Carbohydr Res, 2005 Jan 17, 340(1), 85 - 90 Structural characterization of the O-polysaccharide antigen of Edwardsiella tarda MT 108; Vinogradov E et al.; Edwardsiella tarda, a Gram-negative bacterium, is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans . The lipopolysaccharide produced by the fish pathogenic strain E . tarda MT 108 was isolated and the structure of its antigenic O-polysaccharide component determined by the application of chemical analyses, high-resolution 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometry . The polysaccharide was found to be a polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), 2-acetamido-2-deoxy-d-galactose (d-GalNAc), d-galactose (d-Gal), l-rhamnose (l-Rha), d-galacturonic acid (d-GalA) and (2S,3R)-threonine (1:1:1:1:1:1) having the structure: Chang Gung Med J, 2004 Sep, 27(9), 701 - 5 Polymicrobial bacteremia caused by Escherichia coli, Edwardsiella tarda, and Shewanella putrefaciens; Wang IK et al.; Edwardsiella tarda, a member of Enterobacteriaceae, is found in freshwater and marine environments and in animals living in these environments . This bacterium is primarily associated with gastrointestinal diseases, and has been isolated from stool specimens obtained from persons with or without clinical infectious diseases . Shewanella putrefaciens, a saprophytic gram-negative rod, is rarely responsible for clinical syndromes in humans . Debilitated status and exposure to aquatic environments are the major predisposing factors for E . tarda or S . putrefaciens infection . A 61-year-old woman was febrile with diarrhea 8 hours after ingesting shark meat, and two sets of blood cultures grew Escherichia coli, E . tarda and S . putrefaciens at the same time . She was successfully treated with antibiotics . We present this rare case of polymicrobial bacteremia caused by E . coli, E . tarda and S . putrefaciens without underlying disease, which is the first found in Taiwan . This rare case of febrile diarrhea with consequent polymicrobial bacteremia emphasizes that attention should always be extended to these unusual pathogens. Protein Eng Des Sel, 2004 Sep, 17(9), 689 - 97 Epub 2004 Nov 05. A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents; Hao J et al.; Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri . Variants from all four generations of evolution have been purified and characterized . The variants show increased thermostability with no loss of catalytic function at room temperature . The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T(50)) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold . In addition, variant 4-43D6 shows increased stability to treatment with organic solvents . DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed. Electrophoresis, 2004 Oct, 25(18-19), 3139 - 44 Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescence; Yu L et al.; High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria . We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (< 10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence . SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells . Remarkably high efficiency (> 1,200,000 theoretical plates/m) was achieved with this rapid and efficient CE method . It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis . Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5 . The limit of detection (LOD) and recovery were found to be 4.2 x 10(4) cells/mL and 70.0%, respectively . This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings . Dev Comp Immunol, 2005, 29(2), 135 - 42 Analysis of a catfish gene resembling interleukin-8: cDNA cloning, gene structure, and expression after infection with Edwardsiella ictaluri; Chen L et al.; Chemokines are important mediators for innate immunity involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci . While almost all chemokines have been identified from mammals, only a handful of fish chemokines have been identified . Here we report molecular cloning, sequence analysis, and expression of a channel catfish gene resembling interleukin-8 (IL-8) . The gene has two alternatively spliced transcripts encoding 114 and 111 amino acids, respectively . The gene has four exons and three introns, typical of the CXC chemokine gene organization . In spite of the structural conservation through evolution, the piscine IL-8 genes showed a much greater sequence divergence than their counterparts among mammals . RT-PCR indicated that both spliced forms were expressed . Expression of the IL-8 like gene was up-regulated 3-5-fold in channel catfish and blue catfish after infection with pathogenic bacteria Edwardsiella ictaluri. J Fish Dis, 2004 Sep, 27(9), 497 - 505 Bacterial enteritis and the development of the larval digestive tract in olive flounder, Paralichthys olivaceus (Temminck & Schlegel); Kim DH et al.; Three bacterial isolates obtained from diseased olive flounder larvae, Paralichthys olivaceus, were identified as Vibrio ichthyoenteri based on the results of phenotypic characterization and 16S rRNA gene sequencing studies . Bacterial enteritis was reproduced in 16 and 22 days post-hatch (dph) larvae by administering brine shrimp nauplii, Artemia salina, dosed with the environmental isolates and reference strains of V . ichthyoenteri . To investigate the effect of the disease on development of the stomach, a pepsin activity assay and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the expression of the pepsinogen gene were performed . Expression of olive flounder pepsinogen was detected from 30-dph larvae and the increased level of pepsin activity coincided with reduced susceptibility to the disease . Growth rates of V . ichthyoenteri, V . anguillarum and Edwardsiella tarda were tested in artificial stomach conditions using HCl and porcine pepsin . All the strains of V . ichthyoenteri were inhibited by low pH conditions which corresponded with an increase in pepsin levels . This suggests that differentiation of the stomach in olive flounder larvae and juveniles, an essential physiological development, also provides the host with a non-immunological defence mechanism. Vaccine, 2004 Sep 3, 22(25-26), 3411 - 8 A conserved 37 kDa outer membrane protein of Edwardsiella tarda is an effective vaccine candidate; Kawai K et al.; An effective vaccine against Edwardsiella tarda has not been reported, one of main reasons is the variation in its serotypes . This study aimed to develop an effective vaccine against different serotypes of E . tarda . A conserved 37 kDa outer membrane protein (OMP) of E . tarda was obtained by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) . It showed comprising several proteins by two-dimensional (2D) PAGE analysis and showed a high similarity to glyceraldehyde-3-phosphate dehydrogenase by N-terminal amino acid sequence analysis . Japanese flounder (Paralichthys olivaceus) vaccinated with the 37 kDa OMP was significantly protected against the infections by different serotypes of E . tarda . A specific antibody was also detected by using ELISA . This study suggests that the 37 kDa OMP is an effective potent vaccine candidate against different serotypes of E . tarda. Mol Microbiol, 2004 Jul, 53(2), 573 - 86 Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis; Rao PS et al.; Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extraintestinal infections in humans . Using a combination of comparative proteomics and TnphoA mutagenesis, we have identified five proteins that may contribute to E . tarda PPD130/91 pathogenesis . Lowered protein secretion, impaired autoaggregation and the absence of six proteins were observed only in three highly attenuated mutants when cultured in Dulbecco's modified eagle medium (DMEM) . Five out of six proteins could be identified by their mass spectra . Three proteins were identified as putative effector proteins (EseB, EseC and EseD) that are homologous to SseB, SseC and SseD of a type III secretion system (TTSS) in Salmonella species . The other two were EvpA and EvpC, homologous to Eip20 and Eip18 in Edwardsiella ictaluri . The complete sequencing and homology studies of evpA-H indicate that similar gene clusters are widely distributed in other pathogens such as Escherichia, Salmonella, Vibrio and Yersinia species with unknown functions . Insertional inactivation and deletion of evpB or evpC led to lower replication rates in gourami phagocytes, and reduced protein secretion and virulence in blue gourami . Complementation of these deletion mutants showed partial recovery in the above three phenotypes, indicating that these genes are vital for E . tarda pathogenesis . The transport of the EvpC protein may not use the TTSS in E . tarda . The expression of EvpA and EvpC as well as EseB, EseC and EseD was temperature dependent (suppressed at 37 degrees C), and disruption of esrB affected their expression . The present study identifies two possible secretion systems (TTSS and Evp) that are vital for E . tarda pathogenesis. J Appl Microbiol, 2004, 97(1), 87 - 92 In vitro inhibitory effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri; Yildirim-Aksoy M et al.; AIM: This study was conducted to evaluate the toxic effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri . METHODS AND RESULTS: Inhibitory effect of various concentrations of gossypol on the growth of E . ictaluri was determined . Bacterial recovery was performed by preincubation of bacteria in medium containing various concentrations of gossypol and subsequent activation of bacteria by inoculating on gossypol-free plates . Concentrations of racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 microg ml(-1) or higher significantly reduced the number of bacterial colonies compared with that of the control . The growth of E . ictaluri was completely inhibited on agar plates supplemented with 3 microg ml(-1), regardless of the forms of gossypol . The inhibitory effect of (+)-gossypol was higher than that of (-)-gossypol or gossypol-acetic acid . Recovery of E . ictaluri was <50% for all three forms of gossypol at concentrations of 5 microg ml(-1) . Bacterial recovery remained relatively constant (6.5%) at gossypol concentrations from 10 to 100 microg ml(-1) . Complete killing of E . ictaluri was not reached at gossypol levels up to 100 microg ml(-1) . CONCLUSION: Gossypol-acetic acid, and (+)- and (-)-optical isomers have anti-bacterial effect against E . ictaluri . The results suggest the action is bacteriostatic rather than bactericidal . SIGNIFICANCE AND IMPACT OF THE STUDY: The therapeutic effect of gossypol against E . ictaluri may be useful in controlling enteric septicaemia of catfish. Fish Shellfish Immunol, 2004 Mar, 16(3), 415 - 28 Immune response induced by N-lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish; Bader JA et al.; A virulent isolate of Edwardsiella ictaluri (AL-93-75), the causative agent of enteric septicaemia of catfish (ESC), was used to derive a lipopolysaccharide-reduced N-lauroylsarcosine outer-membrane protein (OMP) fraction vaccine . The OMP fraction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to whole-cell lysate, purified lipopolysaccharide (LPS) and a crude cell-wall fraction . The OMP fraction contained less than 2% (W/V) LPS . SDS-PAGE showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa . Channel catfish, Ictalurus punctatus, were vaccinated with 12.5 microg/100 microl OMP and immunogenicity was confirmed by subsequent Western blots . Blots showed that 97, 80, and 19 kDa proteins were immunogenic . Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21 days post injection . OMP concentrations of 3.13, 6.25, 12.5, 25, and 50 microg/100 microl total protein were tested for protective immunity . Marginal protection by relative percent survival (RPS) was only seen for fish injected with 12.5 microg/100 microl with RPSs between 55-67.5% . A booster dose of 12.5 microg/100 microl OMP did not significantly enhance protection. Dev Comp Immunol, 2004 Jun, 28(7-8), 769 - 80 Sequence analysis and expression of a CXC chemokine in resistant and susceptible catfish after infection of Edwardsiella ictaluri; Baoprasertkul P et al.; Chemokines represent a superfamily of chemotactic cytokines involved in recruitment, activation and adhesion of a variety of leukocyte types to inflammatory foci . We cloned and sequenced the cDNA of a CXC chemokine that is most similar to CXCL10 from channel catfish and blue catfish . Sequence analysis of PCR amplicons from a single F1 hybrid catfish indicated that channel catfish and blue catfish may have a multigene family for the CXC chemokine . The catfish CXC chemokine was expressed in a wide range of tissues including head kidney, spleen, liver, gill, skin, stomach, and intestine, but not in the muscle . Fish challenged with intracellular bacterium Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), showed dramatically elevated levels of the CXC chemokine expression, as quantified with real time RT-PCR . Differential expression profiles were observed between resistant and susceptible channel catfish strains and blue catfish . Blue catfish were characterized by only modest induction in comparison to the drastic elevation of the CXC chemokine in channel catfish. J Vet Pharmacol Ther, 2004 Feb, 27(1), 1 - 6 Pharmacokinetics of sulfadimethoxine and ormetoprim in a 5:1 ratio following intraperitoneal and oral administration, in the hybrid striped bass (Morone chrysops x Morone saxitalis); Bakal RS et al.; Selected pharmacokinetic parameters for sulfadimethoxine and ormetoprim, administered in a 5:1 ratio, via the oral and intraperitoneal (i.p.) routes were determined in the hybrid striped bass (Morone chrysops x Morone saxitalis) . Plasma concentrations of both drugs were determined by high-performance liquid chromatography . A first-order one-compartment model adequately described plasma drug disposition . The elimination half-lives for sulfadimethoxine following i.p . and oral administration were 26 and 10.5 h, respectively . The half-lives for ormetoprim administered via i.p . and oral routes were 7.5 and 3.9 h, respectively . Cmax for sulfadimethoxine via the i.p . and oral routes were calculated to be 27.7 (+/-9.0) microg/mL at 3.6 h and 3.2 (+/-1.2) microg/mL at 1.2 h, respectively . Cmax for ormetoprim via the i.p . route was calculated to be 1.2 (+/-0.5) microg/mL at 9.1 h and 1.58 (+/-0.7) microg/mL at 5.7 h for the oral route . The oral availability of sulfadimethoxine relative to the i.p . route was 4.6%, while the oral availability of ormetoprim relative to the i.p . route was 78.5% . Due to the nonconstant ratio of these drugs in the plasma of the animal, the actual drug ratio to use for determining minimum inhibitory concentration (MIC) is unclear . Using the ratio of the total amount of each drug that is absorbed as a surrogate for the mean actual ratio may be the best alternative to current methods . Using this ratio as determined in these studies, (2.14:1 sulfadimethoxine:ormetoprim) to determine the MICs the single 50 mg/kg oral dose of the 5:1 combination of sulfadimethoxine and ormetoprim appears to provide plasma concentrations high enough to inhibit the growth of Yersinia ruckeri, Edwardsiella tarda, and Escherichia coli. Mar Biotechnol (NY), 2002 Jun, 4(3), 338 - 44 Enhanced bacterial disease resistance of transgenic channel catfish Ictalurus punctatus possessing cecropin genes; Dunham RA et al.; The cecropin B gene from the moth Hyalophora cecropia, driven by the cytomegalovirus promoter, was transferred to the channel catfish Ictalurus punctatus . Transgenic individuals (P1) were mated to produce individuals (F1) that exhibited enhanced disease resistance and survival when challenged with pathogenic bacteria . During the epizootic of Flavobacterium columnare in an earthen pond, the percentage of transgenic individuals containing preprocecropin B construct that survived (100%) was significantly greater (P <0.005) than that of nontransgenic controls (27.3%) . Also, when challenged in tanks with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish, the percentage of transgenic individuals containing catfish IG leader cecropin B construct that survived (40.7%) was significantly greater (P <0.01) than that of nontransgenic controls (14.8%) . There were no pleiotropic effects of the transgenes, and growth rates of the transgenic and nontransgenic siblings were not different (P > 0.05) . Inheritance of the transgene by the F1 generation, 20.2% to 30.7% was typical of that in studies with transgenic channel catfish. Appl Environ Microbiol, 2004 Jan, 70(1), 621 - 4 Sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method; Savan R et al.; Here we report a rapid and sensitive method (using loop-mediated isothermal amplification {LAMP}) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder . A set of four primers was designed, and conditions for the detection were optimized for the detection of E . tarda in 45 min at 65 degrees C . No amplification of the target hemolysin gene was detected in other related bacteria . When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible . We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish . This is the first report of the application of LAMP for the diagnosis of a fish pathogen. J Infect Chemother, 2003 Dec, 9(4), 341 - 3 Puerperal intrauterine infection caused by Edwardsiella tarda; Mikamo H et al.; Edwardsiella tarda infection is uncommon and has been reported to be associated with pet reptiles and the ingestion of raw fish . The majority of the infections occur as gastrointestinal disorders in immunocompromised hosts . We report a case of puerperal intrauterine E . tarda infection in a patient with no history of predisposing illness or of the recent ingestion of raw fish; this is the first such report . The patient complained of high fever, lower abdominal pain, and increasing amounts of malodorous genital discharge; these signs and symptoms responded well to intravenous antimicrobial chemotherapy. Biochem Biophys Res Commun, 2004 Jan 2, 313(1), 28 - 36 Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes; Ourth DD et al.; The channel catfish (Ictalurus punctatus) is extensively used in aquaculture in the Southeast US and is susceptible to many bacterial infections acquired from its pond environment . Research is needed to better understand the defensive response and innate immunity of channel catfish against fish pathogens like Edwardsiella ictaluri and Aeromonas hydrophila . The main objectives were purification and characterization of an innate antimicrobial factor isolated from catfish leucocytes that has both bactericidal and antiviral activities . Oxygen-independent mechanisms of innate immunity for killing microorganisms have not been identified in leucocytes of channel catfish . Leucocytes were separated from catfish blood, and granule extracts were obtained by homogenization, centrifugation, and extraction with 10% acetic acid . The granule extracts were further purified by gel filtration chromatography . Bactericidal assays against the two fish pathogens and SDS-PAGE analysis were done on the isolated antimicrobial factor . Determination of antiviral activity of the factor was done by in vitro tissue culture using herpes simplex virus-type 1 . Mass spectrometry analyses were done for molecular weight (655 Da), purity, and structural characterization of the innate non-peptide antimicrobial factor. J Vet Diagn Invest, 2003 Nov, 15(6), 576 - 9 In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol; McGinnis A et al.; In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC) . Twelve different E . ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing . These isolates originated from channel catfish (Ictalurus punctatus) infected with E . ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi . Seven hundred sixty-seven additional cultures of E . ictaluri were obtained from channel catfish infected experimentally with E . ictaluri . In some of these experimental infections, FFC was used for treatment . These cultures of E . ictaluri were identified by morphological and biochemical tests . Kirby-Bauer zones of inhibition (in mm) for FFC against E . ictaluri were determined using standard methods . The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E . ictaluri isolates and arbitrarily selected experimental cultures . The zones of inhibition for FFC tested with E . ictaluri ranged from 31 to 51 mm . The MIC for FFC tested with E . ictaluri was consistently 0.25 microg/ml . Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro. Toxicol Pathol, 2003 Nov-Dec, 31(6), 689 - 97 Safety of Aquaflor (florfenicol, 50% type A medicated article), administered in feed to channel catfish, Ictalurus punctatus; Gaikowski MP et al.; Aquaflor, a feed premix containing the broad spectrum antibacterial agent florfenicol (50% w/w), is being developed for use to control enteric septicemia (ESC) in channel catfish Ictalurus punctatus caused by the gram-negative enterobacterium Edwardsiella ictaluri . The recommended dose of Aquaflor to control ESC is 10 mg/kg body weight (BW)/day for 10 days . The study objective was to determine the safety of Aquaflor administered in feed to channel catfish at doses of 0 (control), 10, 30, and 50 mg/kg BW/day for 20 consecutive days . Parameters evaluated included daily mortality, behavioral (appetite, distribution, flight/fright response), and water chemistry observations, initial and terminal weight measurements, and gross and microscopic pathology . Medicated feed consumption was 67-86% of target with group mean doses of 8.5 mg/kg BW/day, 24.6 mg/kg BW/day, and 34.9 mg/kg BW/day . There were no mortalities or clinically observable changes noted at any of the dose levels tested . Aquaflor-related changes were limited to the food consumption and histopathology data . Although Aquaflor-related decreased feed consumption was noted in the 30 and 50 mg/kg BW/day groups, there were no differences in fish growth among the treatment groups . Aquaflor-related histopathology findings were limited to a histomorphologically evident dose-dependent decrease in hematopoietic/lymphopoietic tissue in the anterior kidneys, posterior kidneys, and spleens of channel catfish. Dis Aquat Organ, 2003 Aug 15, 56(1), 43 - 8 CpG motif in synthetic ODN primes respiratory burst of olive flounder Paralichthys olivaceus phagocytes and enhances protection against Edwardsiella tarda; Lee CH et al.; Effects of synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN) on respiratory burst activity of olive flounder (Paralichthys olivaceus) head-kidney phagocytes and on protection against lethal infection with Edwardsiella tarda were investigated . Phagocytes precultured with a CpG ODN showed significantly higher chemiluminescence (CL) responses than phagocytes precultured with guanosine-phosphate-cytidine (GpC) ODN or culture medium alone (control) at all concentrations . Supernatants produced from leucocytes, which were pulsed with CpG ODN, induced significantly higher respiratory burst activity than supernatants produced by GpC ODN or culture medium alone . In an in vivo experiment, respiratory burst activities of the head kidney phagocytes in the groups injected either 0.25 or 0.5 microg fish(-1) of CpG ODN were significantly higher than those in the groups injected with GpC ODN or HBSS (control) at 3, 5 and 7 d after injection . The groups of fish injected with 0.25 or 0.5 microg of CpG ODN showed higher survival rates (83.3%) than groups treated with GpC ODN (33.3%) and a control group (8.3%) after challenge with E . tarda . The present in vitro and in vivo experiments have demonstrated the ability of synthetic CpG ODN to increase phagocyte respiratory burst activity and disease resistance in olive flounder. FEMS Microbiol Lett, 2003 Sep 12, 226(1), 127 - 33 Identification of a 19.3-kDa protein in MRHA-positive Edwardsiella tarda: putative fimbrial major subunit; Sakai T et al.; The hemagglutinating properties of Edwardsiella tarda isolated from fish were investigated . Hemagglutination of E . tarda was not inhibited by D-mannose but was strongly inhibited by fetuin and N-acetylneuraminic acid . Extraction of hemagglutinating activity from bacterial cells was achieved using n-octyl-beta-D-thioglucoside (NOTG), and the NOTG extracts were fractionated by sucrose density gradient ultracentrifugation . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fractions revealed that a 19.3-kDa protein band appeared in the fractions exhibiting highest hemagglutinating activity . In an immunoblot analysis of NOTG extracts from 18 strains of E . tarda, the 19.3-kDa protein was detected only in the extracts possessing hemagglutinating activity . The predicted amino acid sequence of a 534-bp gene encoding the 19.3-kDa protein was identical to fimbrial subunit (FimA) of E . tarda by FASTA homology search . These findings suggest that fimbriae are implicated in the hemagglutination of E . tarda. Fish Shellfish Immunol, 2003 Oct, 15(4), 333 - 40 Comparative sensitivity of different serological tests for seromonitoring and surveillance of Edwardsiella tarda infection of Indian major carps; Swain P et al.; Different serological tests viz . indirect ELISA, indirect blocking ELISA, competitive ELISA and serum agglutination tests were evaluated to detect antibodies against Edwardsiella tarda in naturally infected fish sera for seromonitoring and epizootiological studies . Approximately 66.6, 62.5, 57.6 and 16.6% of the field sera samples were found to be positive by indirect ELISA, competitive ELISA, indirect blocking ELISA and serum agglutination test, respectively . The percentage of serum samples positive for E . tarda antibodies in serum agglutination, competitive ELISA and indirect blocking ELISA, when compared with indirect ELISA, were 33.3, 83.6 and 66.6%, respectively, but its use was restricted due to the requirement of several conjugates against different fish species and the difficulty in assaying large numbers of serum samples from different fish species in a limited time to enable seromonitoring of the disease prevalence . No significant difference (P<0.05) in the mean optical density value was found in indirect and competitive ELISA . Although the competitive ELISA was slightly less sensitive than the indirect ELISA, it could accommodate a large number of serum samples with one anti-rabbit conjugate, and the need for different fish conjugates as required in indirect ELISA was eliminated . As in medical and veterinary practices, these tests can now be used in aquaculture practices for seromonitoring and study of pre-exposure of Indian major carps to pathogens in enzootic areas. Microbiology, 2003 Sep, 149(Pt 9), 2635 - 44 A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda; Srinivasa Rao PS et al.; Edwardsiella tarda causes haemorrhagic septicaemia in fish and gastro- and extra-intestinal infections in animals including humans . Resistance to phagocyte-mediated killing is one of the virulence factors of Ed . tarda . The authors' previous studies using TnphoA transposon mutagenesis indicated that katB mutants derived from the strain PPD130/91 are at least 1.6 log higher in LD50 values than the wild-type strain . These findings suggest the involvement of catalase (KatB) in Ed . tarda pathogenesis . In this study, experiments were conducted to characterize the contribution of KatB to Ed . tarda infection . Zymographic analyses indicated that the 22 Ed . tarda strains examined expressed three different types of catalase-peroxidases (Kat1-3) based on their mobility in non-denaturing polyacrylamide gels . KatB (Kat1), the major catalase enzyme, was expressed in eight out of 22 Ed . tarda strains, and was commonly found in virulent strains except AL9379 . AL9379 has a mutated katB, which has a base substitution and a deletion that translate into stop codons in the catalase gene . KatB produced by PPD130/91 was located in both periplasmic and cytoplasmic fractions and was constitutively expressed in various growth phases . Kinetics studies indicated that the catalase provided resistance to H2O2- and phagocyte-mediated killing . Infection kinetics studies of katB mutant 34 in gourami fish demonstrated its inability to survive and replicate in phagocyte-rich organs and this prevented the dissemination of infections when compared to the wild-type . Complementation of catalase mutants restored the production of catalase, and led to an increase in the resistance to H2O2- and phagocyte-mediated killing, and a decrease in LD50 values . This study has identified and characterized a major catalase gene (katB) that is required for resistance to H2O2- and phagocyte-mediated killing in Ed . tarda . The results also suggest that catalase may play a role as a virulence factor in Ed . tarda pathogenesis. J Fish Dis, 2003 Jul, 26(7), 415 - 21 A comparative study of Edwardsiella ictaluri parent (EILO) and E . ictaluri rifampicin-mutant (RE-33) isolates using lipopolysaccharides, outer membrane proteins, fatty acids, Biolog, API 20E and genomic analyses; Arias CR et al.; The biological properties of Edwardsiella ictaluri RE-33 rifampicin-mutant and its parent strain EILO were analysed . RE-33 is an avirulent isolate used as a modified live vaccine against enteric septicaemia of catfish . Electrophoretic analysis of lipopolysaccharide (LPS) patterns showed high homology between both isolates . Further characterization of LPS by immunoblotting revealed the main differences in LPS composition . The RE-33 isolate lacks the high molecular weight bands of LPS (HMW-LPS) . Outer membrane protein analysis also showed some immunological differences between RE-33 and the EILO parent strain . Only two fingerprinting techniques, fatty acid composition analysis and Biolog phenotypic profiles, were able to discriminate between both isolates. Int J Antimicrob Agents, 2003 Jul, 22(1), 67 - 9 Combination effects of cephalexin and gentamicin on Edwardsiella tarda and Streptococcus iniae; Lim JH et al.; The objective of this study was to determine the in vitro activity of cephalexin-gentamicin combination by a microbroth chequerboard technique against clinical isolates of Edwardsiella tarda and Streptococcus iniae . Gentamicin was shown more susceptible than cephalexin against both bacteria . The effect of cephalexin-gentamicin combination against both bacteria represented additive interaction . The combination even showed synergic interaction (22%) against E . tarda, with a FIC index of <0.5 as a borderline . No antagonism for cephalexin-gentamicin combination was observed for any bacterial strain. Microbiology, 2003 Jun, 149(Pt 6), 1409 - 21 The Edwardsiella ictaluri O polysaccharide biosynthesis gene cluster and the role of O polysaccharide in resistance to normal catfish serum and catfish neutrophils; Lawrence ML et al.; Edwardsiella ictaluri, the causative agent of enteric septicaemia of catfish (ESC), expresses long O polysaccharide (OPS) chains on its surface . The authors previously reported the construction of an isogenic Ed . ictaluri OPS mutant strain and demonstrated that this strain is avirulent in channel catfish . This paper reports the cloning of the Ed . ictaluri OPS biosynthesis gene cluster and identification of the mutated gene in the OPS-negative strain . The sequenced region contains eight complete ORFs and one incomplete ORF encoding LPS biosynthesis enzymes . The mutated gene (designated wbiT) was similar to other bacterial galactose-4-epimerases . Glycosyl composition analysis indicated that wild-type Ed . ictaluri OPS contains higher amounts of galactose and N-acetylgalactosamine than the OPS mutant strain, which correlated well with predicted functions of the genes identified in the OPS biosynthesis cluster . The OPS mutant had a relatively small, but significant, decrease in its ability to survive in normal catfish serum compared to wild-type Ed . ictaluri, but it retained the ability to resist killing by catfish neutrophils. Pediatrics, 2003 Mar, 111(3), e296 - 8 Maternal colonization and neonatal sepsis caused by Edwardsiella tarda; Mowbray EE et al.; A case of neonatal sepsis caused by Edwardsiella tarda, a bacterium usually associated with freshwater ecosystems, is described . The infant's mother was immersed in lake water during the sixth month of pregnancy and had vaginal and gastrointestinal colonization with the same strain of E tarda as the infant at the time of delivery . This case suggests that maternal exposures to contaminated bodies of water during pregnancy may represent a risk to newborns. Infect Immun, 2003 Mar, 71(3), 1343 - 51 Functional genomics approach to the identification of virulence genes involved in Edwardsiella tarda pathogenesis; Srinivasa Rao PS et al.; Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans . Here, we report the identification of 14 virulence genes of pathogenic E . tarda that are essential for disseminated infection, via a genome-wide analysis . We screened 490 alkaline phosphatase fusion mutants from a library of 450,000 TnphoA transconjugants derived from strain PPD130/91, using fish as an infection model . Compared to the wild type, 15 mutants showed significant decreases in virulence . Six mutants had insertions in the known virulence-related genes, namely, fimA, gadB, katB, pstS, pstC, and ssrB . Some mutants corresponded to known genes (astA, isor, and ompS2) that had not been previously shown to be involved in pathogenesis, and three had insertions in two novel genes . In vivo infection kinetics experiments confirmed the inability of these attenuated mutants to proliferate and cause fatal infection in fish . Screening for the presence of the above-described virulence genes in six virulent and seven avirulent strains of E . tarda indicated that seven of the genes were specific to pathogenic E . tarda . The genes identified here may be used to develop vaccines and diagnostic kits as well as for further studying the pathogenesis of E . tarda and other pathogenic bacteria. Comp Immunol Microbiol Infect Dis, 2003 May, 26(3), 199 - 211 High antigenic cross-reaction among the bacterial species responsible for diseases of cultured freshwater fishes and strategies to overcome it for specific serodiagnosis; Swain P et al.; Antigenic sharing among the most commonly bacterial pathogens such as Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens of Indian major carps has been studied using immunological reactions such as cross-agglutination, disc diffusion and indirect enzyme linked immunosorbent assay (ELISA) . The data were analysed using statistical analysis (SAS), version 6.12 . The results showed high antigenic similarities among the bacterial whole cells, whole cell lysates, somatic 'O' antigens, lipopolysaccharides (LPS) and extracellular products (ECP) . However, few or no similarities were observed in ECP components of <20kD . The present study indicates a need to develop differential diagnostic methods based on serology by choosing the highly specific less cross-reactive ECP antigen. Mol Plant Microbe Interact, 2003 Jan, 16(1), 53 - 64 Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion; Bladergroen MR et al.; Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation . The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea . A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea . We have sequenced a 33-kb region around the phage-transductable Tn5 insertion . The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus . Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens . Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain . Other proteins show similarity to proteins involved in type III protein secretion . Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5 . The production of these proteins in the wild-type strain is temperature dependent . Sequencing of two of these proteins revealed that their first 20 amino acids are identical . This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V . cholerae . Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems . Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen . Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype . These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants. Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 137 - 40 {Studies on the lactate oxidase producing conditions by Edwardsiella tarda}; Xu P et al.; A bacterium producing lactate oxidase was re-screened from five strains based on previous researches . The lactate oxidase activity was the highest in strain L1 and this stain was chosen as the enzyme source . Morphological and physiological studies revealed that the bacterial strain L1 belongs to the Edwardsiella tarda Biogroup I . This stain is different from the reported strains Mycobacterium and Pediococcus, which produce lactate oxidase . The enzyme producing conditions were studied in shaking cultures, and the effects of initial pH, riboflavin, lactate and ammonia sulphate concentrations on the production were carried out respectively . The bacteria resource of enzyme is significant to pyruvate production by enzymatic method, and to the enzyme assay of lactate for medical diagnosis, and the application of enzyme electronic probe. Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 736 - 40 {Detection of pathogenic Edwarsiella tarda}; Xiong Q et al.; 25 Edwardsiella tarda(Et) strains had been detected both on their viruslent factor Excellular Product (ECP), including the hemolysin and extracellular protease (ECPase), and on their pathogenicity to mice and Xiphophorus helleri . ECP was detected by Dot-ELISA with rabbit antiserum against ECP of reference strain JEL4 . The results showed that the animal pathogenicity of Et had good correlation with its hemolysin other than with ECPase . The agreement between Dot-ELISA of JEL4 ECP and pathogenicity to animal was up to 100% . It was desirable to establish a detecting method, which only need detect the ECP with plate assay (PA) and Dot-ELISA, but needn't have animal experiment . Furthermore it is possible to develop a diagnosis kit of application to simplify the detecting procedure of pathogenic Et. Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 630 - 4 {Analys of outer membrane protein and drug-resistant of Edwardsiella tarda}; Huang X et al.; The SDS-PAGE profiles of out membrane protein(OMP) of 27 Et strains showed diverse patterns and they could be divided into six types . Between 21 pathogenic strains and 6 non-pathogenic strains there were obviously difference . The pathogenic strains isolated from China belong to E type and were simary to ATCC 15947 . The OMP bands of pathogenic strains were more and denser than those of the non-pathogenic strains, and the strains from same souce had the identical patters . Interestingly, the non-pathogenic strains from different sources also had the almost same OMP profiles . What's more, they were also high resistant to some antibiotics such as Smx, Gem Tet and so on, while the 18 pathogenic strains were sensitivity to these. Wei Sheng Wu Xue Bao, 1998 Oct, 38(5), 336 - 40 {Internalization and replication of Edwardsiella tarda in HEp-2 cells}; Ma X et al.; It is demonstrated that Edwardsiella tarda possesses the ability to invade cultured epithelial HEp-2 cells by both lysis-counting assay and thin-section electron microscopy . Among fifteen strains of E . tarda, 6 strains internalized into HEp-2 cells and located mainly in vesicles . After internalization, the bacteria replicated in the host cells and then released into medium . Pretreatment of the HEp-2 cells with various concentration of cytochalasin D (0.1-5.0 micrograms/ml) or cytochalasin B (2.5-10.0 micrograms/ml) significantly reduced the amount of internalized bacteria in a dose-dependent manner . While pretreatment of HEp-2 cells with 0.25-100 mumol/L colchicine did not show any effect on the invasiveness of E . tarda . This strongly indicates that microfilaments are required for the internalization of E . tarda into HEp-2 cells and that microtubules are not involved in the entrance of E . tarda into HEp-2 cells. Dis Aquat Organ, 2002 Nov 22, 52(2), 93 - 107 Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection; Moore MM et al.; An Edwardsiella ictaluri expression library was screened for clones expressing antigenic E . ictaluri proteins using anti-E . ictaluri serum, which resulted in the isolation of 32 clones . The clones were partially characterized and 4 were selected for complete analysis . Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimensional PAGE, Western blotting, and DNA sequencing were used to analyze expressed antigenic proteins and encoded genes . Sequence analysis identified 4 putative open reading frames (ORFs) in the insert of Clone 4d6, which corresponded to antigenic acidic proteins of 55, 20 and 18 kDa expressed by both the clone and E . ictaluri cells . The predicted gene products of these ORFs were similar to several products of the imp locus of Rhizobium leguminosarum bv . trifolii . The imp locus of R . leguminosarum contains 14 genes that encode proteins involved in a putative temperature-dependent protein secretion system . In addition there was significant amino acid identity for a variety of hypothetical proteins from R . solanacearum, Ps . aeruginosa, A . tumefaciens, Y . pestis, and Salmonella typhimurium . Overlapping inserts of Clones 1.4, 5d2, and 5d3 encoded ORFs similar to Escherichia coli partial genes serA and pgk, and complete genes rpiA, iciA, yggE, yggB and fda . These genes encode D-3-phosphoglycerate dehydrogenase (serA), ribose 5-phosphate isomerase (rpiA), a specific inhibitor of chromosomal initiation of replication (iciA), a hypothetical protein (yggE), a protein involved in responses to osmotic stress (yggB), fructose 1,6-bisphosphate aldolase (fda), and phosphoglycerate kinase (pgk) . Cloned antigenic E . ictaluri proteins of 33, 27, 35 and 45 kDa appeared to be products of the ORFs similar to yggE, rpiA, iciA, and fda respectively . All the cloned antigenic proteins were recognized by antiserum from catfish that had recovered from enteric septicemia of catfish (ESC), indicating that these antigens are expressed during the infectious process . The cloned antigenic proteins were subsequently evaluated as subunit vaccines for protection against wild-type E . ictaluri . All vaccine treatments were protective against E . ictaluri in catfish, but results were inconclusive due to high levels of cross-reactive protection afforded by the E . coli host strain of the cloning vector. J Gen Appl Microbiol, 1998 Oct, 44(5), 311 - 316 Specific probiotic characterization of Weissella hellenica DS-12 isolated from flounder intestine; Cai Y et al.; A total of 199 microorganisms were isolated from the intestinal contents of flounder (Paralichthys olivaceus) in a fish farm in Seoul, Korea . Among these strains, DS-12 was selected as a candidate for flounder probiotics because of its excellent exhibition of antimicrobial activity against fish pathogens such as edwardsiella, pasteurella, aeromonas, and vibrio, and initiate growth in 10% NaCl, 10% bile, and in broth at pH 3 for 90 min . This strain was Gram-positive, and catalase-negative coccoid rods that produced gas from glucose and formed more than 90% of lactate as the D(-) isomer . This organism is positioned at a cluster in the genus Weissella on the phylogenetic tree based on 16S rRNA sequences, which were assigned to Weissella hellenica on the basis of DNA-DNA relatedness . However, the type strain of W . hellenica JCM 10103(T) had no antibacterial activity against the fish pathogenic bacteria and was found to be quite different from the DS-12 strain in some sugar fermentation patterns of alpha-methyl-D-glucoside, esculine, cellobiose, melibiose, D-raffinose, and D-turanose, being especially unable to grow at 15 and 35 degrees C in 7% NaCl and 10% bile . The results obtained in the present study demonstrated that the type strain of W . hellenica had no probiotic characteristics, but the strain DS-12 could be used as a specific probiotic for flounder. J Med Food, 2002 Fall, 5(3), 159 - 69 Chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review; Mine Y et al.; Passive immunization by oral administration of specific antibodies has been an attractive approach against gastrointestinal (GI) pathogens in both humans and animals . Recently, laying chickens have attracted considerable attention as an alternative source of antibodies for the prevention and treatment of infectious GI diseases . After immunization, the specific antibodies (called IgY) are transported to the egg yolk, from which the IgY then can be separated without sacrificing chickens . A chicken usually lays about 280 eggs in a year, and egg yolk contains 100-150 mg of IgY per yolk, suggesting that more than 40 g of IgY per year can be obtained from each chicken through eggs . IgY is also an alternative to antibiotics for treatment of enteric antibiotic-resistant pathogens . Oral administration of IgY has proved successful for treatment of a variety of GI infections, such as bovine and human rotaviruses, bovine coronavirus, Yersinia ruckeri, enterotoxigenic Escherichia coli, Salmonella spp., Edwardsiella tarda, Staphylococcus, and Pseudomonas . The IgY technology offers great future opportunities for designing prophylactic strategies against infectious GI diseases in humans and animals . However, there is still controversy regarding the stability of IgY through the GI tract . Finding an effective way to protect the antibodies from degradation in the GI tract would open the door for significant advances in IgY technology and nutraceutical applications. Dis Aquat Organ, 2002 Oct 4, 51(3), 161 - 7 Edwardsiella ictaluri invasion of IEC-6, Henle 407, fathead minnow and channel catfish enteric epithelial cells; Skirpstunas RT et al.; Invasion of Edwardsiella ictaluri into cultured mammalian, fish and enzymatically harvested catfish enteric epithelial cells is described . Gentamicin survival assays were used to demonstrate the ability of this catfish pathogen to invade IEC-6 (origin: rat small intestinal epithelium), Henle 407 (origin: human embryonic intestinal epithelium), fathead minnow (FHM, minnow epithelial cells) and trypsin/pepsin-harvested channel catfish enteric epithelial cells . Invasion of all cell types occurred within 2 h of contact at 26 degrees C, in contrast to Escherichia coli DH5 alpha, which did not invade cells tested . Eight Edwardsiella ictaluri isolates from diseased catfish and the ATCC (American Type Culture Collection) strain were evaluated for invasion efficiency using FHM cells . All isolates were invasive, but at differing efficiencies . Invasion blocking assays using chemical blocking agents were performed on a single isolate (LA 89-9) using IEC-6 epithelial cells . Preincubation of IEC-6 cells with cytochalasin D (microfilament depolymerizer) and monodansylcadaverine (blocks receptor-mediated endocytosis) significantly reduced invasion by E . ictaluri, whereas exposure to colchicine (microtubule depolymerizer) had no effect on bacterial internalization . Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E . ictaluri by IEC-6 epithelial cells . Invasion trials using freshly harvested cells from the intestine of the natural host, Ictalurus punctatus, show that invasion occurs, but at a low efficiency . This is possibly due to loss of outer membrane receptors during enzymatic cell harvest . This study provides the first documentation of the invasion of cultured mammalian and fish cells by E . ictaluri, and identifies possible mechanisms used for intracellular access . Additionally, the study describes several functional in vitro invasion models using commercially available cell lines as well as cells from the natural host (channel catfish, I . punctatus). J Microbiol Methods, 2003 Feb, 52(2), 209 - 20 Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare; Bader JA et al.; A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set . The primer set amplified a specific 1193-bp DNA fragment from F . columnare strains but not from related bacteria, F . psychrophilum, F . aquatile, F . branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E . tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli . The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used) . DNA was extracted by a boiled-extraction method or by commercial column purification . The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells . Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F . columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish . The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al . {Fish Pathol . 29 (1994) 271.} and that of Bader and Shotts {J . Aquat . Anim . Health 10 (1998) 311.} . The new primer set is as good or better than the previously published primer sets for detecting F . columnare in all samples and under all conditions tested . Fish Shellfish Immunol, 2002 Aug, 13(2), 133 - 40 Bath immunisation of spawn, fry and fingerlings of Indian major carps using a particulate bacterial antigen; Swain P et al.; Larval mortality in Indian major carps is one of the major problems encountered in the pond culture system . The present investigation was carried out to investigate the proper age, duration of exposure, and optimum bacterin concentration for vaccinating rohu (Labeo rohita) and catla (Catla catla) at their early stages with a formalin killed Edwardsiella tarda bacterin suspension . The development of immunological competence was recorded with spawn of rohu and catla of 3 weeks of age exposed to a bacterin at a concentration 10(9) cfu ml(-1) for 15 min, where it persisted up to 4 weeks post vaccination . They showed significant resistance against challenge with virulent E . tarda bacteria . Significant antibody titre could be recorded in advanced fries and fingerlings exposed to 10(9) cfu/ml(-1) bacterin concentration for 45 and 60 min, respectively. Infect Immun, 2002 Nov, 70(11), 6475 - 80 Comparative proteomic analysis of extracellular proteins of Edwardsiella tarda; Tan YP et al.; A comparison of extracellular proteins of virulent and avirulent Edwardsiella tarda strains revealed several major, virulent-strain-specific proteins . Proteomic analysis identified two of the proteins in the virulent strain PPD130/91 as flagellin and SseB, which are virulence factors in bacterial pathogens . PCR amplification and DNA sequencing confirmed the presence of the genes that encode these proteins . Our results clearly demonstrated the potency of the proteomic approach in identifying virulence factors. Curr Protein Pept Sci, 2000 Jul, 1(1), 75 - 89 Serratia type pore forming toxins; Hertle R; The Serratia marcescens hemolysin represents a new type of hemolysin and has been studied in great molecular detail with regard to structure, activation and secretion . It has nothing in common with the pore forming toxins of E . coli type (RTX toxins), the Staphylococcus aureus alpha-toxin or the thiol activated toxin of group A beta-hemolytic streptococci (Streptolysin O) . Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins . The S . marcescens hemolysin proteins ShlB and ShlA exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Edwardsiella tarda and Erwinia chrysantemi . Furthermore, sequence motifs present in ShlA and Shlb have been shown to be important for activity and secretion of the S . marcescens hemolysin . Thus, the S . marcescens hemolysin forms the prototype of a new class of hemolysins and of a new secretory mechanism . The uniqueness of this new mechanism is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor . New data implicate a conformational change in ShlA during activation . In addition, ShlA not only forms pores in erythrocytes but also in fibroblasts and epithelial cells . The cytotoxic action of ShlA is mainly determined by lysis of infected cells in vitro . In sublytic doses, as will normally be the situation in vivo, ShlA exerts additionally effects which are currently under investigation . The knowledge of the structure, activation, secretion and mode of action of S . marcescens hemolysin has implications for proteins, related in sequence or in mode of secretion and activation. Vet Microbiol, 2002 Oct 2, 89(1), 29 - 39 Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans; Nucci C et al.; It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin . We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E . tarda from humans and fish from several countries . All isolates were negative for all genes investigated by PCR . Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min . All isolates adhered and invaded in these tests . Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin. J Zoo Wildl Med, 2002 Mar, 33(1), 62 - 5 First report of a parasitic copepod (Pennella balaenopterae) infestation in a pinniped; Dailey MD et al.; An infestation by the parasitic copepod Pennella balaenopterae was found in a stranded, 8-mo-old, female northern elephant seal (Mirounga angustirostris) . Diagnosis was based on the finding of the cephalothoraxes of 14 adult female copepods from three subcutaneous sites . Bacteria cultured from lesion exudate included Arcanobacterium phocae, Escherichia coli, Edwardsiella tarda, an Enterococcus sp., and Proteus mirabilis . The lesions were drained and irrigated with chlorhexidine, and the seal was treated with a subcutaneous injection of ivermectin . The seal recovered and was released after 43 days. J Food Prot, 2002 Jul, 65(7), 1146 - 51 Influence of catfish skin mucus on trisodium phosphate inactivation of attached Salmonella Typhimurium, Edwardsiella tarda, and Listeria monocytogenes; Kim J et al.; This study examined the antimicrobial effectiveness of trisodium phosphate (TSP) on Edwardsiella tarda, Listeria monocytogenes, and Salmonella Typhimurium attached to catfish skin with and without mucus . Salmonella Typhimurium and E . tarda attached more readily to catfish skin than did L monocytogenes . At high inoculum levels (10(7) CFU/ml), TSP treatments (at 2 to 6%) for 10 min reduced bacterial counts of E . tarda by >2.5 to >3.3 log10 CFU per skin sample for firmly attached cells and by 3.5 to 3.6 log10 CFU per skin sample for loosely attached cells . Counts of L . monocytogenes declined by 0.6 to >1.8 log10 CFU per skin sample for firmly attached cells and by 1.2 to 2.2 log10 CFU per skin sample for loosely attached cells . Counts of Salmonella Typhimurium were reduced by 3.6 to >3.8 log10 CFU per skin sample for firmly attached cells and by 3.5 to >3.8 log10 CFU per skin sample for loosely attached cells . Overall, counts of firmly attached bacteria on TSP-treated skins with mucus were higher than counts on skin without mucus . Firmly attached L . monocytogenes was more resistant to TSP than was firmly attached Salmonella Typhimurium or E . tarda . The presence of mucus on skins slightly decreased the antimicrobial effect of TSP Significant (P < 0.05) reduction in the numbers of all three bacteria can be achieved by treatment with 6% TSP for 10 min. Indian J Exp Biol, 2001 Dec, 39(12), 1311 - 3 Edwardsiella tarda endotoxin as an immunopotentiator in Singhi, Heteropneustes fossilis fingerlings; Das BK et al.; Endotoxin of E . tarda grown in brain heart infusion broth at 30 degrees C for 18 hr was extracted by differential centrifugation . Fingerlings of H . fossilis (weighing 1-2 g) were allowed for hyperosmotic infiltration in the endotoxin at the rate of 0,2,4,8,16 and 20 mg/l . Mortality varied from 20-50% at 2 to 20 mg/ml . Toxin treated fishes were challenged 21 days post treatment with the same E . tarda strain containing 2.1 x 10(9) CFU/ml . There was 80% mortality in the control group whereas only 20% mortality in toxin treated group at 2 mg/l concentration after challenge with homologous E . tarda . Subsequently a second challenge of E . tarda was given to the survivors of fish one month after first challenge using same concentration where no mortality could be observed . It was concluded that the endotoxin could enhance percentage of survival against E . tarda infection in Singhi. Fish Shellfish Immunol, 2002 Jan, 12(1), 1 - 16 The effect of dietary immunomodulation upon Edwardsiella tarda vaccination in healthy and immunocompromised Indian major carp (Labeo rohita); Sahoo PK et al.; In order to determine the impact on disease resistance of four dietary immunomodulators viz., beta-1,3 glucan, levamisole, vitamins C and E, in an important farmed Indian major carp species, rohu (Labeo rohita Ham.), fish were fed diets containing various levels of these substances during a 60 day trial . Aflatoxin B1 (AFB,) at 125 mg kg(-1) body weight was injected intraperitoneally (i.p.) into fish to induce an immunosuppressive state on the first day of the experiment in some individuals . The fish were vaccinated against formalin-killed Edwardsiella tarda vaccine on day 30 of the experiment . Specific immunity, as measured by bacterial agglutination titre and disease resistance against E . tarda, was determined at the end of the trial . The results demonstrate that all the four immunomodulators were capable of significantly (P<0.05) increasing specific immunity and reducing mortality in immunocompromised fish but failed to enhance specific immunity and protection in healthy fish . The increased bacterial agglutination titre by beta-1,3 glucan, and reduced mortality losses by both beta-1,3 glucan and levamisole were marked in healthy vaccinated fish compared with their controls . Similarly, all four substances significantly reduced the mortality rates in immunocompromised and healthy unvaccinated fish . Out of these four substances, glucan was recorded to be the most effective immunomodulator in rohu . The present results suggest that the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions could increase their resistance to infection by reducing mortality rates and offer economic benefits. Yi Chuan Xue Bao, 2001, 28(12), 1162 - 7 {Primary analysis and sequencing the hemolytic relative gene of Edwardsiella tarda}; Gao DQ et al.; Hemolysin is an important pathogenic agent of Edwardsiella tarda (ET) . A fragment containing hemolytic gene, of which sequence is 4,264 bp, was cloned from ET-12 chromosome by shutgun method . It has no similarity to the hemolysin genes reported, of which 424 bp of open read frame (ORF) 3 has 68% similarity to the hemolysin regulatory gene of S . typhi (slyA) . The subclone which has complete ORF3 was hemolytic, and the other of which Kanamycin gene was inserted in Pvu I of ORF3 was no hemolytic . It proved the gene was in chromosomes of ET-12 and the other ET strains by hybridization in situ and Southern blot . As recombination plasmid with the gene entered nonhemolytic ET by electroporation, no hemolytic phenomenon was observed . Conclusion was that the gene wasn't the hemolysin gene and was hemolytic relative gene. Fish Shellfish Immunol, 2001 Nov, 11(8), 683 - 95 Effect of dietary beta-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton); Sahoo PK et al.; The immunostimulant beta-1,3 glucan was fed at 0.1% in feed for 7 days to healthy and aflatoxin B1 (AFB1)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial . The effects of AFB1, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied . A single intraperitoneal injection of AFB1 at 1.25 mg kg(-1) body weight) caused a significant (P < 0.05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan . Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control . Feeding of glucan to AFB1-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A . hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB1 exposed fish . Although feeding of glucan was able to increase specific immunity, all measured through haemagglutination titre against sheep red blood cells, and bacterial (E . tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen. Infect Immun, 2001 Sep, 69(9), 5689 - 97 Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates; Srinivasa Rao PS et al.; Edwardsiella tarda is responsible for hemorrhagic septicemia (edwardsiellosis) in fish and also causes diseases in higher vertebrates such as birds, reptiles, and mammals, including humans . Interactions of E . tarda with blue gourami phagocytes were studied by light microscopy as well as by adherence, intracellular replication, and superoxide anion assays . Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bacteria could adhere to and survive and replicate within phagocytes, while only opsonized virulent strains replicated within the phagocytes . Furthermore, only avirulent E . tarda elicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish phagocytes . TnphoA transposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA+) fusion mutants from a total of 182,000 transconjugants derived from E . tarda PPD130/91 . Five of these mutants induced more ROI production in phagocytes than the wild-type strain . Two mutants had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain . Sequence analysis revealed that three of these mutants had insertions at sequences having homology to PhoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens . These three independent mutations might have changed the cell surface characteristics of the bacteria, which in turn induced phagocytes to produce increased ROIs . Sequences from two other mutants had no homology to known genes, indicating that they may be novel genes for antiphagocytic killing . The present study showed that there are differences in the interactions of virulent and avirulent E . tarda organisms with fish phagocytes and PhoA+ fusion mutants that could be used successfully to identify virulence genes . The information elucidated here would help in the development of suitable strategies to combat the disease caused by E . tarda. Biochim Biophys Acta, 2001 Jul 30, 1520(1), 35 - 44 Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein; Hikima J et al.; In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene . The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns . A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme . Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes . In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene . A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene . Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda . Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria . This is the first report of a g-type lysozyme gene other than for reported avian species. Antimicrob Agents Chemother, 2001 Aug, 45(8), 2245 - 55 Natural antibiotic susceptibilities of Edwardsiella tarda, E . ictaluri, and E . hoshinae; Stock I et al.; The natural antibiotic susceptibilities to 71 antibiotics of 102 Edwardsiella strains belonging to E . tarda (n = 42), E . ictaluri (n = 41), and E . hoshinae (n = 19) were investigated . MICs were determined using a microdilution procedure according to NCCLS criteria and German standards . All edwardsiellae were naturally sensitive to tetracyclines, aminoglycosides, most beta-lactams, quinolones, antifolates, chloramphenicol, nitrofurantoin, and fosfomycin . Edwardsiella species were naturally resistant to macrolides, lincosamides, streptogramins, glycopeptides, rifampin, fusidic acid, and oxacillin . Although slight species-dependent differences in natural susceptibilities to some antibiotics (e.g., macrolides and cefaclor) were seen, differences in natural susceptibility affecting clinical assessment criteria were only seen with benzylpenicillin . Whereas E . tarda was naturally resistant to benzylpenicillin, E . hoshinae was naturally sensitive . Natural sensitivity and resistance to this penicillin were found among the strains of E . ictaluri . The observed oxacillin sensitivity of E . ictaluri was attributed to the failure of the species to grow at higher salt concentrations found in oxacillin-containing microtiter plates . The present study describes a database concerning the natural susceptibility of Edwardsiella species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these microorganisms. Comp Immunol Microbiol Infect Dis, 2001 Jul, 24(3), 143 - 9 Immunosuppressive effects of aflatoxin B1 in Indian major carp (Labeo rohita); Sahoo PK et al.; Aflatoxin B1 (AFB1) is a potent immunomodulator in endotherms . The experiment was carried out to study the immunosuppressive nature of AFB1 in one ectothermic species of Indian major carp . Graded levels (0, 1.25, 5.00 mg/kg of body weight) of purified AFB1 were intraperitoneally (i.p.) injected into rohu (Labeo rohita) fingerlings weighing 30-50 g, and the fish were observed for a period of 90 days . At the end of the trial, blood samples were collected from the control group as well as the AFB1 injected fish and were screened for nitroblue tetrazolium (NBT) assay, serum total protein, albumin, globulin, albumin-globulin ratio (A:G), serum bactericidal activity and bacterial agglutination titre against Edwardsiella tarda . The aflatoxin-treated fish revealed a reduction of total protein, globulin levels, bacterial agglutination titre, NBT and serum bactericidal activities, as well as an enhanced A:G ratio without change in albumin concentration, irrespective of dose levels of toxin treatment, when compared to the control group . Thus, AFB1 proved to be immunosuppressive in rohu even at the lowest dose (1.25 mg/kg body weight) of toxin treatment . This could be of economic significance in intensive culture systems of rohu. Dev Comp Immunol, 2001 Jun-Jul, 25(5-6), 439 - 45 Expression of Japanese flounder c-type lysozyme cDNA in insect cells; Minagawa S et al.; Lysozyme is a widely distributed hydrolase which likely plays an important role in bio-defense systems . In the current study, we constructed a baculovirus expression system for the c-type lysozyme cDNA of Japanese flounder (Paralichthys olivaceus) and evaluated the activity of the recombinant protein . This activity was estimated, by turbidimetric assay, to be 7.7U/mg, a value five times higher than that of hen egg white (HEW) c-type lysozyme examined under the same conditions . The optimum pH and temperature for the lytic activity of the Japanese flounder c-type recombinant lysozyme were found to be 5.0-6.5 and 40 degrees C, respectively . Two groupings for fish lysozyme activity are proposed; the first has an optimum pH of approximately 6.0 and the second an optimum pH of above 8.0 . Flounder c-type lysozyme was found to possess little lytic activities against Edwardsiella tarda and Streptococcus sp . This latter characteristic may help explain the fact that these two bacterial species are responsible for significant disease problems in cultured Japanese flounder. Plasmid, 2001 Jan, 45(1), 52 - 6 Sequencing and analysis of the Edwardsiella ictaluri plasmids; Fernandez DH et al.; To determine possible functions of the Edwardsiella ictaluri plasmids, pEI1 and pEI2, we analyzed the sequence of both plasmids . Plasmid pEI1 is 4807 bp, with 51% G + C, and 23 possible open reading frames of 40 amino acids or greater . Plasmid pEI2 is 5643 bp, with 51% G + C, and 24 possible reading frames . Database searches indicated that pEI1 contains an insertion element and a ROM analog . In addition, pEI1 possesses an open reading frame with strong homology to SlrP, SspH1, and SspH2 of Salmonella typhimurium and IpaH of Shigella flexnari, which have leucine-rich repeat regions and are components of type III secretory systems . pEI2 has a frame with weak homology to Spa15 of S . flexnari 5 and InvB of S . sonnei and S . typhimurium, which are also type III secretory system components, three origins of replication, a Rep analog, and a multimer resolution site . Clin Infect Dis, 2001 May 15, 32(10), 1430 - 3 Epub 2001 Apr 17. Myonecrosis caused by Edwardsiella tarda: a case report and case series of extraintestinal E . tarda infections; Slaven EM et al.; Edwardsiella tarda is an unusual human pathogen . It is primarily associated with gastrointestinal disease, although recent reports of extraintestinal disease are broadening the current understanding of the clinical spectrum of E . tarda . A series of 11 cases of extraintestinal E . tarda infection is presented, including the first reported case of myonecrosis in an immunocompetent patient . Wound infections were the most common manifestation, and 3 of 5 patients with infected wounds had been exposed to a marine environment . One patient had bacteremia, and the remaining 5 patients developed abscesses that required surgical drainage . Four patients had E . tarda isolated in pure culture, including the patient with myonecrosis . Although it is often difficult to ascertain the contribution of E . tarda to infection when it is isolated as part of a mixed culture, this case series suggests that E . tarda is singularly capable of causing limb- and life-threatening infections. Comp Biochem Physiol B Biochem Mol Biol, 2000 Dec, 127(4), 525 - 32 Characterization of Japanese eel immunoglobulin M and its level in serum; Uchida D et al.; Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized . Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790,000; it contained an equimolar heavy chain and light chain with molecular weights of 72,000 and 25,000, respectively . While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains . Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by D-mannose and could be abolished by alpha-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses . The average IgM concentration in the sera of the healthy eels was 3.4 mg ml(-1); it amounted to 10.3% of the total serum protein. FEMS Microbiol Lett, 2001 Jan 15, 194(2), 239 - 43 Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish; Ling SH et al.; The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus . An immersion challenge model was utilized to mimic natural infection conditions in fish . Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E . tarda (PPD130/91) by histological and infection kinetics studies . On the other hand, avirulent E . tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days. Microbiology, 2001 Feb, 147(Pt 2), 449 - 57 Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity; Mathew JA et al.; Edwardsiella tarda is a Gram-negative bacterium that causes a systemic infection, edwardsiellosis, in fish . The virulence factors of this pathogen and its genetic determinants have not been systematically examined . In this study, TnphoA transposon mutagenesis was used to construct a library of 440 alkaline phosphatase (PhoA(+)) fusion mutants from a total of 400000 transconjugants derived from Ed . tarda PPD130/91 . This library included genes for secreted and membrane-associated proteins normally involved in virulence . The library was screened for four virulence factors: siderophore production, motility, serum resistance and catalase production . Eight mutants deficient in one or more of these phenotypes were grouped into four classes . They were further characterized for their stimulation of reactive oxygen intermediate production by fish phagocytes, for their adhesion to and internalization into EPC (epithelioma papillosum of carp) cells, and for attenuation of virulence in blue gourami . Mutants 2A and 34 were highly attenuated in fish, with LD(50) values about 10 times higher than for the wild-type . These strains had mutations in the genes encoding arylsulfate sulfotransferase (mutant 2A) and a catalase precursor protein (mutant 34) . One hyperinvasive/adhesive mutant and four pst mutants that were pleiotropic and slightly attenuated in fish were also isolated. Med J Aust, 2000 Sep, 173(5), 256 - 9 Topically acquired bacterial zoonoses from fish: a review; Lehane L et al.; The main pathogens acquired topically from fish (through spine puncture or open wounds) are Aeromonas hydrophila, Edwardsiella tarda, Erysipelothrix rhusiopathiae, Mycobacterium marinum, Streptococcus iniae, Vibrio vulnificus and Vibrio damsela . S . iniae has recently emerged as a public health hazard associated with aquaculture, and M . marinum often infects home aquarium hobbyists . With the expansion of aquaculture and popularity of recreational fishing in Australia, medical practitioners can expect to see more infections of this nature . Diagnosis and treatment may be difficult, especially in view of emerging antibiotic resistance in fish pathogens. Dis Aquat Organ, 2000 Sep 28, 42(3), 227 - 31 Infection with Edwardsiella tarda causes hypertrophy of liver cells in the Japanese flounder Paralichthys olivaceus; Miwa S et al.; To study the direct cause of liver enlargement in the Japanese flounder Paralichthys olivaceus infected with Edwardsiella tarda, the fish were challenged with E . tarda and reared without feeding . The liver of fish exposed to the bacteria was markedly enlarged compared to that of the controls while no severe histopathological change appeared in the organ during the experiments . No notable difference was observed in the crude fat, glycogen, and water content of the liver between challenged and control fish . The size of liver cells and nuclei of the challenged fish was apparently larger than that of the controls . Analysis of crude DNA in the liver suggested that the number of liver cells of starved control fish significantly decreased during the experiment while that of the challenged fish was maintained at a level of the initial control . RNA/DNA ratio of the liver of challenged fish clearly increased while it decreased in the control fish during the experiment . These observations suggest that liver enlargement of flounder infected with E . tarda, at least in the early stage of infection, is not a result of any readily observable histopathological changes and that E . tarda infection causes hypertrophy of the cells, as well as preventing decrease in liver cell number. Fish Shellfish Immunol, 2000 Aug, 10(6), 475 - 87 Survivability and immune responses after challenge with Edwardsiella ictaluri in susceptible and resistant families of channel catfish, Ictalurus punctatus; Camp KL et al.; Diseases in catfish farming are prevalent and costly, particularly the bacterial disease Enteric Septicemia of Catfish . Considerable research has focused on different aspects of this disease, including the biology of the causative agent, Edwardsiella ictaluri . However, no satisfactory treatment or preventive has resulted from these efforts . One solution is to increase the natural disease resistance of the fish through genetic selection . Recent research has demonstrated that genetic factors influence resistance to infection in mammals as well as fish . Selective breeding for disease resistance in channel catfish is ongoing, however differences in defence mechanisms among E . ictaluri challenged strains and families are only now being investigated . Antigen-specific as well as non-specific immune responses of full-sib families of channel catfish to laboratory challenge with E . ictaluri have been investigated . Both resistant and sensitive families produce a humoral response as specific antibody, but there were no differences found in the level of specific antibody produced . The sensitive family produced a slightly higher percentage of B lymphocytes in mononuclear cell preparations from peripheral blood, while the resistant family had a higher percentage of T lymphocytes in those preparations . The most significant observation was that the resistant family produced more macrophage aggregations in the spleen and posterior kidney throughout the infection than the sensitive family . Neither family produced stress-associated amounts of cortisol. Int J Hyg Environ Health, 2000 Mar, 203(1), 77 - 82 Rapid identification of Enterobacteriaceae using a novel 23S rRNA-targeted oligonucleotide probe; Bohnert J et al.; The aim of this study was to rapidly identify bacteria of the family of Enterobacteriaceae using fluorescent in situ hybridization (FISH) . A comparative sequence analysis was carried out and a 23S rRNA signature sequence for Enterobacteriaceae was identified . A 23S rRNA-targeted oligonucleotide probe (EBAC1790) was constructed and subsequently tested against 40 reference strains . Nearly all of the Enterobacteriaceae used in this study yielded positive results with EBAC1790, except for Edwardsiella tarda (ATCC 15947) . None of the non-Enterobacteriaceae reference strains gave positive signals with the probe . The possibility of a rapid detection of Enterobacteriaceae in groundwater was demonstrated using colony hybridization. Nahrung, 2000 Jun, 44(3), 201 - 6 Multi-functional biopolymer prepared by covalent attachment of galactomannan to egg-white proteins through naturally occurring Maillard reaction; Nakamura S et al.; Protein-polysaccharide conjugate was prepared as a functional biopolymer using protein and polysaccharide via a Maillard-type reaction . Ovalbumin and lysozyme were conjugated with galactomannan under controlled heating and humidity conditions . The antioxidant effect of ovalbumin and the antimicrobial activity of lysozyme were enhanced by the glycosylation . The emulsifying properties of the egg protein were also significantly improved by the modification . The increase in lipid affinity due to the conjugation resulted in the enhancement of the radical scavenging ability of ovalbumin . The effectiveness of lysozyme and its glycosylated derivative in restricting the activity of a Gram-negative pathogen, Edwardsiella tarda in fish was also investigated. Dev Comp Immunol, 2000 Dec, 24(8), 741 - 50 Characterization and expression of an EB1 protein in Ictalurus punctatus neutrophils; Qian Y et al.; We have identified an EB1 gene (CfEB1) and protein in channel catfish neutrophils . The complete cDNA sequence is 1725 bp and the putative protein is composed of 258 amino acids . Western blot analysis of channel catfish neutrophil cell membrane with a monoclonal antibody (14I) yielded a 28 kD protein band with the protein preparation . Aside from neutrophils only a small percentage of other cells tested expressed detectable amounts of EB1 as determined by flow cytometry . Furthermore, EB1 expression increased after phorbol dibutyrate stimulation of neutrophils or incubation of catfish neutrophils with Edwardsiella ictaluri . Nonpermeabilized, fixed catfish neutrophils demonstrated immunofluorescent staining with 14I indicating that EB1 is apparently externally oriented in catfish neutrophils . This is the first report of the external orientation of the EB1 molecule . Because of its increase after stimulation and detection on cell membranes, EB1 may participate in catfish neutrophil cell regulation, signal transduction, or cell membrane changes necessary for phagocytosis. Microbiology, 2000 Jan, 146 ( Pt 1), 7 - 19 Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models; Ling SH et al.; Edwardsiella tarda is a fish pathogen that causes systemic infections in many food and ornamental fish . E . tarda PPD130/91 and PPD125/87 were selected as representatives of the virulent and avirulent groups, respectively, from eight fish isolates, and transformed with plasmids encoding either green fluorescent protein (pGFPuv) or blue fluorescent protein (pBFP2) . Two host models were used to study the invasion pathway of E . tarda in vitro and in vivo . Epithelioma papillosum of carp (EPC) was used as the first model . Virulent and avirulent E . tarda strains were found to adhere to and invade EPC cells . Interactions between E . tarda and host cells examined under confocal microscopy and intracellular growth were followed at different time points . Bacterial internalization of PPD130/91 and PPD125/87 involved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization . Confocal studies revealed co-localization of polymerized actin with bacteria . Staurosporine, a protein kinase C inhibitor, accelerated internalization of PPD125/87, whereas PD098059, a mitogen-activated protein kinase (MAPK) kinase inhibitor prevented internalization of PPD130/91 . In the second model, blue gourami were infected with E . tarda intramuscularly . Mortalities were observed in PPD130/91(pGFPuv)-infected fish with high bacterial numbers detectable in all organs . PPD125/87(pBFP2)-infected fish did not die and the bacterial population decreased over time . Mixed infections comprised of both PPD130/91(pGFPuv) and PPD125/87(pBFP2), where inoculum size was similar to the single infections, caused mortalities in fish . High bacterial populations were noted only in the fish body muscle . The PPD125/87(pBFP2) population in the fish decreased after 5 d . The number of PPD130/91(pGFPuv) also decreased in the fish organs, except for continued high growth in the body muscle . Histology revealed necrosis of the tissue (body muscle and liver) and fluorescent bacteria in fish that were infected with PPD130/91(pGFPuv) but not with PPD125/87(pBFP2) . This study showed that fluorescent proteins are a useful tool for investigating bacterial host cell infection, and information elucidated here sheds new light on the interactions between E . tarda and its hosts. Onderstepoort J Vet Res, 2000 Sep, 67(3), 201 - 4 Aerobic intestinal flora of wild-caught African dwarf crocodiles Osteolaemus tetraspis; Huchzermeyer FW et al.; Intestinal contents were collected from wild-caught African dwarf crocodiles (Osteolaemus tetraspis) in 1993 and 1995 which were slaughtered at urban markets in the Congo Republic . The samples were kept frozen and brought back to Onderstepoort for aerobic culture . Out of 29 specimens, 33 species of bacteria and 20 species of fungi were isolated . The bacteria included three isolates of Salmonella and eight isolates of Escherichia coli, most of the latter being rough strains . The flora of individual specimens contained 1-5 bacterial and 0-5 fungal species . Neither Aeromonas hydrophila nor Edwardsiella tarda were isolated from any of the samples. J Infect, 1999 Mar, 38(2), 124 - 6 Edwardsiella tarda bacteraemia--complicated by acute pancreatitis and pyomyoma; Yang CH et al.; Edwardsiella tarda (E . tarda) has recently become recognized as a pathogen in humans . Here we report a new case of E . tarda bacteraemia complicated by acute pancreatitis and pyomyoma . A 46-year-old female came to our emergency room complaining of sudden onset of left upper quadrant pain and vomiting for the previous few hours after drinking three bottles of wine . An abdominal computed tomography (CT) scan revealed multiple biliary stones, acute pancreatitis with extensive inflammatory change, and a large uterine myoma . Fever, watery diarrhoea, and mild suprapubic discomfort with vaginal spotting were noted soon after admission . The patient's blood cultures yielded E . tarda and symptoms subsided after antibiotic therapy . Fever and severe suprapubic pain with rebound tenderness developed 12 days later . Repeat abdominal CT scan revealed an enlarged uterine myoma with central necrosis . The patient subsequently underwent anterior total hysterectomy and bilateral salpingo-oophorectomy, revealing a uterine myoma with infarction and abscess formation . The patient recovered uneventfully and was discharged 1 week later. Adv Vet Med, 1999, 41, 523 - 37 Development and use of modified live Edwardsiella ictaluri vaccine against enteric septicemia of catfish; Klesius PH et al.; The present study showed that E . ictaluri RE-33 vaccine does not cause ESC but does stimulate protective immunity . The RE-33 vaccinates were protected against ESC for at least 4 months following a single bath immersion in a low number of E . ictaluri RE-33 without booster vaccination . Antibody responses are weak after RE-33 vaccination . Edwardsiella ictaluri RE-33 vaccine presents no risk or hazard to catfish . RE-33 vaccine will prevent ESC caused by most isolates of E . ictaluri in catfish . We recently obtained from USDA, Animal Plant Health Inspection Service (APHIS), and the state veterinarians of Alabama and Mississippi, approval to field test the RE-33 vaccine in young catfish . About 2-3 million 10- to 30-day-old channel catfish in Alabama and Mississippi have been vaccinated since June 1997 with no adverse effects of vaccination. J Vet Pharmacol Ther, 1998 Oct, 21(5), 364 - 8 Pharmacokinetics of oxytetracycline in the red pacu (Colossoma brachypomum) following different routes of administration; Doi AM et al.; Oxytetracycline (OTC) pharmacokinetics were studied in the red pacu (Colossoma brachypomum) following intravenous (i.v.) and intramuscular (i.m.) administration at a dose of 5 mg/kg body weight . OTC plasma concentrations were determined by high-performance-liquid-chromatography (HPLC) . A non-compartmental model was used to describe plasma drug disposition after OTC administration . Following i.m . administration, the elimination half-life (t1/2) was 62.65 +/- 1.25 h and the bioavailability was 49.80 +/- 0.01% . After i.v . administration the t1/2 was 50.97 +/- 2.99 h, the Vd was 534.11 +/- 38.58 mL/kg, and CI(b) was 0.121 +/- 0.003 mL/min x kg . The 5 mg/kg i.v . dose used in this experiment resulted in up to 48 h plasma concentrations of OTC above the reported MIC values for some strains of fish pathogens such as Aeromonas hydrophila, A . liquefaciens, A . salmonicida, Cytophaga columnaris, Edwardsiella ictaluri, Vibrio anguillarium, V . ordalii, V . salmonicida and Yeersinia ruckeri . These MIC values are below the susceptible range (4 microg/mL) listed by the National Committee for Clinical Laboratory Standards (NCCLS) as determined by the NCCLS susceptibility interpretive criteria. Mol Microbiol, 1998 Jun, 28(6), 1269 - 81 The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells; Mecsas J et al.; Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops . After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm . Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways . In contrast to this active inhibition of phagocytosis by Yersinia, other pathogens such as Salmonella, Shigella, Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways . We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens . We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen . We then measured the level of bacterial invasion . Preinfection with Yersinia inhibited invasion of Edwardsiella, Shigella and Listeria, but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion . We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways . We also speculate that YopE affects the rho pathway. Dis Aquat Organ, 1998 May 14, 33(1), 19 - 24 In vivo modulation of innate resistance to Edwardsiella ictaluri with a phosphatase inhibitor; Evans DL et al.; Catfish were treated with the protein phosphatase inhibitor sodium orthovanadate (vanadate) and challenged with the pathogen Edwardsiella ictaluri to investigate the relationship between the in vivo immunoregulatory effects of tyrosine and serine phosphatases on nonspecific modulation of resistance to bacterial infections . Two different infection protocols were used: fish were pretreated by immersion in vanadate and subsequently infected (by immersion) with 1 LD100 E . ictaluri, or fish were injected (intraperitoneally, i.p.) with bacteria and simultaneously treated (by immersion) with 25 microM vanadate . In the absence of vanadate, both infection models produced fulminant infection by 10 or 6 d, respectively . Zero to 48 h treatment with vanadate (by immersion) prior to infection produced 17 to 100% survival of infected fish . In addition to augmentation of inmate immunity, vanadate enhanced acquired immunity to this pathogen . Fish which had vanadate-induced resistance to primary infection were 'immune' to secondary challenge with a LD100 of E . ictaluri . Experiments were done to determine the mechanism(s) of the altered innate resistance . Catfish were injected (i.p.) with E . ictaluri and simultaneously treated (by immersion) with 25 microM vanadate . Assays were done to measure nonspecific cytotoxic cell (NCC) activity at 0, 24, 48, 72 and 96 h post-infection/vanadate treatment . Increased NCC activity at 48 to 96 h post-infection appeared to correlate with resistance to bacterial related mortality . These data indicated that in vivo vanadate treatment of catfish significantly increased resistance to otherwise fulminant E . ictaluri infections . This effect coincided with the initiation of resistance to secondary infections without additional vanadate treatments . Vanadate-modulated resistance in catfish may be associated with augmented NCC activity. Mol Mar Biol Biotechnol, 1998 Jun, 7(2), 138 - 44 Molecular cloning of a novel interferon regulatory factor in Japanese flounder, Paralichthys olivaceus; Yabu T et al.; A complementary DNA library was constructed in lambda ZAP II using messenger RNA from the leukocytes of some heterocloned Japanese flounder, Paralichthys olivaceus, that had been artificially infected with Hirame rhabdovirus (HRV) . A cloned flounder interferon regulatory factor (designated fIRF) cDNA was found to be 1746 bp in length, with an open reading frame of 297 amino acids . The overall amino acid sequence of fIRF had approximately 40% identity with the previously reported avian and mammalian IRF-1s and IRF-2s . The fIRF sequence was most similar to that recorded for the chicken IRF-1 . Amino acid sequence identities between the DNA-binding domain of the fIRF and that of both chicken IRF-1 and chicken IRF-2 were 72.3% . The DNA-binding domain of fIRF contained the repeated tryptophan motif that is characteristic of members of the IRF family . The mRNA of fIRF was detected in various tissues by reverse transcription-polymerase chain reaction (RT-PCR) . The fIRF was transcribed mainly in the intestine, ovary, muscle, liver, heart and spleen, while it was minimally transcribed in the brain and kidney . When Japanese flounder were injected with HRV, the relative expression of fIRF mRNA was found to increase and peak 3 days after injection . The quantities of the fIRF mRNA increased to levels that were 7.5-fold higher than those of noninjected fish . In addition, when Japanese flounder were injected with Edwardsiella tarda, the expression of fIRF mRNA showed increases 2, 3, and 4 days after injection . The quantities of the fIRF mRNA on those days represented approximately 6-, 15-, and 14-fold increases, respectively, over the levels in noninjected fish. Med J Aust, 1998 May 4, 168(9), 443 - 4 Septic arthritis of the knee caused by Edwardsiella tarda after a catfish puncture wound; Ashford RU et al.; Anglers are wary of catfish as their sharp fin spines may cause injury and, in some species, envenomation . We describe another complication of catfish spine injury--septic arthritis caused by Edwardsiella tarda . We believe this is the first report of this organism causing septic arthritis. FEMS Microbiol Lett, 1998 Apr 15, 161(2), 317 - 23 Edwardsiella tarda induces plasma membrane ruffles on infection of HEp-2 cells; Phillips AD et al.; Interaction of two clinical Edwardsiella tarda isolates with HEp-2 cells was investigated . By electron microscopy we observed at 1 h post infection that E . tarda induced formation of extensive plasma membrane projections resembling membrane ruffles . The ruffles did not coincide with adhering bacteria . Only few invading bacteria were seen . Vacuolated nuclear membrane was occasionally observed . Three hours post infection, E . tarda induced a contact-dependent cell lysis, revealing the host cell cytoskeleton and nucleus . Only one of the E . tarda strains was seen residing within the host cell remains . The results indicate that E . tarda-induced membrane ruffles may involve a distinct mechanism of bacterial pathogenesis. Southeast Asian J Trop Med Public Health, 1997 Sep, 28(3), 669 - 72 Edwardsiella tarda bacteremia and septic arthritis in a patient with diabetes mellitus; Osiri M et al.; Edwardsiella tarda is an uncommon pathogen in the family Enterobacteriaceae which usually infects patients with underlying diseases . Its habitats include fresh water, a variety of animals and human feces . We report a case of E . tarda bacteremia and septic arthritis with underlying diabetes mellitus, the first found in Thailand. Vet Clin North Am Food Anim Pract, 1998 Mar, 14(1), 101 - 12 Food borne microbial pathogens of cultured aquatic species; Greenlees KJ et al.; This article provides a broad overview of microbial pathogens associated with marine and fresh water aquatic animals, including Vibrio species, Escherichia coli, Streptococcus iniae, Salmonella species, and Edwardsiella tarda . Historically, cultured fish were not considered important vectors of human pathogens . This situation is changing, partly due to increasing animal densities as a consequence of a rapidly growing industry and partly due to increasing awareness by health care providers of pathogens in aquatic species that may result in human illness . Concerns facing the industry are also discussed along with possible solutions. Mol Mar Biol Biotechnol, 1997 Dec, 6(4), 339 - 44 Characterization and expression of c-type lysozyme cDNA from Japanese flounder (Paralichthys olivaceus); Hikima J et al.; Lysozyme is a widely distributed enzyme located in the serum, skin mucus, and other organs of fish, which is responsible for catalyzing the hydrolysis of the cell walls of most bacteria . A c-type of lysozyme cDNA was cloned from a kidney cDNA library of the Japanese flounder (Paralichthys olivaceus) . The cDNAs consisted of 612 bp, which coded for 143 amino acid residues . The deduced amino acid sequence of Japanese flounder c-type lysozyme possessed 72.9%, 57.4%, and 65.4% identities with rainbow trout, chicken, and human c-type lysozymes, respectively . Comparison of the c-type lysozymes showed that the catalytic residues, the residues binding to sugar chains, and cysteine residues were completely conserved . Northern blot analysis indicated that the c-type lysozyme gene is apparently transcribed in the head kidney, posterior kidney, spleen, brain, and ovary of healthy flounder . When flounder were experimentally infected with Edwardsiella tarda, quantities of the c-type lysozyme mRNA increased in the head kidney, spleen, and ovary of the flounder. FEMS Microbiol Lett, 1997 Dec 1, 157(1), 123 - 9 Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19; Sendra RM et al.; An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels . The enzyme-linked immunosorbent assay specificity was confirmed after testing A . hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V . furnisii, V . damsela, Yersinia ruckerii and Edwardsiella tarda . The detection limits for A . hydrophila O:19 cells were around 10(4)-10(5) cells/well . Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods . With this methodology A . hydrophila O:19 was successfully detected in infected eels and water samples . We described two subgroups within the serogroup O:19 (Guinee and Jansen system), one of them presents a 50 kDa outer membrane protein as a strong thermostable antigen which is not present in the other group. Infect Immun, 1997 Nov, 65(11), 4642 - 51 Attenuation, persistence, and vaccine potential of an Edwardsiella ictaluri purA mutant; Lawrence ML et al.; In this study, an adenine-auxotrophic strain of Edwardsiella ictaluri was constructed and its virulence, tissue persistence, and vaccine efficacy were evaluated . A clone containing the purA gene was isolated from an E . ictaluri genomic library, sequenced, and shown to have an overall sequence identity of 79.3% at the nucleotide level and 85.7% at the amino acid level with the Escherichia coli purA gene . The cloned E . ictaluri purA gene was mutated by deleting a 598-bp segment of the gene and inserting the kanamycin resistance gene from Tn903 into the gap . The delta purA::Km(r) gene was subcloned into the suicide plasmid pGP704, and the resulting plasmid was used to deliver the modified gene into a virulent strain of E . ictaluri by conjugation . Homologous recombination replaced the chromosomal purA gene with the mutated gene to create an adenine-auxotrophic strain (LSU-E2) . Compared to wild-type E . ictaluri, LSU-E2 was highly attenuated by the injection, immersion, and oral routes of exposure . By the injection route, LSU-E2 had a 50% lethal dose (LD50) that was greater than 5 logs10 higher than the LD50 for wild-type E . ictaluri . In a tissue persistence study, LSU-E2 was able to invade channel catfish by the immersion route and persist in internal organs for at least 48 h . Channel catfish that were vaccinated with a single immersion dose of LSU-E2 had mortality significantly lower (P < 0.01) following a wild-type E . ictaluri challenge than that of nonvaccinated fish. Vet Immunol Immunopathol, 1997 Sep, 58(2), 181 - 90 Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish; Shoemaker CA et al.; The role of peritoneal macrophages in immunity to enteric septicemia of catfish (ESC) after infection with live Edwardsiella ictaluri was investigated . Channel catfish macrophage-mediated bacteriocidal activity was dependent on the macrophage:bacteria ratio . Ratios of 1:1 to 1:12 exhibited significant differences (P < or = 0.05) in killing between macrophages from immune fish when compared to killing by macrophages from susceptible fish at 2.5 h . At 5 h, macrophages from immune fish were capable of effective killing (83.3%) at a 1:24 effector:target ratio, whereas macrophages from susceptible fish killed significantly (P < or = 0.05) less (56.9%) . Macrophage bacteriocidal activity was significantly greater (P < or = 0.05) in macrophages from individual immune fish (93.4%) compared to macrophages from individual susceptible fish (85.4%) . The kinetics of macrophage killing showed a linear increase in bacteriocidal activity from 1 to 3 h . Opsonization with immune serum enabled macrophages from immune fish to kill bacteria more effectively (93.8 vs . 75.9%) at 2.5 h . Opsonization of E . ictaluri with immune serum significantly suppressed the killing ability of macrophages from susceptible fish (46.2%) at 2.5 h . The results suggest that macrophages from fish immune to ESC had a greater capacity to kill E . ictaluri than macrophages from susceptible fish especially when E . ictaluri were opsonized with anti-E . ictaluri antibody. Vet Immunol Immunopathol, 1997 Sep, 58(2), 171 - 80 Quantification of immunoglobulin in the serum and mucus of channel catfish at different ages and following infection with Edwardsiella ictaluri; Zilberg D et al.; An enzyme-linked immunosorbent assay was developed to quantify immunoglobulin (Ig) in the serum and mucus of channel catfish (Ictalurus punctatus) . The capture antibody (Ab) was a monoclonal Ab to channel catfish Ig and the detector was a polyclonal goat serum against channel catfish Ig . Detectable levels of channel catfish Ig were 0.1 microgram ml-1 and coefficients of intra- and interassay variations were 20% and 15%, respectively . Serum Ig concentration was found to increase from 0.36 +/- 0.25 to 0.49 +/- 0.26 mg ml-1 between 3 and 15 months of age . In contrast, mucus Ig concentration slightly decreased from 0.55 +/- 0.52 to 0.45 +/- 0.82 ng cm-2 of skin between 3 and 15 months of age . Mucus Ig concentrations were highest immediately caudal to gill covers and between pectoral to anal fins, at concentrations of 0.45 +/- 0.82 and 0.34 +/- 0.67 ng cm-2, respectively . The concentration of mucus Ig between anal and caudal fins and on the ventral skin between gill covers and pectoral fin was 0.18 +/- 0.42 and 0.09 +/- 0.14 ng cm-2, respectively . The kinetics of Ig production following infection with Edwardsiella ictaluri showed that serum Ig concentrations markedly increased (20.92 +/- 32.75 mg ml-1) at 13 days post-infection . The increase in mucus Ig concentrations occurred 14 days later (0.80 +/- 1.91 ng cm-2) . Individual variation in both serum and mucus Ig production increased considerably during the course of infection (+/-0.23 to +/-32.75 mg ml-1 for serum Ig and +/-0.1 to +3.41 ng cm-2 for mucus Ig) . The results suggest that E . ictaluri infection causes higher Ig production than that found in non-infected fish of the same age . Ab response to E . ictaluri was detected in the serum but not in the mucus of infected fish . Our results show differences in serum and mucus Ig concentrations with age and after infection with E . ictaluri. Infect Immun, 1997 Sep, 65(9), 3924 -