Journal of Aquatic Animal Health For Peer Review Only Exophiala angulospora causes systemic mycosis in Atlantic halibut (Hippoglossus hippoglossus) −−−−−− A Case Report Journal: Journal of Aquatic Animal Health Manuscript ID: UAAH-2014-0011 Manuscript Type: Communication Keywords: Pathology < Culture, Disease and Parasites, Coldwater < Culture URL: http://mc.manuscriptcentral.com/jaah Email: [email protected] Page 1 of 19 Journal of Aquatic Animal Health 1 Exophiala angulospora causes systemic mycosis in Atlantic halibut ( Hippoglossus 2 hippoglossus ) −−−−−− A Case Report 3 4 5 David P. Overy 6 Department of Pathology and Microbiology, University of Prince Edward Island, 550 7 University ForAvenue Charlottetown, Peer PEI, Review Canada, C1A 4P3, andOnly Nautilus Biosciences 8 Canada, Duffy Research Center (NRC-INH), 550 University Ave., Charlottetown, Prince 9 Edward Island, Canada, C1A 4P3 10 11 David Groman and Jan Giles 12 Aquatic Diagnostic Services, University of Prince Edward Island, 550 University Ave., 13 Charlottetown, Prince Edward Island, Canada, C1A 4P3 14 15 Stephanie Duffy 16 Nautilus Biosciences Canada, Duffy Research Center (NRC-INH), 550 University Ave., 17 Charlottetown, Prince Edward Island, Canada, C1A 4P3 18 19 Mellisa Rommens 20 Aquaculture Solutions, 1475 Peter Rd., Emyvale, Prince Edward Island, Canada, 21 C0A1Y0 22 23 Gerald Johnson 24 Department of Pathology & Microbiology, University of Prince Edward Island, 550 25 University of Prince Edward Island, Prince Edward Island, Canada, C1A 4P3 26 27 28 29 ∗ Corresponding author: [email protected] 30 1 URL: http://mc.manuscriptcentral.com/jaah Email: [email protected] Journal of Aquatic Animal Health Page 2 of 19 31 <A> Abstract 32 33 Filamentous black yeasts from the genus Exophiala are ubiquitous, opportunistic 34 pathogens causing both superficial and systemic mycoses in warm- and cold-blooded 35 animals. Infections by black yeasts have been reported relatively frequently in a variety 36 of captive and farmed freshwater and marine fish. In November 2012, moribund and 37 recently deadFor farm raised Peer Atlantic halibut Review (Hippoglossus hippoglossus Only ) were necropsied 38 to determine the cause of death. Histopathology revealed that three of five fish were 39 affected by a combination of an ascending trans-ureter granulomatous mycotic nephritis, 40 necrotizing histiocytic encephalitis, and in one fish the addition of a fibrogranulomatous 41 submucosal branchitis. Microbial cultures of kidney using selective mycotic media 42 revealed pure growth of a black pigmenting septated agent. Application of molecular and 43 phenotypic taxonomy methodologies determined that all three isolates were genetically 44 consistent with Exophiala angulospora . This is the first report of Exophiala 45 angulospora as the causal agent of systemic mycosis in Atlantic halibut. 46 2 URL: http://mc.manuscriptcentral.com/jaah Email: [email protected] Page 3 of 19 Journal of Aquatic Animal Health 47 <A> Introduction and Clinical History 48 49 Exophiala spp. are ubiquitous fungi, historically isolated from a variety of environmental 50 substrates such as soil and sediment, decaying wood and plant material, human hair and 51 nails and drinking water (Iwatsu et al. 1991; Domsch et al. 2007; de Hoog et al. 2011). 52 These filamentous black yeasts are also opportunistic pathogens causing both superficial 53 and systemicFor mycoses Peer in warm- and Reviewcold-blooded animals (UijthofOnly et al. 1997; de Hoog 54 et al. 2011; Gjessing et al. 2011). De Hoog and collaborators suggest that animals with 55 moist skin are more susceptible to infection by filamentous black yeasts, where the ability 56 of the pathogen to assimilate alkylbenzenes and accumulate melanin within the hyphae 57 and conidia are purported to be general virulence factors for these agents (de Hoog et al., 58 2011). Black yeast infections in aquarium and farmed fish as well as amphibians are 59 relatively frequent. An accurate estimation of the magnitude of the disease outbreaks has 60 been difficult to ascertain due to the infrequent and happenstance nature of disease 61 reports, in spite of the fact that outbreaks of this mycotic disease have been reported to 62 result in severe losses. (de Hoog et al. 2011). 63 64 Although first isolated and described from drinking water (Iwatsu et al. 1991), Exophiala 65 angulospora is also an opportunistic pathogen of both freshwater and marine fish. 66 Exophiala angulospora has been reported to cause mycosis in aquaria-maintained weedy 67 sea dragons (Phyllopteryx taeniolatus ) in the United States (Nyaoke et al. 2009). This 68 pathogen has also been isolated from the freshwater whitefish Stenodus leucichtys in 69 southern Russia (de Hoog et al. 2011) and was determined to be the cause of systemic 70 mycosis in farmed Atlantic cod (Gadus morhua ) from Norway (Gjessing et al. 2011). 71 Necropsy of moribund cod revealed chronic multifocal inflammation in the internal 72 organs, consisting of dematiaceous fungal hyphae surrounded by distinct layers of 73 inflammatory cells. Gjessing and co-workers noted that the fungal infection was not 74 eliminated by this inflammatory response in the cod, resulting in a systemic infection and 75 mortality. 76 3 URL: http://mc.manuscriptcentral.com/jaah Email: [email protected] Journal of Aquatic Animal Health Page 4 of 19 77 In this Case Report we describe the diagnostic evaluation of Atlantic halibut 78 (Hippoglossus hippoglossus ) which were submitted on multiple occasions from a grow- 79 out population of approximately 4000 fish, during November of 2012. Necropsies and 80 diagnostic testing on both fresh dead and moribund fish harvested from the slowest 81 growing group in the population were undertaken at the Aquatic Diagnostic Services Unit 82 of the Atlantic Veterinary College, University of Prince Edward Island, Canada. Fish 83 had been rearedFor in 5 'Peer by 12' fiberglass Review tanks ( water temperature Only 12.1- 12.2 ° C, O 2 = 84 105-119 % saturation, N 2 < 96% saturation ) and were being fed dry commercial fish feed 85 ( 6mm Europa 18 – Skretting Feeds). The mean weight of the three mortalities was 227 86 grams. Clinical observations of affected fish, as reported by the on-site clinician, were 87 limited to reduced appetite and loss of equilibrium. Cumulative mortality during the 88 previous two months was five to 10 times higher than normally expected, ranging from 89 0.8 to 1.5 % per month as compared to the normal rate of < 0.1 % per month. Diagnostic 90 investigation into the cause of death revealed that the halibut were suffering from a 91 systemic mycotic infection caused by Exophiala angulospora . To the authors’ 92 knowledge this is the first report of a clinical myosis due to Exophiala angulospora in 93 farm reared Atlantic halibut. 94 95 96 <A> Methods 97 <B> Necropsy 98 Live fish were euthanized using an overdose of a saturated solution of benzocaine 99 dissolved in 95% ethanol. All fish were subsequently examined for gross external and 100 internal lesions, assessed for sex determinations and content of the alimentary tract. The 101 gills of all live fish were examined by wet mounts by phase contrast microscopy. 102 103 <B> Histopathology: 104 The following tissues were selected from each of five fish at the time of necropsy, 105 trimmed approximately 1 cm in diameter and placed into 10% Neutral Buffered 106 Formalin (1 : 10 ratio of tissue to formalin): gill, brain, eye, kidney, spleen, heart, liver, 107 nares, gonad, esophagus, stomach, intestine, pyloric caeca, pancreas and body wall. 4 URL: http://mc.manuscriptcentral.com/jaah Email: [email protected] Page 5 of 19 Journal of Aquatic Animal Health 108 Fixation was completed by holding all samples on a shaker for a minimum of 48 hours 109 prior to routine histology processing. For tissues which contained bone and/or cartilage 110 (cranium, eye, body wall, gill), decalcification was undertaken by immersion for a further 111 24 hours in a commercially available decalcification fluid ( CAL-EX II ® Fisher 112 Chemical ). Sections from all tissues were subsequently trimmed into cassettes, 113 processed to wax blocks, sectioned to 5 µm, mounted/dried onto glass slides and stained 114 by the HematoxylinFor andPeer Eosin (H&E) Review method (Luna, L.G. 1968).Only Selected tissues were 115 recut and stained by the Periodic Acid-Schiff (PAS) technique (Luna, L.G. 1968). 116 Stained slides were examined using a Lieca DM2500 light microscope and, where 117 appropriate, digital images were captured with a top mounted PixeLink PL-B625 digital 118 microscope camera using the PixeLINK® µScope Standard v3.6 software. 119 120 <B> Bacteriology 121 Phenotypic Identification. − A sterile swab of the kidney was provided at necropsy for 122 routine bacteriology. Swabs were streaked onto Blood Agar and Blood Agar containing 123 2 % NaCl, and held at both 22°C and 15°C for a minimum of seven days. Any bacterial 124 growth deemed significant was sub-cultured onto new media and subsequently 125 indentified using routing biochemical and immunologic methodologies (American 126 Fisheries Society-Fish Health Section 2010) 127 128 <B> Mycology 129 Phenotypic Identification .− A sterile swab of the kidney was provided at necropsy for 130 routine mycology. Swabs were streaked out onto Sabouraud’s agar and incubated at 131 room temperature ( ~22°C ) and monitored daily for fungal outgrowth. Obtained axenic 132 fungal isolates were maintained on potato dextrose and Sabouraud’s agar at 22 °C for a 133 two month period. Micromorphology was visualized from cellotape slide mounts 134 prepared in lactic acid observed under 40x and 100x magnification using Phase Contrast 135 microscopy on a Leica DME microscope. Digital photomicrographs were obtained using 136 a Leica EC3 camera (Leica Microsystems, Switzerland) and micromorphological 137 measurements were made from digital photomicrographs using the Leica LAS EZ 138 software (v2.1.0). 5 URL: http://mc.manuscriptcentral.com/jaah Email: [email protected] Journal of Aquatic Animal Health Page 6 of 19 139 140 DNA Extraction and PCR Amplification.