Author: marina02

Susceptibility of different genetic lines of rainbow trout (Oncorhynchus mykiss) to infection with Flavobacterium psychrophilum

Introduction: Rainbow trout fry syndrome (RTFS) caused by Flavobacterium psychrophilum is one of the most serious diseases affecting farmed rainbow trout. Breeding of resistant fish strains is considered a promising strategy to reduce economical losses caused by the disease. This work was aimed at testing resistance of three genetically distinct lines of rainbow trout against experimental infection with F. psychrophilum.

Methodology: Three lines of juvenile rainbow trout (0.40 ± 0.13 g) were used for the experiment. Lines 2 and 3 were marketed as containing QTL associated with resistance to RFTSand resistance to RTFS/white spot disease (ichthyophthiriasis) respectively. Line 1 from the same supplier was without QTL associated with resistance traits. Fish were divided into six experimental groups. After acclimatization, fish were infected with two different strains of Flavobacterium psychrophilum by 2h bath in bacterial suspension (2.10⁷ CFU/ml). Three groups (lines 1, 2 and 3) were infected with the strain 560/7 and three groups (lines 1, 2 and 3) with the strain 6739/7. Mortality was monitored throughout the experiment and expressed as cumulative mortality rate. All fish that died during the experiment were examined pathoanatomically and cultivation from spleen, kidney and brain was performed. Experiment was finished four weeks after the experimental infection. The experiment was repeated twice and each experiment was performed in duplicate.

Results: Mortality started five days after the experimental infection. Massive systemic infection with F. psychrophilum was detected in all dead fish. Mortality varied considerably depending on the F. psychrophilum strain used. In fish infected with strain 560/7, cumulative mortality rate was more than three times higher than in fish infected with strain 6739/7. Surprisingly, highest mortality was found in line 2; line 3 appeared to be more resistant. Line 1 without QTL associated with resistance showed the lowest mortality.

Conclusions:Infectious disease outbreak and outcome is conditioned by multiple factors, the pathogen being only one of them. Enhanced resistance to one pathogen may come at the cost of reduced resistance to other factors which can contribute to disease development. Moreover, fish can show significant differences in resistance to various bacterial strains, even of the same species.

Acknowledgments: This work was supported by the Ministry of Agriculture of the Czech Republic, project No QK QK 21010030

1. PAPEŽÍKOVÁ, IVANA, 1University of Veterinary Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Department of Ecology & Diseases of Zoo , Presenter
2. Vaibarová, Věra, 1University of Veterinary Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Department of Ecology & Diseases of Zoo , Author
3. Pojezdal, Ľubomír, 3Veterinary Research Institute Brno, Department of Infectious Diseases and Preventive Medicine, Hudcova 296/70, 621 00 Brno, Cze, Author
4. Matějíčková, Kateřina, 3Veterinary Research Institute Brno, Department of Infectious Diseases and Preventive Medicine, Hudcova 296/70, 621 00 Brno, Cze, Author
5. Mikulíková, Ivana, 1University of Veterinary Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Department of Ecology & Diseases of Zoo , Author
6. Palíková, Miroslava, 1University of Veterinary Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Department of Ecology & Diseases of Zoo , Author
7. Toulová, Ivona, 1University of Veterinary Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Department of Ecology & Diseases of Zoo , Author
8. Lepková, Zuzana, 1University of Veterinary Sciences Brno, Faculty of Veterinary Hygiene and Ecology, Department of Ecology & Diseases of Zoo , Author

Experimental infection with Edwardsiella tarda in African catfish (Clarias gariepinus)

Introduction: Edwardsiella tarda is a facultative anaerobic, intracellular, gram-negative, motile bacterium, that affects a wide range of fish species, including African catfish (Clarias gariepinus), a species that has become perspective aquaculture species thanks to rapid growth and tolerance to environmental extremes. Outbreaks of edwardsiellosis can cause significant economic losses in aquaculture. The aim of the present study is to establish a challenge model of experimental infection with E. tarda in C. gariepinus for further research into pathogenesis and immune response.

Methodology: The experiment was conducted in three phases. The first phase aimed to test the feasibility of infection via two different routes: intraperitoneal injection and immersion bath. It included five groups, each of ten fish, which were exposed to E. tarda either by intraperitoneal injection (10⁴ and 10⁵ CFU/mL) or immersion bath (10⁶, 10⁷, and 10⁸ CFU/mL). During the experiment, fish behaviour, disease symptoms, and mortality were monitored. In the dead fish, head kidney cultivation was performed. The second phase was to confirm the successful infection of C. gariepinus with E. tarda using the immersion bath method and focused on optimizing the bacterial concentration for the immersion challenge. Three groups, each of ten fish, were exposed to immersion bath with concentrations of 10⁶, 10⁷, and 10⁸ CFU/mL. During the experiment, fish behaviour, disease symptoms and mortality were monitored. Due to zero mortality and the disappearance of disease symptoms, three fish were sampled for bacterial cultivation from the head kidney. The third phase included two different immersion concentrations (10⁸ and 2×10⁸ CFU/mL)and two different immersion durations (1 and 2 hours). This phase included five experimental groups with 15 fish per tank. The experiment was performed in duplicate. Fish were monitored daily, and on day 10 post-infection, four fish from each tank were sampled. Hematological and immunological parameters were analysed from blood samples, and necropsy was performed to assess gross pathological changes.

Results and conclusions: The results confirmed that E. tarda infection could be induced via immersion exposure at high doses (10⁸ and 2×10⁸ CFU/mL). The course of the disease was influenced by both the bacterial dose and duration of the immersion bath. This experiment provides a practical and reproducible method for future studies on host-pathogen interactions, vaccine development, and immune responses in C. gariepinus.

Acknowledgements: This research was funded by the project FVHE/Pikula/2025ITA22 and project NaCeBiVet TN02000017.

1. Toulová, Ivona, University of Veterinary Sciences Brno; Mendel University in Brno, Czech Republic, Presenter
2. Vaibarová, Věra, University of Veterinary Sciences Brno, Czech Republic, Author
3. Papežíková, Ivana, University of Veterinary Sciences Brno; Mendel University in Brno, Czech Republic, Author
4. Novotná, Hana, University of Veterinary Sciences Brno; Mendel University in Brno; Veterinary Research Institute, Czech Republic, Author
5. Mikulíková, Ivana, University of Veterinary Sciences Brno; Mendel University in Brno, Czech Republic, Author
6. Lepková, Zuzana, University of Veterinary Sciences Brno, Brno, Czech Republic, Author
7. Palíková, Miroslava, University of Veterinary Sciences Brno; Mendel University in Brno, Czech Republic, Author

LactoTruite, a project to increase the resilience of trout farms towards the emergence of lactococcosis in Brittany.

Introduction

The aquaculture sector must develop strategies to adapt towards the consequences of global warming, which could lead to emergence or re-emergence of infectious diseases favored by an increase in water temperature. Lactococcosis is a bacterial disease that has been known for years to affect fish farms of the Mediterranean countries, with severe clinical signs at water temperature above 17°C. In 2023, Brittany trout farms were for the first time strongly affected by a major lactococcosis outbreak, generating up to 50% of mortality in some farms and an associated economic loss of several millions of euros.

Aims

The aim of this project is to bring short and mid-terms solutions to the fish farmers to understand and combat the disease, thereby reducing its future impact.

Methodology

The objectives of the first task will be to understand how the disease develops in fish farms, identify bacterial isolates, investigate virulence factors and their antibiotic resistance profiles, and develop reliable diagnostic tools.

The second task aim to optimize the vaccinal and therapeutic (antibiotherapy) protocols. The goal will be to establish vaccinal scheme suitable for each life stage of the trout and to develop therapeutic protocols more effective. Experimental in vivo assays will be developed to conduct vaccination trials as well as optimized antibiotic therapy schemes.

The third task will evaluate the heritability of genetic resistance of rainbow trout against lactococcosis as well as against furonculosis and Infectious Pancreatic Necrosis disease, which are both important trout diseases. Genetic correlations between these three resistance traits and growth traits will be calculated, allowing to estimate survival gains obtainable through trout selection. Additionally we will investigate the immune resistance mechanisms that may be shared across various bacterial infections to which trout are known to be susceptible. This preliminary study will assess the feasibility of conduction genetic selection based on the fish’s immune robustness.

Conclusion

At the end of the project, we will have both a better knowledge of lactococcosis epidemiology – in connection with climate evolution – and several concrete tools to detect or prevent its emergence. The project will help preserving the trout farming sector and maintaining its competitiveness.

The European Maritime and Fisheries Fund (EMFF) of the Brittany Region funds this collaborative project (N°0076650).

1. François, Yoannah, SYSAAF, Presenter
2. Baron, Sandrine, ANSES, Author
3. Tardy, Florence, ANSES, Author
4. Moreau, Emmanuelle, Oniris, Author
5. Oberle, Kenny, Oniris, Author
6. Rostang, Antoine, Oniris, Author
7. Desgranges, Alexandre, Milin Nevez, Author
8. Picchi, Nicolas, Les aquaculteurs bretons, Author
9. Charles, Dominique, Les aquaculteurs bretons, Author
10. Blanchard, Yannick, ANSES, Author
11. Morin, Thierry, ANSES, Author
12. Danion, Morgane, ANSES, Author
13. D’Ambrosio, Jonathan, SYSAAF, Author
14. Besson, Mathieu, SYSAAF, Author
15. Haffray, Pierrick, SYSAAF, Author

Phagocytosis and respiratory burst of Atlantic salmon (Salmo salar) head-kidney leukocytes in response to Renibacterium salmoninarum exposure

Introduction: In recent years there has been an increase in reports of Bacterial Kidney Disease (BKD) in the Norwegian salmon farming industry, a disease caused by infection by the facultatively intracellular bacterium Renibacterium salmoninarum. BKD results in reduced fish welfare and economic losses and presents a challenging problem for the industry due to a lack of effective treatment options. Further, the development of prophylactic measures is hampered by a lack of knowledge concerning the bacterium and pathogenesis. Here we present initial results of how R. salmoninarum affect phagocytosis and respiratory burst in Atlantic salmon (Salmo salar) after in vitro exposure.
Methodology: Phagocytosis was assessed in in vitro exposed (live R. salmoninarum or formalin-inactivated R. salmoninarum) head-kidney leukocytes by flow cytometry. Respiratory burst was similarly assessed by exposure of salmon leukocytes to live or formalin-inactivated bacteria, and activity was quantified by measuring conversion of di-hydro rhodamine (DHR) to fluorescent rhodamine (RHO) by flow cytometry.
Results: A significant increase in respiratory burst activity was observed in leukocytes exposed to live R. salmoninarum in comparison to unstimulated controls and leukocytes exposed to inactivated bacteria. No significant difference in phagocytic activity was measured in response to exposure of either live or inactivated R. salmoninarum.
Conclusions: BKD is an increasing problem in the aquaculture industry of Norway and other parts of the world, but the lack of treatment options leaves the industry poorly equipped to respond to outbreaks. Investigations into host-pathogen interactions will form the basis for understanding pathogenesis and the future development of prophylactic measures.

1. TOLLAKSVIK, THOMAS, UNIVERSITY OF BERGEN, Presenter
2. Myklebust, Maren, UNIVERSITY OF BERGEN, Author
3. Risnes, Ingrid, UNIVERSITY OF BERGEN, Author
4. Haugland, Gyri, UNIVERSITY OF BERGEN, Author
5. Rønneseth, Anita, UNIVERSITY OF BERGEN, Author

Disinfection efficacy of Chlorhexidine Gluconate Against Pathogenic Bacteria Infecting Salmonids

HW Kim*, ES Lee, JY Yoon, SR Kwon

Sunmoon University, Asan, South Korea

Introduction

The recent introduction of Atlantic salmon (Salmo salar) aquaculture in South Korea underscores an urgent need for effective bacterial disease control strategies. Such diseases persist as significant and costly problems in aquaculture, exacerbated by antibiotic overuse and the resulting emergence of resistant strains. To reduce reliance on conventional antibiotics, we investigated the efficacy and safety of chlorhexidine gluconate as an alternative disinfectant for Atlantic salmon. This broad-spectrum, non-antibiotic disinfectant has a lower risk of resistance development, making it a promising candidate for controlling bacterial pathogens.

Methodology

Atlantic salmon (mean length 17.6 ± 1.3 cm) were exposed to chlorhexidine gluconate at 4, 8, and 16 ppm for 3 days to evaluate toxicity. To assess bactericidal efficacy, five bacterial species commonly isolated from domestic aquaculture were tested. Each bacterium was suspended in sterilized seawater to a turbidity of 0.5 McFarland, and 25 μL of this suspension was added to seawater containing chlorhexidine gluconate (1 to 32 ppm) or to an untreated control. After 24 h of shaking incubation at 27 °C, colony-forming units (CFUs) were counted.

Results

No mortality was observed in Atlantic salmon exposed to 4, 8, or 16 ppm chlorhexidine gluconate, indicating that these concentrations are safe for Atlantic salmon. At 4 ppm, Streptococcus iniae, Vibrio harveyi, and Vibrio scophthalmi were completely eliminated. At this concentration, Aeromonas salmonicida and Edwardsiella piscicida were reduced by 83.3% and 97.4%, respectively. At 8 ppm, all species were eliminated except A. salmonicida, which was reduced by 94.7%. At 16 ppm, all species were eliminated except A. salmonicida, which was reduced by 99.1%.

Conclusion

Chlorhexidine gluconate effectively eliminated or significantly reduced the tested aquaculture pathogens at concentrations that were non-toxic to Atlantic salmon. These findings indicate that adding chlorhexidine gluconate to rearing water could provide a simple, practical method for bacterial control in Atlantic salmon aquaculture in South Korea.

1. Kim, Hyunwoo, Sunmoon University, Presenter
2. Lee, Eun Sup, Sunmoon University, Author
3. Park, Ji-Yoon, Sunmoon University, Author
4. Kwon, Se Ryun, Sunmoon University, Author

Evaluation of Imoviral supplement effects on acute-phase response in Sparus aurata after Vibrio anguillarum infection

Introduction

Research on food additives to boost immune responses in marine organisms is crucial for aquaculture. This study evaluated the efficacy of Imoviral® complex in Sparus aurata, a dietary supplement with exclusively natural extracts such as uncaria (Uncaria tomentosa), shiitake (Lentinula edodes), beta-glucan and blackcurrant (Ribes nigrum), whose immunostimulant and analgesic properties have been demonstrated.

Methodology

An acute phase response (APR) was experimentally induced in Sparus aurata by intraperitoneal (IP) injection of Vibrio anguillarum. One hundred fish (12.96 ± 0.96g) obtained from a fish farm were randomly assigned to 5 experimental groups (in duplicate). After the feeding period, an experimental IP infection with V. anguillarum was performed and the APR was evaluated at different time points i.e., 1, 24, 72 and 168 hours post infection (hpi). The IP infection has been performed with a virulent suspension of V. anguillarum or PBS (control). RNA samples have been collected from spleen tissues at different time points. The immune response was analysed by examining the gene expression of pro-inflammatory cytokines (IL-1β, TNF-α) antimicrobial peptides (defensin, hepcidin), antioxidant enzymes (CAT, SOD, GST, Cu-Zn-SOD), anti-inflammatory cytokines TGF-β and Interleukin-10. Experimental procedures have been performed at Centre for Experimental Fish Pathology, University of Messina, accredited for use of aquatic organisms for experimental purposes (DM n.39/ March/2006).

Results

The results showed a significant increase in the expression of TNF-α, IL-1β and antimicrobial peptides in the early stages of infection in fish fed with Imoviral®, suggesting an enhanced immune response compared to control groups. Fish fed with Imoviral® showed higher gene expression of TNF-α and il-10 in the first 72 hpi. In contrast, in the control groups, the expression of these genes was less pronounced. Antimicrobial peptide gene expression analysis revealed a marked modulation of hepcidin and defensin, with significantly higher levels in fish fed commercial diets than those fed Imoviral®. Fish infected with V. anguillarum and fed commercial diets showed upregulated expression of antioxidant genes, which varied according to time and group. TgFb showed temporal variations within groups. The study found no significant differences in weight gain or growth rates between groups, indicating Imoviral® supplementation did not harm fish development.

Conclusion

In conclusion, Imoviral? showed to enhance the immune response of S. aurata to V. anguillarum infection, suggesting its potential to boost disease resistance in aquaculture without affecting growth performances. These findings warrant further research into the underlying immune mechanisms in marine species.

1. CAPPARUCCI, FABIANO, UNIVERSITY OF MESSINA, Presenter
2. Natale, Sabrina, UNIVERSITY OF MESSINA, Author
3. Savoca, Serena, UNIVERSITY OF MESSINA, Author
4. Bragason, Birkir Thor, Institute for Experimental Pathology, University of Iceland, Author
5. Giannetto, Alessia, UNIVERSITY OF MESSINA, Author
6. Marino, Fabio, UNIVERSITY OF MESSINA, Author
7. Iaria, Carmelo, UNIVERSITY OF MESSINA, Author

Turkish Yerisina ruckeri isolate characterisation by MLVA genotyping and comparative genomics

Introduction

In the last decade, the incidence of Enteric red mouth (ERM) disease has increased in Turkish farmed rainbow trout despite vaccination and other disease control measures. Genotyping and genomic characterisation of Yersinia ruckeri isolates are thus important in order to understand the epidemiology and as a basis for developing improved vaccines against problematic local strains of the bacterium.

Methodology

Herein, we use the MLVA method to genotype 35 Turkish isolates. It is a high-tech variant of the variable number tandem repeat (VNTR) analysis which utilizes multiplex-PCR with several fluorescence labelled probes in combination with subsequent capillary electrophoresis analysis of PCR-amplicons on a sequencing platform. Minimum spanning trees (MST) were generated with GeneMapper 5 whereas cluster analysis was performed in BioNumerics. On the basis of MLVA-results, three isolates were selected for full genome sequencing on both the Illumina and Pacbio platforms. Genome assembly was done with Spades and draft genomes were thereafter analysed using various bioinformatics and public resources including the virulence factor database (VFDB).

Results and conclusions

Three older Turkish Y. ruckeri isolates and more recent isolates from four different rainbow trout farms were motile and thus biotype 1. Moreover, these isolates clustered within MLVA clonal complex 2, a serotype O1 lineage associated with rainbow trout farming internationally. They belonged, more specifically, to clonal complex 2C previously found to dominate in continental Europe. Some degree of farm-specific clustering was observed, with one farm displaying a higher degree of diversity. Preliminary results from the analysis of one sequenced genome (isolate YR12) revealed the presence of previously reported virulence genes like the inverse autotransporter invasins YrInv and YrIlm, several hemolysin and enterobactin-related genes as well as genes encoding bacterial type II, III and IV secretion systems.

1. KARATAŞ STEINUM, SÜHEYLA, İSTANBUL UNIVERSITY, Presenter
2. TURGAY, EMRE, İSTANBUL UNIVERSITY, Author
3. GULLA, SNORRE, NORWEGIAN VETERINARY INSTITUTE, Author
4. YARDIMCI, REMZİYE EDA, İSTANBUL UNIVERSITY, Author
5. COLQUHOUN, DUNCAN, NORWEGIAN VETERINARY INSTITUTE, Author
6. STEINUM, TERJE MARKEN, İSTANBUL UNIVERSITY, Author

PHYLOGENETIC AND GENOMIC ANALYSIS OF Photobacterium damselae subsp. piscicida REVEALS GEOGRAPHICAL DIVERSITY BASED ON VIRULENCE PLASMIDS

Photobacterium damselae subsp. piscicida (Pdp) is the causative agent of photobacteriosis, an endemic disease in Mediterranean aquaculture with special relevance in sea bass and sea bream farms. This work focuses on the phylogenetic and genomic analysis of Pdp strains isolated from sea bass and sea bream in the North Atlantic and Mediterranean areas in different spatio-temporal contexts, and its comparison with selected complete genomes of Pdp strains from Australia and Japan.

The comparative analysis revealed a synteny of >99% in the overall genome (chromosomes and plasmids), reflecting a high genomic conservation despite the notable presence of mobile elements in this subspecies. This characteristic is mainly due to the adaptation of these strains to a pathogenic lifestyle that is highly dependent on the host. The phylogenetic analysis showed the existence of two large clades differentiated mainly by the geographical origin of the isolates. This makes it easier to distinguish between strains isolated in Europe (North Atlantic and Mediterranean), and those from Oceania and the Pacific.

The divergences were found mainly in the virulence plasmids described in this pathogen. In plasmids pPHDP10 and pPHDP70, encoding the pore-forming toxin Aip56 and the siderophore piscibactin respectively, sequence differences were attributed to the presence/absence of insertion sequences (IS). The complexity of these differences increased markedly in pPHDPT3 plasmid. This is a newly described virulence plasmid characterized by the presence of genes encoding the type III secretion system (T3SS) and a lysozyme inhibitor. Noteworthy, this plasmid exhibited a high unstability under in vitro conditions, being easily cured after a few passages in the laboratory.

Although the high unstability of pPHDPT3 has caused its absence in most of the available genomes, the comparative genomic analysis of the available sequences allowed us to distinguish a hypervariable central region of pPHDPT3 that contrasts with the general synteny of the two chromosomes. A higher number of pPHDPT3 sequences will clarify inter-strain differences and facilitate the development of a molecular typing scheme to guide strain selection for development of control measures.

1. Vila-Fajardo, Pablo, Aquatic One Health Research Center (ARCUS), Universidade de Santiago de Compostela, Spain, Author
2. V. Barca, Alba, Aquatic One Health Research Center (ARCUS), Universidade de Santiago de Compostela, Spain, Presenter
3. Fraga-Pampín, Soraya, Aquatic One Health Research Center (ARCUS), Universidade de Santiago de Compostela, Spain, Author
4. Christofilogiannis, Panos, AQUATRECK ANIMAL HEALTH S.L A Relva s/n. Torneiros, 36410 O Porriño. Pontevedra (Spain), Author
5. R. Osorio, Carlos, Aquatic One Health Research Center (ARCUS), Universidade de Santiago de Compostela, Spain, Author

Increase in bacterial gastroenteritis cases in salmonids at Finnish fish farms

Introduction: Rainbow trout gastroenteritis (RTGE) was diagnosed in Finland for the first time in 2010, and edwardsiellosis from farmed whitefish in 2000. In recent years, intestinal infections caused by these pathogens have been increasing in Finland. The climate in Finland has experienced a significant warming trend, and climate change will increase the frequency of prolonged heatwaves, leading to higher water temperatures. This rise in temperature can have significant effects on aquatic ecosystems by creating optimal growth conditions for bacteria such as Edwardsiella spp., while also increasing the susceptibility of salmonids to bacterial diseases.

Methods: In this study, we recorded frequency of edwardsiellosis and RTGE outbreaks in Finnish fish farms from samples examined by the Finnish Food Authority (FFA). Studied samples were obtained between years 2015 and 2024. Studied fish were examined by bacterial cultivation from spleen and kidney and selectively from skin lesions. Suspected colonies of Edwardsiella sp. in the primary bacterial cultures on blood agar were re-cultivated and identified earlier using biochemical tests, and more often since 2020 using MALDI-TOF MS for genus identification, and since 2014 enterobacterial repetitive intergenic consensus (ERIC)-PCR method for Edwardsiella species identification. Within routine pathological examinations, when gastroenteritis was suspected macroscopically, samples were taken from the intestines of rainbow trout and examined microscopically or by in-house PCR to diagnose the RTGE. Water temperature, co-infections and fish farming system were analyzed as possible factors to increased gastrointestinal cases in salmonids.

Results: The frequency of RTGE outbreaks have varied over the years, with cases appearing intermittently. RTGE infections were detected in six fish farms in 2022, nine in 2023, and five in 2024. In Finland, the fish species in which edwardsiellosis has been detected are rainbow trout (Oncorhynchus mykiss) and whitefish (Coregonus lavaretus). Before 2023, edwardsiellosis was sporadically detected in fish samples. However, in both 2023 and 2024, cases were confirmed in six fish farms during warm water periods (14°C–24.5°C), and mostly in Eastern Finland. Detailed analysis of the regional distribution of edwardsiellosis and RTGE outbreaks in Finland, and other disease-associated factors will be further discussed.

Conclusions: Intestinal bacterial infections in fish, including edwardsiellosis and RTGE, have been increased in Finnish fish farms in recent years. Warmer summers and water temperatures may have contributed to this rise. Additionally, advancements in diagnostic methods from biochemical tests to MALDI-TOF MS and PCR have likely improved the sensitivity and accuracy of disease detection.

1. Enbom, Tuulia, Finnish Food Authority, Presenter
2. Eriksson-Kallio, Anna Maria, Finnish Food Authority, Author
3. Rask, Marjukka, Finnish Food Authority, Author
4. Korkea-aho, Tiina, Finnish Food Authority, Author

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

1. BROCCA, GINEVRA, Aquatic Diagnostic Services, Atlantic Veterinary College, University of Prince Edward Island, Canada, Author
2. QUAGLIO, FRANCESCO, Department of Comparative Biomedicine and Nutrition, University of Padova, Italy, Author
3. CAFFARA, MONICA, Department of Veterinary Medical Sciences,University of Bologna, Italy, Author
4. BIGNAMI, GIORGIA, Department of Veterinary Medical Sciences,University of Bologna, Italy, Author
5. PIROLLO, TERESA, Department of Veterinary Medical Sciences,University of Bologna, Italy, Author
6. GUSTINELLI, ANDREA, Department of Veterinary Medical Sciences,University of Bologna, Italy, Presenter