Identification of third stage larvae of strongyles and molecular diagnosis of Strongylus vulgaris in the feces of Thoroughbred horses kept in training centers in Rio de Janeiro, Brazil.

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals.

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.

Precision of cyathostomin luminal worm counts: Investigation of storage duration and fixative.

Essentially all grazing horses are infected with cyathostomin parasites. Adult cyathostomins reside in the large intestine of the horse and larval stages encyst within intestinal mucosa. Manual worm collection from aliquots of intestinal content is the current gold standard for retrieval and enumeration of luminal parasites, however, no research has been conducted to standardize specific parameters for processing and storage of samples. The aims of this study were (1) to evaluate the precision of current standard operating procedures for enumeration of luminal adult cyathostomin populations, (2) investigate the influence of chosen fixative, either 70 % ethanol or 10 % buffered formalin, as well as storage duration, immediately post necropsy vs. stored for eight weeks, on the magnitude and precision of worm counts, and (3) compare the luminal count magnitude between the three intestinal segments (cecum, ventral colon, dorsal colon). Ten miniature horses were enrolled in this study for euthanasia and necropsy over a four-week period. Luminal worm counts were conducted for 2 % aliquots of the cecum, ventral colon, and dorsal colon and samples were allocated to the two fixatives and the two storage durations. Precision was evaluated by coefficient of variation (CV) and was 13.04 % for total cyathostomin counts. Mean CV for large intestinal segments ranged from 15.31 % to 52.50 % irrespective of fixative used or storage duration. cecum worm counts were significantly lower compared to the ventral colon (p = 0.008) and dorsal colon (p = 0.01). Fixative and storage duration were not statistically associated with count precision or magnitude. This study demonstrated moderate to high precision estimates for luminal cyathostomin worm counts but did not identify any effects of fixative and storage duration within the framework of the study. This is the first study to determine cyathostomin worm count precision, and results will be useful for power analyses in the future.

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.

Reviving a tradition: The Development of Strongylus vulgaris in larval culture.

All horses are susceptible to the equine gastrointestinal parasite, Strongylus vulgaris, which is known to cause significant disease and death. The parasite undergoes development from the egg through the first (L1), second (L2) and third (L3) larval stages outside the horse. The L3 is the infective stage. The universally available technique for detection of S. vulgaris larvae is the larval culture method. This requires a 10-14 day culture period to induce development from egg to L3, followed by Baermannization and identification of the L3s to genus and/or species. It is unknown if the culture duration is necessary or ideal for S. vulgaris identification. The purpose of this study was to perform daily examinations of known S. vulgaris positive fecal samples in coproculture. Fresh feces were collected from a horse known to be shedding S. vulgaris eggs. A total of 140 cultures were set up using 10 g of feces. Cultures remained at room temperature and moistened every other day. Every day, 10 samples were examined, and all larvae were identified to stage, genus/species, and enumerated. Throughout the study, L1, L2, and L3 stages were observed, and S. vulgaris, Strongylus edentatus, Triodontophorus spp., and cyathostomin L3s were identified. Third stage larvae were observed on Day 5, and the mean number of L3s significantly increased on Day 10 (P < .001), and declined thereafter. Strongylus vulgaris was first observed on Day 6 with a mean count of 4.1 (95 % CI: 1.1, 7.1) S. vulgaris larvae, accounting for 4.1 % (95 % CI:1.8, 7) of the total L3s observed. The number of S. vulgaris larvae was significantly higher on Day 10 with a mean of 156.8 (95 % CI: 120.7, 192.9) S. vulgaris larvae (P < .001), and the proportion was also significantly higher with S. vulgaris comprising 50 % (95 % CI: 45.9, 54.8) (P = .006) of the total larvae. However, after 10 days, the mean number of S. vulgaris larvae declined, as did the proportion of S. vulgaris larvae compared to the total number of larvae. Using the described methods, it is possible to identify S. vulgaris as early as 6 days, and the optimal period is 10 days to detect the maximum number of S. vulgaris.

Diagnosing Strongylus vulgaris in pooled fecal samples.

Strongylus vulgaris is the most pathogenic intestinal helminth parasite infecting horses. The migrating larvae in the mesenteric blood vessels can cause non-strangulating intestinal infarctions, which have a guarded prognosis for survival. Infections are typically diagnosed by coproculture, but a PCR test is available in some countries. While it is ideal to test horses individually, many veterinarians and clients wish to pool samples to reduce workload and cost of the diagnostic method. The purpose of this study was to determine if pooling of fecal samples would negatively impact diagnostic performance of the coproculture and the PCR for determination of S. vulgaris infection. Ten horses with strongylid eggs per gram (EPG) >500 and confirmed as either S. vulgaris positive or negative were selected as fecal donors. Eight pools with feces from five horses were created with 0%, 10 %, 20 %, 30 %, 40 %, 50 %, 80 %, and 100 % S. vulgaris positive feces. From each pool, 20 subsamples of 10 g each were collected and analyzed. Half of these samples were set up for coproculture and the other half for PCR. All pools containing 50 % or greater S. vulgaris positive feces were detected positive by both PCR and coproculture. In the pools with less than 50 % S. vulgaris positive feces, the PCR detected 33 positive samples compared to 24 with the coproculture. Three samples from the 0% pool were detected as low-level PCR positives, but this could be due to contamination. These results indicate that diagnosing S. vulgaris on pooled samples is reliable, when at least 50 % of the feces in a pool are from S. vulgaris positive animals. Since S. vulgaris remains relatively rare in managed horses, however, some diagnostic sensitivity is expected to be lost with a pooled sample screening approach. Nonetheless, pooled sample screening on farms could still be considered useful under some circumstances, and the PCR generally performed better at the lower proportions of S. vulgaris positive feces.

Salivary Cortisol, Equine Characteristics, and Management Factors Associated With Strongyle-Type Egg Shedding of Ohio Horses.

Identification of factors associated with parasite infestation in horses could reduce frequency of anthelmintic administration and slow development of anthelmintic resistance. The study aim was to evaluate management factors, equine characteristics, and equine salivary cortisol concentrations for association with strongyle-type egg shedding levels. As immunocompromised horses appear to be more susceptible to parasite infestation, it was hypothesized that salivary cortisol concentration could be associated with parasite egg shedding. Saliva and fecal samples were collected from 200 horses across the state of Ohio. Equine management questionnaires were administered to barn managers. Fecal egg counts were performed with a modified Stoll method, and saliva samples were analyzed for salivary cortisol with an enzyme-linked immunosorbent assay (ELISA). A total of 24 variables were tested for association with fecal egg count results (dichotomized with 50 eggs per gram as a cutoff) using chi-square test of independence, Fisher's exact test, independent t-test, or Mann Whitney U test. The sample of horses was partitioned into tertiles based on salivary cortisol concentrations (i.e., low <0.052 µg/dL, medium 0.052-0.0712 µg/dL, and high >0.0712 µg/dL). Variables with P < .30 were analyzed for association with fecal egg count with multiple logistic regression. The final logistic regression model (P< .05) included horse age, horse salivary cortisol concentration, and pasture mowing frequency. Older horses had higher adjusted odds of having <50 EPG. Pasture mowing frequency of at least once per week increased the adjusted odds of <50 EPG. Horses with cortisol concentrations in the highest tertile had increased adjusted odds of having <50 EPG.

A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses.

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

Cytokine responses to various larval stages of equine strongyles and modulatory effects of the adjuvant G3 in vitro.

AIMS: To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites. METHODS AND RESULTS: EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL-4, IL-5 and IL-13 was induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL-5 and IL-9 (exsheated L3 and L4) and IFN-γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL-4, IL-5 and IL-10 while concomitantly enhancing the expression of IFN-γ. CONCLUSION: The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.

Strongylus vulgaris: Infection rate and molecular characterization from naturally infected donkeys at Sadat City, Egypt.

Strongylus vulgaris has high pathogenicity to equines. It causes aneurysm and thrombosis in the arteries particularly an anterior mesenteric artery, that is fatal to equines. In this study, we aimed to diagnose microscopically the natural infection of donkeys with Strongylus vulgaris from Sadat City, Minoufiya Governorate, Egypt. Fecal egg culture was used after the diagnosis of strongyle eggs to identify the species. Hematological and biochemical parameters were assessed. Adult worms were collected after post mortem examination of the infected animal. The sequence of ITS-2 was used to confirm the species of the parasite. The infection rate was 15.85% using the microscopical examination. The larval culture confirmed the infection with strongyle eggs as Strongylus vulgaris larvae. The sequence of ITS-2 was highly identical (about 95%) to sequences from Germany, China, and Turkey and occurred in the same genetic clade with the sequence from Germany. In conclusion, the study presented the diagnosis, the changes in the hematological and biochemical parameters in the infected animals, and the genetic characterization of Strongylus vulgaris from Sadat City, Minoufiya Governorate Egypt for the first time.

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.

Diagnostic performance of McMaster, Wisconsin, and automated egg counting techniques for enumeration of equine strongyle eggs in fecal samples.

Fecal egg counts are the cornerstone of equine parasite control programs. Previous work led to the development of an automated, image-analysis-based parasite egg counting system. The system has been further developed to include an automated reagent dispenser unit and a custom camera (CC) unit that generates higher resolution images, as well as a particle shape analysis (PSA) algorithm and machine learning (ML) algorithm. The first aim of this study was to conduct a comprehensive comparison of method precision between the original smartphone (SP) unit with the PSA algorithm, CC/PSA, CC/ML, and the traditional McMaster (MM) and Wisconsin (MW) manual techniques. Additionally, a Bayesian analysis was performed to estimate and compare sensitivity and specificity of all five methods. Feces were collected from horses, screened with triplicate Mini-FLOTAC counts, and placed into five categories: negative (no eggs seen), > 0 - ≤ 200 eggs per gram (EPG), > 200 - ≤ 500 EPG, > 500 - ≤ 1000 EPG, and > 1000 EPG. Ten replicates per horse were analyzed for each technique. Technical variability for samples > 200 EPG was significantly higher for MM than CC/PSA and CC/ML (p <  0.0001). Biological variability for samples> 0 was numerically highest for CC/PSA, but with samples > 200 EPG, MM had a significantly lower CV than MW (p =  0.001), MW had a significantly lower CV than CC/PSA (p <  0.0001), CC/ML had a significantly lower CV than both MW and SP/PSA (p <  0.0001, p =  0.0003), and CC/PSA had a significantly lower CV than CC/SP (p =  0.0115). Sensitivity was> 98 % for all five methods with no significant differences. Specificity, however, was significantly the highest for CC/PSA, followed numerically by SP/PSA, MM, CC/ML, and finally MW. Overall, the automated counting system is a promising new development in equine parasitology. Continued refinement to the counting algorithms will help improve precision and specificity, while additional research in areas such as egg loss, analyst variability at the counting step, and accuracy will help create a complete picture of its impact as a new fecal egg count method.