A novel O-serotype in Tenacibaculum maritimum strains isolated from cultured sole (Solea senegalensis)

Tenacibaculum maritimum was consistently isolated from tenacibaculosis outbreaks affecting sole farms in Portugal and south of Spain in the last two years. These new T. maritimum isolates could not be assigned within the two major serotypes (O1 and O2) already described. Rabbit antiserum was prepared against one Portuguese sole isolate to examine the antigenic relationships between the isolates from sole using microtitre agglutination tests, dot blot assay and immunoblotting of lipopolysaccharides. Serological characterization of the recent sole isolates demonstrated that they belong to a novel O-serotype named O3, allowing us to extend the serological scheme for this fish pathogen. This information is useful for epizootiological and vaccination studies. *Corresponding author’s email: mpaetjlb@usc.es Tenacibaculosis or flexibacteriosis is recognized as an important infectious disease in marine fish since 1979 (Hikida et al., 1979; McVicar and White, 1979). The presence of the etiological agent Tenacibaculum maritimum (formerly Flexibacter maritimus) was first demonstrated by Bernardet et al. (1990) and since then, this pathogen has spread among a wide variety of host species, producing significant losses in cultured fish such as turbot (Scophthalmus maximus), Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus kisutch), sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata) (Devesa et al., 1989; Pazos et al., 1993; Bernardet et al., 1994; Ostland et al., 1999) and, lately, in sole (Solea solea and S. senegalensis) (Cepeda and Santos, 2002; Avendaño-Herrera et al., 2004). In previous work we analysed the antigenic diversity of T. maritimum from sole, gilthead sea bream and turbot, which allowed us to propose a O-serotyping scheme for T. maritimum composed by two major serotypes (O1 and O2) mainly associated with the host species (AvendañoHerrera et al., 2004). However, since the middle of 2003, new outbreaks of tenacibaculosis have occurred in sole farmed in Portugal and south of Spain. These new T. maritimum isolates could not be assigned within the serotypes already described. We report here the existence of a Bull. Eur. Ass. Fish Pathol. , 25(2) 2005, 71 new serotype within this fish pathogen, which was proposed as serotype O3. Twelve T. maritimum strains isolated from sole in Portugal and south of Spain were used in the present study. The bacterial strains were confirmed as T. maritimum using the PCRbased analysis described by Toyama et al. (1996). The bacteria were routinely cultivated on F. maritimus Medium (FMM) (Pazos et al., 1996) at 24°C for 72 h. Stock cultures were maintained frozen at –70°C in Criobille tubes (AES Lab., France). The antigenic analysis were carried out using the thermostable antigens of each strain obtained after heat killing the bacterial suspensions (109 cells ml-1) in phosphate buffered saline (pH 7.4) at 100°C for 60 min, washed once in the same saline solution and maintained at 4°C until required. The Portuguese sole strain ACC13.1 was selected and used to obtain immune serum in rabbit according to the methods described by Sørensen and Larsen (1986). The serological analysis were performed with the unabsorbed and absorbed rabbit serum. For the absorption, the serum against isolate ACC13.1, as well as antisera against strains PC503.1 from sole (serotype O1) and PC424.1 from turbot (serotype O2) (Avendaño-Herrera et al., 2004), were mixed with the antigens of the heterologous isolates and incubated overnight at 4°C. This process was repeated twice to ensure a complete absorption of common antigens. The agglutination titers of the antiserum ACC13.1 were measured against the suspensions of O-antigens from the homologous and heterologous strains (Stevenson and Daly, 1982). The serological characterization of the twelve T. maritimum isolates was performed employing the dot blot assays as described by Cipriano et al. (1985) with the unabsorbed and absorbed antiserum n o i t a n i t u l g g a e r t i t o r c i M y a s s a t o l b t o D i t n a m u r e s h t i W i t n a m u r e s h t i W n e g i t n A 1 . 3 0 5 C P ) 1 O ( a 1 . 4 2 4 C P ) 2 O ( 1 . 3 1 C C A ) 3 O ( 1 . 3 0 5 C P ) 1 O ( 1 . 4 2 4 C P ) 2 O ( 1 . 3 1 C C A ) 3 O ( d e b r o s b A b