DNA hybridization assays for the detection of Piscirickettsia salmonis in salmonid fish

Since it was first recorded in 1989, Piscirickettsia salmonis has been a constraint on the Chilean salmon farming industry (Bravo & Campos 1989; Fryer, Lannan, Garcés, Larenas & Smith 1990). This obligate, intracellular, Gram-negative bacterium and the disease it causes, known as salmon rickettsial septicaemia (Cvitanich, Gárate & Smith 1991) or piscirickettsiosis (Fryer, Lannan, Giovannoni & Wood 1992), causes major losses in salmon farming in Chile (Smith, Pizarro, Ojeda, Contreras, Oyanedel & Larenas 1999). Laboratory diagnosis of P. salmonis is based largely on a presumptive detection using Giemsa stained smears or on pathogen isolation by cell culture (Fryer et al. 1990). Confirmative diagnosis is by indirect fluorescent antibody test (Lannan, Ewing & Fryer 1991; OIE 2000), immunohistochemistry (Alday-Sanz, Rodger, Turnbull, Adams & Richards 1994; OIE 2000), and/or polymerase chain reaction (PCR) (Mauel, Giovannoni & Fryer 1996; OIE 2000; House & Fryer 2002). The interest in the use of DNA-based techniques for the diagnosis/detection of fish and shellfish pathogens, particularly those that are difficult to culture, has been steadily increasing. The present communication reports on two of these approaches, dot-blot hybridization (DBH) and in situ hybridization (ISH). Two sets of primers (PS2S-PS2AS and PS2SPS3AS) were used as specific DNA probes against P. salmonis according to information reported by Mauel et al. (1996). Unless otherwise indicated procedures were performed at room temperature. Appropriate non-infected samples were included as controls for both DBH and ISH. For hybridization detection, the primers were directly labelled with 11-digoxigenin-dNTPs (Roche Molecular Biochemicals, Mannheim, Germany) by PCR amplification. For the DBH assay, a phenolic extraction of nucleic acids was made from samples of kidney, liver and spleen of P. salmonis (SLGO-95 strain)-infected rainbow trout, Oncorhynchus mykiss (Walbaum), and from the supernatant of infected CHSE-214 cells. The tissue samples were obtained from an infectivity trial performed in our laboratory. Briefly, the samples were homogenized and digested with a lysis buffer (280 mm Tris, 80 mm EDTA, 45 mm SDS, 685 mm NaCl, 1.3% b-mercaptoethanol) and Proteinase K (0.2 mg mL; GibcoBRL, Carlsbad, CA, USA), incubated at 50 C for 1 h and subjected to a phenol/chloroform extraction. Extracted DNAs were denatured at 95 C for 5 min, quenched on ice for 3 min and then treated with 2x SSC (standard saline citrate 20x: 300 mm NaCl, 30 mm Na citrate, pH 7.0) and blotted onto positively charged nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membranes and nucleic acids were cross-linked by UV radiation for 3 min and treated with a Journal of Fish Diseases 2004, 27, 431–433