COMPARATIVE SPATIAL TRANSCRIPTOMICS OF Aeromonas veronii INFECTION IN EUROPEAN SEABASS SPLEEN
Introduction
Aeromonas veronii is an important emerging bacterial pathogen of European seabass (Dicentrarchus labrax) aquaculture in the Mediterranean and is associated with substantial morbidity and mortality (Smyrli et al., 2017). A characteristic lesion of the disease is the formation of splenic abscesses, localized inflammatory foci containing host immune cells and viable bacteria. These structured lesions offer an excellent system for studying infection biology in situ. Within the Cure4Aqua project we used a seabass A. veronii challenge model to combine gross pathology, histology, scanning electron microscopy (SEM) and high-resolution spatial transcriptomics. The aim was to compare infected and uninfected spleen tissue and to define the spatial organization of the host response inside and around infection-associated lesions.
Materials and Methods
Forty-five healthy European seabass of approximately 130 g were distributed into three 350 L tanks. One group served as an uninfected control, while two groups were exposed by immersion for 2.5 h to 105 CFU mL-1 of two A. veronii strains: a typical motile, pigment-producing strain (PDB) and a non-motile, non-pigmented strain (NS). Fish were monitored for clinical signs, and moribund fish were euthanized with an overdose of MS-222. Organs were collected for bacteriology, pathology, histology, SEM and spatial transcriptomics. Histology was performed at Pathovet AG, SEM at the University of Crete and HCMR, and spatial transcriptomic analyses at the Functional Genomics Center Zurich. Spatial transcriptomics was performed on spleen sections using the Visium HD 3' platform (10x Genomics). Infected spleen sections containing visible lesions were compared with an uninfected control spleen with normal histology.
Results
Both A. veronii strains produced typical disease lesions within 7 days post-challenge, including epidermal haemorrhages, and abscesses in various internal organs such as spleen, liver kidney and brain. Bacteria were re-isolated from affected fish and confirmed by PCR. Histology showed severe splenic pathology in infected fish, while the uninfected control spleen showed normal tissue organization. SEM confirmed the presence of bacterial cells within splenic lesions and showed bacteria in close association with host phagocytic cells, supporting the interpretation that abscesses represent active host-pathogen interaction sites.
Spatial transcriptomics resolved infected spleen tissue into distinct molecular domains rather than a uniform organ-wide response. Lesion-associated regions showed strong enrichment of inflammatory, macrophage/myeloid, phagocytic, lysosomal and complement-related signatures. As examples, infected spatial domains included markers consistent with acute inflammation and cytokine signaling (il1b, tnfa, cxcl8), complement activation (c3), phagocyte activation and antimicrobial defense (nos2, irg1), tissue remodeling (mmp9), acute-phase response (saa) and cellular stress (hsp70). These markers supported active innate immune recognition, bacterial clearance mechanisms and local inflammatory activation. Surrounding tissue showed additional signatures associated with tissue stress, extracellular-matrix remodeling, stromal organization and repair. In contrast, the uninfected control spleen provided a spatial baseline of normal splenic architecture, without the strong focal inflammatory and remodeling domains observed in infected tissue.
Comparative analysis of infected and control spleens indicated that A. veronii infection reorganizes the splenic microenvironment into localized infection niches. These niches contained inflammatory and phagocytic cores surrounded by regions of metabolically active and remodeling tissue. The spatial data also showed that important disease-related signals were concentrated in specific tissue areas and would be partly diluted in whole-organ analyses. This spatial resolution is particularly useful for fish health studies because it connects molecular immune responses directly with lesion morphology and tissue damage.
Figure 1. Left: spleen of a fish challenged with Aeromonas veronii NS with the characteristic lesions. Right: The spatial transcriptomic map of the same sample
Discussion
This study demonstrates that A. veronii infection in European seabass spleen is spatially organized. The combination of pathology, histology, SEM and spatial transcriptomics showed that splenic abscesses are not simple areas of tissue destruction, but structured infection microenvironments where bacteria, phagocytic cells, inflammatory pathways and tissue-remodeling responses interact. The comparison with an uninfected control spleen was important because it separated normal splenic organization from infection-associated molecular changes. For aquaculture, these findings are relevant because they provide tissue-architecture-level markers of disease progression and host response. Spatially resolved inflammatory, phagocytic, complement and remodeling signatures could be used in future challenge studies to evaluate disease severity, compare bacterial strains and assess whether interventions reduce lesion-associated pathology. By linking molecular signatures with histological and ultrastructural evidence, this approach offers a practical platform for understanding aeromonad disease mechanisms and for supporting targeted health-management strategies in Mediterranean seabass aquaculture.
Acknowledgment
The study has been funded by the European Union's Horizon Europe research and innovation program, and the State Secretariat for Education, Research and Innovation (SERI) of the Swiss Federal Department of Economic Affairs, Education and Research (EAER), under grant agreement No. 101084204 Cure4Aqua. neither the European Union nor the granting authority can be held responsible for them. We also would like to acknowledge technical supports from the institute of Pathology and Molecular Pathology (University Hospital Zurich) for the help with the tissue sections for spatial transcriptomics.
References
Smyrli, M., Prapas, A., Rigos, G., Kokkari, C., Pavlidis, M., & Katharios, P. (2017). Aeromonas veronii infection associated with high morbidity and mortality in farmed European seabass Dicentrarchus labrax in the Aegean Sea, Greece. Fish Pathology, 52(2).