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Applied and Environmental Microbiology, October 2003, p. 6268-6271, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6268-6271.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

An Alternative Efficient Procedure for Purification of the Obligate Intracellular Fish Bacterial Pathogen Piscirickettsia salmonis

Vitalia Henríquez, María Verónica Rojas, and Sergio H. Marshall*

Laboratorio de Genética e Immunología Molecular, Instituto de Biología, Universidad Católica de Valparaíso, Valparaíso, Chile

Received 4 February 2003/ Accepted 4 August 2003

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.

* Corresponding author. Mailing address: Laboratorio de Genética e Immunología Molecular, Instituto de Biología, Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile. Phone: 56 32 273119. Fax: 56 32 596703. E-mail: smarshal{at}ucv.cl.

Applied and Environmental Microbiology, October 2003, p. 6268-6271, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6268-6271.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mauel, M. J, Ware, C., Smith, P. A. (2008). Culture of Piscirickettsia salmonis on enriched blood agar. jvdi 20: 213-214 [Abstract] [Full Text]  


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