Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

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.

Catfish lymphocytes expressing CC41-reactive leukocyte immune-type receptors (LITRs) proliferate in response to Edwardsiella ictaluri infection in vitro.

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.

Insights into the dynamics of memory, effector and apoptotic cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

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.

A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, leukocyte immune type receptors (LITRs) represent a multigene family that encodes Ig superfamily proteins that mediate activating or inhibitory signaling. In this study, we demonstrate the use of mAb CC41 to monitor viral cytotoxic responses in catfish and determine that CC41 binds to a subset of LITRs on the surface of catfish clonal CTLs. Homozygous gynogenetic catfish were immunized with channel catfish virus (CCV)-infected MHC-matched clonal T cells (G14D-CCV), and PBL were collected at various times after immunization for flow cytometric analyses. The percentage of CC41(+) cells was significantly increased 5 d after primary immunization with G14D-CCV and at 3 d after a booster immunization as compared with control fish only injected with G14D. Moreover, CC41(+) cells magnetically isolated from the PBL specifically killed CCV-infected targets as measured by (51)Cr release assays and expressed messages for CD3γδ, perforin, and at least one of the CD4-like receptors as analyzed by RNA flow cytometry. When MLC effector cells derived from a G14D-CCV-immunized fish were preincubated with CC41 mAb, killing of G14D-CCV targets was reduced by ∼40%, suggesting that at least some LITRs have a role in target cell recognition and/or cytotoxicity. The availability of a LITR-specific mAb has allowed, to our knowledge for the first time, functional characterization of LITRs in an autologous system. In addition, the identification of an LITR subset as a cytotoxic cell marker will allow for more effective monitoring of catfish immune responses to pathogens.

Identification of SHIP-1 and SHIP-2 homologs in channel catfish, Ictalurus punctatus.

Src homology domain 2 (SH2) domain-containing inositol 5'-phosphatases (SHIP) proteins have diverse roles in signal transduction. SHIP-1 and SHIP-2 homologs were identified in channel catfish, Ictalurus punctatus, based on sequence homology to murine and human SHIP sequences. Full-length cDNAs for catfish SHIP-1 and SHIP-2 (IpSHIP-1 and IpSHIP-2) were obtained using 5' and 3' RACE protocols. Catfish SHIP molecules share a high degree of sequence identity to their respective SHIP sequences from diverse taxa and both are encoded by single copy genes. IpSHIP-1 and IpSHIP-2 transcripts were expressed in all catfish tissues analyzed except for skin, and IpSHIP-1 message was more abundant than IpSHIP-2 message in lymphoid tissues. Catfish clonal B, cytotoxic T, and macrophage cell lines also expressed message for both molecules. IpSHIP-1 and IpSHIP-2 SH2 domains were expressed as recombinant proteins and were both found to be bound by cross-reacting rabbit anti-mouse SHIP-1 pAb. The anti-mouse SHIP-1 pAb also reacted with cell lysates from the cytotoxic T cell lines, macrophages and stimulated PBL. SHIP-1 is also phosphorylated at a conserved tyrosine residue, as shown by immunoprecipitation studies.

Small subunit ribosomal RNA sequence links the myxospore stage of Henneguya mississippiensis n. sp. from channel catfish Ictalurus punctatus to an actinospore released by the benthic oligochaete Dero digitata.

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.

18S rRNA gene sequencing identifies a novel species of Henneguya parasitizing the gills of the channel catfish (Ictaluridae).

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.

Channel catfish cytotoxic cells: a mini-review.

The use of allogeneic and autologous lymphoid cell lines has facilitated studies of cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cells in channel catfish. Naïve catfish leukocytes were shown to spontaneously kill allogeneic cells and virally-infected autologous cells without the need for prior sensitization, and allogeneic cytotoxic responses were greatly enhanced by in vitro alloantigen stimulation. Both catfish CTL and NK-like cells have been successfully cloned from these alloantigen-stimulated cultures, and represent the first cytotoxic cell lines derived from any ectothermic vertebrate. These cloned cytotoxic cells contain granules and likely induce apoptosis in sensitive targets via a putative perforin/granzyme mechanism. In addition, some catfish CTL clones may also kill targets by an additional mechanism, possibly by Fas/FasL-like interactions. Importantly, these cytotoxic cells do not express the marker for catfish nonspecific cytotoxic cells (NCCs), and thus represent cell types distinct from NCCs. The use of monoclonal antibodies against the catfish F and G immunoglobulin light chain isotypes revealed the presence of a putative Fc receptor for IgM (Fc mu R) on some catfish NK-like cells that appears to 'arm' these cells with surface IgM. In addition, a potentially important monoclonal antibody (CC41) developed against catfish NK-like cells was found to recognize an approximately 150kDa molecule on the surface of catfish cytotoxic cells. These studies clearly demonstrate that catfish possess an array of different cytotoxic cells. The availability of various cloned cytotoxic cell lines should enable unambiguous functional studies to be performed in ways not currently possible with any other fish species.