Abstract [Objectives] This study aimed to determine the infection pathway and target organs of Streptococcus agalactiae in GIFT strain of Nile tilapia， thus providing theoretical basis for the breeding of disease??resistant tilapia and development of S. agalactiae vaccines. [Methods] GIFT strain of Nile tilapia was inoculated by S. agalactiae through intraperitoneal injection， oral gavage and in vitro immersion. The gill， spleen， liver and small intestine tissues of infected tilapia were collected for pathomorphological observation. Immunohistochemical localization was conducted using rabbit anti??S. agalactiae serum to identify the distribution pattern of S. agalactiae in various tissues of tilapia and its target organs via different infection pathways. [Results] GIFT strain of Nile tilapia could be infected by S. agalactiae via three artificial inoculation modes. Specifically， pathological changes occurred at 2 h post??inoculation in intraperitoneal injection and oral gavage groups， whereas tilapia in in vitro immersion group showed pathological changes at 5 h post??inoculation， and the lesion intensity in in vitro immersion group was slighter than that in intraperitoneal injection and oral gavage groups. Immunohistochemical localization indicated that the appearance time of positive signals in intraperitoneal injection group demonstrated an order of spleen?úliver and gill?úsmall intestine； positive signals in oral gavage group appeared in the order of small intestine?úgill and spleen?úliver； the appearance time of positive signals in in vitro immersion group showed an order of gill?úspleen?úliver and small intestine. [Conclusions] GIFT strain of Nile tilapia could be infected by S. agalactiae via intraperitoneal injection， oral gavage and in vitro immersion. The corresponding positive signals for pathogen infection were preferentially present in the spleen， intestine and gill tissues. Thus， preventing S. agalactiae contamination in aquaculture water and food sources is an effective measure to control the outbreak of S. agalactiae infections in tilapia under natural aquaculture conditions.
　　Key words GIFT strain of Nile tilapia； Streptococcus agalactiae； Pathogenic pathway； Target organ； Immunohistochemistry
　　Streptococcus agalactiae is a conditional pathogen widely distributed in nature and can harm different fish species in aquacultures， including tilapia （Oreochromis niloticus）， pompano （Trachinotus ovatus）， channel catfish （Ietalurus punetaus） and grouper （Epinephelus sp.）[1-5]. Since 2009， with the large??scale promotion of intensive aquaculture pattern of tilapia， its diseases have become increasingly serious. Especially， S. agalactiae infections have gradually increased with high mortality and rapid spread， but no complete cure has yet been found. Therefore， it is urgent to carry out research on the breeding of disease??resistant tilapia strains to ensure healthy and sustainable development of tilapia industries. Mechanisms of the immune response of tilapia against S. agalactiae infections should be clarified for breeding tilapia resistant to S. agalactiae. 　　At present， studies about the mechanism of immune response to pathogenic bacteria in fishes have been reported. Skirpstunas and Baldwin[6] found that Edwardsiella ictaluri invades Ietalurus punetaus through intestinal epithelial cells， causing intestinal sepsis. Cotter et al.[7] reported that Streptococcus iniae first infects the gastrointestinal tissue of tilapia followed by entering the blood circulation system via local diffusion， and finally enters the nerve central system and causes various symptoms such as meningitis. Tu et al.[8] confirmed that Aeromonas hydrophila infects Cyprinus carpio through the intestinal tract； pathogens can be isolated from the intestinal tract at 5 weeks post??infection. However， Guo et al.[9] found that pathological changes first appeared in the kidney of Anguilla anguilla after Aeromonas hydrophila infection， and then Aeromonas hydrophila spread through the circulatory system to the whole body and caused diseases. Zhang[10] detected mRNA transcription levels of IL??1??， IFN and TNF in Danio rerio before and after Vibrio parahaemolyticus infection by real??time fluorescent quantitative PCR and found that expression levels of three inflammatory cytokines were closely related to the infection mode， which increased first and then decreased with the prolongation of infection time. Peatmana et al.[11] confirmed that the first target organ for Flavobacterium cloumnare infection in Ietalurus punetaus is the gill. Ji[12] reported that Notch1a may inhibit TLR signaling pathway via inhibiting the irak family and NF??B family genes in the Notch signaling pathway， and negatively regulates the expression of nfkbiaa， cxcl18b， cxcr3， il11r， c3a， cfb， myhb， myh11a， serpinf2a， ctss2 and ctslb in zebrafish at 2 h after Vibrio parahaemolyticus infection， thereby regulating the natural immune response mechanism of zebrafish.
　　Currently， domestic research on S. agalactiae infections in tilapia mainly focuses on the isolation and identification of pathogenic bacteria[13-14]， virulence factor screening[15-16]， medical treatment[17-18] and vaccine development[19-20]. However， there are few reports on the pathogenesis of S. agalactiae and pathological changes in tilapia after S. agalactiae infection.
　　In this study， GIFT strain of Nile tilapia was inoculated by S. agalactiae via intraperitoneal injection， oral gavage and in vitro immersion. The gill， small intestine， spleen， and liver tissues of infected tilapia were collected for pathomorphological observation. Moreover， immunohistochemical localization was performed using rabbit anti??S. agalactiae serum to identify the distribution pattern of S. agalactiae in various tissues of tilapia and its target organs via different infection pathways， aiming at providing the theoretical basis for breeding disease??resistant GIFT strain of Nile tilapia and developing vaccines against S. agalactiae. 　　Materials and Methods
　　Materials
　　Experimental individuals of GIFT strain of Nile tilapia were obtained from the National Tilapia Farm in Nanning， Guangxi. SPF adult rabbits were purchased from the Experimental Animal Center of Guangxi Medical University. S. agalactiae strain HN016 was provided by Fish Disease Control Research Department， Guangxi Academy of Fishery Sciences. HN016 strain was thawed， directly inoculated onto chicken blood medium， and cultured at 30 ?? for 18-24 h. A single colony was randomly selected for Gram staining to detect whether it carried bacteria. Subsequently， a single colony was randomly selected， inoculated into 200 ml of tryptone soy broth medium， incubated at 32 ?? for 24 h on a shaker， diluted to 1??107 CFU/ml with sterile saline， and store at 4 ?? before use.
　　Inoculation modes of S. agalactiae
　　GIFT strain of Nile tilapia was inoculated by S. agalactiae through intraperitoneal injection， oral gavage and in vitro immersion. The infected tilapia individuals were cultured in a 3 m3 water vat and the water temperature was controlled at 30-31 ??. These tilapia individuals were fed twice a day with puffed bait and water was renewed every 3 days （1/2）. After inoculation， the incidence or death of tilapia was observed and recorded every 1 h. Diseased and dead individuals were removed in time.
　　Pathomorphological observation
　　The liver， spleen， gill and small intestine tissues of tilapia infected by S. agalactiae were collected and fixed by Bouin??s solution. After gradient dehydration， paraffin embedding， sectioning and HE staining[21]， pathological changes in different tissues were observed under a microscope.
　　Preparation of rabbit anti??S. agalactiae serum
　　After rejuvenation， HN016 bacterial liquid was inactivated with 0.08% formaldehyde in thermostatic oscillator （28 ??， 300 r/min） overnight， adjusted to a concentration of 5??108 CFU/ml in PBS， and mixed evenly with Freund??s complete adjuvant （1?? 1） to obtain a mixture of immunogens against S. agalactiae strain HN016. Five SPF adult rabbits were selected and injected subcutaneously with the mixture of immunogens against S. agalactiae strain HN016 at different points into the back， 0.1-0.2 ml per point， 2.0 ml per rabbit. After initial immunization， these rabbits were immunized every three weeks in accordance with the above method， three times in total. During the immunization period， adequate feed and water was provided. After three immunizations， blood was collected from the middle auricular artery of rabbit. The blood samples were placed slantwise in a 37 ?? incubator for a period of time （no more than 1 h）， stored in a refrigerator at 4 ?? overnight， and centrifuged at 3 500 r/min for 30 min at 4 ??. The upper??layer serum was collected as rabbit anti??S. agalactiae serum， and stored at -80 ?? before use. 　　Immunohistochemical observation
　　The embedded tissue sections were deparaffinized and hydrated followed by antigen modification and three??step DAB staining[9， 22]. After dehydration with gradient ethanol， clearing with xylene and mounting with neutral balsam， the sections were observed under a microscope.
　　Results and Analysis
　　Pathomorphological observation results
　　Histopathological sections indicated that all these three infection modes， intraperitoneal injection， oral gavage and in vitro immersion， allowed entry of S. agalactiae into Nile tilapia. Specifically， GIFT strain of Nile tilapia in intraperitoneal injection and oral gavage groups showed lesions at 2 h post??inoculation， whereas tilapia in in vitro immersion group had lesions after 5 h of infection. Moreover， lesion intensity in in vitro immersion group was slighter in comparison to that in intraperitoneal injection and oral gavage groups. After artificial infection by S. agalactiae， the pathological changes in gill， spleen， liver and small intestine tissues of tilapia were recorded as follows：
　　Gills： At 2 h post??inoculation， GIFT strain of Nile tilapia in intraperitoneal injection and oral gavage groups showed significant pathological changes in the gill （Fig. 1??A and Fig. 1??B）， including basal epithelial cell proliferation， mucous cell hypersecretion， inflammatory cell infiltration， partial gill lamellae thickening， necrosis and decomposition of respiratory epithelial cells and columnar cells， and edema dispersion in the base and gill cartilage. At 5 h post??inoculation， tilapia in intraperitoneal injection and oral gavage groups showed obvious clinical symptoms； especially， there were marked pathological changes in the gill， including basal epithelial cell proliferation in a large amount that filled the gap between gill lamellaes， necrosis and decomposition of respiratory epithelial cells and columnar cells in most gill lamellaes， mucous cell hypersecretion and mass inflammatory cell infiltration （Fig. 1??C and Fig. 1??D）. GIFT strain of Nile tilapia in in vitro immersion group showed significant pathological changes in the gill at 5 h post??inoculation （Fig. 1??E and Fig. 1??F）， including necrosis and decomposition of respiratory epithelial cells and columnar cells in some gill lamellaes， edema dispersion in the base and gill cartilage， basal epithelial cell proliferation in a large amount that filled the gap between gill lamellaes， and gill lamellae thickening. 　　Spleen： At 2 h post??inoculation， GIFT strain of Nile tilapia in intraperitoneal injection and oral gavage groups began to show significant pathological changes in the spleen， including an increase in hemosiderin， diffuse infiltration of macrophages， and blood vessel thinning. At 5 h post??inoculation， spleen nodules were thickened and increased； a large number of cells showed vacuolar degeneration with dissolution and disappearance of nuclei； splenic blood vessels were narrowed or disappeared， and blood cells decreased （Fig. 2??A and Fig. 2??B）. GIFT strain of Nile tilapia in in vitro immersion group showed slight pathological changes in the spleen at 5 h post??inoculation， which had significant pathological changes at 8 h post??inoculation， including mass deposition of hemosiderin， blood vessel narrowing， mass proliferation and diffuse infiltration of macrophages， and infiltration of a large amount of red blood cells around the central artery （Fig. 2??C）.
　　Agricultural Biotechnology 2018
　　Liver： GIFT strain of Nile tilapia in intraperitoneal injection group showed remarkable pathological changes in the liver at 2 h post??inoculation， which were characterized by telangiectasia and hyperemia， severe deformation or necrosis and disordered arrangement of hepatocytes， disappearance of nuclei， and even large areas of necrotic foci （Fig. 3??A）. GIFT strain of Nile tilapia in oral gavage group showed significant pathological changes in the liver at 5 h post??inoculation， including obvious congestion of capillaries， vacuolar degeneration or necrosis of stem cells， light staining， dissolution or disappearance of cytoplasm and nuclei of some hepatocytes， and distinct necrotic foci formed by some hepatocytes （Fig. 3??B）. At 5 h post??inoculation， GIFT strain of Nile tilapia in in vitro immersion group showed vacuolar degeneration and capillary congestion in the liver； at 8 h post??inoculation， hepatocytes had vacuoles with lightly stained cytoplasm and nuclei （Fig. 3??C）； there was severe cavitation in some areas， and vacuoles in the right lobe of the liver were significantly more than that in the left lobe， but the overall lesion intensity was significantly lower than that in intraperitoneal injection and oral gavage groups at the same time.
　　Small intestine： At 2 h post??inoculation， GIFT strain of Nile tilapia in intraperitoneal injection and oral gavage groups showed significant pathological changes in the small intestine， including intestinal villi fracture， exfoliated upper epithelial cells and homogenized and red??stained protein exudates in the intestinal lumen， goblet cell enlargement， and dissolution or disappearance of cytoplasm and nuclei of some submucosal cells. At 5 h post??inoculation， the small intestine of tilapia in intraperitoneal injection and oral gavage groups showed epithelial cell exfoliation in large areas， disappearance of striated border， dissolution and disappearance of cytomembrane of absorptive cells， fracture， uneven staining or coagulative necrosis of myenteric smooth muscle fibers， interstitial broadening between lamina propria and submucosa with mild edema， and mass inflammatory cell infiltration （Fig. 4??A and Fig. 4??B）. At 8 h post??inoculation， GIFT strain of Nile tilapia in in vitro immersion group had a small amount of fractured intestinal villi in the small intestine （Fig. 4??C）； at 12 h post??inoculation， the pathological changes began to aggravate， which were characterized by exfoliated upper epithelial cells and homogenized and red??stained protein exudates in the intestinal lumen， goblet cell enlargement， dissolution or disappearance of cytoplasm and nuclei of some submucosal cells， and myenteric fiber fracture （Fig. 4??D）. 　　Immunohistochemical observation results
　　Immunohistochemical observation revealed that positive signals for pathogen infection in GIFT strain of Nile tilapia appeared initially in gill lamellaes and basal epithelial cells as well as the surrounding capillaries （Fig. 5??A and Fig. 5??B）. Early positive signals in the spleen mainly appeared in phagocytes that engulfed S. agalactiae and the surrounding regions after their rupture and necrosis； with the prolongation of infection time， obvious positive signals were also observed in splenic arteries and edematous vascular walls （Fig. 5??C and Fig. 5??D）； the white pulp of the spleen also showed strong positive signals， suggesting that the rapidly proliferating S. agalactiae had caused pathological changes in the target organs. Positive signals in the liver were mainly concentrated in the hepatic sinusoids and the surrounding regions after phagocyte rupture； moreover， a small number of positive signals appeared in the surrounding hepatocytes and sinusoidal wall cells of necrotic hepatic sinusoids （Fig. 5??E and Fig. 5??F）. in addition， positive signals could be detected in the connective tissues， serosa and submucosa of the intestine （Fig. 5??G and Fig. 5??H） as well as in some lamina propria； besides， positive signals were mainly concentrated in capillaries that contained phagocytes.
　　After artificial inoculation of S. agalactiae into GIFT strain of Nile tilapia via intraperitoneal injection， oral gavage and in vitro immersion， dynamic location and distribution of positive signals for pathogen infection in the gill， spleen， liver and small intestine tissues of tilapia were analyzed. As shown in Table 1， positive signals for pathogen infection were found in four organs of tilapia at 2 h post??inoculation in intraperitoneal injection and oral gavage groups. Specifically， the strongest positive signals in intraperitoneal injection group were found in the spleen， while that in oral gavage group appeared in the small intestine. In in vitro immersion group， positive signals were observed in the gill and spleen of tilapia at 5 h post??inoculation， and those in the gill were much stronger. According to the appearance time and intensity of positive signals for pathogen infection， the appearance time of positive signals in intraperitoneal injection group demonstrated an order of spleen?úliver and gill?úsmall intestine； positive signals in oral gavage group appeared in the order of small intestine?úgill and spleen?úliver； the appearance time of positive signals in in vitro immersion group showed an order of gill?úspleen?úliver and small intestine. 　　Discussions
　　Pathological changes in various organs of GIFT strain of Nile tilapia infected by S. agalactiae
　　S. agalactiae is the main pathogen in the process of tilapia culture， with the characteristics of rapid spread， high mortality， and difficult to treat. So far， the specific pathway and target organ through which S. agalactiae invades tilapia has not been clarified yet. In this study， GIFT strain of Nile tilapia was artificially inoculated by S. agalactiae via intraperitoneal injection， oral gavage and in vitro immersion. Based on observations of pathological changes in the gill， spleen， liver and small intestine tissues， it was found that tilapia infected by S. agalactiae showed extensive congestion and hemorrhage in various organs. The gill is the main organ for gas exchange between blood circulation and the external environment in fishes[23]. Pathological changes in the gill were mainly characterized by mass proliferation of epithelial cells and basal cells in gill lamellaes， accompanied by degeneration or even necrosis. Moreover， most of the gill lamellaes were thickened and stuck， which hindered the oxygen carrying capacity， resulted in a serious reduction or even loss of oxygen utilization efficiency in blood circulation， and caused anoxic dysfunction of various organs， thereby accelerating the death of diseased fishes[24]. Tilapia infected by S. agalactiae showed spleen enlargement and hemorrhage with mass deposition of hemosiderin， indicating that a large number of red blood cells in the splenic blood vessels were destroyed[25-26]； the spleen had serious hemolysis that blocked its hematopoietic function； moreover， the immune resistance of tilapia was reduced， which accelerated the proliferation of pathogenic bacteria in tilapia infected. The liver is an important digestive organ of fishes， which not only plays an active role in protein synthesis， maintenance of blood glucose balance and regulation of fat metabolism， but also exerts detoxification and defense functions[27]. In this study， it was found that tilapia infected by S. agalactiae showed degeneration， necrosis and disordered arrangement of hepatocytes， which indicated that the metabolic function of the liver was impaired， resulting in insufficient energy supply to the body， thereby affecting its immune function. The intestine is an important part of the digestive system in fishes. In this study， it was found that the contents of the small intestine of tilapia infected by S. agalactiae were emptied and there was a large amount of water accumulated in the intestine. Pathological observation indicated that most of the epithelial cells were degenerated and necrotic， which suggested that the digestion and absorption functions of tilapia were affected， resulting in reduced feeding ability or even apastia， eventually leading to insufficient energy supply. 　　Dynamic distribution of S. agalactiae in GIFT strain of Nile tilapia infected
　　At present， there have been a large number of reports on the distribution pattern of pathogens in various tissues and organs after invasion by immunohistochemistry. Xia et al.[22] located Vibrio anguillarum in the liver and gastrointestinal tissues of flounder after artificial inoculation via immunohistochemical ABC method. Guo et al.[9] analyzed the distribution pattern of Aeromonas hydrophila in the liver， kidney and intestine of European eel （Anguilla anguilla） at different stages after artificial inoculation by immunohistochemical localization techniques. In this study， immunohistochemistry was employed to detect signals for pathogen infection in the gill， small intestine， spleen， and liver tissues of tilapia infected by S. agalactiae at different time after artificial inoculation. The invasion process of S. agalactiae via three inoculation modes was analyzed based on the appearance time and intensity of positive signals. According to the results， the appearance time of positive signals in intraperitoneal injection group demonstrated an order of spleen?úliver and gill?úsmall intestine； positive signals in oral gavage group appeared in the order of small intestine?úgill and spleen?úliver； the appearance time of positive signals in in vitro immersion group showed an order of gill?úspleen?úliver and small intestine. In the pre??test， GIFT strain of Nile tilapia was inoculated by high??concentration S. agalactiae via in vitro immersion. The results showed that although the pathogen could invade tilapia， no obvious symptoms or death was observed. It is speculated that the gill is the initial target organ of S. agalactiae invasion in tilapia under natural aquaculture conditions， and feeding foods harboring S. agalactiae is the main pathogenic pathway.
　　Conclusion
　　The GIFT strain of Nile tilapia could be infected by S. agalactiae via intraperitoneal injection， oral gavage and in vitro immersion. The corresponding positive signals for pathogen infection were preferentially present in the spleen， intestine and gill tissues. Therefore， preventing S. agalactiae contamination in cultured water bodies and food sources is an effective measure to prevent and control the outbreak of S. agalactiae infections in tilapia under natural aquaculture conditions.
　　References
　　[1] JAFAR QA， SAMEER AZ， SALWA AM， et al. Molecular investigation of Streptococcus agalactiae isolates from environmental samples and fish specimens during a massive fish kill in Kuwait Bay[J]. Pakistan Journal of Biological Sciences， 2008， 11（21）：2500-2504. 　　[2] AMAL MN， ZAMRI??SAAD M， IFTIKHAR AR， et al. An outbreak of Streptococcus agalactiae infection in cage??cultured golden pompano， Trachinotus lochii （Lacepede）， in Malaysia[J]. Journal of Fish Diseases， 2012， 35（11）：849-852.
　　[3] LIU X， AO QW， TAN Y， et al. Dynamic distribution of pathogenic bacteria in GIFT infected artificially with Streptococcus agalactiae[J]. Journal of Southern Agriculture， 2015， 46（9）：1715-1719.（in Chinese）
　　[4] GUO CM， YUAN C， ZHU SY， et al. Quantitative identification of differential proteins in Streptococcus agalactiae piscine strain and bovine strain using iTRAQ[J]. Jiangsu Journal of Agricultural Sciences， 2017， 33（4）：868-873.（in Chinese）
　　[5] ZHANG DF， YUAN W， KE XL， et al. Molecular characteristics and transmission of Streptococcus agalactiae in a major tilapia culturing area of China[J]. Journal of Fishery Sciences of China， 2017， 24（3）：606-614.（in Chinese）
　　[6] SKIRPSTUNAS RT， BALDWIN TJ. Edwardsiella ictaluri invasion of IEC??6， Henle 407， fathead minnow and channel catfish enteric epithelial cells[J]. Diseases of Aquatic Organisns， 2002， 51（3）：161-167.
　　[7] COTTER PD， HILL C， ROSS RP. Bacteriocins： Developing innate immunity for food[J]. Nature Reviews， Microbiology， 2005， 3（10）：777-788.
　　[8] TU FP， CHU WH， ZHUANG XY， et al. Effect of oral immunization with Aeromonas hydrophila ghosts on protection against experimental fish infection[J]. Letters in Applied Microbiology， 2010， 50（1）：13-17.
　　[9] GUO SL， FENG JJ， XIONG J， et al. Immunohistochemistry distribution and pathological characteristics of Aeromonas hydrophila in several organs of European eel （Anguilla anguilla） after artificial infection[J]. Journal of Huazhong Agricultural University， 2011， 30（4）：494-499.（in Chinese）
　　[10] ZHANG YH. Pathogenicity and innate immune response to Vibrio parahaemolyticus infection in the zebrafish[D]. Shanghai??Shanghai Ocean University， 2012.（in Chinese）
　　[11] PEATMANA E， LI C， PETERSON BC， et al. Basal polarization of the mucosal compartment in Flavobacterium columnare susceptible and resistant channel catfish （Ictalurus punctatus）[J]. Molecular Immunology， 2013， 56（4）：317-327.
　　[12] JI C. Transcriptome analysis of Notch1a in innate immune response in zebrafish （Danio rerio） larvae challenged by Vibrio parahaemolyticus[D]. Shanghai： Shanghai Ocean University， 2017.（in Chinese）
　　[13] KE J， ZHAO F， LUO L， et al. Isolation， identification and pathogenicity of pathogenic bacteria of fulminant disease of tilapia in Guangdong Province[J]. Journal of Guangdong Ocean University， 2010， 30（3）：22-27.（in Chinese） 　　[14] GUO YJ， ZHANG DF， FAN HP， et al. Molecular epidemiology of Streptococcus agalactiae isolated from tilapia in southern China[J]. Journal of Fisheries of China， 2012， 36（3）：399-406.（in Chinese）
　　[15] DENG YQ， WANG KY. Research progress on fish stress Streptococcus agalactiae disease[J]. China Animal Husbandry & Veterinary Medicine， 2016， 43（9）：2490-2495.（in Chinese）
　　[16] FANG W， LIANG YH， NING D， et al. Molecular epidemiology of Streptococcus agalactiae in tilapia in Guangdong[J]. Acta Scientiarum Naturlium Universitatis Sunyatseni， 2016， 55（2）：97-101.（in Chinese）
　　[17] HUA YN， CHEN H， ZHANG QZ. In vitro efficacy of aqueous extracts of 100 Chinese herbal medicines against Streptococcus agalactiae from tilapia[J]. Ecological Science， 2015， 34（3）：26-30.（in Chinese）
　　[18] LIANG ZL， MA YP， MA JY， et al. Antimicrobial activities of 82 kinds of Chinese herbal medicines against the pathogen of Streptococcus from tilapia in vitro[J]. Guangdong Agricultural Sciences， 2016， 43（10）：128-133.（in Chinese）
　　[19] LI LP， WANG R， LIANG WW， et al. Biological characteristics and immunization of live attenuated Streptococcus agalactiae strain for tilapia with parent[J]. Southwest China Journal of Agricultural Sciences， 2015， 28（5）：2316-2322.（in Chinese）
　　[20] WANG R， LI LP， HUANG T， et al. Efficacy of tilapia streptococcal disease live attenuated vaccine and inactivated vaccine antigen DNA tracer and protection rate comparison delivered by oral administration[J]. Southwest China Journal of Agricultural Sciences， 2015， 28（1）：423-428.（in Chinese）
　　[21] WANG J， WANG B， LI JT， et al. Fluorescent histological observation of zabrafish （Danio rerio） gonad[J]. Journal of Southern Agriculture， 2011， 42（4）：437-440.（in Chinese）
　　[22] XIA YJ， HUANG WQ， JIANG GL. Histopathology and immunohistochemistry of flounder infected by Vibrio anguillarum[J]. Marine Sciences， 2000， 24（10）：41-44.（in Chinese）
　　[23] HUANG JL. Study on etiology， pathology of tilapia Streptococcus agalactiae disease and on the prokaryotic expression of cpsE gene[D]. Ya??an： Sichuan Agricultural University， 2012. （in Chinese）
　　[24] AZAD IS， AL??MARZOUK A， JAMES CM， et al. Outbreak of natural Streptococcosis in hatchery produced silver pomfret （Pampus argenteus Euphrasen） larvae in Kuwait[J]. Aquaculture， 2012， 330-333：15-20.
　　[25] CHEN CY， CHAO CB， BOWSER PR. Comparative histopathology of Streptococcus iniae and Streptococcus agalactiae??infected tilapia[J]. Bulletin of the European Association of Fish Pathologists， 2007， 27（1）：2-9.
　　[26] ZHU JL， QI ZL， LI DY， et al. Pathological changes in tilapia （Oreochromis niloticus） naturally infected by Streptococcus iniae[J]. Journal of Fisheries of China， 2014a， 38（5）：722-730.（in Chinese）
　　[27] ZHU JL， ZOU ZY， LI DY， et al. Pathological changes in tilapia （Oreochromis niloticus） naturally infected by Streptococcus agalactiae[J]. Journal of Fisheries of China， 2014b， 38（11）：1937-1944.（in Chinese）