Isolation and Identification of Nematode-Infecting Microsporidia.
Curr Protoc
; 4(5): e1035, 2024 May.
Article
em En
| MEDLINE
| ID: mdl-38727641
ABSTRACT
Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 Gathering samples Support Protocol 1 Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2 Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3 DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4 Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2 Bleach-synchronizing nematodes.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Microsporídios
/
Nematoides
Limite:
Animals
Idioma:
En
Revista:
Curr Protoc
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Canadá