Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis.

Comparative pathogenesis of piscirickettsiosis in Atlantic salmon (Salmo salar L.) post-smolt experimentally challenged with LF-89-like and EM-90-like Piscirickettsia salmonis isolates.

Piscirickettsiosis (SRS) is the most prevalent bacterial disease in Chilean salmon aquaculture and is responsible for high economic losses. The aim of this study was to comparatively characterize the pathogenesis of SRS in post-smolt Atlantic salmon during the early and late stages of infection with Piscirickettsia salmonis LF-89-like (PS-LF-89) and EM-90-like (PS-EM-90) using a cohabitation challenge. The pathogenesis of cohabitant fish infected with the two isolates was relatively different due to cohabitant fish infected with PS-EM-90 showing higher cumulative mortality and shorter time until death compared with PS-LF-89 fish. PS-LF-89 caused an SRS infection characterized by kidney and liver lesions, whereas PS-EM-90 caused systemic and haemorrhagic disease characterized by kidney, liver, heart, brain, skeletal muscle and intestine lesions. Decreased serum concentration of total proteins and albumin as well as increased serum ALT, AST and creatinine levels in fish infected with both isolates confirmed that changes in liver and kidney function occurred during infection. Tissue damage, expressed as an SRS histoscore, showed a strong positive correlation with the bacterial load expressed as abundance of P. salmonis 16S rRNA transcripts in the livers and kidneys of fish affected with either isolate, but the correlation was significantly higher in fish infected with PS-EM-90. The results contribute to improving the understanding of the bacteria-host interaction.

Apoptosis inhibition of Atlantic salmon (Salmo salar) peritoneal macrophages by Piscirickettsia salmonis.

To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post-exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post-clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC-1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.

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.

Piscirickettsia-like organisms as a cause of acute necrotic lesions in Colombian tilapia larvae.

Rickettsial organisms are well-known fish pathogens in both natural and culture environments. This study reports an outbreak of disease in red tilapia larvae caused by piscirickettsia-like organisms (PLOs), which lasted from June until October 2009. Severe mortality was recorded almost exclusively in larvae and postlarvae aged 1-22 days old. Although clinical or gross findings were not evident in diseased fish, histopathology revealed severe necrosis of the epidermis and gill epithelium, with concomitant changes in the underlying skeletal muscle as being the most relevant microscopic lesions. Although PLOs were visible with the routine hematoxylin eosin technique, they were better observed with Giemsa and toluidine blue stains. Transmission electron microscopy revealed that the bacterium was located within the cytoplasm and phagolysosoma-like structures of epithelial cells from the gills and the skin. The bacteria measured 0.9 ± 0.2 µm × 2.1 ± 0.6 µm and had a double cell membrane (the outer one having undulating projections), with variable electron-dense and electron-lucent areas. Ultrastructurally, abundant myelin figures surrounded the microorganisms within host cell cytoplasm. Results indicated that Piscirickettsia-like organisms can cause massive epithelial cell damage associated with concomitant alteration of the electrolyte balance.

A piscirickettsiosis-like syndrome in cultured Nile tilapia in Latin America with Francisella spp. as the pathogenic agent.

In 2004, cultured Nile tilapia Oreochromis niloticus in several Latin America farms began to succumb to a disease similar to the piscirickettsiosis-like syndrome previously reported in tilapia in Taiwan and the United States. Mortality increased during 2005; reductions in tilapia biomass ranged from 5% to 80% in individual ponds and averaged 50% overall. All ages of fish have been involved. Clinical signs include lethargy, loss of appetite, petechia, exophthalmia, and abnormal swimming behavior. Gross lesions have included splenomegaly, renomegaly, and numerous white nodules observed in the spleen, kidney, testes, heart, ovaries, and occasionally the liver. A previously unreported black granulomatous lesion was reported in up to 30% of the fillets. Histologically, granulomatous infiltrates were observed in the kidney, spleen, liver, testes, ovary, and choroid gland, and rarely in the brain and heart. A small pleomorphic bacterium was observed in Giemsa-stained blood smears and spleen imprints. The bacterium did not grow on standard microbiological media and has not been isolated in cell culture. We obtained a near-complete 16S ribosomal DNA sequence with high similarity to Francisella spp. sequences previously identified in tilapias Oreochromis spp. (Taiwan), Atlantic cod Gadus morhua (Norway), and three-line grunts Parapristipoma trilineatum (Japan).

Persistence of Piscirickettsia salmonis and detection of serum antibodies to the bacterium in white seabass Atractoscion nobilis following experimental exposure.

White seabass Atractoscion nobilis surviving experimental exposure to Piscirickettsia salmonis harbored the bacterium for periods up to at least 123 d post injection (dpi). Intraperitoneal injections of juvenile white seabass with 1.26 x 10(2) TCID50 P. salmonis fish(-1) resulted in a 29% cumulative mortality over a 27 d period. Both molecular and histologic methods provided evidence for persistence of the bacterium in fish sampled sequentially from the surviving population. Throughout the period of acute mortality, the bacterium was detected in all impression smears of liver tissue stained with Giemsa and was reisolated in cell cultures from all dead fish sampled. Polymerase chain reaction (PCR) assays detected P. salmonis-specific DNA in 13.3 to 50% of the fish sampled at time points between 28 and 123 dpi, while cell culture reisolation was largely ineffective in detecting the bacterium. An enzyme-linked immunosorbent assay (ELISA) detected serum anti-P. salmonis antibodies in 48 of 59 white seabass exposed to P. salmonis but not in fish which were not exposed to the bacterium. At the end of the 4 mo experiment, microscopic lesions consisting of single to multiple and coalescing granulomas were found in liver and kidney tissues of 9 of 10 fish examined from the exposure group, while no lesions were detected in the fish from the control group. Immunohistochemical staining with anti-P. salmonis polyclonal antibodies detected bacterial antigens in some but not all granulomas examined from the exposure group at 4 mo. This study demonstrates that P. salmonis may persist among white seabass following infection, and thus provide a potential reservoir of infection contributing to transmission both within and between fish species in the marine environment.

Confirmation of Piscirickettsia salmonis as a pathogen in European sea bass Dicentrarchus labrax and phylogenetic comparison with salmonid strains.

European sea bass Dicentrarchus labrax from the Mediterranean were diagnosed with a severe encephalitis. Rickettsia-like organisms (RLOs) were associated with brain lesions in routine paraffin sections. These were found to share common antigens with the Piscirickettsia salmonis type-strain, LF-89, by indirect fluorescent antibody test (IFAT) and by immunohistochemistry (IHC). In addition, we compared the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) with those published for P. salmonis strains and found that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. Furthermore, we showed that the SBPLO possessed at least 2 ITS regions, 1 of which contained tRNA genes.

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis.

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.