PCR-RFLP Detection and Genogroup Identification of Piscirickettsia salmonis in Field Samples

Abstract

Piscirickettsia salmons, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the P. salmonis genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the P. salmonis genogroups. Using an in silico analysis of the P. salmonis 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 P. salmonis strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the P. salmonis genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation.

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

“…The same dichotomous distribution of samples was observed using both methodologies and found both genogroups are widely distributed among the different fish hosts. This finding differs from previous reports reporting that EM-90 genogroups are responsible for most Piscirickettsiosis cases (Saavedra et al, 2017;Aravena et al, 2020). Therefore, we recommend using a higher number of field isolates and epidemiological studies to take different hosts into account.…”

“…The disease has important economic impacts which has encouraged to many researchers to develop diverse strategies for the molecular characterization of the major clusters of strains associated to field disease outbreaks. In this sense, diverse studies based on the 16S rRNA gene analysis (sequencing, PCR-RFLP) (Mauel et al, 1999;Mandakovic et al, 2016;Aravena et al, 2020), phenotypic characterization (Otterlei et al, 2016), MLST (Isla et al, 2019) and comparative genomics (Bohle et al, 2014;Bravo and Martinez, 2016;Nourdin-Galindo et al, 2017) have indicated the existence of LF-89 and EM-90 genogroups containing epidemiologically relevant P. salmonis stains. Both genogroup isolates show differential susceptibility to florfenicol, oxytetracycline, and quinolones (Saavedra et al, 2017).…”

“…The existence of two genetic groups was reinforced after a comparative genomic analysis of 19 fully sequenced P. salmonis isolates (Nourdin-Galindo et al, 2017). 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) (Isla et al, 2019;Aravena et al, 2020). Significant differences in genomic and phenotypic traits between P. salmonis genogroups highlight the need to provide alternative methodologies for genotyping.…”

“…Therefore, there is a need for new genotyping strategies for microorganisms. Others have proposed methods based on PCR-RLFP(Aravena et al, 2020) and MLST(Isla et al, 2019); however, these methodologies are qualitative and only indicate the presence or absence of the pathogen in a sample. Previous reports have used relative qPCR to measure the…”

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

“…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 ).…”