DAO 108:11-21 (2014)  -  DOI: https://doi.org/10.3354/dao02696

Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication

Marte Kaldestad1, Gyri T. Haugland1, Anita Rønneseth1, Heidrun I. Wergeland1,*, Ole Bent Samuelsen1,2 

1Department of Biology, University of Bergen, Bergen High-Technology Centre, PO Box 7803, 5020 Bergen, Norway
2Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway
*Corresponding author:

ABSTRACT: The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.


KEY WORDS: Francisella noatunensis subsp. noatunensis · Intracellular replication · Antibacterials · Leucocytes · HPLC · Flow cytometry · Atlantic Cod · Gadus morhua


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Cite this article as: Kaldestad M, Haugland GT, Rønneseth A, Wergeland HI, Samuelsen OB (2014) Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication. Dis Aquat Org 108:11-21. https://doi.org/10.3354/dao02696

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