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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.

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Intensive aquaculture has increased the severity and frequency of fish diseases. Given the functional importance of gut microbiota in various facets of host physiology, modulation of this microbiota is a feasible strategy to mitigate emerging diseases in aquaculture. To achieve this, a fundamental understanding of the interplay among fish health, microbiota, and invading pathogens is required. This mini-review focuses on current knowledge regarding the associations between fish diseases, dysbiosis of gut microbiota, and immune responses. Furthermore, updated research on fish disease from an ecological perspective is discussed, including colonization resistance imposed by commensals and strategies used by pathogens to overcome resistance. We also propose several directions for future research, such as exploration of the causal links between fish diseases and specific taxa, and identification of universal gut microbial biomarkers for rapid disease diagnosis.

In this study, we investigated the immune responses against Mycobacterium gordonae in ginbuna crucian carp. Cumulative mortality of ginbuna injected with 2.0 × 107 CFU of M. gordonae was 50% at 170 days post-infection. CD4-1, CD8α, T-bet and IFNγ2 gene expression levels were significantly upregulated in ginbuna injected with 1.9 × 108 CFU of M. gordonae at 21 and 28 days post-infection. The CD4-2 level did not change during the experiment. Granulomatous responses consisted of central macrophage accumulation and surrounding lymphocytes, and Ziehl-Neelsen-positive bacteria were observed in the trunk kidney of the challenged fish. Immunohistochemistry using anti-ginbuna IFNγs and anti-ginbuna CD4-1 polyclonal antibody revealed that the marginal lymphocytes were positive for CD4-1, and the IFNγ-producing cells surrounded the mycobacterial cell-laden phagocytes. These results suggest that CD4-1+ cells and IFNγ2 play important roles in the granulomatous inflammation against Mycobacterial infections in teleosts.

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 ).

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.

Transcriptome analysis is a powerful tool that enables a deep understanding of complicated physiological pathways, including immune responses. RNA sequencing (RNA-Seq)-based transcriptome analysis and various bioinformatics tools have also been used to study non-model animals, including aquaculture species for which reference genomes are not available. Rapid developments in these techniques have not only accelerated investigations into the process of pathogenic infection and defense strategies in fish, but also used to identify immunity-related genes in fish. These findings will contribute to fish immunotherapy for the prevention and treatment of bacterial infections through the design of more specific and effective immune stimulants, adjuvants, and vaccines. Until now, there has been little information regarding the universality and diversity of immune reactions against pathogenic infection in fish. Therefore, one of the aims of this paper is to introduce the RNA-Seq technique for examination of immune responses in pathogen-infected fish. This review also aims to highlight comparative studies of immune responses against bacteria, based on our previous findings in largemouth bass (Micropterus salmoides) against Nocardia seriolae, gray mullet (Mugil cephalus) against Lactococcus garvieae, orange-spotted grouper (Epinephelus coioides) against Vibrio harveyi, and koi carp (Cyprinus carpio) against Aeromonas sobria, using RNA-seq techniques. We demonstrated that only 39 differentially expressed genes (DEGs) were present in all species. However, the number of specific DEGs in each species was relatively higher than that of common DEGs; 493 DEGs in largemouth bass against N. seriolae, 819 DEGs in mullets against L. garvieae, 909 in groupers against V. harveyi, and 1471 in carps against A. sobria. The DEGs in different fish species were also representative of specific immune-related pathways. The results of this study will enhance our understanding of the immune responses of fish, and will aid in the development of effective vaccines, therapies, and disease-resistant strains.

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.

The objective of the present study was to evaluate the occurrence of pathogens and diseases in laboratory fish over a 10-year period at the Centre for Experimental Fish Pathology of Sicily, University of Messina. This report also emphasizes the adverse effects of subclinical infections on research endpoints, as well as the importance of animal health with respect to welfare. Infections in fish used for research can alter experimental outcomes, increase the variability of data, and impede experimental reproducibility. For this purpose, 411 diseased fish of different species (out of a total of 2820 fish) that belonged to four marine species ( Dicentrarchus labrax, Sparus aurata, Argyrosomus regius and Mugil cephalus) and to four fresh water species ( Danio rerio, Carassius auratus, Xiphophorus variatus and Poecilia reticulata) were examined in this study. Our results showed that mycobacteriosis and myxosporidiosis were the most important diseases found in our research fish, and the results represent a useful tool to obtain wider knowledge on the incidence of various diseases in different fish species. Further studies in this field are necessary to improve knowledge on the state of the health of fish used for research.

Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease (BCWD), also referred to as rainbow trout fry syndrome (RTFS), a disease with great economic impact in salmonid aquaculture. Despite this, to date, not many studies have analyzed in depth how the immune system is regulated during the course of the disease. In the current study, we have studied the transcription of several immune genes related to T and B cell activity in the skin of rainbow trout (Oncorhynchus mykiss) naturally infected with F. psychrophilum in a farm located in Lake Titicaca (Peru). The levels of expression of these genes were tested and compared to those obtained in asymptomatic and apparently healthy rainbow trout. In the case of symptomatic fish, skin samples containing characteristic ulcerative lesions were taken, as well as skin samples with no lesions. Our results pointed to a significant local up-regulation of IgD, CD4, CD8, perforin and IFNγ within the ulcerative lesions. On the other hand, no differences between the levels of expression of these genes were visible in the spleen. To confirm these results, the distribution of IgD+ and CD3+ cells was studied through immunohistochemical techniques in the ulcerative lesions. Our results demonstrate a strong local response to F. psychrophilum in rainbow trout in which IgD and T cells seem to play a major role.

The aims of this study were to determine the prevalence and phylogenetic relationship of motile Aeromonas spp. that might be pathogenic species for rainbow trout in infected/mix infection cases (based upon different outbreaks on fish farms). A total of 99 motile Aeromonas isolates (and three reference strains) were analysed that were isolated from four different fish species in different sizes of fish (0.1-3,000 g), different months and water temperatures (6.1-21.2 degrees C). The biochemical characteristics of the isolates were determined using conventional tests and a rapid test kit. Additionally, molecular identification was performed using the gyrB housekeeping gene region and with glycerophospholipid-cholesterol acyltransferase polymerase chain reaction (GCAT-PCR). The sequencing results obtained from the gyrB gene region were deposited in the GenBank database, and phylogenetic relationships were determined with the BioNumerics 7.6 database. Nearly half of the Aeromonas isolates that were isolated from rainbow trout showing signs of disease were determined to be possible infectious agents. Aeromonas species exhibit biochemical variability for many characters, so some Aeromonas species tested negative for GCAT-PCR despite that this test was created especially for Aeromonas identification. The phylogenetic tree based upon gyrB contained 10 different phylogroups that were based on 96% cut-off value in gyrB gene region. Ministry of Food, Aqriculture and Animal Husbandry (TAGEM/14/AR-GE/26)

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.

Aquaculture is increasing rapidly to meet global seafood demand. Some hydroid populations have been linked to mortality and health issues in finfish and shellfish, but their dynamics in and around aquaculture farms remain understudied. In the present work, two experiments, each with 36 panels, tested colonization (factors: depth, season of immersion) and succession (factors: depth, submersion duration) over one year. Hydroid surface cover was estimated for each species, and data were analyzed with multivariate techniques. The assemblage of hydrozoans was species-poor, although species richness, frequency and abundance increased with time, paralleling the overall increase in structural complexity of fouling assemblages. Submersion duration and season of immersion were particularly important in determining the species composition of the assemblages in the succession and colonization experiments, respectively. Production of water-borne propagules, including medusae, from the hydroids was observed from locally abundant colonies, among them the well-known fouling species Obelia dichotoma, potentially representing a nuisance for cultured fish through contact-driven envenomations and gill disorders. The results illustrate the potential importance of fouling hydroids and their medusae to the health of organisms in the aquaculture industry.

Background Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture fish species and cause high lethality and economic loss. Current methods of controlling this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. Piscidins with broad spectrum antibacterial, antifungal and antiviral activities were found to have potent activity against C. irritans. Little, however, has been understood about the killing mechanisms of piscidins in parasites. Results In total, 57.12, 50.44, 55.86 and 47.87 million raw reads were generated from untreated theront and trophont, and piscidin (Lc-pis) treated theront and trophont libraries, respectively. After de novo assembly, 966,609 unigenes were generated with an average length of 420 bp: among these, 618,629 unigenes showed identity with sequences in one or more databases, with some showing to be significantly manipulated by Lc-pis treatment. The species classification showed that more than 25.8% unigenes from trophonts were homologous to the large yellow croaker (Larimichthys crocea) and less than 3.8% unigenes from theronts were matched. The homologous unigenes demonstrated that the tissue from host could exist in trophonts and might be transported to parasite via vesicular transports. Our analysis showed that regulatory transcripts were involved in vesicular trafficking. Among transcripts induced by Lc-pis, most genes up-regulated in treated and untreated theronts were involved in cell migration and apoptosis related pathways. Few transcripts were found to be down-regulated in treated and untreated trophonts related to cell structure and migration after treatment. Conclusions This is the first transcriptome analysis of C. irritans exposed to Lc-pis, which enhanced the genomic resources and provided novel insights into molecular mechanisms of ciliates treated by cationic antimicrobial peptide. Our comprehensive transcriptome analysis can facilitate the identification of potential drug targets and vaccines candidates for controlling this devastating fish pathogen. Electronic supplementary material The online version of this article (10.1186/s12864-018-4565-5) contains supplementary material, which is available to authorized users.

Understanding ecological and epidemiological factors driving pathogen evolution in contemporary time scales is a major challenge in modern health management. Pathogens that replicate outside the hosts are subject to selection imposed by ambient environmental conditions. Increased nutrient levels could increase pathogen virulence by pre-adapting for efficient use of resources upon contact to a nutrient rich host or by favouring transmission of fast-growing virulent strains. We measured changes in virulence and competition in Flavobacterium columnare, a bacterial pathogen of freshwater fish, under high and low nutrient levels. To test competition between strains in genotype mixtures, we developed a quantitative real-time PCR assay. We found that a virulent strain maintained its virulence and outcompeted less virulent strains independent of the nutrient level and resource renewal rate while a less virulent strain further lost virulence in chemostats under low nutrient level and over long-term serial culture under high nutrient level. Our results suggest that increased outside-host nutrient levels might maintain virulence in less virulent strains and increase their contribution to epidemics in aquaculture. The results highlight a need to further explore the role of resource in the outside-host environment in maintaining strain diversity and driving evolution of virulence among environmentally growing pathogens.

Certain types of marine fish, eaten raw or undercooked, may serve as a source of human infection with these parasites., The Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum) is the causative agent of the third most common fish-borne cestodosis among humans. Although most of the nearly 1,000 cases among humans have been reported in South America (Peru, Chile, and Ecuador), cases recently imported to Europe demonstrate the potential for spread of this tapeworm throughout the world as a result of global trade of fresh or chilled marine fish and travel or migration of humans. We provide a comprehensive survey of human cases of infection with this zoonotic parasite, summarize the history of this re-emerging disease, and identify marine fish species that may serve as a source of human infection when eaten raw or undercooked.

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 .

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.

This study presents the helminthofauna of digenean parasites of Acestrorhynchus falcirostris in the state of Amazonas (Brazil). Eight species belonging to four families were recovered: Metacercariae of Austrodiplostomum compactum (Diplostomidae) and Clinostomum marginatum (Clinostomidae) and mature specimens of Bellumcorpus majus, Prosorhynchoides carvajali, Rhipidocotyle santanaensis (Bucephalidae); Prosthenhystera obesa (Callodistomidae); Genarchella genarchella (Derogenidae) and Phyllodistomum spatula (Gorgoderidae). Examining specimens belonging to the genus Bellumcorpus collected from A. falcirostris, were observed a great variation in the size and shape of testes, which also could be observed in the type specimens of Bellumcorpus majus and B. schubarti. Considering the similar morphology and morphometric characteristics of both species, in this paper B. schubarti is considered synonym of B. majus. Acestrorhynchus falcirostris is a new host records for all these species of digenean, except to A. compactum and C. marginatum. Prosorhynchoides carvajali is referred for the first time in Brazil. Resumo Esse estudo apresenta a fauna helmintológica de Digenea parasitos de Acestrorhynchus falcirostris no estado do Amazonas. Oito espécies pertencentes a quatro famílias foram estudadas: Metacercarias de Austrodiplostomum compactum (Diplostomidae) e Clinostomum marginatum (Clinostomidae) e espécimes adultos de Bellumcorpus majus, Prosorhynchoides carvajali, Rhipidocotyle santanaensis (Bucephalidae); Prosthenhystera obesa (Callodistomidae); Genarchella genarchella (Derogenidae) e Phyllodistomum spatula (Gorgoderidae). Examinando espécimes pertencentes ao gênero Bellumcorpus coletados de A. falcirostris, foi observada uma grande variação no tamanho e forma dos testículos, que também pode ser observada nos espécimes tipo de Bellumcorpus majus e B. schubarti. Considerando a morfologia e características morfométricas similares de ambas as espécies, neste artigo, B. schubarti é considerado sinônimo de B. majus. Acestrorhynchus falcirostris representa um novo hospedeiro para todas essas espécies de Digenea, exceto para A. compactum e C. marginatum. Prosorhynchoides carvajali é referido pela primeira vez no Brasil.

Aim: Over the last decade, Citrobacter species have been responsible for infections in fish and many species and also new Citrobacter species have been identified. In this study, molecular identifications and the phenotypic and genotypic antimicrobial-resistance characteristics of atypical and typical Citrobacter species were determined. Materials and methods: Seven Citrobacter isolates were investigated from rainbow trout of different lengths with signs of disease. Biochemical characteristics were determined using conventional tests and rapid test kits; moreover, molecular identifications were conducted with 16S rRNA and the gyrB housekeeping gene region. The sequencing results obtained from the gyrB gene region were deposited in the GenBank database and compared with isolates from different countries that were registered in the database. Resistances to florfenicol, sulfonamides, and tetracycline antimicrobials were determined using the broth micro dilution method, and molecular resistance genes against these antimicrobials were identified. All detected resistance genes were confirmed by sequence analyses. Results: It was determined that three of the Citrobacter species with biochemical characteristics were atypical and showed oxidase-positive reactions. All the Citrobacter species were identified as Citrobacter sp. using the 16S rRNA gene; three isolates were identified as Citrobacter gillenii and four as Citrobacter sp. based on gyrB gene sequence analysis. Some isolates were found in the same group as other countries' isolates in the GenBank database, while isolates with high identities were found in different genogroups. All isolates were found to be phenotypically resistant to sulfamethoxazole and susceptible to tetracycline; these isolates' resistance genes included sulI, tetA, tetB, and tetD. Gıda Tarım ve Hayvancılık Bakanlığı - TAGEM/14/AR-GE/26 Scientific Researcher Department through KUAP(V)-2014/7

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.

This study evaluated the parasite fauna of farmed hybrid surubim (Pseudoplatystoma reticulatum x P. corruscans) and the host-parasite-environment relationship in two fish farms located in Mato Grosso do Sul, Central Brazil, South America. A total of 120 hybrids from two different farms, 60 in each season (30 in the hot and 30 in cold season) were examined during a year. Water quality was weekly measured to evaluate the interaction among environmental conditions and parasitism. Histopathology was used to observe the effects of the parasites and environment on the fish gills. The ciliate protozoan Ichthyophthirius multifiliis and the monogeneans (Ameloblastella sp., Amphocleithrium paraguayensis, Vancleaveus ciccinus, V. fungulus and V. janacauensis) were the most prevalent parasites detected in both seasons in both farms, with prevalence above 80%. It was stated that parasites did not cause important damage in the health status of the hybrid surubim. These results might be related to general good management practices and environmental quality implemented by the fish farmers. The presence of uncommon monogenean parasites to this hybrid compared to their parents causing an environmental and ecological concern is here discussed. Resumo Este estudo avaliou a fauna de parasitos do surubim híbrido cultivado (Pseudoplatystoma reticulatum x P. corruscans) e a relação hospedeiro-parasito-ambiente em duas pisciculturas localizadas no Estado do Mato Grosso do Sul, região Centro-Oeste, Brasil. Um total de 120 híbridos de duas fazendas, 60 em cada estação (30 na estação quente e 30 na fria), foram examinados durante um ano. A qualidade da água foi medida semanalmente para avaliar a interação entre as condições ambientais e o parasitismo. Histopatologia foi usada para observar os efeitos dos parasitos e do ambiente nas brânquias dos peixes. O protozoário ciliado Ichthyophthirius multifiliis e Monogenea (Ameloblastella sp., Amphocleithrium paraguayensis, Vancleaveus ciccinus, V. fungulus e V. janacauensis) foram os parasitos mais prevalentes detectados em ambas estações nas duas fazendas, com prevalências acima de 80%. Observou-se que os parasitos não causaram danos ao estado de saúde do surubim híbrido. Esses resultados estão relacionados às boas práticas de manejo e qualidade ambiental implementada pelos produtores. É discutida a presença incomum de Monogenea para esse híbrido, comparado com seus progenitores, podendo causar preocupação ambiental e ecológica.

We describe a hernia of the swim bladder, with a concurrent mycotic granulomatous inflammation, and carcinoma of the swim bladder in a wild mullet ( Mugil cephalus) referred for an exophytic dorsal mass. Grossly, the mass was white, soft, and composed of multiple cystic gas-containing chambers connected by a funnel-shaped tissue segment to the coelomic swim bladder. Histologically, the mass was characterized by cysts of variable size, multifocally contiguous with the subepithelial rete mirabile, supported by abundant fibrous tissue. The skin covering the herniated swim bladder was focally ulcerated and replaced by abundant granulation tissue in which multiple scattered granulomas centered on pigmented fungal hyphae were observed. These granulomas were also seen in the remaining coelomic portion of the swim bladder as well as in the spleen, perivisceral pancreas, and peritoneal adipose tissue; the fungus was molecularly identified as Cladosporium spp. Focally, arising from the herniated swim bladder epithelium, an unencapsulated poorly demarcated, moderately cellular neoplasm, composed of islands, lobules, and acini of neoplastic epithelium, was found.

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 (

A redescription of Philometra globiceps (Rudolphi 1819), the type species of Philometra Costa 1845 (Philometridae), is provided on the basis of specimens collected from the gonads, stomach, pyloric caeca, intestine and gallbladder of the marine fish Uranoscopus scaber Linnaeus (Uranoscopidae, Perciformes) caught in the Ionian Sea off Ugento, southern Italy. Light microscopical and scanning electron microscopical examinations (latter used for the first time in this species) of the specimens revealed some previously unreported morphological features, such as the location of submedian pairs of cephalic papillae of external circle on four elevated cuticular lobes in gravid females, the presence of amphids, genital papillae and phasmids in males, the lamellate structure of the distal end of gubernaculum appearing as a dorsal protuberance in lateral view and the structure of the male caudal end. A taxonomically important feature of P. globiceps is the details in the dorsal transverse lamella-like structures on the distal end of the gubernaculum, by which this species can be separated from other gonad-infecting species of this genus. Apparently, P. globiceps is a specific parasite of U. scaber and all previous records of this species from hosts belonging to other fish families are evidently based on misidentifications.

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.

We isolated a Mycobacterium sp. resembling Mycobacterium marinum and M. ulcerans from diseased striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. This isolate may represent an undescribed Mycobacterium species, based on phenotypic characteristics and comparative 16S rRNA gene sequence.

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.

The genus Carnobacterium belongs to the lactic acid bacteria and Carnobacterium maltaromaticum is commonly found in modified atmosphere packed and vacuum packed fish and meat products as well as in live fish. This species has been described as a fish pathogenic organism but human clinical isolates have only been obtained at one occasion. To investigate the virulence potential we sequenced the entire genome of strain ATCC 35586, isolated from a diseased salmon. When comparing the translated gene products of ATCC 35586 to those of Gram positive bacterial pathogens and probiotics as well as the related Carnobacterium sp. AT7 we identified a range of putative virulence genes including genes encoding products involved in adhesion to fibronectin and collagen, capsule synthesis, cell wall modification, iron scavenging mechanisms, haemolysis, invasion and resistance to toxic compounds. Of particular interest was the presence of internalin encoding gene homologues to some of those found in Listeria spp. and Lactobacillus plantarum. Furthermore, the ATCC 35586 strain possesses a gene encoding a product similar to the central Listeria monocytogenes transcriptional regulator PrfA, that in this organism controls virulence gene expression by binding to conserved DNA binding sites. Based on the consensus DNA sequence of this binding site, we identified a total of 65 genes in the ATCC 35586 genome that in the upstream region carry a PrfA binding motif. Among these is one of the internalin encoding genes; two genes encoding products involved in capsule biosynthesis as well as various genes encoding products with metabolic functions. In contrast to L. monocytogenes, the ATCC 35586 strain did not encode other PrfA dependent virulence factors such as listeriolysin O, phospholipases A and B, ActA, listeriolysin O, zinc metallo protease and internalins A and B. In conclusion, C. maltaromaticum ATCC 35586 carries putative virulence genes that may explain its reported ability to infect fish. The findings of this study give no reason for concern regarding human health by the presence of this species in food products.

WOS: 000292802800008 PubMed: 21848040 The aim of the study was the phenotypic and molecular characterization of Yersinia (Y.) ruckeri strains, the causative agent of Enteric Red mouth Disease (ERM), by antibiotyping, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of whole cell proteins. For this aim, a total of 97 Y ruckeri isolates were analyzed. The isolates were distinguished into ten antibiotypes and six phenotypes according to their resistance properties and whole cell protein profiles, respectively. Also, a glycoprotein band of approximately 25.5 kDa was observed in all Y ruckeri strains tested. In all strains, six different RAPD types were observed. In conclusion, Y ruckeri strains isolated from rainbow trout of fish farms in Turkey showed variation according to their phenotypic and genotypic characteristics, and the use of these three typing techniques in double and triple combinations could be more useful for discriminating the strains.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:42:24Z No. of bitstreams: 1 S1519-69842008000200025.pdf: 172084 bytes, checksum: 8a723bd768d3c60f32061903ee166a40 (MD5) Made available in DSpace on 2013-08-22T18:42:24Z (GMT). No. of bitstreams: 1 S1519-69842008000200025.pdf: 172084 bytes, checksum: 8a723bd768d3c60f32061903ee166a40 (MD5) Previous issue date: 2008-05-01 Made available in DSpace on 2013-09-30T17:41:55Z (GMT). No. of bitstreams: 2 S1519-69842008000200025.pdf: 172084 bytes, checksum: 8a723bd768d3c60f32061903ee166a40 (MD5) S1519-69842008000200025.pdf.txt: 27773 bytes, checksum: 6b9e52d86ca2a45de1cc719f0f761483 (MD5) Previous issue date: 2008-05-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:12:37Z No. of bitstreams: 2 S1519-69842008000200025.pdf: 172084 bytes, checksum: 8a723bd768d3c60f32061903ee166a40 (MD5) S1519-69842008000200025.pdf.txt: 27773 bytes, checksum: 6b9e52d86ca2a45de1cc719f0f761483 (MD5) Made available in DSpace on 2014-05-20T13:12:37Z (GMT). No. of bitstreams: 2 S1519-69842008000200025.pdf: 172084 bytes, checksum: 8a723bd768d3c60f32061903ee166a40 (MD5) S1519-69842008000200025.pdf.txt: 27773 bytes, checksum: 6b9e52d86ca2a45de1cc719f0f761483 (MD5) Previous issue date: 2008-05-01 Flavobacterium columnare é o agente etiológico da columnariose em peixes de água doce, ocasionando enfermidade na pele e nas brânquias, provocando freqüentemente um grande número de mortalidade. O objetivo deste estudo foi o isolamento e a caracterização de Flavobacterium columnare em peixes tropicais no Brasil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) e cascudo (Hypostomus plecostomus) foram examinados externamente com relação a sinais característicos de columnariose, como manchas acinzentadas na cabeça, região dorsal e pedúnculo caudal dos peixes. A amostragem compreendeu a coleta de 50 exemplares de peixes, representando as quatro diferentes espécies escolhidas para este estudo. Amostras para o isolamento foram obtidas através de raspado com swab estéril das lesões e do rim dos peixes clinicamente diagnosticados como acometidos por columnarios e imediatamente semeados em meios de culturas artificiais (líquido e sólido) próprios para o estudo de Flavobacterium segundo Carlson e Pacha (1968). No meio líquido, houve o desenvolvimento de microrganismos que observados em gota pendente apresentaram a forma de bacilos finos, longos, móveis por deslizamento. Através da coloração de Gram, apresentaram morfologia de bacilos finos, Gram negativos, agrupados em colunas. em meio sólido, as colônias eram pequenas, cinza-amareladas, com borda em forma de raiz. No total, foram obtidos quatro isolamentos: 01 cepa de Brycon orbignyanus; 01 cepa de Piaractus mesopotamicus; 01 cepa de Colossoma macropomum; e 01 cepa de Hypostomus plecostomus. A caracterização bioquímica das amostras, como absorção do vermelho Congo, produção de flexirrubina, produção de H2S e redução do nitrato, sugere que os isolamentos poderiam ser classificados como Flavobacterium columnare. Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare. Universidade Estadual Paulista Centro de Aquicultura Universidade de São Paulo Laboratório de Saúde Animal Centro de Pesquisa e Gestão de Recursos Pesqueiros Continentais Universidade Estadual Paulista Centro de Aquicultura

The response of rainbow trout (Oncorhynchus mykiss, Walbaum) towards probiotics present in the feed was investigated by examining the proteome of serum as a measure of the acute phase response (APR). Proteomic analysis by two-dimensional electrophoresis (2D) concurrently with mass spectrometry was used to detect APR related proteins in rainbow trout serum following feeding with probiotics Aeromonas sobria GC2 and Bacillus sp. JB-1. Three candidate proteins increased following use of GC2, and were putatively identified as NADH dehydrogenase, dystrophin and mKIAA0350. Conversely, one of the proteins, which were induced following use of JB-1 was identified as transferrin.

The matrinxã, Brycon amazonicus, is one of the main species being used for aquaculture and fishery activities in the Amazonian region, yet little is known about the parasites that cause its diseases. The present study monitored parasite fauna infesting an intensive husbandry system in a stream channel. The collections were carried out monthly from September, 1997 to January, 1998. Water quality parameters, fish removal procedures, as well as biometry and anaesthetic data were observed in order to monitor the management conditions. Of the 80 sampled fish, 71 (89%) were infested with four Monogenoidea species (Anacanthorus spiralocirrus, Jainus amazonensis, Tereancistrum kerri and Trinibaculum brazilensis), 12 (15%) one of Nematoda Spirocamallanus inopinatus, and three (3.75%) with the Copepoda Ergasilus bryconis. Monogenoidea presented the highest prevalence and intensity of infestation. The Kruskall-Wallis non-parametric variance analysis showed a significant difference (P < 0.05) in the monthly parasite indexes of A. spiralocirrus, J. amazonensis and T. brazilensis, however no significant difference (P > 0.05) was detected for T. kerri. Evaluation of the management conditions pointed out that monthly capture anaesthesia and hauling procedures may have been responsible for some of the clinical signals such as loss of scales, erosion and no pigments on the caudal fin and cornea opacity, which were the most frequently found injuries in husbandry.

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

International audience; Experimental infections and lethal dose 50% (LD$_{50}$) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 °C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 °C, revival and subsequent in vivo passages in fish. Experiments were performed 1, 8 and 23 months after storing the bacteria. Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages. In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage. In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 °C were found significantly attenuated. Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD$_{50}$. It was concluded that virulent strains of F. psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 °C were reliable methods for long-term preservation of virulence.

The aim of this work was to study the structure of the parasite communities of Digeneans of 2 families of Teleost fishes (Sparidae and Labridae) of the Mediterranean sea. We tried to quantify the importance of both the microhabitat requirements of the parasite species and the effect of host biological factors on the parasite communities. We applied, for the first time in parasite community studies, the Canonical Correspondence Analysis (CCA) to analyse (i) the spatial distribution of parasite species within the digestive tract of the hosts; (ii) the host's biological factors (such as diet, host length, gregariousness and abundance) that may influence this spatial distribution of parasite species. Our results showed that potential microhabitats were vacant in the 2 host families studied revealing a lack of niche saturation because either there was little inter- and/or intraspecific competition or there were enough available space and resources within the host. Our results also indicated that the position of the parasite in the digestive tract is much more important than host biological factors for the structure of parasite community. Finally, we highlight the potential use of the CCA method for controlling for phylogenetic constraints in multi-species analyses.

Bacterial gill disease (BGD) (causative agent: Flavobacterium branchiophilum) has been a persistent problem in early-rearing salmonids in the Ontario Ministry of Natural Resources (OMNR) fish hatchery system. Retrospective epidemiological investigations of BGD diagnoses and treatments in OMNR fish hatcheries during the period 1991–2001 were conducted using University of Guelph Fish Health Laboratory and OMNR central office data. All investigations were conducted at the lot-level, which is the major within-hatchery-level of population aggregation. Survivorship of BGD diagnosis in early-rearing lots within seven individual hatcheries ranged from 84.2 to 100%; within individual species groups, survivorship was lowest (84.6%) in brook trout (Salvelinus fontinalis) lots. Annual risk percentages (cumulative incidence) for BGD diagnosis within hatchery and species groups varied considerably among years. Multivariable proportional hazards survival analysis indicated that the species brook trout, and the Spring (March–May) season, were significantly associated with treatment for BGD. Combined, these results emphasize the importance of hatchery, species, and time on the development of BGD. Future observational research on this disease must consider these factors in their design and analysis.

Early-rearing salmonids in Ontario Ministry of Natural Resources (OMNR) fish hatcheries have been consistently affected by bacterial gill disease (BGD) (causative agent: Flavobacterium branchiophilum) for many years. Separate retrospective epidemiological investigations of BGD treatments at two OMNR fish hatcheries (Hatcheries A and B) for the 1999 production year were conducted using on-site hatchery records. Both investigations were carried out at the rearing unit-level, with early-rearing (

Background Animal behavioral responses have been recently established as a suitable tool for detecting contaminants in the environment for risk assessment in situ. In this study, we observed movement behavior of zebrafish (Danio rerio) before and after infection with Edwardsiella tarda CK41 for 3 days until death. Methods Infection status of zebrafish was confirmed through PCR and colonization assay as time progressed and lesion development in the tails of zebrafish was also examined. Movement behaviors in response to bacterial infection were patterned by self-organizing map (SOM) based on movement parameters, including speed (mm/s), acceleration (mm/s2), stop duration (t), stop number (n), locomotory rate (mm/s), turning rate (rad/s), and meander (rad/mm). Results According to SOM result, clusters were identified firstly according to time and secondly according to infection. Two movement patterns were observed in the early period of infection: one group with minimum turning rate and meander (i.e., stiff movement) and the other group with maximum strop number. Late infection was characterized by long stop duration. Conclusion SOM was suitable for extracting complex behavioral data and thus can serve as a referencing system for diagnosing disease development in order to reveal the mechanism of the infection process. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0579-2) contains supplementary material, which is available to authorized users.

Background The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters. Results The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus. Conclusions Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile.

Background: Swim bladder inflammation (SBI) is an important disease of common carp fingerlings in Central Europe. In the 1980s, its etiology was ascribed to multicellular proliferative stages of the myxozoan parasite Sphaerospora dykovae (formerly S. renicola). S. dykovae was reported to proliferate in the blood and in the swim bladder prior to the invasion of the kidney, where sporogony takes place. Due to the presence of emerging numbers of proliferative myxozoan blood stages at different carp culture sites in recent years we analysed cases of SBI, for the first time, using molecular diagnostics, to identify the myxozoan parasites present in diseased swim bladders. Methods: We amplified myxozoan SSU rDNA in a non-specific approach and compared the species composition in swim bladders at culture sites where carp demonstrated 1. No signs of SBI, 2. Minor pathological changes, and 3. Heavy SBI. Based on DNA sequences, we determined the localisation and distribution of the most frequent species by in situ hybridisation, thereby determining which myxozoans are involved in SBI. Results: Large multicellular myxozoan swim bladder stages characterised heavy SBI cases and were identified as S. dykovae, however, blood stages were predominantly represented by Sphaerospora molnari, whose numbers were greatly increased in carp with mild and heavy SBI, compared with SBI-free fish. S. molnari was found to invade different organs and cause inflammatory changes also in the absence of S. dykovae. One site with mild SBI cases was characterised by Buddenbrockia sp. infection in different organs and a general granulomatous response. Conclusions: We provide evidence that the etiology of SBI can vary in relation to culture site and disease severity and that emerging numbers of S. molnari in the blood represent an important co-factor or precondition for SBI.