鱼类无乳链球菌病的研究进展

无乳链球菌是常见的人兽共患病原菌,常导致新生儿肺炎、脑膜炎、败血症及奶牛乳腺炎。近年来,在水生动物体内也频繁分离到该菌,尤其是自2007年以来,该菌每年都在中国广东、海南、福建、广西等罗非鱼主要养殖区暴发流行,严重威胁着罗非鱼养殖业的健康发展。作者综述了当前国内外对鱼类无乳链球菌病的病原学、流行病学、症状、病理变化、毒力因子（荚膜多糖、CAMP因子、表面免疫原性蛋白、α蛋白、C5a肽酶）和防控等方面的研究进展,以期为中国鱼类无乳链球菌病的防控提供参考。     

鱼类无乳链球菌病

无乳链球菌亦称为B组链球菌,是一种在自然界广泛存在的革兰阳性菌,是人类的重要病原之一,也是鱼类的重要病原菌。近年来,无乳链球菌成为鱼类链球菌病的主要病原之一,鱼类感染无乳链球菌的常见临床症状包括眼球突出、腹部肿胀、脊骨弯曲、鳍条基部出血等,发病率和死亡率高,常造成严重的经济损失,危害着水产养殖业的健康发展。论文对鱼类无乳链球菌病的研究进行了总结,对鱼类无乳链球菌病病原、致病机理进行综述,介绍了鱼类无乳链球菌病常见的诊断方法,以及现阶段针对鱼类无乳链球菌病的疫苗开发情况,旨在进一步丰富、完善鱼类无乳链球菌病的研究资料,为更好的防治鱼类无乳链球菌病提供参考。     

罗非鱼无乳链球菌生物学特性与致病力研究

本试验分离纯化1株致使罗非鱼具有链球菌发病症状的病原菌,药敏试验结果表明,该菌对氯霉素等10种药物敏感。PCR扩增该菌的cfb基因,将扩增片段测序后与GenBank上登录的罗非鱼无乳链球菌的cfb基因序列进行比对。同时对该病原菌的生物学特性进行研究,包括培养时间和pH对该菌生长的影响;培养温度对其致病力的影响;探讨了不同攻毒方式(腹腔、胸腔和背部肌肉注射)与鱼发病快慢的关系;并对不同规格(100和10 g左右)的罗非鱼对该菌的易感性进行分析。试验结果表明,该病原菌为无乳链球菌(Streptococcus agalactiae);该菌的最适培养时间为11.5 h,最适培养pH为7.5;腹腔、胸腔和背部肌肉注射后,其发病时间几乎一致,但胸腔注射的试验鱼死亡速度最慢,背部肌肉注射是较安全可行的攻毒方式;33℃培养的无乳链球菌攻毒的罗非鱼发病最快,死亡最集中;100 g的试验鱼比10 g的试验鱼更易感染。     

三黄连散预防罗非鱼链球菌病临床应用研究

在开展三黄连散对人工感染罗非鱼无乳链球菌病预防效果研究的基础上,进一步验证以每千克饲料中添加13.2 g的三黄连散为推荐剂量,在生产中大面积应用的临床效果及安全性。设立三黄连散试验组、中药对照组和空白对照组3个组,每组300尾罗非鱼,连续用药7 d后,每组随机取30尾,测定增重率、器官指数、血液生理生化指标和肝脏抗氧化指标。用药后第8天,采用同居感染的方法感染鱼群,于感染后第14天计算保护率。结果表明,给药7 d后,与空白对照组相比,三黄连散试验组罗非鱼的增重率明显提高,器官指数和多项肝脏抗氧化指标均明显升高,血液生理生化指标总体无明显异常,预防无乳链球菌感染的保护率为43.67%。综上提示,三黄连散能够提高罗非鱼机体抵抗力,降低无乳链球菌感染罗非鱼的死亡率。     

罗非鱼无乳链球菌EsxA基因克隆与原核表达

Ⅶ型分泌系统(T7SS)是近年来发现的分泌系统,分泌两种胞外蛋白,EsxA基因编码的ESAT6蛋白和EsxB基因编码的CFP-10蛋白。分泌蛋白具有良好的免疫原性,促进细菌从巨噬细胞吞噬体逃逸、影响巨噬细胞凋亡及裂解细胞等生物学功能,与致病性密切相关。以罗非鱼源无乳链球菌为模板,克隆到294bp的EsxA基因,将目的序列克隆到pMD18-T载体,经测序,目的序列与无乳链球菌EsxA基因同源率在99%以上。EsxA基因克隆到pET32a载体中,重组载体在28℃、0.5mmol/L IPTG诱导条件下表达量最大,可溶性表达。将纯化的ESAT6蛋白免疫Balb/c鼠,成功制备ESAT6蛋白鼠多克隆抗体,经Western blot,ESAT6蛋白具有良好的反应原性。研究结果为进一步进行无乳链球菌ESAT6蛋白的免疫学功能研究奠定了基础。     

牦牛乳房炎病原无乳链球菌的分离鉴定与耐药性分析

本试验通过无菌采集西藏地区牦牛乳汁,进行细菌分离、纯化及PCR鉴定;同时对分离的菌株进行药物敏感性研究,以期在生产实践中对该病原的防治提供参考.结果显示,纯化所得到的细菌为革兰氏阳性链球菌,经生化试验及PCR鉴定为无乳链球菌;药敏试验结果显示分离菌株对青霉素类、氨基糖苷类和氯霉素类等大部分药物敏感.本研究为西藏地区牦牛无乳链球菌引起的乳房炎的有效防治提供参考.     

奶牛乳房炎病原分离鉴定及其治疗方法研究

为进一步制定奶牛乳房炎的防治措施,对天水地区6个不同奶牛场50份乳房炎奶样,进行病原分离、鉴定和药敏试验,共分离得到3类106株细菌,分别为：金黄色葡萄球菌、表皮葡萄球菌、腐生葡萄球菌、溶血葡萄球菌、无乳链球菌、乳房链球菌、停乳链球菌、大肠杆菌、沙门菌、沙雷菌和变形杆菌。金黄色葡萄球菌检出率最高,为24.5%。同时比较了不同治疗方法对奶牛乳房炎的治疗效果,认为中西医结合治疗乳房炎,其治愈率高,见效快。     

奶牛乳房炎无乳链球菌活菌数与吸光度之间相关性研究

为了建立一种通过吸光度测定能够快速准确检测奶牛乳房炎疫苗中无乳链球菌抗原浓度的方法,本试验将血平板培养的无乳链球菌用生理盐水将菌苔洗下,稀释成不同浓度的细菌悬浮液,然后用平板计数法测定不同浓度菌液的活菌数,同时测定其相应的吸光度,进而研究二者之间的相关性,建立标准曲线。结果表明,无乳链球菌的活菌数与其吸光度呈直线线性相关,无乳链球菌菌液浓度与吸光度之间的回归方程为y＝7.5861x－0.0805,R²＝0.9735,通过测定细菌悬浮液的吸光度,可以快速计算出该菌的菌液浓度。笔者对发酵罐培养的5批无乳链球菌菌液进行了吸光度测定和传统平板计数方法的比较及验证,证明了吸光度测定法优于平板计数法,具有简单、快速、准确的特点。     

光合细菌对动物的营养作用及对动物疾病的防治作用

光合细菌是一类有益的微生物,它在动物的营养及疾病防治方面可以发挥重要作用。本文综述了我们试验的光合细菌对家畜动物如牛、猪、羊、肉狗、兔的增重和水产动物如淡水鱼、对虾的增产作用,对哺乳期动物的采食和泌乳的影响及对动物疾病尤其是胃肠道疾病的防治作用。光合细菌可以作为一种优质安全的营养保健型饲料添加剂进行推广,预计可具有可观的经济效益和社会效益。     

Piscirickettsiosis and piscirickettsiosis-like infections in fish: a review

Piscirickettsia salmonis was the first "rickettsia-like" bacteria to be recognized as a pathogenic agent of fish. Since the first reports of piscirickettsiosis emerged from Chile in the late 1980s, Piscirickettsia-like bacteria have been recognized with increasing frequency in a variety of fish species, from both fresh and saltwaters around the world. Although the first reported incidents of Piscirickettsia were in salmonids, Piscirickettsia-like bacteria are now being frequently associated with disease syndromes in non-salmonid fish. Mortalities have occurred in white seabass (Atactoscion noblis), black seabass (Dicentrarchus sp.), tilapia (Oreochromis, Tilapia and Sarotherodon spp.) and blue-eyed plecostomus (Panaque suttoni). Piscirickettsiosis and piscirickettsiosis-like diseases have affected aquaculture productivity, profitability, the species of fish compatible with commercial rearing, and transportation of fish from site to site. Piscirickettsiosis and syndromes caused by similar bacteria are an emerging disease complex that will increasingly inhibit fish production.     

PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China

Large-scale streptococcal outbreaks occurred continuously in tilapia farms of China from 2009 to 2011. The objective of this study was to characterize the prevalent strains of tilapia streptococci from the main cultured areas of China through species specific PCR and pulse field gel electrophoresis (PFGE). A total of 105 prevalent strains were isolated from Guangdong, Guangxi, Hainan and Fujian provinces between 2006 and 2011, 85 of which were identified as Streptococcus agalactiae while the rest were all identified as Streptococcus iniae. The prevalent stains in 2006 and 2007 were S. iniae (94.7%, 18/19), with S. agalactiae account for only 5.3% (1/19); The prevalent strains in 2009 and 2011 however changed to S. agalactiae (97.7%, 84/86), with only 2.3% (2/86) was S. iniae. Of these 105 strains, a total of 13 PFGE types (A-M) were characterized, among which D, F, G and K genotypes were predominant, accounting for 81.90% (86/105). The cluster analysis of PFGE electropherograms separated S. iniae and S. agalactiae to two distinctive branches, 20 strains of S. iniae exhibiting 3 types of PFGE band patterns with a similarity of 94.8-100%, and the 85 strains of S. agalactiae producing 10 types of PFGE band patterns with a similarity between 48.4% and 100%. Data suggested that the prevalent strains of tilapia streptococci in China have shifted from the former (before 2008) dominant strains of S. iniae to the current (2009-2011) dominant strains of S. agalactiae. Moreover, PFGE genotypes of the prevalent strains demonstrated geographic differences and temporal changes.     

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。     

牛源性无乳链球菌血清型分布及抗生素耐药性研究

本研究旨在查明牛源性无乳链球菌血清型分布及对常见抗生素的耐药情况,指导临床合理用药。对从中国部分地区奶牛场采集的临床型乳房炎病牛乳中分离鉴定出78株无乳链球菌地方菌株,制备沉淀反应抗原及6株标准血清型无乳链球菌单因子血清抗体,采用环状沉淀试验,对78株无乳链球菌地方菌株进行了血清学分型鉴定;同时采用K-B纸片法测定了这些菌株对抗生素的耐药情况。结果表明,引起奶牛乳房炎的无乳链球菌血清型主要为X型(60.26%),其次为Ⅲ型(10.26%)、R型(7.69%)、Ⅱ型(7.69%)和Ⅰb型(5.13%),Ⅰa型尚未发现。无乳链球菌对目前临床上使用的大部分抗生素,如头孢唑啉、头孢噻肟、丁胺卡那霉素、卡那霉素、庆大霉素、四环素、强力霉素、氟苯尼考、多黏菌素B、环丙沙星、氟哌酸和头孢他啶/棒酸均较敏感;但对氨苄青霉素、链霉素、恩诺沙星、阿莫西林/棒酸和复方新诺明,有一定的耐药性,其耐药率达50%~100%。本研究对进一步研制有效的药物及疫苗,指导临床合理用药具有重要的意义。     

Streptococcus agalactiae and Streptococcus difficile 16S-23S intergenic rDNA: genetic homogeneity and species-specific PCR

Streptococcus difficile is a non-hemolytic Gram-positive bacterial coccus that causes septicemia and meningoencephalitis in farmed tilapia (Oreochromis sp.) and rainbow trout (Oncorhynchus mykiss). Recent studies have demonstrated S. difficile to be a group B, type Ib streptococcus with a whole cell protein electrophoretic profile indistinguishable from S. agalactiae and a biochemical profile similar to that observed for other group B, type Ib streptococci isolated from fish and frogs. The aim of this study was to expand on these findings by comparative nucleic acid sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacers of S. difficile and S. agalactiae. The 97.7% sequence homology identified in these studies supports the taxonomic relationship of these two organisms. The sequence data generated were also used to construct a pair of species-specific PCR primers for use in molecular detection and identification schemes.     

菌丝霉素分泌型丁酸梭菌防治罗非鱼链球菌病的应用研究

为解决罗非鱼链球菌感染难题,本研究在抗菌肽菌丝霉素分泌型丁酸梭菌制剂的基础上,对其进行了抑菌活性、稳定性和罗非鱼养殖应用研究。结果发现:表达产物菌丝霉素对常见致病菌如大肠杆菌、沙门氏菌、金黄色葡萄球菌、蜡样芽孢杆菌和猪链球菌均有较好的抑菌活性。选用米糠作为菌丝霉素分泌型丁酸梭菌的保护剂载体,此制剂可在常温保存一年。通过上述的应用基础试验,初步建立一套菌丝霉素分泌型丁酸梭菌制剂养殖应用方案,此方案可显著降低罗非鱼链球菌病发病率58.6%以上。本研究为丁酸梭菌制剂在防治罗非鱼链球菌病应用方面提供了参考,此制剂有望替代抗生素,促进罗非鱼养殖业的持续健康发展。     

宁夏吴忠地区奶牛临床型乳房炎主要病原菌的分离鉴定及耐药性分析

为了解宁夏吴忠地区奶牛乳房炎的主要病原菌及其耐药情况,采集56份罹患乳房炎的奶牛乳样进行病原菌分离,并对分离菌进行了生化鉴定、16S rRNA基因序列分析、药敏试验以及耐药基因检测.结果表明,该地区的主要病原菌为大肠埃希氏菌和无乳链球菌,检出率分别为23.2％和14.3％.药敏试验结果表明,大肠埃希氏菌主要对头孢他啶、...     

Histopathology of Hybrid Tilapias Infected witha Biotype of Streptococcus iniae

Abstract

A systemic streptococcal disease caused by a biotype of Streptococcus iniae affecting hybrids of Nile tilapia Tilapia nilotica × blue tilapia T. aurea at a commercial aquaculture facility in central Texas was investigated. Histological analysis revealed a general septicemia with the occurrence of cellular infiltration and numerous cocci in most organ systems. Observations included cellular infiltration of the eye, meningitis, meningeal granuloma, numerous foci of infection, and massive cellular infiltration in the kidneys. Bacteria were seen in the subcapsular capillaries of the liver, and some liver tissue was granulomatous. Splenic sinuses contained numerous cocci, and both epicarditis and myocarditis was observed in the heart tissue of some specimens. Cocci were free in the plasma of infected fish and often phagocytized by macrophages.     

Streptococcus agalactiae mastitis: a review.

Streptococcus agalactiae continues to be a major cause of subclinical mastitis in dairy cattle and a source of economic loss for the industry. Veterinarians are often asked to provide information on herd level control and eradication of S. agalactiae mastitis. This review collects and collates relevant publications on the subject. The literature search was conducted in 1993 on the Agricola database. Articles related to S. agalactiae epidemiology, pathogen identification techniques, milk quality consequences, and control, prevention, and therapy were included. Streptococcus agalactiae is an oblique parasite of the bovine mammary gland and is susceptible to treatment with a variety of antibiotics. Despite this fact, where state or provincial census data are available, herd prevalence levels range from 11% (Alberta, 1991) to 47% (Vermont, 1985). Infection with S. agalactiae is associated with elevated somatic cell count and total bacteria count and a decrease in the quantity and quality of milk products produced. Bulk tank milk culture has, using traditional milk culture techniques, had a low sensitivity for identifying S. agalactiae at the herd level. New culture methods, using selective media and large inocula, have substantially improved the sensitivity of bulk tank culture. Efficacy of therapy on individual cows remains high. Protocols for therapy of all infected animals in a herd are generally successful in eradicating the pathogen from the herd, especially if they are followed up with good udder hygiene techniques.     

Nisin对奶牛乳房炎主要致病菌的体外抗菌作用研究

从杭州郊区奶牛场的乳房炎病例中分离到金黄色葡萄球菌和无乳链球菌各20株。药敏试验表明，50％以上的金黄色葡萄球菌菌株对苯唑青霉素、氟哌酸、复方新诺明和链霉素耐药；乳酸链球菌素（Nisin）在体外对无乳链球菌和青霉素耐药和敏感的金黄色葡萄球菌均有抗菌作用。采用微量TTC法测定牛奶中Nisin的体外抗菌作用，结果表明Nisin在牛奶中对两种乳房炎致病菌也有抗菌作用，但略低于在普通肉汤培养基的作用。     

青海地区奶牛无乳链球菌的分离与鉴定

无乳链球菌是导致奶牛乳房炎的常见病原菌之一,为了掌握青海地区无乳链球菌的流行情况及常用药物的敏感性,从而为本地区更好的预防与治疗奶牛乳房炎提供保障。本试验在青海地区16个奶牛场共采集258份乳房炎乳样,经革兰氏染色与绵羊鲜血琼脂培养基培养纯化,获得疑似链球菌137株;随后经玻片法血浆凝固酶试验、CAMP试验、生化试验与动物致病性试验对无乳链球菌进行鉴定,结果显示,137株链球菌中含有无乳链球菌42株,分离率为30.7%;经K-B纸片扩散法对无乳链球菌耐药性检测结果显示,该地区无乳链球菌主要对头孢类高度敏感,对氧氟沙星、磺胺类、氨苄青霉素与阿米卡星中度敏感。