A competitive enzyme-linked immunosorbent assay specific for murine hepcidin-1: correlation with hepatic mRNA expression in established and novel models of dysregulated iron homeostasis.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including ß-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

Combination of biomarkers for diagnosis of acute kidney injury after cardiopulmonary bypass.

Novel acute kidney injury (AKI) biomarkers offer promise of earlier diagnosis and risk stratification, but have yet to find widespread clinical application. We measured urinary α and π glutathione S-transferases (α-GST and π-GST), urinary l-type fatty acid-binding protein (l-FABP), urinary neutrophil gelatinase-associated lipocalin (NGAL), urinary hepcidin and serum cystatin c (CysC) before surgery, post-operatively and at 24 h after surgery in 93 high risk patient undergoing cardiopulmonary bypass (CPB) and assessed the ability of these biomarkers alone and in combination to predict RIFLE-R defined AKI in the first 5 post-operative days. Twenty-five patients developed AKI. π-GST (ROCAUC = 0.75), lower urine Hepcidin:Creatine ratio at 24 h (0.77), greater urine NGAL:Cr ratio post-op (0.73) and greater serum CysC at 24 h (0.72) best predicted AKI. Linear combinations with significant improvement in AUC were: Hepcidin:Cr 24 h + post-operative π-GST (AUC = 0.86, p = 0.01), Hepcidin:Cr 24 h + NGAL:Cr post-op (0.84, p = 0.03) and CysC 24 h + post-operative π-GST (0.83, p = 0.03), notably these significant biomarkers combinations all involved a tubular injury and a glomerular filtration biomarker. Despite statistical significance in receiver-operator characteristic (ROC) analysis, when assessed by ability to define patients to two groups at high and low risk of AKI, combinations failed to significantly improve classification of risk compared to the best single biomarkers. In an alternative approach using Classification and Regression Tree (CART) analysis a model involving NGAL:Cr measurement post-op followed by Hepcidin:Cr at 24 h was developed which identified high, intermediate and low risk groups for AKI. Regression tree analysis has the potential produce models with greater clinical utility than single combined scores.

Hepcidin and ferritin levels as markers of immune cell activation during septic shock, severe COVID-19 and sterile inflammation.

Introduction: Major clinically relevant inflammatory events such as septic shock and severe COVID-19 trigger dynamic changes in the host immune system, presenting promising candidates for new biomarkers to improve precision diagnostics and patient stratification. Hepcidin, a master regulator of iron metabolism, has been intensively studied in many pathologies associated with immune system activation, however these data have never been compared to other clinical settings. Thus, we aimed to reveal the dynamics of iron regulation in various clinical settings and to determine the suitability of hepcidin and/or ferritin levels as biomarkers of inflammatory disease severity. Cohorts: To investigate the overall predictive ability of hepcidin and ferritin, we enrolled the patients suffering with three different diagnoses - in detail 40 patients with COVID-19, 29 patients in septic shock and eight orthopedic patients who were compared to nine healthy donors and all cohorts to each other. Results: We showed that increased hepcidin levels reflect overall immune cell activation driven by intrinsic stimuli, without requiring direct involvement of infection vectors. Contrary to hepcidin, ferritin levels were more strongly boosted by pathogen-induced inflammation - in septic shock more than four-fold and in COVID-19 six-fold in comparison to sterile inflammation. We also defined the predictive capacity of hepcidin-to-ferritin ratio with AUC=0.79 and P = 0.03. Discussion: Our findings confirm that hepcidin is a potent marker of septic shock and other acute inflammation-associated pathologies and demonstrate the utility of the hepcidin-to-ferritin ratio as a predictor of mortality in septic shock, but not in COVID-19.

Greater increase in urinary hepcidin predicts protection from acute kidney injury after cardiopulmonary bypass.

BACKGROUND: Acute kidney injury (AKI) is a common and serious complication of cardiopulmonary bypass (CPB) surgery. Hepcidin, a peptide hormone that regulates iron homeostasis, is a potential biomarker of AKI following CPB. METHODS: We investigated the association between post-operative changes in serum and urinary hepcidin and AKI in 93 patients undergoing CPB. RESULTS: Twenty-five patients developed AKI based on the Risk, Injury, Failure, Loss, End-stage kidney disease (RIFLE) criteria in the first 5 days. Serum hepcidin, urine hepcidin concentration, the urinary hepcidin:creatinine ratio and fractional excretion of hepcidin in urine rose significantly after surgery. However, urine hepcidin concentration and urinary hepcidin:creatinine ratio were significantly lower at 24 h in patients with RIFLE-Risk, Injury or Failure compared to those without AKI (P = 0.0009 and P < 0.0001, respectively). Receiver operator characteristic analysis showed that lower 24-h urine hepcidin concentration and urinary hepcidin:creatinine ratio were sensitive and specific predictors of AKI. The urinary hepcidin:creatinine ratio had an area under the curve for the diagnosis of RIFLE ≥ risk at 24 h of 0.77 and of 0.84 for RIFLE ≥ injury. Urinary hepcidin had similar predictive accuracy. Such predictive ability remained when patients with early creatinine increases were excluded. CONCLUSIONS: Urinary hepcidin and hepcidin:creatinine ratio are biomarkers of AKI after CPB, with an inverse association between its increase at 24 h and risk of AKI in the first five post-operative days. Measuring hepcidin in the urine on the first day following surgery may deliver earlier diagnosis and interventions.

Urine hepcidin has additive value in ruling out cardiopulmonary bypass-associated acute kidney injury: an observational cohort study.

INTRODUCTION: Conventional markers of acute kidney injury (AKI) lack diagnostic accuracy and are expressed only late after cardiac surgery with cardiopulmonary bypass (CPB). Recently, interest has focused on hepcidin, a regulator of iron homeostasis, as a unique renal biomarker. METHODS: We studied 100 adult patients in the control arm of a randomized, controlled trial http://www.clinicaltrials.gov/NCT00672334 who were identified as being at increased risk of AKI after cardiac surgery with CPB. AKI was defined according to the Risk, Injury, Failure, Loss, End-stage renal disease classification of AKI classification stage. Samples of plasma and urine were obtained simultaneously (1) before CPB (2) six hours after the start of CPB and (3) twenty-four hours after CPB. Plasma and urine hepcidin 25-isoforms were quantified by competitive enzyme-linked immunoassay. RESULTS: In AKI-free patients (N = 91), urine hepcidin concentrations had largely increased at six and twenty-four hours after CPB, and they were three to seven times higher compared to patients with subsequent AKI (N = 9) in whom postoperative urine hepcidin remained at preoperative levels (P = 0.004, P = 0.002). Furthermore, higher urine hepcidin and, even more so, urine hepcidin adjusted to urine creatinine at six hours after CPB discriminated patients who did not develop AKI (area under the curve (AUC) receiver operating characteristic curve 0.80 [95% confidence interval (95% CI) 0.71 to 0.87] and 0.88 [95% CI 0.78 to 0.97]) or did not need renal replacement therapy initiation (AUC 0.81 [95% CI 0.72 to 0.88] 0.88 [95% CI 0.70 to 0.99]) from those who did. At six hours, urine hepcidin adjusted to urine creatinine was an independent predictor of ruling out AKI (P = 0.011). Plasma hepcidin did not predict no development of AKI. The study findings remained essentially unchanged after excluding patients with preoperative chronic kidney disease. CONCLUSIONS: Our findings suggest that urine hepcidin is an early predictive biomarker of ruling out AKI after CPB, thereby contributing to early patient risk stratification.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as ß-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in ß-thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

Maternal serum hepcidin is low at term and independent of cord blood iron status.

OBJECTIVES: Hepcidin is the key regulator of iron homeostasis. The aims of this study were to determine serum hepcidin concentrations and reference ranges in pregnant women and cord blood of newborns at term and to evaluate the associations between hepcidin concentrations and iron status parameters. METHODS: A total of 191 pregnant women-newborn pairs were studied in Kuopio University Hospital, Finland. The measured parameters were serum hepcidin, ferritin, transferrin receptor, transferrin saturation, red cell indices, and erythropoietin. RESULTS: The hepcidin concentration in pregnant women was significantly lower than in cord blood at term [geometric mean concentration (GMC) (95% confidence intervals) in pregnant women 10.7 ng/mL (8.5-13.4 ng/mL) vs. GMC of cord blood hepcidin 69.3 ng/mL (55.3-86.8 ng/mL), P<0.001, adjusted analysis of variance]. Hepcidin was undetectable in 12% of mothers. Hepcidin concentration in pregnant women was the lowest in those who had the lowest iron status. However, maternal hepcidin concentration was not associated with cord blood hepcidin or iron status markers. Hepcidin concentration in cord blood was associated with cord blood iron status, but not with maternal iron status. CONCLUSIONS: At term pregnancy, hepcidin concentrations are very low, allowing maximal availability of iron for the fetus. Maternal and cord blood hepcidin levels were independently associated with either maternal or cord blood iron status.

Evaluation of Streptococcus iniae killed bacterin and live attenuated vaccines in hybrid striped bass through injection and bath immersion.

Streptococcus iniae poses a serious threat to finfish aquaculture operations worldwide. Stringent regulatory standards limit the use of antibiotics to treat S. iniae infections; improved vaccination strategies are thus of great interest. We investigated the potential for efficient, non-injectable batch vaccination via the use of live attenuated vaccines. Three attenuated S. iniae strains with genetic mutations eliminating the production of virulence factors--capsular polysaccharide (delta cpsD), M-like protein (delta simA), and phosphoglucomutase (delta pgmA)--were evaluated in parallel with an adjuvanted, formalin-killed, whole-cell S. iniae bacterin. Juvenile hybrid striped bass (HSB; Morone chrysops x M. saxatilis) were vaccinated through intraperitoneal (i.p.) injection or bath immersion and held for 800 degree-days prior to challenge with a lethal dose of the virulent wild-type (WT) S. iniae parent strain. The delta cpsD, delta pgmA, and bacterin vaccines provided the highest level of vaccination safety (0% mortality), whereas the delta simA mutant, although it caused 12 to 16% vaccination-related mortality, was the only vaccine candidate to provide 100% protection in both i.p. and immersion delivery models. Our studies demonstrate the efficacy of live attenuated vaccines for prevention of S. iniae infection, and identify immersion delivery of live vaccines as an attractive option for use in commercial aquaculture settings.

Clinical Immunoassay for Human Hepcidin Predicts Iron Deficiency in First-Time Blood Donors.

BACKGROUND: Serum markers currently used as indicators of iron status have clinical limitations. Hepcidin, a key regulator of iron homeostasis, is reduced in iron deficiency (ID) and increased in iron overload. We describe the first CLIA-validated immunoassay with excellent accuracy and precision to quantify human serum hepcidin. Its diagnostic utility for detecting ID in first-time blood donors was demonstrated. METHODS: A monoclonal competitive ELISA (C-ELISA) was developed for the quantitation of human hepcidin and validated according to CLIA guidelines. Sera from nonanemic first-time blood donors (n = 292) were analyzed for hepcidin, ferritin, transferrin, and serum iron. Logistic regression served to determine the utility of hepcidin as a predictor of ID. RESULTS: The C-ELISA was specific for human hepcidin and had a low limit of quantitation (4.0 ng/mL). The hepcidin concentration measured with the monoclonal C-ELISA was strongly correlated with a previously established, extensively tested polyclonal C-ELISA (Blood 2008;112:4292-7) (r = 0.95, P < 0.001). The area under the receiver operating characteristic curve for hepcidin as a predictor of ID, defined by 3 ferritin concentration thresholds, was >0.9. For predicting ID defined by ferritin <15 ng/mL, hepcidin <10 ng/mL yielded sensitivity of 93.1% and specificity of 85.5%, whereas the same hepcidin cutoff for ferritin <30 ng/mL yielded sensitivity of 67.6% and specificity of 91.7%. CONCLUSION: The clinical measurement of serum hepcidin concentrations was shown to be a potentially useful tool for diagnosing ID.

Host immune response and acute disease in a zebrafish model of Francisella pathogenesis.

Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebrafish were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebrafish following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebrafish mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-1beta (IL-1beta), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebrafish to heat-killed bacteria demonstrated that the significant induction of IL-1beta was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebrafish share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach.

Low preoperative hepcidin concentration as a risk factor for mortality after cardiac surgery: a pilot study.

OBJECTIVE: Hepcidin regulates iron absorption and recycling and is central to host defense, protection from reactive iron species, and a biomarker of iron-related pathophysiology. We assessed the value of hepcidin measured preoperatively for the prediction of in-hospital mortality and renal outcomes. METHODS: We studied 100 adult patients undergoing cardiac surgery in the control arm of a randomized, controlled trial. Plasma and urine were sampled before induction of anesthesia, and hepcidin-25 was quantified by competitive enzyme-linked immunoassay. Renal outcomes were acute kidney injury defined by risk, injury, failure, loss of function, end-stage renal disease (RIFLE) classification and need for renal replacement therapy. Variables with the potential to influence hepcidin expression were investigated. RESULTS: Low preoperative hepcidin concentration in urine (median, 15.3 ng/mL; 25-75 percentiles, 0-129.1) and plasma (median, 49.2 ng/mL; 25th-75th percentile, 0-52.2) predicted mortality (area under the curve-receiver operating characteristic [AUC-ROC] for urine hepcidin, 0.89; 95% confidence interval, 0.73-0.99; cutoff, 130 ng/mL; sensitivity, 73%; specificity, 100%; and AUC-ROC for plasma hepcidin, 0.90; 95% confidence interval, 0.80-0.99; cutoff, 55 ng/mL; sensitivity, 83%; specificity, 100%). Survivors had median preoperative hepcidin concentrations of 325.3 ng/mL (25th-75th percentile, 120-770.1 ng/mL) in urine and 113.1 ng/mL (25th-75th percentile, 77.7-203.1 ng/mL) in plasma. Preoperative serum creatinine did not predict mortality (AUC-ROC, 0.50; 95% confidence interval, 0.10-0.94). Furthermore, preoperative urine, plasma hepcidin, and serum creatinine did not distinguish patients requiring postoperative renal replacement therapy from those without (urine: AUC-ROC, 0.62; 95% confidence interval, 0.38-0.86; plasma: AUC-ROC, 0.63; 95% confidence interval, 0.34-0.91; serum creatinine: AUC-ROC, 0.61; 95% confidence interval, 0.22-0.99). Preoperative renal function and hemoglobin did not correlate with hepcidin indices whereas plasma markers of inflammation did. CONCLUSIONS: Low preoperative hepcidin concentration might be a risk factor for in-hospital mortality. Findings should be validated in larger patient cohorts with a greater number of events.

Effectiveness of aquaflor (50% florfenicol) to control mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass.

We conducted a field trial to evaluate the effectiveness of Aquaflor (50% florfenicol) for controlling mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass (female white bass Morone chrysops X male striped bass M. saxatilis). Bacterial samples collected from moribund fish representing a reference population were presumptively identified microbiologically and were later confirmed to be S. iniae by biochemical characterization and polymerase chain reaction. The trial comprised a 1-d acclimation period, 10-d treatment period, and 14-d posttreatment period. During the treatment period, Aquaflor-medicated feed was administered to treated tanks (N = 3) at a target dose of 10 mg of florfenicol x kg of fish(-1) x d(-1), and nonmedicated feed was administered to control tanks (N = 3). At the end of the posttreatment period, mean (+/- SD) cumulative mortality in treated tanks (9 +/- 11%) was significantly (P = 0.040) less than that in control tanks (52 +/- 13%). Analysis of medicated feed samples revealed that treated tanks had received an actual dose of 8.3 mg florfenicol x kg fish(-1) x d(-1) (83% of target). No florfenicol was detected in control feed samples. Although the actual florfenicol dose administered to treated tanks was less than the target dose, the trial was accepted by the U.S. Food and Drug Administration Center for Veterinary Medicine as demonstrating the efficacy of Aquaflor to control mortality associated with S. iniae in cultured sunshine bass populations.

Iron-deficiency anemia in Castleman disease: implication of the interleukin 6/hepcidin pathway.

In addition to occasional autoimmune hemolytic anemia, unexplained iron-deficiency anemia has been reported in childhood Castleman disease (CD). The recent discovery of hepcidin has regenerated the research on iron metabolism. This hormone is a key regulator of iron homeostasis, mainly by inhibiting intestinal iron absorption. Liver expression of hepcidin increases in response to interleukin 6 (IL-6). With chronic overproduction of IL-6 as a hallmark, CD could be an interesting human model for studying the contribution of the IL-6/hepcidin pathway in the pathogenesis of anemia of chronic disease. We report here the case of a 16-year-old boy with chronic iron-deficiency anemia (plasma ferritin: 19 µg/L; plasma iron: 2.2 µmol/L; negative bone marrow Perls' Prussian blue stain), inflammatory syndrome (C-reactive protein: 108 mg/L), and growth retardation for the previous 2 years. Diagnostic workup revealed a large mesenteric mass corresponding to localized CD of mixed histologic type. Resection of the tumor resulted in complete resolution of iron-deficiency anemia and inflammatory syndrome. Parallel variations of plasma IL-6, C-reactive protein, and hepcidin concentrations, together with tumor immunohistochemistry, strongly suggested that IL-6 synthesized by the tumor caused both the inflammation and iron deficiency through enhancement of hepcidin production by the liver. The results of this unique case study (1) explain the mechanism of iron deficiency observed in some children with CD, (2) confirm in vivo the regulatory effect of IL-6 in human hepcidin production, and (3) suggest that iron deficiency is a causal link between IL-6 and anemia of chronic disease.

Testosterone suppresses hepcidin in men: a potential mechanism for testosterone-induced erythrocytosis.

CONTEXT: The mechanisms by which testosterone increases hemoglobin and hematocrit are unknown. OBJECTIVE: The aim was to test the hypothesis that testosterone-induced increase in hematocrit is associated with suppression of the iron regulatory peptide hepcidin. PARTICIPANTS: Healthy younger men (ages 19-35 yr; n = 53) and older men (ages 59-75 yr; n = 56) were studied. METHODS: Weekly doses of testosterone enanthate (25, 50, 125, 300, and 600 mg) were administered over 20 wk, whereas endogenous testosterone was suppressed by monthly GnRH agonist administration. Blood and serum parameters from each individual were measured at wk 0, 1, 2, 4, 8, and 20. Longitudinal analyses were performed to examine the relationship between hepcidin, hemoglobin, hematocrit, and testosterone while controlling for potential confounders. RESULTS: High levels of testosterone markedly suppressed serum hepcidin within 1 wk. Hepcidin suppression in response to testosterone administration was dose-dependent in older men and more pronounced than in young men, and this corresponded to a greater rise in hemoglobin in older men. Serum hepcidin levels at 4 and 8 wk were predictive of change in hematocrit from baseline to peak levels. CONCLUSION: Testosterone administration is associated with suppression of serum hepcidin. Greater increases in hematocrit in older men during testosterone therapy are related to greater suppression of hepcidin.

Phenotypic and genotypic analysis of Flavobacterium psychrophilum isolates from Ontario salmonids with bacterial coldwater disease.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome. BCWD has a considerable economic impact on aquaculture operations in Ontario, Canada, and our limited understanding of the population structure and epidemiology of F. psychrophilum isolates is an impediment to the development of improved management strategies. Seventy-five 16S rRNA gene and gyr polymerase chain reaction positive isolates of F. psychrophilum that had been collected over a 16-year period from farmed salmonids with tail rot, necrotic myositis, and osteochondrosis were characterized morphologically, biochemically, and genotypically. Although the isolates were homogeneous by preliminary biochemical and phenotypic characterization, two distinct biovars were found by API ZYM testing. As well, four restriction pattern types were detected by 16S rRNA polymerase chain reaction -- restriction fragment length polymorphism analysis and there was a significant (P < 0.001) correlation between biovar I and digestion with MaeIII and between biovar II and digestion with MnlI or no site (P < 0.05). Further heterogenity was detected by sequence analysis of a 194 bp stem loop 3 region of rRNA. Nine sequence types were identified; 40/46 biovar I isolates were sequence type "a", while 21/32 biovar II isolates belonged to either sequence type "c" or "d". More than one biovar and genotype was identified among the strains recovered from separate fish sampled from three groups of rainbow trout (Oncorhynchus mykiss) experiencing BCWD mortality events. No association was found between genotype or biovar and type of disease. Taken together, these data suggest that F. psychrophilum from Ontario can be grouped into two major lineages based on biovar and 16S rRNA polymorphisms, and although three major strain types were most frequently isolated in this study, it appears that the population of F. psychrophilum with pathogenic potential is quite heterogeneous.

Bacterial-binding activity and plasma concentration of ladderlectin in rainbow trout (Oncorhynchus mykiss).

Soluble, defense lectins bind conserved microbial patterns leading to pathogen opsonization, enhanced phagocytosis and activation of complement. These immune functions, however, vary widely among individuals due to genetic and acquired differences affecting binding capacity or plasma concentration. Most evidence for the defensive function of soluble lectins is based on mammals, but several functionally homologous, but less well-characterized, lectins have been identified in fish. In this study, we compared binding of rainbow trout plasma ladderlectin to relevant, intact bacterial targets. A polyclonal antiserum raised against a synthetic peptide identical to the 20 N-terminal amino acids of the reduced 16 kDa rainbow trout ladderlectin subunit was used to detect plasma ladderlectin in immunoblots and indirect enzyme-linked immunosorbent assay (ELISA). Ladderlectin binding to Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, Yersinia ruckeri and Pseudomonas sp. was detected by PAGE and immunoblots of saccharide elutions from intact bacteria incubated in the presence of normal trout plasma. Although plasma concentrations of immunoreactive ladderlectin were low in the majority of trout, significant (P < 0.0001) variation between individual fish was observed in two separate populations. In addition, one population demonstrated a subset of individuals whose ladderlectin levels were approximately seven-fold higher than the population median. These findings indicate that rainbow trout have variable amounts of plasma ladderlectin capable of binding to the surfaces of several relevant bacterial targets.

Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development.

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the DeltaPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the DeltaPGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.

Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections.

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.

Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge.

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) x striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges. Peptide sequence homology to human hepcidin and other predicted hepcidins, indicated that the peptide is a new member of the hepcidin family. Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A predicted prepropeptide (85 amino acids) consists of three domains: a signal peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21 amino acids). The gene has two introns and three exons. A TATA box and several consensus-binding motifs for transcription factors including C/EBP, nuclear factor-kappaB, and hepatocyte nuclear factor were found in the region upstream of the transcriptional start site. In white bass liver, hepcidin gene expression was induced 4500-fold following challenge with the fish pathogen, Streptococcus iniae, while expression levels remained low in all other tissues tested. A novel antimicrobial peptide from the gill, bass hepcidin, is predominantly expressed in the liver and highly inducible by bacterial exposure.