Antibacterial Effects of Chinese Herbal Medicines against Streptococcus iniae in Oreochromis niloticus

In this study, the antibacterial effects of Chinese herbal medicines against Streptococcus iniae in Oreochromis niloticus were investigated by drug sensitiv- ity test. The results indicated that Streptococcus in/ae was extremely sensitive to Calla Chinensis, Fructus Hippophae and Fructus Murne, highly sensitive to Sappan Lignum, Pericarpium Granati, Folium Eucalypti and Radix Scutellariae, moderately sensitive to Rh/zoma Coptidis, Radix et Rhizoma Rhei, Flos Caryophyllata, Cortex phellodendri and Rabdosia serra （ Maxim. ） Hara, and insensitive to Herba Portulacae , Herba Houttuyniae , Polygonum hydropiper L. , Herba Menthae , Radix Glycyrrhizae , Pericarpium Citri Reticulatae , Herba Andrographitis and Rhizoma Acori Tatarinowii. Chinese medicinal herbs which Streptococcus iniae was extremely sensitive or moderately sensitive to were selected and prepared into three Chinese herbal medicine formulae for medicated bath of Oreochromis niloticus infected arti- ficially with Streptococcus iniae. The results showed that medicated bath elicited therapeutic effects on infected Oreochromis niloticus. Formula II exhibited the best therapeutic effect with an effective percentage of 90%.

Variation in the immunoglobulin levels in turbot(Scophthalmus maximus) after vaccination with Streptococcus iniae

A pathogenic bacterium （S636）, identified as Streptococcus iniae, was isolated from turbot （Scophthalrnus maximus） in 2005. We immunized turbot with formalin-killed S. iniae four times （on days 1, 14, 21, and 28） by intraperitoneal inoculation. After each vaccination, we obtained serum samples and isolated the lymphocytes from the peripheral blood, spleen, pronephros, and mesonephros. We measured surface Ig-positive （sIg＋） lymphocytes and serum antibody levels from these organs using flow cytometry and enzyme-linked immunosorbent assay （ELISA）, respectively, using monoclonal antibodies against turbot immunoglohulin. We confirmed that the antibody reacted with both the surface and plasma Ig by confocal laser scanning microscopy and electron microscopy. The percentage of sIg＋ in the lymphocytes increased following each successive vaccination. The mean percentage increased from 31.96% （control） to 37.49%, 38.36%, 42.9%, and 51.63% in the peripheral blood; from 27.09% to 36.63%, 36.81%, 39.28%, and 46.0% in the spleen; from 22.2% to 28.99%, 29.21%, 32.83%, and 41.58% in pronephros; and from 18.12% to 22.17%, 22.45%, 25.69%, and 31.68% in the mesonephros. The ELISA results were consistent with these results. Both the total and specific antibody levels increased with each vaccination. The mean OD value of the specific antibody assay increased from 0.094, to 0.269, 0.283, 0.333, and 0.421; for total antibody the mean OD value increased from 0.133, to 0.292, 0.323, 0.413, and 0.527.

Analysis of Biological Activity and Immunogenicity of Extracellular Products from Streptococcus iniae

[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. Cu^2＋ , Ca 2＋ , K^＋ and Mg^2＋ exhibited an inhibitory effect on ECPase activity, whereas Fe^3＋ , Co^2＋ and Mn^2＋ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Two-dimensional 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 anti- S. iniae was used for Western-blot 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 .

牛蛙背部肿胀病的病原菌分离与鉴定及药敏实验

在广东广州番禺某牛蛙养殖场发现较为特殊的疾病且传染率和死亡率较高,为查明其致病原,从患病牛蛙(Rana catesbeiana)中抽取其背部的积液和对其他病变部位如肝、脾等进行细菌的分离纯化后得到两株优势菌,然后将两株优势菌进行16S rRNA基因测序、进化树构建、革兰氏染色细菌和生理生化实验进行鉴定和分析,结果显示一株菌株GPY923为嗜水气单胞菌(Aeromonas hydrophila),另一株菌株GPY924为海豚链球菌(Streptococcus iniae),进行人工感染后表明其具有较强的致病性。药敏试验结果显示嗜水气单胞菌对丁胺卡那、红霉素、头孢他啶、头孢呋辛、青霉素和氟苯尼考敏感,对多种药物耐药;海豚链球菌对林可霉素和头孢呋辛耐药,对头孢他啶中度敏感,其余药物均敏感。

2株海豚链球菌的分离鉴定及药敏特性分析

从河北省石家庄市平山县某养殖场患病鲟鱼和井陉县某养殖场患病鲟鱼体内分别分离获得一株革兰氏阳性菌,对2株菌的形态特征、生理生化特性及16S rDNA序列进行分析,鉴定均为海豚链球菌(Streptococcus iniae);采用微量肉汤稀释法对2菌株分别进行药敏试验,获得各抗生素对菌株的最小抑菌浓度(MIC),发现由井陉患病鲟鱼分离获得的菌株JX-1对硫酸新霉素、甲砜霉素、盐酸多西环素、磺胺甲氧嘧啶钠、磺胺甲噁唑+甲氧苄啶高度敏感,对氟苯尼考中度敏感,对恩诺沙星、氟甲喹耐药;由平山患病鲟鱼分离获得的菌株PS-3对甲砜霉素、盐酸多西环素、磺胺间甲氧嘧啶钠、磺胺甲噁唑+甲氧苄啶高度敏感,对硫酸新霉素、氟苯尼考中度敏感,对恩诺沙星、氟甲喹耐药。2菌株的药敏特性差异主要表现在JX-1对硫酸新霉素中度敏感,PS-3对硫酸新霉素高度敏感。