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. 2017 Jun 27;7:4265. doi: 10.1038/s41598-017-04069-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Evaluation of the bactericidal activity of hepcidin in various serum samples against test strains by inhibition zone assay. (A and B) Detection of the inhibition zones for E. coli (A) and A. hydrophila (B) strains by unstimulated serum (a1 and b1), A. hydrophila DNA (1 μg per fish) stimulated serum (a2 and b2), A. hydrophila DNA stimulated serum neutralized with diluted (1:1,000) anti-hepcidin Ab (a3 and b3), and A. hydrophila DNA stimulated serum neutralized with diluted (1:200) anti-hepcidin Ab (a4 and b4), scale bars = 1 cm. (C and D) Measurement of the corresponding diameters of the inhibition zones (mm) from the inhibition zone assays by using serum samples with various treatments against E. coli (C) and A. hydrophila (D) strains. The labels a1 to a4 and b1 to b4 under the horizontal axis of C and D are in accordance with A and B. The annotations “A. hydrophila DNA+/−” and “Hepcidin Ab dilutions” under the labels represent that the E. coli (a1 to a4) and A. hydrophila (b1 to b4) were treated with the serum derived from A. hydrophila DNA stimulated (+) or unstimulated (−) fish, which were in combination with neutralizing the serum with anti-hepcidin Ab at different dilutions (1:1000 and 1:200) or without treatment with the anti-hepcidin Ab (N/A, not applicable). Data are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.