Identification of immunoreactive protein encoding genes of the fish pathogen Piscirickettsia salmonis and evaluation of their use in genetic vaccination

Since the emergence of Piscirickettsia salmonis in the late 1980's, it has become a major disease issue in the salmonid aquaculture and no effective control measures are currently available. As a potential vaccination strategy, genetic or DNA vaccines offer great promise. They function by carrying the gene coding for a protective antigen on a plasmid into the host cells where the antigen is produced by the cells themselves. These vaccines mimic the pathway by which antigens from intracellular pathogens are normally presented to the immune system. To develop a DNA vaccine for P. salmonis , genes coding for potentially protective antigens had to be identified although very little is known about the genetic makeup of this pathogen. The most appropriate way to identify protective genes was by generating an expression library of the entire genome of P. salmonis and screening this library with anti-P. salmonis rabbit antiserum. To obtain large amounts of P. salmonis for genomic DNA extraction and antigen production, the growth in culture of P. salmonis had to be optimized. To follow the course of infection of P. salmonis in CHSE-214 cell culture a sandwich ELISA (s-ELISA) was developed using rabbit anti-P. salmonis antiserum to capture P. salmonis and monoclonal 10E6.6C5 for detection. This s-ELISA proved to be both sensitive and practical. The optimum time to harvest P. salmonis culture was determined to be 10 days post inoculation, with the fastest increase in antigen levels between day 6 and 7. P. salmonis genomic DNA without contaminating CHSE-214 DNA was obtained by treating purified P. salmonis with DNaseI to remove CHSE-214 DNA before P. salmonis lysis and DNA purification. Genomic DNA was used to generate an expression library in the λZapExpress vector. By screening approximately 18,000 clones, 41 immuno-reactive clones were identified. Inserts from these clones were amplified by PCR and sequenced. Ten open reading frames (ORFs) were assigned putative functions by comparing deduced amino acid sequences to sequences available in Genbank. Five clones were selected for showing homologies to promising targets for the immune system. Putative functions of these five ORFs were: (1) the 17 kDa surface antigen from the spotted fever group rickettsia; (2) transposase; (3) ATP binding cassette type transporter; (4) Preprotein translocase subunit SecA; and (5) amino acid transporter. (Abstract shortened by UMI.).