The infections of Gram-positive Streptococcus-like bacteria in fish played an important role to causing heavy economic losses of Taiwan aquaculture industry, especially during the high temperature seasons. Echymotic hemorrhages were found on the body surface of diseased fish. Within the visceral organs revealed the characteristic changes of bacteremia in the most of cases. The identification of the associated bacterial species has not been carried out as yet. A total of 86 isolates was collected from the infected fish for investigation. We made reference to 16S rRNA and designed a pair of primers for detection of the agents. API 20 STREP, API 50 CHL and NaCl tolerance test were simultaneously performed for double check. The results showed that four species of Streptococcus-like pathogens were identified. The frequency was shown as follows: Streptococcus agalactiae (44 isolates), Streptococcus iniae (23 isolates), Streptococcus difficile (16 isolates) and Lacotococcus garvieae (3 isolates). Both Streptococcus agalactiae and Streptococcus difficile mainly attacked the freshwater fish, and most of the marine fish were infected by Streptococcus iniae. In addition, Lacotococcus garvieae was found only in the grey mullet (Mugil cephalus). In order to take less time comsuption for the identification of Streptococcus spp., we designed species-specific primers for Streptococcus agalactiae and Streptococcus iniae. The specificity of multiplex PCR was assessed with a panel of 24 reference strains and the collected isolates of Streptococcus agalactiae and Streptococcus iniae. The results showed PCR products derived from amplification were observed only from target strains, indicating the species-specific primers could serve as a diagnostic tool for the broth Streptococci. Despite the 16S rRNA sequencing and biochemical characteristics, both the isolates of Streptococcus agalactiae and Streptococcus difficile had a high similarity. However, the colonies of Streptococcus difficile adhered strongly on the medium with a g hemolytic reaction. It was quite different from the b hemolytic Streptococcus agalactiae, which was lack of adhesive ability. All the isolates were sensitive to cephalexin, florfenicol, chloramphenicol and doxycycline, which could be recommended as reference drug to treatment.

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