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Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL-1) and IVM at 1.1 μm (1 μg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Parascaris spp. infect foals worldwide and foals typically shed eggs in the feces from about three to six months of age, upon which natural immunity is incurred. High levels of anthelmintic resistance of Parascaris spp. are a global concern, and further understanding egg shedding patterns and fecal egg counting (FEC) data variability is of high importance. The aims of this study were to monitor Parascaris spp. egg shedding in untreated foals during 12-23 weeks of age, estimate sources of data variability, and assess precision of two ascarid FEC techniques. Fecal samples were collected weekly from 11 foals born in 2022, from May through November (29 weeks). Six subsamples were extracted from each weekly sample to determine 30 FECs between two techniques: a McMaster technique and an Automated Egg Counting System (AECS). Mixed linear modeling was carried out with age, sex, birth month, seasonality, spring- or summer-born foals, and egg counting technique as explanatory variables. Ascarid FECs were associated with age (p < 0.001), seasonality (p < 0.001), and technique (p < 0.001). The McMaster technique was more precise with a mean coefficient of variation (CV) of 34.57% and a 95% confidence interval (CI) of 30.80%- 38.30% compared to the CV for the AECS, which was 42.22% (CI: 37.70%-46.70%). Seasonality accounted for the highest proportion of variance (PV) of all covariates, but differences in PVs for covariates existed between techniques with foal age and subsample contributing more variance to the McMaster, and individual foal and seasonality contributing more to the AECS. Subsamples and replicate counts accounted for less than 1% of the total data variance. The results highlighted substantial differences in PVs between the two techniques at the subsample (AECS: 57.14%; McMaster: 77.51%) and replicate count levels (AECS: 42.86%; McMaster: 22.49%). While differences in precision were observed between the two FEC techniques, they were negligible in the data set, as the overwhelming majority of the data variability in ascarid FECs was attributed to individual foal, seasonality, and foal age., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Parasitic nematodes, including large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarids, Parascaris spp., are important parasites of juvenile horses and the first ascarids to develop widespread anthelmintic resistance. The microbiota has been shown to be an important factor in the fitness of many organisms, including parasitic nematodes, where endosymbiotic Wolbachia have been exploited for treatment of filariasis in humans., Methods: This study used short-read 16S rRNA sequences and Illumina sequencing to characterize and compare microbiota of whole worm small intestinal stages and microbiota of male and female intestines and gonads. Diversity metrics including alpha and beta diversity, and the differential abundance analyses DESeq2, ANCOM-BC, corncob, and metagenomeSeq were used for comparisons., Results: Alpha and beta diversity of whole worm microbiota did not differ significantly between groups, but Simpson alpha diversity was significantly different between female intestine (FI) and male gonad (MG) (P= 0.0018), and Shannon alpha diversity was significantly different between female and male gonads (P = 0.0130), FI and horse jejunum (HJ) (P = 0.0383), and FI and MG (P= 0.0001). Beta diversity (Fig. 2B) was significantly different between female and male gonads (P = 0.0006), male intestine (MI) and FG (P = 0.0093), and MG and FI (P = 0.0041). When comparing organs, Veillonella was differentially abundant for DESeq2 and ANCOM-BC (p < 0.0001), corncob (P = 0.0008), and metagenomeSeq (P = 0.0118), and Sarcina was differentially abundant across four methods (P < 0.0001). Finally, the microbiota of all individual Parascaris spp. specimens were compared to establish shared microbiota between groups., Conclusions: Overall, this study provided important information regarding the Parascaris spp. microbiota and provides a first step towards determining whether the microbiota may be a viable target for future parasite control options., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anthelmintic resistance among cyathostomin parasites is a wide-spread problem. The parasite control guidelines written by the American Association of Equine Practitioners (AAEP) encourages the preservation of anthelmintic efficacy by reducing treatment frequency, using targeted deworming, and implementing environmental management practices. While there is knowledge regarding parasite management practices of affluent horse farms in the United States, surveys rarely explore the rural and underserved regions. The purpose of this study was to observe the management practices of horse farms in rural regions Kentucky, including working Amish farms, and determine factors associated with strongyle prevalence. A total of 160 horses among 38 owners from 28 different farms were enrolled in this study. A questionnaire survey regarding equine information, farm management, and deworming history was performed with each owner. Fecal samples were collected to determine fecal egg counts, perform coprocultures for subsequent strongyle larvae identification, and Strongylus vulgaris specific PCR. Serum samples were collected for the S. vulgaris antibody specific ELISA. The mean number of deworming treatments given in the last year was 2.1 with a 95% confidence interval of 1.9-2.3 with ivermectin being the most common active used. Statistical analysis showed horses treated within the last three months with a macrocylic lactone (ML) drug had significantly lower egg counts than horses treated with a ML 7-9 months ago (p = .0005). Despite the AAEP recommendations to reduce the overall number of treatments by using a surveillance-based approach and to no longer rotate treatments, only 17 horses reportedly had a fecal sample submitted for a fecal egg count and 65 horses were dewormed in a rotational manner. Horses whose owners utilized an informative deworming source (i.e., veterinarian, internet, magazine, local feed store) also had significantly lower counts (p = .0026). All coprocultures were negative for S. vulgaris while five horses were PCR positive. Interestingly, 95 horses tested ELISA positive for S. vulgaris. The strongyle egg counts of the working Amish horses were not significantly different from the other horses in this study and deworming practices including the use of efficacious drugs and low treatment frequencies were in accordance with the AAEP guidelines. This study was the first to summarize deworming management practices of rural regions in Kentucky, including a working Amish community. Overall, horse owners employed deworming practices recommended by the AAEP, however rotational deworming is still commonly implemented and fecal egg counts are rarely used., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Strongylus vulgaris is the most pathogenic helminth parasite of horses, causing verminous endarteritis with thromboembolism and infarction. A serum enzyme-linked immunosorbent assay (ELISA) has been validated for detection of antibodies to an antigen produced by migrating larvae of this parasite. The aim was to evaluate ELISA responses to anthelmintic treatment in cohorts of naturally infected horses. Fifteen healthy horses harboring patent S. vulgaris infections were turned out for communal grazing in May 2013 (day 0). On day 55, horses were ranked according to ELISA titers and randomly allocated to the following three groups: no treatment followed by placebo pellets daily; ivermectin on day 60 followed by placebo pellets daily; or ivermectin on day 60 followed by daily pyrantel tartrate. Fecal and serum samples were collected at ∼28-day intervals until study termination on day 231. Increased ELISA values were observed for the first 53 days following ivermectin treatment. Titers were significantly reduced 80 days after ivermectin treatment. Horses receiving daily pyrantel tartrate maintained lower ELISA values from 137 days post ivermectin treatment until trial termination. These results illustrate that a positive ELISA result is indicative of either current or prior exposure to larval S. vulgaris infection within the previous 5 months.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)