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Tenacibaculosis is a fish disease that limits the culture of a variety of marine fish species of commercial value in the world. The genus Tenacibaculum includes several species, and their discrimination is of clinical interest in order to improve the management of an outbreak of the disease. In this study, a novel proteomic approach based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was evaluated for the identification and differentiation of Tenacibaculum species. The peak mass lists derived from MALDI-TOF-MS analysis were examined for the detection of potential biomarkers, similarity and cluster analysis and principal component analysis (PCA). Culture media used for bacterial growth did not affect the mass fingerprints. Eight genus-specific peaks were found in all the Tenacibaculum species analysed. Moreover, at least one species-specific peak was found in the species Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum dicentrarchi, Tenacibaculum litoreum and Tenacibaculum ovolyticum. These peaks could serve as biomarkers for the rapid identification of these bacterial fish pathogens. The cluster and PCA clearly separated the species T. maritimum, T. soleae, T. dicentrarchi and T. ovolyticum in different clusters. However, species of Tenacibaculum discolor and Tenacibaculum gallaicum were difficult to distinguish based on their protein fingerprints. To our knowledge, this is the first study that deals with the characterization and determination of biomarkers of Tenacibaculum species by MALDI-TOF mass spectrometry. This approach proved to be an effective and reliable technique for the discrimination of the Tenacibaculum species; therefore, it could be integrated as a routine diagnostic tool in microbiological laboratories.

This paper describes the predicted structure for the cps loci involved in capsule biosynthesis for Streptococcus parauberis serotypes III, IV, and V. Based on the specific serotype regions I, II, and III, a multiplex PCR protocol (mPCR) was designed to differentiate the main serotypes causing fish diseases. A real-time PCR method (qPCR) is also described to identify S. parauberis of serotype III in bacterial cultures and fish tissues. In silico and in vitro analyses revealed that both methods have a 100% specificity. The mPCR assay was optimized for the detection of S. parauberis strains of subtypes Ia (amplicon size 213 bp), subtypes Ib and Ic (both amplicon size 303 bp), serotype II (amplicon size 403 bp), and serotype III (amplicon size 130 bp) from bacterial cultures. The qPCR assay was optimized for the identification and quantification of S. parauberis serotype III strains in bacterial cultures and fish tissues. This assay achieved a sensitivity of 2.67 × 10 2 gene copies (equivalent to 3.8 × 10 −9 ng/μl) using pure bacterial cultures of S. parauberis serotype III and 1.76 × 10 2 gene copies in fish tissues experimentally and naturally infected with S. parauberis of the serotype III. The specificity and sensitivity of the protocols described in this study suggest that these methods could be used for diagnostic and/or epidemiological purposes in clinical diagnostic laboratories. Key Points• Structure of loci cps for S. parauberis of serotypes III, IV and V was described.• mPCR to differentiate S. parauberis serotypes causing disease in fish was optimized.• qPCR assay to quantify strains of S. parauberis serotype III in fish tissues.

In the present study, the potential of serological methods, the repetitive extragenic palindromic PCR (REP-PCR) and the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) for the typing of the species Tenacibaculum maritimum, Tenacibaculum soleae and Tenacibaculum discolor was evaluated. Moreover, molecular and proteomic techniques were used to assess variability among strains belonging to different serotypes, as well as isolated from different host species and geographical areas. Slide agglutination and dot-blot assays demonstrated the lack of immunological relationships among Tenacibaculum species analyzed. The serotype O1 was predominant within T. maritimum isolates regardless of the fish species or geographical area. Two serotypes were distinguished within T. soleae isolates and at least one within T. discolor strains. Species- and strain-specific profiles were obtained from the analysis of T. maritimum, T. soleae and T. discolor by REP-PCR and ERIC-PCR, demonstrating their potential as diagnostic tools. The genotyping analysis using both techniques showed genetic variability among the strains of each fish pathogenic Tenacibaculum species analysed. However, Tenacibaculum strains isolated from different host species or geographical areas or belonging to different serotypes produced REP and ERIC profiles with high similarity. Analysis by MALDI-TOF-MS of the T. maritimum strains could not detect any serotype-identifying biomarkers. Serotype-specific mass peaks were found for the serotypes O1 and O2 of T. soleae and for the serotype O1 of T. discolor. However, no relationships between the proteomic profiles and the source of isolation of the strains were obtained for any of the Tenacibaculum species analysed in this study.

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