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.

Prevalence and distribution of Renibacterium salmoninarum, causative agent of bacterial kidney disease, in wild trout fisheries in Colorado.

Detections of Renibacterium salmoninarum in Colorado USA fish hatcheries have become more frequent in recent years, including one disease outbreak that originated with a wild broodstock. Our objectives were to document the prevalence and distribution of R. salmoninarum in Colorado's wild trout fisheries, investigate variables that influence that distribution, and evaluate the effectiveness of common testing methods on non-anadromous trout. We sampled wild trout across Colorado and tested kidney tissue with enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), nested polymerase chain reaction (nPCR), and direct fluorescent antibody test (DFAT). Screening with ELISA showed high prevalence of R. salmoninarum among fish populations, but antigen levels were low. No clinical disease was observed in any of the fish sampled despite the antigen of R. salmoninarum being common. Antigen levels measured by ELISA increased in smaller streams with lower historic fish stocking rates. Brook trout Salvelinus fontinalis had the highest prevalence of the bacterium among fish species and highest ELISA antigen levels. The distribution of brook trout in the smallest streams may help explain the patterns of R. salmoninarum across the landscape. The most effective assays for screening wild trout were qPCR and ELISA; DFAT was inconsistent for bacterial levels encountered in wild trout and generally uninformative. Additionally, qPCR and ELISA can provide quantitative information about bacteria levels. The bacterium R. salmoninarum is ubiquitous in Colorado trout fisheries but is generally found at low levels. Active infections are rare and overt bacterial kidney disease appears more common in Colorado hatcheries than in wild fish.

Proteomic analysis reveals Renibacterium salmoninarum grown under iron-limited conditions induces iron uptake mechanisms and overproduction of the 57-kDa protein.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen, is the causative agent of bacterial kidney disease, a chronic, progressive and granulomatous infection that threatens farmed and wild salmonids worldwide. Pathogenic R. salmoninarum colonizes tissues and invades the host through cell surface-associated and secreted proteins. While correlations between iron acquisition genes and virulence have been demonstrated in vitro, these mechanisms have not undergone proteomic characterization. The present study applied a proteomic approach to elucidate the differences between the virulent Chilean R. salmoninarum H-2 strain and the type strain ATCC 33209T . Analyses were conducted under normal (control) and iron-limited conditions (DIP) emulating the host environment. Interestingly, strain H-2 apparently responded better to the iron-limited condition-for example, only this strain presented a significantly enriched iron ion homeostasis pathway. Furthermore, key virulence factors related to an iron-limited environment were more abundant in strain H-2. Importantly, the lack of iron favoured the expression of the 57-kDa protein in strain H-2, the principal virulence factor for R. salmoninarum. Our findings can be employed in the design and development of treatments targeted to iron uptake mechanisms (e.g. siderophore synthesis or haem uptake), which represents a promising therapeutic approach for treating this persistent fastidious bacterium.

Assessing the presence and spread of Renibacterium salmoninarum between farmed and wild fish in Sweden.

Bacterial kidney disease (BKD) can be a devastating bacterial infection in salmonids, and it is present in aquaculture throughout the world. BKD is caused by the Gram-positive facultative intracellular bacterium Renibacterium salmoninarum (R. salmoninarum) that is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and internal signs such as organ swelling, granulomas, petechiae and ascites. In Sweden, BKD accounts for a significant income loss in aquacultures due to expensive decontamination of the facility and increased disease susceptibility for the immunocompromised fish leading to higher mortality rates. In addition, uncontrolled spread in aquaculture may threaten the survival of wild fish populations. The aim of our study was to investigate the prevalence of R. salmoninarum in wild salmonids caught in Swedish waters where net pen farms with a recent history of BKD are present. Four rivers with at least one BKD-positive or recently BKD-positive farm were selected. In addition, we evaluated the use of environmental DNA (eDNA) for surveillance and monitoring of ongoing infections at these locations. In total, 1058 fish were sampled from four different river systems, and of them 52 (4.9%) were positive for R. salmoninarum by antigen ELISA. Surprisingly, these fish were not evenly distributed between the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high rate of 17% R. salmoninarum-positive samples in wild salmonids in this area. This number is far above what was expected and clearly shows the risk with an open farming system as well as the importance of effective health monitoring programmes to avoid an uncontrolled spread of the disease. The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analysed were PCR positive for R. salmoninarum (2 of 38) and those were collected where no ELISA positive fish were identified. In addition to water, sediment samples were collected under a net pen farm that had recently slaughtered all fish due to ongoing R. salmoninarum infections. Sediment samples are more promising than water as 4 of 5 samples at one farming facility where positive for R. salmoninarum. Thus, sediment samples may be valuable for monitoring potential ongoing BKD in farms, without the need to sacrifice valuable fish.

Comparative pan-genomic analysis of 51 Renibacterium salmoninarum indicates heterogeneity in the principal virulence factor, the 57 kDa protein.

Renibacterium salmoninarum, a Gram-positive intracellular pathogen, is the causative agent of bacterial kidney disease (BKD), the impacts of which are high mortalities and economic losses for the salmon industry. This study provides novel analyses for the whole-genome sequences of 50 R. salmoninarum isolates and the reference strain ATCC 33209 using a pan-genomic approach to elucidate phylogenomic relationships and identify unique and shared genes associated with pathogenicity and infection mechanisms. Genome size varied from 3,061,638 to 3,155,332 bp; gene count from 3452 to 3580; and predicted coding sequences from 3402 to 3527. Comparative analyses revealed an open, but approaching closed, pan-genome. The pan-genome analysis recovered 4064 genes, with a core genome containing 3306 genes. Phylogenetic analysis of R. salmoninarum showed high genomic homogeneity, apart from one isolate obtained from Salmo trutta in Norway. All genomes presented the 57-kDa protein (p57). Strain ATCC 33209 and the Chilean isolates H-2 and DJ2R presented two copies of the msa gene, while the remaining isolates had one copy. The pan-genome analysis further identified differences in the number of copies and length of the signalling peptide for p57, the principal virulence factor reported for this bacterium. This heterogeneity could be associated with the secretion levels of p57, potentially influencing virulence. Additionally identified were numerous common genes related to iron uptake, the stress response and regulation, and cell signalling-all of which constitute the pathogenic repertoire of R. salmoninarum. This investigation provides information that is applicable in future studies for identifying therapeutic targets and/or for designing new strategies (e.g., vaccines) to prevent BKD infections in salmon farming.

Non-lethal sampling for the detection of Renibacterium salmoninarum by qPCR for diagnosis of bacterial kidney disease.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill-/cloacal swabs collected in vivo for real-time PCR (qPCRgc ), allow a sensitive and specific detection of Rs. The sensitivity of qPCRgc was estimated to 97.8% (credible interval (ci) 93.8%-100%) compared to 98.3% (ci 92.7%-100%) and 48.8% (ci 38.8%-58.8%) of kidney samples for qPCR (qPCRk ) and ELISA (ELISAk ) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCRgc instead of ELISAk will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions.

Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of Renibacterium salmoninarum.

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a Kd of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses.

Expression and Characterization of a Novel Cold-Adapted Chitosanase from Marine Renibacterium sp. Suitable for Chitooligosaccharides Preparation.

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni-NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0-50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20-30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

Evidence for the existence of extracellular vesicles in Renibacterium salmoninarum and related cytotoxic effects on SHK-1 cells.

Extracellular vesicles (EVs) in bacteria have been implicated in invasive and, through enzymes, infective processes. One Gram-positive bacterium lacking any EV research, despite having commercial impacts on the aquaculture industry, is Renibacterium salmoninarum. We addressed this gap in knowledge by utilizing scanning electron microscopy to provide the first reported evidence for the production of EVs by R. salmoninarum strain H-2. Dispersive light scattering detected that the EVs were heterogeneous in size, and the protein compositions were similar to the bacterial membrane and contained the virulent protein factors p22 and p57. The EVs additionally had a concentrated negative charge compared with R. salmoninarum H-2, as determined by Z potential. Finally, these particles seemed to play a role in host invasion in vitro in the salmon head kidney cell line, as demonstrated by the occurrence of a cytotoxic effect within the first 48 hr post-infection. Higher EV concentrations (i.e. 52.6 µg/ml) were more toxic than R. salmoninarum H-2. This information serves as a foundation to develop and test possible uses for R. salmoninarum EVs in salmon aquaculture, inspiring future advances against bacterial kidney disease.

Proposed protocol for performing MIC testing to determine the antimicrobial susceptibility of Renibacterium salmoninarum in Chilean salmon farms.

Surveillance of antibiotic resistance is of paramount importance for animal welfare and production. Despite aquaculture being a main source of animal protein, studies on antibiotic susceptibility in fish pathogens are scarce. Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD), is one of the most common bacterial pathogens affecting salmon farming. In this work, we present an analysis of susceptibility patterns using determinations of minimum inhibitory concentration (MIC) for 65 field isolates, which were collected over seven years (2013-2019) from Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) farms across southern Chile. The MIC protocol described by the Clinical Laboratory Standards Institute (CLSI) was used, but with microdilution instead of macrodilution and eight instead of four days of incubation. Two laboratories independently conducted analyses to provide data on the epidemiological cut-off values for R. salmoninarum to florfenicol, oxytetracycline and erythromycin. By using two calculation methods, our results provide evidence for an evolving subpopulation of non-wild-type isolates for the macrolide erythromycin, which is consistent with the respective treatment frequencies prescribed against BKD. Contrasting with what was expected, R. salmoninarum isolates were most susceptible to florfenicol and oxytetracycline, both of which are widely used antibiotics currently used in the Chilean salmon industry. The presented findings can serve as a reference for national or international antibiotic surveillance programmes, for both MIC interpretation and to identify emerging resistance to the conventional drugs used in BKD management. Finally, our results indicate that an 8-day incubation period for establishing MIC values of R. salmoninarum should be considered in a future revision of the CLSI guidelines.

Profiling the transcriptome response of Atlantic salmon head kidney to formalin-killed Renibacterium salmoninarum.

Renibacterium salmoninarum is a Gram-positive, intracellular bacterial pathogen that causes Bacterial Kidney Disease (BKD) in Atlantic salmon (Salmo salar). The host transcriptomic response to this immune-suppressive pathogen remains poorly understood. To identify R. salmoninarum-responsive genes, Atlantic salmon were intraperitoneally injected with a low (5 × 105 cells/kg, Low-Rs) or high (5 × 107 cells/kg; High-Rs) dose of formalin-killed R. salmoninarum bacterin or phosphate-buffered saline (PBS control); head kidney samples were collected before and 24 h after injection. Using 44K microarray analysis, we identified 107 and 345 differentially expressed probes in response to R. salmoninarum bacterin (i.e. High-Rs vs. PBS control) by Significance Analysis of Microarrays (SAM) and Rank Products (RP), respectively. Twenty-two microarray-identified genes were subjected to qPCR assays, and 17 genes were confirmed as being significantly responsive to the bacterin. There was an up-regulation in expression of genes playing putative roles as immune receptors and antimicrobial effectors. Genes with putative roles as pathogen recognition (e.g. clec12b and tlr5) or immunoregulatory (e.g. tnfrsf6b and tnfrsf11b) receptors were up-regulated in response to R.salmoninarum bacterin. Also, chemokines and a chemokine receptor showed opposite regulation [up-regulation of effectors (i.e. ccl13 and ccl) and down-regulation of cxcr1] in response to the bacterin. The present study identified and validated novel biomarker genes (e.g. ctsl1, lipe, cldn4, ccny) that can be used to assess Atlantic salmon response to R. salmoninarum, and will be valuable in the development of tools to combat BKD.

Renibacterium salmoninarum and Mycobacterium spp.: two bacterial pathogens present at low levels in wild brown trout (Salmo trutta fario) populations in Austrian rivers.

BACKGROUND: Renibacterium salmoninarum and Mycobacterium sp. are important bacterial pathogens of fish. R. salmoninarum is the causative agent of bacterial kidney disease, a Gram-positive bacterium mostly known for causing chronic infections in salmonid fish, while multiple species belonging to the Mycobacterium genus have been associated with mycobacteriosis in fish as well as in human. The objective of this study was to determine the prevalence of these two bacterial pathogens in populations of wild brown trout (Salmo trutta fario) in four rivers (Kamp, Wulka, Traun and Ybbs) in Austria. RESULTS: A total of 457 kidney samples were examined for both bacterial agents using nested and conventional PCR as well as bacterial cultivation on KDM-2, histological examination and immunohistochemistry. Molecular evidence showed an estimated prevalence level of 0.94% for R. salmoninarum in 2017 while the bacterium could not be detected in 2018 and histology showed signs consistent with a low-level chronic inflammation in the kidney of infected fish. Similarly, no fish were found positive for Mycobacterium in 2017 but in 2018, the prevalence was found to be 37.03% in the Kamp river (4.08% across all rivers). The sequencing data confirmed that these fish carried Mycobacterium sp. although the precise species of Mycobacterium could not be ascertained. CONCLUSIONS: This survey constitutes the first insight into the prevalence rate of R. salmoninarum and Mycobacterium sp. in wild brown trout (Salmo trutta fario) populations in Austria. Both of these pathogens were only detected in the summer months (June and July), which might suggest that the stress linked to increased water temperature could act as stressor factor and contribute to the outbreak of these diseases. The age of the fish might also play a role, especially in the case of Mycobacterium sp. as all the infected fish were in their first summer (June).

First report of bacterial kidney disease in coho salmon Oncorhynchus kisutch in Russia.

This paper describes the first case of bacterial kidney disease (BKD) to be identified in coho salmon Oncorhynchus kisutch in Russia. The fish in question was caught in Lake Bolshoi Vilyui on the Kamchatka Peninsula. The diseased fish had foci of granulomatous inflammation in the kidneys. The diagnosis was confirmed by isolating the bacterium Renibacterium salmoninarum from kidney tissue in pure culture, and by determining the partial 16S RNA gene sequence of the isolate. This is the first detection of this pathogen in the genus Oncorhynchus in Russia, and detection of BKD in coho salmon indicates that the pathogen is present in the natural fish populations of Kamchatka. Therefore, it will be necessary to conduct screening studies of mature salmon selected for artificial reproduction, for the presence of BKD signs and asymptomatic infection with R. salmoninarum, which will allow us to estimate the prevalence of the pathogen.

Effects of Hatchery Rearing Density, Handling, and Nutrition on Renibacterium salmoninarum Infection Prevalence in Juvenile Chinook Salmon.

Bacterial kidney disease, caused by Renibacterium salmoninarum (RS), is a chronic and often fatal disease of salmonid species, and can be particularly harmful to hatchery-reared Chinook Salmon Oncorhynchus tshawytscha. A considerable amount of research has focused on the prevention of vertical and horizontal transmission; however, a comparatively little amount has investigated factors that increase the prevalence of RS infection in captive environments. We evaluated the effects of three common hatchery conditions (handling, nutrition level, and rearing density) on RS infection prevalence. Fish were sampled at 30-d and 60-d postexposure to RS. Of 577 juveniles examined, 65 (11.27%) had anterior kidneys infected with RS. Using a logistic mixed model analysis, we found effects of nutrition level (P = 0.018), handling (P = 0.010), and sampling period (P = 0.003) on the prevalence of RS. The interactions of nutrition and handling (P = 0.008) and nutrition and time (P < 0.001) were also significant. When fed a standard-nutrition diet, proportionately fewer fish were infected with RS when not handled (7.16% versus 0.04%; P = 0.003). Fish in the standard-nutrition group also had a lower prevalence of RS during the second sampling period (4.08% versus 0.08%, respectively; P < 0.001). When not handled, rearing with standard nutrition (11.50% versus 0.04%; P = 0.004) resulted in a reduction in prevalence of RS infection. Additionally, nonhandled fish had a much lower prevalence of RS infection during the second sampling period (2.66% versus 0.21%; P = 0.009). While density did not affect the prevalence of RS infection (P = 0.145), fish reared at a higher density had lower RS infection when not handled (16.48% versus 0.84%, P = 0.004). For fish at a higher density, the RS prevalence was lower during the second sampling period (10.57% versus 1.40%; P = 0.002). Our results suggest that hatchery managers can reduce RS infection prevalence by maintaining an adequate nutritional regime as recommended by the manufacturer. Additionally, the prevalence of RS may be reduced if managers decrease handling of hatchery-reared Chinook Salmon if exposed to RS.

Renibacterium salmoninarum iron-acquisition mechanisms and ASK cell line infection: Virulence and immune response.

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron-acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT-qPCR to assess the effects of normal and iron-limited conditions on iron-uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H-2 and the type strain DSM20767T . Further evaluated was the in vitro immune-related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron-limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H-2 than DSM20767T . These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF-α, IL-1ß, TLR1 and INF-γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron-limited conditions, especially H-2. These observations indicate that iron-acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron-uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.

Transcriptome profiling of lumpfish (Cyclopterus lumpus) head kidney to Renibacterium salmoninarum at early and chronic infection stages.

Renibacterium salmoninarum causes Bacterial Kidney Disease (BKD) in several fish species. Atlantic lumpfish, a cleaner fish, is susceptible to R. salmoninarum. To profile the transcriptome response of lumpfish to R. salmoninarum at early and chronic infection stages, fish were intraperitoneally injected with either a high dose of R. salmoninarum (1 × 109 cells dose-1) or PBS (control). Head kidney tissue samples were collected at 28- and 98-days post-infection (dpi) for RNA sequencing. Transcriptomic profiling identified 1971 and 139 differentially expressed genes (DEGs) in infected compared with control samples at 28 and 98 dpi, respectively. At 28 dpi, R. salmoninarum-induced genes (n = 434) mainly involved in innate and adaptive immune response-related pathways, whereas R. salmoninarum-suppressed genes (n = 1537) were largely connected to amino acid metabolism and cellular processes. Cell-mediated immunity-related genes showed dysregulation at 98 dpi. Several immune-signalling pathways were dysregulated in response to R. salmoninarum, including apoptosis, alternative complement, JAK-STAT signalling, and MHC-I dependent pathways. In summary, R. salmoninarum causes immune suppression at early infection, whereas lumpfish induce a cell-mediated immune response at chronic infection. This study provides a complete depiction of diverse immune mechanisms dysregulated by R. salmoninarum in lumpfish and opens new avenues to develop immune prophylactic tools to prevent BKD.

Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence.

Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.

HIGH PREVALENCE OF CIRCULATING ANTIBODIES TO RENIBACTERIUM SALMONINARUM IN SPAWNING ONCORHYNCHUS SPP. FROM LAKE MICHIGAN, USA.

Bacterial kidney disease, caused by Renibacterium salmoninarum, threatens salmonids worldwide. Following devastating mortality episodes in Oncorhynchus spp. in Lake Michigan, US, in the 1980s and infection rates >90%, pathogen prevalence has steadily declined to <5% over three decades in the three state-managed stocks. In this study, we sought to determine if the declining infection rates were associated with heightened circulating antibodies in state-managed Oncorhynchus spp. residing in the Lake Michigan watershed. A single-dilution, indirect enzyme-linked immunosorbent assay (ELISA) was modified to detect circulating antibodies against R. salmoninarum. Baseline values were delineated from naive chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss). The assay was first used to assess primary antibody production over a 4-wk period in chinook salmon experimentally infected with R. salmoninarum. Mean antibody response was detected as early as 2 wk postinfection and continued to increase to the end of the observation period. The modified ELISA was then used to detect antibodies in serum samples collected from feral adult chinook salmon, coho salmon (Oncorhynchus kisutch), and steelhead trout (O. mykiss) returning to spawn at Lake Michigan weirs in 2009 and 2013. Results demonstrated that about 80% of feral Oncorhynchus spp. had measurable titers of circulating antibodies to R. salmoninarum. The relative ease and reasonable costs of this modified ELISA makes it a valuable serosurveillance tool for assessing the humoral immune status of feral salmonid populations.

Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1ß, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Atlantic Salmon Pre-smolt Survivors of Renibacterium salmoninarum Infection Show Inhibited Cell-Mediated Adaptive Immune Response and a Higher Risk of Death During the Late Stage of Infection at Lower Water Temperatures.

Bacterial kidney disease (BKD) is widespread in many areas of the world and can cause substantial economic losses for the salmon aquaculture industry. The objective of this study was to investigate the pathophysiological response and gene expression profiles related to the immune response at different water temperatures and to identify the best immunopathological biomarkers to define a phenotype of resistance to BKD. The abundance of msa transcripts of R. salmoninarum in the head kidney was significantly higher in infected fish at 11°C. R. salmoninarum induced significantly more severe kidney lesions, anemia and impaired renal function at 11°C. In addition, the expression pattern of the genes related to humoral and cell-mediated immune responses in infected fish at 11 and 15°C was very similar, although R. salmoninarum induced a significantly greater downregulation of the adaptive immune response genes at the lower water temperature. These results could be due to a suppressed host response directly related to the lowest water temperature and/or associated with a delayed host response related to the lowest water temperature. Although no significant differences in survival rate were observed, fish infected at the lowest temperature showed a higher probability of death and delayed the mortality curve during the late stage of infection (35 days after infection). Thirty-three immunopathological biomarkers were identified for potential use in the search for a resistance phenotype for BKD, and eight were genes related specifically to the adaptive cell-mediated immune response.