Characterization of a Major Outer-Membrane Antigen of Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri is the cause of enteric septicemia of catfish. A monoclonal antibody (MAb AA224) was used to identify a specific and predominant outer-membrane antigen of E. ictaluri. The MAb AA224 was produced by conventional cell fusion technology with spleen cells from mice immunized with an affinity-purified antigen. The affinity-purified antigen was obtained by immunoaffinity chromatography of an E. ictaluri extract with immunoaffinity purified immunoglobin from sera of channel catfish Ictalurus punctatus immune to E. ictaluri as a result of natural infection. The immunoaffinity-purified antigen was used for immunization and identification of the hybridoma producing MAb AA224 by enzyme-linked immunosorbent assay. The predominant antigen was purified by immunoaffinity chromatography with MAb AA224 as the immunoadsorbent. Immunoblotting and high-pressure liquid chromatography were used to determine that the relative sizes of the predominant antigens are 60 and 36 kilodaltons. Immunoelectron microscopy with MAb AA224 conjugated with colloidal gold localized the predominant antigen on the surface of the outer membrane of E. ictaluri     

Fish-Eating Birds as Potential Vectors of Edwardsiella ictaluri

Abstract

Intestinal and rectal smears from 137 birds (4 snowy egrets Egretta thula, 22 great egrets Casmerodius albus, 30 great blue herons Ardea herodias, and 81 double-crested cormorants Phalacrocorax auritus) were examined by indirect fluorescent antibody test for the presence of Edwardsiella ictaluri. Edwardsiella ictaluri was detected in 53% of the birds sampled. Rectal samples from eight birds were placed in a special antibiotic broth for isolation of viable E. ictaluri. Two of these samples produced colonies of viable E. ictaluri, and the identity of these colonies was confirmed biochemically and serologically.     

Biochemical,Biophysical, and Serological Homogeneity of Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish, which, during the past 5 years, has become the most serious infectious disease problem of cultured channel catfish Ictalurus punctatus. We compared 40 isolates of E. ictaluri from different geographical regions and host fish species. From the biophysical tests, a pH of 7.0–7.5 and a temperature of 25–30°C were optimum growth conditions for all E. ictaluri isolates. All isolates grew well in media with an NaCl concentration of 0.5% or less, but none of the E. ictaluri isolates grew in media with a concentration of 2.0 or 5.0% NaCl. Biochemically, 42 out of 46 tests gave the same reaction for all 40 isolates. The only observed differences were in gas production at 25°C, the o-nitrophenylbeta-D-galactopyranoside test, ornithine decarboxylation, and D-mannose utilization. Serologically, identical agglutinin titers (1:80) to E. ictaluri-specific rabbit antisera were observed, and all isolates cross-agglutinated with four different antisera. Based on the biophysical, biochemical, and serological reactions of 40 isolates of E. ictaluri, identification of distinct strains was not possible, although some were slightly different biotypically.     

Specificity of Humoral Responses of Convalescent Channel Catfish to Edwardsiella ictaluri

Abstract

Serum samples from 15 yearling channel catfish Ictalurus punctatus convalescing after experiencing enteric septicemia of catfish were distributed into three representative serum pools, each containing equal volumes of serum from five fish. Serologic recognition of each pooled serum sample against Edwardsiella ictaluri and Escherichia coli whole cells and against secretory antigen (exoantigen) derived from E. ictaluri was measured by enzyme-linked immunosorbent assays (ELISA). Serum samples were purified by affinity chromatography with a heterologous Ra,-mutant lipopolysaccharide that was derived from rough Salmonella typhimurium TV 119 and was covalently bound to an agarose matrix. Removal of antibodies recognizing the lipopolysaccharide by cross-reactive affinity purification caused significant decreases in serologic recognition of E. ictaluri (P < 0.10) and E. coli (P < 0.01) whole cells; however, serologic recognition of the E. ictaluri-specific exoantigen was not significantly decreased. These results indicate that serologic recognition of the exoantigen is highly specific and that cross-reactive immune responses recognizing homologous gram-negative core antigens will not cause false-positive test results when the specific capture ELISA is used to detect exposure to E. ictaluri     

Edwardsiella ictaluri as the causative agent of mortality in cultured Nile tilapia

Edwardsiella ictaluri was consistently isolated from the spleens, livers, and head kidneys of diseased Nile tilapia Oreochromis niloticus from a farm experiencing mortality events in several culture ponds. We describe the first published outbreak of E. ictaluri-induced edwardsiellosis in Nile tilapia. Pure cultures of the isolated bacteria were characterized both biochemically and molecularly. Biochemical analysis was performed using the API-20E and RapID One systems, and antimicrobial susceptibility was determined by the broth microdilution method. Molecular analysis involved sequencing of the 16S rRNA gene, species-specific real-time polymerase chain reaction (PCR), and PCR-mediated genomic fingerprinting (rep-PCR). Pairwise sequence analysis of the 16S rRNA gene identified the case isolates to be a 100% match to E. ictaluri cultured from channel catfish in the southeastern United States. However, rep-PCR analysis identified the case isolates to be genetically different from representative strains isolated from disease outbreaks in cultured channel catfish in Mississippi. Infectivity challenges (intraperitoneal injection and immersion) demonstrated that a representative E. ictaluri strain isolated from tilapia was pathogenic to naive tilapia, reproducing clinical signs and mortality, thereby establishing Koch's postulates.     

Edwardsiella ictaluri as the Causative Agent of Mortality in Cultured Nile Tilapia

Abstract

Edwardsiella ictaluri was consistently isolated from the spleens, livers, and head kidneys of diseased Nile tilapia Oreochromis niloticus from a farm experiencing mortality events in several culture ponds. We describe the first published outbreak of E. ictaluri–induced edwardsiellosis in Nile tilapia. Pure cultures of the isolated bacteria were characterized both biochemically and molecularly. Biochemical analysis was performed using the API-20E and RapID One systems, and antimicrobial susceptibility was determined by the broth microdilution method. Molecular analysis involved sequencing of the 16S rRNA gene, species-specific real-time polymerase chain reaction (PCR), and PCR-mediated genomic fingerprinting (rep-PCR). Pairwise sequence analysis of the 16S rRNA gene identified the case isolates to be a 100% match to E. ictaluri cultured from channel catfish in the southeastern United States. However, rep-PCR analysis identified the case isolates to be genetically different from representative strains isolated from disease outbreaks in cultured channel catfish in Mississippi. Infectivity challenges (intraperitoneal injection and immersion) demonstrated that a representative E. ictaluri strain isolated from tilapia was pathogenic to naïve tilapia, reproducing clinical signs and mortality, thereby establishing Koch's postulates.

Received August 30, 2011; accepted January 30, 2012     

Communications: Specificity of the Channel Catfish Antibody to Edwardsiella ictaluri

Abstract

The specificity of channel catfish Ictalurus punctatus serum antibody to Edwardsiella ictaluri was characterized by microtiter agglutination assay. There was no correlation between antibody titer to Aeromonas hydrophila and antibody titer to E. ictaluri in wild or feral channel catfish. Anti-E. ictaluri antibodies in naturally infected channel catfish were not removed by adsorption by nine other species of bacteria found in the channel catfish intestine and fish ponds. Channel catfish immunized with nine other species of bacteria did not develop substantial antibody titer to E. ictaluri. The antibody response of channel catfish to E. ictaluri is highly specific, and the microtiter agglutination test is a specific indicator of previous exposure to E. ictaluri     

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretic and Immunochemical Characterization of Lipopolysaccharide of Edwardsiella ictaluri from Channel Catfish

Abstract

Lipopolysaccharide (LPS) was purified from 40 isolates of Edwardsiella ictaluri by two methods: (1) enzyme digestion and hot aqueous phenol extraction and (2) enzyme digestion and gel exclusion chromatography. Purified LPS was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblot analysis. Both methods of purification yielded smooth LPS as evidenced by a ladderlike pattern of more than 40 LPS bands. With silver staining, both low- and high-molecular-mass LPS bands were seen. Lower-molecularmass LPS stained more intensely than higher-molecular-mass LPS bands, indicating a preponderance of lower-molecular-mass LPSs. Lipopolysaccharide bands from the 40 isolates migrated similarly within SDS-PAGE gels, indicating a high degree of structural similarity among the isolates examined. The ladderlike array was more easily seen with immunoblot analysis than with silver staining of SDS-PAGE gels. Additionally, immunoblot analysis revealed a high degree of antigenic similarity among the 40 isolates.     

Transmission of Edwardsiella ictaluri from Infected,Dead to Noninfected Channel Catfish

Abstract

Enteric septicemia of catfish (ESC) was transmitted horizontally from channel catfish Icialurus punctatus that had died from Edwardsiella ictaluri infection to contact channel catfish during 2 d of habitation in a tank. The contact channel catfish became positive for E. ictaluri antibody, became infected with this bacterium, and had signs of ESC and died within 12 d postexposure. Edwardsiella ictaluri was recovered from 24 of the 30 contact channel catfish that died from ESC, as well as from 9 of the 25 tested contact survivors. The cannibalizing of E. ictaluri-infected fish, or the shedding of E. ictaluri from dead fish, or both, were shown to be mechanisms of horizontal transmission of ESC among channel catfish.     

Cool Weather Feeding Influences Responses of Channel Catfish to Edwardsiella ictaluri Challenge

Abstract

A pond study was conducted from November 1 through April 30 with young (age-0; average size, 43 g) and market-size (age-2; average size, 660 g) channel catfish Ictalurus punctatus to compare three management regimens: No feeding, partial feeding (no feeding in December, January, and February), and continuous feeding according to fish size and water temperature. Weight change, feed conversion ratio, and responses to experimental challenge with Edwardsiella ictaluri were evaluated. No significant difference in weight gain occurred between partially fed and continuously fed fish in either age-group; average weight increase for fed age-0 fish was 99% and for fed age-2 fish was 38%. The nonfed age-0 fish lost 12.3% and the nonfed age-2 fish lost 7% of their initial weight. Feed conversion ratios were significantly lower in partially fed fish than in continuously fed fish in both age-groups. Mortality from E. ictaluri challenge among age-0 fish was significantly higher in the nonfed fish, but among the age-2 fish, mortality was significantly lower in the nonfed fish. No difference in mortality rate occurred between partially fed and continuously fed fish in either age-group. Starvation induced lower antibody production against E. ictaluri antigen in the age-0 fish but higher antibody production in the age-2 fish. Phagocytic index was lower in nonfed fish than in fed fish from both age-groups. This study indicates no benefit from feeding age-0 and age-2 channel catfish during December, January, or February if feeding is reintroduced in March and continued through April. Although starvation was immunosuppressive in small channel catfish, it enhanced resistance to bacterial infection in larger fish. However, more research information is needed before reduced feeding can be recommended to enhance resistance of channel catfish to E. ictaluri.     

Effect of Common Aquaculture Chemicals against Edwardsiella ictaluri and E. tarda

Abstract

Edwardsiellosis is an important bacterial infection of freshwater and marine fishes. Edwardsiella ictaluri causes enteric septicemia of catfish, and E. tarda causes emphysematous putrefactive disease of catfish and fish gangrene in various species; these diseases have considerable economic effects on the aquaculture industry. In addition, E. tarda is an important zoonotic pathogen. Thus, the reduction or elimination of these pathogens from an aquarium or aquaculture facility is imperative. This study examined a variety of commercially available chemicals for their ability to reduce or eliminate E. ictaluri and E. tarda from the aquatic environment. The various concentrations of chemicals were tested in vitro in microcentrifuge tubes with a known concentration of bacteria at room temperature. In this study, ethyl alcohol (30, 50, or 70%), benzyl-4-chlorophenol/phenylphenol (1%), sodium hypochlorite (50, 100, 200, or 50,000 mg/L), n-alkyl dimethyl benzyl ammonium chloride (1:256), povidone iodine (50 or 100 mg/L), glutaraldehyde (2%), and potassium peroxymonosulfate/sodium chloride (1%) were effective disinfectants, as each reduced or eliminated the number of detectable organisms within 1 min of contact time. However, neither Chloramine-T (15 mg/L) nor formalin (250 mg/L) substantially reduced bacterial counts even after 60 min of contact time.     

Modified Live Edwardsiella ictaluri Vaccine,AQUAVAC-ESC,Lacks Multidrug Resistance Plasmids

Abstract

Plasmid-mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish Ictalurus punctatus infected with the bacterium. Due to the identification of multidrug resistance plasmids in aquaculture and their potential clinical importance, we sought to determine whether the modified live E. ictaluri vaccine strain in AQUAVAC-ESC harbors such plasmids, so that the use of this vaccine will not directly contribute to the pool of bacteria carrying plasmid-borne resistance. Antimicrobial sensitivity testing of the E. ictaluri parent isolate and vaccine strain demonstrated that both were sensitive to 15 of the 16 antimicrobials tested. Total DNA from each isolate was analyzed by polymerase chain reaction (PCR) using a set of 13 primer pairs specific for conserved regions of the IncA/C plasmid backbone, and no specific products were obtained. PCR-based replicon typing of the parent isolate and vaccine strain demonstrated the absence of the 18 commonly occurring plasmid incompatibility groups. These results demonstrate that the vaccine strain does not carry resistance to commonly used antimicrobials and provide strong support for the absence of IncA/C and other commonly occurring plasmid incompatibility groups. Therefore, its use should not directly contribute to the pool of bacteria carrying plasmid-borne resistance. This work highlights the importance of thoroughly investigating potential vaccine strains for the presence of plasmids or other transmissible elements that may encode resistance to antibiotics.

Received May 6, 2011; accepted July 22, 2011     

A Real-Time Polymerase Chain Reaction Assay of the Bacterium Edwardsiella ictaluri in Channel Catfish

Abstract

A rapid (4.5-h) and sensitive assay based on polymerase chain reaction (PCR) was developed to facilitate the early detection of Edwardsiella ictaluri in channel catfish Ictalurus punctatus. A 129-base-pair fragment of a sequence specific to E. ictaluri was amplified with both standard and real-time (quantitative) PCR. The sensitivity of detection was determined to be as low as the equivalent of 2.5 cells in DNA samples from both E. ictaluri cells and mixtures of blood from noninfected catfish and E. ictaluri cells. Infection levels (as determined by real-time PCR) in blood from experimentally challenged fish were compared with brain–heart-infusion-cultured bacterial colony counts to assess the accuracy of the PCR assay. The PCR-based detection level (the equivalent of 10⁵–10⁸ cells/mL) was comparable to that of traditional culturing techniques (10⁶–10⁷ cells/mL). In future applications, this assay will be applied in a comprehensive breeding program to select channel catfish that are resistant to enteric septicemia of catfish.     

Olfactory Organ of Channel Catfish as a Site of Experimental Edwardsiella ictaluri Infection

Abstract

Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), is one of the most important pathogens to infect channel catfish Ictalurus punctatus. Although the full pathogenesis of E. ictaluri is unclear, the olfactory organ is thought to be a site of entry. We have examined the effects of applying E. ictaluri directly into the olfactory capsule of channel catfish. Olfactory organs of 30 experimental fish were exposed to E. ictaluri for 1 h (1 mL, 1 × 10⁶ colony-forming units/mL). Live fish were sampled at 1, 24, 48, and 72 h, and days 5 and 14 postinfection, and their olfactory organs were examined by light and electron microscopy. Damage, including loss of sensory cilia and microvilli from the olfactory mucosal surface, was observed at 1 h postinfection. Degeneration of olfactory receptors and supporting cells was evident by 24 h postinfection. The nonsensory region also showed signs of degeneration, such as columnar cells lacking cilia. Electron microscopic immunocytochemistry confirmed the presence of E. ictaluri on the mucosal surface and within the epithelium. Host leukocytes responded to bacteria by migrating through the olfactory epithelium into the interlamellar lumen and phagocytosing organisms, but phagocytosed E. ictaluri did not appear to be destroyed. Our results indicate that during initial stages of infection channel catfish olfactory epithelium is vulnerable, and E. ictaluri can enter the host through the olfactory organ. It is also possible that host phagocytic cells serve as a vehicle for the systemic dissemination of E. ictaluri     

Chemotactic and Chemokinetic Responses of Channel Catfish Macrophages to Exoantigen from Edwardsiella ictaluri

Abstract

The ability of Edwardsiella ictaluri to attract macrophages of channel catfish Ictalurus punctatus was investigated. Exoantigen from E. ictaluri was tested for macrophage chemotactic activities both in vitro and in vivo. The exoantigen was chemotactic and chemokinetic for macrophages in vitro. Peritoneal injection of 750 μg of exoantigen protein into normal (E. ictaluri-free) channel catfish induced a marked increase in macrophage accumulations at 24 and 48 h. Neutrophil accumulation did not occur at the injection sites. Edwardsiella ictaluri exoantigen attracts macrophages, and this attraction may play an important role in macrophage responses during E. ictaluri infections.     

Decreased Resistance to Edwardsiella ictaluri Infection in Channel Catfish Intraperitoneally Administered an Oil Adjuvant

Abstract

The effects of intraperitoneal injection of squalene, an oil adjuvant, on nonspecific mortality of channel catfish Ictalurus punctatus and on their resistance to experimental Edwardsiella ictaluri infection were studied. Yearling channel catfish were assigned to control (N = 22) or squalene (N = 25) treatment groups, and mortality was monitored for 14 d following treatment. On day 14 both groups were infected with E. ictaluri, the causative agent of enteric septicemia of catfish, and mortality was monitored for an additional 11 d. Before infection, mortality did not differ between groups. After E. ictaluri infection, fish that received squalene were at a substantially higher risk of dying than control fish (relative risk after squalene treatment = 6.86). These results suggest that intraperitoneal administration of squalene, although not directly toxic, decreased resistance to E. ictaluri infection.     

Effect of common aquaculture chemicals against Edwardsiella ictaluri and E. tarda

Edwardsiellosis is an important bacterial infection of freshwater and marine fishes. Edwardsiella ictaluri causes enteric septicemia of catfish, and E. tarda causes emphysematous putrefactive disease of catfish and fish gangrene in various species; these diseases have considerable economic effects on the aquaculture industry. In addition, E. tarda is an important zoonotic pathogen. Thus, the reduction or elimination of these pathogens from an aquarium or aquaculture facility is imperative. This study examined a variety of commercially available chemicals for their ability to reduce or eliminate E. ictaluri and E. tarda from the aquatic environment. The various concentrations of chemicals were tested in vitro in microcentrifuge tubes with a known concentration of bacteria at room temperature. In this study, ethyl alcohol (30, 50, or 70%), benzyl-4-chlorophenol/phenylphenol (1%), sodium hypochlorite (50, 100, 200, or 50,000 mg/L), n-alkyl dimethyl benzyl ammonium chloride (1:256), povidone iodine (50 or 100 mg/L), glutaraldehyde (2%), and potassium peroxymonosulfate/sodium chloride (1%) were effective disinfectants, as each reduced or eliminated the number of detectable organisms within 1 min of contact time. However, neither Chloramine-T (15 mg/L) nor formalin (250 mg/L) substantially reduced bacterial counts even after 60 min of contact time.     

Use of a Mini-Transposon to Study Chondroitinase Activity Associated with Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), is the leading cause of bacterial disease in commercially raised channel catfish Ictalurus punctatus. Little work has been conducted at a genotypic level to determine potential virulence characteristics, but the production of chondroitin sulfatase is a suspected virulence factor. Using transpositional mutagenesis, we created stable E. ictaluri mutants that are deficient in chondroitinase activity. Channel catfish were challenged by injection with E. ictaluri transposon mutant MI15. None of the catfish challenged with the mutant died or showed signs of ESC. These fish were held for 2 weeks and then challenged by injection with the known virulent parent strain of E. ictaluri. The challenged naive control fish showed clinical signs of and a mortality rate consistent with ESC, whereas catfish that had been injected with MI15 prior to challenge with the parent strain were resistant to disease. This work represents a preliminary study to suggest a possible role of chondroitin sulfatase activity in the virulence of E. ictaluri.     

Effect of Temperature and Dissolved Oxygen Concentration on Edwardsiella ictaluri in Experimentally Infected Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were experimentally infected with Edwardsiella ictaluri by immersion exposure. After clinical disease ran its course for 52 d, the surviving fish were exposed to one of the following environmental regimes in troughs: 25°C with aeration, 25°C with no aeration, or variable temperature (18–23°C) with no aeration. After 29 d of exposure to the environmental regimes, various organs and tissues of the fish were assayed to determine the effects of these conditions on E. ictaluri concentrations (colony-forming units/mL of tissue sample). The concentrations of this pathogen were significantly (P < 0.05) higher in all tissues (trunk kidney, liver, head kidney, blood, spleen, gallbladder, muscle, brain, and gonad) 52 d postinfection than 29 d after exposure to any of the environmental regimes (81 d postinfection). Fish exposed to a near-normal concentration of dissolved oxygen (6.4 mg/L) and a constant temperature of 25°C had E. ictaluri concentrations that were significantly (P < 0.01) lower than those offish exposed to a low oxygen concentration (2.6 or 1.8 mg/L) and either a constant or a variable temperature.