Ricardo Enríquez

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.

Serum amyloid A: a typical acute-phase reactant in rainbow trout?

Acute serum amyloid A (A-SAA) has been considered a major acute-phase reactant and an effector of innate immunity in all vertebrates. The work presented here shows that the expression of A-SAA is strongly induced in a wide variety of immune-relevant tissues in rainbow trout, either naturally infected with Flavobacterium psychrophilum or challenged with lipopolysaccharide (LPS) or CpG oligonucleotides (CpG ODN). Nevertheless, A-SAA was undetectable by Western blot either in the plasma or in high-density lipoprotein (HDL) of infected or challenged fish, using either an anti-mouse SAA1 IgG or an anti-trout A-SAA peptide serum, which recognise both the intact recombinant trout A-SAA and fragments derived from it. However, the anti-peptide serum was the immunoreactive in all primary defence barriers and in mononuclear cells of head kidney, spleen and liver. These findings reveal that, unlike mammalian SAA, trout A-SAA does not increase significantly in the plasma of diseased fish, suggesting it is more likely to be involved in local defence.

Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile.

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, a severe disease causing high mortalities in salmonids. This bacterium has been previously identified and isolated in all cultivated salmonids in Chile and worldwide, including Salmo salar, Oncorhynchus kisutch, and O. mykiss, in addition to being found in non-salmonid species such as Dicentrarchus labrax and Atractoscion nobilis. In this study, the 16S rRNA gene and intergenic spacer ITS-1 of P. salmonis were amplified by PCR from DNA samples extracted from the native Chilean fish species Eleginops maclovinus, Odontesthes regia, Sebastes capensis, and Salilota australis. Analysis of the 16S rRNA sequences from O. regia demonstrated a close phylogenetic relationship with the 16S rRNA gene in the Chilean EM-90 strain. The 16S rRNA sequences from E. maclovinus, S. capensis, and S. australis were related to the Chilean LF-89 sequence and Scottish strains. To confirm these findings, analysis of P. salmonis ITS-1 sequences obtained from the 4 sampled native species demonstrated a high degree of identity and a close phylogenetic relationship with Chilean P. salmonis sequences, including LF-89 and EM-90. These results suggest a strong relationship between the nucleotide sequences from the 16S rRNA and ITS-1 genes amplified from native fish with those sequences described in the first P. salmonis strains to be identified and isolated in Chile.

First identification of Francisella noatunensis subsp. orientalis causing mortality in mexican tilapia Oreochromis spp

Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

New immunomodulatory role of neuropeptide Y (NPY) in Salmo salar leucocytes

Abstract Neuropeptide Y (NPY) plays different roles in mammals such as: regulate food intake, memory retention, cardiovascular functions, and anxiety. It has also been shown in the modulation of chemotaxis, T lymphocyte differentiation, and leukocyte migration. In fish, NPY expression and functions have been studied but its immunomodulatory role remains undescribed. This study confirmed the expression and synthesis of NPY in S. salar under inflammation, and validated a commercial antibody for NPY detection in teleost. Additionally, immunomodulatory effects of NPY were assayed in vitro and in vivo. Phagocytosis and superoxide anion production in leukocytes and SHK cells were induced under stimulation with a synthetic peptide. IL‐8 mRNA was selectively and strongly induced in the spleen, head kidney, and isolated cells, after in vivo challenge with NPY. All together suggest that NPY is expressed in immune tissues and modulates the immune response in teleost fish. HighlightsS. salar immune cells express and synthetize a NPY‐like molecule under pro‐inflammatory conditions.The NPY peptide by itself may induce leukocytes priming and its activation, increasing the innate immune response in fish.NPY induces IL‐8 overexpression and supports a cellular immune response in activated leukocytes in vitro and in vivo.