Program

In mammals, interleukin (IL)-17A and IL-17F are hallmark inflammatory cytokines, which are expressed by Th17 cells, and play key roles in protection against infection and intestinal mucosal immunity. Although, fish IL-17A and IL-17F homologs named as IL-17A/F have been identified, their functional aspects, especially in intestinal mucosal immunity are still poorly understood. In this study, IL17A/F1-mutated (IL17AF1-KO-) medaka (Oryzias latipes) was established using CRISPR/Cas9 system, and a 7-bp deletion was confirmed in IL-17A/F1 gene of IL17AF1-KO-medaka. To understand the role of IL-17A/F1, RNA-Seq analysis in the intestine of IL17AF1-KO-medaka was performed using a MiSeq next generation sequencer. In the intestine of IL17AF1-KO-medaka, expression levels of immune related genes [e.g., c-type lysozyme, g-type lysozyme, transferrin A, C1q-like protein, peptidoglycan-recognition protein 2 and interleukin(IL)-1β] and digestive enzyme genes (e.g., chymotrypsin-like protease, and carboxypeptidase A, and carboxyl ester lipase) were significantly decreased compared to those of the wild-type (WT) medaka. Expression of antimicrobial peptide and cytokine genes such as two lysozymes, transferrin A and IL-1β was confirmed by the quantitative real-time PCR. These results suggest that IL-17A/F1 could play an important role in regulation of antimicrobial peptide and digestive enzyme production in the intestine of medaka.

Bacterial flagellum is essential to their motility. Flagellum consists of several different flagellin proteins. Flagellin protein consists of flagella fiber containing D1, D2 and D3 domains and the D1 domain is recognized by a pattern recognition receptor, Toll-like receptor 5 (TLR5) to activate innate immune system. In this study, we examined potential of two flagellins from Edwardsiella tarda and Escherichia coli to induce immune response differently in the Japanese flounder (Paralichthys olivaceus). The activation sites in the D1 domain of E. tarda flagellin were not conserved with those of E. coli flagellin, which possesses a potential to induce immune responses mediated by mammalian TLR5. Over-expressing DNA plasmids encoding the two flagellin genes (pEtFliC and pEcFliC) and TLR5-soluble gene (pTLR5S) were constructed, and intramuscularly injected to the Japanese flounder. The expression of inflammatory cytokine gene (e.g., Interlukine-1β) was differently induced in the flounder injected with pEtFliC + pTLR5S and that of pEcFliC + pTLR5S compared to the flounder injected with the empty vector. The results suggested that D1 domain sequence of E. tarda flagellin could involve in inflammatory response in the Japanese flounder.

Endonucleases are enzymes that cleave phosphodiester bonds within a polynucleotide chain. In previous study, mRNA levels of endonuclease domain-containing 1 gene (Jf_ENDOD1) were up-regulated after intraperitoneal injection of Edwardsiella tarda formalin killed cells (FKC) in Japanese flounder Paralichthys olivaceus. Furthermore, DNase activity were enhanced after the FKC injection in the kidney and spleen.
In order to reveal the molecular properties of Jf_ENDOD1, the effect of divalent metal ions and temperature on the enzymatic activities were assessed using the recombinant protein (rJf_ENDOD1). In addition, to show the in vivo roles of the molecule, changes of Jf_ENDOD1 protein levels in the kidney after E. tarda-FKC injection was analyzed by Western blotting.
DNase activity of rJf_ENDOD1 was affected by chelating with EDTA but not EGTA, suggesting that the activity is dependent on Mg2+ but not Ca2+. The optimum temperature of rJf_ENDOD1 was 55 ℃, and the activities were not observed at 5 ℃ and 65 ℃. The Jf_ENDOD1 protein was detected in the kidney of healthy fish. The protein levels were decreased at 12 hours post injection, while mRNA level of Jf_ENDOD1 was remarkably up-regulated at 12 hpi. In addition, there was no difference in the DNase activity among experimental groups. Thus, further studies will be needed to reveal the relationship between transcription and translation of Jf_ENDOD1.

Insects attract to be a source of fish feed that produce various useful substances. Our previous study has identified a novel polysaccharide from silkmoth (Antherea yamamai) pupae that can activate innate immune response of mammalian macrophages, and we named this polysaccharide “Silkrose”. It has been confirmed that Silkrose prevents infectious diseases of teleost fishes, but the protection mechanism has not been uncovered yet. In this study, we elucidate defence mechanism of Silkrose by analysing comprehensive gene expression profile of medaka (Oryzias latipes) in Edwardsiella tarda infection.

Since the efficacy of Silkrose for preventing infectious diseases had not been confirmed in medaka, we first established challenge study system, in which E. tarda was infected to medaka by immersion after feeding the diets with or without Silkrose (1 and 10 ng/g) for 7 days. The survival rate percentages were 70 and 80% in 1 and 10 ng/g Silkrose groups respectively, in contrast to 45% in control group after 8 days post challenge. For infection rates on kidney, fishes in Silkrose group were less positive than the fishes in the control group. Therefore, the addition of Silkrose in fish feed was resulted in significant increases in protection for bacterial disease through preventing infection.

To understand the gene expression profile, DNA microarray was performed before and after challenge. Of total 36398 probes, 2930 probes exhibited differential expression patterns between Silkrose and control groups (upregulated: 836, downregulated: 2094). Focusing on representative genes that are known to involved in innate and acquired immunity and intercellular barrier function of mucosal epithelia, gene responses on E. tarda infection were more rapid in Silkrose group than control group.

Our result not only provides comprehensive gene expression profile in bacterial pathogen infection in teleost fish, but also provides evidence to understand protection mechanism from infectious pathogen by immunostimulative polysaccharide.

The rapid expansion of the global tilapia production is due in large part to the intensification of culture systems made possible with the use of commercial pelleted feeds. Intensification of culture systems often leads to increased incidence of disease. Functional feeds are the new generation of aquafeeds with additional function, usually with health-promoting properties beyond their nutritional value. Our research into the use of organic acids as a functional feed additive in tilapia feeds as an alternative to the use of harmful antibiotics will be presented.

Four 20-meter diameter plastic circular floating cages (2 replicates per treatment) in a tilapia farm were used in the study. All male Nile tilapia fingerlings (~25 g) were stocked at a density of 60,000 fish per cage. The tilapia feeds, containing no added organic acids or 2% of a prototype organic acids blend were produced and purchased from Cargill Ltd., Malaysia. With the exception of the added organic acids, both feeds were similarly formulated. Two to four weeks before the final harvest, 85 large fish were randomly sampled using a dip net from each cage. All fish were dissected, tissues excised and fecal samples extracted from the terminal segment of the tilapia gut.

Results indicated that dietary organic acids did not significantly (P>0.05) affect growth and biological indices but a general trend of improved nutrient utilization efficiency was observed. Reduced antibiotic use was registered during the culture period when fish were fed organic acid-added feeds. Decreased total viable bacteria and increased lactic acid bacteria counts in the gut of tilapia fed organic acids-added feeds was observed. Tilapia fed organic acids-added feeds had significantly (P<0.05) enhanced serum lysozyme activity. Functional feed additives such as organic acids can be used as an immuno-stimulant to offer a more environmentally friendly strategy for intensive tilapia farming.

In the last decade, medicinal plants have been used for treatment and control fish pathogens in aquaculture. Rosemary is a new candidate plant for elimination of monogenean parasites. In this study, we investigated the histological effects of rosemary treatment on monogeneans and also regeneration activity within damaged gills. The effects of rosemary extracts on organs during immersion and oral administration were examined histopathologically. In addition, we investigated the pharmacokinetic activity of 1,8-cineole, a major bioactive component in rosemary.
Doses of 1, 10 and 50 g/L (safe doses for fish) rosemary aqueous extract were used for parasite infected and uninfected fish in immersion treatments of 30 and 60 min. Gill regeneration was examined at 10 days, 1, 3, 6 and 12 months after treatment. At 50 g/L extract treatment, parasites were eliminated from the host's gills. Moreover, gill fusion, mucus secretion and hyperplasia activity recovered completely after 3 months. In a oral experiment, uninfected fish were fed with 10 ml/100 g (extract/feed) ethanol extract and 100 ml/100 g aqueous extract for 20 days. Gill, intestine midsection, liver and caudal kidney were investigated by histopathology. Uninfected and control samples were not significantly different in histopathology. The pharmacokinetic activity of 1,8-cineole in blood and mucus were analyzed by GC-MS. 1,8-cineole was detected in blood and mucus. Following oral administration, 1,8-cineole level in blood peaked at 60 min. The elimination half-lives of 1,8-cineole ranged from 120 to 720 min in fish blood.
These results suggest that rosemary extract would be usable for fish health. Treatment and control of monogenean parasitic diseases appear plausible. Furthermore, 1,8-cineole is apparently anthelmintic via host blood and mucus.

The project was conducted with Nile tilapia, Oreochromis niloticus, at APTA/Pólo - Centro Leste/UPD de Pirassununga/SP - Brazil and aimed to evaluate the parameters of growth performance, microbiology, hematology, immunology, food digestibility, digestive enzymes with [Bacillus subtilis - (1,6x10¹⁰UFC g^-1) and Bacillus licheniformis - (1,6x10¹⁰UFC g^-1)] included in the diet of Nile tilapia at concentrations of 0.00; 0.02 0.04 and 0.08% of fish fed, conducting with experimental design interely randomized with four treatment and four replicates. The inclusion in the probiotic diet induced large changes in the intestinal microbiota of tilapia species richness and values of habitability values in the three portions of the intestinal tract (P<0.05). The changes observed in this study reflect increases in the genetic variability of the intestinal microbiota of these specimens Diets containing 0.04% and 0.08% of probiotic presented higher weight gain when compared to the control group and fish that received 0.02%. The fish that received 0.08% of probiotic in diet had high values of CHCM when compared to the treatment that received 0.02 and 0.04% of inclusion in the diet, however there was no difference between the fish of control group. Statistical differences in thrombocyte values were observed between the control treatment and the 0.04% inclusion treatment. No differences were observed in the phagocytic activity of the macrophages, NBT assay of blood, lysozyme serum, glucose serum, fed digestibility and in the production of digestive enzymes among the treatments tested. The inclusion of the probiotic Bioplus 2BC^® in the diet of Nile tilapia resulted in better weight gain, increase in the number of thrombocytes in the blood stream and modified the intestinal microbiota increasing the values of habitability and species richness.

Application of microalgae to strengthen the resistance the virus disease in shrimp would be another alternative approach to control the disease. Experiment was conducted to examine the effect of extracellular products of microalgae against covert mortality nodavirus (CMNV) in Pacific white shrimp (Litopenaeus vannamei). A completely randomized design with three replications was performed. There were six treatments as follows: (1) 2.6% NaCl, (2) CMNV + Amphora coffeaeformis, (3) CMNV + Spirulina platensis, (4) CMNV + Tetraselmis sp., (5) CMNV + Nanochloropsis spp. (6) CMNV + Thalassiosira wessflogii and (7) CMNV only. The extracellular products was mixed with CMNV (LD50) in a ratio of 1:1 and incubated at room temperature for 2 hr. Shrimps have a mean body weight (MBW) 10 g were injected with 0.2 ml of each mixture solution. The cumulative mortality of each group was observed for 14 days and expressed in terms of relative percent survival (RPS). The highest RPS (50.00%) was observed in shrimps injected with Spirulina platensismixed with CMNV. Application of the extracellular products of S. platensis in enhancing the resistance of CMNV in L. vannamei is considered.

Weissella sp. causes weissellosis in rainbow trout in China, Brazil, and the USA, which can result in high mortality for market-sized fish and significant economic loss. Several methods have been employed to prevent the outbreak of this disease. We investigated the therapeutic appication of a novel bacteriophage of Weissella sp. for the first time. The phage was collected from cultured Weissella sp. isolated from the kidney of diseased fish, and named phage PWc. It belonged to the family Siphoviridae and possessed an isometric head (approximately 65 nm in diameter) and a flexible, non-contractile tail of 170-180 nm in length. The latent time and burst size of the phage were approximately 25 min and 16 PFU/infected cell, respectively. It was relatively stable at pH ranging from 3 to 10 with 60% survival at 60°C for 1 h. The phage was observed to have a wide host range and lysed all 36 Weissella sp. strains tested. Measurement of its antibacterial activity at different temperatures revealed that it could be used for therapy at 15 to 30°C. The protein profile and whole genome sequence were also obtained, and the phage was found to possess a circular genome consisting of 38,783 bp and 54 ORFs. No significant similarity was detected between the phage’s genome and bacterial and viral genomes in GenBank. This is the first study to investigate a bacteriophage virulent to a newly emerging pathogen that causes an infectious disease in rainbow trout. The various features of the phage identified in this study suggest that it could be used for therapy in the future.

The two biosubstances of α-mangostin (a natural xanthonoid) and gartanin (a naturally-occurring derivative of xanthone) from mangosteen (Garcinia mangostana L.) have been reported as the novel bioactive compounds in human as food supplement, herbal cosmetics and pharmaceuticals. In this study, we were extracted from mangosteen pericarp and purified by HPLC fractionation. We investigated for their in vitro antifungal activities against saprolegniasis fungal species; i.e. Aphanomyces invadans, Achlya bisexualis and Saprolegnia diclina. Fungistatic effect was determined by agar disc diffusion and microscopic techniques. Fungicidal effect of hyphae was determined by immersion and observing for the presence of fungal growth. It resulted that α-mangostin and gartanin exhibited fungistatic effects on mycelium growth of A. invadans at concentrations of 125 and 250 ppm respectively, and S. diclina at concentrations of 125 ppm. Whereas A. bisexualis was inhibited at concentrations of 250 ppm. It also showed that inhibition efficacy gradually increased upon higher concentration. The abnormal hypha growth, with short branch and dense, was observed at the mycelium edge of inhibition zone. Fungicidal effect of zoospore was also investigated at concentrations of 125, 250, 500, 1000 ppm. Zoospore growth was observed by agar plate culture and SEM technique. Therefore, α-mangostin and gartanin are to be candidate a novel effective antifungal bio-substance that instead of antibiotics in Saprolegniasis fungi treatment. The pharmaceutical form of α-mangostin and gartanin for aquatic animals will be developed. Our result will be been high impact for safety and organic aquaculture.

The extensive use of tetracycline as prophylactic and therapeutic agent in aquaculture, results in the occurrence of antibiotic resistant bacteria and Antibiotic Resistant Genes (ARGs) in the aquaculture environment. The aim of the study was determined the genetic determinants which responsible for tetracycline resistance in isolated tetracycline resistance bacteria in aquaculture farms in Sri Lanka. In this study, 42 Tetracycline (TET) resistant bacteria strains (MIC ≥60 µg/ml) were isolated from 16 aquaculture sites. They were examined for the presence of selected tet genes (tet A, tet B, tet M, tet S ) using Polymerase Chain Reaction (PCR) method. Bacillus and Staphylococcus were the most dominant bacterial genera recorded for both OTC and TET resistance. Acinetobacter sp., Achromabacter sp., Staphylococcus sp., Micrococcus sp. were also identified as for TET and OTC. Out of 42 bacteria isolates one or more tet genes were detected in 30 strains (71%). However 12 bacteria strains were not positive (29%) for selected tet genes. It was found that, out of 30 tet gene positive bacteria, 21 bacterial strains were positive for tet (M), where 14 strains were positive for tet (A) gene, 11 bacterial strains for tet (S) and 8 bacterial strain for tet (B) gene respectively. The results reveled that, tet M (70%) was the most determinant gene, followed by tet A (47%), tet S (37%) and tet B (27%). Therefore, the results of the present study showed that bacterial isolates from aquaculture sources in Sri Lanka harbor a variety of tetracycline resistance genes which implies an urgent need to have monitoring system limit and monitor antibiotic usage in aquaculture as this may lead to problems in the efficiency of antibiotics which used to treat fish diseases and eventually to production losses at fish farms.

Key words:- Tetracycline (TET); Oxytetracycline (OTC); Antibiotic Resistance Genes (ARGs)

Photobacterium damselae subsp. piscicida (Pdp) is the causative agent of acute fish pasteurellosis, a very serious bacterial septicemia. This disease was observed in yellowtail Seriola quinqueradiata (in Japan), perch Morone americanus, striped bass M. saxatilis (in the USA), gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax (in Europe). To understand the propagation mechanism of drug resistant genes, virulence factors and infection mechanism of Pdp, it is very important for establishment of genetic basis on the complete genome. In this study, complete genome sequences of two strains of Pdp isolated in Japan (OT-51443 strain) and the USA (91-197 strain) were determined and compared between them. Genomic DNA was extracted from Pdp strains (OT-51443 and 91-197 strains), which were isolated from yellowtail and hybrid striped bass (Morone sp.). Nucleotide sequences of Pdp were determined using PacBio-RS-II sequencing platform (Pacific Biosciences). The sequencing data was assembled using HGAP2 version3, Falcon and PBcR, and the gaps were manually closed by the conventional PCR method. Genome annotation was completed using the Prokka pipeline and Rapid Annotations using Subsystems Technology (RAST) to identify protein-coding sequences (CDS), tRNA and rRNA. While OT-51443 genome was composed of two circular chromosomes (Ch.1: 3.13Mb, Ch.2: 1.01Mb) and five plasmids, 91-197 genome contained two circular chromosomes (Ch.1: 3.18Mb, Ch.2: 1.05Mb) and two plasmids. In Japanese strain OT-51443, Ch.1 encoded 3,164 CDSs, 129 tRNAs and 44 rRNAs, and Ch.2 encoded 1,429 CDSs and 3 tRNAs. In contrast, Ch.1 encoded 3,211 CDSs, 129 tRNAs and 49 rRNAs, and Ch.2 encoded 1,496 CDSs and 3 tRNAs in the US strain 91-197. Difference between both strains was found in CDSs encoding in the chromosomes and in number of the plasmids.

In recent years, light emitting diode technology has been applied to the inactivation of pathogens, especially those affecting humans. The purposes of this study were to assess the effect of blue LED (405 and 465nm) on antimicrobial activity of seven fish bacterial pathogens, scuticociliate Miamiensis avidus and viral hemorrhagic septicaemia virus (VHSV) using blue LED irradiation. Some pathogenic bacterial species were not completely inactivated by exposure of 465 nm wavelength, but all the pathogens were killed by 405 nm. In particular, P. damselae, V. anguillarum and E. tarda, and S. iniae, A. salmonicida and S. parauberis were more susceptible to 405 and 465 nm, respectively. M. avidus and VHSV were also inactivated by blue light irradiation, and, in particular, we demonstrated that M. avidus was killed by apoptosis, Irradiation of blue LEDs led to decrease viable pathogens in rearing water of a tank containing infected fish (i.e., olive flounder or fancy carp). Also, 405 and 465 nm LED reduced mortality of fish cohabitated with infected fish compared to control. However, these blue LEDs did not appear to induce adverse effects on fish, which were confirmed by histopathologic examination.

Edwardsiella spp. consist of three species: Edwardsiella hoshinae, E. ictaluri and E. tarda. Of these, E. ictaluri and E. tarda are well known bacterial pathogens affecting many different fish species, including eels, olive flounder and catfish. Recent studies showed that E. tarda harbored at least two more new species, E. anguillarum and E. piscicida, based on average nucleotide identity. In this study, we performed comparative genome analysis using hitherto sequenced genomes of strains belonging to the genus Edwardsiella. Twenty seven hitherto sequenced genomes consisted of total of 9307 protein-encoding genes, only 16% of the genes (1447) were core genes, and the remaining 84.5% were dispensable and singleton genes within the genus. Pan development plot analysis showed that E. piscicida and E. anguillarum had open pan-genome, indicating that Edwardsiella spp. are adaptable to a variety of environments, and it is expected that the genes shared by species may be related to adaptation ability and species specificity. The phylogeny using the pan-genome and the core genes showed clear lineage between species within the genus. In particular, based on phylogenetic analysis using core genes, E. anguillarum and E. piscicida were more closely related to E. ictaluri than E. tarda. This study shows that strains that have been identified as E. tarda were re-identified as E. anguillarum and E. piscicida, and pan-genome analysis would provide very valuable information on epidemiology.

The present study evaluated the benefits of dietary administration of host-derived probiotics Lactococcus lactis WFLU12 (at 10⁹ CFU g^-1 feed for 8 weeks) in olive flounder with a group fed with the basal diet serving as control. Results showed that the growth and natural infection rate were significantly improved in probiotic groups. By conjunction with hitherto-sequenced L. lactis genomes, the comparative synteny analysis indicated that strain WFLU12 has been evolved through change of gene ordering on a chromosome which may indicate long-term colonization of the strain in the gastrointestinal tract. Interestingly, the singleton genes extracted from strain WFLU12 provided insights into their additional properties functioning in antagonistic activity, adhesion and resistance ability, which might enhance its survival capacity and then can secrete beneficial compounds at optimal levels as advantages of host-derived probiotic bacteria. Whilst study of metabolome unraveled that the better growth performance in probiotic group may related to the significant increase of intestinal metabolites levels which were dominated in lipid metabolism relatives involving in the improvement of fat digestibility and feed utilization. Together, the increase of coenzymes including ascorbic acid, riboflavin, pyridoxine, and nicotinamide in fish fed with probiotic might enhance many process of oxidases and reductases in the enzymatic breakdown of energy-yielding nutrients. Some metabolites increased significantly in serum seem to be connected to its yields in intestine, and are being candidate markers (i.e. citrulline, cystathionine) for studying the growth performance in fish. Genes of WFLU12 encoding functions involved in the increase of metabolites in intestine. In conclusion, the genome features facilitate the strategy of host-derived probiotic development in modern aquaculture while the metabolome analysis is critical for clarifying the influence of probiotic on the metabolomics profiles, homeotastis, as well as understanding their contribution to fish health and welfare.