Unidentified intracellular bacteria causing severe disease outbreak in farmed gilthead sea bream (Sparus aurata).

INTRODUCTION Mediterranean mariculture is increasingly challenged by emerging transmissible diseases, primarily caused by pathogenic bacteria such as Lactococccus garvieae, Aeromonas veronii and Rickettsia-like organisms (RLO) among others. This report describes the diagnostic process undertaken to identify the etiological agent responsible for a severe mortality outbreak in gilthead sea bream (Sparus aurata) farmed in the Mediterranean. In March 2025, an outbreak occurred in a pre-growing sector of an inland facility, affecting juveniles with an average weight of 1-10 g. Affected fish exhibited nonspecific clinical signs such as lethargy and disorexia, followed by the appearance of whitish patches on the skin over the lateral musculature. These lesions progressed into erosive/ulcerative wounds and eventually into widespread proliferative skin alterations. Cumulative mortality reached nearly 100% in some tanks, while adjacent tanks showed either no mortality or only minimal losses. The outbreak was successfully managed with Florfenicol. METHODOLOGY Fish were sampled from tanks with the highest mortality and submitted to the involved diagnostic labs as properly refrigerated fresh samples and specimens fixed in 10% neutral buffered formalin for histopathological evaluation. Histological slides were stained with hematoxylin and eosin, Gram stain, and Giemsa stain. All fresh samples underwent parasitological and bacteriological examination according to standard diagnostic procedures. Additionally, skin samples from affected fish were frozen at -20°C and then subjected to molecular analyses. RESULTS AND DISCUSSION Both parasitological and bacteriological examinations yielded negative results. Histological analysis revealed demarcated areas of ulceration of the body walls at various stages of progression, characterized by a marked lymphohistiocytic infiltrate. Along with mixed extracellular bacterial clusters, mainly located on the surface of ulcerated lesions, numerous Gram-negative, intracellular spheroidal bodies were consistently detected within the affected body walls in all fish and interpreted as intracellular bacteria. However, molecular assays targeting RLOs were negative Outbreaks involving intracellular bacteria with similar clinical features have been previously reported in European sea bass (Dicentrarchus labrax) by Athanassopoulou et al. (1998). Although the histological findings suggested a severe infection by intracellular bacteria morphologically consistent with RLO, the lack of molecular confirmation indicate that the causative agent of the disease may belong to a different taxonomic group. Further investigations are required to identify these enigmatic microorganisms with the goal of implementing effective prevention and control strategies to reduce the risk of future outbreaks. Athanassopoulou et al., 1999: First incidence of Rickettsia-like infections in cultured sea bass (D. labrax). In: Proceedings 9th International EAFP Conference, Rhodes, Greece.