Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48 h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and + 38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.

Effect of Flavobacterium psychrophilum on the neuroendocrine response of rainbow trout (Oncorhynchus mykiss) in a time course experiment.

The aim of this study was to examine the effects of Flavobacterium psychrophilum, a pathogen that is economically important in the aquaculture sector, on the neuroendocrine response of Oncorhynchus mykiss during a time course experiment with sampling at 0.5, 1, 2, 6, 10, and 30 days post injection (dpi). In the brain, serotonin (5HT) content increased in the infected group at all the measured time points, a similar pattern was observed for 5-hydroxyindole-3-acetic acid (5HIAA). Infected fish presented an increase in brain dopamine levels on day 0.5 and 1 dpi. A non-significant variation in noradrenaline levels was observed on all treatment days. Foregut 5-HT and 5-HIAA content in the infected group presented the highest 5-HT concentrations with 248.6 and 983.5 ng/g tissue at 0.5 dpi respectively. Midgut 5-HT and 5-HIAA levels presented the highest 5-HT concentrations, 486.9 ng/g tissue and 1006.4 ng/g tissue respectively, at the beginning of the experiment (0.5 dpi). 5-HT levels in the hindgut presented the highest concentrations with 233.9 ng/g tissue at 0.5 dpi, while 5-HIAA presented the highest concentrations, 690.5 ng/g tissue, at the same time point. After injection with F. psychrophilum the neuroendocrine response in rainbow trout was tissue dependent. Brain levels of 5HT and 5HIIA indicate that the neuroendocrine response increased together with dopamine following intramuscular infection. These increases are in line with reports from other authors, indicating an early response of catecholamines as neurotransmitters to stressful stimulus. In addition the intestinal response was also increased, implying that there could be a possible relationship between the serotonergic system at the intestinal level and the immune system.

High doses of Francisella noatunensis induces an immune response in Eleginops maclovinus.

Francisella noatunensis subsp. noatunensis, the etiological agent of Francisellosis, affects a large number of farmed species such as Salmo salar. This species coexists with several native species in the same ecosystem, including Eleginops maclovinus. Our objective was to evaluate the susceptibility, presence of clinical symptoms, and the ability of Eleginops maclovinus to respond to Francisella infection. For this, healthy individuals were inoculated with 1.5 × 101, 1.5 × 105, and 1.5 × 1010 bact/µL of Francisella by intraperitoneal injection, subsequently the fish were sampled on days 1, 3, 7, 14, 21, and 28 post injection (dpi). At the end of the experiment, no mortality, nor internal and external clinical signs were observed, although in the high dose anaemia was detected. Additionally, bacteria were detected in all three doses, however there was replication at day 28 only in the liver in the high dose. Analysis of gene expression by qPCR showed that the spleen generated an immune response against infection from day 1 dpi, however at day 7 dpi most of the genes suffered repressed expression; observing over expression of the genes C3, NLRC3, NLRC5, MHCI, IgM. In contrast, expression in the anterior kidney did not vary significantly during the challenge. IgM quantification showed the production of antibodies in the medium and high doses. This study provides new knowledge about Francisella infection and the long-lasting and specific immune response generated by Eleginops maclovinus. It also demonstrates its susceptibility to Francisellosis where there is a difference in the immune response according to the tissue.

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico.

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S-1 and S-2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP-PCR and ERIC-PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 104 and 106  CFU per fish with the S-1 isolate, while 100% mortality rates were recorded in zebrafish at 102 and 104  CFU per fish with the S-2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.

Comparative genome analysis of two Streptococcus phocae subspecies provides novel insights into pathogenicity.

Streptococcus phocae is a beta-hemolytic, Gram-positive bacterium that was first isolated in Norway from clinical specimens of harbor seal (Phoca vitulina) affected by pneumonia or respiratory infection, and in 2005, this bacterium was identified from disease outbreaks at an Atlantic salmon farm. A recent comparative polyphasic study reclassified Streptococcus phocae as subsp. phocae and subsp. salmonis, and there are currently two S. phocae NCBI sequencing projects for the type strains ATCC 51973T and C-4T. The present study compared these genome sequences to determine shared properties between the pathogenic mammalian and fish S. phocae subspecies. Both subspecies presented genomic islands, prophages, CRISPRs, and multiple gene activator and RofA regulator regions that could play key roles in the pathogenesis of streptococcal species. Likewise, proteins possibly influencing immune system evasion and virulence strategies were identified in both genomes, including Streptokinases, Streptolysin S, IgG endopeptidase, Fibronectin binding proteins, Daunorubicin, and Penicillin resistance proteins. Comparative differences in phage, non-phage, and genomic island sequences may form the genetic basis for the virulence, pathogenicity, and ability of S. phocae subsp. salmonis to infect and cause disease in Atlantic salmon, in contrast to S. phocae subsp. phocae. This comparative genomic study between two S. phocae subsp. provides novel insights into virulence factors and pathogenicity, offering important information that will facilitate the development of preventive and treatment measures against this pathogen.

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 µm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 µm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 µm), it did facilitate growth at lower iron concentrations (10 µm and 1 µm). An exception was the H-2 isolate, which only grew with 100 µm of iron sulphide. In turn, 100 µm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

Isolation, Characterization and Virulence Potential of Tenacibaculum dicentrarchi in Salmonid Cultures in Chile.

In this study, we isolated, identified and characterized isolates of Tenacibaculum dicentrarchi in Atlantic salmon (Salmo salar) farmed in Chile for the first time. In 2010 and 2014, mortalities were observed in Atlantic salmon (average weight 25-30 and 480-520 g, respectively) at an aquaculture centre in Puerto Montt, Chile. Severe tail rots, frayed fins and, in some cases, damaged gills were detected. Wet smear analyses of these lesions revealed a high occurrence of Gram-negative, filamentous bacteria. Microbiological analysis of infected gill and tail tissues yielded six bacterial isolates. All were identified as T. dicentrarchi through polyphasic taxonomy, which included phenotypic characterization, 16S rRNA sequencing and multilocus sequence typing. The latter method revealed a close relationship of the Chilean genotype with the T. dicentrarchi type strain and two Norwegian Atlantic cod (Gadus morhua) isolates. The pathogenic potential of the TdChD05 isolate was assessed by challenging Atlantic salmon and rainbow trout (Oncorhynchus mykiss) for one hour, which resulted in mean cumulative mortality rates of 65% and 93%, respectively, as well as clinical signs 14 days post-challenge. However, challenged Coho salmon (Oncorhynchus kisutch) presented no mortalities or clinical signs of infection. These findings indicate that the geographical and host distribution of T. dicentrarchi is wider than previously established and that this bacterium may have negative impacts on salmonid cultures.

Bacterial lipopolysaccharide induces rainbow trout myotube atrophy via Akt/FoxO1/Atrogin-1 signaling pathway.

Lipopolysaccharide (LPS) is considered as a powerful inducer of muscle atrophy in higher vertebrates due to skeletal muscle cell recognition of the endotoxin and a consequent activation of catabolic signaling pathways. In contrast, there is no evidence of LPS directly inducing skeletal muscle atrophy in lower vertebrates, such as fish. For years it has been assumed that fish are resistant to LPS, mainly due to differences in the key features of toll-like receptor (TLR) signaling pathways when compared with mammals. In this study, we report that the stimulation of cultured rainbow trout (Oncorhynchus mykiss) myotubes with LPS (100 ng/ml) resulted in a transient decrease in the pAkt/Akt ratio, a subsequent reduction in the pFoxO1/FoxO1 ratio, and a significant increase in atrogin-1 transcript expression. Preincubation with polymyxin B, an LPS-neutralizing agent, and 740 Y-P, an agonist of p85-PI3K, blocked the effects of LPS. Additionally, LPS treatment induced an increase in protein ubiquitination and a reduction in myotube diameter, both of which are associated with muscular atrophy that is not observed under polymyxin B and 740 Y-P pretreatments. Finally, rainbow trout myotubes expressed the genes tlr1, tlr3, tlr5m, tlr8a1, tlr8a2, tlr9, and tlr22, with significantly increased expressions of tlr5m and tlr9 under LPS stimulation. These results indicate that LPS is an inducer of fish skeletal muscle atrophy and suggest that TLR5M and TLR9 may play important roles in detecting LPS, which supports for the first time the hypothesis that LPS is a direct inducer of skeletal muscle atrophy in teleost species.

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.

Cell-surface properties of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chilean farms.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture, but the factors determining its virulence have not yet been elucidated. In this work, cell-surface-related properties of the isolates responsible for outbreaks in Atlantic salmon were investigated. We also briefly examined whether pathogenicity against fish varied for V. ordalii strains with differing cell-surface properties. Hydrocarbon adhesions indicated the hydrophobic character of V. ordalii, although only 4 of 18 isolates induced haemagglutination in Atlantic salmon erythrocytes. A minority of the studied isolates (6 of 18) and the type strain ATCC 33509T produced low-grade biofilm formation on polyethylene surface after 2 h post-inoculation (hpi), but no strains were slime producers. Interestingly, V. ordalii isolates showed wide differences in hydrophobicity. Therefore, we chose 3 V. ordalii isolates (Vo-LM-03, Vo-LM-18 and Vo-LM-16) as representative of each hydrophobicity group (strongly hydrophobic, relatively hydrophobic and quasi-hydrophilic, respectively) and ATCC 33509T was used in the pathogenicity studies. All tested V. ordalii strains except the type strain resisted the killing activity of Atlantic salmon mucus and serum, and could proliferate in these components. Moreover, all V. ordalii isolates adhered to SHK-1 cells, causing damage to fish cell membrane permeability after 16 hpi. Virulence testing using rainbow trout revealed that isolate Vo-LM-18 was more virulent than isolates Vo-LM-03 and Vo-LM-16, indicating some relationship between haemagglutination and virulence, but not with hydrophobicity.

PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.

Infectivity study of Streptococcus phocae to seven fish and mammalian cell lines by confocal microscopy.

Streptococcus phocae is a beta-haemolytic bacterium that causes systemic infections in Atlantic salmon, Salmo salar L., cultured in southern Chile and also in seals. In this study, the host-pathogen interaction between S. phocae and seven types of cell lines (fish and mammalian) was examined using an indirect fluorescent antibody and confocal microscopy (CM). Chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), salmon head kidney (SHK-1) and Atlantic salmon kidney were used as the fish cell lines, while human cervix epithelial adenocarcinoma (HeLa), African green monkey kidney fibroblast (Cos-7) and mouse leukaemic monocyte macrophage (Raw 264.7) were included as mammalian cell lines. Streptococcus phocae type strain ATCC 51973(T) and isolates LM-08-Sp and P23 were selected as representatives from the salmon and seal host, respectively. For the CM examination, monolayers seeded on round coverslips were studied at 2- and 20-h post-inoculation (pi). The results showed that there is no common infectivity pattern between the three S. phocae strains at 2-h pi and the cell lines tested, regardless of the source of isolation (seal or salmon). All S. phocae strains could internalize and were found inside the fish and mammalian cell cytoplasm after 20-h pi. Regardless of the cells studied (fish or mammal) and incubation (2 and 20 h), S. phocae was never observed inside the nuclei. Seal and salmon isolates showed the highest number of bacteria entering into the primate cell lines (HeLa and Cos-7) from 2-h pi, while ATCC 51973(T) was not found outside or inside the HeLa and Cos-7 cells.

Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis .

Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cell--culture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRAL-SRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines.

Flavobacterium chilense sp. nov. and Flavobacterium araucananum sp. nov., isolated from farmed salmonid fish.

Three Gram-staining-negative non-endospore-forming strains were isolated from farmed fish in Chile: one (LM-09-Fp(T)) from a rainbow trout (Oncorhynchus mykiss) and the others (LM-19-Fp(T) and LM-20-Fp) from two Atlantic salmon (Salmo salar). Phylogenetic analyses based on 16S rRNA gene sequences indicated that all three isolates belonged to the genus Flavobacterium. In these analyses, strain LM-09-Fp(T) appeared most closely related to the type strains of Flavobacterium chungangense (98.5 % sequence similarity), Flavobacterium glaciei (98.2 %), Flavobacterium aquidurense (97.6 %), Flavobacterium saccharophilum (97.6 %) and Flavobacterium hercynium (97.6 %). The 16S rRNA gene sequences of strains LM-19-Fp(T) and LM-20-Fp were found to be identical and most similar to the corresponding sequences of the type strains of Flavobacterium aquidurense (98.6 %), Flavobacterium frigidimaris (98.5 %), Flavobacterium hercynium (97.9 %), Flavobacterium saccharophilum (97.7 %) and Flavobacterium pectinovorum (97.7 %). For each of the three novel strains, menaquinone (MK-6) was the predominant respiratory quinone and the major compounds in the polar lipid profile were phosphatidylethanolamine, an unidentified aminolipid, phosphatidylserine and two or three unknown lipids. The fatty acid profile of each strain, which comprised major amounts of iso-C(15:0), C(15:0) and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH) as well as smaller amounts of various hydroxylated fatty acids (e.g. iso-C(16:0) 3-OH, iso-C(17:0) 3-OH, C(16:0) 3-OH and C(15:0) 3-OH), indicated that each belonged to the genus Flavobacterium. Based on their physiological and biochemical characteristics and the results of DNA-DNA hybridizations, which showed relatively low levels of relatedness between the novel strains and the most closely related Flavobacterium species, strain LM-09-Fp(T) ( = LMG 26360(T) = CCM 7940(T)) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium chilense sp. nov. is proposed, and strains LM-19-Fp(T) ( = LMG 26359(T) = CCM 7939(T)) and LM-20-Fp ( = LMG 26331) represent a second novel species within the same genus, for which the name Flavobacterium araucananum sp. nov. is proposed.