Phenotypic and genotypic characterization of Streptococcus spp. isolated from tilapia (Oreochromis spp.) cultured in river-based cage and earthen ponds in Northern Thailand.

Streptococcus spp. are major pathogenic bacteria associated with massive mortality in tilapia. This study investigated the phenotypic and genotypic characterization of Streptococcus agalactiae (GBS) and Streptococcus iniae (S. iniae) isolated from tilapia in river-based floating cage and earthen pond farms in northern Thailand. Isolates were identified by biochemical and molecular analyses. Capsular typing, enterobacterial repetitive intergenic consensus polymerase chain reaction and multilocus sequence typing were performed to investigate the genetic relatedness. Six and one isolates were confirmed as GBS and S. iniae, respectively. All Streptococcus spp. isolates were obtained from 4 river-based cage farms (4/33), while samples collected from earthen pond farms (N = 28) were negative for streptococcosis. All GBS with serotype Ⅲ and sequence type (ST) 283 was observed. The ß-haemolytic GBS isolates were resistant to five antimicrobials, while the S. iniae was susceptible to all antimicrobials. This study indicates both GBS and S. iniae are the major bacterial pathogens responsible for streptococcosis infection in farmed tilapia of northern Thailand with GBS as dominant species. This survey highlights that the river-based cage farms seriously impact on the healthy development of the tilapia industry.

Identification and screening of effective protective antigens for channel catfish against Streptococcus iniae.

Vaccination is a potential approach for prevention and control of disease in fish. The use of genetically engineered vaccines is an effective method and a green intervention to control bacterial infection in aquaculture. However, efforts to develop these vaccines are limited by the lack of conserved protective antigens. In this study, three candidate immunogens (Srr, NeuA, and Hsp) of the pathogenic Streptococcus iniae strain DGX07 isolated from diseased channel catfish were identified and analyzed. Molecular cloning, expression, and purification of candidate antigen genes were carried out to obtain the candidate immunogens in the form of recombinant subunit vaccines. Western blotting was performed to evaluate immunogenicity in vitro and channel catfish were vaccinated by intraperitoneal injection and the specific antibody titers and relative percent of survival were determined to evaluate immune protection in vivo. The results showed that these three candidate immunogens were expressed correctly as recombinant proteins fused with His tags, with molecular weights of 70 kDa for Srr, 86 kDa for NeuA, and 51 kDa for Hsp, respectively. Moreover, each immunogen was predicted to be located either extracellularly or on the surface of S. iniae, and were able to offer protection against S. iniae infection in the form of recombinant subunit vaccines with adjuvant ISA763, especially Srr, with a relative percent of survival of 70% for Srr, 55% for NeuA, and 50% for Hsp, respectively.

Rapid MALDI biotyper-based identification and cluster analysis of Streptococcus iniae.

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either "no reliable identification" or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.