Comparative Genomics of Typical and Atypical Aeromonas salmonicida Complete Genomes Revealed New Insights into Pathogenesis Evolution.

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55% ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies' taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

A Novel Marine Pathogen Isolated from Wild Cunners (Tautogolabrus adspersus): Comparative Genomics and Transcriptome Profiling of Pseudomonas sp. Strain J380.

Cunner (Tautogolabrus adspersus) is a cleaner fish being considered for utilized in the North Atlantic salmon (Salmo salar) aquaculture industry to biocontrol sea lice infestations. However, bacterial diseases due to natural infections in wild cunners have yet to be described. This study reports the isolation of Pseudomonas sp. J380 from infected wild cunners and its phenotypic, genomic, and transcriptomic characterization. This Gram-negative motile rod-shaped bacterium showed a mesophilic (4-28 °C) and halotolerant growth. Under iron-limited conditions, Pseudomonas sp. J380 produced pyoverdine-type fluorescent siderophore. Koch's postulates were verified in wild cunners by intraperitoneally (i.p.) injecting Pseudomonas sp. J380 at 4 × 103, 4 × 105, and 4 × 107 colony forming units (CFU)/dose. Host-range and comparative virulence were also investigated in lumpfish and Atlantic salmon i.p. injected with ~106 CFU/dose. Lumpfish were more susceptible compared to cunners, and Atlantic salmon was resistant to Pseudomonas sp. J380 infection. Cunner tissues were heavily colonized by Pseudomonas sp. J380 compared to lumpfish and Atlantic salmon suggesting that it might be an opportunistic pathogen in cunners. The genome of Pseudomonas sp. J380 was 6.26 megabases (Mb) with a guanine-cytosine (GC) content of 59.7%. Biochemical profiles, as well as comparative and phylogenomic analyses, suggested that Pseudomonas sp. J380 belongs to the P. fluorescens species complex. Transcriptome profiling under iron-limited vs. iron-enriched conditions identified 1159 differentially expressed genes (DEGs). Cellular metabolic processes, such as ribosomal and energy production, and protein synthesis, were impeded by iron limitation. In contrast, genes involved in environmental adaptation mechanisms including two-component systems, histidine catabolism, and redox balance were transcriptionally up-regulated. Furthermore, iron limitation triggered the differential expression of genes encoding proteins associated with iron homeostasis. As the first report on a bacterial infection in cunners, the current study provides an overview of a new marine pathogen, Pseudomonas sp. J380.

CD10+ Cells and IgM in Pathogen Response in Lumpfish (Cyclopterus lumpus) Eye Tissues.

Lumpfish (Cyclopterus lumpus), a North Atlantic "cleaner" fish, is utilized to biocontrol salmon louse (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar) farms. Lumpfish require excellent vision to scan for and eat louse on salmon skin. The lumpfish eye immune response to infectious diseases has not been explored. We examined the ocular response to a natural parasite infection in wild lumpfish and to an experimental bacterial infection in cultured lumpfish. Cysts associated with natural myxozoan infection in the ocular scleral cartilage of wild adult lumpfish harbored cells expressing cluster of differentiation 10 (CD10) and immunoglobulin M (IgM). Experimental Vibrio anguillarum infection, which led to exophthalmos and disorganization of the retinal tissues was associated with disruption of normal CD10 expression, CD10+ cellular infiltration and IgM expression. We further describe the lumpfish CD10 orthologue and characterize the lumpfish scleral skeleton in the context of myxozoan scleral cysts. We propose that lumpfish develop an intraocular response to pathogens, exemplified herein by myxozoan and V. anguillarum infection involving novel CD10+ cells and IgM+ cells to contain and mitigate damage to eye structures. This work is the first demonstration of CD10 and IgM expressing cells in a novel ocular immune system component in response to disease in a teleost.

Aeromonas salmonicida infection kinetics and protective immune response to vaccination in sablefish (Anoplopoma fimbria).

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6-8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

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.

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.

Colibacillosis in a New Zealand white rabbit (Oryctolagus cuniculus).

Colibacillosis is a disease caused by Escherichia coli in a variety of animals, including humans. Rabbit colibacillosis is infrequent or with an incipient description in Chile. Here, we describe an E. coli case in a white New Zealand rabbit at an animal facility in Santiago, Chile. Necropsy, histology, bacteriology, and 16S sequencing indicated an E. coli systemic infection. Phylogenetic analysis suggested that this E. coli J305 isolate is closely related to Shigella spp.

Genome Sequence of Vibrio VPAP30, Isolated from an Episode of Massive Mortality of Reared Larvae of the Scallop Argopecten purpuratus.

We report here the 5.167-Mbp draft genome sequence of Vibrio VPAP30, isolated from an Argopecten purpuratus larval culture. Vibrio VPAP30 is the etiological agent of a vibriosis outbreak causing a complete collapse of a larval culture of the scallop A. purpuratus, which occurred in a commercial hatchery in Chile.

Pectenotoxins and yessotoxin from Arica Bay, North Chile as determined by tandem mass spectrometry.

Lipophilic phycotoxins were measured by tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) in size-fractionated plankton samples taken at five stations in Arica Bay, northern Chile in the southern summer 2007/2008. Pectenotoxins-2 (PTX-2), -11 (PTX-11), -2 seco acid (PTX-2sa) and yessotoxin (YTX) were identified by comparison of retention times and collision-induced mass spectra of certified standards and field sample extracts. This is the first report of PTXs and YTX from planktonic samples in Chilean coastal waters.

Parasitic castration of Eurhomalea lenticularis (Bivalvia: Veneridae) by a digenetic trematode: quantitative histological analysis.

The clam Eurhomalea lenticularis may be parasitized by digenean trematodes of the family Plagiorchidae, specifically in the gonads (parasitic castration). A quantitative histological analysis of the parasitized gonads demonstrated a significant decrease in gonadal area, in the size of individual acini, and in the numbers of differentiated germ cells compared to unparasitized clams. Castration may be caused by mechanical compression due to trematode sporocyst growth. However, the uniform loss of germ cells in areas without sporocysts suggests that a more generalized mechanism is responsible. We suggest that parasitic castration has a primary effect on the host's neuroendocrine and gametogenic systems that regulate gamete production.