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.

Herd-prevalence of Coxiella burnetii(Q fever) antibodies in dairy cattle farms based on bulk tank milk analysis

Objective:To determine the prevalence of Coxiella burnetii(C.burnetii) antibody positive randomly selected dairy herds in southeast Iran(Kerman).Methods:Bulk tank milk samples were collected randomly from 44 sufficiently large commercial dairy herds,included near 12 000 dairy cattle,in Kerman(The largest province of Iran),southeast Iran.The samples were tested for antibodies against C.burnetii using the commercial CHEKIT(?) Q fever antibody ELISA Test Kit(Idexx,Liebefeld-Bern,Switzerland).Results:The prevalence of positive,negative and intermediate herds were 45.4%,43.2%and 11.4%,respectively.Conclusions:The result supports the hypothesis of high prevalence and endemic pattern of Q fever in Iran.This investigation highlights the importance of further studies on Q fever in Iran.

Characterizations of Chinese isolates of Coxiella burnetii in the com1 gene sequence

Objective: To know some genetical characterizations of Coxiella burnetii Chinese isolates by comparing the coml gene sequence. Methods: com1 gene sequences of Chinese isolates were amplified, se-quenced, and analyzed by comparing our result and the previous published data. Results: Three different com1 sequences were identified in 7 Chinese isolates. Sequence comparison indicated that the isolates harboring the QpRS plasmid could be defined as a new group and, in addition, the isolates carrying the same plas-mid type showed similar com1 gene sequence. Conclusion: Study suggests that the classification of the group based on the coml gene sequence is highly associated with the plasmid type of the isolates and, however, little related to disease forms and geographical origins of the isolates.

Growth Yields of Four <i>Coxiella burnetii</i>Isolates in Four Different Cell Culture Lines

Although Coxiella burnetii is considered to be an obligate intracellular bacterium and grows in embryonated eggs, laboratory animals and cell culture, recently it has been grown in cell-free media and on agar plates. This current study was conducted to compare four cell lines for their yield of C. burnetii. Four different isolates of C. burnetii (Henzerling, Arandale, Cumberland and Timony) were grown in DH82, L929, Vero and XTC-2 cell lines. The DH82 and XTC-2 cells lines produced the highest C. burnetii yield which was slightly less than the yields achieved in recently published studies using cell free media. The Arandale isolate of C. burnetii produced a significantly higher yield in DH82 cells compared to XTC-2 cells (P 0.03).

PCR法对鸡卵黄中Coxiella burnetii菌的测定

【目的】通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii(C.b)Com1基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义。【方法】提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物。【结果】用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Com1基因,用此方法可测出鸡卵中Coxiella burnetii Com1基因达104-106个,阳性率为5%?22%;对阳性鸡卵Com1基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物。【结论】由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源。

The potential utility of nested PCR for investigation of Coxiella burnetii in Iranian snails

Objective:To detect the prevalence of Coxiella burnetii(C.burnetii)in two species of snails consisted of Lymnaea palustris(L.palustris)and Pomacea canaliculata(P.canaliculata)by using nested PCR method in Chaharmahel Va Bakhtiari Province which is located in the southwest of Iran.Methods:A total of 160 snail samples consisted of 100 L.palustris and 60 P.canaliculata were collected from 4 rice paddy fields in the southwest of Iran between June and August 2014.Snails'DNA was extracted by a genomic DNA purification kit according to the manufacturer's instructions.Detection of the presence of C.burnetii's DNA was carried out by using a nested PCR assay with[specific primers outer membrane protein 1(OMP1)-OMP2 and OMP3-OMP4]targeting the com1 gene.Results:In this study,a total of 160 snail samples were tested and 15(9.37%)samples were found positive for C.burnetii,15 samples were positive from the L.palustris and there were no positive samples from P.canaliculata.Conclusions:Snails are kind of gastropods which seem to be harmless in life,but these small gastropods can be very dangerous for farmers,especially in humid climates.Also,C.burnetii in snails showed that this bacterium can be a factor of transmission of contamination to human beings and animals.

Protein array of Coxiella burnetii probed with Q fever sera

Coxiella burnetii is the etiological agent of Q fever.To identify its major seroreactive proteins,a subgenomic protein array was developed.A total of 101 assumed virulence-associated recombinant proteins of C.burnetii were probed with sera from mice experimentally infected with C.burnetii and sera from Q fever patients.Sixteen proteins were recognized as major seroreactive antigens by the mouse sera.Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera.Notably,HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera.These results suggest that these seven major seroreactive proteins,particularly HspB,are potential serodiagnostic and subunit vaccine antigens of Q fever.

宏基因组二代测序技术辅助诊断中枢神经系统贝氏柯克斯体感染性血管炎1例

Q热是由贝氏柯克斯体感染引起的人畜共患病,临床表现多样且无特异性,贝氏柯克斯体颅内感染罕见,经常被误诊和漏诊,导致部分患者预后不佳。此文报告1例宏基因组二代测序(mNGS)技术辅助诊断的中枢神经系统贝氏柯克斯体颅内感染性血管炎病例,提示mNGS技术在Q热快速诊断中起到重要作用,通过早期诊断,精准治疗,明显改善患者预后。在此基础上回顾国内外相关文献,总结贝氏柯克斯体颅内感染的临床表现和诊治经验,供国内外同行参考。

羊贝氏柯克斯体TaqMan荧光定量PCR检测方法的建立及应用

为建立检测贝氏柯克斯体(Coxiella burnetii)实时荧光定量PCR方法,根据GenBank上收录的C.burnetii RSA439株com1基因序列(CP040059.1)保守区设计特异性引物和探针,并构建重组质粒及优化反应条件,以期建立一种检测贝氏柯克斯体的Taq Man荧光定量PCR方法。结果显示,该方法所建立标准曲线线性关系良好;敏感性试验结果显示,最低检出限为2.45×10^(2) copies/μL,与PCR最低检出限为2.45×10^(4) copies/μL相比较,灵敏度提高了100倍;对羊支原体、羊流产衣原体、羊布鲁氏菌等均无交叉反应,批内和批间重复性试验结果稳定,变异系数均小于3%,说明特异性、重复性好;利用该方法对收集的126份样品进行检测,结果显示样品阳性率为9.52%,常规PCR检测阳性率为7.14%,符合率为90.47%。该方法可为贝氏柯克斯体临床上快速检测及防控提供技术支持。

山羊源贝氏柯克斯体抗体间接ELISA检测方法的建立与应用

为建立山羊源贝氏柯克斯体(Coxiella burnetii)抗体间接ELISA检测方法,对贝氏柯克斯体的com1基因进行克隆及原核表达,以com1纯化蛋白作为抗原,建立间接ELISA检测方法,并对临床收集的血清样本进行检测。结果显示,重组蛋白大小为27 ku,经Western blot鉴定com1蛋白反应原性良好;ELISA检测方法抗原包被量为4μg/mL,血清的稀释倍数为1∶100,酶标二抗稀释倍数为1∶2500,阴阳临界值为0.278。用该方法对流产衣原体、山羊支原体、牛支原体阳性血清进行检测,均为阴性;当C.burnetii阳性血清稀释至2048倍时检测结果仍为阳性;批内、批间变异系数均小于10%。用该方法对陕西省部分羊场收集到的307份血清样本进行检测,样本阳性率为9.12%。成功建立了一种检测贝氏柯克斯体抗体的间接ELISA检测方法,这为C.burnetii引起的Q热的实验室诊断和流行病学调查提供了技术支持。

基于CRISPRi的贝氏柯克斯体dotB基因沉默

目的更简便快捷地研究贝氏柯克斯体基因功能。方法基于CRISPRi原理构建携带化脓链球菌dCas9的重组质粒,将靶向贝氏柯克斯体dotB基因的sgRNA序列插入重组质粒中并将质粒经电转化导入贝氏柯克斯体,利用脱水四环素(aTC)诱导dCas9表达以抑制贝氏柯克斯体dotB基因的转录,从而建立基于CRISPRi系统的贝氏柯克斯体基因沉默技术。结果在aTC诱导下,该CRISPRi系统中dCas9可正常表达,dotB在转录水平和蛋白水平均被抑制,dotB表达抑制后贝氏柯克斯体胞内生长繁殖水平降低。结论成功建立起基于CRISPRi的贝氏柯克斯体的基因沉默技术,为研究贝氏柯克斯体特定基因的生物学功能研究提供了技术支撑。

贝氏柯克斯体感染致心内膜炎1例

贝氏柯克斯体是导致Q热的病原体。Q热是一种人畜共患病,在我国报道较少。Q热临床表现多样,最严重的表现为血培养阴性的心内膜炎,如果未能及时识别并适当地抗菌治疗,会延误患者病程,严重者甚至死亡。本文介绍1例心内膜炎病例,患者血培养阴性,应用基因测序而诊断。本文报告该病例流行病学、临床表现、诊断和治疗过程。

基于贝氏柯克斯体急性期抗原A的间接ELISA方法建立及应用

为开发快速有效的Q热血清学诊断技术,以贝氏柯克斯体急性期抗原A(acute disease antigen A,AdaA)为诊断靶标分子,构建了基于该重组抗原的间接ELISA方法,完成了来自福建某牛场的200份临床牛血清样本检测,并对检测结果进行了评估。结果表明:以商品化ELISA试剂盒ID Screen■Q热为金标准,建立的间接ELISA检测特异性为94.4%(152/161),检测敏感性为84.6%(33/39),与感染牛的常见病原体如衣原体、布氏杆菌、副结核杆菌无交叉。应用该诊断方法检测发现,福建某牛场约有21%(42/200)的牛为Q热阳性。该结果证实,基于AdaA的间接ELISA方法具有较高的特异性及敏感性,有良好的应用价值,可以用于牛Q热的血清学监测。

云南玉龙鼠类携带贝氏柯克斯体的基因分型研究

为更好地了解云南省鼠类中贝氏柯克斯体Coxiella burnetii的流行情况,以便制定针对Q热的预防和控制措施,本研究采用巢式PCR方法检测云南省玉龙县捕获的鼠类携带贝氏柯克斯体的情况,并对阳性样本的贝氏柯克斯体进行多间隔区序列分型(multispacer sequence typing,MST)和多位点可变数目的串联重复分析(multilocus variable number tandem repeats analysis,MLVA)。结果显示,在玉龙共捕获到49只野生鼠类,其中24只携带贝氏柯克斯体(48.98%,24/49)。基因分型结果显示阳性样本携带贝氏柯克斯体的MST型别可分为MST16和一种新的MST基因型别,但未能获得最终的MLVA基因型别。本研究表明云南玉龙的鼠类携带新的贝氏柯克斯体MST基因型别,可为我国Q热的溯源提供一定的理论依据和数据支撑。

检测贝氏柯克斯体的实时荧光定量PCR

目的采用新型TaqMan-MGB探针建立检测贝氏柯克斯体的实时荧光定量PCR(real-timequantitativePCR)方法。方法根据贝氏柯克斯体特异的23SrRNA插入基因序列设计引物和探针,以克隆的23SrRNA插入基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900型)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.997);与套式PCR相比较,荧光定量PCR检测敏感性是其100倍。用荧光定量PCR检测其它相关立克次体,检出结果均为0。用荧光定量PCR检测贝氏柯克斯体感染的小鼠脾脏标本,脾脏中贝氏柯克斯体的感染量与感染过程具有相关性。结论本研究建立的检测贝氏柯克斯体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合样本中微量贝氏柯克斯体DNA的检测,其定量检测可用于动物实验中的贝氏柯克斯体感染程度的分析。

多重PCR检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体

为了建立同步检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体感染的多重PCR方法,根据GenBank上具有属间特异性的布鲁菌.Bp26基因、鹦鹉热衣原体23S rRNA基因和贝纳氏柯克斯氏体IS1111a基因,利用Primer premier 5.0软件各设计1对特异性引物。通过优化PCR反应体系和扩增条件,建立了能够同时检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体的多重PCR方法。该方法具有较好的特异性和可重复性,对3种基因单重PCR检测敏感性均达到3.1×10~2拷贝·反应^-1,对3种病原基因同步检测的敏感性能达到3.1×10~3拷贝·反应^-1。利用该方法对采自不同流产牛的抗凝全血、血清、流产胎儿及奶液等172份临床样品进行检测,检测到布鲁菌感染阳性样品53份,鹦鹉热衣原体感染阳性样品2份,贝纳氏柯克斯氏体感染阳性样品10份,且检测到布鲁菌和鹦鹉衣原体混合感染阳性样品2份,布鲁菌和贝纳氏柯克斯氏体混合感染阳性样品2份,尚未检测到3种病原混合感染的阳性样品。结果表明,建立的多重PCR方法可以用来对奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体进行同步、快速、灵敏的检测。

氯仿-甲醇提取贝氏柯克斯体残存组分Q热疫苗的免疫原性及免疫保护性研究

目的实验评价氯仿-甲醇提取贝氏柯克斯体残存组分(CMR)疫苗的免疫特性及免疫保护性。方法采用国内分离贝氏柯克斯体新桥株制备CMR疫苗,用CMR免疫Balb/c小鼠;采用间接免疫荧光法检测CMR免疫小鼠血清特异性抗体水平和用淋巴细胞增殖试验评价CMR疫苗体外刺激小鼠脾细胞增殖的能力;以贝氏柯克斯体攻击免疫小鼠和用贝氏柯克斯体特异的荧光定量PCR检测感染小鼠血和脾脏样本。结果CMR免疫第4周起,小鼠血清中检出高水平的特异性抗体,CMR加氢氧化铝佐剂免疫小鼠的血清特异性抗体显著高于未加佐剂组。CMR在体外刺激正常小鼠和免疫小鼠脾淋巴细胞增殖水平显著高于对照组,WCV则抑制正常脾淋巴细胞增殖。三种剂量(30μg、10μg、1μg/只)CMR免疫小鼠血和脾脏样本贝氏柯克斯体含量显著低于未免疫组,以30μg组及加佐剂免疫小鼠的样本含菌量更显著低于对照组。结论采用我国分离株制备的CMR疫苗能诱导机体产生高效特异性体液免疫和细胞免疫应答,使机体抵抗大剂量的贝氏柯克斯体感染,具有良好的免疫原性和免疫保护性;氢氧化铝佐剂能显著增强CMR疫苗的免疫保护效能。

两种Q热疫苗的脂肪酸分析

目的比较分析灭活贝氏柯克斯体全细胞(WC)和氯仿-甲醇提取WC的残存组分(CMR)Q热疫苗的脂肪酸种类及含量差异。方法制备WC和CMR,采用高压气相色谱检测WC和CMR脂肪酸种类及相对含量。结果WC经氯仿-甲醇回馏处理后得到的不溶性组分CMR。气相色谱分析发现CMR较WC减少了11种脂肪酸,其中4种为不饱和脂肪酸,4种为支链脂肪酸,70%为C原子数≥18的较长链的脂肪酸。另外,两种疫苗的支链脂肪酸与直链脂肪酸、不饱和脂肪酸与饱和脂肪酸的比值也明显不同。结论灭活贝氏柯克斯体疫苗经氯仿-甲醇提取后,除去了它的多种脂肪酸或减少某些脂肪酸含量,这些脂肪酸的消除与减少可能是CMR疫苗诱导的局部和全身不良反应消失或减轻的主要原因。

Q热立克次体中国株的16S-23SrDNA间区序列分析

用PCR扩增了 7株中国分离株 (七医、新桥、雅安、李、YS 8、YS 9和YH 1 1 )和2株国际参考株 (Henzerling和Grita)的 1 6S 2 3SrDNA基因间区 ,对扩增产物进行了序列分析。结果发现它们仅在少数几个碱基位上有不同。在第 6 0位上 3个云南分离株YS 8、YS 9、YH 1 1和Grita株与Stein等报道的序列 (包括九里、其它国际参考株和一些法国临床分离株 )的碱基序列一致 ,为“C” ;其他 5株为“A” ,此位点的不同可能与适应不同的地理环境相关。另外 ,YS 9株在第 8和 2 0 8位、新桥株在第 43 2位也分别出现缺失 ,可能与适应不同的动物宿主或在实验室传代情况不同有关。

半套式PCR和DNA探针技术检测Q热立克次体

目的 建立半套式PCR和DNA探针技术由于检测Q热立克次体。方法 依据已知的Q热立克次体的 16S和 2 3SrRNA基因及其间区的序列 ,设计 3条引物 ,建立了扩增 16S 2 3SrRNA基因间区的半套式PCR方法 ,并将扩增片段制成探针 ,建立了斑点杂交技术。结果 10株Q热立克次体分离株均可扩增出 5 72bp的PCR条带 ,而对照菌都为阴性 ,此半套式PCR方法的灵敏度高达 1pg ;用九里株扩增片段标记后制成的探针 ,与 10个Q热立克次体分离株的全DNA呈阳性杂交 ,对照菌为阴性 ,此杂交方法的灵敏度为 0 .5ng。结论 基于Q热立克次体 16S 2 3SrRNA基因间区的半套式PCR和DNA探针技术具有较好的特异性和灵敏度 ,可联合应用于Q热的病原学诊断。