124, Bull. Eur. Ass. Fish Pathol., 34(4) 2014 NOTE Mycobacterium marinum Argyrosomus regius M. L. Avsever 1 2, M. Z. Günen 1, 1, S. Eskiizmirliler 1, B. I. Didinen 3, 4, G. Erdal 1 and M. Özden 1 1 Bornova Veterinary Control Institute, Fish Diseases National Reference Laboratory (NRL), Izmir, Turkey; 2 Ege University, Faculty of Medicine, Department of Medical Microbiology, Izmir, Turkey; 3 Suleyman East Campus, 32260, Cünür- Isparta, Turkey; 4 Bornova Veterinary Control Institute, Veterinary Biological Products Control Department, Izmir, Turkey Abstract laboratory with anorexia, weight loss and mortality as symptoms. Necropsy revealed grey-white In histopathology, coalescing tubercles were observed on the same tissues. Mycobacterium marinum sequenced and recorded in GenBank as Mycobacterium marinum. Argyrosomus regius, nean and the Black Sea, as well as on the Atlantic ; Quemener, 2002). Meagre, mariculture as long as appropriate husbandry methods are practiced (Poli et al., 2001; Roo et Elkesh et al., 2013). Global meagre production is reported to have increased rapidly; although it 2013). in chinook salmon (Oncorhynchus tshawytscua Salmo salar) (Brocklebank et al., 2003), Argentinian silver-
Figure 1. Granulomas in spleen Mycobacterium marinum. side (Odonthestes bonariensis striped snakehead (Channa striata) (Chinabut et al., 1990), turbot (Scophthalmus maximus) (Dos Santos et al., 2002), European seabass (Dicentrarchus labrax) (Colorni, 1992), striped bass (Morone saxatilis drum (Sciaenops ocellatus mycobacteriosis in meagre. Fish mycobacteriosis, also known as piscine tuberculosis, is a chronic, progressive (zoonotic) Mycobacterium caused by Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae (Talaat in mycobacteriosis is classical granulomatous these lesions are observed as epithelioid mac- liver and kidney, as disease progresses they spread to all organs (Jacobs et al., 2009). - - thead sea bream (Sparus aurata seabass (Dicentrarchus labrax, mullet (Mugil cephalus, around the cages. Diseased meagre showed lethargy, weakening, swelling and dermal lesions. The necrop- lesions in the liver, kidney and spleen (Figure 1). There were no macroscopic disease signs in Internal organ samples (spleen, liver, kidney)
Figure 2. Mycobacterium spp. in reverse hybridization assay M. marinum). hexadecylpyridinium chloride (HPC, detergent) - was discarded. Slides were then prepared the Ziehl-Neelsen (ZN) method. For primary isolation, the homogenates was inoculated on Löwenstein-Jensen media and BACTEC MGIT 960 liquid media. While Löwenstein-Jen- growth, BACTEC MGIT 960 were evaluated with optic readers. Inoculated media were incu- 2 were obtained Figure 3. Mycobacterium marinum KC2 in reverse hybridization assay. signal in the same week. carried out with biotin conjugated primers. Amplicons with biotin were hybridized with The isolates were researched on a species level with reverse hybridization based GenoType Mycobacterium -
a b Figure 4. ZN x 1000(b). made visible with enzymes and chromogenic according to the data supplied by the manu- M. marinum and M. ulcerans. The isolates were determined to be M. marinum being photochromogenic (yellow pigment). Reverse hybridization Mycobacterium spp. are shown in Figure 2 and M. marinum KC2 in Figure 3. M. marinum Double sided sequence analysis was carried out by BM Laboratory Systems in Macrogen M. marinum. tity with M. marinum strain and. with hematoxylin and eosin (H&E) and Zie- Granulomas with necrosis in the centers, sur- this, Ziehl-Nielsen staining revealed positive in the Ziehl-Neelsen staining (Figure 4b). wild gilthead sea bream, European seabass and species (Chinabut et al., 1990) and this may be a possibility in this case. The bacteria could also have been introduced with the original
dermal lesions, exophthalmos and grey-white granulomas in various organs, especially liver, spleen and kidney were common symptoms in - 2011; Jeronimo et al., 2013). In this study, sections showed tubercle above containing typical ZN positive bacteria in the center or around the coagulation ne- (Dos Santos et al., 2002; Jeronimo et al., 2013) and in Atlantic salmon osis. Gauthier et al. (2003) also reported a severe M. marinum. As a result, clinical, pathological, bacteriologi- in cultured meagre caused by M. marinum was were negative may also show that population M. marinum References Adams A, Thompson KD, McEwan H, Chen Mycobacterium Journal of Aquatic Animal Health 8 (Oncorhynchus tshawytscha Walbaum) in Australia. Journal of Fish Biology 10(6) Annual Review of Fish Diseases 5 Brocklebank J, Raverty S and Robinson J (2003). in British Columbia. The Canadian Veterinary Journal 44(6) BP, Fletcher ZJ, Drobniewski FA and to Mycobacterium chelonae salmon Salmo salar. Diseases of Aquatic Organisms 33, Chinabut S, Limsuwan C and Chanratchakool P (1990). Mycobacteriosis in the snakehead, Channa striatus (Fowler). Journal of Fish Diseases 13 Colorni A (1992). A systemic mycobacteriosis in the European sea bass Dicentrarchus labrax cultured in Eilat (Red Sea). Israeli Journal of Aquaculture-Bamidgeh 44 Colorni A, Avtalion R, Knibb W, Berger Dicentrarchus labrax Mycobacterium marinum and treated with streptomycin and garlic (Allium sativum) extract. Aquaculture 160 Dos Santos NMS, Do Vale A, Sousa MJ and Scophthalmus maximus. Diseases of Aquatic Organisms 52(1) Elkesh A, Kantham KPL, Shinn AP, Crumlish M and Richards RH (2013). Systemic nocardiosis in a Mediterranean population Argyrosomus regius Asso Journal of Fish Diseases 36(2), 141-149. Frerichs GN (1993). Mycobacteriosis: nocardiosis. In: Bacterial Diseases of Fish
Bull. Eur. Ass. Fish Pathol., 34(4) 2014, 129 (ed. by V Inglis, RJ Roberts, NR Bromage), Gauthier DT, Rhodes MW, Vogelbein WK, Kator mycobacteriosis in striped bass Morone saxatilis. Diseases of Aquatic Organisms 54, Ichthyological Bulletin 65 Hatai KA, Lawhavinit O, Kubota SS, Toda Odonthestes bonariensis. Fish Pathology 23(3), Journal of Wildlife Diseases 23(3), Jacobs JM, Stine CB, Baya AM and Kent ML Journal of Fish Diseases 32(2), 119-130. Jeronimo D, Barbosa A, Ramos M, Marques J, Moreira M, Sousa J, Cruze C and Saraiva A (2013). Granulomas caused by Mycobacterium Scopthalmus maximus Mediterranean Marine Science 14(2), 424-431. Manual of histologic staining methods of the Armed Forces Institute of Pathology, 3 rd edn. McGraw-Hill, New York. Argyrosomus regius), as an emerging species in Mediterranean aquaculture. Studies F, Zampacavallo G and Gilmozzi M (2001). The meagre (Argyrosomus regius), a new 1. Morphological, merchantable and nutritional traits in a commercial wide sizerange. European Aquaculture Society Special Publication 29, 209-210 A (2000). Mycobacteriosis: detection and species. Fish Veterinary Journal 5 Quemener L (2002). La maigre commun (Argyrosomus regius) Biologie, peche, marche et potential aquacole. Editions Roo J, Hernández-Cruz C M, Borrero C, Schuchardt D and Fernández-Palacios H sequence on meagre (Argyrosomus regius; Aquaculture 302, Talaat AM, Reimschuessel R and Trucksis polymerase chain reaction and restriction enzyme analysis. Veterinary Microbiology 58 Benedenia sciaenae van Argyrosomus regius). Acta parasitologica Turcica/Turkish Society for Parasitology 31(1) Vogel H (1958). animals. American Review of Tuberculosis and Pulmonary Diseases 77, 823-838. Zerihun M A, Hjortaas MJ, Falk K and Colquhoun DJ (2011). Immunohistochemical Journal of Fish Diseases 34(3)