A study of the antigens produced by adult Fasciola hepatica maintained in vitro

Summary Metabolic antigen of adult Fasciola hepatica was prepared by concentrating the culture medium in which 100 adult flukes had been maintained in vitro for 7 days. Serological reactions between this antigen and sera from fluke-infected rabbits, rats and sheep were studied using immunodiffusion in agar and enzyme linked immunosorbent assay. The used medium contained a number of antigenic components and their various reactions with the sera from these different hosts were investigated.     

Type I hypersensitivity reactions in intestinal mucosae from rats infected with Fasciola hepatica

Type I hypersensitivity reactions in the intestinal tract of sensitized animals may contribute to resistance to reinfection with Fasciola hepatica. Colonic mucosae isolated from previously infected rats were voltage clamped in Ussing chambers. Antigen was prepared as a crude homogenate from adult liver fluke. Assay of serum antibodies against fluke antigen confirmed sensitization. Antigen challenge evoked a rapid onset, transient inward current in sensitized but not in control preparations. Chloride secretion accounted for at least part of the response since the loop diuretic bumetanide reduced the effect of antigen by 61%. Anti-rat IgE mimicked the response to antigen and desensitized tissues to subsequent antigen challenge. Local synthesis of eicosanoids may mediate the response to antigen since the cyclo-oxygenase inhibitor piroxicam reduced the response by 76%. In contrast, mepyramine which is a histamine receptor antagonist did not alter the ion transport response evoked by antigen. Tetrodotoxin reduced the response to antigen by 53% implicating intrinsic neurons within the lamina propria as effector cells in the responses of this tissue to antigen. We propose that antigen stimulation of electrogenic chloride movement and consequent fluid secretion in vivo may contribute to a local effector mechanism in prevention of reinfection of previously sensitized hosts.     

Vaccination against Fasciola hepatica infection using a Schistosoma mansoni defined recombinant antigen,Sm14

Fasciola hepatica is the causative agent of fasciolosis in many areas in America, Europe, Africa, Asia and Australia. There is an urgent need for improved methods to control the parasite's transmission. We describe the use of an experimental vaccine based on a recombinant antigen cloned from another parasite, Schistosoma mansoni (Sm14), that induces high levels of cross protection in mice against both S. mansoni and F. hepatica. Sheep and mice vaccinated with Sm14 were significantly protected against challenge infection with metacercariae of Fasciola hepatica and were completely free of the histopathological hepatic damage related to liver fluke infection. The vaccine will provide a valuable new tool to aid in transmission control of this economically important disease.     

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     

Fasciola hepatica: rapid switching of stage-specific antigen expression after infection

Early developmental stages of the trematode parasite Fasciola hepatica were collected from the peritoneal cavity and liver of mice during a ten day infection period. Using one dimensional SDS-PAGE, differences in protein expression profiles were observed in stages collected on the same day post-infection in different physiological locations and also in juvenile parasites collected from the same location on different days post-infection. Four rat monoclonal antibodies were raised against the parasite using lymph nodes draining infected tissues. Three monoclonal antibodies, FY3-1, FY3-2 and FY4-7, were generated using cells from the mesenteric lymph node of recently challenged immune rats, while FY1-6 was derived from hepatic lymph node cells of a chronically infected rat. The epitope recognized by FY3-2 appeared to be carbohydrate in nature and was present on the surface of newly excysted juveniles. Immunoblots revealed that the antigens recognized by FY3-1, FY3-2 and FY4-7 were only expressed for two days after infection. In contrast, FY1-6 recognized epitopes expressed across all developmental stages screened. The rapid changes in protein and antigen expression observed during the early stages of infection may assist the parasite to evade the host immune response.     

Fasciola hepatica: immunoprecipitation analysis of biosynthetically labelled antigens using sera from infected sheep

The antigenicity of the biosynthetically labelled somatic and excretory/secretory proteins of adult Fasciola hepatica was investigated over 20 weeks of an infection with F. hepatica in sheep. The antibody response was initially detected by ELISA 2 weeks after infection, and was sustained at this level for the remainder of the infection. Immunoprecipitation analysis indicated that a large number of proteins were recognized by the sheep, with several dominant antigens occurring in the 29-31 kilodalton molecular weight range. No differences were found between the antigens recognized by sheep in the early or late stages of infection suggesting that there are similarities between the antigens of the immature and mature forms of the parasite.     

Fasciola hepatica: changes in tegument during killing of adult flukes surgically transferred to sensitized rats

Summary Adult Fasciola hepatica were placed intraperitoneally into rats which had been sensitized by an oral infection of 20 metacercariae given 3–5 weeks previously. Implanted flukes were recovered at intervals during the next 24 h. Wax embedded sections of flukes and surface impressions of attached cells were examined by light microscopy (LM). Flukes were also examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In all stages, post transplantation, increased secretory activity was noted at the tegu-mental surface both by the blebbing of membrane and the formation of microvilli. Monitoring of the surface by SEM revealed attachment of very few cells (less than 100/sq mm) in the first hour. By TEM some of these were found to be eosinophils. At 1.5 h local palisading of cells, mainly eosinophils, occurred. Many of these degranulated at a distance from the parasite. Between 2 and 4 h many more neutrophils and macrophages were found, with fewer eosinophils. In the period up to 4 h the tegumental syncytium became charged with abnormally high concentrations of secretory granules and the surface membrane was maintained often enclosing apparently damaged areas. No cells penetrated the syncytium at this stage. Between 4 and 12 h the number of secretory granules in the tegument declined. Around 12 h the remaining granules contributed to the formation of large irregular microvilli. It was at this stage that neutrophils, macrophages and occasional eosinophils moved into the syncytium and began to lift it from beneath.     

Killing of newly excysted juveniles of Fasciola hepatica in sensitized rats

Newly excysted juvenile Fasciola hepatica were injected intraperitoneally into previously sensitized rats. They were recovered at various intervals post-injection and examined by light and electron microscopy. The challenge flukes were rapidly coated with peritoneal cells which, in the early stages, were mainly eosinophils. The eosinophils adhered closely to the flukes and degranulated on to their surface releasing cytochemically detectable peroxidase. Vacuoles formed in the tegument of the flukes beneath the adherent eosinophils and these increased in size until they spanned the width of the tegument, thus destroying its continuity. From 4 h post-injection some of the flukes had lost their teguments and were surrounded by phagocytic cells, particularly neutrophils; at this stage they were judged to be dead. During the first five minutes post-injection degranulating mast cells were associated with the challenge flukes. Their role in eosinophil chemotaxis is discussed as are the possible mechanisms of eosinophil adherence and degranulation. Using an anti-C₃ fluorescein conjugate, it was demonstrated that C₃ was not bound to the surface of challenge flukes either in vitro or in vitro in immune serum.     

The immune response of regional lymph nodes during the early stages of Fasciola hepatica infection in cattle

In this study we examined regional immune responses to Fasciola hepatica infection in the natural ruminant host. Naïve cattle and those pre‐exposed to a drug‐abbreviated infection were subsequently challenged and lymph nodes extracted at slaughter. In vitro proliferation and cytokine production by mononuclear cells isolated from hepatic and mesenteric lymph nodes were measured after culture with whole fluke antigen (WFA). Hepatic lymph node cells had a significantly greater response to parasite antigen than mesenteric lymph node cells (P < 0·02), although there was no difference in the magnitude of the proliferative response between naïve and pre‐exposed challenged cattle. Mononuclear cells from hepatic lymph nodes produced interferon gamma, interleukin 2 and interleukin 4 after culture with parasite antigen, indicative of a mixed, T helper type 0, response. Comparison of the hepatic node response to a variety of F. hepatica antigens showed that proliferation was lower after culture with cathepsin‐L, than with a high molecular weight fraction, WFA or excretory–secretory antigen. Cell culture supernatant fluid from unstimulated hepatic lymph node cells showed an IgG1 response to antigens of 48, 52–70, 82, 96 and 120–190 kDa on Western blot in pre‐exposed, but not naïve, challenged animals.     

Fasciola hepatica in the rat: immune responses associated with the development of resistance to infection

F. hepatica infections were established in rats and immune responses were monitored during primary and challenge infections. Antibody levels peaked at 3 weeks post-primary infection and at 6 days post-challenge infection. No significant correlation was found between antibody titre and number of flukes recovered at autopsy. Immunoblotting revealed a limited number of immunogenic polypeptides. When antibodies from these reactive bands were eluted and tested by IFA they all gave identical binding patterns: on juvenile fluke sections tegumental syncytium, tegumental cells and gut cells were labelled, while on adult sections the same antibodies labelled gut cells, reproductive tissue, excretory ducts and flame cells. This suggested that these tissues shared a common epitope or range of epitopes. A pronounced eosinophilia was observed throughout the infection period studied and infected liver sections showed massive cellular infiltration. Histochemical and immunocytochemical investigation of infected liver revealed the presence of large numbers of eosinophils, neutrophils, lymphocytes and phagocytes. The implications of these findings, to an understanding of concomitant immunity in the rat are discussed.     

肝片吸虫GST真核表达载体构建及重组蛋白活性分析

目的构建肝片吸虫谷胱甘肽S-转移酶（GST）的真核表达载体,研究重组蛋白的免疫原性。方法以构建好的重组质粒pET30a-FhGST为模板,利用PCR技术扩增肝片吸虫谷胱甘肽S-转移酶基因（GST）,连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-GST,转染Hela细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况。结果重组质粒pEGFP-GST在Hela细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应。结论肝片吸虫GST真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有生物学活性,可做为分子疫苗的候选进行进一步的研究。     

肝片吸虫组织蛋白酶L1基因cDNA序列的克隆与分析

目的　研制肝片吸虫的候选DNA疫苗。　方法　根据国际发表的相关序列设计引物,在引物端加酶切位点,应用RTPCR方法。　结果　成功地将肝片吸虫组织蛋白酶L1(FheCL1)基因cDNA序列克隆进真核表达载体pcDNA3.1中。对所克隆的FheCL1基因序列及其推导的氨基酸序列进行分析,发现其与已发表序列相比,核苷酸序列有4.3%的差异;推导的氨基酸序列有6.9%的差异。两者蛋白质二级结构主要有3个区域的区别,磷酸化位点有10个不同,但共有一由20个疏水氨基酸残基组成的保守区域。　结论　实验构建的pcDNA3.1FheCL1重组质粒是一种新型的抗肝片吸虫DNA疫苗候选重组质粒;肝片吸虫可能存在不同的亚种,两者的FheCL1基因编码的第1～20个氨基酸残基可能组成膜螺旋。     

Humoral responses in mice following vaccination with DNA encoding glutathione S-transferase of Fasciola hepatica: effects of mode of vaccination and the cellular compartment of antigen expression

The humoral responses in mice following vaccination with DNA constructs encoding Fasciola hepatica glutathione S-transferase (GST) have been evaluated. GST47 cDNA was subcloned into two DNA vaccine vectors, VR1012 and VR1020, which direct expression to the cytoplasmic and extracellular compartments, respectively. Expression was confirmed by transfection into COS 7 cells. Groups of mice were vaccinated with these constructs, by either intramuscular injection with the VR1012-or VR1020-based constructs, or intradermal vaccination (with a gene gun) with the VR1020-based construct. Vaccination with the construct designed for secretion resulted in an increased humoral response compared to vaccination with the nonsecretory construct. The level of the total humoral response after vaccination with the secretion construct was not dependent on the route of vaccination. However, the isotype profile of the response differed between the groups; intramuscular vaccination with the construct directing cytoplasmic expression yielded an immuoglobulin (Ig)G2a dominant (Th1-type) response, intradermal vaccination with the secretory construct a IgG1/IgE dominant (Th2-type) response, and intramuscular vaccination with the secretory construct a mixed isotype response. These results demonstrate that the immunogenicity of a DNA vaccine based on Fasciola GST, as well as the isotype of the response against GST, is determined by the mode of vaccine administration.     

Biochemical characterization and localization of Fasciola hepatica 26-28 kDa diagnostic coproantigen

We have previously reported the usefulness of a 26-28 kDa coproantigen of Fasciola hepatica for diagnosis of infection. In this study, the 26-28 kDa coproantigen was biochemically characterized with the aid of monoclonal antibodies (MoAb) in an effort to better understand the biology of the antigen. Differential staining of chromatographically-purified 26-28 kDa coproantigen on SDS-PAGE, under reducing and non-reducing conditions, indicated that the coproantigen was a monomeric, highly glycosylated glycoprotein. Alkaline treatment of the purified coproantigen resulted in an 8 kDa protein core which still contained the epitope recognized by the MoAb. No protease activity was associated with the 26-28 kDa coproantigen. The coproantigen could be cleaved by trypsin without altering the reactive epitope recognized by the MoAb, but was resistant to pepsin digestion. Further, the coproantigen was stable under several different storage conditions. Indirect immunofluorescence on tissue sections of adult flukes indicated that the coproantigen was present in gut cells and tegument. Taken together these results confirm the stability of the 26-28 kDa coproantigen and its usefulness in diagnostic tests for F. hepatica infections.     

Fasciola hepatica: comparison of immature and mature immunoreactive glycoproteins

A comparison of the 35S-methionine metabolically labelled immunoreactive glycoproteins of immature and mature F. hepatica was carried out by one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of rabbits infected for 3 weeks reacted much more strongly with glycoproteins of immature flukes than with glycoproteins of mature flukes as compared to sera of rabbits infected for 9 weeks. Several of the immunoreactive glycoproteins were also released by immature F. hepatica into the culture medium. At least one was a component of the T1 type granules. Analysis of the in vitro translation products of mature F. hepatica indicated that the initial humoral immune response of rabbit hosts may be directed against carbohydrate moieties.     

Fasciola hepatica: development of monoclonal antibodies and their use to characterize a glycocalyx antigen in migrating flukes

Summary Using mice harbouring early Fasciola hepatica infections, six monoclonal antibodies were prepared against a tegumental antigen present in T1 granules and glycocalyx of flukes. Blocking tests indicated that all monoclonals bound the same T1 epitope (or epitopes in close proximity on the antigen molecule), but this was not the determinant recognized by sheep and cattle. Localization of antibody binding at light and electron microscope levels showed that T1-type antigen also occurred in metacercarial tegument and in glycocalyx of gut cells and excretory ducts in juvenile and adult flukes. This indicates that the natural host-antibody response to F. hepatica may be to one antigen early in the infection. Protein A-gold labelling of monoclonal treated fluke sections revealed that the epitope was probably a polypeptide, unmodified by glycosylation in Golgi bodies. When isolated by immunoadsorption and separated electrophoreti-cally under reducing conditions T1-type antigen was found to consist of a polypeptide mol. wt. 50000, possibly linked to smaller entities mol. wt. 25–40 000. Tissue-specific variations in the antigen molecule might be conferred by linkage of different polypeptides or carbohydrate side-chains to an antigenic core polypeptide. A component of T1-type antigen was found to have mol. wt. of 25000, possibly resembling a polypeptide of mol. wt. 24000 from Schistosoma mansoni tegument.     

Flow cytometry analysis of leukocytes response in rats immunized and infected with Fasciola hepatica

Flow cytometry analysis of leukocytes response in rats immunized and infected with Fasciola hepatica. In the present experiment the differences in blood leucocytes between rats immunised intranasally with cDNA encoding F. hepatica GST, challenged with the parasite (group 1) and non-immunised infected rats (group 2) were compared. The number of leukocytes were estimated by flow cytometry on the 1, 5, 9 week after infection. Changes in the level of blood lymphocytes and monocytes were similar, with an increasing tendency in both groups. The level of eosinophils decreased to the 9th week after infection in both groups, however the number of these cells was seven times higher in control rats than in immunised rats.     

The heterolgous protection of rats against a challenge with Fasciola hepatica by prior infection with the nematode Nippostrongylus brasiliensis

Previous infection of rats with Nippostrongylus brasiliensis was shown to result in protection against an oral challenge with Fusciola hepatica metacercariae but not against an intraperitoneal challenge with newly excysted juvenile (NEJ) flukes. The timing of the challenge was important and a double infection with the nematode gave more consistent results than a single. Resistance appeared to be associated with a prior induction of intestinal eosinophilia. Sera from these resistant rats, however, failed to induce eosinophil adherence to NEJ flukes in vitro.     

Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni

Free radicals have previously been shown to kill the immature stages of the trematode, Schistosoma mansoni but their effect on newly excysted juvenile (NEJ) flukes of Fasciola hepatica has not been established. Using acetaldehyde and xanthine oxidase to chemically generate reactive oxygen intermediates (ROI), up to 61% of NEJ were killed but only when exposed to high levels of ROI. At low concentrations of acetaldehyde and xanthine oxidase as sources of reactive oxygen intermediates, only 6-29% of NEJ were killed compared with 70-92% of schistosomula. Incubation with lipopolysaccharide (LPS)-stimulated rat peritoneal lavage cells (PLCs) killed only 7-15% of NEJ whereas 78-87% of schistosomula were killed under the same conditions by a mechanism dependent on the production of reactive nitrogen intermediates. Relative to immature and adult parasites, NEJ expressed 2.5-20-fold lower levels of superoxide dismutase and glutathione S-transferase but no catalase activity was detected. Incubation of NEJ with inhibitors of peroxidases and glutathione metabolism increased the mean killing of NEJ by LPS-stimulated rat PLCs to 40-75%. These results demonstrate that, in comparison to schistosomula of S. mansoni, NEJ of F. hepatica are relatively resistant to killing by free radicals and this resistance could, in part, be due to the activity of oxidant scavenger enzymes of NEJ.     

Protection to Fasciola hepatica in the gut mucosa of immune rats is associated with infiltrates of eosinophils,IgG1 and IgG2a antibodies around the parasites

We investigated the immune effector mechanisms that underlie protection against F. hepatica in the gut wall of immune rats, using (immuno)histochemistry. In the lamina propria of immune Wistar rats, four weeks after oral infection, frequencies of IgE-positive cells, eosinophils and mucosal mast cells were significantly increased, compared with naïve rats. These factors represent the traditional effector mechanisms against helminths. No significant differences were detected between the two groups in frequencies of IgM-, IgG2a-, IgG1- and IgA- positive cells, CD4- and CD8-positive cells, NK cells, macrophages, neutrophils or goblet cells. Upon challenge of immune rats with F. hepatica in an ex vivo gut segment, NEJs that migrated through the (sub)mucosa were coated with IgG1 and IgG2a antibodies and surrounded by eosinophils. No IgE or IgA antibodies were detected on the parasites. The onset of these immune effector responses, two h after challenge, was related to the expression of protection. These results suggest that NEJs are killed by an eosinophil-mediated cytotoxic response involving IgG antibodies. These antibodies were not produced in the intestine, but infiltrated the gut upon challenge. The observed immune effector responses were not restricted to the site where the primary infection is located, namely the small intestine, but were also detected in the large intestine. The presence of the protective immune mechanisms in two other rat strains demonstrates the pivotal importance of these responses, irrespective the genetic background of the host.