Piscirickettsia salmonis : a Gram‐negative intracellular bacterial pathogen of fish

The Piscirickettsia salmonis genome was screened to evaluate potential flagellarelated open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.

Section: Introductionmentioning

confidence: 99%

Expression of flagellin and key regulatory flagellar genes in the non-motile bacterium Piscirickettsia salmonis

Carril

Gómez²,

Marshall³

2017

“…Indeed, the re-isolation of TRLO in culture from the liver of affected fish during challenges and also the routine direct isolation of TRLO in culture from the liver of sea cage-farmed fish verifies the consistently hepatotropic nature of TRLO. However, unlike in piscirickettsiosis-affected fish, the pale gills (anaemia), cream-coloured focal subcapsular nodules in the liver and swollen grey kidneys (Birrel et al 2003, Fryer & Hedrick 2003, Rozas & Enriquez 2014 were not seen in TRLO-injected fish. Histopathological findings in TRLO-injected fish such as necrotizing hepatitis have also been seen in farmed Atlantic salmon naturally infected with TRLO in Tasmania (DPIPWE unpubl.…”

Section: Discussionmentioning

confidence: 99%

“…It is also culturable in Xenopus laevis (Daudin) XTC-2 and fall army worm Spodoptera frugiperda Sf21 cells (Birkbeck et al 2004a). For many years, P. salmonis was thought to be obligately intracellular (Fryer et al 1992, Almendras & Fuentealba 1997, Mauel & Miller 2002, Fryer & Hedrick 2003, but using specialised enriched media (Mauel et al 2008, Mikalsen et al 2008, Gómez et al 2009, Otterlei et al 2016), the bacterium is now considered fastidious and facultatively intracellular. The development of these new media provided an ideal opportunity to attempt to isolate TRLO in a cell culture-independent fashion.…”

mentioning

confidence: 99%

Isolation of Tasmanian Rickettsia-like organism (RLO) from farmed salmonids: identification of multiple serotypes and confirmation of pathogenicity

Morrison¹,

Young²,

Knowles³

et al. 2016

Atlantic salmon Salmo salar L. farmed in south-east Tasmania, Australia, are susceptible to infection by the Tasmanian Rickettsia-like organism (TRLO), a Gram-negative bacterium. Here, we report the first isolation of TRLO from south-east Tasmania in pure culture and show that the bacterium is culturable on both specialised enriched agar and in cell culture using the CHSE-214 cell line. In vitro cultured TRLO was used to reproducibly elicit disease in Atlantic salmon parr held in fresh water. In inoculated fish, TRLO was observed intracytoplasmically in peripheral blood leucocytes, suggesting that these cells are responsible for haematogenous dispersal of the bacterium within the host. Fish with experimentally induced disease presented with gross and histopathological changes similar to TRLO-infected fish at commercial marine farms. TRLO was also isolated in culture from farmed Atlantic salmon in the Tamar River and Macquarie Harbour production areas in Tasmania, both of which have no history of TRLO-associated disease. These TRLO isolates appear to be serologically distinct from each other as well as from isolates obtained from south-east Tasmania, linking each serotype to a specific geographical location within Tasmania. Despite the lack of clinical evidence of TRLO-linked disease in fish grown in the Tamar River and Macquarie Harbour, experimental infection trials demonstrably showed the pathogenic potential of these TRLO serovars. Together, these data provide evidence that TRLO is a fastidious, facultative intracellular bacterium and confirm TRLO as a pathogen of Atlantic salmon, causing a disease designated Tasmanian salmonid rickettsiosis.KEY WORDS: Rickettsia-like organism · TRLO · Atlantic salmon · Salmo salar · Culture · Pathogenicity · Tasmania Resale or republication not permitted without written consent of the publisherDis Aquat Org 122: [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] 2016 RLO (Khoo et al. 1995, Athanassopoulou et al. 2004, Corbeil et al. 2005, Wu et al. 2005 or P. salmonis-like organism (Antonio et al. 2000, Chen et al. 2000a,b, Mauel et al. 2003 until their taxonomic status can be formally resolved. This includes the Tasmanian RLO (TRLO), a bacte rium detected in Atlantic salmon Salmo salar L. farmed on the south-east coast of Tasmania, Austra lia (Corbeil et al. 2005).TRLO is an intracellular, Gram-negative coccoid bacterium that emerged in the summer of 2001 (Department of Primary Industries, Parks, Water and Environment [DPIPWE] unpublished data). Since then, TRLO has been implicated in minor sporadic outbreaks of disease which have typically coincided with the annual peak in water temperature. The disease is characterised by a high morbidity rate which results in significant production loss through reduced feed intake but relatively low mortality, less than 10% of affected stock. During outbreaks, affected fish are commonly co-infected with the Tasmanian Atlantic salmon reovirus (TSRV; Zainathan et al. 2015), and it is not known wh...

“…However, disease challenges caused by various intracellular fish pathogenic bacteria like Francisella noatunensis, Renibacterium salmoninarum and Piscirickettsia salmonis (Gutenberger et al 1997, Fryer & Hedrick 2003, Birkbeck et al 2011) make such analyses highly relevant in order to develop effective treatment strategies.…”

Section: Discussionmentioning

confidence: 99%

Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication

Kaldestad¹,

Haugland²,

Rønneseth³

et al. 2014

The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.