Fig 1.
Effects of T. gondii infection on rat gut microbiota diversity.
(A) Rarefaction curves showing the relationship between gene richness and sequencing depth, indicating that microbial gene diversity reaches a plateau with increased sequencing effort. (B–C) Raincloud plots depicting richness and Shannon diversity index in T. gondii-infected and uninfected rats. Individual sample values are shown as dots, density plots represent the distribution, and box plots summarize the median and interquartile range. Statistical significance was determined using the Wilcoxon rank-sum test: *P < 0.05; **P < 0.01; ***P < 0.001. (D–E) Principal coordinate analysis (PCoA) based on Bray–Curtis distances depicting differences in microbial community composition across infection stages. Ellipses represent 95% confidence intervals. Box plots adjacent to PCoA axes display sample scores for PCoA1 and PCoA2, with boxes indicating medians and interquartile ranges, and whiskers extending to 1.5 × the IQR. Overall and pairwise differences in community structure were assessed using PERMANOVA, with effect sizes shown in the upper right scatter plot. P-values were calculated using the adonis test with 1,000 permutations. LI: large intestine; SI: small intestine.
Fig 2.
Composition of intestinal microbial communities in the small intestine of rats before and after T. gondii infection.
(A–B) Bar plots showing taxonomic composition at the phylum and species levels across groups before and after infection. (C) Box plots depicting the relative abundance of major bacterial phyla, including Bacillota, Actinomycetota, Pseudomonadota, Bacteroidota, and Verrucomicrobiota, at different infection stages. (D) Box plots illustrating changes in the relative abundance of representative bacterial species, including Lactobacillus johnsonii, Limosilactobacillus reuteri, Lactobacillus intestinalis, Rothia nasimurium, and Streptococcus salivarius, following T. gondii infection. Statistical significance was determined using the Wilcoxon rank-sum test: *P < 0.05; **P < 0.01.
Fig 3.
Composition of intestinal microbial communities in the large intestine of rats before and after T. gondii infection.
(A–B) Bar plots illustrating taxonomic composition at the phylum and species levels across different groups before and after infection. (C) Box plots depicting the relative abundance of major bacterial phyla, including Verrucomicrobiota, Actinomycetota, Pseudomonadota, Thermodesulfobacteriota and Spirochaetota, at different infection stages. (D) Box plots illustrating changes in the relative abundance of representative bacterial species, including Lactobacillus johnsonii, Liqilactobacillus murinus, Akkermansia muciniphila and Limosilactobacillus reuteri, following T. gondii infection. Statistical significance was assessed using the Wilcoxon rank-sum test: * P < 0.05; **P < 0.01.
Fig 4.
Functional analysis of the intestinal microbial community following T. gondii infection.
(A) Heatmap of log₁₀(TPM + 1) transformed abundances of CAZymes significantly enriched by LEfSe (Linear Discriminant Analysis (LDA) score > 2). Rows represent samples, grouped by treatment and intestinal segment, while columns denote CAZy families grouped by class. Asterisks (*) indicate a CAZy families with significant differences (LDA score > 2). The top annotation bar plot shows the magnitude and direction of enrichment, colored by the enriched group. (B) Bar plots significantly enriched CAZymes in each group. Pie charts illustrate predicted substrate-type proportions for control-enriched CAZymes in the small and large intestine. (C) Violin plots representative metabolic pathways in the small and large intestine across infection stages. Statistical significance was assessed by the Wilcoxon rank-sum test: *P < 0.05; **P < 0.01.
Fig 5.
Untargeted serum metabolomics analysis before and after T. gondii infection.
(A–B) Pie charts illustrating the classification of metabolites detected in rat serum under positive and negative ionization modes. (C) Violin plot of metabolite expression distributions across samples. The central line denotes the median; box edges indicate the 25th and 75th percentiles; whiskers extend to the 10th and 90th percentiles. The outer shapes represent the kernel density estimation. (D–I) Volcano plots and bar charts of differential serum metabolites between groups: (D–E) control (CON) vs. acute infection (AI); (F–G) CON vs. chronic infection (CI); (H–I) AI vs. CI. Volcano plots display log2-transformed fold change on the x-axis and –log₁₀ P-value (Student’s t-test) on the y-axis. Vertical dashed lines mark |log2FC| = 1 (twofold change), and the horizontal red line marks P = 0.05. Point colors indicate significance, while the point size corresponds to the variable importance in projection (VIP) score from OPLS-DA.
Fig 6.
KEGG pathway enrichment analysis of differential serum metabolites.
Each dot represents a metabolic pathway. The x-axis shows the pathway impact score, while the y-axis indicates–log₁₀ (P value). Dot size reflects the impact score, and the color gradient from yellow to red corresponds to increasing statistical significance. Results are shown for (A) control (CON) vs. acute infection (AI), (B) CON vs. chronic infection (CI), and (C) AI vs. CI.
Fig 7.
Correlation network between intestinal bacterial species and differential serum metabolites.
Species–metabolite associations are shown at the species level. Solid lines indicate positive correlations, and dashed lines indicate negative correlations. Edge thickness reflects the correlation strength (Spearman’s correlation coefficient), while node size denotes connectivity (number of associations). Results are shown for (A) the small intestine. (B) the large intestine.