Epidemiology of cercarial stage of trematodes in freshwater snails from Chiang Mai province,Thailand

Objective:To investigate the epidemiological situation of cercarial trematodes infection in freshwater snails from different water resources in Chiang Mai province,Thailand.Methods:The snail specimens were collected from 13 districts of Chiang Mai province during April 2008 to February 2012.The prevalence of cercarial infection in snails was investigated using the crushing method.The drawing was done with the help of a camera lucida for the morphological study.Results:A total of 2479 snail individuals were collected and classified into 7 families,11 genera,and 14 species,Among them,8 snails species were found to be infected with an overall prevalence of 17.27%(428/2479),which infected with nine groups of cercariae;gymnocephalous cercaria,strigea cercaria,megalurous cercaria,monostome cercaria,parapleurolophocercous cercaria(Haplorchis cercaria),pleurolophocercous cercaria,furcocercous cercaria(Transversotrema cercaria),xiphidiocercaria,and virgulate cercaria.The parapleurolophocercous cercaria was found to be the dominant type among the cercarial infection in the snails(64.25%).Conclusions:The various species of snails found in the research location act as the intermediate hosts for the high prevalence of parasitic infection of many species of mammals.This work will provide new information on both the distribution and first intermediate host of trematodes.

AUTORADIOGRAPHIC STUDY OF THE ATTRITION OF MIGRATING SCHISTOSOMULA IN THE SKIN OF MICE II.MICE CHALLENGED WITH NORMAL S.MANSONI CERCARIAE AFTER IMMUNIZATION WITH HIGHLY IRRADIATED CERCARIAE

The migratory pattern and attrition of75Se-labelled S.mansoni challenge cercariae in CF1mice immunized 0,1,3, or 5 times with 12kR-or 48kR-irradiated cercariae were investigatedby compressed organ autoradiography.Mice were percutaneously inoculated with 500-1000 cer-cariae for each immunization and with a known count of approximately 90 cercariae at adifferent site for challenge.The skin,lungs,liver,other organs,and catrcass were processedfor autoradiography.Hepatic and mesenteric perfusions for worm collection were alsosampled from day 19 through 34.In naive tnice,25% of challenge cercariae died in theskin.Skin attrition of challenge cercariae in mice immunized with 12kR-irradiated cercariaewas 43% with 1 immunization,54% with 3,and 58% with 5,whereas in mice immunized with48kR-irradiated cercariae,skin,attrition was48% with 1,63% with 3,and 76% with 5 immunni-zations.In all immunized mice,a high percentage of migrating sehistosomula died in the skinwithin 24 hours of a challenge infection.Utilizing autoradiography,it was found that thesilver foci created by single labelled worms were difficult to differentiate from those of pairedworms.This study documents the induction of acquired resistance against a challenge infectionin the skin by vaccination with highly irradiated cercariae,with the degree of attemtation ofimmunizing cercariae and the number of immunizations being closely related to the attritiotn.rate in the skin.Finally,we discuss the contradictory views concerning the site of attritiotn ofchallenge cercariae.

Effects of albendazole,artesunate,praziquantel and miltefosine,on Opisthorchis viverrini cercariae and mature metacercariae

Objective:To explore larvicidal effects of anthelmintic drugs on Opisthorchis viverrini(O.viverrini) for alternative approach to interrupting its cycle for developing a field-based control program.Methods:The larvicidal activities of albendazole(A1),artesunate(Ar),praziquantel(Pzq) and miltefosine(Mf) on O.viverrini cercariae and mature metacercariae were investigated.Lethal concentrations(LC_(50) and LC_(95)) of these drugs were determined.Mature metacercariae previously exposed to various concentrations of the drugs were administered to hamsters.Worms were harvested 30 d post infection and worm recovery rates calculated.Al,Ar,Pzq and Mf produced morphological degeneration and induced shedding tails of cercariae after 24 h exposure.Results:The LC_(50) and LC_(95) of Al,Ar,Pzq and Mf on cercariae were 0.720 and 1.139,0.350 and 0.861,0.017 and 0.693,and 0.530 and 1.134 ppm,respectively.LC_(50) and LC_(95) of Ar on mature metacercariae were 303.643 and 446.237 ppm and of Mf were 289.711 and 631.781 ppm,respectively but no lethal effect in Pzq-and Al-treated groups(up to 1 ppt).No worms were found in hamsters administered Pzq-treated metacercariae.The adult worms from Al-treated metacercariae were significantly bigger in size compared to the control group(P<0.05).Fecundity and body width were greater in adults from Mf-treated metacercariae compared to the control group(P<0.05).Conclusions:The larvicidal effects of these drugs were high efficacy to O.viverrini cercariae but lesser efficacy to metacercariae.It should be further studied with the eventual aim of developing a field-based control program.

Antigen presenting cells may be able to distinguish between normal and radiatedSchistosoma japonicum cercaria:anin vitro observation

Objective： To observe the discrepancies of responses induced by Schistosoma japonicum （S. japonicum） normal cercaria antigen （NCA） and ultraviolet （UV） -radiation-attenuated cercaria antigen （UVACA） in an in vitro system. Methods： S. japonicum cercariae were collected and UVACA and NCA were prepared. Mouse macro- phage model cells （RAW 264.7） were treated with medium, NCA （40 μg/mL） or UVACA （40 μg/mL） in the presence or absence of recombinant mouse interferon gamma （rmIFN-γ; 4 ng/mL） for 48 h. Cell surface staining and flow cytometry were used to assess the major histocompatibility complex （MHC） Ⅱ expression, and data were expressed as mean fluorescence intensities （MFI）. Interleukin （IL） -10, IL-6 and prostaglandin E2 （PGE2） in cell culture supernatant were evaluated by commercial enzyme-linked immunosorbent assays. Results： NCA significantly suppressed IFN-7-induced MHC Ⅱ expression on RAW 264.7 cells. In the presence of 1FN-7, NCA significantly promoted IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells. In the presence of IFN-γ, UVACA significantly promoted IL-10 but not IL-6 and PGE2 secretion from RAW 264.7 cells and showed no effect on IFN-γ-induced MHC Ⅱ expression. Compared with UVACA, NCA significantly suppressed IFN-γ-induced MHC Ⅱ expression and significantly promoted IL-6, PGE2 and IL-10 secretion from RAW 264.7 cells. Conclusion： RAW 264.7 cells respond differently to NCA and UVACA. NCA can significantly suppress IFN-γ-induced MHC Ⅱ expression and significantly promote IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells compared with UVACA.

Ultraviolet-attenuated cercariae ofSchistosoma japonicum fail to effectively induce a Th1 response in spite of up-regulating expression of cytotoxicity-related genes in C57BL/6 mice

Objective： To better understand the reason that Schistosoma japonicurn （S. japonicum） ultraviolet （UV）- radiated cercariae could not induce high level of protection in C57BL/6 mice. Methods： Microarray technology was performed to investigate the gene transcription profile in skin draining lymph nodes （sdLNs） at 1 w after exposure to attenuated cercariae （AC） or normal cercariae （NC） of S. japonicum in C57BL/6 mice. The expressions of some representative genes were further confirmed by real-time PCR. Subsequently, the expressions of Th1/Th2 cytokine genes, cytotoxicity-related genes, as well as co-stimulator genes in spleens from AC-vaccinated and NC- infected mice were analyzed by real-time PCR at w 3 and 6 post-exposure. Results： The gene expressions of Th1 cytokines, including interferon-y （IFN-γ）, interleukin （IL）-12 and tumor necrosis factor-α （TNF-α） in the sdLNs were significantly lower in AC-vaccinated mice than in NC-infected mice. Furthermore, the gene expressions of Th1- and Th2- cytokines, including IFN-γ, IL-12, TNF-α, IL-4 and IL-10, in the spleens from AC-vaccinated mice showed little changes at w 3 and 6 post-vaccination. In addition, cytotoxicity-related molecules including granzyme A, granzyme B, granzyme K, perforin 1 and Fas L were up-regulated from the early stage of vaccination, and peaked at the 3rd w after vaccination with UV-AC. Conclusion： UV-AC of S. japonicum could not ef- fectively induce a Thl response in C57BL/6 mice, which may be an explanation for the low protection against parasite challenge, and the role played by up-regulated expression of cytotoxicity-related genes in mice needs to be further investigated.

Characterization of randomly amplified polymorphic DNA (RAPD) fragments revealing clonal variability in cercariae of avian schistosome <i>Trichobilharzia szidati</i>(Trematoda: Schistosomatidae)

Recently we applied randomly amplified polymorphic DNA (RAPD) fingerprinting to detect clonal variability among individual cercariae within daughter sporocysts and rediae of 10 digenean trematodes (Platyhelminthes: Trematoda). The most variable RAPD patterns were obtained for Schistosomatidae representative-avian schistosome Trichobilharzia szidati. In this work, 50 polymorphic DNA fragments of approximately 300-1500 bp from RAPD patterns of individual T. szidati cercariae were cloned and sequenced. As a result genomic DNA sequences (total length of approximately 41,000 bp) revealing clonal variability in T. szidati cercariae were obtained and analyzed. The analysis indicated that these sequences contained tandem, inverted and dispersed repeats as well as regions homological to retroelements of two human parasites, Schistosoma mansoni and S. japonicum. Tandem and inverted repeats constituted 8.9% and 22.1% respectively, while the percentage of dispersed repeats was 21.0%. The average content of these components was 41.7% with the average AT content being 59.0%. About 40% of sequences included regions ranging in length from 96 to 1005 bp which displayed amino acid homology with open reading frame pol products of S. mansoni and S. japonicum retroelements: non-long terminal repeat retrotransposons (nLTRs, 76%), long terminal repeat retrotransposons (LTRs, 14%), and Penelope-like elements (PLEs, 10%). Most of these regions (86.4%) contained frameshifts, gaps, and stop-codons. The largest portion of them was homological to nLTRs of the RTE clade (67%). The number of sequences homologous to the members of CR1 lineage was 7 times smaller (9%). Homology with LTRs of Gypsy/Ty3 and BEL clades was revealed in 5% and 9% of cases respectively. We assume that the repetitive elements including retroelement-like sequences described in the current study may serve as the source of clonal variability detected previously in T. szidati and other digenean trematodes. Such genome regions rapidly accumulate mutations and thus may play an important functional role in the life history of the species.

Prevalence of cercarial infections in freshwater snails and morphological and molecular identification and phylogenetic trends of trematodes

Objective:To investigate the prevalence of cercarial infections in freshwater snails from several water sources in Nakhon Nayok,Nonthaburi,and Pathum Thani provinces of Central Thailand,and to reconstruct a phylogenetic tree for improved understanding of the relationships in the cercarial stage.Methods:The snail specimens were collected from 34 total sampling sites and investigated for cercarial infections using the crushing method.The cercarial specimens were classified and used for the phylogenetic tree analysis using the Internal Transcribed Spacer 2(ITS2).Results:A total of 1921 snail specimens were classified into five families and seven species.The results showed that four snail species were identified as intermediate hosts of the larval stages of trematodes,with an overall prevalence of infection of 2.45%(47/1921).The infected snail specimens included five groups of the cercarial type:cercariaeum cercariae,echinostome cercaria,megalurous cercaria,parapleurolophocercous cercaria,and xiphidiocercariae.This is particularly true of xiphidiocercariae,which was found to be the dominant type among cercarial infections in bithyniid snails by approximately 38.00%.With regard to molecular identification,the phylogenetic tree was reconstructed using the neighbor-joining method with 10000 bootstraps and separated the trematodes into three clades:Echinostomatoidea,Microphalloidea and Opisthorchioidea.Conclusions:The study reveals a high prevalence of cercarial infection for each cercarial type and maturation into a definite trematode genus and delineates morphological characteristics and evolutionary trends among each larval trematode in Nakhon Nayok,Nonthaburi and Pathum Thani provinces.In addition,the ITS2 sequence data of cercariae could be used to examine classification of these species at the family level.

IN VIVO AND IN VITRO ENCYSTMENT OF ECHINOCHASMUS LILIPUTANUS CERCARIAE AND BIOLOGICAL ACTIVITY OF THE METACERCARIAE

Aim To explore if Echinochasmus liliputanus cercariae can develop into metacercariae both invivo and in vitro and biological activity of the metacercariae,then to determine the effects of silver ni-trate on eereariae encystation in vitro.Methods Cereariae of Echinochasmus liliputanus from Bellamyaaeruginosa snails treated with or without silver nitrate were used to infected goldfish,the second inter-mediate host,or to incubated in many different solutions for 24 h to record the encystation rates.Themetacercariae formed both in vivo and in vitro were then used to infected New Zealand rabbits to testtheir infectivity to its definitive hosts or to exeyst in 0.1% sodium deoxycholate excystation medium at37℃ for 1 h.Results In vivo encystment of cercariae occurred in the gills of goldfish.However,thecercariae were also able to encyst in vitro in Locke’s solution,NaCl solution,artificial gastric juice orhuman gastric juice with eneystation rates of 74.28 %,44.94 %,8.37 % and 10.79 %.0.7×-1.2×Locke’s or 0.7%-1.2% NaCl solution was shown to be appropriate for in vitro encystment to occurwithin 24 hour,however,full- strength Locke’s solution was shown to be optimal.The one- day- oldencysted metacereariae formed in vivo showed 88.53 % exeystation when treated in 0.1% sodium de-oxycholate exeystation medium at 37℃ for 1h.The metacercariae formed in vitro,however,showed88.60% and 84.95% excystation for normal and abnormal ones respectively.While abnormal cysts atroom temperature usually die within 10 days,about 70% normal cyst,both in vivo and in vitro,canstill excyst after stored in 0.5×Locke’s at 4℃ for 3 mouths.Cysts formed in vivo and in vitro were e-qually infective to rabbits.1 uM silver nitrate had a dramatic effect on the cercariae encysting in vitro.When treated with silver nitrate,the cereariae encystation rates decreased to 16.25% in Locke’s solu-tion and 6.69% in NaCl solution,however,the encystment was largely restored wben the cercariaewere washed to remove Ag+.Conclusion The finding of E.liliputanus cercariae encysting in vitro,especially in human gastric juice,might be helpful in elucidating mechanisms of the definitive hosts in-fected by the cercariae directly.The encystment of the cercariae in vitro could be inhibited when thecercariae were treated with 1 uM silver nitrate.As silver nitrate binds to the papillae,especially to theciliated papillae,on the cercarial surface,it is suggested that papillar chemoreceptors may be involved inencystment of the cercariae.

C-6膜粘附水中日本血吸虫尾蚴技术的研究

在实验室和现场水面,应用C-6膜多点抽样粘取方法检测水中尾蚴。获蚴率达40％,高于现用的尼龙绢条粘取沉淀法约20倍,在现场用现有方法未查获尾蚴的水面,调查钉螺阳性率为零或灭螺后的水面,本法可在一些环境查获尾蚴。本法操作简便,当天可有检测结果,费用仅约为哨鼠法的1％,利于广泛应用。

血吸虫对吡喹酮抗药性的研究 Ⅲ曼氏血吸虫吡喹酮敏感株与抗性株尾蚴对吡喹酮的反应性

目的 比较曼氏血吸虫吡喹酮敏感株与抗性株尾蚴阶段对吡喹酮的反应性 ,旨在运用敏感株与抗性株反应性间的差异 ,建立现场监测吡喹酮抗性的方法。方法 将各虫株尾蚴分别暴露于10 - 4、10 - 5、6× 10 - 7、4× 10 - 7m ol/ L吡喹酮溶液中 ,0、2 0、4 0、6 0、80、10 0 min后 ,解剖镜下观察尾蚴的泳动、收缩和断尾率的变化。结果 尾蚴暴露于 10 - 4m ol/ L吡喹酮中即刻停止泳动、沉底并伴有强直性收缩 ;5 min后开始出现体与尾部不协调的快速蠕动 ,尾蚴体区的后端与尾部的前端发生分离即断尾 ,敏感株断尾尾蚴明显多于抗性株 ;暴露于 10 - 5mol/ L吡喹酮中 4 0、6 0、80 m in和 10 0 min后 ,敏感株尾蚴的断尾率分别为 2 8.2 %、5 2 .7%、6 7.5 %和 78.0 % ;抗性株分别为 11.3%、2 8.6 %、39.3%和 4 5 .5 %。暴露于 4× 10 - 7m ol/ L中 80 min和 10 0 min后 ,敏感株尾蚴的断尾率为 10 .3%和 17.0 % ;抗性株为 0 .5 %和 1.1%。结论 曼氏血吸虫吡喹酮抗性株和敏感株尾蚴阶段对吡喹酮反应性存在非常显著的差异。提示 ,将尾蚴移入 4× 10 - 7mol/ L吡喹酮溶液中 80 - 10 0 min,镜下观察其断尾率 ,作为曼氏血吸虫对吡喹酮敏感性的检测方法 ,可用于螺体内吡喹酮抗性虫株的现场监测。

长效涂肤剂“防蚴灵”预防血吸虫尾蚴感染的研究

目的 观察“防蚴灵”涂肤剂预防血吸虫尾蚴感染的效果。方法 用氯硝柳胺加透皮促进剂制成 1% - 2 %浓度的“防蚴灵”,对感染前 1、2、3、4、5、6、7d涂肤的小白鼠和家兔分别进行预防血吸虫尾蚴感染的试验 ,并与对照组比较。结果 小鼠在感染前 1- 4 d,涂 1%防蚴灵 1次 ,其减虫率为 10 0 % ,在感染前 5 - 7d涂药 1次 ,减虫率为 99.7% - 88.1%。家兔感染前 3- 7d涂 2 %的防蚴灵1次 ,减虫率为 86 .4 % - 80 .1%。结论 涂肤剂“防蚴灵”

环介导等温扩增技术检测日本血吸虫尾蚴的实验研究

目的建立一种检测日本血吸虫尾蚴的方法。方法采用环介导等温扩增技术(LAMP):使用基因释放剂快速提取尾蚴基因组DNA,设计4条扩增尾蚴钙结合蛋白基因的LAMP引物,进行LAMP反应,以华支睾吸虫为阴性对照,LAMP产物经显色、电泳鉴定。用细吸管在解剖镜下分别吸取20、10、5、1条尾蚴进行LAMP反应,检测其敏感性。结果尾蚴检测管经显色后呈绿色(阳性),对照组呈棕色(阴性)。尾蚴LAMP产物电泳后呈LAMP特征性梯状条带,对照组无扩增产物。LAMP可检测到尾蚴的最低数量为1条。结论LAMP方法敏感、特异、简便,可用于检测日本血吸虫尾蚴。

日本血吸虫尾蚴cDNA文库的构建及分析

目的 构建日本血吸虫尾蚴 c DNA文库。 方法 从日本血吸虫尾蚴中提取总 RNA,运用“SMART c D-NA文库构建试剂盒”构建文库。 结果 所建文库的初始滴度为 1.8× 10 7pfu/ ml,经 1次扩增后的滴度为 2 .5× 10 7pfu/ ml,插入子的平均长度为 1.0 75 kb,重组率为 94 .4 % ,并从该文库中调出了日本血吸虫保守基因 TPI和 JF2的全长 c DNA片段。 结论 构建了日本血吸虫尾蚴 c

氯硝柳胺悬浮剂杀灭日本血吸虫尾蚴的实验研究

目的观察25％氯硝柳胺悬浮剂对日本血吸虫尾蚴的杀灭效果,确定杀蚴有效剂量,为氯硝柳胺悬浮剂现场灭蚴提供依据。方法杀蚴实验:用25％氯硝柳胺悬浮剂配制氯硝柳胺基质浓度分别为10、5、1、0．5、0．1、0．05、0．01 mg／L溶液,每个浓度组分别取药液0．3 ml置于48孔培养板中,后加入活尾蚴20-50条,解剖镜下观察尾蚴的存活。小鼠感染实验:将200条活尾蚴移入到30 ml的水体中用微型喷雾器均匀喷洒不同浓度氯硝柳胺溶液1 000、100、10、1、0．1、0．01 mg／L于水体表面,喷洒量为药液在水体扩散后整个水体中的终浓度分别为11．6、1．16、0．116、 1．16×10-2、1．16×10-3、1．16×10-4mg／L。喷药后10 min采用浸尾法感染小鼠30 min,小鼠感染 35 d后解剖,观察鼠的虫负荷。结果尾蚴在浓度为10 mg／L和5 mg／L溶液中1 min、1 mg／L和 0．5 mg／L中2 min及0．1 mg／L溶液中30 min死亡率均为100％;在0．05 mg／L和0．01 mg／L溶液中60 min,未见尾蚴死亡。小鼠感染实验显示,经1 000、100、10、1、0．1 mg／L溶液作水面喷洒后,小鼠感染虫负荷显著低于对照组。结论氯硝柳胺杀蚴的有效浓度为0．1 mg／L,随有效浓度的降低,其杀蚴效果逐渐减弱,<0．1 mg／L时,对尾蚴无杀灭作用;用浓度>0．1 mg／L氯硝柳胺悬浮剂对水体表面进行喷洒可起到水体表面灭蚴的作用。

2012年基于感染性水体哨鼠监测的血吸虫病风险区域预警

目的利用哨鼠监测血吸虫病高风险区域的水体感染情况，并根据监测结果作出预警。方法在湖南、湖北、江西、安徽、江苏、云南和四川等7个省47个县（市、区）选择72个易感水域监测点，分别于2012年6～7月和9月投放1组小鼠，每组20只，分成2笼，固定悬浮于水面，每次测定2d，每天4h，现场测定后哨鼠带回实验室饲养35d后，在实验室解剖观察血吸虫感染情况。结果2012年6～7月和9月在7个省47个县（市、区）设立了72个监测点，共投放哨鼠3283只，回收并解剖哨鼠3062只，剖检率为93-3％；共检出6个阳性点，其中2个阳性点在2次监测中均发现阳性哨鼠；阳性点总出现率为8.3％（6／72），其中6～7月份和9月份的阳性点出现率分别为8．3％（6／72）和2．8％（2／72）；发现阳性哨鼠33只，哨鼠总感染率为1．08％（33／3062），检获血吸虫成虫1085条，阳性鼠平均虫荷为32．9条／鼠；6个阳性点主要分布在湖南（4个）和江西（2个），其中2个阳性点所在的县分别出现本地感染的急性血吸虫病例或异地报告疑似当地感染的急性血吸虫病例，针对6个阳性点均发出了预警并作出了现场处置。结论2012年全年共发现6处高风险水域。监测区域的血吸虫病感染风险范围较2010年显著缩小，但部分区域的血吸虫病感染风险程度仍然较高。

水温对日本血吸虫尾蚴的逸出及其感染力的影响

目的 实验观察水温对日本血吸虫尾蚴的逸出及其感染力的影响。方法 将一定量感染性钉螺分别置于 1、5、10、15、2 0、2 5、30、35℃和 4 0℃水中逸蚴 4 h,观察各水温组钉螺逸蚴率和逸蚴量 ;将一定量尾蚴分别移入 1、10、2 0、30℃和 4 0℃水中 ,后采用接触法感染小鼠 30 min,观察各水温组鼠感染率和虫负荷。结果 在温度为 1～ 4 0℃的水体中 ,云南和江苏感染性钉螺均能逸出尾蚴 ,其中云南螺逸蚴的最适水温为 2 0～ 35℃ ,江苏钉螺则为 15～ 35℃。在温度为 1～ 4 0℃水中 ,江苏虫株尾蚴对各水温组鼠的感染率均为 10 0 .0 % ,云南虫株尾蚴对各水温组鼠的感染率均 >83.3%。结论 在温度为 1～ 4 0℃的水体中 ,日本血吸虫尾蚴均能从感染性钉螺中逸出并能感染终宿主 ;

血吸虫对吡喹酮抗药性的研究 Ⅻ曼氏血吸虫吡喹酮敏感株和抗性株雌雄尾蚴对吡喹酮敏感性的比较

目的比较曼氏血吸虫吡喹酮敏感株、抗性株雌性与雄性尾蚴对吡喹酮敏感性的差异,为探索血吸虫对吡喹酮抗性产生机制提供线索。方法分别用曼氏血吸虫吡喹酮敏感株、抗性株感染鼠粪便中的虫卵孵化毛蚴,以单只毛蚴感染单只光滑双脐螺,建立单性别血吸虫尾蚴系;以W1特异性序列为引物,采用直接PCR法鉴别出单性别系尾蚴的性别,分别将敏感株与抗性株的雌、雄尾蚴暴露于不同浓度的吡喹酮溶液中,经一定时间后观察并计算尾蚴的断尾率。结果曼氏血吸虫尾蚴暴露于10-4、10-5、6×10-7mol/L和4×10-7mol/L吡喹酮溶液中100min,吡喹酮敏感株雄性尾蚴的断尾率分别为66.7%、75.8%、43.5%和21.7%,雌性的断尾率分别为29.3%、27.9%、12.1%和7.6%,雄性尾蚴的断尾率显著高于雌性尾蚴(P均<0.05);而抗性株雄性尾蚴的断尾率分别为43.3%、39.4%、25.4%和6.9%,雌性尾蚴的断尾率分别为47.0%、38.9%、26.3%和6.3%,两者间差异无统计学意义(P均>0.05)。曼氏血吸虫尾蚴分别暴露于10-4、10-5、6×10-7mol/L和4×10-7mol/L吡喹酮溶液中80min,敏感株雄性尾蚴的断尾率分别为54.4%、68.6%、42.1%和16.1%,抗性株雄性尾蚴的断尾率分别为30.2%、34.4%、20.1%和2.8%,敏感株显著高于抗性株(P均<0.05)。结论曼氏血吸虫雌雄尾蚴对吡喹酮的敏感性存在差异,雄性尾蚴敏感性高于雌性。曼氏血吸虫吡喹酮抗性的产生与雄虫对吡喹酮的敏感性降低有关。

日本血吸虫97kDa DNA疫苗与致弱尾蚴疫苗诱导免疫应答特征的比较研究

目的 对比观察日本血吸虫大陆株副肌球蛋白基因疫苗 (Sjc97DNA)与紫外线致弱尾蚴 (UVC)疫苗免疫C5 7BL 6小鼠诱导的抗感染保护力及免疫应答特征。 方法 以Sjc97DNA核酸疫苗经后腿胫前肌免疫C5 7BL 6小鼠共 2次 ,每次间隔 3wk ,末次免疫后 3wk攻击感染日本血吸虫尾蚴 ;UVC疫苗接种同种小鼠后 5wk攻击感染上述等量尾蚴。均于攻击感染后 7wk计数虫负荷及肝卵负荷。并设空质粒对照及感染对照组。用ELISA分析免疫鼠攻击感染前后血清特异性IgG、IgA及亚型抗体水平 ,以及脾淋巴细胞体外诱生的细胞因子水平。 结果 Sjc97DNA疫苗及UVC疫苗免疫小鼠均诱生出以Th1型免疫应答为主的IL 2、IFN γ及特异性抗AWA、SEAIgG2a、IgG2b亚型及IgA抗体 ,UVC疫苗组小鼠各细胞因子及抗体水平均显著高于Sjc97DNA疫苗组 ,但两疫苗组均未测及IL 4。攻击感染后 ,Sjc97DNA疫苗组的减虫率 3 6.3 %、减卵率 42 .4% ,明显低于UVC疫苗组的 66.9%和 75 .6%。攻击感染后 7wk ,两疫苗组小鼠Th2型免疫应答虽有所增强 ,但仍以Th1型免疫应答占优势 ;而空质粒对照组和感染对照组小鼠则以Th2型免疫应答为主。 结论 核酸疫苗与紫外线致弱尾蚴疫苗均能诱导产生抗感染免疫保护力 ,致弱尾蚴疫苗的免疫保护力高于Sjc97DNA。两疫苗诱导的抗感染?

血吸虫对吡喹酮抗药性的研究 Ⅺ日本血吸虫中国大陆现场分离株虫卵毛蚴尾蚴对吡喹酮的敏感性

目的测定日本血吸虫中国大陆主要流行区现场分离株虫卵、毛蚴和尾蚴阶段对吡喹酮的敏感性,为建立日本血吸虫吡喹酮敏感性检测技术提供基础。方法分别从中国湖南、湖北、江西、安徽、江苏和云南6省日本血吸虫病流行区病人粪便标本中分离虫卵,建立日本血吸虫现场分离虫株;采用实验室已有的3株曼氏血吸虫株为对照。将各虫株虫卵分别孵育于5×10-6、10-6、5×10-7、10-7mol/L吡喹酮溶液中24h后移至清水孵化,观察虫卵的孵化率。将各虫株毛蚴分别暴露于5×10-6、10-6、5×10-7、10-7mol/L吡喹酮溶液中,0、1、5min后观察比较毛蚴的运动及形态学变化。将各虫株尾蚴分别暴露于10-5、6×10-7、4×10-7、10-7mol/L吡喹酮溶液中,0、20、40、60、80、100min后,解剖镜下观察尾蚴的泳动、收缩和断尾率的变化。并将结果与曼氏血吸虫株进行比较。结果经10-6,5×10-7、10-7mol/L吡喹酮溶液中24h后,日本血吸虫虫卵的孵化率分别为0.52%、11.90%和49.15%,曼氏血吸虫分别为4.17%、31.37%和92.53%。当暴露于10-6mol/L吡喹酮1min后,日本血吸虫毛蚴的变形率为100.00%,曼氏血吸虫为55.73%;当分别暴露于5×10-7、10-7mol/L吡喹酮5min后,日本血吸虫毛蚴的变形率为96.82%和21.80%,曼氏血吸虫毛蚴为21.80%和0。当暴露于10-5mol/L吡喹酮中40min,日本血吸虫尾蚴的断尾率为96.75%,曼氏血吸虫为28.30%;暴露于4×10-7mol/L吡喹酮中100min,日本血吸虫尾蚴的断尾率为95.82%,曼氏血吸虫为11.40%;当暴露于10-7mol/L吡喹酮中80min,日本血吸虫尾蚴的断尾率为29.65%,曼氏血吸虫尾蚴为0。结论日本血吸虫各现场分离株间在虫卵、毛蚴和尾蚴阶段对吡喹酮的敏感性无明显差异,但均明显高于曼氏血吸虫。研究提示,将毛蚴移入5×10-7mol/L吡喹酮溶液中1min,镜下观察其变形率,可作为日本血吸虫对吡喹酮敏感性的观察指标,可用于现场判断病人化疗失败的原因是否是由于吡喹酮不敏感株产生所引起。将尾蚴移入4×10-7mol/L吡喹酮溶液中80～100min,镜下观察其断尾率,可用于螺体内虫株吡喹酮敏感性监测。

紫外线减毒日本血吸虫尾蚴和童虫免疫小鼠的抵抗力研究

本文报告用２５条、５０条、１５０条和３００条紫外线减毒日本血吸虫尾蚴钻皮免疫或减毒童虫皮下注射免疫均能诱导小鼠产生有效的抗攻击感染抵抗力。以２５条免疫量为例，尾蚴钻皮及童虫皮下注射后的减成虫率分别为７２．１％和７０．４％，肝脏减卵率分别为６９．４％和５７．４％，小肠组织减卵率分别为７６．６％和８０．５％，免疫后的有效保护期为７．５个月。