Francisella noatunensis subsp. noatunensis replicates within Atlantic cod (Gadus morhua L.) leucocytes and inhibits respiratory burst activity.

Francisella noatunensis subsp. noatunensis, causing granulomatosis in cod, has been shown to reside within cod immune cells, mainly within monocytes and macrophages. In the present study, we analysed the ability of the bacterium to replicate within adherent cells isolated from head kidney by in vitro infection of leucocytes. Two different technical approaches for flow cytometry analyses were performed for detection of intracellular bacteria. The presence of the wild type was assessed after identification by intracellular binding of specific antibodies to the pathogen. The other way was to use green fluorescent protein (GFP) transformed bacterium for infection studies allowing direct measurements of fluorescence from infected cells. By both methods we found an increase in fluorescence in infected cells, verifying bacterial replication, both after 4 and 28 h post infection in leucocytes isolated from head kidney (HKL). The GFP transformed bacterium was similar to the wild type in growth and infectivity pattern, showing that it can be a valuable tool for further studies of infection routes and pathology. Further, F. noatunensis subsp. noatunensis was found to inhibit respiratory burst activity, a potent pathogen killing mechanism, in cod leucocytes, but not in such cells from salmon. Our findings may indicate that inhibition of respiratory burst during Francisella infection is a key to its intracellular existence. This strategy seems to be conserved through evolution as it is also observed during infections in higher vertebrates caused by bacteria within the Francisella genus. The results presented here, showing the intracellular existence of Francisella, its replication within leucocytes and the inhibitory effect on respiratory burst, strongly support that these factors contribute to disease and pathology in infected cod. The intracellular replication shown in the present study might contribute to explain the problems of obtaining protective vaccines against Francisella and effective antibiotic treatment of infected fish.

Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes.

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.