Immunological and molecular diagnostic techniques in fish health: present and future prospectus.

Fish health management is critical to aquaculture and fisheries as it directly affects sustainability and productivity. Fish disease diagnosis has taken a massive stride because of advances in immunological and molecular diagnostic tools which provide a sensitive, quick, and accurate means of identifying diseases. This review presents an overview of the main molecular and immunological diagnostic methods for determining the health of fish. The immunological techniques help to diagnose different fish diseases by detecting specific antigens and antibodies. The application of immunological techniques to vaccine development is also examined in this review. The genetic identification of pathogens is made possible by molecular diagnostic techniques that enable the precise identification of bacterial, viral, and parasitic organisms in addition to evaluating host reactions and genetic variation associated with resistance to disease. The combination of molecular and immunological methods has resulted in the creation of novel techniques for thorough evaluation of fish health. These developments improve treatment measures, pathogen identification and provide new information about the variables affecting fish health, such as genetic predispositions and environmental stresses. In the framework of sustainable fish farming and fisheries management, this paper focuses on the importance of these diagnostic techniques that play a crucial role in protecting fish populations and the aquatic habitats. This review also examines the present and potential future directions in immunological and molecular diagnostic techniques in fish health.

Detection of Acipenser European Iridovirus (AcIV-E) in Sturgeon Farms in Northern Italy between 2021-2023.

Sturgeon farming is rapidly expanding in Europe, where Italy ranks first in farmed caviar production. A major threat to sturgeon health in captivity is infection with Acipenser European Iridovirus (AcIV-E), a viral disease definitively identified in 2016. Here we present data on the occurrence of AcIV-E in 482 sturgeons (age ≤ 12 months, species of the genus Acipenser and the species Huso huso) collected from sturgeon farms in northern Italy between January 2021 and December 2023. The health status of each specimen was determined by necroscopy and virological assay. Virological analysis was performed on gill samples and real-time PCR specific to the MCP gene of the iridovirus viral capsid. Molecular analysis revealed positivity to the virus in 204 samples (42.68% of the total), while anatomopathological examination of nearly all fish with positive real-time PCR disclosed swollen abdomen, hepatic steatosis, splenomegaly, and increased gill volume. Two challenges to timely diagnosis are the absence of pathognomonic symptoms and the inability to isolate the virus on cell monolayers. Continuous and widespread health monitoring is therefore crucial for disease management and to effectively control spread of the virus.

Botulism in fish: a review.

Published information about fish botulism is scant. We review here the current literature on fish botulism. Freshwater fish are susceptible to botulism. Only anecdotal evidence exists about possible botulism cases in saltwater fish. With only a few exceptions, the etiology of all cases of fish botulism reported is Clostridium botulinum type E, although fish are sensitive to, and may carry, various C. botulinum types. Clinical signs of botulism in fish include loss of equilibrium and motion, abducted opercula, open mouths, dark pigmentation, and head up/tail down orientation in which attempts to swim result in breaching the surface of the water. Dark pigmentation is thought to be associated with acetylcholine imbalance in botulinum neurotoxin (BoNT)-affected fish. Rarely, but similar to the situation in other animal species, fish can recover from botulism. Fish botulism can cause secondary outbreaks of the disease in birds, as botulism-affected fish stand out from normal fish, and are selectively preyed upon by fish-eating birds, which thus become intoxicated by the BoNT present in sick fish. The source of BoNT in fish has not been definitively confirmed. Fish may ingest C. botulinum spores that then germinate in their digestive tract, but the possibility that fish ingest preformed BoNT from the environment (e.g., dead fish, shellfish, insects) cannot be ruled out. The presumptive diagnosis of botulism in fish is established based on clinical signs, and as in other species, confirmation should be based on detection of BoNT in intestinal content, liver, and/or serum of affected fish.

Application and development of a TaqMan-based real-time PCR assay for rapid detection of snakehead vesiculovirus.

Snakehead vesiculovirus (SHVV) is one of the primary pathogens responsible for viral diseases in the snakehead fish. A TaqMan-based real-time PCR assay was established for the rapid detection and quantification of SHVV in this study. Specific primers and fluorescent probes were designed for phosphoprotein (P) gene, and after optimizing the reaction conditions, the results indicated that the detection limit of this method could reach 37.1 copies, representing a 100-fold increase in detection sensitivity compared to RT-PCR. The specificity testing results revealed that this method exhibited no cross-reactivity with ISKNV, LMBV, RSIV, RGNNV, GCRV, and CyHV-2. Repetition experiments demonstrated that both intra-batch and inter-batch coefficients of variation were not higher than 1.66%. Through in vitro infection experiments monitoring the quantitative changes of SHVV in different tissues, the results indicated that the liver and spleen exhibited the highest viral load at 3 poi. The TaqMan-based real-time PCR method established in this study exhibits high sensitivity, excellent specificity, and strong reproducibility. It can be employed for rapid detection and viral load monitoring of SHVV, thus providing a robust tool for the clinical diagnosis and pathogen research of SHVV.

Diagnosis, isolation and description of a novel amnoonvirus recovered from diseased fancy guppies, Poecilia reticulata.

The guppy, Poecilia reticulata, is one of the most common cultured ornamental fish species, and a popular pet fish highly desired by hobbyists worldwide due to its availability of many brilliantly coloured fish of many varieties. The susceptibility of guppies to diseases presents a remarkable concern for both breeders and hobbyists. In this study, we report the emergence of disease in fancy guppies caused by a previously uncharacterized virus in the USA. This virus was isolated from moribund guppies in two separate outbreaks in California and Alabama, from December 2021 to June 2023. The infected guppies presented with acute morbidity and mortality shortly after shipping, displaying nonspecific clinical signs and gross changes including lethargy, anorexia, swimming at the water surface, gill pallor, mild to moderate coelomic distension and occasional skin lesions including protruding scales, skin ulcers and hyperaemia. Histological changes in affected fish were mild and nonspecific; however, liver and testes from moribund fish were positive for Tilapia lake virus (TiLV), the single described member in the family Amnoonviridae, using immunohistochemistry and in situ hybridization, although the latter was weak. A virus was successfully recovered following tissue inoculation on epithelioma papulosum cyprini and snakehead fish cell lines. Whole genome sequencing and phylogenetic analyses revealed nucleotide and amino acid homologies from 78.3%-91.2%, and 78.2%-97.7%, respectively, when comparing the guppy virus genomes to TiLV isolates. Based on the criteria outlined herein, we propose the classification of this new virus, fancy tailed guppy virus (FTGV), as a member of the family Amnoonviridae, with the name Tilapinevirus poikilos (from the Greek 'poikilos', meaning of many colours; various sorts, akin to 'poecilia').

Diagnostic performance of cross-priming amplification-based lateral flow assay (CPA-LFA) and real-time PCR for koi herpesvirus (KHV) detection.

Epizootics of Koi herpesvirus (KHV) cause mass mortality in koi carp (Cyprinus rubrofuscus) and common carp (Cyprinus carpio) worldwide. Rapid and accurate virus detection technology is crucial for preventing pathogen spread and minimizing damage. Although several diagnostic assays have been developed for KHV, the analytical and diagnostic performance of the detection methods has not been evaluated. In this study, we developed and validated the diagnostic performance of two molecular diagnostic assays, cross-priming amplification-based lateral flow assay (CPA-LFA) and TaqMan probe-based real-time polymerase chain reaction (PCR). To detect KHV, primers and probe were designed based on the thymidine kinase (TK) genes. The detection limits of developed CPA-LFA and real-time PCR assays were determined to be 675.69 copies/µL and 8.384 copies/µL, respectively. The diagnostic sensitivity and specificity of the developed assay were determined using fish samples (n = 179). CPA-LFA was found to be 93.67% and 100%, respectively, and real-time PCR was found to be 100% and 100%, respectively. Therefore, the newly developed CPA-LFA and real-time PCR assays accurately and rapidly detect KHV. CPA-LFA is particularly suitable for point-of-care diagnosis because of its simple diagnostic process, and real-time PCR analysis is most suitable for precise diagnosis because it can detect low viral loads.

Rapid and sensitive detection of large yellow croaker iridovirus by real-time RPA and RPA-LFD.

Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/µL, while the detection limit of RPA-LFD was 101 copies/µL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.

Rapid differentiation of infectious salmon anemia virus avirulent (HPR0) from virulent (HPRΔ) variants using multiplex RT-qPCR.

Infectious salmon anemia virus (ISAV; Isavirus salaris) causes an economically important disease of Atlantic salmon (Salmo salar L.). ISA outbreaks have resulted in significant losses of farmed salmon globally, often with a sudden onset. However, 2 phenotypically distinct variants of ISAV exist, each with divergent disease outcomes, associated regulations, and control measures. ISAV-HPRΔ, also known as ISAV-HPR deleted, is responsible for ISA outbreaks; ISAV-HPR0, is avirulent and is not known to cause fish mortality. Current detection methodology requires genetic sequencing of ISAV-positive samples to differentiate phenotypes, which may slow responses to disease management. To increase the speed of phenotypic determinations of ISAV, we developed a new, rapid multiplex RT-qPCR method capable of 1) detecting if a sample contains any form of ISAV, 2) discriminating whether positive samples contain HPRΔ or HPR0, and 3) validating RNA extractions with an internal control, all in a single reaction. Following assay development and optimization, we validated this new multiplex on 31 ISAV strains collected from North America and Europe (28 ISAV-HPRΔ, 3 ISAV-HPR0). Finally, we completed an inter-laboratory comparison of this multiplex qPCR with commercial ISAV testing and found that both methods provided equivalent results for ISAV detection.

Non-lethal detection of Eubothrium crassum (Cestoda) in farmed Atlantic salmon, Salmo salar, using anal swabs and real-time PCR.

Detection of intestinal parasites in fish typically requires autopsy, resulting in the sacrifice of the fish. Here, we describe a non-lethal method for detecting the tapeworm Eubothrium crassum in fish using anal swabs and real-time PCR detection. Two assays were developed to detect cytochrome oxidase I (COI) mitochondrial DNA and 18S ribosomal DNA sequences of E. crassum, respectively. The assays were tested on swab samples from confirmed pathogen free Atlantic salmon (Salmo salar L.) and on samples from farmed Atlantic salmon, where the presence and intensity of parasites had been established through autopsy. The COI assay was shown to be specific to E. crassum, while the 18S assay also amplified the closely related E. salvelini, a species infecting Arctic charr (Salvelinus alpinus L.) in freshwater. The COI assay detected E. crassum in all field samples regardless of parasite load while the 18S assay failed to detect the parasite in two samples. The results thus demonstrates that this non-lethal approach can effectively detect E. crassum and can be a valuable tool in assessing the prevalence of infection in farmed salmon, aiding in treatment decisions and evaluating treatment effectiveness.

Confirmatory test of active IHHNV infection in shrimp by immunohistochemistry and IHHNV-LongAmp PCR.

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection.

Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

Selection and characterization of ssDNA aptamer targeting Macrobrachium rosenbergii nodavirus capsid protein: A potential capture agent in gold-nanoparticle-based aptasensor for viral protein detection.

The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.

Isolation, in vitro, and in vivo pathogenicity test of Tilapia lake virus (TiLV) and development of a prognostic semi-quantitative lesion scoring system for differentiating clinical/subclinical infection in farmed tilapia (Oreochromis niloticus L.).

Tilapia lake virus ('TiLV-MH-2022') was recently recovered from the naturally infected farmed tilapia. Reverse transcription-polymerase chain reaction (RT-PCR) using segment 1 specific primers, followed by Sanger sequencing, confirmed the infection. The pairwise sequence homology of segment 1 showed its close relationship with the previous isolates. The virus was successfully detected from the mucus, which emphasised the possibility of non-invasive screening of tilapia on a large scale. The virus inoculum prepared from the infected tissues was tested for in vivo and in vitro pathogenicity. Around 100-140 nm-sized electron-dense virus particles were observed in the infected OnlL cells. Based on the onset of symptoms and lesions, all RT-PCR-positive fish were categorised into two groups, 'clinical' and 'subclinical'. A lesion-scoring technique was developed for assessing the pathogenicity of the virus isolate. The external and internal gross lesions and histopathological alterations in the critical organs of the fish, such as the brain, kidney, gills, and liver, were assessed on a scale of 0 (no gross lesion) to 5 (most severe lesions). Overall lesion score was significantly high in the clinical and subclinical groups for gross and histopathology, respectively. This study is the first such attempt to standardise a semi-quantitative lesion scoring technique for TiLV infection, which establishes a clinical relevance and prognostic ability to distinguish between the apparent and inapparent infection.

Comparison of specificity and sensitivity of diagnostic methods for Enteromyxum leei and Enteromyxum fugu detected from cultured tiger puffer, Takifugu rubripes in Korea.

Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real-time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real-time and two-step PCR for early diagnosis and follow-up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one-step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.

Development and application of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid visual detection of anguillid herpesvirus 1.

Eel (Anguilla sp.) is an important freshwater-cultured species with high economic value in China. Anguillid herpesvirus 1 (AngHV-1) has been proven to be the pathogen of "mucus sloughing and haemorrhagic septicaemia disease" in eels, resulting in significant mortality and substantial losses to the eel industry. Current diagnostic methods for detecting AngHV-1 are limited to laboratory-based tests, for example, conventional end-point PCR and qPCR. Therefore, there is an urgent need to develop an accurate, rapid, and simple detection method for on-site diagnosis of AngHV-1. In this study, we developed a recombinase-aided amplification combined lateral flow dipstick (RAA-LFD) assay for the detection of AngHV-1. The RAA-LFD assay can be performed within a temperature range of 18-45°C, with a reaction time of just 10 min for amplification. Importantly, the established RAA-LFD assay exhibited no reactivity with other common aquatic viral pathogens, indicating its high specificity. The limit of detection for this method is 102 copies of AngHV-1, which is more sensitive than the established conventional end-point PCR method similarly targeting ORF95. Clinical detection of the diseased samples demonstrated that the accuracy of RAA-LFD was significantly higher than that of the conventional end-point PCR. In conclusion, the developed RAA-LFD assay has proven to be a convenient, rapid, sensitive, and reliable tool for on-site diagnosis of AngHV-1. This advancement will be invaluable for the prevention and control of AngHV-1 in the eel farming industry.

[Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish].

OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS: Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.

Diagnosis of piscine francisellosis in Largemouth Bass from a public display exhibit in north-central Florida, USA.

OBJECTIVE: The Largemouth Bass Micropterus salmoides is an important freshwater fish that is native to the southeastern United States and is cultured for conservation, food, and for the sports fishing industry. Francisella orientalis is a globally distributed bacterial pathogen of warmwater fish species and is associated with granulomatous inflammation and high mortalities. Outbreaks of piscine francisellosis in the United States have been reported in only a few fish species. This study describes three case presentations of francisellosis in Largemouth Bass from a public display system in north-central Florida. Additionally, laboratory-controlled immersion challenges using an F. orientalis isolate from tilapia Oreochromis spp. evaluate susceptibility of Largemouth Bass fingerlings to F. orientalis infection and mortality through this exposure route. METHODS: Necropsy, histologic examination, immunohistochemistry, bacterial recovery and culture, and quantitative polymerase chain reaction were used as diagnostic tools to evaluate both the affected display fish and the immersion-challenged fingerlings. RESULT: Although the display fish and immersion-challenged fingerlings presented with nonspecific clinical signs, gross and histological changes were indicative of granulomatous disease. Immunohistochemical and molecular testing methods confirmed F. orientalis infection in affected fish. CONCLUSION: The three case presentations described here mark the first reporting of naturally occurring piscine francisellosis in Largemouth Bass that were held in a public display exhibit. Additionally, causality was proven in the Largemouth Bass fingerlings through the immersion challenges. These findings demonstrate susceptibility through immersion-based exposure and assert that francisellosis should be considered among the list of differential diagnoses for Largemouth Bass with granulomatous disease.

ISOTHERMAL RECOMBINANT POLYMERASE AMPLIFICATION AND CRIPSR(CAS12A) ASSAY DETECTION OF RENIBACTERIUM SALMONINARUM AS AN EXAMPLE FOR WILDLIFE PATHOGEN DETECTION IN ENVIRONMENTAL DNA SAMPLES.

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon (Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum, is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20-40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.