Rapid, automated quantification of Haemonchus contortus ova in sheep faecal samples.

Haemonchus contortus is one of the most pathogenic nematodes affecting small ruminants globally and is responsible for large economic losses in the sheep and goat industry. Anthelmintic resistance is rampant in this parasite and thus parasite control programs must account for drug efficacy on individual farms and, sometimes, whether H. contortus is the most prevalent trichostrongylid. Historically, coproculture has been the main way to determine the prevalence of H. contortus in faecal samples due to the inability to morphologically differentiate between trichostrongylid egg types, but this process requires a skilled technician and takes multiple days to complete. Fluoresceinated peanut agglutinin (PNA) has been shown to specifically bind H. contortus and thus differentiate eggs based on whether they fluoresce, but this method has not been widely adopted. The ParasightTM System (PS) fluorescently stains helminth eggs in order to identify and quantify them, and the H. contortus PNA staining method was therefore adapted to this platform using methodology requiring only 20 min to obtain results. In this study, 74 fecal samples were collected from sheep and analyzed for PNA-stained H. contortus, using both PS and manual fluorescence microscopy. The percentage of H. contortus was determined based on standard total strongylid counts with PS or brightfield microscopy. Additionally, 15 samples were processed for coproculture with larval identification, and analyzed with both manual and automated PNA methods. All methods were compared using the coefficient of determination (R2) and the Lin's concordance correlation coefficient (ρc). ParasightTM and manual PNA percent H. contortus results were highly correlated with R2 = 0.8436 and ρc = 0.9100 for all 74 fecal samples. Coproculture versus PS percent H. contortus were also highly correlated with R2 = 0.8245 and ρc = 0.8605. Overall, this system provides a rapid and convenient method for determining the percentage of H. contortus in sheep and goat fecal samples without requiring specialized training.

A diverse microbial community and common core microbiota associated with the gonad of female Parascaris spp.

The microbiome plays an important role in health, where changes in microbiota composition can have significant downstream effects within the host, and host-microbiota relationships can be exploited to affect health outcomes. Parasitic helminths affect animals globally, but an exploration of their microbiota has been limited, despite the development of anti-Wolbachia drugs to help control infections with some filarial nematodes. The equine ascarids, Parascaris spp., are considered the most pathogenic nematodes affecting juvenile horses and are also the only ascarid parasite to have developed widespread anthelmintic resistance. The aim of this study was to characterize the microbiota of this helminth, focusing on the female gonad, determine a core microbiota for this organ, identify bacterial species, and show bacterial localization to the female gonad via in situ hybridization (ISH). A total of 22 gonads were isolated from female Parascaris spp. collected from three foals, and 9 female parasites were formalin-fixed and paraffin-embedded for ISH. Next-generation sequencing was performed using V3-V4 primers as well as the Swift Amplicon™ 16S+ ITS Panel. Overall, ten genera were identified as members of the Parascaris spp. female gonad and twelve bacterial species were identified. The most prevalent genus was Mycoplasma, followed by Reyranella, and there were no differences in alpha diversity between parasites from different horses. Specific eubacteria staining was identified in both the intestine and within the gonad using ISH. Overall, this study provided in-depth information regarding the female Parascaris spp. microbiota and was the first to identify the core microbiota within a specific parasite organ.

The microbial community associated with Parascaris spp. infecting juvenile horses.

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.

The equine ascarids: resuscitating historic model organisms for modern purposes.

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.

The effect of analyst training on fecal egg counting variability.

Fecal egg counts (FECs) are essential for veterinary parasite control programs. Recent advances led to the creation of an automated FEC system that performs with increased precision and reduces the need for training of analysts. However, the variability contributed by analysts has not been quantified for FEC methods, nor has the impact of training on analyst performance been quantified. In this study, three untrained analysts performed FECs on the same slides using the modified McMaster (MM), modified Wisconsin (MW), and the automated system with two different algorithms: particle shape analysis (PSA) and machine learning (ML). Samples were screened and separated into negative (no strongylid eggs seen), 1-200 eggs per gram of feces (EPG), 201-500 EPG, 501-1000 EPG, and 1001+ EPG levels, and ten repeated counts were performed for each level and method. Analysts were then formally trained and repeated the study protocol. Between analyst variability (BV), analyst precision (AP), and the proportion of variance contributed by analysts were calculated. Total BV was significantly lower for MM post-training (p = 0.0105). Additionally, AP variability and analyst variance both tended to decrease for the manual MM and MW methods. Overall, MM had the lowest BV both pre- and post-training, although PSA and ML were minimally affected by analyst training. This research illustrates not only how the automated methods could be useful when formal training is unavailable but also how impactful formal training is for traditional manual FEC methods.

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.

The importance of anthelmintic efficacy monitoring: results of an outreach effort.

Anthelmintic resistance in equine cyathostomin parasites is widespread. A surveillance-based parasite control program using fecal egg counts (FECs) and fecal egg count reduction tests (FECRTs) to decrease anthelmintic use and monitor treatment efficacy is recommended. The purpose of this study was to examine shifts in equine parasite control program management practices via a short course presented by the Penn State Extension, and to highlight how data collected from these programs is useful for monitoring anthelmintic efficacy on a large scale. Horse owners were enrolled after participating in a short course and filled out questionnaire surveys about their parasite management programs pre and post study, horse information, and farm information. FECs were performed at three time points, and horses above a 300 strongyle eggs per gram cut-off were treated with pyrantel pamoate, fenbendazole, or ivermectin. Two weeks post-treatment, FECRTs were performed to determine treatment efficacy, which included 29 farms with 513 individual treatments. Prior to the study, only 30.6% of farms used FECs, but after the study, 97.3% of farms said they would use FECs in the future. Horses were given an average of 4.1 anthelmintic treatments per year before the study, and post study 89.2% of farms were able to reduce the number of anthelmintic treatments used. Fenbendazole was effective on zero farms, pyrantel pamoate on 7.4% of farms, and ivermectin on 92.9% of farms. This outreach project helped generate information about anthelmintic efficacy levels, causing a shift in practices on participating farms, and collected useful anthelmintic resistance data.

Correlation between fecal egg count, presence of Strongylus vulgaris, and body score of feral horses on Fort Polk, Louisiana.

Approximately 700 feral horses, dubbed "trespass horses" by the United States Army, occupy Fort Polk, Louisiana and the surrounding Kisatchie National Forest. These horses are considered a nuisance and hazard, and the military is seeking to remove the horses via adoption. The aim of this research was to evaluate the fecal egg count (FEC), body condition score (BCS), and the presence of Strongylus vulgaris within this previously unstudied horse population prior to removal. The feral horse data was compared to domestic horses living on a single farm in the same area. A modified McMaster FEC, Henneke body scoring via photography, and PCR were used to evaluate 10 domestic horses and 28 feral horses. A significantly higher FEC was identified for feral horses when compared to domestic horses (p = 0.004), and 69.2% of feral horses were positive for S. vulgaris while all domestic horses tested negative. Additionally, no correlation was found between FEC and BCS for domestic (p = 0.213) or feral (p = 0.099) horses, and no association was found between FEC and S. vulgaris presence (p = 0.21) or BCS and S. vulgaris presence (p = 0.52). This study provides insight into S. vulgaris and strongyle prevalence in a previously unstudied group of horses and indicates a need for anthelmintic treatment and monitoring of the feral horses once they are adopted.