A visceral mycosis in farmed rainbow trout (Oncorhynchus mykiss) caused by Neopyrenochaeta submersa

A mycotic infection manifesting as abdominal distension with free serous fluid accumulation in the coelomic cavity is documented in farmed rainbow trout. Histological examination using PAS and silver staining revealed the presence of numerous fungal hyphae in the spleen and gastrointestinal wall. The isolated fungus was sterile and identified by using phylogenetic analysis based on four loci as Neopyrenochaeta submersa. This is the first time this fungus has been reported as pathogen.


Introduction
Protection of fish in intensive culture against infections of bacterial or mycotic origin is among the key preventive measures within the range of the key principles of veterinary health management to minimize the morbidity of the fish. Routine use of antibiotics and their combinations, which all affect the immunity mechanisms, results in an increasing incidence of opportune mycotic infections. In immunodeficient hosts they may lead to a severe manifest illness in the form of generalized infection, induced either through activation of a latent infection from an endogenous infection source, or through transfer of an exogenous pathogen to the host from a source in the external environment. Colonization of the mucous membrane of the digestive tract is a significant endogenous source of the mycotic agent and a risk factor underlying the rise of disseminated mycosis. In salmonid aquaculture there is a trend of increasing incidence of visceral mycotic infections in farmed rainbow trout. The spectrum of primary etiological agents of serious health disorders also expanded in the last years [1][2][3].
Coelomycetous fungi are relatively rare and poorly studied human and animal pathogens with genera Phoma, Pyrenochaeta and Neoscytalidium being the most commonly reported pathogens [4]. Valenzuela-Lopez et al. [5] proposed a new genus Neopyrenochaeta and new family Neopyrenochaetaceae encompassing several taxa previously included in Pyrenochaeta. Pathogenic Pyrenochaeta spp. were mostly accommodated in other genera such as Neocucurbitaria (N. keratinophila, N. unguis-hominis), Medicopsis (M. romeroi) and Nigrograna (N. mackinnonii) [4,5]. Thus, current members of the genus Neopyrenochaeta are predominantly saprophytes and plant pathogens which are non-pathogenic for human and animals. As far as we know, the only exception is the report of swim bladder infection in pacific salmon caused by Neopyrenochaeta acicola (syn. Pyrenochaeta acicola) [6]. Other cases of infection in fish due to coelomycetous fungi are mostly restricted to the genera Phoma or Phaeophleospora [7,8].
This report describes the first case of infection by a recently described species of Neopyrenochaeta, N. submersa, isolated from a young, farmed rainbow trout, Oncorhynchus mykiss Walbaum. The fungal pathogen was identified using morphological and molecular data. To our knowledge, this is the first case of infection due to this fungal species in animals.

Case presentation
The disease occurred in one of the fifteen rainbow trout subjected to post-mortem examination during a routine health check on a commercial fish farm (404 m above sea level in the watershed of the River Odra, Czech Republic). The fish were reared in concrete tanks and kept at a density of 100 kg m -3 . The water had the following physical and chemical characteristics: temperature 7 • C, dissolved O 2 9 mg L -1 , pH 7.1, total hardness 6.5 • N, chemical oxygen demand (COD Mn ) 2.7 mg L -1 , nitrites (NO 2 -) 0.062 mg L -1 and nitrates (NO 3 -) 5.9 mg L -1 . The fish had a standard length of 115 mm and was fed dry pellets containing 40% crude protein and 14% crude fat. Clinical signs included abdominal distension (Fig. 1a), and at necropsy the coelomic cavity contained serous fluid. Microscopic wet mount examination of the spleen and intestine contents revealed abundant fungal hyphae. Histological examination showed foci of PAS positive fungal hyphae in the spleen and numerous fungal hyphae invading the gastrointestinal wall ( Fig. 1b-e), no fungal elements were found in gill, liver, kidney, heart, and brain. Histological findings showed the fungus' tendency to penetrate the surrounding tissue and induce systemic mycosis. A fungal strain was isolated in pure culture from the spleen and intestine on Sabouraud's agar at 24 • C. Bacteria were not observed in the lesion material and were not cultivated from samples inoculated on Blood agar (Columbia Agar Base; Merck), trypticase soya agar (Oxoid Ltd), Mueller-Hinton agar and Anacker-Ordal agar. Parasitological investigation revealed only minor infestation by Gyrodactylus bohemicus (the intensity of infestation on fins was three specimens). Subcultures of isolated fungal species were grown on malt extract agar (MEA), potato-dextrose agar (PDA) and oatmeal agar (OA) at 10, 15, 20, 25 and 30 • C (Fig. 2a). Colonies very similar on all media: greyish brown, velvety, centrally raised to umbonate with regular margins. Colony diameters after 14 days were as follows: 5-7 mm (10 • C), 10-13 mm (15 • C), 16-19 mm (20 • C), 7-9 mm (25 • C), no growth at 30 • C. The fungus remained sterile even after prolonged cultivation of 2 months and only dark-pigmented vegetative hyphae were observed (Fig. 2b-d).
No sporulation was induced on water agar or V8-agar supplemented with sterile banana leaves or grains. Similarly, the fungus remained sterile when incubating some cultures under near-ultraviolet light (12 h light, 12 h dark) [9]. BLAST similarity search with ITS region of rDNA showed affinity of the fungus to the species of Neopyrenochaeta. Additional phylogenetic marker genes used in the taxonomy of Phoma-like genera [5] were amplified as described previously [7] and phylogenetic tree was constructed (Fig. 3). The fungus was identified as Neopyrenochaeta submersa. The sequences were deposited to the GenBank database under following accession numbers: OK329892 (ITS), OK329942 (LSU), OK329943 (SSU), OK334131 (tub2) and OK334130 (rpb2). The fungus was deposited into the Culture Collection of Fungi, Charles University, Department of Botany, Prague, Czech Republic as CCF 5741, and Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands as CBS 148472.

Discussion
The coelomycetous fungi involved in mycoses are poorly known due to complexity of their identification and relatively low frequency of infections. However, they are responsible for a large variety of clinical entities from superficial to deep and systemic mycoses [11]. Previously reported fish infections were mostly attributed to coelomycete genus Phoma and reviewed by Ř ehulka et al. [7]. Other coelomycete genera are rarely reported as fish pathogens [8]. Until now, only single study reported genus Neopyrenochaeta as animal pathogen, namely, N. acicola has been reported in fish [6]. Marchenko [6] ranked N. acicola among seven species of fungi that caused swim bladder mycosis in a farmed fingerling pacific salmon, especially pink salmon (Oncorhynchus gorbuscha) and chum salmon (Oncorhynchus keta). Histologically, fungal hyphae were found in the posterior kidney and in some cases protruding into the lumen of intestine.
The infection of spleen, such as found in our study, is relatively common in fish. The spleen is often a harbor for infection, threatening to spread to other organs, including the gut. We think that the administration of quinoline-based wide-spectrum antibiotics might cause changes in the intestinal microflora, facilitating the propagation of the fungus, which then extensively colonized the intestine and grew into adjacent tissues.
In this study, we extended the spectrum of coelomycetous fungi pathogenic for fish. Although our attempts to find characteristic fruiting bodies in isolated fungus were not successful, phylogenetic analysis clearly identified it as a recently described species of Neopyrenochaeta, N. submersa, described from plant debris in freshwater in Spain [12]. Problems with induction of sporulation in colelomomycetous fungi are relatively common and make their identification based on phenotype challenging or impossible [11]. In these cases, the use of molecular techniques is the only possibility to achieve reliable identification.

Consent
Authors have obtained written and signed consent to publish the case report from legal guardian. Necessary certificate was obtained by the first author (Act No. 167, registration number 0155/2000-V 3). The research conducted in this work was supported by internal grant "Research on systemic fish diseases IV" (project No. IGS 201708/2017) that were approved by the committee DKRVO, MK000595. A copy is available upon request.

Declaration of competing interest
None. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3. Best-scoring maximum-likelihood tree based on combined data from ITS and LSU rDNA regions, β-tubulin (tub2) and RNA polymerase II second largest subunit (rpb2) genes, showing the relationships of case isolate of Neopyrenochaeta submersa CCF 5741 to other Neopyrenochaeta species. The dataset was partitioned, models were selected (ITS: TNe + G4; LSU: K2P + I; tub2: TNe + G; rpb2: TIM2e + I + G4) and tree constructed using IQ-TREE v. 1.6.12 [10]. Only bootstrap support ≥70% are shown; lower supports are designated by a dash; the ex-type strains are designated by a letter "T".