内蒙阿盟斑点热群立克次体Ha—91株的分离与鉴定

我们于1991年从内蒙阿盟亚东璃眼蜱分离到一株斑点热群(SFG)立克次体,命名为Ha-91株。免疫荧光法及蛋白免疫印迹分析表明Ha-91株立克次体与西伯利亚立克次体抗原性相近,但不完全相同;明显不同于其它种班点热群立克次体。这是首次证实亚东璃眼蜱为SFG立克次体的贮存宿主,也是首次从内蒙发现抗原性与西伯利亚立克次体不完全相同的SFG立克次体。     

Q热立克次体基因文库的构建

Q热立克次体七医株用6种限制性平端内切酶部分酶切,电泳回收0.2—7kb大小DNA片段,EcoRI位点甲基化,加上EcoRI接头,与去磷酸化λgtll DNA-EcoRI臂连接,经体外包装后感染E.coli Y1090,建成含2.6×10~6重组子的表达型基因文库。随机选择的噬菌体DNA经EcoRI酶切鉴定,重组子含大小不等的插入DNA片段。     

Q热立克次体红斑技术

本文根据Q热立克次体在鸡胚成纤维细胞空泡酸性环境中大量繁殖的特点,建立了Q热立克次体红斑技术,它出斑早,稳定并易于观察。用该技术研究单克隆及多克隆抗体对立克次体的体外中和作用表明,Ⅱ相抗体能使Ⅱ相Q热立克次体红斑减数,而Ⅰ相抗体反而使Ⅰ相病原体红斑增数,但用间接法试验则Ⅰ相病原体也出现红斑减数。红斑技术提供了一种快速分离与鉴定Q热立克次体的新方法。     

广东连平县存在新的蜱传斑点热立克次体

目的对采自广东省连平县的3种硬蜱进行蜱传斑点热立克次体检测分析。方法斑点热立克次体的ompA基因片段扩增并测定扩增片段的DNA序列。结果20只蜱分为17组,其中15组PCR检测阳性。对4个测序成功的标本进行聚类分析,证实其中3个序列单独聚为一类,与引起Flinders岛蜱传斑点热的弗诺立克次体(R.honei)、非洲蜱咬热的非洲立克次体(R.africa)、未定名斑点热的斯洛伐克立克次体(R.slovaca)以及西伯利亚立克次体BJ-90株等亲缘关系较近,另一序列与我国长白山地区检测到的JL-02具有较高的同源性。结论本研究提示该地区除了已证实的西伯利亚斑点热外,还存在新的蜱传斑点热。     

黑龙江逊克地区森林革蜱斑点热立克次体DNA的检测

目的 调查黑龙江逊克地区蜱传斑点热自然疫源地,发现该地区蜱携带斑点热立克次体的种类。方法 采用斑点热立克次体ompA和gltA基因特异的PCR,检测该地区森林革蜱的DNA样本,并对扩得阳性产物进行测序和聚类分析。结果 从60只森林革蜱中检测有14只扩得斑点热立克次体ompA和gltA基因片段,阳性率为23.33%。随机选择2只蜱的阳性片段进行测序,二者同源性为100%,ompA基因序列与Rickettsia sp.JL-02同源性为99.30%, 与Rickettsia raoultii为99.18%。结论 黑龙江省逊克地区森林革蜱携带与Rickettsia sp.JL-02株亲缘关系相近的斑点热群立克次体。     

我国Q热立克次体脂多糖的SDS-PAGE和单克隆抗体免疫印迹分析

用SDS-PAGE对5株Q热立克次体LPS进行分析,发现七医、新桥和Ys8等国内分离的3个Ⅰ相株与国外Ⅰ相参考株Henzerling的LPS图象相似,而国外Ⅱ相参考株Grita的LPS图象与它们有明显的差异。我国分离株之间的SDS-PAGE分析也显示多糖结构的差异,新桥株与Henzerling株的LPS图象最相似,具有最完整的带型,七医株和Ys8株则与新桥株有所不同,特别是在蛋白酶K消化后的WCP电泳中均有顶部区带下移现象,七医株63和59K带较为细小,而Ys8株则无30K而有40K带等,有类似Grita株之处。免疫印迹发现,用Ⅰ相McAbB_5与LPS作免疫印迹,Ys8株和Grita株显示类似在多克隆抗体免疫印迹中的指纹状印迹(在17.5K下),而七医株仅显一14K带,新桥和Henzerling两株则无印迹。用Ⅰ相McAb与蛋白酶K消化后的WCP作免疫印迹的结果与上述基本一致。     

套式PCR和DNA探针技术检测Q热立克次体

目的 建立套式PCR（NPCR）和DNA探针技术用于检测Q热立克次体。方法 依据已知的Cb 23s rRNA基因序列,设计两对引物用于建立套式PCR技术,并将扩增片段制成探针进行斑点杂交。结果 9株Cb分离株均可扩增出阳性产物带,而对照菌均为阴性,扩增灵敏度可达0.1pg Cb DNA,实验感染第6天的豚鼠血、小鼠血、小鼠脾脏均可扩增出阳性带;用七医株扩增片段制成的探针与9株Cb分离株的全DNA呈阳性杂交,对照菌为阴性,探针杂交灵敏度为0.5ng Cb DNA,感染的豚鼠血、小鼠血、小鼠脾脏均可呈阳性反应。结论 我们的NPCR和DNA探针技术具有很好的特异性和灵敏度,可将二者联合用于Q热的病原学诊断。     

对黑龙江立克次体和远东蜱传斑点热调查研究的思考

斑点热立克次体病是由斑点热群立克次体(spotted fever group rickettsiae SFGR) 引起的蜱、螨传播的人兽共患病.该病广泛分布于世界各地.     

四川石渠县牦牛源蜱感染斑点热群立克次体分子流行病学调查

目的 了解四川省石渠县牦牛体表寄生蜱的种类及其斑点热群立克次体的感染情况。方法 采集牦牛体表的蜱,经形态学初步鉴定后,提取蜱总DNA,PCR扩增蜱16S rRNA基因及斑点热群立克次体ompA、ompB基因,并对扩得阳性产物进行测序和构建进化树分析,从而确定蜱及其携带斑点热群立克次体的种类。结果 在石渠县4个乡共采集到蜱818只,其中西藏革蜱占78.97%(646/818)、青海血蜱占21.03%(172/818)。在818只蜱中有408只扩得斑点热群立克次体ompA、ompB基因,总阳性率为49.8%。经比对分析, ompA基因总共得到4条序列(uncultured Rickettsia sp.shiqu1、R.raoultii.shiqu2、R.raoultii.shiqu3和R.raoultii.shiqu4),ompB基因也得到4条序列(uncultured Rickettsia sp.shiqu1、R.raoultii.shiqu2、R.raoultii.shiqu3和R.raoultii.shiqu4)。经遗传进化分析显示ompA基因的uncultured Rickettsia sp.shiqu1与我国青海未定种的Uncultured Rickettsia sp.(MG228270)亲缘关系最近;ompB基因的uncultured Rickettsia sp.shiqu1与韩国未定种的Rickettsia sp.(KC888953)和Candidatus Rickettsia longicornii(MG906675)亲缘关系最近;ompA和ompB基因的R.raoultii.shiqu2-4与对人有致病性的劳氏立克次体(R.raoultii)的亲缘关系最近。结论 首次在牦牛体表寄生的西藏革蜱中检出劳氏立克次体。石渠县存在西藏革蜱和青海血蜱,蜱传劳氏立克次体感染率较高并且具有感染人的风险。     

云南首次从患者体内分离到基孔肯雅病毒

本文报告1987年从云南西双版纳地区97份急性发热期病人血中分离到一株基孔肯雅病毒,并在分离到病毒患者恢复期血清中查到中和抗体,指数为316。首次证实云南有基孔肯雅轻型病例及自然疫源地存在。     

贝氏柯克斯体实时荧光定量PCR方法的建立及对云南鼠标本检测

目的 建立贝氏柯克斯体荧光定量PCR方法并应用于云南省鼠标本的检测.方法 根据贝氏柯克斯体ISlllla基因设计引物和探针,以克隆的ISlllla基因片断(485 bp)作标准DNA模板,建立实时荧光定量PCR检测方法,并与普通巢式PCR方法进行比较.采用2种方法对云南玉溪自然界采集的鼠各类标本进行检测分析,计算2种方法的检出率.结果 实时荧光定量PCR标准曲线的循环阈值(cycle threshold,Ct)与模板拷贝数呈良好的线性关系(r=-0.999),重复性测试Ct变异系数(coefficient of variation,CV)为0.25%～3.98%,灵敏度为巢式PCR的10倍.以其他立克次体和常见病原菌共26种DNA为模板进行特异性检测,结果均为阴性.实时荧光定量PCR检测鼠血、肝脏、脾脏中的贝氏柯克斯体,阳性率分别为32.31%、61.29%、85.19%;巢式PCR法检测相应标本阳性率分别为27.69%、11.29%、74.07%;间接免疫荧光试验检测鼠血清中贝氏柯克斯体IgG抗体阳性率为26.15%.结论 本实验建立的实时荧光定量PCR比巢式PCR敏感,且具有良好的特异性,可用于人群和动物贝氏柯克斯体感染监测及临床标本的检测.     

Coxiella burnetii,the causative agent of Q fever in Saudi Arabia:molecular detection from camel and other domestic livestock

Objective:To detect Coxiella burnetii(C.burnetii)DNA in clinical specimens from camel,goats,cattle and sheep in the Kingdom of Saudi Arabia.Methods:A total of 367 clinical samples including blood,milk,faeces and urine were collected from different livestock and subjected to PCR amplification using primers which amplify transposon-like region and transposase gene.Results:Positive amplification from both regions was obtained from camel,goats and cattle but not from sheep.A percentage of 10.8%samples yielded positive PCR amplification from both blood and milk,where 15 of 139 blood and 16 of 148 milk samples were positive.Faeces and urine showed higher percentages of positive samples reaching 40.8%and 23.8%respectively.Conclusions:The preferred route of shedding in camel appeared to be the faeces followed by urine,while that of goats appeared to be the faeces and that of the cattle appeared to be the milk.     

首次从北京市腹泻病人粪便中检出致病性弧菌

本文报告1986年夏季从北京市腹泻病例中首次检出致病性弧菌。经形态、生长表现和生物学性状检查,确定一株为弗尼斯弧菌,它具有与河弧菌极为相似的特性,主要通过对葡萄糖产酸产气加以区别。另一株为麦契尼可夫弧菌,它对氧化酶和硝酸盐还原反应均阴性。两菌都不产生肠毒素,豚鼠眼角膜试验阴性,但对乳鼠有较强的致死作用,并对L929小鼠成纤维细胞有明显的粘附表现。对常用的抗菌药物均较敏感。     

国内首次从猪中检出假结核耶氏菌的报告

本文报告检验福建省市售28种食品标本764份,其中猪肉标本占177份。结果从猪肠中分离出一株假结核耶氏菌。该株根据形态学、血清学及生化的特征,鉴定为血清Ⅲ型,具有毒力,含有VW抗原,且能使小白鼠致死。这证明猪可自然携带假结核耶氏菌,是该病的传染源。     

Full Genome Sequencing of Three Sedoreoviridae Viruses Isolated from Culicoides spp. (Diptera,Ceratopogonidae) in China

Sedoreoviridae is a family of viruses belonging to the order Reovirales and comprises six genera, two of which, Orbivirus and Seadornavirus, contain arboviruses that cause disease in humans and livestock. Areas such as Yunnan Province in southwestern China, have high arboviral activity due in part to warm and wet summers, which support high populations of biting flies such as mosquitoes and Culicoides. Three viral isolates previously obtained from Culicoides collected at cattle farms in Shizong County of Yunnan Province, China, between 2019 and 2020 were completely sequenced and identified as Banna virus (BAV) genotype A of Seadornavirus and serotypes 1 and 7 of epizootic hemorrhagic disease virus (EHDV) of Orbivirus. These results suggest that Culicoides tainanus and C. orientalis are potential vectors of BAV and EHDV, respectively, and represent the first association of a BAV with C. tainanus and of an arbovirus with C. orientalis. Analysis using VP9 generally agreed with the current groupings within this genus based on VP12, although the classification for some strains should be corrected. Furthermore, the placement of Kadipiro virus (KDV) and Liao ning virus (LNV) in Seadornavirus may need confirmation as phylogenetic analysis placed these viruses as sister to other species in the genus.     

山东恙虫病立克次体生物学及免疫学性质的研究

本文报告了山东恙虫病立克次体人株生物学及免疫学性质的初步研究。所研究3株中的1株感染小鼠发病不典型,极少致死;另两株感染小鼠发病典型,小鼠呈规律性的死亡,易适应于鸡胚,对小鼠毒力LD_(50)为3.4。感染小鼠之IgG抗体出现于接种后的第13天,于接种后的第3～4周抗体水平达峰值。上述分离株与Karp毒株间存在较强的交叉保护作用。酶免疫染色血清学反应结果指出,与Gilliam株很一致,可能属Gilliam同一血清型。     

Non-Vitamin K Antagonist Oral Anticoagulants in Adult Congenital Heart Disease

Non-vitamin K antagonist (VKA) oral anticoagulants (NOACs) have several advantages over VKAs that render them an attractive option for adults with congenital heart disease (CHD). Efficacy and safety data specific to the adult CHD population are emerging. Herein, we synthesize the growing literature regarding NOACs in adults with CHD and attempt to identify subgroups for which it appears reasonable to extrapolate data from populations without CHD. Small observational studies suggest that NOACs are safe and effective in selected adults with CHD. NOACs are contraindicated in patients with a mechanical valve, in those with mitral or tricuspid valve stenosis with enlarged and diseased atria, with or without a mitral or tricuspid bioprosthesis, and after recent cardiac surgery (< 3 months). There is currently insufficient evidence to recommend NOACs in patients with a Fontan circulation or cyanotic CHD. Growing literature supports the use of NOACs in patients without CHD who have various forms of valvular heart disease. Therefore, when an indication for oral anticoagulation is established, it appears reasonable to consider a NOAC instead of a VKA in adults with CHD lesions analogous to isolated mitral regurgitation, tricuspid regurgitation, or aortic regurgitation or stenosis. The NOAC agent selected and the prescribed dose should be tailored according to bleeding risk, body weight, renal function, and comedications, especially antiepileptic drugs. The decision to initiate a NOAC should be shared between the patient and care provider. Large-scale research studies are required to further assess safety and efficacy in selected patient subgroups.     

Ventricular function curves from the cardiac response to angiographic contrast: a sensitive detector of ventricular dysfunction in coronary artery disease

Fifty cardiac catheterizations were performed in 44 patients undergoing evaluation for coronary artery disease. Ventricular function curves (VFC) were constructed by plotting the left ventricular end-diastolic pressure (LVEDP) and stroke work index (SWI) before and three to four minutes after a standard left ventricular angiogram. In an attempt to delineate the mechanism that produces changes in the post-angiogram LVEDP and SWI, 13 individuals (Group A) with no evidence of cardiac disease were compared to 14 patients with severe coronary artery disease (Group B). Cardiac output and LVEDP increased in both groups after angiogram. The increase in cardiac output was less and the increase in LVEDP greater in Group B. V_max. did not change significantly after angiogram in either group. Other measures of contractility ($\text{dp}\text{dt}$ max., peak Vce, and dp/dt/40 mm. developed pressure) changed appropriately for the large changes in preload seen after angiogram.Thirty-seven studies in patients with coronary artery disease demonstrated that VFC obtained from the cardiac response to contrast are more sensitive than resting LVEDP or ejection fraction in detecting left ventricular abnormality.VFC can be obtained from the ventricular response to angiographic contrast because of the increase in venous return produced by the hyperosmotic effect of contrast. Depressed curves occur in patients with coronary artery disease because of their stiff ventricles and not because of depression of myocardial contractility.     

Adenylate cyclase activity in human parathyroid tissues: Reduced sensitivity to suppression by calcium in parathyroid adenomas as compared with normal glands form normocalcemic subjects or noninvolved glands from hyperparathyroid subjects

To examine whether alterations in parathyroid adenylate cyclase might be associated with glandular hyperfunction, we compared enzyme activity in membranes from 7 normal glands with activity from 18 abnormal and 5 noninvolved glands from patients with primary hyperparathyroidism. Compared with the normal glands, the specific enzyme activity after full stimulation with guanyl-5′yl imidodiphosphate was significantly decreased in both hyperplastic and noninvolved glands from the hyperparathyroid subjects. While the enzyme activity of all tissues could be suppressed by calcium, a twofold higher calcium concentration was required for comparable suppression of the enzyme from adenomas as compared with normal or noninvolved glands. Alterations in the adenylate cyclase complex of hyperplastic parathyroid glands may explain, in part, the elevated “set point” for calcium homeostasis in primary hyperparathyroidism.