Parasite dynamics in untreated horses through one calendar year.

BACKGROUND: Horses are host to a plethora of parasites. Knowledge of the seasonality of parasite egg shedding and transmission is important for constructing parasite control programs. However, studies describing these patterns are sparse, and have largely been conducted only in the United Kingdom. This study evaluated strongylid egg shedding patterns and transmission dynamics of Strongylus vulgaris in naturally infected and untreated mares and foals through one calendar year in Kentucky, USA. The study also investigated the existence of a peri-parturient rise (PPR) in strongylid egg counts in foaling mares and collected information about Strongyloides westeri and Parascaris spp. in the foals. METHODS: This study was conducted from January to December 2018. A herd of 18 mares, one stallion, and 14 foals born in 2018 were followed throughout the year. Sera and feces were collected biweekly from all horses, and worm burdens enumerated in 13 foals at necropsy. An S. vulgaris ELISA antibody test was run on all serum samples. Fecal egg counts were determined for all horses, and coproculture and qPCR assay were employed to test for the presence of S. vulgaris in the mature horses. Data were analyzed using the proc glimmix procedure in the SAS 9.4 software program. RESULTS: We found a general lack of seasonality in strongylid egg shedding throughout the year among the mature horses, and no PPR was demonstrated. Shedding of S. vulgaris eggs displayed a higher abundance during the spring, but findings were variable and not statistically significant. Anti-S. vulgaris antibody concentrations did not display significant fluctuations in the mature horses, but evidence of passive transfer of antibodies to the foals was demonstrated, and foals assumed their own production of antibodies starting at approximately 20 weeks of age. Overall, colts shed higher numbers of strongylid, ascarid, and S. westeri eggs than fillies. CONCLUSIONS: This study demonstrated a lack of seasonality in strongylid egg shedding for the study population, which is in stark contrast to previous studies conducted elsewhere. This strongly suggests that more studies should be done investigating these patterns under different climatic conditions.

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.

The pelvic flexure separates distinct microbial communities in the equine hindgut.

As hindgut fermenters, horses are especially dependent on the microbiota residing in their cecum and large intestines. Interactions between these microbial populations and the horse are critical for maintaining gut homeostasis, which supports proper digestion. The current project was motivated to determine if any features of the fecal microbiota are informative of the microbial communities from the cecum, ventral colon, or dorsal colon. Digesta from the cecum, ventral colon, dorsal colon and feces were collected from 6 yearling miniature horses. Microbial DNA was isolated and the microbiota from each sample was characterized by profiling the V4 region of the 16S rRNA. Principal coordinate analysis of the beta diversity results revealed significant (p = 0.0001; F = 5.2393) similarities between the microbial populations from cecal and ventral colon and the dorsal colon and fecal samples, however, there was little overlap between the proximal and distal ends of the hindgut. These distinct population structures observed in our results coincide with the pelvic flexure, which itself separates intestinal compartments with distinct roles in digestive physiology. An indicator species analysis confirmed the population differences, supported by the identification of several microbial families characteristic of the compartments upstream of the pelvic flexure that were not represented following it. Our data suggest that the fecal microbiota is not informative of the proximal hindgut but can provide insight into communities of the distal compartments. Further, our results suggest that the pelvic flexure might be an important anatomical landmark relative to the microbial communities in the equine large intestine.

Precision and spatial variation of cyathostomin mucosal larval counts.

Cyathostomins are pervasive parasites of equids across the world. Larval stages encyst in the mucosa of the cecum, ventral and dorsal colon and can induce an inflammatory response leading to larval cyathostominosis, a life-threatening generalized typhlocolitis. Mucosal digestion is the only gold standard procedure for identifying and quantifying all larval stages. There is a lack of standardization of this technique and several aspects are ambiguous, such as precision of the method and the possibility of spatial variation of mucosal larval counts. The aim of this study was to estimate precision for enumeration of early third stage larvae (EL3) and late third stage/fourth stage (LL3/L4) larvae and investigate spatial variation of encysted counts within large intestinal organs. Six naturally infected and untreated horses aged 2-5 years were euthanized as part of an anthelmintic efficacy study, and the cecum (Cec), ventral colon (VC) and dorsal colon (DC) were collected. Each organ was rinsed, weighed, and visually separated into 3 equally sized sections. Two 5% tissue samples were collected from each section, a total of six replicates per organ. The mucosae were digested, and 2% examined under the microscope for presence of EL3 and LL3/L4 stage larvae. Overall, 59 % of the harvested larvae were EL3s, and 41 % were LL3/L4s. The ventral colons represented 45 % of the total organ weight, and contributed 37 and 41 % of the EL3s and LL3/L4s harvested, respectively. The Cec, representing only 27 % of the weight contributed 23 % of EL3s and 47 % of LL3/L4s. The DC represented 28 % of the total organ weight, and 28 % and 12 % of the total EL3s and LL3/L4s, respectively. Coefficients of variation varied from 33 to 183 % for EL3 counts and 38-245% for LL3/L4 counts. There were no statistically significant associations between EL3 counts and either organ or location. For LL3/L4 counts there were no statistically significant differences between the three locations within organs (p = 0.1166), but the DC had significantly lower counts than the other two organs (p < 0.0001). Increasing the number of mucosal replicates from each organ improved estimation, but required a considerably increased workload. In conclusion, mucosal larval cyathostomin counts are highly variable, complicating their use for treatment efficacy estimation.

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.

Encysted cyathostomin larval counts: Mucosal digestion revisited.

Cyathostomins are pervasive equine parasites in horses across the world, and larval stages are known to cause the deadly disease larval cyathostominosis. The mucosal digestion technique is widely used for enumeration of encysted larval stages. Previous studies have investigated the spatial variation of encysted larvae, however current protocols lack a description of a standardized area from which to take the tissue sample. This study sought to evaluate spatial variation in encysted cyathostomin larval counts among the large intestinal organs and their subsections. Following humane euthanasia, ceca, ventral, and dorsal colons were harvested from 8 foals (aged 4-8 months) raised in an anthelmintic naïve parasitology research herd. Each organ was weighed and separated into 3 equal sections by length: the orad, intermediate, and aborad portions. From each of those sections, two 5% weight tissue samples were collected and digested to quantify the early third stage larvae (EL3) and late third stage larvae/fourth stage larvae (LL3/L4). A mixed model statistical analysis was carried out to evaluate for differences of larval counts among the different organs, sections, and the interaction term between the organs and sections. There were significant differences among organs (P < 0.0001), with the ceca having higher counts than the ventral and dorsal colons. However, there were no significant differences among the three defined organ sections (P = 0.1076). Coefficients of variation (CV) were all calculated to be greater than 1, suggesting a high level of variability among the samples; the least amount of variation can be found in the cecal data with a CV of 1.4024 compared with the ventral colon's 1.529845 and dorsal colon's 3.339135 within the respective organ. The following sections had the highest mean counts of encysted larvae: intermediate cecum, orad ventral colon, and aborad dorsal colon. Though only a portion of the results were significant, trends were observed and these should be investigated further in future studies and potentially employed in larvicidal efficacy evaluations.

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 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.