Agent Of Bacterial Kidney Disease Research Articles

Optimizing culture medium to facilitate rapid Renibacterium salmoninarum growth

This study presents significant improvements in the culturing of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salmonids. Our goal was to improve R. salmoninarum cultivation and its detection in salmon farming by adjusting to 1.5%wv−1 NaCl into kidney disease medium-2 (KDM2), which significantly expedited growth without further effects from varying NaCl concentrations. The reductions in incubation periods of cultivation, this approach enhances early and accurate bacterial and disease surveillance, enabling prompt intervention. The modified medium significantly accelerated R. salmoninarum growth, achieving an optical density at 600 nm of 0.5 within 5–6 days post-incubation, marking a notable improvement over traditional KDM2. Exploring a range of NaCl concentrations in solid and liquid media revealed distinct growth patterns of R. salmoninarum across various strains. Among these, 1.5%wv−1 NaCl proved highly effective in promoting rapid pathogen proliferation while preserving essential components roles in the medium, with an average of 75% of reduction in incubation times in plate agar culture media. Strains isolated from salmon organs in the Los Lagos and Magallanes region exhibited high pathogenicity, leading to 80–100% mortality within 30 days, underscoring the wide-ranging efficacy of NaCl in enhancing growth, irrespective of geographic origins. Moreover, the modified media, enriched with NaCl, demonstrated no compromise in the pathogenicity of R. salmoninarum strains. These results highlight the potential of NaCl supplementation, supporting its addition in the media of R. salmoninarum. This inclusion could expedite biomass growth within shortened timeframes, stimulate microbial aggregates, and enhance bacterial multiplication within the medium. These findings affirm the utility of KDM2-NaCl in enhancing R. salmoninarum effective cultivation and detection, offering cost-effective strategies for managing infections and supporting targeted interventions in the salmon industry.

Diagnosis of Renibacterium salmoninarum infection in harvested Atlantic salmon (Salmo salar L.) on the east coast of Canada: Clinical findings, sample collection methods and laboratory diagnostic tests.

Chronic subclinical infection with the aetiological agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, presents challenges for the clinical management of disease in farmed salmonids and for prevalence estimation. Harvested salmon sampled at processing plants provide the opportunity to describe subclinical outcomes of BKD using gross necropsy observations and diagnostic test results in farmed Atlantic salmon (Salmo salar L.) populations that are apparently healthy (i.e. alive at harvest) but naturally exposed to R. salmoninarum infection. Sampling of farmed salmon (Population A, n=124 and Population B, n=160) was performed immediately post-slaughter as fish were being processed at a plant in New Brunswick, Canada. Populations were selected based on planned harvests from sites with histories of recent exposure events related to clinical BKD as evidenced by the site veterinarian's diagnosis of mortality attributable to BKD: One site (Pop A) had recently increasing mortalities attributed to BKD, and the other site (Pop B) had ongoing low-level mortalities with BKD pathology. As expected with the different exposure histories, Pop A had a higher percentage (57.2%) of R. salmoninarum culture-positive kidney samples compared with similar fish samples in Pop B (17.5%). Diagnosis of R. salmoninarum by gross granulomatous lesions in internal visceral organs, bacterial culture and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using different swab transport methods, and molecular detection methods (quantitative PCR, qPCR) were compared. Agreement of culture-positive percentages at the sample level was moderate (kappa: 0.61-0.75) among specimens collected using different kidney sampling methods in Pop A and Pop B. The highest proportion of R. salmoninarum-positive cultures occurred when kidney tissues were transported to the laboratory and inoculated directly onto agar using a swab (94% of cultures from Pop A and 82% from Pop B when fish were positive by any culture method). Fish with cumulative lesion scores (severity of granulomatous lesions in 3 different visceral organs) of >4 were all culture positive, and when compared with non-lesioned fish, had substantially higher odds of being culture positive: Pop A: odds ratio (OR)=73, 95% confidence interval (CI) (7.91, 680.8); Pop B: OR=66, 95% CI (6.12, 720.7). Our study found that onsite postmortem examinations with severity scores of gross granulomatous lesions were predictive of positive culture results for R. salmoninarum, and they were a useful proxy for assessing prevalence in apparently healthy populations with subclinical infection.

Comparison of injection and immersion challenges of Renibacterium salmoninarum strains in rainbow trout (Oncorhynchus mykiss).

Renibacterium salmoninarum is a pathogenic gram-positive bacterium and the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in rainbow trout (Oncorhynchus mykiss). The CK-90 strain was previously isolated in steelhead trout (O. mykiss) from Dworshak National Fish Hatchery (NFH) in 1990. It was intracoelomically injected (100 μL) at a high dose containing 4.80 x 106 CFU g fish-1 (OD525 =1.779) and a low dose containing 6.86 x105 CFU g fish-1 (OD525 = 1.077), or fish were immersed in a solution containing 2.21 x 105 CFU g fish-1 (OD525 =0.886) of R. salmoninarum. The ATCC 33739 strain, originating from brook trout (Salvelinus fontinalis), was also included and intracoelomically-injected at 3.58 x 105 CFU/g (OD525 =1.431) to discern differences in virulence between the strains. Clinical signs of BKD manifested approximately ten days post-challenge, and mortalities began 19 days post-challenge. To confirm infection and quantify R. salmonirarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected post-challenge. Injection-challenged rainbow trout with CK-90 (both high and low groups) exhibited significantly higher mortality (P<0.001) than fish injected with ATCC 33739 or those exposed to CK-90 via immersion challenge. The R. salmoninarum antigen, p57-kDa, was confirmed via ELISA. Antigen load for fish injected with the CK-90 strain (high dose: OD405 =0.71, low dose: OD405 =0.66) was significantly higher than for fish injected with the ATCC 33739 strain (OD405 =0.34). It was found that the CK-90 strain (both high and low doses) was more virulent than ATCC 33739 strain, which exhibited no mortalities over 28 days. Although there were no mortalities during the 28-day trial, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in ATCC 33739 fish. Further, the immersion challenge methodology for these R. salmoninarum strains was ineffective in inducing mortalities at the abovementioned doses.

Method for lineage typing of epidemic Renibacterium salmoninarum in Chilean salmon farms.

Renibacterium salmoninarum (Rs) is the etiological agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. Although the whole genome of Rs (~3.1 million nucleotides) is highly conserved, genomic epidemiology analyses have identified four sub-lineages from Chilean isolates. A total of 94 Rs genomes from the BIGSdb aquaculture database were aligned and compared using bioinformatics tools, identifying 2199 independent single-nucleotide polymorphisms (SNPs) spread along the genome. A detailed analysis of the distribution of the SNPs showed five local zones of a length in the range of 10-15 kbp that should be used to unambiguously identify a specific sub-lineage. Based on the Rs type strain DSM 20767T , we designed multiplex PCR primers that produce specific amplification products which were further sequenced by the Sanger method to obtain the genotype of the sub-lineage. For the genetic typing, we evaluated 27 Rs isolates recovered from BKD outbreaks from different fish species and regions of Chile. Based on the findings reported here, we propose the PCR approach as a valuable tool for the rapid and reliable studying of the relationships between Rs isolates and the different sub-lineages without requiring the sequencing of the entire genome.

Genomic and proteomic aspects of p57 protein from Renibacterium salmoninarum: Characteristics in virulence patterns.

Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.

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

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.

Prevalence and distribution of Renibacterium salmoninarum in non-salmonid fishes from Laurentian Great Lakes and inland habitats

Renibacterium salmoninarum (RS), the causative agent of bacterial kidney disease, has been a serious threat to salmonid health in the Laurentian Great Lakes. Despite its wide spread presence in the Great Lakes basin, little is known about RS ecology and the potential role of non-salmonid species as one of the pathogen’s reservoirs. This information is of paramount importance to fishery managers in order to better understand RS distribution in the different biotic components of the Great Lakes watershed. In this study, non-salmonid species from lakes Michigan and Huron, and from 13 inland waters of the Great Lakes watershed were collected from 1999 to 2008. Out of 380 fish sampled from lakes Michigan and Huron, 42 (11.05%) tested positive for RS as determined by the nested polymerase chain reaction. Prevalence was lower in Lake Huron (5.71%) compared to Michigan (20.74%), but the difference was not statistically significant. Prevalence of RS was not found to be significantly different between species or sites; however, when species were grouped into demersal vs. pelagic categories, significant differences (P < 0.01) in prevalence were observed. Out of 607 fish sampled from inland waters, 111 (18.28%) tested positive for RS as determined by the sandwich enzyme-linked immunosorbent assay. Infection prevalence was highly variable across species and among localities. Our results indicate that many non-salmonid species can harbor this bacterium without progression to disease and may become a reservoir for infection.

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.

Renibacterium salmoninarum-The Causative Agent of Bacterial Kidney Disease in Salmonid Fish.

Renibacterium salmoninarum is one of the oldest known bacterial pathogens of fish. This Gram-positive bacterium is the causative agent of bacterial kidney disease, a chronic infection that is mostly known to infect salmonid fish at low temperatures. Externally, infected fish can display exophthalmia as well as blebs on the skin and ulcerations alongside haemorrhages at the base of the fins and alongside the lateral line. Internally, the kidney, heart, spleen and liver can show signs of swelling. Granulomas can be seen on various internal organs, as can haemorrhages, and the organs can be covered with a false membrane. Ascites can also accumulate in the abdominal cavity. The bacterium is generally cultivated on specialized media such as kidney disease medium-1 (KDM-1), KDM-2 and selective kidney disease medium (SKDM), and a diagnostic is performed using molecular tools such as PCRs or real-time quantitative PCRs (RT-qPCRs). Several virulence mechanisms have been identified in R. salmoninarum, in particular the protein p57 that is known to play a role in both agglutination and immunosuppression of the host’s defense mechanisms. Control of the disease is difficult; the presence of asymptomatic carriers complicates the eradication of the disease, as does the ability of the bacterium to gain entrance inside the eggs. Bacterin-killed vaccines have proven to be of doubtful efficacy in controlling the disease, and even more recent application of a virulent environmental relative of R. salmoninarum is of limited efficacy. Treatment by antibiotics such as erythromycin, azithromycin and enrofloxacin can be effective but it is slow and requires prolonged treatment. Moreover, antibiotic-resistant strains have been reported. Despite being known for a long time, there is still much to be discovered about R. salmoninarum, notably regarding its virulence mechanisms and its vaccine potential. Consequently, these gaps in knowledge continue to hinder control of this bacterial disease in aquaculture settings.

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

BackgroundRenibacterium 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.ResultsA 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.ConclusionsThis 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).

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-1β, 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.

Genomic epidemiology of the commercially important pathogen Renibacterium salmoninarum within the Chilean salmon industry.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), which is a commercially important disease of farmed salmonids. Typing by conventional methods provides limited information on the evolution and spread of this pathogen, as there is a low level of standing variation within the R. salmoninarum population. Here, we apply whole-genome sequencing to 42 R. salmoninarum isolates from Chile, primarily from salmon farms, in order to understand the epidemiology of BKD in this country. The patterns of genomic variation are consistent with multiple introductions to Chile, followed by rapid dissemination over a 30 year period. The estimated dates of introduction broadly coincide with major events in the development of the Chilean aquaculture industry. We find evidence for significant barriers to transmission of BKD in the Chilean salmon production chain that may also be explained by previously undescribed signals of host tropism in R. salmoninarum. Understanding the genomic epidemiology of BKD can inform disease intervention and improve sustainability of the economically important salmon industry. This article contains data hosted by Microreact.

Genome wide association analysis for bacterial kidney disease resistance in a commercial North American Atlantic salmon (Salmo salar) population using a 50 K SNP panel

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), an important disease of commercial Atlantic salmon causing substantial economic loss. Genetic improvement programs have been developed for many aquaculture species for a variety of economically important traits, and selective breeding has been proposed for low to moderate heritability disease traits to increase disease resistance. The aim of this study was to perform a genome wide association (GWA) analysis to identify the genomic architecture of BKD resistance and detect SNP markers associated with BKD resistance in a commercial population of Atlantic salmon held in seawater. We experimentally challenged 652 post smolts from 63 families with R. salmoninarum through intraperitoneal injection. A binary trait, overall challenge survival, and a quantitative trait, day of death, were recorded for each challenged individual. DNA extraction and 50 K SNP genotyping was performed for 576 individuals. Fish and loci that did not pass quality control and fish with >10% European ancestry were removed leaving 507 post smolts genotyped for 44,527 SNPs. GWA for survival and time to death was conducted using a mixed model approach using the relationship matrix to account for population and family structure and a Cox proportional hazards model, respectively. A single marker on Ssa08 was significantly associated with survival at the chromosome wide level. One marker located on Ssa04 was significantly associated with time to death at the chromosome wide level. The two significant SNPs explained a small percentage of the phenotypic variance and were located on different chromosomes, 4.0% (Ssa08) and 5.3% (Ssa04), indicating a polygenic trait.

Identifying copy number variation of the dominant virulence factors msa and p22 within genomes of the fish pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, an important disease of farmed and wild salmonid fish worldwide. Despite the wide spatiotemporal distribution of this disease and habitat pressures ranging from the natural environment to aquaculture and rivers to marine environments, little variation has been observed in the R. salmoninarum genome. Here we use the coverage depth from genomic sequencing corroborated by real-time quantitative PCR to detect copy number variation (CNV) among the genes of R. salmoninarum. CNV was primarily limited to the known dominant virulence factors msa and p22. Among 68 isolates representing the UK, Norway and North America, the msa gene ranged from two to five identical copies and the p22 gene ranged from one to five copies. CNV for these two genes co-occurred, suggesting they may be functionally linked. Isolates carrying CNV were phylogenetically restricted and originated predominantly from sites in North America, rather than the UK or Norway. Although both phylogenetic relationship and geographical origin were found to correlate with CNV status, geographical origin was a much stronger predictor than phylogeny, suggesting a role for local selection pressures in the repeated emergence and maintenance of this trait.

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330μm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200μm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100μm), it did facilitate growth at lower iron concentrations (10μm and 1μm). An exception was the H-2 isolate, which only grew with 100μm of iron sulphide. In turn, 100μm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R.salmoninarum infections. We challenged Chinook salmon with R.salmoninarum at 12°C for 2weeks and then divided the fish into three temperature groups (8, 12 and 15°C). Fish in the 8°C group had significantly higher R.salmoninarum-specific mortality, kidney R.salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15°C. There was a trend towards suppressed bacterial load and shedding in the 15°C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12°C groups but not for the 15°C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R.salmoninarum.

Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444–2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ∼40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.