Development of a Multiplex PCR Assay for Genotyping the Fish Pathogen Piscirickettsia salmonis Through Comparative Genomics

Abstract

Piscirickettsia salmonis is a bacterial pathogen that severely impact the aquaculture in several countries as Canada, Scotland, Ireland, Norway, and Chile. It provokes Piscirickettsiosis outbreaks in the marine phase of salmonid farming, resulting in economic losses. The monophyletic genogroup LF-89 and a divergent genogroup EM-90 are responsible for the most severe Piscirickettsiosis outbreaks in Chile. Therefore, the development of methods for quick genotyping of P. salmonis genogroups in field samples is vital for veterinary diagnoses and understanding the population structure of this pathogen. The present study reports the development of a multiplex PCR for genotyping LF-89 and EM-90 genogroups based on comparative genomics of 73 fully sequenced P. salmonis genomes. The results revealed 2,322 sequences shared between 35 LF-89 genomes, 2,280 sequences in the core-genome of 38 EM-90 genomes, and 331 and 534 accessory coding sequences each genogroup, respectively. A total of 1,801 clusters of coding sequences were shared among all tested genomes of P. salmonis (LF-89 and EM-90), with 253 and 291 unique sequences for LF-89 and EM-90 genogroups, respectively. The Multiplex-1 prototype was chosen for reliable genotyping because of differences in annealing temperatures and respective reaction efficiencies. This method also identified the pathogen in field samples infected with LF-89 or EM-90 strains, which is not possible with other methods currently available. Finally, the genome-based multiplex PCR protocol presented in this study is a rapid and affordable alternative to classical sequencing of PCR products and analyzing the length of restriction fragment polymorphisms.

Piscirickettsia salmonis Produces a N-Acetyl-L-Homoserine Lactone as a Bacterial Quorum Sensing System-Related Molecule

“…For all experiments, P. salmonis colonies were grown in AUSTRAL-SRS broth (ASB) at 18°C for 120 h and 75 rpm ( Yañez et al., 2012 ). The identity of each isolate was confirmed by phenotyping procedures and PCR assays ( Karatas et al., 2008 ; Isla et al., 2021 ). Bacterial cell density was measured using the BacLight™ LIVE/DEAD Viability Kit (Invitrogen™, USA).…”

Why Does Piscirickettsia salmonis Break the Immunological Paradigm in Farmed Salmon? Biological Context to Understand the Relative Control of Piscirickettsiosis

“…( 46 ) showed that P. salmonis isolated in different parts of the world, including the reference strain LF-89, are closely related to each other (over >99% similarity in their 16S, ITS and 23S genes), but the Chilean isolate EM-90 is unique and genetically divergent from the others. Later, these findings were confirmed with more evidence from evaluations of their genomic structures and phylogenetic relationships to support the existence of two genogroups of P. salmonis , LF-89 and EM-90 ( 44 , 47 – 52 ). P. salmonis pangenome LF-89 and EM-90 show 148 and 273 unique proteins, respectively ( 44 ), and different geographical distribution patterns and susceptibilities of salmon species have been described ( 53 ).…”

“…This notion has also been experimentally supported by studies based on multilocus sequence typing (MLST) and PCR amplification of 16S rRNA genes followed by restriction fragment length polymorphism (PCR-RFLP) typing ( 51 , 54 ). Although different real-time TaqMan ® PCR assays have been used for several years by private diagnostic laboratories to identify and discriminate between LF-89 and EM-90 isolates in the Chilean salmon industry, a multiplex PCR protocol to differentiate both genogroups has recently been reported ( 52 ). P. salmonis can survive ≥120 h and replicate in cell cultures enriched with Atlantic salmon macrophages ( 55 ).…”

Piscirickettsia salmonis forms a biofilm on nylon surface using a CDC Biofilm Reactor

“…Two P. salmonis strains were studied: the LF-89 (ATCC VR-1361) wild type and the EM-90-like (Ps007) strain. The identity of each isolate was confirmed via phenotyping and polymerase chain reaction (PCR) assays (Isla et al, 2021;Karatas et al, 2008). Cultures were grown on tryptone soy with haemoglobin (Austral-TSHem) agar plates at 18°C for 10 days (Yáñez et al, 2013) or in AUSTRAL-SRS broth (ASB) at 18°C for 120 h with moderate agitation (75 rpm) (Yañez et al, 2012).…”

“…Cultures were grown on tryptone soy with haemoglobin (Austral-TSHem) agar plates at 18°C for 10 days (Yáñez et al, 2013) or in AUSTRAL-SRS broth (ASB) at 18°C for 120 h with moderate agitation (75 rpm) (Yañez et al, 2012). The identity of each isolate was confirmed by phenotyping and PCR assays (Isla et al, 2021;Karatas et al, 2008).…”