Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

Abstract

The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

Search and analysis of genes involved in antibiotic resistance in Chilean strains of Piscirickettsia salmonis

“…), and Chilean strains IBM‐009 isolated from O. mikiss (Similar to LF‐89, Mandakovic et al . ) and IBM‐034 isolated from O. mikiss (similar to EM‐90, Mandakovic et al . ), were analysed using the bioinformatics tool GenDB, after having sequenced them en masse covering close to 85% with Roche 454 and Illumina technology.…”

“…) and IBM‐034 isolated from O. mikiss (similar to EM‐90, Mandakovic et al . ), were analysed using the bioinformatics tool GenDB, after having sequenced them en masse covering close to 85% with Roche 454 and Illumina technology. The putative antibiotic resistance genes found using this software were compared between strains, by using BLAST and comparing these genes against NCBI databases in order to see the presence or absence of these different analysed strains.…”

Addressing viral and bacterial threats to salmon farming in Chile: historical contexts and perspectives for management and control

“…This information was applied to characterize the bacterial population present in the marine environment of southern Chile and to make comparisons against strains of mainly European and American origins (Mandakovic et al . ; Otterlei et al . ).…”

“…Mbp, three plasmids, 2850 protein coding genes, 56 tRNAs and six copies of 5S-16S-23S rRNA. This information was applied to characterize the bacterial population present in the marine environment of southern Chile and to make comparisons against strains of mainly European and American origins(Mandakovic et al 2016;Otterlei et al 2016).Originally, the disease caused by this bacterium was described under various names, the most common being salmonid rickettsial septicaemia or piscirickettsiosis, the name recognized by the World Organization for Animal Health. Piscirickettsiosis, an infectious disease, is considered to be the main cause of mortalities in the salmon industry in Chile, affecting a wide variety of salmon species, such as Atlantic salmon, rainbow trout, Chinook salmonReviews in Aquaculture (2019) 11, 299-324 © 2019 The Authors.…”

Metagenome complexity and template length are the main causes of bias in PCR‐based bacteria community analysis

“…To reveal the microbial community structures in an environmental sample, the 16S rRNA gene fragments is often amplified by PCR with universal primers directed against a sequence common to all microorganisms. The products can be analyzed by cloning and sequencing, restriction fragment length polymorphism (RFLP) analysis , denaturing gradient gel electrophoresis (DGGE) , terminal restriction fragment length polymorphism (T‐RFLP) , and other methods .…”