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

Author
item Russo, Riccardo
item Shoemaker, Craig
item Panangala, Victor
item Klesius, Phillip

Submitted to: Fish and Shellfish Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/12/2009
Publication Date: 5/24/2009
Citation: Russo, R., Shoemaker, C.A., Panangala, V.S., Klesius, P.H. 2009. In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri. Fish and Shellfish Immunology. 26: 543-552.

Interpretive Summary: The enteric septicemia of channel catfish (ESC) caused by Edwardsiella ictaluri, accounts for considerable economic loss to the catfish farming industry world wide. Recent attempts to control ESC through prophylactic immunization have shown that live attenuated E. ictaluri vaccines are efficacious in protecting channel catfish (Ictalurus punctatus). Several studies have alluded that protective immunity in channel catfish against E. ictaluri is largely mediated by a cellular immune response with humoral antibodies having a secondary function. In the present study we used red fluorescent E. ictaluri to examine the interaction between channel catfish macrophage and E. ictaluri in vaccinated and non-vaccinated catfish. The results of this study showed that only macrophages from vaccinated fish were efficient in killing E. ictaluri, meanwhile in non-vaccinated fish E. ictaluri was able to survive and replicate within macrophages. A positive correlation between the amounts of reactive oxygen species (ROS) and nitric oxide produced by macrophages and the killing efficiency of macrophages was observed, even if E. ictaluri was able to survive inside macrophages in the presence of ROS and nitric oxide. Similar results have been documented for other pathogenic bacteria such as Salmonella spp.. Overall, this study supports the importance of macrophage mediated immunity against E. ictaluri showing that macrophages from vaccinated fish are more efficient in killing E. ictaluri. All examined parameters, such as ROS production, nitric oxide production, and killing efficiency, reflected that vaccinated fish have an augmented immune response compared to non-vaccinated fish.

Technical Abstract: Macrophages from modified live vaccinated and non-vaccinated catfish were used in in vitro and in vivo studies with red fluorescent Edwardsiella 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 6 hours, 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 necropsies effectuated in the first 48 hours 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 modified live vaccinated fish are activated and responsible for rapid clearance of infection upon re-exsposure to virulent E. ictaluri.