Swim Bladder Mycosis in Farmed Rainbow Trout Oncorhynchus Mykiss Caused by Phoma Herbarum and Experimental Verification of Pathogenicity

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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 fingerling rainbow trout from a raceway culture system. At necropsy, the gross lesions included a thickened swim bladder wall, and the posterior portion of the swim bladder was enlarged due to massive 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 inflammatory 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 serosanguinous fluid. Histopathological examination showed severe hepatic congestion, sinusoidal dilatation, 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 taxonomic 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

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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 clinical 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 Rˇ ehulka et al.: Phoma herbarum mycosis in rainbow trout 239 ehulka et al. (2018) ehulka et al. (2018) ˇ ˇ Norway Poppe et al. (1991) Germany Lehmann et al. (1999) Czech Republic R Czech Republic R Finland Aho et al. (1988) USA Blazer & Wolke (1979) USA Blazer & Wolke USA Camus et al. (2015) USA Faisal et al. (2007) Australia Reuter et al. (2003) ; Australia Nyaoke et al. (2009) USA Marty & Heintz (2010) ; Russia Marchenko (1985) ; Russia Marchenko (1988) ; Russia Marchenko (1985) ; Russia Marchenko (1985) ; Russia Marchenko (1985) ; Russia Marchenko (1985) ; Russia Marchenko (1985) Australia Bowater et al. (2003) Australia Bowater et al. (2003) Australia Bowater et al. (2003) USA Blaylock et al. (2001) USA Blaylock et al. (2001) ; rainbow trout ; Chinook USA Ross et al. (1975) Country Reference UK Bruno (1989) Stenotomus versicolor Japan Hatai et al. (1986) Japan Miura et al. (2010) Sillaginodes punctata Epinephelus lanceolatus ; scup Phyllopteryx taeniolatus Phycodurus eques Oncorhynchus tshawytscha Cromileptes altivelis Cromileptes altivelis Cromileptes altivelis Salmo salar Salmo salar Salmo salar Oncorhynchus keta Oncorhynchus keta Oncorhynchus keta Oncorhynchus keta Oncorhynchus keta Oncorhynchus keta Oncorhynchus keta Salmo salar Oncorhynchus kisutch Lutjanus campechanus Lutjanus campechanus Oncorhynchus gorbuscha Oncorhynchus gorbuscha Oncorhynchus gorbuscha Oncorhynchus gorbuscha Oncorhynchus gorbuscha Oncorhynchus gorbuscha Oncorhynchus gorbuscha Oncorhynchus gorbuscha Hemigrammus pulcher Hemigrammus pulcher Oncorhynchus tshawytscha Gadus morhua Plecoglossus altivelis Plecoglossus altivelis chum salmon chum salmon leafy seadragon Oncorhynchus mykiss ) Pink salmon Barramundi cod Baltic salmon ) Pink salmon Atlantic salmon Table 1. Review of swim bladder infections in fish caused by fungi and oomycetes Table (Nectriaceae) Pink salmon (Mycosphaerellaceae) Pretty tetra (Cladosporiaceae) Red snapper (Cladosporiaceae) Barramundi cod Pyrenochaeta acicola sp. (Didymellaceae) Atlantic salmon herbarum (= (Aspergillaceae) Red snapper (Ophiocordycipitaceae) Pink salmon Acremonium kiliense Scopulariopsis brumptii) (Herpotrichiellaceae) seadragon Weedy (Herpotrichiellaceae) Queensland grouper (Pleosporaceae) Pink salmon var. var. (Sympoventuriaceae) Barramundi cod (= (= (Herpotrichiellaceae) Pretty tetra Phoma (Pythiaceae) Ayu (Nectriaceae) Pink salmon (Didymellaceae) Coho salmon Paecilomyces farinosus) and (= sp. (Herpotrichiellaceae) Cod sp. (Didymellaceae) Ayu (incertaes sedis, Hypocreales) chum salmon (Neopyrenochaetaceae) chum salmon (Cordycipitaceae) (Microascaceae) (Cordycipitaceae) chum salmon

salmon King George whiting

chum salmon Atlantic salmon

Pink salmon chum salmon Chinook salmon Undetermined fungal species Pink salmon Volutella salmonis Volutella Tolypocladium inflatum Tolypocladium Ochroconis humicola Cladosporium cladosporioides Phoma Pythium flevoense Sarocladium kiliense Phoma herbarum Isaria farinosa Microascus brumptii Phaeophleospora hymenocallidicola Neopyrenochaeta acicola Exophiala xenobiotica Penicillium corylophilum Exophiala angulospora Exophiala pisciphila Cladosporium sphaerospermum Exophiala Alternaria consortialis Phoma leveillei Fusarium avenaceum Pathogen (family) Fish species affected Akanthomyces lecanii (= Lecanicillium lecanii) 240 Dis Aquat Org 138: 237–246, 2020

amplified with the primers NS1 and NS24 (White et al. dry pelleted feed containing 40% crude protein (N- 1990). A partial β-tubulin gene fragment was ampli fied content × 6.25) and 14% crude fat. using the primers Ben2f (Hubka & Kolarˇík 2012) and At necropsy, gross lesions included an enlarged Bt2b (Glass & Donaldson 1995), and a partial fragment swim bladder, especially in the posterior portion, with of the rpb2 gene, encoding the second largest RNA an extremely thickened swim bladder wall causing a polymerase subunit, was amplified using the primers great reduction of the lumen. The posterior portion of RPB2-5F2 (Sung et al. 2007) and fRPB2-7cR (Liu et al. the swim bladder was enlarged, dome-shaped and 1999). The PCR protocol and DNA sequencing used in firm; it was approximately 15 mm. A microscopic wet this study were described by Hubka et al. (2018), and mount examination of the swim bladder contents PCR product purification was performed as described revealed abundant septate fungal hyphae, and a his- by Réblová et al. (2016). The obtained DNA sequences tological examination showed PAS-positive mycelia were compared with those derived from the ex-type or in the lumen and wall of the swim bladder. Filamen- reference strains and were deposited into the Euro- tous and septate hyphae penetrated and effaced the pean Nucleotide Archive (ENA) database: LR215836 swim bladder wall. Polymorphonuclear cells were (ITS rDNA), LR215838 (LSU rDNA), LR215837 (SSU associated with the fungi (Fig. 1a). In addition, rDNA), LR215839 (β-tubulin) and LR215840 (rpb2) melanized fungal hyphae protruded into the lumen (study accession number PRJEB31231). of the swim bladder (Fig. 1b,c). Histological examination of the posterior swim bladder revealed that the swim bladder muscle was affected by expansion 2.5. Mycological studies of inflammatory cells (Fig. 1d). No parasites or bacte- ria were observed in the examined tissues. The fun- The case isolate was grown on MEA (Oxoid), gus was not observed in other organs. A fungal strain potato carrot agar (PCA) (Fassatiová 1986) and potato (CCF 5740) was isolated in pure culture on MEA. dextrose agar (PDA) (Burgess et al. 1988) at 15, 20, 25, 30, 33 and 37°C in the dark. Colour codes and names followed the conventions described by Kornerup & 3.2. Experimental infection of fish Wanscher (1967). The methods used to observe the micromorphology and imaging were described by Gross clinical signs of fungal infection were first ob- Hubka et al. (2015). The strain was deposited into the served after 4 d, including weakened reflex responses, Culture Collection of Fungi (CCF; Charles Univer- lethargy and weakness, and the fish stayed near the sity, Prague) under accession number CCF 5740. upper edge of the tank. Necropsies of the 3 fish that died on Days 14, 17 and 19 and 2 diseased fish examined on Days 10 and 11 of the experiment revealed 3. RESULTS extensive congestion and haemorrhages under the serosa (Fig. 2a). Histological examination showed se- 3.1. Case description vere congestion in the liver and sinusoidal dilatation, activation of Kupffer cells, leukostasis and regressive The mycosis described in this study was identified changes with foci of selective necrosis (Fig. 2b,c). in 1 of 10 rainbow trout that were caught by random H&E-, PAS- and GMS-stained sections demonstrated selection in a raceway culture on a commercial fish the presence of hyphal foci in the liver, pyloric caeca, farm (404 m above sea level in the watershed of the spleen, kidney and heart (Fig. 2c−g). Mild interstitial River Odra, Czech Republic) and were subjected to myocarditis was observed in the heart (Fig. 2h). post mortem examination during a regular preven- Granulomas were not observed in any sections. tive routine health check that included clinical, para- Within the splenic paren chyma, the hyphae were sitic, bacterial, mycological and histological exami- strongly melanized (Fig. 2i). nations. The fish were without apparent clinical signs suggesting infection. Fish were reared in concrete tanks (8 × 0.8 × 8 m) and kept at a density of 100 kg 3.3. Molecular identification m−3. The water had the following physical and chemical characteristics: temperature 7°C; dissolved O2 Using the BLAST similarity search, the ITS region of −1 11 mg l ; pH 7.1; total hardness 5°N; CODMn 3.2 mg the case isolate was matched to that of the reference −1 − −1 − −1 l ; NO2 0.067 mg l ; and NO3 6.9 mg l . The strain of Phoma herbarum CBS 615.75 (KF251212) examined fingerling had an LS of 87 mm and was fed and the ex-type of Didymella boeremae CBS 109942 Rˇ ehulka et al.: Phoma herbarum mycosis in rainbow trout 241

Fig. 1. Spontaneous infection in Oncorhynchus mykiss caused by Phoma herbarum. (a) Mass of fungal hyphae in swim bladder wall permeated by infiltrating cellular inflammation with abundant polynuclear leukocytes. (b) Melanized fungal hyphae (inset shows detailed image) protruding into the lumen of swim bladder. (c) Detail of fungal hyphae and inflated cells. (d) Expan- sion of the swim bladder muscle by inflammatory cells, macroscopically manifesting itself as a tumoral focus in the posterior part of the swim bladder. Haematoxylin and eosin stain (a–d), Fontana-Masson (b, inset). Scale bars = (a,b) 50 µm, (b, inset) 10 µm, (c) 20 µm, (d) 100 µm

(FJ426982). The LSU region was identical to that of brown (colour code 6F5 as per Kornerup & Wanscher P. herbarum CBS 615.75 (EU754186) and several other 1967) with lighter, brownish yellow (5C2) areas with members of the family Didymellaceae, similar to the aerial mycelia and margins that were filamentous results obtained for the SSU rDNA region, which also and submerged; the reverse was black with reddish has only limited discriminatory power between mem- brown (8E8) sectors, and yellowish brown (5E5) bers of the family Didymellaceae. A partial β-tubulin areas were observed in the marginal portions. gene sequence showed 99.4% similarity (322/324 bp) Colonies grown on PDA at 25°C after 2 wk were floc- to P. herbarum CBS 615.75 (FJ427133), while other cose, centrally raised and white in the centre due to species showed lower than 95% similarity. The simi- abundant aerial mycelia, with pale orange (5A3) in larity of the rpb2 sequence to the P. herbarum CBS the margins that were delicately filamentous and 615.75 (EU874863) was 99.7% (948/951 bp), while submerged; the reverse was black with reddish other species showed <90% similarity. brown (8E8) sectors in the centre, and pale orange (5A3) areas were observed in the marginal portions. Colonies grown on PCA at 25°C after 2 wk were 3.4. Mycological studies uncoloured, flat, submersed (except the central portion) with few aerial mycelia, orange white (5A2) in Colonies of the isolate CCF 5740 grown on MEA at the centre, and the reverse was similar to the obverse 25°C after 2 wk were floccose, centrally raised, dark (Fig. 3a−d). Growth rates at different temperatures 242 Dis Aquat Org 138: 237–246, 2020

Fig. 2. Experimental infection of Oncorhynchus mykiss with Phoma herbarum strain CCF 5740. External signs of infected rainbow trout and histological section 16 d after intraperitoneal injection with spore suspension. (a) Extensive hyperaemic area with subcapsular haemorrhage (arrow) in liver and other visceral organs and the presence of serosanguinous fluid in the abdominal cavity. (b) Leukostasis in the liver. (c) Degenerative changes around hyphae in the liver. (d) Massive proliferation of fungal hyphae in the pyloric caeca with an inflammatory reaction. (e) Invasive proliferation of fungal hyphae into the spleen parenchyma. (f) Septate and budding fungal hyphae in the kidney. (g) A fragment of the fungal hyphae in the myocardium. (h) Non-suppurative interstitial myocarditis. (i) Melanized hyphae in the spleen parenchyma. Haematoxylin and eosin (b,c,h,i), Periodic acid-Schiff (d,g), Grocott’s methenamine silver stain (e,f). Scale bars = (b,c,d,e,h) 50 µm, (f,g,i) 20 µm Rˇ ehulka et al.: Phoma herbarum mycosis in rainbow trout 243

Fig. 3. Macromorphology and micromorphology of Phoma herbarum CCF 5740. (a) Colonies incubated for 14 d on malt extract agar (MEA) at different temperatures; from the left to right: 15, 20, 25 and 30°C, lower row shows reverse of colonies. (b) Detail of colony on MEA at 20°C with red and brown-black sectors; dark sector contains conidiomata. Colonies incubated for 14 d at 25°C on (c) potato dextrose agar and (d) potato carrot agar. (e,f) Section of conidiomata releasing conidia. (g) Peridium with textura angularis. (h−j) Conidiogenous cells. (k) Hyaline conidia in phase contrast. Scale bars = (e−f) 100 µm, (g) 10 µm, (h−k) 2 µm 244 Dis Aquat Org 138: 237–246, 2020

on MEA/PDA/PCA after 7 d (in mm) were as follows; Favourable conditions for the occurrence of fungal 15°C: 22−24/27−29/23−25; 20°C: 34−35/36−38/35−37; infections include the continuous ingestion of spores 25°C: 34−35/38−40/38−40; and 30°C: 18−23/18−22/ that may be present in feed. In addition to the case 20−22. At 33°C, only germination was observed described in this study, we repeatedly isolated (2−4 mm), while no growth was observed at 37°C on Phoma herbarum in feed pellets, where the fungus any of the tested media. likely finds a suitable substrate to grow (fish meal, Following is a description of the micromorphology: meat-bone meal, soy extrudate, wheat extrudate, vegetative hyphae hyaline; conidiomata pycnidial milk powder and whey powder). A representative (Fig. 3e,f), most abundantly produced on MEA at strain identified by DNA sequencing (ENA accession 25°C, solitary, globose to subglobose, glabrous, semi- numbers LR745530−LR745534) is available in the immersed to immersed, typically 120−330 µm in dia - CCF collection of fungi (CCF 4483). The possibility meter, pycnidial wall of textura angularis (Fig. 3g), that P. herbarum is transmitted by contaminated light brown to black-brown; conidiogenous cells feed, leading to a clinical effect, was conclusively phialide (Fig. 3h−j), hyaline; conidia ellipsoidal demonstrated by Ross et al. (1975), who fed a diet (Fig. 3k), smooth-walled, aseptate, 3.5−5 (4.4 ± 0.4; comprising granules mixed with a suspension of fun- mean ± SD) × 1.5−2 (1.7 ± 0.1) µm. Our observations gal spores to Chinook salmon fry. In the fish that died and measurements are in accordance with the after 18 d, the anal area musculature was inflamed, descriptions of P. herbarum listed in the monographs and the intestinal contents and visceral organs were on this genus (Boerema et al. 2004, Chen et al. 2015). filled with masses of mycelia. In our study, unlike in the study published by Ross et al. (1975), only the swim bladder was infected 4. DISCUSSION without the subsequent observable clinical signs described by those authors (swollen and haemor- Fungal infections of swim bladders have primarily rhagic vents, lateral compression of the abdominal been described in marine salmonids and freshwater area, extensive haemorrhaging of the caudal fin, species. An overview of the swim bladder mycoses petechiae occurring on the lateral and ventral body and fungal pathogens is presented in Table 1. The surfaces and hyperaemic areas in the striated mus- swim bladder environment offers optimum condi- cle). We did not observe any signs of extensive in - tions for the growth of a wide spectrum of fungal spe- flammatory reaction in the surrounding tissues. The cies. Fungal penetration of the swim bladder into sur- inflamed musculature of the anal area described by rounding tissues has been described in multiple Ross et al. (1975) in experimentally infected chinook studies (Ross et al. 1975, Blaylock et al. 2001, Faisal et salmon fry was also missing in our experiments. al. 2007; see also Table 1). However, there have been However, Ross et al. (1975) fed their fish a diet con- no clear explanations as to whether the swim bladder sisting of granules mixed with an aqueous suspen- is the primary site of infection. sion of fungal conidia. Faisal et al. (2007) and Ross et Wood (1979) proposed that infection of the swim al. (1975) described multiple rounded areas of muscle bladder may occur during the swim-up stage when it softening, protruded haemorrhagic vents and abdom- is initially filled with air. This is in agreement with inal swelling in 2 outbreaks of systemic mycosis in histopathological findings made by several authors hatchery-reared chinook salmon fingerlings. In com- who identified aspiration via the pneumatic duct as a parison with the above authors, we can add 2 more probable route of swim bladder infection (Ross et al. clinical signs observed during spontaneous infec- 1975, Poppe et al. 1991, Miura et al. 2010). The tions: extremely thickened swim bladder wall with a pathogen may develop first in the lumen of the swim massive presence of hyphae and with intensive cellu- bladder and later penetrates through the wall of the lar inflammatory response, and in particular, inflam- swim bladder into the kidneys, gastrointestinal tract, mation and hyperplasia of the muscle. Experimentally abdominal cavity, surrounding musculature and infected fish showed marked extensive con gestion eventually to the exterior through the abdominal wall and haemorrhage in the liver and other visceral (Ross et al. 1975, Hatai et al. 1986, Faisal et al. 2007). organs and the presence of serosanguinous fluid in The use of the pneumatic duct as the route of infec- the abdomen, which are not among the clinical signs tion is supported by the results of our histological of moribund fish described by Ross et al. (1975). Find- examination, together with the descriptions by Bruno ings in our study could be associated with the in tra - (1989) and Lehmann et al. (1999), who observed fun- peritoneal route of infection, which is unlikely to gal hyphae in the swim bladders of young fish. occur in natural scenarios. Rˇ ehulka et al.: Phoma herbarum mycosis in rainbow trout 245

Similar to infection by Cephalotheca sulfurea, LITERATURE CITED fungal elements in the fish in our experiment were Aho R, Koski P, Salonen A, Rintamäki P (1988) Fungal swim- not surrounded by granulomas, similar to findings bladder infection in farmed Baltic salmon (Salmo salar L.) by Ross et al. (1975) and Nyaoke et al. (2009), who caused by Verticillium lecanii. Mycoses 31: 208−212 proposed that the absence of granuloma formation Ali EH, Hashem M, Al-Salahy MB (2011) Pathogenicity and or significant host inflammatory response to Exo - oxidative stress in Nile tilapia caused by Aphanomyces laevis and Phoma herbarum isolated from farmed fish. phiala spp. infection could be a result of the host’s Dis Aquat Org 94:17−28 inadequate or deficient immunologic response. Bennett A, Ponder MM, Garcia-Diaz J (2018) Phoma infec- Absence of granulomas could be associated with tions: classification, potential food sources, and their the ability of the hyphae of the fungus to produce clinical impact. Microorganisms 6: 58 granulocyte inhibitors, as described for Candida Blaylock R, Overstreet RM, Klich M (2001) Mycoses in red snapper (Lutjanus campechanus) caused by two deu ter - species by Smail et al. (1988). With respect to the o mycete fungi (Penicillium corylophilum and Cladospo- detection of melanins, it should be noted that they rium sphaerospermum). Hydrobiologia 460:221−228 act as a virulence factor (Jacobson 2000, Feng et al. Blazer VS, Wolke RE (1979) An Exophiala-like fungus as the 2001, Nosanchuk & Casadevall 2003), able to inter- cause of a systemic mycosis of marine fish. J Fish Dis 2: 145−152 cept the free radicals and hypochlorite produced by Boerema GH, de Gruyter J, Noordeloos ME, Hamers MEC phagocytic cells (Jacobson 2000, Feng et al. 2001, (2004) Phoma identification manual: differentiation of Szaniszlo 2006), and that melanin-synthesizing specific and infra-specific taxa in culture. CABI Publish- fungi are able to cause disseminated infection ing, Wallingford related to the resistance of melanin to oxidative Bowater RO, Thomas A, Shivas RG, Humphrey JD (2003) Deuteromycotic fungi infecting barramundi cod, Cro - damage caused by host phagocytic mechanisms. mileptes altivelis (Valenciennes), from Australia. J Fish With its ability to disseminate in the host and Dis 26:681−686 induce systemic infection, P. herbarum is a species Bruno D (1989) Observations on a swim bladder fungal that can seriously affect host health. This issue was infection of farmed Atlantic salmon, Salmo salar L. Bull Eur Assoc Fish Pathol 9:7−8 confirmed in trials conducted by Ali et al. (2011), Burgess LW, Liddell CM, Summerell BA (1988) Laboratory who examined pathogenicity and oxidative stress manual for Fusarium research: incorporating a key and in Nile tilapia Oreochromis niloticus caused by P. descriptions of common species found in Australasia. herbarum isolated from farmed fish. Their study University of Sydney, Sydney Burton TO, Meyers TR, Starkey NS, Follett JE (2004) Exper- revealed that P. herbarum infection of fish signifi- imental transmission of the fungus Phoma herbarum to cantly increased oxidative stress, particularly in Chinook salmon. J Aquat Anim Health 16:251−257 gills, and induced a marked drop in the levels of Camus A, Berliner A, Hyatt M, Hatcher N, Clauss T (2015) most antioxidants. Exophiala xenobiotica aerocystitis in a Queensland In summary, based on our findings, the patho- grouper Epinephelus lanceolatus (Bloch). 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This work was financially supported by Molecular cloning and characterization of WdPKS1, a the Ministry of Culture of the Czech Republic through insti- gene involved in dihydroxynaphthalene melanin biosyn- tutional financing of long-term conceptual development of thesis and virulence in Wangiella (Exophiala) dermati- the research institution (the Silesian Museum, MK000100595) tidis. Infect Immun 69: 1781−1794 and an internal grant of the Silesian Museum No. IGS Garcia-Hermoso D, Valenzuela-Lopez N, Rivero-Menendez 201708/2017. We thank H. Šerá of the Pathology Depart- O, Alastruey-Izquierdo A and others (2019) Diversity of ment (and departmental head: E. Sehnálková MD) of the coelomycetous fungi in human infections: a 10-y experi- Silesian Hospital in Opava for technical assistance in histo- ence of two European reference centres. Fungal Biol 123: logical preparations. Mycological studies were supported by 341−349 the project BIOCEV (CZ.1.05/1.1.00/02.0109) provided by Glass NL, Donaldson GC (1995) Development of primer sets the Czech Republic Ministry of Education, Youth and designed for use with the PCR to amplify conserved genes Sports, and the European Research Development Fund; and from filamentous ascomycetes. Appl Environ Microbiol Charles University Research Centre program No. 204069. 61: 1323−1330 246 Dis Aquat Org 138: 237–246, 2020

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Editorial responsibility: Dave Rotstein, Submitted: October 13, 2019; Accepted: February 18, 2020 Olney, Maryland, USA Proofs received from author(s): April 3, 2019