Lactococcus garvieae and Lactococcus petauri are Gram-positive bacteria species causing fish lactococcosis, considered as one of the major threats for rainbow trout (Oncorhynchus mykiss) farming in Europe and worldwide. Therefore, novel analytical methods should be explored to rapidly detect the etiological agent. Droplet digital PCR (ddPCR) is a highly sensitive technique for absolute quantification of nucleic acids. The aim of this study was to develop and validate a ddPCR assay to detect and quantify the presence of L. garvieae/petauri DNA in rainbow trout head kidney, cloacal swabs and in rearing water. L. garvieae/petauri DNA dilutions obtained from pure bacterial cultures and from 89 field samples (tissue, swabs, water) were considered for the ddPCR analysis and parallel quantitative PCR (qPCR) analyses. The detection limit of ddPCR was found to be 1.6 DNA copy number for L. garvieae and 1.7 for L. petauri, showing one order of magnitude higher sensitivity than qPCR. Considering field samples, ddPCR had a 4.49% higher detection capacity for positive cases than qPCR. The ddPCR method validated in this study shows specificity for L. garvieae/petauri, high sensitivity in the detection of low bacterial loads and can be used as surveillance tool both on potentially infected trout and environmental samples, as rearing water or sediment.

The blue crab (Callinectes sapidus) is an invasive alien species in the Mediterranean Sea, posing threats to biodiversity, fisheries, and aquaculture. Climate change has worsened these challenges, influencing the distribution of bacterial species, including Shewanella species, which are sensitive to changes in temperature and salinity. In this study, 300 blue crabs were sampled between June and October 2024 from the Sacca di Goro (Northern Adriatic Sea, Italy) to investigate the prevalence of Shewanella species in their haemolymph. The prevalence was found to be 7% (21/300), with species such as S. mesophila, S. algae, S. cowelliana, and S. baltica identified, particularly in the months of September and October. Molecular techniques, including MALDI-TOF MS and rpoB gene amplification, were used to identify isolates. Antibiotic susceptibility testing (AST) revealed a trend of resistance to beta-lactam antibiotics. A network analysis was also conducted to examine the global trends of Shewanella research in relation to humans, animals, and the marine environment. While proper cooking eliminates the risk to consumers, handling without personal protective equipment can increase exposure, particularly for vulnerable individuals such as those who are elderly or immunocompromised. Mild symptoms are observed in children. Further studies, particularly with a One Health approach, are crucial to better understand the transmission dynamics and evolving antibiotic resistance of Shewanella species.

Lactococcus garvieae has been considered for a long time the only causal agent of lactococcosis. In recent years, different papers reported the involvement of other two bacterial species: Lactococcus petauri and Lactococcus formosensis. A different host tropism has been described for these species where L. garvieae and L. petauri are predominant species in fish and humans’ infections, while L. formosensis in bovine. L. garvieae has been reported as rare infectious agent in dog. This paper represents the first isolation of L. petauri in two domesticated dog cases from urine and skin samples, respectively. The recovered L. petauri has been identified using PCR and sequencing based on Internal Transcribe Spacer (ITS) and phylogenetic analysis showed that it belongs to the L. petauri cluster with a 100% of identity with sequences previously reported from fish isolates while there were differences with L. petauri isolated from urinary tract infection from humans. L. petauri in human infection has been considered not necessarily deriving from the ingestion of contaminated food but rather as an opportunistic pathogen colonization intestinal tract. Differences among virulotypes have been reported for humans and dogs, and a comparison was also made between the virulotyping of L. petauri and L. garvieae in dogs. The antimicrobial pattern showed susceptibility for the election treatment molecules. These data contribute to our understanding of the host trophism of this species which was misclassified for long time and provide new data on its virulence factors and antimicrobial resistance.

Reptiles are usually asymptomatic carriers of Salmonella, with the manifestation of typical clinical signs of acute forms in adult and non-immunocompromised animals being considered exceptions. In the present case, an adult male corn snake (Pantherophis guttatus) was found dead due to septic shock 48 h after consuming a feeder mouse purchased online. The snake’s tissue samples and faeces were cultured for bacteria isolation. Microbiological examinations of the snake and mouse livers revealed the presence of Salmonella enterica subsp. enterica serovar Midway. A whole-genome analysis of these two isolates showed a high correlation between them: they belonged to the strain type ST-357 for the classic MLST scheme and to the strain type ST 171322 for the cgMLST scheme. Also, a virulence gene analysis revealed the presence of stdB and STM3026 genes. This report conveys a case of food-borne salmonellosis in a pet snake, transmitted from a feeder mouse, likely responsible for the snake’s death due to septic shock. It highlights the relevance of feeder mice as a source of Salmonella infections in snakes and the associated risks to human health.

The major histocompatibility complex (MHC) consists of genes involved in immune response and molecular discrimination between self and non-self. MHC genes are the most polymorphic in vertebrates. The origin and maintenance of polymorphism in MHC genes in populations is still unresolved. Mechanisms such as sexual selection and heterozygote advantage have been suggested as explanations for this high variability. In this study, a farmed population of rainbow trout (Oncorhynchus mykiss) characterized by the presence of specific MHC class IIB gene haplotypes at a frequency higher (30%) than that expected from random matings was investigated. Therefore, it was hypothesized that disassortative matings occur with an adaptive advantage for females, resulting in improved reproductive performance when mated with individuals with similar MHC haplotypes. Genetic analyses of the breeders were performed to define the MHC haplotypes and to perform specific matings. The effect of mating was evaluated by analyzing the survival rate of the offspring at various stages of incubation until swim-up. The reproductive performance of the offspring derived from specimens with similar haplotypes showed a better survival trend during the first life stages and reduced malformations. The results obtained are in contrast with the heterozygous advantage theory, therefore it was hypothesized, as for other salmonid species, the presence of a positive selection towards locally adapted MHC genes that promotes reproduction between genetically similar individuals.

Conservation genetic research is essential for the management and recovery of endangered taxa. However, the invasive collection of biological material for DNA analysis is controversial. From an ethical perspective, non-destructive sampling methods leave the aquatic specimen alive and less invasive procedures minimize stress on the animals. DNA can be obtained from fish using minimally invasive techniques such as buccal swabs. Here we evaluated the performance of buccal swabs for long-term storage of DNA obtained from brown trout (Salmo trutta). The buccal swabs were stored at room temperature and cut into pieces, one part of which was used for extraction of an aliquot and the others were stored as a “biobank” of biological material. The elapsed time from sampling to molecular analysis was one and half year. The amplification of three different DNA targets was tested to assess the effectiveness of the extraction: mitochondrial DNA (the D-LOOP region), nuclear DNA (the LDH gene) and microsatellite DNA at multiple loci. The results showed high quantification (mean value: 281.84±72.4 ng/µL), indicating that DNA could be effectively extracted from the buccal swabs. Our study results suggest that buccal swabs for long-term storage of DNA at room temperature are promising for use in field conservation studies.

Parthenogenesis, or virgin birth, describes a mode of reproduction where an egg develops into an offspring without fertilization, and is observed across various vertebrate taxa, excluding mammals. Obligate parthenogenesis, found in around 100 vertebrate species and 1000 invertebrate species, is relatively rare. Conversely, facultative parthenogenesis, where females can reproduce both sexually and parthenogenetically, is observed in some vertebrates, including elasmobranchs. Notably, this phenomenon in elasmobranchs is mainly documented in captivity, allowing for detailed long-term observation. Specifically, this study reports the first case of facultative parthenogenesis in the common smooth-hound shark Mustelus mustelus, a species classified by IUCN as endangered. Here we show that the juvenile M. mustelus were born through parthenogenesis, exhibiting homozygosity at each genetic marker, consistent with terminal fusion automixis. Remarkably, this finding reveals that parthenogenesis can occur annually in these sharks, alternating between two females, and conclusively excludes long-term sperm storage as a cause. Consequently, this enhances our understanding of parthenogenesis in elasmobranchs and highlights the reproductive flexibility of M. mustelus. Overall, these results contribute to our broader understanding of reproductive strategies in elasmobranchs, which could inform conservation efforts for endangered species.

Lactococcus petauri is a recently described species of the genus Lactococcus. It was reported as an etiological agent of piscine lactococcosis together with Lactococcus garvieae. L. garvieae was already described as an opportunistic pathogen in human infections, with a potential zoonotic role. This paper represents the first report of a human urinary tract infection caused by L. petauri. A 91-year-old man was admitted to the emergency department for a femur fracture consequent to a domestic accident. The fracture was reduced by surgery and a catheterized specimen urine culture revealed a high bacterial load sustained by Gram-positive cocci, identified by Vitek 2 compact as L. garvieae, and subsequently as L. petauri through Internal Transcribed spacer 16S-23S r-RNA amplification. The number of L. petauri infections in humans is expected to rise in the near future mainly due to diagnostic improvement. A dedicated survey on L. garvieae and L. petauri infections in humans should be performed to better understand their role as pathogens and as zoonotic agents.

Italian rainbow trout (Oncorhynchus mykiss) farms are periodically affected by outbreaks of lactococcosis caused by Lactococcus garvieae, and rearing basins reveal simultaneously asymptomatic and symptomatic fish. The present study, as part of an "in field" sanitary survey, was designed to describe the expression of immune related genes in infected versus healthy fish, as well as to explore a Near Infrared Spectroscopy (NIR) as a novel approach to discriminate between them. Measurements/sampling were performed in a farm of Northern Italy and included two groups of fish (150-300 g), sharing the same basin: asymptomatic (n = 10) and symptomatic (n = Fingerling (n = 5, 15 g) were also sampled from the farm internal hatchery and considered as L. garvieae-free reference group. NIR spectra were collected from the abdominal region of the fish through the SCiO molecular device. Spleen and head kidney were sampled for bacterial detection (PCR). Head kidney was also subjected gene expression analysis (qPCR). Seven out of nine (7/9) symptomatic trout were L.garvieae positive (bacterial DNA) while all the asymptomatic (10/10) and fingerling (2/2) were L. garvieae negative. SCiO scanning provided two different spectral populations, almost coinciding with the two fish groups considered (asymptomatic symptomatic). Among cytokines, IL-1 beta showed significant up-regulation in symptomatic group compared asymptomatic group (P < 0.05) while IL-8 showed very high (P < 0.01) and high significant (P < 0.05) ferences in its up-regulation in symptomatic group compared to asymptomatic group and fingerling, respectively. IL-10 showed a tendency of significant over-expressed in symptomatic fish compared to fingerling (P < 0.1). TCR-beta showed significant (P < 0.05) down-regulation in symptomatic fish compared to asymptomatic ones and MHC-II resulted in down-regulation in a very significant (P < 0.01) manner in symptomatic trout compared fingerling and asymptomatic. Concerning immunoglobulin genes expression, the level of mRNA transcript of was significantly higher in symptomatic trout compared to fingerling (P < 0.05) and tended to be higher asymptomatic trout compared to fingerling (P < 0.1). No significant differences were observed for IL-6, TNF-alpha, TLR5, MHC-I and IgM. In this study, a NIR based approach was used for the first time in the field of fish thology/immunology with the aim of differentiating between healthy and diseased fish. Moreover, our results immune related genes modulation highlighted how the outcome of a L. garvieae infection might be influenced a specific gene expression pattern, which is crucial in the mode of action of trout immune system against garvieae.

Food supplements are a category of products perceived safe and therefore commonly used by different categories of consumers without any particular attention or precaution. However, health risks associated with the consumption of supplements containing undeclared substances cannot be excluded. A variety of analytical methods are used to control supplement quality composition, but usually, these procedures are complex and time-consuming. Here, we report the results of a simple and fast liquid chromatography-tandem mass spec-trometry (LC-MS/MS) method, to detect and quantify simultaneously different categories of active molecules, such as biogenic ammines and natural alkaloids that at high doses can produce negative health effect in con-sumers. Three categories of products intended for body weight loss, energy boosting, and erectile dysfunction treatment, purchased through e-commerce sites and from local supermarkets, were analyzed (n = 91). The caffeine, synephrine, agmatine sulfate, yohimbine, phenethylamine, and icariin were correctly separated and identified with good precision (RSD < 20%) and recovery (89-109%). The identification and quantification of the analytes in real samples highlighted that the 26% of the samples were not compliant with labeling, confirming that frauds are very common also in the natural supplements market. This LC-MS/MS method could be easily used to test natural supplements in order to check the correct labeling and to protect consumers from potential health risks and food frauds.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

Lactococcosis, mainly sustained by the warm-water bacterium Lactococcus garvieae, is a disease associated with evident mortalities and economic lossed. Main disease signs are commonly bilateral exophthalmia, hyperpigmentation in the body, haemorrhages around the eye area, opercula, and mouth region, swollen abdomen, and anal prolapse. An effective monitoring system of fish diseases relies on rapid techniques for the timely management of infection outbreaks. We evaluated the potential of the SCiO sensor, a portable near infrared spectroscope, to assess in field the health status of rainbow trout. The study sample was composed of 20 symptomatic and 20 asymptomatic rainbow trout obtained from an aquaculture farm and submitted to molecular detection of L. garvieae and SCiO examination. In order to detect presence or absence of septicaemia condition, blood smears were prepared from peripheral blood. Part of the symptomatic individuals (9/20) showed septicaemia, whereas no bacteria were found in the asymptomatic individuals. PCR assay of bacterial DNA was positive in spleen and kidney of 19/20 symptomatic and in 1/20 asymptomatic individuals. Bacteriological analysis revealed L. garvieae in 18/20 symptomatic samples and in 2/20 asymptomatic samples. The SCiO sensor was found to be able to discriminate between healthy and sick fish [sensibility 0.95 (0.75–1.00 I.C. 95%), specificity 0.80 (0.56–0.94 I.C. 95%)]. By virtue of rapid acquisition, low cost and use on site, the SCiO was considered as a useful diagnostic tool for monitoring L. garvieae infection in rainbow trout.

European sea bass (Dicentrarchus labrax L.) is one of the most economically important fish species in the Mediterranean Sea area. Despite strict requirements regarding indications of production method (wild/farmed), incorrect labelling of sea bass is a practice still frequently detected. The aim of this study was to evaluate the capabilities of two techniques, Near-InfraRed (NIR) spectroscopy and mass spectrometry, to discriminate sea bass according to the production method. Two categories were discriminated based on the docosahexaenoic and arachidonic fatty acid ratio by using a Direct Sample Analysis (DSA) system integrated with a time-of-flight (TOF) mass spectrometer. The cut-off value of 3.42, of fatty acid ratio, was able to discriminate between the two types of fish with sensitivity and specificity of 100%. It was possible to classify fish production by using multivariate analysis with portable NIR. The results achieved by the developed validation models suggest that this approach is able to distinguish the two product categories with high sensitivity (100%) and specificity (90%). The results obtained from this study highlight the potential application of two easy, fast, and accurate screening methods to detect fraud in commercial sea bass production.

The first case of infection of Streptococcus iniae in Adriatic sturgeon (Acipenser naccarii) was recently reported in a raceway system located in Northern Italy. A second episode of infection in sturgeons with absence of mortality and evident clinical signs, was registered in November 2020 in the same farm and is reported in this study. Histopathological changes observed in infected organs are described. The strains isolated in the two episodes were compared using molecular analysis based on PCR, phylogeny and virulence factors analysis. Not all the major virulence factors were detected for the two strains; in particular the strains 78697, isolated in November, lacks cpsD, compared to the strains 64844, isolated in September. Moreover, genetic variations were reported for lctO and pmg genes. These findings let us hypothesize a different virulence of the strains in accordance with clinical findings related to the sturgeons.

Infectious diseases place an economic burden on aquaculture and a limitation to its growth. An innovative approach to mitigate their impact on production is breeding for disease resistance: selection for domestication, family-based selection, marker-assisted selection, and more recently, genomic selection. Advances in genetics and genomics approaches to the control of infectious diseases are key to increasing aquaculture efficiency, profitability, and sustainability and to reducing its environmental footprint. Interaction and co-evolution between a host and pathogen can, however, turn breeding to boost infectious disease resistance into a potential driver of pathogenic change. Parallel molecular characterization of the pathogen and its virulence and antimicrobial resistance genes is therefore essential to understand pathogen evolution over time in response to host immunity, and to apply appropriate mitigation strategies.

Extra virgin olive oil is the highest quality olive oil mainly due to its beneficial constituents and nutritional properties. However, olive oil adulteration is a common fraudulent practice by deliberate mislabelling of less expensive oil categories and admixing expensive olive oils with low oils. To protect consumers from such commercial frauds, an easy and fast method to detect the real composition of oil is needed. For this study we used direct sampling analysis (DSA) coupled with a high-resolution mass spectrometer (AxION2 TOF Perkin Elmer) to analyse the fatty acid composition of three types of edible oil: extra virgin olive oil, refined olive oil and seed oil (EVOO, ROO, and SO respectively) to find a marker that could distinguish between them. Good precision in repeatability and reproducibility (RSD% < 15%) was obtained. The fatty acid ratio between the oleic acid/oleic acid dimer was able to distinguish EVOO from the other two types of oil, while the ratio between linoleic and oleic acid was found to discriminate refined oil from seed oil. The development of an easy, fast and cost-effective method can help to limit commercial frauds, increase the number of controlled samples, and enhance food control along the commercial chain.

Because the world's wild fish stocks are limited and the market demand is increasing, fish farming has become an alternative food source and a way to reduce costs for consumers. The sale of farmed as wild fish is a fraudulent practice; it is, therefore, important to find new and alternative tools that can help in the fight against fraud to protect consumers and to ensure food traceability. The proteomic profiles of farmed and wild fish differ. With this study we wanted to identify liver protein markers via two-dimensional electrophoresis that would allow us to distinguish wild from farmed gilthead seabream. The liver samples from 32 gilthead seabream, wild and farmed, were stored at -80 degrees C before protein extraction. The samples were subjected to two-dimensional electrophoresis to detect qualitative and quantitative differences. Proteomic analysis showed a protein spot (molecular weight of similar to 34 kDa and isoelectric point of similar to 6.9) only in the samples from the wild gilthead seabream; liquid chromatography-tandem mass spectrometry identified the spot as ubiquitin. Ubiquitin could be a valid marker to differentiate wild from farmed gilthead seabream; it could be used to ensure continuous monitoring throughout the entire commercial chain and to fight commercial fraud.

Background: Fiore Sardo PDO is one of the oldest Mediterranean hard cheeses, exclusively produced in Sardinia (Italy) from raw whole ewe’s milk. Some manufacturers, improperly, submit raw milk to heat-treatment. Aim of this study was to evaluate the proteomic profile of Fiore Sardo PDO and to investigate possible differences between cheese made from raw milk and from heat-treated milk. Materials and methods: Starting from the same ovine bulk milk, eight cheese wheels of Fiore Sardo PDO were produced, four from raw milk and four from heat-treated milk. The subsequent production steps were the same for both types of cheese as was sampling at different ripening times. Samples were analyzed by Urea-PAGE electrophoresis. Afterwards, blind analysis of 32 cheese samples, produced by 17 different cheesemakers was performed to evaluate the method’s analytical performance. Results: Urea-PAGE analysis showed the presence of a protein band only in cheese made from raw milk, regardless of cheese ripening time. Protein identification analysis by liquid chromatography mass spectrometry identified two different proteins in the band, alpha-S1 and alpha-S2 casein. Blind analyses conducted to verify the analytical performance of the method showed that it could be a useful tool for the protection of this typical agri-food product. Conclusion: We developed a simple, robust, and economic method for discriminating between Fiore Sardo PDO cheese made from raw milk with up to 24 months of maturation and Sardinian sheep milk cheese made from heattreated milk.