An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads

Julia M. Maritz, Krysta H. Rogers, Tara M. Rock, Nicole Liu, Susan Joseph, Kirkwood M. Land, Jane M. Carlton

Research output: Research - peer-reviewArticle

Abstract

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.

LanguageEnglish (US)
Pages1-14
Number of pages14
JournalMicrobial Ecology
DOIs
StateAccepted/In press - May 24 2017

Fingerprint

Trichomonadida
sewage
ribosomal RNA
protists
protist
sampling
DNA
animals
methodology
animal
method
protocol
Giardia lamblia
Animalia
Cryptosporidium parvum
Toxoplasma gondii
aquatic environment
eukaryotic cells
vertebrates
genetic markers

Keywords

  • 18S rRNA amplicon sequencing
  • Environmental sequencing
  • Protist
  • Sewage
  • Trichomonad
  • Zoonoses

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Ecology
  • Soil Science

Cite this

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads. / Maritz, Julia M.; Rogers, Krysta H.; Rock, Tara M.; Liu, Nicole; Joseph, Susan; Land, Kirkwood M.; Carlton, Jane M.

In: Microbial Ecology, 24.05.2017, p. 1-14.

Research output: Research - peer-reviewArticle

Maritz JM, Rogers KH, Rock TM, Liu N, Joseph S, Land KM et al. An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads. Microbial Ecology. 2017 May 24;1-14. Available from, DOI: 10.1007/s00248-017-0996-9
Maritz, Julia M. ; Rogers, Krysta H. ; Rock, Tara M. ; Liu, Nicole ; Joseph, Susan ; Land, Kirkwood M. ; Carlton, Jane M./ An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads. In: Microbial Ecology. 2017 ; pp. 1-14
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