湖南目平湖钉螺血吸虫病原生物控制资源调查及感染试验

目的虽然一种贝类可作数种吸虫的中间宿主,但在各吸虫种类都存在的一自然环境中,它们共同贝类宿主的一个体通常只携带一种吸虫幼虫期。作者利用这一规律在湖南汉寿目平湖血吸虫病区进行媒介钉螺的调查,查到钉螺感染有日本血吸虫(Schistosoma japonicum)、外睾类吸虫(Exorchis)、斜睾类吸虫(Plagiorchis)、侧殖类吸虫(Asymphylodora)和背孔类吸虫(Notocotylus)5种吸虫的幼虫期。其中对生物控制媒介钉螺有利用价值的外睾类吸虫,其钉螺感染率为3.298%(96/2911),终宿主鲶鱼(Parasilurus asoyus)的感染率为99.31%(434/437)、平均每尾鲶鱼的感染强度为115.4条。先用外睾类吸虫虫卵感染钉螺后再感染日本血吸虫毛蚴,切片观察不同时间的实验螺,后侵入的血吸虫幼虫,全部受损害不能发育。     

钉螺感染目平外睾吸虫的分泌物及其对杀灭不同时间再感染日本血吸虫幼虫的进一步观察

目的 湖北钉螺(Oncomelania hupensis)先感染目平外睾吸虫(Exorchis mupingensis)虫卵后,再间隔不同时间感染日本血吸虫(Schistosoma japonicum)毛蚴,观察螺体分泌物的强度对血吸虫幼虫损害和被杀灭情况的关系。方法 钉螺感染目平外睾吸虫后分别于21 d、37 d、55 d、70 d和85 d再感染血吸虫毛蚴。钉螺经双重感染后4-82 d, 作钉螺整体连续埋蜡切片、染色制片和全片观察,并记录血吸虫幼虫残体数。结果与结论 单独感染外睾吸虫的钉螺,和两吸虫感染间隔时间为21-85 d的钉螺,螺体都产生大量血淋巴细胞和分泌物,它们会围攻再侵入的日本血吸虫早期幼虫并侵入其体内,血吸虫幼虫结构发生异常、停止发育直至死亡。两种吸虫双重感染的间隔时间愈长,螺体血淋巴细胞数随时间增加而逐渐减少;而螺体分泌物不断增多,并见于血吸虫幼虫残骸内,螺体攻击血吸虫幼虫的效力愈强。这现象在单独感染日本血吸虫的钉螺体内未见到。     

日本血吸虫幼虫在钉螺及感染外睾吸虫钉螺发育的比较

湖北钉螺先感染外睾吸虫后再感染日本血吸虫,血吸虫幼虫在其体内被击杀(唐崇惕等,2008;2009),本文用日本血吸虫毛蚴分别接触已感染外睾吸虫21d的钉螺和阴性钉螺,观察血吸虫幼虫在此2组实验钉螺体内发育情况。从感染外睾吸虫钉螺再感染血吸虫的28个(73.7%)阳性螺,查获4～82d血吸虫幼虫共300条(侵入率26.74%),全部虫体结构异常停留在早期母胞蚴阶段。从单独感染血吸虫的25粒(69.4%)阳性螺,查获5～61d正常血吸虫母胞蚴67条(侵入率13.96%)和许多不同发育期子胞蚴,感染后75d阳性螺含血吸虫成熟子胞蚴和尾蚴。单独感染血吸虫钉螺的血淋巴细胞增生情况与双重感染外睾吸虫和血吸虫的钉螺存在差异。     

基于COI基因片段的拟钉螺种类鉴定初探

目的探讨COI基因片段在拟钉螺种类鉴定中的作用。方法收集来自福建省内多个地区的疑似拟钉螺标本,用形态学方法进行物种鉴定后,取出螺肉提取基因组DNA,通过PCR的方式扩增COI基因序列片段并测序,通过序列比对及系统进化分析鉴定其物种。结果共采集到标本13份,通过形态学方法将样本鉴定为福建境拟钉螺属。所有标本经实验扩增到长度为515~598bp的COI基因片段。13份标本中11株的COI基因片段与γ拟钉螺属最为相似(88.96%~97.82%),另外2株分别与拟钉螺属下的武鸣拟钉螺(87.08%)及新拟钉螺属下的开放新拟钉螺(88.55%)最为相似。在属水平上,13份标本中仅有1份的形态学鉴定结果与基因鉴定结果吻合,其余12份样本的形态学鉴定结果均与基因鉴定不同,两种方法的鉴定结果存在较大差异。结论通过COI基因检测可快速对拟钉螺亚科作出准确判断,但对属水平的鉴定的准确性还需要进一步的研究。     

检测日本血吸虫感染性钉螺PCR方法的建立

目的 建立灵敏、特异的检测日本血吸虫感染性钉螺的PCR方法。 方法 根据日本血吸虫18S小亚基单位核糖体核酸（18S-rRNA）基因设计1对引物，建立检测日本血吸虫感染性钉螺的PCR方法。测定PCR产物的DNA序列，稀释血吸虫毛蚴DNA进行PCR方法的灵敏性试验，扩增单尾尾蚴感染的钉螺DNA进行交叉反应试验，并根据不同稀释度的感染性钉螺DNA的扩增结果来验证PCR的群体检测效果。 结果 PCR扩增日本血吸虫感染性钉螺，得到了与靶DNA片段位置相同的产物，测序片段长度为469 bp，与靶DNA相同且序列一致，并将序列登录GenBank(注册号：DQ442999)。扩增阴性钉螺无产物出现。灵敏性试验提示，PCR可检出日本血吸虫毛蚴DNA的最低浓度为40 pg/μl；交叉反应试验显示，PCR方法不能扩增出单尾尾蚴感染钉螺的DNA；群体检测试验表明，PCR可检出感染性钉螺提取的DNA最高稀释度为1∶640。 结论 初步建立的检测日本血吸虫感染性钉螺的PCR方法灵敏、特异且具有良好的群体检测效果。     

安徽省宁国市有螺无病地区钉螺对日本血吸虫易感性的研究

目的研究安徽省有钉螺无血吸虫病流行地区(简称"有螺无病地区")钉螺对日本血吸虫的易感性,为制定该类地区血吸虫病防治策略提供依据。方法采集宁国市有螺无病地区钉螺400只,随机分为2组,每组200只,以本省日本血吸虫毛蚴在实验室内进行钉螺群体感染,两组钉螺与毛蚴比例分别为1∶20和1∶40,感染时间为4小时,感染时的温度为24～28℃。以同样方法感染该省血吸虫病流行区宣城市有螺有病地区钉螺作为对照。感染后将钉螺置于室内常温下饲养60天,观察有螺无病地区和有螺有病地区钉螺感染率和死亡情况。结果在1∶20(钉螺/毛蚴)组和1∶40(钉螺/毛蚴)组中,有螺无病地区钉螺均未能成功感染日本血吸虫毛蚴,而有螺有病区钉螺感染率分别为5.89%(2/51)和16.67%(7/42),且两组中有螺有病区钉螺感染率的差异具有统计学意义(χ2=4.15,P<0.05)。观察期结束时,在1∶20(钉螺/毛蚴)组中,有螺无病地区钉螺与有螺有病地区钉螺死亡率分别为77%(154/200)和74.5%(149/200),两地钉螺死亡率无显著性差异(χ2=0.661,P>0.05);在1∶40(钉螺/毛蚴)组中,两地钉螺死亡率分别为81%(162/200)和78%(156/200),死亡率差异无统计学意义(χ2=0.507,P>0.05)。结论尽管本研究中有螺无病地区钉螺未能成功感染日本血吸虫毛蚴,但今后仍需加强有螺无病地区输入性血吸虫病传染源的监测工作。     

叶巢外睾吸虫感染钉螺对钉螺体内日本血吸虫发育的影响

目的　探讨在日本血吸虫中间宿主湖北钉螺体内叶巢外睾吸虫和日本血吸虫的对抗性。　方法　通过叶巢外睾吸虫和日本血吸虫对湖北钉螺的双重感染 ,计算血吸虫的感染率和尾蚴逸出量 ,常规石蜡切片观察钉螺组织学变化。　结果　钉螺在感染血吸虫 37d后再感染外睾吸虫 ,经一定时间后检查发现 ,血吸虫的感染率为 5 2 9% ,显著低于同时单独感染日本血吸虫对照组的感染率 (75 9% ) ;先感染叶巢外睾吸虫 ,经 10、 32、6 0、 10 0和 12 0d不同的时间间隔后再感染日本血吸虫的钉螺 ,血吸虫感染率分别为 6 4 %、 6 6 7%、 6 5 2 %、5 6 4 %和 5 7 1% ,而单独感染日本血吸虫对照组钉螺血吸虫感染率为 90 5 % ,经统计检验 ,各双重感染实验组血吸虫与对照组的感染率间差异具有显著或非常显著性意义 (P <0 0 5或P <0 0 1)。对各试验组及对照组钉螺逸出尾蚴试验发现 ,试验组钉螺血吸虫尾蚴逸出量均显著低于对照组的逸出量。组织学观察发现各双重感染组钉螺消化腺萎缩 ,消化腺盲囊间隙只有少量血吸虫子胞蚴 ,血吸虫子胞蚴皱缩、变形及不规则 ,且子胞蚴中只含稀疏的尾蚴胚球 ,有的子胞蚴中已无胚球 ;而单独感染血吸虫的对照组中血吸虫均发育到成熟的子胞蚴或尾蚴。　结论　钉螺体内叶巢外睾吸虫和日本血吸虫之间存     

重组酶聚合酶扩增技术快速检测日本血吸虫核酸方法的建立

目的应用重组酶聚合酶扩增(RPA)技术,建立一种敏感、特异、简便且快速的日本血吸虫核酸检测方法。方法选择Sj28S核糖体基因片段为靶序列,用Primer Premier 5软件设计特异性引物,建立RPA扩增反应,并进行优化以确定最佳反应条件。提取不同虫期日本血吸虫,以及曼氏血吸虫、埃及血吸虫、单尾尾蚴感染钉螺、华支睾吸虫、大片形吸虫、卫氏并殖吸虫和牛带绦虫等基因组DNA,评价所建立方法的特异性和敏感性,并对不同混合比例(1∶10、 1∶50、 1∶100、 1∶250、 1∶500、 1∶1 000、 1∶2 000)的阳性和阴性钉螺基因组DNA的检出性能和重复性进行评价。结果建立的RPA方法可特异地扩增出日本血吸虫216 bp大小的目的基因片段。RPA的最佳反应条件为39℃、 20 min。该方法与曼氏血吸虫、埃及血吸虫、单尾尾蚴感染钉螺、华支睾吸虫、大片形吸虫、卫氏并殖吸虫和牛带绦虫及阴性钉螺基因组DNA均无交叉反应。针对日本血吸虫成虫基因组的最低检出限为100 fg/μl,针对重组质粒的最低检出限为100拷贝/μl,且血吸虫不同虫期的基因组DNA样本均能被准确检出。应用建立的RPA方法检测不同混合比例钉螺DNA混合样品,结果显示,最低检出比例为1∶1 000。重复性试验结果显示,5次检测结果完全一致,无假阴性和假阳性。结论建立了日本血吸虫RPA检测方法 ,该法敏感性高、特异性好、简便快速,有望用于血吸虫感染的快速检测及风险监测。     

基于重组酶聚合酶扩增的曼氏血吸虫核酸可视化检测技术的建立及初步评价

目的 结合重组酶聚合酶扩增技术(RPA)和侧流层析试纸条(LFD)，建立一种快速、便捷的曼氏血吸虫核酸可视化检测方法，并初步评价其检测效能。方法 以曼氏血吸虫细胞色素c氧化酶亚基1(SmCOX1)基因为靶序列，利用Primer Primer 5软件结合手工辅助，设计特异性引物和探针并进行筛选，建立曼氏血吸虫核酸LFD-RPA检测方法。制备不同浓度(1 ng/μl、100 pg/μl、10 pg/μl、1 pg/μl、100 fg/μl、10 fg/μl、1 fg/μl、0.1 fg/μl)的曼氏血吸虫成虫基因组DNA和含有不同拷贝数浓度(10~5、10~4、10~3、10~2、10~1、10~0、10^-1拷贝/μl)的SmCox1重组质粒DNA，评价所建立方法的敏感度。以曼氏血吸虫成虫、日本血吸虫成虫、埃及血吸虫虫卵、感染曼氏血吸虫双脐螺(阳性双脐螺)和阴性双脐螺、感染日本血吸虫钉螺(阳性钉螺)和阴性钉螺、华支睾吸虫成虫、大片形吸虫成虫、卫氏并殖吸虫成虫基因组DNA为模板，评价所建立方法的特异性。将30只雌性BALB/c小鼠随机分为40尾感染组、80尾感染组和健康...     

血水草生物碱杀螺、灭蚴作用的研究

目的研究血水草生物碱(ECA)杀灭钉螺及日本血吸虫尾蚴的作用。方法观察实验室及现场浸泡杀螺效果;钉螺接触药物上爬情况;浸泡杀螺卵效果;浸杀尾蚴防护血吸虫感染的作用。结果浸泡72h钉螺死亡率100％的血水草生物碱溶液的浓度及温度分别为:1.25mg/L,30℃;2.5 mg/L、5 mg/L、10 mg/L均为25℃。水温 26℃～28℃现场灭螺10mg/L,72h钉螺死亡率84％;20 mg/L,72h死亡率达92％。5mg/L溶液螺卵浸泡72h孵出率为0％～10％。5mg/L ECA溶液明显抑制钉螺上爬,20mg/L时钉螺静止不动。结论血水草有杀灭钉螺、螺卵及日本血吸虫尾蚴的作用,是一种有研究前景的植物杀螺、灭蚴剂。     

异盘并殖吸虫第一中间宿主的发现

在广西那坡县异盘并殖吸虫流行区，从采集的拟钉螺（种名待定）体内首次检获子雷蚴和尾蚴，感染率为０．１１％（４／３５３０）。经用异盘并殖吸虫毛蚴感染正常的拟钉螺获得成功，其子雷蚴和尾蚴与自然感染者一致，证实该地拟钉螺是该虫自然界第一中间宿主。     

Study on Schistosoma sinensium in Fang district, Chiangmai province, Thailand

A survey on human infection and possible natural definitive host of Schistosoma sinensium was carried out in Fang District, Chiangmai Province, North Thailand, where Tricula bollingi snails which harbour cercariae of S. sinensium inhabits. Stool examination of the people in the two villages along the stream, where T. bollingi were found, was by formalin-ether concentration technique and by Stoll's method. The stools were found to be negative for S. sinensium eggs. Field rats were also trapped and examined for the presence of S. sinensium. Adult worms and eggs of S. sinensium were found in the mesenteric veins and livers, respectively, of the field rats, Rattus rattus, captured in the rice fields along the stream.     

基于18S rDNA基因序列探讨几种食源性吸虫系统发生关系

目的 研究几种食源性吸虫间的系统发生关系.方法 PCR扩增华支睾吸虫和东方次睾吸虫18S rDNA片段并测序,从GenBank检索其他食源性吸虫和日本血吸虫的18S rDNA序列,利用CLUSTAL X软件比对后,用MEGA 5.0软件计算食源性吸虫间的遗传距离,并用邻接法、最大似然法和最大简约法构建系统发生树.结果 共获得12种食源性吸虫序列,12种吸虫间遗传距离从0.001到0.083,系统发生树显示后睾科与异形科先聚在一起,然后与并殖科和双腔科聚成一支,片形科单独聚成一支.结论 后睾科与异形科有较近的亲缘关系.

Abstract：

Objective To discuss the phylogeny of several food-borne trematodes.Methods The 18S rDNA partial sequences of Clonorchis sinensis and Metorchis orientalis were specifically amplified by PCR and then sequenced,the corresponding sequences of other food-borne trematodes were obtained from GenBank.After aligned by CLUSTAL X program all sequences were used to calculate the genetic distance and construct the phylogenic trees using neighbor-joining method,maximum parsimony method and maximum likelihood method in MEGA5.0 program.Results The 18S rDNA panial sequences of 12 species of food-bome trematode were obtained.The genetic distances among 12 trematodes were from 0.001 to 0.083.phylogenic trees showed that Opisthorchiidae and Hererophyidae polymerize first,then form a clader with Paragonimidae and Dicrocoeliidae,Fasciolidae form another clader.Conclusion Opisthorchiidae is closer to Hererophyidae in evolution.     

中国不同种拟钉螺CO1基因序列差异分析及其系统学初探

目的研究中国拟钉螺线粒体CO1基因的差异并初步探讨其系统发生。方法收集4省7地拟钉螺标本,抽提基因组DNA,PCR扩增线粒体CO1基因,扩增产物纯化后进行序列测定,所测序列用Kimura双参数法计算遗传距离,NJ和UPGMA法构建系统进化树。结果PCR扩增得到大小约700bp的片段。遗传距离显示:台湾邱氏拟钉螺与湖北钉螺滇川亚种亲缘关系最近距离为0.124,而与其余6种拟钉螺遗传距离较远,遂将其分为两组。6种拟钉螺组内距离为0.127,两组间距离为0.179。两种方法构建进化树拓扑结构基本一致。台湾邱氏拟钉螺与湖北钉螺滇川亚种聚为一支,其余6种拟钉螺位于另一支。结论台湾邱氏拟钉螺应归入湖北钉螺,钉螺与拟钉螺属单源进化。中国不同种拟钉螺CO1基因存在差异。     

滇西横断山区家畜体表蜱类调查及鉴定

目的 调查滇西横断山区家畜体表蜱的种类。方法 采集家畜体表寄生的蜱虫,经形态学鉴定后,用PCR法扩增蜱虫样本的16S rRNA、12S rRNA、COI、ITS2的基因片断,测序后进行序列分析。结果 共采集成虫蜱样本1 874只,经形态学鉴定为1科、1属(扇头蜱属Rhipicephalus)、4种,其中微小扇头蜱(R. microplus)1 637只(占87.35%)),镰形扇头蜱(R. haemaphysaloides)218只(11.63%),短小扇头蜱(R. pumilio)11只(0.59%),血红扇头蜱(R. sanguineus)8只(0.43%)。样品分子鉴定结果与形态学鉴定结果一致。系统发育树分析显示,微小扇头蜱Y2 16S rRNA、12S rRNA、COI、ITS2的基因序列分别与来自印度(EU918188)、贵州(KC503259)、马来西亚(KM246873)、贵州(KC503274)的微小扇头蜱在同一分支上,而与以往云南发现的微小扇头蜱不在同一分支;镰形扇头蜱Y5的 16S rRNA、12S rRNA和COI基因序列分别与来自泰国(KC170743)、台湾(DQ003005)和湖南(KM083593)的镰形扇头蜱在同一分支上;短小扇头蜱Y6和Y01 的ITS2基因序列与来自澳大利亚的短小扇头蜱(AF271282)在同一分支上。结论 滇西横断山区家畜体表蜱以微小扇头蜱为优势种,短小扇头蜱为云南境内首次发现。     

Lymnaea stagnalis (Linnaeus, 1758) snails' infection to trematoda larval stages in Shahrekord city's springs

ObjectiveTo determine the Lymnaea stagnalis snails' infection with trematodes larval stage in one of the springs of the Shahrekord city in Chahar Mahal and Bakhtiari.MethodsTo determine the snail infection to trematodes larval stages, the snails were caught from the field, and transferred to the Parasitology Department of Razi Vaccine and Serum Research Institute. Then stimulating of snails by light, tubing and squashing of them were used for detection and identification of the isolated cercariae.ResultsOf 400 collected snails from the referred springs, 350 of them identified as Lymnaea stagnalis. Observed cercariae were identified and classificated as order Plagiorchis, family Plagiorchiidae and genus Opisthioglyphe and Plagiorchis.ConclusionsIn Chahar Mahal and Bakhtiari Province, due to having more than 10% of water content of country, ecological conditions can play important role to develop sensitive snail especially Lymnaeidae and be considered as a critical and suitable habitat for them.     

Cercarial infection in Paludomus petrosus,freshwater snail in Pa La-U Waterfall

Paludomus petrosus, the freshwater snails found in Pa La-U Waterfall, were examined for cercarial infection of trematodes. The snails were collected every other month from April, 2001 to February, 2002. Collections were taken from two sampling stations. The counts per unit of time' method was used for collection of the snails. The density of snails was highest in June 2001, and the highest of parasite infection rate was in February 2002. Four types of cercariae were found in the snails: Xiphidiocercariae, Amphistome, Furcocercous cercariae type I, and Furcocercous cercariae type II. Xiphidiocercariae were found in April 2001 to February 2002. Amphistome, Furcocercous cercariae type I and Furcocercous cercariae type II were found in February 2002.     

Scanning electron microscopic study of the tegumental surface of the hybrid schistosome between Schistosoma mekongi and S. japonicum-like (Malaysian)

Hybridization experiments between the two non-sibling species of schistosomes, Schistosoma mekongi in man and S. japonicum-like (Malaysian) in rodents, were carried out. Two laboratory-bred snail species, Tricula aperta (beta race), the snail host of S. mekongi and Robertsiella kaporensis, the snail host of S. japonicum-like (Malaysian), were used for the production of cercariae. Cross mating between S. mekongi and S. japonicum-like (Malaysian) were achieved in the laboratory by the usual procedure of exposing snails to single miracidia of each species, then exposing mice to cercariae emanating from two snails only, each infected with a different species. Hybrid eggs and miracidia were used to infect snails of both species. The resultant F1 cercariae were used to infect mice. It was shown in this study that the attempt to cross these two species of schistosomes could be achieved in the laboratory, but the results provided very low yield of hybrid worms and eggs. F1 hybrid adult worms from S. mekongi male and S. japonicum-like (Malaysian) female were obtained and examined for the microtopography of the tegument by scanning electron microscopy. The tegumental surface of the hybrid male schistosome resembled the male parent, S. mekongi, with a few characters which resembled the male, S. japonicum-like (Malaysian). The surface tegument of the hybrid male worm was characterized by the presence of highly-branched and perforated ridges interspersed with a large number of papillae all over the body surface with the heaviest concentration on the middle portion of the body. There were four types of papillae present; the pleomorphic papillae; the cratered papillae, with or without cilia; the hemispherical sensory papillae with cilia; and the fungiform papillae. Spines were absent on the body surface except in the oral and ventral suckers and in the gynecophoral canal. The tegument lining the gynecophoral canal was characterized by the presence of low ridges with scattered papillae with small number of short spines in the posterior portion of the canal. In contrast to the male, the female hybrid worm had numerous spines all over the body surface with the most concentration in the posterior region. Among the spines were low perforated ridges. Two types of papillae were present in the female hybrid; the cratered papillae, with or without cilia, and the hemispherical papillae.     

Identification of snails infected with schistosomes by ELISA employing monoclonal antibodies: Schistosoma mansoni in laboratory snails (Biomphalaria glabrata) and in field snails (Biomphalaria pfeifferi) from Kenya

An enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibodies was used for detecting Schistosoma mansoni antigens in hemolymph of laboratory snails (Biomphalaria glabrata) in Kenya. Infected laboratory snails shedding cercariae were differentially identified by ELISA from uninfected snails with 100% sensitivity and specificity. Prepatent infections were detected by ELISA from 2 weeks after exposure to miracidia. Thus, ELISA revealed infection 3 weeks before maximal patency was reached (5-6 weeks post-exposure). Infected field snails (B. pfeifferi) shedding cercariae were differentially identified by ELISA, with 100% sensitivity and specificity, from uninfected field snails and from snails naturally infected with other trematodes (echinostomes and strigeids). Prepatent infections with S. mansoni were readily identified by ELISA in field snails. A case is demonstrated where infection rate, as determined by shedding test alone, was 9.8%, whereas the combined figure of prepatent and patent infection rates was 22.9%