Bacterial Granulomatous Diseases in Teleost Fish from Sardinian Aquaculture and Innovative Strategies for Mycobacteriosis Prevention and Vaccination

Bacterial granulomatous diseases pose a significant challenge to fish health, particularly in aquaculture systems, where environmental conditions and pathogen exposure contribute to disease outbreaks. Through a multidisciplinary approach that combined histopathological, microbiological, and molecular tools, the study investigated the prevalence and etiology of granulomatous diseases affecting most farmed fish species in Sardinia, including Argyrosomus regius (meagre), Sparus aurata (gilthead seabream), Dicentrarchus labrax (European seabass), and Mugilidae (mullets). Particular attention was given to non-tuberculous mycobacteria (NTM), also referred to as atypical or environmental mycobacteria, which cause a granulomatous chronic disease in fish (Mycobacteriosis) that is also an important zoonosis. Histopathological analysis revealed a high prevalence of granulomas, with meagre being the most affected fish species (94%), followed by mullets (93%), seabream (42%), and seabass (30%). Granulomas were associated mostly with parasitic infection, especially in mullets (91%), and with Mycobacterium chelonae only in a few cases in meagre (13%) and in mullets (5%). No bacteria were associated to granulomas in seabream and seabass. In meagre, the presence of granulomas was almost due to Systemic Granulomatosis (SG), a chronic disorder of unknown origin that shares histological features with fish mycobacteriosis, although nutritional/metabolic etiology is favored. An additional study was conducted to validate commercial antibodies including anti-pancytokeratin, anti-GFAP, anti-vimentin, anti-desmin, and anti-S-100. Immunohistochemical and Western blot analyses confirmed the cross-reactivity of anti-panCK (clone AE1/AE3) and anti-GFAP antibodies in Sparus aurata, Dicentrarchus labrax, Argyrosomus regius, and other fish species offering valuable tools for detecting epithelial and glial cells in skin and brain tissues. Furthermore, a key aspect of this research was the development of preventive strategies and vaccination against mycobacterial infections in fish. A first study assessed the susceptibility of mycobacterial biofilms to commonly used aquaculture disinfectants. Using the Minimal Biofilm Eradication Concentration (MBEC) assay® system, biofilms of Mycobacterium chelonae, M. salmoniphilum, M. arcueilense, and M. marinum were evaluated and subsequently challenged with disinfectants, highlighting significant species-specific variations in biofilm resistance. The second study assessed the potential of a mutant M. chelonae as a live-attenuated vaccine for fish mycobacteriosis in Carassius auratus (goldfish). Two in vivo trials, including intraperitoneal injection and oral delivery, were conducted to assess the vaccine's immunogenicity and safety, with gene expression analysis targeting interleukin-12 (IL-12) and interferon-gamma (IFN-γ). The vaccine demonstrated promising results against M. marinum, stimulating a robust immune response, and causing no adverse effects in vaccinated fish. Overall, this research contributes to a more comprehensive understanding of bacterial granulomatous diseases in farmed fish and provides practical insights into improved diagnostic approaches, vaccination strategies, and biofilm management. These findings support sustainable aquaculture practices in Sardinia and beyond, offering valuable tools for fish mycobacteriosis prevention and control in the aquaculture industry.