The present study was conducted to research the antibacterial effects of 7 common fishery drugs and their combinations against pathogenic Edwardsiella ictaluri from channel catfish in vitro.The minimum inhibitory concentrations and the minimal bactericidal concentrations of 7 fish drugs were tested by the method of doubling dilution in test tube.On the basis of preliminary screening,antibacterial effecst of 5 fish drugs against Edwardsiella ictaluri were compared and the optimal parameters were optimized,respectively,by using the circle of inhibiting bacterium of 1,5,10 and 20 times MICs as parameters,MIC regarded as a unit.Then,the best parameter was applied to compare the inhibiting effects of 5 fishery drugs in single and their combinations.The results showed that the MIC and MBC of ETSP was the minimum,which were 3.2 and 6.4 μg/mL,respectively,and that of GCHBH were 12.8 and 25.6 μg/mL,respectively,but CSP and NSP had no inhibitory effects on it,even at the concentration of 1638.4 μg/mL.The bacteriostasis circle under 5 times concentration of MIC was the best parameter.Under the parameter,Edwardsiella ictaluri was the most sensitive to ETSP,and followed by GCHBH,EP,FP and DHP.The combination of EP and ETSP had the best antibacterial effect.

In this report,we use the recombinant tilapia Hsp70(tHsp70)from Pichia yeast to prepared non-covalent HSP70-antigen complexes with thallus and extracellular bacteria products(ECP)of Streptococcus iniae,and investigated their effects on cellular immune response of tilapia by detection of Proliferation and related immune gene expression of peripheral blood lymphocyte in vitro.Our results demonstrated that tHsp70 could form complexes with thallus and ECP of tilaplia in vitro.In addition,tHsp70(10 μg/mL)and all tHsp70(10 μg/mL)-thallus complexes enhanced the proliferation of tilapia peripheral blood lymphocytes(P0.01).Similarly,thallus alone also enhanced the proliferation of tilapia peripheral blood lymphocytes(P0.05).By contrast,ECP alone exhibited some inhibitory effect on the proliferation of tilapia peripheral blood lymphocytes(P=0.056).However,the complex of tHsp70 at 10,but not at 100 and 1 μg/mL,with ECP reduced the inhibitory effect of ECP on the proliferation of tilapia peripheral blood lymphocytes.Moreover,tHsp70(10 μg/mL)alone or tHsp70(10 μg/mL)-antigen complexes could significantly influence the expression of immune related genes(PGK,MMP9,ICER,MHCⅡ)in tilapia peripheral blood lymphocyte compare to antigen alone and medium control groups.The study indicated that tHsp70 have the immunoadjuvant and the immunopotentiator function,and provided experimental data for development the tHsp70 based vaccine against tilapia streptococcal diseases.

An outbreak of a poxvirus-like infection in a population of 20,500 50-day-old soft shell turtles (Pelodiscus sinensis) at a culture farm in Pingtung County, southern Taiwan, occurred from late November to early December 2008. A mortality rate of approximately 44% was recorded and the clinical findings included anorexia, lethargy, swollen necks and several unusual vesicles and bullas were present on the legs and shell skin. When the surviving turtles reached the age of 11 months (October 2009), another outbreak of this disease occurred in the same pond, resulting in a mortality rate of approximately 20%. In both outbreaks, histopathological examination revealed typical acute lesions, such as ballooning degeneration and acute cell swelling of keratinocyte 5 of the epidermis. Furthermore, tiny eosinophilic cytoplasmic inclusion bodies were observed in the vacuoles. Sloughed necrotic epidermis, inflammatory cells, and a predominance of heterophils were found to have infiltrated the area of the exposed underlying dermis. In addition, patches of skin ulceration and deep layer inflammation were present, and secondary bacterial infection was suspected.Six soft shell turtles were challenged with a 0.45 mu m filtered virus suspension and showed clinical signs of anorexia and lethargy. The observed cutaneous lesions, 14 days post-challenge were similar to those reported on the farm and included medium to large size vesicles, white and circular thin-layered pustules and sloughed necrotic epidermis over the legs, head and shell skin. In addition, numerous white pock lesions on the CAM of embryonated soft shell turtle eggs which were also inoculated with the virus suspension were observed 3 days post-challenge. A transmission electron microscopy study; conducted from both farmed and experimentally infected turtles revealed enveloped, ovoid-shaped viral particles, measuring approximately 239 nm in diameter and 483 nm in length. According to its morphology and size, the virus was very similar to a poxvirus. Therefore, we provisionally named this agent, "soft shell turtle poxvirus-like virus".

In order to study factors that influence the separation and culture of tilapia peritoneal macrophages,and to recognition the characteristic of tilapia peritoneal macrophages.In the study,We separated macrophages from peritoneal cavity of Tialpia(Oreochromis niloticus♀×Oreochromis aureus♂)after intraperitoneal injected with squalene in the condition of difference growth temperature;The cell characteristic was observed by screening cell medium and Wright's staining.The results show the macrophages were easily obtained in the condition of 19~25℃,Separation result was obviously affected when water temperature was aboved 28℃;It was a optimization period for separation between 48-72 h after injected squalene;Serum of tilapia play an important role in the tilapia peritoneal macrophages invitro cultrue and fetal bovine serum was not fit;The characteristic of tilapia peritoneal macrophage was similar to mammalian macrophage,irregularly shaped with a low nucleus to cytoplasm ratio,adherent to plastic and glass,able to form multi-nucleated giant.The work laid a foundation for the further research of immunological function about tilapia peritoneal macrophage.

The research aimed to investigate the antibacterial effects of 28 antibiotics and 10 Chinese herbal medicines to the pathogen of Streptococcus iniae disease from tilapia by disc agar diffusion and microdilution procedure. The results showed that Streptococcus iniae were highly sensitive to Cefaclor, Clarithromycin, Cefalotin, Cefobid, Ceftriaxone, Cefradine, Albamycin, Cephaloridine Ⅴ, Roxithromycin, Cefuroxime and Cephalexin, and which were resistant to Kanamycin, Aztreonam, Cefixime and Lyncomycin. In the meantime, the results showed that 10 Chinese herbal medicine water extracts had inhibitory effect on Streptococcus iniae to different degree. The antibacterial effect of Rhizoma Coptidis was better than the other nine kinds with the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) to Streptococcus iniae were 3.75 mg/mL and 7.50 mg/mL respectively, and which inhibitory circle exceeded 20 mm; Flos Lonicerae, Scutellaria baicalensis, Radix et Rhizoma Rhei, Forsythia suspense and Cortex Phellodendri chinesis had to some extent inhibition of the bacterium with MIC of 7.50～15.0 mg/mL, and which inhibitory circle of the five medicine extracts exceeded 10 mm, but Folium Isatidis, Herba Andrographitis, Herba Taraxaci and Radix Gentianae had no inhibition of the bacterium with MIC of more than 240 mg/mL.

采用琼脂扩散法和二倍稀释法测定了29种抗生素和10种中草药及其组合对致病性鮰爱德华氏菌(Edwardsiella ictaluri)的抑菌作用。结果表明:病原菌对29种抗菌类药物中的头孢克洛、复达欣、头孢噻吩、先锋必、氨曲南、恩诺沙星、头孢拉定、头孢克肟、阿莫西林、新生霉素、先锋Ⅴ、氧氟沙星、呋喃妥因、头孢呋肟、头孢氨苄和羧苄青霉素等16种敏感,对克拉霉素、菌必治、红霉素、林可霉素、多粘菌素B、罗红霉素等6种表现出耐药性。10种中草药制剂对鮰爱德华氏菌均有不同程度的抑菌作用,其中黄芩的抑菌作用最强,它对鮰爱德华氏菌的最低抑菌浓度和最低杀菌浓度分别为3.75mg/mL和7.50mg/mL,抑菌圈平均直径达29.17±1.60mm;连翘、大黄、蒲公英、金银花、黄柏有较强的抑菌作用,MIC为7.50～15.0mg/mL,抑菌圈平均直径均在20mm以上;而龙胆草表现出较弱的抑菌作用。试验中所选15组由2种中草药组合的复方制剂,大部分具有协同作用,少数药物配伍后可能因拮抗而抵消作用。

为筛选罗非鱼链球菌疫苗免疫前后白细胞表达变化基因及探讨鱼类白细胞免疫机理,利用抑制性差减杂交(SSH)技术,以链球菌疫苗免疫前后罗非鱼白细胞cDNA为材料,构建了罗非鱼免疫前后正、负2个cDNA差减文库,并采用地高辛(DIG)标记差减文库cDNA作为探针,进行差异表达片段的筛选鉴定.结果显示:2个文库重组率均在90 %以上,插入片段在300～900 bp,接头连接效率超过50 %,差减效率非常高效,阳性率均超过70 %.杂交筛选后正、负文库各挑选30个阳性克隆进行测序组装聚类,正、负文库各获得16和18条非冗余EST(unigene),所得序列经GenBank同源性分析,正、负文库各有10和11条unigene获得功能注释,且各有6和7条unigene没有同源性匹配,为未知新基因.

The present experiment was conducted to detect and analyze the expressions of hsp70 gene in brain and spleen of tilapia infected with Streptococcus using real-time fluorescence quantitative PCR,the 2-△△CT method and 7500 Software v2.0.1.The results showed no significant changes in the expression of hsp70 mRNA in brain after 48h infection,while the expression of hsp70 was increased in spleen significantly and was 5.5 times higher than that of the control after 24h infection,which suggested that hsp70 may play an important role in immune response of tilapia infected with Streptococcus.These results laid down the foundation for further research on the function of hsp70 in tilapia infected with Streptococcus.

采用琼脂扩散法和二倍稀释法测定了31种抗生素和10味中草药及组合对致病性细爱德华氏菌(Edwardsiella ictaluri)的抑菌作用.结果表明:31种抗菌类药物中,病原菌对头孢克洛、复达欣、头孢噻吩、先锋必、氟哌酸、环丙沙星、氨曲南、恩诺沙星、头孢拉定、头孢克肟、阿莫西林、新生霉素、先锋V、氧氟沙星、呋喃妥因、头孢呋肟、头孢氨苄和羧苄青霉素18种药物敏感,对克拉霉素、菌必治、红霉素、林可霉素、多粘菌素B、罗红霉素6种药物表现耐药性.10种中草药水提取物对鮰爱德华氏菌均有不同程度的押菌作用,其中黄芩水提物的抑菌作用最强,其对鮰爱德华氏菌的最低抑菌浓度和最低杀菌浓度分别为3.75 mg/mL和7.50 mg/mL.抑茵圈平均直径达29.17±1.60 mm;连翘、大黄、蒲公英、金银花、黄柏有较强的抑菌作用,MIC为7.50～15.0 mg/mL,抑菌圈平均直径均在20 mm以上;而龙胆草表现出较弱的抑菌作用.

The serum immunoglobulins(IgM) of tilapia(Oreochromis niloticus) and channel catfish(Ictalurus punctatus) were purified using rProtein A Sepharose affinity chromatography,a one-step purification method.The purified IgM was applied to rabbit for preparation of anti-IgM sera.The results showed that the IgMs from two kinds of fish were well separated from each serum and were obtained at high purity by the rProtein A Sepharose affinity chromatography.The molecular weights of tilapia’s heavy chain and light chain was 88.0 and 21.0 kD,respectively.While the molecular weight of channel catfish’s heavy chain was 101.0 kD.Sera anti-IgM of Oreochromis niloticus and Ictalurus punctatus were prepared by repeatedly immunizing the New Zealand rabbits with purified IgM,and the antibody activity of anti-sera was tested by Western Blotting.The titers of anti-sera reached 1∶32000 and 1∶16000,respectively,when detected by indirect ELISA method.The Western Blotting results showed that there was a band nearby 88.0 and 101.0 kD for rabbit anti-sera of tilapia and channel catfish,respectively,which proved that the obtained anti-sera had immune activity.

Healthy tilapia Oreochromis niloticus averaging(100±10)g in body weight were vaccinated by killed Streptococcus iniae cells as immunogen via oral administration,immersion,and intraperitoneal injection.The white blood cell count(WBC),differential leucocyte count(DLC),phagocytes activities,bacteriolytic activities,lysozyme activities,superoxide dismutase(SOD) activities and antibody activity(OD450 nm) were determined in the blood collected from the caudal veins of three fish in each group randomly sampled 0,3,7,10,14,and 21 days after immunization.The test fish were challenged by intraperitioneal injection(IP)(1.0×107 colony-forming units(cfu)/fish)of S.iniae 22 days after immunization.The tilapia immunized with vaccine showed significantly higher WBC,and the amount of lymphocyte,neutrophilic granulocyte and monocyte than those in the control group(P<0.05).There were significantly higher serum bacteriolytic activities,lysozyme activities and antibody activity(OD450 nm) in the vaccinated fish than those in the fish in the control group(P<0.05) while the immunized fish showed no significant differences in the superoxide dismutase(SOD) activities compared with the control group(P>0.05).The higher relative percent survival(RPS) was found in the fish immunized by IP and immersion,and the highest immune ability was observed in the tilapia treated by IP.

Eight streptococcuses were isolated from diseased tilapias in farms in Nanning.The results showed that 6 isolated strains were S.iniae,the other 2 strains were S.agalactiae by biochemistry identification,and drug sensitivity test provided useful information for clinic.

鉴于海豚链球菌已成为我国罗非鱼养殖发病死亡最主要的病原菌,本研究利用临床分离菌株对低剂量(1.5 x105 CFU·mL-1)攻毒耐过罗非鱼进行再次攻毒试验,筛选到1株具有很好交叉免疫保护的疫苗候选菌株CMS005,甲醛灭活制备成疫苗分别通过注射、浸泡及口服免疫罗非鱼.室内水族箱免疫试验中,注射、浸泡和口服三种免疫途径各自的最佳免疫保护率分别为90.5%、61.9%和14.3%;池塘小网箱免疫试验结果为100%、86.1%和66.7%.数据分析显示,相同剂量注射组免疫保护率明显高于浸泡免疫组与口服免疫组;免疫剂量对浸泡免疫和口服免疫效果的影响大于注射免疫;同时加强免疫可显著提高浸泡免疫与口服免疫效果;超声波处理也可提高浸泡免疫效果.生产小试结果表明,未免疫组罗非鱼月累计死亡率在6%～15%之间,而免疫组罗非鱼月累计死亡率不到O.5%;注射免疫和口服免疫效果相当,免疫7d和15d后均可完全控制罗非鱼海豚链球菌病的继续发生;疫苗发生的免疫保护力可持续2～3个月.上述结果表明,所研制的预防罗非鱼海豚链球菌病疫苗具有很好免疫效果及生产应用前景.

The immune effect of a vaccine prepared with formalin killed strain CMS005 selected from 3 typical strains of clinic S.iniae in tilapia vaccinated by injection,bath,and oral respectively were studied.The immune experiment showed that the best immune protective rate of three methods were 90.5%,61.9% and 14.3% respectively under laboratory experiment in tank,and the results were 100%,86.1% and 66.7% respectively in small cage in pool.The data analysis revealed the vaecinate dose had greater influence on immune efficacy of bath group than injection and oral group,and the second vaccination can significantly improve the immune efficacy of bath and oral groups.In addition,ultrasonic treatment can improve the protective efficacy of bath group.The field experiment displayed that the S.iniae disease was controlled after vaccinated 7 and 15 days by injection and oral vaccination;the accumulative mortality of non vaccinated group was between 4%～16% in a month,whereas the vaccinated group did not reach 0.5%;the protection duration of the vaccine could last 2～3 months.

In recent years,Streptococcus iniae infected farmed tilapia causes economic losses in our country and the world.To set up a method detecting S.iniae in tilapia,this paper designed primers CM1/CM2 based on the sequences of S.iniae.At the same time,the tests which amplified the specific DNA fragment,optimized the PCR reaction condition,sensitiveness and special were detected.The different detected material was compared and 9 clinical samples were detected at one time.The result indicated that the CM1/CM2 primers set only amplified a specific DNA fragment from S.iniae but not from 9 strains common pathology bacteria of fisheries.It can detect bacterial cells in 20-30,and the PCR reaction also can directly detect the S.iniae from brain,liver,kidney,spleen of infected tilapia.On the other hand,the detection results of clinical strains were consistent with the Phylogenist Analysis based on 16S rRNA gene sequences.The method makes up for the disadvantage of traditional method that can not detect the species between the bacteria and reduce the detected time and the cost.So,the method detecting S.iniae will have a good future for application.

通过饱和硫酸铵盐析结合 Sephadex G-200柱层析的方法,纯化制备了健康非免疫状态下奥尼罗非鱼的免疫球蛋白,进行变性还原条件下的聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹试验对血清的免疫球蛋白进行初步分析,发现SDS-PAGE电泳条件下血清重链分子量为85 kD,轻链分子量为30 kD.如果罗非鱼血清免疫球蛋白在自然状态与其他硬骨鱼类一样也为四聚体,那么其总分子量的理论值应为920 kD.

An epidemic disease consecutively happened in cage-cultured channel catfish(Ictalurus punctatus) was recorded in Guangxi during 2006 to 2007.The disease was widely spread with high infectious and lethal feature,exophthalmia,disorientation,unilateral eyes turbidity,and large systemic bleeding in later stage,often 30% to 100% mortality.We isolated seven representative bacterial strains named as CMS003-CMS005,CMS009-CMS012 from the diseased fish.The challenge test of these isolated bacteria showed highly pathogenic to the fish.The challenged fish presented the similar external signs to natural infected catfish,such as exophthalmia and disorientation.These seven isolates were identified as Streptococcus iniae by the conventional morphological,physiological and biochemical characteristics,specific PCR diagnosis and 16S rRNA phylogenetic evolution of gene sequence analysis method,indicating that Streptococcus iniae was the main pathogen of cage cultured channel catfish.

A pair of specific primers was designed and synthesized according to 16S RNA gene sequences of Aeromonas hydrophila from GenBank.The specific DNA fragment of an Aeromonas hydrophila strain separated from dead disease hypophthalmichthys molitrix was amplified by using PCR.The PCR reaction condition was optimized and the specificity and sensitivity of this PCR method were tested.The results of specificity test indicated that this primer could amplify 680 bp specific DNA fragment from Aeromonas hydrophila and it had no cross-reactivity with Vibrio anguillarum,Vibrio alginolyticus,Vibrio parahaemolyticus,Aeromonas sobria,Edwardsiella tarde,Edwardsiella ictaluli and Streptococcus iniae.The sensitivity test indicated that 10pg DNA of Aeromonas hydrophila could be detected with this method.Therefore,the PCR method established in this study could be used to detect Aeromonas hydrophila of hypophthalmichthys molitrix rapidly,and it had important significance on the effective diagnosis and control of Aeromonas hydrophila.

Edwardsiella ictaluri as a causative pathogen of enteric septicemia of channel catfish(Ictalurus punctatus)(ESC) is the most important disease affecting the channel catfish aquaculture industry.Good management practices including rapid and sensitive detection of the pathogen are necessary for effective control of the disease.We designed primers CM3/CM4 based on the sequences of E.ictaluri published in the NCBI to amplify the specific DNA fragment of E.ictaluri.The PCR reaction was optimized and clinical samples from 4 places of Guangxi were detected.Besides,the specific test,sensitive test and comparison of different materials for PCR amplification were also carried out.The results indicated that the CM3/CM4 primers set amplified a specific 276 bp DNA fragment from the pathogen,but not from the common bacteria in fisheries,Pseudomonas fluorescent biotypeⅠ,Aeromonas punctata sub.,Vibrio anguillarum,Aeromonas sobria,Aeromonas punctata f.intertinalis,Flavobacterium columnare,Aeromonas hydrophila,Vibrio fluvialis,Edwardsiella tarde and Streptococcus iniae.The minimal bacterial number the PCR can detect was below as few as 12 cells of Edwardsiella ictaluri.The PCR was also effective in detecting the bacterium from inoculated tissue of brain,liver,kidney and spleen.Furthermore,the materials including lawn and liquid culture were directly amplified in this PCR.The results of clinical strains by PCR were consistent with the phylogenetic analysis results base on 16S rRNA gene sequence and the biochemistry.