Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus)

Abstract

Leinbach, Israel L.; Klasner, Ina H.; Kaluna, Lisa M.; Snook, Kirsten A.; Howe, M. Kathleen; Jacquier, Steven H.; Lange, Ingo; Atkinson, Abigail L.; Deane, Ashley R.; Niebuhr, Chris N.; and Siers, Shane R., "Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus)" (2019). AbstractAngiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Nematocidal Effects of a Coriander Essential Oil and Five Pure Principles on the Infective Larvae of Major Ovine Gastrointestinal Nematodes In Vitro

“…However, this assay seems to be impractical especially when evaluating numerous compounds or concentrations [24]. More throughput assays were developed and used in non-parasitic nematodes (C. elegans) to provide a more convenient means to determine the worm viability after treatment with drugs [55]. In this study, we used three fluorescent strains (Sytox green, PI and C 12 -resazurin) to identify dead/live larvae and tested their feasibility for testing compounds examined in this study.…”

In vitro efficacy of anthelmintics on Angiostrongylus cantonensis L3 larvae

“…The use of propidium iodide in establishing the death of A. cantonensis larvae was recently demonstrated (Jarvi et al, 2019). In this study, the treatment plates were imaged every 24 h for 30 days using the Operetta.…”

“…Angiostrongylus cantonensis L3 were isolated based on their unique and continuous 'Q' and 'S' movements (Lv et al, 2009) from wild P. martensi (semi-slug) collected from east Hawai'i island, and stock solutions were prepared as described in Jarvi et al (2019). Since A. cantonensis L3 are isolated from multiple wild P. martensi there may be some level of temporal or slug-to-slug variation.…”

“…Although, 2% (v/ v) DMSO is considered as cytotoxic in mammalian cell culture studies (Singh et al, 2017), we found that A. cantonensis L3 can tolerate up to 2% (v/v) DMSO without adverse effects, as we have evaluated various concentrations of DMSO on L3 and compared it with H 2 O controls (see results section). A 2.5% (v/v) propidium iodide (Biotium, Fremont, CA, USA) solution was prepared with 10× penicillin-streptomycin (Omega Science, Tarzana, CA, USA) in dH 2 O for differential staining of dead larvae (Jarvi et al, 2019). All solutions were stored at −20°C and shielded from light.…”

“…We developed a differential staining technique based on propidium iodide penetration as the indicator of death, combined with the nematode's natural autofluorescence at 525 nm (Jarvi et al, 2019). Using this assay, we quantify the in vitro efficacy of the most widely prescribed anthelmintics: albendazole, pyrantel pamoate, diethylcarbamazine, levamisole, piperazine, niclosamide, praziquantel, ivermectin and moxidectin on A. cantonensis L3 isolated from Parmarion martensi (semi-slugs) from East Hawai'i Island.…”