七种常用渔药及组合对罗非鱼致病性海豚链球菌的体外抗菌试验

探讨了7种常用抗菌渔药对罗非鱼致病性海豚链球菌的体内外抗菌作用。采用二倍稀释法测定了7种渔药对致病菌的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）;在初筛基础上以MIC为单位,1×MIC、5×MIC、10×MIC和20×MIC下平板抑菌圈直径为参数,分别比较所筛选5种药物抗菌效果并优化最佳参数;利用优化的最佳参数比较5种药物单独用药与联合用药对海豚链球菌的抑菌效果;并通过拌饵投喂给药方式对罗非鱼进行5种药物治疗性给药试验。结果表明,抗菌先锋对海豚链球菌的MIC和MBC最小,分别为6.4、12.8μg/mL,其次是伯乐立康,均为12.8μg/mL;而鱼康和肠炎烂鳃灵即使在1638.4μg/mL下也无抗菌效果。5倍MIC下抑菌圈直径为本试验筛选抗菌药物的最佳参数。5 MIC时,海豚链球菌对伯乐立康和抗菌先锋敏感,对菌必清、菌毒康和海鱼安中度敏感。菌毒康与海鱼安的联合抗菌效果最佳。鱼体内抗菌试验结果表明,抗菌先锋和伯乐立康的治疗有效率超过75%,达到了理想的治疗效果,是防治罗非鱼海豚链球菌病的首选药物。     

三黄连散对罗非鱼无乳链球菌的体外抑菌效果观察

为开发三黄连散于水产养殖防治细菌性疾病的应用,通过常规检查和PCR技术对试验菌株进行罗非鱼无乳链球菌鉴定后,检测三黄连散对该菌的体外抑菌效果。结果试验菌株为罗非鱼无乳链球菌,血清型为Ia型、II型、dIts型。三黄连散对该菌的最小抑菌浓度(MIC)为15.625mg/mL,最小杀菌浓度(MBC)为31.25mg/mL;1/2×MIC和1×MIC能抑制对数生长期的菌体分裂,2×MIC能抑制细菌进入对数生长期。本研究表明:三黄连散对罗非鱼无乳链球菌有良好的体外抑菌效果,可为其临床应用提供依据。     

高速逆流色谱分离黄连生物碱及其抑菌活性初探

为了研究黄连生物碱的抑菌活性,采用高速逆流色谱(HSCCC)法分离黄连生物碱,K-B法检测其抑菌活性,测其对敏感菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果显示：从黄连中分离出黄连碱、掌叶防己碱、小檗碱、表小檗碱,提取率分别为44.41%、36.72%、68.54%、18.33%;对金黄色葡萄球菌的抑菌圈分别为10.00、29.50、21.00和12.50 mm;对无乳链球菌分别为11.40、27.50、14.00和15.50 mm;对粪肠球菌、大肠杆菌O157等均无效;黄连碱、表小檗碱对金黄色葡萄球菌、无乳链球菌的MIC和MBC均分别为136.36、272.73μg/mL;小檗碱对金黄色葡萄球菌的MIC和MBC分别为62.18、136.36μg/mL,对无乳链球菌的MIC和MBC分别为136.36、272.73μg/mL;掌叶防己碱对金黄色葡萄球菌、无乳链球菌的MIC和MBC均分别为62.18、136.36μg/mL。结果表明：用HSCCC制备生物碱,省时、方便,4种生物碱对金黄色葡萄球菌和无乳链球菌作用明显,对其余试验菌无效。     

9种抗菌药物对禽多杀性巴氏杆菌体外抗菌活性研究

采用试管二倍稀释法，测定了9种抗菌药物对禽多杀性巴氏杆菌的体外抗菌活性（MIC和MBC）。结果表明氨苄西林、阿莫西林和诺氟沙星对禽多杀性巴氏杆菌的MIC均为0．125μg／mL，诺氟沙星MBC为0．5μg／mL，氨苄西林和阿莫西林的MBC都为1．0μg／mL。氟苯尼考、替米考星和卡那霉素对禽多杀性巴氏杆菌的MIC分别为：0．5、2和2μg／mL。     

单味中草药对罗非鱼维氏气单胞菌的体外抑菌试验

为了研究了20种单味中草药对罗非鱼维氏气单胞菌的体外抑菌情况,试验采用琼脂扩散法和试管二倍稀释法测定抑菌圈大小及最小抑菌浓度(MIC)。结果表明:罗非鱼维氏气单胞菌对五味子、五倍子、黄连等5种药物极度敏感,对黄柏、苏木、石榴皮等5种药物高度敏感,对薄荷、公丁香、鱼腥草等5种药物中度敏感;其中黄连、乌梅、沙棘等5种药物对罗非鱼维氏气单胞菌有较好的抑菌效果,MIC在15.63~62.50 mg/mL之间;黄柏、白头翁、石榴皮3种药物的MIC均为125 mg/mL;苏木、黄芩、薄荷3种药物的MIC均为250 mg/mL;公丁香、鱼腥草、夏枯草等5种药物对该菌抑菌效果比较差,MIC均为500 mg/mL。说明五味子、五倍子、黄连等5种中草药可用来防治罗非鱼维氏气单胞菌。     

功劳叶提取物的体外抗菌活性研究

采用水提法和乙醇回流加热法分别提取功劳叶,应用牛津杯法和试管二倍稀释法分别检测功劳叶提取物对试验菌的抑菌圈直径、最低抑菌浓度(MinimumInhibitory Consistency,MIC)和最低杀菌浓度(Minimum Bactericidal Consistency,MBC)。结果显示,功劳叶醇提物金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为20.69 mm、15.40 mE;MIC值分别是31.3 mg/mL、125 mg/mL;MBC值分别是62.5 mg/mL、500 mg/mL。功劳叶水提物对金黄色葡萄球菌的抑菌圈13.54mm,其MIC值、MBC值分别是62.5 mg/mL、500 mg/mL,对大肠杆菌抗菌活性较弱,其MIC值为500 mg/mL,MBC500 mg/mL。功劳叶不同提取物对细菌的抗菌活性有所差异,功劳叶醇提物对金黄色葡萄球菌和大肠杆菌均由不同程度的抑菌或杀菌能力。     

裸花紫珠颗粒对罗非鱼源无乳链球菌体外抑菌活性研究

无乳链球菌是导致罗非鱼链球菌病的主要病原菌。为探讨裸花紫珠颗粒对无乳链球菌体外抑菌活性，采用肉汤二倍稀释法测定裸花紫珠颗粒对无乳链球菌的最低抑菌浓度(MIC)和最小杀菌浓度(MBC),同时进行标准无乳链球菌和临床分离无乳链球菌对常用抗菌药的敏感性试验。结果表明，裸花紫珠颗粒对罗非鱼源无乳链球菌的最低抑菌浓度(MIC)为10.000 mg/mL,最小杀菌浓度(MBC)为20.000 mg/mL;药敏试验结果显示临床分离株无乳链球菌对四环素、环丙沙星、氟苯尼考的敏感性降低。该结果为下一步开展裸花紫珠对罗非鱼临床保护试验研究奠定了基础。     

杂种鹅掌楸叶提取物的抗菌作用

为了探讨杂种鹅掌楸叶不同极性部位提取物对大肠埃希菌、沙门菌、金黄色葡萄球菌和链球菌的体外抗菌效果,通过乙醇回流提取制得鹅掌楸醇提物,采用系统溶剂提取法萃取得到鹅掌楸叶醇提物的不同部位,并通过琼脂平板法和微量肉汤稀释法检测各部位的抗菌作用。结果显示:杂种鹅掌楸叶石油醚、氯仿、乙酸乙酯提取物对沙门菌、金黄色葡萄球菌、链球菌有不同程度的抗菌作用,其中氯仿部位作用最强,抑菌圈分别为18.0、16.0及17.0 mm,最小抑菌浓度(MIC)分别为0.625、0.312 5及0.625 mg/mL,最低杀菌浓度(MBC)分别为1.25、0.312 5及1.25 mg/mL。各部位提取物对大肠埃希菌无抗菌作用,正丁醇、水部位提取物对测试的4种菌均无抗菌作用。研究表明杂种鹅掌楸叶提取物对沙门菌、金黄色葡萄球菌和链球菌有不同程度抗菌作用,其中氯仿部位提取物有较强抗菌作用。     

野生苦菜不同部位抑菌作用研究

为了探明野生苦菜不同部位(根、茎、叶及全草)对巴彦淖尔地区常见环境致病菌(大肠杆菌、金黄色葡萄球菌和链球菌菌株)的抑制作用,试验采用中药打孔法及试管二倍稀释法进行体外抑菌试验,测定抑菌圈直径及最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果表明:苦菜叶和全草对大肠杆菌、金黄色葡萄球菌及链球菌三种常见环境致病菌抑菌效果良好,其中苦菜叶的抑菌圈直径分别为12. 3 mm、9. 1 mm、8. 5 mm;苦菜全草的抑菌圈直径分别为11. 2 mm、10. 4 mm、9. 3 mm。苦菜叶对三种环境致病菌的MIC分别为31. 3 mg/mL、125. 0 mg/mL、250. 0 mg/mL,MBC分别为31. 3 mg/mL、250. 0 mg/mL、500. 0 mg/mL;苦菜全草对三种环境致病菌的MIC分别为62. 5 mg/mL、125. 0 mg/mL、250. 0mg/mL,MBC分别为62. 5 mg/mL、250. 0 mg/mL、250. 0mg/mL。说明野生苦菜可以用于防控当地常见环境致病菌所导致的疾病。     

乳酸恩诺沙星对温和气单胞菌的体外抗菌后效应

为研究乳酸恩诺沙星对温和气单胞菌(A.sobria)的体外药效学,采用二倍稀释法测定了乳酸恩诺沙星对温和气单胞菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),在此基础上探究在温和气单胞菌的不同生长时期加入乳酸恩诺沙星后对其生长的影响,研究乳酸恩诺沙星不同药物浓度(2×MIC、4×MIC、8×MIC)作用对温和气单胞菌的杀菌动力学和抗菌后效应(PAE)。结果显示,乳酸恩诺沙星对温和气单胞菌的MIC和MBC分别为0.39μg/mL和1.56μg/mL,乳酸恩诺沙星对温和气单胞菌有较强的杀菌作用,其PAE与药物浓度呈正相关。     

Immunoproteomic analysis of the antibody response obtained in Nile tilapia following vaccination with a Streptococcus iniae vaccine

Streptococcus iniae is one of the most economically important Gram-positive pathogens in cultured fish species worldwide. The USDA-ARS Aquatic Animal Health Research Unit developed a modified (contains concentrated culture supernatant) S. iniae bacterin that has been demonstrated to be efficacious, and protection is mediated by specific anti-S. iniae antibodies. Although effective, the specific vaccine components important for efficacy are not known. In the present study, an immunoproteomic approach was utilized to identify whole-cell lysate proteins of S. iniae that stimulated specific antibody production in Nile tilapia (Oreochromis niloticus) following vaccination. Groups of tilapia were vaccinated by intraperitoneal injection with the modified S. iniae bacterin or were mock-vaccinated, and at 30 d post-vaccination sera samples were obtained from individual fish. Vaccination of tilapia with the S. iniae vaccine stimulated significantly elevated specific antibody responses against proteins of the bacterium and passive immunization of tilapia with this serum demonstrated the antibodies were highly protective. Whole-cell lysate proteins of S. iniae were separated by 2D-PAGE and were probed with a pooled serum sample from vaccinated tilapia. A total of eleven unique immunogenic proteins were positively identified by mass spectrometry. Based on research conducted on homologous proteins in other Streptococcus spp., antibodies specific for three of the identified proteins, enolase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase, are likely involved in protection from streptococcosis caused by S. iniae.     

Development of a PCR assay for Streptococcus iniae based on the lactate oxidase (lctO) gene with potential diagnostic value

Streptococcus iniae is a well-known pathogen of both fish and humans that is difficult to identify by conventional biochemical tests. A PCR assay based on the lactate oxidase (lctO) gene of S. iniae was developed for the rapid and specific detection and identification of this pathogen from different sources. The PCR assay had a detection limit of 62-31 cells, and 25 pg of DNA per PCR reaction mixture. The PCR was also effective in detecting the bacterium from inoculated tissue homogenates, suggesting its potential use for a rapid and accurate diagnosis of S. iniae infections.     

Prevalence of Streptococcus iniae in tilapia, hybrid striped bass, and channel catfish on commercial fish farms in the United States

OBJECTIVE: To determine the prevalence of Streptococcus iniae in tilapia (Oreochromis spp), hybrid striped bass (Morone chrysops X M saxatilis), and channel catfish (Ictalurus punctatus) on commercial fish farms in the United States. ANIMALS: 1,543 fish (970 tilapia, 415 hybrid striped bass, and 158 channel catfish). PROCEDURES: The dry-swab technique was used for collection of specimens for streptococcal isolation. Specimens were shipped by overnight delivery and processed by use of standard bacteriologic techniques. RESULTS: Streptococcus iniae was not isolated from market-size channel catfish. Prevalence in tilapia and hybrid striped bass was 37 of 970 (3.81%) and 30 of 415 (7.23%), respectively. Prevalence by farm ranged from 0.0 to 27.4% for tilapia and 0.0 to 21.6% for hybrid striped bass. In tilapia, prevalence was lowest in market-size and nursery fish (4 of 239 [1.67%] and 3 of 339 [0.88%], respectively), with an increase in prevalence for fish in the grow-out stage (30 of 337 [7.96%]). For hybrid striped bass, prevalence was lowest in nursery and market-size fish (3 of 96 [3.12%] and 1 of 47 [2.12%], respectively) and highest in fish in the grow-out stage (26 of 272 [9.56%]). Prevalence in market-size tilapia and hybrid striped bass was 5 of 286 (1.75%). CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study do not support the contention that S iniae is a serious public health threat associated with commercially raised fish; rather, it represents a limited risk for older or immunocompromised people who incur puncture wounds while handling and preparing fish.     

Strain-associated virulence factors of Streptococcus iniae in hybrid-striped bass

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Development of in vitro models of S. iniae virulence may provide insight to the pathogenesis of infection in vivo. Three S. iniae strains (K288, 94-426, and 29178) were tested for virulence in a hybrid-striped bass (HSB) model using intraperitoneal injection. S. iniae strains K288 and 94-426 caused high levels of mortality in HSB (lethal dose 2x10(5)CFU) while strain 29178 was avirulent even upon IP challenge with 1000-fold higher inocula. In vitro assays were developed to test for the presence of characteristics previously associated with virulence in other species of pathogenic Streptococcus in animals and humans. In vitro differences relevant to virulence were not detected for beta-hemolysin activity, sensitivity to antimicrobial peptides, or adherence and invasion of epithelial cell layers. However, in whole-blood killing assays, the pathogenic strains were resistant to blood clearance, while 29178 was cleared (P<0.001) and more sensitive to complement (P<0.001). The avirulent strain 29178 was most efficiently phagocytosed and was most susceptible to intracellular killing (P<0.01) by the carp leukocyte cell line (CLC). When exposed to reactive oxygen species, strain 29178 was most susceptible. When the oxidative burst of CLC cells was inhibited, intracellular survival of 29178 was rescued fivefold, while no significant enhancement in survival of K288 or 94-426 was detected. Our results indicate that resistance to phagocytosis, oxidative killing, and associated phagocytic clearance is a significant factor in S. iniae virulence.     

In vitro study of adherence, invasion, and persistence of Streptococcus iniae in fibroblastic-like fish cell line SAF-1

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Gentamicin protection assays were used to investigate the ability of different S. iniae strains to invade and adhere to fibroblastic-like fish cell line SAF-1. All strains tested were detected intracellularly using both techniques, with variable internalization degrees between strains. The experiments carried out at 4°C demonstrated that active cell metabolism is necessary for bacterial internalization. Intracellular bacteria were detected for up to 3 d with a round morphology and were stained with 4',6-diamidino-2-phenylindole (DAPI), indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that S. iniae is capable of adhering, entering, and surviving within fibroblastic cells, which may be important for the persistence and establishment of a carrier state.     

Identification and expression profile of multiple genes in Nile tilapia in response to formalin killed Streptococcus iniae vaccination

Twenty-eight expressed sequence tags (ESTs) were isolated from a Nile tilapia (Oreochromis niloticus) vaccinated vs non-vaccinated subtractive library at 12-h post injection of a formalin killed Streptococcus iniae ARS-98-60 vaccine. The 28 ESTs were classified in terms of their putative functions. Half of the ESTs identified were unknown proteins. Of the remaining half ESTs, 17% have putative functions in protein biosynthesis and 11% have putative functions in immunity, energy production, and signal transduction, respectively. Immunity-related ESTs identified included high density lipoprotein-binding protein vigilin, immunoglobulin heavy chain, and QM-like protein. Quantitative PCR revealed that one EST (cytochrome c oxidase subunit II) was highly upregulated (1825 ± 336 fold) in vaccinated fish compared to that in non-vaccinated fish. Of the remaining 27 ESTs, nine were significantly (P<0.05) upregulated (<20 fold) in vaccinated fish. The nine significantly upregulated genes included five unknown or hypothetical proteins and four known proteins (high density lipoprotein-binding protein vigilin, QM-like protein, ribosomal protein S13, and ribosomal protein L5). The upregulation of these genes induced by killed S. iniae vaccines suggest that they might play important role in Nile tilapia defense against S. iniae infection.     

Effectiveness of aquaflor (50% florfenicol) to control mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass

We conducted a field trial to evaluate the effectiveness of Aquaflor (50% florfenicol) for controlling mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass (female white bass Morone chrysops X male striped bass M. saxatilis). Bacterial samples collected from moribund fish representing a reference population were presumptively identified microbiologically and were later confirmed to be S. iniae by biochemical characterization and polymerase chain reaction. The trial comprised a 1-d acclimation period, 10-d treatment period, and 14-d posttreatment period. During the treatment period, Aquaflor-medicated feed was administered to treated tanks (N = 3) at a target dose of 10 mg of florfenicol x kg of fish(-1) x d(-1), and nonmedicated feed was administered to control tanks (N = 3). At the end of the posttreatment period, mean (+/- SD) cumulative mortality in treated tanks (9 +/- 11%) was significantly (P = 0.040) less than that in control tanks (52 +/- 13%). Analysis of medicated feed samples revealed that treated tanks had received an actual dose of 8.3 mg florfenicol x kg fish(-1) x d(-1) (83% of target). No florfenicol was detected in control feed samples. Although the actual florfenicol dose administered to treated tanks was less than the target dose, the trial was accepted by the U.S. Food and Drug Administration Center for Veterinary Medicine as demonstrating the efficacy of Aquaflor to control mortality associated with S. iniae in cultured sunshine bass populations.     

Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination

Streptococcus iniae has become one the most serious aquatic pathogens in the last decade causing high losses in farmed marine and freshwater finfish in warmer regions. Although first identified in 1976 from a captive Amazon freshwater dolphin, from which it derives its name, disease outbreaks had most likely been occurring for several decades in marine aquaculture in Japan. S. iniae is globally distributed throughout warm water finfish aquaculture. In common with other encapsulated beta-haemolytic streptococci and in direct contradiction to the phenomenal success story of bacterial vaccines in finfish aquaculture, control of S. iniae by vaccination has met with limited success. Thus, antibiotic usage is the current practice for reducing mortality and consequent economic loss. Vaccine failure appears to result in part from serotypic variation and, whilst 2 serotypes have been named, variation would appear to be more complex. S. iniae also has zoonotic potential, with human infections identified in the USA, Canada, and throughout Asia. In humans, infection is clearly opportunistic with all cases to date associated with direct infection of puncture wounds during preparation of contaminated fish, and generally in elderly or immunocompromised individuals. Significant progress has been made in terms of research into pathogenic mechanisms of S. iniae, with recent research elucidating the role of capsule in virulence for fish through antiopsonic activity. In light of this recent coverage in the literature, the present review centres on areas of direct veterinary interest including identification, epidemiology, therapy and prevention in farmed finfish. Clearly as the prevalence of S. iniae and associated economic losses continue to increase, further work towards developing a reliable vaccine is essential. This would appear to require a much better understanding of cell-surface variability amongst S. iniae isolates.     

A third report of "golf ball disease" in an Amazon River dolphin (Inia geoffrensis) associated with Streptococcus iniae.

An Amazon River dolphin (Inia geoffrensis) developed a dermatologic syndrome characterized by the occurrence of slow-growing, nodular, s.c. abscesses. Initial biopsies, cultures, and cytologic analysis of needle aspirates from the abscesses indicated steatitis with probable secondary, gram-negative bacterial infection. Treatment with dietary vitamin E supplement and broad-spectrum antibiotics yielded minimal improvement. Subsequent cultures revealed Streptococcus iniae in addition to several gram-negative bacteria. Vigorous surgical management of the abscesses, including lancing, debridement, and irrigation, combined with antimicrobial therapy specific for Streptococcus and gram-negative organisms, and improvement of the animal's diet and environmental water quality led to gradual recovery. When the animal was ill, it demonstrated an inflammatory leukogram and transient uremia. Streptococcus iniae is a serious pathogen of aquacultured fishes and humans and should be included in the differential diagnosis of chronic dermatopathy in river dolphins. Specific antimicrobial therapy, excellent water quality, surgical management of abscesses, and adherence to sanitary protocols should be observed in cases of suspected S. iniae infection in dolphins.