INFLUENCE OF WATER TEMPERATURE TO STREPTOCOCCOSIS CAUSED BY STREPTOCOCCUS AGALACTIAE IN TILAPIA (OREOCHROMIS SP.)

Abstract

Streptococcus agalactiae is one of the most important pathogen affecting aquaculture business worldwide. Various warm-water fish species were reported to be infected by S. agalactiae but one of the most vulnerable species is tilapia (Oreochromis sp.). Presently, tilapia farming has been recognized as the most valuable fresh-water cultured fish industry in Thailand. Streptococcosis associated with S. agalactiae infection can produce high mortalities up to 70% according to acute septicemia condition within only short period as 1-2 weeks. However, the susceptibility to the infection can be affected by several factors such as fish size, stocking density, pH and temperature. Currently, the information obtained from both field and experimental studies were mentioning on the closely relationship between an increasing of temperature and occurrence of streptococcosis and its virulence. Generally, shifting of water temperature not only affect to the fish physiology but also to the pathogenic organisms. Therefore, the main objective of this study is to elucidate the effect of temperature to the tilapia and S. agalactiae by studying their physiological responses in parallel and observing the outcome of disease in different temperature condition. Streptococcus were obtained from the fish and environmental samples (such as mud and pond water) in tilapia farms in Thailand during 2009 to 2012. Samples were collected during both disease outbreak and disease-calming situation. The relative percent recovery of streptococcus from environmental samples was 13-67%. Totally 60 isolates of streptococcus were found and all of them were identified as β-hemolytic S. agalactiae using standard biochemical assays and species-specific PCR. Molecular serotyping of S. agalactiae revealed that 59 isolates belong to type Ia while only 1 isolates were type III. Additional genotypic analyses (infB allelic assay, virulence genes profiling and RAPD) of S. agalactiae suggested a large diversity among S. agalactiae strains which is tend to be geographically dependent. Comparison of genotypic characteristic with other strains of S. agalactiae isolated from mammalian host showed distinct genetic of fish-originated streptococcus apart from human and bovine strains. Experimental infection via intraperitoneal injection of S. agalactiae to Nile tilapia revealed that both fish and environmental strains were pathogenic, with 6.25-7.56 log CFU LD50. Due to this LD50 analysis, we select for the highest virulence strain to conduct the experiment in the next step. An increasing of cultured temperature of S. agalactiae tremendously enhances in vitro pathogenicity as expected. Comparison between S. agalactiae cultured in 35 ⁰C and 28 ⁰C difference temperature suggested that the bacterium can growth faster, producing more hemolysin, surface-capsular polysaccharide and tolerate to tilapia whole blood when high temperature condition was applied. The expression analysis of putative virulence genes by qPCR also revealed the concordance result as up-regulation of 9 genes (from 13 genes included in the test) in 35 ⁰C condition comparing with 28 ⁰C. Among these virulence genes, three were exhibited massive up-regulation with more than 15 to 40 folds increased. Theses 3 genes comprised of cylE, cfb and PI-2b which encoded for β-hemolysin/cytolysin, CAMP factor and pili backbone, respectively. Susceptibility of S. agalactiae infected Nile tilapia reared in hot (35 ⁰C) and normal (28 ⁰C) water temperature were determined from experimental infection analysis. The accumulated mortalities over 14 days period was 85% and 45% for hot and normal condition, respectively. Higher numbers of streptococcus were enumerated from the brain of infected tilapia kept in hot condition with 1000 times higher than normal condition. Abundant up-regulation of inflammatory related genes (COX-2, IL-1β and TNF-α) had been detected since 6 hpi until 96 hpi, which indicated for massive inflammation of the fish in high temperature condition. In conclusion, our study suggested that the increasing of S. agalactiae pathogenicity due to the high temperature condition can leads to massive inflammatory response of infected Nile tilapia and producing acute mortality.