Molecular analysis of the salmon pathogen Piscirickettsia salmonis
Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a disease causing significant losses in salmon aquaculture in Chile and which has also been isolated in salmonids in several other countries. This investigation was designed to prepare a recombinant library of P. salmonis DNA in a phage lambda vector. P. salmonis LF-89 was grown in CHSE-214 and RTG-2 tissue culture cells and morphological changes monitored by light microscopy and staining. Cytoplasmic vacuoles were visible in infected cells by 5 days post-infection and these expanded in size until cells burst, releasing bacteria into the culture fluid. Within 14-17 days the complete cell monolayer showed cytopathic effects and cells detached from the culture surface. Bacteria were collected from large volumes of culture fluid by centrifugation, resuspension and brief homogenisation it disperse the material, low speed centrifugation to remove large cellular debris, followed by purification of bacteria from cell debris by centrifugation on a Percoll density gradient. DNA was extracted from the bacteria, partially digested with EcoRI and ligated into ExCell. The library appeared to contain approximately 1.79 x 105 recombinants/g DNA, and the recombinant library was screened with rabbit antiserum to purified P. salmonis and antisera raised in fish. No reactive clones were detected in screening with antisera, or with oligonucleotide probes against both P. salmonis 16S rRNA gene and Legionella pneumophila Mip gene. A representative selection of recombinant phage was analysed. None of these recombinant phage inserts could be released with EcoRI, the restriction enzyme used in preparation of the library. Release of inserts with restriction enzymes flanking the EcoRI site was successful and several inserts, ranging in size from 0.4 to 3.5kb were detected. Sequence analysis of two inserts showed that the expected EcoRI site in pExCell recombinants was corrupted to the sequence CCATTC or GCGTTC. Limited sequence analysis of inserts revealed very high identity with E. coli gene sequences in the databases, and little similarity with rickettsial sequences.