High temperature is a main cause to result in the outbreak of tilapia streptococcal disease. However, the underlying mechanisms are not well understood. In this study, we first confirmed that tilapia infected with Streptococcus agalactiae (S. agalactiae) had a higher mortality at high temperature (35 °C) than that at normal temperature (28 °C). Subsequently, the effects of high temperature on gene expression pattern of S. agalactiae and intestinal microbiota of tilapia were respectively detected by RNA-seq and 16S rDNA sequencing. RNA-seq identified 357 differentially expressed genes (DEGs) in S. agalactiae cultured at 28 °C and 35 °C. GO and KEGG analysis showed that these DEGs were highly involved in metabolic processes, including glucose, lipid and amino acid metabolisms, which indicates that S. agalactiae have stronger vitality and are likely to be more infectious under high temperature. Microbiota analysis revealed that high temperature could influence the bacterial community composition of tilapia intestine, accompanied by changes in intestinal structure. Compared to feed at 28 °C, the total bacterial species as well as pathogens, such as norank_f__Rhizobiales_Incertae_Sedis, Pseudorhodoplanes, Ancylobacter, in tilapia intestine were significantly increased at 35 °C, which may weaken the immune resistance of tilapia. Taken together, our results suggest that high temperature evoked tilapia susceptible to S. agalactiae should be the combined effect of enhanced S. agalactiae metabolism and dysregulated tilapia intestinal microbiota.

Streptococcus agalactiae has a serious negative impact on tilapia aquaculture, and rapid detection of trace S. agalactiae in aquaculture waters and timely and effective sterilization measures could significantly reduce the probability of its outbreak in tilapia farming. Here, we established a fluorescence quantitative detection method of S. agalactiae in tilapia aquaculture waters based on the Streptococcus agalactiae CAMP factor (cfb) (GU217532.1) sequence of its CAMP factor. The results showed that the Ct value and logarithm of plasmid copy number presented a good linear relationship (y=3.49x+38.78; R²=0.997) in the plasmid concentration range of 1.5710²1.5710⁹ copies/μL, and the lowest concentration for sensitive detection of the established method was 1.5710² copies/μL. The method exhibited high specificity for the detection of S. agalactiae and generated negative results when using the DNA of Aeromonas veronii, Staphylococcus epidermidis, Aeromonas hydrophila, and Edwardsiella tarda as templates for qPCR. Intra- and inter-group repeated experiments produced variation coefficients lower than 2, indicating high stability and specificity of the method. The method was then used to detect S. agalactiae in two tilapia aquaculture waters. The results showed that the concentration of S. agalactiae was 12 copies/mL and 2 copies/mL in the two water samples, respectively. The method can directly detect trace S. agalactiae in the aquaculture water and facilitate the diagnosis, prevention, and control of S. agalactiae in tilapia aquaculture, which will greatly reduce the probability of large-scale outbreak of S. agalactiae and economic loss.