Infectious salmon anaemia virus (ISAV) can cause disease and mortality in Atlantic salmon (Salmo salar). The virus is endemic in several regions including the Atlantic coast of Canada. Lumpfish (Cyclopterus lumpus) are increasingly utilised as cleaner fish to control sea lice levels in Atlantic salmon farms. With welfare concerns for both species, and the possibility of escaped fish, the objective of this study was to determine the susceptibility of lumpfish to ISAV. Lumpfish were exposed to two ISAV isolates (high and low virulence) through a cohabitation challenge (i.e., exposure to infected salmon) and through an intra-peritoneal challenge. Naïve salmon were exposed simultaneously to the same isolates tested to verify their virulence. In the cohabitation challenge, ISAV infection progressed as expected in the naïve salmon. ISAV was detected in a small number of lumpfish gill samples, but the lack of detection in other organs, e.g., heart, kidney, spleen, liver, and blood, suggests environmental contamination. Although two lumpfish blood samples tested positive in the later stages of the challenge, all other disease indicators, including a lack of anaemia, did not support potential for virus replication in lumpfish. Based on both exposure experiments, lumpfish do not appear to be susceptible to ISAV.
Health monitoring based on post-mortem examination is essential for the management of endangered animal species. This is especially true for reintroduced species living in small populations with low genetic diversity, such as the Eurasian lynx (Lynx lynx) in Switzerland. Thanks to systematic post-mortem examinations, the Institute for Fish and Wildlife Health (FIWI), University of Bern, has acquired a comprehensive view of the lynx health in Switzerland. This study provides an updated overview of the causes of morbidity and mortality in the Eurasian lynx in Switzerland from 2000 to 2022. A total of 346 necropsied lynx (found dead, euthanized, or culled) were included in this study, and a cause of death (COD) was identified in 318 of them (91.9%). Overall, the main COD was blunt trauma (n = 183, 52.9% - largely vehicular collision). Starvation, resulting from the separation of dependent juveniles from their mother, was the second most frequent COD (n = 63, 18.2%). Fatal infectious diseases were relatively low (n = 32, 9.2%). However, we documented some significant pathogens such as canine distemper virus (CDV) and metastrongyloid nematodes. Illegal killing was confirmed in 23 cases (6.6%). Of note, illegal killing is likely underestimated in this study, given that radio-collared lynx were found to be proportionally more often illegally killed than the unmonitored ones found by chance. Furthermore, most individuals were found to be affected at least by one non-specific, mild to moderate inflammatory process of unknown origin, such as interstitial pneumonia (n = 59) or interstitial nephritis (n = 25). Additionally, cardiac changes of variable severity were observed in 125 lynx, and severe soft tissue mineralization was detected in 10 individuals. The frequency of these findings warrants further investigation. Thus, this study confirms the importance of systemic post-mortem examination and general health surveillance of free-ranging Eurasian lynx in Switzerland, in support of translocation projects, conservation of the species, and to provide a better understanding of their pathologies.
Piscirickettsiosis is the most prevalent bacterial disease affecting Chilean aquaculture and responsible for the majority of mortality in salmonids. Currently, large quantities of antibiotics, predominantly florfenicol, are used in the Chilean aquaculture industry, and sub-MIC concentrations of this antibiotic, similar to what occurs in the marine environment, have been shown to induce biofilm formation on both biotic and abiotic surfaces when sub-MIC doses of florfenicol, raising concerns about the emergence of antibiotic-resistant bacterial strains. Thus, the aim of this study was to evaluate whether in vitro sub-MIC concentrations of florfenicol induce the expression of genes associated with biofilm formation and antibiotic resistance in the biofilm-embedded P. salmonis. Interestingly, in vitro analyses showed that sub-MIC dilutions of antibiotic significantly modulated the expression of an efflux pump acrAB and the two-component systems cpxAR, and qseBC, as well as the antibiotic resistance-associated genes tclor/tflor and t.flor in the biofilm-embedded P. salmonis isolates tested. Thus, this study highlights the negative consequences of the extensive use of antibiotics in aquaculture, which can promote biofilm formation in marine bacterial pathogens, potentially facilitating the spread of resistance genes among different bacterial species in the aquatic environment and increasing the risk of reinfection within culture systems.
Cell line misidentification or contamination undermines the reliability of research outcomes. In this study, we verified the species origin of commonly used fish cell lines, with a particular focus on the grass carp ovarian cell line (GCO). RNA-seq data from 146 samples of 11 widely used fish cell lines were collected from public databases (NCBI-SRA) and were mapped to the reference genomes of eight species. GCO RNA-seq samples from three Chinese laboratories showed a mapping rate of 21.64% ± 3.61% to the grass carp genome, but 89.81% ± 4.83% to the fathead minnow genome. Further transcriptomic similarity analysis revealed that these GCO RNA-seq samples clustered more closely with cell lines originating from fathead minnow. Targeted analysis of the COI gene amplification region confirmed that nearly all reads from GCO RNA-seq samples mapped exclusively to the fathead minnow COI amplicon. Collectively, these results indicate that GCO may have undergone cross-contamination or species misidentification. This study raises doubts about the true species origin of the GCO cell line and proposes that COI DNA barcoding can be used to discriminate the species origin of fish cell lines.
Harmful algal blooms (HABs) are a threat to fish welfare, occurring suddenly and unexpectedly causing significant consequences for fish and salmon farmers worldwide. Norwegian farmers have been facing this challenge at irregular intervals since the very beginning of the industry. This report describes the events on the first fish farm affected by the spring HAB of 2025 in northern Norway. Specifically at the production site Fornes, which at the time of the strike had more than 1 million Atlantic salmon (Salmo salar) with an average weight above 3 kg in its sea cages. First signs of the event were reduced fish appetite and cloudy water appearance, followed by changes in fish behaviour and acute mortality of 39.5%. Water samples showed dominance of the phytoplankton species Phaeocystis pouchetii, followed by Chrysochromulina leadbeateri. Necropsy of the fish and histopathological lesions on the gills and liver supported that the mortality was caused by the algae. The farm's contingency plan was enforced immediately when the acute high mortality was observed. Including, but not limited to, feed withdrawal followed by emergency harvest of the whole production site. The dramatic incidence at Fornes highlights the urgent need for monitoring and early-warning systems for HABs, as well as further development of suitable mitigation strategies and contingency plans to minimise the effect of HABs once they have been forecasted or detected.
Hepcidin is a cysteine-rich peptide that functions both as an antimicrobial peptide and a central regulator of iron homeostasis in vertebrates. Structurally, it consists of an N-terminal signal peptide, a propeptide region and a C-terminal bioactive mature peptide. The canonical mature hepcidin contains eight conserved cysteine residues that form four disulfide bonds, stabilising a characteristic β-sheet hairpin-like structure. In the Antarctic toothfish (Dissostichus mawsoni), which inhabits extremely cold marine environments, a unique hepcidin variant was identified possessing only four cysteine residues, designated dm_4cyshep. To evaluate the antimicrobial function of the dm_4cyshep peptide, the minimum inhibitory concentration (MIC) assays and flow cytometry analyses were performed. The peptide demonstrated binding affinity and bactericidal activity against a range of Gram-positive and Gram-negative bacteria, with distinct MIC values observed for different strains. Furthermore, overexpression of dm_4cyshep in a hepcidin-deficient zebrafish model markedly reduced systemic iron accumulation, indicating a conserved role in iron regulation. These results demonstrate that despite the reduced number of cysteine residues, dm_4cyshep retains both antimicrobial and iron-modulatory functions, underscoring its potential as an immune effector molecule. This study enhances the understanding of the structural and functional diversity of fish hepcidins and identifies a promising candidate for improving disease resistance in aquaculture.
Edwardsiellosis, a disease caused by Edwardsiella anguillarum, poses a serious threat to red seabream (Pagrus major) aquaculture. To understand the transmission cycle of E. anguillarum in aquaculture environments, we analysed the release dynamics of the pathogen and fish-derived materials from red seabream experimentally infected via intraperitoneal injection, immersion or cohabitation. Environmental DNA (eDNA) concentrations of E. anguillarum in rearing water increased as infection progressed and showed positive correlations with red seabream eDNA across all infection methods. Size-fractionation demonstrated that although > 50% of E. anguillarum eDNA was in the fraction < 3.0 μm but ≥ 0.4 μm, 30%-40% was also detected in the 3- and 20-μm fractions. These larger fractions contained 80% of red seabream eDNA. Furthermore, viable E. anguillarum and its DNA were detected in external tissues of surviving fish (gill filaments, skin mucus and distal intestine), with no significant differences in quantities between immersion and cohabitation groups. These results collectively suggest that E. anguillarum released from infected red seabream enters the surrounding water together with fish-derived materials, with multiple external tissues contributing to this release, and further suggest that a portion of the bacteria may persist in the environment attached to tissue particles.
Mycobacteriosis is a significant disease in fish husbandry, with negative impacts on production facilities. Moreover, some Mycobacterium spp. that infect fish have zoonotic potential. Morbidity and mortality events at hybrid tilapia (Oreochromis spp.) farms in Trinidad and Tobago (T&T) associated with mycobacterial infections prompted a broader surveillance investigation across the hybrid tilapia farms within the country. Of the 168 specimens examined by polymerase chain reaction (PCR), 42% were Mycobacterium spp. positive. Among the mycobacteria-positive specimens, clinical signs (lethargy and loss of equilibrium) were observed in 96% (68 of 71), of which gross external abnormalities were evident in 7% (5 of 68) and internal lesions (granulomas) in 22% (15 of 68). Specimens that were mycobacteria-negative showed no clinical signs, gross abnormalities or lesions. Sequence analysis of a partial 723-bp rpoB (region V) fragment confirmed the presence of a consortium of uncharacterised Mycobacterium spp. in 18 hybrid tilapia specimens. These findings suggest that potentially novel species are circulating within and causing clinical and subclinical disease in the local farmed hybrid tilapia population in T&T. The study also revealed that over the period 2020-2021, 75% (9 of 12) of the major hybrid tilapia production facilities in T&T were infected with mycobacteria. This represents the first documentation of a piscine mycobacteriosis outbreak in hybrid tilapia in the Caribbean.
Edwardsiella anguillarum causes Edwardsiellosis in fish, resulting in severe internal organ lesions and substantial economic losses in aquaculture. The limited efficacy of antibiotics in treating this disease, coupled with concerns regarding resistance and side effects, has driven interest in exploring fish vaccines. Various vaccine candidates derived from E. anguillarum, including inactivated cells and protein subunits, have shown potential. Here, we developed a vaccine against E. anguillarum in milkfish, and its efficacy was evaluated through vaccination challenge trials using milkfish as the target species. The formalin-killed vaccine (FKC) in combination with an adjuvant exhibited superior efficacy, achieving a relative percent survival (RPS) of 83.4%. Furthermore, the agglutination titers indicated a robust immune response in the FKC + Adjuvant group, suggesting a potent and sustained antibody response. Additionally, serum lysozyme activity was significantly higher in vaccinated fish than in controls. Immune-related gene expression analysis in spleen and head kidney revealed that IL-1β peaked at 14 days post-vaccination, indicating early pro-inflammatory response, while MHC-II showed progressive elevation, suggesting sustained antigen presentation and humoral immunity. Overall, this study underscores the effectiveness of the FKC + Adjuvant combination in enhancing immune response and protecting against bacterial pathogens in aquaculture, presenting a promising strategy for disease control.
The Polydora complex is a globally widespread polychaete parasite known for secreting acidic substances to bore tunnels into oyster shells. This process leads to the formation of dark brown crusts on the inner shell surface, a condition commonly referred to as "black shell disease". Not only does this disease degrade the quality of shellfish, but it also causes substantial mortalities, with the Hong Kong oyster (Crassostrea hongkongensis) being one of its primary hosts. In this study, an improved Deeplabv3+ semantic segmentation model integrated with an attention mechanism was developed to identify and classify the severity of Polydora-induced disease in oysters. First, through systematic data collection and enhancement techniques, a comprehensive dataset of 4590 shellfish disease images was constructed, fully meeting the training requirements of convolutional neural networks (CNNs). Second, transfer learning was employed by leveraging pre-trained model weights, which validated the suitability of the self-constructed dataset for model training. Comparative analysis of multiple segmentation models confirmed Deeplabv3+ as the optimal baseline, achieving a mean Intersection over Union (MioU) of 88.76% and an average precision of 94%. To further enhance performance, the Deeplabv3+ model was upgraded by incorporating the Convolutional Block Attention Module (CBAM), a dual-module mechanism integrating channel and spatial attention. Compared to the original model, the improved version exhibited significant performance gains: MioU increased by 1.19%, average precision rose by 1.78%, and mFscore improved by 0.68%. Finally, an efficient and user-friendly web-based system was designed for grading and identifying Polydora disease severity. This system can accurately segment the diseased regions on the inner oyster shell, calculate their proportion relative to the total inner shell area, and precisely grade the disease severity, thereby providing robust technical support for the monitoring and prevention of oyster black shell disease.
Phosphorus (P) deficiency in aquacultured fish has become increasingly prevalent due to stricter environmental regulations and reduced dietary P inputs. P-deficient fish have been reported to exhibit morphometric alterations, notably in the ribs and neural/hemal spines; however, whether these deformities represent true pathological manifestations remains uncertain. This study re-examined skeletal tissues of juvenile rainbow trout Oncorhynchus mykiss using alternative protocols. Fish were fed either a P-deficient or a control diet for 1.5 months. Following formalin fixation, muscular tissues were enzymatically digested, and the skeletons were stained with alizarin red, alcian blue and aniline blue for detailed examination. P-deficient fish exhibited only minor rib deformities but showed extensive non-calcified regions across the skeleton, teeth, fins and scales. These findings were further confirmed by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS). In contrast, heating the skeletons (70°C or 100°C) markedly exacerbated skeletal deformities in P-deficient fish but not in control fish. These results indicate that bending deformities previously reported as clinical signs of P deficiency are not evident in intact fish but become conspicuous only after heating during analytical procedures. The expansion of non-calcified skeletal regions may serve as a sensitive diagnostic indicator of P deficiency in farmed fish.
Cyprinid herpesvirus 2 (CyHV-2) is a major pathogen causing high mortality in farmed goldfish and crucian carp, for which effective prophylactic or therapeutic treatments are currently limited. Lauric acid (LA) and glycerol monolaurate (GML), representative of a medium-chain fatty acid (MCFA) and its corresponding monoglyceride, respectively, have been reported to possess antiviral and immunomodulatory properties. This study systematically evaluated the anti-CyHV-2 effects of LA and GML through in vitro assays, transcriptomic profiling, and in vivo experiments. In vitro, both LA and GML significantly reduced viral copy numbers in gibel carp caudal fin (GiCF) cells, attenuated virus-induced cytopathic effects (CPE), and suppressed intracellular viral replication. Transcriptomic analysis revealed that LA and GML induced widespread alterations in host signalling pathways, with significant enrichment of pathways related to steroid biosynthesis, ECM-receptor interaction, protein digestion and absorption, and cell survival-associated signalling. Quantitative PCR analysis further demonstrated that the expression levels of insr, akt2, pdk1, lamtor3, and hsp90b were significantly upregulated, whereas inpp4b expression was downregulated. These results further validate that host cell metabolic processes, stress responses, and signal transduction pathways are substantially modulated following LA and GML treatment. The in vivo protective efficacy of GML was further assessed. In goldfish infected with CyHV-2, GML administration significantly increased survival rate and mitigated histopathological damage in the gills, liver, spleen, and kidney. At 3 days post-infection (dpi), expression levels of pro-inflammatory cytokines il-1β, il-6, and tnf-α were significantly lower in the GML-treated group compared to the virus-infected control group, whereas the anti-inflammatory cytokine il-10 was significantly upregulated. By 7 dpi, differences in inflammatory cytokine expression between groups had diminished, suggesting that GML not only exerts direct antiviral activity but may also modulate host immune responses during the early stage of infection. Collectively, these findings provide a theoretical basis for the practical application of LA and GML and propose a novel strategy for developing safe, effective, and environmentally friendly anti-CyHV-2 agents.
Immersion anaesthesia is widely used in fish handling and research, yet optimization of protocols requires not only effective immobilization but also consideration of internal physiological responses. This study evaluated the effects of immersion-administered lidocaine (60 and 80 mg/L) as an adjuvant to clove essential oil (CEO; 100 μL/L) on anaesthesia induction and recovery in juvenile Oreochromis niloticus under tightly controlled temperature (27.5°C ± 0.3°C). Behavioural endpoints included loss of equilibrium (LOE), time to an operational anaesthetic plane and recovery time; opercular respiratory rate was assessed as an internal physiological indicator. In the main experiment, adding lidocaine to CEO did not significantly alter LOE, time to anaesthetic plane or recovery time relative to CEO alone. In contrast, respiratory-rate trajectories differed significantly among treatments during both induction and recovery, indicating protocol-dependent modulation of ventilatory dynamics despite similar time-based anaesthetic endpoints. A complementary preliminary experiment showed that lidocaine alone (60 or 80 mg/L) did not induce LOE or anaesthetic plane within a predefined 5-min exposure window, although it produced clear dose-dependent changes in respiratory rate relative to water and ethanol controls. Together with qualitative observations of reduced behavioural activation, these findings are compatible with a possible calming or sedation-like effect of lidocaine, although this was not formally measured and should be considered a hypothesis. Overall, lidocaine did not improve the operational speed of a CEO-based immersion anaesthetic protocol in Nile tilapia, but it did modulate physiological responses relevant to protocol refinement. These results highlight the importance of integrating both behavioural and internal physiological indicators, together with strict thermal control, when evaluating immersion anaesthesia protocols in fish.
Ectoparasites that penetrate host skin can act as biological or mechanical vectors for pathogens and, in some cases, serve as reservoirs. Crustacean ectoparasites of fish are potential vectors of pathogens, which is especially relevant for obligate pathogens (e.g., Aeromonas salmonicida) with limited seawater survival. Sea lice (Lepeophtheirus salmonis), affecting Atlantic salmon, cause dermal damage and can facilitate secondary infections, resulting in economic losses. While the physical impact of sea lice is well known, their role in pathogen transmission is less clear. The gut bacterial microbiome of lice collected over four months from a salmon farm in Ireland was analysed using Illumina MiSeq and Oxford Nanopore Technologies (ONT) PromethION sequencing for comparison. Illumina and Nanopore sequencing identified 15 and 24 genera of known fish pathogens, respectively. Moreover, Nanopore data revealed up to 15 putative pathogenic species, including Tenacibaculum maritimum, T. dicentrarchi and Vibrio anguillarum, causative agents of tenacibaculosis and vibriosis. The results of this study provide a gut bacterial microbiome characterisation of L. salmonis in a commercial aquaculture setting and demonstrate the potential of sea lice to act as pathogen vectors or reservoirs. These findings have important implications for pathogen surveillance, management, and prevention strategies in salmon aquaculture.
Tilapia Lake Virus (TiLV) is an emerging pathogen that poses a significant threat to global tilapia aquaculture, leading to substantial economic losses. The development of rapid, sensitive, and specific diagnostic methods is crucial for disease control and prevention. In this study, TiLV-infected tilapia brain cell line (TiB cells) was used as the target for screening specific aptamers via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. The affinity, specificity, stability, cytotoxicity, and antiviral activity of the obtained aptamers were systematically analysed through flow cytometry, confocal laser scanning microscopy, real-time quantitative PCR, and cell viability assays. This study successfully screened and obtained two aptamers, LH-1 and LH-2. Both aptamers specifically recognize and bind to TiLV-infected TiB cells while showing no cross-reactivity with normal cells or cells infected with other viruses. Their secondary structures exhibit typical stem-loop configurations, with dissociation constants (Kd) reaching nanomolar levels at 506.9 nM and 326.4 nM, respectively, and they demonstrate no cytotoxicity toward host cells. Aptamer LH-2 is capable of detecting as few as 1 × 103 cells/mL within 5 min at temperatures ranging from 4°C to 28°C. The detection capability of LH-2 for TiLV infection was consistent with that of RT-qPCR, as validated in vivo. These results demonstrate that LH-2 possesses significant potential to be developed as a core recognition component for on-site rapid detection kits. This study provides critical molecular tools and a theoretical foundation for the rapid diagnosis of TiLV and the development of novel targeted prevention and control strategies. It strongly advances the practical application of aptamer technology in the precise prevention and control of aquatic diseases.
To identify enriched pathogens and characterise the viral community associated with epidemic disease outbreaks in the freshwater mussel Hyriopsis cumingii, we performed metatranscriptomic sequencing combined with VirID-driven RNA-dependent RNA polymerase (RdRP) mining and phylogenetic analysis using hepatopancreas and intestinal samples from six severely infected individuals. Clinical observations were consistent with hallmark features of epidemic outbreaks. The sequencing yielded 86.2 Gb of raw data, of which 97.1% passed quality control, resulting in 77.7 Gb of high-quality clean data. Taxonomic annotation identified 182 viral species, predominantly unclassified viruses (45% Transcripts Per Million, TPM), followed by members of the phyla Lenarviricota (28%) and Uroviricota (17%). Phylogenetic analysis of RdRP sequences revealed 13 viral supergroups, with the Picorna-Calici supergroup showing the highest abundance (26.2% of annotated viruses) and reaching a prevalence of 39.3% in sample HcAV3. Notably, 89.6% of the identified viral RdRPs exhibited less than 70% amino acid identity to known viral sequences, highlighting the presence of extensive "viral dark matter" in this host species. This study establishes the first viral profile associated with epidemic disease in H. cumingii, providing a baseline for further etiological research on this high-mortality aquaculture disease.
Hypermelanosis associated with chromatophore hyperplasia was documented in an incidental specimen of Terapon jarbua (Forsskål, 1775) collected from a semi-intensive brackish-water culture pond used for Asian sea bass (Lates calcarifer) production. Gross examination revealed prominent, irregular, and partially coalescent hypermelanotic patches distributed along the lateral body surface and fins, accompanied by localized skin ulceration, whereas the internal organs appeared macroscopically normal. Radiographic assessment indicated that the lesions were restricted to soft tissues, with no evidence of skeletal involvement. Microscopically, the lesions were characterized by marked disruption of normal chromatophore architecture including extensive melanophore proliferation and alterations in associated chromatophore populations. Histopathological examination showed dense dermal melanin deposition, hypertrophic melanophores and disorganization in the dermal connective tissue framework. Fontana-Masson staining confirmed the presence of actively synthesized melanin within the affected tissues. Transmission electron microscopy further revealed abundant melanosomes at different stages of maturation within melanophores, together with cytoplasmic crowding and peripheral nuclear displacement. Bacteriological screening of skin lesions and internal organs on tryptic soy agar supplemented with 2% NaCl yielded no culturable bacterial growth under the incubation conditions tested. These findings are consistent with localized melanophore hyperplasia affecting the chromatophore system. This case highlights the occurrence of significant pigmentation abnormalities in a non-target species inhabiting aquaculture environments and underscores the importance of continued health surveillance and documentation of unusual phenotypic conditions in brackish-water culture systems.
Cell lines are essential tools for studying animal physiology and immunopathology, reducing the use of live animals and supporting the 3Rs principle of reduction, refinement, and replacement. The Senegalese sole (Solea senegalensis), a relevant species for Spanish aquaculture diversification, lacks established in vitro models. We developed SsB-1, the first brain-derived cell line from this species, providing a valuable platform for studying virus-host interactions. SsB-1 exhibits glial features, stable morphology, and high susceptibility to several betanodavirus (NNV) genotypes, enabling detailed analysis of neurotropism and virulence mechanisms. Its selective permissiveness to NNV and limited replication of infectious pancreatic necrosis virus (IPNV), spring viremia of carp (SVCV), and rhabdovirus eel virus European X (EVEX) highlight its versatility. The ability of some viruses to enter cells without triggering cytopathic or antiviral responses also points to new research avenues for potential reservoir roles in flatfish. Overall, SsB-1 offers a robust, ethical, and versatile model to advance virological and immunological research in support of sustainable marine aquaculture.
The objective of this study was to evaluate welfare and health effects following single and repeated non-lethal blood sampling from the caudal vasculature of Atlantic salmon. Two experiments were conducted at three different temperatures: a 6-week freshwater experiment with fish weighing 50-100 g, undergoing up to four blood withdrawals, and a 4-week seawater experiment with fish weighing 300-450 g, undergoing up to three blood withdrawals. Observed clinical signs included dark pigmentation of the skin and vascular disturbances posterior to the puncture site. Histological evaluations revealed injuries to the vasculature, vertebral column, nervous tissue and skeletal muscle. At 12°C and 15°C, fish displayed strong capacity for tissue regeneration and haematological recovery, but also an elevated risk of acute mortality. At 5°C, acute mortality was minimal, but recovery time was approximately twice as long compared with higher temperatures. Mortality rates and clinical findings decreased with increasing fish weight. These findings demonstrate that non-lethal caudal blood sampling poses a welfare risk in salmon weighing 450 g or less. The risk factors identified here may inform future risk assessments and support the development of welfare-compatible guidelines for non-lethal blood sampling in Atlantic salmon.
Listeria monocytogenes (LM) is one of the eight species within the Listeria genus and one of the most lethal foodborne pathogens. Accurate and rapid detection is crucial for preventing infections caused by this bacterium. Nanobodies, with the advantages of strong affinity, high specificity, low off-target effects, excellent thermal stability, and good solubility, have emerged as a novel reagent for pathogen detection. InlG (Internalin G), a surface-anchored virulence protein accessible on the bacterial surface, was selected as the target antigen to ensure high specificity and effective antibody binding. This study expressed and purified the inlG protein by prokaryotic expression. High-specificity nanobodies targeting the inlG protein were screened using phage display technology and were then used as capture antibodies to establish an indirect sandwich ELISA detection method. The established indirect sandwich ELISA assay demonstrates excellent reproducibility (intra-assay CV: 2.39% and inter-assay CV: 2.78%) and high specificity, with slight cross-reaction with Escherichia coli, and no significant cross-reaction with other food-borne pathogens. The limit of detection (LOD) was 1.0 × 105 CFU mL-1 and limit of quantitation (LOQ) was 3.0 × 105 CFU mL-1 (S/N = 10, CV ≤ 10%). Spiked recovery rates ranged from 96.0% to 107.5% in five food matrices (beef, fish, milk, soft cheese, and deli turkey), with an RSD ≤ 4.68%. Furthermore, these nanobodies targeting InlG may serve as penicillin for controlling L. monocytogenes infections, and chemical residues benzylpenicilloic acid (BPNLA) risk, addressing the challenge of antibiotic resistance.