Vol. 138: 237–246, 2020 DISEASES OF AQUATIC ORGANISMS Published online April 9 https://doi.org/10.3354/dao03464 Dis Aquat Org Swim bladder mycosis in farmed rainbow trout Oncorhynchus mykiss caused by Phoma herbarum and experimental verification of pathogenicity Jirˇí Rˇ ehulka1, Alena Kubátová2, Vit Hubka2,3,* 1Department of Zoology, Silesian Museum, 746 01 Opava, Czech Republic 2Department of Botany, Faculty of Science, Charles University, 128 01 Prague 2, Czech Republic 3Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology of the Academy of Sciences of the Czech Republic, v. v. i., 142 20 Prague 4, Czech Republic ABSTRACT: In this study, spontaneous swim bladder mycosis was documented in a farmed finger- ling rainbow trout from a raceway culture system. At necropsy, the gross lesions included a thick- ened swim bladder wall, and the posterior portion of the swim bladder was enlarged due to mas- sive hyperplasia of muscle. A microscopic wet mount examination of the swim bladder contents revealed abundant septate hyphae, and histopathological examination showed periodic acid- Schiff-positive mycelia in the lumen and wall of the swim bladder. Histopathological examination of the thickened posterior swim bladder revealed muscle hyperplasia with expansion by inflam- matory cells. The causative agent was identified as Phoma herbarum through morphological an - alysis and DNA sequencing. The disease was reproduced in rainbow trout fingerlings using intra - peritoneal injection of a spore suspension. Necropsy in dead and moribund fish revealed ex tensive congestion and haemorrhages in the serosa of visceral organs and in liver and abdominal serosan- guinous fluid. Histopathological examination showed severe hepatic congestion, sinusoidal dilata- tion, Kupffer cell reactivity, leukostasis and degenerative changes. Fungi were disseminated to the liver, pyloric caeca, kidney, spleen and heart. Although infections caused by Phoma spp. have been repeatedly reported in fish, species identification has been hampered by extensive taxo- nomic changes. The results of this study confirmed the pathogenicity of P. herbarum in salmonids by using a reliably identified strain during experimental fish infection and provides new know - ledge regarding the course of infection. KEY WORDS: Experimental infection · Spontaneous infection · Rainbow trout fingerling · Histopathology · Swim bladder · Coelomycetous fungi Resale or republication not permitted without written consent of the publisher 1. INTRODUCTION The pigmented coelomycete fungal species Phoma herbarum belongs to the order Pleosporales (Asco - The development of aquaculture with intensive mycota, Dothideomycetes) and the family Didymel- approaches for fish production promotes the occur- laceae. This family encompasses cosmopolitan fungi rence of various infectious diseases. Apart from inva- distributed in a broad range of environments, with sions by parasites and bacterial or viral epizootics, an most being plant pathogens (Chen et al. 2015, 2017). increasing number of fungal diseases have been iden- In addition to being a plant saprophyte/pathogen, tified. Mycotic infections have been observed in in - P. herbarum has been documented as a causative tensively cultured rainbow trout (Hubka et al. 2014, agent of onychomycosis in humans (Tullio et al. 2010) Rˇ ehulka et al. 2016). and is frequently observed in human clinical samples *Corresponding author: [email protected] © Inter-Research 2020 · www.int-res.com 238 Dis Aquat Org 138: 237–246, 2020 (Punithalingam 1979, Valenzuela-Lopez et al. 2018, the isolation of fungi were made with inoculation Garcia-Hermoso et al. 2019). Other reports typically loops onto malt extract agar (MEA; Oxoid), and the refer to other Phoma-like pathogens without exact samples were incubated in the dark at 24°C. identification to a species or even a generic level. Phoma infections of the swim bladder have been described in 16 fish species, particularly marine spe- 2.2. Design of experimental fish infection cies (Table 1), with infections of individual fish and outbreaks primarily occurring in the fry and finger- Rainbow trout weighing 30−40 g and standard lings of salmonid fish. body length (LS) of 114−130 mm were used. All fish In general, identification of Phoma-like species were acclimated to laboratory conditions for 2 mo, represents a dilemma to mycologists. Modern phylo- fed dry pellets and remained in good health. After genetic studies have shown that this genus is highly anaesthesia with Menocain (3-aminobenzoic acid polyphyletic and includes species that are in at least ethyl ester sodium hydrogen sulphate) at a concen- 6 families within the order Pleosporales. After taxo- tration of 0.06 g l−1 (Král 1988), 5 fish (experimental nomic rearrangements, the genus Phoma became group) were intraperitoneally injected (into the flank monotypic and includes 1 species, P. herbarum, while halfway between the pectoral and ventral fins) with a the remaining species were transferred to other gen- saline suspension of spores (0.2 ml of sterile 0.85% era (Chen et al. 2015, 2017). Phoma-like fungal iden- physiological saline water [PSW] with 1.6 × 103 fungal tification remains difficult, and in most cases the aeti- spores) prepared from 3 wk old colonies cultivated ological agent is only labelled as Phoma sp. without on MEA. Five fish (control group) were similarly further specification (Bennett et al. 2018). injected with 0.2 ml of PSW. The experiments were The infections caused by P. herbarum in salmonid carried out at a water temperature of 16°C in 150 l fish were first described by Ross et al. (1975), but the tanks. The continuously aerated and filtered water original isolates are not available for comparative had the following physical and chemical characteris- studies. Similarly, with the exception of that of Faisal tics: pH 7−7.3; water hardness 7−9°N; dissolved et al. (2007), other studies that reported Phoma spe- oxygen content 8−9 mg l−1; chemical oxygen demand −1 + −1 − cies as fish pathogens did not use molecular methods (CODMn) 5−7 mg l ; NH4 0.4−0.6 mg l ; NO2 −1 − −1 for identification, making definitive identification of 0.04−0.07 mg l ; and NO3 5−7 mg l . the aetiological agent questionable. The purpose of this study was to describe the clini- cal signs and histopathological changes in sponta- 2.3. Histology neous and experimental infections by P. herbarum in rainbow trout Oncorhynchus mykiss fingerlings in Gill, liver, kidney, spleen, heart and brain tissue freshwater aquaculture. Another aim was to verify samples were fixed in Davidson’s alcohol/formalde- the pathogenicity of P. herbarum by using a reliably hyde/acetic solution, embedded in paraffin and sec- identified fungal strain. To this end, we performed a tioned into 5 µm thick slices. The histological sections detailed morphological examination of the aetiologi- were stained with haematoxylin and eosin (H&E), cal agent and obtained sequences of the rDNA Gram’s stain, Giemsa, periodic acid-Schiff (PAS) re - region as well as 2 protein-coding loci (β-tubulin and agent and Grocott’s methenamine silver (GMS) rpb2 genes), which are crucial for the reliable identi- method, and the Fontana-Masson technique. fication of Phoma-like species. Previously reported cases of swim bladder mycoses are summarized, and routes of infection are discussed. 2.4. Molecular studies An ArchivePure DNA yeast and Gram2 + kit 2. MATERIALS AND METHODS (5PRIME) was used to isolate genomic DNA as described by Hubka et al. (2016) from 7 d old colonies 2.1. Bacteriology and isolation of fungi grown on MEA (Oxoid). The internal transcribed spacer (ITS) rDNA region was amplified with the Blood agar (Columbia Blood Agar Base, Merck), primers ITS5 (White et al. 1990) and ITS4S (Kretzer et tryp ticase soy agar (Oxoid) and Cytophaga agar al. 1996); the large subunit (LSU) rDNA region was (Pacha & Ordal 1967) served as the media for bacte- amplified with the primers NL1 and NL4 (O’Donnell riological examinations. Microbiological smears for 1993); and the small subunit (SSU) rDNA region was Table 1. Review of swim bladder infections in fish caused by fungi and oomycetes Pathogen (family) Fish species affected Country Reference Akanthomyces lecanii (= Lecanicillium lecanii) Baltic salmon Salmo salar Finland Aho et al. (1988) (Cordycipitaceae) Alternaria consortialis (Pleosporaceae) Pink salmon Oncorhynchus gorbuscha; Russia Marchenko (1985) chum salmon Oncorhynchus keta Cladosporium cladosporioides (Cladosporiaceae) Barramundi cod Cromileptes altivelis Australia Bowater et al. (2003) Cladosporium sphaerospermum (Cladosporiaceae) Red snapper Lutjanus campechanus USA Blaylock et al. (2001) R Exophiala sp. (Herpotrichiellaceae) Cod Gadus morhua; scup Stenotomus versicolor USA Blazer & Wolke (1979) ˇ King George whiting Sillaginodes punctata Australia Reuter et al. (2003) ehulka etal.: Exophiala angulospora (Herpotrichiellaceae) Weedy seadragon Phyllopteryx taeniolatus; Australia Nyaoke et al. (2009) leafy seadragon Phycodurus eques Exophiala pisciphila (Herpotrichiellaceae) Pretty tetra Hemigrammus pulcher Czech Republic Rˇ ehulka et al. (2018) Exophiala xenobiotica (Herpotrichiellaceae) Queensland grouper Epinephelus lanceolatus USA Camus et al. (2015) Fusarium avenaceum var. herbarum (Nectriaceae) Pink salmon Oncorhynchus gorbuscha; Russia Marchenko (1985) Phoma herbarum chum salmon Oncorhynchus keta Isaria farinosa (= Paecilomyces farinosus) Atlantic salmon Salmo salar UK Bruno (1989) (Cordycipitaceae) Atlantic