PCR诊断犬感染细粒棘球绦虫方法检出日期的确定

目的确定犬细粒棘球绦虫感染PCR检测的窗口期。方法家犬8只,口服喂饲细粒棘球绦虫原头节,感染后40 d,收集犬粪,用PCR方法检测粪便中的目的DNA。结果6只犬获得细粒棘球绦虫重度感染,感染后21 dPCR检出目的DNA片段。结论犬重度感染细粒棘球绦虫后粪便PCR检测的窗口期为20 d。     

基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立

目的建立一种安全、快速、高效的细粒棘球绦虫(Echinococcus granulosus)的诊断方法——环介导等温扩增方法(loop-mediated isothermal amplification,LAMP)。方法根据细粒棘球绦虫线粒体细胞色素c氧化酶亚基2(cytochrome c oxidase subunit 2,cox2)基因序列,设计4条LAMP引物,建立LAMP检测方法,采用LAMP法和常规PCR法检测细粒棘球绦虫、泡状带绦虫(Cysticercus tenuicollis)、扩展莫尼茨绦虫(Moniezia expansa)、羊曲子宫绦虫(Thysaniezia ovilla)、中点无卵黄腺绦虫(Avitellina centripunctata)、鞭毛线虫(flagella nematodes)、肝片吸虫(Fasciola hepatica)、多房棘球绦虫(E.multilocularis)和捻转血矛线虫(Haemonchus contortus)等9种寄生虫的DNA以验证LAMP法的特异性;分别用LAMP法和常规PCR法检测梯度稀释的含cox2基因片段的细粒棘球绦虫标准质粒后,比较两者的敏感性。采用LAMP、PCR和夹心ELISA法检测50份犬粪样,计算细粒棘球绦虫cox2基因的阳性率,评价所建立的LAMP法检测效果。结果LAMP法的特异性验证结果表明,仅在以细粒棘球绦虫DNA为模板的反应体系中出现特异性产物。LAMP法在细粒棘球绦虫标准质粒浓度为16.09 ag/μl时仍可见梯状条带,而常规PCR法仅可检测到浓度为16.9 fg/μl以上的质粒,LAMP法灵敏性是常规PCR法的1 000倍。对50份犬粪中细粒棘球绦虫DNA的检测结果显示,PCR、LAMP和夹心ELISA法检测结果相同,阳性率为8.0%(4/50)。结论基于细粒棘球绦虫线粒体cox2基因的LAMP检测方法操作方便、特异性较强、敏感性较高,适于细粒棘球绦虫感染犬的快速检测。     

基于线粒体ND6基因检测犬感染细粒棘球绦虫的粪便PCR方法

目的 旨在了解动物包虫病流行区内犬细粒棘球绦虫的感染情况。方法 本研究利用Qiagen DNeasy Powersoil试剂盒对犬粪提取DNA,并从细粒棘球绦虫线粒体全基因组中筛选出完整线粒体ND6基因作为靶基因,建立一种可对犬粪中细粒棘球绦虫同时进行检测及基因分型的PCR方法。结果 该法特异性很高,仅能扩增出细粒棘球绦虫的目的条带而对照组均无条带扩增。其灵敏度达到4 pg的DNA含量。当犬人工感染约50 000只原头蚴时,该法最早可以扩增出感染第13 d粪便中的ND6目的基因。同时,对40份随机采自包虫病流行区的待检犬粪进行PCR检测,共有6份样品可扩增出目的条带且其基因型均属G1型,其检测及分型结果均与经典基因分型依据(COX1基因片段)的结果一致。结论 本研究建立的粪便PCR方法具有很高的特异性和灵敏度,并可用于检测犬的细粒棘球绦虫早期感染情况。对于PCR检测阳性者,其产物经测序分析后也可用于细粒棘球绦虫的基因分型及种群遗传结构的分析研究。     

家犬粪便中的细粒棘球绦虫DNA PCR检测方法的建立

目的 建立检测家犬粪便中细粒棘球绦虫DNA的PCR方法，为开展流行病学监测提供检测手段。方法 犬粪经液氮反复冻溶后提取DNA，PCR扩增编码细粒棘球绦虫12srDNA检测该靶DNA片段（255bp）。结果 10只细粒棘球绦虫感染犬，8只（体内成虫数〉80条）阳性，2只（体内成虫数分别为4条和5条）阴性。4只未感染家犬粪样及其他3种绦虫DNA样本均为阴性。结论 初步建立了灵敏、特异的检测家犬粪便中细粒棘球绦虫的PCR方法。     

环介导等温扩增技术(LAMP)检测棘球绦虫感染犬粪DNA的研究

目的 建立快速检测棘球绦虫感染犬粪便的DNA检测方法一环介导等温扩增技术(loop-mediated isothermal amplification.LAMP).方法 针对细粒棘球绦虫线粒体DNA Cox1基因6个位点特异性的设计4条LAMP引物,利用LAMP法和普通PCR方法同时检测棘球绦虫、肥胖带绦虫以及犬肠道内...     

环介导等温扩增技术(LAMP)检测棘球绦虫感染犬粪DNA的研究Ⅱ

目的 为有效地控制预防流行区犬科动物的棘球绦虫感染情况,建立一种快速检测棘球绦虫感染犬的粪便DNA检测方法—环介导等温扩增技术(loop-mediated isothermal amplification. LAMP)。方法 针对细粒棘球绦虫线粒体DNA ND2基因6个位点特异性的设计4条LAMP引物,利用LAMP法和普通PCR方法同时检测棘球绦虫、肥胖带绦虫以及犬肠道内的其它寄生虫DNA来验证LAMP方法的特异性;将转入ND2基因片段的质粒作为标准阳性对照,并做梯度稀释后同时用 LAMP 和 PCR 方法进行检测比较两者的敏感性。此外,将采集的46份犬粪便标本提取DNA,分别运用LAMP和犬尸体剖检进行感染犬的初步检测评估。结果 E.g mtDNA ND2基因的LAMP引物能够鉴别多房棘球绦虫和细粒棘球绦虫这两个相近的种属,并不与其它待检寄生虫发生交叉反应,且最低检测限度为4×10¹拷贝(灵敏度比普通PCR高出10³倍)。在初步对46份犬粪便DNA检测结果中 ND2 LAMP 引物显示出较好的灵敏度和特异度,χ²检验结果该方法与犬尸体剖检方法的诊断效果无统计学差异。结论 本研究初步建立了LAMP技术检测细粒棘球绦虫感染犬的新方法,在各项特异度、灵敏度和样本检测中展现出较好的效果。该方法不需要昂贵的仪器设备和繁琐的电泳分析过程,有望成为流行区和基层兽医站细粒棘球绦虫感染犬检测的新方法。     

粪抗原检测法诊断犬细粒棘球绦虫感染的研究Ⅲ.双抗体夹心ELISA法检测粪抗原诊断家犬细粒棘球绦虫感染的现场评价

本文报道用槟榔碱驱虫法对检测粪抗原诊断家犬细粒棘球绦虫感染的应用价值进行现场比较评价的结果.73只受试犬中有16只对槟榔碱无反应.在导泻成功的57只犬中粪抗原阳性者11只阳性率19.2%.其中驱虫阴性的38只犬中粪抗原阳性者仅2只(5.3%).在10只驱出细粒棘球绦虫的家犬中8只粪抗原阳性.5只驱出泡状带绦虫的犬中粪抗原阳性者1只,但反应强度低.粪抗原的反应强度与细粒棘球绦虫的感染强度呈正相关(r=0.94).证明本法可用于家犬细粒棘球绦虫感染的调查.     

粪抗原检测法诊断犬细粒棘球绦虫感染的研究Ⅲ.双抗体夹心ELISA法检测粪抗原诊断家犬细粒棘球绦虫感染的现场评价

本文报道用槟榔碱驱虫法对检测粪抗原诊断家犬细粒棘球绦虫感染的应用价值进行现场比较评价的结果。７３只受试犬中有１６只对槟榔碱无反应。在导泻成功的５７ 只犬中粪抗原阳性者１１只阳性率１９．２％ 。其中驱虫阴性的３８只犬中粪抗原阳性者仅２只（５．３％ ）。在１０只驱出细粒棘球绦虫的家犬中８只粪抗原阳性。５ 只驱出泡状带绦虫的犬中粪抗原阳性者１ 只,但反应强度低。粪抗原的反应强度与细粒棘球绦虫的感染强度呈正相关（ｒ＝ ０．９４）。证明本法可用于家犬细粒棘球绦虫感染的调查。     

现场应用粪抗原检测法诊断犬细粒棘球绦虫感染的效果评价

目的 评价粪抗原检测法在包虫病流行病学监测中的实际应用价值。方法 家犬在采集粪便后，进行槟榔碱导泻，应用粪抗原检测法检测粪样中的细粒棘球绦虫抗原，并与槟榔碱导泻法的驱虫结果比较，同时分析犬肠道中寄生的其它蠕虫对粪抗原检测结果的影响。结果 在294只槟榔碱导泻成功的家犬中，45只检出细粒棘球绦虫，粪抗原阳性犬共46只。二者的符合率为97．8％。在249只未检出细粒棘球绦虫的家犬中，粪抗原阳性者1只。结论 粪抗原检测法诊断犬细粒棘球绦虫感染具有较高的敏感性和特异性。可作为包虫病常规监测方法推广应用。     

现场应用粪抗原检测法诊断犬细粒棘球绦虫感染的效果评价

目的　评价粪抗原检测法在包虫病流行病学监测中的实际应用价值。　方法　家犬在采集粪便后 ,进行槟榔碱导泻 ,应用粪抗原检测法检测粪样中的细粒棘球绦虫抗原 ,并与槟榔碱导泻法的驱虫结果比较 ,同时分析犬肠道中寄生的其它蠕虫对粪抗原检测结果的影响。　结果　在 2 94只槟榔碱导泻成功的家犬中 ,4 5只检出细粒棘球绦虫 ,粪抗原阳性犬共 4 6只。二者的符合率为 97.8%。在 2 4 9只未检出细粒棘球绦虫的家犬中 ,粪抗原阳性者 1只。　结论　粪抗原检测法诊断犬细粒棘球绦虫感染具有较高的敏感性和特异性。可作为包虫病常规监测方法推广应用。     

Specific antibody responses in dogs experimentally infected with Echinococcus granulosus

Six dogs reared helminth-free were divided into 2 groups. Four dogs were infected per os with 200,000 protoscoleces each of Echinococcus granulosus and 2 were kept as uninfected controls. All the dogs were kept together until 32 days after infection, when 1 infected dog was killed, its intestine removed and the contents examined to confirm that the infection with E. granulosus had been successful. The remaining 3 infected dogs were transferred to high security housing and their feces inspected daily to establish the time infections became patent. The infected and control dogs were bled every 5 days for 75 days from the time of infection and the sera were stored at -70 degrees C. Sera were tested by the enzyme-linked immunosorbent assay (ELISA) for antibodies to E. granulosus scolex excretory/secretory (ES) antigen, protoscolex antigen and oncosphere antigen. Antibodies to scolex ES antigen and protoscolex antigen were detected in the sera of infected dogs within 2 weeks of infection. Antibody titers rose rapidly and remained at a high level until the dogs were killed 75 days after infection. Antibodies in these sera did not cross react with antigens prepared from Taenia ovis, T. hydatigena, T. pisiformis, Ancylostoma caninum, Trichuris vulpis and Toxocara canis.     

小鼠感染细粒棘球蚴早期TGF-β1表达分析

目的观察细粒棘球蚴早期感染BALB/c小鼠TGF-β1的表达情况。方法用细粒棘球蚴感染BALB/c小鼠,分别于感染后第1、3、5、7、9、12d处死,取其脾细胞和外周血,采用酶联免疫法检测外周血细胞因子TGF-β1的含量,采用qRT-PCR法检测TGF-β1基因的表达量。结果实验鼠Eg感染后第1~12d,外周血TGF-β1的含量为2 695.79~3 055.09ng/ml,与对照组比较差异有统计学意义(P<0.05);TGF-β1mRNA相对表达量为0.029~0.060,与对照组比较差异有统计学意义(P<0.05)。TGF-β1及其mRNA表达量均有随着感染时间延长呈增高的趋势。结论TGF-β1在小鼠细粒棘球蚴感染早期表达增加,可能有利于细粒棘球蚴的免疫逃逸。     

Copro-diagnosis of Echinococcus granulosus infection in dogs by amplification of a newly identified repeated DNA sequence

Diagnosis of Echinococcus granulosus infection in dogs by detecting adult worms recovered post mortem or purged from the intestines after treatment with arecoline is not suitable for mass screening. Large-scale diagnosis by detection of copro-antigens is useful but only with relatively high intensity infections, and only by genus. To provide a more sensitive and specific diagnosis, a polymerase chain reaction (PCR) assay was developed, that amplified a target repeated sequence (EgG1 Hae III) newly identified in the genome of the common sheep strain of E. granulosus. This repeated sequence consists of approximately 6,900 copies, arranged in tandem, in groups of 2-6 repeats. The corresponding primers used in the PCR easily detected a single egg with no cross-amplification of DNA from closely related cestodes, including E. multilocularis and Taenia spp. Fecal samples from naturally infected dogs, with 2-10,000 E. granulosus worms at necropsy, were all PCR positive, while E. multilocularis or Taenia spp. positive controls as well as non-endemic controls were all PCR negative. This copro-PCR assay was demonstrated to be 100% specific and also detected all necropsy-positive E. granulosus-infected dogs. It is suggested that this copro-PCR assay has the potential for pre-mortem diagnosis of E. granulosus infection even in areas where E. granulosus and E. multilocularis are co-endemic.     

The epidemiology of Echinococcus granulosus in Great Britain. V. The status of subspecies of Echinococcus granulosus in Great Britain

Twenty-five years ago Williams and Sweatman suggested that in Great Britain there are two subspecies of Echinococcus granulosus--E. granulosus granulosus and E. granulosus equinus. Echinococcus granulosus granulosus does not mature either in foxes or in horses: E. granulosus equinus will mature in either. The prepatent period of E. granulosus granulosus in the definitive host is about 42 days while that of E. granulosus equinus is about 70 days. Each subspecies has a characteristic morphology. More recently, in the course of seven experiments, dogs, red foxes (Vulpes vulpes crucigera), arctic foxes (Alopex lagopus), badgers (Meles meles), domestic ferrets and domestic cats have been infected with protoscoleces derived from hydatid cysts of human, equine and ovine hosts from different regions of England and Wales. Transmissions to horses and sheep were always succeeded by the development of viable hydatid cysts; transmissions to dogs and foxes, by the development of gravid adults. The prepatent period in both dogs and foxes was invariably about 70 days (or longer) and the morphology of all the adult parasites in these definitive hosts was characteristic of E. granulosus equinus. All the evidence derived from these experiments supports the view that there is, in Great Britain, but one subspecies of E. granulosus. That one subspecies is E. granulosus equinus. It infects dogs, red foxes, arctic foxes (experimentally), cats (experimentally), humans, sheep, goats, horses, donkeys, pigs, cattle, roe deer and reindeer (in Scotland). Attempts to transmit E. granulosus to badgers and domestic ferrets were unsuccessful. Of 123 cats infected with protoscoleces of horse origin, one gravid adult parasite was recovered from one animal.     

Evaluation of three PCR assays for the identification of the sheep strain (genotype 1) of Echinococcus granulosus in canid feces and parasite tissues

The performance of 3 PCR assays for the identification of the G1 sheep genotype of Echinococcus granulosus was evaluated using tissue and canid fecal samples. The "Dinkel" and "Stefani?" primers were the most sensitive in detecting E. granulosus DNA in feces of necropsied dogs (73.7% and 100%, respectively). The "Abbasi" primers detected 52.6% of E. granulosus infected dogs but were the most species-specific, cross-reacting only with Echinococcus shiquicus (tissue 90.9%; feces 75%). The Stefani? primers were the least specific (tissue, 27.3%; feces, 25%) for E. granulosus. The Dinkel primers also showed inter-species cross-reactivity (tissue, 63.6%; feces, 100%) but were found to be strain-specific for the E. granulosus G1 sheep genotype. Improvement of PCR tests for Echinococcus species and subspecific variants should rely on the use of less-conserved genes and development of protocols that improve the quality and quantity of DNA extracted from feces.     

Identification of protein components of Echinococcus granulosus protoscolex antigens for specific serodiagnosis of E. granulosus infection in dogs

Proteins of Echinococcus granulosus protoscolex excretory/secretory or deoxycholate solubilized somatic antigens were radiolabelled with 125I and immunoprecipitated with sera from dogs naturally or experimentally infected with E. granulosus and various control dog sera. Analysis of immunoprecipitates was performed using one- and two-dimensional gel electrophoresis to identify antigenic protein components specific for E. granulosus. Using both electrophoretic techniques, a basic component of Mr 27,000 and an acidic component of Mr 94,000 were defined in both excretory/secretory and somatic protoscolex antigens, and were specifically identified by 95% and 62% of 21 sera from E. granulosus-infected dogs, respectively. An abundant component of Mr 35,000 was identified by 100% of these dogs, parts of which were E. granulosus specific. Results of this study should allow identification of specific recombinant antigens for routine serodiagnosis of E. granulosus infection in dogs.