The Mechanism Of Influence Of Different Water Temperature On Streptococcus Agalactiae Infection In Tilapia

Tilapia is an important freshwater farmed fish.It has excellent biological characteristics and can survive in poor quality water.Tilapia is also one of the best fish breeds in southern China.However,with the increase of the stocking density of fish and water quality deterioration in aquaculture,diseases caused by Streptococcus sp.,Flabobacterium columnare,Aeromonas hydrophila and Edwardsiella ictaluri have severely restricted the development of tilapia breeding industry.Among them,Streptococcicosis caused by Streptococcus iniae and Streptococcus agalactiae infection bring the most serious damage to tilapia culture.Tilapia disease was less and mainly caused by S.iniae before 2008.S.agalactiae became the most serious pathogenic bacteria of tilapia after a large outbreak of Streptococcicosis in tilapia in Hainan province,2009.Studies showed that the pathogenicity of S.agalactiae to tilapia was closely related to water temperature.When the water temperature exceeded 32°C,it was easy causing epidemic and outbreak of tilapia Streptococcus disease.The influence of temperature on the pathogenic mechanism of S.agalactiae on tilapia is not known.The research will illustrate the pathogenesis mechanism of S.agalactiae to tilapia mediated by temperature through investigating S.agalactiae dynamic change under different temperature and the effect of temperature on virulence gene expression of S.agalactiae.The main results are as follows:1.The dynamic distribution and elimination of S.agalactiae in tilapia under different water temperature.The plate method for counting viable bacteria was used.Nile tilapia was intraperitoneally injected with S.agalactiae.Vitro isolation,culture and counting of S.agalactiae were carried out for three experimental tilapia groups under 25 °C,29 °C and33 °C.The temporal variation of S.agalactiae in brain,liver,spleen and kidney were analyzed.The results showed that the growth rate of S.agalactiae was faster with the higher water temperature.The removal rate of S.agalactiae was faster with the higher peak concentration.It is speculated that the water temperature can affect the proliferation rate and survival time of S.agalactiae in tilapia.The peak concentrations in spleen were significantly higher than other organizations in all experimental groups.It is speculated that the spleen is the key target organ of S.agalactiae infection.2.The quantitative analysis of S.agalactiae in the tissues of tilapia under differentwater temperature.The fluorescence quantitative polymerase chain reaction(PCR)method was used to detect the change pattern of bacteria in liver,brain,intestine,gill,spleen,head kidney and body kidney of tilapia during different infection period of experimental groups at25 °C,low temperature-rising,29 °C,33 °C and high temperature-descending.Results showed that the S.agalactiae was detected in seven different organs after two hours.It is speculated that the proliferation of S.agalactiae in tilapia is fast.The reproduction of S.agalactiae in tilapia was faster and the fatality rate for tilapia was higher with the higher water temperature.The rapid rise and fall of temperature can also significantly affect the proliferation rate of S.agalactiae and the fatality rate of tilapia.It is speculated that the temperature is the key factor that affects the reproduction rate of S.agalactiae in tilapia.The peak concentration of S.agalactiae in head kidney,intestine,and spleen in the constant temperature group at 33 °C was significantly higher than the same tissue in other experimental groups.It is speculated that the head kidney,intestines and spleen are the key target organs of S.agalactiae infection.These experiments build the foundation for the in-depth study of the influence of temperature on the pathogenesis of Tilapia's Streptococcicosis.3.Effects of different water temperature on the virulence gene expression of S.agalactiaeThe growth rate of S.agalactiae under different water temperature was compared.The results showed S.agalactiae in vitro growth accelerated with the rising of temperature.The time of achieving logarithmic growth period was also shortened.The temperature could affect the proliferation rate of S.agalactiae.The virulence genes expression level of cfb,cylE,hylB,fbsA,bibA,pi-2b of S.agalactiae were detected by fluorescence quantitative PCR at different water temperature.The results showed that the expression of multiple virulence genes of S.agalactiae was affected by temperature.After the S.agalactiae was cultured in the stationary phase,the relative expression level of the virulence genes such as cfb,fbsA,bibA,pi-2b,and cylE increased with the increase of water temperature,and the relative expression level of the virulence gene hylB decreased.It is speculated that the temperature can affect the infection of tilapia by affecting the expression of virulence genes in S.agalactiae.