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 × 10copies/μL, which was 100-fold more sensitive than PCR (1.97 × 10copies/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.

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 TCID assay at 7 days post-infection (dpi), reached 10 ± 10 TCID/mL for LMBV, 10· ± 10 TCID/mL for ISKNV, and 10· ± 10 TCID/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.

Cestode infestations in cultured fish have resulted in significant economic problems associated with international trade. Pharmacological treatments containing albendazole (ABZ) represent a promising alternative for controlling these infestations. This study evaluated the in vitro efficacy of ABZ against the metacestode Parvitaenia cochlearii, a parasite of the Pacific fat sleeper (Dormitator latifrons), by exposing the parasites to different concentrations (200, 600, 800, and 1000 mg/L). The median lethal concentration (LC50) was determined at 10 and 15 min of exposure. Results showed that ABZ was 100% effective in killing P. cochlearii, achieving complete mortality at 15 min with a concentration of 600 mg/L, and at 30 min with 200 mg/L. The LC50 value decreased over time, with 618.91 mg/L at 10 min and 260.32 mg/L at 15 min, indicating a cumulative effect of ABZ on metacestode tissues. In conclusion, ABZ is an effective treatment against cestode infestations, and further evaluation under experimental and commercial scales is recommended to establish control protocols in aquaculture systems.

Goldfish (Carassius auratus) are among the most widely cultured ornamental fish. Skeletal deformities and muscular lesions have been reported in cyprinids, but their aetiology and pathology remain unclear. Advanced diagnostic tools, including radiography, ultrasonography and computed tomography, provide valuable insights when combined with histopathology. This study aimed to characterise clinical, radiological, ultrasonographical, computed tomographical and histopathological features of goldfish with hump-like lesions and muscular cavities caused by Myxobolus lentisuturalis in two farms, and to compare disease manifestations between populations using advanced imaging plus histology. From January 2023 to March 2024, ~10% of fish from two commercial goldfish farms in Tehran Province showed clinical signs. The first group had only hump-like lesions behind the head without exudate, whereas the second also had large muscular cavities with whitish exudate. Thirty goldfish (15 per farm) were sampled. Digital radiography, B-mode ultrasonography, computed tomography (CT/3D-CT), wet smears and histopathological sections were prepared. In the first stock, bilateral crescent-shaped humps containing Myxobolus lentisuturalis spores were observed, with radiographs revealing increased lesion density and obscured swim bladder margins, and ultrasonography indicating muscular hypertrophy with elevated echogenicity. In the second stock, hump-like lesions accompanied by exudates, cavitations, and severe muscular dystrophy were detected, supported by radiographic and CT evidence of deformity, cavitation, and asymmetry. This study demonstrated that M. lentisuturalis infection in goldfish can present with distinct clinical and pathological manifestations, ranging from localised hump-like lesions to extensive muscular cavitation and deformity. The integration of radiography, ultrasonography, CT imaging, and histopathology provided a comprehensive diagnostic approach for characterising disease progression. These findings highlight the importance of advanced imaging modalities in ornamental fish medicine and contribute to a better understanding of myxozoan-associated lesions in cyprinids.

Grass carp (Ctenopharyngodon idellus) is a vital species in China's aquaculture industry but is highly susceptible to grass carp haemorrhagic disease (GCHD) caused by the grass carp reovirus (GCRV). GCRV infection in Ctenopharyngodon idellus kidney cells (CIK) induces reactive oxygen species (ROS) overproduction, triggering oxidative stress. Catalase (Cat) is a crucial antioxidant enzyme that decomposes hydrogen peroxide (HO) into HO and O. In this study, we investigated and characterised the roles of CiCat in grass carp. CiCat is expressed ubiquitously in all examined tissues. Furthermore, when challenged with GCRV, its expression levels are significantly upregulated. Fluorescence analysis revealed that CiCat exhibits a widespread cellular distribution, while GCRV infection enhances its aggregation and co-localisation with mitochondria (Mit) and endoplasmic reticulum (ER). Overexpression of CiCat eliminates both HO and GCRV-induced ROS, decreases the expression of antioxidant enzymes and promotes GCRV replication. Further research showed that overexpression of interferon regulatory factor-7 (IRF-7) inhibits GCRV replication, but CiCat reduces IRF-7 expression. In summary, CiCat promotes GCRV replication by reducing CiIRF-7 expression.

Turbot (Scophthalmus maximus ) is an economically important farming fish in China. Turbot circovirus (TCV), a recently discovered member of the Circovirus genus within the Circoviridae family, has emerged as a significant threat to the turbot aquaculture industry in China. This virus is the causative agent of turbot acute hemorrhagic disease (TAHD), resulting in substantial economic losses in recent years. As a novel pathogen, the development of more accurate and efficient detection methods for TCV remains an urgent need. In this study, the specific probe and primers were designed based on the consensus sequence of the cap gene of TCV to develop a TaqMan probe-based real-time PCR method for virus detection. The specificity of the assay was validated using turbot reddish body iridovirus (TRBIV), viral nervous necrosis virus (VNNV), the cap gene of European catfish (Silurus glanis ) circovirus (CfCV) and European eel (Anguilla anguilla ) circovirus (EeCV), and no cross-reaction was observed, indicating the high specificity of the established method. The detection limit of this assay was 10 copies per reaction, with an efficiency of 93.7%, a regression squared value of 0.999, and a linear range from 10 copies to 10 copies of TCV DNA. The assay demonstrated high specificity, sensitivity and reproducibility, with both intra- and inter-assay coefficients of variation less than 2.0%. Then 390 clinical samples suspected of TAHD collected from 2019 to 2022 were tested using this method, and all the samples tested positive for TCV. In this study, we established a TaqMan qPCR-based method for TCV detection, which could be applied to epidemiological investigations and pathogenic studies of TCV.

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 × 10 to 5 × 10 copies/μL, with a standard curve of Y = -3.3132X + 39.142, a correlation coefficient of R = 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 × 10 to 2 × 10 copies/μL, with a standard curve of Y = -3.2327X + 45.502, a correlation coefficient of R = 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 R = 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.

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.

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 (ID). 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 ID 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.

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 10 to 10 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.

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.

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.

Aeromonas hydrophila-induced bacterial sepsis is a major threat to aquaculture, characterised by excessive inflammation, ferroptosis (an iron-dependent lipid peroxidation-driven cell death) and oxidative damage, which collectively lead to high mortality. Taurine (TAU) and glutathione (GSH) have been demonstrated to have potential therapeutic efficacy against ferroptosis-associated pathologies through redox modulation. However, their combined effects and underlying mechanisms in alleviating A. hydrophila infection remain unclear. In this study, we investigated the protective roles of TAU, GSH and their combination in vitro using yellow catfish (Pelteobagrus fulvidraco) macrophages and grass carp (Ctenopharyngodon idella) kidney (CIK) cells and in vivo using yellow catfish challenged with A. hydrophila. The results showed that TAU and GSH, either alone or in combination, alleviated oxidative stress by significantly reducing reactive oxygen species (ROS) accumulation and inhibited nuclear factor-κB (NF-κB) pathway activation. This led to downregulated expression of pro-inflammatory cytokines (IL-1β and TNF-α) and upregulated anti-inflammatory cytokines (IL-10 and TGF-β), accompanied by alleviated ferroptosis. In vivo, dietary supplementation with TAU (10 g/kg) or GSH (350 mg/kg) alone improved survival rates (51.7% and 38.3%, respectively), reduced tissue bacterial loads and protected intestinal and hepatic tissues by preserving mucosal barrier function. However, the TAU + GSH combination unexpectedly decreased survival (28.3%) due to excessive immunosuppression (overproduction of IL-10 and TGF-β) and impaired mucosal barrier, which exacerbated pathogen colonisation. Together, our findings demonstrate that TAU and GSH alleviate A. hydrophila-induced oxidative stress by reducing mitochondrial ROS overproduction and disrupting the ROS/NF-κB signalling pathway, thereby attenuating inflammatory cytokine storms and ferroptosis. These results provide novel insights into the pathological mechanisms of bacterial sepsis in fish and develop sustainable strategies to improve disease resistance in aquaculture.

The gill parasite Sparicotyle chrysophrii poses a significant threat to gilthead sea bream (Sparus aurata) aquaculture in the Mediterranean, causing considerable mortality and economic losses. As traditional chemotherapeutic treatments, like formalin, face growing regulatory restrictions, there is an urgent need for alternative control strategies. This study screened the in vitro antiparasitic activity of 16 phytogenic active ingredients (AIs), including essential oils (EOs) and commercial feed additives, against adult S. chrysophrii. Worms were exposed to a range of concentrations of each compound, and mortality was assessed over 24 h. Lethal dose 50% (LD) values were calculated and categorised by short-, mid-, and long-term efficacy. Several AIs, such as Cinnamomum zeylanicum, Origanum vulgare, Thymus vulgaris, and the commercial formulations Arotec-G and OA + HE+EO, demonstrated rapid and strong toxicity, with low LD values observed within 2 h. In addition to lethality, characteristic morphological damage was detected in exposed parasites, indicating a direct and severe parasiticidal effect. These results highlight the high efficacy of various AIs at concentrations significantly lower than those used in standard formalin baths. Overall, the study identifies several promising candidates for the development of alternative treatments against S. chrysophrii, providing a solid scientific basis for future in vivo validation and integration into sustainable parasite control programs in marine aquaculture.

Myxoid neurothekeoma, also known as nerve sheath myxoma, is a rare benign peripheral nerve sheath tumour primarily reported in mammals, with scarce documentation in teleost fishes. This study reports a confirmed case of myxoid neurothekeoma in the Indian oil sardine (Sardinella longiceps ), a commercially significant marine species along India's southeast coast. An adult female specimen exhibiting a fleshy, dome-shaped, slightly pale brownish mass on the dorsal head was examined grossly and subjected to histopathological analysis using haematoxylin and eosin, Alcian blue and Masson's trichrome staining. Immunohistochemical staining for S100 protein confirmed the neural origin of the tumour cells. Microscopic analysis revealed a well-circumscribed lobular neoplasm composed of spindle to stellate cells within a myxoid stroma rich in acid mucopolysaccharides. Tumour lobules were separated by thin collagenous septa, and widespread S100 positivity supported Schwann cell lineage. The lack of mitotic activity, cytological atypia or invasive features supported a diagnosis of benign myxoid neurothekeoma. This case expands the current understanding of peripheral nerve sheath tumours in marine teleosts and contributes to the broader characterisation of rare neoplasms in aquatic species.

An adult female striped burrfish from a public display aquarium, mixed species recirculating system presented for an exophytic yellow-orange mass on the dorsal head suggestive of neoplasia. The mass was surgically excised twice, adding cryosurgical ablation during the second procedure, but recurred both times post-operatively. After approximately 9 months, euthanasia was elected due to lesion progression. Histopathologic evaluation of surgical biopsy samples and tissues collected at necropsy suggested chromatophoromas. Immunohistochemistry using SOX10, Melan-A and PNL-2 antibodies produced no labelling of tumour cells. Ultrastructurally, neoplastic cells contained poorly formed pterinosomes and vesicles consistent with either xanthophoroma, erythrophoroma or a mixed xanthoerythrophroma. Pigment analysis using absorbance spectrophotometry and gas chromatography mass spectrophotometry identified the primary pigment as xanthopterin with low amounts of the carotenoid tunaxanthin, confirming the diagnosis of xanthophoroma, a rare subtype of chromatophoroma in fish.

Isolating leukocytes from small-bodied fish presents significant technical challenges, especially in species such as grouper, which exhibited rapid blood coagulation. Attaining high purity and quality leukocyte recovery in such species is often hindered by clot formation and erythrocyte contamination. Conventional methods using anticoagulants, buffy coat extraction, and density gradient centrifugation are typically labor-intensive and often yielded inconsistent results. In this study, a streamlined and efficient whole blood lysis protocol for isolating leukocytes from grouper fingerling is described. This protocol offers improvements in blood handling, cell recovery, and cryopreservation. The optimized method significantly reduces processing time, preserves cellular integrity, and facilitates downstream applications in fish immunological studies. Overall, this protocol presented a cost-effective and scalable solution for leukocyte isolation in teleost fish, particularly grouper species.

While nephrocalcinosis (kidney stones) is uncommon in wild teleost fish, various environmental and nutritional factors could lead to its occurrence in aquacultured fish. This study presents the first documented case of kidney stones in aquacultured Brazilian sardine (Sardinella brasiliensis). During necropsy, eighteen hard, white kidney stones were found in the posterior kidney, with an average diameter of 3.71 mm and a total length of 16.8 mm. Morphological analysis revealed stones of different sizes and shapes, including elongated and irregular structures. This discovery enhances our understanding of pathological conditions in S. brasiliensis and underscores the importance of further research into the causes, prevalence and potential implications for fish health and fisheries sustainability.

Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance.