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

Scare, J.A.; Dini, Pouya; Norris, Jamie K.; Steuer, Ashley E.; Scoggin, Kirsten E.; Gravatte, Holli S.; Howe, Daniel K.; Nielsen, Martin K.

doi:10.1017/s0031182020000189

Cited by 11 publications

(19 citation statements)

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“…To our knowledge, no in vivo treatment experiments have been performed in this parasite to date and only two studies have reported the expression of genes after in vitro drug exposure of adult Parascaris spp. [33,34].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…Only one member of the CYP family (PgR020_g037) was differentially expressed in this study, with a downregulation after exposure to IVM (10 − 11 M). This finding is in contrast to the upregulation of the CYP family member PgR071_g005 in P. univalens in vitro exposed to IVM and oxibendazole (33). Upregulation of CYP genes has also been seen in susceptible strains of H. contortus after in vitro exposure to IVM (26).…”

Section: Discussionmentioning

confidence: 62%

“…These varying results are most likely due to the differences in study design between the two experiments, such as different drug concentrations and drug exposure times. Scare et al (33) also allowed a 24 h acclimatization period for the worms in tissue culture media before drug exposure, whereas we started the drug exposure at 0 h. These two studies show that despite similar aims experimental design can affect differential expression considerably and thus also highlight the potential for future studies to identify important genes involved in drug interactions not found in either study.…”

Section: Discussionmentioning

confidence: 85%

“…The equine roundworm Parascaris spp. is resistant to several classes of anthelmintic drugs, but regardless of this few studies focusing on the mechanisms involved have been published (33,34,45).…”

Section: Discussionmentioning

confidence: 99%

“…To our knowledge only two reports have studied the expression of genes after in vitro drug exposure of adult Parascaris spp. (33,34).…”

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confidence: 99%

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

Martin

Dube

Lindsjö

et al. 2020

Preprint

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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 is 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 end 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 (p < 0.05 and fold change ≥ 2) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes up- 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 up- 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: This is the first time the expression of these genes has been described in P. univalens and future work should be focused on characterising these candidate genes further to explore their potential involvement in drug metabolism and anthelmintic resistance.

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The equine ascarids: resuscitating historic model organisms for modern purposes

Cain

Nielsen

2022

Parasitol Res

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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.

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

Cited by 11 publications

References 41 publications

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

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

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

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