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.     

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.     

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.     

Molecular characterization of three L-type lectin genes from channel catfish, Ictalurus punctatus and their responses to Edwardsiella ictaluri challenge

L-type lectins have a leguminous lectin domain and can bind to high-mannose type oligosaccharides. In the secretory pathway, L-type lectins play crucial roles in selective protein trafficking, sorting and targeting. Three L-type lectins were cloned in the channel catfish, Ictalurus punctatus, the 53 kDa endoplasmic reticulum ER-Golgi intermediate compartment protein (ERGIC-53), the vesicular integral protein of 36 kDa (VIP36) and VIP36-like. Phylogenetic analysis indicated that the catfish genes are orthologous to their counterparts in other species. Southern blot analysis demonstrated that all three L-type lectin genes are likely single-copy genes in the catfish genome. Analysis of expression in healthy tissues using quantitative real time RT-PCR indicated that all three genes are expressed widely in all tested tissues, but with strong tissue preference of expression: ERGIC-53 was found to be abundantly expressed in the liver, VIP36 was found to be abundantly expressed in the head-kidney, whereas VIP36-like was found to be abundantly expressed in the brain. Upon infection with Edwardsiella ictaluri, expressions of the three genes all had significant up-regulation in the head-kidney, but had distinct expression patterns: ERGIC-53 was gradually induced with the highest expression 7 days after challenge in the head-kidney, but was down-regulated in the liver, spleen, and brain. VIP36 was highly induced in the head-kidney, and 3 days after challenge in the brain, but was not up-regulated in any other tissues or timepoints after challenge. Expression levels of the catfish VIP36-like gene appeared to also respond to infection, albeit with differing patterns among the tested tissues. Taken together, our results indicate that all three L-type lectin genes may be involved in the immune responses of catfish after infection with E. ictaluri.     

Survivability and immune responses after challenge with Edwardsiella ictaluri in susceptible and resistant families of channel catfish, Ictalurus punctatus

Diseases in catfish farming are prevalent and costly, particularly the bacterial disease Enteric Septicemia of Catfish. Considerable research has focused on different aspects of this disease, including the biology of the causative agent, Edwardsiella ictaluri. However, no satisfactory treatment or preventive has resulted from these efforts. One solution is to increase the natural disease resistance of the fish through genetic selection. Recent research has demonstrated that genetic factors influence resistance to infection in mammals as well as fish. Selective breeding for disease resistance in channel catfish is ongoing, however differences in defence mechanisms among E. ictaluri challenged strains and families are only now being investigated. Antigen-specific as well as non-specific immune responses of full-sib families of channel catfish to laboratory challenge with E. ictaluri have been investigated. Both resistant and sensitive families produce a humoral response as specific antibody, but there were no differences found in the level of specific antibody produced. The sensitive family produced a slightly higher percentage of B lymphocytes in mononuclear cell preparations from peripheral blood, while the resistant family had a higher percentage of T lymphocytes in those preparations. The most significant observation was that the resistant family produced more macrophage aggregations in the spleen and posterior kidney throughout the infection than the sensitive family. Neither family produced stress-associated amounts of cortisol.     

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection

An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E. ictaluri proteins using anti-E. ictaluri serum, which resulted in the isolation of 32 clones. The clones were partially characterized and 4 were selected for complete analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes. Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E. ictaluri cells. The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv. trifolii. The imp locus of R. leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system. In addition there was significant amino acid identity for a variety of hypothetical proteins from R. solanacearum, Ps. aeruginosa, A. tumefaciens, Y. pestis, and Salmonella typhimurium. Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda. These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk). Cloned antigenic E. ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively. All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process. The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E. ictaluri. All vaccine treatments were protective against E. ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E. coli host strain of the cloning vector.     

Pathology associated with Edwardsiella ictaluri in catfish, Ictalurus punctatus Rafinesque, and Danio devario (Hamilton-Buchanan, 1822)

The pathology associated with infections of Edwardsiella ictaluri in a new host, the danio, is described and compared to that observed in the channel catfish. In catfish the acute stage was characterized by petechial haemorrhage of the jaw, ventral body surface, and at the base of the fins. In chronic cases a characteristic finding was an erosion of skin and muscle overlying the skull, exposing bone and occasionally the brain. Histologically there was a diffuse granulomatous inflammation of the olfactory bulb and the telencephalon. In the catfish the infection was often systemic, involving intestine, liver, spleen, and occasionally kidney. In the danio no gross lesions were observed. Infected fish exhibited an erratic, spinning-type swimming behaviour. Histologically, lesions usually were confined to the brain. They consisted of an acute, primarily granulocytic inflammation of the medulla oblongata (rhomboencephalon). Only one fish showed evidence of a systemic infection.     

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.     

Gastrointestinal torsion in a channel catfish (Ictalurus punctatus)

A case of gastrointestinal torsion with dilatation in a farm-raised channel catfish (Ictalurus punctatus) was examined at the Thad Cochran National Warmwater Aquaculture Center (Stoneville, Mississippi, USA). The affected fish was a gravid female broodfish, which displayed pale gills and a markedly distended abdomen. Internal examination revealed that the gastrointestinal tract and ovaries were rotated around each other four times in a counterclockwise direction as viewed in right lateral recumbency. The catfish had a markedly distended gastrointestinal tract, pale liver, hypoplastic spleen, hypoplastic swim bladder, and high volume of ascitic fluid. Blood analysis indicated multiple abnormalities, including severe anemia and metabolic acidosis. The etiology of the torsion was uncertain; however, the presence of a hypoplastic swim bladder most likely allowed for increased movement of the gastrointestinal tract and ovaries. When examining cases of abdominal distention in fish, gastrointestinal torsion can be considered among the differential diagnoses.     

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.     

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly three decades ago ^1,2, so far, to the best of these authors'' knowledge, no method has been developed to allow for the in situ visualization of the bacteria in histological sections.While bacterial localization has been determined in vivo in previous studies using plate counts ³, radiometric labeled ⁴, or bioluminescent bacteria ⁵, most of these studies have only been performed at the gross organ level, with one exception ⁶. This limitation is of particular concern because E. ictaluri has a complex infection cycle ^1,7, and it has a variety of virulence factors ^8,9. The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi ¹⁰, which is in the same taxonomic family.Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.¹¹.Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium.This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.Download video file.^{(33M, mov)}     

Cortisol-induced hematologic and immunologic changes in channel catfish (Ictalurus punctatus)

1. Intravenous injections of physiologic doses of cortisol resulted in both hematologic and immunologic changes in channel catfish peripheral blood leucocytes. These changes mimicked those seen when catfish were acutely stressed by handling and transport. 2. Eighteen hours after the administration of cortisol, decreases in the number of circulating lymphocytes and concomitant increases in the number of circulating neutrophils were observed, i.e. to the same levels seen previously in stressed fish. 3. Functional analysis of peripheral blood leucocytes from cortisol-injected fish indicated that the remaining lymphocytes were no longer capable of responding to mitogenic stimuli. 4. This suppression of mitogenic stimuli was not seen when peripheral blood leucocytes were cultured in vitro with physiologic doses of cortisol. 5. This latter observation suggests that the cortisol alone was probably not directly responsible for the loss of responsiveness but possibly acted in vivo as an initiator of other events that eventually resulted in the observed immunosuppression.     

Histology of cultured channel catfish, Ictalurus punctatus (Rafinesque)

Dissolved oxygen and un-ionized ammonia concentrations were monitored in 12 0.04 ha earthen ponds stocked with 10 000 channel catfish, Ictalurus punctatus ,/ha. Gill, liver and trunk kidney tissue samples were removed periodically for histological examination. Total ammonia and dissolved oxygen levels were in the ranges reported for catfish culture at this level of intensity. Average un-ionized ammonia nitrogen concentrations ranged from 20 to 67 μgh⁻¹ and average daily maxima ranged from 63 to 183 μgh⁻¹. Gill lesions characteristic of un-ionized ammonia exposure were common in fish from all ponds.     

Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus)

Aim: To develop a method for conducting pulsed-field gel electrophoresis (PFGE) on Flavobacterium columnare, to use PFGE to characterize F. columnare channel catfish isolates, and to determine whether variation in pathogenic potential exists in F. columnare isolates from channel catfish. Methods and Results: On the basis of PFGE-derived profiles, similarity dendrograms constructed for more than 30 F. columnare isolates showed two major genetic groups with more than 60% similarity. Channel catfish fingerlings challenged with PFGE group A isolates by bath immersion had significantly higher average mortalities (>60%) than fish challenged with PFGE group B isolates (<9%). However, abrasion and skin mucus removal made channel catfish fingerlings susceptible to disease caused by group B isolates following immersion exposure. Conclusion: Our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, and that isolates in PFGE group A isolates tend to be more pathogenic to immunocompetent channel catfish fingerlings than PFGE group B isolates. Significance and Impact of the Study: PFGE is a potentially useful tool for determining whether F. columnare isolates are more likely to be primary or secondary pathogens. Pathogenesis research for columnaris disease in catfish should focus on pathogenic isolates from PFGE group A.     

Ultrastructure of the liver in channel catfish Ictalurus punctatus (Rafinesque)

Livers of 38 channel catfish were studied by light and electron microscopy and the morphology of hepatocytes, endothelial cells, Kupffer cells, fat-storing cells, macrophages, exocrine pancreatic cells, and epithelium of bile pre-ductules and ducts was described, Hepatocytes contained large peri-canalicular bodies which showed acid hydrolase activity. A comparison between catfish liver and that of other teleosts was made. The applicability of the findings to pathobiological studies of teleost liver is described.     

Immune hypersensitivity in the channel catfish, Ictalurus punctatus (Rafinesque)

Channel catfish, Ictalurus punctatus , exhibited passive cutaneous anaphylaxis (PCA) when sensitized with Flexibacter columnaris antiserum produced in channel catfish (the first record of PCA in any fish), but not when sensitized with homologous antiserum against channel catfish virus. Channel catfish did not give delayed skin hypersensitivity responses after immunization with either channel catfish virus or F. columnaris .