Richard K. Cooper

Pathogenesis of gram-negative bacterial infections in warmwater fish

Abstract Knowledge concerning the pathogenesis of many bacterial diseases in fish is limited, especially in those diseases that occur in warmwater species. This limited knowledge base is due to the relative recent emergence of warmwater fish culture as a major industry in many parts of the world, and to the previous economic insignificance of warmwater aquaculture and the bacterial pathogens affecting warmwater species. This article is an overview of the important gram-negative pathogens of warmwater fish, including members of the genera Aeromonas, Edwardsiella, Pasteurella, Pseudomonas, and Vibrio. The current knowledge of the pathogenesis of these organisms is emphasized, including: the source of the pathogen, its preferred site and method for attaching to and penetrating the host, its adaptations for surviving the host immune system, and its strategies for obtaining nutrients required for proliferation and growth. Although information for many of these pathogens is limited, the intent of this article is to provide a baseline for the development of future research projects. Increases in worldwide aquaculture production will result in a demand for knowledge about the pathogenesis of bacterial pathogens in warmwater fish, because of its importance in making health management decisions, in deciding on treatment regimens, and in the development of vaccines.

Transposon Mutagenesis Used to Study the Role of Complement Resistance in the Virulence of an Avian Escherichia coli Isolate

The role of complement resistance in the virulence of an avian Escherichia coli isolate was examined with transposon mutagenesis. A suicide plasmid containing a kanamycin-encoding mini-transposon was used to transform a virulent complement-resistant avian E. coli isolate. A less resistant mutant was identified that contained a transposon insertion in a plasmid and in the chromosome. This loss of complement resistance was associated with a drop in virulence in an embryo assay. No other phenotypic changes were detected in the mutant. These results suggest that complement resistance is associated with the virulence of this organism.

Use of a Mini-Transposon to Study Chondroitinase Activity Associated with Edwardsiella ictaluri

Abstract Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), is the leading cause of bacterial disease in commercially raised channel catfish Ictalurus punctatus. Little work has been conducted at a genotypic level to determine potential virulence characteristics, but the production of chondroitin sulfatase is a suspected virulence factor. Using transpositional mutagenesis, we created stable E. ictaluri mutants that are deficient in chondroitinase activity. Channel catfish were challenged by injection with E. ictaluri transposon mutant MI15. None of the catfish challenged with the mutant died or showed signs of ESC. These fish were held for 2 weeks and then challenged by injection with the known virulent parent strain of E. ictaluri. The challenged naive control fish showed clinical signs of and a mortality rate consistent with ESC, whereas catfish that had been injected with MI15 prior to challenge with the parent strain were resistant to disease. This work represents a p...

Biochemical and Conjugation Studies of Romet-Resistant Strains of Aeromonas salmonicida from Salmonid Rearing Facilities in the Eastern United States

Abstract Strains of Aeromonas salmonicida (n = 585) were collected from covertly infected and diseased salmonid hosts from 12 hatcheries in the eastern United States. Strains and sites were selected because of their potential for harboring antimicrobial resistance, in particular, to Romet™. Resistance to Romet was displayed by 315 strains (53.8%), which were isolated from all six host species sampled at 10 of 12 sites. Thirty of the resistant strains (9.5%) from five sites had no zone of inhibition, whereas the other strains had either confluent growth or resistant colonies within a zone of inhibition. Fifty-one resistant strains, representing each of the three resistance phenotypes, were selected for biochemical and antimicrobial comparisons with Romet-sensitive strains. All were confirmed to be A. salmonicida, and no characteristic biochemical phenotypes were found to be associated with resistance to Romet. Differential resistances between resistant and sensitive strains were detected to the antimicrobi...

Comparison of Plasmids Isolated from Romet-30-Resistant Edwardsiella ictaluri and Tribrissen-Resistant Escherichia coli

Abstract Edwardsiella ictaluri, the etiological agent of enteric septicemia of channel catfish (ESC) is the leading cause of bacterial disease in commercially raised channel catfish Ictalurus punctatus. The only drug approved by U.S. Food and Drug Administration for use against ESC is Romet-30. Recently, several isolates were obtained that had a naturally occurring resistance to Romet-30. On further characterization these isolates were shown to possess a 55-kilobase (kb) plasmid that encodes resistance to the drug. We compared Romet-30-resistant E. ictaluri and a Tribrissen-resistant strain of Escherichia coli (strain 1898) isolated from equine cystitis. Antimicrobial profiles, plasmid screening, restriction digest, and Southern blot analysis indicated that the two plasmids are very similar. The resistance afforded to the E. coli was encoded by a 55-kb plasmid. Each of the R plasmids conferred resistance to Romet-30, tetracycline, and Terramycin (oxytetracycline). The R plasmid from E. coli strain 1898 wa...

Laser pressure catapulting followed by B actin gene identification in Japanese quail macrochromosomes and microchromosomes using teflon-coated coverslip slides

Laser microdissection of individual mammalian chromosomes (> 2 µm) has been achieved though the use of a microscope slide coated with a polyethylene naphthalate (PEN) membrane. Although these slides have proved sufficient for larger chromosomes, they are insufficient for small chromosomes (< 1 µm). We have developed a new type of slide which allows laser microdissection of single Japanese quail microchromosomes (0.5 µm) and macrochromosomes (3–4 µm). To test the usefulness of these slides, a Japanese quail single nucleus, a macrochromosome, and a microchromosome were collected with Laser pressure catapulting, the B‐actin gene was PCR amplified, and sequenced. The resulting PCR product was confirmed by nucleotide sequencing to be B‐actin. These newly developed slides were shown to facilitate the laser microdissection of both Japanese quail macrochromosomes and microchromosomes.

Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting

Transgenic avian bioreactors produce therapeutic recombinant proteins in egg white. To date, however, methods for transgenic modification of the avian genome or determining transgenic status of individual birds are scarce. The dual, but interrelated, goals of this research were to: (1) develop a method of detecting stable DNA insertion into Japanese quail; and (2) provide a method for gene location on avian chromosomes. We created Teflon-coated coverslip slides to facilitate laser pressure catapulting of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail, containing germline incorporation of proinsulin, were identified by isolation of chromosomes using laser microdissection and laser pressure catapulting. Subsequent amplification of each chromosome identified 2–5 chromosomes with the proinsulin transgene inserted. Nucleotide sequencing of each chromosomal insertion was identical to the proinsulin portion of the original vector. By applying laser pressure catapulting and PCR of individual chromosomes, we were able to determine that the transgene correctly inserted into avian chromosomes and that the majority of the insertions occurred within microchromosomes. Because many potential therapeutic transgenes have similar or nearly identical nucleotide sequence to the host’s native gene, laser microdissection and subsequent analysis may be required for detailed documentation of transgene expression before proceeding with transgenic protein production.