Infectious Diseases In Fish Research Articles

The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.

The effects of essential oils and their major compounds on fish bacterial pathogens - a review.

The increased resistance of fish pathogens to conventional treatments has lead researchers to investigate the antibacterial properties of natural resources, such as essential oils (EOs) of plants, in an effort to find products that are less harmful to the environment. The objective of this review is to provide an overview of the studies, invivo and invitro, that addressed the use of EOs and their major compounds as antimicrobial agents in fish, to identify the best EOs and compounds to investigate considering feasibility of application and suggest possible future studies. To date, studies suggest that the use of EOs in the prevention and/or treatment of infectious diseases in fish may be a promising strategy to reduce the use of conventional antibiotics in aquaculture, since several EOs effectively reduce or avoid the effects of bacterial infections in fish. The use of EOs through nanotechnology delivery systems, especially in dietary supplementation experiments, is promising. This form of application of the EOs allows a potentiation and targeting of the desired effect of the EOs and also allows the protection of EOs active constituents against enzymatic hydrolysis, deserving further study.

Probiotic Strains Used in Aquaculture

Aquaculture is the fastest growing animal food producing agricultural sector in the world which has been constrained by several factors. Infectious disease is a key limitations to the sustainable development of aquaculture sector. This paper reviewed the application and efficiency of probiotic microorganisms in aquaculture to prevent infectious diseases and their effects on fish immunology. Information was collected from different secondary sources like journals, reports, articles and electronic media and arranged chronologically. The study reveals that, currently, many probiotic products extracted from different species of bacteria including Bacillus sp., Lactobacillus sp., Carnobacterium sp., Enterococcus sp. and a yeast Saccharomyces cerevisiae are commercially available for use in aquaculture. Probiotics have gained popularity in aquaculture as an alternative to the antibiotic in the prevention of infectious diseases of fish because probiotics are eco-friendly. This review also shows that, probiotics have significant effect on growth rate, feed utilization capacity and the immune performance of finfish, shrimp, prawns and crabs in aquaculture. Probiotics are able to interfere with colonization of harmful pathogens and to improve the water quality through decomposition of uneaten food materials, nitrate and other organic materials.

Current knowledge of metabolomic approach in infectious fish disease studies.

The approaches of transcriptomic and proteomic have been widely used to study host-pathogen interactions in fish diseases, and this is comparable to the recently emerging application of metabolomic in elucidating disease-resistant mechanisms in fish that gives new insight into potential therapeutic strategies to improve fish health. Metabolomic is defined as the large-scale study of all metabolites within an organism and represents the frontline in the 'omics' approaches, providing direct information on the metabolic responses and perturbations in metabolic pathways. In this review, the current research in infectious fish diseases using metabolomic approach will be summarized. The metabolomic approach in economically important fish infected with viruses, bacteria and nematodes will also be discussed. The potential of the metabolomic approach for management of these infectious diseases as well as the challenges and the limitations of metabolomic in fish disease studies will be explored. Current review highlights the impacts of metabolomic studies in infectious fish diseases, which proposed the potential of new therapeutic strategies to enhance disease resistance in fish.

Induction of Toll-like receptor (TLR) 2 and Myeloid88-dependent TLR-Signaling Response to Aeromonas Hydrophila Infections in Gouramy (Osphronemus Gouramy)

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns associated with microbial pathogens (PAMP) and induce antimicrobial immune responses. Here we report the expression of TLR2, Myeloid differentiation primary response gene 88 (Myd88) and Interleukin 1beta (IL1β) from the Gouramy an economically freshwater fish in Indonesia. Upon stimulation with Aeromonas hydrophila, the TLR2 expression was obviously up-regulated in spleen and liver, Myd88 expression up-regulated in spleen and kidney, and IL1β expression was up-regulated in a spleen, kidney, and liver. The highest expression of TLR2 was in liver or hepatopancreas, MyD88 in kidney and IL1β was in the spleen. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish and indicated its important role in immune surveillance of various organs during the pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish and will be helpful in developing preventive measures against infectious diseases in fish.

Avian Egg Yolk Antibodies (IgY) and their Potential Therapeutic Applications for Countering Infectious Diseases of Fish and Aquatic Animals

Avian Egg Yolk Antibodies (IgY) and their Potential Therapeutic Applications for Countering Infectious Diseases of Fish and Aquatic Animals

Importance of prebiotics in aquaculture as immunostimulants. Effects on immune system of Sparus aurata and Dicentrarchus labrax.

Infectious diseases in fish represent a major problem for the aquaculture field as they produce extensive damages and loss. Over the last few years, with increased development of the aquaculture industry, different methods have been used to contrast these pathologies. Common interest has led to the use of components (as additives in diets) that could contrast diseases without causing any negative impact on the environment. These components are represented by prebiotics, probiotics, and plant extracts. In this review, the effects of prebiotics are described. Prebiotics are indigestible fibres fermented by gut enzymes and commensal bacteria, whose beneficial effects are due to the by-products generated from fermentation. The influence of pre-biotics on the immune system of fish is called immunosaccharides. Mannanoligosaccharides (MOS), Fructooligosaccharides (FOS) and Inulin act at different levels in the innate immune response. For example, through phagocytosis, lysozyme activity, and the complement system activity, an increase in fish growth and an amelioration of their health status is brought about. In this review, the use of prebiotics in aquaculture, such as immunostimulants, has been highlighted: particularly in two teleost fish species, Sparus aurata and Dicentrarchus labrax. The results demonstrate that the road is still long and further studies are required, but the use of prebiotics, individually or coupled together, can open the doors to pioneering a new model of alternative components to antimicrobial agents.

Detection and Quantification of 41 Antibiotic Residues in Gilthead Sea Bream (Sparus aurata) From Aquaculture Origin, Using a Multiclass and Multi-residue UHPLC-MS/MS Method

Aquaculture is one of the worldwide strategic development fields, and its importance is evident in its significant worldwide growth in the last decades. This growth is associated with the implementation of intensive and semi-intensive production methods, with the use of antibiotics in order to prevent the emergence and spread of infectious diseases in fish. This practice constitutes a real public health problem, not only due to the presence of antimicrobial residues in edible tissues, which can cause allergic reactions in hypersensitive individuals, but also due to the emergence of bacterial resistance. Consequently, the Regulatory Agencies have established maximum residue limits (MRLs). In the present study, a validated multiclass multi-residue ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem methodology was used for the determination of 41 antibiotics from seven different classes—sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol—in 29 samples of gilthead sea bream of aquaculture origin, purchased in Portugal. The analysed samples showed that, in eight of them, antibiotic residues were present, three being of doxycycline—antibiotic for which no MRL is established—that was detected in concentrations ranging from 0.35 to 0.61 μg kg−1. Other antibiotics were also detected and quantified and their concentrations were below the MRL established by the European legislation.

Detection and quantification of 41 antibiotic residues’ in Gilthead sea bream (Sparus aurata) from aquaculture origin, using a multiclass and multi-residue UHPLC-MS/MS method

Aquaculture is one of the worldwide strategic development fields, and its importance is evident in its significant worldwide growth in the last decades. This growth is associated with the implementation of intensive and semi-intensive production methods, with the use of antibiotics in order to prevent the emergence and spread of infectious diseases in fish. This practice constitutes a real public health problem, not only due to the presence of antimicrobial residues in edible tissues, which can cause allergic reactions in hypersensitive individuals, but also due to the emergence of bacterial resistance. Consequently, the Regulatory Agencies have established maximum residue limits (MRLs). In the present study, a validated multiclass multi-residue ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem methodology was used for the determination of 41 antibiotics from seven different classes—sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol—in 29 samples of gilthead sea bream of aquaculture origin, purchased in Portugal. The analysed samples showed that, in eight of them, antibiotic residues were present, three being of doxycycline—antibiotic for which no MRL is established—that was detected in concentrations ranging from 0.35 to 0.61 μg kg−1. Other antibiotics were also detected and quantified and their concentrations were below the MRL established by the European legislation.

Results of Surveillance Studies of Infectious Fish Diseases in Freshwater Aquaculture of Ukraine

Aim. To investigate the epizootic state of fi sheries in Ukraine; to study the biological specifi cities of viral and bacterial isolates of fi sh in freshwater aquaculture. Methods. The epizootic state of fi sheries was defi ned ac- cording to the surveillance plan for fi sheries, virological (biosampling of sensitive fi sh species, virus isolation on sensitive passaged cell cultures), serological (enzyme immunoassay, virus neutralization test using sensitive passaged cell cultures) and molecular-biological (reverse transcriptase polymerase chain reaction − RTPCR) methods of investigation were used. The pathogenicity of the isolated bacteria was studied in the biosample. The identifi cation was performed using Bergey’s Manual. The express-identifi cation of bacteria was performed using the standardized test-system API 20E Bio Merieux (France). Results. The IPNV isolates of rainbow trout were fi rst isolated in the fi sheries of different forms of ownership in the western regions of Ukraine (Volyn, L’viv, Transcarpathian, Chernivtsi regions). It was demonstrated that different age groups of carp are infested with the virus in the fi sheries of L’viv, Donetsk, Chernihiv, Kyiv, and Odesa regions which testifi es to a wide spread of the virus in Ukraine. Out of fi sh infectious diseases the red spot-like disease and the swim bladder infl ammation of carp, the diseases of young trout and sturgeon were detected in the investigated fi sheries of Ukraine. Conclusions. The epizootic data were used to estimate the condition of the fi sheries in Ukraine in terms of fi sh infectious diseases. An infectious pancreatic necrosis virus, new for Ukraine, was revealed. It was found to affect rainbow trout (Oncorhynchus mykiss , Walbaum, 1792) and the spread of SVCV in carp fi sheries was demonstrated. As for bacterial fi sh diseases, the decrease in the epizootic situation was described along with considerable extension of the range of species of bacterial pathogens of fi sh. Annual systematic monitoring and measures of preventing the introduction of the agents of infectious diseases are the guarantee of protection of the specialized fi sheries of Ukraine.

효모표면표출(YSD) 기법을 이용한 참돔 이리도바이러스(RSIV) 외피단백질의 발현

The red seabream iridovirus (RSIV), which belongs to the iridoviridae, causes infectious fish diseases in many Asian countries, leading to considerable economic losses to the aquaculture industry. Using the yeast surface display (YSD) technique, a new experimental system was recently developed for the detection and identification of a variety of marine viruses. In this study, a coat protein gene of RSIV was synthesized based on the nucleotide sequence database and subcloned into the yeast expression vector, pCTCON2. The expression of viral coat proteins in the yeast strain, EBY100, was detected by flow cytometry and Western blot analysis. Finally, they were isolated from the yeast surface through a treatment with β-mercaptoethanol. The data suggests that the YSD system can be a useful method for acquiring coating proteins of marine viruses.

Therapeutic potential of CpG-ODN 1668 against scuticociliatosis in olive flounder (Paralichthys olivaceus)

Abstract Scuticociliatosis caused by Miamiensis avidus is one of the major diseases in cultured olive flounder ( Paralichthys olivaceus ) in Korea. However, until recently, no effective chemotherapy or vaccine has been developed. Oligodeoxynucleotides containing unmethylated deoxycytidyl-deoxyguanosine motifs (CpG-ODNs) have been shown to be effective in enhancing innate immune responses and preventing infectious diseases in fish. However, it has not been experimented whether CpG-ODNs can be used as a therapeutic tool against infectious diseases in fish. In the present study, we evaluated the therapeutic potential of CpG-ODN 1668 against scuticociliatosis in olive flounder for the first time. The group of fish administered CpG-ODN 1668 at 3 days after the infection showed clearly higher survival rates than the groups of fish administered GpC-ODN 1720 or PBS, indicating a high therapeutic potential of CpG-ODN 1668 against scuticociliatosis. However, the treatment efficacy of CpG-ODN 1668 was diminished when administered at 6 days post-infection, and no treatment effect was found when administered at 9 days after the infection. These results suggest that early diagnosis through continuous monitoring and early treatment with CpG-ODN 1668 are essential for increase of treatment efficacy. In conclusion, the present study demonstrated for the first time the possible use of CpG-ODN 1668 in treatment of scuticociliatosis in olive flounder.

INFECTION OF SEA LAMPREY WITH AN UNUSUAL STRAIN OFAEROMONAS SALMONICIDA

The invasion of the Laurentian Great Lakes by the fish-parasitic sea lamprey has led to catastrophic consequences, including the potential introduction of fish pathogens. Aeromonas salmonicida is a bacterial fish pathogen that causes devastating losses worldwide. Currently, there are five accepted subspecies of Aeromonas salmonicida: A. salmonicida subsp. salmonicida, masoucida, smithia, achromogenes, and pectinolytica. We discuss the discovery of an isolate of A. salmonicida that is pathogenic to rainbow trout (Oncorhynchus mykiss) and exhibits unique phenotypic and molecular characteristics. We examined 181 adult sea lamprey (Petromyzon marinus) from the Humber River (Lake Ontario watershed) and 162 adult sea lamprey from Duffins Creek (Lake Ontario watershed) during the spring seasons of 2005-11. Among those, 4/343 (1.2%) sea lamprey were culture positive for A. salmonicida, whereby biochemical and molecular studies identified three of the isolates as A. salmonicida subsp. salmonicida. The remaining isolate (As-SL1) recovered from Humber River sea lamprey was phenotypically more similar to A. salmonicida subsp. salmonicida than to the four other A. salmonicida subspecies. However, unlike A. salmonicida subsp. salmonicida, As-SL1 was sucrose positive, produced an acid-over-acid reaction on triple-sugar iron medium and did not amplify with A. salmonicida subsp. salmonicida specific primers. Phylogenetic analysis based on partial stretches of the 16S rRNA and DNA gyrase subunit B genes further confirmed that the As-SL1 isolate was not A. salmonicida subsp. masoucida, smithia, achromogenes, or pectinolytica. Based on our analyses, the As-SL1 isolate is either an unusual strain of A. salmonicida subsp. salmonicida or a novel A. salmonicida subspecies. The four A. salmonicida isolates that were recovered from sea lamprey were pathogenic to rainbow trout in experimental challenge studies. Our study also underscores the potential role of sea lamprey in the ecology of infectious fish diseases.

Diseases In The Aquarium Fishes: Challenges And Areas Of Concern: An Overview

Progress over the past years has revealed much strength of the ornamental fish as an alternative model for the environmental sideline in the aquaculture both in fresh water and marine waters. These include low rearing costs, an earlier life stage. Aquarium fish are one of the largest groups of pets in the world. There is an increasing demand for the veterinary services that are related to the ornamental fish. Early clinical signs in many infectious and noninfectious diseases of fish are manifested by the skin. More diseases in fish have been described and have been associated with the research for the future has been discussed. This article also describes the more common environmental and pathogen related dermatologic diseases of the aquarium fishes. Disease prevention, control and various treatment methods also have been discussed. International Journal of Environment, Volume-2, Issue-1, Sep-Nov 2013, Pages 127-146 DOI: http://dx.doi.org/10.3126/ije.v2i1.9216

Linking fish diseases and animal welfare in aquaculture – A study of the turbot herpesvirus

Infectious diseases of fish are of major concern of the aquaculture industry, impacting upon economic growth, sustainability and financial viability. In Germany there has been a paucity of research in this area, and while German marine aquaculture is still in its infancy, the problems which this sector faces nationally in dealing with e.g. disease outbreaks, are similar to those encountered worldwide. The Geomar Helmholtz Institute for Ocean Research in Kiel, Germany, is ideally located to conduct studies of marine fish within the environment of the North and Baltic Sea. Our newly founded Aquaculture and Fish Disease group comprises experts in immunology, physiology, ecology and diseases of fish, as well as larval biology and evolutionary ecology. Our research focuses on species that are important for German and European marine Aquaculture, including sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. The overall aim of our research is to maximize the productivity and sustainability of aquaculture through research-led improvements in disease characterization, prevention and diagnostics and animal welfare. By understanding the factors that contribute to disease outbreaks, such as variations in water temperature and quality, and the genetic background and stresses faced by farmed fish, we hope to devise new methods of predicting when infections will occur, and minimize their impact. Of particular interest is the way in which stress can affect the immune response of fish in aquaculture at each stage of their life history. Characterization of changes which occur in immune parameters following infection would facilitate identification of infections in aquaculture at an early stage, such that appropriate treatment can be begun before mortalities occur. We also plan to evaluate the potential efficacy of novel tools including siDNA and immunostimulation in disease treatment and prevention. In this context we are especially interested in the herpesvirus of turbot (Herpesvirus scophthalmi) and aim to characterize its influence on both its host and aquaculture as a whole. We will report some of our recent advances in this field.

Immune Suppression by Neonicotinoid Insecticides at the Root of Global Wildlife Declines

of infectious diseases in honey bees, fish, amphibians, bats and birds in the past two decades have coincided with the increasing use of systemic insecticides, notably the neonicotinoids and fipronil. A link between insecticides and such diseases is hypothesised. Firstly, the disease outbreaks started in countries and regions where systemic insecticides were used for the first time, and later they spread to other countries. Secondly, recent evidence of immune suppression in bees and fish caused by neonicotinoids has provided an important clue to understand the sub-lethal impact of these insecticides not only on these organisms, but probably on other wildlife affected by emerging infectious diseases. While this is occurring, environmental authorities in developed countries ignore the calls of apiarists (who are most affected) and do not target neonicotinoids in their regular monitoring schedules. Equally, scientists looking for answers to the problem are unaware of the new threat that systemic insecticides have introduced in terrestrial and aquatic ecosystems. Journal of Environmental Immunology and Toxicology 2013; 1:3-12

Evolutionary Mechanisms of Microbial Genomes 2012

What is the driving force in the course of microbial genome evolution? What is the mechanism for distinguishing self-genome from others? These fundamental questions remain elusive although rigorous studies are underway by using comparative genomics. The special issue “Evolutionary mechanisms of microbial genomes” has been launched in 2011 and presented 11 original papers. Here, this new version in 2012 presents 10 papers (one review and nine research articles). Two papers are presented in phylogenomics. K. Oshima et al. revealed a close relationship of Aquificales to Thermotogales based on the whole-genome comparison in “Phylogenetic position of Aquificales based on the whole genome sequences of six Aquificales species.” An extensive and elaborate review of fish pathogenic bacteria has been presented by P. S. Sudheesh et al. in “Comparative pathogenomics of bacteria causing infectious diseases in fish.” Two papers are presented on subjects related to evolution of base composition in genomes. H. Nishida et al. in “Genome signature difference between Deinococcus radiodurans and Thermus thermophilus” observed distinct tetranucleotide frequencies between the genomes of D. radiodurans and T. thermophilus, potentially reflecting different evolutionary backgrounds of the two species after divergence from common ancestor. H. Nishida in “Comparative analyses of base compositions, DNA sizes, and dinucleotide frequency profiles in archaeal and bacterial chromosomes and plasmids” reported lower GC content (by up to ~10%) of plasmids compared to their host chromosomes and higher correlation of GC content and chromosome size in bacteria than in archaea. Two papers are presented about horizontal gene transfer in genome evolution. M. Jalasvuori in “Vehicles, replicators, and intercellular movement of genetic information: Evolutionary dissection of a bacterial cell” discussed a hypothesis that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve toward possessing horizontally moving replicators of various types. V. S. Pylro et al. described horizontal gene transfer events of the gene dszC involved in the cleavage of carbon-sulfur bonds in “Detection of horizontal gene transfers from phylogenetic comparisons.” An article about DNA mutation is presented by Y. Shiwa et al. in “Whole-genome profiling of a novel mutagenesis technique using proofreading-deficient DNA polymerase δ.” They compared mutations created by the chemical mutagen ethyl methanesulfonate (EMS) and the proofreading-deficient DNA polymerase δ and found that the mutations created by the proofreading-deficient DNA polymerase δ generated more diverse amino acid substitution patterns than those by EMS. Three papers are presented on subjects related to metabolic pathway. H. Nishida in “Comparative analyses of homocitrate synthase genes of ascomycetous yeasts” described gene duplications of the homocitrate synthase which have occurred multiple times during evolution of the ascomycetous yeasts. H. Nishida and M. Nishiyama in “Evolution of lysine biosynthesis in the phylum Deinococcus-Thermus” reported that bacterial lysine biosynthesis genes of the common ancestor of the Deinococcus-Thermus phylum used the α-aminoadipate pathway instead of the diaminopimelate pathway. K. Ueda et al. in “Dispensabilities of carbonic anhydrase in Proteobacteria” analyzed the distribution of carbonic anhydrase (CA) in proteobacteria, compared CA-retaining and CA-deficient genomes, and found absence of coding sequence in some strains and frame shifts in others. In closing this introduction to the special issue, we would like to express our full appreciation to all the authors and reviewers for their enormous efforts that have made the timely completion of our assignment successful. We sincerely hope that this special issue will stimulate further the investigation of evolutionary mechanisms of microbial genomes. Hiromi Nishida Shinji Kondo Hideaki Nojiri Ken-ichi Noma Kenro Oshima

Comparative pathogenomics of bacteria causing infectious diseases in fish.

Fish living in the wild as well as reared in the aquaculture facilities are susceptible to infectious diseases caused by a phylogenetically diverse collection of bacterial pathogens. Control and treatment options using vaccines and drugs are either inadequate, inefficient, or impracticable. The classical approach in studying fish bacterial pathogens has been looking at individual or few virulence factors. Recently, genome sequencing of a number of bacterial fish pathogens has tremendously increased our understanding of the biology, host adaptation, and virulence factors of these important pathogens. This paper attempts to compile the scattered literature on genome sequence information of fish pathogenic bacteria published and available to date. The genome sequencing has uncovered several complex adaptive evolutionary strategies mediated by horizontal gene transfer, insertion sequence elements, mutations and prophage sequences operating in fish pathogens, and how their genomes evolved from generalist environmental strains to highly virulent obligatory pathogens. In addition, the comparative genomics has allowed the identification of unique pathogen-specific gene clusters. The paper focuses on the comparative analysis of the virulogenomes of important fish bacterial pathogens, and the genes involved in their evolutionary adaptation to different ecological niches. The paper also proposes some new directions on finding novel vaccine and chemotherapeutic targets in the genomes of bacterial pathogens of fish.

Potential of auxotrophic Edwardsiella tarda double-knockout mutant as a delivery vector for DNA vaccine in olive flounder (Paralichthys olivaceus)

To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×107CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×109CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish.

Induction of toll-like receptor (TLR) 2, and MyD88-dependent TLR- signaling in response to ligand stimulation and bacterial infections in the Indian major carp, mrigal (Cirrhinus mrigala)

Toll-like receptor 2 (TLR2) is a member of TLR family. It recognizes a wide range of bacteria and their products, and is involved in inducing innate immune responses. In this article, we reported inductive expression of TLR2 and myeloid differentiation primary response gene 88 (MyD88)-dependent signaling in the Indian major carp, mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) genes by quantitative real-time PCR (qRT-PCR) revealed constitutive expression of these genes in all embryonic developmental stages, indicating their involvement in embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by qRT-PCR showed their wide distribution in various organs and tissues. Highest expression of TLR2 was in gill, MyD88 in liver and TRAF6 was in kidney. Inductive expression of TLR2, MyD88 and TRAF6 genes were observed following peptidoglycan (PGN)-treatment, and Streptococcus uberis and Aeromonas hydrophila infections. Expression of interleukin (IL)-8 and TNF-α in various organs were significantly enhanced by PGN-treatment and bacterial infections, and were closely associated with TLR2 induction. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish, and indicated it's important role in immune surveillance of various organs during pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish, and will be helpful in developing preventive measures against infectious diseases in fish.