Histone-like protein: a novel method for measuring stress in fish

We assessed the effect of chronic stress using a group of potent, broad-spectrum antimicrobial polypeptides, called histone-like proteins (HLPs), which appear to be an important component of non-specific immunity in channel catfish Ictalurus punctatus skin. An enzyme-linked immunosorbent assay (ELISA) was developed to measure the predominant HLP (HLP-1) in channel catfish skin. Catfish were then exposed to a chronic stress consisting of overcrowding and elevated ammonia. Healthy unstressed fish had consistently high HLP-1 levels, but fish that had been stressed for 1 wk had significantly depressed HLP-1 levels; HLP-1 levels declined further in fish stressed for 3 or 4 wk. The time-dependent decline in HLP-1 levels was not accompanied by any gross signs of disease. In contrast to HLP-1 levels, antibacterial activity in the skin was significantly greater in fish stressed for 1 wk compared with unstressed fish; in addition, antibacterial activity was the same in fish that were unstressed or stressed for 3 or 4 wk. This suggests that other antibiotics besides HLP-1 may be induced in the skin, especially during early stages of stress, that may compensate for depressed HLP-1 levels. Our results indicate that chronic stress has a significant suppressive effect on HLP-1 levels in channel catfish skin. The reduction of HLP-1 in the absence of clinical signs of disease, combined with evidence that its levels are not affected by the acute stressors of capture or sampling, suggests that HLP levels may be a promising indicator for monitoring fish health.     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.     

Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host

Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.     

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus).

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.     

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus)

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.     

Response of toll-like receptors, lysozyme, and IGF-I in back-cross hybrid (F1 male (blue x channel) x female channel) catfish challenged with virulent Edwardsiella ictaluri

Responses of toll-like receptors (TLR3 and TLR5), lysozyme, and insulin-like growth factor-I (IGF-I) to experimental challenge with virulent Edwardsiella ictaluri were measured in back-cross hybrid (F1 male (blue x channel) x female channel) catfish. The resistance levels to E. ictaluri and host response mechanisms of back-cross hybrids are unknown. Fish were challenged with virulent E. ictaluri and sampled pre-challenge, 2 h and 2, 5, 8, 14, and 21 days post-challenge. Levels of mRNA expression of two toll-like receptors (TLR3 and TLR5) in liver, kidney, spleen, and stomach, plasma lysozyme activity, and circulating IGF-I levels were measured at each timepoint. Throughout challenge, TLR3 was expressed at higher levels than TLR5 in liver (P=0.0011) and kidney (P=0.0007) whereas TLR5 was more highly expressed than TLR3 in stomach (P=0.0032). TLR3 was upregulated in comparison to non-exposed controls in liver (P=0.0015) and stomach (P<0.0001) on day 14 and TLR5 was upregulated in liver (P=0.0175) on days 2 through 8. Plasma lysozyme activity peaked on day 5 (P<0.001) and IGF-I levels significantly decreased on days 2 through 14 (P<0.0001). TLR expression patterns suggest that both TLR3 and TLR5 may play a role in host response to bacterial challenge. Plasma lysozyme activity also increased and circulating IGF-I decreased in response to the presence of the pathogen.     

Ichthyophthirius multifiliis as a potential vector of Edwardsiella ictaluri in channel catfish

There is limited information on whether parasites act as vectors to transmit bacteria in fish. In this trial, we used Ichthyophthirius multifiliis and fluorescent Edwardsiella ictaluri as a model to study the interaction between parasite, bacterium, and fish. The percentage (23-39%) of theronts fluorescing after exposure to E.?ictaluri was significantly higher than control theronts (~?6%) using flow cytometry. Theronts exposed to E.?ictaluri at 4?×?10(7) CFU?mL(-1) showed a higher percentage (~?60%) of fluorescent theronts compared to those (42%) exposed to 4?×?10(3) CFU?mL(-1) at 4?h. All tomonts (100%) carried the bacterium after exposure to E.?ictaluri. Edwardsiella ictaluri survived and replicated during tomont division. Confocal microscopy demonstrated that E.?ictaluri was associated with the tomont surface. Among theronts released from tomonts exposed to E.?ictaluri, 31-66% were observed with attached E.?ictaluri. Sixty percent of fish exposed to theronts treated with 5?×?10(7) E.?ictaluri?mL(-1) were positive for E.?ictaluri at 4?h as determined by qPCR or fluorescent microscopy. Fluorescent E.?ictaluri were observed on trophonts in skin and gill wet mounts of dead fish. This study demonstrated that Ich could vector E.?ictaluri to channel catfish.     

Mortality and pathology in brown bullheads Amieurus nebulosus associated with a spontaneous Edwardsiella ictaluri outbreak under tank culture conditions

Brown bullheads Amieurus nebulosus (family Ictaluridae) are commonly used as a sentinel of environmental contamination. These fish are not generally cultured under laboratory conditions and little is known about their disease susceptibility. Here we report an outbreak of disease due to Edwardsiella ictaluri in a laboratory population of tank-reared, wild-caught brown bullheads. The isolate was positively identified as E. ictaluri using standard bacteriological substrate utilization tests and a monoclonal antibody specific for this bacterium. This pathogen causes a significant disease in channel catfish Ictalurus punctatus and is associated with disease in other ictalurid and non-ictalurid fishes. It appears that E. ictaluri is also a significant pathogen in brown bullheads and produces clinical signs and lesions similar but not identical to those observed in channel catfish. Since commercial sources of bullheads for laboratory tank studies are not available, precautions should be taken to prevent potential E. ictaluri disease outbreaks from wild-caught bullheads intended for laboratory research.     

Plasmid and serological differences between Edwardsiella ictaluri strains.

Several studies have shown that isolates of Edwardsiella ictaluri obtained from infected channel catfish in the southeastern United States harbor two cryptic plasmids, designated pCL1 (5.7 kb) and pCL2 (4.9 kb). These isolates appear to be serologically homogeneous. To extend these studies, we focused our analyses on two isolates of nonictalurid origin. Plasmid analyses of a danio isolate showed that it harbored plasmids which were similar if not identical to pCL1 and pCL2. This strain was also serologically indistinguishable from those isolated from channel catfish. In contrast, a green knife fish (GNF) isolate harbored four plasmids with relative mobilities of 6.0, 5.7, 4.1, and 3.1 kb. Southern blot analyses indicated that only the 5.7- and 4.1-kb plasmids strongly hybridized under high-stringency conditions to probes specific for pCL1 and pCL2, respectively. The GNF isolate showed minimal reactivity when reacted with polyclonal antiserum prepared against a channel catfish isolate. However, polyclonal antiserum to the GNF isolate strongly reacted with the GNF isolate in both surface fluorescence and agglutination reactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of cell lysates showed that the protein banding patterns of the strains compared were similar. However, Western blots of proteinase K-digested cell extracts showed that O antigen of the GNF isolate was antigenically distinct from the O antigen of the other isolates. These studies indicate that there are different serotypes of E. ictaluri and suggest that plasmid and serological analyses of future isolates of E. ictaluri can be used to determine whether structurally distinct strains are emerging in major channel catfish aquaculture areas.     

Flavobacterium columnare chemotaxis to channel catfish mucus

Flavobacterium columnare is a Gram-negative pathogen of many species of wild and cultured fish. Isolates from diseased channel catfish belong to either genomovar I or II. Genomovar II isolates were found to be more virulent than genomovar I isolates. The objective of the present study was to determine whether differences exist in the chemotactic response of these genomovars to mucus obtained from the skin, gills and intestines of healthy channel catfish using the capillary chemotaxis assay. Mucus from the skin and gill induced a greater chemotactic response by F. columnare than mucus from the intestine. Sixty percent of mucus from the skin of individual catfish yielded a positive chemotactic response from F. columnare. Finally, skin mucus induced a greater chemotactic response in genomovar II F. columnare than in genomovar I F. columnare isolates. The data indicate that mucus from channel catfish results in a chemotactic response by F. columnare. This positive chemotactic response may be an important first step for F. columnare colonization of channel catfish skin or gills. Although the role that chemotaxis plays in the virulence of F. columnare is not fully defined, the chemotactic response of genomovar ll isolates suggests that chemotaxis is associated with virulence.     

Differences in mortality, growth, lysozyme, and toll-like receptor gene expression among genetic groups of catfish exposed to virulent Edwardsiella ictaluri

Survivorship to ESC (enteric septicemia of catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility is not well-understood. We assessed the effect of pathogen challenge with Edwardsiella ictaluri on five genetic groups of catfish by measuring both phenotypic response (mortality, pathogen levels, specific growth rate), and three measures of immune response, including lysozyme activity and mRNA expression of two toll-like receptors (TLR3 and TLR5). Both mortality and pathogen loads, in addition to non-specific immune response, consistently ranged from the least susceptible Blue catfish (24%, 3.4 x 10(2)+/-9.3 x 10(1)cell-equivalents/mg, 13.2+/-3.2U/mL tissue, respectively) to the most susceptible 103 channel catfish (65%, 1.1x10(4)+/-6.4 x 10(3)cell-equivalents/mg tissue, 67.3+/-28.7U/mL, respectively). Similarly, specific growth rate was reduced in exposed fish, compared to non-exposed controls, only in the most susceptible genetic groups (P=0.0051). Trends in mRNA expression levels were apparent in each tissue type for both genes. In kidney, differences were evident in expression of both TLR3 and TLR5 mRNA between strains early and late in challenge (P<0.01). TLR5 mRNA showed significant downregulation in all strains on days 1 and 4 (P=0.0001). In spleen, all strains had elevated levels of TLR3 (P=0.0050) and TLR5 mRNA (P<0.0001) only 1day post-exposure. In stomach, only one strain (103 x RR) showed upregulation (P=0.0063) throughout challenge. The relationship of phenotypic (mortality and growth) and immune responses measured here, suggests that variation in susceptibility to ESC is a function of differences in innate immune response. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols for channel catfish.     

Edwardsiella ictaluri invasion of IEC-6, Henle 407, fathead minnow and channel catfish enteric epithelial cells

Invasion of Edwardsiella ictaluri into cultured mammalian, fish and enzymatically harvested catfish enteric epithelial cells is described. Gentamicin survival assays were used to demonstrate the ability of this catfish pathogen to invade IEC-6 (origin: rat small intestinal epithelium), Henle 407 (origin: human embryonic intestinal epithelium), fathead minnow (FHM, minnow epithelial cells) and trypsin/pepsin-harvested channel catfish enteric epithelial cells. Invasion of all cell types occurred within 2 h of contact at 26 degrees C, in contrast to Escherichia coli DH5 alpha, which did not invade cells tested. Eight Edwardsiella ictaluri isolates from diseased catfish and the ATCC (American Type Culture Collection) strain were evaluated for invasion efficiency using FHM cells. All isolates were invasive, but at differing efficiencies. Invasion blocking assays using chemical blocking agents were performed on a single isolate (LA 89-9) using IEC-6 epithelial cells. Preincubation of IEC-6 cells with cytochalasin D (microfilament depolymerizer) and monodansylcadaverine (blocks receptor-mediated endocytosis) significantly reduced invasion by E. ictaluri, whereas exposure to colchicine (microtubule depolymerizer) had no effect on bacterial internalization. Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E. ictaluri by IEC-6 epithelial cells. Invasion trials using freshly harvested cells from the intestine of the natural host, Ictalurus punctatus, show that invasion occurs, but at a low efficiency. This is possibly due to loss of outer membrane receptors during enzymatic cell harvest. This study provides the first documentation of the invasion of cultured mammalian and fish cells by E. ictaluri, and identifies possible mechanisms used for intracellular access. Additionally, the study describes several functional in vitro invasion models using commercially available cell lines as well as cells from the natural host (channel catfish, I. punctatus).     

Effects of GH on immune and endocrine responses of channel catfish challenged with Edwardsiella ictaluri

The effects of GH on immune and endocrine responses to channel catfish challenged with the bacterium Edwardsiella ictaluri were examined. Catfish (11.7+/-1.0 g) treated with recombinant bovine growth hormone (rbGH) and challenged with E. ictaluri experienced similar mortality as control-exposed fish. Plasma activity of lysozyme was higher (P<0.01) in rbGH-exposed fish. Compared to day 0 controls (non-exposed fish), IGF-I levels decreased (P<0.05) in challenged fish while levels were similar (P>0.10) between treatments. Abundance of GH receptor (GHR) mRNA tended to decrease (P=0.055) in liver of challenged fish while toll like receptor 5 (TLR5) mRNA increased (P<0.05) in liver compared to d 0 controls. An increase in lysozyme may suggest GH enhances a nonspecific immune response. A decrease in GHR mRNA and plasma IGF-I suggests a downregulation of the somatotropic axis in response to disease. The increase in TLR5 mRNA suggests that TLR5 may play a role in host response to bacterial challenge. While exogenous rbGH may play a stimulatory role to increase lysozyme levels, there was no apparent effect of rbGH on mortality to E. ictaluri.     

Molecular responses of ceruloplasmin to Edwardsiella ictaluri infection and iron overload in channel catfish (Ictalurus punctatus)

Ceruloplasmin is a serum ferroxidase that carries more than 90% of the copper in plasma and has documented roles in iron homeostasis as well as antioxidative functions. In our previous studies, it has been shown that the ceruloplasmin gene is strongly up-regulated in catfish during challenge with Edwardsiella ictaluri. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the ceruloplasmin gene from channel catfish, determine its genomic organization, profile its patterns of tissue expression, and establish its potential for physiological antioxidant responses in catfish after bacterial infection with E. ictaluri and iron treatment. The genomic organization suggested that the catfish ceruloplasmin gene had 20 exons and 19 introns, encoding 1074 amino acids. Exon sizes of the catfish ceruloplasmin gene were close to or identical with mammalian and zebrafish homologs. Further phylogenetic analyses suggested that the gene was highly conserved through evolution. The catfish ceruloplasmin gene was mapped to both the catfish physical map and linkage map. The catfish ceruloplasmin gene was mainly expressed in liver with limited expression in other tissues, and it was significantly up-regulated in the liver after bacterial infection alone or after co-injection with bacteria and iron-dextran, while expression was not significantly induced with iron-dextran treatment alone.     

Effect of temperature on the immune system of channel catfish (Ictalurus punctatus)--II. Adaptation of anterior kidney phagocytes to 10 degrees C.

1. Anterior kidney phagocytes from channel catfish (Ictalurus punctatus) exposed to 10 and 24 degrees C from 1 day to 5 weeks were assayed for phagocytic ability and respiratory burst activity to Aeromonas hydrophila and Edwardsiella ictaluri. 2. The results of this study indicated that phagocytosis in channel catfish remained partially functional at low temperature without adaptation, although partial suppression was observed. 3. Adaptation to low temperature did lead to an improvement in the respiratory burst which would imply improved bacterial killing ability. 4. Our study suggests that phagocytosis may play a significant role in preventing disease at low environmental temperature.     

Signature-tagged mutagenesis of Edwardsiella ictaluri identifies virulence-related genes, including a salmonella pathogenicity island 2 class of type III secretion systems

Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the Salmonella pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type E. ictaluri bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.     

Genome sequence of Edwardsiella ictaluri 93-146, a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish

Edwardsiella ictaluri is the cause of extensive mortalities and economic losses to the channel catfish industry of the southeast United States. Here we report the complete genome of Edwardsiella ictaluri 93-146. Whole-genome sequence analysis of E. ictaluri provides a tool for understanding the genomic regions specific to the species and the Edwardsiella genus.     

Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri

Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.