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.     

Evaluation of Two Aryl-Fluoroquinolones against Bacterial Pathogens of Channel Catfish

Abstract

The in vitro and in vivo efficacies of two aryl-fluoroquinolones, A-56619 and A-56620, against two bacterial pathogens of channel catfish Ictalurus punctatus were determined The minimum inhibitory concentrations (MIC) of A-56619, A-56620, oxytetracycline, nalidixic acid, spectinomycin, ampicillin, doxycycline, and ormetoprim-sulfadimethoxine against 10 isolates of both Edwardsiella ictaluri and Aeromonas sobria were determined by the agar-dilution method. The in vivo efficacies of A-56619 and A-56620 were determined by bacterial challenge of channel catfish with E. ictaluri. Fish were fed a prepared diet that supplied 12.5, 25.0, or 50.0 mg of active drug per kilogram body weight per day. In vitro tests showed that all strains of E. ictaluri and A. sobria were sensitive to A-56619 and A-56620. The two aryl-fluoroquinolones demonstrated a lower mean MIC against these organisms than any of the other antibiotics tested. Significant reductions in mortality occurred within all channel catfish groups treated with these two chemicals as compared with controls. Mortality from E. ictaluri infection of fish given feed medicated with A-56619 and A-56620 ranged from 4 to 12%. Mortality among unmedicated controls ranged from 20 to 68%. No significant difference in mortality among dosage levels was demonstrated.     

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     

A Real-Time Polymerase Chain Reaction Assay for Quantification of Edwardsiella ictaluri in Catfish Pond Water and Genetic Homogeneity of Diagnostic Case Isolates from Mississippi

Abstract

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri–specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.

Received October 20, 2010; accepted June 13, 2011     

Comparison of Two Biochemical Test Systems with Conventional Methods for the Identification of Bacteria Pathogenic to Warmwater Fish

Abstract

One hundred seven Aeromonas spp., 26 Edwardsiella ictaluri, 6 E. tarda, 12 Plesiomonas shigelloides, and 6 Pseudomonas spp. (131 piscine isolates and 26 reference isolates) were studied with 36 biochemical tests from the Minitek system, 20 tests from the API 20E system, and corresponding standard tube tests. Isolates were incubated at 25°C. Arginine dihydrolase, ornithine decarboxylase, mannose, and citrate showed less than 95% agreement between the Minitek system and the tube tests. Arginine dihydrolase, lysine decarboxylase, nitrite reductase, Voges-Proskauer, and citrate showed less than 95% agreement between the API 20E system and the tube tests. The 26 reference isolates were examined with the three systems and were incubated at both 25 and 37°C. There were no major differences between tests run at 25 and 37°C except with nine Aeromonas spp. that did not grow well at 37°C. Both the Minitek and API 20E systems will reproduce standard biochemical tube test results with at least 95% accuracy when used to test warmwater fish pathogens incubated at 25°C. However, the numerical identification databases for both the Minitek and API 20E systems were not usable for identifying fish pathogens.     

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.     

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     

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.     

Distribution of Edwardsiella ictaluri in California

Abstract

Wild and domestic populations of channel catfish Ictalurus punctatus were examined to determine the distribution of the disease called enteric septicemia of catfish (ESC) in California. The causative agent of ESC, Edwardsiella ictaluri, was isolated from five separate sites in California. Two of these isolations were from rectal swabs of asymptomatic fish, confirming that a carrier state may exist. Normal-appearing fish with serum antibody titer to E. ictaluri were commonly found in domestic channel catfish populations, suggesting that many fish become infected but recover. Wild channel catfish with antibody to E. ictaluri were also found in major reservoirs and water distribution canals. Edwardsiella ictaluri appears to be widely distributed within California.     

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.     

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.     

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     

Susceptibility of Six Bacterial Pathogens of Channel Catfish to Six Antibiotics

Abstract

Two hundred eight bacterial isolates from diseased channel catfish Ictalurus punctatus were screened for susceptibility to apramycin, enrofloxacin, cephalothin, oxytetracycline, sulfadimethoxine-ormetoprim, and tilmicosin by the Kirby-Bauer disk diffusion method. Bacteria evaluated were Edwardsiella ictaluri, E. tarda, Aeromonas sp., A. hydrophila, A. sobria, and Pseudomonas sp. All bacteria were most susceptible to enrofloxacin (99.0%) and apramycin (97.6%), but only 86.5% were susceptible to sulfadimethoxine-ormetoprim, 84.1%o to oxytetracycline, and 75.5% to cephalothin. Edwardsiella ictaluri, E. tarda, A. hydrophila, and A. sobria were 100% susceptible to enrofloxacin. Aeromonas sp., E. ictaluri and E. tarda were 100% susceptible to apramycin. Resistance was detected in all bacterial species to cephalothin, oxytetracycline, sulfadimethoxine-ormetoprim, and tilmicosin. Testing E. ictaluri against sulfadimethoxine-ormetoprim gave larger zones of inhibition on Mueller-Hinton medium than on brain-heart infusion agar.     

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.     

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     

Survival and Antibody Response of Channel Catfish,Blue Catfish,and Channel Catfish Female × Blue Catfish Male Hybrids after Exposure to Edwardsiella ictaluri

Abstract

Juvenile Norris strain channel catfish Ictalurus punctatus, blue catfish I. furcatus, and Norris strain channel catfish female × blue catfish male hybrids were challenged with Edwardsiella ictaluri by bath immersion or intraperitoneal injection (high or low dose) in aquaria. Survival (%) after bath immersion was highest for blue catfish (89.5 ± 2.8), intermediate for hybrids (73.8 ± 6.7), and lowest for channel catfish (62.0 ± 4.2). Prechallenge antibody levels to E. ictaluri, measured by enzyme-linked immunosorbent assay, were negative (mean ± SE optical density [OD] = 0.010 ± 0.003). Postchallenge antibody response for blue catfish (OD = 0.132 ± 0.045) was significantly lower than that of channel catfish (OD = 0.350 ± 0.045), whereas the response of the channel × blue catfish F₁ hybrids (OD = 0.263 ± 0.051) was intermediate and not significantly different from either parental species. Intraperitoneal injections of E. ictaluri resulted in significant mortality only in channel catfish (88.3 ± 2.6% survival) and were sublethal to hybrid catfish and blue catfish with 100.0% and 99.3 ± 0.4% survival, respectively. Antibody responses after the injection challenge were significantly different among catfish groups and injection dose with no group × dose interaction. Antibody responses after the injection challenge were consistent with the immersion challenge, and means of high and low challenge doses were lowest in blue catfish (OD = 0.061 ± 0.014), intermediate in hybrids (OD = 0.187 ± 0.014), and highest in channel catfish (OD = 0.272 ± 0.014). For all fish groups combined, the high injection challenge dose resulted in higher antibody levels (OD = 0.206 ± 0.011) than low injection challenge dose (OD = 0.140 ± 0.012). Overall results indicate greater resistance to E. ictaluri and lower antibody response in blue catfish, and show the potential to identify molecular markers linked with disease resistance and introgression of resistance genes from blue catfish into 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.     

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     

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.     

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.