An alternative efficient procedure for purification of the obligate intracellular fish bacterial pathogen Piscirickettsia salmonis

Abstract

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.

FIG. 1.

Equilibrium iodixanol preformed step gradient. Whitish band in the upper third is purified P. salmonis.

FIG. 2.

Microscopic characterization by direct immunofluorescence of the material banded on the iodixanol gradient. Magnification, ×100. (A) FITC-labeled anti-P. salmonis (iodixanol purified). (B) Commercial FITC-labeled anti-P. salmonis.

FIG. 3.

Specific PCR amplification for P. salmonis rDNA. Primers against the ITS region of the rDNA operon were used. Lanes: 1, 160 ng/μl; lanes 2 to 6, serial dilutions from 10⁻¹ to 10⁻⁵, respectively. L, 100-bp ladder.

FIG. 4.

Semiquantitative and competitive PCR amplifications. Primers against fish mitochondrial DNA were used to evaluate the purity of P. salmonis DNA. Lanes 1 to 7, CHSE-214 DNA; lanes 8 to 14, P. salmonis DNA; lanes 15 to 20, P. salmonis DNA mixed with CHSE-214 DNA in a 1:1 genome size ratio; lanes 21 to 27, E. coli DNA (JM109); lanes 28 to 32, E. coli DNA mixed with CHSE-214 DNA. L, 100-bp ladder.

FIG. 5.

Bacterial number estimation by confocal microscopy of CHSE-214 infected cells. (A) Phase contract microscopy. (B) Fluorescence microscopy, commercial FITC-labeled anti-P. salmonis antibody.