Identification of two interleukin 17 receptor C (IL-17RC) genes and their binding activities to three IL-17A/F ligands in the Japanese medaka, Oryzias latipes.

In mammals, interleukin (IL)-17 receptor C (IL-17RC) and IL-17RA mediate IL-17A and IL-17F signaling to produce mucin, antimicrobial peptides, and maintain healthy intestinal flora. However, IL-17RC signaling in fish remains unclear. In this study, three il17rc transcripts (il17rca1, il17rca2, and il17rcb) from the Japanese medaka (Oryzias latipes) were cloned; il17rca1 and il17rca2 mRNAs were alternatively spliced from il17rca pre-mRNA as transcript variants. The il17rca and il17rcb genes were located on chromosomes 7 and 5, respectively. Teleost clades containing medaka il17rca and il17rcb clustered separately from the tetrapod clade. In adult tissues, il17rca1 expression was significantly higher than il17rca2 and il17rcb. Conversely, il17rcb expression was significantly higher in embryos and larvae. These expression patterns changed following infection with Edwardsiella piscicida and Aeromonas hydrophila. Furthermore, an immunoprecipitation assay using recombinant IL-17RCs and rIL-17A/Fs suggested that, in teleosts, three ligands could function in signaling through two IL-17RCs.

Characterization and expression analysis of tandemly-replicated asc genes in the Japanese medaka, Oryzias latipes.

ASC is a component of the inflammasome playing crucial roles in the inflammatory response. In mammals, ASC induces pyroptosis and inflammatory cytokine production. In this study, three asc genes (asc1, asc2, and asc3) from the Japanese medaka (Oryzias latipes) were identified and characterized. These asc genes were tandem replicates on chromosome 16, and their exon-intron structures differed between them. All three ASCs conserved the pyrin and caspase-recruitment domains, which are important for inflammasome formation. In phylogenetic analysis, all ASCs clustered with those of other teleosts. The asc1 expression levels were significantly higher in several organs than those of asc2 and asc3, suggesting that asc1 may act as a dominant asc in the Japanese medaka. Expression of the three asc genes showed different patterns during Aeromonas hydrophila and Edwardsiella piscicida infections. Furthermore, their expression was adequately down-regulated in the medaka fin-derived cells stimulated with ATP for 12 h, while asc2 expression was statistically up-regulated after nigericin stimulation for 24 h. Moreover, the expression of asc2 and asc3 was significantly higher in the skin of ASC-1-knockout medaka than in that of the wild type medaka during A. hydrophila infection.

Study on Streptococcus agalactiae infection in Javanese medaka (Oryzias javanicus Bleeker, 1854) model.

This study determines the median lethal dose, and describes the clinico-pathological changes and disease development following Streptococcus agalactiae infection in Javanese medaka model. Javanese medakas were infected with S. agalactiae via intraperitoneal (IP) from 104 to 108 CFU/mL, and immersion (IM) route from 103 to 107 CFU/mL. The LD50-240h and clinico-pathological changes of the fish was determined until 240 h post infection (hpi). Next, the disease development was determined for 96 hpi in the fish following IP and IM infection at 103 CFU/mL and 104 CFU/mL, respectively. The LD50-240h of S. agalactiae in Javanese medaka was lower following IP injection (4.5 × 102 CFU/mL), compared to IM route (3.5 × 103 CFU/mL). The clinical signs included separating from the schooling group, swimming at the surface of water column, lethargy, erratic swimming pattern, corneal opacity and exophthalmia. Histopathological examinations revealed generalized congestion in almost all internal organs, particularly in liver and brain, while the kidney displayed tubular necrosis. Both IP and IM routes showed significant positive correlation (p < 0.05) between the CFU/g of S. agalactiae in the fish tissue and fish deaths. Moreover, the lesions for histopathological scoring in selected organs following IP and IM challenges were also reflecting the CFU/g and fish deaths. This study indicates the capability of Javanese medaka as a model organism in study of streptococcosis development.

Antifungal activities of combined treatments of irradiation and essential oils (EOs) encapsulated chitosan nanocomposite films in in vitro and in situ conditions.

Cellulose nanocrystals (CNCs) reinforced chitosan based antifungal films were prepared by encapsulating essential oils (EOs) nanoemulsion. Vapor phase assays of the chitosan-based nanocomposite films loaded with thyme-oregano, thyme-tea tree and thyme-peppermint EO mixtures showed significant antifungal activity against Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium chrysogenum, reducing their growth by 51-77%. Combining the bioactive chitosan films loaded with thyme and oregano EOs produced ~2 log reduction in fungal growth in inoculated rice during 8 weeks of storage at 28 °C. The bioactive films showed a slow release (26%) of volatile components over 12 weeks of storage. Sensorial evaluation of rice samples packed with the bioactive films showed no significant change in odor, taste, color and general appreciation compared with untreated rice. Incorporation of cellulose nanocrystals (CNCs) with the chitosan matrix played an important role in stabilizing the physicochemical and release properties of the nanocomposite films. In addition, combining the bioactive chitosan films with a dose of 750 Gy of ionizing radiation showed significantly higher antifungal and mechanical properties than treatment with the bioactive film or irradiation alone.

The effects of fish gender on susceptibility to acute Streptococcus agalactiae infection in Javanese medaka Oryzias javanicus.

This study describes the susceptibility of different fish gender following acute Streptococcus agalactiae infection by using Javanese medaka Oryzias javanicus as test fish. The fish were grouped into four groups, which were: (1) all-male; (2) all-female; (3) mixed-gender (1 male: 1 female ratio); and (4) control non-infected (1 male: 1 female ratio). The fish in group 1, 2 and 3 were intraperitoneally exposed to 5.4 × 108 CFU/mL of S. agalactiae, while for group 4, the fish were exposed using sterile broth. The main clinical signs and histopathological changes of infected Javanese medaka were commonly observed in S. agalactiae infected fishes. However, no difference on clinical signs and histopathological changes of fish in group 1, 2 and 3 were noticed. The Javanese medaka mortality in group 1, 2 and 3 were observed from 4 h post infection (hpi) to 6 hpi, with the cumulative mortality from 3% to 30%. Then, the mortality increased at 12 hpi, with the range from 53% to 80%. However, 100% of the infected fish dead at 24 hpi. No clinical sign, histopathological change and fish mortality recorded in group 4. Generally, the clinical signs, mortality patterns, cumulative mortality and histopathological changes of Javanese medaka infected by S. agalactiae did not show any difference between the all-male, all-female and mixed-gender groups. This indicates that the susceptibility of fish to S. agalactiae infection is not influenced by their gender.

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa.

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C4-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

Immunostaining of UVA-induced DNA damage in erythrocytes of medaka (Oryzias latipes).

Some authors have recently reported that UVA induces double-strand breaks (DSBs) in DNA. Only a few researchers have reported on the induction of DSBs upon UVA exposure, as measured using the Comet assay and γ-H2AX as markers of DSB formation. In the present study, we have investigated for the first time the dose-dependent induction of DSBs by UVA in medaka (Oryzias latipes) erythrocytes. Adult female medaka fish were exposed to UVA for 15, 30, and 60min/day for three continuous days; an unirradiated control group was kept in the same laboratory conditions. At 0h and 24h after UVA exposure, blood was collected to detect DNA damage and repair. The number of γ-H2AX foci was higher than the control value at 0h after UVA exposure and decreased within a 24h. the comet assay showed that DNA repair began during the recovery period. These findings confirm our pervious findings of genotoxic effects after UVA exposure in medaka erythrocytes and suggest that the replication-independent formation of UVA-induced DSBs is mediated through the generation of reactive oxygen species. In conclusion, these results suggest that DNA damage and repair occur after UVA exposure in medaka fish. UVA is the main component of solar UV radiation and is used for artificial UV exposure. Our results may have implications for skin cancer research.

The development of cellular immune defence in marine medaka Oryzias melastigma.

Environmentally induced alterations of the immune system during sensitive developmental stages may manifest as abnormalities in immune organ configuration and/or immune cell differentiation. These not only render the early life stages more vulnerable to pathogens, but may also affect the adult immune competence. Knowledge of these sensitive periods in fish would provide an important prognostic/diagnostic tool for aquatic risk assessment of immunotoxicants. The marine medaka Oryzias melastigma is an emerging seawater fish model for immunotoxicology. Here, the presence and onset of four potentially sensitive periods during the development of innate and adaptive cellular immune defence were revealed in O. melastigma: 1.) initiation of phagocyte differentiation, 2.) migration and expansion of lymphoid progenitor cells, 3.) colonization of immune organs through lymphocyte progenitors and 4.) establishment of immune competence in the thymus. By using an established bacterial resistance assay for O. melastigma, larval immune competence (from newly hatched 1dph to 14dph) was found concomitantly increased with advanced thymus development and the presence of mature T-lymphocytes. A comparison between the marine O. melastigma and the freshwater counterpart Oryzias latipes disclosed a disparity in the T-lymphocyte maturation pattern, resulting in differences in the length of T-lymphocyte maturation. The results shed light on a potential difference between seawater and freshwater medaka in their sensitivity to environmental immunotoxicants. Further, medaka immune system development was compared and contrasted to economically important fish. The present study has provided a strong scientific basis for advanced investigation of critical windows for immune system development in fish.

Three-dimensional visualization of green fluorescence protein-labelled Edwardsiella tarda in whole Medaka larvae.

The invasive fish pathogen Edwardsiella tarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryzias latipes) were immersion-infected with E. tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light-sheet fluorescence microscopy. Confocal microscopy revealed GFP-labelled E. tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light-sheet microscopy additionally showed GFP-labelled E. tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E. tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E. tarda then enters the blood vessels to access organs, including the air bladder and heart.

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene.

AIMS: Isolation and full sequence analysis of ColE-type plasmid, which carries the qnrS2 gene. METHODS AND RESULTS: Quinolone resistance (qnrS2) gene-carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer-walking approach. The total sizes of these plasmids (pAQ2-1 and pAQ2-2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2-type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1-type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. CONCLUSIONS: Two plasmids were assumed to be the same plasmid, and this identification of a plasmid-mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first finding of the ColE-type plasmid carrying the qnrS2 gene.

Mycobacterium ulcerans causes minimal pathogenesis and colonization in medaka (Oryzias latipes): an experimental fish model of disease transmission.

Mycobacterium ulcerans causes Buruli ulcer in humans, a progressive ulcerative epidermal lesion due to the mycolactone toxin produced by the bacterium. Molecular analysis of M. ulcerans reveals it is closely related to Mycobacterium marinum, a pathogen of both fish and man. Molecular evidence from diagnostic PCR assays for the insertion sequence IS2404 suggests an association of M. ulcerans with fish. However, fish infections by M. ulcerans have not been well documented and IS2404 has been found in other mycobacteria. We have thus, employed two experimental approaches to test for M. ulcerans in fish. We show here for the first time that M. ulcerans with or without the toxin does not mount acute or chronic infections in Japanese Medaka "Oryzias latipes" even at high doses. Moreover, M. ulcerans-infected medaka do not exhibit any visible signs of infection nor disease and the bacteria do not appear to replicate over time. In contrast, similar high doses of the wild-type M. marinum or a mycolactone-producing M. marinum "DL" strain are able to mount an acute disease with mortality in medaka. Although these results would suggest that M. ulcerans does not mount infections in fish we have evidence that CLC macrophages from goldfish are susceptible to mycolactones.

Identification of differentially expressed genes and quantitative expression of complement genes in the liver of marine medaka Oryzias melastigma challenged with Vibrio parahaemolyticus.

The innate immune system of fish is the primary defense against acute diseases. The marine medaka Oryzias melastigma has been shown to be a potential marine fish model for ecotoxicology, but little is known about the innate immune system of this small fish. In this study, suppression subtractive hybridization (SSH) was used to identify differentially expressed immune genes in the liver of O. melastigma infected with Vibrio parahaemolyticus. Among the 396 genes identified, based on NCBI BLAST search of the 1279 sequenced clones in the SSH libraries, 38 (9.6%) were involved in the immune process. Besides, genes involved in biological regulations (5.6%); cellular metabolism (24.7%); general response to stimuli (4.8%); cellular component organization (2.3%); signal transduction (2.5%) and transport process (2.8%) were also obtained. Ten complement component genes involved in four activation pathways were quantified (using q-PCR) and exhibited different patterns of transcription between the control and challenged individuals. The results reported upon here support the feasibility of developing O. melastigma as a marine model fish to understand the basic biological processes related to immune function and for immunotoxicological research. Findings of this study established a genetic platform for studying immune function using O. melastigma.

Expression of common fluorescent reporters may modulate virulence for Mycobacterium marinum: dramatic attenuation results from Gfp over-expression.

Mycobacterium marinum is an established surrogate pathogen for Mycobacterium tuberculosis because of its strong conservation of thousands of orthologous genes, lower risk to researchers and similar pathology in fish. This pathogen causes TB-like chronic disease in a wide variety of fish species. As in human TB, the microbe grows within the host macrophages, can mount life-long chronic infections and produces granulomatous lesions in target organs. One of the fish species known to manifest chronic "fish TB" is the small laboratory fish, Japanese ricefish (medaka; Oryzias latipes). Our laboratory is currently characterizing the disease progression in medaka using fluorescent reporter systems that are introduced into engineered strains of M. marinum. While conducting these studies we observed differences in growth, plasmid stability, and virulence depending on which fluorescent reporter construct was present. Here, we describe large negative effects on virulence and organ colonization that occurred with a commonly used plasmid pG13, that expresses green fluorescent protein (Gfp). The studies presented here, indicate that Gfp over-expression was the basis for the reduced virulence in this reporter construct. We also show that these negative effects could be reversed by significantly reducing Gfp expression levels or by using low-expression constructs of Rfp.

Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes).

An accumulating body of research indicates there is an increased cancer risk associated with chronic infections. The genus Mycobacterium contains a number of species, including M. tuberculosis, which mount chronic infections and have been implicated in higher cancer risk. Several non-tuberculosis mycobacterial species, including M. marinum, are known to cause chronic infections in fish and like human tuberculosis, often go undetected. The elevated carcinogenic potential for fish colonies infected with Mycobacterium spp. could have far reaching implications because fish models are widely used to study human diseases. Japanese medaka (Oryzias latipes) is an established laboratory fish model for toxicology, mutagenesis, and carcinogenesis; and produces a chronic tuberculosis-like disease when infected by M. marinum. We examined the role that chronic mycobacterial infections play in cancer risk for medaka. Experimental M. marinum infections of medaka alone did not increase the mutational loads or proliferative lesion incidence in all tissues examined. However, we showed that chronic M. marinum infections increased hepatocellular proliferative lesions in fish also exposed to low doses of the mutagen benzo[a]pyrene. These results indicate that chronic mycobacterial infections of medaka are acting as tumor promoters and thereby suggest increased human risks for cancer promotion in human populations burdened with chronic tuberculosis infections.

Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract.

Antibiotics are commonly employed in most fish aquacultures to prevent disease. One major risk in this practice is that antibiotic-resistant pathogens may be selected. Therefore, we wanted to examine the feasibility of producing an economical, non-antibiotic alternative. The microalga Nannochloropsis oculata is an essential phytoplankton used as live feed for fish larvae. We attempted to culture N. oculata in a way that would provide an organism against bacterial pathogenic infection. To test this idea, we constructed an algae-codon-optimized bovine lactoferricin (LFB) fused with a red fluorescent protein (DsRed) driven by a heat-inducible promoter, which is a heat shock protein 70A promoter combined with a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2' promoter from Chlamydomonas reinhardtii. After electroporation, we examined 491 microalgal clones and generated two stable transgenic lines, each expressing a stable transgene inheritance for at least 26 months. This was confirmed by the positive detection of the mRNA transcript and the protein of LFB-DsRed produced by the transgenic microalgae. To test the efficacy of the antimicrobial peptide LFB, medaka fish (Oryzias latipes) were adapted from freshwater to seawater and were fed with the transgenic algae by oral-in-tube delivery method. Bacterial infection with 1 x 10(5)Vibrio parahaemolyticus per fish was induced 6h thereafter by oral-in-tube delivery as well. For medaka fish fed with 1 x 10(8) transgenic algae per fish, the average survival rate after a 24-h period of infection was much higher than that of medaka fed with wild-type algae (85+/-7.1% versus 5+/-7.1%). This result suggests that medaka fish fed with the LFB-containing transgenic microalgae will have bactericidal defense against V. parahaemolyticus infection in its digestive tract.

In ovo exposure to o,p -DDE affects sexual development but not sexual differentiation in Japanese medaka (Oryzias latipes).

Despite being banned in many countries, dichlorodiphenyltrichloroethane (DDT) and its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) continue to be found in fish tissues at concentrations of concern. Like o,p -DDT, o,p -DDE is estrogenic and is believed to exert its effects through binding to the estrogen receptor. The limited toxicologic data for o,p -DDE suggest that it decreases fecundity and fertility of fishes. We conducted an egg injection study using the d-rR strain of medaka and environmentally relevant concentrations of o,p -DDE to examine its effects on sexual differentiation and development. The gonads of exposed fish showed no evidence of sex reversal or intersex. However, other gonad abnormalities occurred in exposed individuals. Females exhibited few vitellogenic oocytes and increased atresia. Male testes appeared morphologically normal but were very small. Gonadosomatic index values for both sexes were lower for exposed fish. Our observations of abnormal female and very small male gonads after in ovo o,p -DDE exposure may be indicative of effects on early endocrine processes important for normal ovarian and testicular development.

Atypical piscine mycobacteriosis in Japanese medaka (Oryzias latipes).

Japanese medaka, (Oryzias latipes), small, freshwater, tropical cyprinodonts, are principally used for toxicologic and carcinogenicity assays, but are finding more applications in developmental genetic and biological research. An increase in mortality began in brood stock of adult medaka that had been shipped and housed separately by sex. Initially, mortality averaged one fish daily and began in females two weeks after they were received. Cohabitation began eight weeks after arrival. After four to six weeks of cohabitation in different spawning aquaria, mortality was observed in males. Clinical signs of disease included loss of scale luster and color, with subsequent blanching of dorsal flank musculature, small raised nodules on various external surfaces, emaciation, fraying of fin tips, and equilibrium disturbances. Histologic examination of affected adults revealed multi-organ granulomatous inflammation with intracellular acid-fast bacilli. Specimens from 46 juvenile medaka that were spawned from affected adults, were submitted for culture and histologic evaluation. Of 18 fish, two had lesions similar to those of adults. The organism isolated from the remaining fish was identified as Mycobacterium fortuitum. Due to atypical rapid progression of disease, spread of M. fortuitum to progeny, and poor prognosis, the entire colony was euthanized.