Piscirickettsia salmonis infection in cultured lumpfish (Cyclopterus lumpus L.).

A Piscirickettsia salmonis infection was diagnosed in lumpfish (Cyclopterus lumpus L.) juveniles held in a marine research facility on the west coast of Ireland. The main clinical signs and pathology included marked ascites, severe multifocal liver necrosis and severe diffuse inflammation and necrosis of the exocrine pancreas and peri-pancreatic adipose tissue. Numerous Piscirickettsia-like organisms were observed by histopathology in the affected organs, and the bacterial species was characterized by molecular analysis. Sequencing of the partial 16S rDNA gene and internal transcribed spacer region showed the lumpfish sequences to be closely related to previously identified Atlantic salmon (Salmo salar L.) sequences from Ireland. To the authors' knowledge, this is the first detection of P. salmonis in lumpfish worldwide. The infection is considered potentially significant in terms of lumpfish health and biosecurity.

Phenotypic and genetic characterization of Piscirickettsia salmonis from Chilean and Canadian salmonids.

BACKGROUND: The study presents the phenotypic and genetic characterization of selected P. salmonis isolates from Atlantic salmon and rainbow trout suffering from SRS (salmonid rickettsial septicemia) in Chile and in Canada. The phenotypic characterization of the P. salmonis isolates were based on growth on different agar media (including a newly developed medium), different growth temperatures, antibiotics susceptibility and biochemical tests. RESULTS: This is the first study differentiating Chilean P. salmonis isolates into two separate genetic groups. Genotyping, based on 16S rRNA-ITS and concatenated housekeeping genes grouped the selected isolates into two clades, constituted by the Chilean strains, while the Canadian isolates form a branch in the phylogenetic tree. The latter consisted of two isolates that were different in both genetic and phenotypic characteristics. The phylogenies and the MLST do not reflect the origin of the isolates with respect to host species. The isolates included were heterogeneous in phenotypic tests. CONCLUSIONS: The genotyping methods developed in this study provided a tool for separation of P. salmonis isolates into distinct clades. The SRS outbreaks in Chile are caused by minimum two different genetic groups of P. salmonis. This heterogeneity should be considered in future development of vaccines against this bacterium in Chile. Two different strains of P. salmonis, in regards to genetic and phenotypic characteristics, can occur in the same contemporary outbreak of SRS.

In sílico identification and characterization of putative Dot/Icm secreted virulence effectors in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis seriously affects the Chilean salmon industry. The bacterium is phylogenetically related to Legionella pneumophila and Coxiella burnetii, sharing a Dot/Icm secretion system with them. Although it is well documented that L. pneumophila and C. burnetii secrete different virulence effectors via this Dot/Icm system in order to attenuate host cell responses, to date there have been no reported virulence effectors secreted by the Dot/Icm system of P. salmonis. Using several annotations of P. salmonis genome, here we report an in silico analyses of 4 putative Dot/Icm effectors. Three of them contain ankyrin repeat domains and the typical conserved 3D structures of this protein family. The fourth one is highly similar to one of the Dot/Icm-dependent effectors of L. pneumophila. Additionally, all the potential P. salmonis effectors contain a classical Dot/Icm secretion signal in their C-terminus, consisting of: an E-Block, a hydrophobic residue in -3 or -4 and an electronegative charge. Finally, qPCR analysis demonstrated that these proteins are overexpressed early in infection, perhaps contributing to the generation of a replicative vacuole, a key step in the neutralizing strategy proposed for the Dot/Icm system. In summary, this report identifies four Dot/Icm-dependent effectors in P. salmonis.

Stocking density and Piscirickettsia salmonis infection effect on Patagonian blennie (Eleginops maclovinus, Cuvier 1830) skeletal muscle intermediate metabolism.

The need to expand aquaculture production has led to other fish to be considered as potential species for culture, such as the sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1830). The aim of this study was to determine the cumulative effect of density and pathogen infection by protein extract of Piscirickettsia salmonis on skeletal muscle metabolism. In a first experiment, specimens were submitted to three different stocking densities: (1) 3.1 kg m(-3), (2) 15 kg m(-3) and (3) 60 kg m(-3), for a period of 10 days. In a second experiment, metabolic changes caused by an infection of P. salmonis protein extract (a single injection of 0.5 µL P. salmonis protein extract g body weight(-1) was inoculated in the fish) and its combined effect with stocking density was assessed during a period of 10 days. This study concludes that stress caused by high stocking density led to the reorganization of some metabolic routes to fulfill skeletal muscle energy needs. Furthermore, infection response by pathogen P. salmonis differed when stocking density increased, suggesting an increase of energy needs with density in skeletal muscle of infected fish.

Evidence of the presence of a functional Dot/Icm type IV-B secretion system in the fish bacterial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE) are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.

Characterization of a novel and genetically different small infective variant of Piscirickettsia salmonis.

We report a novel genetically different small infective variant of the fish pathogen Piscirickettsia salmonis (sP.s). sP.s variants were recovered both from ageing post-infected CHSE-214 culture cells as well as from naturally infected fish. The ITS region of sP.s variants, although sharing a common core sequence, is different from the ITS of the prototype strain LF-89 from which they originate. Thus, sP.s can be selectively amplified with sequence-specific discriminatory set of PCR primers. Transcriptionally, sP.s are fully active as shown by reverse transcription PCR analysis. Immunologically, sP.s is specifically recognized by antibodies against standard P. salmonis. Structurally, atomic force microscopy shows that sP.s. is well below (<0.2microm) the standard range size described for this pathogen (0.5-1.5microm). Functionally, although sP.s is infective their in vitro progeny is a hundred percent identical to the LF-89 prototype strain. In summary sP.s, represent selectable infective variants of the LF-89 strain and not new strains, probably resulting from a survival strategy of the bacteria in response to limiting growth conditions. In this frame, sP.s could be responsible of horizontal infection of fish in the field.