Biochemical Liver Functions and Molecular Identification of Fasciola hepatica from Experimentally Infected Rat Model

The current study was performed to evaluate the liver function status as well as molecular characterization of the recovered worms in rats experimentally infected with F. hepatica. Sixteen male Wister rats aged 30 days were randomly allocated into two groups (n = 8). The first group was infected orally with 15 viable encysted metacercaria of F. hepatica per animal. The other group was kept non-infected (control group). At zero time (before infection), the 2nd, 4th, 6th, 8th, 10th, 12th and 14th weeks post-infection (WPI), blood and serum samples were collected via puncture of retro-orbital plexus of veins from each rat. Serum enzyme level (AST and ALT) and total protein were measured, and the serum protein profile was carried out using agarose gel electrophoresis. During the period of the experiment, serum ALT and AST activities and serum total globulins significantly increased while serum total proteins and albumin markedly decreased in the infected group. On the 14th WPI, the data of the electropherogram showed that globulin fractions (α1-, β- and γ-globulin) levels were significantly increased while α2-globulin was markedly decreased in the infected group. The molecular analysis confirmed the amplification of the ITS1, ITS2 and NDI genes of F. hepatica recovered from the infected liver of rats with amplicon sizes of 630, 510 and 560 bp, respectively. Sequencing of the amplified ITS gene resulted in the determination of 3 strains (PP108836, PP108837, and PP108838). Also, analysis of the ITS2 gene resulted in obtaining 3 isolates under the accession numbers (PP109065, PP109066, and PP109067). In conclusion, fasciolosis in the rat model is suitable for routine experimental infections and caused a pronounced liver dysfunction with discharging of the Fasciola eggs in the faeces and the development of adult stages in the bile ducts. Furthermore, molecular techniques are a sensitive tool for the identification and characterisation of the Fasciola parasite.

Changes of the Levels of Blood NO and TNF-αConcentrations in Water Buffaloes and Goats Infected with Fasciola hepatica

Changes of concentrations were studied in water buffaloes and goats infected with Fasciola he-patica on blood NO(nitric oxide) and TNF-α(tumor necrosis factor-α). Twenty healthy male castrated water buffaloes of 2 - 3 years old and weighing 300 - 500 kg as well as six goats were confirmed free of fasciolosis by fecal examination and Dot-ELISA. Two studies were conducted using the water buffaloes. In the first experiment, 8 water buffaloes were randomly divided into control group (n = 3) and infection group (n = 5). Each buffalo in the infected group received orally 60 metacercariae of F. hepatica per day for 20 days (total 1 200 metacercariae) to produce a chronic infection. In the second experiment, 12 water buffaloes were randomly divided into infected (n=9) and control group (n = 3). Each buffalo in the infected group was given a single oral dose of 1 600 metacercariae to produce an acute infection. The 6 goats were randomly divided into two infected groups and a control group. The sheep in two infections received a single oral dose of 200 and 500 metacercariae respectively, the control group remained uninfected. Blood NO and TNF-a concentrations of the test animals were measured by a reductive enzyme assay and RIA, respectively. Blood NO concentration in both acutely and chronically infected water buffaloes progressively increased from week 3 post-infection and was significantly greater than that of the control group (P<0. 05) at the 5th week (acute infection) and 7th week (chronic infection), and remaining at higher concentration for the remaining period of the studies. Blood TNF-αconcentrations in both chronically and acutely infected water buffaloes also increased after infection. In the goat experiment, plasma NO concentrations in both infection groups increased from week 3 after infection, and remained higher than that of the control group until the end of the experiment. TNF-α concentrations in goats in infection group 1 and 2 gradually increased after infection and were significantly greater than those of the control group from the 9th to 11th week and from the 11th to 15th week respectively.

Clinical presentation and management of Fasciola hepatica infection:Single-center experience

AIM:To identify the characteristic clinical,laboratory and radiological findings and response to treatment in patients with fascioliasis.METHODS:Patients who were diagnosed with Fasciola hepatica infection were included in this prospective study.Initial clinical,laboratory and radiological findings were recorded.All patients were followed until a complete response was achieved or for 6 mo after treatment discontinuation.RESULTS:Fasciola hepatica infection was diagnosed in 30 patients(24 females;mean age:42.6 years) between January 2008 and February 2011.Twentytwo(73%) patients had hepatic phase fascioliasis,5 patients had biliary phase,and 3 patients had biliary phase associated with acute pancreatitis.Of the 8 patients with biliary phase fascioliasis,2 patients displayed features that overlapped with both hepatic and biliary phase.Abdominal pain and right upper abdominal tenderness were the most prominent signs and symptoms in all patients.Eosinophilia was the most prominent laboratory abnormality in both patients with hepatic and biliary phase(100% and 50%,respectively).Multiple nodular lesions like micro-abscesses on abdominal computerized tomography were the main radiological findings in patients with hepatic phase.Small linear filling defects in the distal choledochus were the main endoscopic retrograde cholangiopancreatography(ERCP) findings in patients with biliary phase.Patients with hepatic phase were treated with triclabendazole alone,and patients with biliary phase were treated with triclabendazole and had live Fasciola hepatica extracted from the bile ducts during ERCP.CONCLUSION:Fasciola hepatica infection should be considered in the differential diagnosis of patients with hepatic or biliary disease and/or acute pancreatitis associated with eosinophilia.

Antifungal,molluscicidal and larvicidal assessment of anemonin and Clematis flammula L.extracts against mollusc Galba truncatula,intermediate host of Fasciola hepatica in Tunisia

Objective: To investigate the potential of anemonin and Clematis flammula(C.flammula) extracts against infective organisms.Methods: The molluscicidal activities of anemonin and C.flammula extracts against Galba truncatula Mull.(Lymnaeidae) and Fasciola hepatica larval stages contaminating this snail in Tunisia were assessed by testing six groups of snails in 250 mL of extracts and aqueous dechlorinated solutions with different concentrations(ranging from 2.5 to 20.0 mg/L) for 48 h.Besides, the antifungal potential of C.flammula leaves and flowers was evaluated by using the diffusion agar and broth dilution methods against four fungal strains: Aspergillus niger, Pythium catenulatum, Rhizoctonia solani and Fusarium phyllophilum.Results: As a result, hexane and ethyl acetate flower extracts exhibited significant molluscicidal activities with LC_(50) median lethal concentrations values of 11.87 and 11.65 mg/L, respectively while LC_(50) value of anemonin was 9.64 mg/L after 48 h exposure.The flower extracts showed a larvicidal effect with a deterioration rate exceeding 35.39% where flower ethyl acetate residue gave a deterioration rate of cercariae close to 97%.Moreover, C.flammula extracts were not noxious to the associated fauna survival.All extracts inhibited the growth of P.catenulatum, the leaves and flowers methanolic extracts had the more important fungicide action with minimum inhibitory concentrations of 1.56 and 3.12 mg/mL together with minimum fungistatic concentrations of 3.12 and 6.25 mg/mL respectively.Only flower extracts were active against Rhizoctonia solani with minimum inhibitory concentrations varying between 0.70 and 1.56 mg/mL and 6.25 mg/mL of minimum fungistatic concentration.Phytochemical tests showed that the antifungal activity may be attributed to the presence of the flavonoids/saponins in the methanolic extracts and the molluscicide effects could be due to the richness of hexane and ethyl acetate extracts on sterols and triterpenoids.Conclusions: This study emphasizes the important molluscicidal and antiparasitic effects of flower ethyl acetate extracts and anemonin compound as well as the considerable antifungal activities of methanolic extracts.These results improve the therapeutic virtues of C.flammula aerial part extracts.

Fasciola hepatica infestation as a very rare cause of extrahepatic cholestasis

Fasciola hepatica,an endemic parasite in Turkey,is still a very rare cause of cholestasis worldwide.Through ingestion of contaminated water plants like watercress,humans can become the definitive host of this parasite.Cholestatic symptoms may be sudden but in some cases they may be preceeded by a long period of fever,eosinophilia and vague gastrointestinal symptoms.We report a woman with cholangitis symptoms of sudden onset which was proved to be due to Fasciola hepatica infestation by an endoscopic retrograde cholangiography.

Does Fasciola hepatica infection modify the response of acute hepatitis C virus infection to IFN-α treatment?

Immunologic response to acute hepatitis C is mainly a Th1 response, whereas fasciolopsiasis is associated with a diverse T-cell response. Interferon-alpha has immunomodulatory effects and enhances Th1 immune response. Fasciola infection could theoretically interfere with the Th1 immune response, even when acquired after an initial response to interferon-alpha treatment for acute hepatitis C virus （HCV） infection. We report here the case of a male patient who acquired Fasciola hepatica infection after an initial response to IFN-alpha therapy with a favorable outcome.

Obtaining the Minimum Lethal Dose against <i>Fasciola hepatica in Vitro</i>Using Plant Extract Hexanes with Fasciolicide Activity and Toxicity Evaluation on CD1 Male Mice

Fascioliasis is a parasitic disease of worldwide distribution affecting mainly cattle and sheep. Its importance lies in the economic losses it produces in the livestock industry. Its control is carried out by using a chemical fasciolicide showing resistance problems and environmental contamination. Looking for an alternative control for this disease the present study was aimed at determining the hexane anti-Fasciola hepatica in the in vitro effect of some plant extracts and the minimum lethal dose of the mentioned extracts. All selected plants were tested in vitro at concentrations of 500, 250, 125 and 50 mg/L):Achilleamillefolium (plumajillo), Artemisiaabsinthium (wormwood), Artemisia mexicana (estafiate), Castelatortuousa (chaparroamargo), Chenopodiumgraveolens (epazote de zorrillo), Gymnospermaglutinosum (popote) Justicia spicigera (muicle), Limpia critridora (cedron), Lippiagraveolens (oregano), Menthapiperita (Mint), Populus alba (alamo) and Thymusvulgaris (thyme). Subsequently proceeded to perform a toxicity study with these fractions in CD1 male mice 10-13 weeks of age, forming groups of 3-5 animals they were administered a single oral dose being (5 mg/kg, 50 mg/kg, 500 mg/kg, 2500 mg/kg and 5000 mg/kg) and were kept under observation 20 days, later were sacrificed and a kidney and liver histology was performed, finding the safety of the extracts. To perform the toxicity study with these fractions, groups of five CD1 male-mice were formed, they were treated with oral doses of 5, 50, 500, 2500 and 5000 mg/kg, administered with a cannule. All mice were kept under observation for 20 days. Finally they were sacrificed to perform histology of the kidney and liver in search of possible side effects. Results show that none of the extracts exhibited that fasciolocide activity for mice CD1 even at the highest dose thereforefinding the safety of the extracts.

肝片吸虫检测方法概述

肝片吸虫是一种人兽共患寄生虫,是目前全世界分布最广泛的吸虫之一,能感染包括人在内的多种哺乳动物,引起肝片吸虫病,不仅严重危害人类健康,还影响畜牧养殖业的发展,造成巨大的经济损失。肝片吸虫因其复杂的生活史和种类广泛的中间宿主和终末宿主,导致其致病机制尚不明确,且肝片吸虫感染引起的动物临床症状与其他肝胆疾病类似,主要表现为急性或慢性肝炎和胆管炎,没有特征性症状,极易被误诊。因此,及时、准确的检测对防治肝片吸虫病尤为重要。目前,肝片吸虫的检测主要依赖病原学、免疫学、分子生物学等手段,论文就肝片吸虫检测方法的研究进展进行综述,分析各项技术的优点与局限性,以期为肝片吸虫病检测方法的研究和发展提供参考。

胆道肝片吸虫病1例报道

通过1例误诊为胆总管结石患者在接受ERCP治疗时,从胆道取出肝片吸虫的诊疗过程,增强医师对该病的认识。

Study on Blood Cell Immune Response in Water Buffaloes Infected Acutely with F. hepatica

Action mechanism of blood cell immune response in water buffaloes against acute infection with F. Hepatica was studied. The results showed that after water buffaloes were infected, the total levels of WBC surpassed control group during whole infection period; Eosinophiles (%) of DC were higher than control group at the 2nd week until 19th week, and then dropped and was close to control group; Neutrophiles(%) was low or significantly lower than control group within the 5 - 16th weeks; The total levels of lymphocytes (%) was lower than control group during the whole infection period; T-lymphocytes (%) dropped significantly, but B-lymphocytes(%) had opposite changes from the first week of infection, and they were close to the control group after 11 weeks; RBC-CR] and RBC-IC rosette rates dropped and rose during 2 - 16 and 2-18 weeks, respectively, and then approached the same between both groups. It was suggested that the violent changes of specific and nonspecific immune responses in water buffaloes with acute F. hepatica infection are related with the mechanism against infection with F. hepatica together.

肝片吸虫CatB9蛋白的分子特征、遗传进化及表达定位研究

为了解肝片吸虫(Fasciolahepatica,Fh)组织蛋白酶B9(CatB9)的生物学功能,该研究对FhCatB9基因进行克隆、表达,并对其免疫原性和在Fh体内的定位进行分析。结果显示:FhCatB9基因长度为1038bp,编码345个氨基酸;编码的蛋白无跨膜区,有信号肽于第19~20氨基酸间。生物信息学分析显示,FhCatB9蛋白含2个N-糖基化位点、7个磷酸化位点、15个线性B细胞抗原表位和CatB的保守结构域,高级结构显示与大鼠CatB酶原相似。系统进化结果显示,Fh和大片吸虫亲缘关系最近,与其他寄生虫亲缘关系较远。SDS-PAGE和Westernblot都在55.3ku处检出目的条带。免疫组化显示,CatB9在Fh的肠道上皮细胞和排泄管上皮细胞均有表达,重组蛋白的获得为Fh血清学检测试剂的研发提供了一个有潜在价值的候选抗原。

人肝片形吸虫病1例报告

1临床资料 患者，女，41岁，因肝区胀痛2个月余入院。2个月前无诱因出现肝区胀痛，伴发热（体温最高38℃），当地医院予抗感染后疼痛病状缓解，体温下降，但此后症状时有反复发作。

肝片吸虫感染对反刍动物胆道粘膜肥大细胞数量的变化

分别对肝片吸虫自然感染的５头黄牛和５头绵羊以及无肝片吸虫感染的５头黄牛和５头绵羊的胆道粘膜肥大细胞（ＭＭＣ）进行了计数观察。结果与某些动物肠道蠕虫感染一样，肝片吸虫在反刍动物胆道的寄生引起了胆道ＭＭＣ的显著增多。有肝片吸虫和无肝片吸虫感染的黄牛胆道ＭＭＣ分别为４３１±１０３／ｍｍ２及１９１±６６／ｍｍ２（Ｐ＜０．０５），而绵羊胆道ＭＭＣ则分别为７０２±３００／ｍｍ２和７５±１３／ｍｍ２（Ｐ＜０．０１）。

肝片形吸虫重组谷胱甘肽-S-转移酶对SD大鼠的免疫原性分析

目的分析肝片形吸虫重组谷胱甘肽-S-转移酶(FhGST)对SD大鼠的免疫原性。方法将已构建重组原核表达质粒pET30a-FhGST转化大肠埃希菌BL21(DE3)宿主菌,以异丙基-β-D-硫代半乳精苷(IPTG)诱导重组蛋白表达十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,蛋白质印迹(Western blotting)分析其免疫反应性。20只SD大鼠随机均分为蛋白免疫组和佐剂对照组,蛋白免疫组以纯化的重组蛋白皮下注射免疫Sd大鼠,抗原免疫剂量为200μg/(只·次),共免疫3次,每次间隔3周。佐剂对照组以PBS代替免疫抗原同法注射。免疫前、每次免疫后和末次免疫后3周、6周鼠尾采血,分离血清。利用间接ELISA法检测免疫大鼠血清IgG抗体水平的动态变化,噻唑兰法(MTT法)检测脾淋巴细胞增殖情况。结果经纯化获得重组FhGST蛋白,SDS-PAGE电泳结果显示,在相对分子质量M_r 31300处出现单一条带。Western blotting分析结果表明,纯化重组FhGST蛋白能识别自然感然肝片形吸虫的山羊阳性血清,在M_r 31 300处可见特异的免疫反应带。重组FhGST蛋白免疫SD大鼠后,可诱导产生特异性IgG抗体,在免疫后第9周,抗体效价达到顶峰(1:89 144),且明显高于佐剂对照组(1:1000)重组蛋白能显著刺激大鼠脾细胞的生长和增殖。结论重组FhGST蛋白能诱导SD大鼠产生特异性免疫应答,具有良好的免疫原性。

水牛慢性感染肝片吸虫后血液免疫功能指标及激素水平的动态变化

将８头经粪便检查和Ｄｏｔ－ＥＬＩＳＡ检测确认无肝片吸虫感染的雄性去势水牛随机分成感染组（ｎ＝５）和对照组（ｎ＝３）。感染组每天每头经口感染６０个囊蚴，连续２０ｄ。结果表明：水牛慢性感染肝片吸虫后，红细胞ＣＲ１花环率从感染后第２周即显著降低，而红细胞免疫复合物花环率则显著升高；白细胞数的总体水平在１９周前高于对照组，其中嗜酸性粒细胞显著增加，而淋巴细胞则有所下降；在淋巴细胞中可见Ｔ细胞比例显著下降，Ｂ细胞比例显著升高，并引起血清白细胞介素－２水平的降低和抗肝片吸虫的ＩｇＧ含量的显著升高。激素变化主要表现为皮质醇水平在感染初期升高，胰岛素在高于对照组的水平上波动，甲状腺激素Ｔ３、Ｔ４水平未见与对照组有明显差异。提示水牛在慢性感染肝片吸虫后，红细胞免疫功能下降，机体主要依靠嗜酸性粒细胞增加和产生特异性的ＩｇＧ抗感染，某些内分泌激素可能参与抗感染的调节机制。

肝吸虫性肝胆疾病22例分析

目的通过对肝吸虫致肝胆疾病的临床分析,增加对该病的认识,减少误诊。方法回顾性分析本院1992年至2012年以肝胆疾病入院后确诊为肝吸虫病患者22例。其中男19例,女3例,平均年龄45.41岁,其中入院诊断为胆囊炎或胆结石8例、肝吸虫病7例,其他7例。结果实验室检查ALT升高者14例(63.63%)、AST升高者16例(72.72%)、TBil升高者11例(50.00%)、GGT升高者17例(77.27%)、嗜酸性粒细胞(E)升高者14例(63.63%)、大便集卵检查确诊10例(45.45%),胆汁涂片确诊7例(31.82%)、肝吸虫抗体检测确诊2例、既往肝吸虫病史者3例;经驱虫及综合治疗后预后良好。结论肝吸虫病具有地方性,临床少见且临床表现无特异性,易与其他肝胆疾病混淆,误诊率高。对于病因不明腹痛、黄疸、肝损害患者应注意询问患者及家人有无特殊饮食史,应注意行相关检查明确诊断,避免延误治疗。

肝片吸虫病的研究进展

肝片吸虫病(fascioliasis hepatica)又称肝蛭病,是由肝片吸虫引起的家畜常见的人畜共患病。该病遍及世界各地,严重阻碍了畜牧养殖业的健康发展,对保障食品安全、公共卫生安全和环境生态安全构成了极大威胁,同时也造成了严重的经济损失。全面掌握肝片吸虫病的相关信息是有效防治肝片吸虫病的基础。文章从该病的流行病学特点、临床表现、诊断方法、研究现状、防治措施及存在问题等方面进行了综述,并提出了展望。

肝片吸虫谷胱甘肽S-转移酶基因的克隆表达及活性分析

目的克隆和表达肝片吸虫谷胱甘肽S-转移酶基因(FhGST)以研究重组蛋白免疫学特性。方法参考Gen-Bank上发表的FhGST基因序列设计一对特异性引物,利用RT-PCR法扩增FhGST基因,将扩增产物克隆入pET-30a(+),构建重组表达载体。经PCR、双酶切和序列测定鉴定后转入E.coliBL21(DE3)宿主菌,经IPTG诱导表达,SDS-PAGE和Western blotting对该重组蛋白进行分析和鉴定。结果成功获得肝片吸虫GST基因全长为657bp的编码序列,SDS-PAGE结果表明重组蛋白分子量为31.3kDa,以包涵体形式表达。Western blotting结果证实重组蛋白能被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能与感染肝片吸虫的绒山羊血清发生反应,表明重组蛋白具有免疫反应性。结论成功的克隆了肝片吸虫谷胱甘肽S-转移酶基因,实现了在原核表达系统中高效表达重组蛋白,并具有良好的免疫学活性。

肝脏寄生虫感染48例临床分析

目的分析48例肝脏寄生虫感染患者的临床特征,提高临床诊断水平和指导治疗。方法搜集2010年4～7月在四川省人民医院感染科住院治疗的肝脏寄生虫感染患者资料,对其流行病学、临床表现、病原学、免疫学、腹部CT影像资料以及治疗进行分析。结果患者均有进食生泥鳅的病史、主要临床表现包括发热、乏力、食欲减退、上腹不适、腹痛、腹泻、肩颈腰背部和四肢酸痛,嗜酸性粒细胞显著升高,腹部增强CT肝包膜下、肝实质内低密度片状影或结节影。泥鳅肌肉组织查见囊蚴;患者血清中囊虫、包虫、肺吸虫、肝吸虫或血吸虫抗体1项或多项阳性。3例粪便自然沉淀法查见类似姜片虫虫卵。口服吡喹酮治疗全部治愈,随访1月以上无复发。结论生食泥鳅可引起肝脏寄生虫感染,本次感染的病原学以肝片吸虫可能性大,结合流行病学、临床、实验室和CT表现可以诊断,吡喹酮治疗安全、有效。

肝片形吸虫重组组织蛋白酶L的免疫反应性和免疫原性分析

目的分析肝片形吸虫(Fasciola hepatica)重组组织蛋白酶L(CatL)的免疫反应性,以及对SD大鼠的免疫原性。方法诱导含重组原核表达质粒pET30a-FhCatL的大肠埃希菌BL21(DE3)宿主菌表达,SDS-PAGE分析表达产物,以感染肝片形吸虫山羊血清为一抗,蛋白质印迹(Western blotting)分析其免疫反应性。20只SD大鼠随机均分为重组蛋白免疫组和佐剂对照组,免疫组大鼠皮下注射纯化的重组FhCatL,200μg/(只·次),共免疫3次,每次间隔3周;对照组用等量PBS与佐剂混合后注射。于第2次和末次免疫前,以及末次免疫后3、6和9周尾静脉采血,分离血清。利用间接ELISA法检测免疫大鼠血清IgG抗体水平,噻唑蓝比色法(MTT法)检测脾淋巴细胞增殖情况。结果经纯化获得重组FhCatL蛋白,相对分子质量(Mr)约42 000。Western blotting分析结果表明,纯化重组蛋白能被感染肝片形吸虫的山羊血清识别。重组FhCatL蛋白免疫SD大鼠后可诱导产生特异性IgG抗体,随着免疫时间的延长抗体水平逐渐升高,于末次免疫后3周抗体效价达到峰值(1∶102 400),明显高于对照组(1∶1 000)。免疫组脾淋巴细胞刺激指数为2.176±0.047,显著高于对照组(1.171±0.032)(P<0.05)。结论重组FhCatL蛋白具有较好的免疫反应性和免疫原性。