Alterations of the Mice Gut Microbiome via Schistosoma japonicum Ova-Induced Granuloma

Abstract

Schistosomiasis, also called bilharziasis, is a neglected tropical disease induced by Schistosoma spp. that causes hundreds of millions of infections. Although Schistosoma ova-induced granulomas commonly cause inflammation, hyperplasia, ulceration, micro abscess formation, and polyposis, the role of the egg granuloma on the gut microbiome remains unclear. To explore the role, gut microbial communities in mice infected with Schistosoma japonicum were surveyed. Female C57BL/6 and BALB/c mice were exposed to cercariae of S. japonicum for 45 and 65 days and then sacrificed. Intestinal contents and feces were collected, DNA was extracted, and high-throughput 16S rRNA gene-based pyrosequencing was used to provide a comparative analysis of gut microbial diversity. The intestinal mucosal tissues were also examined. Histopathologic analysis demonstrated that the basic structure of the colonic mucosa was damaged by ova-induced granuloma. Regarding the gut microbiome, 2,578,303 good-quality sequences were studied and assigned to 25,278 Operational Taxonomic Units (OTUs) at a threshold of 97% similarity. The average number of OTUs for C57BL/6 and BALB/c were 545 and 530, respectively. At the phylum level, intestinal microbial communities were dominated by Firmicutes, Bacteroidetes, Proteobacteria, and Verrucomicrobia. Infection with S. japonicum modified bacterial richness in the fecal associated microbiota. Exposure significantly modified bacterial community composition among different groups. At the phylogenetic levels, LEfSe analysis revealed that several bacterial taxa were significantly associated with the S. japonicum-infected mice. The present results suggest that egg granulomas in the intestine influence differentiation of the gut microbial community under pathophysiological conditions. This result suggests that intestinal microbiome-based strategies should be considered for early diagnosis, clinical treatment, and prognosis evaluation of schistosomiasis.

Keywords: 16S rRNA; Schistosoma japonicum; egg granulomas; gut microbiome; operational taxonomic units.

Figure 1

Microscopy visualization and pathological analysis of C57BL/6 and BALB/c mice with Schistosoma japonicum infection. For C57BL/6 and BALB/c mice, feces from groups Ctrl and AP at day 45 and CP on day 65 were obtained. The liver, spleen, intestine and feces from groups Ctrl and AP at day 45 and CP on day 65 were obtained. The blue arrow represents an S. japonicum egg. The red circle represents a small intestinal lesion. The egg granulomas of S. japonicum from the intestinal mucosa are indicated by the black frame.

Figure 2

Variation of bacterial community structure. The relative abundance of bacterial phyla and families present in the gut of C57BL/6 and BALB/c mice. Data shown represent the top 10 most abundant phyla and families, whereas low abundance and unclassified OTUs were grouped in “Other.” Sample names refer to samples as described in Table 1. (A) Bacterial community structure variation at the phylum level in C57BL/6 mice; (B) Bacterial community structure variation at the phylum level in BALB/c mice; (C) Bacterial community structure variation at the family level in C57BL/6 mice; (D) Bacterial community structure variation at the family level in BALB/c mice. (E) Taxonomic heatmap of different groups at the genus level. Characteristic relative abundances of various genera present in the Control (C57.Ctrl, BA.Ctrl), Acute phase (C57.AP, BA.AP), and Chronic phase (C57.CP, BA.CP). The portrait and landscape stand for different group and species annotation information, respectively. The left pattern indicates a clustering tree at the level of genus. The above profile displays clustering trees among different groups. Each column represents a unique subject.

Figure 3

Bacterial community comparison of different groups between uninfected mice and infected mice. Outliers were plotted as individual points. (A) Alpha diversity analysis based on the Chao1 index among groups in different mouse strains. (B) Alpha diversity analysis based on the observed species index among groups in different mouse strains. (C) Beta diversity analysis based on weighted UniFrac in different mouse strain.

Figure 4

Phylogenetic relationships and Species annotation of Operational Taxonomic Units (OTUs). The inner band shows genera colored by OTUs, the next band shows the relative abundance of OTUs, and the outer band shows the annotation reliability distribution of OTUs. Overall abundance and the magnitude of the difference among genera are indicated by bars.

Figure 5

Two-dimensional principal coordinates analysis (PCoA) plot of unweighted (A) and weighted (B) UniFrac distance matrices for both intestinal contents and feces samples from C57BL/6 and BALB/c mice during infection. The bacterial community of the feces and intestinal contents from C57BL/6 mice in group C57.Ctrl, C57.AP, and C57.CP and from BALB/c mice in group BA.Ctrl, BA.AP, and BA.CP are shown. Sample names refer to samples as described in Table 1.

Figure 6

LEfSe identified the most differentially abundant taxa in different groups of C57BL/6 and BALB/c mice. Only taxa meeting an LDA significance threshold >4 are shown in the figures. (A) Histogram of linear discriminant analysis (LDA) score distribution among the six groups; (B) Histogram of LDA score distribution among three groups of C57BL/6 mice; (C) Histogram of LDA score distribution among three groups of BALB/c mice; (D) Histogram of LDA score distribution between the C57.Ctrl and C57.AP group; (E) Histogram of LDA score distribution between the C57.AP and C57.CP group; (F) Histogram of LDA score distribution between the BA.Ctrl and BA.AP group; (G) Histogram of LDA score distribution between the BA.AP and BA.CP group; (H) Histogram of LDA score distribution between the C57.AP and BA.AP groups. (I) Histogram of LDA score distribution between the C57.CP and BA.CP groups.

Figure 7

Cladogram of the most differentially abundant taxa in different groups of C57BL/6 and BALB/c mice. (A) Cladogram of the most differentially abundant taxa in six groups of C57BL/6 and BALB/c mice; (B) Cladogram of the most differentially abundant taxa in different groups of C57BL/6 mice; (C) Cladogram of the most differentially abundant taxa in different groups of BALB/c mice; (D) Cladogram of the most differentially abundant taxa between C57.Ctrl and C57.AP; (E) Cladogram of the most differentially abundant taxa between C57.AP and C57.CP; (F) Cladogram of the most differentially abundant taxa between BA.Ctrl and BA.AP; (G) Cladogram of the most differentially abundant taxa between BA.AP and BA.CP; (H) Cladogram of the most differentially abundant taxa between C57.AP and BA.AP; (I) Cladogram of the most differentially abundant taxa between C57.CP and BA.CP.