The genus Edwardsiella comprises a genetically distinct taxon related to other members of the family Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cationic antimicrobial peptides (CAMPs) is a specific property of the genus Edwardsiella. In particular, Edwardsiella ictaluri, an important pathogen of the catfish (Ictalurus punctatus) aquaculture and the causative agent of a fatal systemic infection, is highly resistant to CAMPs. E. ictaluri mechanisms of resistance to CAMPs are unknown. We hypothesized that E. ictaluri lipopolysaccharide (LPS) plays a role in both virulence and resistance to CAMPs. The putative genes related to LPS oligo-polysaccharide (O-PS) synthesis were in-frame deleted. Individual deletions of wibT, gne and ugd eliminated synthesis of the O-PS, causing auto-agglutination, rough colonies, biofilm-like formation and motility defects. Deletion of ugd, the gene that encodes the UDP-glucose dehydrogenase enzyme responsible for synthesis of UDP-glucuronic acid, causes sensitivity to CAMPs, indicating that UDP-glucuronic acid and its derivatives are related to CAMP intrinsic resistance. E. ictaluri OP-S mutants showed different levels of attenuation, colonization of lymphoid tissues and immune protection in zebrafish (Danio rerio) and catfish. Orally inoculated catfish with O-PS mutant strains presented different degrees of gut inflammation and colonization of lymphoid tissues. Here we conclude that intrinsic resistance to CAMPs is mediated by Ugd enzyme, which has a pleiotropic effect in E. ictaluri influencing LPS synthesis, motility, agglutination, fish gut inflammation and virulence.

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.

The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri , a host-restricted pathogen of channel catfish ( Ictalurus punctatus ) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δ fur mutant is attenuated and immune protective in zebrafish ( Danio rerio ) and catfish ( Ictalurus punctatus ), triggering systemic immunity. We conclude that an E. ictaluri Δ fur mutant could be an effective component of an immersion-oral vaccine for the catfish industry.

Salmonid rickettsial septicemia (SRS), caused byPiscirickettsia 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 (excepthampa), 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 underlyingP. salmonis-triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen.

Regulation of the synthesis of Vi polysaccharide, a major virulence determinant inSalmonella entericaserotype Typhi, is under the control of two regulatory systems,ompR-envZandrscB-rscC, which respond to changes in osmolarity. Some serotype Typhi strains exhibit overexpression of Vi polysaccharide, which masks clinical detection of lipopolysaccharide O antigen. This variation in Vi polysaccharide and O antigen display (VW variation) has been observed since the initial studies of serotype Typhi. In this study, we report thatrpoSplays a role in this increased expression in Vi polysaccharide. We constructed a variety of isogenic serotype Typhi mutants that differed in their expression levels of RpoS and examined the role of therpoSproduct in synthesis of Vi polysaccharide under different osmolarity conditions. Vi polysaccharide synthesis was also examined in serotype Typhi mutants in which the native promoter of therpoSwas replaced by anaraCP_BADcassette, so that the expression ofrpoSwas arabinose dependent. The RpoS⁻strains showed increased syntheses of Vi polysaccharide, which at low and medium osmolarities masked O antigen detection. In contrast, RpoS⁺strains showed lower syntheses of Vi polysaccharide, and an increased detection of O antigen was observed. During exponential growth, whenrpoSis unstable or present at low levels, serotype Typhi RpoS⁺strains overexpress the Vi polysaccharide at levels comparable to those for RpoS⁻strains. Our results show that RpoS is another regulator of Vi polysaccharide synthesis and contributes to VW variation in serotype Typhi, which has implications for the development of recombinant attenuatedSalmonellavaccines in humans.