Immune strategies of silver pomfret (Pampus argenteus) infected with Nocardia seriolae at different infection stages

Nocardia seriolae, a widespread opportunistic pathogen, has the ability to infect various economically farmed fishes, leading to prolonged disease courses and high mortality rates. In our current study, we focused on the silver pomfret (Pampus argenteus) and examined the liver, spleen, and kidney tissues at three specific N. seriolae infection phases: control (0 day), L group (infection for 5 days), and H group (infection for 15 days). Utilizing HE staining, we observed typical granulomatous nodules displaying stratified concentric layers, including caseous necrotic cells, epithelioid cells and multinucleated giant cells, fibroblasts and fibrocyte envelopes. The TUNEL assay revealed a significant presence of apoptotic cells concentrated within the renal nodules. Following the infection, there was a noticeable increase in AKP, ACP, and LZM levels in all tissues, while LPS exhibited a consistent decline. Through combined analysis of WGCNA and KEGG pathways derived from the tissue transcriptome, we identified several highly enriched pathways, such as the PI3K-Akt signaling pathway and p53 signaling pathway, in all three tissues. Key DEGs from these pathways underwent further investigation through RT-qPCR. In comparison to the control group, the L group exhibited up-regulation of genes associated with biosynthesis catabolism, whereas genes related to immune response, apoptosis, and ion transport displayed down-regulation. Interestingly, the H group showcased a stark reversal of this trend. To validate these DEGs expressions in vitro, we established a Pampus argenteus spleen cell line (PaS). By evaluating the invasion metrics of NS into PaS, we determined an optimal invasion threshold of 105 CFU/ml. The TUNEL assay demonstrated a notable presence of apoptotic signals in the 5 h and 7 h treatment groups. JC-1 assays indicated a gradual decline in membrane potential starting from the 3-h mark. As the duration of treatment increased, RT-qPCR and IHC findings highlighted an initial up-regulation followed by a subsequent down-regulation of genes associated with biosynthesis catabolism. In contrast, genes related to innate immunity displayed up-regulation, whereas genes related to adaptive immunity and ion transport exhibited an initial surge followed by a gradual decrease. Additionally, genes inhibiting apoptosis showed down-regulation. In conclusion, these findings provide valuable insights into the apoptosis mediated by N. seriolae in piscine hosts. These effects are potentially attributed to the increased expression of ion transport-related genes. Moreover, our study sheds light on the intricate molecular mechanisms underlying piscine immunological responses throughout the entire infection timeline, offering promising opportunities for disease control.