piscirickettsia salmonis
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2021 ◽  
Vol 12 ◽  
Author(s):  
Xi Xue ◽  
Albert Caballero-Solares ◽  
Jennifer R. Hall ◽  
Navaneethaiyer Umasuthan ◽  
Surendra Kumar ◽  
...  

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis, is one of the most devastating diseases of salmonids. However, the transcriptomic responses of Atlantic salmon (Salmon salar) in freshwater to an EM-90-like isolate have not been explored. Here, we infected Atlantic salmon parr with an EM-90-like isolate and conducted time-course qPCR analyses of pathogen load and four biomarkers (campb, hampa, il8a, tlr5a) of innate immunity on the head kidney samples. Transcript expression of three of these genes (except hampa), as well as pathogen level, peaked at 21 days post-injection (DPI). Multivariate analyses of infected individuals at 21 DPI revealed two infection phenotypes [lower (L-SRS) and higher (H-SRS) infection level]. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling using a 44K salmonid microarray platform. We identified 1,636 and 3,076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups compared with the control group, respectively (FDR = 1%). Gene ontology term enrichment analyses of SRS-responsive genes revealed the activation of a large number of innate (e.g. “phagocytosis”, “defense response to bacterium”, “inflammatory response”) and adaptive (e.g. “regulation of T cell activation”, “antigen processing and presentation of exogenous antigen”) immune processes, while a small number of general physiological processes (e.g. “apoptotic process”, development and metabolism relevant) was enriched. Transcriptome results were confirmed by qPCR analyses of 42 microarray-identified transcripts. Furthermore, the comparison of individuals with differing levels of infection (H-SRS vs. L-SRS) generated insights into the biological processes possibly involved in disease resistance or susceptibility. This study demonstrated a low mortality (~30%) EM-90-like infection model and broadened the current understanding of molecular pathways underlying P. salmonis-triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen.


2021 ◽  
Author(s):  
Robert Mukiibi ◽  
Carolina Peñaloza ◽  
Alejandro Gutierrez ◽  
José M. Yáñez ◽  
Ross D. Houston ◽  
...  

Salmon rickettsial septicaemia (SRS), caused by the intracellular bacteria Piscirickettsia Salmonis, generates significant mortalities to farmed Atlantic salmon, particularly in Chile. Due to its economic importance, a wealth of research has focussed on the biological mechanisms underlying pathogenicity of P. salmonis, the host response, and genetic variation in host resistance. DNA methylation is a fundamental epigenetic mechanism that influences almost every biological process via the regulation of gene expression and plays a key role in the response of an organism to stimuli. In the current study, the role of head kidney and liver DNA methylation in the response to P. salmonis infection was investigated in a commercial Atlantic salmon population. A total of 66 salmon were profiled using reduced representation bisulphite sequencing (RRBS), with head kidney and liver methylomes compared between infected animals (3 and 9 days post infection) and uninfected controls. These included groups of salmon with divergent (high or low) breeding values for resistance to P. salmonis infection, to examine the influence of genetic resistance. Head kidney and liver showed organ-specific global methylation patterns, but with similar distribution of methylation across gene features. Integration of methylation with RNA-Seq data revealed that methylation levels predominantly showed a negative correlation with gene expression, although positive correlations were also observed. Methylation within the first exon showed the strongest negative correlation with gene expression. A total of 911 and 813 differentially methylated CpG sites were identified between infected and control samples in the head kidney at 3 and 9 days respectively, whereas only 30 and 44 sites were differentially methylated in the liver. Differential methylation in the head kidney was associated with immunological processes such as actin cytoskeleton regulation, phagocytosis, endocytosis and pathogen associated pattern receptor signaling. We also identified 113 and 48 differentially methylated sites between resistant and susceptible fish in the head kidney and liver respectively. Our results contribute to the growing understanding of the role of methylation in regulation of gene expression and response to infectious diseases, and in particular reveal key immunological functions regulated by methylation in Atlantic salmon in response to P. salmonis.


2021 ◽  
Vol 9 (12) ◽  
pp. 2516
Author(s):  
Javiera Ortiz-Severín ◽  
Julia I. Tandberg ◽  
Hanne C. Winther-Larsen ◽  
Francisco P. Chávez ◽  
Verónica Cambiazo

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.


2021 ◽  
Vol 12 ◽  
Author(s):  
Javiera Ortiz-Severín ◽  
Camila J. Stuardo ◽  
Natalia E. Jiménez ◽  
Ricardo Palma ◽  
María P. Cortés ◽  
...  

Extensive use of antibiotics has been the primary treatment for the Salmonid Rickettsial Septicemia, a salmonid disease caused by the bacterium Piscirickettsia salmonis. Occurrence of antibiotic resistance has been explored in various P. salmonis isolates using different assays; however, P. salmonis is a nutritionally demanding intracellular facultative pathogen; thus, assessing its antibiotic susceptibility with standardized and validated protocols is essential. In this work, we studied the pathogen response to antibiotics using a genomic, a transcriptomic, and a phenotypic approach. A new defined medium (CMMAB) was developed based on a metabolic model of P. salmonis. CMMAB was formulated to increase bacterial growth in nutrient-limited conditions and to be suitable for performing antibiotic susceptibility tests. Antibiotic resistance was evaluated based on a comprehensive search of antibiotic resistance genes (ARGs) from P. salmonis genomes. Minimum inhibitory concentration assays were conducted to test the pathogen susceptibility to antibiotics from drug categories with predicted ARGs. In all tested P. salmonis strains, resistance to erythromycin, ampicillin, penicillin G, streptomycin, spectinomycin, polymyxin B, ceftazidime, and trimethoprim was medium-dependent, showing resistance to higher antibiotic concentrations in the CMMAB medium. The mechanism for antibiotic resistance to ampicillin in the defined medium was further explored and was proven to be associated to a decrease in the bacterial central metabolism, including the TCA cycle, the pentose-phosphate pathway, energy production, and nucleotide metabolism, and it was not associated with decreased growth rate of the bacterium or with the expression of any predicted ARG. Our results suggest that nutrient scarcity plays a role in the bacterial antibiotic resistance, protecting against the detrimental effects of antibiotics, and thus, we propose that P. salmonis exhibits a metabolic resistance to ampicillin when growing in a nutrient-limited medium.


Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2399
Author(s):  
Rodrigo Zuloaga ◽  
Phillip Dettleff ◽  
Macarena Bastias-Molina ◽  
Claudio Meneses ◽  
Claudia Altamirano ◽  
...  

Salmonid rickettsial septicemia (SRS) is the major infectious disease of the Chilean salmonid aquaculture industry caused by Piscirickettsia salmonis. Intensive farming conditions generate stress and increased susceptibility to diseases, being skeletal muscle mainly affected. However, the interplay between pathogen infection and stress in muscle is poorly understood. In this study, we perform an RNA-seq analysis on rainbow trout myotubes that are pretreated for 3 h with cortisol (100 ng/mL) and then infected with P. salmonis strain LF-89 for 8 h (MOI 50). Twelve libraries are constructed from RNA samples (n = 3 per group) and sequenced on Illumina HiSeq 4000. A total of 704,979,454 high-quality reads are obtained, with 70.25% mapped against the reference genome. In silico DETs include 175 total genes—124 are upregulated and 51 are downregulated. GO enrichment analysis reveals highly impacted biological processes related to apoptosis, negative regulation of cell proliferation, and innate immune response. These results are validated by RT-qPCR of nine candidate transcripts. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression of ahpC and 23s compared to infection. In conclusion, for the first time, we describe a transcriptomic response of trout myotubes infected with P. salmonis by inducing apoptosis, downregulating cell proliferation, and intrinsic immune-like response that is differentially regulated by cortisol.


Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 847
Author(s):  
Hernán D. Cortés ◽  
Fernando A. Gómez ◽  
Sergio H. Marshall

Piscirickettsia salmonis, the etiological agent of Piscirickettsiosis, is a Gram-negative and facultative intracellular pathogen that has affected the Chilean salmon industry since 1989. The bacterium is highly aggressive and can survive and replicate within fish macrophages using the Dot/Icm secretion system to evade the host’s immune response and spread systemically. To date, no efficient control measures have been developed for this disease; therefore, the producers use large amounts of antibiotics to control this pathogen. In this frame, this work has focused on evaluating the use of saponins from Quillaja saponaria as a new alternative to control the Piscirickettsiosis. It has been previously reported that purified extract of Q. saponaria (PQSE) displays both antimicrobial activity against pathogenic bacteria and viruses and adjuvant properties. Our results show that PQSE does not present antimicrobial activity against P. salmonis, although it reduces P. salmonis infection in an in vitro model, promoting the phagosome–lysosome fusion. Additionally, we demonstrate that PQSE modulates the expression of IL-12 and IL-10 in infected cells, promoting the immune response against the pathogen and reducing the expression of pathogen virulence genes. These results together strongly argue for specific anti-invasion and anti-intracellular replication effects induced by the PQSE in macrophages.


2021 ◽  
Vol 12 ◽  
Author(s):  
Adolfo Isla ◽  
J. Eduardo Martinez-Hernandez ◽  
Héctor A. Levipan ◽  
Denise Haussmann ◽  
Jaime Figueroa ◽  
...  

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.


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