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.

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions.

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.

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.

The chemokinome superfamily: II. The 64 CC chemokines in channel catfish and their involvement in disease and hypoxia responses.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. Based on the arrangement of the first four cysteine residues, they are classified into CC, CXC, C and CX3C subfamilies. In this study, a complete set of 64 CC chemokine ligand (CCL) genes was systematically identified, annotated, and characterized from the channel catfish genome. Extensive phylogenetic and comparative genomic analyses supported their annotations, allowing establishment of their orthologies, revealing fish-specific CC chemokines and the expansion of CC chemokines in the teleost genomes through lineage-specific tandem duplications. With 64 genes, the channel catfish genome harbors the largest numbers of CC chemokines among all the genomes characterized to date, however, they fall into 11 distinct CC chemokine groups. Analysis of gene expression after bacterial infections indicated that the CC chemokines were regulated in a gene-specific and time-dependent manner. While only one member of CCL19 (CCL19a.1) was significantly up-regulated after Edwardsiella ictaluri infection, all CCL19 members (CCL19a.1, CCL19a.2 and CCL19b) were significantly induced after Flavobacterium columnare infection. In addition, CCL19a.1, CCL19a.2 and CCL19b were also drastically up-regulated in ESC-susceptible fish, but not in resistant fish, suggesting potential significant roles of CCL19 in catfish immune responses. High expression levels of certain CC appeared to be correlated with susceptibility to diseases and intolerance to hypoxia.

Identification and characterization of a ß-defensin gene involved in the immune defense response of channel catfish, Ictalurus punctatus.

Antimicrobial peptides are small peptides that play important roles in a host's innate immune response. As an important antimicrobial peptide, ß-defensin widely distribute in mammals, insects and plants with broad-spectrum antimicrobial activity. In this study, the ß-defensin gene of the channel catfish, Ictalurus punctatus, was cloned, sequenced, and subjected to a bioinformatic analysis. The ß-defensin gene of the channel catfish contains three exons and two introns, and encodes a precursor peptide consisting of two domains: a signal peptide of 24 amino acid residues and a mature peptide of 43 amino acid residues. The mature peptide is estimated to have a molecular mass of 7.1kDa and a theoretical isoelectric point of 8.21. Channel catfish ß-defensin (ccBD) has six conserved cysteine residues, forming three disulfide bridges at C1-C5, C2-C4, and C3-C6, and a ß-sheet in the predicted three-dimensional structure. A phylogenetic analysis suggests that ccBD belongs to the type 1 ß-defensins. Real-time quantitative PCR showed that channel catfish ß-defensin transcripts are constitutively expressed in various tissues in healthy fish, with highest expression in the skin. The expression of ccBD in vivo increased significantly in the head kidney (2.9-fold), gill (2.2-fold), and skin (6.6-fold) at 48h after bacterial (Edwardsiella ictaluri) challenge. In vitro, lipopolysaccharide (LPS), a bacterial mimic, induced significant changes in ccBD expression in leukocytes from the spleen (3.4-fold) and head kidney (3.9-fold) 24h after stimulation. Chemically synthesized ccBD displayed marked inhibitory activity against a broad range of bacteria. These results suggest that ccBD is involved in the innate antibacterial defenses of the channel catfish.

Imaging flow cytometry and GST pulldown assays provide new insights into channel catfish leukocyte immune-type receptor-mediated phagocytic pathways.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) control various innate immune cell effector responses including the phagocytic process. This large immunoregulatory receptor family also consists of multiple receptor-types with variable signaling abilities that is dependent on their inherent or acquired tyrosine-containing cytoplasmic tail (CYT) regions. For example, IpLITR 2.6b associates with the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor molecule IpFcRγ-L, and when expressed in mammalian cells it activates phagocytosis using a similar profile of intracellular signaling mediators that also regulate the prototypical mammalian Fc receptor (FcR) phagocytic pathway. Alternatively, IpLITR 1.1b contains a long tyrosine-containing CYT with multifunctional capabilities including both inhibitory and stimulatory actions. Recently, we demonstrated that IpLITR 1.1b activates a unique phagocytic pathway involving the generation of multiple plasma membrane extensions that rapidly capture extracellular targets and secure them on the cell surface in phagocytic cup-like structures. Occasionally, these captured targets are completely engulfed albeit at a significantly lower rate than what was observed for IpLITR 2.6b. While this novel IpLITR 1.1b phagocytic activity is insensitive to classical blockers of phagocytosis, its distinct target capture and engulfment actions depend on the engagement of the actin polymerization machinery. However, it is not known how this protein translates target recognition into intracellular signaling events during this atypical mode of phagocytosis. Using imaging flow cytometry and GST pulldown assays, the aims of this study were to specifically examine what regions of the IpLITR 1.1b CYT trigger phagocytosis and to establish what profile of intracellular signaling molecules likely participate in its actions. Our results show that in stably transfected AD293 cells, the membrane proximal and distal CYT segments of IpLITR 1.1b independently regulate its phagocytic activities. These CYT regions were also shown to differentially recruit various SH2 domain-containing intracellular mediators, which provides new information about the dynamic immunoregulatory abilities of IpLITR 1.1b. Overall, this work further advances our understanding of how certain immunoregulatory receptor-types link extracellular target binding events to the actin polymerization machinery during a non-classical mode of phagocytosis.

Expression of immune genes in skin of channel catfish immunized with live theronts of Ichthyophthirius multifiliis.

The objective of this study was to evaluate differential expression of innate and adaptive immune genes, including immunoglobulin, immune cell receptor, cytokine, inflammatory protein, toll-like receptors (TLR) and recombination-activating gene (RAG) in skin from channel catfish, Ictalurus punctatus after immunization with live theronts of Ichthyophthirius multifiliis (Ich) by intraperitoneal injection. The immunized catfish showed significantly higher survival rate (95%) than those of mock-immunized control fish (0% survival) after the theront challenge. The gene expression of innate immune system, such as cytokines (IL-1ß type a, IL-1ß type b, IFN-γ, TGF1-ß and TNF-α) and inflammatory proteins (NF-kB and iNOS 2), showed significant upregulation at day 1 (D1) post-immunization. Expression of TLR genes exhibited a rapid increase from hour 4 (h4) to D10 post-immunization. Genes of the adaptive response, such as the cell receptor MHC I, CD8+ , CD4+ and TCR-α, showed upregulation at D1, D6 and D10. The TCR-ß expression increased rapidly at h4 and remained upregulated until D10. Immunoglobulin IgM upregulation was detected from h4 until D2 while IgD expression was increased from D1 until D10. Rapid upregulation of innate and adaptive immune genes in skin of catfish following live theront vaccination was demonstrated in this study ultimately resulting in significant protection against Ich infection.

Expression of Bcl-2 genes in channel catfish after bacterial infection and hypoxia stress.

Bcl-2 proteins are of vital importance in regulation of apoptosis, and are involved in a number of biological processes such as carcinogenesis and immune responses. Bcl-2 genes have been well studied in mammals, while they are not well investigated in teleost fish including channel catfish, the major aquaculture species in the United States. In this study, we identified 34 bcl-2 genes from the channel catfish genome, and verified their identities by conducting phylogenetic and syntenic analyses. The expression profiles of the bcl-2 genes in response to bacterial infections (Edwardsiella ictaluri and Flavobacterium columnare) and hypoxia stress were determined by performing meta-analysis using the existing RNA-Seq datasets. Differential expressions of bcl-2 genes were observed after bacterial infections and hypoxia treatment, including 22 bcl-2 genes after E. ictaluri infection, 22 bcl-2 genes after F. columnare infection, and 19 bcl-2 genes after hypoxia stress. Overall, the expression of the pro-apoptotic bcl-2 genes were repressed after bacterial infection and hypoxia stress, indicating that bcl-2 genes are potentially involved in the stress response by reducing cell apoptosis. Some bcl-2 genes, such as bcl2b, mcl1a, bmf1, and bnip3, showed different expression pattern during the E. ictaluri and F. columnare infection, suggesting the difference in the pathogenicity of diseases. This work presented the first systematic identification and annotation of bcl-2 genes in catfish, providing essential genomic resources for further immune and physiological studies.

Expression profile analysis of two cathepsin S in channel catfish (Ictalurus punctatus) mucosal tissues following bacterial challenge.

Cathepsin S belongs to the papain family of cysteine protease, and is considered to play key roles in immune responses after bacterial challenge. However, despite the recognized importance of Cathepsin S in immunity, no studies have systematically characterized Cathepsin S in catfish. In this regard, here, we characterized the Cathepsin S gene family in channel catfish, and investigated their expression patterns following two different Gram-negative bacterial challenge. In the present study, two Cathepsin S genes (ctss and ctssa) were captured in channel catfish. In comparison to other species, the catfish Cathepsin S genes are highly conserved in their structural features. Phylogenetic analysis indicated the strongest phylogenetic relationship with zebrafish, which is consistent with their evolutional relationships. Tissue distribution analysis revealed that Cathepsin S genes were ubiquitously expressed in catfish tissues. Following bacterial infection, the Cathepsin S genes were significantly up-regulated at most time-points in mucosal surfaces, with an acute response post Edwardsiella ictaluri infection. Obviously, the expression profiles were quite distinct between two Cathepsin S genes, across the tissues and between pathogens, suggesting that Cathepsin S genes may exert disparate roles in mucosal immune responses. Our findings here, provide early insight into the immune functions of Cathepsin S in catfish; however, further studies are needed to determine the mechanisms of Cathepsin S for antigen presentation during inflammatory processes and innate host defense.

Septin genes in channel catfish (Ictalurus punctatus) and their involvement in disease defense responses.

Septins are an evolutionarily conserved family of GTP-binding proteins. They are involved in diverse processes including cytokinesis, apoptosis, infection, neurodegeneration and neoplasia. In this study, through thorough data mining of existed channel catfish genomic resources, we identified a complete set of 15 septin genes. Septins were classified into four subgroups according to phylogenetic analysis. Extensive comparative genomic analysis, including domain and syntenic analysis, supported their annotation and orthologies. The expression patterns of septins in channel catfish were examined in healthy tissues and after infection with two major bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. In healthy channel catfish, most septin genes were ubiquitously expressed and presented diversity patterns in various tissues, especially mucosal tissues, proposing the significant roles septin genes may play in maintaining homeostasis and host immune response activities. After bacterial infections, most septin genes were regulated, but opposite direction in expression profiles were found with the two bacterial pathogens: the differentially expressed septin genes were down-regulated in the intestine after E. ictaluri infection while generally up-regulated in the gill after F. columnare infection, suggesting a pathogen-specific and tissue-specific pattern of regulation. Taken together, these results suggested that septin genes may play complex and important roles in the host immune responses to bacterial pathogens in channel catfish.

The Src tyrosine kinase Lck binds to CD2, CD4-1, and CD4-2 T cell co-receptors in channel catfish, Ictalurus punctatus.

The binding of the lymphocyte specific protein tyrosine kinase (Lck) to T cell co-receptors is required for T cell development and activation. In mammals, Lck initiates signal transduction by binding to CD4 and CD8 co-receptors and phosphorylating ITAMs in the cytoplasmic tail of the CD3 molecules and the ζ chains. In addition, Lck can also bind to the adhesion molecule CD2 and trigger T cell activation. In this study, Lck and CD2 homologs were identified and characterized in channel catfish, Ictalurus punctatus. Lck and CD2 mRNAs were specifically expressed by clonal T cell lines, including both CD4(+) and CD4(-)CD8(-) CTL lines, and in mixed lymphocyte cultures (MLC). Western blot analyses using anti-trout Lck and anti-human p-Lck antibodies demonstrated that Lck protein is expressed in catfish clonal CTL and is phosphorylated at a conserved tyrosine residue. Because of the lack of CD8(+) CTL lines as well as the absence of CD8 message in MLC, we performed magnetic bead binding assays to correlate CD2, CD4, and CD8 co-receptor expression with Lck binding ability. Recombinant Lck reproducibly bound to CD2, CD4-1, and CD4-2, but not to CD8α or CD8ß. These data provide one possible explanation for the apparent low numbers of CD8(+) CTL and the presence of CD4(+) and CD4(-)CD8(-)CD2(+) CTL in catfish.

A dietary dairy/yeast prebiotic and flaxseed oil enhance growth, hematological and immunological parameters in channel catfish at a suboptimal temperature (15°C).

Channel catfish raised in the southern United States require two growing seasons to reach market size. Growing seasons are separated by a cool period of about 3 months when feed intake and growth are greatly reduced. A cool-weather feeding strategy to improve feed intake, growth or health of catfish might improve survival and reduce the time needed to achieve market size. We conducted a feeding trial with channel catfish at a suboptimal temperature (15°C) to determine the effects of supplementing diets with either a dairy/yeast prebiotic or flaxseed oil (high in 18:3n-3) compared with a control with soybean oil (high in 18:2n-6). The trial was conducted in recirculating systems with 1140-l tanks containing 100 fish each (mean initial weight 61.4 g±0.43 s.e.m.). A 28%-protein basal diet was supplemented with 20 g/kg cellulose and 20 g/kg soybean oil (SBO, control), 20 g/kg cellulose and 20 g/kg flaxseed oil (FLAX) or 20 g/kg of a dairy/yeast prebiotic and 20 g/kg soybean oil (PREB). Fish were fed once daily to satiation and weighed every 3 weeks to track growth. Hematology, non-specific immune responses, proximate and fatty acid composition of muscle were determined to assess diet effects. Catfish-fed FLAX or PREB had higher weight gain, feed consumption and lysozyme activity than fish fed SBO. Total n-3 fatty acids in muscle were higher in fish fed SBO or FLAX than those fed PREB. Total n-6 long-chain polyunsaturated acids were higher in muscle of fish fed PREB than those fed SBO. Fatty acids in the PREB and SBO diets were similar, so the PREB appeared to increase elongation and desaturation of n-6 fatty acids in muscle. Flaxseed oil and the dairy/yeast prebiotic both have potential to increase catfish performance at a low temperature.

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.

Expression of tumor suppressor genes in channel catfish after bacterial infections.

Tumor suppressor genes are negative regulators of tumor formation. While their anti-tumor functions have been well studied, they have been found to be also involved in immune responses and innate immunity. In this study, 21 tumor suppressor genes in channel catfish (Ictalurus punctatus) were characterized. Phylogenetic and syntenic analyses allowed annotation of all 21 catfish tumor suppressor genes. The expression profiles of the 21 catfish tumor suppressor genes were determined using the RNA-Seq datasets. After Edwardsiella ictaluri infection, expression of five of the 21 tumor suppressor genes was up-regulated at 3 days in the intestine, and four of the 21 genes were up-regulated in the liver 14 days post-infection. With Flavobacterium columnare infection, seven genes were up-regulated in the gill at 48 h post-infection. These results expanded our knowledge on the tumor suppressor genes in teleosts, setting a foundation for future studies to unravel functions of tumor suppressor genes in response to stresses, particularly after bacterial disease infections.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERß2 and are functionally modulated by estrogens.

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERß2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERß2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17ß-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

Comparative transcriptional analysis reveals distinct expression patterns of channel catfish genes after the first infection and re-infection with Aeromonas hydrophila.

To determine whether transcriptional levels of channel catfish (Ictalurus punctatus) genes are differentially regulated between a first infection with Aeromonas hydrophila and a re-infection, suppression subtractive hybridization (SSH) was performed in this study using anterior kidney cDNA after the re-infection as tester. Of the 96 clones isolated from the SSH library, 28 unique expressed sequence tags (ESTs) were obtained, of which eight were confirmed to be slightly but significantly (P < 0.05) more up-regulated by the re-infection at 6 h post infection (hpi). Expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the eight ESTs were significantly (P = 0.016) more up-regulated by the first infection, with a major peak at 3 hpi. A total of 96 genes reported in literature to be up-regulated by bacterial infections were selected and subjected to expression analysis at 3 hpi. Of the 96 selected genes, 19 were found to be significantly (P < 0.05) induced by A. hydrophila after the first infection and the re-infection. The 19 genes belonged to the following five main categories: 1) toll-like receptor (TLR2, TLR3, TLR5, TLR21); 2) antimicrobial peptide (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, cathepsin D, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1ß, interleukin-10, tumor necrosis factor α, chemokine CXCL-10); 4) signaling proteins (cadherin EGF LAG seven-pass G-type receptor 1, very large inducible GTPase 1, arginine deiminase type 2, lymphokine-activated killer T-cell originated protein kinase); 5) lysozyme (lysozyme c). Overall, the total 27 genes (8 ESTs plus the 19 selected genes) were significantly (P < 0.001) more induced by the first infection. Peaked expression of lysozyme c and serum lysozyme activity after the first infection were seen at 24 hpi, whereas that after the re-infection were seen at 12 hpi, suggesting that both innate and adaptive immunity were involved in the defense against the re-infection of A. hydrophila.

Basal polarization of the mucosal compartment in Flavobacterium columnare susceptible and resistant channel catfish (Ictalurus punctatus).

The freshwater bacterial pathogen, Flavobacterium columnare, infects a variety of ornamental and farmed fish species worldwide through mucosal attachment points on the gill and skin. While previous studies have demonstrated a chemotactic response of F. columnare to fish mucus, little is known about how host gill mucosal molecular and cellular constituents may impact rates of adhesion, tissue invasion, and ultimately, mortality. Here, we describe the use of RNA-seq to profile gill expression differences between channel catfish (Ictalurus punctatus) differing in their susceptibility to F. columnare both basally (before infection) and at three early timepoints post-infection (1 h, 2 h, and 8 h). After sequencing and de novo assembly of over 350 million 100 base-pair transcript reads, between group comparisons revealed 1714 unique genes differentially expressed greater than 1.5-fold at one or more timepoints. In the large dataset, we focused our analysis on basal differential expression between resistant and susceptible catfish as these genes could potentially reveal genetic and/or environmental factors linked with differential rates of infection. A number of critical innate immune components including iNOS2b, lysozyme C, IL-8, and TNF-alpha were constitutively higher in resistant catfish gill, while susceptible fish showed high expression levels of secreted mucin forms, a rhamnose-binding lectin previously linked to susceptibility, and mucosal immune factors such as CD103 and IL-17. Taken together, the immune and mucin profiles obtained by RNA-seq suggest a basal polarization in the gill mucosa, with susceptible fish possessing a putative mucosecretory, toleragenic phenotype which may predispose them to F. columnare infection.

Effects of dietary sweet potato leaf meal on the growth, non-specific immune responses, total phenols and antioxidant capacity in channel catfish (Ictalurus punctatus).

BACKGROUND: Traditional energy sources in catfish diets have become costly, and economical alternatives are needed. Sweet potato leaves are underutilised agricultural by-products that provide energy and substantial amounts of phenols, which affect animal and human health. There is little information on the effects of these compounds on catfish, or the capacity of catfish to accumulate dietary phenols. Catfish enriched with phenols have marketing potential as functional foods. This study investigated the effects of diets with sweet potato leaf meal (SPLM) on growth performance, health and total phenolic compounds in catfish. RESULTS: SPLM was substituted for wheat middlings in three diets fed to groups of juvenile catfish for 10 weeks. Weight gain, feed conversion, survival, alternative complement activity and lysozyme activity were similar among diets. Haematocrit was lower in fish fed diets with SPLM, but within the normal range. Total phenols and antioxidant capacity in the whole body were similar among treatments. CONCLUSION: SPLM was an effective energy source for catfish up to the maximum level tested (230 g kg(-1) diet). SPLM did not enhance total phenols in catfish, but there were no apparent antinutritional effects of the meal on catfish growth, health or survival.

Channel catfish (Ictalurus punctatus) protein disulphide isomerase, PDIA6: molecular characterization and expression regulated by bacteria and virus inoculation.

Protein disulfide isomerases (PDIs) are thought to aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Currently, increasing evidence suggests PDIs play an important role in host cell invasion and they are relevant targets for the host immune response. However the roles of specific PDIs in teleosts are little known. Here, we characterized the Protein disulfide isomerase family A, member 6 (PDIA6) from channel catfish, Ictalurus punctatus (named as ccPDIA6). The catfish ccPDIA6 gene was homologous to those of other vertebrate species with 13 exons and 12 introns. The consensus full-length ccPDIA6 cDNA contained an ORF of 1320 bp encoding a putative protein of 439 amino acids. It had a 19 amino acid signal peptide and two active thioredoxin-like domains. Sequence of phylogenic analysis and multiple alignments showed that ccPDIA6 was conserved throughout vertebrate evolution. Southern blot analysis suggested the presence of one copy of the ccPDIA6 gene in the catfish genome. Tissue distribution shows that ccPDIA6 was expressed in all examined tissues at the mRNA level. When using the aquatic zoonotic pathogens such as Edwardsiella tara, Streptococcus iniae, and channel catfish reovirus （CCRV） to challenge channel catfish, ccPDIA6 expression was significant changed in immune-related tissues such as head kidney, intestine, liver and spleen. The results suggested that ccPDIA6 might play an important role in the immunity of channel catfish. This is the first report that the PDI gene may be involved in fish host defense against pathogen infection.

The effect of adjuvant and microbial challenge on the expression of antimicrobial polypeptides in channel catfish (Ictalurus punctatus).

While antimicrobial polypeptides (AMPPs) are increasingly recognized as one of the most important components of innate immunity, there is very little information in vertebrates that documents their upregulation to levels that are microbicidal in vivo. Here we demonstrate that intraperitoneal injection of either Freund's complete adjuvant (FCA) or live Tetrahymena pyriformis (a parasitic ciliate) upregulated AMPP expression in channel catfish skin. FCA induced significant upregulation of total antibacterial activity, anti-Edwardsiella ictaluri activity (the fraction of antibacterial activity active against E. ictaluri), and HLP-1 (the major AMPP in channel catfish skin). Tetrahymena induced a similar upregulation, except that HLP-1 was not significantly induced and the response appeared to be more transient than FCA immunostimulation. AMPP levels were increased up to five-fold higher than resting levels and levels expressed were well within concentrations known to be inhibitory to many important pathogens in vitro. These results provide encouragement that AMPP upregulation may be a promising tool in aquaculture for enhancing the resistance of fish to disease.