柱状黄杆菌胶体金免疫层析快速检测方法的建立

为建立一种通过胶体金免疫层析技术快速检测柱状黄杆菌的方法,试验采用柠檬酸三钠还原法制备粒径为20 nm的胶体金颗粒,将其标记纯化的抗柱状黄杆菌单克隆抗体(McAb)制备出金标抗体结合垫。纯化的兔抗柱状黄杆菌多克隆抗体(PcAb)和羊抗鼠IgG分别包被在硝酸纤维素膜的检测线(T)与质控线(C)上,制备出胶体金免疫层析试纸条,并对试纸条的灵敏度、特异性及稳定性进行测定。结果显示,该试纸条检测灵敏度为1×10³ CFU,检测时间为3.5 min,制备的试纸条与迟钝爱德华氏菌、大肠杆菌、嗜水气单胞菌、鳗弧菌、溶藻弧菌、副溶血弧菌、哈维氏弧菌均无交叉反应,且稳定性好。本研究首次成功建立了柱状黄杆菌胶体金快速检测方法,所制备的试纸条具有灵敏、特异、稳定、快速等优点,可用于柱状黄杆菌的检测。     

应用ＥＬＩＳＰＯＴ试验测定重组卡介苗诱导的Ｔ细胞应答

应用免疫磁性分离技术去除免疫小鼠脾脏细胞中ＣＤ４^ 或ＣＤ8^ Ｔ细胞后，在酶免疫斑点试验中证实表达大肠杆菌ＭalE蛋白的重组卡介苗（rBCG.MalE)诱导的Ｔ细胞应答是ＣＤ４^ t细胞依赖的对MalE、PPD持异Ｔ细胞应答的动态分析结果表明，，rBCG.MalE、ＢＣＧ诱导的特异ＣＤ４^ T细胞应答存在Ｔh1/Th2平衡转换现象，即起始阶段为Ｔh1应答，一段时间后出现Ｔh2应答，并逐步形成Ｔh1/Th２混合应答。这些结果，分为枝杆菌Ｔ细胞应答规律提供了新的认识，同时，亦表明ＢＣＧ是优良的外源抗原表达与运送载体。     

牛轮状病毒TaqMan实时荧光RT-PCR 快速检测方法的建立

本研究按照牛轮状病毒(BRV)结构蛋白VP6基因序列,设计合成引物和探针,经各反应条件的优化,建立了BRV TaqMan实时荧光定量RT-PCR技术。对BRV进行了特异性、敏感性和重复性试验。结果表明,TaqMan实时荧光RT-PCR最低可检测到100个拷贝病毒RNA;与牛病毒性腹泻病毒(BVD)、猪瘟病毒(CSFV)、牛结核杆菌(MB)和牛传染性鼻气管炎病毒(IBRV)不发生交叉反应;所制作的标准曲线在10²~10⁹拷贝/μL浓度范围内有极好的线性关系且线性范围宽,相关系数为0.997;与常规的RT-PCR相比,该方法具有快速、特异、敏感、重复性好、可同时检测大量样品等优点。可对样品中微量BRV进行准确检测,对BRV的诊断有重要意义。     

应用实时荧光定量TaqMan RT-PCR检测口蹄疫病毒

按照口蹄疫病毒(FMDV)聚合酶3D基因序列,设计合成了引物和探针,经各反应务件的优化,建立了实时荧光定量RT-PcR技术,对细胞培养物、水泡液、水泡皮及分泌物、血液中的FMDV进行了特异性检测和敏感性试验。结果,用300nmol／L的引物浓度和200nmol／L探针浓度,获得的CT值较小,而△Rn最大;可检测到相当于9．1TCID50的病毒RNA;与VSV和其他水泡性病毒不发生交叉反应;制作的标准曲线中各浓度范围内有极好的线性关系,且线性范围宽,相关系数为0．984;组内和组间试验重复性的变异系数(CV)分别为5．4％和6．7％;与常规RT-PCR相比较,该方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点。     

A Real-Time Polymerase Chain Reaction Assay of the Bacterium Edwardsiella ictaluri in Channel Catfish

Abstract

A rapid (4.5-h) and sensitive assay based on polymerase chain reaction (PCR) was developed to facilitate the early detection of Edwardsiella ictaluri in channel catfish Ictalurus punctatus. A 129-base-pair fragment of a sequence specific to E. ictaluri was amplified with both standard and real-time (quantitative) PCR. The sensitivity of detection was determined to be as low as the equivalent of 2.5 cells in DNA samples from both E. ictaluri cells and mixtures of blood from noninfected catfish and E. ictaluri cells. Infection levels (as determined by real-time PCR) in blood from experimentally challenged fish were compared with brain–heart-infusion-cultured bacterial colony counts to assess the accuracy of the PCR assay. The PCR-based detection level (the equivalent of 10⁵–10⁸ cells/mL) was comparable to that of traditional culturing techniques (10⁶–10⁷ cells/mL). In future applications, this assay will be applied in a comprehensive breeding program to select channel catfish that are resistant to enteric septicemia of catfish.     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

实时荧光定量RT-PCR检测猪水疱病病毒的研究

按照猪水疱病病毒结构蛋白VP1基因序列,设计合成引物和探针,经各反应条件的优化,建立了实时荧光定量RT-PCR技术,对猪水疱病病毒进行了特异性、敏感性和重复性试验。结果表明,实时荧光TaqManRT-PCR可检测到每个反应相当于1×102拷贝病毒RNA;与FMDV、VSV等其他水疱性病毒不发生交叉反应;制作的标准曲线中各浓度范围内有极好的线性关系且线性范围宽,相关系数为0.993;与常规的RT-PCR比较,该方法具有快速、特异、敏感、重复性好、可同时检测大量样品等优点。     

Potassium permanganate elicits a shift of the external fish microbiome and increases host susceptibility to columnaris disease

The external microbiome of fish is thought to benefit the host by hindering the invasion of opportunistic pathogens and/or stimulating the immune system. Disruption of those microbial communities could increase susceptibility to diseases. Traditional aquaculture practices include the use of potent surface-acting disinfectants such as potassium permanganate (PP, KMnO₄) to treat external infections. This study evaluated the effect of PP on the external microbiome of channel catfish and investigated if dysbiosis leads to an increase in disease susceptibility. Columnaris disease, caused by Flavobacterium columnare, was used as disease model. Four treatments were compared in the study: (I) negative control (not treated with PP nor challenged with F. columnare), (II) treated but not challenged, (III) not treated but challenged, and (IV) treated and challenged. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze changes in the external microbiome during the experiment. Exposure to PP significantly disturbed the external microbiomes and increased catfish mortality following the experimental challenge. Analysis of similarities of RISA profiles showed statistically significant changes in the skin and gill microbiomes based on treatment and sampling time. Characterization of the microbiomes using 16S rRNA gene pyrosequencing confirmed the disruption of the skin microbiome by PP at different phylogenetic levels. Loss of diversity occurred during the study, even in the control group, but was more noticeable in fish subjected to PP than in those challenged with F. columnare. Fish treated with PP and challenged with the pathogen exhibited the least diverse microbiome at the end of the study.     

Use of vaccination against enteric septicemia of catfish and columnaris disease by the U.S. catfish industry

Vaccination is an effective strategy used for the protection of food animals against infectious diseases. A 2010 U.S. Department of Agriculture questionnaire examined U.S. catfish industry use (in 2009) of two commercial vaccines that provide protection against enteric septicemia of catfish (ESC) and columnaris disease, catfish producers' opinions regarding the percentage of vaccinated fish they expect to be protected, and producers' general expectations regarding survival of vaccinated fish compared with unvaccinated fish. During 2009, 9.7% of the total fingerling operations used one or both vaccines; 12.3% of the total industry fry production was vaccinated against ESC, and 17.0% was vaccinated against columnaris disease. Of the producers who grew food-sized catfish to harvest, 6.7% used vaccinated catfish. The farms that did not use vaccinated fish for grow out had a mean size of 63.4 water surface hectares (156.6 water surface acres). The operations that used vaccinated fish were larger (mean size = 206.6 water surface hectares, or 510.6 water surface acres). The producers that stocked ESC-vaccinated fish for grow out represented 19.0% of the total water surface area of food fish production; producers that stocked columnaris-vaccinated fish represented 16.6% of the total area. Of the producers that stocked ESC-vaccinated catfish, 41.9% thought that survival was better in vaccinated fish than in unvaccinated fish; of the producers that stocked columnaris-vaccinated catfish, 46.2% thought that vaccinated fish displayed better survival. However, 37.5% of producers that used the ESC vaccine and 39.7% of producers that used the columnaris vaccine did not know whether vaccination improved survival rates. When all producers were asked about their expectations regarding the percentage of vaccinated fish that would be protected from disease, 52.4% responded that they expected 100% of their fish to be protected. More producer information about reasonable expectations regarding vaccine efficacy, the conditions under which immunosuppression and vaccine failure can occur, and assessment of vaccine performance may result in increased use of vaccination as a tool for the catfish industry.     

Determination of Phylogenetic Relationships of Flavobacterium psychrophilum (Flexibacter psychrophilus), Flavobacterium columnare (Flexibacter columnaris), and Flexibacter maritimus by Sequence Analysis of 16S Ribosomal RNA Genes Amplified by Polymerase Chain Reaction

Abstract

The nearly complete small subunit 16S ribosomal RNA (rRNA) gene sequence amplified by polymerase chain reaction was determined for Flavobacterium psychrophilum (formerly Flexibacter psychrophilus) by using automated nucleotide sequencing. The sequence was found to be 1,465 base pairs (bp) in length, a size consistent with previously determined sequences for 14 other bacterial species from various taxa, including the yellow-pigmented bacteria. Sequence signatures confirmed that this organism was a member of the bacterial division Bacteroides–Flavobacterium. Parsimonious and additive phylogenetic trees were constructed with homology, and pairwise evolutionary distances were used to estimate phylogenetic relationships. Data show that F. psychrophilum, F. columnare, and Flexibacter maritimus are closely related, have a common descent, and represent a distinct group within the division Bacteroides–Flavobacterium. This group also included other organisms from the genera Flavobacterium and Cytophaga. Further, Flavobacterium aquatile, the type species for the genus Flavobacterium, was also determined to be a member of this “Flavobacterium–Cytophaga–Flexibacter subcomplex.” This supports previous assertions that the type strain of Flavobacterium should be changed to a more representative species such as Flavobacterium breve.     

Phenotypic and genetic relationships of so-called Moraxella (Pasteurella) anatipestifer to the Flavobacterium/Cytophaga group

So-called Moraxella (or Pasteurella) anatipestifer and members of the Flavobacterium/Cytophaga group exhibit remarkable common features: lack of flagellation, low guanine + cytosine content of the chromosomal DNA, production of menaquinones and branched-chain fatty acids, absence of carbohydrate fermentation, and similar patterns of hydrolytic enzymes. Using the renaturation method of DNA:DNA hybridization two urease-negative European isolates and the urease-positive type strain (which was isolated in the United States) of M. P. anatipestifer were shown to have about 85% of their genome DNA base sequences in common; they may represent two subspecies. The type strain of this species was neither measurably related to the type species of the genus Moraxella nor to selected members of the family Pasteurellaceae (Pohl 1981). On the other hand, low but significant degrees of DNA binding between selected strains of so-called M. anatipestifer, Cytophaga marinoflava, Flavobacterium meningosepticum, F. odoratum and F. pectinovorum were observed. On the basis of these findings the transfer of the so-called M. anatipestifer to the Flavobacterium/Cytophaga group (family Cytophagaceae) is proposed. More detailed investigations are required to establish its relationship at the genus level.     

应用新技术捆裹青贮燕麦草的品质评定

应用捆裹青贮这一新技术青贮燕麦草 ,经感观评定、PH值、乳酸菌数测定、NH3—N TN %比值测定等项目分析 ,对捆裹青贮燕麦草的品质综合评定 ,结果是 :捆裹青贮燕麦草的优良品率为80 %以上。     

Genomic Typing of Canine Parvovirus Circulating in the State of Rio de Janeiro,Brazil from 1995 to 2001 Using Polymerase Chain Reaction Assay

In this study, the genomic types of canine parvovirus (CPV) circulating in the State of Rio de Janeiro, Brazil, from 1995 to 2001, were investigated using the polymerase chain reaction assay (PCR). A total of 78 faecal samples from gastroenteritic puppies, confirmed as positive for canine parvovirus by haemagglutination/haemagglutination inhibition tests or virus isolation in cell culture (MDCK), were examined. The viral DNA was extracted from faecal samples using a combination of phenol– chloroform and silica–guanidine thiocyanate methods. PCR was carried out with differential pairs of primers to distinguish the old (CPV-2) and new types of virus (CPv-2a or CPV-2b). Specific amplicons were observed for all samples using the primer pair P2ab, which detects CPV-2a and CPV-2b. Seventy-six from a total of 78 samples (97%) were considered as CPV-2b because of their reaction with the primer pair P2b. Thirty samples (30/78) were from previously vaccinated puppies and in 15 of them the enteritis symptoms began from 1 to 12 days after vaccination. PCR confirmed the infection by wild virus (CPV-2b) in 5 of these 15 puppies who had received old-type vaccines. Our results show that CPV-2b was the prevalent type circulating in the State of Rio de Janeiro from 1995 to 2001.     

城郊型畜牧业物化生态资源的价值核算

分析了城郊地区畜牧业以消耗和生成的水、肥、气等农业物质资源为形式的物化生态服务功能。消耗水资源包括畜禽饮水和栏舍冲水2部分,产生肥力资源以粪便中有机质和N、P、K等养分来源为主,影响大气资源主要是消耗O2、释放CO2、排放CH4的过程。以佛山市2006年畜牧业为例,消耗水资源和破坏大气资源形成负价值,增加肥料资源产生正价值,每年生态服务价值共计186．73万元。     

城郊型畜牧业物化生态资源的价值核算

分析了城郊地区畜牧业以消耗和生成的水、肥、气等农业物质资源为形式的物化生态服务功能.消耗水资源包括畜禽饮水和栏舍冲水2部分,产生肥力资源以粪便中有机质和N、P、K等养分来源为主,影响大气资源主要是消耗O2、释放CO2、排放CH4的过程.以佛山市2006年畜牧业为例,消耗水资源和破坏大气资源形成负价值,增加肥料资源产生正价值,每年生态服务价值共计186.73万元.     

Studies on the Contamination of Products Produced by Rendering Plants

Studies on the bacterial contamination in rendered product and the environment of five rendering plants were carried out. From a total of 180 samples examined, total bacterial and anaerobic spore counts were conducted on 135.

Plants with melter systems produced a sterile product which was recontaminated before reaching the finished stage. Two plants with continuous rendering systems did not achieve sterilization of the product during the heating process. Spore forming organisms regularly survived heating in the continuous rendering system.

Salmonellae were isolated from samples collected in four of the five plants under study. Pathogenic Clostridia, especially Cl. novyi, Cl. septicum and Cl. perfringens were present in samples from all plants. Other pathogens found were Staphylococci, Streptococci, Corynebacteria and Pasteurella.

     

管碟法测定吉他霉素含量的不确定度评定

本实验建立管碟法测定吉他霉素含量不确定度的评定方法。通过建立数学模型,应用测量不确定度评定与表达理论,分析不确定度来源,对各个不确定度分量进行评估,计算扩展不确定度,得到测定结果的不确定度报告。本实验的测定结果可表示为（1636.2±11.1）U/mg（k=2）,测量不确度的主要来源为溶液的制备过程。     

用CNCPS评定反刍动物几种常用粗饲料营养价值的研究

分别从黑龙江省的哈尔滨市、大庆市、八五三农场、红卫农场和青冈县等地采集反刍动物常用粗饲料5类14种25个样品,进行常规营养成分分析,并应用康奈尔净碳水化合物-蛋白质体系(CNCPS)对碳水化合物和蛋白质成分进行了分类分析.结果表明,不同类别的粗饲料各种营养成分具有一定的规律性,通过CNCPS可对反刍动物常用粗饲料进行较细致的评定,能更好地反映饲料的特性,为粗饲料的科学利用提供了基础参数.     

Kinetics of the wattle reaction to human plasma

Human plasma produced cutaneous basophil hypersensitivity when injected into chicken wattles. The kinetics of the response development were affected by presensitization, whether or not complete Freund's adjuvant was used. Presensitized chickens developed their maximum response significantly sooner (12.6 hr earlier) than the controls.     

紫外分光光度法测定维生素B1注射液含量的不确定度评价

为建立紫外分光光度法测定维生素B_1注射液含量的不确定度评价方法,利用数学模型分析溶液配置过程和仪器测定过程的不确定度来源,并对各分量进行评价,最后计算了扩展不确定度并给出测量不确定度报告。维生素B_1注射液含量测定结果可表示为(93.9±0.9)%(k=2)。测量结果不确定度的主要来源为紫外分光光度计和小容量量器。