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Background: Current methods for obtaining fecal egg counts in horses are often inaccurate and variable depending on the analyst's skill and experience. Automated digital scanning of fecal sample slides integrated with analysis by an artificial intelligence (AI) algorithm is a viable, emerging alternative that can mitigate operator variation compared to conventional methods in companion animal fecal parasite diagnostics. Vetscan Imagyst is a novel fecal parasite detection system that uploads the scanned image to the cloud where proprietary software analyzes captured images for diagnostic recognition by a deep learning, object detection AI algorithm. The study describes the use and validation of Vetscan Imagyst in equine parasitology., Methods: The primary objective of the study was to evaluate the performance of the Vetscan Imagyst system in terms of diagnostic sensitivity and specificity in testing equine fecal samples (n = 108) for ova from two parasites that commonly infect horses, strongyles and Parascaris spp., compared to reference assays performed by expert parasitologists using a Mini-FLOTAC technique. Two different fecal flotation solutions were used to prepare the sample slides, NaNO 3 and Sheather's sugar solution., Results: Diagnostic sensitivity of the Vetscan Imagyst algorithm for strongyles versus the manual reference test was 99.2% for samples prepared with NaNO 3 solution and 100.0% for samples prepared with Sheather's sugar solution. Sensitivity for Parascaris spp. was 88.9% and 99.9%, respectively, for samples prepared with NaNO 3 and Sheather's sugar solutions. Diagnostic specificity for strongyles was 91.4% and 99.9%, respectively, for samples prepared with NaNO 3 and Sheather's sugar solutions. Specificity for Parascaris spp. was 93.6% and 99.9%, respectively, for samples prepared with NaNO 3 and Sheather's sugar solutions. Lin's concordance correlation coefficients for VETSCAN IMAGYST eggs per gram counts versus those determined by the expert parasitologist were 0.924-0.978 for strongyles and 0.944-0.955 for Parascaris spp., depending on the flotation solution., Conclusions: Sensitivity and specificity results for detecting strongyles and Parascaris spp. in equine fecal samples showed that Vetscan Imagyst can consistently provide diagnostic accuracy equivalent to manual evaluations by skilled parasitologists. As an automated method driven by a deep learning AI algorithm, VETSCAN IMAGYST has the potential to avoid variations in analyst characteristics, thus providing more consistent results in a timely manner, in either clinical or laboratory settings., Competing Interests: Declarations Ethics approval and consent to participate No IACUC or IRB approval was needed for this study design, in accordance with Texas Tech University’s IACUC policies. They were consulted in regard to this study and set up. Consent for publication All authors consent to publication in Parasites and Vectors. Competing interests Ashley Steuer is now employed by Zois, however, at the time of study design and conduct, was under the employ of Texas Tech University., (© 2024. The Author(s).)

Background: Parascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors., Methods: Live adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS)., Results: Proteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations., Conclusions: These findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection., (© 2024. The Author(s).)

Anthelmintic resistance is reported in equine nematodes with increasing frequency in recent years, and no new anthelmintic classes have been introduced during the past 40 years. This manuscript reviews published literature describing anthelmintic resistance in cyathostomins, Parascaris spp., and Oxyuris equi with special emphasis on larvicidal efficacy against encysted cyathostomin larvae and strongylid egg reappearance periods (ERP). Resistance to benzimidazoles and pyrimidines is highly prevalent in cyathostomin populations around the world, and macrocyclic lactone resistance has been documented in cyathostomins in recent years as well. Two recent studies have documented resistance to the larvicidal regimen of fenbendazole, whereas the larvicidal efficacy of moxidectin is variable, but with no evidence of a reduction from historic levels. In the 1990s, ERP estimates were 8-10 and 12-16 weeks for ivermectin and moxidectin, respectively, while several studies published after year 2000 found ERPs to be 5 weeks for both compounds. This is a clear change in anthelmintic performance, but it remains unclear if this is due to development of anthelmintic resistance or selection for other biological traits leading to a quicker resumption of strongylid egg shedding following anthelmintic treatment. Macrocyclic lactone resistance is common in Parascaris spp. around the world, but recent reports suggests that resistance to the two other classes should be monitored as well. Finally, O. equi has been reported resistant to ivermectin and moxidectin in countries representing four continents. In conclusion, multi-drug resistance is becoming the norm in managed cyathostomin populations around the world, and a similar pattern may be emerging in Parascaris spp. More work is required to understand the mechanisms behind the shortened ERPs, and researchers and veterinarians around the world are encouraged to routinely monitor anthelmintic efficacy against equine nematodes., (Copyright © 2022 The Author. Published by Elsevier Ltd.. 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).)

The equine ascarids, Parascaris spp., are important nematode parasites of juvenile horses and were historically model organisms in the field of cell biology, leading to many important discoveries, and are used for the study of chromatin diminution. In veterinary parasitology, Parascaris spp. are important not only because they can cause clinical disease in young horses but also because they are the only ascarid parasites to have developed widespread anthelmintic resistance. Despite this, much of the general biology and mechanisms of anthelmintic resistance are poorly understood. This review condenses known basic biological information and knowledge on the mechanisms of anthelmintic resistance in Parascaris spp., highlighting the importance of foundational research programs. Although two variants of this parasite were recognized based on the number of chromosomes in the 1870s and suggested to be two species in 1890, one of these, P. univalens, appears to have been largely forgotten in the veterinary scientific literature over the past 100 years. We describe how this omission has had a century-long effect on nomenclature and data analysis in the field, highlighting the importance of proper specimen identification in public repositories. A summary of important basic biology, including life cycle, in vitro maintenance, and immunology, is given, and areas of future research for the improvement of knowledge and development of new systems are given. Finally, the limited knowledge regarding anthelmintic resistance in Parascaris spp. is summarized, along with caution regarding assumptions that resistance mechanisms can be applied across clades., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Equine ascariosis, caused by Parascaris spp., is a worldwide endoparasitic disease affecting young horses in particular. Despite the great number of horses reared in Italy, large-scale epidemiological surveys dealing with ascariosis prevalence in the country are not reported in the current literature. For this reason, the present survey aims to describe, for the first time, the spread and infestation of Parascaris spp. in a large population of Italian horses (6896 animals) using faecal egg counts, and further to identify risk factors associated with ascarid egg shedding., Methods: Individual rectal faecal samples collected during routine veterinary examinations were used and Parascaris spp. prevalence was tested against the animal's age, sex, housing conditions, geographic provenance as well as the respective sampling season., Results: Among the examined stables, 35.8% showed at least one horse to be positive for Parascaris spp. eggs and an overall prevalence of 6.3% was found. Ascariosis rates tended to decrease significantly with age and, proportionally, 80.0% of the recorded Parascaris spp. eggs were found in 0.7% of the examined animals. Statistically significant differences among prevalence rates were found between the different geographic areas of provenance and prevalence was found to be higher in horses reared outdoors compared to those raised indoors. Analysis of data based on sex and season did not show any significant differences. Despite the lower prevalence found compared to other European countries, ascariosis was concluded to represent a significant health challenge for horses reared in Italy, especially foals. Age (foals and yearlings) and outdoor rearing were identified to be significant risk factors for Parascaris spp. egg shedding. Furthermore, the relevance of the infected horses over 6 years of age should not be underestimated as these represent a significant source of contamination for younger animals., Conclusions: The development of improved treatment protocols based on regular faecal examination combined with follow-up assessment of the efficacy of integrated action plans would prove beneficial in regard to animal health and anthelmintic resistance reduction in the field.

Horses in Iceland have been isolated for more than 1,000 yr but still harbor a similar range of gastrointestinal parasites as do horses across the world. The long isolation of the horses and their parasites presumably means that no resistance genes have been introduced into the Parascaris spp. population. It is therefore of particular interest to investigate the efficacy of ivermectin on Parascaris spp. infecting Icelandic foals. Potential treatment failure of ivermectin in Iceland will add substantial new information on how resistance can arise independently. This study aimed to determine the efficacy of subcutaneous injection of ivermectin for the treatment of Parascaris spp. infection in foals and to identify the Parascaris species present in the west and north of Iceland. A fecal egg count reduction (FECR) test (FECRT) was performed on 50 foals from 8 farms, including an untreated control group of 6 foals, from September to November 2019. The foals were between 3 and 5 mo of age at the start of the study and had not previously been treated with anthelmintic drugs. Each foal was treated subcutaneously with off-label use of Ivomec® injection 10 mg/ml or Noromectin® 1% at a dose of 0.2 mg/kg. The FECR for each farm was calculated in 2 ways, by the eggCounts package in R and by the Presidente formula (FECRT). Both calculation methods resulted in efficacy levels between 0% and 80.78%, indicating ivermectin resistance on all farms. We also confirmed, by karyotyping, that the species of equine ascarid present in the west and north of Iceland is Parascaris univalens. This study provides evidence for treatment failure of ivermectin against P. univalens infection in foals. Since Icelandic horses have been isolated on the island for more than 1,000 yr, this implies that resistance alleles have developed independently in the Icelandic Parascaris population. The actual clinical impact of ivermectin resistance is unknown but another drug of choice should be considered to treat Parascaris infection in foals in Iceland., (© American Society of Parasitologists 2021.)

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.

Macrocyclic lactones are the most widely used drugs for the control of gastrointestinal nematodes of horses in Argentina. Ivermectin and moxidectin are used as broad spectrum anthelmintics and although there are several international reports on the resistance of Parascaris spp., the resistance status of the local nematode population is largely unknow. This report informs a case of suboptimal efficacy to both drugs to control Parascaris spp in foals in central Argentina. In February 2018, routine fecal parasite egg counts showed a moderate-high number of Parascaris spp eggs (mean = 680 eggs per gram of feces) in foals treated approximately one month before with moxidectin. Upon suspicion of resistance of this parasite to the macrocyclic lactones, 24 of these animals were selected for a fecal egg count reduction test (FECRT). Twelve foals were treated with ivermectin and the remaining 12 animals with moxidectin. Two weeks after treatment, the FECRT was 48.1% and 34.8% for moxidectin and ivermectin respectively (25% of the animals increased the number of eggs in feces after treatment). Five days later, the administration of fenbendazole resulted in a FECRT = 100%. The monitoring of the status of susceptibility or resistance in each establishment is critical for the design of control programs based on rational and sustainable use of anthelmintics., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gastrointestinal parasites, Parascaris sp. and strongyles, are common in young horses worldwide and control of these parasites is challenged by increasing anthelmintic resistance. Our aim was to identify risk factors for these infections as well as to assess the efficacy of fenbendazole (dose 7.5 mg/kg) and pyrantel embonate (dose 19 mg/kg) against Parascaris sp. We also evaluated association between owner observed symptoms and patent infections with these parasites. Fecal samples were collected from 367 young horses in Finland and a questionnaire study was conducted. Fecal egg counts were performed by Mini-FLOTAC® method. Univariable logistic regression models using patent infection status (Yes/No), separately for Parascaris sp. and strongyle infections as an outcome were run initially to screen potential risk factors collected by the questionnaire. After the initial screening, multiple logistic regression models were constructed and run to account for correlated data structure, risk factors and potential confounders simultaneously. Two significant risk factors for a patent Parascaris sp. infection were found: breeding farm size (p = 0.028) and frequency of horse movements (p = 0.010). Horses originating from large breeding farms were more likely (OR = 2.47, 95% confidence interval (CI) 1.10-5.51) to shed Parascaris sp. eggs upon relocation to training stables compared to horses originating from small breeding farms. Horses living in farms with frequent horse movements to other premises had higher odds (OR = 3.56, 95% CI: 1.35-9.39) of a patent Parascaris sp. infection compared to farms with less frequent horse movements. Risk factors for patent strongyle infection included age (p < 0.001) and season (p = 0.017). Horses were less likely (OR = 0.27, 95% CI: 0.10 - 0.66) to shed strongylid eggs during the spring compared to the winter. Horses excreting over 200 ascarid eggs per gram were included in the anthelmintic efficacy trial. A mean FECR less than 90% was interpreted as presence of anthelmintic resistance. The mean FECR was 98.5% (95% CI: 95.8-100) and 68.0% (95% CI: 52.7-83.3) in the fenbendazole (n = 31) and pyrantel (n = 26) treatment groups, respectively. In conclusion, we identified two new risk factors for patent Parascaris sp. infection; breeding farm size and frequency of horse movements. Reduced efficacy of pyrantel against Parascaris sp. was observed for the second time in Europe. A relatively high Parascaris sp. prevalence in yearlings (34%) and two-year-olds (20%) was observed, which has not been reported earlier. An association between symptoms and a patent Parascaris sp. infection was observed in foals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Macrocyclic lactone-resistant Parascaris have been reported throughout the world. In part, the drug resistant phenotype is hypothesized to be associated with ATP-binding cassette transporters known as P-glycoproteins. In many systems, P-glycoproteins efflux drugs out of cells thereby precluding drug binding to target receptors. Parascaris may evade macrocyclic lactone-mediated death by effluxing drugs away from target receptors in the nervous system. Alternatively, P-glycoprotein expression in the gut or body wall could prevent penetration of drugs into the body of the parasite altogether. In the present study, we evaluate expression of Peq-pgp-11 and Peq-pgp-16 using a novel multiple nucleic acid hybridization method. This method allowed for visualization of individual mRNA transcripts within fixed tissue sections of Parascaris adults. Our investigation revealed expression of Peq-pgp-11 and Peq-pgp-16 in the intestine, body wall, nerves, lateral cords, and reproductive tissues of male and female parasites. These results suggest that Pgp could efflux drugs locally at the level of parasite neuronal tissue as well as at sites of entry for drugs such as the hypodermis and intestine. The multiple nucleic acid hybridization method could be useful for providing tissue context for gene expression in a variety of nematode parasites., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A previously described model for the dynamics of the parasitic stages of Parascaris spp. was modified to include eggs outside the host and the genetics of anthelmintic resistance before being used to address questions regarding the development of resistance. Three broad questions were addressed; i) How sustainable is the current common practice of treating foals monthly for their first year of life (i.e. 12 treatments/year)? ii) Does the timing of treatments have an effect on resistance development? (i.e. do certain treatments select for resistance more strongly than others?), and iii) How sustainable is the currently recommended strategy of targeting ascarid infections in foals with two treatments applied during the first five months of life? A range of variations within these broad questions were considered, such as the value in rotational deworming, whether larvicidal treatments are more selective for resistance, and whether combination anthelmintics should be introduced. Twelve anthelmintic treatments at monthly intervals resulted in the development of resistance to all the anthelmintics used, regardless of how they were used, indicating that such intensive treatment frequency is unlikely to be sustainable. The timing of a single annual treatment influenced resistance development with treatments at 3 and 4 months of age being more selective than treatments at other times. Treatments administered to foals older than 6 months of age did not select for resistance within the timeframe of these simulations. Treatments with activity against migrating third stage larvae (ivermectin and a programme of 5 daily treatments with fenbendazole) were more selective for resistance than those which only killed worms in the intestine. Restricting the number of treatments to young foals to two, administered at 2 and 5 months of age slowed the development of resistance by allowing a small contribution from susceptible genotype worms to subsequent generations. If the interval between treatments was reduced, resistance developed more rapidly demonstrating the importance of allowing some susceptible worms to reach patency before the second treatment is administered. Under a reduced treatment schedule with a clearly defined 'refugium' of susceptibility, the use of effective actives in combination appears to offer advantages for delaying resistance development. The model offers insights into more sustainable drug use strategies and has identified some priority questions for future research., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Literature documenting the growth and development of Parascaris spp. infections was used to develop a model describing worm dynamics in the young horse. The model incorporates four main variables; the rate at which larvae migrate through host tissues to return to the small intestine, the proportion of migrating larvae which succeed in returning to the small intestine, the rate of growth in size of maturing and adult worms and the survival rate of maturing and adult worms. In addition, the number of eggs laid each day by adult female worms is calculated as a function of worm size (length) and is used to calculate faecal egg output of the foal. Published data describing the rate of migration through host tissues, and the growth of worms following their return to the small intestine, was used to derive relationships describing these processes. However, only limited data exists relating the survival of migrating larvae and mature worms in the intestine to host age and experience of infection. Therefore, relationships and coefficients describing these variables were modified so that output aligned with published experimental results. As a consequence, the model has not yet been evaluated against an independent data set, and so remains as the best 'current hypothesis' for the dynamics of this parasite. Hopefully, future experiments designed to test specific assumptions and outputs of the model will lead to a better understanding of the biology of this important parasite. For example, the most influential variable in determining model output is the survival rate of worms in the small intestine. In the model, worm survival declines in response to both the increasing age of the horse and the increasing cumulative length of worms in the intestine (used as a proxy for crowding). Given the importance of this variable to model behaviour and the paucity of experimental data on this topic this would seem a priority for future study. Initial experiments using the model suggest that a single anthelmintic treatment, administered soon after patency of initial infection, may effectively control environmental contamination with Parascaris spp. eggs while allowing a small 'refugia' of susceptibility to delay the emergence of anthelmintic resistance. Further evaluations of the practicality of this approach may be worthwhile., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Reasons for Performing Study: Parascaris spp. infections can lead to life-threatening small intestinal impactions in foals. Currently available diagnostic techniques cannot estimate the magnitude of an ascarid burden, and hence identify foals potentially at risk of developing impactions., Objectives: To describe and evaluate an ultrasonographic transabdominal scoring technique for monitoring of ascarid burdens in foals and to perform a cost-benefit analysis of the application of this technique., Study Design: A transabdominal ultrasonographic technique was validated against ascarid worm counts from 10 foals aged 162-294 days. In a treatment trial, 15 foals were randomly allocated to 3 treatment groups: ivermectin, oxibendazole and no treatment. Blinded ultrasound examinations were performed daily for 5 consecutive days following treatment. Foals were examined ultrasonographically twice by the same investigator, and by different investigators for intra- and interobserver agreement evaluation. Cost-benefit analyses identified threshold values for the probability of ascarid impactions above which the screening method becomes cost-effective., Methods: The ultrasound technique used 3 locations along the ventral midline. An ascarid scoring system was established that assessed the magnitude of ascarid burden ranging from 1-4. The method was validated against worm burdens of 10 worms and above with calculation of diagnostic specificity, sensitivity, and predictive values. Treatment trial data were evaluated statistically using mixed model analysis. Kappa values were generated for intra- and interobserver agreement., Results: Two consecutive examinations were found to detect worm burdens >10 ascarids reliably. Ascarid scores declined in response to both anthelmintic treatments, although differences were not statistically significant. Kappa values indicated fair to moderate intra- and interobserver agreements. The majority of cost-benefit analyses indicated that ultrasound examinations are cost effective when the probability of ascarid impactions is above a range of 0.0001-0.0082 (i.e. 1 in 10,000 to 8 in 1000 foals)., Conclusions: The ultrasonographic screening techniques can be a useful tool for monitoring ascarid burdens in foals., (© 2015 EVJ Ltd.)