Amplicon sequencing allows differential quantification of closely related parasite species: an example from rodent Coccidia (Eimeria).
Amplicon sequencing
Body condition
Eimeria
Microbiome
Parasite infection
Parasite quantification
qPCR
Journal
Parasites & vectors
ISSN: 1756-3305
Titre abrégé: Parasit Vectors
Pays: England
ID NLM: 101462774
Informations de publication
Date de publication:
17 Jun 2023
17 Jun 2023
Historique:
received:
24
03
2023
accepted:
03
05
2023
medline:
19
6
2023
pubmed:
18
6
2023
entrez:
17
6
2023
Statut:
epublish
Résumé
Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended. We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population. We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance. We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.
Sections du résumé
BACKGROUND
BACKGROUND
Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended.
METHODS
METHODS
We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population.
RESULTS
RESULTS
We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance.
CONCLUSIONS
CONCLUSIONS
We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.
Identifiants
pubmed: 37330545
doi: 10.1186/s13071-023-05800-6
pii: 10.1186/s13071-023-05800-6
pmc: PMC10276917
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
204Subventions
Organisme : DFG
ID : 440909536
Organisme : Humboldt University Berlin
ID : Open Access Publication Fund
Organisme : Humboldt University Berlin
ID : Open Access Publication Fund
Organisme : Humboldt University Berlin
ID : Open Access Publication Fund
Organisme : German Foundation of Scientific Research (DFG)
ID : 285969495/HE 7320/2-1
Informations de copyright
© 2023. The Author(s).
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