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Upon infection, B lymphocytes develop clonal responses. In teleost fish, which lack lymph nodes, the kinetics and location of B cell responses remain poorly characterized. Fish pronephros is the site of B cell differentiation and the main niche for persistence of plasma cells. In this study, we undertook the analysis of the rainbow trout IgHμ repertoire in this critical tissue for humoral adaptive immunity after primary immunization and boost with a rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We used a barcoded 5′ RACE-cDNA sequencing approach to characterize modifications of the IgHμ repertoire, including VH usage in expressed V(D)J rearrangements, clonal diversity, and clonotype sharing between individual fish and treatments. In the pronephros, our approach quantified the clonotype frequency across the whole IgH repertoire (i.e., with all VH), measuring the frequency of Ag-responding clonotypes. Viral infection led to extensive modifications of the pronephros B cell repertoire, implicating several VH subgroups after primary infection. In contrast, only modest changes in repertoire persisted 5 mo later, including VHSV-specific public expansions. The IgM public response implicating IgHV1-18 and JH5, previously described in spleen, was confirmed in pronephros in all infected fish, strongly correlated to the response. However, the distribution of top clonotypes showed that pronephros and spleen B cells constitute distinct compartments with different IgH repertoires. Unexpectedly, after boost, the frequency of anti-VHSV clonotypes decreased both in pronephros and spleen, raising questions about B cell circulation. A better monitoring of B cell response kinetics in lymphoid tissues will be an essential step to understand B memory and plasmocyte formation mechanisms in fish.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

Assessment of fish health is one of the efforts of farmers in minimizing losses due to disease. Rapid tests on fish health can be done through blood observations. This study aimed to determine the blood glucose profile of koi carp due to ectoparasite infestation from the level of blood glucose. The results showed that reported parasites from Blitar’s koi carp were Trichodina, Dactylogyrus, Gyrodactylus, Myxobolus, Thelohanellus, Ichthyophthirius, and Argulus. Trichodina showed the highest prevalence (100%) in this case while Thelohanellus was the highest intensity level (93.8±16.3). The results of blood glucose level measurement based on parasite infestation levels showed no significant difference (p>0.05) though the health problems caused by parasites in light, medium or heavy infestation. This research also indicated that the blood glucose profile could be used as a rapid method to detect fish health caused by parasites. We suggest that other variables such as nutritional status, life stage or feeding must be conducted to ensure the glucose role in parasite identification as a rapid method for the future work. Resumo A avaliação da saúde dos peixes é um dos esforços dos produtores para minimizar as perdas por doenças. Testes rápidos de saúde de peixes podem ser feitos por meio de observações de sangue. Este estudo teve como objetivo determinar o perfil glicêmico de peixes “koi”, devido à infestação de ectoparasitos a partir do nível de glicemia. Os resultados mostraram que os parasitas relatados de peixes “koi” de Blitar foram Trichodina, Dactylogyrus, Gyrodactylus, Myxobolus, Thelohanellus, Ichthyophthirius e Argulus. Trichodina apresentou a maior prevalência (100%) neste caso, enquanto Thelohanellus foi o maior em nível de intensidade (93,8±16,3). Os resultados da medição do nível de glicose no sangue, com base nos níveis de infestação parasitária, não mostraram diferença significativa (p>0,05), apesar dos problemas de saúde causados por parasitas em infestação leve, média ou pesada. Esta pesquisa também indicou que o perfil de glicose no sangue pode ser usado como um método rápido, para detectar a saúde dos peixes causada por parasitas. Este estudo também sugere que outras variáveis, como estado nutricional, estágio de vida ou alimentação, devem ser conduzidas para garantir o papel da glicose na identificação do parasita como um método rápido para trabalhos futuros.

Highlight Research: It has been found that many fish died with wounds from farmers in Blitar, Indonesia. The mucosal smear test from mucosal gills of C. carpio infected with Myxobolus . In the blood smear test of C. carpio , there was no myxobolus found in the blood. In gill organ testing using SEM that Myxobolus is found in gills ( C. carpio ). The shape of Myxobolus resembles an imperfect ball with a hollow in the middle. Abstract The biggest problem that is often considered to be an obstacle to Common carp culture is the emergence of disease attacks. One type of disease that often attacks the seeds of Cyprinus carpio is Myxobolus (a systemic parasite that can cause harm to fish farming). The aim of this study was to determine the molecular expression through the smear test on C. carpio gills, to determine the image of the gill organs of C. carpio using the SEM test, and to determine the description of the spores of Myxobolus sp. Data were analyzed using descriptive methods. Descriptive method used was comparative descriptive comparing molecular expression in the test of gill mucosal smear of fish using a light microscope and gill organ testing using Scanning Electron Microscope (SEM) on C. carpio infected by Myxobolus sp. In this study, the results showed that in C. carpio infected with true Myxobolus found the presence of Myxobolus in the mucosal smear test and SEM test on gill tissue, but not found in the blood smear test.

A primary problem affecting the sustainable development of aquaculture is fish skin diseases. In order to prevent the outbreak of fish diseases and to provide prompt treatment to avoid mass mortality of fish, it is essential to detect and identify skin diseases immediately. Based on the YOLOv4 model, coupled with lightweight depthwise separable convolution and optimized feature extraction network and activation function, the detection and identification model of fish skin disease is constructed in this study. The developed model is tested for the diseases hemorrhagic septicemia, saprolegniasis, benedeniasis, and scuticociliatosis, and applied to monitor the health condition of fish skin in deep-sea cage culture. Results show that the MobileNet3-GELU-YOLOv4 model proposed in this study has an improved learning ability, and the number of model parameters is reduced. Compared to the original YOLOv4 model, its mAP and detection speed increased by 12.39% and 19.31 FPS, respectively. The advantages of the model are its intra-species classification capability, lightweight deployment, detection accuracy, and speed, making the model more applicable to the real-time monitoring of fish skin health in a deep-sea aquaculture environment.

The treatment of bacterial diseases in aquaculture is done using antibiotics, their applications has resulted in contamination and bacterial resistance. Natural extracts are a potential alternative as an antimicrobial, they have demonstrated effectiveness in their use aimed at treating conditions. The purpose of this study was to evaluate the antimicrobial activity of Lemna minor extracts against Pseudomonas fluorescens with different solvent for extraction. Methanol, chloroform and hexane were used. Subsequently, the safety assessment of the extracts in Danio rerio embryos and larvae was performed to validate as ecologically harmless. Antibacterial activity was detected in three extracts with significant differences (p = 0.001). Hexane extract had the highest antibacterial activity, followed by chloroform and methanol extracts. The three extracts have differences with respect to the control, between times and concentrations tested (p = 0.001). Minimum inhibitory concentration values (MIC) at 24 h methanolic extract ME 0.05 µg mL−1. In embryos and larvae increased safety of the LC50 methanolic extract was evidenced followed by the hexane and chloroform extract. No morphological or tissue changes were observed in embryos and larvae. The hexane extracts of L. minor had a greater bactericidal effect against P. fluorescens and are functional because of their antibacterial activity, but methanolic extract is more safety in embryos and larvae of D. rerio, making it a potential alternative for use in the treatment and control of septicemia in fish.

Piscine novirhabdovirus = Viral Hemorrhagic Septicemia Virus (VHSV) first appeared in the Laurentian Great Lakes with large outbreaks from 2005 to 2006, as a new and novel RNA rhabdovirus subgenogroup (IVb) that killed >30 fish species. Interlude periods punctuated smaller more localized outbreaks in 2007, 2010, and 2017, although some fishes tested positive in the intervals. There have not been reports of outbreaks or positives from 2018, 2019, or 2020. Here, we employ a combined population genetics and phylogenetic approach to evaluate spatial and temporal evolutionary trajectory on its G‐gene sequence variation, in comparison with whole‐genome sequences (11,083 bp) from a subset of 44 individual isolates (including 40 newly sequenced ones). Our results show that IVb (N = 184 individual fish isolates) diversified into 36 G‐gene haplotypes from 2003 to 2017, stemming from two originals (“a” and “b”). G‐gene haplotypes “a” and “b” differed by just one synonymous single‐nucleotide polymorphism (SNP) substitution, remained the most abundant until 2011, then disappeared. Group “a” descendants (14 haplotypes) remained most prevalent in the Upper and Central Great Lakes, with eight (51%) having nonsynonymous substitutions. Group “b” descendants primarily have occurred in the Lower Great Lakes, including 22 haplotypes, of which 15 (68%) contained nonsynonymous changes. Evolutionary patterns of the whole‐genome sequences (which had 34 haplotypes among 44 isolates) appear congruent with those from the G‐gene. Virus populations significantly diverged among the Upper, Central, and Lower Great Lakes, diversifying over time. Spatial divergence was apparent in the overall patterns of nucleotide substitutions, while amino acid changes increased temporally. VHSV‐IVb thus significantly differentiated across its less than two decades in the Great Lakes, accompanied by declining outbreaks and virulence. Continuing diversification likely allowed the virus to persist at low levels in resident fish populations, and may facilitate its potential for further and future spread to new habitats and nonacclimated hosts., This study analyses the evolutionary trajectory of a novel virus over its 17‐year history, examining the dynamics of its host relationships and its patterns across geographic space and evolutionary time. We sequence its entire genome and discern that the virus has significantly changed, with its original genotypes disappearing and being replaced in a quasispecies trajectory. It also has become less virulent, appearing to adapt for long‐term persistence at low levels in the host populations.

This study aims to create a computer based intelligent system to detect freshwater fish disease. Problems often faced by fish farmers, especially freshwater fish, are fish diseases that begin with signs such us wounds on the surface of the fish’s body, bleeding on the gills, fish’s stomach which suddenly enlarges and is filled with fluid, the release of fins or the tail of the fish which results in the death of cultivated fish, thereby reducing freshwater fish production. The expert system developed to implement the forward chaining method which starts with tracking the symptoms experienced by fish to determine the preventive action that must be taken by bringing the symptoms to the system. System development in this study uses extreme programming (XP) where the flexibility of this method supports all changes that occur when developing an expert system to detect fish diseases. The output of the research carried out in the form of an expert system that provides information about freshwater fish diseases, the cause and how to overcome them.

Bacterial fish pathogens are one of the key challenges in the aquaculture industry, one of the fast-growing industries worldwide. These pathogens rely on arsenal of virulence factors such as toxins, adhesins, effectors and enzymes to promote colonization and infection. Translocation of virulence factors across the membrane to either the extracellular environment or directly into the host cells is performed by single or multiple dedicated secretion systems. These secretion systems are often key to the infection process. They can range from simple single-protein systems to complex injection needles made from dozens of subunits. Here, we review the different types of secretion systems in Gram-negative bacterial fish pathogens and describe their putative roles in pathogenicity. We find that the available information is fragmented and often descriptive, and hope that our overview will help researchers to more systematically learn from the similarities and differences between the virulence factors and secretion systems of the fish-pathogenic species described here.

Simple Summary Catfish aquaculture is a prominent agricultural sector for foodfish production in the Southern United States. Catfish producers often experience high-level mortality events due to bacterial pathogens. In many instances, co-infections caused by multiple bacterial fish pathogens are isolated during diagnostic cases. These bacterial–bacterial interactions may alter the infection dynamics, and many of these mechanisms and interactions remain unclear. Furthermore, these co-infections may complicate disease management plans and treatment strategies. The current review provides an overview of the prevalent bacterial pathogens in catfish culture and previously reported instances of co-infections in catfish and other production fish species. Abstract Catfish production is a major aquaculture industry in the United States and is the largest sector of food fish production. As producers aim to optimize production yields, diseases caused by bacterial pathogens are responsible for high pond mortality rates and economic losses. The major bacterial pathogens responsible are Edwardsiella ictaluri, Aeromonas spp., and Flavobacterium columnare. Given the outdoor pond culture environments and ubiquitous nature of these aquatic pathogens, there have been many reports of co-infective bacterial infections within this aquaculture sector. Co-infections may be responsible for altering disease infection mechanics, increasing mortality rates, and creating difficulties for disease management plans. Furthermore, proper diagnoses of primary and secondary pathogens are essential in ensuring the correct treatment approaches for antimicrobials and chemical applications. A thorough understanding of the interactions and infectivity dynamics for these warm water bacterial pathogens will allow for the adoption of new prevention and control methods, particularly in vaccine development. This review aims to provide an overview of co-infective pathogens in catfish culture and present diagnostic case data from Mississippi and Alabama to define prevalence for these multiple-species infections better.

Indonesia become a maritime country with large of ocean area and beach line with marine species diversity reaches 37% of the world’s fish. This makes Indonesia have aquaculture potential that supported by an appropriate climate. However, the challenge of viral diseases caused by Infectious Spleen and Kidney Necrosis Virus (ISKNV) and Viral Nervous Necrosis (VNN) would hamper marine aquaculture and causing huge economic losses. Therefore, it is necessary to carry out early detection methods that are efficient, fast, precise and accurate to identifying these viral disease. Here we developed a multiplex-PCR (mPCR) a method that can detect simultaneously of the ISKNV and VNN. The results of this method showed high sensitivity and specificity by using MCP and CP gene target primers to detect ISKNV and VNN, respectively. From a total of 353 samples of seawater fish examined by mPCR, positive results following of single infection of ISKNV and VNN were 14 and 2, respectively. We also found that 18 grouper fish was co-infection with these viruses. From these results, it shows that the mPCR method developed has efficiency, faster and high accuracy. This founding was possible to be applied in laboratory testing or early detection system in the field for ISKNV and VNN.

This study aimed to evaluate the protective effects of four autochthonous bacteria isolated from juvenile sole (Solea senegalensis) intestine as dietary probiotic supplement against bacterial pathogen infection and handling/transport stressors. Growth performance and immune responses were evaluated after 85 days of feeding trial. Sole (IBW = 16.07 ± 0.11 g) were fed six experimental diets, a control diet (CTRL, without the dietary probiotic supplementation), and five diets supplemented with probiotic bacteria: PB1 (Shewanella hafniensis), PB2 (Enterococcus raffinosus), PB3 (Shewanella hafniensis + Arthrobacter soli), PB4 (Pseudomonas protegens + Arthrobacter soli) and PB5 (Shewanella hafniensis + Arthrobacter soli + Enterococcus raffinosus). All bacteria were selected based on their in vitro antimicrobial activity. After the growth trial, fish were submitted to a stress factor (transport) and then each dietary group was divided in two additional groups: non-infected (placebo) and infected with Photobacterium damselae subsp. piscicida. Immune and antioxidant responses were evaluated at day 10 post-infection. In infection trial A, fish were infected on the same day of transport, whereas in trial B fish were infected after a 7-day recovery from the transport stress. At the end of the feeding trial, fish fed with PB2 and PB4 showed lower final body weight when compared with the other dietary groups. Respiratory burst activity and nitric oxide production were not affected by probiotic supplementation. Fish fed with PB5 presented lower peroxidase activity compared to CTRL. Lysozyme and alternative complement pathway activity (ACH50) showed no significant differences between treatments. The innate immune responses were significantly affected after handling stress and bacterial infection. In trial A, the ACH50 levels of infected fish were significantly lower than the placebo groups. On the other hand, in trial B fish infected with Pdp demonstrated higher ACH50 levels when compared to placebos. Peroxidase levels were strongly modulated by bacterial infection and handling stress. In trials A and B, infection had a clear downgrade effect in peroxidase levels. Lipid peroxidation, catalase, glutathione S-transferase and glutathione reductase were altered by both bacterial infection and transport. Overall, dietary probiotic supplementation did not influence growth performance of sole. The immune and oxidative defenses of sole responded differently to infection depending on the probiotic and the synergy between pathogen infection and transport. © 2018 The current study was partially supported by the project PROBIOSOLEA (reference 13551), co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN) , through the European Regional Development Fund . The current study was also supported by the Research Line INSEAFOOD of the project INNOVMAR-Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035 ), funded by the Northern Regional Operational Programme (NORTE 2020) through the ERDF. B. Costas was supported by FCT, Portugal ( IF/00197/2015 ).

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis. The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish (Danio rerio) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod (Gadus morhua L.) and Nile tilapia (Oreochromis niloticus). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Streptococcus agalactiae is an invasive multi-host pathogen that causes invasive diseases mainly in newborns, elderly, and individuals with underlying health complications. In fish, S. agalactiae causes streptococcosis, which is characterized by septicemia and neurological signs, and leads to great economic losses to the fish farming industry worldwide. These bacteria can be classified into different serotypes based on capsular antigens, and into different sequence types (ST) based on multilocus sequence typing (MLST). In 2015, serotype III ST283 was identified to be associated with a foodborne invasive disease in non-pregnant immunocompetent humans in Singapore, and the infection was related to raw fish consumption. In addition, a serotype III strain isolated from tilapia in Brazil has been reported to be resistant to five antibiotic classes. This specific serotype can serve as a reservoir of resistance genes and pose a serious threat to public health. Thus, new approaches for the control and treatment of S. agalactiae infections are needed. In the present study, 24 S. agalactiae serotype III complete genomes, isolated from human and fish hosts, were compared. The core genome was identified, and, using bioinformatics tools and subtractive criteria, five proteins were identified as potential drug targets. Furthermore, 5,008 drug-like natural compounds were virtually screened against the identified targets. The ligands with the best binding properties are suggested for further in vitro and in vivo analysis.

Salmon gill poxvirus (SGPV) can cause serious gill disease in Atlantic salmon (Salmo salar L.) and represents a significant problem to aquaculture industries in Northern Europe. Here, a single-tube multi-locus variable-number tandem-repeat (VNTR) analysis (MLVA) genotyping assay, targeting eight VNTR loci, was developed for studying the epizootiology of SGPV. Through MLVA typing of SGPV positive samples from 180 farmed and wild Atlantic salmon in Northern Europe, the first molecular population study of this virus was undertaken. Comparison of resulting MLVA profiles by cluster analysis revealed considerable micro-diversity, while only a limited degree of specific clustering by country of origin could be observed, and no clustering relating to the severity of disease outbreaks. Phylogenetic analysis, based on genomic data from six SGPV specimens (three Norwegian, one Scottish, one Faroese and one Canadian), complemented and corroborated MLVA by pointing to a marked transatlantic divide in the species, with one main, relatively conserved, SGPV lineage as predominant in Europe. Within certain fjord systems and individual freshwater salmon smolt farms in Norway, however, discrete MLVA clustering patterns that prevailed over time were observed, likely reflecting local predominance of specific SGPV sub-lineages. MLVA typing was also used to refute two suspected instances of vertical SGPV transmission from salmon broodstock to offspring, and to confirm a failed disinfection attempt in one farm. These novel insights into the previously undocumented population structure of SGPV provide important clues, e.g., regarding the mechanisms underlying spread and recurrence of the virus amongst wild and farmed salmon populations, but so far no indications of more or less virulent SGPV sub-lineages have been found. The MLVA scheme represents a highly sensitive genotyping tool particularly well suited for illuminating SGPV infection routes, and adds to the relatively low number of MLVA protocols that have so far been published for viral species. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed within a single working day. Resulting MLVA profiles can be readily shared and compared across laboratories, facilitating rapid placement of samples in an international ezpizootiological context.

The treatment of bacterial diseases in aquaculture is done using antibiotics, their applications has resulted in contamination and bacterial resistance. Natural extracts are a potential alternative as an antimicrobial, they have demonstrated effectiveness in their use aimed at treating conditions. The purpose of this study was to evaluate the antimicrobial activity of Lemna minor extracts against Pseudomonas fluorescens with different solvent for extraction. Methanol, chloroform and hexane were used. Subsequently, the safety assessment of the extracts in Danio rerio embryos and larvae was performed to validate as ecologically harmless. Antibacterial activity was detected in three extracts with significant differences (p = 0.001). Hexane extract had the highest antibacterial activity, followed by chloroform and methanol extracts. The three extracts have differences with respect to the control, between times and concentrations tested (p = 0.001). Minimum inhibitory concentration values (MIC) at 24 h methanolic extract ME 0.05 µg mL−1. In embryos and larvae increased safety of the LC50 methanolic extract was evidenced followed by the hexane and chloroform extract. No morphological or tissue changes were observed in embryos and larvae. The hexane extracts of L. minor had a greater bactericidal effect against P. fluorescens and are functional because of their antibacterial activity, but methanolic extract is more safety in embryos and larvae of D. rerio, making it a potential alternative for use in the treatment and control of septicemia in fish.

Background The majority of infectious diseases of cultured fish is caused by bacteria. Rapid identification of bacterial pathogens is necessary for immediate management. The present study developed a custom Main Spectra Profile (MSP) database and validate the method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of fish bacterial pathogens. Streptococcus agalactiae, Streptococcus iniae, Aeromonas hydrophila, Aeromonas veronii, and Edwardsiella tarda obtained from diseased fish were used as representative bacterial pathogens in this study. Bacterial peptides were extracted to create a Main Spectra Profile (MSP), and the MSPs of each bacterial species was added into the MALDI Biotyper database. Fifteen additional isolates of each bacterial species were tested to validate the utilized technique. Results The MSPs of all field isolates were clearly distinguishable, and the MSPs of the same species were clustered together. The identification methodology was validated with 75 bacterial isolates. The reliability and specificity of the method were determined with MALDI Biotyper log score values and matching results with 16 s rDNA sequencing. The species identification using the public MALDI Biotyper library (Bruker MALDI Biotyper) showed unreliable results (log score 2.115, and a 100% matching result. Conclusion This study demonstrates an effective identification of fish bacterial pathogens when a complete custom-made MSP database is applied. Further applications require a broad, well-established database to accommodate prudent identification of many fish bacterial pathogens by MALDI-TOF MS.

Vibriosis caused byListonella anguillarumwas reported in several fish species from both fresh and saltwater conditions. This pathogen causes disease in rainbow trout, sea bass, and sea bream in Turkey, however, it has not been reported from meagre (Argyrosomus regius) before. Great loss of meagre was observed in the Recirculated Aquaculture System at the Faculty of Fisheries of Izmir Katip Celebi University, which had been transferred from a commercial hatchery for a nutrition experiment. Clinical signs of vibriosis were observed in infected fish, i.e. haemorrhage in the anal area and pectoral fins, mostly as tail ulcers. Petechial haemorrhages in the muscle, liver, peritoneal membranes and pyloric caeca were determined by necropsy. A Gram-negative, rod-shaped, motile bacterium was isolated, showing a positive reaction to oxidase, catalase and gelatin tests, and being sensitive to O/129. Biochemical identification tests and PCR amplifications identified the bacterium asListonella anguillarum. In slide agglutination test with antiL. anguillarumO1 (ATCC43305) serum, all isolates were positive. The isolated bacteria was resistant to oxytetracycline, sensitive to enrofloxacin, flumequine, phosphomycin, furozulidone, kanamycin and oxolinic acid. In this study, the isolated bacteria from meagre were determined asListonella anguillarumO1 with biochemical, moleculer identification and agglutination tests.

Streptococcus parauberis is an alpha-hemolytic gram positive coccoid bacterium belonging to the Streptococcaceae family. This bacterium cause streptococcosis is a major disease in cultured fish due to the intensification of aquaculture and causes significant economic losses in fish farm industry. In our study, we isolated a total of 37 lactic acid bacteria from wild fish (Sander lucioperca, Carassius gibelio, Cyprinus carpio) of Lake Eğirdir and cultured fish (Oncorhynchus mykiss, Walbaum) of farm in Turkey. For the isolation of bacteria phenotypic and biochemical characteristics of the 37 isolates obtained from the colonies grown on MRS, TSA and M17 agar characterized by determining colony morphology, cell morphology, motility, gram staining and the production of cytochrome oxidase and catalase. Further biochemical characteristics were determined using conventional tests according to Bergey’s Manual of Systematic Bacteriology. From the isolated lactic acid bacteria 25 of were identified as S. parauberis, 2 of as Vagococcus sp., 2 of as Lactococcus garvieae and 5 of as Lactococcus lactis by culture-based, biochemical test and 16Sr RNA gene sequencing techniques but 3 of them couldn’t (F65, F49 and F50) identified. In this study, the main objective of us is to identify S. parauberis with conventional and 16Sr RNA gene sequencing techniques from wild fish in Lake Eğirdir and in cultured fish in fish farm of Egirdir-Turkey.

As they integrate into recipient food webs, invasive exotic species may influence the population dynamics of native parasites. Here we assess the potential impact of the Eurasian round goby (Neogobius melanostomus) on the abundance of eyeflukes of the genus Diplostomum, which are common parasites in fishes of the St. Lawrence River (Canada). Analyses of data collected over nearly two decades revealed that the infection levels in three native fish [spottail shiner (Notropis hudsonius), golden shiner (Notemigonus crysoleucas) yellow perch (Perca flavescens)] declined sharply throughout the St. Lawrence River after the introduction of the goby. At two sites where data were collected at regular time intervals, declines of Diplostomum spp. in spottail shiners occurred within two years of the goby's first recorded appearance, with prevalence dropping as much as 77–80% between pre-invasion and post-invasion periods. Furthermore, in localities where gobies remained scarce, infection in native species did not change significantly over time. Altogether, these observations suggest that gobies play a role in the eyefluke collapse. The decline in populations of the main definitive host (ring-billed gulls, Larus delawarensis) and changes in hydrology during periods of parasite recruitment were not strongly supported as alternate explanations for this phenomenon. Since other snail-transmitted trematodes with similar life cycles to Diplostomum spp. did not show the same decreasing pattern, we conclude that eyeflukes did not decline as a result of snail depletion due to goby predation. Rather, we suggest that gobies acted as decoys, diluting the infection. As Diplostomum spp. occurred at lower abundance in gobies than in native fish hosts, the replacement of native fish with exotic gobies in the diet of gulls might have played a part in reducing parasite transmission. In contrast to the typically negative impact of invasions, the goby-induced decline of this pathogen may have beneficial effects for native fishes., Graphical abstract Image 1, Highlights • Fish infection by eyeflukes sharply declined in the St. Lawrence River. • Eyeflukes collapse occurred after the establishment of the invasive round goby. • Eyeflukes remained abundant in areas not invaded by the round goby. • A round goby-induced dilution effect is the most strongly supported explanation. • Decline of this pathogenic parasite may have beneficial effects for native fishes.

Saprolegniasis, caused by Saprolegnia infection, is one of the most common diseases in freshwater fish. Our study aimed to determine the epidemiological characteristics of saprolegniasis in Chinese regions of high incidence. Saprolegnia were isolated and identified by morphological and molecular methods targeting the internal transcribed spacer (ITS) ribosomal DNA (rDNA) and building neighbor-joining (NJ) and maximum parsimony (MP) phylogenetic trees. The ITS sequences of eight isolated strains were compared with GenBank sequences and all strains fell into three clades: CLADE1 (02, LP, 04 and 14), CLADE2 (S1), and CLADE3 (CP, S2, L5 and the reference ATCC200013). Isolates 02 and LP shared 80% sequence similarity with S. diclina, S. longicaulis, S. ferax, S. mixta, and S. anomalies. Further, isolates 04 and 14 shared 80% similarity with S. bulbosa and S. oliviae. Finally, extremely high ITS sequence similarities were identified between isolates S1 and S. australis (100%); CP and S. hypogyna (96%); and S2, L5, ATCC200013 and S. salmonis (98%). This research provides insights into the identification, prevention and control of saprolegniasis pathogens and the potential development of effective drugs.

Background Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. Results In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar. Conclusions Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.

Made available in DSpace on 2021-06-25T10:45:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-25 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Among the antimicrobial peptides (AMPs), which act as natural antibiotics in preventing the colonization of pathogens, LL-37 is noteworthy. It is a human cathelicidin characterized as a broad-spectrum antibiotic, with chemotactic and immunomodulatory properties. This study aimed to evaluate the antimicrobial activity and therapeutic effectiveness of peptide LL-37 against streptococcosis caused by Streptococcus agalactiae in Nile tilapia and to assess some of its potential adverse effects (in vivo haematological, biochemical, and immunological profiles) in the fish. After in vitro cytotoxicity studies of LL-37 against Nile tilapia red blood cells (RBCs) and antimicrobial susceptibility testing (minimum inhibitory concentration and minimum bactericidal concentration, MIC and MBC, respectively) against Aeromonas hydrophila and S. agalactiae, fish were divided into six experimental groups. G1 and G2 were challenged by intraperitoneal injection with S. agalactiae and received a single oral dose of LL-37 (15 mg/kg bw) and florfenicol (FFC) (15 mg/kg bw), respectively. G3 was challenged with S. agalactiae and received a single intraperitoneal dose of LL-37 (15 mg/kg bw). The other groups were controls for infection (G4) and antimicrobial administration (G5 and G6). LL-37 showed weak hemolytic activity against Nile tilapia RBCs (24.4% lysis at 625 μg/mL) and strong in vitro antimicrobial activity against S. agalactiae (MIC and MBC = 31.25 μg/mL). No significant effects were observed in the haematological, biochemical, and immunological profiles of fish medicated with LL-37 or FFC, compared to the control fish. Twenty days after the challenge, survival rates were 33.38% (G1), 38.51% (G2), 23.12% (G3), 25% (G4), and 100% (G5 and G6). Despite the strong in vitro antimicrobial activity, both antimicrobials were ineffective to control streptococcosis caused by S. agalactiae in Nile tilapia. These findings suggest that administration of LL-37 (15 mg/kg bw) is safe for Nile tilapia but not effective to control streptococcosis. Graduate Program in Agricultural and Livestock Microbiology São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences Universidade Zambeze (UniZambeze) Faculdade de Ciências Agrárias Post-graduate Program in Aquaculture Laboratory of Microbiology and Parasitology of Aquatic Organisms São Paulo State University (Unesp) Aquaculture Center of Unesp Department of Biochemistry and Technological Chemistry Institute of Chemistry São Paulo State University (Unesp) Registro Experimental Campus São Paulo State University (Unesp), Registro Graduate Program in Agricultural and Livestock Microbiology São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences Post-graduate Program in Aquaculture Laboratory of Microbiology and Parasitology of Aquatic Organisms São Paulo State University (Unesp) Aquaculture Center of Unesp Department of Biochemistry and Technological Chemistry Institute of Chemistry São Paulo State University (Unesp) Registro Experimental Campus São Paulo State University (Unesp), Registro

Cutaneous microbiota play an important role in protecting fish against pathogens. Aphanomyces infection causes epizootic ulcerative syndrome (EUS) in fish, and by perturbing the integrity of the cutaneous microbiota, increases the potential for infection by pathogenic bacteria. However, whether the composition of the cutaneous microbiota is altered in fish with EUS, and if so, which species are changed and how this might influence infected fish, is still largely unclear. Considering the importance of cutaneous microbiota in maintaining host health, we hypothesized that Aphanomyces infection significantly enhances the presence of certain bacterial pathogens in the cutaneous microbiota and causes cutaneous dysbacteriosis. To test this hypothesis, we compared the cutaneous microbiota compositions of hybrid snakehead (Channa maculata♀ × Channa argus♂) with and without Aphanomyces infection using Illumina Miseq sequencing of the 16S rRNA gene. Our results showed that the cutaneous microbiota of hybrid snakehead were significantly altered subsequent to EUS infection and that the numbers of potentially pathogenic bacteria classified into the genera Anaerosinus, Anaerovorax, Dorea, and Clostridium were significantly enhanced in the cutaneous microbiota of hybrid snakehead with EUS, whereas bacteria classified into the genera Arthrobacter, Dysgonomonas, Anoxybacillus, Bacillus, Solibacillus, Carnobacterium, Lactococcus, Streptococcus, Achromobacter, Polynucleobacter, Vogesella, and Pseudomonas were significantly reduced. These results imply that treatment for EUS should not only take into consideration the control of Aphanomyces reproduction but should also focus on regulating the cutaneous microbiota of infected fish.

International audience; Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum. At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STS), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, 5T275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species. IMPORTANCE Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

In this study we describe the anti- Trichodina effects of teflubenzuron (TFB) for Oreochromis niloticus and for Piaractus mesopotamicus . We also evaluated the acute toxicity, for both species, by using TFB in the concentrations of 700.0, 800.0, 900.0 and 1000.0 mg L −1 and a control, without the drug. To assess the efficacy of TFB against Trichodina spp., we used the concentrations of 30.0 or 50.0 mg L −1 for one hour exposure in tilapia, and the concentration of 30.0, 50.0 and 80.0 mg L −1 for one hour and 50 mg L −1 for two hours exposures in pacu. Teflubenzuron did not present significant toxicity in either species, with LC50;48h > 1000.0 mg L −1 . The drug effectiveness was observed against four identified Trichodina species: T. magna, T. heterodentata , T. compacta and T. centrostrigeata , with 87.9% parasite reduction with one hour exposure to 50.0 mg L −1 TFB on O. niloticus and 96.1% with two hours exposure to 50.0 mg L −1 TFB on P. mesopotamicus . Teflubenzuron is a drug with potential to be used in Brazilian aquaculture; it attends to important requirements, such as low toxicity and high efficacy in controlling Trichodina spp. infection in O. niloticus and P. mesopotamicus .

This study aimed to evaluate the current status on the use of probiotics, disinfectants and antimicrobials in hatcheries, nurseries and grow-out farms producing Pangasius catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam. Semi-structured interviews were conducted with 83 aquaculture enterprises (15 hatcheries, 32 nurseries and 36 grow-out farms). Farmers reported use of a total of 24 different antimicrobials, e.g. for treatment of bacillary necrosis and motile aeromonad septicaemia, and a variety of disinfectants, probiotics and nutritional supplements. In contrast to small-scale farmers, all large-scale grow-out farmers studied were certified and therefore had higher levels of formal education and specialized aquaculture training to diagnose and treat diseases. All farmers prepared their own medicated feed with a high risk of treatment failure, negative environmental impact from released antimicrobials and resistance development. Small-scale farmers were at particular occupational health risks when handling antimicrobials and other chemicals, e.g. mixing medicated feed with bare hands. There is an urgent need to improve knowledge and use innovative approaches, e.g. private-public partnerships, to assure a prudent use of chemicals, to improve capacity and access to disease diagnosis, particularly for small-scale grow-out farmers and nurseries. Efforts to control use of antimicrobials in aquaculture should be coordinated with the livestock and human health sectors taking an One-Health approach.

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.

In recent years, with the advent of next-generation sequencing along with the development of various bioinformatics tools, RNA sequencing (RNA-Seq)-based transcriptome analysis has become much more affordable in the field of biological research. This technique has even opened up avenues to explore the transcriptome of non-model organisms for which a reference genome is not available. This has made fish health researchers march towards this technology to understand pathogenic processes and immune reactions in fish during the event of infection. Recent studies using this technology have altered and updated the previous understanding of many diseases in fish. RNA-Seq has been employed in the understanding of fish pathogens like bacteria, virus, parasites, and oomycetes. Also, it has been helpful in unraveling the immune mechanisms in fish. Additionally, RNA-Seq technology has made its way for future works, such as genetic linkage mapping, quantitative trait analysis, disease-resistant strain or broodstock selection, and the development of effective vaccines and therapies. Until now, there are no reviews that comprehensively summarize the studies which made use of RNA-Seq to explore the mechanisms of infection of pathogens and the defense strategies of fish hosts. This review aims to summarize the contemporary understanding and findings with regard to infectious pathogens and the immune system of fish that have been achieved through RNA-Seq technology.

The Kievka Bay is one of the cleanest regions on the Far East coast of Russia. There are no anthropogenic impacts on the coastal area, and thus the influence of pollutants on the state of fish health is minimized. Histopathological examination of black plaice was performed to study the background level of morbidity and the occurrence of pathological alterations in tissues of fi shes from this ecologically clean region. Different pathological alterations were detected in fi shes sampled. The most surprising was a disease of black plaice, where necrotic changes were found in the gonads of 28% of the males, and the destructive changes were absent in females. In the gills, pathogenic microorganisms were detected, which induced strong hyperplasia of the respiratory epithelium, fusion of lamellas, and necrosis of pillar cells, in 28% of the individuals. Diseases of the liver were recorded for two fishes that comprised 8% of the total number, in one case – granulomatous inflammation. Besides that some fishes had a diffuse necrosis in the liver. In the kidneys of all sampled fish, parasites were present in the lumen of the renal duct, very likely a moderately pathogenic species, as the consequences of parasitism were insignificant and for a smaller part of fishes – moderate. Thus, different pathological alterations were found in all investigated organs, and, in most cases, it was possible to establish their occurrence with parasitism.

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.

Yersinia ruckeri is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of Y. ruckeri but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to Y. ruckeri virulence. This review summarizes the knowledge on Y. ruckeri virulence factors. Understanding the molecular pathogenicity of Y. ruckeri will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.

The use of plants for herbal medicine in the Indonesian aquaculture is still poorly known. The present study aimed to provide an inventory of the plants used by fish farmers, establishing their respective ethnobotanic importance and identifying the variables that determine the use and the choice of these plants in fish health management. A survey based on a semi-directive questionnaire was conducted using a representative sample of fish farmers (n = 504 from 176 villages) from the province of West Java. Of these fish farmers, 46% [41%, 50%; CI95%] of them use plants in their farms and 79 species of plants belonging to 36 families have been identified. Most of these plants were common plants used also traditionally in human pharmacopeia. Four categories of plant use were identified namely: improvement of water quality; reduction of fish stress; increase of fish resistance to pathogens; and treatment of fish diseases (when an outbreak occurred). In order to appraise the significance of plant usage, the following ethnobotanic parameters were determined: the Use Value (UV), the Fidelity Level (FL) and the Informant Consensus Factor (ICF). The Use Value (UV) was generally low for plants except for Carica papaya which reached the highest UV scores. The majority of the plants were used according to personal experience of the fish farmers and the knowledge related to herbal therapy appeared variable among fish farmers. Only 26 species of plants had a UV > 0.025. The highest Fidelity Level (FL) value was obtained for C. papaya. The Informant Consensus Factor (ICF) of each plant usage was relatively high for all four categories of use – ranging from 0.78 to 0.88 – but the same plant may have several therapeutic indications. The use of plants, as well as their variety and number, depended not only on fish species, production systems and production areas but also on social characteristics such as the professional experience of fish farmers and their ethnic origin. To our knowledge, this is the first ethnobotanic survey specifically applied to aquaculture. It was able to identify the species of plants used in the West Java Province, and how they are used. It also highlighted the significance of traditional Indonesian herbal therapy in aquaculture. The use of several plants in aquaculture is reported for the first time in this study.

Background Significant increases in the bacterial resistance to various antibiotics have been found in fish farms. Non-antibiotic therapies for infectious diseases in aquaculture are needed. In recent years, light-emitting diode technology has been applied to the inactivation of pathogens, especially those affecting humans. The purpose of this study was to assess the effect of blue light (wavelengths 405 and 465 nm) on seven major bacterial pathogens that affect fish and shellfish important in aquaculture. Results We successfully demonstrate inactivation activity of a 405/465-nm LED on selected bacterial pathogens. Although some bacteria were not fully inactivated by the 465-nm light, the 405-nm light had a bactericidal effect against all seven pathogens, indicating that blue light can be effective without the addition of a photosensitizer. Photobacterium damselae, Vibrio anguillarum, and Edwardsiella tarda were the most susceptible to the 405-nm light (36.1, 41.2, and 68.4 J cm−2, respectively, produced one log reduction in the bacterial populations), whereas Streptococcus parauberis was the least susceptible (153.8 J cm−2 per one log reduction). In general, optical density (OD) values indicated that higher bacterial densities were associated with lower inactivating efficacy, with the exception of P. damselae and Vibrio harveyi. In conclusion, growth of the bacterial fish and shellfish pathogens evaluated in this study was inactivated by exposure to either the 405- or 465-nm light. In addition, inactivation was dependent on exposure time. Conclusions This study presents that blue LED has potentially alternative therapy for treating fish and shellfish bacterial pathogens. It has great advantages in aspect of eco-friendly treating methods differed from antimicrobial methods.

The expanding aquaculture industry continues to encounter major challenges from highly contagious viruses. Control and eradication measures for lethal and economically damaging notifiable viral diseases involve ‘stamping out’ policies and surveillance strategies. Mass-culling of stock and restricted movement of fish and fish products, used to control the spread of notifiable diseases, has considerable impacts on the trade of fish products. Although effective, these measures are expensive and ethically complex and could possibly be reduced by emulating innovative vaccination strategies used by the terrestrial livestock industry. DIVA (differentiating infected from vaccinated animal) strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced during infection can be used to distinguish from those induced by vaccination. The potential and feasibility of DIVA vaccination in aquaculture is explored here with reference to DIVA strategies applied in higher vertebrates. Three economically important notifiable viruses, causing major problems in three different cultured fish industries, are considered. The increased availability and application of sophisticated biotechnology tools has enabled improved prophylaxis and serological diagnosis for control of viral haemorrhagic septicaemia in rainbow trout, infectious salmon anaemia in Atlantic salmon and koi herpesvirus disease in carp. Improving the specificity of serological diagnostics in aquaculture in conjunction with suitable vaccines could enable the application of DIVA strategies, but the immunological variation between different fish species and contrasting pathobiological characteristics of different viruses determines the feasibility and potential of such DIVA approaches for aquaculture industries.

The majority of mesophilic waterborne species of the black yeast genus Exophiala (Chaetothyriales) belong to a single clade judging from SSU rDNA data. Most taxa are also found to cause cutaneous or disseminated infections in cold-blooded, water animals, occasionally reaching epidemic proportions. Hosts are mainly fish, frogs, toads, turtles or crabs, all sharing smooth, moist or mucous skins and waterborne or amphibian lifestyles; occasionally superficial infections in humans are noted. Cold-blooded animals with strictly terrestrial life styles, such as reptiles and birds are missing. It is concluded that animals with moist skins, i.e. those being waterborne and those possessing sweat glands, are more susceptible to black yeast infection. Melanin and the ability to assimilate alkylbenzenes are purported general virulence factors. Thermotolerance influences the choice of host. Exophiala species in ocean water mostly have maximum growth temperatures below 30 °C, whereas those able to grow until 33(– 36) °C are found in shallow waters and occasionally on humans. Tissue responses vary with the phylogenetic position of the host, the lower animals showing poor granulome formation. Species circumscriptions have been determined by multilocus analyses involving partial ITS, TEF1, BT2 and ACT1.

Early-rearing salmonids in Ontario, Canada government fish hatcheries have been persistently affected by bacterial gill disease (BGD), and outbreaks at these locations have often been associated with high morbidity and mortality. The causative agent of BGD, Flavobacterium branchiophilum, is ubiquitous in fresh water, and outbreaks of BGD are considered to be associated with deleterious environmental conditions. This paper summarizes a 14-month rearing unit-level prospective nested matched case–control investigation at six Ontario government hatcheries (raising a total of six different salmonid species) to identify, and quantify the effects of, important predictors of BGD outbreaks. Ongoing husbandry data were collected on all early-rearing (

Vibrio harveyi, which now includes Vibrio carchariae as a junior synonym, is a serious pathogen of marine fish and invertebrates, particularly penaeid shrimp. In fish, the diseases include vasculitis, gastro-enteritis and eye lesions. With shrimp, the pathogen is associated with luminous vibriosis and Bolitas negricans. Yet, the pathogenicity mechanisms are imprecisely understood, with likely mechanisms involving the ability to attach and form biofilms, quorum sensing, various extracellular products including proteases and haemolysins, lipopolysaccharide, and interaction with bacteriophage and bacteriocin-like substances.

Bacteriophages have been proposed as an alternative to antibiotic usage and several studies on their application in aquaculture have been reported. This review highlights progress to date on phage therapies for the following fish and shellfish diseases and associated pathogens: hemorrhagic septicemia (Aeromonas hydrophila) in loaches, furunculosis (Aeromonas salmonicida) in trout and salmon, edwardsiellosis (Edwardsiella tarda) in eel, columnaris disease (Flavobacterium columnare) in catfish, rainbow trout fry syndrome or cold water disease (Flavobacterium psychrophilum) in trout and salmon, lactococcosis (Lactococcus spp.) in yellowtail, ulcerative skin lesions (Pseudomonas aeruginosa) in freshwater catfish, bacterial hemorrhagic ascites disease (Pseudomonas plecoglossicida) in ayu fish, streptococcosis (Streptococcus iniae) in flounder, and luminescent vibriosis (Vibrio harveyi) in shrimp. Information is reviewed on phage specificity, host resistance, routes of administration, and dosing of fish and shellfish. Limitations in phage research are described and recommended guidelines are provided for conducting future phage studies involving fish and shellfish.

Background Betanodaviruses are the causative agents of Viral Encephalopathy and Retinopathy (VER). To date, more than 50 species have proved to be susceptible and among them, those found in genus Epinephelus are highly represented. Clinical disease outbreaks are generally characterized by typical nervous signs and significant mortalities mainly associated with aquaculture activities, although some concerns for the impact of this infection in wild fish have been raised. In this study, the authors present the first documented report describing an outbreak of VER in wild species in the Mediterranean basin. Case presentation In late summer - early winter 2011 (September-December), significant mortalities affecting wild Dusky grouper (Epinephelus marginatus), Golden grouper (Epinephelus costae) and European sea bass (Dicentrarchus labrax) were reported in the municipality of Santa Maria di Leuca (Northern Ionian Sea, Italy). The affected fish showed an abnormal swimming behavior and swollen abdomens. During this epizootic, five moribund fish showing clear neurological signs were captured and underwent laboratory investigations. Analytical results confirmed the diagnosis of VER in all the specimens. Genetic characterization classified all betanodavirus isolates as belonging to the RGNNV genotype, revealing a close genetic relationship with viral sequences obtained from diseased farmed fish reared in the same area in previous years. Conclusion The close relationship of the viral sequences between the isolates collected in wild affected fish and those isolated during clinical disease outbreaks in farmed fish in the same area in previous years suggests a persistent circulation of betanodaviruses and transmission between wild and farmed stocks. Further investigations are necessary to assess the risk of viral transmission between wild and farmed fish populations, particularly in marine protected areas where endangered species are present.

11 pages, 4 figures, Jellyfish are of particular concern for marine finfish aquaculture. In recent years repeated mass mortality episodes of farmed fish were caused by blooms of gelatinous cnidarian stingers, as a consequence of a wide range of hemolytic, cytotoxic, and neurotoxic properties of associated cnidocytes venoms. The mauve stinger jellyfish Pelagia noctiluca (Scyphozoa) has been identified as direct causative agent for several documented fish mortality events both in Northern Europe and the Mediterranean Sea aquaculture farms. We investigated the effects of P. noctiluca envenomations on the gilthead sea bream Sparus aurata by in vivo laboratory assays. Fish were incubated for 8 hours with jellyfish at 3 different densities in 300 l experimental tanks. Gill disorders were assessed by histological analyses and histopathological scoring of samples collected at time intervals from 3 hours to 4 weeks after initial exposure. Fish gills showed different extent and severity of gill lesions according to jellyfish density and incubation time, and long after the removal of jellyfish from tanks. Jellyfish envenomation elicits local and systemic inflammation reactions, histopathology and gill cell toxicity, with severe impacts on fish health. Altogether, these results shows P. noctiluca swarms may represent a high risk for Mediterranean finfish aquaculture farms, generating significant gill damage after only a few hours of contact with farmed S. aurata. Due to the growth of the aquaculture sector and the increased frequency of jellyfish blooms in the coastal waters, negative interactions between stinging jellyfish and farmed fish are likely to increase with the potential for significant economic losses, The present work was developed within the framework of the EU- funded projects MED-JELLYRISK, (www.jellyrisk.eu) (ENPI CBCMED programme, grant no. I-A/1.3/098), and VECTORS, (http://www.marine-vectors.eu) (Vectors of Change in Oceans and Seas Marine Life, Impact on Economic Sectors, grant no. 266445). Moreover, CERES project, (http://ceresproject.eu) (Climate Change and European Aquatic Resources, grant no. 678193) belonging to European Union’s Horizon 2020 research and innovation programme has also partially supported this research

During a survey of freshwater fishes from Turkey two species of Acanthocephala, one of them new, were found. Pomphorhynchus tereticollis (Pomphorhynchidae) is reported at 24% prevalence in 37 Cobitis bilseli (Cobitidae) from Lake Beysehir, Konya, for the first time. The eoacanthoacaphalan Triaspiron aphanii gen. n. et sp. n. (Quadrigyridae), at a prevalence of 90%, is described from 29 Aphanius mento (Cyprinodontidae), from Kirkgoz Springs, Antalya. The new genus most closely resembles Raosentis Datta, 1947, both having a small spindle shaped trunk, and Acanthogyrus Thapar, 1927, both having a proboscis armature of three circles of hooks. Triaspiron differs from Raosentis in proboscis shape, cylindrical not globular, proboscis armature, three circles, a total of 16 hooks in all, not four circles, a total of 26-30 hooks in all, and trunk spination, two fields of spines in the anterior field with spines arranged in up to 40 circular rows, not a single field with 9-17 rows of spines. Triaspiron differs from Acanthogyrus in having fewer proboscis hooks, 16 compared with 18-24, arranged in three circles, one anterior and two posteriorly placed, with an unarmed region between, not three circles of hooks evenly spaced, and two fields of trunk spines, not one.

Bu çalışmada, iç su balıkları yetiştiriciliğinin en yoğun yapıldığıbölgelerden biri olan Muğla'nın Fethiye bölgesindeki gökkuşağı alabalıkçiftliklerinde en sık karşılaşılan hastalıklardan biri olan Hareketli AeromonasSeptiseminin etkeni olan Aeromonas hydrophila, A. veronii, A. caviae ve A.sobria türlerinin gen bölgelerine bakılarak Polimeraz Zincir Reaksiyonu(PCR) yöntemi ile tespiti amaçlanmıştır.Alabalık çiftliklerinden hastalık belirtisi gösteren balık örneklerihastalık olduğunda, steril ortamlarda ve soğuk zincir koşullarına uyularaklaboratuara getirilmiştir. Balıkların farklı dokularından örnek alınmış vebesiyerlerine ekilmiştir. Hem ekilen hem de direk alınan örneklerdenhastalıkla alakalı genlerin tespiti için bakteriyel DNA izolasyonu yapılmıştır.Bakterilerin belirlenmesi için seçilen gen bölgeleri A. hydrophila için Aero veOmpTS, A. veronii için tet, A.sobria için AH ve A. caviae için F3-B3 genbölgeleridir. Bu bölgeleri belirlemek için uygun PCR protokolleri seçilmiş veher bir örnek için uygulanmıştır. PCR uygulaması sonucuda örneklerelektroforezde yürütülmüş ve incelenmiştir.Bu çalışmanın sonucu olarak hastalık belirtisi gösteren balıklarınkaraciğer ve böbreklerinden izole edilen A. hydrophila, A.caviae, A. veronii, veA. sobria bakterileri ile referans bakterilerin PCR yöntemi ile çoğaltılmasısonucu elde edilen görüntüler eşleşmiştir. Klinik bulgularla desteklenen PCRbulgusu bize Hareketli Aeromonas Septisemi' yi doğrulamıştır. In this thesis, we have studied bacterial fish disease as MotileAeromonas Septicemia caused by A. hydrophila, A. veronii, A. caviae and A.sobria in Rainbow trout at Mugla (Fethiye) region.Fish samples have been collected and carried out in cold and sterileconditions from rainbow trout farm as the fish showes symptones ofdiseases. The bacterial samples have been collected from different tisues andincubated in steril medium. DNA isolates was made from those samples todetect pathogen gene related to Motile Aeromonas Septicemia. We haveselected Aero gene and OmpTS gene from A. hydrophila, tet gene from A.veronii, F3-B3 gene from A. caviae and AH gene from A. sobria to detectbacteria. PCR was optimised and samples were electrophoresed.As a result of this study, A. hydrophila, A. veronii, A. caviae and A.sobria has seen as positive PCR results in liver and kidney tissue. 86

Title (Bahasa Indonesia): Pemetaan penyakit ikan di Provinsi Sulawesi Utara This study aims to determine fish diseases and the causative pathogens, and to map the spread of the diseases in fish farms in North Sulawesi province. The organisms studied are freshwater and salt water fish. Fish samples were taken randomly both healthy fish and fish showing clinical signs of infections. Fish samples were randomly taken, 3 fishes from each sampling location, and only 1 fish farm was selected from each regency/city. Salt water fish were taken only from regency/city that has fish farm. To support the fish disease data, some water quality parameters were also measured in situ such as: temperature, pH, dissolved oxygen (DO), brightness and ammonia concentration. Results showed that the fish samples were infected with pathogenic parasites, bacteria and viruses. The bacteria were Vibrio carchariae, Vibrio mimicus, Vibrio damsel, Vibrio alginolyticus, Aeromonas hydrophilla, Pseudomonas sp, Vibrio sp, Aeromonas sp, Flexibacter sp, Pseudomonas flourescens. A. hydrophilla is the dominant species of bacteria in all freshwater fish samples from all sampling locations. The parasites were Dactylogirus sp, Trichodina sp, Ichthyopthirius multifiliis, Argulus sp, Lernea sp. Parasites Trichodina sp is the dominant parasite species found. The viruses were VNN virus (Viral Nervous Necrosis) KHV virus (Koi Herpes Virus). Virus NN virus was found only in grouper cultured in the floating net of Talengen village, district Sangihe Islands. Penelitian ini bertujuan untuk mengetahui jenis-jenis penyakit ikan dan patogen penyebabnya serta memetakan penyebaran penyakit ikan di lokasi budidaya yang ada di provinsi Sulawesi Utara. Sampel yang digunakan adalah ikan air tawar dan air laut. Sampel ikan diambil secara acak baik ikan yang sehat maupun ikan yang memperlihatkan gejalah terinfeksi penyakit. Tiga ekor ikan sampel diambil dari setiap lokasi, dimana dipilih 1 lokasi budidaya di tiap kabupaten/kota. Sampel ikan air laut hanya diambil pada kabupaten/kota yang memiliki lokasi budidaya air laut. pengukuran beberapa parameter kualitas air juga dilakukan secara in situ seperti: suhu, pH, oksigen terlarut (DO), kecerahan dan kandungan amoniak. Hasil penelitian memperlihatkan bahwa sampel ikan yang diperiksa terinfeksi pathogen parasit, bakteri dan virus. Jenis bakteri patogen yang ditemukan adalah: Vibrio carchariae, Vibrio mimicus, Vibrio damsel, Vibrio alginolyticus, Aeromonas hydrophilla, Pseudomonas sp, Vibrio sp, Aeromonas sp, Flexibacter sp, Pseudomonas flourescens. Bakteri Aeromonas hydrophilla merupakan jenis bakteri yang paling dominan yang ditemukan di semua sampel ikan air tawar di lokasi-lokasi budidaya tempat pengambilan sampel. Jenis-jenis parasit yang ditemukan adalah: Dactylogirus sp, Trichodina sp, Ichthyopthirius multifiliis, Argulus sp, Lernea sp. Jenis parasit yang dominan adalah Trichodina sp. Jenis virus yang ditemukan adalah: virus VNN (Viral Nervous Necrosis), virus KHV (Koi Herpes Virus). Virus VNN ditemukan hanya pada sampel ikan kerapu yang dibudidayakan di jaring apung Kampung Talengen, Kabupaten Kepulauan Sangihe.

Correspondance: jean-francois.bernardet@jouy.inra.fr; International audience; Members of most Chryseottacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseottacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas

Whereas it is well documented that the genus Acinetobacter is associated with antibiotic resistant bacteria and human clinical infections, there are very few studies to date that report it as an emerging new pathogen for fish. In the present study, Acinetobacter lwoffii was isolated from diseased rainbow trout (Oncorhynchus mykiss) from Iranian farms, in the Khuzestan Province by both biochemical tests and polymerase chain reaction (PCR)-based methods and further confirmed as the causative agent by infectivity experiment. Uni or bilateral exophthalmia, blackening of the skin, abdominal distension, hemorrhages around the mouth cavity and eyes, basal fine, skin, gills and in internal organs were the main clinical signs in the affected fish. Phylogenetic analysis of the 16S rRNA sequence of MD77 revealed a 99% homology with that of A. lwoffii (GenBank accession no. KR856323.1) from Iran. Histopathological changes of challenged rainbow trout including focal necrosis of liver cells (hepatocyte), pyknotic nucleus and karyolysis of hepatocyte, hyperemia, fatty acid composition change of liver, infiltration of inflammatory cells, sinusoidal dilatation and congestion. The drug resistance of isolates and minimal inhibitory concentration (MIC) was examined and showed that A. lwoffii was multiresistant to 8 of the antimicrobial agents tested.

The Astacus leptodactylus specimens were collected from four sites of Aras reservoir, North-West of Iran and examined for the epibionts during 2009. Protozoan epibionts from ciliophora (one genus and seven species) and tracheophyta were isolated from the cuticular surface of different body parts of narrow-claw crayfish, A. leptodactylus. Seasonal prevalence of infestation was determined in 394 individuals of A. leptodactylus. The facultative ciliate Tetrahymena pyriformis was identified on the gills and gill haemocoel with 0.5% prevalence. Furthermore, epibiont fouling organisms such as Epistylis chrysemidis (52.3%); Vorticella similis (45.9%); Cothurnia sieboldii (68.5%); Pyxicola annulata (66%); Chilodonella spp. (0.5%); Zoothamnium intermedium (57.1%); Opercularia articulate (20.6%) and Podophrya fixa (8.6%) were also isolated from 13 body parts of A. leptodactylus. The presence of Chilodonella infestation is the first record of this genus on freshwater crayfish species. The comparison of biometrical data of the epibionts showed no significant differences in prevalence of seasonal infestation between sampling sites. The current work represents the first documentation for the presence of protozoan epibionts on A. leptodactylus in Aras Reservoir, Iran.