Charalampos Kotzamanidis

Lactococcosis is a bacterial warm water disease whose occurrence is enhanced by climate change. It induces chronic stress and immunodepression in trout, thereby enhancing host susceptibility to bacterial pathogens. Lesions are typically systemic, with involvement of hematopoietic organs and of central nervous system. Clinical signs include lethargy and early anorexia, abnormal swimming behaviour, exophthalmia, cutaneous, visceral and ocular haemorrhages, ascites and splenomegaly. As conventional preventive measures are becoming less effective, complementary, long‑term strategies aimed at improving host resistance. In this context, selective breeding of broodstock for disease resistance is regarded as an effective adaptive measure to mitigate climate related health risks in aquaculture. Two complementary approaches were used to identify genetic markers associated with lactococcosis. The first was a candidate gene approach based on the MHC class II gene on 739 rainbow trout reared in three Mediterranean countries: Italy, Greece and Türkiye. Different single nucleotide polymorphisms of the MHC class II gene were found to have a protective role. Nevertheless, the most interesting results came from the haplotype-level analysis of the gene. Haplotype A126T130A131 emerged as a promising genetic marker associated with resistance in populations affected by both Lactococcus garvieae or Lactococcus petauri as the causative agents of the disease. The second approach, applied to a subset of 271 fish, combined genome-wide case-control association (GWAS) with a detection of selection signatures (SS). The functional analysis of genes located in chromosomal regions harbouring significant signals in the association study revealed several GO terms associated with neuronal development, vision, infection and disease response and platelet development. Ten SNPs were identified in common by GWAS and SS. The incorporation of molecular markers into marker‑assisted or genomic selection programmes may contribute to the development of trout stocks better adapted to future environmental conditions, reducing the impact of lactococcosis and improving animal welfare while decreasing reliance on chemotherapeutic treatments. Further studies aimed at validating these associations across different environmental conditions and genetic backgrounds are essential to support their implementation at a commercial level.

This study presents the first comprehensive molecular characterization of Escherichia coli producing extended-spectrum beta-lactamases (ESBL-Ec) in surface waters in Albania, focusing on the Shkumbini river. Antimicrobial resistance (AMR) in aquatic ecosystems poses a significant threat to public health, yet data from Albania remain scarce. Thirty water samples were collected from six locations near Elbasan between September 2022 and February 2024. Following the WHO Tricycle protocol, 52 ESBL-Ec isolates were recovered and characterized for antimicrobial susceptibility, biofilm formation, resistance genotypes and clonal relatedness via pulsed-field gel electrophoresis (PFGE). ESBL-Ec was detected in 80% of the samples analyzed, with 94.2% of the isolates classified as multidrug-resistant (MDR). High resistance frequencies were observed for ampicillin (98.1%) and cefotaxime (86.5%), while 7.7% of the isolates displayed colistin resistance associated with the mcr-3 gene. The bla_CTX-M-1 genotype was the most prevalent (57.7%), and almost half of the isolates harbored multiple ESBL genes. Phylogroup A (46.2%) predominated, followed by the high-risk extraintestinal lineages B2 (23.1%) and D (11.5%). PFGE revealed high genetic heterogeneity, with 51 distinct pulsotypes indicating multiple sources of contamination, such as untreated municipal, agricultural and industrial waste. Additionally, 55.8% of the isolates were capable of forming biofilms. These results highlight the critical role of the Shkumbini river as a reservoir for highly resistant pathogens and emphasize the urgent need for integrated environmental surveillance and improved wastewater management in Albania.

Snails depend entirely on innate immunity, primarily mediated by hemolymph amoebocytes and hyalinocytes, which are responsible for pathogen recognition, phagocytosis and immune signaling. While amoebocyte functions are well established, the role of hyalinocytes and the origin of hemolymph cells remain unclear. Hyalinocytes proliferate rapidly during pathogenic infections associated with increased mortality and inflammation. This study investigated snail dysbiosis following oral administration and hemocoel injection of the intracellular Listeria monocytogenes SN3 and the extracellular Pseudomonas baetica 7A snail pathogens and the symbiotic effect of the probiotic Lactiplantibacillus plantarum Sgs14. Chemotaxis and necrosis assays revealed that snail pathogens increased amoebocyte and hyalinocyte numbers and induced cell necrosis, whereas the probiotic elicited the so-called “normal” inflammatory response without cell necrosis. Histological analysis showed increased cell accumulation within hepatopancreatic cavities without significant connective tissue damage. Immunohistochemistry indicated elevated expression of the LGR5 receptor homologue in hepatopancreatic cavity cells following pathogenic treatments, while no expression was detected in hemolymph cells. Hemocoel injection of bacteria or hepatopancreatic cell suspensions altered hemolymph cell populations, causing short-term increase in amoebocytes, hyalinocytes, and dead cells. In vitro and in vivo assays demonstrated that SN3-activated hyalinocytes adhered to infected amoebocytes and induced necrosis, highlighting their potential cytotoxic role. Flow cytometry and immunofluorescence revealed upregulation of immune marker homologues (CD16, CD49b, IFN-γ, IL-17A), mainly in hyalinocytes, following pathogenic challenge. These findings identify the hepatopancreas as a primary hemopoietic organ in snails and demonstrate pathogen-specific immune modulation, elucidating the mechanisms underlying snail responses and the functional activation of hyalinocytes as cytotoxic effectors.

This study presents the first comprehensive molecular characterization of Escherichia coli producing extended-spectrum beta-lactamases (ESBL-Ec) in surface waters in Albania, focusing on the Shkumbini river. Antimicrobial resistance (AMR) in aquatic ecosystems poses a significant threat to public health, yet data from Albania remain scarce. Thirty water samples were collected from six locations near Elbasan between September 2022 and February 2024. Following the WHO Tricycle protocol, 52 ESBL-Ec isolates were recovered and characterized for antimicrobial susceptibility, biofilm formation, resistance genotypes and clonal relatedness via pulsed-field gel electrophoresis (PFGE). ESBL-Ec was detected in 80% of the samples analyzed, with 94.2% of the isolates classified as multidrug-resistant (MDR). High resistance frequencies were observed for ampicillin (98.1%) and cefotaxime (86.5%), while 7.7% of the isolates displayed colistin resistance associated with the mcr-3 gene. The bla_CTX-M-1 genotype was the most prevalent (57.7%), and almost half of the isolates harbored multiple ESBL genes. Phylogroup A (46.2%) predominated, followed by the high-risk extraintestinal lineages B2 (23.1%) and D (11.5%). PFGE revealed high genetic heterogeneity, with 51 distinct pulsotypes indicating multiple sources of contamination, such as untreated municipal, agricultural and industrial waste. Additionally, 55.8% of the isolates were capable of forming biofilms. These results highlight the critical role of the Shkumbini river as a reservoir for highly resistant pathogens and emphasize the urgent need for integrated environmental surveillance and improved wastewater management in Albania.

The present study investigated the impact of aestivation of free-range snails on the intestinal microflora and its potential to protect the host from infection by pathogenic bacteria. Bacterial strains were isolated just before and at the end of aestivation from the gut of free-range Cornu aspersum aspersum snails from the island of Crete (Greece). Α total of 31 BA (Before Aestivation) and 25 EA (End Aestivation) isolates, were tested for their in vitro probiotic properties. Principal component analysis (PCA) and a machine learning model showed that BA isolates exhibited a different pattern of in vitro properties from that of EA isolates, and that all the EA isolates, except one, were presumptive probiotics, while among BA only isolates BA2Ae and BA5An were presumptive probiotics. 16s rRNA sequencing analysis at species-level revealed that most BA isolates belong to potentially snail pathogens, while EA isolates belonged to the genera of Citrobacter, Lactiplantibacillus and Enterococcus, that belong to normal intestinal microflora and presumptive probiotics. Injection of selected isolates in snails resulted in increased immune activity such as chemotaxis and phagocytosis. Food administration of the strains, E. malodoratus BA5An and L. plantarum EA2An, exhibiting the highest immunomodulatory activity, showed further probiotic properties since symbiosis achieved by enhancing some immune humoral responses, as well as rebiosis by inhibiting the dysbiosis induced by the pathogenic strain Listeria monocytogenes SN3. Each probiotic strain exhibited a different pattern of immunomodulatory activity against the pathogen.

Background: Multi-epitope vaccines have become the preferred strategy for protection against infectious diseases by integrating multiple MHC-restricted T-cell and B-cell epitopes that elicit both humoral and cellular immune responses against pathogens. Computational methods address various aspects independently, yet their orchestration is technically challenging, as most bioinformatics tools are accessible through heterogeneous interfaces and lack interoperability features. The present work proposes a novel framework for rationalized multi-epitope vaccine design that streamlines end-to-end analyses through an integrated web-based environment. Results: VaccineDesigner is a comprehensive web-based framework that streamlines the design of protective epitope-based vaccines by seamlessly integrating computational methods for B-cell, CTL, and HTL epitope prediction. VaccineDesigner incorporates single-epitope prediction and evaluation as well as additional analyses, such as multi-epitope vaccine generation, estimation of population coverage, molecular mimicry, and proteasome cleavage. The functionalities are transparently integrated into a modular architecture, providing a single access point for rationalized, multi-epitope vaccine generation in a time- and cost-effective manner. Conclusions: VaccineDesigner is a web-based tool that identifies and evaluates candidate B-cell, CTL, and HTL epitopes and constructs a library of multi-epitope vaccines that combine strong immunogenic responses, safety, and broad population coverage. The source code is available under the academic license and freely accessible.

Acinetobacter baumannii has emerged as a major pathogen responsible for healthcare-associated infections, particularly in intensive care units, contributing to significant morbidity and mortality due to its multidrug resistance and ability to persist in clinical environments. This study aimed to investigate the phenotypic and genomic characteristics of all multidrug-resistant A. baumannii isolates collected between January and June 2022 from two tertiary care hospitals in Thessaloniki, Greece. A total of 40 isolates were included. All isolates exhibited resistance to colistin; however, none harbored the mcr-1 to mcr-9 genes, as confirmed by polymerase chain reaction (PCR). PCR-based screening for virulence-associated genes revealed high prevalence rates of basD (100%), pld (95%), csuE (87.5%), and bap (77.5%). In contrast, ompA and pglC were not detected. Twitching motility ranged from 2 to 50 mm, with 25% of the isolates classified as non-motile and 20% as highly motile. Swarming motility was observed in all strains. Additionally, all isolates demonstrated positive α-hemolysis, suggesting a potential virulence mechanism involving tissue damage and iron acquisition. Pulsed-field gel electrophoresis (PFGE) revealed significant genomic diversity among the isolates, indicating a low likelihood of patient-to-patient or clonal transmission within the hospital setting. These findings highlight the complex relationship between antimicrobial resistance and virulence in clinical A. baumannii isolates and emphasize the urgent need for robust infection control strategies and continued microbiological surveillance.

This article documents the first outbreak of aeromoniasis caused by Aeromonas veronii in farmed European seabass (Dicentrarchus labrax) along the Greek Ionian Sea coast. In late spring 2024, commercially sized fish exhibited anorexia, hemorrhages, and ulcers on the skin, accompanied by elevated morbidity and mortality rates. The outbreak spread rapidly across local farms in Sagiada Bay, reaching its peak in late summer, and extending into the Astakos Gulf, southern in the Ionian Sea. The postmortem examination revealed hemorrhages, organomegaly, abscess formation, and granulomatous inflammation. Aeromonas veronii was isolated from all examined individuals in nutrient media and confirmed by biochemical and molecular methods. Whole genome sequencing and phylogenetic analysis demonstrated genetic homogeneity among two strains from two different areas along the Ionian Sea and a close evolutionary relationship with other Aeromonas veronii strains from the Aegean Sea. Although genetically similar, the isolates exhibited differences in phenotypic and biochemical characteristics, indicating regional variability. The present study provides an overview of the pathology, clinical characteristics and progression of aeromoniasis in Ionian Sea aquaculture, highlighting the need for continued monitoring, in-depth genomic and phenotypic assessment, and the design of region-specific preventive strategies, including autogenous vaccines, for effective disease management.

Piscine lactococcosis poses a significant threat to a wide range of cultured and wild fish populations worldwide, typically presenting as acute haemorrhagic septicemia with high morbidity and mortality. Although Lactococcus garvieae was historically considered the sole causative agent of piscine lactococcosis, recent studies have identified L. petauri and L. formosensis as additional, highly pathogenic species. In this study, we developed a novel TaqMan‐based multiplex qPCR assay for the simultaneous detection and differentiation of L. garvieae , L. petauri and L. formosensis , following a pangenome analysis of the publicly available genomes of these bacterial species. The assay demonstrated high sensitivity and specificity across 156 bacterial isolates obtained from various cultured fish species and geographical locations between 2008 and 2024, as well as against a panel of non‐target bacteria. It also successfully detected target pathogens in 146 field tissue samples, including tissues preserved in 70% ethanol, formalin‐fixed paraffin‐embedded (FFPE) tissues and tissues fixed on FTA cards. Compared to classical bacteriology, the multiplex qPCR assay yielded higher detection rates and enabled precise identification of the causative species of piscine lactococcosis. Overall, the multiplex qPCR assay developed in this study provides a reliable, rapid, highly sensitive and species‐specific molecular approach for diagnosing piscine lactococcosis, contributing to better surveillance and management of the disease in aquaculture.

This research evaluated the impact of incorporating dried olive pulp (OP) into the feed of laying hens on the fatty acid profile, cholesterol, triglyceride, total phenolic, oleuropein and hydroxytyrosol content, and health lipid indices of eggs produced by mid- (39 weeks) and late-laying (59 weeks) birds. Over a 36-week trial, 300 eggs from 180 Isa-Brown hens, assigned to three dietary groups with different OP levels (CON, OP4 and OP6), were analyzed. OP reduced egg cholesterol, with significant effects in late-age eggs (p < 0.05). In mid-age hens, the OP6 eggs had higher total phenolics than the controls (p < 0.05) and more PUFAs than the other groups (p < 0.05). The concentration of total phenolics, cholesterol, n3 PUFAs and % fat increased with hen age (p < 0.05), while triglycerides and oleuropein decreased (p < 0.05). With increasing hen age, the SFAs in the OP eggs decreased (p < 0.05) and the MUFAs increased (p < 0.05). Eggs from older hens had higher nutritional value, as indicated by the lower n6/n3 PUFA ratio, lower AI and TI indices, and higher h/H ratio (p < 0.05). Overall, dietary OP supplementation improved the nutritional quality of eggs, suggesting potential health benefits. Our results also highlighted eggs from older hens as a valuable source of high-quality fats.