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Viral-Infected Change of the Digestive Tract Microbiota Associated With Mucosal Immunity in Teleost Fish

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Viral-Infected Change of the Digestive Tract Microbiota Associated With Mucosal Immunity in Teleost Fish

Shuai Dong et al. Front Immunol.

Abstract

The digestive tract is a unique series of organs that is inhabited by a range of commensal microbes while also exposed to an overwhelming load of dietary antigens. It is widely known that mammals have evolved complex and efficient immune strategies to protect the mucosa of the digestive tract. However, in the early vertebrates, the roles of mucosal immune defense and microbial communities in the different segments of the digestive tract are not well-understood. Here, we constructed a bath infection model with infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Importantly, following viral infection, we found that the IHNV distribution and the reactions of immune-related genes had similar trends that decreased across the digestive tract. Hematoxylin and eosin (H & E) and alcian blue (A & B) staining of the trout digestive tract showed that the pathological changes only occurred in the buccal and pharyngeal mucosal tissues. Moreover, the increased diversity of the microbial community was only detected in the buccal mucosa through 16S rRNA gene sequencing, suggesting that the magnitude of the immune response and microbial community changes are related to the IHNV load and the original microbial diversity. In addition, the loss of digestive tract dominant species and increased colonization of opportunistic bacteria were discovered in the buccal mucosal surface indicating that a secondary bacterial infection occurred in this mucosal tissue.

Keywords: digestive tract; infectious hematopoietic necrosis virus; microbiota; mucosal immunity; rainbow trout (Oncorhynchus mykiss).

Figures

Figure 1
Figure 1
The detection of IHNV and immune-related genes in the buccal mucosa, pharynx, stomach, foregut, midgut, hindgut, head kidney, and spleen of trout at 7 days after infection with IHNV. (A) The histogram showed the loads of IHNV in different tissues at 7 dpi (n = 6 fish per group). Data are representative of three independent experiments (mean ± SD). (B) Heat map illustrates results from quantitative real-time PCR of mRNAs for immune-related genes in virus-challenged fish vs. control fish measured at 7 dpi with IHNV in the buccal mucosa, pharynx, stomach, foregut, midgut, hindgut, head kidney and spleen of rainbow trout (n = 6 fish per group). Color value: log2 (fold change). The mRNA expression levels of Mx1 (C), IFNAR (D), RIG-I (E) were detected at 7 days after infection with IHNV by qRT-PCR (n = 6 per group). Control vs. Infected: *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student's t-test). Data are representative of three independent experiments (mean ± SD). BM, buccal mucosa; Pha, pharynx; Sto, stomach; FG, foregut; MG, midgut; HG, hindgut; HK, head kidney; Sp, spleen.
Figure 2
Figure 2
Pathological changes in the digestive tract of rainbow trout following IHNV infection. (A) Histological examination (H & E) of the digestive tract (including buccal mucosa, pharynx, stomach, foregut, midgut, and hindgut) from control fish and experimental fish infected with IHNV after 7 days (n = 6 fish per group). The thickness of buccal mucosa epidermis (B) and pharyngeal villus epidermis (C) of control and infected fish (n = 6 fish per group). The length-width ratio of stomach villus (D), foregut villus (E), midgut villus (F), and hindgut villus (G) of control and infected fish (n = 6 fish per group). BM, buccal mucosa; Pha, pharynx; Sto, stomach; FG, foregut; MG, midgut; HG, hindgut; OC, oral cavity; EP, epidermis; LP, lamina propria; SM, submucosa; PC, pharyngeal cavity; SC, stomachic cavity; EC, enteric cavity. The red line with arrowheads represent the thickness of mouth epidermis and pharyngeal villus epidermis. The red line indicates the length or width of stomach villus, foregut villus, midgut villus and hindgut villus. Scale bars, 50 μm. Control vs. Infected: *p < 0.05, ***p < 0.001, ns, not significant, unpaired Student's t-test. Data are representative of three different independent experiments (mean ± SEM).
Figure 3
Figure 3
Changes in the abundance and diversity of trout digestive tract microbiota community in response to IHNV infection. (A) Richness and diversity of bacterial community in trout buccal mucosa, pharynx, stomach, foregut, midgut and hindgut from control and infected groups (n = 4 fish per group). Richness and diversity of the digestive tract bacterial community was measured using Chao mean and Shannon index, respectively. Error bars represent standard error of mean (SEM). Mann-Whitney test was conducted between groups with significance level. Control vs. Infection: *p < 0.05. (B) The bacterial community composition in different samples. The relative abundance of bacteria annotated in phylum level. Bar chart of the mean relative abundance in phylum was presented to describe the details. BM, buccal mucosa; Pha, pharynx; Sto, stomach; FG, foregut; MG, midgut; HG, hindgut. (C) Principal coordinate analysis (PCoA) with weighted UniFrac distance matrix for all the 56 taxonomic profiles from trout following IHNV infection. Each symbol represents one sample.
Figure 4
Figure 4
Significantly changed microbes in trout digestive tract after infection with IHNV. Description of top 20 significantly changed microbes that were significant different in the buccal mucosa (A), pharynx (B), stomach (C), foregut (D), midgut (E), and hindgut (F) between control and infected groups.
Figure 5
Figure 5
Pheatmap package of R (version 3.4.4) was used to picture the heat map to illustrate the abundance changes of Pseudomonas, Clostridiales, Bacteroidales, and Escherichia-Shigella in IHNV-challenged and control group. The abundance values were centered and scaled in the row direction.
Figure 6
Figure 6
The transcription level of antimicrobial genes in different tissues. The mRNA expression levels of IL1β (A), IL8 (B), CATH1 (C), CATH2 (D), HP (E), and TLR5S (F) were detected at 7 day after infection with IHNV by qPCR (n = 6 per group). BM, buccal mucosa; Pha, pharynx; Sto, stomach; FG, foregut; MG, midgut; HG, hindgut; HK, head kidney; Sp, spleen. Control vs. Infected: *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student t-test). Data are representative of three independent experiments (mean ± SD).

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