Bacterial pathogens of freshwater fish: environmental survival and evolution of virulence

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The bacterium Yersinia ruckeri infects rainbow trout and other fish, moving from the aquatic environment into the host animal through gills, skin and digestive tract and persisting in organs of the fish. Enteric redmouth disease appears when carrier fish are stressed by water quality, crowding, or handling. Not all isolates are identical, and the diversity affects their ability to infect different fish species and to persist and spread in the aquatic environment. We will identify genetic markers that can be used to clearly differentiate strains of Yersinia ruckeri, based on the genes involved in synthesizing their major surface antigen (lipopolysaccharide) and virulence-related genes. Sets of PCR primers will allow laboratories to quickly characterize isolates and help us understand distribution of strain types in fish populations. Our approach to finding the markers is genomic subtractive hybridization, in which single-stranded DNA isolated from two strains is mixed and allowed to reanneal to double-strands. DNA fragments from a tester strain that do not find a match, even in an excess of DNA from the second (driver) strain will be cloned and used to screen panels of bacterial strains collected from many sources. Fish responses to pathogen strains would ideally be studied in fish challenge experiments, but high individual variability in fish responses and the effect of stress mean that experiments require large numbers of fish. Single-celled protozoans can be useful as a model to indicate bacterial genes that affect virulence. Protozoa, such as amoeba, are voracious feeders on bacteria but some resist digestion. Resistant bacteria are often pathogens, perhaps because the characteristics that allow them to resist amoeba are also effective against host macrophages. We will use cultured amoeba as a way to compare bacterial strains (and mutants constructed in the studied genes) for survival. Research on amoeba-Yersinia ruckeri interactions can also indicate how amoeba in an aquatic environment serve as a reservoir for pathogen, allowing survival and transmission to new fish hosts. This can provide new ways of testing for significant fish pathogens in the environment and in aquaculture recirculation systems.