Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

PACAP binds conserved receptors and modulates cytokine gene expression and protein secretion in trout cell lines.

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1ß and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1ß protein from previously accumulated transcripts or the cleavage of accumulated IL-1ß precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

Innate and adaptive immune response of Rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri.

Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1ß and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study.

Diploid and triploid Chinook salmon (Oncorhynchus tshawytscha) exhibit differential immunological responses to acute thermal stress.

Exposure to temperatures outside of a fish's optimal range results in suppression of the immune system, ultimately leaving aquaculture stocks susceptible to disease outbreaks. This effect is exacerbated in triploid fishes, which demonstrate greater susceptibility to stress than their diploid counterparts. This study investigates the impacts of acute heat stress on the abundance of immune transcripts and proteins in diploid and triploid Chinook salmon (Oncorhynchus tshawytscha), an important finfish crop. This study also demonstrates that acute heat stress induces significant increases in the abundance hsp70, hsp90 and il1b transcripts in the head kidneys, gills and heart ventricles of both diploid and triploid Chinook salmon. Widespread dysregulation of antigen-presentation transcripts was also observed in fish of both ploidies. These results suggest that acute heat stress activates acute-phase responses in Chinook salmon and dysregulates antigen presentation, potentially leaving fish more susceptible to infection. At the protein level, IL-1ß was differentially expressed in the head kidney and ventricles of diploid and triploid salmon following heat shock. Differential expression of two tapasin-like proteins in diploid and triploid salmon subjected to heat shock was also observed. Altogether, these data indicate that diploid and triploid Chinook salmon respond differently to acute thermal stressors.

Intermediary metabolic response and gene transcription modulation on the Sub-Antarctic notothenioid Eleginops maclovinus (Valenciennes, 1930) injected with two strains of Piscirickettsia salmonis.

Pathogen interactions with cultured fish populations are well studied, but their effects on native fishes have not been characterized. In Chile, the disease caused by bacterial species Piscirickettsia salmonis represents one of the main issues and is considered to be one of the important pathogens in the field of aquaculture. They have been found to infect native fish. Therefore, it is necessary to understand the impact of P. salmonis on native species of local commercial value, as well as the potential impact associated with the emergence of antibiotic-resistant strains of P. salmonis. Due to this purpose, the native fish Eleginops maclovinus was used in our study. Fish were randomly distributed in tanks and intraperitoneally inoculated with two strains of P. salmonis. No mortality was recorded during the experiment. Cortisol, glucose and total α-amino acid levels increased in fish injected with AUSTRAL-005 strain compared to sham-injected and LF-89-inoculated fish. Moreover, results showed an increase in the activity of carbohydrates and lipids metabolism in liver; and an increase in the carbohydrates, lipids and total α-amino acid metabolism in muscle after injection with AUSTRAL-005. Our results suggest that P. salmonis modulates the physiology of E. maclovinus and the physiological impact increase in the presence of the antibiotic-resistant strain AUSTRAL-005.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes.

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post-infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.

The effects of Pediococcus acidilactici MA18/5M on growth performance, gut integrity, and immune response using in vitro and in vivo Pacific salmonid models.

Microbial management is central to aquaculture's efficiency. Pediococcus acidilactici MA18/5M has shown promising results promoting growth, modulation of the immune response, and disease resistance in many fishes. However, the mechanisms through which this strain confers health benefits in fish are poorly understood, particularly in Pacific salmonid models. Briefly, the aims of this study were to i) assess the protective effects of P. acidilactici MA18/5M by examining gut barrier function and the expression of tight junction (TJ) and immune genes in vitro and in vivo, and ii) to determine the protective effects of this strain against a common saltwater pathogen, Vibrio anguillarum J382. An in vitro model of the salmonid gut was employed utilizing the cell line RTgutGC. Barrier formation and integrity assessed by TEER measurements in RTgutGC, showed a significant decrease in resistance in cells exposed only to V. anguillarum J382 for 24 h, but pre-treatment with P. acidilactici MA18/5M for 48 h mitigated these effects. While P. acidilactici MA18/5M did not significantly upregulate tight junction and immune molecules, pre-treatment with this strain protected against pathogen-induced insults to the gut barrier. In particular, the expression of ocldn was significantly induced by V. anguillarum J382, suggesting that this molecule might play a role in the host response against this pathogen. To corroborate these observations in live fish, the effects of P. acidilactici MA18/5M was evaluated in Chinook salmon reared in real aquaculture conditions. Supplementation with P. acidilactici MA18/5M had no effect on Chinook salmon growth parameters after 10 weeks. Interestingly, histopathological results did not show alterations associated with P. acidilactici MA18/5M supplementation, indicating that this strain is safe to be used in the industry. Finally, the expression pattern of transcripts encoding TJ and immune genes in all the treatments suggest that variation in expression is more likely to be due to developmental processes rather than P. acidilactici MA18/5M supplementation. Overall, our results showed that P. acidilactici MA18/5M is a safe strain for use in fish production, however, to assess the effects on growth and immune response previously observed in other salmonid species, an assessment in adult fish is needed.

Comparative Genomic Analysis of Virulent Vibrio (Listonella) anguillarum Serotypes Revealed Genetic Diversity and Genomic Signatures in the O-Antigen Biosynthesis Gene Cluster.

Vibrio anguillarum is the most frequent pathogen affecting fish worldwide. The only known virulent strains of V. anguillarum are serotypes O1, O2, and O3. Genetic differences between the serotypes that could shed insight on the evolution and serotype differences of this marine pathogen are unknown. Here, we fully sequenced and characterized a strain of V. anguillarum O1 (J382) isolated from winter steelhead trout (Oncorhynchus mykiss irideus) in British Columbia, Canada. Koch's postulates using the O1 strain were replicated in naïve lumpfish (Cyclopterus lumpus) and compared to O2. Phenotypic and genotypic comparisons were conducted for serotypes O1, O2, and O3, using biochemical tests and bioinformatic tools, respectively. The genome of V. anguillarum O1 (J382) contains two chromosomes (3.13 Mb and 1.03 Mb) and two typical pJM1-like plasmids (65,573 and 76,959 bp). Furthermore, V. anguillarum O1 (J382) displayed resistance to colistin sulphate, which differs from serotype O2 and could be attributed to the presence of the ugd gene. Comparative genomic analysis, among the serotypes, showed that intra-species evolution is driven by insertion sequences, bacteriophages, and a different repertoire of putative ncRNAs. Genetic heterogeneity in the O-antigen biosynthesis gene cluster is characterized by the absence or the presence of unique genes, which could result in differences in the immune evasion mechanisms employed by the respective serotypes. This study contributes to understanding the genetic differences among V. anguillarum serovars and their evolution.

Relative expression and validation of Aeromonas salmonicida subsp. salmonicida reference genes during ex vivo and in vivo fish infection.

The genus Aeromonas is found worldwide in freshwater and marine environments and has been implicated in the etiology of human and animal diseases. In fish, among Aeromonas species, A. salmonicida causes massive mortality and great economic losses in marine and continental aquaculture species. Currently, several aspects of the clinical signs and pathogenesis of this Gram-negative bacterium have been described; however, determination of an appropriate reference gene is essential to normalize cellular mRNA data remain unknown. Here we evaluate the stability of seven candidate reference genes to be used for data normalization during ex vivo and in vivo experiments conducted in Atlantic cod, Atlantic salmon, and lumpfish. To assess this, raw Ct values obtained were evaluated by using geNorm, NormFinder, BestKeeper, Delta Ct comparison, and the comprehensive ranking, through the bioinformatic open-access portal RefFinder. We determined that fabD and era were most suitable reference genes in Atlantic cod primary macrophages, hfq and era in Atlantic salmon primary macrophages, rpoB and fabD in lumpfish head kidney samples, and hfq and era in lumpfish spleen. Our study demonstrates that use of multiple reference genes and its validation before measurements helps to minimize variability arising in qPCR studies that evaluate A. salmonicida gene expression in fish tissues. Overall, this study provided with an expanded list of reliable reference genes for A. salmonicida gene expression using qPCR during fish infection studies.

Aeromonas salmonicida subsp. salmonicida Early Infection and Immune Response of Atlantic Cod (Gadus morhua L.) Primary Macrophages.

In contrast to other teleosts, Atlantic cod (Gadus morhua) has an expanded repertoire of MHC-I and TLR components, but lacks the MHC-II, the invariant chain/CD74, and CD4+ T cell response, essential for production of antibodies and prevention of bacterial infectious diseases. The mechanisms by which G. morhua fight bacterial infections are not well understood. Aeromonas salmonicida subsp. salmonicida is a recurrent pathogen in cultured and wild fish, and has been reported in Atlantic cod. Macrophages are some of the first responders to bacterial infection and the link between innate and adaptive immune response. Here, we evaluated the viability, reactive oxygen species (ROS) production, cell morphology, and gene expression of cod primary macrophages in response to A. salmonicida infection. We found that A. salmonicida infects cod primary macrophages without killing the cod cells. Likewise, infected Atlantic cod macrophages up-regulated key genes involved in the inflammatory response (e.g., IL-1ß and IL-8) and bacterial recognition (e.g., BPI/LBP). Nevertheless, our results showed a down-regulation of genes related to antimicrobial peptide and ROS production, suggesting that A. salmonicida utilizes its virulence mechanisms to control and prevent macrophage anti-bacterial activity. Our results also indicate that Atlantic cod has a basal ROS production in non-infected cells, and this was not increased after contact with A. salmonicida. Transmission electron microscopy results showed that A. salmonicida was able to infect the macrophages in a high number, and release outer membrane vesicles (OMV) during intracellular infection. These results suggest that Atlantic cod macrophage innate immunity is able to detect A. salmonicida and trigger an anti-inflammatory response, however A. salmonicida controls the cell immune response to prevent bacterial clearance, during early infection.

Identification and validation of reliable Aeromonas salmonicida subspecies salmonicida reference genes for differential gene expression analyses.

Aeromonas salmonicida subsp. salmonicida is a Gram-negative, facultative intracellular pathogen of a wide range of freshwater and marine fish species. A. salmonicida is the causative agent of furunculosis, an immunosuppressive disease that typically progresses to septicemia. Several aspects of A. salmonicida pathogenesis has already been described, but fundamental genetic aspects of the psychrophilic lifestyle of this bacterium remain unknown. Reverse transcription quantitative real-time polymerase chain reaction (qPCR) is a precise molecular technique used to detect very slight changes in gene expression. The appropriate choice of reference genes is essential for accurate normalization of qPCR gene expression data. Despite the available abundance of validated reference genes for mesophilic pathogens, a broad list of validated reference genes for A. salmonicida is not available. Here, we evaluated seven A. salmonicida reference genes under different culture conditions, including different growth phases, iron-limited and iron-supplemented conditions, and thermal stress. We determined that hfq maintained the most stable expression, followed by era, recA, rpoB, 16S, fabD, and gapA. The results of this study provided with an expanded list of reliable reference genes for A. salmonicida gene expression studies using qPCR.

Effects of Vitamin D2 (Ergocalciferol) and D3 (Cholecalciferol) on Atlantic Salmon (Salmo salar) Primary Macrophage Immune Response to Aeromonas salmonicida subsp. salmonicida Infection.

Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D2 and D3 through the diet. In mammals, vitamin D forms are involved in mineral metabolism, cell growth, tissue differentiation, and antibacterial immune response. Vitamin D is an essential nutrient in aquafeeds for finfish. However, the influence of vitamin D on fish cell immunity has not yet been explored. Here, we examined the effects of vitamin D2 and vitamin D3 on Salmo salar primary macrophage immune response to A. salmonicida subspecies salmonicida infection under in vitro conditions. We determined that high concentrations of vitamin D2 (100,000 ng/ml) and D3 (10,000 ng/ml) affect the growth of A. salmonicida and decrease the viability of S. salar primary macrophages. In addition, we determined that primary macrophages pre-treated with a biologically relevant concentration of vitamin D3 for 24 h showed a decrease of A. salmonicida infection. In contrast, vitamin D2 did not influence the antibacterial activity of the S. salar macrophages infected with A. salmonicida. Vitamin D2 and D3 did not influence the expression of canonical genes related to innate immune response. On the other hand, we found that A. salmonicida up-regulated the expression of several canonical genes and suppressed the expression of leukocyte-derived chemotaxin 2 (lect-2) gene, involved in neutrophil recruitment. Primary macrophages pre-treated for 24 h with vitamin D3 counteracted this immune suppression and up-regulated the transcription of lect-2. Our results suggest that vitamin D3 affects A. salmonicida attachment to the S. salar primary macrophages, and as a consequence, the A. salmonicida invasion decreased. Moreover, our study shows that the positive effects of vitamin D3 on fish cell immunity seem to be related to the lect-2 innate immunity mechanisms. We did not identify positive effects of vitamin D2 on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D3, cholecalciferol, induced anti-bacterial innate immunity pathways in Atlantic salmon primary macrophages, suggesting that its utilization as a component of a healthy aquafeed diet in Atlantic salmon could enhance the immune response against A. salmonicida.

Draft Genome Sequence of the Type Strain Aeromonas salmonicida subsp. salmonicida ATCC 33658.

Here, we report the draft genome sequence of the type strain Aeromonas salmonicida subsp. salmonicida ATCC 33658 isolated from Salmo salar The size of the genome is 4,728,143 bp with a G+C content of 58.5%. The A. salmonicida subsp. salmonicida ATCC 33658 genome lacks essential virulence genes that were likely lost during genomic rearrangements.