Abstract
Sturgeon aquaculture is particularly important since wild populations are significantly affected due to overfishing, habitat destruction, and pollution.1 In the Pacific Northwest, White Sturgeon (Acipenser transmontanus) culture is a multi-million-dollar industry. The production of globally recognized, high-quality caviar makes this product one of the few aquaculture-generated exports for the United States. Veronaea is a small genus of saprobic fungi found in soil and on plant material, belonging to the family Herpotrichiellaceae, order Chaetothyriales. Veronaea botryosa is the etiologic agent of fluid belly; one of the most important emergent diseases in sturgeon aquaculture and has also been associated with disease in other aquatic animals as well as humans.2,3 Despite its impact on the caviar industry, there are no commercially available therapeutants against systemic V. botryosa infections and little is known regarding its disease pathogenesis. Additionally, there is currently very little published data regarding antifungal susceptibility in vivo or in vitro, and at present there are no known efficacious chemotherapeutants or vaccines available. Natamycin, also known as pimaricin, is a macrolide polyene antifungal agent produced by the bacterium Streptomyces natalensis. It targets ergosterol in the cell wall of fungi and has been used for food preservation and treatment of fungal infections in over 150 countries. As a food additive, levels beneath 40 mg/kg in the finished product are considered safe for human consumption.4 In the present study, we established microbroth kinetic protocols, based on turbidity measurements, to analyze the growth characteristics of V. botryosa in seven nutrient media using modified protocols from Meletiadis, et al.5 In vitro growth of V. botryosa was optimized when spores were inoculated in Potato or Sabouraud Dextrose base (SBD) media incubated at 25°C. The generated protocol was then used to test the susceptibility of nine different V. botryosa isolates with natamycin, following protocols from Meletiadis, et al.6 SBD and RPMI containing 1, 2, 4, 8, 16, 32 mg/ml natamycin were inoculated with purified V. botryosa spores and growth curves were generated using a 96-well plate reader over a 120-hour period at 25°C. A cubic smoothing spline model compared the generated areas under the curve (AUC) for the different treatments in the different broth media. All concentrations of natamycin significantly lowered the AUC when compared to the respective positive controls (p<0.05). However, at least 4 and 16 mg/ml of natamycin were needed to decrease the AUC by 70% for fungal growth in SBD and RPMI media respectively. This information can be used to investigate susceptibility of pathogenic fungus to antimicrobials and disinfectants as well as support future therapeutic protocols against emerging fungal diseases like fluid belly.
* Presenting author
Literature Cited
1. Monterey Bay Aquarium. 2016. White sturgeon. www.montereybayaquarium.org/animal-guide/fishes/white-sturgeon. Accessed 2018 Jan 10.
2. Steckler NK, Yanong RP, Pouder DB, Nyaoke A, Sutton DA, Lindner JR, Wickes BL, Frasca S Jr, Wolf JC, Waltzek TB. 2014. New disease records for hatchery-reared sturgeon. II. Phaeohyphomycosis due to Veronaea botryosa. Dis Aquat Organ. 111(3):229–238.
3. Sang H, Zheng XE, Kong QT, Zhou WQ, He W, Lv GX, Shen YN, Liu WD. 2011. A rare complication of ear piercing: a case of subcutaneous phaeohyphomycosis caused by Veronaea botryosa in China. Med Mycol. 49(3):296–302.
4. FAO/WHO Food Standards: Codex alimentarius. 2017. Food additive details. www.fao.org/gsfaonline/additives/details.html?id=208. Accessed 2018 Jan 10.
5. Meletiadis J, Meis JF, Mouton JW, Verweij PE. 2001. Analysis of growth characteristics of filamentous fungi in different nutrient media. J Clin Microbiol. 39(2):478–484.
6. Meletiadis J, te Dorsthorst DT, Verweij PE. 2003. Use of turbidimetric growth curves for early determination of antifungal drug resistance of filamentous fungi. J Clin Microbiol. 41(10):4718–4725.