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Abstract [Objectives]This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae.
[Methods]S. iniae was incubated with brain heart infusion agar medium （BHIA + 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS， centrifuged， and filtered through microporous filtering film to collect extracellular products （ECPs）.
[Results]Extracellular proteinase （ECPase） of S. iniae exhibited amylase， protease， lecitinase， gelatinase， lipase activities and hemolytic activity but had no urease activity. EDTA， DTT and PMSF could reduce ECPase activity to 72.4%， 77.6% and 72.4%， respectively. Cu2+， Ca2+， K+ and Mg2+ exhibited an inhibitory effect on ECPase activity， whereas Fe3+， Co2+ and Mn2+ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Twodimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs， and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected， and the molecular weights mainly ranged between 26-95 kDa. The mouse antiS. iniae was used for Westernblot analysis of ECPs， and the results showed that there were four proteins， with molecular weights of 26， 37， 95， and 97 kDa， respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.
[Conclusions]This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae.
Key words Streptococcus iniae； Extracellular products； Heat stability； Mortality rate
Streptococcus iniae is a betahemolytic Grampositive bacterium， which was first isolated in 1976 from an Amazon freshwater dolphin， Inia geoffrensis， with skin ulcers[1]. Since the 1990s， there has been a sharp rise in the incidence of streptococcal disease caused by S. iniae in fishes. At present， S. iniae has been considered as an important pathogenic bacterium causing illness and death of a large number of farmed and wild fishes such as tilapia， American redfish， channel catfish， Siganus， rainbow trout， Paralichthys， wild Siganus chrysospilos， striped grouper and Synodus variegatus[2-8]， which has also been identified as a pathogen that can cause human inflammatory soft tissue infections[9-10]. Extracellular products （ECPs） are the metabolic products released by pathogenic bacteria during growth and reproduction and serve as one of the main components for invasion of pathogenic bacteria. A large number of studies have shown that ECPs are one of the most important pathogenic factors in many fish diseases[11-14]. However， studies on ECPs from S. iniae have rarely been reported in China. Shin et al.[15]used ECPs from S. iniae as a vaccine to find antigens. In this study， ECPs were extracted from S. iniae to analyze their biological activity and physicochemical properties， aiming at providing an important theoretical basis for the investigation of the pathogenic mechanism of ECPs， development of subunit vaccines and immunological control of streptococcal diseases.
Materials and Methods
Materials
Strains
Streptococcus iniae strain ZJMX04 was isolated and identified from diseased genetically improved farmed tilapia （GIFT） in Guangxi[16]and preserved in Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals.
Experimental animals
Genetically improved farmed tilapia （GIFT） was purchased from Maxie Aquafarm in Zhanjiang City， Guangdong Province， which was temporarily raised at the Donghai Island Breeding Base of Guangdong Ocean University. Eightweekold male SPF mice were donated by Guangzhou Winsun Pharmaceutical Co.， Ltd.
Reagents
IPG Drystrip （pH 4-7）， IPG Buffer （pH 4-7）， protease inhibitor， ribozyme inhibitor， Coomassie Brilliant Blue R250， urea， thiourea， and iodoacetamide were purchased from GE Healthcare Co.， Ltd.； CHAPS was purchase from MD Corporation； acrylamide， methylene bisacrylamide， TEMED， and dithiothreitol （DTT） were purchased from AMRESCO Inc.； prestained protein Marker was purchased from MBI Inc.； SDS was purchased from MBCHM； Freunds complete adjuvant and Freunds incomplete adjuvant were purchased from Sangon Biotech （Shanghai） Co.， Ltd.； DAB chromogenic kit was purchased from Wuhan Boster Biological Technology Co.， Ltd.； HRPlabeled goat antimouse IgG was purchased from Wuhan Boster Biological Technology Co.， Ltd.； PVDF membrane was purchased from PALL Corporation.
Preparation of ECPs from S. iniae
ECPs of S. iniae were prepared according to the reference[15]with slight modifications as follows： 1 ml of activated S. iniae bacterial liquid was coated on brain heart infusion agar plate （containing 4% calf serum） medium that was covered with cellophane and cultured at 30 ℃ for 60 h； bacterial liquid was rinsed with PBS （pH 7.4） and centrifuged at 2 000 r/m for 30 min at 4 ℃； the supernatant was filtered through a 0.22 μm microporous filtering film， and concentrated by a concentration tube （Amicon）. The protein content was determined by the Bradford method. The collected ECPs of S. iniae were stored in a -20 ℃ refrigerator. Analysis of the characteristics of ECPs from S. iniae
Qualitative analysis of S. iniae ECPase activity
S. iniae ECPase activity was analyzed in accordance with the reference[12]： seven types of sterile agar plates （pH 7.5）， containing casein （1.0%）， skim milk （1.0%）， gelatin （1.0%）， soluble starch （0.2%）， egg yolk （2.5%）， Tween 80 （1.0%）， urea （2.7%） and phenol red， were separately prepared with distilled water. The plates were punched with holes and added with 20 μl of ECPs； plates added with an equal volume of saline （0.85%） were used as a negative control. After incubation for 48 h at 30 ℃， casein agar plates and skim milk agar plates were added with trichloroacetic acid solution （10%， W/V）； gelatin agar plates were added with acidic mercuric chloride solution （0.1 mol/L）； starch agar plates were added with Lugols iodine solution. The presence of transparent rings （gelatin， casein， skim milk， starch plates） or absence of transparent rings （egg yolk， Tween 80 plates） or turning red （urea plates） suggested positive reactions.
Hemolysis test of ECPs
The hemolysis test was conducted by diffusion method with rabbit blood agar plates. The plates were punched with holes and added with 20 μl of ECPs； plates added with an equal volume of saline （0.85%） were used as a negative control. After incubation for 48 h at 30 ℃， the presence or absence of hemolysis were observed. The hemolytic titer of ECPs against tilapia erythrocytes was determined as follows： ECPs were diluted exponentially with PBS on 96well microplates， added with an equal amount of 1% tilapia erythrocytes， incubated at 37 ℃ for 1 h， and stored overnight in a -4 ℃ refrigerator. The highest dilution rate of ECPs that allows lysis of 50% of the red blood cells was identified as the hemolytic titer.
Detection of ECPase
ECPase was detected according to the method proposed by Henry et al.[17]with slight modifications as follows： 0.5 g of azocasein （Sigma） was dissolved in 100 ml of 0.05 mol/L TrisHCl buffer （pH 8.0）； 100 μl of azocasein was mixed with 100 μl of different dilutions of ECPs， diluted with TrisHCl buffer to 500 μl， incubated at 37 ℃ for 30 min， added with 400 μl of 10% （W/V） trichloroacetic acid and placed at room temperature for 30 min to terminate the reaction； the mixture was centrifuged at 12 000 r/min for 5 min； the precipitated protein was removed， whereas the supernatant was transferred to another centrifuge tube and added with 1 000 μl of 0.525 mol/L NaOH for chromogenic reaction. OD442 value was determined. An enzyme activity unit was defined as the amount of the enzyme that increased 0.01 OD442 every 30 min. Effect of temperature on ECPase activity
ECPs of S. iniae were incubated with the substrate （azocasein） for 30 min at 4， 15， 25， 37， 45， 55， 65， 75， 85， and 100 ℃， respectively. ECPase activity at different temperatures was determined with enzyme activity at 4 ℃ as a control （100% relative enzyme activity）.
Heat stability
ECPs of S. iniae were incubated for 30 min at 4， 15， 25， 37， 45， 55， 65， 75， 85， and 100 ℃， respectively， immediately immersed in an icewater bath， and added with the substrate. The changes in ECPase activity were detected with enzyme activity at 4 ℃ as a control （100% relative enzyme activity）.
Effect of inhibitors on ECPase activity
ECPs of S. iniae were incubated with different inhibitors for 60 min at 37 ℃. The changes in ECPase activity were detected. The final concentrations of different inhibitors were shown in Table 1.
Effect of pH on ECPase activity
PBS buffer was adjusted to pH 3-10 with 1 mol/L HCl or 1 mol/L NaOH， added with 0.1 ml of ECPs， incubated at 4 ℃ for 1 h， and added with the substrate （azocasein） that was prepared in distilled water. The changes in ECPase activity were detected with the highest enzyme activity as a control （100% relative enzyme activity）.
Analysis of ECPs by SDSPAGE
ECPs were added with an equal volume of 2× electrophoresis loading buffer and boiled for 3-5 min. The Lamemmli discontinuous SDSPAGE system was used with 5% concentration gel and 12% separation gel. The initial voltage at room temperature was 80 V， which was increased to 120 V when the sample was on the separation gel. When the bromophenol blue reached the edge of the separation gel， the gel was removed for Coomassie brilliant blue G250 staining， followed by decolorization.
Analysis of ECPs by twodimensional electrophoresis
Twodimensional electrophoresis was performed according to the operation manual of GE Healthcare with slight modifications.
Hydration
For 7 cm pH 4-7 strips， each strip was loaded with 120 μg of protein and soak for 12 h at 20 ℃.
Primary isoelectric focusing electrophoresis
①100 V 1 h， 200 V 1 h， 300 V 30 min， 1 000 V 1 h， 5 000 V 2 h， 5 000 V 2 h to the completion of isoelectric focusing. ② After completion of isoelectric focusing， IPG strips were quickly removed and placed in equilibrium solution A （6 mmol/L urea， 2% SDS， 50 mmol/L TrisHCl pH 8.8， 30% glycerol， 0.2% DTT， a trace of bromophenol blue） and equilibrium solution B （6 mmol/L urea， 2% SDS， 50 mmol/L TrisHCl pH 8.8， 30% glycerol， 3% iodoacetamide， a trace of bromophenol blue） for 15 min， respectively. Secondary vertical SDSPAGE electrophoresis
The wellbalanced 7 cm strip was transferred to a small electrophoresis tank for secondary electrophoresis with 12% polyacrylamide gel. The voltage was controlled at 80 V for 30 min and turned to 180 V until bromophenol blue line reached the bottom of the gel. The test was repeated 3 times.
Coomassie brilliant blue staining
The gel was peeled off from the glass plate， soaked in fixative （10% glacial acetic acid， 40% methanol）， stained in staining solution （10% glacial acetic acid， 45% methanol， 0.1% Coomassie brilliant blue R250） for 4 h or overnight， and decolorized in decolorization solution （5% glacial acetic acid， 20% methanol） for 3 h.
Pathogenicity of ECPs to tilapia
By using the modified Karbers method[18]， tilapia was injected intraperitoneally. Fishes with an average body weight of 10 to 15 g were divided into five groups， 10 individuals per group. Each fish in the control group was injected with 0.1 ml of 0.85% sterile saline. All the fishes were observed for 10 d and the number of deaths was recorded.
Analysis of immunogenicity of ECPs
Preparation of immune serum
After incubation for 24 h， S. iniae bacterial liquid was added into 0.4% formalin and inactivated at 28℃ with shaking at 150 r/min. The inactivated broth was centrifuged and the precipitate was collected. Finally， the collected cells were diluted with sterile PBS to immunize mice. The immunization was conducted for four weeks. In the first week， inactivated vaccine was mixed completely with Freunds complete adjuvant （1∶1） for immunization by multipoint subcutaneous injection （0.1 ml per point）； in other three weeks， inactivated vaccine was mixed completely with Freunds incomplete adjuvant （1∶1） for immunization with the same injection method. Four weeks later， blood samples were collected from the eyeballs and placed at room temperature for 3-4 h. Solidified blood samples were placed at 4 ℃ overnight for blood clot retraction， which were centrifuged at 4 500 r/min for 10 min the next day. The supernatant was collected as the serum and stored at -80 ℃. The titer was determined by DotELISA method[19].
Westernblot analysis
The prepared ECPs were subjected to SDSPAGE electrophoresis. Protein transfer was performed for about 2 h at 4℃， 100 V by using a BIORAD protein transfer apparatus according to the conventional method. Westernblot was conducted with 1∶100 mouse antiserum as the primary antibody and 1∶1 000 HRPlabeled goat antimouse IgG as the secondary antibody. Results and Analysis
Analysis of biological activity of ECPs from S. iniae
Qualitative analysis of enzyme activities of S. iniae ECPs
ECPs have the activities of five enzymes including gelatinase， protease， amylase， lecitinase and lipase （Fig. 1）. The results showed that ECPs exhibited the highest activity of gelatinase （the diameter of transparent ring reached 25 mm）， followed by activities of lipase， amylase and protease （the diameter of transparent ring reached 22， 15 and 10 mm， respectively）； ECPs exhibited the lowest activity of lecitinase （the diameter of transparent ring reached 9 mm）. However， urease activity was not detected.
Effect of temperature on ECPase activity
ECPase activity was determined using casein as the substrate. As shown in Fig. 2， ECPase activity of S. iniae increased gradually when the temperature was 25 ℃， reached the peak level when the temperature was 55 ℃， and declined gradually when the temperature continued to rise.
According to the heat stability， ECPase of S. iniae was relatively stable to heat. Specifically， ECPase activity of S. iniae remained at a relatively stable level when the temperature was below 55 ℃ and began to decline when the temperature was higher than 55 ℃. Analysis of variance showed that there were significant differences between treatments.
Effect of inhibitors on ECPase activity
As shown in Table 1， EDTA， DTT and PMSF could reduce ECPase activity to 72.4%， 77.6% and 72.4%， respectively. Cu2+， Ca2+， K+ and Mg2+ exhibited an inhibitory effect on ECPase activity， whereas Fe3+， Co2+ and Mn2+ could activate ECPase activity. Specifically， Mn2+ had the most significant activating effect on ECPase activity of S. iniae.
Effect of pH on ECPase activity
As shown in Fig. 3， ECPase activity of S. iniae was relatively weak under acidic conditions， increased significantly when pH was greater than 7， and reached the peak level when pH was up to 9. Moreover， at pH 10， ECPase activity of S. iniae was relatively high.
The results of primary isoelectric focusing electrophoresis and secondary vertical SDSPAGE electrophoresis were shown in Fig， 4 and Fig.5. There are about 12 main bands of ECPs， and the molecular weights mainly ranged between 28-68 kDa （Fig. 4）. ECPs were isolated by secondary electrophoresis at pH 4-7. About 120 protein spots were detected by the software， and the molecular weights mainly ranged between 26-95 kDa. Westernblot analysis of ECPs
According to DotELISA determination results， the titer of mouse antiS. iniae was 1∶500. The mouse antiS. iniae was used for Westernblot analysis of ECPs. As shown in Fig. 6， there were four main immuno strips， and the molecular weights were 26， 37， 95， and 97 kDa， respectively.
Pathogenicity of ECPs to tilapia
As shown in Table 2， at the initial stage after the injection of ECPs， several tilapia individuals exhibited slow action， loss of appetite and reduced balance capacity， while several individuals swam to the surface of the water or stayed in the bottom of the water. As the disease progressed， tilapia fins and tails exhibited hyperemia. There were erosive substances in the abdominal cavity and unevenly colored liver in serious cases. As could be seen from Table 2， ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.
Conclusion and Discussion
Pathogenicity of ECPs
ECPs play an important role in the pathogenic process of aquatic animal pathogens. So far， it has been found that ECPs of various aquatic animal pathogens， such as Vibrio parahaemolyticus， Vibrio alginolyticus， Vibrio vulnificus and Aeromonas hydrophila， have remarkable pathogenicity[12， 20-22]. The present study indicated that ECPs from S. iniae had amylase， protease， lecitinase， gelatinase， lipase activities and hemolytic activity. Previous studies have shown that gelatinase and protease could decompose collagen and other protein components， while lipase could decompose lipids， and the combined action of gelatinase， protease and lipase could destroy membranous structure， which leads to extensive injury in tissue structure， thus causing degeneration and death， resulting in the reduction or loss of functions of various tissues and organs or even death. Moreover， hemolysin could lyze red blood cells， leading to a decrease in the number of red blood cells in blood and a reduction in oxygen carrying capacity， thereby causing anoxia and accelerating the death of animals[23]. In the present study， ECP inoculation of tilapia indicated that ECPs were highly pathogenic to tilapia， which further confirmed that ECPs played an important role in the pathogenic process of S. iniae. Fuller et al.[24]and Locke et al.[25]elaborated the pathogenic effect of hemolysin S from S. iniae. However， the exact roles of other components of ECPs from S. iniae during the pathogenesis remain to be investigated. Effect of environmental conditions on ECPase activity
The study found that ECPs of S. iniae had relatively high enzyme activity under appropriate conditions. The optimal temperature for ECPs was 55 ℃. ECPs exhibited high heat stability and ECPase activity was relatively high under alkaline conditions. Bisno et al.[26]reported that the amount of ECPase secreted by group A streptococcus was closely related to environmental conditions， such as culture time， nutrient level， pH， and temperature. These environmental conditions were important to antiphagocytosis of group A streptococcus. So far， a large number of studies have investigated the effect of environmental conditions on ECPase of Vibrio alginolyticus， Vibrio anguillarum and Aeromonas hydrophila. The results showed that ECPase activity was closely related to the environmental conditions[12-13， 21].
Electrophoretic analysis and immunogenicity assay of ECPs
In this study， the main bands of ECPs of S. iniae were analyzed by one and twodimensional electrophoresis. The results of onedimensional electrophoresis showed that there are about 12 main bands of ECPs， and the molecular weights mainly ranged between 28-68 kDa； the results of twodimensional electrophoresis showed that the molecular weights mainly ranged between 26-95 kDa， and 120 protein spots were detected at pH 4-7. Shin et al.[15]analyzed the protein spots of ECPs of S. iniae cultured for different time by twodimensional electrophoresis technique. The results showed that ECPs of S. iniae cultured for 60 h had more protein spots， and 150 protein spots were found at pH 4-7. The differences might be related to different strains. Zhang et al.[27]analyzed ECP profiles of virulent and avirulent strains of S. suis by twodimensional electrophoresis technique and found that the number of protein spots of ECPs might by affected by various factors， such as sample preparation， sample processing， and electrophoresis conditions. In addition， ECP profiles suggested that 50 protein spots were present only in the avirulent strains but not in the virulent strains， which indicated that there might be differences in the electrophoresis patterns between different strains.
Moreover， the immunogenicity of ECPs of S. iniae was analyzed by mouse antiS. iniae. The results showed that there were four proteins， with molecular weights of 26， 37， 95， and 97 kDa， respectively. Shin et al.[15]detected the immunogenicity of ECPs by S. iniae vaccine and found that ECPs of S. iniae had multiple antigenic spots at pH 4-7， with molecular weights of 27， 35， 37， 42 and 45 kDa， which was similar to the molecular weights of main proteins in the present study. However， largemolecularweight antigen proteins were present in this study， whereas most proteins analyzed by Shin et al.[15]exhibited small molecular weights， which might be related to pH for twodimensional electrophoresis or different strains. The determination of immunogenicity of ECPs from S. iniae laid a theoretical foundation for the research and development of ECP subunit vaccines and the control of aquatic animal diseases. Which proteins served as immunogenic proteins in the present study and whether these proteins could be used to develop new vaccines against S. iniae remain to be studied. References
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Editor： Chunmei WU Proofreader： Xinxiu ZHU
[Methods]S. iniae was incubated with brain heart infusion agar medium （BHIA + 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS， centrifuged， and filtered through microporous filtering film to collect extracellular products （ECPs）.
[Results]Extracellular proteinase （ECPase） of S. iniae exhibited amylase， protease， lecitinase， gelatinase， lipase activities and hemolytic activity but had no urease activity. EDTA， DTT and PMSF could reduce ECPase activity to 72.4%， 77.6% and 72.4%， respectively. Cu2+， Ca2+， K+ and Mg2+ exhibited an inhibitory effect on ECPase activity， whereas Fe3+， Co2+ and Mn2+ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Twodimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs， and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected， and the molecular weights mainly ranged between 26-95 kDa. The mouse antiS. iniae was used for Westernblot analysis of ECPs， and the results showed that there were four proteins， with molecular weights of 26， 37， 95， and 97 kDa， respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.
[Conclusions]This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae.
Key words Streptococcus iniae； Extracellular products； Heat stability； Mortality rate
Streptococcus iniae is a betahemolytic Grampositive bacterium， which was first isolated in 1976 from an Amazon freshwater dolphin， Inia geoffrensis， with skin ulcers[1]. Since the 1990s， there has been a sharp rise in the incidence of streptococcal disease caused by S. iniae in fishes. At present， S. iniae has been considered as an important pathogenic bacterium causing illness and death of a large number of farmed and wild fishes such as tilapia， American redfish， channel catfish， Siganus， rainbow trout， Paralichthys， wild Siganus chrysospilos， striped grouper and Synodus variegatus[2-8]， which has also been identified as a pathogen that can cause human inflammatory soft tissue infections[9-10]. Extracellular products （ECPs） are the metabolic products released by pathogenic bacteria during growth and reproduction and serve as one of the main components for invasion of pathogenic bacteria. A large number of studies have shown that ECPs are one of the most important pathogenic factors in many fish diseases[11-14]. However， studies on ECPs from S. iniae have rarely been reported in China. Shin et al.[15]used ECPs from S. iniae as a vaccine to find antigens. In this study， ECPs were extracted from S. iniae to analyze their biological activity and physicochemical properties， aiming at providing an important theoretical basis for the investigation of the pathogenic mechanism of ECPs， development of subunit vaccines and immunological control of streptococcal diseases.
Materials and Methods
Materials
Strains
Streptococcus iniae strain ZJMX04 was isolated and identified from diseased genetically improved farmed tilapia （GIFT） in Guangxi[16]and preserved in Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals.
Experimental animals
Genetically improved farmed tilapia （GIFT） was purchased from Maxie Aquafarm in Zhanjiang City， Guangdong Province， which was temporarily raised at the Donghai Island Breeding Base of Guangdong Ocean University. Eightweekold male SPF mice were donated by Guangzhou Winsun Pharmaceutical Co.， Ltd.
Reagents
IPG Drystrip （pH 4-7）， IPG Buffer （pH 4-7）， protease inhibitor， ribozyme inhibitor， Coomassie Brilliant Blue R250， urea， thiourea， and iodoacetamide were purchased from GE Healthcare Co.， Ltd.； CHAPS was purchase from MD Corporation； acrylamide， methylene bisacrylamide， TEMED， and dithiothreitol （DTT） were purchased from AMRESCO Inc.； prestained protein Marker was purchased from MBI Inc.； SDS was purchased from MBCHM； Freunds complete adjuvant and Freunds incomplete adjuvant were purchased from Sangon Biotech （Shanghai） Co.， Ltd.； DAB chromogenic kit was purchased from Wuhan Boster Biological Technology Co.， Ltd.； HRPlabeled goat antimouse IgG was purchased from Wuhan Boster Biological Technology Co.， Ltd.； PVDF membrane was purchased from PALL Corporation.
Preparation of ECPs from S. iniae
ECPs of S. iniae were prepared according to the reference[15]with slight modifications as follows： 1 ml of activated S. iniae bacterial liquid was coated on brain heart infusion agar plate （containing 4% calf serum） medium that was covered with cellophane and cultured at 30 ℃ for 60 h； bacterial liquid was rinsed with PBS （pH 7.4） and centrifuged at 2 000 r/m for 30 min at 4 ℃； the supernatant was filtered through a 0.22 μm microporous filtering film， and concentrated by a concentration tube （Amicon）. The protein content was determined by the Bradford method. The collected ECPs of S. iniae were stored in a -20 ℃ refrigerator. Analysis of the characteristics of ECPs from S. iniae
Qualitative analysis of S. iniae ECPase activity
S. iniae ECPase activity was analyzed in accordance with the reference[12]： seven types of sterile agar plates （pH 7.5）， containing casein （1.0%）， skim milk （1.0%）， gelatin （1.0%）， soluble starch （0.2%）， egg yolk （2.5%）， Tween 80 （1.0%）， urea （2.7%） and phenol red， were separately prepared with distilled water. The plates were punched with holes and added with 20 μl of ECPs； plates added with an equal volume of saline （0.85%） were used as a negative control. After incubation for 48 h at 30 ℃， casein agar plates and skim milk agar plates were added with trichloroacetic acid solution （10%， W/V）； gelatin agar plates were added with acidic mercuric chloride solution （0.1 mol/L）； starch agar plates were added with Lugols iodine solution. The presence of transparent rings （gelatin， casein， skim milk， starch plates） or absence of transparent rings （egg yolk， Tween 80 plates） or turning red （urea plates） suggested positive reactions.
Hemolysis test of ECPs
The hemolysis test was conducted by diffusion method with rabbit blood agar plates. The plates were punched with holes and added with 20 μl of ECPs； plates added with an equal volume of saline （0.85%） were used as a negative control. After incubation for 48 h at 30 ℃， the presence or absence of hemolysis were observed. The hemolytic titer of ECPs against tilapia erythrocytes was determined as follows： ECPs were diluted exponentially with PBS on 96well microplates， added with an equal amount of 1% tilapia erythrocytes， incubated at 37 ℃ for 1 h， and stored overnight in a -4 ℃ refrigerator. The highest dilution rate of ECPs that allows lysis of 50% of the red blood cells was identified as the hemolytic titer.
Detection of ECPase
ECPase was detected according to the method proposed by Henry et al.[17]with slight modifications as follows： 0.5 g of azocasein （Sigma） was dissolved in 100 ml of 0.05 mol/L TrisHCl buffer （pH 8.0）； 100 μl of azocasein was mixed with 100 μl of different dilutions of ECPs， diluted with TrisHCl buffer to 500 μl， incubated at 37 ℃ for 30 min， added with 400 μl of 10% （W/V） trichloroacetic acid and placed at room temperature for 30 min to terminate the reaction； the mixture was centrifuged at 12 000 r/min for 5 min； the precipitated protein was removed， whereas the supernatant was transferred to another centrifuge tube and added with 1 000 μl of 0.525 mol/L NaOH for chromogenic reaction. OD442 value was determined. An enzyme activity unit was defined as the amount of the enzyme that increased 0.01 OD442 every 30 min. Effect of temperature on ECPase activity
ECPs of S. iniae were incubated with the substrate （azocasein） for 30 min at 4， 15， 25， 37， 45， 55， 65， 75， 85， and 100 ℃， respectively. ECPase activity at different temperatures was determined with enzyme activity at 4 ℃ as a control （100% relative enzyme activity）.
Heat stability
ECPs of S. iniae were incubated for 30 min at 4， 15， 25， 37， 45， 55， 65， 75， 85， and 100 ℃， respectively， immediately immersed in an icewater bath， and added with the substrate. The changes in ECPase activity were detected with enzyme activity at 4 ℃ as a control （100% relative enzyme activity）.
Effect of inhibitors on ECPase activity
ECPs of S. iniae were incubated with different inhibitors for 60 min at 37 ℃. The changes in ECPase activity were detected. The final concentrations of different inhibitors were shown in Table 1.
Effect of pH on ECPase activity
PBS buffer was adjusted to pH 3-10 with 1 mol/L HCl or 1 mol/L NaOH， added with 0.1 ml of ECPs， incubated at 4 ℃ for 1 h， and added with the substrate （azocasein） that was prepared in distilled water. The changes in ECPase activity were detected with the highest enzyme activity as a control （100% relative enzyme activity）.
Analysis of ECPs by SDSPAGE
ECPs were added with an equal volume of 2× electrophoresis loading buffer and boiled for 3-5 min. The Lamemmli discontinuous SDSPAGE system was used with 5% concentration gel and 12% separation gel. The initial voltage at room temperature was 80 V， which was increased to 120 V when the sample was on the separation gel. When the bromophenol blue reached the edge of the separation gel， the gel was removed for Coomassie brilliant blue G250 staining， followed by decolorization.
Analysis of ECPs by twodimensional electrophoresis
Twodimensional electrophoresis was performed according to the operation manual of GE Healthcare with slight modifications.
Hydration
For 7 cm pH 4-7 strips， each strip was loaded with 120 μg of protein and soak for 12 h at 20 ℃.
Primary isoelectric focusing electrophoresis
①100 V 1 h， 200 V 1 h， 300 V 30 min， 1 000 V 1 h， 5 000 V 2 h， 5 000 V 2 h to the completion of isoelectric focusing. ② After completion of isoelectric focusing， IPG strips were quickly removed and placed in equilibrium solution A （6 mmol/L urea， 2% SDS， 50 mmol/L TrisHCl pH 8.8， 30% glycerol， 0.2% DTT， a trace of bromophenol blue） and equilibrium solution B （6 mmol/L urea， 2% SDS， 50 mmol/L TrisHCl pH 8.8， 30% glycerol， 3% iodoacetamide， a trace of bromophenol blue） for 15 min， respectively. Secondary vertical SDSPAGE electrophoresis
The wellbalanced 7 cm strip was transferred to a small electrophoresis tank for secondary electrophoresis with 12% polyacrylamide gel. The voltage was controlled at 80 V for 30 min and turned to 180 V until bromophenol blue line reached the bottom of the gel. The test was repeated 3 times.
Coomassie brilliant blue staining
The gel was peeled off from the glass plate， soaked in fixative （10% glacial acetic acid， 40% methanol）， stained in staining solution （10% glacial acetic acid， 45% methanol， 0.1% Coomassie brilliant blue R250） for 4 h or overnight， and decolorized in decolorization solution （5% glacial acetic acid， 20% methanol） for 3 h.
Pathogenicity of ECPs to tilapia
By using the modified Karbers method[18]， tilapia was injected intraperitoneally. Fishes with an average body weight of 10 to 15 g were divided into five groups， 10 individuals per group. Each fish in the control group was injected with 0.1 ml of 0.85% sterile saline. All the fishes were observed for 10 d and the number of deaths was recorded.
Analysis of immunogenicity of ECPs
Preparation of immune serum
After incubation for 24 h， S. iniae bacterial liquid was added into 0.4% formalin and inactivated at 28℃ with shaking at 150 r/min. The inactivated broth was centrifuged and the precipitate was collected. Finally， the collected cells were diluted with sterile PBS to immunize mice. The immunization was conducted for four weeks. In the first week， inactivated vaccine was mixed completely with Freunds complete adjuvant （1∶1） for immunization by multipoint subcutaneous injection （0.1 ml per point）； in other three weeks， inactivated vaccine was mixed completely with Freunds incomplete adjuvant （1∶1） for immunization with the same injection method. Four weeks later， blood samples were collected from the eyeballs and placed at room temperature for 3-4 h. Solidified blood samples were placed at 4 ℃ overnight for blood clot retraction， which were centrifuged at 4 500 r/min for 10 min the next day. The supernatant was collected as the serum and stored at -80 ℃. The titer was determined by DotELISA method[19].
Westernblot analysis
The prepared ECPs were subjected to SDSPAGE electrophoresis. Protein transfer was performed for about 2 h at 4℃， 100 V by using a BIORAD protein transfer apparatus according to the conventional method. Westernblot was conducted with 1∶100 mouse antiserum as the primary antibody and 1∶1 000 HRPlabeled goat antimouse IgG as the secondary antibody. Results and Analysis
Analysis of biological activity of ECPs from S. iniae
Qualitative analysis of enzyme activities of S. iniae ECPs
ECPs have the activities of five enzymes including gelatinase， protease， amylase， lecitinase and lipase （Fig. 1）. The results showed that ECPs exhibited the highest activity of gelatinase （the diameter of transparent ring reached 25 mm）， followed by activities of lipase， amylase and protease （the diameter of transparent ring reached 22， 15 and 10 mm， respectively）； ECPs exhibited the lowest activity of lecitinase （the diameter of transparent ring reached 9 mm）. However， urease activity was not detected.
Effect of temperature on ECPase activity
ECPase activity was determined using casein as the substrate. As shown in Fig. 2， ECPase activity of S. iniae increased gradually when the temperature was 25 ℃， reached the peak level when the temperature was 55 ℃， and declined gradually when the temperature continued to rise.
According to the heat stability， ECPase of S. iniae was relatively stable to heat. Specifically， ECPase activity of S. iniae remained at a relatively stable level when the temperature was below 55 ℃ and began to decline when the temperature was higher than 55 ℃. Analysis of variance showed that there were significant differences between treatments.
Effect of inhibitors on ECPase activity
As shown in Table 1， EDTA， DTT and PMSF could reduce ECPase activity to 72.4%， 77.6% and 72.4%， respectively. Cu2+， Ca2+， K+ and Mg2+ exhibited an inhibitory effect on ECPase activity， whereas Fe3+， Co2+ and Mn2+ could activate ECPase activity. Specifically， Mn2+ had the most significant activating effect on ECPase activity of S. iniae.
Effect of pH on ECPase activity
As shown in Fig. 3， ECPase activity of S. iniae was relatively weak under acidic conditions， increased significantly when pH was greater than 7， and reached the peak level when pH was up to 9. Moreover， at pH 10， ECPase activity of S. iniae was relatively high.
The results of primary isoelectric focusing electrophoresis and secondary vertical SDSPAGE electrophoresis were shown in Fig， 4 and Fig.5. There are about 12 main bands of ECPs， and the molecular weights mainly ranged between 28-68 kDa （Fig. 4）. ECPs were isolated by secondary electrophoresis at pH 4-7. About 120 protein spots were detected by the software， and the molecular weights mainly ranged between 26-95 kDa. Westernblot analysis of ECPs
According to DotELISA determination results， the titer of mouse antiS. iniae was 1∶500. The mouse antiS. iniae was used for Westernblot analysis of ECPs. As shown in Fig. 6， there were four main immuno strips， and the molecular weights were 26， 37， 95， and 97 kDa， respectively.
Pathogenicity of ECPs to tilapia
As shown in Table 2， at the initial stage after the injection of ECPs， several tilapia individuals exhibited slow action， loss of appetite and reduced balance capacity， while several individuals swam to the surface of the water or stayed in the bottom of the water. As the disease progressed， tilapia fins and tails exhibited hyperemia. There were erosive substances in the abdominal cavity and unevenly colored liver in serious cases. As could be seen from Table 2， ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.
Conclusion and Discussion
Pathogenicity of ECPs
ECPs play an important role in the pathogenic process of aquatic animal pathogens. So far， it has been found that ECPs of various aquatic animal pathogens， such as Vibrio parahaemolyticus， Vibrio alginolyticus， Vibrio vulnificus and Aeromonas hydrophila， have remarkable pathogenicity[12， 20-22]. The present study indicated that ECPs from S. iniae had amylase， protease， lecitinase， gelatinase， lipase activities and hemolytic activity. Previous studies have shown that gelatinase and protease could decompose collagen and other protein components， while lipase could decompose lipids， and the combined action of gelatinase， protease and lipase could destroy membranous structure， which leads to extensive injury in tissue structure， thus causing degeneration and death， resulting in the reduction or loss of functions of various tissues and organs or even death. Moreover， hemolysin could lyze red blood cells， leading to a decrease in the number of red blood cells in blood and a reduction in oxygen carrying capacity， thereby causing anoxia and accelerating the death of animals[23]. In the present study， ECP inoculation of tilapia indicated that ECPs were highly pathogenic to tilapia， which further confirmed that ECPs played an important role in the pathogenic process of S. iniae. Fuller et al.[24]and Locke et al.[25]elaborated the pathogenic effect of hemolysin S from S. iniae. However， the exact roles of other components of ECPs from S. iniae during the pathogenesis remain to be investigated. Effect of environmental conditions on ECPase activity
The study found that ECPs of S. iniae had relatively high enzyme activity under appropriate conditions. The optimal temperature for ECPs was 55 ℃. ECPs exhibited high heat stability and ECPase activity was relatively high under alkaline conditions. Bisno et al.[26]reported that the amount of ECPase secreted by group A streptococcus was closely related to environmental conditions， such as culture time， nutrient level， pH， and temperature. These environmental conditions were important to antiphagocytosis of group A streptococcus. So far， a large number of studies have investigated the effect of environmental conditions on ECPase of Vibrio alginolyticus， Vibrio anguillarum and Aeromonas hydrophila. The results showed that ECPase activity was closely related to the environmental conditions[12-13， 21].
Electrophoretic analysis and immunogenicity assay of ECPs
In this study， the main bands of ECPs of S. iniae were analyzed by one and twodimensional electrophoresis. The results of onedimensional electrophoresis showed that there are about 12 main bands of ECPs， and the molecular weights mainly ranged between 28-68 kDa； the results of twodimensional electrophoresis showed that the molecular weights mainly ranged between 26-95 kDa， and 120 protein spots were detected at pH 4-7. Shin et al.[15]analyzed the protein spots of ECPs of S. iniae cultured for different time by twodimensional electrophoresis technique. The results showed that ECPs of S. iniae cultured for 60 h had more protein spots， and 150 protein spots were found at pH 4-7. The differences might be related to different strains. Zhang et al.[27]analyzed ECP profiles of virulent and avirulent strains of S. suis by twodimensional electrophoresis technique and found that the number of protein spots of ECPs might by affected by various factors， such as sample preparation， sample processing， and electrophoresis conditions. In addition， ECP profiles suggested that 50 protein spots were present only in the avirulent strains but not in the virulent strains， which indicated that there might be differences in the electrophoresis patterns between different strains.
Moreover， the immunogenicity of ECPs of S. iniae was analyzed by mouse antiS. iniae. The results showed that there were four proteins， with molecular weights of 26， 37， 95， and 97 kDa， respectively. Shin et al.[15]detected the immunogenicity of ECPs by S. iniae vaccine and found that ECPs of S. iniae had multiple antigenic spots at pH 4-7， with molecular weights of 27， 35， 37， 42 and 45 kDa， which was similar to the molecular weights of main proteins in the present study. However， largemolecularweight antigen proteins were present in this study， whereas most proteins analyzed by Shin et al.[15]exhibited small molecular weights， which might be related to pH for twodimensional electrophoresis or different strains. The determination of immunogenicity of ECPs from S. iniae laid a theoretical foundation for the research and development of ECP subunit vaccines and the control of aquatic animal diseases. Which proteins served as immunogenic proteins in the present study and whether these proteins could be used to develop new vaccines against S. iniae remain to be studied. References
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