Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi (Lates calcarifer)

Abstract

Bacterial infections are a major contributor to losses in aquaculture production. Diagnosis of these bacterial diseases is based on sacrificing fish and isolating the infectious agent from the internal organs. Infections lead to an inflammatory response in the host that is characterized by changes in leukocyte profile and activity. We hypothesized that the profile of circulating peripheral blood leukocytes and markers of their activity, such as reactive oxygen species production, would change following an event of bacterial infection in fish. We therefore developed a flow cytometry-based methodology for analyzing blood leukocytes in barramundi (Lates calcarifer), then used it to compare healthy fish to those infected with a common pathogenic bacterium, Vibrio harveyi. The initial step of the analysis relied on the effective separation of erythrocytes and leukocytes, and two methods were tested: one based on hypotonic lysis of red blood cells, and the other involving stain-based differentiation using carboxyfluorescein succinimidyl ester (CFSE). Fluorescence-activated cell sorting analysis revealed significant differences in leukocyte parameters of infected and control fish using the stain-based approach vs. hypotonic lysis. Changes in blood leukocytes included an increase in the proportion of monocytes and decrease in that of lymphocytes in infected fish. Furthermore, cell size and granularity of the different leukocyte populations changed over time in infected fish as compared to controls. Monitoring peripheral blood leukocytes provides a non-lethal analysis of inflammation that allows repeated sampling of individual fish over time. This is beneficial given the high variability between individual fish. The use of CFSE-staining for leukocyte isolation proved to be advantageous over a method based on lysis of erythrocytes.