WOS: 000385990400003Mycobacterium marinum is the most important fish tuberculosis agent. Turkey is an important Mediterrenean country in terms of sea bass, sea bream and trout production. In this work, a total of 1050 fish samples (300 sea bass, 300 sea bream, 450 trout, respectively) taken randomly from 20 sea bass and sea bream (No:1-20), and 15 trout (No: 21-35) farms, were analysed using a combination of culture-based and molecular (Reverse Hybridisation) methods for Mycobacterium spp. The majority of samples were negative for mycobacteriosis. However, isolates that were positive by Reverse Hybridisation for M. marinum were isolated and identified from 60 fish (30 sea bass and 30 sea bream samples) from two cages of one farm in the Milas/Mugla province (Southeastern part of the Aegean Sea). This is the first M. marinum isolation from cultured sea bass and sea bream in Turkey

Avsever, M. L.

Eskiizmirliler, S.

Korun, J.

Türe, M.

Çamkerten, I.

Çavuşoğlu, C.

Mycobacterium marinum is the most important fish tuberculosis agent. Turkey is an important Mediterranean country in terms of sea bass, sea bream and trout production. In this work, a total of 1050 fish samples (300 sea bass, 300 sea bream, 450 trout, respectively) taken randomly from 20 sea bass and sea bream (No:1-20), and 15 trout (No: 21-35) farms, were analysed using a combination of culture-based and molecular (Reverse Hybridisation) methods for Mycobacterium spp. The majority of samples were negative for mycobacteriosis. However, isolates that were positive by Reverse Hybridisation for M. marinum were isolated and identified from 60 fish (30 sea bass and 30 sea bream samples) from two cages of one farm in the Milas/Mugla province (Southeastern part of the Aegean Sea). This is the first M. marinum isolation from cultured sea bass and sea bream in Turkey

Avsever, Meriç Lütfi

Çavuçoglu, C.

Eskiizmirliler, Sefa

Türe, Mustafa

Korun, Jale

Çamkerten, İlker

Aksaray University Institutional Repository

First isolation of Mycobacterium marinum from sea bass (Dicentrarchus labrax) and gilthead sea bream (Spams auratus) cultured in Turkey

Bull. Eur. Ass. Fish Pathol., 36(5) 2016, 193* Corresponding author’s email: lutfiavsever@gmail.comFirst isolation of Mycobacterium marinum from sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus auratus) cultured in TurkeyM. L. Avsever1*, C. Çavuşoğlu2, S. Eskiizmirliler3, M. Türe4, J. Korun5 and İ. Çamkerten11Aksaray University, Eskil Vocation of High School, Laboratory and Veterinary Sciences, 68100 Aksaray, Turkey; 2Ege University, Faculty of Medicine, Department of Medical Microbiology, Section of Mycobacteria, 35010 Izmir, Turkey; 3Bornova Veterinary Control Institute, Fish Diseases National Reference Laboratory (NRL), 35010 Izmir, Turkey; 4Central of Fisheries Research Institute, Fish Disease Laboratory, 61250 Trabzon, Turkey; 5Akdeniz University, Faculty of Fisheries, Department of Fish Disease, 07058 Antalya, TurkeyAbstractMycobacterium marinum is the most important fish tuberculosis agent. Turkey is an important Medi-terrenean country in terms of sea bass, sea bream and trout production. In this work, a total of 1050 fish samples (300 sea bass, 300 sea bream, 450 trout, respectively) taken randomly from 20 sea bass and sea bream (No:1-20), and 15 trout (No: 21-35) farms, were analysed using a combination of culture-based and molecular (Reverse Hybridisation) methods for Mycobacterium spp. The major-ity of samples were negative for mycobacteriosis. However, isolates that were positive by Reverse Hybridisation for M. marinum were isolated and identified from 60 fish (30 sea bass and 30 sea bream samples) from two cages of one farm in the Milas/Muğla province (Southeastern part of the Aegean Sea). This is the first M. marinum isolation from cultured sea bass and sea bream in Turkey.IntroductionAquaculture is an emerging sector within the Turkish economy. According to the data sup-plied by the The State of World Fisheries and Aquaculture (FAO, 2010), Turkey has shown the third highest rate of increase of aquaculture production, after China and India. In 2013, there were 2.353 aquaculture production premises, with 1935 freshwater and 418 marine sites. Sea bass and sea bream farms are predominantly located alongside the Mugla and Izmir coast of the Aegean Sea, especially in Milas. Other production sites are located in Trabzon, Ordu and Samsun provinces in the Black Sea Region; Hatay, Antalya and Mersin provinces in the Mediterrenean Region and Çanakkale and Balıkesir in the Marmara Region. Trout farms are spread throughout Turkey (Anonymous, 2015). Infectious disease is an important issue 194, Bull. Eur. Ass. Fish Pathol., 36(5) 2016which hinders the fast growth rate of the aqua-culture sector (Aiello and Mays, 2006).Mycobacterium marinum is a recognized fish pathogen (Austin and Austin, 1987; Colorni, 1992), that can also infect mammals, includ-ing humans (Smith and Willett, 1980). Initially isolated from sea bass (Dicentrarchus labrax) cul-tured in the Red Sea in 1990 (Colorni, 1992), the agent has since been reported from more than 20 fish species (Frerichs, 1993). It has also recently been recovered from a Turkish species, meagre (Argyrosomus regius) (Avsever et al., 2014). Mycobacterium marinum may cause local (Knibb et al., 1993) or systemic (Frerichs, 1993) infec-tions in fish. Skin lesions are predominant in local infections, whereas in systemic infections stunted growth and tubercule formation is com-monplace, especially in internal organs such as the spleen and kidneys (Avsever et al., 2014). Secondary infections and an increase in mor-tality may be seen in both cases. Even when death is averted; weight loss and deformed appearence lower the market value of fish. An-tibiotic treatment is not considered to be eco-nomic or practical. The main methods of disease control and management include marketing the fish as fillets, following harvest, disinfecting the cages after depopulation and fallowing for a year. Mycobacterium spp., can maintain their viability in the organic debris layer on the cages (Colorni, 1992; Frerichs, 1993; Ucko and Colorni, 2005; Wayne and Kubica, 1986). Mycobacterium marinum is a threat to human health. The agent can cause granulomas on the surface of hands and in tendons following contact with infected fish or contaminated water (Williams and Riordan, 1973; Ucko and Colorni, 2005). As symptoms and blood tests are mostly non-specific, diagnosis is generally delayed and this leads to a series of complications such as tendon rupture, osteomyelitis, loss of motion and even amputation (Barton et al., 1997; Lee et al., 2004). There are no previous reports on M. marinum isolation from sea bass and sea bream cultured in Turkey. Although Avsever et al. (2014) iso-lated M. marinum from meagre cultured in the Milas region of Turkey. The main method for M. marinum diagnosis is isolation (Koneman et al., 1992) and identi-fication is mostly carried out with molecular methods. Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) are used successfully for M. marinum identification (Smith and Willett, 1980; Talaat et al., 1997). However, Reverse Hybridisation (RH) is a rapid and accurate identification method (Posteraro et al.,1998; Sanguinetti et al., 1998).We report the first isolation of M. marinum from cultured sea bass and sea bream in Turkey.Materials and methodsThirty five fish farms were chosen from a list of premises supplied by the General Directo-rate of Fisheries and Aquaculture, Ministry of Food, Agriculture and Livestock. A total of 1050 harvest size (200-400g) cultured fish (300 sea bass, 300 sea bream, 450 trout samples) were sampled at random from farms (Table 1). Also, 40 wild fish samples (14 grey mullet, 12 sea bass, 4 sea bream) taken from the proximity of the cages that contained M. marinum positive fish were also included in the study. All the samples were taken in the summer (June and July). Sea Bull. Eur. Ass. Fish Pathol., 36(5) 2016, 195Table 1. Sample information and study results.Provinces Number of farms/Number of fish RegionNumber of fish Positive for M. marinum Sea bass/bream farms and number of fish samplesMuğla (Milas) 12 (Farm no: 1-12) /360* Southern Aegean60 (30 sea bass, 30 sea bream; in two cages of one farm- Farm no:1-) İzmir 4 (Farm no: 12-16/120*Northern Aegean -Aydın 1 (Farm no: 17)/30*Çanakkale 1 (Farm no: 18) /30*Trabzon 1 (Farm no: 19) /30* Black Sea -Mersin 1 (Farm no: 20) /30* Mediterrenean -Total sea bass/bream: 600*Trout farms and number of fish samplesTrabzon 1 (Farm no: 21) /30Black Sea -Artvin 1 (Farm no: 22) /30Rize 2 (Farm no: 23-24) /60Ordu 1 (Farm no: 25) /30Tokat 1 (Farm no: 26) /30Gümüşhane 1 (Farm no: 27) /30Sivas 1 (Farm no: 28) /30Mersin 1 (Farm no: 29) /30 Mediterrenean -İzmir  6 (Farm no: 30-35) /180 Aegean -Total trout: 450* Half sea bass, half sea bream196, Bull. Eur. Ass. Fish Pathol., 36(5) 2016water temperatures during the sampling were as follows: Aegean Sea (25 ±2oC), Mediterrenean (28 ±2oC), Black Sea (23 ±2oC ), Marmara sea (24 ±2oC), respectively. Freshwater temperatures during trout sampling were 15 oC (±4 °C).Isolation of Mycobacterium spp.Internal organ samples (spleen, liver, kidney) of fish were decontaminated first with hexa-decylpyridinium chloride (HPC). For this, the pellets formed after centrifugation were re-suspended in 10 mL 2% (w/v) HPC (Sigma) and incubated for 5 h at room temperature (21°C). Following incubation, the samples were centrifuged (2500 x g, 15 min) and the pellets resuspended in 1 mL PBS. A slide was prepared from this PBS solution, stained using Ziehl-Neelsen method and examined for the existence of acid-resistant bacilli. For primary isolation inoculations were made on Löwenstein-Jensen (L-J) media and BACTEC MGIT 960. Inocu-lated media were incubated for 28 days at 30 oC (Avsever et al., 2014; Koneman, 1992; Posteraro et al., 1998; Sanguinetti et al., 1998). Identification with Reverse-Hybridisation methodColonies taken from the L-J medium were identified with a Reverse Hybridisation based GenoType Mycobacterium assay CM/AS (Hain Lifescience/ Germany). For this purpose, hy-bridisation of biotin conjugated amplicons from PCR with species-specific oligonucleotides were carried out on nitrocellulose bands. DNA/DNA hybrids were visualised with enzymes and chro-mogenic substrates. Resulting patterns were evaluated according to the data supplied by the manufacturer (Avsever et al., 2014; Posteraro et al., 1998; Sanguinetti et al., 1998).Results and discussionMycobacteriosis was not detected in any of the fish collected from 34 of the 35 farm samples. However, it was detected at apparent high frequency from 30 sea bass and 30 sea bream samples from two cages of one farm (100%) in Milas. In these fish, skin lesions with varying severity (Figure 1), cachexia and pale coloring were observed. In necropsy, tubercules were observed in the internal organs (Figure 2, 3). Inoculations from these fish resulted in bacterial colony formation on L-J media by three weeks post-inoculation. These samples were positive in the BACTEC MGIT 960 system. Isolates were identified using Reverse Hybridisation and all gave the patterns 1, 2, 3, 10, 15 which are specific to M. marinum and M. ulcerans (Figure 4). As isolates were photochromogenic (Figure 5), they were concluded to likely be M. marinum (M. ulcerans is not photochromogenic). Sampling data and study results are supplied in Table 1.Clinical findings of piscine mycobacteriosis vary with the host and enviromental condi-tions; however, lethargy, inappetence and exophthalmia are typical general symptoms (Avsever et al., 2014). In this study, 60 of the fish examined and found to be positive for M. marinum had skin lesions of varying severity, paleness, cachexia and tubercule formation in internal organs. These findings are paral-leled to fish tuberculosis symptoms (Austin and Austin, 1987).Detection of M. marinum in the sea bass/ sea bream in the Milas region was completed after a previous study by Avsever et al. (2014) regard-ing the isolation of M. marinum from meagre in the same region. There were about two km between the farms containing M. marinum posi-Bull. Eur. Ass. Fish Pathol., 36(5) 2016, 197Figure 1. Severe skin lesion from a M. marinum positive sea bass.Figure 2. Enlargement of spleen and tubercule formation from a sea bass with M. marinum.Figure 3. Enlargement of spleen and tubercule formation from a sea bream positive for M. marinum.198, Bull. Eur. Ass. Fish Pathol., 36(5) 2016tive meagre samples (Avsever et al., 2014) to those with M. marinum positive Sea bass and Sea bream samples (in this work). The short period of time between these two reports is an indication of the spread of the disease. In con-trast, the bacterium was not isolated from 300 fish samples from the Milas region (Southern Aegean) or 180 samples from the Northern part of the Aegean Sea. This might be an indica-tion that the disease is limited to a part of the Milas region. However, as the Milas region is Figure 5. M. marinum colony grown on L-J medium (Sea bass, primary isolation). Photochomogenic properties present in M. marinum and absent in M. ulcerans.an important center for mariculture, there are a high number of stocks at risk. In this study, no M. marinum isolates were obtained from sea bass or sea bream samples taken from the Mediterrenean or Black Sea regions and trout samples taken from all of Turkey.The ‘gold standard’ method for M. marinum diagnosis is isolation. Molecular methods are generally employed for the identification and confirmation of the agent. In this work, reverse Figure 4. Reverse Hybridisation test, 1, 2, 3, 10, 15 patterns specific to M. marinum and M. ulcerans.Bull. Eur. Ass. Fish Pathol., 36(5) 2016, 199hybridisation, which is a rapid and accurate method, was used for identification (Avsever et al., 2014; Posteraro et al., 1998; Sanguinetti et al., 1998). For treatment of M. marinum infections, kana-mycin, streptomycin, isoniazid and rifampin combination, doxycycline or minocycline with sulphasoxazole can be used. However, treat-ment is expensive and time consuming. Also, it is not possible to eradicate M. marinum from a population with antibiotic therapy. Still, treat-ment might be an option for valuable brood-stocks (Aiello and Mays, 2006; Andrews et al., 1988). Filleting fish, following a harvest of the stocks, disinfecting the cages and leaving them fallow for a year are recommended methods for disease control. Mycobacterium spp., can remain viable in the organic debris layer on the cages even after disinfection. For this reason, it is necessary to clean this layer. Avsever et al. (2014) previously did not isolate microbacteriosis agents from wild fish caught from outside the M. marinum positive meagre cages and stated that population density was an important cause of morbidity for this disease. In this work, wild fish were caught from outside the cages of M. marinum positive fish and again no M. marinum was isolated in them.As a result of this work, M. marinum was iso-lated for the first time from sea bass and sea bream stocks in Turkey; zoonotic characteristics of the disease were emphasised and protection/control measures for the disease were discussed.ReferencesAiello SE and Mays A (2006). The Merck veterinary manual. 8th ed. Whitehouse Station, NJ: Merck and Co; 1998. Fish health management: bacterial disease [monograph online]. Available at: http://www.merckvetmanual.com/mvm/index.jsp?cfile=htm/b c/170414.htm. Accessed 28 Jun 2006.Andrews C, Exell A and Carrington N (1988). The Manual of Fish Health New Jersey: Tetra Press.Anonymous (2015). Names and locations of aquaculture facilities in Turkey. Access: http://www.tarim.gov.tr/BSGM/Belgeler/Icerikler/su_urunleri_tesisleri2015.pdf.Austin B and Austin DA (1987). Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. Chapter 12, Ellis Harword Ltd., NewYork.Avsever ML, Çavuşoğlu C, Günen MZ, Yazıcıoğlu Ö, Eskiizmirliler S, Didinen BI, Tunalıgil S, Erdal G and Özden M (2014). The first isolation of Mycobacterium marinum from cultured Argyrosomus regius. Bulletin of the European Association of Fish Pathogolists 34(4), 124-129.Barton A, Bernstein J, Struythers K and O’Neill T (1997). Mycobacterium marinum causing septic arthritis and osteomyelitis. British Journal of Rheumatology 36, 1207-1209.Colorni A (1992). A systemic mycobacteriosis in the European sea bass Dicentrarchus labrax cultured in Eilat (Red Sea). Israeli Journal of Aquaculture - Bamidgeh 44, 75-81.Food and Agrculture Organisation (FAO) (2010). The State of World Fisheries and Aquaculture. Rome. Frerichs GN (1993). Acid-fast fish pathogens, p.217-233. In V. Inglis, R. J. Roberts, and N. R. Bromage (ed.), Bacterial diseases of fish. 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Polymerase chain reaction-reverse cross-blot hybridisation assay in the diagnosis of sporotrichoid Mycobacterium marinum infection. British Journal of Dermatology 139 (5), 872-6.Sanguınetti M, Posteraro B, Ardito F, Zanetti S, Cingolani A, Sechi L, De Luca A, Ortona L and Fadda G (1998). Routine use of PCR reverse cross-blot hybridisation assay for rapid identification of Mycobacterium species growing in liquid media. Journal of Clinical Microbiology 36, 1530-1533.Smith DT and Willett HP (1980). Other mycobacterium species, p.674-698. In W. K. Joklik, H. P. Willet, and D. B. Amos (ed.), Zinsser microbiology, 17th ed. Appleton-Century-Crofts, New York, N.Y.Talaat MA, Reimschuessel R and Trucksis M (1997). Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis. Veterinary Microbiology 58, 229-237. Ucko M and Colorni A (2005). 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English

First isolation of Mycobacterium marinum from sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus auratus) cultured in Turkey

Cavusoglu, C.

Ture, M.

Camkerten, I.

Ege University Institutional Repository

First isolation of Mycobacterium marinum from sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus auratus) cultured in Turkey

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Ege University Institutional Repository