Identification and characterization of a major Schistosoma mansoni glycoprotein antigen cross-reactive with Fasciola hepatica

A major surface antigen of Schistosoma mansoni has been identified and characterized as a glycoprotein of 66,000 molecular weight (Mr) and isoelectric point of 6.2-6.1 (SM66-GP) by use of a monoclonal antibody. The antigen was expressed by schistosome eggs, cercariae, larvae, and adults, and was recognized by sera of schistosome infected hosts. Direct immunofluorescence microscopy showed the antigen was distributed in a uniform pattern on the entire worm surface. Indirect immunofluorescence microscopy revealed that it was present in the parenchymal tissue of immature and mature Fasciola hepatica, in the gut of the mature fluke, and in embryonated fasciola eggs. The cross-reactive F. hepatica epitope recognized was expressed on a polypeptide of Mr 220,000.     

A Schistosoma mansoni surface glycoprotein cross-reactive with a T1 antigen of Fasciola hepatica

An antigen recognized by monoclonal antibody 306B3/5 and present on the surface of S. mansoni was expressed by F. hepatica in the pattern typical of T1 antigens. A single polypeptide of molecular weight (Mr) 160,000 was precipitated from metabolically labeled concanavalin A-binding F. hepatica glycoproteins, whereas multiple polypeptides ranging from Mr greater than 200,000 to Mr 45,000 were precipitated from metabolically labeled glycoproteins of male S. mansoni. The polypeptides of both species were also precipitated by sera of infected hosts, and may account for some of the serological cross-reactivity between S. mansoni and F. hepatica in immunodiagnostic assays. These antigens may also represent potentially immunoprophylactic reagents.     

Successful vaccination against murine Schistosoma mansoni infection with a purified 12 Kd Fasciola hepatica cross-reactive antigen

A 12 Kd antigen was isolated from Fasciola hepatica adult worm extracts by gel filtration in Sephadex G-50 and ion exchange chromatography using DEAE Sephadex A-120. Mice immunized with this Fasciola-derived 12 Kd antigen developed antibodies to Schistosoma mansoni adult worm extracts, demonstrating its cross-reactivity with schistosomes. Vaccination of mice with microgram amounts of the antigen in Freund's adjuvant induced up to 77% reduction in worm burdens after challenge with S. mansoni cercariae. F. hepatica 12 Kd degraded by proteinase K to lower molecular weight polypeptides which still retain their antigenicity as determined by the enzyme-linked immunoelectrotransfer blot technique. Treatment with either Endoglycosidase H, neuraminidase, or dithiothreitol had no effect on its mobility in sodium dodecyl sulfate polyacrylamide gels, or in its recognition by antibody, suggesting the absence of carbohydrate moieties or disulphide bonds in relation to its antigenic determinants and also suggesting that the antigen is a pure polypeptide. These studies establish that a molecularly defined cross-reactive component of one parasitic trematode (F. hepatica) induces resistance to challenge infection with another parasitic trematode (S. mansoni). Its polypeptide nature makes recombinant DNA technology an alternative for the manufacture of a vaccine.     

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.     

Demonstration of a common antigen between Schistosoma mansoni and Fasciola hepatica

The presence of a common antigen between Schistosoma mansoni eggs and Fasciola hepatica adult worms was demonstrated by utilizing in an anti-S. mansoni adult worm antiserum. Although not one of the three major serologic S. mansoni egg antigens, its complete cross-reactivity suggests that serologic tests done with these crude antigenic extracts will result in many false-positive cases in areas where both parasites are endemic.     

Acquired resistance to Fasciola hepatica in cattle using a purified adult worm antigen

Calves were immunized twice in 4 weeks with a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen (denoted FhSmIII(M)) isolated from F. hepatica adult worm extracts by antibody affinity chromatography and challenged 7 weeks later with F. hepatica metacercariae. Flukes were recovered at 16 weeks of infection at which time the immunized calves had 55% less F. hepatica than the controls. All of the immunized calves developed high antibody levels of FhSmIII(M), detectable in the ELISA, by 4 weeks after a single immunization. By 9 weeks of infection with F. hepatica the immunized calves had lower sorbitol dehydrogenase levels than the unimmunized, F. hepatica-infected control calves, indicating less liver damage in the vaccinated group. These studies demonstrate that subcellular F. hepatica macromolecules cross-reactive and cross-protective against S. mansoni also have the potential to serve as vaccines in cattle exposed to this parasitic disease.     

肝片形吸虫感染宿主细胞免疫的研究进展

肝片形吸虫病是一种危害严重的人兽共患病,肝片形吸虫主要感染反刍动物及包括人类在内的哺乳动物,是一个严重威胁健康、影响经济发展的公共卫生问题。目前治疗肝片形吸虫病的药物少、疗效有限。大量研究表明,肝片形吸虫受宿主免疫应答的调控,本文对肝片形吸虫病的发病机制、肝片形吸虫主要参与诱导的几种机体细胞免疫反应及相关的研究进展进行综述.以期为肝片形吸虫病的临床治疗提供资料。     

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low-dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL-4 and IL-5, and low levels of IFN-gamma and IL-2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high-dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL-4 defective (IL-4-/-) C57BL/6 mice infected with 5 metacercariae produced less IFN-gamma and more IL-5 compared to their wild-type C57BL/6 counterparts, suggesting that IL-4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN-gamma and IL-2 was completely suppressed in the high-dose infection and this was also observed in IL-4-/- mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL-4 and IL-5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.     

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.     

Cell mediated immunity to liver fluke antigens during experimental Fasciola hepatica infection of cattle

Summary Cell mediated immunity (CMI) to Fasciola hepatica antigens was detected by lymphocyte proliferation and interleukin-2 (IL-2) production tests in cattle during the first 4 weeks following liver fluke infection. From the fifth week of infection onwards peripheral blood lymphocytes were unresponsive to fluke antigens by these in vitro tests. Investigations into the cause of this unresponsiveness found no evidence to suggest a selective loss of the IL-2 producing lymphocyte sub-population or that macrophages were responsible for the suppression or that antigen responsive cells were being sequestered in the spleen and mesenteric lymph nodes. Tests carried out on culture supernatants demonstrated the production during this unresponsive period of factors capable of suppressing in vitro responses to PHA. Although further tests failed to show antigen specific suppressor factors the presence of MHC restricted suppressor factors could not be ruled out. The early and transient appearance of CMI during F. hepatica infection of cattle indicates that delayed type hypersensitivity is unlikely to be important in protective immunity in cattle.     

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: 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.     

Radiation-resistant acquired immunity of vaccinated mice to Schistosoma mansoni

Vaccination of mice with attenuated cercariae of Schistosoma mansoni induces specific acquired resistance to challenge infection. This resistance is immunologically-mediated, possibly via a delayed-type hypersensitivity. Studies of parasite migration have shown that the protective mechanism operates most effectively in the lungs of vaccinated mice. We have probed the mechanism by exposing mice to 500 rads of gamma radiation before challenge infection. Our results show that the effector mechanism operative against challenge larvae is resistant to radiation. In contrast, classical immune responses are markedly suppressed by the same treatment. While leukocyte populations in the blood fall dramatically after irradiation, numbers of cells recoverable by bronchoalveolar lavage are unaffected. We suggest that vaccination with attenuated cercariae establishes populations of sensitized cells in the lungs which trigger the mechanism of resistance when challenge schistosomula migrate through pulmonary capillary beds. Although the cells may be partially disabled by irradiation, they remain responsive to worm antigens and thereby capable of initiating the elimination mechanism. This hypothesis would explain the radiation resistance of vaccine-induced immunity to S. mansoni.     

Fasciola hepatica:Sp2/0 (helminth:myeloma) hybridoma expressing parasite antigen

Cells from adult Fasciola hepatica were fused with cells from a murine BALB/c myeloma Sp2 line. The hybrid cells were grown in HAT (hypoxanthine, aminopterin, and thymidine) medium, cloned and subcloned, and shown to express parasite antigen for 1 year after fusion. Expression of parasite antigen was demonstrated by the following: 2 histogram flow cytometric analyses, in which a population of hybrid cells in the population of 7 month cultured hybrid cells showed 57% more fluorescence when treated with an anti-F. hepatica serum followed by anti-rabbit immunoglobulin G coupled to fluorescein isothiocyanate as compared with the same hybrid cells washed and treated with normal rabbit serum; Sp2 myeloma cells treated with an anti-F. hepatica serum or normal rabbit serum followed by fluorescein-labeled anti-rabbit IgG had the same negative fluorescence; BALB/c mice immunized with PBS-washed cells from a subclone of these hybridomas developed anti-F. hepatica antibodies (shown by the Falcon assay screening test enzyme-linked immunosorbent assay); and antibodies recognized an F. hepatica antigenic polypeptide of 57,000 Mr in a Western immunoblot. These helminth:myeloma hybrids expressed murine host markers, further confirming the hybrid nature of this cell line. F. hepatica cells alone, like their Sp2 fusion partners, die in HAT supplemented medium by 9 days of culture. F. hepatica:Sp2 hybridomas have been grown continuously in HAT medium for greater than 1 year.     

Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen.

Paramyosin (Sm97), a 97-kDa myofibrillar protein identified by the unusually monospecific antibody response induced by intradermal vaccination of mice with a complex soluble worm antigen preparation (SWAP) of adult Schistosoma mansoni administered with bacillus Calmette-Guérin (BCG), was purified and tested for its capacity to protect mice against challenge infection. When administered intradermally with BCG at total doses of only 4-40 micrograms per mouse, both the native molecule and a recombinant expression product containing approximately 50% of the whole protein were found to confer significant resistance (26-33%) against challenge infection, while 2 mg of unfractionated SWAP was required to induce similar levels of protection. In addition, paramyosin was shown to stimulate T lymphocytes from vaccinated mice to produce lymphokines [e.g., gamma interferon (IFN-gamma)] that activate macrophages to kill schistosomula. Neither schistosome myosin nor a heterologous paramyosin from a different invertebrate genus were protective, indicating a requirement for specific epitopes in the immunization. That the protection induced by paramyosin involves a T-cell-mediated mechanism was supported by the failure of anti-paramyosin antibodies to passively transfer significant resistance to infection to recipient mice. Lymphocytes from mice vaccinated with paramyosin were found to produce IFN-gamma in response to living schistosomula, suggesting that during challenge infection of vaccinated hosts, paramyosin (a nonsurface antigen) may elicit a protective T-cell response as a consequence of its release from migrating parasite larvae. Paramyosin-depleted SWAP was also found to be protective as well as stimulatory for T lymphocytes from SWAP-vaccinated mice, indicating that other antigens in this preparation may have immunoprophylactic potential. In summary, these results (i) suggest that the induction of T-cell-dependent cell-mediated immunity against soluble nonsurface antigens may be an effective strategy for immunization against multicellular parasites and (ii) in the case of schistosomes, identify paramyosin as a candidate vaccine immunogen in this category.     

Sm480: a high molecular weight Schistosoma mansoni antigen associated with protective immunity

Rabbit antisera were raised against an antigen present in Schistosoma mansoni adult worms and eggs, and were shown to yield a single immunoprecipitin arc in immunoelectrophoresis and immunodiffusion against S. mansoni egg and worm antigen extracts. The antisera conferred partial but significant protection (22–30%) against a S.  mansoni challenge when transferred to mice five and six days after the mice had been infected percutaneously with 200 cercariae. The egg and the worm forms of the antigen were immunologically cross-reactive, but the egg antigen possessed peptidolytic activity that could be inhibited with serine protease inhibitors. In indirect immunofluorescence the rabbit antisera reacted with surfaces of cercariae, five-day old lung-stage schistosomula, miracidia and praziquantel-treated adult worms. Gel-filtration chromatography demonstrated a relative molecular size of approximately 480 kDa for both the egg and worm forms of the antigen, and lectin-affinity chromatography indicated both were glycosylated. The serine protease activity and large relative molecular size of egg Sm480 were confirmed by a combination of radiolabelling with tritiated di-isopropyl fluorophosphate, immunoelectrophoresis and polyacrylamide gel electrophoresis.     

Living Fasciola hepatica in biliary tree: a case report

Here we report a rare case of living Fasciola hepatica in biliary tract. The patient was in acute phase of infection and treated successfully with 10 mg/kg oral triclabendazole after the fluke was extracted using endoscopic retrograde cholangiopancreatography (ERCP).