Abstract. Introduction. 136, Bull. Eur. Ass. Fish Pathol., 33(5) 2013

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1 136, Bull. Eur. Ass. Fish Pathol., 33(5) 2013 Renibacterium salmoninarum Abstract During winter 2010/2011, a country-wide survey was conducted on salmonids that were primarily Oncorhynchus mykiss) and brown trout ( Renibacterium salmoninarum using R. salmoninarum R. salmoninarum Introduction Bacterial kidney disease (BKD), a slowly pro- Renibac terium salmoninarum Atlantic salmon in Scotland (Mackie et al., 1933). where wild or cultured salmonids are present - salmonids and it may cause high morbidity and mortality in susceptible species, especially 1969; MacLean & Yoder, 1970; Mitchum et al., 1979; Banner et al., 1986; Chambers et al., 2008), including naturally spawning populations that have never been supplemented with hatchery R. salmoninarum are constantly exposed to bacteria shed into the & Sherman, 1981; Hastein & Lindstad, 1991). In Slovenia, BKD occurrence is related mostly to ling (Thymallus thymallus R. salmoninarum was introduced, R. salmoninarum DNA was detected in cultured grayling, brown trout and brook trout (Salvelinus fontinalis).

2 Bull. Eur. Ass. Fish Pathol., 33(5) 2013, 137 R. salmoninarum in order to get an insight into the situation regarding R. salmoninarum - Material and methods From December 2010 to March 2011, a total R. salmoninarum. On corhynchus mykiss) and 90 brown trout (Salmo rivers and streams covering almost the whole both rainbow and brown trout were sampled, while only rainbow or only brown trout were sampled at 9 and 4 locations, respectively. Fish mined by counting the natural growth rings on and dissected. - and diluted (1:10) DNA template. The DNA R. salmoninarum-positive grayling kidneys R. salmoninarum culture was used as a positive control. R. salmoninarum primers Rs1 PCR products (501 bp) were analyzed by electrophoresis in 1.5% agarose gels stained with ethidium bromide. were subjected to real-time PCR assay using the TaqMan exogenous internal positive control (Applied Biosystems, USA) in order to monitor TaqMan Universal PCR Master Mix (Applied containing the primers and VIC-labelled probe IPC DNA (Applied Biosystems, USA; diluted - Real-Time PCR System (Applied Biosystems, Results and discussion annual production between 5 and 100 tons. In consumption, it is orientated mainly towards the health problem in cultured grayling which is

3 138, Bull. Eur. Ass. Fish Pathol., 33(5) Figure 1.

4 Bull. Eur. Ass. Fish Pathol., 33(5) 2013, 139 Table 1. Sampling site O (mg/l) T (ºC) Rainbow 2 trout nm not measured Brown trout Total 1 nm nm Kokra Poljanska Sora nm nm Sava Bohinjka Sava Dolinka Lubija Trnavica Tesnica Sromljica Capot Kanomljica nm Reka Studena nm nm Lubnica Besnica Polanski potok nm nm Bistrica Framski potok nm nm Krka

5 140, Bull. Eur. Ass. Fish Pathol., 33(5) 2013 grayling populations were never deliberately R. salmoninarum. R. salmoninarum was usually per- R. salmoninarum same period were positive. In addition, 95 salmonids (85 rainbow trout, 6 brook trout and 4 R. salmoninarum DNA was detected in brown trout (n=2) and brook trout (n=2). Freshwater trout on R. salmoninarum in salmonids was carried covering almost entire country, were selected season and water temperature (Ortega et al., 1995). As rainbow trout and brook trout are deemed to be relatively resistant to R. salmoni narum (Mitchum & Sherman, 1981; Starliper et al., 1997), this could partially explain the absence R. salmoninarum in outbreak, Ortega et al. (1995) reported that the 3+ (mean weight 271 g and mean length 27.8 cm). Thus, we could have detected R. salmoni narum in Slovenia. It has also been hypothesized that R. salmoninarum disease, but can eliminate the pathogen under certain conditions (Bruno 1986; Lovely et al., 1994). study were sampled in winter when the mean - changes in temperature take place, i.e. spring and autumn (Ortega et al., 1995). This is the time when epizootics usually appear, but most mortalities occur at higher temperatures, although the reverse has also been reported (Sanders et al., 1978). According to Ortega et al. (1995), the R. salmoninarum in winter was less than 10% as opposed to 80% in autumn. Furthermore, the results should be viewed also is reputed to be the most sensitive test available R. salmoninarum detection (Miriam et al., types. However, inhibitory compounds and

6 Bull. Eur. Ass. Fish Pathol., 33(5) 2013, 141 the results in this study, DNA samples were R. salmoninarum detection taken into account when interpreting the results. - - kidney tissue was not described but the results Despite lacking more data on R. salmoninarum seems that in Slovenia, similarly as in England (Chambers et al., 2008), BKD might be more between R. salmoninarum R. salmoninarum al., 2008). situation regarding R. salmoninarum R. salmoninarum rapid diagnostic methods, should be continued salmonid populations, especially in endangered grayling. Acknowledgements data were kindly provided by colleagues at References Banner CR, Long JJ, Fryer JL and Rohovec Renibacterium salmoninarum Journal of Fish Diseases 9, Brown LL, Iwama GK, Evelyn TPT, Nelson polymerase chain reaction (PCR) to detect Renibacterium salmoninarum within individual salmonid eggs. Diseases of Aquatic Organisms 18, Bruneau NN, Thorburn MA and Stevenson Aeromonas salmonicida, Renibacterium salmoninarum, Ontario salmonid populations. Journal of Aquatic Animal Health 11, between 1976 and Aquaculture and Fishery Management 17,

7 142, Bull. Eur. Ass. Fish Pathol., 33(5) 2013 bacterial kidney disease (BKD) in Scotland between 1990 and Diseases of Aquatic Organisms 59, Chambers E, Gardiner R and Peeler EJ (2008). Renibacterium salmoninarum rainbow trout, Oncorhynchus mykiss selected river catchments in England and Wales between 1998 and Journal of Fish Diseases 31, Earp BJ, Ellis CH and Ordal EJ (1953). Kidney disease in young salmon. Washington, 1, Evelyn TPT, Hoskins GE and Bell GR (1973). an apparently wild salmonid in British Columbia. Journal of the Fisheries Research Board of Canada 30, Evenden AJ, Grayson TH, Gilpin ML and Munn CB (1993). Renibacterium salmoninarum and jigsaw. Annual Review of Fish Diseases 3, Hastein T and Lindstad T (1991). Diseases in wild and cultured salmon: possible interaction. Aquaculture 98, disease (BKD) in grayling (T. thymallus). (EAFP), pp. 9. problems in grayling (Thymallus thymallus Linnaeus, 1758) culture. Seventh Pathologists (EAFP), pp. 68. Renibacterium salmoninarum in wild Arctic charr and brown trout in Iceland. Journal of Fish Biology 53, Renibacterium salmoninarum Atlantic salmon. Journal of Aquatic Animal Health 6, MacLean DG, Yoder WG (1970). Kidney disease among Michigan salmon in The Progressive Fish Culturist 32, WH (1997). PCR and probe-pcr assays to monitor broodstock Atlantic salmon (Salmo salar Renibacterium salmoninarum. Journal of Clinical Microbiology 35, Mitchum DL, Sherman LE and Baxter GT Salvelinus fontinalis), brown trout ( ), and rainbow trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 36, Mitchum DL and Sherman LE (1981). Canadian Journal of Fisheries and Aquatic Sciences 38, Ortega C, Múzquiz JL, Docando J, Planas E, Alonso JL and Simón MC Veterinary Research 26, Pascho RJ, Chase D and McKibben CL (1998). linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid Journal of Veterinary Diagnostic

8 Bull. Eur. Ass. Fish Pathol., 33(5) 2013, , Pippy JHC (1969). Kidney disease in juvenile Atlantic salmon (Salmo salar) in the Margaree River. Journal of the Fisheries Research Board of Canada 26, Sanders JE, Pilcher KS and Fryer JL (1978). bacterial kidney disease in coho salmon (Oncorhynchus kisutch), sockeye salmon (O. nerka), and steelhead trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 35, Souter BW, Dwilow AG and Knight K (1987). Renibacterium salmoninarum in wild Arctic charr Salvelinus alpinus and lake trout S. namaycush Canada. Diseases of Aquatic Organisms 3, Renibacterium salmoninarum to salmonids. Journal of Aquatic Animal Health 9, 1 7.