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.

Half of 'Micrococcus spp.' cases identified by conventional methods are revealed as other life-threatening bacteria with different drug susceptibility patterns by 16S ribosomal RNA gene sequencing.

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required.

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.

Induction of cyclooxygenase-2 signaling by Stomatococcus mucilaginosus highlights the pathogenic potential of an oral commensal.

Stomatococcus mucilaginosus is an oral commensal that has been occasionally reported to cause severe infections in immunocompromised patients. There is no information about the pathogenic role of S. mucilaginosus in airway infections. In a cohort of 182 subjects with bronchiectasis, we found that 9% were colonized with S. mucilaginosus in their lower airways by culture growth from bronchoalveolar lavage. To address the pathogenic potential of S.mucilaginosus, we developed a murine model of S. mucilaginosus lung infection. Intratracheal injection of S. mucilaginosus in C57BL/6 mice resulted in a neutrophilic influx with production of proinflammatory cytokines, chemokines, and lipid mediators, mainly PGE2 with induction of cyclooxygenase-2 (COX-2) in the lungs. Presence of TLR2 was necessary for induction of COX-2 and production of PGE2 by S. mucilaginosus. TLR2-deficient mice showed an enhanced clearance of S. mucilaginosus compared with wild-type mice. Administration of PGE2 to TLR2(-/-) mice resulted in impaired clearance of S. mucilaginosus, suggesting a key role for COX-2-induced PGE2 production in immune response to S. mucilaginosus. Mechanistically, induction of COX-2 in macrophages was dependent on the p38-ERK/MAPK signaling pathway. Furthermore, mice treated with S. mucilaginosus and Pseudomonas aeruginosa showed an increased mortality compared with mice treated with PA103 or S. mucilaginosus alone. Inhibition of COX-2 significantly improved survival in mice infected with PA103 and S. mucilaginosus. These data provide novel insights into the bacteriology and personalized microbiome in patients with bronchiectasis and suggest a pathogenic role for S. mucilaginosus in patients with bronchiectasis.

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.

Development of protection in rainbow trout (Oncorhynchus mykiss, Walbaum) to Vibrio anguillarum following use of the probiotic Kocuria SM1.

Probiotic Kocuria SM1, which was part of the allochthonous gut microbiota of rainbow trout, was administered to rainbow trout (Oncorhynchus mykiss) fingerlings as dietary supplement at approximately 10(8) cells g(-1) for two weeks. The fish were challenged with Vibrio anguillarum and innate immunity determined over the following 5 weeks. The probiotic-fed fish survived (mortalities = 10-28%; P < 0.05) better than the controls (mortalities = 73-92%) for 5 weeks after stopping administration of Kocuria SM1, although the relative percent survival (RPS) declined steadily from 87 to 36% by the end of the experiment. A significant (P < 0.05) effect on the immune parameters was observed in fish within 3 weeks after stopping administration of Kocuria SM1 in comparison with the controls, i.e. enhancement of the respiratory burst (OD(550nm)) from 0.051 +/- 0.014 to 0.067 +/- 0.009, lysozyme 605 +/- 185 units ml(-1) to 872 +/- 114 units ml(-1), total protein 19.8 +/- 2.0 mg ml(-1) to 23.8 +/- 0.8 mg ml(-1) at 2 weeks, and leukocrit 2.9 +/- 1.8% to 5.1 +/- 1.1%, peroxidase (OD(550nm)) 0.23 +/- 0.02 to 0.37 +/- 0.11 and bacterial killing activities (percentage of surviving bacteria = 82 +/- 12% to 57 +/- 4%) at 3 weeks. Generally, the levels reduced progressively towards the end of experiment.

Genes associated with an effective host response by Chinook salmon to Renibacterium salmoninarum.

An effective host response to Renibacterium salmoninarum, the etiologic agent of bacterial kidney disease, is poorly characterized. Using suppression subtractive hybridization, we exploited the difference in early host response in the pronephros of fish challenged by an attenuated strain (MT239) or a virulent strain (ATCC 33209) of R. salmoninarum. Among the 132 expressed sequence tag (EST) clones that were sequenced, 20 were selected for expression analysis at 24 and 72h after challenge. ESTs matching two interferon inducible genes (IFN-inducible GBP and VLIG1), the ligand GAS6, and the kinase VRK2 were upregulated in fish exposed to MT239, but downregulated or unchanged in fish exposed to 33209. A second group of ESTs matching genes involved in apoptosis (caspase 8) and immune function (IkappaBalpha, p47(phoX), EMR/CD97) were more slowly upregulated in fish exposed to 33209 compared to fish exposed to MT239. The ESTs displaying elevated expression in MT239-exposed fish may represent important cellular processes to bacterial challenge, and may be useful indicators of an effective host response to R. salmoninarum infection.

Renibacterium salmoninarum p57 antigenic variation is restricted in geographic distribution and correlated with genomic markers.

The 57 kDa protein (p57) is an important diagnostic antigen that is implicated in the pathogenesis of salmonid bacterial kidney disease. Little is known about the nature and extent of antigenic variation in p57. Previously, we reported that p57 produced by Renibacterium salmoninarum Strain 684 contains a mutation that disrupts monoclonal antibody (MAb) 4C11 binding. In the present study, we examined MAb binding to a panel of 23 additional R. salmoninarum isolates obtained from diverse geographic locations to examine the prevalence of this variant and whether additional variability exists within other p57 epitopes. Six p57-specific MAbs (4C11, 4D3, 3H1, 4H8, 4D10 and 1A1) were used to probe dot and western blots to determine the relative expression, size and cellular association of p57. Full-length p57 was produced by all isolates, and for each isolate, the protein was associated with the bacterial cell surface. The epitopes recognized by 4 MAbs, 4D3, 4H8, 3H1 and 1A1, were conserved among all strains tested. The 4C11 epitope was absent in 5 of 8 strains originating from Norway, while the 4D10 epitope was partially disrupted in one isolate from British Columbia, Canada. The 5 Norwegian antigenic-variant strains appeared to be clonally related as they shared the following characteristics: one tandem repeat in the ETRA locus, a Sequovar-4 16-23S rRNA intervening DNA sequence, a larger XhoI fragment in the msa1 5' region, and absent msa3 gene. These results indicate that limited antigenic and genomic variation exists between strains and this variation appears geographically restricted in distribution.

An immune-complex glomerulonephritis of Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Chinook salmon from New Zealand were shown to have a generalized membranous glomerulonephritis that was most severe in large fish. Marked thickening of the glomerular basement membrane was the most consistent lesion, with the presence of an electron-dense deposit beneath the capillary endothelium.Severely affected glomeruli also had expansion of the mesangium and loss of capillaries,synechiae of the visceral and parietal epithelium and mild fibrosis of Bowmans capsule. Chinook salmon from British Columbia, Canada with bacterial kidney disease caused by Renibacterium salmoninarum had similar histological lesions. They also had thickened glomerular basement membranes that were recognized by rabbit antiserum to rainbow trout immunoglobulin. This was true only when frozen sections of kidney were used and not formalin-fixed tissue. An attempt to experimentally produce a glomerulopathy in rainbow trout by repeated immunization with killed R. salmoninarum was not successful. Case records from the Fish Pathology Laboratory at the University of Guelph over a 10-year period revealed that a range of species were diagnosed with glomerulopathies similar to those seen in Chinook salmon. The majority of these cases were determined to have chronic inflammatory disease. This report has identified the presence of immunoglobulin within thickened basement membranes of Chinook salmon with glomerulonephritis and supports the existence of type III hypersensitivity in fish.

A novel C1q-domain-containing protein from razor clam Sinonovacula constricta mediates G-bacterial agglutination as a pattern recognition receptor.

Complement component 1q (C1q) with a characteristic C1q globular domain is an important pattern recognition molecule in the classical complement systems and plays a major role in the crosslinking between innate immunity and specific immunity in vertebrates. In this study, a homologous gene encoding typically C1q domains was obtained from the razor clam Sinonovacula constricta (designated ScC1qDC) by rapid amplification of the cDNA end. The full-length cDNA of ScC1qDC was 1225 bp in length with a 5'UTR of 258 bp, a 3'UTR of 223 bp, and an open reading frame of 744 bp encoding a polypeptide of 247 amino acids containing a typical C1q globular domain. The mRNA transcripts of ScC1qDC were constitutively transcribed in all examined tissues with higher expression in the hepatopancreas. Time-course expression analysis indicated that ScC1qDC was significantly up-regulated both in hepatopancreas and gills after Vibrio parahaemolyticus challenge. The recombinant ScC1qDC (rScC1qDC) displayed high binding activities to various pathogen-associated molecular patterns, including LPS, PGN, and MAN. Recombinant ScC1qDC showed no agglutinating activity to Gram-positive bacterium of Micrococcus luteus but showed obvious activities towards all the three examined Gram-negative bacteria. All our results indicated that ScC1qDC might be served as a pattern recognition receptor and promoted Gram-negative bacteria agglutination during the pathogen challenge.

Use of ELISA to monitor bacterial kidney disease in naturally spawning chinook salmon.

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum (Rs) is a serious problem among Pacific Northwest salmon hatcheries and has raised concerns that salmon reared in hatcheries may spread BKD to natural populations. In order to monitor the potential spread of this disease to salmon spawning in nature, a method must be available to collect and analyze tissues from naturally spawning salmon. Kidney tissue analyzed by enzyme-linked immunosorbent assay (ELISA) is the standard method to detect the presence of Rs in salmon sampled in hatcheries. In this study, we tested the validity of using ELISA on kidney tissue collected from intact carcasses recovered on the spawning grounds to monitor BKD in naturally spawning populations by comparing ELISA optical density (OD) values from kidney tissue that was subjected to conditions that simulated decomposition in a carcass and collection during a spawning ground survey with samples freshly collected from salmon at a hatchery. Mean ELISA OD levels were 1.060 for the samples prepared by the normal preparation and 1.115 for samples prepared by simulating spawning ground survey collection. There was no significant difference in mean ELISA OD between the 2 sample preparations and the relationship did not significantly differ from 1:1 (slope = 0.946). This demonstrates that BKD prevalence in natural populations can be monitored using ELISA conducted on samples from intact carcasses recovered on spawning ground surveys. This will be an important tool for monitoring the effect of hatchery supplementation on naturally spawning salmon populations.

A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha.

The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37 degrees C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57- and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.

Alpha-globulin-like antigens in bacteria.

Seventy-two strains of bacteria of the families Bacillaceae, Enterobacteriaceae and Micrococcaceae were investigated for antigens resembling Gm (1), Gm (2) and Inv (1) human immunoglobulin group factors. Antigens similar to these factors were encountered in some strains of the genus Bacillus. Staph. epidermidis strains had antigenic structures resembling Gm (1) and Inv (1) factors, and strains of Enterobacteriaceae structures resembling Gm (2) and Inv (1). Occurrence among various species of bacteria of antigens resembling human gamma-globulin group factors suggests immunization by bacterial infection as one of the causes of presence of anti-Gm and anti-Inv antibodies in human beings.

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines.

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.

Relationship between antigen concentration and bacterial load in Pacific salmon with bacterial kidney disease.

Using data collected to test spawning female Pacific salmon (Oncorhynchus kisutch and O. tshawytscha for the presence and severity of bacterial kidney disease (BKD), a mathematical model of the relationship between bacterial load and antigen concentration in tissues and ovarian fluid is developed. Renibacterium salmoninarum, the causative agent of BKD, secretes large amounts of a 57 kDa protein ('p57'), its major soluble antigen, which eventually breaks down or is otherwise removed from free circulation. Bacterial load and soluble antigen concentration in tissues are strong indicators of fish health, while in ovarian fluid they are predictors of the success of offspring. Model results indicate either an exponentially increasing antigen removal rate or an exponentially decreasing per-bacterium antigen secretion rate with increasing antigen concentration. Possible mechanisms underlying the observed relationship include a nonlinear increasing autolytic rate of the 'p57' antigen and a bacterium-antigen interaction threshold which prevents bacterial antigen secretion.

[Reactivity to bacterial peptidoglycans in a phagocytosis system. 1. The range of immunological specificity of Staphylococcus aureus peptidoglycan]. / Reaktivnost' k bakterial'nym peptidoglikanam v sisteme fagotsitoza. 1. Diapazon immunologicheskoi spetsifichnosti peptidoglikana zolotistogo stafilokokka.

The antigenic features of S. aureus peptidoglycan (PG) were studied in the reaction of stimulation of oxygen-dependent neutrophil metabolism, mediated by the IgG opsonins of normal human serum. The study was carried out at different taxonomic levels: the species (S. aureus), the genus (Staphylococcus), the family (Micrococcaceae), as well as in relation to remote taxons (organisms belonging to the families Streptococcaceae, Enterobacteriaceae, Neisseriaceae, to the genus Corynebacterium). All S. aureus strains were identical with respect to the specificity of their PG, essentially differing from other bacteria in this regard. After the removal of antibodies to different PG the effectiveness of PG opsonization decreased by 10.4-44.7%. Such decrease was most pronounced in experiments with the PG of streptococci (S. pyogenes, S. faecalis, S. salivarius) and Micrococcus luteus.

Rothia dentocariosa induces TNF-alpha production in a TLR2-dependent manner.

Previous work suggested that Rothia dentocariosa is associated with periodontal inflammatory disease. However, little is known about the pathogenicity of this bacterium. To characterize host response to this bacterium, we measured (via ELISA) the amount of TNF-α in the culture supernatant following the stimulation of THP-1 cells (a human acute monocytic leukemia cell line) with R. dentocariosa cells (ATCC14189 and ATCC14190). Exposure to bacterial cells induced the production of TNF-α in a dose-dependent manner. The bacterial induction of TNF-α in THP-1 cells was mediated by the Toll-like receptor 2 (TLR2), as demonstrated by gene-specific knockdown via siRNA, which successfully suppressed TLR2 expression and significantly inhibited the production of TNF-α in the culture supernatant. To confirm the role of TLR2, we examined TLR2-dependent NF-κB activation by R. dentocariosa cells in a distinct cell line. Specifically, HEK293 cells were transiently cotransfected with the human TLR2 gene and an NF-κB-dependent luciferase-encoding reporter gene. The bacterial cells induced NF-κB activation in the transfected HEK293 cells in a dose-dependent manner. In contrast, bacterial cells failed to induce NF-κB activation in cells transfected with pEF6 control vector. Taken together, these results suggest that R. dentocariosa induces host TNF-α production by a TLR2-dependent mechanism.

Zygosity at the major histocompatibility class IIB locus predicts susceptibility to Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

Major histocompatibility (MH) class II genes play an important role in the vertebrate immune response. Here, we investigate the relationship between Atlantic salmon (Salmo salar) MH class IIB zygosity and susceptibility to Renibacterium salmoninarum, the causal agent of bacterial kidney disease. By combining DNA sequences from the salmon MH class IIB gene with quantitative ELISA data on R. salmoninarum antigen levels, we found that MH class IIB homozygotes were significantly more susceptible to R. salmoninarum than heterozygotes. These findings are discussed in the context of current evolutionary theory.

Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides.

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.