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.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Henríquez V,Rojas MV,Marshall SHdoi
10.1128/aem.69.10.6268-6271.2003keywords:
subject
Has Abstractpub_date
2003-10-01 00:00:00pages
6268-71issue
10eissn
0099-2240issn
1098-5336journal_volume
69pub_type
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更新日期:1997-03-01 00:00:00
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