Journal of fish diseases最新文献

A spleen-derived cell line was established from the spleen of hybrid snakehead (♀Channa argus × ♂Channa maculata) (abbreviated as CAMsp), a species of considerable economic importance in China's freshwater aquaculture, which is severely impacted by the hybrid snakehead rhabdovirus (HSHRV), largemouth bass ranavirus (LMBV), and infectious spleen and kidney necrosis virus (ISKNV). The establishment of a stable in vitro culture system is imperative for the effective isolation, identification, and study of fish viruses. CAMsp, generated through trypsin digestion, had successfully undergone over 80 passages since its initial culture. This cell line exhibited rapid proliferation in Leibovitz's-15 medium (L-15) supplemented with 10% fetal bovine serum at 28°C, achieving monolayer formation within 24 h at a passage ratio of 1:3. Chromosomal analysis of CAMsp at the 60th passage identified a chromosome count of 42; the chromosome number in hybrid snakehead somatic cells is 44, 45, or 46, suggesting chromosomal alterations. Inoculation of CAMsp monolayers with HSHRV, LMBV, and ISKNV resulted in characteristic cytopathic effects (CPE), including cell rounding, aggregation, and eventual detachment. Transmission electron microscopy (TEM) confirmed viral replication and revealed extensive cytopathological changes within the infected cells, demonstrating the susceptibility of the CAMsp cell line to all three viruses. Viral titers, determined by TCID₅₀ assay at 7 days post-infection (dpi), reached 10^9.25 ± 10^0.36 TCID₅₀/mL for LMBV, 10⁶·¹⁵ ± 10^0.25 TCID₅₀/mL for ISKNV, and 10⁸·³³ ± 10^0.12 TCID₅₀/mL for HSHRV, indicating efficient viral propagation in this cell line. The CAMsp cell line serves as a valuable model for studying certain fish viruses, virus-host interactions, and disease prevention strategies.

Tilapia parvovirus (TiPV) is an emerging pathogen associated with high mortality rates in farmed tilapia, highlighting the urgent need for rapid and accurate diagnostic tools. In this study, we established an RPA-CRISPR/Cas12a detection system targeting the TiPV NS1 gene. The assay conditions were systematically optimised, including 15-min RPA amplification at 39°C, with reagent concentrations of 200 nM Cas12a, 250 nM crRNA and 200 nM ssDNA reporter. Specificity tests showed no cross-reactivity with other tilapia pathogens (TiLV, S. agalactiae) and other aquatic pathogens (LMBRaV, YcCV, GCRV II, WSSV, CyHV-2, SVCV). Sensitivity evaluation revealed a limit of detection (LoD) of 1.97 × 10¹copies/μL, which was 100-fold more sensitive than PCR (1.97 × 10³copies/μL). Clinical validation with 20 tilapia samples demonstrated a 50% positive detection rate for RPA-CRISPR/Cas12a, 15% higher than PCR (35%). This integrated method combines the advantages of RPA and CRISPR-based signal transduction, offering a field-applicable solution for TiPV monitoring in resource-limited aquaculture environments.

The formation of extracellular traps (ETosis) is an innate immune mechanism in shrimp against pathogens. Microorganisms are entrapped in extruded DNA fibres co-localised with antimicrobial peptides and eventually killed. However, as a cell death mechanism, its strict regulation is essential as excessive formation of ETs may cause detrimental effects to the host by contributing to disease pathophysiology. Here, we investigated the ability of freeze-dried Lactiplantibacillus plantarum (FD-LAB), previously reported to enhance shrimp immunity against pathogenic infections, to regulate ETosis. In an ex vivo setup, gill cells and circulating hemocytes were pre-exposed to FD-LAB and were then stimulated with Vibrio parahaemolyticus. Results showed that V. parahaemolyticus alone induced ETosis in both gill cells and circulating hemocytes, while FD-LAB alone did not. However, when cells were pre-exposed to FD-LAB prior to stimulation with V. parahaemolyticus, no ETosis occurred. Similarly, changes in reactive oxygen species (ROS) production coincided with the formation of ETs, thus signifying that FD-LAB may regulate ETosis in shrimp gill cells and circulating hemocytes possibly by dampening ROS production. These results present a novel means to regulate ETosis and indicate that FD-LAB may enhance shrimp immunity while also acting on immune regulation.

Tilapia Lake Virus (TiLV) is a significant threat to global tilapia aquaculture, highlighting the need for rapid and accurate diagnostic methods to manage outbreaks and minimise economic losses. This study presents the development and partial validation of a one-pot assay integrating RT-LAMP with the CRISPR/Cas12b system for sensitive and specific TiLV detection. This assay amplifies viral RNA using RT-LAMP, while CRISPR/Cas12b enables a real-time detectable signal. Targeting a conserved region in TiLV segment four, the assay achieves results within 75 min at 62°C, with easy visualisation using a portable fluorescence viewer. It demonstrated high sensitivity, with a 95% limit of detection of 79.6 copies (95% CI: 48-132 copies), and high specificity, with no cross-reaction to other fish RNA or DNA viruses. Based on a validation panel of 261 samples from 9 source populations, the assay exhibited 92% diagnostic sensitivity (95% CI: 87%-96%) and 100% diagnostic specificity (95% CI: 97%-100%). When assessed as a non-lethal sample, gills provided a reliable and less invasive alternative despite lower viral loads compared to internal organs. Therefore, this partially validated one-pot assay is potentially practical for enhancing TiLV detection and disease management in aquaculture systems, especially in field settings and resource-limited laboratories.

Amyloodinium ocellatum causes epizootics in marine fish hatcheries. Frozen, wild fish, often used as food in aquaculture, could introduce the disease. We investigated the parasite's ability to survive, reproduce and infect a host after freezing. Infected gills were frozen at -20°C for either 0, 24, 36, 48, or 72 h. Parasite viability was assessed first by placing thawed infected gills into tanks with naïve fish. Gills of the exposed fish were examined on day 0 and on days 4 and 7 post-exposure. Tomonts from infected gills in all freeze durations produced infections. However, dinospore production decreased and infections took longer to establish in fish exposed to tomonts from the 72 h treatment. Second, eight tomonts from each freeze duration were placed in 5 mL of 25 ppt artificial seawater at 22°C in individual wells of a 12-well plate and monitored for hatching. Ninety seven percent of tomonts from all treatments hatched. Tomonts frozen for up to 72 h survived, reproduced and infected fish, but dinospore production decreased as freeze duration increased. Extrapolation from the rate of reduction in dinospore production over the freeze durations tested suggests that a freeze duration of 237 h could inactivate all tomonts.

This study presents the first description and characterisation of Lactococcus garvieae isolated from diseased European sea bass (Dicentrarchus labrax) farmed in Spain. During autumn 2023, two fish farms were affected by infectious outbreaks causing moderate cumulative fish mortality (5%-10%). Diseased fish showed clinical signs of haemorrhagic septicaemia, compatible with lactococcosis. Standardised screening tests revealed the presence of Gram-positive cocci in the kidney, spleen and brain of the diseased fish, and negative results for parasites and viruses. Bacterial cultures recovered from the internal organs of all diseased fish were identified as Lactococcus garvieae by phenotypic, genetic, immunological and proteomic analyses. Strains were sensitive to oxytetracycline, florfenicol and erythromycin and resistant to trimethoprim sulfamethoxazole. In addition, representative isolates were virulent to juvenile sea bass and Senegalese sole (Solea senegalensis) after intracoelomic challenge with doses ranging from 10⁶ to 10⁷ CFU/fish. SDS-PAGE and immunoblotting analyses, using rabbit serum anti L. garvieae CECT 5274 and serum from trout immunised with a bivalent oil-based vaccine against L. garvieae-Yersinia ruckeri, showed that the current strains of sea bass shared some antigenic proteins with strains of L. garvieae from other hosts and with the reference strain of L. petauri DSM104842. Our overall results confirm the presence of this emerging pathogen in Spanish marine aquaculture and suggest that commercially available lactococcosis vaccines could confer some protection to sea bass, helping to prevent this new threat.

Infectious spleen and kidney necrosis virus (ISKNV) is a significant threat to global aquatic food security by causing large-scale mortality in the aquaculture of tilapia (Oreochromis niloticus) and mandarin fish (Siniperca chuatsi). ISKNV is a genogroup of Megalocytivirus pagrus1, along with RSIV and TRBIV, and their recent listing as WOAH-notifiable diseases highlights the need to assess spread pathways to prevent exotic pathogen incursions. An albino rainbow shark (Epalzeorhynchos frenatum) challenge model was used to evaluate the risk of ISKNV introduction from the trade in frozen seafood products by determining viability after freezing and the median infectious dose (ID₅₀). Six donor fish were injected with ISKNV with tissues collected after clinical signs appeared and used immediately or stored at -20°C for 7 days. Challenge inocula were prepared consisting of snout, eyes, and brain (Pool A; representative of head-on eviscerated fish products), caudal skin and muscle (Pool B; representative of skin-on fillet only products) or peritoneal viscera (Pool C; positive control group). Fish were challenged by bath immersion or intraperitoneal injection and sampled for ISKNV detection by qPCR at morbidity/death, or on Day 14. Negative control fish all survived without detection of ISKNV. All tissue pools including 'skin-on fillet only' caused infection and disease via IP injection or immersion, whether used fresh or frozen, showing ISKNV remains infectious after 7 days at -20°C. The model estimated an ID₅₀ of 42 ISKNV genome equivalents (95% CI: 19-98). This study is the first to investigate the potential for ISKNV spread via frozen fish products, a commodity frequently traded in international markets. The findings provide evidence to inform import risk assessments and highlight the need for further investigation into spread pathways involving uncooked, frozen fish products.

Streptococcus parauberis is recognised as a major pathogen in marine aquaculture species. This pathogen has been reported in Platichthys stellatus aquaculture farms in South Korea. To investigate the causative agent responsible for mass mortality, monthly disease monitoring was conducted at three aquaculture farms in Pohang. A total of 31 S. parauberis isolates were identified using Spa primers. Additionally, to confirm the pathogenicity of S. parauberis , starry flounder were intraperitoneally injected with strain AD112410, which resulted in a dose-dependent mortality pattern. Whole genome sequencing was conducted on strain AD112410 isolated from starry flounder and compared with that of other S. parauberis strains isolated from olive flounder. All S. parauberis isolates were classified as serotype Ia and exhibited γ-hemolysis. PCR detection revealed the presence of the tet(S) resistance gene in all farms, while erm(B) was absent only in farm C. Virulence genes (gapC and hasC) were found in all farms. All isolates showed resistance to tetracycline and oxytetracycline, while susceptibility to erythromycin and clindamycin depending on the presence of erm(B). These findings not only demonstrate an association between S. parauberis and mass mortality in starry flounder, but also indicate a correlation among serotype, antibiotic resistance genes, and antibiotic susceptibility.

The Carpione rhabdovirus strain 2023 (CAPRV2023) is an emerging viral pathogen that has caused significant morbidity and mortality in cultured golden pompano (Trachinotus ovatus) in China over recent years. There is an urgent need for a rapid and accurate method for the quantitative detection of CAPRV2023. In this study, we developed a TaqMan probe-based quantitative PCR assay targeting the N gene of CAPRV2023 and systematically evaluated its specificity, sensitivity, repeatability, and reproducibility of the assay. Both plasmid DNA, in vitro-transcribed RNA standards, and in vitro-transcribed RNA mixed with RNA extracted from CAPRV2023-negative golden pompano tissues were used as templates to evaluate assay performance under both optimal and biologically relevant conditions. Using plasmid DNA standards, the assay exhibited excellent linearity over a broad range of 2 × 10¹ to 5 × 10⁹ copies/μL, with a standard curve of Y = -3.3132X + 39.142, a correlation coefficient of R² = 0.9993, an amplification efficiency of 100.37%, and a detection limit of 20 copies per reaction. In vitro-transcribed RNA standards demonstrated robust linearity over the range of 2 × 10² to 2 × 10⁹ copies/μL, with a standard curve of Y = -3.2327X + 45.502, a correlation coefficient of R² = 0.9968, and a detection limit of 200 copies per reaction. Notably, when the in vitro-transcribed RNA standards were combined with RNA extracted from CAPRV2023-negative golden pompano tissues, the assay maintained similar performance, yielding a standard curve of Y = -3.2182X + 45.445, a correlation coefficient of R² = 0.9967, and the same detection limit of 200 copies per reaction. These results indicate that the presence of background tissue RNA does not significantly interfere with assay accuracy or sensitivity. Specificity test revealed that the assay exhibits no cross-reactivity with common bacterial or viral agents present in various aquatic organisms. The assay demonstrated high reproducibility and repeatability, with intra-assay and inter-assay coefficients of variation (CVs) below 2.5%. Field sample detection yielded a significantly higher detection rate compared to the conventional PCR assay. The newly developed TaqMan qPCR assay provides a robust diagnostic tool for the efficient and accurate quantitative detection of CAPRV2023 in field samples and surveillance programs.