Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids.

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl(2)) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO(4)) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5+/-0.2 x 10(4) transformants ng(-1) plasmid DNA) than with the CaCl(2) (8.1+/-6.1 x 10(-1) transformants ng(-1) plasmid) and the 'one-step transformation' protocol (2.5+/-2.7 transformants ng(-1) plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl(2) and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.

In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri.

Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from -0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly (p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly (p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila.

A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 x 10(2) and 2.5 x 10(5) cells g(-1) of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques.

Trinucleotide GAA repeats dictate pMGA gene expression in Mycoplasma gallisepticum by affecting spacing between flanking regions.

The pMGA genes of the avian respiratory pathogen Mycoplasma gallisepticum encode a family of hemagglutinins that are subject to phase variation. A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site. The length of the repeat region varies at a high frequency due to changes in the number of repeat units. Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12. To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats. The modified promoter region was fused to a promoterless lacZ gene and transformed into M. gallisepticum by using transposon Tn4001 as a vector. Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor beta-galactosidase activity. For the transformants of M. gallisepticum containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished. For the transformants containing the reporter with an addition of 10- or 12-bp linkers, lacZ was expressed only when eight GAA repeats were present. These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression.