Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish.

BACKGROUND: The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters. RESULTS: The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus. CONCLUSIONS: Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile.

Genomics of Re-Emergent Aeromonas salmonicida in Atlantic Salmon Outbreaks.

Furunculosis, caused by Aeromonas salmonicida, poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent A. salmonicida outbreaks in Atlantic salmon (Salmo salar). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent A. salmonicida and provides novel insights into their virulence, antibiotic resistance, and population structure.

Isolation, Functional Characterization and Transmissibility of p3PS10, a Multidrug Resistance Plasmid of the Fish Pathogen Piscirickettsia salmonis.

Antibiotic resistance is a major public health concern due to its association with the loss of efficacy of antimicrobial therapies. Horizontal transfer events may play a significant role in the dissemination of resistant bacterial phenotypes, being mobilizable plasmids a well-known mechanism. In this study, we aimed to gain insights into the genetics underlying the development of antibiotic resistance by Piscirickettsia salmonis isolates, a bacterial fish pathogen and causative agent of salmonid piscirickettsiosis, and the main target of antibiotics used in Chilean salmon farming. We provide experimental evidence that the plasmid p3PS10, which harbors multidrug resistance genes for chloramphenicol (cat2), tetracyclines [tet(31)], aminoglycosides (sat1 and aadA1), and sulfonamides (sul2), is carried by a group of P. salmonis isolates exhibiting a markedly reduced susceptibility to oxytetracycline in vitro (128-256 µg/mL of minimal inhibitory concentration, MIC). Antibiotic susceptibility analysis extended to those antibiotics showed that MIC of chloramphenicol, streptomycin, and sulfamethoxazole/trimethoprim were high, but the MIC of florfenicol remained at the wild-type level. By means of molecular cloning, we demonstrate that those genes encoding putative resistance markers are indeed functional. Interestingly, mating assays clearly show that p3PS10 is able to be transferred into and replicate in different hosts, thereby conferring phenotypes similar to those found in the original host. According to epidemiological data, this strain is distributed across aquaculture settings in southern Chile and is likely to be responsible for oxytetracycline treatment failures. This work demonstrates that P. salmonis is more versatile than it was thought, capable of horizontally transferring DNA, and probably playing a role as a vector of resistance traits among the seawater bacterial population. However, the low transmission frequency of p3PS10 suggests a negligible chance of resistance markers being spread to human pathogens.

First Complete Genome Sequence of Piscine Orthoreovirus Variant 3 Infecting Coho Salmon (Oncorhynchus kisutch) Farmed in Southern Chile.

We report here the complete genome of an isolate of piscine orthoreovirus variant 3 sequenced from a moribund coho salmon with jaundice that was reared in a seawater farm in southern Chile. The genome consists of 23,627 bp, including 10 segments that range from 1,052 bp (segment S4) to 4,014 bp (segment L1).

The Genome Sequence of an Oxytetracycline-Resistant Isolate of the Fish Pathogen Piscirickettsia salmonis Harbors a Multidrug Resistance Plasmid.

The amount of antibiotics needed to counteract frequent piscirickettsiosis outbreaks is a major concern for the Chilean salmon industry. Resistance to antibiotics may contribute to this issue. To understand the genetics underlying Piscirickettsia salmonis-resistant phenotypes, the genome of AY3800B, an oxytetracycline-resistant isolate bearing a multidrug resistance plasmid, is presented here.

First Complete Genome Sequence of Tenacibaculum dicentrarchi, an Emerging Bacterial Pathogen of Salmonids.

Tenacibaculum-like bacilli have recently been isolated from diseased sea-reared Atlantic salmon in outbreaks that took place in the XI region (Región de Aysén) of Chile. Molecular typing identified the bacterium as Tenacibaculum dicentrarchi. Here, we report the complete genome sequence of the AY7486TD isolate recovered during those outbreaks.

Draft Genome Sequence of the Fish Pathogen Yersinia ruckeri Strain 37551, Serotype O1b, Isolated from Diseased, Vaccinated Atlantic Salmon (Salmo salar) in Chile.

We sequenced the genome of a motile O1b Yersinia ruckeri field isolate from Chile, which is causing enteric redmouth disease (ERM) in vaccinated Atlantic salmon (Salmo salar). The draft genome has 3,775,486 bp, a G+C content of 47.1%, and is predicted to contain 3,406 coding sequences.

Comparative Genome Analysis of Two Isolates of the Fish Pathogen Piscirickettsia salmonis from Different Hosts Reveals Major Differences in Virulence-Associated Secretion Systems.

Outbreaks caused by Piscirickettsia salmonis are one of the major threats to the sustainability of the Chilean salmon industry. We report here the annotated draft genomes of two P. salmonis isolates recovered from different salmonid species. A comparative analysis showed that the number of virulence-associated secretion systems constitutes a main genomic difference.

Selection of marker genes using whole-genome DNA polymorphism analysis.

Molecular markers serve to assign individual samples to specific groups. Such markers should be easily identified and have a high discrimination power, being highly conserved within groups while showing sufficient variability between the groups that are to be distinguished. The availability of a large number of complete genomic sequences now enables the informed selection of genes as molecular markers based on the observed patterns of variability. We derived a new scoring system based on observed DNA polymorphic differences, and which uses the Bayes theorem as adapted by Wilcox. For validation, we applied this system to the problem of identifying individual species within a prokaryotic (Vibrio) and a eukaryotic (Diphyllobothrium) genus for validation. Top-scoring candidates genes Chromosome segregation ATPase and ATPase-subunit 6 showed better discrimination power in Vibrio and Diphyllobothrium, respectively, as compared to standard molecular markers (recA, dnaJ and atpA for Vibrio, and 18s rRNA, ITS and COX1 for Diphyllobothrium).

Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV).

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use.