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. 2022 Jun 30:13:872896.
doi: 10.3389/fimmu.2022.872896. eCollection 2022.

Isolation, Identification, and Investigation of Pathogenic Bacteria From Common Carp (Cyprinus carpio) Naturally Infected With Plesiomonas shigelloides

Huijie Chen  1   2 , Yuanli Zhao  1 , Kuangxin Chen  1   3 , Yulai Wei  1   3 , Hongrui Luo  1 , Yongming Li  1 , Fei Liu  1   3 , Zuoyan Zhu  1 , Wei Hu  1 , Daji Luo  1   2   3
Affiliations

Isolation, Identification, and Investigation of Pathogenic Bacteria From Common Carp (Cyprinus carpio) Naturally Infected With Plesiomonas shigelloides

Huijie Chen et al. Front Immunol. .

Abstract

Various bacterial diseases have caused great economic losses to the high-density and intensive aquaculture industry; however, the pathogenic mechanism underlying the large-scale challenged to caused by many bacteria remain unclear, making the prevention and treatment of these diseases difficult. In the present study, we isolated a bacterial strain from Cyprinus carpio having a typical bacterial disease and named it Cc2021. Through subsequent morphological observations, a regression challenge, biochemical identification, and 16S rRNA gene sequence analysis, we determined Cc2021 to be Plesiomonas shigelloides. Subsequently, we comprehensively investigated the pathogenicity of P. shigelloides in C. carpio through a regression challenge and assessed the underlying the pathogenic mechanism. Mortality results revealed that P. shigelloides is highly pathogenic and infects various tissues throughout the body, resulting in edema of the liver, spleen, and body and head kidneys. Histopathological analysis revealed obvious inflammation, bleeding, and necrosis in the intestine, spleen, and head kidney. The body's immune tissues actively produce complement C3, superoxide dismutase, and lysozyme after a challenge to resist bacterial invasion. With regard to the underlying pathogenesis of P. shigelloides, comparative transcriptome analysis revealed 876 upregulated genes and 828 downregulated genes in the intestine of C. carpio after the challenge. Analysis of differentially expressed unigenes revealed the involvement of major immune pathways, particularly the TNF signaling pathway, interleukin (IL)-17 signaling pathway, and Toll-like receptor signaling pathway. The present study provides new valuable information on the immune system and defense mechanisms of P. shigelloides.

Keywords: bacteria identification; comparative transcriptome; histopathological analysis; natural infection; pathogenicity; tissue bacterial load.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Symptoms of infected C carpio. (A) The abdominal scales of infected C carpio fell off, and the abdomen was red and swollen. (B) Red head of infected C carpio. (C) Swollen abdomen of infected C carpio. (D) The abdominal scales of infected C carpio fell off. The abdomen became red, with abscess-like fluid flowing out of it. (E) The visceral tissue was diffuse, and the liver was enlarged and pale. (F) The abdominal cavity of C carpio had a lot of yellowish fluid after dissection.
Figure 2
Figure 2
Identification of the pathogen’s morphological and molecular characteristics. (A) (a) Growth characteristics of isolates in BHI medium. (b) Gram staining results of isolates. (c) Negative staining results of isolates. (B) Phylogenetic tree based on the partial 16S rRNA gene sequences included in the expanded MicroSeq 500 library; the algorithm used to construct the tree was the unweighted pair group method using averages (UPGMA). (C) Phylogenetic tree based on the partial gyrB gene sequences included in the expanded MicroSeq 500 library; the algorithm used to construct the tree was UPGMA.
Figure 3
Figure 3
Tissue edema and bacterial load in C. carpio after P. shigelloides challenge. (A–D) The dry/wet ratio of the liver, spleen, body kidney, and head kidney of C. carpio 0–3 days after P. shigelloides challenge. (E) Bacterial load in C. carpio tissues after P. shigelloides challenge.
Figure 4
Figure 4
Histopathological changes in C carpio tissues after P. shigelloides challenge. (A) Infected liver. (B) Infected head kidney. (C) Infected spleen. (D) Infected intestine. Pictures of typical symptoms are shown. Scale bar of A, B, and C = 20 μm. Scale bar of D = 50 μm.
Figure 5
Figure 5
(A) Changes in complement C3 levels in the serum of C carpio with time after P. shigelloides challenge. (B) Changes in LZM activity in tissues of C carpio with time after P. shigelloides challenge. (C) Changes in SOD activity in tissues of C carpio with time after P. shigelloides challenge. "*" means P < 0.05.
Figure 6
Figure 6
Differentially expressed genes. (A) Heatmap showing the expression of differentially expressed genes in each sample. (B) Volcano map showing the expression of differentially expressed genes in each group of samples. (C) GO enrichment analysis was performed for differentially expressed genes, and three categories of the top 10 immune-related pathways were selected for display. (D) KEGG enrichment analysis was performed for differentially expressed genes, and the top 10 immune-related pathways were selected for display.
Figure 7
Figure 7
Relative mRNA expression levels of IL-1β, Hsp70, TLR5a, MCH II, CD22, and IL-8 in C carpio tissues after P. shigelloides challenge. mRNA expression levels of IL-1β (A), Hsp70 (B), TLR5a (C), MCH II (D), CD22 (E), and IL-8 (F) in the blood, spleen, head kidney, and intestine of C carpio at 0, 1, 3, 5, and 7 days after P. shigelloides challenge. The β-actin gene was used as a reference gene, and the data of day 0 were used for comparison with those of days 1, 3, 5, and 7 in order to analyze the differences. Data are presented as the means ± SEs (n = 4). Statistical analysis was performed using unpaired Student’s t-test (*P < 0.05 and **P < 0.01).
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