Oliva, Harold

Oliver, Cristian

Sanhueza, Constanza

Valdebenito, Samuel

Valenzuela, Karla

Yáñez, Alejandro

English

University of Maine

INTERACTION, INTRACELLULAR REPLICATION AND CYTOPATHIC EFFECT OF A HIGHLY PATHOGENIC CHILEAN ISOLATE OF PISCIRICKETTSIA SALMONIS IN SHK-1 CELLS. Constanza Sanhueza 1,6, Cristian Oliver 1,2,5, Karla Valenzuela 1,3, Harold Oliva 6, Samuel Valdebenito 6, Alejandro Yáñez 1,4,5 1Department of Biochemistry and Microbiology, Universidad Austral de Chile, Valdivia, Chile.  2Department of Biological Sciences, Universidad Andrés Bello, Santiago, Chile. 3Microbiology and Immunology Department, Dalhousie University, Halifax, Nova Scotia, Canada. 4Austral-OMICS, Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile 5Interdisciplinary Center for Aquaculture Research INCAR, Concepcion, Chile 6 Veterquímica S.A., Laboratory of Research and Development, Santiago, Chile.  ABSTRACT Piscirickettsia salmonis is a Gram-negative, facultative intracellular bacterium, which is the etiologic agent of Piscirickettsiosis, a systemic infection of multiple organs and tissues among as kidney, liver, spleen, brain, intestine, ovaries, and gills in several salmonids species such as Rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch), causing high mortality and serious economic losses for fish farming in the south of Chile. P. salmonis is able to infect, evade the immune response and replicate inside of several fish cell lines such as RTS-11, SHK-1 and CHSE-214. However, there are no reports concerning the infection and lifestyle of P. salmonis inside SHK-1 cell line. Thus, the aim of this study was to characterize, in a time-course, the infection and intracellular lifestyle of P. salmonis in SHK-1 fish cell line.    SHK-1 cells were cultured at 20ºC in 75 cm2 flask, in Leibovitz’s L-15 medium supplemented with 10% FBS. P. salmonis AUSTRAL-005 isolate and LF-89 type strain were grown in AUSTRAL-SRS broth at 18°C with moderate agitation (50 rpm) until logarithmic phase. SHK-1 cells (2x104 cell/well) or (3x105 cells/well) were seeded onto 8-well chamber slides (Lab-Tek) or in 24-well culture plates with L-15 medium supplemented with 2% FBS for confocal microscopy or transmission electron microscopy (TEM), respectively. Then, monolayers containing adherent cells were infected with P. salmonis at multiplicity of infection (MOI) 20 bacteria per cell. For confocal microscopy, infected and control cells were fixed in 4% (wt/vol) freshly paraformaldehyde for 15 min at room temperature (RT) at the following post-infection (pi) times: 5, 15, and 30 min, 1, 2, 3, 4, and 6 h. P. salmonis was stained using an oligoclonal antibody anti-P. salmonis (BIOS Chile S. A). Additionally, BrdU was used to evaluate the intracellular replication. Samples were analyzed with an Olympus FluoView™ FV1000 confocal laser scanning microscope. For TEM, infected and control SHK-1 cells were fixed for 2 h at RT in 2.5% (vol/vol) freshly glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Finally, samples were observed in a JEOL JEM-1230 transmission electron microscope at 80kV.   Results showed that P. salmonis was found attached to the plasma membrane as early as 15 min pi, interacting tightly with the plasma membrane. Finally at 3 h pi and longer, P. salmonis was detected inside discrete cytoplasmic vacuoles. After 4 days pi and longer, SHK-1 cells shown a high number of actively replicating bacteria labelled with BrdU inside the vacuole. Interestingly, outer membrane vesicles were detected near to P. salmonis inside the vacuole.  KEYWORDS Piscirickettsia salmonis, SHK-1, BrdU, infectivity, intracellular replication  §Corresponding author. Tel.: +56 9 97480467; +56 63 2221332 E-mail address: ayanez@uach.cl     

INTERACTION, INTRACELLULAR REPLICATION AND CYTOPATHIC EFFECT OF A HIGHLY PATHOGENIC CHILEAN ISOLATE OF PISCIRICKETTSIA SALMONIS IN SHK-1 CELLS

https://digitalcommons.library.umaine.edu/cgi/viewcontent.cgi?article=1268&amp;context=isfsi

INTERACTION, INTRACELLULAR REPLICATION AND CYTOPATHIC EFFECT OF A HIGHLY PATHOGENIC CHILEAN ISOLATE OF PISCIRICKETTSIA SALMONIS IN SHK-1 CELLS

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