A comprehensive survey of channel catfish Toll-like receptors (TLRs) was undertaken following a genomic PCR approach based on degenerate primers. Twenty different TLRs were identified in channel ...catfish. Channel catfish TLR sequences were characterized by phylogenetic analysis based on their conserved Toll/interleukin-1 receptor domain and by in-depth analysis of leucine-rich repeat (LRR) motifs of the ligand binding extracellular domain (ECD). The catfish have representatives of all the TLR types defined in vertebrates with the exception of TLR6, TLR10, TLR11, TLR12, TLR13, TLR15, TLR23, and TLR24. Additionally, two new types were discovered: TLR25 and TLR26. TLR25 is also present in cyprinids, cichlids, plecoglossids, and adrianichthyids, suggesting its presence early in fish evolution. To date, TLR26 was found only in channel catfish. Like TLR18–23, TLR25 and TLR26 were not found in any other vertebrate classes and appear to be fish specific. Data mining using the catfish TLR sequences revealed that in addition to ictalurids and cyprinids, TLR4 is also present in salmonids. TLR19 and TLR20 were both found in ictalurids, cyprinids, and salmonids, demonstrating a wider range than previously known. The LRR structure within ECDs appeared generally well conserved. TLR7 demonstrated a very high identity to human TLR7 strongly suggesting that ligand specificity maybe conserved. Finally, expression profiling confirmed that most TLRs are widely expressed in a diversity of tissues and revealed marked differences of expression level.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The CD28-B7 interaction is required to deliver a second signal necessary for T-cell activation. Additional membrane receptors of the CD28 and B7 families are also involved in immune checkpoints that ...positively or negatively regulate leukocyte activation, in particular T lymphocytes. BTLA is an inhibitory receptor that belongs to a third receptor family. Fish orthologs exist only for some of these genes, and the potential interactions between the corresponding ligands remain mostly unclear. In this work, we focused on the channel catfish (
Ictalurus punctatus
), a long-standing model for fish immunology, to analyze these co-stimulatory and co-inhibitory receptors. We identified one copy of
cd28
,
ctla4
,
cd80/86
,
b7h1
/
dc, b7h3
,
b7h4
,
b7h5,
two
btla
, and four
b7h7
genes. Catfish CD28 contains the highly conserved mammalian cytoplasmic motif for PI3K and GRB2 recruitment, however this motif is absent in cyprinids. Fish CTLA4 share a C-terminal putative GRB2-binding site but lacks the mammalian PI3K/GRB2-binding motif. While critical V-domain residues for human CD80 or CD86 binding to CD28/CTLA4 show low conservation in fish CD80/86, C-domain residues are highly conserved, underscoring their significance. Catfish B7H1/DC had a long intracytoplasmic domain with a P-loop-NTPase domain that is absent in mammalian sequences, while the lack of NLS motif in fish B7H4 suggests this protein may not regulate cell growth when expressed intracellularly. Finally, there is a notable expansion of fish B7H7s, which likely play diverse roles in leukocyte regulation. Overall, our work contributes to a better understanding of fish leukocyte co-stimulatory and co-inhibitory receptors.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Monoclonal antibodies (mAbs) CC34 and CC41 recognize overlapping subsets of leukocyte immune-type receptors (LITRs). The mAb CC34 was raised against the clonal TS32.15 cytotoxic T cell line and the ...mAb CC41 was raised against the clonal NK cell line TS10.1. In this study, an in vitro model was developed to monitor CC34- and CC41-reactive cells in response to Edwardsiella ictaluri infection. Briefly, head kidney leukocytes and peripheral blood lymphocytes (PBL) were isolated from individual catfish and labeled with CellTrace Violet and CellTrace FarRed dye, respectively. Head kidney-derived macrophages were infected with E. ictaluri and then cocultured with autologous PBL. The combined cell cultures were then analyzed using flow cytometry. A significant increase in CC41 staining was observed in the PBL population at 2, 5 and 7 days after culture, which suggest that LITRs are involved in cell-mediated immunity to E. ictaluri.
•Development of an assay to measure cellular responses against intracellular infection.•Cell labeling monitors distinct populations in autologous mixed leukocyte reactions.•E. ictaluri infected head kidney macrophages stimulate LITR expressing lymphocytes.•CC41+ lymphocytes proliferate in response to E. ictaluri infected macrophages.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this work, we describe the complete repertoire of channel catfish, Ictalurus punctatus, IFNs and IFN receptor genes. Based on multiple genomic and transcriptomic resources we identified 16 type I ...IFN genes, which represent the six type I IFN subgroups previously defined in salmonids (a-f.) No representatives of subgroup h previously only found in percomorphs were identified. An expansion in copy numbers of subgroup d IFN genes was of particular interest, as this has not been reported in other fish species to date. Furthermore, we confirmed the presence of two type II ifn genes encoding orthologs of IFNγ and the teleost-specific IFNγRel. Six homologs of IFN type I receptor genes were found in an array that shows conserved synteny with human chromosome 21. Three homologs of type II IFN receptor genes were also identified. These type I and type II receptor sequences are compatible with the dual type I IFN receptors, and the potentially more complex type II IFN receptors described in teleosts. Our data provide a comprehensive resource for future studies of channel catfish innate antiviral immunity.
•Sixteen interferon type I and two type II genes are present in channel catfish.•The 16 type I interferons belong to six subgroups (a, b, c, d, e, f).•Interferon subgroup d is expanded in channel catfish with 5 copies.•Six homologs of interferon type I and three of type II receptor genes are found in channel catfish.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been ...characterized molecularly using sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. However, only two of these have confirmed life cycles that involve the oligochaete Dero digitata as the definitive host. During a health screening of farm-raised channel catfish, several fish presented with deformed primary lamellae. Lamellae harbored large, nodular, white pseudocysts 1.25 mm in diameter, and upon rupturing, these pseudocysts released Henneguya myxospores, with a typical lanceolate-shaped spore body, measuring 17.1 ± 1.0 μm (mean ± SD; range = 15.0–19.3 μm) in length and 4.8 ± 0.4 μm (3.7–5.6 μm) in width. Pyriform-shaped polar capsules were 5.8 ± 0.3 μm in length (5.1–6.4 μm) and 1.7 ± 0.1 μm (1.4–1.9 μm) in width. The two caudal processes were 40.0 ± 5.1 μm in length (29.5–50.0 μm) with a spore length of 57.2 ± 4.7 (46.8–66.8 μm). The contiguous SSU rRNA gene sequence obtained from myxospores of five excised cysts did not match any Henneguya sp. in GenBank. The greatest sequence homology (91 % over 1,900 bp) was with Henneguya pellis, associated with blister-like lesions on the skin of blue catfish Ictalurus furcatus. Based on the unique combination of pseudocyst and myxospore morphology, tissue location, host, and SSU rRNA gene sequence data, we report this isolate to be a previously unreported species, Henneguya bulbosus sp. nov.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Channel catfish, Ictalurus punctatus, leukocyte immune-type receptors (LITRs) constitute a large family of paired, immunoregulatory receptors unique to teleosts. A role for LITRs in phagocytosis has ...been proposed based on studies in mammalian cell lines; however, LITR-mediated phagocytosis has not been examined in the catfish model. In this study, we use two anti-LITR monoclonal antibodies, CC41 and 125.2, to contrast the effects of crosslinking subsets of inhibitory and activating LITRs. Briefly, LITRs expressed by catfish γδ T cells, αβ T cells, and macrophage cell lines were crosslinked using mAb-conjugated fluorescent microbeads, and bead uptake was evaluated by flow cytometry and confirmed by confocal microscopy. A clear difference in the uptake of 125.2- and CC41-conjugated beads was observed. Crosslinking LITRs with mAb 125.2 resulted in efficient bead internalization, while mAb CC41 crosslinking of inhibitory LITRs resulted predominantly in a capturing phenotype. Pretreating catfish macrophages with mAb CC41 resulted in a marked decrease in LITR-mediated phagocytosis of 125.2-conjugated beads. Overall, these findings provide insight into fish immunobiology and validate LITRs as regulators of phagocytosis in catfish macrophages and γδ T cells.
•Monoclonal antibody 125.2 binds activating Leukocyte Immune-Type Receptors (LITRs).•Crosslinking 125.2-reactive LITRs mediates phagocytosis in T cells and macrophages.•Crosslinking CC41-reactive LITRs inhibits phagocytosis by catfish macrophages.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. ...Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 μm (range = 40.7–61.6 μm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 μm (14.4–19.3 μm) in length and 5.0 ± 0.3 μm (4.5–5.5 μm) in width. The two polar capsules were 6.2 ± 0.4 μm (5.8–7.0 μm) long and 5.0 ± 0.3 μm (4.5–5.5 μm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 μm (22.9–40.6 μm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4 % similarity (100 % coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The actinospore diversity of infected Dero digitata was surveyed (May 2011) from a channel catfish (Ictalurus punctatus) production pond in the Mississippi Delta region for the elucidation of unknown ...myxozoan life cycles. At present, only 2 myxozoan life cycles have been molecularly confirmed in channel catfish, linking the actinospore stage from an aquatic oligochaete (D. digitata) and the myxospore stage from the catfish. In this study D. digitata (n = 2,592) were isolated from oligochaetes collected from the bottom sediment of a channel catfish production pond. After 1 wk of daily observation, a total of 6 genetically different actinospore types were observed. The collective groups were classified as 2 aurantiactinomyxons, 2 helioactinomyxons, 1 raabeia, and 1 triactinomyxon. Overall prevalence of myxozoan infections in the isolated oligochaetes was 4.4%. Actinospores were photographed and measured for morphological characterization. Four previously undescribed actinospore types were identified and characterized molecularly and morphologically. Phylogenetic analysis revealed the raabeia and one of the helioactinomyxon (type 1) actinospores were closely related to the group of myxozoans known to parasitize ictalurids in North America. To date, no myxospores have been linked to the newly sequenced actinospores reported in this survey. The morphological and molecular data generated from this study will assist in the identification of myxospore counterparts for these actinospore stages and aid in the elucidation of unknown myxozoan life cycles in closed production systems.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood ...leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.
•Teleost anti-viral cytotoxic T cells are heterogeneous.•Four catfish anti-viral clonal cytotoxic T cell lines were established.•Two cell lines preferentially lysed channel catfish virus-infected syngeneic targets.•One clonal γδ T cell line exhibits broad target cell recognition.•None of these T cell lines expressed CD8, however three expressed CD4-like molecules.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this study, we used the channel catfish model clonal TS32.15 alloantigen-specific cytotoxic T cell (CTL) line to examine the dynamics of memory CTL expansion and senescence in teleosts. Although ...TS32.15 has been routinely cultured to study catfish CTL responses and killing mechanisms, little is known about the dynamics of the CTLs in these cultures. Here we show that this cell line consists of small non-cytotoxic T cells and larger granular effector T cells and that their ratios vary with time after stimulation. Small CTLs, when exposed to their irradiated targets, replicate and differentiate to morphologically distinct cytotoxic effectors, which do not replicate. After lysing target cells, or with prolonged absence of stimulation, the effector cells transition to a non-cytolytic senescent stage or become apoptotic. In addition, we demonstrate that natural IgM in catfish serum binds lipids, including PIP2, on early apoptotic CTLs, and that these IgM+ CTL can be cleared by catfish head kidney-derived macrophages.
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•Catfish clonal cytotoxic T cell line contains cells of three differentiation stages.•Small memory-like CTLs replicate following stimulation with specific antigen.•Large effector CTLs kill target cells, but do not replicate.•After killing their targets, effector CTLs become small and apoptotic.•Catfish natural IgM binds lipids on annexin V positive catfish T cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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