Long live the worms: methods for maintaining and assessing the viability of intestinal stages of <i>Parascaris</i> spp. <i>in vitro</i>

Scare, J.A.; Steuer, Ashley E.; Shaffer, Carrie L.; Mousley, Angela; Nielsen, Martin K.

doi:10.1017/s0031182018002019

Cited by 6 publications

(14 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This study consisted of two parts. Part 1 was to perform in vitro drug exposures at varying concentrations and observe the worm responses using a motility-based scoring system (Scare et al ., 2018). Part 1 ended with the determination of the sub-lethal concentration for each drug type and exposure length where worm viability decreased approximately ⩾25%.…”

Section: Methodsmentioning

confidence: 99%

“…and more commonly studied nematodes ( Haemonchus contortus , Caenorhabditis elegans ), inadequate knowledge of genes (HKGs) and how they might vary between parasite stages and drug exposures, how the drugs enter the parasite (orally or transcuticularly), and the mode of action of anthelmintic drugs and involvement of drug metabolizing enzymes. While the challenges seem endless, recent and current studies are making progress by establishing in vitro maintenance protocols for adult (Scare et al ., 2018) and larval stages (personal communication, Eva Tydén), availability of a partially annotated genome (Wang et al ., 2017), identification of p-glycoproteins in various tissues (Chelladurai and Brewer, 2019) and preliminary evidence of genes involved with drug resistance and drug exposure (Janssen et al ., 2013; personal communications Alexander Gerhard, Nicolas Lamassiaude and Frida Martin).…”

Section: Introductionmentioning

confidence: 99%

“…Only recently has an in vitro maintenance and viability assessment method been tested and established for intestinal stages of Parascaris spp. (Scare et al ., 2018), and this provides a basis for establishing an in vitro anthelmintic exposure method while monitoring the phenotypic and transcriptomic responses.…”

Section: Introductionmentioning

confidence: 99%

“…The purpose of this study was to aid in overcoming some of the previously mentioned challenges by (1) establishing a method for in vitro anthelmintic exposure of adult Parascaris spp. using different concentrations of anthelmintic drugs, (2) reporting the phenotypic responses to anthelmintic exposure in terms of worm viability over time using a previously established objective scoring system (Scare et al ., 2018), and (3) providing a preliminary examination of transcriptomic responses of drug-susceptible adult Parascaris spp. to ivermectin (IVM), an ML drug to which resistance is widely established on managed horse farms, and to oxibendazole (OBZ), a drug that remains effective.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…After 24 h incubation, worm viability was assessed according to the scoring system developed by Scare et al [36] to evaluate the effects of the different drug concentrations on viability. The worms in each container were observed and viability scores between 2 (movement only when stimulated with forceps) and 6 (seven or more whole body movements without stimulation) were given for each container.…”

Section: Viability Scoring and Dissectionmentioning

confidence: 99%

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

et al. 2020

View full text Add to dashboard Cite

Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10 −9 M, 10 −11 M, 10 −13 M), pyrantel citrate (10 −6 M, 10 −8 M, 10 −10 M), thiabendazole (10 −5 M, 10 −7 M, 10 −9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ − 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10 −9 M of ivermectin. Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

show abstract

The equine ascarids: resuscitating historic model organisms for modern purposes

Cain

Nielsen

2022

Parasitol Res

View full text Add to dashboard Cite

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.

show abstract

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro

Cited by 6 publications

References 31 publications

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

The equine ascarids: resuscitating historic model organisms for modern purposes

Contact Info

Product

Resources

About