Schistosoma mansoni infection causes consistent changes to the fecal bacterial microbiota of mice across and within sites
Description
Schistosomiasis is a neglected tropical disease caused by several blood fluke species in the genus Schistosoma. Schistosoma mansoni enters the mammalian host via skin penetration and sexually reproduces in mesenteric vasculature. Schistosoma mansoni eggs penetrate both serosal and epithelial intestinal barriers, and are excreted with feces. Consequent physical damage or immune responses may disrupt normal gut microbiota. Reported microbiome composition may vary considerably with both biotic and abiotic factors, including differences between study sites (e.g. microbial milieu of animal facilities and technicians), and study organisms (e.g. dietary background of experimental mice). In this study, we evaluate the fecal bacterial microbiome of mice in three experiments at two locations in order to assess Schistosoma-associated microbiome composition changes, as well as potential effects of study location on microbiome composition. When analyzing overall composition and diversity, the largest visible differences were between institutions. However, there were clear and specific changes due to schistosome infection and these specific changes were reproducible, regardless of the institution at which the experiment was performed.
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Data collection This study includes data from 3 independent experiments: 1 at UGA and 2 at ODU. Female BALB/c mice were infected intraperitoneally with 30-35 Schistosoma mansoni cercariae (Sm+) or left uninfected to serve as age-matched controls (Sm-). Fresh fecal pellets were collected at 0, 4, and 10 weeks post infection (wpi) and stored at -80C until use. DNA was isolated from feces with Qiagen's DNeasy PowerSoil kit, and diluted to 5 ng/ul in nuclease free water. Libraries of the 16S rRNA V3-V4 region were prepared with the Illumina MiSeq System, according to manufacturer's instructions. Briefly, (1) the DNA was amplified by PCR using the universal primer set and Roche's KAPA HiFi Hotstart Readymix, (2) PCR products purified with Beckman Coulter's AmPure XP beads, (3) barcodes were added by PCR with Illumina Nextera XT Index primers, (4) barcoded PCR products were again cleaned up, (5) products were quantified with Biotium's AccuBlue, then (6) equimolar amounts were pooled. Libraries were loaded onto the Illumina MiSeq machine at 10pM with a 15% PhiX concentration as a control. Data analysis Sequence files (.fastq) were analyzed using Quantitative Insights Into Microbial Ecology (QIIME2) software. Trimming, read filtration, and generation of Amplicon Sequence Variants (ASVs) was performed using DADA2 implemented within QIIME2. Forward and reverse paired-end reads were left-truncated at 240 bp based on evaluation of per-base quality charts. Chimaera filtration was performed at 2-fold overabundance (--p-min-fold-parent-over-abundance 2). The reads were trimmed for the amplicon PCR primers using default settings before merging. Bacterial sequences were annotated using the SILVA 138.1 database. Based on alpha-rarefaction curves of Shannon index, samples with fewer than 7500 reads were excluded (2 samples were lost post- filtration). Taxonomy-based filtration was performed to remove reads identified as chloroplast, mitochondria, and unassigned Amplicon Sequence Variants. All filtering processes were conducted prior to any downstream analyses. We uploaded the R script used to get the data analyzed for alpha and beta diversities as well as the source files for running the code.