentatus) occurred in few domestic horses and trotters. Except for C. minutus and C. nassatus, which decreased with age, and C. catinatum and S. vulgaris, which increased, no specific trends were observed with respect to horse age. Taken together, these results are broadly consistent with data obtained before the introduction of selective targeted treatment in Sweden in 2007. All in all, our results suggest that this treatment strategy has not led to a significant change in strongyle nematode community structure in Swedish horses. The study also confirms that nemabiome analysis in combination with diversity index analysis is an objective method to study strongyle communities in horses.

Validation of a serum ELISA test for cyathostomin infection in equines.

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection.

Detection of SNPs and benzimidazole resistance in strongyle nematode eggs of horses by allele-specific PCR.

This study was conducted to determine single nucleotide polymorphisms (SNPs) and the benzimidazole (BZ) resistance in strongyle nematode egg populations in horses using molecular techniques. A total of 200 fecal samples were collected from horses in 26 farms in two provinces (Kayseri and Nevsehir) of the Central Anatolia Region of Türkiye between May and August 2022. The flotation method was used to detect strongyle nematode eggs in the fecal samples of the horses. Afterward, strongyle nematode eggs were collected, and the allele-specific polymerase chain reaction (AS-PCR) technique was used to detect the BZ resistance. BZ-susceptible and BZ-resistant PCR products were sequenced to determine single nucleotide polymorphisms (SNPs) in the ß-tubulin isotype 1 gene. The strongyle nematode eggs were determined in 85 (42.5%) out of 200 fecal samples. AS-PCR detected 50.58% (43/85) BZ-resistant (homozygous resistant) and 36.4% (31/85) BZ-susceptible (homozygous susceptible) genes in the strongyle eggs. Both BZ-resistant and BZ-susceptible genes (heterozygous) were determined in 11 samples. BZ-resistant and BZ-susceptible allele frequencies were determined as 57.0% (48.5/85) and 43.0% (36.5/85), respectively. SNPs were detected only in codon 200 of the ß-tubulin isotype 1 gene in four sequenced isolates of the two resistant and two susceptible isolates. This study is the first molecular report on BZ resistance in strongyle nematode eggs in horses in Türkiye. The widespread prevalence of BZ-resistant alleles in equine strongyle nematodes shows the requirement for the immediate usage of other anthelmintics instead of the BZ group drugs for the effective management and control of equine strongyle nematodes.

Occurrence and control of equine strongyle nematode infections in Prince Edward Island, Canada.

Widespread overuse of anthelmintics has produced a growing population of intestinal parasites resistant to control measures. A paradigm shift in equine parasite control is warranted to prevent continued resistance development and maintain equine health. Small strongyles, which are ubiquitous in horses, are currently the most important intestinal parasites of adult horses. Sustainable management programs consider the variation in egg shedding by individual horses, and varied risks associated with age, use, density, climate, and environment. To develop regional recommendations for Prince Edward Island (PEI), Canada, it is important to first characterize strongyle egg shedding patterns and parasite management practices in use. Study objectives were to conduct a cross-sectional observational survey and risk factor analysis of parasite control programs, strongyle egg shedding and Strongylus vulgaris serology. A total of 339 horses from 40 PEI farms were sampled. Mean farm size was 8 horses and ranged from 2 to 30. Mean horse age was 11.6 years (Std. Dev. =7.2) and ranged from 2 months to 32 years. Mean fecal egg count (FEC) was 322 eggs per gram (EPG) (Std. Dev. =648). On average, 32% (Std. Dev. =16%) of horses shed 80% of strongyle eggs across 32 eligible farms. When considering all horses (n = 313) as one large herd, 18.7% of horses shed 80% of strongyle eggs. Use of FEC was identified in 4.6% of horses at 15% (n = 6) of PEI farms. Reported deworming intervals included 37.4% (n = 123) every 2-3 months and 58% (n = 191) every 3-4 months. Positive S. vulgaris titers were identified in 60% of horses (n = 200). Univariate analysis revealed that months since last deworming, age, and body condition score (BCS) were associated with strongyle shedding. The estimated odds of being in the high FEC category (>500 EPG) was 1.4 (95% CI, 1.1-1.8) times higher when last deworming increased by one month. Under-conditioned (BCS <4.5) horses had 3.6 (95% CI, 1.2-10.6) times odds of being in the higher FEC category than over-conditioned horses. Non-racing horses had 5.4 times odds of having a positive S. vulgaris titer than racehorses. This cross-sectional observational study is the first to report on the occurrence, risk factors and control of equine strongyle nematode infections in PEI, Canada. We conclude that the 80:20 rule can be used to develop control recommendations in PEI. Very few farms in PEI currently use FEC to guide parasite management. These findings provide a basis for future client education and investigations aimed at providing region specific recommendations.

Patterns of variation in equine strongyle community structure across age groups and gut compartments.

BACKGROUND: Equine strongyles encompass more than 64 species of nematode worms that are responsible for growth retardation and the death of animals. The factors underpinning variation in the structure of the equine strongyle community remain unknown. METHODS: Using horse-based strongyle community data collected after horse deworming (48 horses in Poland, 197 horses in Ukraine), we regressed species richness and the Gini-Simpson index upon the horse's age, faecal egg count, sex and operation of origin. Using the Ukrainian observations, we applied a hierarchical diversity partitioning framework to estimate how communities were remodelled across operations, age groups and horses. Lastly, strongyle species counts collected after necropsy (46 horses in France, 150 in Australia) were considered for analysis of their co-occurrences across intestinal compartments using a joint species distribution modelling approach. RESULTS: First, inter-operation variation accounted for > 45% of the variance in species richness or the Gini-Simpson index (which relates to species dominance in communities). Species richness decreased with horse's age (P = 0.01) and showed a mild increase with parasite egg excretion (P < 0.1), but the Gini-Simpson index was neither associated with parasite egg excretion (P = 0.8) nor with horse age (P = 0.37). Second, within-host diversity represented half of the overall diversity across Ukrainian operations. While this is expected to erase species diversity across communities, community dissimilarity between horse age classes was the second most important contributor to overall diversity (25.8%). Third, analysis of species abundance data quantified at necropsy defined a network of positive co-occurrences between the four most prevalent strongyle genera. This pattern was common to necropsies performed in France and Australia. CONCLUSIONS: Taken together, these results show a pattern of ß-diversity maintenance across age classes combined with positive co-occurrences that might be grounded by priority effects between the major species.

Shortened egg reappearance periods of equine cyathostomins following ivermectin or moxidectin treatment: morphological and molecular investigation of efficacy and species composition.

Macrocyclic lactones have been the most widely used drugs for equine parasite control during the past four decades. Unlike ivermectin, moxidectin exhibits efficacy against encysted cyathostomin larvae, and is reported to have persistent efficacy with substantially longer egg reappearance periods. However, shortened egg reappearance periods have been reported recently for both macrocyclic lactones, and these findings have raised several questions: (i) are egg reappearance period patterns different after ivermectin or moxidectin treatment? (ii) Are shortened egg reappearance periods associated with certain cyathostomin species or stages? (iii) How does moxidectin's larvicidal efficacy affect egg reappearance period? To address these questions, 36 horses at pasture, aged 2-5 years old, were randomly allocated to three treatment groups: 1, moxidectin; 2, ivermectin; and 3, untreated control. Strongylid fecal egg counts were measured on a weekly basis, and the egg reappearance period was 5 weeks for both compounds. Strongylid worm counts were determined for all horses: 18 were necropsied at 2 weeks post-treatment (PT), and the remaining 18 at 5 weeks PT. Worms were identified to species morphologically and by internal transcribed spacer-2 (ITS-2) rDNA metabarcoding. Moxidectin and ivermectin were 99.9% and 99.7% efficacious against adults at 2 weeks post treatment, whereas the respective efficacies against luminal L4s were 84.3% and 69.7%. At 5 weeks PT, adulticidal efficacy was 88.3% and 57.6% for moxidectin and ivermectin, respectively, while the efficacy against luminal L4s was 0% for both drugs. Moxidectin reduced early L3 counts by 18.1% and 8.0% at 2 or 5 weeks, while the efficacies against late L3s and mucosal L4s were 60.4% and 21.2% at the same intervals, respectively. The luminal L4s surviving ivermectin treatment were predominantly Cylicocyclus (Cyc.) insigne. The ITS-2 rDNA metabarcoding was in good agreement with morphologic species estimates but suggested differential activity between moxidectin and ivermectin for several species, most notably Cyc. insigne and Cylicocyclus nassatus. This study was a comprehensive investigation of current macrocyclic lactone efficacy patterns and provided important insight into potential mechanisms behind shortened egg reappearance periods.

Anthelmintic resistance of horse strongyle nematodes to fenbendazole in Lithuania.

BACKGROUND: Control of strongyle infections presents a global challenge for horse practitioners due to the development of anthelmintic resistance (AR), however comprehensive information on AR in Lithuania is still lacking. The aim of this study was to assess the current situation of fenbendazole (FBZ) AR in horses at stable level in Lithuania. RESULTS: Faecal samples from 121 horses from six stables were examined using the Mini-FLOTAC method. Of these, 89 horses met the inclusion criteria that included strongyle faecal egg counts (FEC) exceeding 200 eggs per gram (EPG). Faecal egg count reduction tests (FECRTs) were performed in these. AR was evaluated at horse stable level based on faecal egg count reduction (FECR) and the lower limit of the 95% credible interval (LLCI) using the Bayesian hierarchical model. This study confirmed that strongylids (Cyathostominae (CYA)) resistant to FBZ are pervasive in Lithuania. FBZ was ineffective in three of the six stables (FECR 77.1-79.0%; 49.8-99.8 LLCI), was suspected to be ineffective in one stable (FECR 93.6%; 85.4-100 LLCI) and was effective (FECR 99.8-100%; 99.8-100 LLCI) in two stables. FEC showed a significant (P < 0.01) difference between the treatment and control groups. Only CYA larvae were detected in larval cultures derived from strongyle-positive faecal samples collected 14 days after treatment of a test group with FBZ. CONCLUSION: This in vivo study showed that resistance to FBZ in the treatment of strongyle nematodes is prevalent in horse stables in Lithuania. These findings should guide the implementation of more sustainable management of strongyle infections in horses in Lithuania.

Helminths and their management in Swiss Army horses: differences between riding horses and pack horses evidence the need of improvement.

INTRODUCTION: Intestinal helminth management in horses has both clinical and epidemiologic relevance, in additional association with anthelmintic resistance. The Swiss Army employs military owned riding horses and privately owned pack horses, which are brought together for service periods up to 12 weeks. We compared husbandry conditions and intestinal helminth management of both groups via questionnaire and analysed faecal samples of 53 riding horses and 130 pack horses using combined sedimentation/flotation, the McMaster method and larval cultures. Riding horses only had cyathostomin infections (prevalence: 60,4 %), while pack horses harboured cyathostomins (71,5 %), Parascaris sp. (6,9 %) and Strongylus vulgaris (1,5 %). Regression models combining faecal sample results with questionnaire data unveiled correlations of husbandry practices with parasite frequencies identifying risk and protective factors. Pasture management, hygiene and deworming practices were highly variable for pack horses, while for riding horses there was an overall concept. This included a selective deworming strategy with faecal egg counts (FECs) of strongyles prior to deworming, applying a threshold of 200 eggs per gram of faeces (epg). Anthelmintic treatments based on FECs, weekly faeces removal on pastures (pastures), the use of macrocyclic lactones and deworming horses regularly were identified as protective factors regarding the 200 epg threshold for strongyle eggs. Accordingly, the mean epg for strongyle eggs between the groups (111 and 539 in riding and pack horses, respectively) was significantly different (p < 0,001). Overall, intestinal helminth management in pack -horses showed room for improvement regarding pasture hygiene, the used anthelmintics and the frequency of deworming, from which all Swiss Army horses would benefit, as they share pastures during their -service, therefore entailing the risk of parasite transmission.

INTRODUCTION: La gestion des helminthes intestinaux chez les chevaux présente une importance clinique et épidémiologique, en association avec la résistance aux anthelminthiques. L'armée suisse emploie des chevaux de selle militaires et des chevaux de bât privés qui sont réunis pour des périodes de service allant jusqu'à 12 semaines. Nous avons comparé la détention animale et la gestion des helminthes intestinaux des deux groupes par le biais d'un questionnaire et analysé les échantillons fécaux de 53 chevaux de selle et 130 chevaux de bât en utilisant la sédimentation/flottation combinée, la méthode McMaster et les cultures larvaires. Les chevaux de selle ne présentaient que des infections à cyathostomes (prévalence: 60,4 %), tandis que les chevaux de bât hébergeaient des cyathostomes (71,5 %), Parascaris sp. (6,9 %) et Strongylus vulgaris (1,5 %). Des modèles de régression combinant les résultats des échantillons fécaux et les données du questionnaire ont révélé des corrélations entre les pratiques de détention animale et la fréquence des parasites, identifiant les facteurs de risque et de protection. La gestion des pâturages, l'hygiène et les pratiques en matière de vermifugation étaient très variables pour les chevaux de bât, tandis que pour les chevaux de selle, il existait un concept unitaire. Ceci comprenait stratégie de vermifugation sélective avec comptage des œufs fécaux (CEF) de strongles avant la vermifugation, en appliquant un seuil de 200 œufs par gramme de fèces (opg). Les traitements anthelminthiques basés sur les CEF, l'enlèvement hebdomadaire des crottins sur le pâturage, l'utilisation de lactones macrocycliques et la vermifugation régulière des chevaux ont été identifiés comme des facteurs de protection concernant le seuil de 200 opg pour les œufs de strongles. En conséquence, l'iog moyen pour les œufs de strongles entre les groupes (111 et 539 chez les chevaux de selle et de bât, respectivement) était significativement différent (p < 0,001). Globalement, la gestion des helminthes intestinaux chez les chevaux de bât a montré qu'il est possible d'améliorer l'hygiène des pâturages, le choix des anthelminthiques et la fréquence des vermifuges, ce dont tous les chevaux de l'armée suisse bénéficieraient, car ils partagent les pâturages pendant leur service, ce qui entraîne un risque de transmission des parasites.

Feasibility of selective anthelmintic therapy to horses in tropical conditions: the Cuban scenario.

For the past several decades, selective anthelmintic therapy (SAT) has been recommended in temperate climate countries for controlling gastrointestinal parasites in horses. However, the feasibility of this approach in tropical climates remains unknown, given the very different parasite transmission patterns and a larger representation of working equids. The aim of this study was to evaluate the bio-economic feasibility of SAT in horses kept under tropical conditions of Camagüey, Cuba. Fecal egg counts were determined from 794 adult horses and used for evaluating three different putative treatment thresholds; > 500 strongylid eggs per gram of feces (EPG); > 1000 EPG; and treatments yielding > 80% of the total herd egg output. These scenarios were evaluated under three treatment frequencies (every 2, 6, and 12 months). The bio-economic feasibility of these nine possible selective anthelmintic therapy scenarios was evaluated taking into account the cost of parasitological tests, and the mean cost of anthelmintic treatment in Camagüey. The majority (96.7%) of the samples tested were positive for strongyle eggs, with a mean of 1549 EPG. The percentage of horses exceeding the cut-off points at the nine scenarios varied between 40.1 and 93.8%. All owners surveyed used extra-label anthelmintic product in their horses on a routine basis. The economic analysis demonstrated that selective therapy generally was much more costly than deworming the entire herd without determining egg counts. However, we consider that the deworming every 6 months of the horses that expel 80% of the eggs in the herd allows a reduction of the treatment intensity without increasing costs, and it can be considered as a viable selective deworming scheme under similar conditions.

Multiple resistance in equine cyathostomins: a case study from military establishments in Rio Grande do Sul, Brazil.

Semi-intensive equine breeding system favors gastrointestinal nematode infections. The treatment of these infections is based on the use of anthelmintics. However, the inappropriate use of these drugs has led to parasitic resistance to the available active principles. The objective of this study was to evaluate the efficacy of the main classes of antiparasitic (ATP) used in control in adult and young animals, including: benzimidazoles (fenbendazole), pyrimidines (pyrantel pamoate), macrocyclic lactones (ivermectin and moxidectin), as well as the combination of active ingredients (ivermectin + pyrantel pamoate). The study was carried out in two military establishments, located in Rio Grande do Sul (RS), from January to December, 2018. The intervals between the treatments of the animals were performed from 30 to 90 days. Coproparasitological evaluations were determined by the egg count reduction in the faeces. Cyatostomine larvae were identified in pre and post-treatment cultures. The results demonstrated the multiple parasitic resistance of cyathostomins to fenbendazole, moxidectin in young animals, and to fenbendazole, pyrantel pamoate in adult animals. Thus, it is necessary to define or diagnose parasitic resistance to assist in the creation of prophylactic parasitic control, using suppressive treatment with ATP associated with integrated alternatives. The progress of parasitic resistance can be slowed.

Eficacy of ivermectin, moxidectin and febendazole in equine in Brazil.

The objectives of the present study were to evaluate the efficacy of three anthelmintic drugs, i.e. fenbendazole, ivermectin and moxidectin; to determine the genera and species of the most abundant strongyles; and to investigate parasite control measures used in herds of horses in the western region of the state of São Paulo, Brazil. This study was onducted between February and December 2013 on 10 farms in this region. Coprological evaluations were conducted for counting the numbers of eggs per gram of feces (EPG) and coprocultures were made in order to identify third-stage larvae (L3) of strongyles. Parasite control measures were investigated by surveying each farm's management and characteristics. A fecal egg count reduction test (FECRT) was used to determine the anthelmintic efficacy based on EPG count results before and after treatment. The FECRT showed that anthelmintic resistance to fenbendazole was present and that ivermectin and moxidectin had high effectiveness in the western region of the state of São Paulo. Identification of L3 revealed that there was high prevalence of cyathostomins among strongyle larvae. The highest prevalence of Strongylus vulgaris occurred on farms where the intervals between deworming were long. The questionnaire applied showed that ivermectin was the drug most used among these farmers.

Characterisation of serum IgG(T) responses to potential diagnostic antigens for equine cyathostominosis.

Cyathostomins are ubiquitous parasitic nematodes of horses. These worms spend substantial periods as intestinal wall stage encysted larvae, which can comprise up to 90% of the total burden. Several million larvae have been reported in individuals. Emergence of these larvae from the gut wall can lead to life-threatening colitis. Faecal egg count tests, increasingly used by horse owners to inform anthelmintic treatments, do not correlate with the intra-host burden of cyathostomins; this represents a key gap in the diagnostic toolbox. Previously, a cyathostomin Gut Associated Larval Antigen was identified as a promising marker for the intra-host stages of infection. Here, cyathostomin Gut Associated Larval Antigen and an additional protein, Cyathostomin Immuno-diagnostic antigen, were investigated to examine their value in providing information on cyathostomin burden. ELISA analyses examined serum IgG(T) responses to recombinant proteins derived from individual cyathostomin species. Receiver Operator Characteristic curve analysis was performed on the ELISA data; proteins with the highest Area Under the Curve values were selected to test protein combinations to investigate which were the most informative in identifying the infection status of individuals. Three cocktail combinations were tested, comprising: (a) Cy-GALA proteins from two species and a Cy-CID protein from a third species (CT3), (b) Cy-GALA proteins from five species (CT5), and (c) all CT5 components, plus a Cy-CID protein from an additional species (CT6). The best predictive values for infection were obtained using CT3 and CT6, with similar values achieved for both. Proteins in CT3 are derived from the most commonly reported species, Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus longibursatus. This combination was selected for future development since it represents a more commercially viable format for a diagnostic test.

Horses grazing with cattle have reduced strongyle egg count due to the dilution effect and increased reliance on macrocyclic lactones in mixed farms.

Strongyle infection is an important issue in horse breeding. It impairs horse health and performance, with young horses being the most sensitive. Strongyle control has long relied on the systematic use of chemical treatments. However, expanding anthelmintic resistance among strongyles calls for alternative options. Mixed grazing is assumed to reduce strongyle load on the pasture as the result of a dilution effect. This has been shown in small ruminants grazing with cattle, but the putative benefits of co-grazing between horses and cattle have not yet been evaluated. Here, we conducted field surveys and face-to-face interviews on 44 farms from two contrasted saddle-horse production areas, Normandy and northern Massif Central, to compare equine strongyle management practices between specialized systems and mixed horse-cattle systems. Our goals were (i) to quantify breeders' awareness of the putative benefits associated with the co-grazing of horses and cattle, (ii) to establish whether mixed farming was associated with different strongyle management strategies and (iii) to test whether strongyle egg excretion was reduced in horses grazed with beef cattle. Every breeder relied on systematic calendar treatments, and only 8 out of the 23 mixed breeders were aware that co-grazing of horses with cattle could be used as part of their strongyle control strategy. Management practices were similar across both systems in Normandy. In Massif Central, mixed breeders formed a distinct cluster from their specialized counterparts: deworming was less frequent and stocking density was higher in mixed farms, while specialized breeders seemed more willing to integrate herd and plot management into control strategies. Faecal egg counts measured in horses from Massif Central were significantly reduced when horses were grazed with cattle. This was the result of an increased reliance on macrocyclic lactones in mixed farms (P < 0.01) and a significant dilution effect (P < 0.01). When considering a subsample of horses treated with macrocyclic lactones only, young horses grazed with cattle had 50% fewer strongyle eggs excreted in their faeces than horses grazed in equine-only pastures (P < 0.01). This is the first evidence of the benefits of mixed grazing with cattle as an alternative to control strongyle infection in horses, although this promising alternative remains largely unknown by horse breeders.

Dealing with double trouble: Combination deworming against double-drug resistant cyathostomins.

An alternative control regimen for drug-resistant parasites is combination deworming, where two drugs with different modes of action are administered simultaneously to target the same parasite. Few studies have investigated this in equine cyathostomins. We previously reported that an oxibendazole (OBZ) and pyrantel pamoate (PYR) combination was not sustainable against a cyathostomin population with high levels of OBZ and PYR resistance. This study consisted of a field study and two computer simulations to evaluate the efficacy of a moxidectin-oxibendazole (MOX-OBZ) combination against the same cyathostomin population. In the field study, anthelmintic treatments occurred when ten horses exceeded 100 eggs per gram. Fecal egg counts and efficacy evaluations were performed every two weeks. The two simulations utilized weather data as well as equine and parasite population parameters from the field study. The first simulation repeated the treatment schedule used in the field study over a 40 year period. The second evaluated efficacies of combination treatments using selective therapy over 40 years. In the field study, efficacies of MOX and both combination treatments were 100%. The egg reappearance period for MOX was 16 weeks, and the two combination treatments were 12 and 18 weeks. The first (46.7%) and last (40.1%) OBZ efficacies were not significantly different from each other. In the simulation study, the combination treatment delayed MOX resistance development compared to when MOX was used as a single active. This occurred despite the low efficacy of OBZ. The second set of simulations identified combination treatments used with selective therapy to be the most effective at delaying MOX resistance. Overall, this study supports the use of combination treatment against drug-resistant cyathostomins, when one of the actives exhibits high efficacy, and demonstrates benefits of this approach despite substantially lowered efficacy of the other active ingredient.

Multiple resistance in equine cyathostomins: a case study from military establishments in Rio Grande do Sul, Brazil / Múltipla resistência de ciatostomíneos de equinos: um estudo de caso em estabelecimentos militares no Rio Grande do Sul, Brasil

Abstract Semi-intensive equine breeding system favors gastrointestinal nematode infections. The treatment of these infections is based on the use of anthelmintics. However, the inappropriate use of these drugs has led to parasitic resistance to the available active principles. The objective of this study was to evaluate the efficacy of the main classes of antiparasitic (ATP) used in control in adult and young animals, including: benzimidazoles (fenbendazole), pyrimidines (pyrantel pamoate), macrocyclic lactones (ivermectin and moxidectin), as well as the combination of active ingredients (ivermectin + pyrantel pamoate). The study was carried out in two military establishments, located in Rio Grande do Sul (RS), from January to December, 2018. The intervals between the treatments of the animals were performed from 30 to 90 days. Coproparasitological evaluations were determined by the egg count reduction in the faeces. Cyatostomine larvae were identified in pre and post-treatment cultures. The results demonstrated the multiple parasitic resistance of cyathostomins to fenbendazole, moxidectin in young animals, and to fenbendazole, pyrantel pamoate in adult animals. Thus, it is necessary to define or diagnose parasitic resistance to assist in the creation of prophylactic parasitic control, using suppressive treatment with ATP associated with integrated alternatives. The progress of parasitic resistance can be slowed.

Resumo O sistema semi-intensivo de criação de equinos favorece infecções por nematoides gastrointestinais. O tratamento dessas infecções é baseado no uso de anti-helmínticos. No entanto, o uso inadequado desses medicamentos levou à resistência parasitária aos princípios ativos disponíveis. O objetivo deste estudo foi avaliar a eficácia das principais classes de antiparasitários (ATP), utilizados no controle em animais adultos e jovens, incluindo: benzimidazois (fenbendazol), pirimidinas (pamoato de pirantel), lactonas macrocíclicas (ivermectina e moxidectina), bem como a combinação de ingredientes ativos (ivermectina + pamoato de pirantel). O estudo foi realizado em dois estabelecimentos militares, localizados no Rio Grande do Sul (RS), de janeiro a dezembro de 2018. Os intervalos entre os tratamentos foram realizados de 30 a 90 dias. As avaliações coproparasitológicas foram determinadas pela redução da contagem de ovos nas fezes. Foram identificadas larvas de ciatostomíneos nas coproculturas pré e pós tratamentos. Os resultados demonstraram a resistência parasitária múltipla dos ciatostomíneos ao fenbendazol, moxidectina em animais jovens, febendazole e pamoato de pirantel em animais adultos. Estabelecer o diagnóstico da resistência parasitária auxiliará na elaboração de um controle parasitário profilático, reduzindo o tratamento supressivo com ATP juntamente com alternativas de controle integrado. Dessa forma, o avanço da resistência parasitária poderá ser retardado.

Managing anthelmintic resistance in cyathostomin parasites: Investigating the benefits of refugia-based strategies.

Selective anthelmintic therapy has been recommended as a sustainable strategy for cyathostomin control in horse populations for several decades. The traditional approach has been to determine strongyle fecal egg counts (FEC) for all horses, with treatment only recommended for those exceeding a predetermined threshold. The aims are to achieve a reduction of overall egg shedding, while leaving a proportion of the herd untreated, which lowers anthelmintic treatment intensity and reduces selection pressure for development of anthelmintic resistance. This study made use of the cyathostomin model to evaluate the influence of treatment strategies with between 1 and 8 yearly treatment occasions, where either 1) all horses were treated, 2) a predetermined proportion of the herd remained untreated, or 3) horses were treated if their FEC exceeded thresholds between 100 and 600 strongyle eggs per gram. Weather data representing four different climatic zones was used and three different herd age structures were compared; 1) all yearlings, 2) all mature horses 10-20 years old, and 3) a mixed age structure of 1-20 years of age. Results indicated a consistent effect of age structure, with anthelmintic resistance developing quickest in the yearling group and slowest among the mature horses. Development of anthelmintic resistance was affected by treatment intensity and selective therapy generally delayed resistance. Importantly, the results suggest that the effects of selective therapy on resistance development are likely to vary between climatic zones and herd age structures. Overall, a substantial delaying of resistance development requires that the average number of treatments administered annually across a herd of horses needs to be about two or less. However, results also indicate that an age-structured prioritisation of treatment to younger horses should still be effective. It appears that a 'one-size-fits-all' approach to the management of anthelmintic resistance in cyathostomins is unlikely to be optimal.

Evaluation of the effectiveness of programs combating the invasions of strongyles (Strongylidae) in horses in selected stables of Western Pomerania

The emergence of drug resistance among parasites is a side effect of the common use of chemotherapeutic agents in horses. Combating parasites typically involves routine deworming treatments twice a year (before and after the grazing season). However, in grazing farming this treatment may be insufficient. The aim of the study was to evaluate the effectiveness of programs to combat the invasion of strongyles (Strongylidae) in horses in the selected stables of Western Pomerania. Coprological examination was conducted in 85 horses from three stables located in Western Pomerania. Horses from all three stables remained in the pastures in the period from spring to late autumn, and deworming treatment was performed twice. The examination of horses has found nematodes of the family Strongylidae. The average prevalence of infection in horses in the studs tested was 36.16% for Strongylinae, and 58.49% for Cyathostominae. Administration of the medication twice did not eradicate the invasion of strongyles in the horses investigated. A significant reduction of strongyle invasion was observed directly after treatment, followed by rapid reinvasion. It appears necessary to monitor the effectiveness of deworming treatments by performing parasitological examination of feces. The participating stables should develop and implement new strategies to combat parasitic invasions in horses that will be adapted to the environmental conditions (the life cycle of parasites).

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.