| Tilapia is one of the important export aquaculture species in Hainan Province.In recent years,streptococcosis caused by Streptococcus agalactiae with high morbidity and high mortality outbreak annually at high temperature period,causing huge economic losses to the tilapia industry and restricting the sustainable healthy development of tilapia aquaculture.Because lacking of effective disease prevent and control methods,studies on pathogenesis are urgently needed to find a new effective way for the prevention and treatment of streptococcosis.Temperature is one of the most important environmental factors that affect the outbreak of tilapia streptococcosis.In order to understand the effect of temperature on the physiological status of host and pathogen,the growth and virulence of two S.agalactiae strains PBSA05 and PBSA52 isolated from sick tilapia and the serum non-specific immune indexes of Nile tilapia at different temperatures were tested.The growth rate of two strains were accelerated with the increase of temperature at 25,30 and 35 ?,the lag-phase was relative short and the concentration of bacteria increased at high temperature.Hemolytic capacity increased with increasing temperature,it was significantly higher at 35? than at 25 ?.The non-specific adhesion to inert substrates and the specific adhesion to the tilapia surface mucus were significantly enhanced with increasing temperature.The activity of hyaluronidase in PBSA05 reached the peak at 30 ?,and was lowest at 25 ?,while the activity of hyaluronidase in PBSA52 was not affected by temperature.After incubation in tilapia whole blood,the survival rates of both strains increased significantly with the increase of culture temperature.The total antioxidant capacity and alkaline phosphatase activity of tilapia decreased with increasing temperature and maximized at 25 ?.Activity of alkaline phosphatase at 35 ? was significantly lower than 25 ?.Activity of lysozyme was significantly higher at 30 ? than at 25 ? and 35 ?,and the activity minimized at 35 ?.There were no significant changes in the activity of superoxide dismutase(SOD)and complement C3 in serum of tilapia at different temperatures.The accumulated mortalities of tilapia challenged with PBSA05 at 25 ? and 35 ? were 38.33%and 81.67%,respectively.For PBSA52,the accumulated mortalities were 17.67%and 71.67%,respectively.The results indicated that the temperature changes affect the physiological state of host and pathogen,and then lead to the difference of the incidence and symptoms.To elucidate the influence of temperature variation on the pathogen from a metabolic perspective,the global metabolomics of 2 pathogenic strains of S.agalactiae from sick tilapia were analyzed at 35 ? and 25 ? using ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)combined with pattern recognition approaches and pathway analysis.A total of 34 overlaped metabolites were identified from the two strains as biomarkers affected by temperature of S.agalactia.The result showed that the metabolic status of S.agalactiae was extensively affected by its culture temperature.Temperature changes affected nucleic acid metabolism,carbohydrate metabolism,amino acid metabolism and lipid metabolism.Metabolic changes were mainly related to the growth and replication,cell wall structure,membrane protein specificity and intracellular redox balance.Based on the results of metabolites contributing to these differences,a large number of nucleotides and their ramifications were markedly elevated at 35?.Various energy substances,components of the cell wall and substances associated with stress regulation such as glyceraldehyde 3-phosphate,pyroglutamic acid,glutamate,D-Alanyl-D-alanine,glycerophosphocholine,dephospho-CoA,and oxidized glutathione increased when the strains were cultured at 35 ?.Additionally,a general decrease in various precursors of capsule,antigen,and virulence protein formation were detected including mannose,maltotriose,N-acetyl-D-glucosamine 6-phosphate,uracil,proline,and citrulline.These metabolic changes indicated that metabolic activity decreased,while adaptive ability to environment and pathogenicity to host increased at high temperature.This study is the first to determine the metabolomic responses of S.agalactiae to temperature,and the results are useful to reveal its pathogenic mechanism and find effective disease control methods.The metabolomics based on gas chromatography-mass spectrometry(GC-MS)was used to compare the serum metabolic profile of tilapia at 25 ? and 35 ?.A total of 278 effective peaks were analyzed by principal component analysis(PCA)and partial least squares discriminate analysis(PLS-DA)models,and samples were separated with different temperatures.From the identified 87 metabolites,17 metabolites were selected change by t-test(p<0.1)and orthogonal partial least squares discriminate analysis(OPLS-DA)(VIP>1.0).Compared the tilapia serum in 35 ? group with these at 25 ? group,the contents of inositol,ribose,N-acetylmannosamine,hydroxylamine,lactic acid,3,6-anhydrogalactose,carbamoyl aspartic acid,glycerol,ketoglutaric acid,glutaric acid,3-hydroxyproline and 3-hydroxymethylglutaric acid were increased,and the contents of xylose,guanosine,oleic acid,palmitoleic acid and acetylgalactosamine were decreased.According to metabolic pathway analysis,a total of 6 metabolic pathways including triglyceride metabolism,phosphoinositide metabolism,conversion of pentose and glucuronic acid,tricarboxylic acid cycle,alanine,aspartic acid and glutamic acid metabolism,and purine metabolism were impacted by temperature change in tilapia serum.High temperature caused disturbance of energy metabolism in tilapia,increased catabolism of amino acids,slowed down protein synthesis and accelerated lipolysis.The effects of exogenous xylose were investigated by intragastric administration and intraperitoneal injection.The results showed that supplementation of xylose by intraperitoneal injection could improve the total antioxidant capacity and alkaline phosphatase activity of tilapia,while the intragastric administration of xylose was no significant effect on these two nonspecific immune parameters.The results provide a reference for improving nonspecific immunity of tilapia and preventing the occurrence of streptococcosis in high temperature season.Metabolomics analysis of tilapia infected with S.agalactiae was carried out by GC-MS.The changes of serum metabolites of tilapia at 25? or 35? after infection were compared respectively.The results showed that the metabolic profile of infected tilapia was naturally separated from the metabolic profile of healthy tilapia,the metabolic response of 35? group was stronger than that of 25 ? group,and carbohydrates and lipids in serum were greatly affected.From the identified 89 metabolites,27 metabolites in 35 ? group and 21 metabolites in 25 ? group were selected as potential biomarkers by foldchange and OPLS-DA(VIP>1.0,Foldchange>1.2 or<0.8).After infection with S.agalactia,6 metabolic pathways of tilapia were impacted at 25 ? and 8 metabolic pathways were impacted at 35?.The metabolic pathways which are commonly affected were arachidonic acid metabolism,glycolysis or gluconeogenesis,and pentose phosphate pathway.The contents of arachidonic acid and fructose 2,6-diphosphate decreased,while the content of 6-phosphate increased in tilapia after infection.Infection of S.agalactiae inhibited the glycolysis of tilapia and enhanced the inflammatory response associated with arachidonic acid metabolism.Tilapia with S.agalactiae were injected with 5 mg and 10 mg of fructose 1,6-diphosphate as a glycolysis accelerator by intraperitoneal injection once a day for five days.The survival rate of tilapia increased from 31.11%to 42.22%and 57.78%,respectively.The results of the study indicate the importance of metabolic pathway regulation during bacterial infection,and functional metabonomics can be used as an effective way to develop drugs and effective methods for hydrobios infectious diseases. |
PDF Full Text Request