Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri and preliminary investigation of its effects on virulence.

Edwardsiella ictaluri is the etiologic agent of enteric septicemia of catfish, which causes substantial losses in catfish aquaculture. To determine pathogen-host interactions, previous studies have used the green fluorescence protein (GFP) gene. Here, the pEI2 plasmid of E. ictaluri isolate I49 was tagged using a Tn10-GFP-kan cassette to create the green fluorescence-expressing derivative I49-gfp. The Tn10-GFP-kan insertion site was mapped by plasmid sequencing to 663 bp upstream of open reading frame 2 and appeared to be at a neutral site in the plasmid. Purification of the pEI2::GFPKan plasmid and mobilization into E. coli resulted in GFP expression. The isolated pEI2::GFPkan plasmid was used to retransform the wild type I49 isolate (ensuring a single Tn10-GFP-kan insertion) and an independent E. ictaluri isolate, S97-73-3. The wild type and the green fluorescent-tagged strains were compared for modulation of pathogenicity in channel catfish Ictalurus punctatus by immersion challenge. A significant reduction in mortalities occurred for the I49GFPkan strain as compared to its isogenic parent, but no difference was observed between the S97-73-3GFPkan strain and the S97-73-3 wild type. This GFP-tagged plasmid will be useful for determining the effects that the pEI2::GFPkan plasmid has on virulence and host-pathogen interactions between E. ictaluri isolates.

Spleen index and mannose-binding lectin levels in four channel catfish families exhibiting different susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri.

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

Expression of leptin-like peptide (LLP) mRNA in channel catfish (Ictalurus punctatus) is induced by exposure to Edwardsiella ictaluri but is independent of energy status.

Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in mammals. In channel catfish, the presence of a partial messenger RNA sequence that encodes a leptin-like peptide (LLP) has been identified and investigated. The objectives of the present studies were to clone and characterize full-length catfish LLP gene, examine tissue expression of LLP mRNA, and determine effects of prolonged fasting and exposure to Edwardsiella ictaluri (E. ictaluri), the bacteria that causes enteric septicemia in catfish, on LLP mRNA expression. Full-length catfish LLP gene was sequenced by genome walking and by 5'- and 3'-RACE. Catfish LLP gene contained three exons with the coding region located in exons 2 and 3. The amino acid sequence of the channel catfish LLP shared very low sequence similarities with leptin of other fish species or the mammalian leptin (24-49%). Using real-time polymerase chain reaction, LLP mRNA expression was detected in various tissues including brain, stomach, spleen, heart, liver, and trunk kidney and was especially high in the liver and trunk kidney. Expression of LLP mRNA in liver and brain was similar between fish that were fasted for 30days and those that received feed daily for 30days (P>0.10). Expression of LLP mRNA was increased in liver, spleen, and trunk kidney within 48h post-exposure to E. ictaluri compared to unexposed fish (P<0.05). Based on the results of the current studies, amino acid sequence of catfish LLP is highly dissimilar to mammalian and fish leptin. Unlike in most mammals, catfish LLP expression is independent of energy status. However, the expression of catfish LLP is increased after exposure to pathogenic bacteria, which is similar to mammals. Further investigations are required to clearly define the biological function and regulation of catfish LLP.

Validation of a whole-body cortisol extraction procedure for channel catfish (Ictalurus punctatus) fry.

We validated a whole-body cortisol extraction technique for channel catfish, Ictalurus punctatus, fry. Three volume enhancement methods were tested: CAL method (zero calibrator A diluent added to lipid extract), PBS method (phosphate buffered saline added to lipid extract), and VO method (food grade vegetable oil added to lipid extract). The volume enhancement extracts were evaluated using a commercial radioimmunoassay kit. Sensitivity, accuracy, precision, reproducibility, and parallelism could not be determined for the PBS method as cortisol levels were not detected in any of the extracted samples. Intra-assay coefficient of variation (CV) for the CAL and VO methods were 7.3 and 8.3%, respectively, while inter-assay CV were 9.6 and 10.6%, respectively. Based on the sensitivity, accuracy, precision, reproducibility, and parallelism results, we conclude that the CAL method is the most appropriate method for volume enhancement of catfish fry lipid extract. Using the CAL method to detect cortisol in catfish fry, fish were stressed daily for 2 weeks. Fry weights were similar throughout the study while whole-body cortisol levels were higher (P < 0.01) in stressed fish after 1 day of stress. These data show the CAL method can effectively measure whole-body cortisol in catfish fry.

Edwardsiella ictaluri encodes an acid-activated urease that is required for intracellular replication in channel catfish (Ictalurus punctatus) macrophages.

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.

Proteomic analysis of head kidney tissue from high and low susceptibility families of channel catfish following challenge with Edwardsiella ictaluri.

A study was performed to compare proteomic profiles of channel catfish from families with high and low susceptibility to Edwardsiella ictaluri following an immersion challenge. Total protein was isolated from head kidney samples, collected at 2 and 6h postexposure, and analyzed by 2-D-gel electrophoresis coupled with peptide mass fingerprint analysis by matrix-assisted laser desorption/ionization and time of flight tandem mass spectrometry. Comparisons were made between proteomic profiles from infected and uninfected fish from high and low susceptibility families. Heat shock protein 90-beta, from the high susceptibility infected family, and fructose-1,6-bisphosphatase 1-like protein, from the low susceptibility infected family, were identified at 2h postexposure. Rab-11-like protein (low susceptibility infected family), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (low susceptibility infected family), glyceraldehyde-3-phosphate dehydrogenase (high susceptibility infected family), and ATP synthase beta subunit (low susceptibility uninfected family) were identified at 6h postexposure. Four proteins, heat shock protein 90-beta, Rab-11, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon, and glyceraldehyde-3-phosphate dehydrogenase, have activities involved in macrophage function or cellular stress responses, while the other two have functions associated with cellular energy production and metabolism. These results demonstrate the potential for use of proteomic techniques in channel catfish research.

Signature-tagged mutagenesis of Edwardsiella ictaluri identifies virulence-related genes, including a salmonella pathogenicity island 2 class of type III secretion systems.

Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the Salmonella pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type E. ictaluri bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.

RecA and RadA proteins of Brucella abortus do not perform overlapping protective DNA repair functions following oxidative burst.

Very little is known about the role of DNA repair networks in Brucella abortus and its role in pathogenesis. We investigated the roles of RecA protein, DNA repair, and SOS regulation in B. abortus. While recA mutants in most bacterial species are hypersensitive to UV damage, surprisingly a B. abortus recA null mutant conferred only modest sensitivity. We considered the presence of a second RecA protein to account for this modest UV sensitivity. Analyses of the Brucella spp. genomes and our molecular studies documented the presence of only one recA gene, suggesting a RecA-independent repair process. Searches of the available Brucella genomes revealed some homology between RecA and RadA, a protein implicated in E. coli DNA repair. We considered the possibility that B. abortus RadA might be compensating for the loss of RecA by promoting similar repair activities. We present functional analyses that demonstrated that B. abortus RadA complements a radA defect in E. coli but could not act in place of the B. abortus RecA. We show that RecA but not RadA was required for survival in macrophages. We also discovered that recA was expressed at high constitutive levels, due to constitutive LexA cleavage by RecA, with little induction following DNA damage. Higher basal levels of RecA and its SOS-regulated gene products might protect against DNA damage experienced following the oxidative burst within macrophages.