Transcriptome Analysis Reveals Effect of Dietary Probiotics on Immune Response Mechanism in Southern Catfish (Silurus meridionalis) in Response to Plesiomonas shigelloides

Simple Summary We probed the effect of a probiotic complex on the immune response caused by Plesiomonas shigelloides infection in the southern catfish. Our study revealed the key genes for this inflammatory response, and the probiotic complex effectively inhibited the expression of key inflammatory factors caused by P. shigelloides, providing basic data for future artificial breeding of the southern catfish and the prevention and control of bacterial diseases caused by P. shigelloides. Abstract To explore whether a probiotic complex composed of Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei can prevent or inhibit the inflammatory response caused by the invasion of Plesiomonas shigelloides in the southern catfish, we screened differentially expressed genes and enriched inflammation-related pathways among a control and three experimental groups and conducted analysis by transcriptome sequencing after a 21-day breeding experiment. Compared with those in the PS (Plesiomonas shigelloides) group, southern catfish in the L-PS (Lactobacillus-Plesiomonas shigelloides) group had no obvious haemorrhages or ulcerations. The results also showed that inflammation-related genes, such as mmp9, cxcr4, nfkbia, socs3, il-8, pigr, tlr5, and tnfr1, were significantly upregulated in the PS group compared with those in the L-PS groups. In addition, we verified six DEGs (mmp9, cxcr4, nfkbia, socs3, rbp2, and calr) and three proteins (CXCR4, NFKBIA, and CALR) by qRT-PCR and ELISA, respectively. Our results were consistent with the transcriptome data. Moreover, significantly downregulated genes (p < 0.05) were enriched in inflammation-related GO terms (lymphocyte chemotaxis and positive regulation of inflammatory response) and immune-related pathways (intestinal immune network for IgA production and IL-17 signalling pathway) in the L-PS vs. the PS group. Our results indicate that the infection of P. shigelloides can produce an inflammatory response, and probiotics could inhibit the inflammatory response caused by P. shigelloides to some extent.


Introduction
The goal of intensive aquaculture is to create the most suitable environment for the growth and development of aquatic organisms at high densities in small facilities by using advanced feeding management methods, achieving a high yield and efficiently breeding the organisms in a relatively short period of time by accelerating their growth rate to increase their production. However, the development of the aquaculture industry is restricted by animal diseases that are caused by the aquatic environment in high-density aquacultural facilities, which are more conducive to the reproduction, growth, and spread of bacteria. Therefore, bacterial diseases have seriously affected the economical profitability of the aquaculture industry [1].
First, we divided the experimental fish into four groups (each with 20 individuals): (1) blank control group (BC), (2) Lactobacillus group (L), (3) Lactobacillus + P. shigelloides group (L-PS), and (4) P. shigelloides group (PS). Feeding probiotic complex: L group and L-PS group were administered a mix of Lactobacillus (1.0 × 10 9 CFU/mL, 0.2 mL) by intragastric administration for 21 d, whereas the BC group and PS group were gavaged with normal saline (0.2 mL) to provide the same management stress. P. shigelloides infection test: After 21 d, the L-PS group and the PS group were injected intraperitoneally with P. shigelloides (1.0 × 10 9 CFU/mL, 0.2 mL), whereas the BC group and the L group were replaced by normal saline (0.2 mL) to provide the same management stress [30][31][32][33][34]. We collected samples 72 h after injecting P. shigelloides. We obtained spleens from each group (3 samples per group), which we immediately snap-froze in liquid nitrogen and stored at −80 • C for subsequent testing.

RNA Extraction, Library Construction, and Sequencing
We extracted the total RNA of the spleens using a mirVana™ miRNA ISOlation Kit (Ambion-1561) (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. We detected the purity (OD260/280), concentration, and absorption peak of the RNA by a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and then examined the quality of the RNA by 1% agarose gel electrophoresis.
We constructed the libraries using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumia, San Diego, CA, USA) according to the manufacturer's instructions. Then, sequencing was completed using the Illumina HiSeq X Ten platform by OE Biotech Co., Ltd. (Shanghai, China), which generated 150 bp paired-end reads.

Functional Annotation
We functionally annotated the transcripts by alignment of the transcripts with the NCBI nonredundant (NR), SwissProt, and clusters of orthologous groups for the eukaryotic complete genome (KOG) databases using Diamond. The threshold takes E-value < 1e-5 to find orthologs. We used the proteins with the highest hits to the transcripts to assign functional annotations.

Validation of DEGs by q-PCR
To test the RNA-Seq results, we selected six DEGs (mmp9, socs3, nfkbia, cxcr4, rbp2, and calr) for qRT-PCR analysis. The specific primers used are listed in Table 1, and we used β-actin and 18S rRNA as internal controls. We incubated reactions at 94 • C for 30 s, Animals 2023, 13, 449 4 of 18 followed by 45 cycles of 94 • C for 5 s and 60 • C for 30 s. We performed melting curve analysis to verify the specific generation of the expected PCR product after the PCR cycles ended. We normalised the expression levels of mRNAs to β-actin, and we calculated 18S rRNA using the 2 −∆∆Ct method [44]. Table 1. Specific primers list.

Validation of DEGs by ELISA
We analysed three proteins (CXCR4, CALR, and NFKBIA) by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions (Enzyme-Free Biologics) to further verify the reliability of the transcriptome data. We drew the standard curve according to the concentration and absorbance values of the standard to obtain the corresponding calculation formula. Then, we entered the absorbance value of each sample into the formula to calculate the secretion of each protein.

Clinical Symptoms
In our experiment, we observed body surface changes in the PS and L-PS groups 72 h after injection of P. shigelloides (Figure 1a). The most obvious symptoms in the PS group were bleeding of the caudal fins and anal fin, and the blackening, moulting, and ulceration of the skin surface; however, we did not find lesions in other parts (Figure 1b). After dissection, we found one case of intestinal redness and swelling; however, no lesions were found in the other individuals ( Figure 1c). In the L-PS group, except for two individuals with hyperaemia of the anal fin base and one with redness and swelling at the injection site, no lesions were found in other individuals. The observations showed that the BC and L groups showed no body surface symptoms during the experimental period. The incidence table is as follows: southern catfish with caudal fin congestion; bleeding, ulceration, skin blackening, moulting, and other symptoms are counted as the disease ( Table 2)

Data Analysis of Transcriptome
From Illumina sequencing, a total of 79.37 Gb clean data were quality-filtered from 89.46 Gb of raw data ( Table 3). The average length of merged transcripts was 1918 bp, and the N50 length was 32598 bp.

Differentially Expressed Genes (DEGs) Regulated by Exposure of P. shigelloides and Lactobacillus
In total, 370 upregulated and 381 downregulated DEGs were detected in the PS group vs. the BC group, 630 upregulated and 583 downregulated DEGs were detected in the L group vs. the BC group, 583 upregulated and 520 downregulated DEGs were detected in the L-PS group vs. the BC group, and 172 upregulated and 220 downregulated DEGs were detected in the L-PS group vs. the PS group ( Figure 2).

Differentially Expressed Genes (DEGs) Regulated by Exposure of P. shigelloides and Lactobacillus
In total, 370 upregulated and 381 downregulated DEGs were detected in the PS group vs. the BC group, 630 upregulated and 583 downregulated DEGs were detected in the L group vs. the BC group, 583 upregulated and 520 downregulated DEGs were detected in the L-PS group vs. the BC group, and 172 upregulated and 220 downregulated DEGs were detected in the L-PS group vs. the PS group ( Figure 2).

Gene Ontology Enrichment Analysis for DEGs
To explore the mechanism of the southern catfish's immune response to P. shigelloides at the genetic level, 751 DEGs were processed for GO enrichment analysis between the BC and PS groups. The top 30 enriched GO terms of upregulated genes are presented in Figure 3a. The immune-related GO terms were significantly (p < 0.05) enriched in the spleens of southern catfish infected with P. shigelloides, such as the inflammatory response, immune system process, and platelet alpha granules.
To explore the mechanism of Lactobacillus supplementation at the genetic level, 392 DEGs were processed for GO enrichment analysis between the L-PS and PS groups.

Gene Ontology Enrichment Analysis for DEGs
To explore the mechanism of the southern catfish's immune response to P. shigelloides at the genetic level, 751 DEGs were processed for GO enrichment analysis between the BC and PS groups. The top 30 enriched GO terms of upregulated genes are presented in Figure 3a. The immune-related GO terms were significantly (p < 0.05) enriched in the spleens of southern catfish infected with P. shigelloides, such as the inflammatory response, immune system process, and platelet alpha granules.
To explore the mechanism of Lactobacillus supplementation at the genetic level, 392 DEGs were processed for GO enrichment analysis between the L-PS and PS groups. Inflammationrelated GO terms were significantly (p < 0.05) enriched, such as lymphocyte chemotaxis, positive regulation of inflammatory response, and interleukin-1 receptor activity. The top 30 enriched GO terms of downregulated DEGs are presented in Figure 3b. In the L group vs. the BC group, significantly upregulated genes (p < 0.05) were enriched in neutrophil homeostasis, regulation of vasoconstriction, and so on (Figure 3c).

KEGG Analysis for DEGs
To examine the pathways affected by P. shigelloides, 751 DEGs were processed for KEGG pathway enrichment analysis between the PS and BC groups. Some immune-related pathways, such as the intestinal immune network for IgA production, IL-17 signalling pathway, and so on, were significantly (p < 0.05) enriched in upregulated DEGs (Figure 4a).
Inflammation-related GO terms were significantly (p < 0.05) enriched, such as lymphocyte chemotaxis, positive regulation of inflammatory response, and interleukin-1 receptor activity. The top 30 enriched GO terms of downregulated DEGs are presented in Figure 3b. In the L group vs. the BC group, significantly upregulated genes (p < 0.05) were enriched in neutrophil homeostasis, regulation of vasoconstriction, and so on (Figure 3c).

KEGG Analysis for DEGs
To examine the pathways affected by P. shigelloides, 751 DEGs were processed for KEGG pathway enrichment analysis between the PS and BC groups. Some immune-related pathways, such as the intestinal immune network for IgA production, IL-17 signalling pathway, and so on, were significantly (p < 0.05) enriched in upregulated DEGs (Figure 4a).
To explore the mechanism of Lactobacillus supplementation at the genetic level, 392 DEGs were processed for KEGG enrichment analysis between the L-PS and PS groups. In the L-PS vs. the PS group, significantly downregulated genes (p < 0.05) were also enriched in the intestinal immune network for IgA production and the IL-17 signalling pathway ( Figure 4b). In the L group vs. the BC group, significantly downregulated genes (p < 0.05) were enriched in the B-cell receptor signalling pathway, which was the same as the results in the L-PS group vs. the PS group (Figure 4c).   In the IL-17 signalling pathway, our results showed that the expression of proinflammatory factors within the range was increased, and the trend between groups, such as-mmp9, il-8, jun, and mmp13, was significantly upregulated (p < 0.05) in the PS group vs. the BC group, and hsp90 was significantly downregulated (p < 0.05) in the PS group vs.  To explore the mechanism of Lactobacillus supplementation at the genetic level, 392 DEGs were processed for KEGG enrichment analysis between the L-PS and PS groups. In the L-PS vs. the PS group, significantly downregulated genes (p < 0.05) were also enriched in the intestinal immune network for IgA production and the IL-17 signalling pathway (Figure 4b). In the L group vs. the BC group, significantly downregulated genes (p < 0.05) were enriched in the B-cell receptor signalling pathway, which was the same as the results in the L-PS group vs. the PS group (Figure 4c).
3.6. Analysis of Immune-Related Pathways 3.6.1. IL-17 Signalling Pathway In the IL-17 signalling pathway, our results showed that the expression of proinflammatory factors within the range was increased, and the trend between groups, such as mmp9, il-8, jun, and mmp13, was significantly upregulated (p < 0.05) in the PS group vs. the BC group, and hsp90 was significantly downregulated (p < 0.05) in the PS group vs. the BC group. In the L-PS group vs. the PS group, mmp9 and mmp13 were significantly downregulated (p < 0.05), and hsp90 was significantly upregulated (p < 0.05), while il-8 expression was downregulated (p < 0.05). In the L group vs. the BC group, the expression of mmp9 and mmp13 was significantly downregulated (p < 0.05) ( Figure 5).

TNF Signalling Pathway
In the TNF signalling pathway, the results showed that the expression of socs3, tnfr1, traf2, and ap-1 was significantly upregulated (p < 0.05) in the PS group vs. the BC group; however, the expression of p38 and creb was significantly downregulated (p < 0.05). In the L-PS group vs. the PS group, the expression of socs3 and mmp9 was significantly downregulated (p < 0.05), while the expression of tnfr1, traf2, ap-1, and jun was downregulated ( Figure 6). In the L group vs. the BC group, p38 expression was significantly upregulated (p < 0.05).

TNF Signalling Pathway
In the TNF signalling pathway, the results showed that the expression of socs3, tnfr1, traf2, and ap-1 was significantly upregulated (p < 0.05) in the PS group vs. the BC group; however, the expression of p38 and creb was significantly downregulated (p < 0.05). In the L-PS group vs. the PS group, the expression of socs3 and mmp9 was significantly downregulated (p < 0.05), while the expression of tnfr1, traf2, ap-1, and jun was downregulated ( Figure 6). In the L group vs. the BC group, p38 expression was significantly upregulated (p < 0.05).

Toll-like Receptor Signalling Pathway
The results showed that several proinflammatory factors, such as tlr5, nfkbia, and ccl3, were significantly (p < 0.05) upregulated in the PS group vs. the BC group. In the L-PS group vs. the PS group, the expression of tlr5, jun, and nfkbia was downregulated; however, p38 expression was significantly (p < 0.05) upregulated. In the L group vs. the BC group, ccl3 expression was significantly (p < 0.05) upregulated, while the expression of rac1, tlr2, and tlr9 was significantly (p < 0.05) downregulated (Figure 7). however, the expression of p38 and creb was significantly downregulated (p < 0.05). In the L-PS group vs. the PS group, the expression of socs3 and mmp9 was significantly downregulated (p < 0.05), while the expression of tnfr1, traf2, ap-1, and jun was downregulated ( Figure 6). In the L group vs. the BC group, p38 expression was significantly upregulated (p < 0.05).

Toll-Like Receptor Signalling Pathway
The results showed that several proinflammatory factors, such as tlr5, nfkbia, and ccl3, were significantly (p < 0.05) upregulated in the PS group vs. the BC group. In the L-PS group vs. the PS group, the expression of tlr5, jun, and nfkbia was downregulated; however, p38 expression was significantly (p < 0.05) upregulated. In the L group vs. the BC group, ccl3 expression was significantly (p < 0.05) upregulated, while the expression of rac1, tlr2, and tlr9 was significantly (p < 0.05) downregulated (Figure 7).

Intestinal Immune Network for IgA Production
In this signalling pathway, the expression of cxcr4, pigr, ccr9, α4β7, and cd40l was significantly upregulated (p < 0.05) in the PS group vs. the BC group. In the L-PS group vs. the PS group, the expression of cxcr4, pigr, ccr9, tcr, and aid was significantly downregulated (p < 0.05) (Figure 8). In the L-PS group vs. the BC group, the expression of ccr9 and cxcr4 had no significant difference.

Intestinal Immune Network for IgA Production
In this signalling pathway, the expression of cxcr4, pigr, ccr9, α4β7, and cd40l was significantly upregulated (p < 0.05) in the PS group vs. the BC group. In the L-PS group vs. the PS group, the expression of cxcr4, pigr, ccr9, tcr, and aid was significantly downregulated (p < 0.05) (Figure 8). In the L-PS group vs. the BC group, the expression of ccr9 and cxcr4 had no significant difference.
In this signalling pathway, the expression of cxcr4, pigr, ccr9, α4β7, and cd40l was significantly upregulated (p < 0.05) in the PS group vs. the BC group. In the L-PS group vs. the PS group, the expression of cxcr4, pigr, ccr9, tcr, and aid was significantly downregulated (p < 0.05) (Figure 8). In the L-PS group vs. the BC group, the expression of ccr9 and cxcr4 had no significant difference.

Validation of DEGs by qRT-PCR
To validate the expression profiles of genes identified through RNA-Seq, we analysed the relative mRNA levels of the following six differentially expressed genes (mmp9, socs3, cxcr4, nfkbia, rbp2, and calr) by qRT-PCR ( Figure 9). Our results showed that the qRT-PCR results and the results obtained through RNA-Seq were consistent ( Figure 10).

Validation of DEGs by qRT-PCR
To validate the expression profiles of genes identified through RNA-Seq, we analysed the relative mRNA levels of the following six differentially expressed genes (mmp9, socs3, cxcr4, nfkbia, rbp2, and calr) by qRT-PCR (Figure 9). Our results showed that the qRT-PCR results and the results obtained through RNA-Seq were consistent ( Figure 10).

Validation of DEGs by ELISA
To further confirm the changes in the spleens, we used ELISA to detect the contents of CXCR4, CALR, and NFKBIA in the spleen tissue homogenate. The ELISA results showed that the concentrations of CXCR4 and NFKBIA proteins were significantly (p < 0.05) upregulated in the PS group compared with the other groups. The concentrations of the CALR protein were significantly (p < 0.05) downregulated between the PS and BC groups. The trends of the three proteins were consistent with the RNA expression and transcriptome data ( Figure 11).

Validation of DEGs by ELISA
To further confirm the changes in the spleens, we used ELISA to detect the contents of CXCR4, CALR, and NFKBIA in the spleen tissue homogenate. The ELISA results showed that the concentrations of CXCR4 and NFKBIA proteins were significantly (p < 0.05) upregulated in the PS group compared with the other groups. The concentrations of the CALR protein were significantly (p < 0.05) downregulated between the PS and BC groups. The trends of the three proteins were consistent with the RNA expression and transcriptome data ( Figure 11).

Discussion
The spleen, an important organ in a fish's immune system, is considered a primordial secondary lymphoid organ where an adaptive immune response is produced [45]. Bacterial diseases are extremely harmful to animals in large-scale aquaculture facilities. Once an infection occurs, these diseases spread rapidly. At present, the most common treatment for bacterial diseases is the use of antibiotics. However, the long-term use of antibiotics will not only pollute the water body but also make the intestinal flora of the fish more resistant, making the treatment of fish diseases more difficult in the future. Therefore, it is of great significance to explore effective and green control methods.

IL-17 Signalling Pathway
IL-17 is a proinflammatory cytokine specifically secreted by helper T cells that can induce the secretion of early immune mediators [46,47]. Its family members, IL-17A and IL-17F, activate the downstream IK-Ba through IL-17R-Act1-TRAF6 and indirectly act on MAPKs to activate AP-1. A sustained inflammatory response can increase the expression of NF-κB, activate downstream chemokines and other effector genes of the NF-κB signalling pathway to increase the expression, and ultimately act on biological processes, such as neutrophil recruitment [48,49]. A high expression of matrix metalloproteinases can also indicate that the body is in a state of inflammation. Matrix metalloproteinase-9 (mmp9) is an important member of the MMP family, also known as gelatinase B [50]. A high expression of mmp9 under pathological conditions reshapes the extracellular matrix through ex-

Discussion
The spleen, an important organ in a fish's immune system, is considered a primordial secondary lymphoid organ where an adaptive immune response is produced [45]. Bacterial diseases are extremely harmful to animals in large-scale aquaculture facilities. Once an infection occurs, these diseases spread rapidly. At present, the most common treatment for bacterial diseases is the use of antibiotics. However, the long-term use of antibiotics will not only pollute the water body but also make the intestinal flora of the fish more resistant, making the treatment of fish diseases more difficult in the future. Therefore, it is of great significance to explore effective and green control methods.

IL-17 Signalling Pathway
IL-17 is a proinflammatory cytokine specifically secreted by helper T cells that can induce the secretion of early immune mediators [46,47]. Its family members, IL-17A and IL-17F, activate the downstream IK-Ba through IL-17R-Act1-TRAF6 and indirectly act on MAPKs to activate AP-1. A sustained inflammatory response can increase the expression of NF-κB, activate downstream chemokines and other effector genes of the NF-κB signalling pathway to increase the expression, and ultimately act on biological processes, such as neutrophil recruitment [48,49]. A high expression of matrix metalloproteinases can also indicate that the body is in a state of inflammation. Matrix metalloproteinase-9 (mmp9) is an important member of the MMP family, also known as gelatinase B [50]. A high expression of mmp9 under pathological conditions reshapes the extracellular matrix through excessive degradation, exposes relevant active sites, destroys the physiological barrier, and aggravates the inflammatory response [51]. Studies have confirmed that the upregulation of mmp9 is closely related to the body's inflammatory response process [52][53][54]. In this study, the mmp9 gene was significantly (p < 0.05) expressed in the PS group, which proved that mmp9 may be involved in the pathogenesis of the inflammatory response caused by P. shigelloides. Combined with the comprehensive analysis of other groups, the expression of mmp9 in the L group and the L-PS group was significantly (p < 0.05) reduced, indicating that the probiotic complex had an inhibitory effect on the expression of this gene; therefore, it could inhibit the inflammatory response to a certain extent.
Neutrophils are the first line of immune defence and are phagocytes that play a crucial role in the early immune response of the host by clearing pathogens [55,56]. il-8 is a member of the α subfamily of chemokines, which acts on neutrophil chemotaxis, promotes T-cell chemotaxis and migration, and strengthens the immune response [57]. In our study, the reason for the significant expression of il-8 in the PS group may be due to the inflammatory response of the southern catfish infected with P. shigelloides, which activates macrophages and promotes the chemotaxis of neutrophils to the site of inflammation. Compared with the BC group, il-8 expression in the L group was significantly (p < 0.05) reduced, indicating that the expression of proinflammatory factors could be inhibited after treatment with the probiotic complex, thereby inhibiting the inflammatory response. Comparing the L-PS group and the PS group, we found that the expression of il-8 was significantly (p < 0.05) reduced, indicating that the feeding of probiotics may enhance the resistance of southern catfish to pathogenic invasion and that the probiotic complex can effectively inhibit the inflammatory response caused by the invasion of P. shigelloides.

TNF Signalling Pathway
The TNF (tumour necrosis factor), also known as TNFα, is a cytokine that can directly kill tumour cells and has no obvious cytotoxicity to normal cells. It is a cell signalling protein involved in systemic inflammation and a cytokine that constitutes the acute phase response [58]. The expression of the downstream gene socs3 in this pathway is also regulated to varying degrees. SOCS3 is one of the important members of the SOCS family proteins. socs3 has been certified to play a key role in immune response in aquatic animals, such as Cynoglossus semilaevis [59], Paralichthys olivaceus [60], and Ictalurus punctatus [61]. In our study, socs3 gene expression was significantly upregulated (p < 0.05) in the PS vs. BC group, indicating that P. shigelloides could induce the expression of socs3. Studies have also shown that socs3 expression can be induced by the lipopolysaccharides of Gramnegative bacteria [62]. In the L-PS vs. PS group, the expression of socs3 was significantly downregulated. We speculated that feeding of the probiotic complex had a protective effect on southern catfish.
The TNF is involved in the innate immune response through two receptors, TNFR1 and TNFR2. In this study, the significant expression of tnfr1 in the PS group showed that infection with P. shigelloides stimulated an immune response and activated the TNFR1 signalling pathway (TNFR1-TRADD-TRAF), which inhibits apoptosis and promotes inflammation. Because TNFα is associated with an acute inflammatory response, it may be the reason for the lack of a significant difference in tnfr1 expression between the L group and the BC group. Compared with the PS group, the expression of tnfr1 in the L-PS group was significantly (p < 0.05) decreased, indicating that long-term feeding of probiotics may effectively respond to the TNFα-mediated inflammatory pathway caused by the invasion of P. shigelloides and inhibit the inflammatory response to a certain extent. The cytoplasmic TIR domain of TLR recruits signal adaptors MyD88, TIRAP, TRAM,  and/or TRIF and activates various kinases (IRAK4, IRAK1, IRAK2, TBK1, and IKKε) and ubiquitin ligases (TRAF6 and pellino 1), thereby activating nuclear factor NF-κB and inducing the expression of inflammatory factors. nfkbia is the gene encoding the NF-κB complex inhibitory protein IκB [63]. There are related studies involving this gene in aquatic animals. nfkbia gene expression in zebrafish juveniles infected with Vibrio parahaemolyticus [64], Lampetra japonica stimulated by dsRNA virus mimetic [65], and Channa argus after infection with Americide nocardia [66] were significantly (p < 0.05) upregulated. In this study, nfkbia expression in the PS group was significantly (p < 0.05) upregulated compared with the BC group, consistent with the findings above, indicating that nfkbia may be involved in the early generation of the innate immune response to P. shigelloides infection in the southern catfish.

Toll-like Receptor Signalling Pathway
In the TLR family, TLR5, the receptor of flagellin, which is related to the pathogenicity of bacteria, has a defensive effect against the invasion of flagella [67]. In our study, the expression of tlr5 in the PS group was significantly (p < 0.05) upregulated, which may be due to the surface structure of bacteria inducing the body to produce a large number of TLR5 receptor proteins to resist bacterial invasion, indicating that P. shigelloides infection can cause TLR5-mediated innate immunity in the southern catfish. The expression of tlr5 in the L group was not significantly (p < 0.05) different from that in the BC group, which may be because probiotics have little effect on tlr5 regulation without pathogenic invasion. Compared with the PS group, the expression of tlr5 in the L-PS group was significantly (p < 0.05) reduced, and we hypothesised that this may have been due to the enhanced immunity and disease resistance of the southern catfish due to the probiotics. In addition, it is possible that the number of probiotics in the body is large and that the produced metabolites inhibit the growth of P. shigelloides. We performed a series of in vitro bacteriostatic experiments and showed that different probiotic metabolite concentrations can inhibit the growth of P. shigelloides to varying degrees.

Intestinal Immune Network for IgA Production
CXCR4 (chemokine receptor 4) is an inflammatory chemokine that acts by binding to its ligand SDF-1 (stromal cell-derived factor-1). The possible mechanism of the SDF-1/CXCR4 axis-mediated inflammatory response is chemotaxis of neutrophils and other inflammatory cells to the site of inflammation [68]. In our study, the expression of cxcr4 in the PS group was significantly (p < 0.05) increased, indicating that the invasion of P. shigelloides caused the southern catfish to be in an inflammatory state. In the L-PS vs. the PS group, the cxcr4 gene expression was significantly (p < 0.05) downregulated. Our results showed that the degree of inflammatory response in the L-PS group was lighter compared with the PS group. In addition, we detected the concentration of CXCR4 protein by ELISA, and the change trend was consistent with the change trend of cxcr4 expression. pIgR (poly-Ig receptor) is involved in the innate immunity of the organism mucosa. pIgR mediates IgA transportation to neutralise extracellular pathogens in mammals [69,70]. To some extent, the expression level of pIgR in mucosal epithelium represents the expression level of IgA antibody in extra mucosal secretions [71]. In teleost fish, pIgR can transport IgM and IgT to mucosal secretions and exert its mucosal immune function [72,73]. Studies have also shown that IgT in fish can mediate the intestinal lumen through pIgR, which is considered a specific immunoglobulin involved in intestinal mucosal immunity, and its function is similar to mammalian IgA [74]. Studies have shown that the expression of pigr in the spleen of Scophthalmus maxima is significantly (p < 0.05) upregulated after infection by Vibrio anguillarum [75]. The expression of pigr in the spleen of zebrafish was also significantly (p < 0.05) upregulated after infection by Streptococcus iniae [76]. In our study, the expression of pigr was significantly (p < 0.05) upregulated in the PS group compared with the BC group, indicating that the invasion of P. shigelloides can lead to the activation of the intestinal immune network for IgA production, upregulating the downstream key gene pigr to promote inflammatory response. The expression of some transcripts of the pigr gene in the L vs. the BC group was downregulated, and pigr gene expression was significantly (p < 0.05) downregulated in the L-PS vs. the PS group. The above results show that the probiotic complex could inhibit the expression of pigr to relieve inflammation.

Other Related Key Genes
Calreticulin (CALR) protein can maintain intracellular calcium homeostasis and assist in protein folding, and plays a significant role in immune regulation, cell phagocytosis, cell proliferation, and apoptosis [77]. In our study, the expression of calr in the L, PS, and L-PS groups was significantly (p < 0.05) lower than the BC group; however, CALR protein concentration in the PS and L-PS groups was lower than that in BC and L groups, probably because the invasion of P. shigelloides could inhibit the function of CALR. RBP2 plays a key role in maintaining the normal DC response, which has functions in phagocytosis, processing, and presenting antigens [78]. In our study, we hypothesised that the downregulation of rbp2 in the PS group may be related to the mechanism of P. shigelloides invasion to evade immunity. MHCI cytotoxic T lymphocytes (CD8+) play a key role in the recognition of antigen peptide-MHC complexes [79]. In this study, mhc I expression was significantly (p < 0.05) downregulated in the PS group. We speculated that it was because P. shigelloides inhibited mhc I expression to evade the body's immune system; however, this conjecture needs to be further verified. Arachidonate 5-lipoxygenase (Alox5) is a membrane-bound protein mainly expressed in polymorphonuclear leukocytes, peripheral blood monocytes, macrophages, and mast cells. It is highly expressed in many diseases [80][81][82]. Inhibition of alox5 expression attenuates lipopolysaccharide (LPS)-induced inflammation [83]. In our study, the expression of alox5 in the L and L-PS groups was significantly (p < 0.05) downregulated, which also showed that the probiotic complex could inhibit the expression of alox5 and block the production of inflammatory mediators (Supplementary Figure S1 [84][85][86]).

Conclusions
In conclusion, in our experiment, we found that P. shigelloides can cause hyperaemia and the festering of fins, while southern catfish fed a probiotic complex were free of related inflammation after the infection of P. shigelloides. Moreover, significantly downregulated genes (p < 0.05) were enriched in inflammation-related GO terms (lymphocyte chemotaxis and positive regulation of inflammatory response) and immune-related pathways (intestinal immune network for IgA production and IL-17 signalling pathway) in the L-PS vs. the PS group. Hence, probiotics could inhibit the inflammatory response caused by P. shigelloides to some extent. Therefore, probiotics are promising alternatives to antibiotics to prevent bacterial diseases in the aquaculture industry.