Exploring the Gastrointestinal "Nemabiome": Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities

PLoS One. 2015 Dec 2;10(12):e0143559. doi: 10.1371/journal.pone.0143559. eCollection 2015.

Abstract

Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal "nemabiome". The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the 'nemabiome' have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions during co-infection, parasite epidemiology, parasite ecology and the response of parasite populations to both drug treatments and control programs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Biota / genetics
  • Cattle / parasitology*
  • Cattle Diseases / parasitology
  • DNA, Helminth / genetics
  • DNA, Ribosomal / genetics
  • Gastrointestinal Tract / parasitology*
  • High-Throughput Nucleotide Sequencing
  • Host-Parasite Interactions
  • Humans
  • Intestinal Diseases, Parasitic / parasitology
  • Intestinal Diseases, Parasitic / veterinary
  • Larva / genetics
  • Nematoda / genetics*
  • Nematoda / isolation & purification
  • Nematoda / pathogenicity*
  • Nematode Infections / parasitology
  • Nematode Infections / veterinary
  • Species Specificity

Substances

  • DNA, Helminth
  • DNA, Ribosomal

Grants and funding

Funding support from Natural Sciences and Engineering Research Council of Canada (NSERC) (Grant number RGPIN/371529-2209), the University of Calgary NSERC-CREATE Host Parasite Interactions (HPI) graduate training program and the Alberta Livestock and Meat Agency (ALMA) (Grant number 2011R024R). The funders provided support in the form of salaries for RWA, EMR, and JSG and research materials, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section. Roy Lewis is employed with Merck Animal Health as a Technical Services Veterinarian part-time. Dr Lewis provided fecal samples for the study but the project was not funded by Merck Animal Health. Merck Animal Health provided support in the form of salary for RL, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the 'author contributions' section.