Pathogen entrance and development of disease during infection of the American channel catfish Ictalurus punctatus by the enterobacterium Edwardsiella ictaluri

Abstract

Since being first reported in the late 1980ies, the Enterobacterium Edwardsiella ictaluri has rose in prevalence to become one of the two most damaging pathogens affecting the channel catfish industry. Despite this significance of the pathogen, understanding of the development of the disease, especially its route of entry into the host and the earlier stages of the infection, is still incomplete.

A series of challenges were conducted using bioluminescent E. ictaluri either by infecting fish through immersion or topical application of the bacteria directly on the intact or abraded epithelium. This showed that abraded fish developed septicemia and died faster than non-abraded ones. Furthermore, results from a co-habitation challenge suggested that the bacterium induced septicemia through the skin instead of becoming water-borne. Finally, a histological technique was developed allowing the determination that the bacteria radiated from the initial skin infection site and penetrated deeper into the tissue as the challenge progressed. These results all suggest that site of abrasion on the skin can act as a route of entrance for the pathogen into the fish, a fact never previously reported.

Transposon mutagenesis was also performed to construct a library of 1728 mutants. Screening of this library allowed us to identify 16 genes which inactivation lead to a decrease in the bacterium ability to colonize the epithelium or cause mortality. Sequencing of these genes allowed the identification of RstA/B, a regulator of invasion genes in Salmonella enterica Typhimurium, a putative ribonuclease, similar to a Shigella protein regulating the expression of adhesin and a protein that constitutes the second member of a newly discovered adhesin family.

Finally, to investigate the development of the infection, fish were infected by bioluminescent E. ictaluri and sampled at various time points. At each time point, nine organs (gills, muscles, intestine, spleen, liver, stomach, heart, head kidney and trunk kidney) were sampled, and their bioluminescence was measured and half of these organs were homogenized, serial diluted, and plate counts determined. This allowed confirmation of a complex disease pathogenesis during ESC involving a period of intense reproduction in the spleen, anterior and posterior kidneys followed by a sharp increase in the levels of bacteria in the blood.