Effects of Feeding Spirulina on Specific and Nonspecific Immune Responses of Channel Catfish

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.     

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.     

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.     

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     

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.     

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.     

Effect of short-term feeding duration of diets containing commercial whole-cell yeast or yeast subcomponents on immune function and disease resistance in channel catfish, Ictalurus punctatus

Juvenile channel catfish, Ictalurus punctatus, were fed diets supplemented with yeast or yeast subcomponents (YYS) as commercial preparations of β‐glucan (MacroGard^® and Betagard A^®), mannan oligosaccharide (Bio‐Mos^® Aqua Grade), or whole‐cell Saccharomyces cerevisiae (Levucell SB20^®) at the manufacturers’ recommended levels. Fish were fed experimental diets for 1 or 2 weeks prior to disease challenge (pre‐challenge feeding periods) and sampled at the end of each feeding period to measure haematological and immune parameters and to determine the effects of dietary YYS on resistance to Edwardsiella ictaluri, the causative agent of enteric septicaemia disease (ESC). Feeding of experimental diets continued for 3 weeks post‐challenge. In channel catfish fed diets supplemented with MacroGard^®, Betagard A^®, or Levucell SB20^®, survival in the 1 week pre‐challenge feeding group and antibody titres in the 2 week feeding group were significantly higher post‐E. ictaluri challenge in relation to catfish fed with the control diet. In fish fed these same three diets, survival to ESC was significantly higher after 1 week vs. 2 weeks feeding, while the antibody response was significantly higher after 2 weeks vs. 1 week. Lysozyme activity was also higher in the 1 week feeding group, but the increased activity was unrelated to diet. Feeding YYS‐supplemented diets for a shorter duration of 1 week prior to challenge may prove beneficial in increasing resistance to ESC in channel catfish. However, we cannot discount that feeding YYS diets during the recovery period may have contributed to ‘glucan overload’ and reduced survival in the 2 week feeding group.     

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.     

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.     

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.     

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     

Immune Response and Resistance of Channel Catfish to Edwardsiella ictaluri Challenge when Fed Various Dietary Levels of Zinc Methionine and Zinc Sulfate

Abstract

The effects of dietary levels of zinc from zinc methionine (ZnM) and zinc sulfate ZnSO₄ on nonspecific immune responses and disease resistance of juvenile channel catfish Ictalurus punctatus (mean initial weight of 3.3 g) were determined. Fish in triplicate aquaria were fed to satiation twice daily for 16 weeks with an egg white basal diet without zinc supplementation or supplemented with 5, 20, and 60 mg zinc/kg as ZnM or 20 and 60 mg zinc/kg as ZnSO₄. Relative percentages of peripheral blood lymphocytes, thrombocytes, and macrophages were not affected by dietary zinc. The relative percentages of neutrophils significantly increased in fish fed 20 and 60 mg Zn/kg as ZnM. Chemotactic responses of macrophages were significantly higher for fish fed the three ZnM-supplemented diets and the 60-mg ZnSO₄. Dietary zinc, however, had no influence on the phagocytic activity of macrophages for Edwardsiella ictaluri. Likewise, neither the source nor the level of dietary zinc provided protection to channel catfish against E. ictaluri. The severity of E. ictaluri infection (number of colony-forming units/g trunk kidney) and the percentage offish that were culture negative for E. ictaluri (15 d postchallenge) were also unaffected by dietary zinc.     

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.     

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.     

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.     

Spleen Index and Mannose-Binding Lectin Levels in Four Channel Catfish Families Exhibiting Different Susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Received November 18, 2011; accepted February 23, 2012     

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     

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.