水体中泼洒复合乳杆菌对尼罗罗非鱼养殖池塘环境、肠道和鳃健康的影响

研究在尼罗罗非鱼养殖池塘中添加复合乳杆菌对养殖池塘环境、尼罗罗非鱼肠道和鳃健康的影响,为乳杆菌在尼罗罗非鱼健康养殖中的应用提供基础。在尼罗罗非鱼养殖池塘水体中泼洒复合乳杆菌(对照组:0;实验组:1.0×104 CFU/mL),实验周期为10周。复合乳杆菌的成分为干酪乳杆菌和酸鱼乳杆菌,比例为1:2.2。监测池塘水质指标、罗非鱼肠道和鳃的免疫及抗氧化指标,并运用高通量测序技术(Illumina MiSeq),比较对照组和实验组水体、底泥、尼罗罗非鱼肠道和鳃黏膜共生菌群落结构差异。结果显示,从第7周开始,实验组水体中的硝酸盐含量始终低于对照组,实验组比对照组降低了72.68%、72.00%、26.27%和21.46%;实验组水体中的总氮含量在从第6周开始低于对照组,分别降低了43.39%、44.64%、15.64%、37.57%和34.49%;实验组水样中的总磷含量从第4周开始低于对照组,分别降低了40.91%、33.33%、50.00%、33.33%、17.39%、39.29%和25.71%。与对照组相比,实验组尼罗罗非鱼肠道的碱性磷酸酶(AKP)活性提高了45.04%,实验组尼罗罗非鱼鳃的总超氧化物歧化酶(SOD)和AKP分别提高了58.26%和60.99%。复合乳杆菌的添加对尼罗罗非鱼养殖池塘水体菌群结构影响较大,并未影响池塘底泥的菌群结构,影响了尼罗罗非鱼肠道和鳃的菌群结构,其中条件致病菌不动杆菌属、泛菌属细菌的相对丰度降低,而有益细菌鲸杆菌属的相对丰度增加。因此,在养殖水体中泼洒适当浓度的复合乳杆菌可以改善池塘水质,提高尼罗罗非鱼肠道和鳃的免疫力、抗氧化能力,同时调节养殖水体、尼罗罗非鱼肠道和鳃的菌群结构。     

尼罗罗非鱼肠道及养殖环境中菌群结构与链球菌病的相关性

为研究尼罗罗非鱼肠道和池塘养殖环境中菌群结构变化与链球菌病暴发的相关性,实验采用16S r DNA高通量测序方法对比分析发病和健康池塘水体、底泥和尼罗罗非鱼肠道菌群的结构特征。结果显示,底泥中微生物多样性最高,水体和肠道中微生物多样性次之;肠道菌群与水体中菌群的相似性较高,而与底泥中菌群相似性较低;发病组的尼罗罗非鱼肠道和池塘底泥中微生物的多样性均低于健康组,而发病组池塘水体中微生物多样性高于健康组。OTU聚类分析发现,发病与健康尼罗罗非鱼肠道微生物差异极显著,大部分健康尼罗罗非鱼肠道微生物样品单独聚为一支。菌群结构组成分析结果表明,虽然发病组和健康组水体或底泥中优势菌群结构组成的差异较小,但在非优势菌群中存在一定差异,发病组水体中变形菌门和梭杆菌门的比例显著高于健康组水体,疣微菌门和浮霉菌门的丰度显著低于健康组水体;发病组池塘底泥中厌氧粘细菌属、甲烷丝菌属和枝芽孢菌属等具有降解有机质和生态修复功能,菌群的丰度显著低于健康组底泥,而具有致病能力的曼氏杆菌属丰度却显著高于健康组底泥。发病组尼罗罗非鱼肠道菌群中链球菌属、分枝杆菌属和曼氏杆菌属等致病菌群的丰度显著高于健康组尼罗罗非鱼肠道,而乳球菌属、鲸杆菌属和红球菌属等益生菌的丰度显著低于健康尼罗罗非鱼肠道。无乳链球菌普遍存在于发病和健康养殖环境,以及尼罗罗非鱼肠道中,无乳链球菌的丰度在发病和健康养殖环境之间无显著差异,但其在发病尼罗罗非鱼肠道中的丰度要显著高于健康组。研究表明,尼罗罗非鱼链球菌病发病池塘水体和底泥中有益菌丰度降低和致病菌丰度的升高反映其养殖环境出现恶化,尼罗罗非鱼链球菌病的暴发与肠道微生态平衡破坏后无乳链球菌丰度增加密切相关,但肠道中无乳链球菌丰度的剧增与养殖水体和底泥中该病原菌的丰度水平之间并无直接关联。     

投喂乙醇假丝酵母对罗非鱼生长、免疫和肠道菌群的影响

为探究乙醇假丝酵母(Candida ethanolica GXU01)在罗非鱼养殖中的益生潜能,为罗非鱼可持续养殖寻找生态友好的饲用添加菌,该研究将乙醇假丝酵母作为饲料添加菌饲喂尼罗罗非鱼(Oreochroms niloticus),通过测定罗非鱼的生长性能、肠道消化酶、血清非特异性免疫指标、肠道微生物群落结构以及用无乳链球菌(Streptococcus agalactiae)对罗非鱼进行攻毒试验,全面评价了乙醇假丝酵母对罗非鱼生长及其免疫力的影响。结果表明,摄食乙醇假丝酵母可以使罗非鱼的生长性能、消化酶活性、血清溶菌酶活性和补体C3含量显著提高(P0.05)。投喂乙醇假丝酵母后,罗非鱼肠道中梭杆菌门、鲸杆菌属(Cetobacterium)和艾克曼菌属(Akkermansia)等有益菌群的丰度显著上调,蓝细菌门大幅减少。攻毒试验中,摄食酵母饲料的罗非鱼成活率增加26.66%。研究表明乙醇假丝酵母对罗非鱼肠道消化能力和免疫抗病能力均有促进作用。     

南美白对虾肠道细菌菌群分析

对健康养殖南美白对虾胃、肝胰腺、肠道、肌肉中的需氧菌群进行研究。结果表明:胃、肝胰腺、肠道、肌肉中的异氧细菌总菌数分别为5.3×105~6.5×107cfu/g、6.6×105~5.2×106cfu/g、1.4×108~4.5×109cfu/g和1.0×104~1.1×105cfu/g。对从其肠道中分离出来的111株菌株进行属(科)的鉴定。这些菌株分属于13个属(科),发光杆菌属、弧菌属、气单胞菌属、肠杆菌科、黄单胞菌属、土壤杆菌属、芽孢杆菌属、棒状杆菌属、小球菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属,还有一株未鉴定的革兰阳性菌。其中优势菌为发光杆菌属、弧菌属,气单胞菌属、肠杆菌科、黄单胞菌属,次优势菌为土壤杆菌属、芽孢杆菌属、棒状杆菌属、小球菌属。     

不同水温下无乳链球菌在尼罗罗非鱼体内的动态分布及其消除规律

本研究以腹腔注射无乳链球菌(Streptococcus agalactiae, WC1535菌株)后的尼罗罗非鱼(Oreochromis niloticus，GIFT strain)为研究对象，研究不同水温下无乳链球菌在尼罗罗非鱼体内的动态分布及消除规律。首先，分析25℃、29℃和33℃3个实验组罗非鱼的感染累积死亡率；其次，对3个实验组罗非鱼进行无乳链球菌的体外分离、培养和计数；然后，分析感染后不同实验组罗非鱼脑、肝脏、脾脏和肾脏组织中无乳链球菌的浓度。结果显示，随着水温的升高罗非鱼的累积死亡率也随之升高，25℃、29℃和33℃组的累积死亡率分别为6.67%、25.56%和78.90%。菌落统计结果显示，随着水温的升高，无乳链球菌在罗非鱼体内的增殖速度加快，同时，单位质量组织(脑、肝脏、脾脏和肾脏)中无乳链球菌的最大载菌量也随之升高。本研究还发现，无乳链球菌在罗非鱼脾脏中的浓度最高，在肾脏中的存活时间最长。综上可知，尼罗罗非鱼感染无乳链球菌后，体内各组织中链球菌的增殖、消除速度均与水温密切相关。本研究为研究无乳链球菌的致病机制奠定基础，也为通过合理调控水温及施药等措施防治尼罗罗非鱼链球菌病的暴发提供科学依据。     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

育肥饲料中虾青素含量对雄性中华绒螯蟹肠道和鳃部可培养优势细菌数量和组成的影响

在育肥饲料中添加虾青素会影响中华绒螫蟹肠道及鳃内的可培养优势菌群以及机体的免疫力,本实验旨在探讨饲料中虾青素含量及养殖水体对其肠道和鳃部菌群的影响。采用不同虾青素含量的饲料(0.00、26.60、41.62、81.37和75.35 mg/kg,分别对应饲料1#~5#)对雄性中华绒螫蟹进行育肥70 d后,利用基础培养基和选择性培养基对其肠道、鳃及养殖水体中的细菌进行传统分离和纯化,所得菌株进行16S rRNA测序,再进行同源性分析后做系统进化树。结果发现,在分离获得的106株细菌(登录号:KU570293~KU570368,KU570370,KU570372~KU570383,KU601302~KU6013 16,KU720553)中,92株为优势菌株,其中,肠道、鳃部和水体各29、32和31株。肠道中的可培养优势细菌属于柠檬酸杆菌属、假单胞菌属和气单胞菌属;而鳃中可培养优势细菌属于柠檬酸杆菌属、芽孢杆菌属、假单胞菌属和气单胞菌属;养殖水体中可培养优势细菌属于芽孢杆菌属、气单胞菌属、柠檬酸杆菌属、假单胞菌属和黄杆菌属。饲料2#组肠道中可培养细菌总数最高(1.06×10~8cfu/g);饲料5#肠道中的潜在致病菌数量最低,但其可培养细菌总数显著高于其他饲料组;饲料3#组肠道和鳃中潜在致病菌数量相对较高。研究表明,饲料中添加不同含量的虾青素能够显著影响雄性中华绒螫蟹肠道和鳃中的菌群构成,但对水体中可培养细菌数量无显著影响。本研究首次对投喂不同含量虾青素饲料的育肥期雄性中华绒螫蟹肠道、鳃及养殖水体可培养的优势细菌数量和组成进行分析,探究了饲料中虾青素含量以及养殖水体与蟹肠道内可培养细菌之间的联系,为中华绒螫蟹营养免疫和菌群调控提供了理论基础。     

红鳍东方鲀稚鱼肠道可培养细菌的多样性

使用2216E平板涂布法从肠道样品分离可培养细菌,通过16S r DNA测序鉴定细菌。所有分离的细菌分为变形菌门(包含γ-变形细菌纲和α-变形细菌纲)和厚壁菌门。其中以γ-变形细菌纲为优势(83.3%)。在属的水平,从肠道中共分离出弧菌属、希瓦氏菌属、假交替单胞菌属、亚硫酸杆菌属、芽孢杆菌属、Aliivibrio、发光杆菌属、科尔韦尔氏菌属8个属,其中弧菌属、希瓦氏菌属和假交替单胞菌属的种类占总数的70%。首次发现亚硫酸杆菌属和科尔韦尔氏菌属作为红鳍东方鲀稚鱼肠道菌群的一部分。     

BALB/c小鼠用于评价尼罗罗非鱼无乳链球菌毒力的研究

实验采用BALB/c小鼠作为实验动物,旨在建立尼罗罗非鱼无乳链球菌毒力测定的BALB/c小鼠模型。BALB/c小鼠经腹腔注射尼罗罗非鱼源无乳链球菌建立感染模型,比较了尼罗罗非鱼源无乳链球菌分别感染尼罗罗非鱼和小鼠的LD_(50)差异,分别测定了不同毒力尼罗罗非鱼无乳链球菌对尼罗罗非鱼和小鼠的毒力。结果显示,小鼠经腹腔注射无乳链球菌,在24 h内出现死亡现象,且对小鼠脑、肝脏、脾脏、肾脏等组织造成损伤。3次测定尼罗罗非鱼无乳链球菌TFJ0901对尼罗罗非鱼和小鼠LD_(50)分别为7.7×10~7、2.2×10~8、3.5×10~9 CFU/mL和405、361、419 CFU/只。将无乳链球菌TFJ0901和THN0901感染尼罗罗非鱼(1.0×10~7 CFU/mL)和小鼠(100 CFU/只),尼罗罗非鱼和小鼠存活率分别为100%、6.7%±5.8%和100%、0,其存活率都具有显著性差异。将无乳链球菌TFJ0901和TFJ-F感染尼罗罗非鱼(3.0×10~8 CFU/mL)和小鼠(2 500 CFU/只),尼罗罗非鱼的存活率分别为73.3%±11.5%和80.0%±10.0%,存活率差异不显著,小鼠存活率分别为13.3%±11.5%和100.0%,存活率具有显著性差异。研究表明,本实验成功建立了BALB/c小鼠作为尼罗罗非鱼源无乳链球菌毒力测定的稳定模型,测定不同毒力的尼罗罗非鱼源无乳链球菌对小鼠毒力与对尼罗罗非鱼毒力一致,且该模型能够区分尼罗罗非鱼模型难以区分的毒力相近的无乳链球菌。     

养殖花鲈（Lateolabrax maculatus）肠道菌群的多样性分析

为探究养殖花鲈(Lateolabrax maculatus)肠道菌群的结构特征,采用Illumina Hiseq2500高通量测序技术,对福建宁德三都澳网箱养殖花鲈的肠道内容物及肠壁菌群16S r DNA V3-V4区进行测序。肠道内容物和肠壁样品分别获得456和293个OTUs(operational taxonomic units),分属22个门120个属。花鲈肠道菌群以厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和梭杆菌门(Fusobacteria)细菌为主要优势菌群,分别占其肠道菌群数量的47.2%、25.8%、14.4%和10.3%。在属分类水平上,发光杆菌属(Photobacterium)、梭菌属(Clostridium)及鲸杆菌属(Cetobacterium)等属细菌是主要类别。对养殖花鲈的肠道内容物与肠壁菌群进行比较分析显示,肠壁菌群多样性略高于肠道内容物。肠道内容物中,发光杆菌属、梭菌属及鲸杆菌属等是主要菌属,其中鲸杆菌属和梭菌属分别占肠道内容物菌群总菌数的17.8%和12.5%,为主要标志性差异种类;在肠壁样品的菌群中,乳球菌属(Lactococcus)、真杆菌属(Eubacterium)、Lachnoclostridium、弓形菌属(Arcobacter)和Flavonifractor为丰度较高的菌属,其中格氏乳球菌(Lactococcus garvieae)为主要标志性物种,占肠壁菌群总数的5.6%。在花鲈肠壁上定殖着格氏乳球菌(L. garvieae)和美人鱼发光杆菌(Photobacterium damselae subsp. damselae)等条件致病菌,因此,在养殖中需要注重饵料及环境的管理,避免致病菌大量增殖引起暴发性鱼病。     

Streptococcosis,Lactococcosis and Enterococcosis are potential threats facing cultured Nile tilapia (Oreochomis niloticus) production

Streptococcosis, lactococcosis and enterococcosis are among the most important bacterial diseases affecting tilapia farms in Kafr Elsheikh governorate, Egypt. A number of clinically diseased fish were collected and submitted to our laboratory during disease outbreak in 2018. They were characterized by nervous swimming behaviour, skin darkness, exophthalmia, ocular opacity and haemorrhages. Necropsy findings were splenomegaly, congestive hepatomegaly, liquefied brain and enteritis. The phenotypic and molecular characterizations of the bacterial strains isolated from naturally infected fish identified three genera of Gram‐positive cocci: Streptococcus agalactiae, Enterococcus faecalis and Lactococcus garvieae. Infectivity trials were conducted in four groups of Nile tilapia inoculated with S. agalactiae Egy‐1, E. faecalis Egy‐1 and L. garvieae Egy‐1 strains and saline. Mortalities, clinical signs and pathological findings were recorded daily 14 days post infection. Experimentally infected tilapia showed similar clinical signs, postmortem lesions, but varied in the severity and experienced high mortalities up to 70% in case of S. agalactiae and L. garvieae infections and 30% in case of E. faecalis infection. Pathological examination of infected tissue sections stained with modified Brown–Brenn and immunohistochemistry revealed an important direct correlation between the distribution of each bacterial isolate and the lesions developed in different organs. Furthermore, the isolates were subjected to profiling against 11 antibiotics, and they showed resistance against several types of antibiotics, which implicate potential risk to human health and emphasize the urgent need for alternate bio‐control strategies to prevent the diseases and the problem of multidrug resistance in aquatic environment.     

Development of attenuated erythromycin‐resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re‐attenuated S. agalactiae (named TFJ‐ery) was developed from a natural low‐virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ‐ery were determined. The results indicated that TFJ‐ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ‐ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ‐ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ‐ery at a dose of 5.0 × 10⁷ CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post‐vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post‐vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ‐ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.     

吉富罗非鱼感染无乳链球菌后肝脏组织在不同时期蛋白质组的表达差异

为研究罗非鱼感染无乳链球菌前后肝脏组织蛋白质的表达变化。本研究以吉富罗非鱼为材料，采用双向电泳技术分析其在无乳链球菌感染胁迫下24 h、48-144 h、12 d与对照组（未感染）肝脏组织蛋白质组的变化，对差异表达蛋白进行质谱分析鉴定。结果显示：吉富罗非鱼在无乳链球菌胁迫下，3个实验组的蛋白质图谱与对照组相比存在显著差异，共有30个蛋白点发生显著改变，其中13个表达上调，17个下调，2个下调蛋白点在感染12 d组消失。通过MALDI-TOF-MS MS/MS质谱分析和数据库检索对这些蛋白质进行了功能分类，发现它们涉及到能量代谢、细胞防御与应激、消化免疫、抗氧化与排毒等许多方面。推测这些蛋白可能在吉富罗非鱼对无乳链球菌胁迫的抗性反应中发挥了重要作用。研究结果为无乳链球菌疫苗的研制及吉富罗非鱼抗病品种选育奠定了基础。     

cel-EIIB蛋白调控罗非鱼无乳链球菌强毒、弱毒株毒力相关基因的表达模式

罗非鱼无乳链球菌纤维二糖-磷酸转移酶系统（cel-PTS）的EIIB蛋白对强毒株毒力影响有限，但对弱毒株毒力似乎存在潜在的影响，但具体机制仍不清晰，有必要弄清该蛋白调控强、弱毒株的毒力相关基因表达模式。在前期研究中，通过同源重组技术，构建了无乳链球菌强毒株cel-EIIB基因缺失株，本研究通过类似的方法，获得弱毒株该基因的缺失株。用无乳链球菌强毒、弱毒株及它们的cel-EIIB缺失株分别感染斑马鱼，结果显示，cel-EIIB缺失后，导致弱毒株毒力明显返强，而强毒株毒力则轻微减弱。qPCR检测发现，cel-EIIB缺失可致cel-PTS系统的cel-EIIA、双组分信号转导系统（TCS）的DltR和CiaH以及毒力基因sodA、cpsD和cpsG在强、弱毒株中呈现相反的表达模式；此外，TCS系统的RgfC、DltS和CsrR以及毒力基因cspA和pavA在强毒突变株中表达未受影响，但在弱毒突变株中的表达却显著上调。研究揭示，EIIB蛋白可能通过调控上述毒力相关基因表达而负调控弱毒菌株的毒力。     

Isolation and molecular characterization of streptococcal species recovered from clinical infections in farmed Nile tilapia (Oreochromis niloticus) in the Philippines

Streptococcosis cause severe losses for global tilapia farming, especially in developing countries. The aim of this study was to identify and characterize streptococci recovered from Nile tilapia farmed in the Philippines. Moribund and apparently healthy fish were sampled from grow-out cages, ponds and hatcheries. Clinical signs observed included exophthalmia, eye opacity, ascites, lethargy, erratic swimming and haemorrhages. Results showed that both Streptococcus iniae and Streptococcus agalactiae were associated with disease in these sites. Consistent with global reports, including those from South-East Asia, S. agalactiae was more widespread than S. iniae. Molecular serotyping of the S. agalactiae isolates identified the serotype Ia and serotype Ib. Histopathological findings were meningitis, meningoencephalitis and septicaemia. Identical virulence profiles were found for all strains of S. iniae, while S. agalactiae strains were separated into virulence profile I and profile II. All strains were susceptible to the tested antibiotics and resistant to oxolinic acid. Only S. agalactiae serotype Ib showed resistance to sulphamethoxazole–trimethoprim. This is the first study from the Philippines to characterize the streptococci involved in disease outbreaks in tilapia aquaculture. Outputs from this study will promote the development of efficacious disease control strategies in tilapia farming for the Philippines and South-East Asia.     

Antimicrobial susceptibility and enrofloxacin resistance of streptococcal bacteria from farmed Nile tilapia,Oreochromis niloticus (Linnaeus 1758) in Thailand

This study examined the antimicrobial susceptibility and mutation(s) in quinolone resistance‐determining regions (QRDRs) in streptococcal pathogens isolated from farmed Nile tilapia Oreochromis niloticus in Thailand. Surveillance of antimicrobial susceptibility in tilapia streptococcal pathogens reveals that Streptococcus agalactiae (n = 97) and Streptococcus iniae (n = 3) from diseased tilapia were susceptible to amoxicillin, florfenicol, sulfamethoxazole/trimethoprim and sulfadimethoxine/ormetoprim, however, only 78 isolates were susceptible to enrofloxacin. Twenty‐two enrofloxacin‐resistant S. agalactiae isolates were further examined for mutations in the QRDRs of gyrA, gyrB, parC and parE genes. Twenty isolates had single base pair changed in the gyrA sequence, C‐242‐T. Point mutations in gyrB, GC‐1135, 1136‐AA and T‐1466‐G, were identified in one isolate. All resistant isolates harboured a mutation in the parC gene, C‐236‐A, while no mutations were observed in the parE gene. The study represented mutations of gyrA and parC genes as marked modification of the enrofloxacin‐resistant S. agalactiae from farmed tilapia. This study is a primary report of the QRDRs mutations associated with fluoroquinolone resistance from streptococcal pathogen in the cultured fish. The phenotypic and genotypic characterization of enrofloxacin resistance S. agalactiae evident in this study has led to an improved regulation of antimicrobial use in Thai aquaculture.     

Comprehensive Investigation of Streptococcosis Outbreaks in Cultured Nile Tilapia,Oreochromis niloticus,and Red Tilapia,Oreochromis sp., of Thailand

Streptococcosis causes economic losses due to mass mortality at all culturing stages in Nile tilapia, Oreochromis niloticus, and red tilapia, Oreochromis sp., farming throughout Thailand. Diseased tilapia collected from outbreak areas during 2003–2012 were examined using histopathological, biochemical, and molecular tools. Infected fish showed clinical signs of septicemia, and bacteria were found in visceral organs. All gram‐positive cocci isolates were negative to catalase and oxidase, and exhibited β‐hemolysis; however, they possessed various biochemical profiles. PCR amplification of the 16S rRNA gene was used for 165 samples, and resulted in identification of 143 (86.67%) with Streptococcus agalactiae and 14 (8.48%) with Streptococcus iniae, and 8 (4.85%) with mixed infection. High similarity (≥98%) of 16S rRNA gene sequences to the reference strain S. agalactiae (accession no. EF092913) and S. iniae ATCC29178 type strain was observed in the typing of S. agalactiae and S. iniae from Thai farmed tilapia. This investigation documented that at least two species of streptococcal bacteria, S. agalactiae and S. iniae, were involved in tilapia streptococcal infection in Thailand. The molecular recognition of the etiologic agents showed that S. agalactiae was the dominant species that cause disease in all culture areas, whereas S. iniae were discovered only in cases from the northeastern and central regions.     

Morphological characterization of the adherence and invasion of Streptococcus agalactiae to the intestinal mucosa of tilapia Oreochromis sp.: An in vitro model

Streptococcosis in tilapia Oreochromis sp. is possibly the most important bacterial disease for fish production worldwide. In Colombia, streptococcosis is caused by Streptococcus agalactiae (GBS), but in other countries, Streptococcus iniae is also involved. Prevention of streptococcosis is required and must be addressed for economic, social, international trade and public health reasons. This research used an in vitro culture of tilapia intestine to detail the intestinal mucosal response once the pathogen contacts the epithelium. We show that S. agalactiae sheds off its capsule to adhere to the epithelium. The bacterium adheres as a single individuum, in groups or in chains and is able to divide on the apical border of enterocytes. GBS adheres at and invades exclusively through the apical portion of the intestinal folds, using the transepithelial route. Once within the cytoplasm of enterocytes, the bacteria continue to divide. On the basolateral side of the epithelium, the microorganisms leave the cells to reach the propria and travel through the microcirculation. No evidence of an immuno‐inflammatory reaction or goblet cell response in the epithelium or the lamina propria was seen during the process of adherence and invasion of the pathogen.     

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.