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1.
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.  相似文献   

2.
Abstract

Administration of various immunostimulants to fish has resulted in enhanced immune responses. The purpose of this study was to determine if feeding Spirulina, a processed form of the blue-green alga Spirulina platensis, enhanced specific and nonspecific immunity and resistance against Edwardsiella ictaluri infection in channel catfish Ictalurus punctatus. Peritoneal phagocytes from fish fed Spirulina showed enhanced phagocytosis to zymosan and increased chemotaxis to E. ictaluri exoantigen. No significant difference in mortality due to E. ictaluri existed between fish fed Spirulina and fish fed a basal diet. No significant difference in antibody titer or in the percentage of fish positive for E. ictaluri antibody was found between the groups after immunization with formalin-killed E. ictaluri. Spirulina-fed fish had significantly higher antibody titers to key hole limpet hemocyanin (KLH) on day 22, and a greater percentage of these fish were positive for KLH antibody on days 15 and 36. Feeding Spirulina enhanced nonspecific cellular immune responses such as chemotaxis and phagocytosis but did not provide protection against infection with E. ictaluri. The use of Spirulina in feed resulted in enhanced antibody responses to KLH, a thymus-dependent antigen, but not to E. ictaluri, a thymus-independent antigen. These results indicate that stimulation of the nonspecific immune system of channel catfish does not provide enhanced protection from E. ictaluri.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
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  相似文献   

6.
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  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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  相似文献   

10.
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.  相似文献   

11.
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  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
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  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
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  相似文献   

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