Isolation of Fasciola hepatica metacercariae by density gradients

Fasciola hepatica metacercariae were purified in high yield, removing contaminating cyst walls and plant material by step gradients consisting of 10 ml of 60% Percoll (density = 1.08 g ml-1) and 10 ml of 50% Metrizamide (density = 1.25 g ml-1). Greater than 90% of the metacercariae applied to the density gradients were recovered. These isolated metacercariae had an in vitro excystment rate of greater than 80%, which was the same excystment rate as metacercariae not subjected to density gradient centrifugation.     

片形吸虫DNA随机扩增多态性分析

为区别从南京市江宁县采集的片形吸虫非典型形态虫体，应用随机扩增多态性DNA（RAPD）技术，对6株片形吸虫总DNA进行了扩增。结果，10条引物中有8条能产生扩增图谱，电泳图谱经聚类分析，与传统的分类结果一致，并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫，也有形态不典型的大片形吸虫。     

The overwintering of Fasciola hepatica eggs under semi-arid and temperate Mediterranean climate (Madrid, Spain).

In this experimental work, conducted over two consecutive years (1988 and 1989), we have analyzed the overwintering possibilities of Fasciola hepatica eggs deposited in winter in a semi-arid and temperate Mediterranean area (Madrid, Spain). Eggs did not survive for more than a week in faeces at environmental moisture conditions. In contrast, over a 75% of the eggs maintained in permanently wet faeces survived winter temperatures, showing no high mortality until important thermic increment from June. The success of overwintering was higher in eggs arrested in wet faeces than included in water (55% "versus" 20% of hatching in spring), and it was unrelated to the deposition time (excretion in early or late winter). The hatching period of overwintered eggs started in May, at the same time as those excreted in early spring. Therefore, overwintering of eggs will have an effect on the level but not on the start of the spring contamination with miracidia. The dry winters of this semi-arid area greatly restrict the overwintering eggs contamination to soils next to streams, ponds or irrigation channels.     

The effect of pre-exposure to Fasciola hepatica or Schistosoma mansoni on challenge infection with Fasciola hepatica

Two groups of 12 and 6 rats were inoculated with Fasciola hepatica and Schistosoma mansoni, respectively. The Schistosoma-inoculated group, as well as 6 Fasciola-inoculated rats and 6 uninfected rats were challenged 8 weeks later with F. hepatica. A control group of 6 rats was left unexposed. Eight weeks after the challenge exposure all rats were necropsied and subjected to post-mortem examination. The number of Fasciola recovered after challenge was lower in both groups of rats primarily infected with F. hepatica or S. mansoni. F. hepatica-induced pathological changes were observed in all infected rats, but were pronounced in the group which was first exposed at the time of challenge of the primarily infected groups. No Schistosoma eggs or adults were detected in Schistosoma-inoculated rats. The results demonstrated that rats primarily infected with F. hepatica acquired resistance against a challenge exposure to the homologous parasite. Also S. mansoni, even without patency, can provide partial protection against F. hepatica infection.     

肝片吸虫蚴虫的形态学及分子学鉴定

为了找到青海地区肝片吸虫(Fasciola hepatica)的主要中间宿主,并对椎实螺(Lymnaeidae snails)体内的蚴虫进行虫种鉴定。对青海部分地区的椎实螺进行压片镜检,结果发现椭圆萝卜螺(Radix swinhoei)内无肝片吸虫蚴虫感染。狭萝卜螺(Radix lagotis)体内发现雷蚴,雷蚴为肝片吸虫的蚴虫,但是感染率较低为10%。青海萝卜螺(Radix cucnnorica)体内发现了雷蚴和尾蚴,并在养殖青海萝卜螺的水中青菜叶子上收集到囊蚴,经初步鉴定均为肝片吸虫的蚴虫,感染率较高为41.89%。主要中间宿主螺为青海萝卜螺。提取蚴虫体内DNA,通过PCR扩增18S rRNA片段并测序。将测序用DNAMAN软件进行比对后发现,与Gen Bank中发布的大片吸虫(Fasciola gigantica)和肝片吸虫的18S rRNA的序列进行比对其相似度分别为99.12%和92.03%。可以进一步确定所收集的蚴虫为肝片吸虫的蚴虫。     

Sero-diagnosis of Fasciola hepatica infections in cattle

An antigen was prepared from metabolic products which were produced by maintaining Fasciola hepatica adults in RPMI tissue culture medium for 24 h. The antigen compared favourably with a soluble extract of adult flukes when used in an enzyme-linked immunosorbent assay (ELISA). Experimentally infected calves were used to evaluate the sensitivity of the test and a survey from areas of both traditionally high and low incidence of the disease was carried out. Evidence is presented which suggests that this metabolic antigen could be used for the sero diagnosis of naturally occurring infections. In addition the use of a microcomputer to read, file and analyse the results enabled a very large number of samples to be processed daily.     

Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR

Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.     

Interaction of Fasciola hepatica with albendazole and its metabolites

Adult Fasciola hepatica recovered from sheep 12 and 24 h after a single oral dose of albendazole (20 mg/kg) contained significant amounts of two oxidized metabolites of albendazole (ABZ), a sulphoxide (SX) and a sulphone (SO), but not ABZ. Flukes incubated in vitro with 10 microM SX or SO contained these metabolites at a level two to three times the level observed in flukes recovered from sheep 24 h after a curative oral dose of ABZ. The concentration of ABZ in flukes was 10-fold greater than either SX or SO after a 24 h in vitro incubation in 10 microM of the respective drug. Flukes exposed to ABZ in vitro contained two-fold higher SX levels than SX-treated flukes due to a combination of spontaneous oxidation in media and fluke-mediated oxidation of ABZ. Measurement of end-products of glucose metabolism following 24 h incubation in 10 microM of either ABZ, SX or SO did not show a significant difference between treated and untreated flukes.     

Fasciola hepatica products induce apoptosis of peritoneal macrophages

Immunomodulatory properties have been described for Fasciola hepatica excretory-secretory products (FhESP), with their interaction with the innate immune cells being crucial during the early stages of infection. Previously, we demonstrated that FhESP induce eosinophil apoptosis. In this work, the ability of FhESP to induce apoptosis of peritoneal macrophages was evaluated. These parasite products were observed to induce apoptosis in peritoneal macrophages stimulated in vitro with FhESP, as well as in cells recovered from infected mice. The ability of FhESP to modify the viability of macrophages by apoptosis induction may constitute a crucial event for extending its survival in the host.