Short report: Diagnosis of human fascioliasis: detection of anti-cathepsin L antibodies in blood samples collected on filter paper.
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We have developed an ELISA for the diagnosis of human fascioliasis based on the detection of IgG4 antibodies to Fasciola hepatica cathepsin LI cysteine protease. Use of this assay in the Bolivian Altiplano, a region with a high prevalence of the disease, was hampered by the reluctance of the indigenous population to provide blood. To overcome this problem, we have investigated the method of collecting small quantities of blood from the finger onto filter paper, followed by the elution of antibodies for use in the diagnostic assay. Serum samples and blood samples collected onto filter paper were obtained from 57 individuals living in the village of Cutusuma in 1987 and from 11 individuals in Chijipata in 1996. Analysis of the IgG4-ELISA results revealed that there is highly significant linear relationship (P < 0.001) between the two methods of sampling. Most importantly, a reliable diagnosis was made with the blood-filter samples from Cutusuma, which had been stored for 10 years at 40 degrees C. While some deterioration of the blood-filter samples from Cutusuma had occurred over the 10-year storage period, no deterioration occurred with the Chijipata samples, which were stored for one year. Therefore, the method of collecting blood onto filter paper should prove useful for large-scale epidemiologic studies on human fascioliasis in the Bolivian Altiplano and in other regions where this disease is prevalent. (+info)
Short report: Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase.
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Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detection of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1). In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this immunodiagnostic test and compared its performance with native CL1. Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA. A highly statistically significant correlation (r2 = 0.751, P < 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins. These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F. hepatica eggs. All seronegative patients were also coprologically negative. Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1. Therefore, recombinant CL1 shows excellent potential for the development of the first standardized assay for the sensitive and specific diagnosis of human fascioliasis. Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade. (+info)
The Northern Bolivian Altiplano: a region highly endemic for human fascioliasis.
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The worldwide importance of human infection by Fasciola hepatica has been recognized in recent years. The endemic region between Lake Titicaca and the valley of La Paz, Bolivia, at 3800-4100 m altitude, presents the highest prevalences and intensities recorded. Large geographical studies involving Lymnaea truncatula snails (malacological, physico-chemical, and botanic studies of 59, 28 and 30 water bodies, respectively, inhabited by lymnaeids; environmental mean temperature studies covering a 40-year period), livestock (5491 cattle) and human coprological surveys (2723 subjects, 2521 of whom were school children) were conducted during 1991-97 to establish the boundaries and distributional characteristics of this endemic Northern Altiplano region. The endemic area covers part of the Los Andes, Ingavi, Omasuyos and Murillo provinces of the La Paz Department. The human endemic zone is stable, isolated and apparently fixed in its present outline, the boundaries being marked by geographical, climatic and soil-water chemical characteristics. The parasite distribution is irregular in the endemic area, the transmission foci being patchily distributed and linked to the presence of appropriate water bodies. Prevalences in school children are related to snail population distribution and extent. Altiplanic lymnaeids mainly inhabit permanent water bodies, which enables parasite transmission during the whole year. A confluence of several factors mitigates the negative effects of the high altitude. (+info)
Ionic mechanisms underlying spontaneous muscle contractions in the liver fluke, Fasciola hepatica.
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Spontaneous contractions of liver fluke muscle were abolished in Ca(2+)-free saline and by 100 microM nifedipine and reduced by 5 mM cadmium chloride, suggesting that they are dependent on extracellular Ca(2+). Caffeine (5 mM) significantly increased contraction amplitude and frequency. Ryanodine (100 microM) failed to block the caffeine response but significantly reduced spontaneous contraction frequency, suggesting that intracellular stores have a functional role. Cyclopiazonic acid (5 microM) had no effect on the caffeine response or spontaneous activity. 3-Isobutyl-1-methylxanthine (IBMX), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate significantly increased spontaneous contractions, which implies that cAMP has a regulatory function in motility. Caffeine, however, produced no measurable increase in cAMP. The caffeine effect was inhibited by cadmium chloride and nifedipine, whereas IBMX-induced increases in amplitude were reduced by cadmium chloride. Thus caffeine and cAMP appear capable of opening plasma membrane Ca(2+) channels, but the involvement of cAMP in caffeine responses has not been proved. (+info)
Immunochemical characterization and diagnostic potential of a 63-kilodalton Schistosoma antigen.
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Schistosoma circulating antigens were used for the detection of active infection. Anti-S. mansoni IgG2a monoclonal antibody (MAb) designated C5C4 was generated. The target epitope of this MAb was detected in adult worms, eggs, and cercariae antigenic extracts of S. mansoni and S. haematobium, had a molecular size of 63 kD, and was not detected in Fasciola hepatica and Ascaris. In addition, a 50-kD degradation product was identified only in the urine of infected individuals. Analysis by high-performance liquid chromatography of the purified antigen demonstrated only one peak. The 63-kD antigen was characterized as a protein containing 40.4% hydrophobic, 7.5% acidic, and 8.8% basic amino acids. The C5C4 MAb was used in a Fast Dot-ELISA for rapid and simple diagnosis of human schistosomiasis. The 63-kD circulating antigen was detected in 92% of urine samples from 330 S. mansoni-infected individuals, with 16% false-positive results among 130 noninfected individuals. (+info)
Fasciola hepatica suppresses a protective Th1 response against Bordetella pertussis.
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Fasciolosis, like other helminth infections, is associated with the induction of T-cell responses polarized to the Th2 subtype. Respiratory infection with Bordetella pertussis or immunization with a pertussis whole-cell vaccine (Pw) induces a potent Th1 response, which confers a high level of protection against bacterial challenge. We have used these two pathogens to examine bystander cross-regulation of Th1 and Th2 cells in vivo and provide evidence of immunomodulation of host T-cell responses to B. pertussis by a concomitant infection with Fasciola hepatica. Mice with a coinfection of F. hepatica and B. pertussis exhibited a Th2 cytokine profile in response to F. hepatica antigens, similar to those infected with F. hepatica alone. By contrast, the Th1 response to B. pertussis antigens was markedly suppressed and the bacterial infection was exacerbated following infection with F. hepatica. Furthermore, an established Th1 response induced in mice by infection with B. pertussis or by parenteral immunization with Pw was also suppressed following infection with F. hepatica. This immunomodulatory effect of B. pertussis-induced responses by F. hepatica infection is significantly reduced, but not completely abrogated, in IL-4 knockout mice. Our findings demonstrate that Th2-inducing parasites can exert bystander suppression of protective Th1 responses to infection or vaccination with a bacterial pathogen and that the modulation is mediated in part by IL-4 and, significantly, is effective at both the induction and effector stages of the Th1 response. (+info)
Fascioliasis: sonographic abnormalities of the biliary tract and evolution after treatment with triclabendazole.
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Diagnosis of infection with the liver fluke Fasciola hepatica is usually difficult. Ultrasonography (US) might be a useful diagnostic alternative, and we assessed the value of sequential US in the diagnosis and monitoring of fascioliasis in 76 patients at baseline and for 60 days after treatment with triclabendazole. At baseline, biliary abnormalities were observed in 52 patients. Crescent-shaped parasites were seen in 11 patients; in 2 cases parasites were spontaneously moving and in 4 patients parasites were motionless. Postprandial examination revealed parasites adhering to the gallbladder wall in a further 5 cases. In 3 further cases, gallbladder contents were mobile but did not sediment downwards after patients changed position. Non-specific abnormalities were: impaired gallbladder contractility (n = 23), gallbladder tenderness (n = 19), debris (n = 6), calculi (n = 5), wall thickening (n = 2) and bile duct dilatation (n = 12). During day 1-7, Fasciola-like crescents in the gallbladder or passing through the bile duct were detected in another 15 patients, impaired gallbladder contractility in 16, gallbladder tenderness in 16, and bile duct dilatation in an additional 28 patients. Thirty-two patients with these US abnormalities experienced colic-like abdominal pain accompanied by increased alkaline phosphatase in 25 cases. During day 30-60, abnormalities regressed completely in 45 patients; 2/6 triclabendazole failures were evident by detection of living parasites. Biliary tract abnormalities are frequently observed by US, but the detection-rate of Fasciola hepatica is disappointingly low despite the parasite's relatively large size. US findings must therefore be interpreted together with other clinical measurements. The visualization of parasites being expelled through the dilated common bile duct allowed the causal interpretation of post-therapeutic abdominal pain and increase of liver enzymes. When triclabendazole is given on suspicion, visualization of worm expulsion and bile duct dilatation by US may be used to confirm diagnosis. (+info)
Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica.
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The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects. (+info)