Mycobacterium ulcerans causes minimal pathogenesis and colonization in medaka (Oryzias latipes): an experimental fish model of disease transmission

Abstract

Mycobacterium ulcerans causes Buruli ulcer in humans, a progressive ulcerative epidermal lesion due to the mycolactone toxin produced by the bacterium. Molecular analysis of M. ulcerans reveals it is closely related to Mycobacterium marinum, a pathogen of both fish and man. Molecular evidence from diagnostic PCR assays for the insertion sequence IS2404 suggests an association of M. ulcerans with fish. However, fish infections by M. ulcerans have not been well documented and IS2404 has been found in other mycobacteria. We have thus, employed two experimental approaches to test for M. ulcerans in fish. We show here for the first time that M. ulcerans with or without the toxin does not mount acute or chronic infections in Japanese Medaka "Oryzias latipes" even at high doses. Moreover, M. ulcerans-infected medaka do not exhibit any visible signs of infection nor disease and the bacteria do not appear to replicate over time. In contrast, similar high doses of the wild-type M. marinum or a mycolactone-producing M. marinum "DL" strain are able to mount an acute disease with mortality in medaka. Although these results would suggest that M. ulcerans does not mount infections in fish we have evidence that CLC macrophages from goldfish are susceptible to mycolactones.

Figure 1. M. ulcerans establishes initial infection in medaka

Whole organ of dissected liver [A] and kidney [B] from 10⁸ CFU MU1615 GFP and MU1615::TN118 GFP infected medaka expressing fluorescent bacteria 1 wk post-infection [inset of wet mount of respective homogenized organ showing fluorescent bacteria]. First two panels show PBS negative control and bacteria-only control slide of colony and wet mount [inset]. Scale bar = 50μm

Figure 2. M. ulcerans is avirulent in medaka

Percent survival of medaka infected with 10⁴ [A] and 10⁸ [B] CFU of MU1615 GFP, MU1615::TN118 GFP, MM1218 and MMDL. [C] Gross morphology of medaka 8wks post infection with PBS [top panel], 10⁸ CFU MU1615 GFP[middle panel] and 10⁸ CFU MM1218 [bottom panel]. Scale bar = 10mm.

Figure 3. Survival of M. ulcerans in medaka is not mycolactone dependant

[A] Percent survival of medaka infected with 10² CFU of MU1615 GFP and MU1615::TN118 GFP. [B] Genome equivalent of MU1615 GFP and MU1615::TN118GFP in medaka infected with 10² CFU bacteria. [C] Genome equivalent of MU1615 GFP and MU1615::TN118GFP in medaka infected with 10⁴ and 10⁸ CFU bacteria. Data are mean log genome forming units [GFU] of MU1615GFP and MU1615::TN118 GFP infected medaka at 1, 8 and 23 wks p.i. R²=0.99

Figure 4. Histopathology of medaka kidney with 10⁴ CFU of MU1615 GFP, MU1615::TN118 GFP, MM1218 and MMDL

All sections were fixed and stained with Ziehl – Neelsen stain [left panels] and hematoxylin and eosin stain [right panels]. [A and B] PBS negative control; [C and D] MU1615 GFP infected medaka showing few intracellular bacteria [C- arrow and inset];little inflammatory response [D]; [E and F] MU1615::TN118 GFP infected medaka showing scattered pockets of intracellular and extracellular bacteria [E– arrow and inset] and diffuse inflammatory response [F]; [G and H] MM1218 infected medaka showing intracellular and extracellular bacteria [G – arrow and inset] and associated granuloma [H]; [I and J] MMDL infected medaka showing AFB bacteria [I] and associated well organized granuloma with necrotic centers [J – arrow]. Data represents fish sacrificed at 8 wks post infection. Scale bars = 50 μm for all panels and 20μm for insets

Figure 5. Analysis of Mycolactone – mediated cytopathicity on the fish macrophages

[A] Cytopathicity on CLC cells, showing ethanol control treated cells [first left]; mycolactone AB treated cells [top panel] and mycolactone F treated cells [bottom panel]. The final concentration of mycolactone added to treated cells was 10μg. Scale bar = 50 μm [B and C] Cytotoxicity measured by LDH release and nucleosome enrichment. Culture supernatants were collected from wells containing CLC cells 24 h after treatment with mycolactone. [B] The amount of LDH released by necrosis was measured using a Cytotox 96 assay kit [Promega]. Data are means and standard deviations of the values obtained from triplicate samples; P=0.7, 0.03, 0.06 and 0.04 for 10ng, 100ng, 1μg and 10μg respectively [Student’s t test]. [C] Apoptosis was assessed at 24 h with the cell death detection enzyme-linked immunosorbent assay kit [Roche] and expressed as fold enrichment of nucleosomes. Data are means and standard deviations of the values obtained from triplicate samples; P=0.06, 0.9, 0.02 and 0.06 for 10ng, 100ng, 1μg and 10μg respectively [Student’s t test].