Comparative pathological study of tilapia naturally infected with Streptococcus agalactiae and virulence gene profiling of isolated strains
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摘要:
在自然感染无乳链球菌(Streptococcus agalactiae)的罗非鱼(Oreochromis niloticus)成鱼、稚鱼和自然携带无乳链球菌的罗非鱼体内分别获得14株、4株和2株无乳链球菌。临床和组织病理学分析显示,罗非鱼成鱼出现无规则游动,脑、眼眶、鳃和鳍条充血,眼球突出、白浊,内脏器官肿大、充血,以肾小管玻璃样变性、脑膜炎和心外膜炎等组织病理学变化为特征;罗非鱼稚鱼体表无明显症状,但部分内脏器官呈现肿大、充血现象,以脾脏血管区出血、肾小管上皮细胞变性、脑组织炎症反应较轻为其主要组织病理学特征。此外,罗非鱼胃固有层内及稚鱼肝脏组织中有大量的嗜酸性粒细胞浸润,可观察到无乳链球菌在成鱼的脑、心脏以及稚鱼肝脏中增殖;自然携带无乳链球菌的罗非鱼临床症状和组织学病变均不明显。PCR检测发现,各无乳链球菌毒力基因谱相同,但自然感染无乳链球菌的罗非鱼成鱼、稚鱼和自然携带无乳链球菌的罗非鱼的病理学损伤差异显著。
Abstract:We isolated 14, 4 and 2 strains of Streptococcus agalactiae from naturally infected adult and juvenile tilapia as well as tilapia naturally carrying S. agalactiae, respectively. The clinical signs and anatomy changes of adult tilapia were as follows: erratic swimming, congestion of brain, eyeballs, gills and fins, exophthalmia, corneal opacity and swelling of visceral organs, which were characterized by histopathological changes with tubular hyaline degeneration, meningitis and epicarditis. The clinical symptoms of juvenile tilapia were not obvious, but some of the internal organs showed swelling and congestion, characterized by main histopathological features of hemorrhage of spleen vascular area, degenerated renal tubular epithelial cells and milder inflammatory response in brain tissue. In addition, eosinophil infiltration was found in the lamina propria of tilapia and in the liver of juvenile fish. It was observed that S. agalactiae proliferated in the brain and heart of adult fish and in the liver of juvenile tilapia, respectively. The clinical symptoms and histological lesions in tilapia carrying S. agalactiae were not obvious. The results of PCR detection show that all the S. agalactiae strains had the same virulence gene profiles, but there were significant differences in pathological damages among adult fish, juvenile fish and tilapia carrying S. agalactiae.
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海陵湾位于粤西阳江海区,为半封闭港湾,内湾风浪平缓,是重要的贝类养殖和网箱养殖基地,也是广东省重要的增殖放流水域;外湾海岸线狭长,海域开阔,拥有国家海滨AAAAA级旅游风景区。不少学者已对海陵湾浮游植物[1]、沉积物[2-4]、水环境[5-6]、抗生素和雄激素来源[7-10]等方面做过相关研究,但关于海陵湾浮游动物的研究极少见报道。浮游动物作为海洋生态系统中重要的中间环节,其下行控制着浮游植物群落的数量,上行则可影响鱼类等的资源量,在海洋生态系统结构和功能中起重要的调控作用[11-12]。此外,浮游动物的生存状况与水环境密切相关,浮游动物群落结构的变化可反映水环境质量状况,因此,研究浮游动物群落结构变化对评价海域水质污染状况也具有重要意义。
近年来,随着海陵湾养殖业和旅游业的发展,养殖废水、生活污水及船坞废水的大量排放,逐渐改变了海陵湾的生态环境,因此有必要定期监测海湾生态环境,以期为海湾的进一步开发利用提供依据。本研究对2015—2016年海陵湾的浮游动物种类组成、优势种、丰度和生物量、生物多样性等群落结构特征及其与主要环境因子的关系进行了分析,以期为保护海陵湾生态环境及维持海洋生物资源的可持续利用提供基础资料。
1. 材料与方法
1.1 样品采集与分析
于2015年2月(冬季)、7月(夏季)、11月(秋季)及2016年4月(春季)分别对阳江海陵湾(111°42.53'E、21°27.95'N—111°57.36'E、21°38.29'N)进行渔业生态资源综合调查(图1),共设置9个调查站位。浮游动物样品使用浅水Ⅰ型浮游生物网自底至表垂直拖网采集并用福尔马林溶液固定保存,再在室内用显微计数和湿质量生物量法分析。
1.2 统计方法
优势度[13]
$$ Y = \left( {{n_i}/N} \right) \times {f_i} $$ Shannon-Wiener多样性指数[14]
$$ H' = - \sum\limits_{i = 1}^S {{P_i}{{\log }_2}{P_i}} $$ 均匀度[15]
$$ J' = H'/\left( {{{\log }_2}S} \right) $$ 其中ni为第i种的总个体数,N为海陵湾所有物种的总个体数,fi为第i种在海陵湾采样点出现的频率,以Y>0.02作为优势种;S为样品中的总种类数,Pi为第i种在全部样品中的比例(Pi=ni/N)。
2. 结果
2.1 种类组成
阳江海陵湾4个季节共发现132种浮游动物及14类浮游幼虫,以桡足类种类数最多 (54种),占总种数的40.91%,其次是水螅水母类 (18种),占总种数的13.64%;端足类和毛颚类各10种,分别占总种数的7.58%;管水母8种,占总种数的6.06%;其余类群种类数均低于6种(表1)。
表 1 浮游动物种类组成Table 1. Species composition of zooplankton类群
group冬季
winter夏季
summer秋季
autumn春季
spring全年
whole year种数
species number占比/%
percentage种数
species number占比/%
percentage种数
species number占比/%
percentage种数
species number占比/%
percentage种数
species number占比/%
percentage原生动物
Protozoa1 3.33 2 8.00 2 2.70 3 3.26 3 2.27 水螅水母类
Hydromedusae8 26.67 2 8.00 9 12.16 10 10.87 18 13.64 管水母类
Siphonophorae1 3.33 1 4.00 1 1.35 8 8.70 8 6.06 栉水母类
Ctenophora2 6.67 1 4.00 1 1.35 1 1.09 2 1.52 异足类
Heteropoda− − − − 1 1.35 2 2.17 2 1.52 翼足类
Pteropoda− − − − 3 4.05 3 3.26 3 2.27 枝角类
Cladocera2 6.67 2 8.00 2 2.70 2 2.17 2 1.52 桡足类
Copepoda9 30.00 10 40.00 32 43.24 29 31.52 54 40.91 介形类
Ostracoda1 3.33 − − − − 3 3.26 3 2.27 糠虾类
Mysidacea− − − − 2 2.70 2 2.17 2 1.52 端足类
Amphipoda2 6.67 − − 2 2.70 8 8.70 10 7.58 磷虾类
Euphausiacea− − 1 4.00 3 4.05 2 2.17 3 2.27 十足类
Decapoda− − 1 4.00 3 4.05 3 3.26 5 3.79 毛颚类
Chaetognatha2 6.67 2 8.00 9 12.16 9 9.78 10 7.58 海樽类
Thaliacea1 3.33 1 4.00 2 2.70 4 4.35 4 3.03 有尾类
Appendiculata1 3.33 2 8.00 2 2.70 3 3.26 3 2.27 合计 total 30 100.00 25 100.00 74 100.00 92 100.00 132 100.00 浮游幼体类
pelagic larva8 9 11 11 14 浮游动物群落种类组成存在季节变化,冬季共发现30种,处于次低水平;夏季种类数最少(25种),占全年出现浮游动物种类数的18.94%;随后,秋季浮游动物种类数增至74种,占全年出现浮游动物种类数的56.06%;次年春季浮游动物种类数继续增多(92种),占全年出现浮游动物种类数的69.70%。
2.2 优势种
调查期间,该海域共出现11种优势种(不包括幼虫),两相邻调查季节间没有出现共同优势种,浮游动物群落存在明显的季节变化。
冬季夜光虫 (Nocitiluca scintillans) 大量繁殖形成该海域唯一优势种,优势度高达0.96,平均丰度为5.40×103 个·m−3;夏季夜光虫消失,取而代之的是桡足类的红纺锤水蚤 (Acartia erythraea) 和瘦尾胸刺水蚤 (Centropages tenuiremis),以及有尾类的长尾住囊虫 (Oikopleura longicauda) 和红住囊虫 (O. rufescens);秋季毛颚类的肥胖软箭虫 (Flaccisagitta enflata)显著增多,桡足类优势种也发生了变化。到次年春季,夜光虫再次出现形成优势,平均丰度为227.12 个·m−3 (表2)。
表 2 浮游动物优势种Table 2. Dominant species of zooplankton季节
season优势种 (优势度Y)
dominant species (dominance Y)冬季 winter 夜光虫 (0.96) 夏季 summer 红纺锤水蚤 (0.03)、瘦尾胸刺水蚤 (0.03)、长尾住囊虫 (0.07)、红住囊虫 (0.05) 秋季 autumn 锥形宽水蚤 Temora turbinate (0.17)、叉胸刺水蚤 Centropages furcatus (0.10)、肥胖软箭虫 (0.18) 春季 spring 夜光虫 (0.25)、鸟喙尖头溞 Penilia avirostris (0.15)、肥胖三角溞 Pseudevadne tergestina (0.33)、软拟海樽 Dolioletta gegenbauri (0.11) 2.3 丰度和生物量的平面分布和季节变化
海陵湾浮游动物年均丰度为1 737.41 个·m−3,冬季 (5 652.85 个·m−3)最高,主要由夜光虫 (5 399.56 个·m−3)的大量增殖引起,丰度呈现湾内高于湾外;春季次之 (平均为814.41 个·m−3),湾外丰度高于湾内;秋季丰度比春季低 (平均469.51 个·m−3),丰度低值区也位于湾内;夏季浮游动物丰度最低 (仅12.88 个·m−3,图2)。除夜光虫外,海陵湾浮游动物丰度以春季最高(587.29 个·m−3),秋季次之(469.51 个·m−3),冬季为253.29 个·m−3,夏季最低(12.88 个·m−3)。
原生动物是冬季浮游动物群落的第一大类群,仅夜光虫丰度就达到5 399.56 个·m−3,主要分布在湾内,在春季也形成优势,丰度平均为227.14 个·m−3,主要密集分布在湾外。桡足类种类最多,但仅在秋季丰度相对较高。枝角类在春季丰度最高,但其他季节均较低(图3)。
浮游动物年均生物量为115.52 mg·m−3,冬季最高(291.88 mg·m−3),夏季最低 (0.81 mg·m−3),秋季(87.28 mg·m−3)略高于春季(82.11 mg·m−3)。生物量各季节的平面分布与丰度相似(图4)。
浮游动物多样性指数(H′)以秋季最高(4.21),冬季最低(0.86)且站位间变化最大,年平均为2.83;均匀度(J′)以夏季最高(0.93),冬季最低(0.21),年平均为0.60 (表3)。
表 3 浮游动物生物多样性Table 3. Biodiversity index of zooplankton季节
season多样性指数 (H′) 均匀度 (J′) 范围
range均值
average范围
range均值
average冬季 winter 0.11~2.32 0.86 0.03~0.58 0.21 夏季 summer 2.98~4.03 3.55 0.84~0.97 0.93 秋季 autumn 3.55~4.58 4.21 0.60~0.94 0.77 春季 spring 2.00~3.58 2.70 0.32~0.88 0.50 2.4 浮游动物与环境因子关系
原生动物与水温呈显著负相关;枝角类与盐度呈极显著负相关,与叶绿素a浓度呈显著负相关,与溶解无机氮呈极显著正相关;浮游动物总丰度与水温呈显著负相关;浮游动物生物量与水温呈极显著负相关,与溶解氧呈显著正相关。
3. 讨论
3.1 群落结构
调查期间,海陵湾共发现浮游动物132种,以桡足类最为丰富,是海湾浮游动物群落中最重要的类群,是中国海湾的一个普遍现象[16-19]。原生动物是海陵湾浮游动物在冬季的唯一优势类群,在浮游动物群落中占有重要的数量地位,而原生动物中又以夜光虫为主,主要分布在湾内海域。夜光虫是偏冷水性的广温广盐性赤潮种,在广东沿岸的低温季节多次形成赤潮[20]。海陵湾夜光虫也仅出现在温度较低的冬春季节。
海陵湾北面为入海河流,南面为高盐南海水,宽阔的湾口使咸淡水交换充分,并主要存在河口低盐类群、广温广盐类群、暖水沿岸类群和暖水外海类群等4种浮游动物生态类群。海陵湾浮游动物群落在冬季以夜光虫占有绝对优势,但夏季消失。夏季,海陵湾处于丰水期,充沛的河水与南海高盐水高度混合,使该季节浮游动物优势种同时存在4种生态类群,显示了海陵湾夏季浮游动物群落的复杂性。秋季,暖水沿岸种、外海暖水种和广温广盐种依然为该季节的优势种,但优势种类发生了变化。春季,夜光虫再次出现,与鸟喙尖头溞 (Penilia avirostris)、肥胖三角溞 (Pseudevadne tergestina)和软拟海樽 (Dolioletta gegenbauri)共同形成优势。显然,除夜光虫在冬春季成为优势种外,其他种类仅在一个季节形成优势,说明海陵湾浮游动物优势种的季节变化明显,群落结构变化大。然而本研究的冬夏季间和秋春季间均跨越了一个季节,这也有可能导致该调查海域浮游动物群落季节变化明显,因此,在以后的研究中应尽量研究邻近季节的浮游动物变化以避免产生误差。
3.2 环境因子的影响
温度是影响浮游动物生长繁殖的重要环境因子[11,21-22]。除夜光虫外,海陵湾浮游动物丰度春季最高,其后依次为秋季和冬季,夏季最低。金琼贝等[22]发现桡足类等某些浮游动物适宜在20~30 ℃水温之间生长,而超过该上限后出现了负增长现象。海陵湾夏季水温平均为29.64 ℃,未超过最适温度上限,说明夏季水温不是导致浮游动物数量减少的主要因素,而海陵湾冬季水温平均为17.73 ℃,低于最适水温下限,也说明在冬季水温是限制浮游动物生长的因素之一。盐度是影响浮游动物生长繁殖的另一重要环境因子[11,21,23-24]。黄加祺和郑重[24]发现沿岸浮游动物可在盐度介于0~31.7的水环境生长,但超过该上限后出现了负增长现象。海陵湾夏季盐度平均为32.53,高于沿岸类群的最适盐度上限,有可能抑制浮游动物的生长。此外,海陵湾夏季雨水充沛,雨水的冲刷和海水的涌入导致该海域水体波动较大,也可能不利于浮游动物的大量繁殖。海陵湾浮游动物种类数的季节变化也与除夜光虫外的浮游动物丰度的季节变化相似,表现为春秋季高于冬夏季,说明海陵湾浮游动物更适合在春秋季生长,冬季主要受温度影响,而夏季则受多种因素共同制约。
海陵湾浮游动物丰度和生物量均在冬季最高,夏季最低。相关分析表明,浮游动物丰度和生物量均与水温呈现显著和极显著的负相关关系,但这种关系在极大程度上受夜光虫影响。已有研究表明,夜光虫生长的最适温度在17~22 ℃[20,25],水温高于25 ℃时夜光虫会大量死亡[26],而室内实验结果也表明夜光虫的生存上限水温约为26 ℃[27]。海陵湾冬、夏、秋、春季的水温分别为17.73 ℃、29.64 ℃、26.06 ℃和23.93 ℃,只有冬季水温在最适温度范围内,适合夜光虫大量繁殖,春季水温略高于最适水温,夜光虫可以生长但丰度不高,夏、秋季节水温均高于26 ℃,在调查中均未发现夜光虫。夜光虫在海陵湾的出现及其丰度与温度密切相关,主要表现为低温季节丰度高,高温季节丰度低,与南海北部海域的报道[28-29]相似。从表4浮游动物与环境因子的相关性也可看出,水温与以夜光虫为主的原生动物类群丰度呈显著的负相关,而与其他类群丰度则不存在显著的相关关系,说明以夜光虫为主的原生动物类群对浮游动物丰度和生物量与水温均呈显著负相关关系的贡献很大,而除夜光虫外,浮游动物可能不与水温呈显著负相关关系,因为除夜光虫外的浮游动物丰度表现为春秋季高于冬夏季,与水温不呈明显的趋势性变化。除温度外,夜光虫的分布还受营养盐的影响。许多研究表明,夜光虫与营养盐呈正相关关系[30-31]。但在本研究中,以夜光虫为主的原生动物与磷酸盐及溶解无机氮均呈不显著的负相关关系。营养盐和夜光虫主要通过浮游植物形成间接关系,可以认为营养盐是夜光虫的“初级养分”[29],但有时浮游植物丰度与营养盐并不成正比关系[32],夜光虫与浮游植物丰度亦如此。冬季S1的夜光虫丰度最高(35 400个·m−3),而叶绿素a仅3.47 mg·m−3,说明在采样前较长一段时间内叶绿素a浓度可能远高于采样时的浓度,随着夜光虫的大量摄食浮游植物急剧减少。此外,Uhlig和Sahling[33]、Umani等[34]发现水体的富营养化并不一定引起夜光虫繁殖,在水体保持稳定富营养化较长的一段时间内往往没有夜光虫出现。因此,夜光虫的大量繁殖并不直接依赖于水体的高营养盐环境,而是多个因素综合作用的结果。
表 4 浮游动物与环境因子的相关关系Table 4. Pearson correlation between zooplankton and environmental factors环境因子
environmental factor原生动物
Protozoa枝角类
Cladocera桡足类
Copepoda毛颚类
Chaetognatha海樽类
Thaliacea幼体类
larvaZA/
(个·m−3)ZB/
(mg·m−3)水温/℃ temperature 0.351* −0.067 −0.148 0.040 −0.076 0.057 −0.354* −0.526** 盐度 salinity 0.047 −0.488** −0.004 −0.025 −0.227 −0.174 0.021 −0.006 ρ(叶绿素 a )/(mg·L−1) Chl a concentration 0.064 −0.354* 0.142 0.045 −0.218 −0.070 0.054 0.256 ρ(溶解氧)/(mg·L−1) DO 0.319 0.003 0.235 0.063 0.175 0.054 0.329 0.558* ρ(磷酸盐)/(mg·L−1) PO4 3--P −0.128 −0.320 −0.052 −0.002 −0.165 −0.063 −0.143 0.080 ρ(溶解无机氮)/(mg·L−1) DIN −0.061 0.513** −0.107 −0.080 0.299 0.091 −0.041 −0.050 注:ZA. 浮游动物丰度;ZB. 浮游动物生物量;*. P<0.05; **. P<0.01Note: ZA. zooplankton abundance; ZB. zooplankton biomass 夜光虫是一种常见的赤潮生物,赤潮爆发时可引起大量海洋生物死亡,如果赤潮发生在养殖区,则会对养殖业造成巨大损失。海陵湾在冬季出现了大量夜光虫,虽丰度暂时还未达到赤潮阈值,但其丰度之高很有可能随时形成赤潮,需时刻监测其发展动态,以及时应对并减少因赤潮带来的损失。
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图 1 罗非鱼脑组织PCR检测结果
M. Maker DL 2 000;阳. 阳性对照;阴. 阴性对照;1~159. 采自开平养殖场;160~166. 采自高州平养殖场;167~170. 采自廉江养殖场;171~174. 采自吴川养殖场;175~180. 采自惠州养殖场;181~188. 采自河源养殖场
Figure 1. PCR detection results of tilapia brain
positive. positive control; negative. negative control; 1−159. from Kaiping farm; 160-166. from Gaozhouping farm; 167−170. from Lianjiang farm; 171−174. from Wuchuan farm; 175−180. from Huizhou farm; 181−188. from Heyuan farm
图 2 鱼感染无乳链球菌的眼观剖检变化
罗非鱼成鱼:A. 眼眶充血、眼球突出(↓),肠道发炎、肠壁变薄、内容物发黄(*);B. 脑充血、出血(↓);C. 肝脏肿大、充血、胆囊肿大(*),脾脏肿大、充血,肾脏肿大(↓)。罗非鱼稚鱼:体表无明显症状;D. 肝脏充血肿大(*),脾脏肿大(*),肠壁变薄、内容物发黄;自然携带无乳链球菌罗非鱼:E. 体表正常;内脏无明显症状。斑马鱼:F. 身体弯曲,眼球浑浊,鳍条和腹部充血、出血(↓)
Figure 2. Change in anatomy of fish infected with S. agalactiae
Adult tilapia: A. orbital congestion and exophthalmos (↓), intestinal inflammation, thinning of intestinal wall and yellowing of contents (*); B. cerebral hyperemia and hemorrhage (↓); C. liver enlargement, congestion, gallbladder enlargement (*), splenomegaly, hyperemia and kidney enlargement (↓). Juvenile tilapia: no obvious symptoms on the body surface; D. hepatic hyperemia (*), splenomegaly (*), thinning of intestinal wall and yellowing of contents; tilapia naturally carrying S. agalactiae: E. no obvious symptoms on the body surface; no visceral symptoms. Zebrafish: F. bent body, corneal opacity, and congestion of fins (↓)
图 4 自然感染无乳链球菌罗非鱼成鱼组织病理学
A. 脑,脑膜炎,脑膜增厚,大量的炎症细胞浸润,脑血管充血;B. 脑,A图
Figure 4. Histopathology of adult tilapia naturally infected with S. agalactiae
A. brain, meningitis, thickening of the meninges, infiltration of a large number of inflammatory cells, cerebral vascular congestion; B. brain,magnified micrograph of the zone in the black frame in A, inflammatory cell aggregation (*), a large number of proliferating S. agalactiae around the blood vessels (↓); C. brain, intravascular microthrombus formation (↓); D. liver, focal inflammatory response, massive accumulation of inflammatory cells (*); E. intestinal, lamina propria congestion (↓), epithelial cells slightly shed; F. gill, gill filament epithelial cell hyperplasia, fusion (↓), sinus congestion (*); G. heart, epicarditis, epicardial thickening, a large number of inflammatory cell infiltration; H. heart, magnified micrograph of the zone in the black frame in G, massive proliferation around the blood vessels S. agalactiae (↓), intravascular inflammatory cell proliferation (*); I. heart, epicardial septic foci, a large number of cells, cell debris (*) and neutrophils in the abscess (↓); J. spleen , hemorrhage (*), splenic artery epithelial cell damage, thrombosis (↓); K. stomach, gastric lamina propria inflammation, vascular congestion (*), a large number of eosinophil infiltration in the inflammation area (↓); L. kidney, kidney tubulous degeneration (↓), renal interstitial hemorrhage (*)
图 5 自然感染无乳链球菌罗非鱼稚鱼组织病理学
A. 脑,脑炎,小胶质细胞聚集,血管充血;B. 脑,A图
Figure 5. Histopathology of juvenile tilapia naturally infected with S. agalactiae
A. brain, encephalitis, microglia accumulation, vascular congestion; B. brain, magnified micrograph of the zone in the black frame in A, massive proliferation of microglia (*) and vascular congestion (↓); C. liver, liver hemorrhage (*), blood vessels a large number of eosinophil infiltration around; D. liver, magnified micrograph of the zone in the black frame in C, large proliferation of S. agalactiae (*) and hyperplastic eosinophils (↓); E. intestinal, intestinal villi shortened (↓), epithelial cell shedding (*); F. gill, gill silk epithelial cell shedding is "sticky"(↓), sinus congestion (*); G. heart; H. spleen, lymphocyte area shrinkage, vascular area bleeding (*); I. stomach, Inflammatory reaction in the lamina propria of the stomach; J. stomach, magnified micrograph of the zone in the black frame in I, a large number of eosinophils (↓) and neutrophil accumulation (*); K. kidney, tubular degeneration, necrosis; L. kidney, magnified micrograph of the zone in the black frame in K, renal tubular epithelial cells degeneration, shedding (*)
图 6 自然携带无乳链球菌的罗非鱼组织病理学
A. 脑;B. 肝脏,肝细胞轻微肿胀;C. 肠道;D. 鳃;E. 心脏;F. 脾脏,脾血窦充血;G. 胃,固有层轻微水肿;H. 肾脏,肾小管上皮轻微变性
Figure 6. Histopathology of tilapia carrying S. agalactiae
A. brain; B. liver, liver cells slightly swollen; C. intestine; D. gill; E. heart; F. spleen, spleen sinus congestion; G. stomach, lamina propria edema; H. kidney, the epithelium of kidney tubules is slightly degenerated.
表 1 样品采集和无乳链球菌菌株分离信息
Table 1 Sample collection and information of separation of S. agalactiae strain
质量/g
mass采样地
sampling city养殖密度/尾·hm–2
breeding density/ind·hm–2发病史
history of disease样品数/尾
number of samples菌株数
number of strains菌株编号
strain No.检出率/%
detection rate≈500 开平市 ≈100 无 159 2 TKP1601-02 1.26 ≈15 高州市 ≈200 爆发 7 4 TGZ1601-04 57.10 ≈500 廉江市 4 4 TLJ1601-04 63.64 吴川市 4 2 TWC1601-02 惠州市 6 0 − 河源市 8 8 TLC1601-08 表 2 引物列表
Table 2 Primers of this study
引物
primer上游引物序列 (5'−3')
forward primer sequence下游引物序列 (5'−3')
reverse primer sequence扩增靶标
amplification target长度/bp
length16S rDNA-F/R AGAGTTTGATCC TGGCTCAG TACGGCTACCTTGTTACGACTT 16S rDNA 1 472 sdi-F/R ATTCTCCTCCTGGCAAAGCC TGACGCTTGGTAGTTGCTGT 16S−23S rDNA 192 fbsA-F/R AGTGTTGGAAATCAAAGTCAAGGT TTCATTGCGTCTCAAACCGC 纤维蛋白结合蛋白A (fbsA) 924 cfb-F/R AACTCTAGTGGCTGGTGCAT CTCCAACAGCATGTGTGATTGC CAMP因子基因 (cfb) 650 dltR-F/R GTCTGAAGGTCCCCAAACCT TGTTACCCAAACGCTCAGGAT 调节蛋白基因 (dltR) 392 ponA-F/R ACAACTTGCTTTGCTCGCTG AGAGCCCTTCTGGCATTGTC 青霉素结合蛋白基因 (ponA) 1 337 hylB-F/R TCCACAACCCGTCACAACAC AACGCGCCCCATATCTACTA 透明质酸酶基因 (hylB) 790 cspA-F/R TGCACGTAACCAGTATCGCA GCACCGAGTTTAACGGCATC 丝氨酸蛋白酶基因 (cspA) 175 sodA-F/R TGATGCGCTTGAGCCACATA GCTTTGATGTAGTTAGGACGAACA 超氧化物歧化酶基因 (sodA) 513 sip-F/R ACAGATACGACGTGGACAGC ACCACGATCTGGCATTGCAT 表面免疫相关蛋白基因 (sip) 1 173 fbsB-F/R AGTTGCGCAAACTTCTGTCC TTTCCGCAGTTGTTACACCG 纤维蛋白结合蛋白B基因 (fbsB) 158 iagA-F/R GCATGGCCATTCCACTGAAG GCTAGCACTCATGGCACCTT 侵袭相关基因 (iagA) 493 scpB-F/R TGCGGCCTTTATCAGTCGAA AACAGTCCCATGATACCCGC C5a肽酶基因 (scpB) 273 bca-F/R TCAAGTTTGGTGCAGCTTCTG TCCGGTACTGACAATACTAACAAT αC蛋白基因 (bca) 616 srr-1-F/R ATGTTGCAGTAAAGCGCTGC GGAAGAGAGTCGTTTTCGGC 富含丝氨酸重复蛋白基因 (srr-1) 727 bibA-F/R TGCATAATATCCAGGTGTAGGCA TGAGAGATTGGGAAGTGGTGC 免疫原性细菌黏附蛋白基因 (bibA) 943 psaA-F/R AGCTGTCACCCTTTTGACCTT TAGGCTTAGGTGCCTGTGCT 肺炎球菌表面抗原A基因 (psaA) 828 lmb-F/R ATTTGTGACGCAACACACGG TCTTGTTTCCGCTTGGAGCA 层黏连蛋白结合蛋白基因 (lmb) 263 spb1-F/R GACATGGGGAGATGGTGGTG AGCTTCTGTGCCCCATTCAA 溶血素Ⅲ (spb1) 652 bac-F/R TGATTCCCTTTTGCTCTGCCA GTTCATGGGAAGCGTTGCTC βC蛋白基因 (bac) 557 pavA-F/R TCGACTTACATTGCCCCACC GGCGGCATCTGTCTTAACCT 纤维蛋白结合蛋白基因 (pavA) 996 cppA-F/R TGCAAATCTTGTCCCTGTGC TCGTACTCGTGCGGTGAATG C3降解蛋白酶基因 (cppA) 387 cylE-F/R ATTCTCCTCCTGGCAAAGCC TGACGCTTGGTAGTTGCTGT β-溶血素/溶细胞素基因 (cylE) 176 表 3 21对毒力基因检测结果
Table 3 Detection results of 21 virulence genes
毒力基因
virulence gene菌株 strain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 fbsA + + + + + + + + + + + + + + + + + + + + cfb + + + + + + + + + + + + + + + + + + + + dltR + + + + + + + + + + + + + + + + + + + + ponA + + + + + + + + + + + + + + + + + + + + hylB + + + + + + + + + + + + + + + + + + + + cspA + + + + + + + + + + + + + + + + + + + + sodA + + + + + + + + + + + + + + + + + + + + sip + + + + + + + + + + + + + + + + + + + + fbsB + + + + + + + + + + + + + + + + + + + + iagA + + + + + + + + + + + + + + + + + + + + scpB – – – – – – – – – – – – – – – – – – – – bca + + + + + + + + + + + + + + + + + + + + srr-1 + + + + + + + + + + + + + + + + + + + + bibA + + + + + + + + + + + + + + + + + + + + psaA + + + + + + + + + + + + + + + + + + + + lmb – – – – – – – – – – – – – – – – – – – – spb1 + + + + + + + + + + + + + + + + + + + + bac + + + + + + + + + + + + + + + + + + + + pavA + + + + + + + + + + + + + + + + + + + + cppA + + + + + + + + + + + + + + + + + + + + cylE + + + + + + + + + + + + + + + + + + + + 注:1−2. TKP1601−TKP1602;3−6. TGZ1601−TGZ1604;7−10. TLJ1601−TLJ1604;11−12. TWC1601−TWC1602;13−20. TLC1601−
TLC1608 -
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