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.
Judicious use of antimicrobials in aquaculture requires reliable antimicrobial susceptibility testing (AST) of bacterial pathogens for resistance surveillance and for advising therapy decisions. To improve AST of aquatic bacterial pathogens such as Vibrio harveyi, the Clinical and Laboratory Standards Institute (CLSI) has standardized methods specific to testing isolates collected from fish and other aquatic animals. However, no criteria, called epidemiological cutoff values (ECVs), exist yet to interpret results when testing V. harveyi with these standard methods. Microbiologists use ECVs to determine whether an isolate has decreased susceptibility to an antimicrobial relative to other isolates of the same bacterium. In this study, we generated minimal inhibitory concentration (MIC) data using 3 independent laboratories that tested 76 isolates with the CLSI standard broth microdilution method at 28°C for 24-28 h against 9 antimicrobials. The resulting MIC data for 6 of the antimicrobials listed below was combined with previously published data (Smith et al. 2023; Dis Aquat Org 155:35-42) and analyzed with the programs Normalized Resistance Interpretation (NRI) and ECOFFinder to calculate potential ECVs. In collaboration with CLSI's Working Group on Aquatic Animals, the potential ECVs were proposed to CLSI's Subcommittee on Veterinary Antimicrobial Susceptibility Testing, which voted to accept the values. These new ECVs will be included in the next edition of the VET04 supplement. The approved ECVs for enrofloxacin, florfenicol, gentamicin, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole are ≤1, ≤2, ≤4, ≤1, ≤1, and ≤0.5/9.5 µg ml-1, respectively. These new interpretive criteria will improve international harmonization regarding monitoring efforts on antimicrobial susceptibility of V. harveyi.
Cyprinid herpesvirus 3 (CyHV-3) is a highly contagious virus that causes high mortalities in common and koi carp worldwide. The molecular detection of this double-stranded DNA virus has been extensively researched. Nonetheless, there are currently no real-time PCR assays available for detecting CyHV-3 mRNA, which could serve as an indicator of active virus replication, aiding in the evaluation of the susceptibility of non-target species. This study describes a probe-based reverse transcription, real-time PCR (RT-qPCR) assay that was designed to detect CyHV-3 mRNA for efficient, high-throughput detection. The assay is highly specific for CyHV-3 mRNA, with no detection in samples from non-infected fish, closely related viruses or CyHV-3 DNA. The analytical sensitivity of the assay was examined using dilutions of a plasmid control and nucleic acid from a CyHV-3-infected cell line, where the CyHV-3 mRNA limit of detection was approximately 1 copy per reaction. Testing of diluted CyHV-3 mRNA demonstrated comparable sensitivity of the RT-qPCR with an existing reverse transcription PCR assay. Progressive monitoring of positive control samples revealed that the assay had a high level of repeatability. The assay was used to provide further evidence that non-target species silver perch and Murray cod were not susceptible to developing disease when experimentally exposed to CyHV-3. The novel RT-qPCR assay is an invaluable tool for detection of the replication phase of CyHV-3.
This study analysed published data on the distributions of minimum inhibitory concentrations of a group of freshwater isolates classified as Aeromonas spp. with the aim of establishing whether they provided any evidence that epidemiological cut-off values set from these data would be unreliable. This group contained 233 isolates and included members of at least 11 species. The standard deviations (SDs) of the wild-type distributions for 10 antimicrobial agents were calculated for this multi-species group using the ECOFFinder and normalised resistance interpretation (NRI) algorithms. These were compared to the SDs of 110 distributions established for individual species published by the European Committee on Antimicrobial Susceptibility Testing. Fifty-one of these distributions had been generated by multiple laboratories and 59 by single laboratories. When the ECOFFinder algorithm was used to calculate the SDs, the mean for the multi-species group was 0.63 log2 µg ml-1, and the 51 individual species and multiple-laboratory groups were 0.68 and 0.65 log2 µg ml-1, respectively. When the NRI algorithm was used, the mean for the multi-species group was 0.79 log2 µg ml-1, and the 51 individual species and multiple-laboratory groups were 0.79 and 0.76 log2 µg ml-1, respectively. These comparisons indicate that the heterogeneity in the susceptibility to antimicrobial agents within the multi-species group of Aeromonas is not significantly different from that recorded for individual species. This analysis, therefore, suggests that epidemiological cut-off values designed to be applied to all members of the genus Aeromonas would not be inherently unreliable.
A novel dinoflagellate is described from bluegill Lepomis macrochirus, rock bass Ambloplites rupestris, largemouth bass Micropterus nigricans, and yellow perch Perca flavescens collected from Lundgren Lake and Townsend Flowage, Wisconsin, USA. A new genus, Dermisichthinium gen. nov., is established for this species, D. pseudosporum sp. nov., which produces white spots grossly similar to those caused by Ichthyophthirius multifiliis. Microscopically, however, the vegetative cysts of D. pseudosporum closely resemble Haidadinium ichthyophilum, a parasite of threespine stickleback Gasterosteus aculeatus. Haidadinium ichthyophilum was collected from Haida Gwaii, British Columbia, Canada, for morphological and molecular comparison. Molecular analysis of the small subunit (18S), large subunit (28S), and internal transcribed spacer rDNA regions supports the novel species description and erection of a new genus. Pairwise comparisons of partial 18S and 28S sequences revealed divergence levels approximately 3 times greater than those among congeneric suessiacean dinoflagellates. Dermisichthinium pseudosporum sp. nov. lacks a 25 bp insertion in 28S unique to H. ichthyophilum, providing a molecular character for distinguishing the 2 species and supporting their placement in separate genera. Phylogenetic analyses consistently place D. pseudosporum sp. nov. and H. ichthyophilum in distinct clades. This study enhances our understanding of parasitic dinoflagellate diversity, underscores the importance of integrating morphological, molecular, and other diagnostic characteristics in their taxonomic classification, and offers valuable diagnostic insight for fish health professionals and parasitologists encountering this unusual group of cyst-forming dinoflagellates.
Ostreid herpesvirus-1 (OsHV-1) is a threat to the global production of Pacific oysters Crassostrea gigas, often resulting in nearly complete mortality in affected stocks. A sentinel monitoring program was conducted between June and October 2020, to characterize OsHV-1 outbreaks in Pacific oysters along the west coast of the USA. Deployment of sentinel oysters at 5 commercial growing locations, coupled with frequent sampling, allowed measurement of the spatial and temporal occurrence of OsHV-1 outbreaks as well as the viral load and pathogenesis of OsHV-1 during C. gigas mortality events. In addition, 2 divergent oyster families were deployed at sites that have historically tested positive for OsHV-1 to measure the effect of oyster genotype on the severity of OsHV-1-induced mortality in the field. Mortality events at California test sites were associated with elevated levels of OsHV-1 in oyster tissue. OsHV-1 was not detected in oysters at Oregon and Washington test sites. In Tomales Bay, California, high variation among replicate culture units was observed in cumulative field survival and peak viral load. A negative relationship was observed between peak OsHV-1 load in oyster tissues and shell height at the time of peak viral load, suggesting larger seed may be less vulnerable during periods of OsHV-1 infection risk. Cumulative survival over the duration of the growing season in Tomales Bay was related to peak viral load and differed by family. These results corroborate previous findings suggesting selective breeding may effectively increase survival of oyster families during OsHV-1 outbreaks along the US west coast.
The microsporidium Ecytonucleospora hepatopenaei (EHP) continues to disrupt farmed shrimp production globally by causing growth retardation, chronic mortality, and enhancing susceptibility to other diseases. While the susceptibility of black tiger shrimp Penaeus monodon and Pacific white shrimp P. vannamei to EHP is well known, the susceptibility of Pacific blue shrimp P. stylirostris to EHP has not been demonstrated. To determine the susceptibility of P. stylirostris to EHP, infectious inoculum was directly injected into the hepatopancreas of specific-pathogen-free (SPF) P. stylirostris. At 17 d post-injection, the developmental stages of the parasite were observed in the EHP-injected P. stylirostris, and EHP was detected by real-time PCR. The EHP-injected P. stylirostris were then divided into 2 groups. In the first group, P. stylirostris (n = 9) were cohabitated with SPF P. vannamei (n = 55), and in the second group, the hepatopancreas was excised from EHP-injected P. stylirostris, homogenized, and fed to SPF P. vannamei (n = 12). Both experimental challenge routes led to the horizontal transmission of EHP from P. stylirostris to P. vannamei, but cohabitation resulted in a stronger infection. In a follow-up study, one group of SPF P. stylirostris was fed EHP-infected P. vannamei tissue, and another group cohabitated with EHP-infected P. vannamei. Both groups of P. stylirostris developed EHP infections. These results clearly provide evidence of both P. stylirostris susceptibility to EHP and the transmission potential of EHP between P. stylirostris and P. vannamei via natural routes such as cohabitation and cannibalism.
Flavobacterium columnare is the etiological agent of columnaris disease in rainbow trout Oncorhynchus mykiss. F. columnare has recently been speciated into 4 independent columnaris disease-causing Flavobacterium species. Several F. columnare isolates from different geographic origins have been tested for variations in virulence and genetic diversity via multilocus sequence analysis of housekeeping genes. These studies have been limited in their ability to differentiate between highly variable and evolving regions of genetic diversity or to reliably predict differences in virulence between strains. To initiate a serotyping scheme, polyclonal antibodies were generated to F. columnare strains CSF-298-10, MS-FC-4 and PSFC-081215-1, and 7 serotypes were identified based on Western blot analysis of 54 F. columnare strains. Genome sequencing identified a 35-40 kb region encoding 32-36 putative lipopolysaccharide (LPS)-related genes that support 7 serotypes. Genes within this region encode molecules associated with the Lipid A, core region and O-antigen. All isolates were derived from columnaris outbreaks in the Columbia River of Washington State and the Snake River region of Idaho (USA). Serotyping and regional genome analyses allow for rapid and focused determination of isolate comparisons for population heterogeneity, LPS evolution and strain tracking. This is of the utmost importance when considering that all of these isolates were derived from columnaris outbreaks within the main US rainbow trout-producing regions. Knowledge of genetic and serotype diversity will help inform development of therapeutic strategies and vaccines.
Edwardsiella ictaluri and E. piscicida are bacterial pathogens that infect a variety of commercially important wild and cultured fish and cause significant economic losses among various farmed fish sectors, especially the catfish industries in the USA and Vietnam. The Clinical and Laboratory Standards Institute (CLSI) offers standard antimicrobial susceptibility testing (AST) methods for these pathogens and others, but internationally harmonized criteria, called epidemiological cutoff values (ECVs), are still needed to identify isolates with decreased antimicrobial susceptibility. To address this need, minimal inhibitory concentration (MIC) data for 9 antimicrobials were generated using the standard broth microdilution AST method at 28 ± 2°C for 44-48 h. The data sets comprise MICs from 101 E. ictaluri isolates from 4 independent laboratories and 115 E. piscicida isolates from 3 laboratories. Aggregated MIC distributions were used to calculate wild-type (WT) cutoff values for the 18 bacteria/antimicrobial combinations. WT cutoff values for E. ictaluri against ampicillin, enrofloxacin, erythromycin, florfenicol, gentamicin, oxolinic acid, oxytetracycline, ormetoprim/sulfadimethoxine, and trimethoprim/sulfamethoxazole were ≤1, ≤0.12, ≤128, ≤2, ≤2, ≤0.5, ≤2, ≤0.5/9.5, and ≤0.25/4.8 µg ml-1, respectively. WT cutoff values for E. piscicida were ≤8, ≤0.12, n/a, ≤2, ≤2, ≤0.25, ≤2, ≤0.12/2.4, and ≤0.12/2.4 µg ml-1, respectively. No E. piscicida WT cutoff value could be calculated for erythromycin. These WT cutoff values provided the CLSI with data needed to approve ECVs which will be included in the next edition of the VET04 supplement. CLSI-approved ECVs resulting from this work will greatly improve antimicrobial resistance surveillance of these bacterial pathogens impacting global aquaculture.
Fan mussels Pinna nobilis across the Mediterranean Sea have been severely impacted by a widespread mass mortality event, largely attributed to Haplosporidium pinnae. While the Sea of Marmara (SoM) has historically served as a refuge for this Critically Endangered species, recent findings suggest the emergence of various pathogens, including haplosporidian parasites, in SoM populations. This study presents the first confirmed case of H. pinnae infection in fan mussels from the southern SoM, with a focus on species-level identification, and contributes to growing concerns about the potential spread of this pathogen into previously unaffected regions. On 23 November 2024, 5 live fan mussels were collected from depths of 2.3-6.5 m in the southern SoM. Histopathological examination revealed structural alterations in the mantle tissue and the presence of plasmodial stages of H. pinnae. Molecular analyses further confirmed the presence of H. pinnae in several samples, exhibiting 100% sequence similarity with isolates from other Mediterranean regions. This study provides essential evidence of infection in the SoM and underscores the need for continued monitoring and conservation efforts for fan mussel populations in the region, especially as pathogens continue to spread across the Mediterranean Sea. The results represent a significant conservation alarm and highlight the urgent need for continued pathogen surveillance, early warning strategies, and robust management interventions in one of the last strongholds of this keystone species.
Melanoma is a highly aggressive neoplasm of melanocytes, commonly studied in mammals but rarely documented in wild fish populations. This study reports the first confirmed case of malignant melanoma in Terapon jarbua (Forsskål, 1775) from the Parangipettai landing center on the southeastern coast of India. Gross examination revealed superficial raised, hyperpigmented lesions primarily distributed along the dorsal and lateral body surfaces. Wet mount analysis showed extensive melanization within the dermal layers of the affected tissue. Histological examination identified pleomorphic melanocytes with dense melanin deposition, architectural disruption of the skin, and features consistent with superficially spreading melanoma. Scanning electron microscopy of melanotic lesions revealed significant alterations to the epidermal surface, with rounded or angular projections and sharply defined crevices. Transmission electron microscopy showed classical ultrastructural abnormalities such as pleomorphism, irregular melanosome aggregation, binucleation, cytoplasmic vacuolation, and necrotic cells with compromised plasma membranes. The presence of melanosomes at various maturation stages indicated hyperactive melanogenic activity. This novel discovery points to the importance of regular tumour surveillance and research into environmental stressors as possible causative agents, adding to the small but increasing body of evidence of neoplastic disease in Indian marine ecosystems.
The amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) has devastated global amphibian biodiversity. Non-amphibian hosts might facilitate Bd spread across the landscape, but our understanding of their role remains severely limited. Several species of invertebrates have been implicated to varying degrees as potential carriers of Bd, but research is needed to understand the impact these invertebrates have on the spread and maintenance of this pathogen in the wild. Detecting Bd presence in field-collected invertebrates is a necessary first step for identifying potential vectors, but methodological comparisons have not been conducted. Detection thresholds of DNA extraction methods for Bd detection from amphibian skin swabs may not be applicable for invertebrate samples, because they may contain lower Bd loads and higher amounts of PCR-inhibitory substances. This study aimed to identify the most cost-effective and reliable method of DNA extraction for detecting Bd DNA in invertebrate samples using qPCR. We compared the effectiveness of 5 commonly used DNA extraction kits (QIAGEN, Zymo, SPINeasy, PrepMan Ultra, and Chelex resin) for Bd detection in homogenised cricket samples spiked with known concentrations of Bd. We found PrepMan Ultra to be the optimal extraction kit for a broad screening of field-collected invertebrate samples, due to its relatively low cost and the ability to detect Bd presence in homogenised cricket samples containing at least 100 zoospore equivalents. However, researchers need to conduct their own cost-benefit analysis when choosing an extraction method to ensure that the method suits their needs.
Nutrient deficiency can cause increased susceptibility to infectious diseases in fish, thus leading to high rates of morbidity and mortality. Thiamine deficiency complex (TDC) in fish can lead to low reproductive success and high mortality rates. Columnaris disease in salmonids, caused by Flavobacterium columnare, has resulted in devastating losses in aquaculture production and wild populations of Pacific salmon particularly associated with climate change and high water temperatures. There is growing awareness that both TDC and columnaris are emerging diseases of salmonids on the west coast of North America; however, it is unknown whether fish that survive from low/intermediate thiamine level eggs will experience latent mortality due to susceptibility to infectious diseases like columnaris. To investigate the interaction of TDC survivors and columnaris, Chinook salmon Oncorhynchus tshawytscha fry reared from either thiamine-deficient (n = 120) or thiamine-replete (n = 120) eggs were challenged with F. columnare using an immersion challenge model of infection, and morbidity/mortality, immune responses, and bacterial load were evaluated. The cumulative mortalities between the treatment groups were significantly different, with the thiamine-deficient, F. columnare-exposed fry ending the challenge with an 80.3% survival rate and the thiamine-replete, F. columnare-exposed fry ending with a 29.03% survival rate (p < 0.0001). Different transcript abundance was detected in gills and spleen of thiamine-deficient and thiamine-replete fry exposed to F. columnare. This study demonstrated that fry reared from eggs low in thiamine have an altered immune response and warrants further studies to better understand interaction with potential pathogens at different life stages.
A new myxozoan species, Henneguya dugarovi sp. nov., was observed in the gills of Amur common carp Cyprinus carpio haematopterus Martens, 1846 (subspecies of common carp), which inhabits the Lake Baikal basin. The parasite was studied on the basis of spore morphology and molecular methods. Cysts were located intralamellarly in the secondary lamellae. Mature spores of H. dugarovi sp. nov. are symmetrical, with 2 unequal caudal appendages, the spore body is obovate in frontal view and lemon-shaped in lateral view. The spores have a total length of 24-42 μm, a mean ± SD body length of 11.7 ± 0.8 μm (range 10.0-13.1 μm), a width of 8.8 ± 0.5 (7.8-10.2) μm and thickness of 7.3 ± 0.5 (5.8-8.0) μm. Two equal polar capsules are pear-shaped, blunt at the posterior end, narrowed anteriorly and widely spaced, 5.3 ± 0.4 (4.4-6.0) μm long and 3.1 ± 0.3 (2.4-4.0) μm wide. Polar tubules are coiled in 7 turns. The infection rate of H. dugarovi sp. nov. was 56%. Phylogenetic analyses showed that this species is grouped with myxosporeans infecting common carp.
Magellanic penguins Spheniscus magellanicus are seasonal migratory seabirds. During the migratory period, these penguins are observed mainly in the seas of Argentina, Uruguay, and Brazil. Studies on mortality events in the species are limited, particularly those comparing free-ranging and captive penguins. This study describes the causes of death and pathological findings in Magellanic penguins using necropsy records from a retrospective study (2011-2021). Records from 164 penguins were analyzed, comprising 95 in captivity, 68 free-ranging, and 1 of undetermined origin. Non-infectious diseases (n = 104) were predominant in free-ranging penguins and included starvation (n = 49), suspected drowning (n = 27), hypothermia secondary to oil pollution (n = 25), trauma (n = 2), and intestinal obstruction (n = 1). Infectious/inflammatory diseases (n = 23) were most frequent among captive populations, including cases of malaria (n = 9), aspergillosis (n = 7), other mycotic infections (n = 3), bacterial infection (n = 2), protozoal meningoencephalitis (n = 1), and granulomatous air sacculitis of unknown origin (n = 1). Juvenile penguins constituted most cases in both non-infectious and infectious categories. There were 37 cases of undetermined diagnoses. Gastrointestinal parasitosis, cutaneous lesions, and pulmonary edema were the main secondary pathologic findings. These results enhance our understanding of the diseases affecting the Magellanic penguins and provide valuable insights for the conservation and maintenance of free-ranging and captive penguins.
We report significant pathological findings from 272 stranding investigations of 20 cetacean species in the Pacific Islands region between 2006 and 2024. Full or partial necropsies of 209 cases (76.8%) resulted in one or more diagnoses associated with death in 137 cases. Natural disease accounted for 62% of stranded animals; approximately half were in poor body condition due to chronic illness. Morbillivirus and Brucella sp. infections caused mortality in 11 species, including striped dolphins and Longman's beaked whales. Toxoplasmosis, of anthropogenic cause in Hawai'i, led to deaths of 2 spinner dolphins and a bottlenose dolphin. Pygmy and dwarf sperm whales, beaked whales and pilot whales showed heavy parasitism by nematodes, cestodes and trematodes. Approximately 12.4% of stranded individuals were perinates/neonates, with 3 cases of dystocia with maternal mortality. Anthropogenic trauma was observed in 29.2% of strandings, including 6 goose-beaked whales with cranial and/or microvascular hemorrhages. Vertebral and skull fractures were attributed to direct vessel strikes for 2 pygmy sperm whales, 2 humpback whale calves, a goose-beaked whale, a spinner dolphin and a striped dolphin. Blast trauma was observed in 3 Fraser's dolphins in an uncommon stranding event. Significant plastic debris and/or fishery debris were found in stomachs of 6 species, with fatal gastric obstruction in a sperm whale and fatal fishhook penetration in a bottlenose dolphin. This study highlights the value of necropsy examinations in a region inhabited by small island-associated populations where carcass recovery rates are low, and cetaceans face an array of natural and anthropogenic threats.
Globally, Marteilia spp. parasites have been associated with significant mass mortality events in populations of commercially important bivalve molluscs, frequently resulting in large-scale fishery collapses and substantial socio-economic impacts. The Wash Estuary, UK, supports several bivalve fisheries, and among these, common cockles Cerastoderma edule have suffered unusually high mortalities since 2008. We investigate potential causes of these mortalities, and confirm infection with M. cocosarum, strongly associated with cockle moribundity, also confirming its presence in archived samples collected in 2009. Molecular and light microscopy screening of samples collected during mortality events in 2021, including healthy (buried) and moribund (weak, unable to bury) cockles, indicated high prevalence of M. cocosarum in moribund cockles (PCR incidence up to 95%) in contrast to healthy cockles (up to 42%), suggesting an association between cockle moribundity and Marteilia infection. Analysis of the full ribosomal RNA array identified consistently different nucleotides between M. cocosarum infections in the Wash (denoted as genotype WE1) and those in Wales (denoted genotype WA1). A total of 83% of infections in the Wash could be identified as M. cocosarum WE1 and 12% as M. cocosarum WA1, with both genotypes recovered from 5% of infected animals. Histopathologically, M. cocosarum WE1 infects the gill, mantle and connective tissues, identical to observations of M. cocosarum infecting Welsh cockles. Ongoing cockle mortalities in the Wash raise concerns regarding the sustainability of this resource ecologically and economically. Additional measures may be required to reduce the spread of this pathogen, noting that its distribution beyond the Wash and Wales is currently unknown.
Infectious disease-related mass mortality events (ID-MMEs) in marine mammals have increased dramatically over the past 30 years, and viruses have been identified as the primary cause. This study validates the development and utility of point-of-care diagnostics using handheld devices. Using real-time quantitative polymerase chain reaction (real-time qPCR), we developed and validated a portable diagnostic method for detecting viruses infecting marine mammals in field applications. Specifically, the Franklin portable thermocycler was used to demonstrate the feasibility of field diagnostics for marine mammal viruses. Portable devices enable rapid point-of-care diagnostics and have high mobility to detect viral transmission and variants in resource-limited settings for early response. This device enables rapid and accurate diagnosis of marine mammal viruses in resource-limited environments, facilitating timely intervention to mitigate the spread of infection to marine ecosystems and other species, including humans. Despite these advances, several challenges remain, including the optimization of DNA extraction for point-of-care diagnostics and obtaining clinical samples. In conclusion, this study highlights the potential value of point-of-care diagnostic tests in response to viral diseases in marine mammals.
Lactococcus garvieae causes lactococcosis in several marine-cultured fish species in Japan. A new L. garvieae serotype III was recently identified in diseased striped jack Pseudocaranx dentex, yellowtail Seriola quinqueradiata, and amberjack S. dumerili. Herein, we report the first isolation of L. garvieae serotype III isolates LC2313 and LC2323 from cage-cultured cobia Rachycentron canadum. Naturally diseased fish exhibited clinical signs of hemorrhagic eyes and a curved spine. An infectivity trial on cobia using the LC2323 isolate revealed 30% mortality by intramuscular injection at doses of 105 or 107 CFU fish-1, while 50-60% mortalities were observed by intraperitoneal injection at the same infectious doses. Both the LC2313 and LC2323 isolates caused up to 100% mortality in yellowtail and amberjack during 14 d of observation. The draft genomes of LC2313 and LC2323 were 1951432 and 1953218 bp, respectively, with G+C contents of 38.9% for both isolates. A total of 1867 coding sequences were predicted for LC2313 and 1872 for LC2323 with 12 phage-related genes each. The core-genome sequence alignment indicated a close relationship between these isolates and the recently identified L. garvieae serotype III from amberjack and striped jack. Taken together, these results demonstrate the pathogenicity of L. garvieae serotype III in susceptible fish species, highlighting the need for awareness regarding disease transmission among fish species.
We tested the antiviral activity of aqueous extract of star anise Illicium verum (AEIV) against striped jack nervous necrosis virus (SJNNV) in vitro as well as in vivo using the Sahul Indian Seabass Spleen (SISS) cell line and Asian seabass Lates calcarifer. Preliminary results from GC-MS profiling and polyphenol content estimation of AEIV revealed the antiviral potential of the extract. Before testing the compound, the extract was assessed for its toxic potential in an in vitro model (SISS cell line) using 4 different concentrations (50, 75, 100 and 200 µg ml-1) of AEIV through MTT and neutral red assays. The tested concentrations were not toxic (≥90% survival). Subsequently, the same concentrations of AEIV were tested for their antiviral potential against SJNNV in SISS cells. The results showed that AEIV at higher concentration (100 and 200 µg ml-1) effectively inhibited SJNNV replication in the SISS cells. RT-PCR and qPCR confirmed the inhibition of the virus. The same concentrations were then used to evaluate their antiviral efficacy in vivo by intraperitoneal injection in Asian seabass. We observed 100% mortality only in the fish injected with SJNNV (control), which confirms that AEIV possesses the potency to control the replication of SJNNV under both in vitro and in vivo conditions. The present study indicates that medication with plant extracts having antiviral activity could be a potentially beneficial alternative to the usage of chemotherapeutics in aquaculture.