炭疽杆菌与类炭疽杆菌鉴别技术的研究近况

炭疽是自然疫源性疾病，为人畜共患的急性、败血性传染病．严重者可引起死亡，对人类危害大，所以发现病例需快速诊断，以便及时采取措施，减少损失．在类症鉴别上，排除类炭疽杆菌很有必要．今就其鉴别技术简述如下：1快速串珠试验此法鉴别结果快，只需1．5～3小时，且方法简便，R需实验室常用器材和药品，无需特殊设备．滤纸片上青霉素的浓度需在200单位以上，炭疽杆菌都能形成串珠，而类炭疽杆菌则无．2美腹抗体试驻发应杆菌呈现田供，而类炭疽杆菌呈明他3运动力检查用＊满法和半固体培养基法，炭植圩首无运动性，而类发疽杆菌有根毛，…     

PCR检测炭疽杆菌的研究

以无毒炭疽芽孢苗(steme菌株)、Ⅱ号炭疽芽孢苗(pasteurⅡ菌株)、A16R菌苗(steme菌株)为试验茵，建立了检测炭疽芽孢杆菌质粒DNA的PCR方法。该方法特异性强，敏感性可达1131个菌／ml(无毒炭疽芽孢菌菌株)和1720个菌／ml(Ⅱ号炭疽芽孢菌菌株)。同时建立了可区分炭疽杆菌强、弱毒的多重PCR方法。     

炭疽杆菌基因检测技术的研究进展

从炭疽杆菌基因组组成、炭疽杆菌基因组的特征及其在基因检测中的应用几方面阐述了炭疽杆菌基因检测技术的研究进展，探讨了分别位于pXO1和pXO2质粒上的主要致病毒素编码基因pagA、lef、cya和参与荚膜合成的主要蛋白编码基因capA、capB和capC的作用，认为这些炭疽杆菌特异基因序列是建立基因检测体系的可靠基础，其标志性序列有早期发现的序列指纹图谱、高度保守基因序列和最新发现的高度特异基因序列，基因检测方法从早期的RFLP、VNTR、单重PCR、多重PCR发展到最新的实时定量PCR，随着标志性序列特异性的增加和检测方法的改进，炭疽杆菌的诊断工作变得更加准确和快捷。     

炭疽芽孢杆菌疫苗研究进展

炭疽病是一种人畜共患的烈性传染病.该病由炭疽杆菌所致,传播迅速,潜伏期短,死亡率高.炭疽杆菌在生物武器中占有重要地位.本文简述了现用疫苗及各国正在开发的新疫苗,探讨了疫苗研发的新思路,客观评价了人用炭疽疫苗的安全性及有效性.     

羊毛皮张和土壤中炭疽杆菌PCR检测方法的建立

建立了检测炭疽芽孢杆菌染色体特异性基因序列(Ba813)的PCR技术，并用于模拟污染的羊毛、皮张和土壤中炭疽芽孢(Sterne菌株、PasteurⅡ菌株)的检测。结果表明，该方法可特异、敏感地检出0．5g羊毛中Sterne菌株的452个芽孢和PasteurⅡ菌株的688个芽孢；0．5g皮张中Sterne菌株的452个芽孢和PasteurⅡ菌株的1032个芽孢；0．5g土壤中Sterne菌株的452个芽孢和PasteurⅡ菌株1376个芽孢。证实PCR可用于羊毛、皮张和土壤等外环境中炭疽杆菌芽孢的检测。     

炭疽杆菌与炭疽热

通过对炭疽杆菌的形态、生长条件的研究,阐明炭疽发病的最主要原因是炭疽毒素。这种毒素一方面可以引起病理生理学变化,导致人和动物死亡,另一方面可以引发对疾病的免疫反应。同时通过研究炭疽病在人群中和动物间的流行特征,以及人和动物炭疽病的临床表现,提出了相应的诊断与防治措施。     

炭疽杆菌与炭疽热

通过对炭疽杆菌的形态、生长条件的研究，阐明炭疽发病的最主要原因是炭疽毒素。这种毒素一方面可以引起病理生理学变化，导致人和动物死亡，另一方面可以引发对疾病的免疫反应。同时通过研究炭疽病在人群中和动物间的流行特征，以及人和动物炭疽病的临床表现，提出了相应的诊断与防治措施。     

炭疽芽孢杆菌PCR鉴定方法的建立

根据炭疽芽孢杆菌3种特异性毒力基因的序列,包括保护性抗原基因(pag)、荚膜基因(cap)和S-层蛋白基因(sap),建立多重PCR鉴定方法。该方法检测11株炭疽芽孢杆菌均为阳性,检测29株非炭疽的其他需氧芽孢杆菌属菌株均为阴性。该方法检测模拟血液样品的灵敏度是2×10~6 CFU/mL(每1mL血液样品最低含有2×10~6细菌),而样品增菌后的检测灵敏度是2×10~2 CFU/mL。制备加入炭疽疫苗菌株芽孢的模拟土壤样品,经增菌培养后,PCR检测灵敏度为3×10~4芽孢/g。另外,还设计了炭疽芽孢外壁结构蛋白基因BclA的鉴定引物,利用该引物的PCR扩增片段长度以及测序结果,能够有效区分Ⅱ号炭疽疫苗菌株和临床分离菌株,本研究能够为炭疽临床诊断和环境监测工作提供技术支持。     

Taqman-MGB探针法检测炭疽杆菌

根据GenBank中炭疽杆菌的主基因组上gyrA基因,毒力因子px01上pagA基因和侵袭因子px02上的CapA基因分别设计引物和对应Taqman-MGB探针,建立了检测炭疽杆菌的实时定量PCR方法。该方法具有良好特异性,仅对炭疽杆菌检测结果为阳性,而与蜡样芽孢杆菌、苏云金杆菌、巨大芽孢杆菌、沙门菌、大肠杆菌、链球菌、铜绿假单孢杆菌无交叉反应,检测灵敏度最低可达100copies/μL。对24份皮毛盲样和120份送检样品检测结果表明建立的定量PCR方法与商业化定量PCR试剂盒的符合率为100%。Taqman-MGB探针方法灵敏、特异、重复性好,可应用于炭疽杆菌的快速诊断与监测。     

炭疽杆菌双毒力因子荧光PCR检测方法的建立及应用

由炭疽杆菌（Bacillus anthracis）引起的炭疽（anthrax）是严重危害人类和家畜健康的重大传染病。为了提高出入境口岸对不明粉未快件样品的查验效率,防范通过包裹夹带生化武器,本研究使用Taq Man探针技术,建立了针对炭疽杆菌两种毒力质粒pOX1和pOX2的双重荧光PCR检测方法,并对该方法的特异性、灵敏性和稳定性进行了试验。结果显示：本研究建立的炭疽杆菌双重荧光PCR检测方法具有较强的特异性,只特异性检出炭疽杆菌pOX1和pOX2,而与金黄色葡萄球菌等19种对照病原菌均无交叉反应;灵敏度与单重荧光PCR方法相近,最低检测限可达10 copies/μL;稳定性好,反应体系低温保存2、4、6、8个月后,检测阳性质粒的Ct值和峰值相差不大。应用该方法检测600份猪牛羊组织样本,均未检出炭疽杆菌阳性核酸。本研究建立的双重荧光PCR检测方法为炭疽杆菌的快速检测提供了技术支撑。     

PRRS遗传相异毒株的防制

猪繁殖和呼吸综合征(PRRS)病毒不断地危害着全世界的养猪业。该病毒因其自身的生物学特点,具有发生广泛遗传变异(突变和重组)而产生新毒株的能力。另一个问题是,尽管广泛采取了将该病毒抵挡在猪场之外的措施,但该病毒天生具有突破当前生物安全措施而感染猪场的能力。     

吉林省1起炭疽突发事件的病原菌分离和鉴定

从采集的羊肉和土壤样品中分离、鉴定炭疽芽孢杆菌,样品制备悬液,热处理杀死非芽孢的细菌,然后涂布PLET琼脂平板进行选择性增菌培养。疑似菌落首先使用炭疽特异性的引物进行PCR鉴定,然后进行噬菌体裂解和青霉素抑制试验的鉴定;鉴定非炭疽的菌落进行16SrDNA的测序分析。从可疑羊肉样品中成功分离、鉴定出炭疽芽孢杆菌,确定了本次突发事件的传染源。从屠宰地点采集的土壤样品中未检出炭疽芽孢杆菌,土壤中分离的疑似炭疽样菌落经16SrDNA测序和比对鉴定为蜡样芽孢杆菌等其他需氧芽孢杆菌属的细菌。     

Molecular detection and characterization of Anaplasma phagocytophilum strains

The zoonotic rickettsial pathogen Anaplasma phagocytophilum has a broad geographic distribution and a high degree of biological and clinical diversity. To investigate the genetic diversity of A. phagocytophilum strains in the Baltic region and Norway, three species of Ixodidae ticks were examined for A. phagocytophilum infection, and two genes of the pathogen genome were analyzed. Analysis of partial 16S rRNA and partial major surface protein (msp4) gene sequences was accomplished through nested PCR and sequence analysis. Strains identified in this study were compared with those originating from other European countries and the United States. Seven 16S rRNA gene variants and fifteen msp4 gene variants of A. phagocytophilum were detected. Nine sequences had unique nucleotide polymorphisms and therefore differed from other A. phagocytophilum sequences previously submitted to GenBank. The present study represents the first molecular characterization of A. phagocytophum strains circulating in Lithuania and describes the strains detected in Ixodes persulcatus ticks in Latvia and Estonia. This is also the first report describing A. phagocytophilum strains isolated from Dermacentor reticulatus ticks.     

Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations

Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.     

免疫层析法快速检测金黄色葡萄球菌的研究

为建立一种快速检测金黄色葡萄球菌的新方法,本研究采用柠檬酸三钠还原法制备胶体金,用胶体金标记鼠抗金黄色葡萄球菌凝集因子A(C1fA)重组蛋白特异性抗体,从抗金黄色葡萄球茵IgG包被于硝酸纤维素膜,制成免疫层析快速检测试纸条.该方法可以在10min内完成对含金黄色葡萄球菌奶样的检测;敏感度为1×104 cfu/mL以上,与PCR方法进行对比检测,相差一个数量级;与大肠杆菌、链球菌、李氏杆菌、巴氏杆茵及蜡样芽孢杆菌均无交叉;稳定性和重复性较好.该试纸条检测金黄色葡萄球菌临床样品表明其具有特异性好,灵敏度较高的特点.     

Time-series models on somatic cell score improve detection of mastitis

Abstract

In-line detection of mastitis using frequent milk sampling was studied in 241 cows in a Danish research herd. Somatic cell scores obtained at a daily basis were analyzed using a mixture of four time-series models. Probabilities were assigned to each model for the observations to belong to a normal “steady-state” development, change in “level”, change of “slope” or “outlier”. Mastitis was indicated from the sum of probabilities for the “level” and “slope” models. Time-series models were based on the Kalman filter. Reference data was obtained from veterinary assessment of health status combined with bacteriological findings. At a sensitivity of 90% the corresponding specificity was 68%, which increased to 83% using a one-step back smoothing. It is concluded that mixture models based on Kalman filters are efficient in handling in-line sensor data for detection of mastitis and may be useful for similar applications to decision support systems.     

人兽共患病炭疽的防治与诊断

卫生专家提示公众：中国自然疫源地分布广泛,炭疽病例时有发生。近五年来，全国每年发病数在,400～1000人．主要集中在贵州、新疆、甘肃．四川、广西，云南等西部地区。今年7月在沈阳也出现过炭疽的疫情。