Pathogenesis of Enteric Septicemia of Channel Catfish,Caused by Edwardsiella ictaluri: Bacteriologic and Light and Electron Microscopic Findings

Abstract

To clarify early events in the pathogenesis of enteric septicemia of catfish, 140 channel catfish Ictalurus punctatus (8–10 months old) were each infected with approximately 1.0 × 10⁹ colony-forming units of Edwardsiella ictaluri by intragastric intubation. Fish were sacrificed at 0, 0.25, 0.5, 1, 3, 6, 12, 24, 48, 72, 96, and 120 h postinfection (PI). Multiple tissue samples at all scheduled sampling times were evaluated by gross observation, light and electron microscopy, and immunohistochemical methods. In addition, at each sampling time, stomach, intestine, trunk kidney, and liver were cultured to quantitate bacteria. Trunk kidney cultures were positive by 0.25 h PI, indicating rapid transmucosal passage. In the intestine, E. ictaluri was seen in contact with the brush border at 0.5 h PI. Also at 0.5 h PI, dilated basilar cells with large intracytoplasmic inclusions were observed adjacent to the basement membrane. From 1 to 3 h PI, occasional necrotic enterocytes were seen on tips of intestinal folds. Proprial leukocytes were rare before 24 h PI but common thereafter. Immunoelectron microscopy showed E. ictaluri in vacuoles within phagocytes as early as 24 h PI in the intestinal mucosa. In other tissues, earliest observed microscopic lesions (48 h PI) consisted of bacteria within vacuoles of phagocytic cells contained within blood vessels. Bacteria were also seen within degenerate vacuoles in enterocytes and hepatocytes at 72, 96, and 120 h PI. This study confirms that E. ictaluri can invade channel catfish within 0.25 h PI by crossing the intestinal mucosa and suggests that the bacterium may have invasion and survival strategies similar to those of other enteroinvasive members of the Enterobacteriaceae.     

Validation of Fermentation and Processing Procedures for the Commercial-Scale Production of a Live,Attenuated Edwardsiella ictaluri Vaccine for Use in Channel Catfish Aquaculture

Abstract

Mortality associated with Edwardsiella ictaluri infection is a serious impediment to the commercial production of fingerling Channel Catfish Ictalurus punctatus. A patented, live, attenuated, orally delivered vaccine has been developed that offers exceptional protection against E. ictaluri infection in both laboratory and small-scale pond trials. Further vaccine development is contingent on the successful completion of large-scale field trials that accurately reflect industry conditions. This current work focuses on the validation of fermentation protocols and the optimization of downstream processing procedures to produce sufficient quantities of vaccine to conduct commercial-scale field trials. Eight vaccine serials were produced from a master seed stock (S97-773-340X2) in a 50-L floor model fermenter over two consecutive years. Following fermentation, cells were harvested, concentrated 10-fold, and cryogenically stored (?74°C). To assess processing protocols and determine shelf life of cryogenically stored vaccine, serials were tested for cell viability and vaccine potency at various intervals over 24 months. There were no significant differences in cell viability between the fresh vaccine and the stored frozen product. All serials provided a high level of protection (77–100% relative percent survival) against E. ictaluri infection in juvenile Channel Catfish and exhibited excellent poststorage viability. This data demonstrates that the live, attenuated, orally delivered vaccine can be stored at ?74°C for at least 2 years with no reduction in cell viability or vaccine potency.

Received May 17, 2016; accepted January 19, 2017 Published online April 4, 2017     

Enteric Septicemia Resistance in Blue Catfish and Three Channel Catfish Strains

Abstract

Seven full-sib families in each of three strains of channel catfish Ictalurus punctatus (mean weight, 10.7 g; SD, 2.6 g) and one family of blue catfish I. furcatus (mean, 10.9 g; SD, 0.4 g) were challenged by immersion with the bacterium Edwardsiella ictaluri, the causative agent of enteric septicemia, to evaluate variation in disease resistance. Blue catfish were resistant to infection and had only 0.7% mortality. Significant (P < 0.001) variation in channel catfish resistance was found among strains, families, and body weights. Red River strain channel catfish were the most resistant (14.9% mortality), followed by Mississippi-select fish (67.1%) and Mississippi-normal fish (72.3%). Mean family mortality of channel catfish ranged from 1.3% in a Red River family to 95.3% in a Mississippi-select family. Although weight had a significant effect on survival, the regression slope of survival on weight was ?0.10 (SE, 0.02). Sources of variation in channel catfish mortality adjusted for weight were 67.3% from strains, 29.7% from families, and 3.0% from replicate tanks. The mechanism for differential resistance presently is unknown. Results indicate considerable potential for reducing disease losses by using E. ictaluri-resistant catfish strains in aquaculture and for genetic improvement in resistance via selective breeding programs.     

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     

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     

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     

Dose Titration of Sarafloxacin (A-56620) against Edwardsiella ictaluri Infection in Channel Catfish

Abstract

Two dose-titration studies were performed with sarafloxacin (A-56620) on channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri. Fish were infected with E. ictaluri by water-bath exposure and subsequently fed rations for 5 d to equal 0, 2, 6, 10, or 14 mg sarafloxacin/kg body weight per day. In trial I, mortality was significantly reduced from 69% for fish receiving no medication to 17% for those receiving 10 or 14 mg sarafloxacin/kg per day (P ≤ 0.01). In trial II, mortality was reduced from 33% of the nonmedicated infected group to 5 and 10% offish receiving 10 and 14 mg sarafloxacin/kg per day, respectively. Fish receiving 2 or 6 mg sarafloxacin/kg per day had intermediate mortality rates.     

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.     

Using Diagnostic Laboratory Records to Monitor Occurrence of Enteric Septicemia of Channel Catfish in Mississippi

Abstract

Diagnostic laboratory information was used to monitor the temporal and spatial occurrence of enteric septicemia of catfish (ESC) in Mississippi. From 1980 through 1988, the Mississippi Cooperative Extension Service (MCES) entered information on 15,649 fish-kill investigations into a computerized data base. Analysis of the records revealed the following trends and patterns. From 1980 through 1985, there was an increase in laboratory submissions and in the ESC proportional morbidity rate (PMR; proportion of diagnosed epizootics that were specific for ESC. However, from 1986 to 1988, although the total yearly number of laboratory submissions had stabilized, there was a decrease in the PMR of ESC cases. During the 9-year interval, 427 producers from 56 counties had ESC diagnosed in their ponds. Twenty-eight producers from four counties were responsible for about 40% of all ESC cases.     

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.     

An aroA Mutant of Edwardsiella ictaluri Is Safe and Efficacious as a Live,Attenuated Vaccine

Abstract

The aroA gene of Edwardsiella ictaluri was cloned and sequenced, and the sequence data were used to construct a deletion–insertion mutation in the aroA gene. The mutated gene was transferred into a virulent, wild-type E. ictaluri strain by conjugation and allelic exchange. Putative aroA mutants were confirmed phenotypically by demonstrating a need for supplementation with aromatic metabolites to support growth in minimal media. The genetic construction was evaluated by using the polymerase chain reaction to amplify appropriate regions of the aroA deletion–insertion, and DNA sequencing of the amplified products confirmed the predicted construction. A selected mutant, LSU-E1, was passed 30 times in nonselective media with no reversion to the wild-type following screening of 1.6 × 10¹¹ colony-forming units. The mutant was demonstrated via injection to be attenuated more than 5 logs₁₀ compared with the wild-type E. ictaluri strain, and it was avirulent by immersion and oral routes. Tissue persistence studies indicated that the mutant maintained the ability to invade following immersion exposure, but no viable cells were detected after 48–72 h. Significant levels of protection from disease were demonstrated following immersion vaccination of channel catfish Ictalurus punctatus.     

Effect of Vitamin E on the Immune Response of Channel Catfish to Edwardsiella ictaluri

Abstract

Three-month-old fingerling channel catfish Ictalurus punctatus were fed purified diets supplemented with ∝-tocopherol acetate to provide 0, 60, and 2,500 mg vitamin E/kg for 180 d. A 30-s immersion bath and an oral booster were used to deliver a bacterin of formalin-killed Edwardsiella ictaluri to half of the fish from each dietary treatment. Resistance of red blood cells to peroxidation was used as an index of antioxidant status. The susceptibility of red blood cells to oxidative hemolysis decreased with increasing levels of dietary vitamin E. Vaccinated and nonvaccinated fish were evaluated for agglutinating antibody titers and macrophage activity. Humoral antibody titers in response to E. ictaluri were significantly (P ≤ 0.05) higher in vaccinated fish than in nonvaccinated fish; however, no such differences in agglutinating antibody titers were detected among any of the dietary treatment groups. Both vaccination and vitamin E significantly enhanced the ability of macrophages to phagocytize virulent E. ictaluri. Results of this study indicate that elevated levels of dietary vitamin E may affect the ability of channel catfish to respond immunologically to bacterial pathogens.     

Preliminary Assessment of the Tolerance and Efficacy of Florfenicol against Edwardsiella ictaluri Administered in Feed to Channel Catfish

Abstract

A tolerance study was conducted to determine the palatability of florfenicol to channel catfish Ictalurus punctatus. Four tanks of fish (20 fish/tank) were assigned to each of five treatments distinguished by the amount of florfenicol given in feed per kilogram of body weight, namely, 0, 10, 20, 40, or 100 mg. Fish were fed at a rate of 2.5% of body weight per day for 10 consecutive days. On day 11, all surviving fish were euthanatized, counted, and weighed as a group. Florfenicol- medicated feed was palatable to fish at doses of 10, 20, 40, and 100 mg for 10 consecutive days. All 400 fish were necropsied and examined by histopathology, and no treatment-related changes were observed. In a separate exploratory efficacy study, four tanks (20 fish/tank) were assigned to each of the following treatments: (1) not challenged with Edwardsiella ictaluri and fed unmedicated feed, (2) challenged with E. ictaluri and fed unmedicated feed, (3) challenged with E. ictaluri and fed florfenicol at 10 mg per kilogram of body weight, (4) challenged and fed florfenicol at 20 mg/kg, and (5) challenged and fed florfenicol at 40 mg/kg. Treatment was initiated the day after inoculation, and feed was administered by hand for five consecutive days at 2.5% of body weight. The cumulative mortality observed over a 17-d period in treatment groups 1–5 was 2.5, 57.5, 0, 1.25, and 1.25%, respectively. All 400 fish were necropsied and examined by histopathology. The results indicate that florfenicol was effective in controlling mortality from enteric septicemia of catfish with no adverse treatment-related tissue changes. Florfenicol was palatable, safe, and efficacious in controlling mortality due to infection by E. ictaluri in channel catfish.     

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.     

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 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.     

Risk Factors Associated with Enteric Septicemia of Catfish on Mississippi Commercial Catfish Farms

Abstract

A gram-negative bacterium, Edwardsiella ictaluri, is the cause of enteric septicemia of catfish (ESC), which is one of the most prevalent bacterial diseases in farm-raised catfish. The objective of this study was to identify risk factors associated with ESC mortalities and are reported by farm personnel. To identify risk factors a catfish management database was developed. The odds ratios (OR) of the final multivariable logistic regression model were: (1) volume of the pond (OR, 0.56), (2) interval from harvest until a mortality event (OR, 1.49), (3) interval from stocking until a mortality event (OR, 0.52), (4) nitrite measured within 14 d of a mortality (OR, 3.49), (5) total ammonia measured within 14 d of a mortality (OR, 20.48), and (6) sum of feed fed for 14 d prior to the disease outbreak (OR, 1.02), all of which were significantly (P ≤ 0.05) associated with ESC occurrence. This study showed that some commonly recorded production variables were associated with ESC outbreaks and if monitored could help identify “at risk” ponds prior to disease outbreaks.

Received September 18, 2013; accepted December 17, 2013     

Lectin Binding Characteristics of the Olfactory Mucosa of Channel Catfish: Potential Factors in Attachment of Edwardsiella ictaluri

Abstract

The olfactory organ is a primary infection site for Edwardsiella ictaluri, the etiologic agent of enteric septicemia of channel catfish Ictalurus punctatus. The olfactory mucosal surface is a major interface between host and pathogen where commonly occurring carbohydrates may act as receptors for bacterial attachment. In this study, d-mannose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, d-galactose, and l-fucose were histochemically localized in the olfactory mucosa of channel catfish by using lectins that preferentially bind these carbohydrates. These lectins were Concanavalin A (ConA), soybean agglutinin (SBA), pokeweed agglutinin (PWA), wheat germ agglutinin (WGA), peanut agglutinin (PNA), and Ulex europaeus agglutinin I (UEA-I), respectively. The olfactory mucosa expressed d-mannose ubiquitously, whereas l-fucose and N-acetylneuraminic acid expression was specific to the apical mucosal surface. The carbohydrates d-galactose, N-acetylgalactosamine, and N-acetylglucosamine were most abundant in the sensory mucosa, specifically olfactory receptor neurons and cells near the basal lamina. Edwardsiella ictaluri was assayed for carbohydrate affinities by colloidal gold immunolocalization and transmission electron microscopy. Of the anti-lectins examined, those against WGA and UEA-I cross-reacted most intensely with Edwardsiella ictaluri, whereas cross-reactivities of anti-ConA, -SBA, and -PNA were more moderate. Double immunofluorescence labeling of experimentally infected catfish showed E. ictaluri adherent to cell surfaces or intercellularly associated with labeled carbohydrate components of the olfactory mucosa. Preincubation of the olfactory mucosa with soluble d-galactose significantly reduced bacterial adhesion compared with controls. Our results indicate a specific pattern of carbohydrates present in the catfish olfactory mucosa and suggest carbohydrates participate in initial E. ictaluri attachment by acting as ligands for pathogen constituents.