Jennifer L. Bellaw

Accuracy and Precision of Mini-FLOTAC and McMaster Techniques for Determining Equine Strongyle Egg Counts

&NA; The use of fecal egg count techniques to indirectly assess intestinal parasite burdens and determine anthelmintic efficacy is common in parasitological research and veterinary practice. The McMaster method is one of the most widely used techniques in veterinary practice, but recently, the Mini‐FLOTAC technique has been introduced as a possible alternative. Studies comparing the two methods in precision and accuracy are needed. This study aimed at evaluating the Mini‐FLOTAC technique for determining equine strongyle egg counts through a two‐part procedure. First, a set of fecal counts was executed using both methods. Next, blind counts were performed on spiked fecal samples with true egg counts at 0, 5, 50, 500, and 1,000 eggs per gram. All counts were performed in triplicates, and each sample was counted using both methods. Mini‐FLOTAC and McMaster had 83.2% and 53.7% precision, respectively. The accuracy was found to be 42.6% and 23.5% for Mini‐FLOTAC and McMaster, respectively. In conclusion, this study found that Mini‐FLOTAC exhibited both higher precision and accuracy than the McMaster technique and appears to be a more reliable alternative. Using a more precise egg‐counting method can help assure that changes in egg counts before and after treatment reflect a genuine reduction and are not due to chance variability. HighlightsA Mini‐FLOTAC technique was validated and compared to McMaster.The Mini‐FLOTAC technique had higher accuracy and precision.The Mini‐FLOTAC technique had higher sensitivity and specificity.

Biphasic appearance of corticated and decorticated ascarid egg shedding in untreated horse foals

Parascaris spp. infects foals worldwide and may cause airway inflammation in addition to small intestinal impaction and rupture. It is observed that acquired immunity eliminates ascarid burdens beginning at about 6 months of age, and current evidence suggests that a single parasite generation propagates in each foal crop. The purpose of this study was to monitor natural parasitic infections in untreated mixed breed horse foals over the course of 0-300 days of age. Fecal samples were collected monthly from all foals born in 2014 (n=13), beginning July 2014 through February 2015. Fecal egg counts (FECs) were performed in triplicates using the Mini-FLOTAC method. The foals were necropsied between 154 and 298 days of age and all intestinal ascarid were collected and identified to stage. Ascarid FECs exhibited a biphasic distribution with an initial peak at 91-120 days of age and, after a steady decline, a second, smaller peak at 241-300 days of age. Numbers of corticated and decorticated ascarid eggs were compared, with decorticated FECs remaining consistently low with a slight increase directly after the first corticated FEC peak. Overall, 4.36% of the total ascarid eggs counted were decorticated. Ascarid FECs showed a sharp peak in September, declined, and then steadily increased beginning in December and continuing through February. Upon necropsy, moderate to high number of ascarid specimens were recovered from foals between 8 and 10 months of age, coinciding with the second peak for the FECs. Eleven of the 13 foals harbored immature ascarid stages indicating a recent reinfection. However, these data demonstrates that apparently a second, smaller wave of infection is present in 8-10 month old foals. It may be of value to monitor egg counts in this age group to make sure that all parasite categories are well controlled.

Development of Strongylus vulgaris-specific serum antibodies in naturally infected foals

Strongylus vulgaris is regarded as the most pathogenic helminth parasite infecting horses. Migrating larvae cause pronounced endarteritis and thrombosis in the cranial mesenteric artery and adjacent branches, and thromboembolism can lead to ischemia and infarction of large intestinal segments. A recently developed serum ELISA allows detection of S. vulgaris-specific antibodies during the six-month-long prepatent period. A population of horses has been maintained at the University of Kentucky without anthelmintic intervention since 1979, and S. vulgaris has been documented to be highly prevalent. In 2012, 12 foals were born in this population, and were studied during a 12-month period (March-March). Weekly serum samples were collected to monitor S. vulgaris specific antibodies with the ELISA. Nine colts underwent necropsy at different time points between 90 and 300 days of age. At necropsy, Strongylus spp. and Parascaris equorum were identified to species and stage and enumerated. Initial statistical findings indicate a significant interaction between foal age and ELISA results (p<0.042). All foals had initial evidence of S. vulgaris-directed maternal antibodies transferred in the colostrum, but then remained ELISA negative during their first three months of life. Foals born in February and March became ELISA positive at about 12 weeks of age, while those born in April and May went positive at about 15 and 21 weeks, respectively. Foal date of birth was significantly associated with ELISA results (p<0.0001). This could be explained by birth date-dependent differences in parasite exposure. One foal remained ELISA-negative throughout the course of 30 weeks during the study. A significant association was found between ELISA values and larval S. vulgaris burdens (p<0.0001) as well as a three-way interaction between S. vulgaris, S. edentatus, and P. equorum burdens (p<0.001). A plateau with a subsequent decline in ELISA values corresponded with S. vulgaris larvae leaving the bloodstream and migrating back to the intestine.

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.

Changes in Serum Strongylus Vulgaris-Specific Antibody Concentrations in Response to Anthelmintic Treatment of Experimentally Infected Foals.

Strongylus vulgaris is the most pathogenic nematode parasite of horses. Its extensive migration in the mesenteric blood vessels can lead to life-threatening intestinal infarctions. Recent work has shown that this parasite is still identified among managed horse populations. A serum enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of migrating larvae of S. vulgaris. Previous work has documented an increase in ELISA values following larvicidal treatment with ivermectin and suggested that the target parasite antigen is primarily produced by the later larval stages. The aim of this study was to experimentally inoculate cohorts of foals with S. vulgaris, and then compare ELISA responses to early or later ivermectin treatments. Fifteen foals were held in confinement and infected orally with ~25 S. vulgaris third-stage larvae on Days 0, 7, 14, and 21. Foals were weaned on Day 43 and turned out to a pasture not previously grazed by horses. Foals remained at pasture continuously until the study was terminated on Day 196. On Day 55, foals were randomly allocated to three treatment groups of five each. Group 1 received ivermectin on Day 56, Group 2 received ivermectin on Day 112, and Group 3 foals served as untreated controls. Serum and fecal samples were collected at 28-day intervals throughout the study. Serum samples were analyzed with the S. vulgaris-specific ELISA and fecal samples were processed for fecal egg counting. The ELISA values of Group 1 foals were significantly lower than Groups 2 or 3 on Days 140–196. Both treated groups exhibited increased ELISA values following ivermectin treatment. Results indicate that the target diagnostic antigen is produced throughout the course of arterial infection with S. vulgaris, but that an early ivermectin treatment can reduce the cumulative antigen produced over the course of an infection.

Objective evaluation of two deworming regimens in young Thoroughbreds using parasitological and performance parameters

Parasitic helminths of equids are capable of causing ill-thrift, clinical disease, and death. Although young horses are the most susceptible to parasitic disease and are the most intensively treated cohort, deworming regimens are rarely evaluated within this age group. This study objectively evaluated the impact of deworming regimen on fecal egg counts (FECs), growth rates, and body-condition scores in young Thoroughbreds. Forty-eight Thoroughbred foals from three central Kentucky farms were randomly allocated to two treatment groups: an interval dose program receiving bi-monthly rotations of pyrantel pamoate and ivermectin and a daily deworming group receiving daily rations of pyrantel tartrate feed additive throughout the study, oxibendazole at two months of age, and moxidectin treatments at 9.5 and 16.5 months of age. Pre- and post-treatment eggs per gram of feces (EPGs) of Parascaris spp. and strongyle family parasites, gel/paste dewormer efficacies, and monthly weights and body condition scores were collected. Ascarid and strongyle FECs were not significantly different between groups but were significantly influenced by horse age with strongyle counts continually increasing and ascarid counts peaking at 4.5 months of age. Reduced strongyle efficacies of ivermectin and moxidectin were observed on two farms with consistently low pyrantel pamoate efficacies on all three farms. Ivermectin also exhibited reduced ascarid efficacy. Average daily gain did not differ significantly between groups and was only significantly influenced by age, mirroring average daily gain reference data for Kentucky Thoroughbreds born in 2013. Body condition scores also did not differ between groups, remaining in the optimal range (5-6) for the duration of the study. Management practices resulting in growth rates matching the reference data and in optimal body condition scores compensate for the negative impacts of parasitism even in cases of reduced drug efficacy. Performance parameters can provide useful information in cases of suboptimal parasite control.

Transmission of some species of internal parasites in horse foals born in 2013 in the same pasture on a farm in Central Kentucky

Abstract The present research is a continuation of studies conducted periodically over 40 years on transmission of natural infections of internal parasites in the same horse herd on pasture (Field 10) on a farm in Central Kentucky. It included 12 mixed light horse foals born in 2013 and euthanatized between July, 2013 and April, 2014 for collection of internal parasites. Parasites found: Gasterophilus intestinalis, Strongyloides westeri, Parascaris equorum, Anoplocephala perfoliata, small strongyles (cyathostomes), Strongylus vulgaris, Strongylus edentatus and Thelazia lacrymalis. Prevalence generally was related to age of the foals. Overall prevalence and number of specimens were lower than in earlier studies except for P. equorum. There were 15 species (much fewer than previously) of small strongyles found and recorded by location in the large intestines. All stages of small strongyles encysted in the mucosa of the large intestine were recovered by artificial digestion and in significantly lower numbers in older foals

Determination of the specific gravity of eggs of equine strongylids, Parascaris spp., and Anoplocephala perfoliata

Given the ever-increasing levels of anthelmintic resistance in livestock parasites globally, it is recommended to use parasite fecal egg counts to make treatment decisions and to evaluate treatment efficacy. The consensus in equine parasitology is to use a flotation medium with a specific gravity (SG) of ≥ 1.20 to float the main parasite egg types of interest in egg counting techniques. However, the density of common equine endoparasite eggs has been sparsely investigated. Equine tapeworm eggs are known to be particularly difficult to determine and count in fecal samples. It is unknown whether this could be because of differences in egg density. The aim of this study was to provide estimates of relative densities for equine ascarid, strongyle, and tapeworm eggs. Six aqueous glucose-salt solutions with specific gravities ranging from 1.06 to 1.16 were made and placed from most to least dense into thirteen 15 mL centrifuge tubes. Concentrated aqueous suspensions of the three types of endoparasite eggs were placed on top of each tube. These tubes were then centrifuged at 800 g for 20 min and each layer of flotation solution was carefully pipetted and transferred to a McMaster egg counting slide. Egg type and count were recorded for each specific gravity layer. Each egg was assigned a specific gravity based on the specific gravity layer it was observed in. In a second trial of this study, five similar flotation media were made ranging from 1.02 to 1.10 and were used in four subsequent replicates. In total between the two trials, the mean egg SGs of Anoplocephala perfoliata (n = 3811), Parascaris spp. (n = 3478), and strongylid type eggs (n = 9291) were 1.0636 (95% confidence interval (CI): 1.0629-1.0642), 1.0903 (95% CI: 1.0897-1.0909), and 1.0453 (95% CI: 1.0448-1.0458), respectively. The three egg types were statistically different from each other (p < 0.0001). This is the first time that the specific gravity of equine strongylid and Anoplocephala perfoliata eggs has been determined. With a tapeworm egg density demonstrated to be between that of strongylids and Parascaris spp., the poor recovery of tapeworm eggs in equine fecal samples must have other explanations.

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.