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.     

Susceptibility of European catfish (Silurus gianis) to Edwardsiella ictaluri

European catfish (Silurus glanis) were tested for their susceptibility to the bacterium Edwardsiella ictaluri. The LD₅₀ of E. ictaluri when injected into European catfish was 5.4 × 10⁶ compared to 7.1 times; 10⁴ for channel catfish (Ictalurus punctatus). E. ictaluri was isolated from dead and moribund European catfish and the bacterium was also detected in kidney smears by an indirect fluorescent antibody (FA) technique. The bacterium was not isolated or detected by FA from surviving fish 15 days after injection. No clinical signs of E. ictaluri infection were noted in European catfish, but these were prevalent in the channel catfish. These experiments indicate that under experimental conditions European catfish are not as susceptible to E. ictaluri as channel catfish.     

Synergies between vaccination and dietary arginine and glutamine supplementation improve the immune response of channel catfish against Edwardsiella ictaluri

Channel catfish was used to investigate the enhancement of vaccine efficacy following dietary supplementation with arginine (ARG, 4% of diet), glutamine (GLN, 2% of diet), or a combination of both. After vaccination against Edwardsiella ictaluri, humoral and cellular immune responses, along with lymphoid organ responses were evaluated. E.?ictaluri-specific antibody titers in plasma were higher (P?相似文献   

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.     

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.     

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.     

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.     

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).     

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.     

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.     

Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare

Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease.     

Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring

Edwardsiella ictaluri is a facultative intracellular bacterium that causes enteric septicemia of catfish (ESC). In this study, we aimed to develop bioluminescent E. ictaluri that can be monitored by noninvasive bioluminescence imaging (BLI). To accomplish this, the luxCDABE operon of Photorhabdus luminescens was cloned downstream of the lacZ promoter in the broad host range plasmid pBBR1MCS4. Edwardsiella ictaluri strain 93-146 transformed with the new plasmid, pAKlux1, was highly bioluminescent. pAKlux1 was stably maintained in E. ictaluri without any apparent effect on growth or native plasmid stability. To assess the usefulness of the bioluminescent strain in disease studies, catfish were infected with 93-146 pAKlux1 by intraperitoneal injection and by bath immersion, and in vivo bacterial dissemination was observed using BLI. This study demonstrated that bioluminescent E. ictaluri can be used for real-time monitoring of ESC in live fish, which should enable observation of pathogen attachment sites and tissue predilections.     

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.     

Edwardsiella comparative phylogenomics reveal the new intra/inter-species taxonomic relationships, virulence evolution and niche adaptation mechanisms

Edwardsiella bacteria are leading fish pathogens causing huge losses to aquaculture industries worldwide. E. tarda is a broad-host range pathogen that infects more than 20 species of fish and other animals including humans while E. ictaluri is host-adapted to channel catfish causing enteric septicemia of catfish (ESC). Thus, these two species consist of a useful comparative system for studying the intricacies of pathogen evolution. Here we present for the first time the phylogenomic comparisons of 8 genomes of E. tarda and E. ictaluri isolates. Genome-based phylogenetic analysis revealed that E. tarda could be separate into two kinds of genotypes (genotype I, EdwGI and genotype II, EdwGII) based on the sequence similarity. E. tarda strains of EdwGI were clustered together with the E. ictaluri lineage and showed low sequence conservation to E. tarda strains of EdwGII. Multilocus sequence analysis (MLSA) of 48 distinct Edwardsiella strains also supports the new taxonomic relationship of the lineages. We identified the type III and VI secretion systems (T3SS and T6SS) as well as iron scavenging related genes that fulfilled the criteria of a key evolutionary factor likely facilitating the virulence evolution and adaptation to a broad range of hosts in EdwGI E. tarda. The surface structure-related genes may underlie the adaptive evolution of E. ictaluri in the host specification processes. Virulence and competition assays of the null mutants of the representative genes experimentally confirmed their contributive roles in the evolution/niche adaptive processes. We also reconstructed the hypothetical evolutionary pathway to highlight the virulence evolution and niche adaptation mechanisms of Edwardsiella. This study may facilitate the development of diagnostics, vaccines, and therapeutics for this under-studied pathogen.     

Structural characterisation and expression analysis of toll-like receptor 2 gene from catfish

Toll-like receptors (TLRs) are important components of innate immunity. They were found to recognise specific structures on pathogens termed pathogen-associated molecular patterns (PAMPs) and utilise conserved signaling pathways to activate pro-inflammatory cytokines and type-1 interferons. In spite of much understanding gained from the mammalian systems, many fish TLRs are unknown. Recent studies in Japanese flounder as well as in zebrafish suggested that the ligand binding and activation of inflammatory responses in fish may be different from and more complex than those found in mammals. In channel catfish, the major aquaculture species in the United States, only partial sequences of TLR3 and TLR5 were reported. As a part of efforts to characterise the innate immune components in channel catfish, here we cloned and sequenced both the cDNA and the gene for TLR2, a receptor believed mostly responsible for recognition of lipopeptides on the surface of most Gram-positive bacteria. However, expression analysis after infection with a Gram-negative bacterium, Edwardsiella ictaluri indicated that TLR2 was modestly down-regulated in the head kidney tissue of blue catfish, and with a similar pattern in the head kidney of channel catfish though the down-regulation in channel catfish was not statistically significant. In the spleen, an insignificant down-regulation was initially observed early after infection, with an increase of TLR expression later after infection. These results suggest the involvement of TLR2 in the responses after the bacterial infection. As LPS is believed to be the major PAMP for Gram-negative bacteria, additional research is warranted to determine the functions and mechanisms of TLR2 in infections of Gram-negative bacteria.     

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.     

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.     

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.