This is the first report of comprehensive functional analysis of an interleukin-10 in bony fish. Quantitative expression analysis of goldfish IL-10 revealed the greatest mRNA levels in the spleen tissues, peripheral blood leukocytes and granulocytes. The stimulation of cells with recombinant goldfish (rg) TNFα2 significantly reduced IL-10 mRNA levels in granulocytes and monocytes of the goldfish. To functionally assess the goldfish IL-10, we generated a recombinant form of the molecule (rgIL-10). The rgIL-10 substantially reduced the expression of TNFα1, TNFα2, IL-1β1, IL-10, CXCL-8, and NADPH oxidase component, p47(phox) in monocytes activated with heat-killed Aeromonas salmonicida and reduced the expression of IFNγ in A. salmonicida-activated splenocytes. Pre-treatment of monocytes with rgIL-10 resulted in substantial reduction of the ROI response of the A. salmonicida or rgIFNγ-primed monocytes. The rgIL-10 bound to goldfish monocytes and induced phosphorylation and nuclear translocation of Stat3. The rgIL-10 also induced rapid and robust increase in the mRNA levels of the goldfish monocyte SOCS-3. Our results indicate that the function of IL-10 is highly conserved through evolution.

Transforming growth factor beta (TGF-beta) is a pleiotropic cytokine with important roles in the regulation of cell proliferation, differentiation, survival, migration, activation and de-activation. It is one of the first cytokines released during an immune response and plays a strong immunomodulatory role in the activation and subsequent de-activation of macrophages and other immune cells. TGF-beta is a highly conserved molecule, and members of the TGF superfamily can be found in organisms as evolutionarily distant as arthropods. In this manuscript, we described the identification of a goldfish TGF-beta molecule, which was highly expressed in the skin, kidney and spleen of the goldfish and its expression was up-regulated in macrophages treated with LPS or recombinant goldfish TNF-alpha. Goldfish TGF-beta shared a high amino acid identity with, and was phylogenetically related to, TGF-beta1 of other teleost fish, birds, amphibians and mammals. Recombinant goldfish TGF-beta (rTGF-beta) induced the proliferation of a goldfish fibroblast cell line (CCL71) in a dose-dependent manner. In addition, rTGF-beta down-regulated the nitric oxide response of TNF-alpha-activated macrophages. This is the first report of teleost TGF-beta function in an ectothermic vertebrate.

Erythropoietin is the principal regulator of erythropoiesis and promotes the survival, proliferation and differentiation of erythroid progenitor cells in mammals. In this study we report on the molecular and functional characterization of erythropoietin from the goldfish. Quantitative expression analysis of goldfish epo revealed the highest mRNA levels in heart, followed by brain, liver, spleen and kidney tissues. There was no marked change of epo expression in goldfish primary kidney macrophage cultures, as progenitor cell to macrophage development progressed, indicating that erythropoietin is not involved in monopoiesis. Recombinant goldfish erythropoietin induced proliferation of progenitor cells in a dose-dependent manner, and up-regulated the expression of erythroid transcription factors gata1 and lmo2 in progenitor cells. Furthermore, recombinant goldfish erythropoietin stimulated erythroid colony formation in a dose-dependent manner and promoted survival of erythroid progenitor cells as colony-forming cells. Our results demonstrate that the function of erythropoietin in the goldfish is similar to that of mammals and suggest a highly conserved mechanism of early erythrocyte development in lower and higher vertebrates.

We identified and characterized two isoforms of tumor necrosis factor-alpha (TNFalpha) from the goldfish, TNFalpha-1 and TNFalpha-2. At the protein level, goldfish TNFalpha-1 and TNFalpha-2 were most homologous to carp TNFalpha-1 and TNFalpha-2, respectively. Phylogenetically, the two goldfish isoforms grouped most closely with the carp TNFalpha isoforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of goldfish TNFalpha-1 and TNFalpha-2 in all tissues with TNFalpha-2 mRNA levels higher than TNFalpha-1 in all tissues examined. A modest up-regulation in expressions of goldfish TNFalpha-1 and TNFalpha-2 in kidney-derived monocytes and significant increase in expression of both isoforms in mature macrophages were observed in response to activation with macrophage-activating factors. TNFalpha-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNFalpha-2) was functionally characterized. The rTNFalpha-2 induced a dose-dependent chemotactic response and enhanced phagocytosis of primary goldfish macrophages. Furthermore, rTNFalpha-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary goldfish macrophages. Our results indicate that goldfish TNFalpha is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the goldfish.

Erythropoietin receptor (EPOR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and survival of erythrocyte progenitor cells. This study reports on the molecular and functional characterization of goldfish EPOR. The identified goldfish EPOR sequence possesses the conserved EPOR ligand binding domain, the fibronectin domain, the class I cytokine receptor superfamily motif (WSXWS) as well as several intracellular signaling motifs characteristic of other vertebrate EPORs. The expression of epor mRNA in goldfish tissues, cell populations and cells treated with recombinant goldfish EPO (rgEPO) were evaluated by quantitative PCR revealing that goldfish epor mRNA is transcribed in both erythropoietic tissues (blood, kidney and spleen) and non-hematopoietic tissues (brain, heart and gill), as well as in immature erythrocytes. Recombinant goldfish EPOR (rgEPOR), consisting of its extracellular domain, dose-dependently inhibited proliferation of progenitor cells induced by rgEPO. In vitro binding studies indicated that rgEPO exists as monomer, dimer and/or trimmer and that rgEPOR exists as monomer and/or homodimer, and when incubated together, formed a ligand-receptor complex. Our results demonstrate that goldfish EPO/EPOR signaling has been highly conserved throughout vertebrate evolution as a required mechanism for erythrocyte development.

Granulocyte colony stimulating factor receptor (GCSFR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and activation of neutrophils and their precursors. In this study we report on the cloning and molecular characterization of goldfish GCSFR. The identified goldfish GCSFR sequence possesses the conserved Ig-like domain, the cytokine receptor homology domain (CRH), three fibronectin domains as well as several intracellular signaling motifs characteristic of other vertebrate GCSFRs. Goldfish gcsfr mRNA was highly expressed in kidney and spleen, and in primary kidney neutrophils. The neutrophils have significantly higher mRNA levels of the transcription factors pu.1 and cebpα, and down-regulated levels of transcription factors important for macrophage development such as egr1 and cjun, compared to progenitor cells from the kidney. The gcsfr mRNA was present in the kidney progenitor cells, albeit at much lower levels compared to the neutrophils, and the expression of gcsfr in progenitor cells was not affected by duration of cultivation. Furthermore, gcsfr mRNA levels were up-regulated in neutrophils after treatment with heat-killed Aeromonas salmonicida A449 or with mitogens. Our results indicate that GCSFR may be a useful marker for fish neutrophils.

Kit ligand and its type III tyrosine kinase receptor Kit promotes the survival, proliferation and differentiation of progenitor cells involved in mammalian myelopoiesis. In this study we report on the molecular and functional characterization of kit receptor A (kita) and kit ligand A (kitla) from the goldfish. Both kita and kitla were ubiquitously expressed in goldfish tissues, with higher mRNA levels observed in the kidney and spleen, the major hematopoietic organs of fish. Furthermore, both kita and kitla expressions decreased in a time-dependent manner in goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, and the highest expressions of both the receptor and ligand were observed in sorted progenitor cell populations. Activation of mature macrophage cultures increased both kita and kitla expressions. Kit ligand A induced chemotactic response, proliferation and survival of PKM cells in a dose-dependent manner, but did not induce differentiation of early PKM cells. These results are consistent with the role of kita and kitla during myelopoiesis of higher vertebrates and suggest a conserved mechanism of macrophage development throughout vertebrates.

Primary kidney macrophage (PKM) cultures derived from goldfish hematopoietic tissues develop from early progenitors to mature macrophages in response to endogenous growth factor(s). When grown in vitro, PKM shift from a proliferative phase, where most of the proliferation and differentiation events take place, to a senescence phase, where there is cessation of proliferation and differentiation events and ultimately cell death through a process of apoptosis. The phenotypic changes of PKM from the proliferative to senescence phase are a reflection of specific changes in gene expression; therefore, comparison of gene expression patterns between the two phases should lead to the identification of macrophage genes directly involved in the positive and negative regulation of hematopoietic events, as well as genes that are modulated downstream from these regulatory points. Differential cross-screening of the proliferative phase PKM cDNA library using proliferative and senescence phase (32)P-labeled cDNA probes identified several differentially expressed genes. Specifically, initial screen of 9200 clones yielded 734 differential primary clones that were isolated and analyzed using a PCR-based secondary screen. The majority of these clone isolates encoded a single transcript as determined by PCR amplification of the primary clones. The secondary screen confirmed the differential expression of 306 clones (3.32% of the total number of screened clones). Two hundred and forty four clones were sequenced; 158 and 86 were preferentially expressed during proliferative and senescence phases, respectively. Several potential candidates of fish macrophage hematopoiesis were identified. These include, for example, zinc finger protein 147, nucleophosmin, 14-3-3 protein, adenine nucleotide translocator 2 (ANT2), granulin, survivin-1, and apoptosis inhibitor-5. In addition, several potential markers of macrophage differentiation and/or function were identified and their expression patterns characterized across three distinct stages of macrophage development in vitro. These include legumain, CD63, interferon-inducible protein, macrosialin (CD68), transcription factor MafB, and the molecular chaperone BiP/GRP78. These analyses will facilitate future characterization of macrophage developmental events by providing a more global perspective of various facets of macrophage hematopoiesis.

Hepcidin is an antimicrobial peptide and an iron regulatory protein that prevents the release of excess iron in the blood. There is evidence suggesting that teleost hepcidin is a major player in antimicrobial defense against various bacteria species, but little is known regarding the effects of teleost hepcidin in protozoan parasitic infections. We examined the role of hepcidin during the course of infection of goldfish with Trypanosoma carassii. Quantitative real-time PCR was used to determine the expression of hepcidin in goldfish immune organs during the course of T. carassii infection. During the acute phase of the T. carassii infection, the mRNA levels of hepcidin were up-regulated in liver and kidney. In contrast, an up-regulation of hepcidin mRNA expression in spleen was observed during the chronic phase of the infection. Furthermore, a synthetic goldfish hepcidin peptide induced trypanosome lysis in vitro, and parasite surface disruption was confirmed by scanning electron microscopy (SEM) analysis. These results suggest that, in addition to well-characterized direct antibacterial activities, teleost hepcidin also exhibits trypanocidal activity.

Tumor necrosis factor alpha (TNFalpha) is a pleotropic cytokine that mediates its effects by binding to one of two TNF receptors, TNF-R1 or TNF-R2. We have recently identified the cDNA sequences of both goldfish TNF-R1 and TNF-R2. In silico analyses revealed conserved cysteine rich domains, predicted docking sites for TNFR-specific downstream signaling factors for both receptors and a conserved death domain for TNF-R1. The expression of these receptors in tissues and various immune cell types was investigated by Q-PCR. The TNF-R2 expression was substantially higher than that of TNF-R1 in all tissues. Both receptors were most robustly expressed in monocytes where as the mRNA levels of TNF-R1 were the lowest in mature macrophages and those of TNF-R2 in peripheral blood leukocytes. Treatment of goldfish macrophages with recombinant goldfish (rg) TNFalpha-2, rgIFN gamma or rgTGF beta differentially altered the expression of both TNF receptors. The rgTNF alpha-2 up-regulated the expression of TNF-R2 but down-regulated the expression of TNF-R1. The rgIFN gamma increased the expression of both TNF receptors while rgTGF beta caused a time-dependent decreases in mRNA of goldfish TNF-R1 and TNF-R2. In vitro binding studies using recombinant TNFalpha-1 and TNFalpha-2 revealed that either isoform was capable of interacting with the recombinant forms of the extracellular domains of either TNF-R1 or TNF-R2. Functional significance of these ligand-receptor interactions was confirmed by experiments showing that goldfish TNF-R1 and TNF-R2 down-regulated the rgTNF alpha-1 or rgTNF alpha-2-primed respiratory burst response of goldfish macrophages.

We report on the identification and functional characterization of HMGB1 of the goldfish. Quantitative analysis indicated the highest expression of goldfish HMGB1 in the brain, with lower mRNA levels in spleen, intestine, kidney, gill and heart. HMGB1 was also differentially expressed in goldfish immune cell populations with highest mRNA levels present in splenocytes and neutrophils. We generated and functionally characterized the recombinant HMGB1 (rgHMGB1). The rgHMGB1 primed the respiratory burst response in monocytes and induced nitric oxide production of primary goldfish macrophages. Treatment of goldfish macrophages with heat-killed Mycobacterium marinum and Aeromonas salmonicida elevated the expression of HMGB1 and resulted in higher HMGB1 protein levels. The rgHMGB1 induced a dose-dependent production of TNFα-2 and IL-1β1 of goldfish macrophages. Furthermore, the dual luciferase reporter assay revealed that goldfish HMGB1 induced the activation of the NF-κB signaling pathway. Our results indicate that goldfish HMGB1 is a critical regulatory cytokine of inflammatory and antimicrobial response of the goldfish.

We report on the cloning and characterization of Trypanosoma carassii 70 KDa heat shock protein (hsp70). T. carassii hsp70 was secreted/excreted into culture medium in vitro and was recognized by sera from infected fish. Recombinant hsp70 (rhsp70) activated goldfish macrophages and stimulated the production of pro-inflammatory cytokines including interferon gamma (IFNgamma), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, (IL)-12 and chemokines CCL-1 and CXCL-8 (IL-8). T. carassii hsp70-induced cytokine expression was abrogated by pronase treatment of macrophages confirming the existence of receptor(s) on goldfish macrophage surface that recognize parasite molecule. Parasite hsp70 also up-regulated the expression inducible nitric oxide synthase (iNOS) isoforms A and B and induced a strong nitric oxide response of goldfish macrophages.

Interferon gamma (IFNgamma) is a highly pleotropic pro-inflammatory and anti-viral cytokine that mediates its effects by binding to a receptor complex composed of interferon gamma receptors 1 and 2 (IFNGR1 and IFNGR2). Using gene synteny analysis, we identified a distinct isoform of the zebrafish IFNGR1. The two zebrafish IFNGR1 called here IFNGR1-1 and IFNGR1-2 were used to identify the respective cDNA sequences of the goldfish IFNGR1-1 and IFNGR1-2. Analysis of protein sequences revealed that all fish IFNGR1 species have potential JAK1 and STAT1 docking sites. Phylogenetically, teleost IFNGR1 proteins grouped separately from those of higher vertebrates. Q-PCR analysis revealed that while the constitutive mRNA levels of the two zebrafish IFNGR1 isoforms were comparable in different tissues examined, the goldfish IFNGR1-1 tissue expression was substantially higher than that of IFNGR1-2. Q-PCR analysis of goldfish immune cell populations revealed highest expression of both receptor isoforms in monocytes. Incubation of goldfish macrophages with recombinant goldfish IFNgamma2 (rgIFNgamma2) up-regulated expression of both IFNGR1-1 and IFNGR1-2, while treatment of cells with rgTNFalpha2 only increased the expression of IFNGR1-1. Treatment with rgTGFbeta resulted in more modest increases in expression of both receptor isoforms only after prolonged treatment. In vitro binding studies indicated that rgIFNGR1-1 bound to rgIFNgamma1 but not rgIFNgamma2, while the rgIFNGR1-2 bound to rgIFNgamma2. Thus, unlike mammals that have a single IFNGR1, cyprinid fish have two distinct IFNGR1 isoforms that preferentially bind corresponding ligands, IFNgamma1 and IFNgamma2, respectively, suggesting that the type II interferon system of these fish species is distinct from that of higher vertebrates.

The expression of genes encoding the acute phase proteins (APP) during the course of Trypanasoma carassii infection in the goldfish was determined using quantitative PCR. Significant changes in the mRNA levels of ceruloplasmin (Cp), C-reactive protein (CRP), transferrin (Tf), hemopexin (Hx) and serum amyloid A (SAA) were observed in the kidney, liver and spleen at various days post infection (dpi). Of the five acute phase protein genes examined, CRP and SAA exhibited the highest expression in the tissues during the acute infection. Cp and Tf were up-regulated throughout the acute course of infection in the liver. During the chronic phase of the infection, APP expression in the liver was similar to that in the non-infected control fish. At 7 dpi, Cp, Tf and Hx were down-regulated in the spleen, and Cp and Tf kidney, but their mRNA levels gradually returned to those of control non-infected fish. In contrast, during the chronic phase of the infection, there was an up-regulation of Cp, Hx and Tf in the spleen, and Tf and SAA in the kidney. The goldfish CRP was cloned and functionally characterized. CRP was differentially expressed in normal goldfish immune cells, with highest expression in monocytes and lowest expression in mature macrophages. A recombinant goldfish CRP (rgfCRP) was generated using prokaryotic expression. rgfCRP enhanced complement-mediated killing of trypanosomes in vitro, and the lysis increased after addition of immune serum. rgfCRP did not affect the production of reactive oxygen and nitrogen intermediates by monocytes and macrophages, respectively.

This is the first report of the identification and molecular characterization of an interleukin-10 receptor 1 in bony fish. By gene synteny analysis, we identified the zebrafish interleukin-10 receptor 1 (IL10R1) and using this IL10R1 sequence, we cloned the goldfish IL10R1 cDNA transcript. The identified fish IL10R1 protein sequences had a putative JAK1 binding site, only one of the two STAT3 binding sites, that are present in all other vertebrates IL10R1 proteins as well as C-terminal serine rich areas, believed to be responsible for the anti-inflammatory properties of IL10R1. Phylogenetically, the fish IL10R1 proteins grouped independently of the amphibian, avian and mammalian IL10R1s. Quantitative gene expression analysis of the IL10R1 of zebrafish and goldfish revealed highest mRNA levels in the spleen tissues. High mRNA levels were also observed in the zebrafish muscle in contrast to low mRNA levels in the muscle of the goldfish. Moderate IL10R1 mRNA levels were seen in most other tissues examined and lowest gene expression was in the liver of both fish species. Goldfish monocytes stimulated with a recombinant goldfish interleukin-10 (rgIL-10) or with heat killed fish pathogens, Aeromonas salmonicida or Trypanosoma carassii, exhibited significantly reduced mRNA levels of the IL10R1. Furthermore, we produced a recombinant form of the goldfish IL10R1 (rgIL10R1) and using in vitro binding studies, demonstrated that the dimerized rgIL-10 specifically interacted with rgIL10R1. Our results suggest that interleukin-10 system has been highly conserved throughout evolution.

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

Macrophage colony-stimulating factor (CSF-1) is the principal regulator of the survival, proliferation, and differentiation of macrophages and their precursors. CSF-1 activity is tightly controlled through mechanisms regulating gene expression of CSF-1 and its membrane-bound receptor (CSF-1R), as well as by receptor-mediated endocytosis, metabolic processing, and inhibition of downstream signaling. Herein we describe a novel mechanism for control of CSF-1 activity. Spontaneously growing goldfish (Carassius auratus L.) macrophages actively produced a soluble form of CSF-1R (sCSF-1R) that appears to compete for ligand binding with membrane CSF-1R. The sCSF-1R transcript encodes only for the ligand-binding portion of the receptor, but is derived from a full-length mRNA species as determined by sequence analysis. Gene expression was associated with decreased proliferation and differentiation of primary macrophages, decreased growth factor activity in culture supernatants, and marked phenotypic changes that culminated in apoptotic cell death. Recombinant sCSF-1R inhibited macrophage proliferation at nanomolar concentrations. Antibodies against recombinant sCSF-1R identified native sCSF-1R in primary macrophage culture supernatants and fish serum, and suggested the presence of endogenous mechanisms temporally regulating sCSF-1R release. This is the first report of a soluble CSF-1R and points to intrinsic mechanisms of hematopoietic control that may be conserved across evolution in discrete self-renewing macrophage populations.

Ghrelin (GRLN) stimulates maturational gonadotropin (LH) secretion in goldfish. This study identified GRLN receptors (GHS-Rs) in goldfish tissues and examined the involvement of voltage-sensitive Ca(2+) channels (VSCCs) in ghrelin action. A partial goldfish GHS-R1a sequence was obtained and expression observed in brain, pituitary, spleen, kidney, heart, gill, ovary, testis, and intestine. Synthetic goldfish GRLN (gGRLN(19)) stimulated LH secretion from dispersed goldfish pituitary cells in column perifusion and increased intracellular Ca(2+) ([Ca(2+)](i)) in identified goldfish gonadotropes. gGRLN(19) did not stimulate LH secretion either in the presence of Ca(2+)-free media, or the L-type VSCC inhibitors nifedipine and verapamil. Similarly, gGRLN(19)-elicited increases in [Ca(2+)](i) were attenuated by Ca(2+)-free media and nifedipine. Furthermore, when LH release and [Ca(2+)](i) were elevated by Bay K8644, gGRLN(19) had no further effect. These results indicate that GHS-R1a is present in goldfish pituitary and Ca(2+) entry through VSCC mediates direct gGRLN(19) action on LH release in goldfish pituitary cells.

We report on the expression analysis and functional characterization of IL-4/13A and IL-4/13B in goldfish. Quantitative analysis indicated the highest expression in the heart, spleen, brain, and kidney, with comparable expression patterns for both IL-4/13A and IL-4/13B. The mRNA levels of IL-4/13A and IL-4/13B in the immune cells examined were highest in macrophage and monocytes. Assessment of spleen mRNA following infection with Trypanosoma carassii, a prominent protozoan pathogen of fish, revealed decrease in IL-4/13B and arginase expression 14 days post infection, followed by an increase in IL-4/13B and arginase-2 at 28 days post infection. Recombinant forms of IL-4/13A and IL-4/13B induced an increase in arginase activity in macrophages in a dose-dependent manner. Recombinant IL-4/13A and IL-4/13B also induced significant increase in mRNA levels of arginase -2 in macrophages at 6, 12, 18 and 24 h after treatment. Furthermore, treatment with both IL-4/13 recombinants interfered with the IFNγ-induced nitric oxide response of macrophages. Our results suggest a conserved role of IL-4/IL-13 in induction of alternative activation phenotype in teleost macrophages.

The nucleotide-binding oligomerization domain proteins Nod1, Nod2 and Nlrx1 are cytoplasmic pathogen recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In this report, goldfish Nod1 (gfNod1), Nod2 (gfNod2) and Nlrx1 (gfNlrx1) genes were cloned and characterized. The full length of gfNod1, gfNod2 and gfNlrx1 were 3234bp, 3129bp and 4900bp, encoding 937, 982 and 1008 amino acids, respectively. The three Nod-like receptors have a NACHT domain and C-terminal leucine rich repeat (LRR) domains. In addition to these, gfNod1 and gfNod2 also had an N-terminal CARD domain (two in gfNod2). Phylogenetic analysis showed that the three NLRs are highly conserved. Quantitative gene expression analysis of the three receptors revealed the greatest mRNA levels in the spleen, and in isolated neutrophils and splenocytes. Furthermore, treatment of goldfish macrophages with LPS, Poly I:C, MDP, PGN, heat-killed Aeromonas salmonicida or Mycobacterium marinum differentially altered the expression of the Nod-like receptors. Our results indicate that Nod-like receptors are functionally highly conserved and that they play a pivotal role in recognition of fish pathogens such as A. salmonicida and M. marinum.