Advancements in rapid diagnostics and genotyping of Piscirickettsia salmonis using Loop-mediated Isothermal Amplification.

Introduction: Piscirickettsia salmonis, the causative agent of Piscirickettsiosis, poses a significant threat to the Chilean aquaculture industry, resulting in substantial economic losses annually. The pathogen, first identified as specie in 1992, this pathogen was divided into two genogroups: LF-89 and EM-90, associated with different phenotypic mortality and pathogenicity. Traditional genotyping methods, such as multiplex PCR, are effective but limited by their cost, equipment requirements, and the need for specialized expertise. Methods: This study validates Loop-mediated Isothermal Amplification (LAMP) as a rapid and specific alternative for diagnosing P. salmonis infections. We developed the first qPCR and LAMP assay targeting the species-conserved tonB receptor gene (tonB-r, WP_016210144.1) for the specific species-level identification of P. salmonis. Additionally, we designed two genotyping LAMP assays to differentiate between the LF-89 and EM-90 genogroups, utilizing the unique coding sequences Nitronate monooxygenase (WP_144420689.1) for LF-89 and Acid phosphatase (WP_016210154.1) for EM-90. Results: The LAMP assays demonstrated sensitivity and specificity comparable to real-time PCR, with additional benefits including rapid results, lower costs, and simplified operation, making them particularly suitable for field use. Specificity was confirmed by testing against other salmonid pathogens, such as Renibacterium salmoninarum, Vibrio ordalii, Flavobacterium psychrophilum, Tenacibaculum maritimum, and Aeromonas salmonicida, with no cross-reactivity observed. Discussion: The visual detection method and precise differentiation between genogroups underscore LAMP's potential as a robust diagnostic tool for aquaculture. This advancement in the specie detection (qPCR and LAMP) and genotyping of P. salmonis represents a significant step forward in disease management within the aquaculture industry. The implementation of LAMP promises enhanced disease surveillance, early detection, and improved management strategies, ultimately benefiting the salmonid aquaculture sector.

Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

Antibiotic discovery against Piscirickettsia salmonis using a combined in silico and in vitro approach.

Piscirickettsia salmonis is one of the main pathogens causing considerable economic losses in salmonid farming. The DNA gyrase of several pathogenic bacteria has been the target of choice for antibiotic design and discovery for years, due to its key function during DNA replication. In this study, we carried out a combined in silico and in vitro approach to antibiotic discovery targeting the GyrA subunit of Piscirickettsia salmonis. The in silico results of this work showed that flumequine (-6.6 kcal/mol), finafloxacin (-7.2 kcal/mol), rosoxacin (-6.6 kcal/mol), elvitegravir (-6.4 kcal/mol), sarafloxacin (-8.3 kcal/mol), orbifloxacin (-7.9 kcal/mol), and sparfloxacin (-7.2 kcal/mol) are docked with good affinities in the DNA binding domain of the Piscirickettsia salmonis GyrA subunit. In the in vitro inhibition assay, it was observed that most of these molecules inhibit the growth of Piscirickettsia salmonis, except for elvitegravir. We believe that this methodology could help to significantly reduce the time and cost of antibiotic discovery trials to combat Piscirickettsia salmonis within the salmonid farming industry.

Genomic and proteomic aspects of p57 protein from Renibacterium salmoninarum: Characteristics in virulence patterns.

Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57 kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.

Long-term survive of Aliarcobacter butzleri in two models symbiotic interaction with Acanthamoeba castellanii.

Aliarcobacter butzleri (formerly known as Arcobacter butzleri) is an emerging food-borne zoonotic pathogen that establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba. Previously, we described that this bacterium acts as an endocytobiont of A. castellanii, surviving for at least 10 days in absence of bacterial replication. Thus, the aim of this study was to evaluate the ability of A. butzleri to survive as a long-term endosymbiont of A. castellanii for 30 days in two models of symbiotic interaction with A. castellanii: (i) endosymbiotic culture followed by gentamicin protection assay and (ii) transwell co-culture assay. The results allow us to conclude that A. butzleri is capable of surviving as an endosymbiont of A. castellanii for at least 30 days, without multiplying, under controlled laboratory conditions. In addition, in the absence of nutrients and as both microorganisms remain in the same culture, separated by semi-permeable membranes, A. castellanii does not promote the survival of A. butzleri, nor does it multiply. Our findings suggest that the greater survival capacity of A. butzleri is associated with their endosymbiont status inside A. castellanii, pointing out the complexity of this type of symbiotic relationship.

Transcriptional analysis of flagellar and putative virulence genes of Arcobacter butzleri as an endocytobiont of Acanthamoeba castellanii.

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

Mechanisms associated with phagocytosis of Arcobacter butzleri by Acanthamoeba castellanii.

Acanthamoeba castellanii is a free-living amoeba widely found in environmental matrices such as soil and water. Arcobacter butzleri is an emerging potential zoonotic pathogen that can be isolated from environmental water sources, where they can establish endosymbiotic relationships with amoebas. The aim of this study was to describe the implication of mannose-binding proteins and membrane-associated receptors of glucose and galactose present in the amoebic membrane, during the attachment of Arcobacter butzleri by blocking with different saccharides. Another objective was to describe the signaling pathways involved in phagocytosis of these bacteria using specific inhibitors and analyze the implication of phagolysosome formation on the survival of Arcobacter butzleri inside the amoeba. We infer that the attachment of Arcobacter butzleri to the amoeba is a process which involves the participation of mannose-binding proteins and membrane-associated receptors of glucose and galactose present in the amoeba. We also demonstrated an active role of protozoan actin polymerization in the phagocytosis of Arcobacter butzleri and a critical involvement of PI3K and RhoA pathways. Further, we demonstrated that the tyrosine kinase-induced actin polymerization signal is essential in Acanthamoeba-mediated bacterial uptake. Through phagolysosomal formation analysis, we conclude that the survival of Arcobacter butzleri inside the amoeba could be related with the ability to remain inside vacuoles not fused with lysosomes, or with the ability to retard the fusion between these structures. All these results help the understanding of the bacterial uptake mechanisms used by Acanthamoeba castellanii and contribute to evidence of the survival mechanisms of Arcobacter butzleri.