THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res • Ljubljana • 2021 • Volume 58 • Number 4 • 121–160458 Volume – S lo v Ve t R es 2 0 21 ; 5 8 (4 ): 12 1 16 0 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK 458 Volume Slov Vet Res • Ljubljana • 2021 • Volume 58 • Number 4 • 121–160 The Scientific Journal of the Veterinary Faculty University of Ljubljana SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Previously: RESEARCH REPORTS OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA Prej: ZBORNIK VETERINARSKE FAKULTETE UNIVERZA V LJUBLJANI 4 issues per year / izhaja štirikrat letno Volume 58, Number 4 / Letnik 58, Številka 4 Reviewing Editorial Board / ocenjevalni uredniški odbor: Antonio Cruz, Institute Suisse du Medicine Equine (ISME), Vetsuisse Fakultat, University of Bern, Switzerland; Gerry M. Dorrestein, Dutch Research Institute for Birds and Exotic Animals, Veldhoven, The Netherlands; Sara Galac, Utrecht University, The Netherlands; Wolfgang Henninger, Veterinärmedizinische Universität Wien, Austria; Nevenka Kožuh Eržen, Krka, d.d., Novo mesto, Slovenia; Louis Lefaucheur, INRA, Rennes, France; Peter O’Shaughnessy, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Scotland, UK; Peter Popelka, University of Veterinary Medicine, Košice, Slovakia; Dethlef Rath, Institut für Tierzucht, Forschungsbericht Biotechnologie, Bundesforschungsanstalt für Landwirtschaft (FAL), Neustadt, Germany; Phil Rogers, Grange Research Centre, Dunsany, Co. Meath, Ireland, Ireland; Alex Seguino, University of Edinburgh, Scotland, UK; Henry Staempfli, Large Animal Medicine, Department of Clinical Studies, Ontario Veterinary College, Guelph, Ontario, Canada; Frank J. M. Verstraete, University of California Davis, Davis, California, US; Thomas Wittek, Veterinärmedizinische Universität, Wien, Austria Address: Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Naslov: Veterinarska fakulteta, Gerbičeva 60, 1000 Ljubljana, Slovenija Tel.: +386 (0)1 47 79 100, Fax: +386 (0)1 28 32 243 E-mail: slovetres@vf.uni-lj.si Sponsored by the Slovenian Research Agency Sofinancira: Javna agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by/tisk: DZS, d.d., Ljubljana, December 2021 Number of copies printed / Naklada: 220 Indexed in/indeksirano v: Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich’s International Periodicals Directory, Science Citation Index Expanded, Journal Citation Reports – Science Edition https://www.slovetres.si/ Editorial Board / uredniški odbor: Editor in Chief / glavna in odgovorna urednica: Klementina Fon Tacer Co-Editor / sourednik: Modest Vengušt Technical Editor / tehnični urednik: Matjaž Uršič Assistant to Editor / pomočnica urednice: Valentina Kubale Dvojmoč Published by / Založila: University of Ljubljana Press / Založba Univerze v Ljubljani For the Publisher / Za založbo: Majdič Gregor, the Rector of the University of Ljubljana / rektor Univerze v Ljubljani Issued by / Izdala: Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani For the Issuer / Za izdajatelja: Breda Jakovac Strain, the Dean of the Veterinary Faculty / dekanja Veterinarske fakultete Editorial Advisers / svetovalca uredniškega odbora: Gita Grecs-Smole for Bibliography (bibliotekarka), Leon Ščuka for Statistics (za statistiko) Vesna Cerkvenik, Robert Frangež, Polona Juntes, Tina Kotnik, Greogor Majdič, Matjaž Ocepek, Ožbalt Podpečan, Ivan Toplak, Milka Vrecl, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani Simon Horvat, Janez Salobir, Biotehnical Faculty University of Ljubljana / Biotehniška fakulteta Univerze v Ljubljani Andraž Stožer, Faculty of Medicine University of Maribor / Medicinska Fakulteta Univerze v Mariboru This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License / To delo je ponujeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna licenca SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2021; 58 (4) Original Research Articles Voga M, Pleterski A, Majdič G. Isolation of live cells from different mice tissues up to nine days after death . . . . . . . . . . . . . . . . . . . . . . . . 125 Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M. The first outbreak of viral encephalopathy and retinopathy in farmed sea bass (Dicentrarchus labrax) in Slovenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Ari HH, Uslu S. Morphology and histology of the Eurasian Lynx (Lynx lynx) planum nasale. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147 Case Report Vergles Rataj A, Bandelj P, Erjavec V, Pavlin D. First report of canine myiasis with sheep nasal bot fly, Oestrus ovis, in Slovenia . . . . ..155 Author Index Volume 58, 2021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Received: 19 September 2020 Accepted for publication: 15 August 2021 Slov Vet Res 2021: 58 (4): 125 – 36 DOI 10.26873/SVR-1155-2021 UDC 57.086:611.013.1:57.085.4:591.86+591.88:599.323.451 Original Research Article Introduction Biological death is an irreversible cessation of circulatory and respiratory functions or irreversible cessation of all functions of the entire brain, including the brain stem (1). On the basis of biological death organ and tissue procurement can be processed. Cells that have been successfully used in donor transplantation procedures already in the eighties, were harvested very shortly, usually within 1 hour post mortem (2-4). In recent years, however, several studies have shown that viable cells can survive in the cadavers for much ISOLATION OF LIVE CELLS FROM DIFFERENT MICE TISSUES UP TO NINE DAYS AFTER DEATH Metka Voga1, Ana Pleterski1, Gregor Majdič1, 2* 1Institute for preclinical sciences, Veterinary faculty, University of Ljubljana, Gerbiceva 60, Ljubljana, 2Institute for physiology, Medical faculty, University of Maribor, Taborska 8, Maribor, Slovenia *Corresponding author, Email: gregor.majdic@vf.uni-lj.si Abstract: Some limited reports suggest that cells can survive in the cadavers for much longer than it was previously thought. In our study we explored how time after death, tissue type (muscle, brain and adipose tissue), storage temperature of cadavers (4 °C or at room temperature) and form of tissue storage (stored as cadavers or tissue pieces in phosphate buffered saline) affect the success of harvesting live cells from mice after death. Cells were isolated from dead tissues and grown in standard conditions. Some cells were used for RNA extraction and RT² Profiler™ PCR Array for cell lineage identification was performed to establish which lineages the cells obtained from post mortem tissues belong to. Results of our study showed that viable cells can be regu- larly isolated from muscle and brain tissue 3 days post mortem and with difficulty up to 6 days post mortem. Viable cells from brain tissue can be isolated up to 9 days post mortem. No cells were isolated from adipose tissue except immediately after death. In all instances viable cells were isolated only when tissues were stored at 4 °C. Tissue storage did not affect cell isolation. Isolated cells were progenitors from different germ layers. Our results show that live cells could be obtained from mouse cadavers several days after death. Key words: mouse; cadaver; stem cells; brain; muscle; adipose tissue longer than it was previously thought. Viable cells were obtained from different mammalian species at different time periods after death. Viable cells were obtained from murine liver up to 27 hours post mortem (5) and from murine inner ear up to 10 days post mortem (6). Neural stem cells from forebrain of one day old rats were obtained up to 6 days after death (7). Silvestre et al. (8) have obtained viable cells from rabbit and pig ears up to 10 days after death. Fibroblast like cells were recovered from refrigerated goat skin up to 41 days post mortem (9) and cattle skin even up to 49 days post mortem (10). Equine tendons yielded viable stem cells up to 72 hours post mortem (11). From human, myogenic cells were isolated up to 17 days post mortem (12). Results of these studies 126 M. Voga, A. Pleterski, G. Majdič suggest that viable cells survive in the cadavers in different tissues for longer time periods after death. It appears in general that viable cells survive in the tissues for longer time after death if tissues or cadavers are stored at 4°C and even longer when stored in liquid nitrogen (13). It is not known what is happening with cells after death. Due to the lack of studies in this area, it is not clear whether cells survive in all tissues or perhaps only in certain niches, neither is certain whether cells in the cadavers remain active or perhaps assume some dormant state. The latter was proposed by Latil et al. (12) who showed that stem cells are enriched in post mortem tissue due to cellular quiescence where cells adopt a reversible dormant state and thus possess a selective survival advantage compared with other cell types. A recent study has shown that also transcriptional activity of genes remains active for at least several hours after death. Appearance of new transcripts was shown in zebra fish and mouse cells up to 48 hours after death. Interestingly, the relative amount of gene transcripts declined gradually after death from the time of death in murine liver, while in murine brain samples, the amount of transcripts actually increased during the first hour after death. Total number of transcripts that increased in murine samples was over 500, suggesting that gene transcription also continues after death (14). However, there are no systematic studies examining the effect of time after death and different storage conditions on success of postmortem cell harvesting. The purpose of our study was therefore to establish the effect of different post mortem time points, tissue type, storage temperature and form of tissue storage on the success of harvesting live cells from mice. Finally, we investigated which lineages the cells obtained from post mortem tissues belong to. Materials and methods Animals In all experiments, adult, 4 to 5 months old, male BALB/C mice were used. Only males were used in this study to reduce the number of animals. Mice were bred at Institute for preclinical sciences with water and food ad libitum in standard conditions (12:12 light dark cycle and room temperature 22 °C). All animal experiments were approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection of the Republic of Slovenia and were carried out according to ethical principles, EU directive (2010/63/EU), and NIH guidelines. Mice were euthanized by CO2. The death was confirmed by cardiac arrest. After death mice were used in two ways: some mice (n = 9) were dissected. Approximately 150 mm3 of adipose tissue, muscle tissue (musculus quadriceps) and brain tissue from hippocampus and subventricular zone was obtained. Dissected tissues were placed in 0.01 M PBS either for storage or for cell isolation at time point 0. Other mice (n = 9) were stored as whole cadavers. Whole cadavers and tissue pieces were stored for 3, 6 and 9 days at 4°C and at room temperature (20–22°C). At given time points cadavers were dissected and the same tissues as those stored in PBS (adipose, muscle and brain tissue from hippocampus and subventricular zone) were obtained. Afterwards tissues both from cadavers and those stored in PBS were further processed. Cell isolation and cell culture Time point 0 was used for determination of a better method for cell isolation. Tissues at time point 0 were either enzymatically digested or pieces of tissue were placed directly into tissue culture plates as tissue explants. For enzymatic digestion, tissues were dissected with scalpel into very small pieces and then incubated at 37 °C overnight in Dulbecco – modified eagle medium (DMEM, Gibco, USA) containing 0,1% collagenase type II (Sigma - Aldrich, DE). The digested tissue was centrifuged at 240 rcf/min for 4 minutes. Supernatant was discarded. Pellet was resuspended in cell culture medium containing DMEM and 20% Fetal Bovine Serum (FBS, Gibco, USA). Cell suspension was plated into 12 – well plates and cultured at 37 °C in a 5 % CO2 incubator. For direct explantion of tissues, tissue pieces were placed into the center of 12 - well plate wells with cell culture medium. Due to considerably better isolation of cells from tissue explants, tissues from time points 3, 6 and 9 were subsequently used only as tissue explants and were cultured at 37 °C in a 5 % CO2 incubator. Cell culture medium was changed every 2–3 days. Explants were observed daily to monitor the appearance of live cells. Isolation of cells from tissue explants at time point 0 served as positive 127Isolation of live cells from different mice tissues up to nine days after death control. All cell experiments were repeated 3 times - 3 different male mice for each time-point (3, 6 or 9 days after death) and storage conditions (4°C or room temperature). RNA isolation RNA isolation was performed from the cells isolated from muscle, hippocampus and subventricular zone tissues. All samples were stored at 4 °C for 3 days after death. After 80 - 90 % confluence was reached, cells were detached using cell scrapper. Cell suspension was removed from the wells and centrifuged at 240 rcf/min for 4 minutes. Pellet of cells was resuspended in DPBS and centrifuged again. Total RNA extraction was carried out using Trizol (Invitrogen, USA) according to manufacturer's protocol. The amount of extracted total RNA was measured by UV spectrophotometer (Thermo Scientific, USA) at 260/280 nm wave length. Reverse transcription quantitative polymer- ase chain reaction Following RNA isolation, reverse transcription quantitative polymerase chain reaction was performed. Reverse transcription was carried out on 2 samples from each tissue source (muscle, hippocampus and subventricular zone). In the first step, 66.5 ng and 100 ng of total RNA of brain and muscle tissue, respectively, was reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit with RNAse Inhibitor (ThermoFisher) according to the manufacturer’s protocol. Negative reverse transcription controls were included. All reactions were conducted in a total volume of 20 μL. Conditions for reverse transcription were as suggested in the manufacturer’s protocol: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes. Due to very low amount of isolated RNA, 100 ng of total RNA of each muscle sample was amplified using RT² PreAMP cDNA Synthesis Kit (Qiagen, USA) prior to quantitative polymerase chain reaction. In the second step RT² Profiler™ PCR Array for mouse cell lineage identification (Qiagen, USA) was performed. All RT² Profiler™ PCR Array amplifications were conducted in a total volume of 25 μL. For brain tissue, 66,5 ng cDNA was used as a template. For muscle tissue, 100 ng cDNA amplified with RT² PreAMP cDNA Synthesis Kit (Qiagen) was used as a template. The amplification was carried out in 96 - well RT2 Profiler PCR array plates (Qiagen) with a Light Cycler 96 (Roche Life Science) using the following program: 50 °C for 2 minutes, 95 °C for 10 minutes, and 45 cycles at 95 °C for 15 seconds, 60 °C for 60 seconds. List of gene symbols and names according to Mouse Cell Lineage Identification RT2 Profiler PCR Array is presented in table 1. Results Cell isolation at time point 0 Dissected tissues (adipose, muscle and brain tissue from hippocampus and subventricular zone) at time point 0 after death were first used for determination of a better method for cell isolation. Considerably fewer cells were obtained from enzymatically-digested tissues that yielded viable cells only occasionally compared to the tissues that were used as explants. Isolation of cells from tissue explants at time point 0 therefore served as positive control. In all four positive control tissues (adipose, muscle and brain tissue from hippocampus and subventricular zone), viable cells were obtained. Cell isolation at 3, 6 and 9 days after death In all instances at all time points cells were isolated only when tissue pieces or cadavers were stored at 4 °C. No cells were obtained from cadavers or tissue pieces when stored at room temperature. Cells from 3, 6 and 9 days after death were first observed 5 to 15 days after tissue explants were placed in the tissue culture, depending on the time of storage. Earlier observation of cell isolation correlated with earlier time point of tissue explants being placed in the tissue culture. Three days after death, viable cells were obtained from muscle tissue (Figure 1), brain tissue - hippocampus (Figure 2) and subventricular zone (Figure 3), but not from the adipose tissue. Cells from all brain tissues and muscle tissue stored as whole cadavers were obtained from all samples whereas cells from muscle tissue stored as tissue pieces were obtained in 1 out of 3 samples. Six days after death, live cells were obtained only occasionally, when stored at 4 °C. Cells from muscle tissue were obtained in 2 out of 3 samples stored as 128 M. Voga, A. Pleterski, G. Majdič Gene symbol Gene name Gene symbol Gene name Alb Albumin Krt14 Keratin 14 Apoh Apolipoprotein H Krt19 Keratin 19 Aqp1 Aquaporin 1 Lefty1 Left right determination factor 1 Bmp4 Bone morphogenetic protein 4 Map3k12 Mitogen-activated protein kinase kinase kinase 12 Ccr5 Chemokine (C-C motif) receptor 5 Miox Myo-inositol oxygenase Cd34 CD34 antigen Mixl1 Mix1 homeobox-like 1 (Xenopus laevis) Cd3e CD3 antigen, epsilon polypeptide Msln Mesothelin Cd79a CD79A antigen (immunoglobu-lin-associated alpha) Myh1 Myosin, heavy polypeptide 1, skeletal muscle, adult Chat Choline acetyltransferase Myh11 Myosin, heavy polypeptide 11, smooth muscle Col10a1 Collagen, type X, alpha 1 Myh7 Myosin, heavy polypeptide 7, cardiac muscle, beta Comp Cartilage oligomeric matrix protein Myl3 Myosin, light polypeptide 3 Cpa1 Carboxypeptidase A1 Nanog Nanog homeobox Ctsk Cathepsin K Neurod1 Neurogenic differentiation 1 Dcn Decorin Neurog2 Neurogenin 2 Dcx Doublecortin Nkx2-2 NK2 transcription factor related, locus 2 (Drosophila) Dnmt3b DNA methyltransferase 3B Nppa Natriuretic peptide type A Dpp4 Dipeptidylpeptidase 4 Olig2 Oligodendrocyte transcription factor 2 Eno1 Enolase 1, alpha non-neuron Otx2 Orthodenticle homolog 2 (Drosophila) Fabp7 Fatty acid binding protein 7, brain Pdgfra Platelet derived growth factor receptor, alpha poly-peptide Fgf5 Fibroblast growth factor 5 Podxl Podocalyxin-like Foxa1 Forkhead box A1 Pou4f2 POU domain, class 4, transcription factor 2 Foxd3 Forkhead box D3 Pou5f1 POU domain, class 5, transcription factor 1 Foxg1 Forkhead box G1 Prom1 Prominin 1 G6pc Glucose-6-phosphatase, catalytic Ptcra Pre T-cell antigen receptor alpha Gad1 Glutamic acid decarboxylase 1 Rcvrn Recoverin Gad2 Glutamic acid decarboxylase 2 Runx1 Runt related transcription factor 1 Galc Galactosylceramidase Ryr2 Ryanodine receptor 2, cardiac Gata1 GATA binding protein 1 Sftpb Surfactant associated protein B Gata2 GATA binding protein 2 Sftpd Surfactant associated protein D Gata6 GATA binding protein 6 Slc17a6 Solute carrier family 17 (sodium-dependent inor-ganic phosphate cotransporter), member 6 Gbx2 Gastrulation brain homeobox 2 Slc17a7 Solute carrier family 17 (sodium-dependent inor-ganic phosphate cotransporter), member 7 Gdf3 Growth differentiation factor 3 Slc2a2 Solute carrier family 2 (facilitated glucose trans-porter), member 2 Gfap Glial fibrillary acidic protein Slc32a1 Solute carrier family 32 (GABA vesicular trans-porter), member 1 Hand1 Heart and neural crest derivatives expressed transcript 1 Smtn Smoothelin Hand2 Heart and neural crest derivatives expressed transcript 2 Sox17 SRY-box containing gene 17 Hes5 Hairy and enhancer of split 5 (Drosophila) Sox2 SRY-box containing gene 2 Hnf4a Hepatic nuclear factor 4, alpha Sox7 SRY-box containing gene 7 Ibsp Integrin binding sialoprotein T Brachyury Igf2 Insulin-like growth factor 2 Tat Tyrosine aminotransferase Ins2 Insulin II Tyr Tyrosinase Itgb4 Integrin beta 4 Zfp42 Zinc finger protein 42 Krt10 Keratin 10 Zic1 Zinc finger protein of the cerebellum 1 Table 1: Gene symbols and names according to Mouse Cell Lineage Identification RT2 Profiler PCR Array 129Isolation of live cells from different mice tissues up to nine days after death whole cadavers and in 1 out of 3 samples stored as tissue pieces. Cells from hippocampus tissue were obtained in 1 out of 3 samples stored as whole cadavers and in 1 out of 3 samples stored as tissue pieces. Cells from subventricular zone were obtained in 1 out of 3 samples stored as whole cadavers. Nine days after death live cells were obtained in one sample from hippocampus tissue of one mouse, stored as a cadaver at 4°C. No difference in cell isolation at any time point was observed in regard whether tissue was obtained from the cadavers, or was stored as tissue pieces in PBS. Results of successful harvesting of cells from tissue explants are presented in table 2. Figure 1: Cell isolation from muscle tissue stored at 4°C for 3 days, 17 days after seeding. A: cells coming out of muscle tissue explant. B: The same image at larger magnification Figure 2: Cell isolation from brain tissue - hippocampus, stored at 4 °C for 3 days, 15 days after seeding. A: cells coming out of hippocampus tissue explant. B: The same image at larger magnification 130 M. Voga, A. Pleterski, G. Majdič Figure 3: Cell isolation from brain tissue - subventricular zone, stored at 4 °C for 3 days, 15 days after seeding. A: cells coming out of brain tissue explant. B: The same image at larger magnification Days Tissue 0 3 6 9 Tissue pieces stored in PBS 4°C Muscle tissue 3/3 3/3 1/3 0/3 Adipose tissue 3/3 0/3 0/3 0/3 Subventricular zone 3/3 3/3 0/3 0/3 Hippocampus 3/3 3/3 1/3 0/3 Room temperature Muscle tissue 0/3 0/3 0/3 Adipose tissue 0/3 0/3 0/3 Subventricular zone 0/3 0/3 0/3 Hippocampus 0/3 0/3 0/3 Stored cadavers 4°C Muscle tissue 1/3 2/3 0/3 Adipose tissue 0/3 0/3 0/3 Subventricular zone 3/3 1/3 0/3 Hippocampus 3/3 1/3 1/3 Room temperature Muscle tissue 0/3 0/3 0/3 Adipose tissue 0/3 0/3 0/3 Subventricular zone 0/3 0/3 0/3 Hippocampus 0/3 0/3 0/3 Table 2: Number of samples from which cells were obtained regarding post mortem time points of tissue explantion, tissue type, storage temperature and form of tissue storage mRNA expression in isolated cells Mouse Cell Lineage Identification RT2 Profiler PCR Array was used to profile the expression of 84 key genes for cellular differentiation with positive PCR controls included. Genes, for which Ct value was 35 or lower, were marked as genes expressed. In muscle tissue, mesoderm germ layer markers Dcn, Gata2, Pdgfra and Runx1 were expressed. Further, Cd79a, Ptcra and Cd34, mesoderm progenitor markers of early B and T cells and muscle stem cells were expressed, respectively. Of mesoderm terminal differentiation markers, genes encoding smooth muscle cells, osteoclasts and 131Isolation of live cells from different mice tissues up to nine days after death GENE ORIGIN GENE SYMBOL Pluripotency markers Pluripotency markers Dnmt3b, Gdf3 (Vgr - 2), Lefty1, Nanog, Podxl, Pou5f1 (Oct4), Zfp42 Germ layers Ectoderm Fgf5, Foxd3, Otx2, Zic1 Neuroectoderm Gbx2, Neurog2 Mesoderm Bmp4, Cd34, Dcn, Gata2, Hand1, Igf2, Mixl1, Pdgfra, Runx1, Brachyury Endoderm Foxa1, Gata1, Gata6, Hnf4a, Sox17, Sox7 Ectoderm progenitors Neuronal Progenitors Fabp7, Hes5, Prom1, Sox2 Immature Neurons Dcx Immature GABA Neurons Gad2, Slc32a1 Limbal Progenitors Eno1, Msln Motor Neuron Progenitors Foxg1, Olig2 Oligodendrocyte Progenitors Nkx2-2, Olig2 Mesoderm progenitors Early Cardiomyocytes Hand2 Early B Cells Cd79a Early T Cells Cd3e, Ptcra Muscle Stem Cells Cd34 Endoderm Progenitors Pancreatic Islet Cells Krt19 Hepatic Stem Cells Apoh, Dpp4, Map3k12 Ectoderm Terminal Differentiation Markers Keratinocytes Krt10, Krt14 Melanocytes Tyr Mature Neurons Neurod1 Cholinergic Neurons Chat GABA Neurons Gad1 Glutamatergic Neurons Slc17a6, Slc17a7 Astrocytes Galc, Gfap Ganglion Cells Pou4f2 Photoreceptor Cells Rcvrn Mesoderm Terminal Differentiation Markers Skeletal Muscle Cells Myh1 Smooth Muscle Cells Myh11, Smtn Cardiomyocytes Myl3, Myh7, Nppa, Ryr2 Osteoblasts Ibsp Osteoclasts Ctsk Chondrocytes Col10a1, Comp Macrophages Ccr5 Endoderm Terminal Differentiation Markers Hepatocytes Alb, G6pc, Tat Cholangiocytes Itgb4 Beta Cells Ins2, Slc2a2 Exocrine Cells Cpa1 Lung Cells Sftpb, Sftpd Proximal Tubule Cells Aqp1, Miox Table 3: Genes expressed in cells isolated from muscle and brain tissue. Genes for which Ct value was 35 or lower, were marked as genes expressed. Genes expressed in cells isolated from muscle tissue are marked in purple. Genes expressed in cells isolated both from muscle and brain tissue are highlighted in blue 132 M. Voga, A. Pleterski, G. Majdič chondrocytes were expressed. In addition to genes of mesodermal lineages, some genes of ectoderm and endoderm lineages were also expressed (Zic1, Gata6, Gad2, Eno1, Apoh, Map3k12, Galc, Rcvrn and Tat). In brain tissue, only expression of two genes was detected. In subventricular zone, gene Eno1, an ectoderm progenitor marker of limbal progenitors was expressed and in hippocampus, gene Rcvrn, an ectoderm terminal differentiation marker of photoreceptor cells was expressed. Results of gene expression from muscle and brain tissue derived cells are presented in table 3. Discussion In the present study, we examined the differences between time after death, storage temperature and form of tissue storage in obtaining live cells from mice post mortem. Results of our study suggest that cells from muscle and brain tissue can be isolated from murine cadavers or tissue pieces up to 3 days post mortem and with difficulty up to 6 days post mortem. Cells from brain tissue can possibly be isolated even up to 9 days post mortem. Several other studies have also shown that cells from brain and muscle tissue can be isolated from cadavers from different species after different post mortem periods. The longest post mortem time period that still allows for cell isolation from muscle tissue was reported by Latil et al. (12) who showed that viable and functional murine skeletal myogenic cells can be isolated up to 14 days post mortem. Interestingly, this is much longer time period in which cells were isolated post mortem compared to our study. But similarly as in our study, it was shown by Xu et al. (7) that neural stem cells of subventricular zone from deceased rats can also be isolated for up to 6 days post mortem, but not longer, in young rats. Time period in which cells were isolated was longer in young in comparison to old rats. In our study adult mice were used. It is possible that mice age in our study shortened the post mortem time periods in which cells were isolated as in most previous studies including study by Latil et al. (12) younger mice were used. Contrary to rodent neuronal stem cells, cells from human brain tissue are reported to be able to survive only up to 36 hours post mortem (15, 16), which suggests that post mortem time periods of neuronal stem cells from rodents might be longer than that of humans. This suggests that for some cell types post mortem time period of cell isolation might be species specific. Contrary, some studies indicate that post mortem time period might also be tissue specific. For example, it was shown by Latil et al. (12) that the longest post mortem time period of cell isolation from human muscle tissue was 17 days, which means it was not only similar to but it even exceeded the time period in which murine cells were isolated. Furthermore, it was shown by Erker et al. (5) that post mortem time periods of isolated hepatocytes do not differ between murine, rhesus macaque monkeys and humans. The lack of studies to compare post mortem isolation of cells from different species and tissues and different methods used in studies hinder speculation whether certain post mortem time period of cell isolation is species or tissue specific. To our knowledge there are no studies in which comparison of different tissues regarding post mortem cell isolation from one species were studied. In our study, no significant difference was observed regarding post mortem time period for isolation of cells from brain and muscle tissue. In addition to brain, muscle and liver tissue, cells from other tissues have also been isolated post mortem, such as human retinal progenitor cells (immediately post mortem) (17), murine vestibular and cochlear stem cells (up to 10 days post mortem) (6), equine ligament stem cells (up to 72 hours post mortem) (11) and bovine skin fibroblast like cells (up to 49 days post mortem) (10). Interestingly there are no reports of adipose tissue derived cells isolated post mortem, although adipose tissue is an excellent source of mesenchymal stem cells/multipotent mesenchymal stromal cells in different species. In our study adipose tissue yielded cells only immediately post mortem, but no cells were isolated at other time points after death, suggesting that cells in adipose tissue do not survive long after death. One possible reason for unsuccessful harvesting of adipose derived cells from mice post mortem in our study could be unsuitable anatomical site from which adipose tissue was obtained, since it was shown that there are intrinsic differences in adipocyte precursor cells from different white fat depots in mice (18) and that the concentrations of adipose derived stem cells from human cadavers differ between sites (19). Several studies have also shown that anatomical site of adipose tissue harvesting from live organisms is an important factor in terms of 133Isolation of live cells from different mice tissues up to nine days after death differentiation potential, viability, yield and stem cell capacity (20–23), although there are no studies available comparing adipose tissue harvesting from different anatomical sites from BALB/C mice. Effect of different anatomical origins of adipose tissue on stem cell features could also reflect in the possibility of cells being harvested post mortem, although no studies are available on this topic. Another reason for unsuccessful obtaining of adipose derived cells could be in the adipose tissue differences between inbred mouse strains, as was shown in a study by Mo et al. (24), who compared the frequency of proliferative stromal cells in 129x1/svj and C57Bl/6J mice. But comparison of adipose derived cell characteristics between BALB/C and other mouse strains is yet to be studied. In the present study, cells from muscle and brain tissues were isolated only when tissue pieces or cadavers were stored at 4°C. No cells were obtained from cadavers or tissue pieces when stored at room temperature, suggesting that low temperatures preserve the cells in cadavers or tissue pieces while higher (room) temperatures promote cell death. In most studies where post mortem cell isolation was examined, cells were harvested immediately after death for donor transplantation procedures (2–4, 25), or after longer periods of time after cadavers were stored at 4°C (5, 7). Interestingly, in some cases, where cells were successfully isolated from murine inner ear and human muscle tissue or arteries long after death, cadavers were kept at room temperature from 6 to 12 hours before they were stored at lower temperatures for later harvesting of cells (6, 12, 13), suggesting that shorter period at room temperature is compatible with obtaining viable cells while longer periods (as in our study) decrease the viability of cells in cadavers. Interestingly, in a recent study fibroblast - like cells were recovered up to 15- and 49-days postmortem from bovine skin stored at 25°C and 4°C, respectively (10). Although we were unable to obtain live cells when cadavers or tissue pieces were stored at room temperature, it seems that in some cases, stem cells can survive even after cadavers are stored for certain time periods at room temperature. In most of the studies where cells were harvested at certain time points after death, animal or human subjects were stored in cadaveric form. In others, cadavers were dissected prior to experiment and tissue pieces were stored. There are, however, no studies in which affect of form of storage on cell isolation was investigated in the same study. In our study, no difference in cell isolation between two forms of storage was observed. To find out which cells were isolated in our study, we used Mouse Cell Lineage Identification RT2 Profiler PCR Array that profiles the expression of 84 key genes for cellular differentiation. Array contains gene markers for specific cell types throughout cellular lineage progression, including pluripotent stem cells, progenitor cells from each of the three germ layers, and terminally differentiated cells. Results from qPCR suggests that from muscle and brain tissue, cells with characteristics of germ layer cells, progenitor cells and terminally differentiated cells were isolated. Cells obtained from brain tissue seem to be ectodermal progenitors and ectodermal terminally differentiated cells. In cells isolated from muscle tissue there was the strongest expression of markers for mesoderm germ layer, mesoderm progenitors and mesoderm terminally differentiated cells. However, cells isolated from muscle tissue also expressed some genes from ectodermal and endodermal lineages, possibly suggesting that heterogenous population of cells was obtained from muscle tissue. In general expression of all markers was low, possibly due to low amount of RNA obtained from the cells, and genes that were expressed in these cells were heterogenous. Therefore, it was not possible to exactly determine the lineage of cells obtained from dead mice, possibly suggesting that different types of cells were obtained, and further studies will be needed to more carefully examine the lineage and characteristic of cells obtained post mortem. Despite the fact that heterogenic lineage population of cells from muscle tissue was isolated, it was concluded that, regardless of their origin, cells can be readily isolated from muscle and brain tissue 3 days post mortem. Although with difficulty, cells from muscle and brain tissue can also be isolated up to 6 days post mortem, and possibly even up to 9 days post mortem from brain tissue. Our results are consistent with results of other studies that showed that cells survive in the dead organisms for longer time after death than it was previously thought. In majority of the studies focusing on post mortem cell isolation, stem cells were obtained. It is presumed that, for example in satellite cells, the lack of oxygen, nutrients, or the 134 M. Voga, A. Pleterski, G. Majdič the Republic of Slovenia and were done according to ethical principles, EU directive (2010/63/EU), and NIH guidelines. All authors declared that they have no competing interests. MV and AP performed the experiments. GM planned the experiments and analyzed the data together with MV and AP. MV and GM drafted the manuscript, which was edited and approved by all authors. References 1. Leming MR, Dickinson GE. Understanding dy- ing, death, and bereavement. 4th. ed. Fort Warth : Harcourt Brace College Publishers, 1998: 518 str. 2. Blazar BR, Lasky LC, Perentesis JP, et al. Suc- cessful donor cell engraftment in a recipient of bone marrow from a cadaveric donor. Blood 1986; 67(6): 1655–60. doi: 10.1182/blood.V67.6.1655.1655 3. Ciancio G. Donor bone marrow infusion in cadaveric renal transplantation. Transplant Proc 2003; 35(2): 871–2. doi: 10.1016/s0041- 1345(02)04034-4 4. Kapelushnik J, Aker M, Pugatsch T, Samuel S, Salvin S. Bone marrow transplantation from a cadaveric donor. Bone Marrow Transplant 1998; 21: 857–8. doi: 10.1038/sj.bmt.1701165 5. Erker L, Azuma H, Lee AY, et al. Therapeutic liver reconstitution with murine cells isolated long after death. Gastroenterology 2010; 139(3): 1019– 29. doi: 10.1053/j.gastro.2010.05.082 6. Senn P, Oshima K, Teo D, Grimm C, Heller S. Robust postmortem survival of murine vestib- ular and cochlear stem cells. J Assoc Res Otolar- yngol 2007; 8(2): 194–204. doi: 10.1007/s10162- 007-0079-6 7. Xu Y, Kimura K, Matsumoto N, Ide C. Iso- lation of neural stem cells from the forebrain of deceased early postnatal and adult rats with pro- tracted post-mortem intervals. J Neurosci Res 2003; 74: 533–40. doi: 10.1002/jnr.10769 8. Silvestre MA, Saeed AM, Cervera RP, Escribá MJ, García-Ximénez F. Rabbit and pig ear skin sample cryobanking: Effects of storage time and temperature of the whole ear extirpated immedi- ately after death. Theriogenology 2003; 59(5/6): 1469–77. doi: 10.1016/s0093-691x(02)01185-8 9. Okonkwo C, Singh M. Recovery of fibrob- last-like cells from refrigerated goat skin up to 41 d of animal death. In Vitro Cell Dev Biol Anim 2015; 51(5): 463–9. doi: 10.1007/s11626-014- 9856-9 presence of extensive necrosis triggers a cellular response in stem cells resulting in their adopting a deeper state of quiescence or dormancy (12). Similarly, the possibility of post mortem neural stem cell isolation is ascribed to low metabolic level of neuronal stem cells and rich vascular bed in subventricular zone and surrounding tissues (7), which could act as a niche for neuronal stem cells (15). Hypoxia, which is prevalent in muscle stem cell niches (27) as well as in niches of other stem cells such as neuronal stem cells (28), is an important factor that contributes to cell viability and regeneration potential that could maintain stem cell viability for unusually long periods in spite of the necrotic microenvironment. Stem cells are thus assumed to be enriched in post mortem tissue due to cellular quiescence where cells adopt a reversible dormant state and thus possess a selective survival advantage compared with other cell types (12). Contrary to these studies we showed, based on gene expression results, that not only stem cells but also terminally differentiated cells survive in post mortem tissues for at least 3 days after death. In conclusion, in this study we showed that (1) cells from murine animals can be isolated from muscle and brain tissue readily 3 days post mortem and with difficulty up to 6 days post mortem. Cells from brain tissue can possibly be isolated even up to 9 days post mortem (2). Compared to muscle and brain tissue no cells were isolated post mortem from adipose tissue except immediately after death (3). In all instances cells were isolated only when tissues were stored at 4 °C. 4) Form of tissue storage does not affect cell isolation (5). Not only stem cells but also terminally differentiated cells seem to survive in post mortem tissues for at least 3 days after death. Acknowledgments This study was supported by ARRS grant P4- 0053 and Metka Voga is supported by ARRS Ph.D. fellowship. We are grateful to Nina Sterman for technical assistance. The datasets supporting the results of this document are contained within the article. Any additional data may be requested to the corresponding author. All animal experiments were approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection of 135Isolation of live cells from different mice tissues up to nine days after death 10. Walcott B, Singh M, Hatti Kaul R. Recovery of proliferative cells up to 15- and 49-day post- mortem from bovine skin stored at 25 °C and 4 °C, respectively. Cogent Biol 2017; 3(1): e1333760. doi: 10.1080/23312025.2017.1333760 11. Shikh Alsook MK, Gabriel A, Piret J, et al. Tissues from equine cadaver ligaments up to 72 hours of post-mortem: a promising reservoir of stem cells. Stem Cell Res Ther 2015; 6: e253. doi: 10.1186/s13287-015-0250-7 12. Latil M, Rocheteau P, Chatre L, et al. Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capac- ity. Nat Commun 2012; 3: e903. doi: 10.1038/ ncomms1890 13. Valente S, Alviano F, Caivarella C, et al. Human cadavermultipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years. Stem Cell Res Ther 2014; 5(1): e8. doi: 10.1186/scrt397 14. Pozhitkov AE, Neme R, Domazet-Loso T, et al. Tracing the dynamics of gene transcripts after organismal death. Open Biol 2017; 7(1): e160267. doi: 10.1098/rsob.160267 15. Palmer TD, Schwartz PH, Taupin P, Kaspar B, Stein SA,Gage FH. Progenitor cells from hu- man brain after death. Nature 2001; 411(6833): 42–3. doi: 10.1038/35075141 16. Schwartz PH, Bryant PJ, Fuja TJ, Su H, O'Dowd DK, Klassen H. Isolation and character- ization of neural progenitor cells from post-mor- tem human cortex. J Neurosci Res 2003; 74(6): 838–51. doi: 10.1002/jnr.10854 17. Klassen H, Ziaeian B, Kirov, II, Young MJ, Schwartz PH. Isolation of retinal progenitor cells from post-mortem human tissue and comparison with autologous brain progenitors. J Neurosci Res 2004; 77(3): 334–43. doi: 10.1002/jnr.20183 18. Macotela Y, Emanuelli B, Mori MA, et al. Intrinsic differences in adipocyte precursor cells from different white fat depots. Diabetes 2012; 61: 1691–9. doi: 10.2337/db11-1753/-/DC1 19. Kishi K, Imanishi N, Ohara H, et al. Distri- bution of adipose-derived stem cells in adipose tis- sues from human cadavers. J Plast Reconstr Aes- thet Surg 2010; 63(10): 1717–22. doi: 10.1016/j. bjps.2009.10.020 20. Prunet-Marcassus B, Cousin B, Caton D, Andre M, Penicaud L,Casteilla L. From heteroge- neity to plasticity in adipose tissues: Site-specific differences. Exp Cell Res 2006; 312(6): 727–36. doi: 10.1016/j.yexcr.2005.11.021 21. Chen L, Peng EJ, Zeng XY, Zhuang QY,Ye ZQ. Comparison of the proliferation, viability, and differentiation capacity of adipose-derived stem cells from different anatomic sites in rabbits. Cells Tissues Organs 2012; 196(1): 13–22. doi: 10.1159/000330796 22. Tsekouras A, Mantas D, Tsilimigras DI, Moris D, Kontos M, Zografos GC. Comparison of the viability and yield of adipose-derived stem cells (ascs) from different donor areas. In Vivo 2017; 31(6): 1229–34. doi: 10.21873/invivo.11196 23. Reumann MK, Linnemann C, Aspera-Werz RH, et al. Donor site location is critical for prolif- eration, stem cell capacity, and osteogenic differ- entiation of adipose mesenchymal stem/stromal cells: Implications for bone tissue engineering. Int J Mol Sci 2018; 19(7): e1868. doi: 10.3390/ ijms19071868 24. Mo J, Srour EF, Rosen ED. The frequency of proliferative stromal cells in adipose tissue varies between inbred mouse strains. J Stem Cells Regen Med 2009; 5(1): 23–9. doi: 10.46582/jsrm.0501005 25. Michalova J, Savvulidi F, Sefc L, Forgacova K, Necas E. Cadaveric bone marrow as potential source of hematopoietic stem cells for transplan- tation. Chimerism 2011; 2(3): 86–7. doi: 10.4161/ chim.2.3.17917 26. Gustafsson MV, Zheng X, Pereira T, et al. Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev Cell 2005; 9(5): 617–28. doi: 10.1016/j.devcel.2005.09.010 27. Mohyeldin A, Garzon-Muvdi T,Quinon- es-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 2010; 7(2): 150–61. doi: 10.1016/j. stem.2010.07.007 136 M. Voga, A. Pleterski, G. Majdič IZOLACIJA ŽIVIH CELIC IZ RAZLIČNIH TKIV MIŠI DO DEVET DNI PO SMRTI M. Voga, A. Pleterski, G. Majdič Izvleček: Nekatere raziskave kažejo, da je preživetje celic v truplih precej daljše, kot je bilo znano do sedaj. V naši raziskavi smo proučevali, kako na uspešnost izolacije živih celic po smrti miši vplivajo različen čas izolacije po smrti, vrsta tkiva (mišično, možgansko in maščobno), temperatura shranjevanja trupel ter oblika shranjenega tkiva (kot koščki tkiv ali kot celi kadavri). Izo- lacija in gojenje celic iz tkiv mrtvih miši sta potekali pod standardnimi pogoji. Da bi ugotovili, katerim celičnim linijam pripadajo izolirane celice, je bil del celic uporabljen za izolacijo RNK in nadaljno uporabo v sistemu identifikacije izvornih celičnih linij z verižno reakcijo s polimerazo v realnem času. Rezultati naše raziskave so pokazali, da je žive celice mogoče izolirati iz mišičnega in možganskega tkiva 3 dni po smrti, pogojno tudi do 6 dni po smrti. Iz možganskega tkiva je bilo žive celice mogoče izolirati tudi do 9 dni po smrti. Iz maščobnega tkiva je bilo celice mogoče izolirati zgolj takoj po smrti, ne pa tudi v kasnejših časovnih interva- lih. V vseh primerih so bile celice izolirane samo v primeru shranjevanja tkiv pri 4°C. Oblika shranjenega tkiva na izolacijo celic ni vplivala. Izolirane celice so pripadale različnim zarodnim plastem. Rezultati raziskave so pokazali, da je žive celice iz mišjih trupel mogoče izolirati tudi več dni po smrti. Ključne besede: miš; truplo; matične celice; možgansko tkivo; mišično tkivo; maščobno tkivo Received: 4 September 2020 Accepted for publication: 7 July 2021 Slov Vet Res 2021: 58 (4): 137 – 45 DOI 10.26873/SVR-1205-2021 UDC 639.21.09:578.27:616.831-002:616-036.2:597.556.331.1 Original Research Article Introduction Viral encephalopathy and retinopathy (VER), also termed as viral nervous necrosis (VNN), is a serious neuropathological disease of more than 50 fish species almost worldwide (1). It occurs mostly in marine environment, but the outbreaks in freshwater fish have also been reported (2, 3, 4). In marine aquaculture, VER is considered one of the most devastating infectious diseases (5), and sea bass (Dicentrarchus labrax) seems to be one of the most commonly and severely THE FIRST OUTBREAK OF VIRAL ENCEPHALOPATHY AND RETINOPATHY IN FARMED SEA BASS (Dicentrarchus labrax) IN SLOVENIA Rosvita Sitar1, Tanja Švara2, Aleksandra Grilc Fajfar3, Sabina Šturm2, Marko Cvetko2, Irena Fonda4, Mitja Gombač2* 1National veterinary institute, Veterinary Faculty, University of Ljubljana, Unit Nova Gorica, Pri hrastu 18, 5000 Nova Gorica, 2Institute of pathology, wild animals, fish and bees, 3Institute of microbiology and parasitology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, 4 Fonda.si d. o. o., Liminjanska cesta 117, 6320 Portorož, Slovenia *Corresponding author, E-mail: mitja.gombac@vf.uni-lj.si Abstract: Viral encephalopathy and retinopathy (VER) is considered a serious disease of several marine fish species, caused by RNA virus belonging to the family Nodaviridae, genus Betanodavirus. The disease is spread almost worldwide and causes significant losses among diseased fish. It is characterised by vacuolation of the central nervous system and the retina. In July 2018, behavioural abnormalities i.e. altered swimming, swirling and vertical floating as well as lethargy and anorexia were observed in farmed sea bass (Dicentrarchus labrax) in the Gulf of Piran (Slovenia), associated with significant mortality. The dis- ease initially occurred in juvenile sea bass, but later market-sized fish also became affected. Diseased fish displayed ocular opac- ity and multifocal skin ulceration on the head. Emaciation in some fish was also evident. Histopathology revealed characteristic vacuolation in the brain and retina. Performing a RT-PCR and RT-qPCR techniques, we have identified and confirmed the pres- ence of betanodavirus nucleic acid in ocular and brain tissues. In addition, concentrations of the causative agent of VER in spleen and kidney did result in significantly higher viral yield than expected. Phylogenetic analysis showed that Slovenian isolate belongs to RGNNV species of betanodaviruses. Based on the clinical signs, gross and typical microscopic lesions and results of molec- ular analyses, we can conclude that farmed sea bass from the Gulf of Piran were affected with VER. To the best of our knowledge, this is the first report of VER in Slovenia. Key words: viral encephalopathy and retinopathy; betanodavirus; sea bass; histopathology; RT-qPCR affected species (6). The disease affects mostly the larval and juvenile stages (1, 7), however, in several fish species, such as sea bass (8, 9) and grouper (Epinephelus septemfasciatus) (10), mass moralities have been also reported in adult and market-sized fish (1). Additionally, clinical signs and mortalities associated with VER were reported in wild fish species (11). The causative agent is small (approximately 25 nm in diameter) spherical non-enveloped RNA virus, belonging to the genus Betanodavirus within family Nodaviridae (1, 12). Based on the phylogenetic analysis of the RNA sequence of the T4 variable region, betanodaviruses have been clustered into four R. Sitar, T. Švara, A. Grilc Fajfar, S. Šturm, M. Cvetko, I. Fonda, M. Gombač138 species, named striped jack nervous necrosis virus (SJNNV), red-spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV) and tiger puffer nervous necrosis virus (TPNNV) (13). It is reported that RGNNV exhibits the widest host range of warm water species (14, 15), including sea bass. Furthermore, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described and reported to infect different fish species in Mediterranean (5, 16, 17, 18). Betanodaviruses have been often detected in apparently healthy wild marine fish (19, 20, 21). VER is characterized by typical changes in swimming pattern associated with affected nervous system (15), such as whirling, spiralling or looping, erratic swimming, lying down on the bottom, keeping vertical positions, lying on their sides or belly up and body curved (8, 9, 10, 22). In addition, lethargy, changes in skin pigmentation, skin erosion in the head region, ocular opacity and exophthalmia have been described (8, 11). Histopathological findings, most commonly characterized by vacuolation and necrosis of nerve cells of the brain, retina and spinal cord, are remarkably consistent among the various affected fish species (15, 23). In this paper we describe the first occurrence of VER in Slovenia, including clinical signs, gross pathology, histopathological lesions and the results of molecular diagnostic procedures. Some epidemiological aspects are also discussed. Case presentation Case history and clinical signs In July 2018, abnormal swimming behaviour associated with heavy mortalities occurred in sea bass reared in floating cages in the Gulf of Piran. Affected fish showed erratic swimming, impulsive movements, swirling, belly up or keeping vertical position with either head or caudal peduncle upside. Some were laying on their sides and body curved. Moreover, lethargy, anorexia, change in skin pigmentation, endo– or exophthalmia, ocular opacity and congestion of the head were observed. The disease initially occurred in older juveniles (125 g) and later market-sized fish became affected. There was no significant mortality observed in younger juveniles (50 g). Sea bream (Sparus aurata) also remained clinically unaffected (Table 1). The marine fish farm concerned is the only one in Slovenia with the annual production of 100 tons and had no history of VER. No vaccination was carried out at that time; the juveniles introduced into the farm in 2017 had already been vaccinated against Listonella anguillarum, but not against other pathogens. In fact, the number of introduced juveniles was higher than in previous years, yet the density was only about 4.7 kg/m3. Otherwise, husbandry practices and epizootiology in the area in the year of the outbreak generally did not differ from previous years. In spring 2018, only sea bream juveniles were introduced into the farm, while the last introduction of sea bass juveniles took place in autumn 2017. At the time of the disease outbreak, the water temperature exceeded 26°C. The outbreak characterized by high losses lasted until the beginning of October 2018. The mortality firstly decreased in the population of juvenile sea bass (125 g) in the middle of September at the water temperature range 23–25°C. About two weeks later at water temperature below 20°C, the disease mitigated in adult sea bass as well (Figure 1). Nevertheless, after the mortality rate in autumn had decreased, abnormal swimming behaviour was still present in some fish and was regularly observed for months. Gross pathology Samples of clinically affected sea bass were collected from various cages of older juveniles Fish species and category Adult sea bass Adult sea bass Juvenile sea bass Juvenile sea bass Juvenile sea bream Juvenile sea bream An average weight on July 1st, 2018 [g] 870 300 125 50 110 20 Cumulative mortality from July 1st to December 31st, 2018 [%] 51 48 45 8 2 4 Table 1: Cumulative mortality, from July 1st to December 31st, 2018, of different fish populations in the fish farm The first outbreak of viral encephalopathy and retinopathy in farmed sea bass in Slovenia… 139 (125 g) and adults (300 g). Seven fish (four older juveniles, three adults) were subjected for necropsy. External examination revealed lesions limited to the head, which consisted of multifocal skin ulceration and congestion, ocular opacity, exophthalmia or endophthalmia (Figures 2a, b). Additionally, one fish was emaciated, and caudal fin erosion was evident in another one (Figures 2a). No lesions were observed with examination of internal organs. 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 N um be r o f d ea d fis h pe r w ee k 24.2 24.7 26.2 27 27 26.7 26.7 28 27 26 25 24 23 22 21 20 19 18 Av er ag e w at er te m pe ra tu re o f t he w ee k (˚ C) The perid of fourteen weeks from July to October 2018 1.7. −7. 7. 8.7. −14 .7. 15.7 .−2 1.7. 22.7 .−2 8.7. 29.7 .−4 .8. 5.8. −11 .8. 12.8 .−1 8.8. 19.8 .−2 5.8. 26.8 .−1 .9. 2.9. −8. 9. 9.9. −15 .9. 16.9 .−2 2.9. 23.9 .−2 9.9. 30.9 .−6 .10. Juvenile sea bass (125g) Adult sea bass (300g) Adult sea bass (870g) 26 23 25.1 25.1 22.8 19.6 18.8 Figure 1: Disease pattern regarding the number of dead fish per week in most affected populations (juvenile sea bass (125 g) and adult sea bass (300 g and 870 g)) of sea bass at different sea temperature (an average weekly temperature) Histopathology Eye and brain samples were fixed in 10% buffered formalin and routinely embedded in paraffin for histopathological examination. Four-μm thick tissue sections were first deparaffinised and then stained with haematoxylin and eosin (HE). Stained sections were examined with a light microscope. Microscopically, characteristic vacuolation in the retina and the brain was observed at different Figure 2: Viral encephalopathy and retinopathy. (a) Gross lesions in- cluded emaciation, ocular opacity and congestion of the head. In one fish, caudal fin erosion was noticed (arrowhead). (b) Close-up of the gross lesions showing ocular opac- ity and congestion on the head. (c) Characteristic vacuolation of the retina. HE. Scale bar: 100 μm; magnification: 100×. (d) Character- istic vacuolation in the neuropil of the brain. HE. Scale bar: 100 μm; magnification: 200× R. Sitar, T. Švara, A. Grilc Fajfar, S. Šturm, M. Cvetko, I. Fonda, M. Gombač140 Figure 3: Agarose gel electrophoresis of RT-PCR positive samples of fish brain (1) and visceral organs (2) tested with F2/R3 primer pair. Reference negative (3) and positive (4) control. The red arrow indicates the expected size for the 427-bp amplicon. M = 100-bp size marker EU236149 JN189922 JN189920 FJ803921 JN189918 KC696562.1 KY354694 DQ116038.1 KX601141.1 MK767010.1 MK766993.1 SLO1-2018 (MW805305) AY510457.1 JN189965 AM085337 FJ789784.1 AY140798.1 EU826183 RGNNV BFNNV TPNNV SJNNV 0.050 98 100 Figure 4: Maximum-likelihood (ML) phylogenetic tree based on partial RNA2 sequences depicting the phylo- genetic relationships within genus Betanodavirus; sub- division of genus Betanodavirus is displayed by labeling the branches with different colors (violet: RGNNV; green: BFNNV; red: TPNNV; blue: SJNNV); ML bootstrap values >60% are reported next to the nodes; scale is shown at the left as substitutions per site. degrees (Figures 2c, d). Brain vacuolation was mostly present in the neuropil and only single vacuoles were found in the neurons. Only in one fish, multifocal mild perivascular lymphocytic infiltrates and gliosis were found in the brain stem. Virological analysis The brain tissue and eyes as well as spleen and kidney were pooled separately and submitted for laboratory viral diagnostics. Fish organ homogenates were screened for betanodavirus by RT-PCR and RT-qPCR following the methods documented by Nishizawa et al. (24) and the World Organisation for Animal Health (OIE) in Manual of Diagnostic Tests for Aquatic Animals (2016) (1). All tissue samples were stored at –75°C for future analysis. Total RNA was extracted from supernatant of the organ homogenates (samples) using QIAamp Viral RNA (Qiagen, Germany). The extraction procedure was performed following the manufacturer’s instructions. The RNA obtained was eluted in RNAse-free water. As a negative control, pool of negative fish tissue was processed alongside the virus isolate. Total RNA was added to a one-step RT-PCR for amplification of the certain genomic region within coat protein gene. A 427 nucleotide (nts) region targeting the T4 variable region of the RNA2 segment from diagnostic cases was amplified (Figure 3). RT-qPCR method was also introduced in the laboratory diagnostics of VER/VNN. A part of the RNA2 segment of viral genome was successfully detected using specific primers and MGB probe following OIE standard protocol (1). Using molecular method RT-PCR we detected the presence of viral nucleic acid of betanodavirus successfully. With the RT-qPCR method, specific viral RNA in clinical samples can be detected quickly and specifically. Ct values obtained ranged from 15.88 (brain and bulbus) to 25.65 (spleen and kidney). Confirmation of the positive results by both molecular methods, specific RT-PCR amplicon length for RNA2 genome region and the Ct values for RT-qPCR, corresponded 100%. The RNA2 segment of Slovenian betanodavirus isolate was sequenced and compared with known isolates from betanodavirus coat protein sequences from different countries and hosts. A partial sequence of coat protein of 286 nts was aligned and used in phylogenetic analysis. Phylogenetic analysis was generated with the program MEGA version 7.0. and employed the Maximum-likelihood method using the Kimura two-parameter model (25) (Figure 4). The significance of the branching order was assessed The first outbreak of viral encephalopathy and retinopathy in farmed sea bass in Slovenia… 141 Table 2: Data related to the 18 betanodavirus isolates investigated in the phylogenetic analysis; abbreviations: unpubl., unpublished; n.d., data not available; t.s., this study by bootstrap resampling of 1000 replicates. Accession numbers of nucleotide sequences for VER/VNN worldwide isolates available at GenBank were cited and listed in Table 2. Based on clinical signs, typical histopatho- logical lesions followed by identification of the causative agent by molecular analysis, VER was diagnosed. Discussion Since late 80’, mass mortalities in farmed sea bass showing abnormal swimming behaviour have been reported from Mediterranean region by several authors (8, 9, 36). In summer 1995, heavy losses associated with nodavirus infection occurred in juvenile and adult sea bass in several marine fish farms in Italy (9) and VER is currently considered to be endemic in Mediterranean basin (5). However, in Slovenia altered swimming behaviour associated with mass mortalities in marine aquaculture fish species had not been observed until recently. Clinical signs as well as gross and histopathological findings in our case were similar to those described by other authors (8, 9, 22, 37, 38). The temperature range at the time of the outbreak in July 2018 was in accordance with an optimal in-vitro growth temperature for betanodavirus species RGNNV at 25–30°C (39), and mortality significantly decreased at water temperature below 20°C. The source of infection in our case is difficult to define, considering that only sea bream was intro- duced into the farm in 2018. Transmission of the disease occurs mainly horizontally through con- taminated water (1), but vertical transmission has also been demonstrated in several fish species (7). Isolate Year of isolation Country of outbreak Reference GenBank accession no. Betanodavirus species GMNNV-Korea unknown Korea Cha et al., unpubl.(26) DQ116038.1 RGNNV 9Gr.A.2012 2012 Greece Bitchava et al., 2019(27) MK767010.1 RGNNV SpPm-IAusc1586.10 2010 Spain Olveira et al., 2013(28) KC696562.1 RGNNV G9508KS 1995 Taiwan Chi et al., 2003(29) AY140798.1 RGNNV 28Gr.A.2013 2013 Greece Bitchava et al., 2019(27) MK766993.1 RGNNV SFRG08/2013BSMu3 2013 Korea Kim et al., unpubl.(30) KX601141.1 RGNNV 570.16.2008c 2008 Italy Panzarin et al., 2012(17) JN189965 RGNNV “1” 2009 Tunisia Chérif et al., 2010(31) FJ789784.1 RGNNV “Redspotted grouper nervous necrosis virus” unknown China Lin et al., unpubl.(32) AY510457.1 RGNNV Sa-I-97c 1997 Italy Toffolo et al., 2007(33) AM085337 RGNNV VNNV/S. aurata/I/425-10/Sep2008 2008 Italy Toffan et al., 2017(5) KY354694 RGNNV SpSa-IAusc156.03c 2003 Larvae 2003 Spain Olveira et al., 2009(16) FJ803921 SJNNV 37.2.2005c 2005 Portugal Panzarin et al., 2012(17) JN189918 SJNNV 250.1.2009c 2009 Cyprus Panzarin et al., 2012(17) JN189920 SJNNV 292.1.2.2009c 2009 Greece Panzarin et al., 2012(17) JN189922 SJNNV BF93Hok unknown Japan Nerland et al., unpubl.(34) EU826138 BFNNV TPKag93 unknown Japan Okinaka, unpubl.(35) EU236149 TPNNV SLO1-2018 2018 Slovenia t.s. MW805305 RGNNV R. Sitar, T. Švara, A. Grilc Fajfar, S. Šturm, M. Cvetko, I. Fonda, M. Gombač142 An additional possibility of transmission of the bet- anodavirus is represented from infected, asymp- tomatic specimens (21, 40). Results of infection trials reported by Castric et al. (41) showed that experimentally infected sea bream with no clinical signs of the disease was able to infect the juvenile sea bass by cohabitation. Furthermore, betanoda- virus was detected in several marine invertebrate species, including Mediterranean mussel (Mytilus galloprovincialis) (42), which is the main cultured mollusc species in Slovenia. Moreover, the mollusc farming area Seča is in the immediate vicinity to the relevant fish farm. Kim et al. (43) confirmed infectivity of either BFNNVs or RGNNVs from shellfish, which may represent a potential risk for transmission of nodaviruses to cultured and wild host species. The ability of the sea bass nodavirus to survive at least one month at 25°C and at least one year at 15°C indicates that once released into the marine environment, it could remain widely spread during either cold or warm seasons (44). Thus, control measures possibly effective in hatch- eries by implementation of proper disinfection pro- cedures followed by introducing of betanodavirus- free broodstock, have limited results in preventing betanodavirus infections of farmed fish exposed to the marine environment in on-growing sites (15). The nucleotide diversity of RGNNV isolates worldwide has been shown to vary depending on host species and environmental conditions (18, 45). Isolates from Italy, Spain, Portugal, Cyprus, Greece and Tunisia represent Mediterranean Basin. Viral isolates collected from certain geographic area are generally similar to each other. In the present study, the most revealing spatial trend was the clear separation of isolates from the same geographical location. According to the genotype and geographical origin, SJNNV isolates within Mediterranean region and Japanese isolate from TPNNV species form two independent clusters. Maximum-likelihood phylogenetic tree based on partial RNA2 sequences determined that the selected isolates within RGNNV species showed spatial correlation. In this study, geographic clustering of the virus isolates from Greece and Italy was observed. It is also important to note that within betanodavirus species RGNNV, closer phylogenetic relatedness of Korean RGNNV isolates with those from Greece and Italy was detected. The determined partial T4 nucleotide sequence of Slovenian isolate showed 88.93 to 100% identity at the nucleotide level to the sequences among selected betanodavirus isolates analysed, highly related to strain RGNNV. The evolutionary analysis showed the phylogenetic relationships of newly characterized Slovenian isolate with the RGNNV species. Interestingly, it also exhibited 100% identity to the virus isolate from China. In this study, the determined partial RNA2 segment sequences representing four major betanodavirus species showed 65.04 to 100% identity at the nucleotide level to the selected sequences of 18 worldwide betanodavirus isolates. In our case high viral load was detected also in spleen and kidney. Retina and central nervous system including the brain and spinal cord are key organs of the infection in which the virus actively replicates. Kidney and spleen are not considered the target organs and therefore not suitable for VER diagnosis, but nevertheless causative agent of the disease can be detected in many organs according to published data (1). Our results revealed that besides bulbus and brain tissue kidney and spleen could also be suitable tissues for analysis. Considering the severity of the disease and based on available data suggesting the immunogenic characteristics of the NNV in sea bass, great effort has been made in vaccine development (46), including attenuated, inactivated, recombinant and DNA vaccines with promising results (47). Recently, an inactivated injectable vaccine against VER caused by RGNNV species for sea bass has been authorised for use in the appointed Mediterranean countries: Spain, Italy, Croatia and Greece (Pharmaq) (48). It is to be administered to fish of a minimum weight of 12 g, and the expected reduce of mortality caused by nodavirus (RGNNV species) in sea bass is up to 12 months post vaccination. In these aspects, we believe that introduction of already vaccinated juveniles into Slovenian marine aquaculture facilities would be strongly recommended. Conclusion VER is one of the most devastating diseases of marine fish species with great impact on marine aquaculture. It is endemic in Mediterranean Basin, but the first outbreak in Slovenia occurred only in 2018. The disease caused increased mortality in juvenile and adult sea bass, which led to final loss of about 50% of affected populations. Phylogenetic analysis of Slovenian RGNNV isolate indicates its The first outbreak of viral encephalopathy and retinopathy in farmed sea bass in Slovenia… 143 close relation to other isolates from Mediterranean Basin. Subsequently, an authorised vaccine for selected Mediterranean countries could reduce the mortality rate and economic losses caused by VER also in sea bass in Slovenia. References 1. Viral encephalopathy and retinopathy. In: OIE manual of diagnostic tests for aquatic animals. 7th ed. Paris : Office International des Epizooties, 2016: Chapter 2.3.12 https://www.oie.int/file- admin/Home/eng/Health_standards/aahm/cur- rent/chapitre_viral_encephalopathy_retinopathy. pdf (June 2020) 2. Bigarré L, Cabon J, Baud M, et al. Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water. J Fish Dis 2009; 32: 667–73. 3. Bovo G, Gustinelli A, Quaglio F, et al. Viral encephalopathy and retinopathy outbreak in fresh- water fish farmed in Italy. Dis Aquat Org 2011; 96: 45–54. 4. Binesh CP. Mortality due to viral nervous ne- crosis in zebrafish Danio rerio and goldfish Caras- sius auratus. Dis Aquat Org 2013; 104: 257–60. 5. Toffan A, Pascoli F, Pretto T, et al. Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterra- nean aquaculture. Sci Rep 2017; 7: e46755. doi: 10.1038/srep46755 6. Toffan A. Viral encephalopathy and retinopa- thy. In: MedAID H2020 project blog. Mediterranean Aquaculture Integrated Development, 2018. http:// www.medaid-h 2020.eu/index.php/2018/09/06/ viral-encephalopathy-and-retinopathy/ (June 2020) 7. Munday BL, Kwang J, Moody N. Betanoda- virus infections of teleost fish: a review. J Fish Dis 2002; 25: 127–42 8. Le Breton A, Grisez L, Sweetman J, Ollevier F. Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax (L.). J Fish Dis 1997; 20: 145–51. 9. Bovo G, Nishizawa T, Maltese C, et al. Viral encephalopathy and retinopathy of farmed marine fish species in Italy. Virus Res 1999; 63: 143–6. 10. Fukuda Y, Nguyen HD, Furuhashi M, Nakai T. Mass mortality of cultured sevenband grouper, Epinephelus septemfasciatus, asso- ciated with viral nervous necrosis. Fish Pathol 1996; 31: 165–70. 11. Vendramin N, Patarnello P, Toffan, et al. Viral encephalopathy and retinopathy in groupers (Epinephelus spp.) in southern Italy: a threat for wild endangered species? BMC Vet Res 2013; 9: e20. doi: 10.1186/1746-6148-9-20 12. Mori KI, Nakai T, Muroga K, Arimoto M, Mushiake K, Furusawa I. Properties of a new vi- rus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 1992; 187: 368–71. 13. Nishizawa T, Furuhashi M, Nagai T, Nakai T, Muroga K. Genomic classification of fish noda- viruses by molecular phylogenetic analysis of the coat protein gene. Appl Environ Microbiol 1997; 63: 1633–6. 14. Low CF, Syarul Nataqain B, Chee HY, Ro- zaini MZH, Najiah M. Betanodavirus: dissection of the viral cycle. J Fish Dis 2017; 40: 1489–96. 15. Doan QK, Vandeputte M, Chatain B, Morin T, Allal F. Viral encephalopathy and retinopathy in aquaculture: a review. J Fish Dis 2017; 40: 717–42. 16. Olveira JG, Souto S, Dopazo CP, Thiéry R, Barja JL, Bandín I. Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment. J Gen Virol 2009; 90: 2940–51. 17. Panzarin V, Fusaro A, Monne I, et al. Mo- lecular epidemiology and evolutionary dynamics of betanodavirus in southern Europe. Infect Genet Evol 2012; 12: 63–70. 18. Toffan A, Panzarin V, Toson M, Cecchettin K, Pascoli F. Water temperature affects pathoge- nicity of different betanodavirus genotypes in ex- perimentally challenged Dicentrarchus labrax. Dis Aquat Org 2016; 119: 231–8. 19. Barker DE, MacKinnon AM, Boston L, et al. First report of piscine nodavirus infecting wild win- ter flounder Pleuronectes americanus in Passama- quoddy Bay, New Brunswick, Canada. Dis Aquat Org 2002; 49: 99–105. 20. Gomez DK, Sato J, Mushiake K, Isshiki T, Okinaka Y, Nakai T. PCR-based detection of bet- anodaviruses from cultured and wild marine fish with no clinical signs. J Fish Dis 2004; 27: 603–8. 21. Giacopello C, Foti M, Bottari T, Fisichella V, Barbera G. Detection of viral encephalopathy and retinopathy virus (VERV) in wild marine fish spe- cies of the South Tyrrhenian Sea (Central Mediter- ranean). J Fish Dis 2013; 36: 819–21. 22. Grotmol S, Totland GK, Thorud K, Hjeltnes BK. Vacuolating encephalopathy and retinopathy R. Sitar, T. Švara, A. Grilc Fajfar, S. Šturm, M. Cvetko, I. Fonda, M. Gombač144 associated with a nodavirus-like agent: a probable cause of mass moirtality of cultured larval and ju- venile Atlantic halibut Hippoglossus hippoglossus. Dis Aquat Org 1997; 29: 85–97. 23. Munday BL, Nakai T. Special topic review: nodaviruses as pathogens in larval and juvenile marine finfish. World J Microbiol Biotechnol 1997; 13: 375–81. 24. Nishizawa T, Mori KI, Nakai T, Furusawa I, Muroga K. Polymerase chain reaction (PCR) ampli- fication of RNA of striped jack nervous necrosis vi- rus (SJNNV). Dis Aquat Org 1994; 18: 103–7. 25. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16: 111–20. 26. Cha SJ, Do JW, Park JW. Coat protein of a Korean isolate of fish nodavirus from grey mullet (Mugil cephalus). GenBank: DQ116038.1. Bethesda : National Center for Biotechnology In- formation, U.S. National Library of Medicine, 30. Jul. 2005. https://www.ncbi.nlm.nih.gov/nuc- core/DQ116038.1 (Feb. 2019) 27. Bitchava K, Chassalevris T, Lampou E, Athanassopoulou F, Economou V, Dovas CI. Oc- currence and molecular characterization of betan- odaviruses in fish and invertebrates of the Greek territorial waters. J Fish Dis 2019; 42: 1773–83. 28. Olveira JG, Souto S, Dopazo CP, Bandin I. Isolation of betanodavirus from farmed turbot Psetta maxima showing no signs of viral enceph- alopathy and retinopathy. Aquaculture 2013; 406/407: 125–30. 29. Chi SC, Shieh JR, Lin SJ. Genetic and an- tigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Dis Aquat Org 2003; 55: 221–8. 30. Kim YC, Jeong HD. Highly frequent identifi- cation of betanodavirus BFNNV as well as RGNNV genotype in shellfish. GenBank: KX575831.1. Bethesda : National Center for Biotechnology In- formation, U.S. National Library of Medicine, 29. Oct. 2016. https://www.ncbi.nlm.nih.gov/nuc- core/KX575831.1 (Feb. 2019) 31. Cherif N, Gagne N, Groman D, Kibenge F, Iwamoto T, Yason C, Hammami S. Complete se- quencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA-dependent RNA polymerase gene. J Fish Dis 2010; 33: 231–40. 32. Lin L, Huang J, Weng S, He J. Partial capsid protein gene of red-spotted grouper nervous necro- sis virus in Guangdong province, P. R. China. Gen- Bank AY510457.1. Bethesda : National Center for Biotechnology Information, U.S. National Library of Medicine, 26. Jul. 2016. https://www.ncbi.nlm. nih.gov/nuccore/AY510457.1 (Feb. 2019) 33. Toffolo V, Negrisolo E, Maltese C, et al. Phy- logeny of betanodaviruses and molecular evolution of their RNA polymerase and coat proteins. Mol Phylogenet Evol 2007; 43: 298–308. 34. Nerland AH, Oevergaard A-C, Patel S, Nishizawa T. Complete sequence of RNA1 and RNA2 from the nodavirus strain BF93Hok isolated from barfin flounder, Verasper moseri at Hokkiado, Japan. GenBank: EU826138. Bethesda : National Center for Biotechnology Information, U.S. Na- tional Library of Medicine, 20. Jul. 2008. https:// www.ncbi.nlm.nih.gov/nuccore/EU826138 (Feb. 2019) 35. Okinaka Y. Comparison among the com- plete genomes of the four types of Betanodaviruses. GenBank: EU236149. Bethesda : National Cen- ter for Biotechnology Information, U.S. National Library of Medicine, 1. Nov. 2009. https://www. ncbi.nlm.nih.gov/nuccore/EU236149 (Feb. 2019) 36. Breul G, Bonami JR, Pepin JF, Pichot. Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea-bass (Dicen- trarchus labrax) larvae and juveniles. Aquaculture 1991; 97: 109–16. 37. Lopez-Jimena B, Garcia-Rosado E, Thomp- son KD, et al. Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in ner- vous and non-nervous organs of European seabass (Dicentrarchus labrax) during the course of an ex- perimental challenge. J Vet Sci 2012; 13: 355–62. 38. Pascoli F, Serra M, Toson M, Pretto T, Tof- fan A. Betanodavirus ability to infect juvenile Eu- ropean sea bass, Dicentrarchus labrax, at different water salinity. J Fish Dis 2016; 39: 1061–8. 39. Iwamoto T, Nakai T, Mori K, Arimoto M, Fu- rusava I. Cloning of the fish cell line SSN-1 for pi- scine nodaviruses. Dis Aquat Org 2000; 43: 81–9. 40. Terlizzi A, Tedesco P, Patarnello P. Spread of pathogens from marine cage aquaculture: a potential threat for wild fish assemblages under protection regimes? In: Carvalho ED, David GS, Silva RJ, eds. Health and environment in aquacul- ture. Rijeka : IntechOpen, 2012: 403–14. https:// www.intechopen.com/books/health-and-environ- ment-in-aquaculture (Feb. 2019) 41. Castric J, Thiéry R, Jeffroy J, de Kinkelin P, Raymond JC. Sea bream Sparus aurata, an The first outbreak of viral encephalopathy and retinopathy in farmed sea bass in Slovenia… 145 asymptomatic contagious fish host for nodavirus. Dis Aquat Org 2001; 47: 33–8. 42. Gomez DK, Baeck GW, Kim JH, Choresca Jr CH, Park SC. Molecular detection of betanoda- viruses from apparently healthy wild marine in- vertebrates. J Invertebr Pathol 2008; 97: 197–202. 43. Kim YC, Kwon WJ, Kim MS, Kim KI, Min JG, Jeong HD. High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red-spotted grouper nervous necrosis virus geno- type in shellfish. J Fish Dis 2018; 41: 233–46. 44. Frerichs GN, Tweedie A, Starkey WG, Rich- ards RH. Temperature, pH and electrolyte sensi- tivity, and heat, UV and disinfectant inactivation of sea bass (Dicentrarchus labrax) neuropathy no- davirus. Aquaculture 2000; 185: 13–24. 45. Vendramin N, Toffan A, Mancin M, et al. Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass, Dicentrarchus labrax (L.). J Fish Dis 2014; 37: 371–83. 46. Miccoli A, Saraceni PR, Scapigliati G. Vac- cines and immune protection of principal Medi- terranean marine fish species. Fish Shellfish Im- munol 2019; 94: 800–9. 47. Gonzales-Silvera D, Guardiola FA, Es- pinosa C, Chavez-Pozo E, Esteban MÁ, Cuesta A. Recombinant nodavirus vaccine produced in bacteria and administrated without purification elicits humoral immunity and protects European sea bass against infection. Fish Shellfish Immu- nol 2019; 88: 458–63. 48. Pharmaq. Pharmaq has received market- ing authorizations (MA) for Nodavirus vaccine for European sea bass in Spain, Italy, Croatia and Greece. Oslo : Pharmaq, 2016. https://www. pharmaq.no/updates/pharmaq-has-rec/ (June, 2020) PRVI IZBRUH VIRUSNE ENCEFALOPATIJE IN RETINOPATIJE PRI GOJENIH BRANCINIH (Dicentrarchus labrax) V SLOVENIJI R. Sitar, T. Švara, A. Grilc Fajfar, S. Šturm, M. Cvetko, I. Fonda, M. Gombač Izvleček: Virusna encefalopatija in retinopatija (VER) je nevarna bolezen številnih vrst morskih rib, ki jo povzroča nevrotropni RNA virus iz družine Nodaviridae, rod Betanodavirus. Bolezen je razširjena skoraj po vsem svetu in povzroča visok pogin okuže- nih rib. Zanjo so značilne vakuole v centralnem živčnem sistemu in retini. Konec julija 2018 so v ribogojnici v Piranskem zalivu pri brancinih opazili nepravilno plavanje, vrtenje in postavljanje v vertikalno smer ter letargijo in neješčnost, brancini so množično poginjali. Bolezen se je najprej pojavila pri mladicah, nato tudi pri konzumnih kategorijah brancinov. Obolele ribe so imele sivo- motna očesna zrkla ter multifokalne kožne razjede na glavi, posamezne so bile shujšane. S histopatološko preiskavo smo ugo- tovili značilne vakuole v možganih in retini. Z molekularnima metodama RT-PCR in RT-qPCR smo potrdili prisotnost nukleinske kisline betanodavirusa v očesnem zrklu in možganih. Koncentracije virusa, ki so bile signifikantno višje od pričakovanih, smo ugotovili tudi v vranici in ledvicah. Na podlagi kliničnih znakov, makroskopskih in tipičnih histopatoloških sprememb ter rezultatov molekularnih preiskav lahko zaključimo, da so gojeni brancini v ribogojnici v Piranskem zalivu zboleli za VER. Opisani izbruh je prvi potrjeni primer te bolezni v Sloveniji. Ključne besede: virusna encefalopatija in retinopatija; betanodavirus; brancin; histopatologija; RT-qPCR Received: 5 July 2020 Accepted for publication: 12 November 2021 Slov Vet Res 2021: 58 (4): 147 – 53 DOI 10.26873/SVR-1356-2021 UDC 599.742.734:591.421:572.7:591.8 Original Research Article Introduction The Euroasian lynx (Lynx lynx) is an endangered species of wild animal (1). The species features of the lynx are medium size, a small head, a prominent ruff of fur, ears tipped with tufts of black hair, and long legs relative to its body length. The literature reports that the most distinguishing anatomical characteristic of these wild animals is the absence of a set upper premolar (2). The planum nasale, which comes from a Latin word meaning the tip of the nose, is a well-studied anatomical and histological structure in various MORPHOLOGY AND HISTOLOGY OF THE EURASIAN LYNX (Lynx lynx) PLANUM NASALE Hasan Hüseyin Ari1,3, Sema Uslu2* 1Departments of Anatomy, 2Departments of Histology and Embryology, Faculty of Veterinary Medicine, Sivas Cumhuriyet University, Sivas, Turkey, 3Departments of Anatomy, Faculty of Veterinary Medicine, Kyrgyz-Turkish Manas University, Bishkek, Kyrgyzstan Republic *Corresponding author, E-mail: semauslu43@hotmail.com Abstract: This study reveals the macroscopic and microscopic structures of the Eurasian lynx planum nasale using materials from three dead females obtained from the Sivas Forestry Branch of Agriculture and Forestry Ministry of the Republic of Turkey. To accomplish the purpose, planum nasale was investigated using macroscopic, histological, and scanning electron microscopy (SEM) techniques. The microscopic examination showed that the planum nasale consists of hairless, moist, glabrous skin and resembles a ship anchor with arm, palm, stock, and sickle parts. The planum nasale’s surface is formed by epidermal plates or epidermal ridges, which were separated from each other by primary and secondary fissures showed in SEM and macroscopic figures. Based on the microscopic examination, the Mercel’s cells and nerve ends are located in the basal sheet of the planum na- sale’s epidermal layers. In addition, the pores situated on the surface of the epidermal ridges and the dense connective bundles were settled in the dermal layers, based on the SEM examination. Key words: Eurasian lynx (Lynx rufus); morphology; nasal plane; planum nasale species (3, 4, 5, 6, 7). This anatomical structure is called the nasal plane (planum nasale) in carnivores and small ruminants but the nasolabial plane (planum nasolabiale) in large ruminants and the rostral plane (planum rostrale) in swine. The nasal plane is macroanatomically formed by glabrous skin and philtrum, different from other regions of the head (8, 9, 10, 11). The skin of the nasal plane includes medial nasal wings, which are differentiated for adaptation to different environments. The surface of the nasal plane skin exhibits a unique morphology that includes papillae or epidermal ridges (4, 5). The anatomical structure consists of the epidermal ridges, which are used as a nasal print (5, 12) because of the individual animal’s characteristics throughout H. H. Ari, S. Uslu148 life (13, 14). Histologically, the planum nasale’s skin consists of an epidermis and a dermis. In addition, Esrah (5) reported that skeleton muscle exists just below the dermis. The morphology of the planum nasale has been compared between various species for many years. Despite several current studies on the anatomical structures of the Eurasian lynx (1, 15), to our knowledge, no anatomical descriptions of the nasal plane in the Eurasian lynx are available, so this study could present the first anatomical findings on the planum nasale obtained using light and scanning electron microscopy. Materials and methods Gross anatomy The three female Eurasian lynx used in this study died from natural causes (cadaver I was 6,9 kg, cadaver II was 7,6 and cadaver III was 7,2 kg in weight, respectively). The animals were obtained from the Republic of Turkey Ministry of Agriculture and Forestry (Sivas branch) and were immediately transported to the Anatomy Department of the Veterinary Faculty of Sivas Cumhuriyet University (16). Then, we bilaterally inserted a plastic cannula into their common carotid artery and cleaned the arterial system of each cadaver with hydrogen peroxide and water solution. We fixed the cadavers with a 10% formalin solution administered via the plastic cannula. The planum nasale of each cadaver was photographed with a Canon EOS 50D camera. The nomenclature used in this study was adopted from the Nomina Anatomica Veterinaria (17). Light microscopy The tissue samples were fixed in buffered formalin for 24 hr, dehydrated in graded alcohol, and embedded in paraplast to create blocks. Serial sections were cut at 5–6 μm thickness. The sections were stained with a modified version of Mallory’s triple stain for general histological examination (18). Scanning electron microscopy The specimens collected from the planum nasale samples were washed in distilled water and 2.5% glutaraldehyde three times (10 min each), dehydrated in an ascending series of ethyl alcohol solutions (50%, 70%, 90%, and 100% alcohol), dried until they reached the critical point using liquid carbon dioxide, and mounted on metal stubs. The samples were coated with a gold- palladium alloy using a sputtering device; then, we examined and photographed the specimens with a scanning electron microscope (Zeiss, Germany) (10kV) at the Advanced Technology Research Centre of Sivas Cumhuriyet University, Sivas (19). Ethical Statement: This study was approved by the Republic of Turkey Ministry of Agriculture and Forestry (Sivas branch) (09.08.2018-72784983- 488.04-176382). Results Gross morphology The planum nasale of the Eurasian lynx is located around the two nostrils and the middle area of the upper lip. Its skin surface is hairless, glabrous, moist, and grayish-black, and it has a dermatoglyphic pattern consisting of epidermal ridges (see Figure 1). It was divided by a philtrum into two equal halves, around the nostril and only the middle area of the upper lip (see Figure 1). The planum nasale of Eurasian lynx was in the form of a sketchy ship anchor shape consisting of arm, palm, shank, stock, and shackle parts (see Figure 1A). The arm of the ship anchor was directed to the dorsum nasi, while the shank was directed toward the ventral of the planum nasale. The arm of the planum nasale (the arrow in Figure 1A) is located dorsally to the tip of the nose, while its palm (“Pa” in Figure 1A) is situated at the dorsolateral wings of the nose, directed medioventrally. A black dorsal border (the arrow in Figure 1A) was evident between the hairy skin of the dorsum nasi and the arm of the planum nasale. The dorsal border of the planum nasale’s arm was concavely directed toward the dorsum nasi. The width of the planum nasale arm dorsally supported the nostril (“Ns” in Figure 1A) and was wider than the planum nasale’s palm. The planum nasale’s palm (“Pa” in Figure 1A) on both sides situated the lateral wings of the nose and the dorsolateral of the planum nasale. The width of the planum nasale’s arm dorsally surrounding the nostril was wider than the planum nasale’s Morphology and histology of the Eurasian Lynx (Lynx lynx) planum nasale 149 palm. The planum nasale palm on either side was situated around the lateral wings of the nose and dorsolateral limited the nostrils. The lateral border of the convex planum nasale palm firstly began from the dorsal side of the nostril, then continued to the medial side, and finally ended at the ventral side. The lateral and dorsal borders of the planum nasale and philtrum (arrowhead in Figure 1A) were dark-colored glabrous skin, unlike the arm, which comprises glabrous light-colored skin in the central portion planum nasale’s arm. The planum nasale’s shank (“Sh” in Figure 1A) is situated in the area between the left and right nostrils. The philtrum (the arrowhead in Figure 1A) extended from the dorsal midline of the planum nasale’s shank to the planum nasale’s shackle and upper lip. The planum nasale’s stock (“St” in Figure 1A) is located mediolaterally in the ventral portion of the planum nasale, and the ventral part of the planum nasale shank narrows dorsally to the upper lip to become the shackle of the planum nasale (“Shk” in Figure 1A). The skin of the planum nasale’s shackle and the upper lip was dark and is divided into two equal halves by the philtrum. The glabrous skin surfaces of the planum nasale have many moist dermal protuberances and grooves Figure 1A: The macroscopic view of Eurasian lynx Pla- num nasale. (A) A photograph of the planum nasale’s anatomical structures: the arm (Ar), palm (Pa), and dorsal border of the planum nasale (arrow); the nostril (Ns); the philtrum (arrowhead); the shank (Sh); and the shackle (Shk) and stock of the planum nasale (St) (the star in Figure 1A), which were wider and darker in the central area of the planum nasale than at the edges of the planum nasale. Histological Examination: The glabrous skin of planum nasale in the Eurasian lynx histologically consisted of the epidermis (Figure 2A) and dermis (Figure 2A) layers. Deep indentations (Dermal papillae) were seen (Figure 2B) between the epidermis and dermal layers. The epidermis, formed by the hairless keratinized squamous Figure 1B: An SEM image of a planum nasale’s surface, with the epidermal ridges (stars), shallow fissures (arrow- head), secondary fissures (the mark), and small pores (stars). Figure 1C: An SEM image of a planum nasale’s surface and section, with a profound groove (arrows), the epider- mis (arrowheads), the dermis (star), and connective bun- dles (X mark) H. H. Ari, S. Uslu150 epithelium, was composed of the basal, spinous, granular, lucid, and corneum strata in the histological preparations stained with triple stain. In the samples, the basal stratum was formed by single layers of cells located on the basement membrane (arrow in Figure 2C). The mechanoreceptor cells were seen in the basal layer. These cells were thought to have endings in the immediate vicinity (in Figure 2C). The granular layer was composed of two to three cell rows, while the spinous layer consisted of three to five polyhedral cell rows (mark in Figure 2C). The stratum corneum was the most superficial on the epidermis; it is thin and has multiple layers of ceratinocytes. The boundaries of the dermis layer, which has deep and superficial layers, were not evident in the triple-stained preparations. Deep in the dermis were more connective fibers than in the superficial areas. In addition, abundant veins and venules were situated (star in Figure 2D–F) in the dermis layer. Figure 2: The histological view of Eurasian lynx planum nasale Morphology and histology of the Eurasian Lynx (Lynx lynx) planum nasale 151 Scanning electron microscopy examination The SEM surface examination showed many differently sized epidermal ridges in the samples (the stars in Figure 1B), separated from one another by shallow fissures (the arrowheads in Figure 1B). The epidermal ridges were divided into more small domes by secondary fissures (the mark in Figure 1B). The shapes of the epidermal ridges differ from each other, both among samples and between the halves of the same animal specimens. Small pores were situated in the middle of the epidermal ridges (the plus in Figure 1B). The philtrum was a profound groove in the middle line of the samples (the arrows in Figure 1B). The SEM depth module examination of the samples showed that both the philtrum and skin epidermal ridges were composed of epidermal layers (the arrowheads in Figure 1C) and that the dermal layers included dense connective bundles with different sizes and directions (the star and X mark in Figure 1C). Discussion The unique anatomical structures of the skin, such as the planum nasale, form through skin differentiation in various regions. The region, as previously described in ruminants (5, 7) and lemurs (3), is an extensive and easily distinguishable patch of moist, glabrous skin around the nostrils in the Eurasian lynx. In addition, the species’ planum nasale has a dark border on the dorsal, lateral, and ventral sides. On close macroscopic inspection, the planum nasale surface of the Eurasian lynx appears to be composed of packed polygonal plates or epidermal ridges, fissures, and a philtrum, as depicted in carnivores (8, 9), camels (5), and lemurs (3). In addition, the shape of the Eurasian lynx’s planum nasale resembles a ship anchor consisting of arm, palm, shank, stock, and shackle parts. The arm and palm are situated dorsally to the nostrils, while the shank and stock parts are located medially and ventrally, respectively, to the nostrils in the Euroasian lynx. As previously described in ruminants (5, 6, 7, 20) and lemurs (3), the philtrum also divides the planum nasale into two halves in the Eurasian lynx. Moreover, as shown by the gross and SEM samples, in the Eurasian lynx, it has been seen that the philtrum continues to the middle of the upper lip and divides the upper lip. In the macroscopic and SEM examinations, we found that the shapes of the epidermal ridges differ between the planum nasale halves from the same individual and among the planum nasale of different individuals. The findings are similar to descriptions reported of primates by Clifford and Witmer (12) and of cattle by Solis and Maala (14). As described by literature (12, 14), the different shapes of the planum nasale’s skin plates may be used for individual identification of the animals as nose prints. In addition, we observed that the pores are situated on some skin plates. The skin plate is split into multiple secondary skin plates with shallow grooves. We observed that the shapes of the skin plates differed from of camels those photographed with SEM microscopy (5). In agreement with findings from the literature (3, 5), we observed that the histological structure of the planum nasale’s skin consisted of the epidermis and dermal layers. However, the hypodermal layer could not be seen in this study. As reported in the literature (3, 5), a wavy border was seen between the epidermis and dermal layers because of the evident epidermis and dermal papillae in the histological samples. In this study, we saw that the basal sheet of the epidermal layer was formed by a single cell layer, as depicted in Lemur catta (3) and camels (5), whereas our findings of Mercel’s cells in the sheets were only similar to the description by Elofsson et al. (3) in Lemur catta. In addition, as reported by previous studies (3, 5), we observed that the granular sheet was thinner than the spinous sheet. The superficial stratum corneum of the planum nasale in the Eurasian lynx is the same as that of camels (5) and Lemur catta (3), in terms of thickness. Our findings, such as dense connective fibers, rich blood vessels, and dermal papillae in the dermal layers, resemble the statements by Eshrah (5) in camels and Elofsson et al. (3) in Lemur catta. Conclusion The nasal plane or planum nasale of the Eurasian lynx was investigated. In this study, we observed that the Eurasian lynx’s planum nasale consists of glabrous, moist, and hairless skin around nostrils and that its gross morphological shape is in the shape of a ship anchor consisting H. H. Ari, S. Uslu152 of arm, palm, shank, stock, and shackle parts. Both the morphological and SEM investigations show that the planum nasale’s skin is divided by primary and secondary fissures and splinted into epidermal ridges with different sizes and shapes. The different surface shapes of the epidermal ridges in the Eurasian lynx are used for individual identification by nose print, as in other animals. In the histological examination, we saw that the planum nasale’s skin is composed of dermis and epidermal layers. The unmyelinated nerve endings located on the basal sheet of the epidermal layers may be evidence of a sensitive structure. In the SEM examination, we saw small pores on the epidermal ridges and dense bundles in the dermal layers. The dense bundles may create resistance against mechanical effects. Acknowledgments We would like to thank the Republic of Turkey Ministry of Forestry (Sivas Branch) for permission to conduct studies on the Eurasian lynx (Lynx lynx). This study was not supported by any foundation. The authors declare that they have no conflict of interest. Statement of Animal Rights all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References 1. Ozgel O, Aykut M. Macro anatomical investi- gation on ossa membri pelvini of Anatolian bobcat Lynx. Pakistan J Zool 2015; 47: 1492–4. 2. Hansen K. Bobcat: master of survival. Oxford : University Press, 2007. 3. Elofsson R, Tuminaite I, Kröger RHH. A com- plex sensory organ in the nose skin of the promi- sisian primate Lemur catta. J Morphol 2015; 276: 649–76. doi: 10.1002/jmor.20363 4. Elofsson R, Tuminaite I, Kröger RHH. A novel ultrastructure on cornicocyte surface of mammalian nasolabial skin. J Mammal 2016; 95: 1288–94. doi: 10.1093/jmammal/gyw112. 5. Eshrah EA. The camel rhinarium: a study re- vealing the presence of the nasal plane in the drom- edary camel (Camelus dromedarius) with special reference to its epidermal structure. Anat Histol Embryol 2017; 46: 65–72. doi: 10.1111/ahe.12238 6. Maya S, Chungath JJ, Ashok N, Lucy KM, Sreeranjini AR, Indu VR. Comparative morpho-his- tology of muzzle in deer and goat. J Indian Vet As- soc 2015; 12: 46–9. 7. Kalita HC, Kalita PC. Comparative gross an- atomical studies on the muzzle of the mithun (Bos frontalis), yak (Bos grunniens) and zebu (Bos indi- cus). Indian J Anim Res 2004; 38: 150–2. 8. Dyce KM, Sack WO, Wensing CJG. Textbook of veterinary anatomy. Saunders. 1996. 9. Getty R. Sisson and Grossman’s the anatomy of the domestic animals. WB Saunders. 1975. 10. König HE, Liebich HG. Veterinary anatomy of domestic mammals: Textbook and colour atlas. Schattuer. 2004. 11. Nickel R, Schummer A, Seiferle E. The vis- cera of the domestic animals. Verlag Paul Parey. 1973. 12. Clifford AB, Witmer LM. Case study in novel narial anatomy: The enigmatic nose of moose (Artiodactyla: Cervidae Alces alces). Jour- nal of Zoology 2004; 262: 339–60. doi: 10.1017/ S0952836903004692 13. Kozma SM. Feasibility of animal nose print identification using two-dimensional image correla- tion [Unpublished master’s dissertation]. San Diego University, 2004. 14. Solis JA, Maala CP. Muzzle printing as a method for identification of cattle and carboas. Philippine Journal of Medicine 1975; 14: 1–4 15. Arı HH, Kuru N, Uslu S, Özdemir Ö. Mor- phological and histological study on the foot pads of the Anatolian bobcats (Lynx lynx). The Anatomical Record, 2018; 301: 932–8. doi: 10.1002/ar.23761 16. Anonymous. Cumhuriyet University was delivered dead Lynx in Sivas in order to make the post-mortem examination. 2016. www.sivas.or- mansu.gov.tr/sivas/Anasayfa/resimliHaber. 17. International Committee on Veterinary Gross Anatomical Nomenclature. Nomina Anatom- ica Veterinaria. 2012. 18. Bancroft JD. Cook HC. Manual of histologi- cal techniques. Livingstone, 1984. 19. Mahdy MAA, Abdalla KEH, Mohamed SA. Morphological study of the hard palate in the Egyp- tian goat (Capra hircus): A scanning electron micro- scopic study. Anatomia, Histologia, Embryologia, 2018; 47: 391–397. doi: 10.1111/ahe12366 20. Metwally MA, Huissieni HB, Kassab AA, Es- hrah EA. Comparative anatomy of the nasal cavity in the buffaloes, camels and donkeys. Journal of Veterinary Medicine Research 2019; 9: 69–75. Morphology and histology of the Eurasian Lynx (Lynx lynx) planum nasale 153 MORFOLOGIJA IN HISTOLOGIJA SMRČKA EVRAZIJSKEGA RISA (Lynx lynx) H. H. Ari, S. Uslu Izvleček: V študiji so opisane makroskopske in mikroskopske strukture smrčka evrazijskega risa, ki je bila opravljena s proučevanjem tkiv treh mrtvih samic, ki so jih pridobili s pomočjo gozdarske podružnice Sivas Ministrstva za kmetijstvo in goz- darstvo Republike Turčije. Strukturo smrčka so raziskovali z uporabo makroskopskih, histoloških metod ter uporabe vrstičnega elektronskega mikroskopa (SEM). Mikroskopska preiskava je pokazala, da smrček sestavlja brezdlaka, vlažna, gola koža, ki po obliki spominja na ladijsko sidro. Površinski del smrčka tvorijo epidermalne plošče ali grebeni, ki jih ločujejo primarne in sekundarne razpoke, vidne na makroskopskih slikah in s pomočjo vrstične mikroskopije. Na histoloških preparatih so bile v bazalni plasti smrčka epidermisa opazne Merkelove celice in živčni končiči. S pomočjo metode SEM so v plasti epidermisa pokazali pore, ki se nahajajo na površini epidermalnih grebenov in snope togega fibrilarnega veziva, ki segajo v plast dermisa. Ključne besede: Evrazijski ris (Lynx rufus); morfologija; nosna ravnina; smrček Received: 22 April 2021 Accepted for publication: 27 July 2021 Slov Vet Res 2021: 58 (4): 155 – 8 DOI 10.26873/SVR-1331-2021 UDC 636.7.09:616.211:616.99:595.772(497.4) Case Report Introduction The sheep ectoparasite, Oestrus ovis L. (Linnaeus 1758) is commonly known as nasal bot fly of sheep, goats, and other wild ruminants (1), although infestations of dogs (2, 3, 4, 5, 6), humans (7) and a cat (8) have also been documented. It has a worldwide distribution, with a preference to warmer climate, where adult flies can be active year-round (9). In Europe, it has a high prevalence in Mediterranean regions, where seroprevalence in sheep can reach from 43.3-91% (10, 11, 12). In Slovenia, the prevalence was assessed at 40% (13). FIRST REPORT OF CANINE MYIASIS WITH SHEEP NASAL BOT FLY, Oestrus ovis, IN SLOVENIA Aleksandra Vergles Rataj*, Petra Bandelj, Vladimira Erjavec, Darja Pavlin 1Veterinary Faculty, University of Ljubljana, cesta V Mestni log 47, SI-1115 Ljubljana, Slovenia *Corresponding author, Email: aleksandra.verglesrataj@vf.uni-lj.si Abstract: First larval stage (L1) of Oestrus ovis was recovered by flushing of the nasal cavity during rhinoscopy in an urban living dog. The dog was taken to the Small animal clinic after an acute onset of sneezing and bilateral nasal discharge. In Eu- rope, there are sporadic reports of nasal myiasis in dogs caused by sheep bot flies, and the overall prevalence of O. ovis is high in Mediterranean countries. Because of its habitat expansion due to climate change, it should be considered as a differential diagnosis when an animal patient presents with signs of rhinitis in areas bordering the Mediterranean climate. This is the first report of a dog infested by sheep nasal bot fly in Slovenia. Key words: Oestrus ovis; sheep bot fly; nasal myiasis; dog; climate change The presence of the parasite in the population can be detrimental to the infested animals (12, 14), as the larval stages of the O. ovis invade sinu-nasal passages (1). If the infestation is severe, the animal may suffer further complication of the respiratory system, such as chronic pneumonia unresponsive to antimicrobial treatment (15). Reports of dogs with O. ovis infestation are rare but show that dogs can harbor all stages of larvae, including mature larval instar (5). Clinical signs in dogs are mild in the early stage of infestation (4) but can become severe if symptoms go unnoticed, even leading to euthanasia of the animal (3). With some exceptions (3), most cases are associated with the dog or cat living on a sheep farm or in an area of high sheep density (4, 5, 8). A. Vergles Rataj, P. Bandelj, V. Erjavec, D. Pavlin156 The aim of this report is to describe the first case of sheep nasal bot fly infestation in a dog in Slovenia with no history of being in contact with sheep or sheep associated areas. Case presentation In August 2018, a spayed 4-year-old West Highland White Terrier female dog living in the city of Ljubljana (latitude 46°03’03’’ N, longitude 14°30’18’’ E) was presented to the Small animal clinic (Veterinary Faculty, University of Ljubljana) with frequent sneezing and nose licking of an acute onset. The owner reported that the clinical signs started after the dog was taken for a walk to a nearby meadow field in the city center. Clinical examination of the dog revealed low grade serous bilateral nasal discharge with no other changes in her clinical status. A complete blood count was performed to rule out infectious diseases, which showed no abnormalities. Based on the suspected diagnosis of a nasal foreign body, a rhinoscopy was performed the same day. Mild oedema and erythema of the mucosal surface with several small moving larvae-like structures was noticed in the nasal cavity (Figure 1). More than 30 larvae-like structures were noted further down the nasopharynx. Retrograde irrigation of the nasal cavity with saline removed the larvae-like structures, which were sent to the Parasitology department of the Veterinary Faculty (University of Ljubljana) for identification. While waiting for a parasitological diagnosis, the dog was treated with ivermectin (Ivomec 1%, Merial) at a dose of 0.03 mg/kg, applied subcutaneously three times at weekly intervals. The owner stated that the clinical signs subsided within the first week of starting treatment and disappeared completely two weeks after the initial diagnosis. The sneezing resolved one day after rhinoscopy and nasal lavage. The parasitological diagnosis of first larval stage (L1) of Oestrus ovis L (Diptera, Oestridae) was made, based on key morphological features (16) observed under light microscopy. The dorsoventrally flattened L1 was approximately 1.2 mm long and 0.4 mm wide. The larval body was divided into 11 segments, with a pair of prominent, dark brown oral hooks on the first segment (Figure 2) and caudal spines on the last segment (Figure 3). Figure 1: Multiple Oestrus ovis L1 in the nasopharynx of the dog during retroflex nasopharyngoscopy Figure 2: Oestrus ovis L1 segmented body with two dark brown oral hooks (arrow) on the first body segment Figure 3: Oestrus ovis L1 caudal spines on the last body segment First report of canine myiasis with sheep nasal bot fly, Oestrus ovis, in Slovenia 157 Discussion Nasal bot fly, Oestrus ovis, causes myasis in small ruminants (1), but other casual hosts, such as humans (7) and carnivores (2-6, 8) have also been reported. The presence of sheep nasal bot flies in Slovenia was recorded in 1997 with a seroprevalence of 40% (13). Since then, several cases have been reported in Slovenia, in which third stage larvae were identified in sheep (unpublished data). In this study, we report the first case of O. ovis L1 infestation in a dog in Slovenia. The dog presented in this case, lives in the city of Ljubljana, which has a pre-alpine climate. As discussed by Zanzani et al (6), climate change may have been an important component in the spread of O. ovis habitat above 45° N latitude. Although there have been sporadic local reports of nasal bot fly infestation in sheep, no study has been done to determine its prevalence since 1997 (13). The 4-year-old dog lived most of its life in an urban environment. According to the owner, the dog had no contact with sheep or areas associated with sheep. However, Ljubljana as a city is still highly accessible to the rural surroundings, where flocks of sheep are present. It is common for dog owners who live in the city to walk their dogs through the livestock pastures and rural areas, especially in the summer. Adult nasal bot flies are very active during the hot summer months (14, 17) and explains the infestation of the dog in August. If the infestation would go unnoticed by the owner, the O. ovis L1 would remain quiescent during the cold winter months and continued its development the following spring (14, 17). This was the case of a dog in the UK, where the dog expelled the mature larva in the spring of 2011, following the presumptive infestation in autumn 2010 (5). Reportedly the infestation of O. ovis in dogs and cats are less likely than in humans (6), where it usually causes ophthalmomyiasis (7, 18). To the authors knowledge, no human cases have been documented in Slovenia to date. However, there have been several cases of human ophthalmomyiasis in Italy and two recent cases in Croatia (7). It is suspected that the incidence of ocular myiasis in humans is underreported (7), as is with canine cases of nasal myiasis (6). Nasal myiasis due to O. ovis infestation should be considered as a differential diagnosis when an animal is presented with signs of rhinitis, sneezing and nasal discharge (3, 4, 6). With climate change the prevalence and distribution of O. ovis may increase and expand (5) to non-Mediterranean regions. In conclusion, it is important to emphasize the role of reporting disease occurrence in both, animals and humans, and determining the prevalence of sheep nasal bot flies in populations previously believed not to be at risk. References 1. Zumpt F. Myiasis in man and animals in the old world: a textbook for physicians, veteri- narians and zoologists. London : Butterworths, 1965. 2. Lucientes J, Ferrer-Dufol M, Andres MJ, Peribanez MA, Gracia-Salinas MJ, Castillo JA. Canine myiasis by sheep bot fly (Diptera: Oestri- dae). J Med Entomol 1997; 34(2): 242–3. 3. Luján L, Vázquez J, Lucientes J, Panero JA, Varea R. Nasal myiasis due to Oestrus ovis in- festation in a dog. Vet Rec 1998; 142(11): 282–3. 4. Heath ACG, Johnston C. Nasal myiasis in a dog due to Oestrus ovis (Diptera: Oestridae). N Z Vet J 2001; 49(4): 164. 5. McGarry J, Penrose F, Collins C. Oestrus ovis infestation of a dog in the UK. J Small Anim Pract 2012; 53(3): 192–3. 6. Zanzani SA, Cozzi L, Olivieri E, Gazzonis AL, Manfred MT. Oestrus ovis L. (Diptera: Oestridae) induced nasal myiasis in a dog from Northern Italy. Case Rep Vet Med 2016; 2016: e5205416. doi: 10.1155/2016/5205416 7. Pupić-Bakrač A, Pupić-Bakrač J, Škara Kolega M, Beck R. Human opthalmomyiasis caused by Oestrus ovis – first report from Croatia and review on cases from Mediterranean coun- tries. Parasitol Res 2020; 119(3): 783–93. 8. Webb SM, Grillo VL. Nasal myiasis in a cat caused by larvae of the nasal bot fly, Oestrus ovis. Aust Vet J 2010; 88(11): 455–7. 9. Sotiraki S, Hall MJR. A review of compara- tive aspects of myiasis in goats and sheep in Eu- rope. Small Ruminant Res 2012; 103(1): 75–83. 10. Dorchies P, Bergueaud JP, Tabouret G, Prevot F, Jacquiet P. Prevalence and larval bur- den of Oestrus ovis (Linné, 1761) in sheep and goat in northern Mediterranean region of France. Vet Parasitol 2000; 88(3/4): 269–73. 11. Scala A, Solinas G, Citterio CV, Kramer H, Genchi C. Sheep oestrosis (Oestrus ovis, Linné A. Vergles Rataj, P. Bandelj, V. Erjavec, D. Pavlin158 1761, Diptera, Oestridae) in Sardinia, Italy. Vet Parasitol 2001; 102(1/2): 133–41. 12. Alcaide M, Reina D, Sánchez-López J, Fron- tera E, Navarrete I. Seroprevalence of Oestrus ovis (Diptera, Oestridae) infestation and associated risk factors in ovine livestock from Southwestern Spain. J Med Entomol 2005; 42(3): 327–31. 13. Brglez J, Polajner V. Oestrosis in sheep. Vet Nov 1997; 23: 393–4. 14. Hall M, Wall R. Myiasis of humans and do- mestic animals. Adv Parasitol 1995; 35: 257–334. 15. Gomez-Puerta LA, Alroy KA, Ticona DS, Lopez-Urbina MT, Gonzalez AE. A case of nasal myiasis due to Oestrus ovis (Diptera: Oestridae) in a llama (Lama glama). Rev Bras Parasitol Vet 2013; 22(4): 608–10. 16. Colwell DD. Larval morphology. In: Colwell DD, Hall MJ, Sholl PJ, eds. The Oestrid flies: biol- ogy, host-parasite relationship, impact and man- agement. Cambridge : CABI Publishing, 2006: 98–122. 17. Cepeda-Palacios R, Angulo Valadez CE, Scholl JP, Ramirez-Orduna R, Jacquiet P, Dor- chies P. Ecobiology of the sheep nose bot fly (Oestrus ovis L.): a review. Revue Méd Vét 2011; 162(11): 503–7. 18. D’Assumpcao C, Bugas A, Heidari A, Sofin- ski S, McPheeters RA. A case and review of ophthal- monyiasis caused by Oestrus ovis in the central val- ley of California, United States. J Investig Med High Impact Case Rep 2019; 7: e2324709619835852. doi: 10.1177/2324709619835852 PRVI PRIMER PASJE MIAZE Z OVČJIM NOSNIM ZOLJEM, Oestrus ovis, V SLOVENIJI A. Vergles Rataj, P. Bandelj, V. Erjavec, D. Pavlin Izvleček: Med rinoskopijo in spiranjem nosne votline, smo pri psu, ki živi v urbanem okolju, ugotovili ličinke prve stopnje (L1) zajedavca Oestru ovis. Lastniki so psa pripeljali na Kliniko za male živali po akutnem izbruhu kihanja in bilateralnega nosnega izcedka. V Evropi so dokumentirani sporadični primeri nosne miaze pri psih zaradi ovčjega nosnega zolja, O. ovis, in skupna prevalenca ovčjega zajedavca je v mediteranskih državah visoka. Zaradi klimatskih sprememb, se habitat nosnih zoljev čedalje bolj širi, za kar je pomembno O. ovis vključiti v seznam diferencialnih diagnoz pri pacientih s kliničnimi znaki rinitisa tudi na področjih, ki mejijo na mediteransko klimo. To je prvi opisan primer infestacije psa z ovčjim nosnim zoljem v Sloveniji. Ključne besede: Oestrus ovis; ovčji nosni zolj; nosna miaza; pes; podnebne spremembe 159Slov Vet Res 2021: 58 (4) AUTHOR INDEX VOLUME 58, 2021 Abo-Salem ME, see Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI..................................55 Ahmed HA, see Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI.............................................55 Aloke C, Igwe ES, Obasi NA, Amu PA, Ogbonnia EC. Anti-diabetic effect of ethanol extract of Co- paifera salikounda (Heckel) against alloxan-in- duced diabetes in rats.....................................63 Amu PA, see Aloke C, Igwe ES, Obasi NA, Amu PA, Ogbonnia EC..................................................63 Arencibia A, see Pérez S, Encinoso M, Morales M, Arencibia A, Suárez-Bonnet A, González-Rodrí- guez E, Jaber JR...........................................111 Ari HH, Uslu S. Morphology and histology of the Eurasian Lynx (Lynx lynx) planum nasale ....147 Arshed M, Nasir S, Hussain T, Babar MI, Imran M. Comparison efficacy of synthetic chemicals and plant extracts for tick.....................................13 Avšič T, see Strašek Smrdel K, Avšič T.................103 Babar MI, see Arshed M, Nasir S, Hussain T, Babar MI, Imran M....................................................13 Bandelj P, see Vergles Rataj A, Bandelj P, Erjavec V, Pavlin D....................................................155 Brem G, see Grilz-Seger G, Mesarič M, Brem G, Cotman M.......................................................77 Brožič A, see Pavlin D, Nemec A, Lampreht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N...........35 Cotman M, see Grilz-Seger G, Mesarič M, Brem G, Cotman M.......................................................77 Cvetko M, see Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M.........137 Čemazar M, see Pavlin D, Nemec A, Lampreht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N.........35 Durmuşoğlu H, see Habeeb GA, Durmuşoğlu H, İlhak Oİ..........................................................47 El-Deeb W, see Kandeel M, El-Deeb W, Fayez M, Ghoneim I.......................................................95 El-Shawarby RM, see Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI..................................55 EL-Shewy EA, see Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI..................................55 Encinoso M, see Pérez S, Encinoso M, Morales M, Arencibia A, Suárez-Bonnet A, González-Rodrí- guez E, Jaber JR...........................................111 Erjavec V, see Vergles Rataj A, Bandelj P, Erjavec V, Pavlin D....................................................155 Fayez M, see Kandeel M, El-Deeb W, Fayez M, Gho- neim I.............................................................95 Fonda I, see Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M.......................137 Garcês A, Soeiro V, Lóio S, Silva F, Pires I. Osteomyeli- tis on the cervical vertebras of a free-living Europe- an hedgehog (Erinaceus europaeus) by Paeniclos- tridium sordellii............................................117 Ghoneim I, see Kandeel M, El-Deeb W, Fayez M, Ghoneim I.......................................................95 Gombač M, see Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M...........137 González-Rodríguez E, see Pérez S, Encinoso M, Morales M, Arencibia A, Suárez-Bonnet A, Gon- zález-Rodríguez E, Jaber JR..........................111 Grilc Fajfar A, see Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M..........137 Grilz-Seger G, Mesarič M, Brem G, Cotman M. Characterisation of coat colour in the Slovenian Posavje horse.................................................77 Habeeb GA, Durmuşoğlu H, İlhak Oİ. The combi- ned effect of sodium lactate, lactic acid and acetic acid on the survivalof Salmonella spp. and the microbiota of chicken drumsticks....................47 Hagag NM, see Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI.............................................55 Heo G-J, see Wimalasena S H M P, Heo G-J...........25 Hussain T, see Arshed M, Nasir S, Hussain T, Babar MI, Imran M..........................................13 Igwe ES, see Aloke C, Igwe ES, Obasi NA, Amu PA, Ogbonnia EC..................................................63 Igwe ES, see Aloke C, Igwe ES, Obasi NA, Amu PA, Ogbonnia EC..................................................63 İlhak Oİ, see Habeeb GA, Durmuşoğlu H, İlhak Oİ......47 Imran M, see Arshed M, Nasir S, Hussain T, Babar MI, Imran M..........................................................13 Jaber JR, see Pérez S, Encinoso M, Morales M, Arencibia A, Suárez-Bonnet A, González-Rodrí- guez E, Jaber JR...........................................111 160 Slov vet Res, Author Index Volume 58, 2021 Kafi ZZ, see Tamai IA, Kafi ZZ.............................85 Kandeel M, El-Deeb W, Fayez M, Ghoneim I. Phar- macokinetics of the long-acting ceftiofur crystal- line-free acid in Arabian she-camels (Camelus dromedarius)...................................................95 Kirkiłło-Stacewicz K, Nowicki W, Wach J. Telen- cephalon vascularity in dog (Canis lupus f. famili aris)..................................................................5 Lampreht Tratar U, see Pavlin D, Nemec A, Lam- preht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N..........................................................35 Lóio S, see Garcês A, Soeiro V, Lóio S, Silva F, Pires I...........................................................117 Majdič G, see Voga M, Pleterski A,.........................125 Mesarič M, see Grilz-Seger G, Mesarič M, Brem G, Cotman M.......................................................77 Morales M, see Pérez S, Encinoso M, Morales M, Arencibia A, Suárez-Bonnet A, González-Rodrí- guez E, Jaber JR...........................................111 Nasir S, see Arshed M, Nasir S, Hussain T, Babar MI, Imran M....................................................13 Nemec A, see Pavlin D, Nemec A, Lampreht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N...35 Nowicki W, see Kirkiłło-Stacewicz K, Nowicki W, Wach J.............................................................5 Obasi NA, see Aloke C, Igwe ES, Obasi NA, Amu PA, Ogbonnia EC............................................63 Pavlin D, Nemec A, Lampreht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N. Palliative jaw-s- paring treatment of a nonresectable canine oral fibrosarcoma using combination of electroche- motherapy with bleomycin and IL-12 gene elect- rotransfer.......................................................35 Pavlin D, see Vergles Rataj A, Bandelj P, Erjavec V, Pavlin D........................................................155 Pérez S, Encinoso M, Morales M, Arencibia A, Su- árez-Bonnet A, González-Rodríguez E, Jaber JR. Comparative evaluation of the Komodo dragon (Varanus komodoensis) and the Green iguana (Igu- ana iguana) skull by three-dimensional compu- ted tomographic reconstruction....................111 Pires I, see Garcês A, Soeiro V, Lóio S, Silva F, Pires I............................................................117 Pleterski A, see Voga M, Pleterski A, Majdič G....125 Ramadan SI, see Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI.............................................55 Sargious MAN, El-Shawarby RM, Abo-Salem ME, EL-Shewy EA, Ahmed HA, Hagag NM, Ramadan SI. Genetic diversityof Egyptian Arabian horses from El-Zahraa Stud based on 14 TKY microsatelli- te markers......................................................55 Serša G, see Pavlin D, Nemec A, Lampreht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N............35 Silva F, see Garcês A, Soeiro V, Lóio S, Silva F, Pires I...........................................................117 Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M. The first outbreak of viral en- cephalopathy and retinopathy in farmed sea bass (Dicentrarchus labrax) in Slovenia..................137 Soeiro V, see Garcês A, Soeiro V, Lóio S, Silva F, Pires I.............................................................117 Staji H, Tamai IA, Kafi ZZ. First report of Paeni- bacillus cineris from a Burmese python (Python molurus bivittatus) with oral abscess................85 Strašek Smrdel K, Avšič T. The detection of Anap- lasma phagocytophilum and Babesia vulpes in spleen samples of red fox (Vulpes vulpes) in Slovenia.........................................................103 Suárez-Bonnet A, see Pérez S, Encinoso M, Morales M, Arencibia A, Suárez-Bonnet A, González-Rod- ríguez E, Jaber JR.........................................111 Šturm S, see Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M.......................137 Švara T, see Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M........................137 Tamai IA, see Staji H, Tamai IA, Kafi ZZ..................85 Tozon N, see Pavlin D, Nemec A, Lampreht Tratar U, Čemazar M, Brožič A, Serša G, Tozon N............35 Uslu S, see Ari HH, Uslu S...............................147 Vergles Rataj A, Bandelj P, Erjavec V, Pavlin D. First report of canine myiasis with sheep nasal bot fly, Oestrus ovis, in Slovenia................................155 Voga M, Pleterski A, Majdič G. Isolation of live cells from different mice tissues up to nine days after death.............................................................125 Wach J, see Kirkiłło-Stacewicz K, Nowicki W, Wach J.............................................................5 Wimalasena S H M P, Heo G-J. The presence of pu- tative virulence determinants, tetracycline and β-lactams resistance genes of Aeromonas species isolated from pet turtles and their....................25 161Comparative evaluation of the Komodo dragon (Varanus komodoensis) and the Green iguana (Iguana iguana) skull by three-dimensional ... SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2021; 58 (4) Original Research Articles Voga M, Pleterski A, Majdič G. Isolation of live cells from different mice tissues up to nine days after death . . . . . . . . . . . . . . . . . . . . . . . . 125 Sitar R, Švara T, Grilc Fajfar A, Šturm S, Cvetko M, Fonda I, Gombač M. The first outbreak of viral encephalopathy and retinopathy in farmed sea bass (Dicentrarchus labrax) in Slovenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Ari HH, Uslu S. Morphology and histology of the Eurasian Lynx (Lynx lynx) planum nasale. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147 Case Report Vergles Rataj A, Bandelj P, Erjavec V, Pavlin D. First report of canine myiasis with sheep nasal bot fly, Oestrus ovis, in Slovenia . . . . ..155 Author Index Volume 58, 2021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159