Oxidative status and changes in the adenosine deaminase activity in experimental host infected with tropical liver fluke, Fasciola gigantica.

Fine tuning of the metabolic, physiological and immunological cues along with interplay between the biomolecules of the host and the parasite could be responsible for the successful establishment of parasitic infections. The present investigation was aimed at evaluating the oxidative status and the level of adenosine deaminase (ADA) in the serum and liver of rabbits experimentally infected with Fasciola gigantica. A significant increase in level of ROS, MDA and 4-HNE along with a decline in the SOD, CAT, GR and GST activity was evident in rabbits experimentally infected with Fasciola gigantica. However, there was an increase in the GPX activity in the sera of infected rabbits. The increased GPX activity and decreased GR activity would have resulted in the depletion of GSH, a key non-enzymatic antioxidant, in the infected animals. The level of GSSG was also found to be higher in the sera and liver tissues of the infected rabbits along with a decline in the GSH/GSSG ratio, indicating a high level of oxidative stress in the infected animals, which also showed a significant increase in the activity of the marker enzymes of liver pathology, AST and ALT. Further, a significant inhibition of the adenosine deaminase (ADA) activity in the infected rabbits was accompanied with the reduction in the level of pro-inflammatory cytokine, IL-6 while the anti-inflammatory cytokine, IL-4 level was significantly elevated. In conclusion, the F. gigantica induced significant oxidative stress as evident from the increased levels of ROS and lipid peroxidation along with the disruption of antioxidant and detoxification cascade ultimately lead to pathogenic and inflammatory responses in the experimental host. Whereas, the altered ADA activity could modulate the host's immune responses toward Th-2 type and would facilitate the successful establishment of flukes within their host, thus indicating that ADA could be exploited as a target for the development of novel anthelmintic drugs against fasciolosis.

Newly excysted juveniles (NEJs) of Fasciola gigantica induce mice liver fibrosis and M2 macrophage-like phenotype in vivo.

Liver flukes of animals are parasitic flatworms of major socioeconomic importance in many countries. Particularly, Fasciola gigantica is a leading cause of production losses to the livestock (mainly sheep and cattle) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Immune responses induced by helminth have been extensively studied, but there is limited information on this aspect by F. gigantica, especially on macrophages induced with this parasite. Studies have shown that host immune responses induced by parasitic infection is greatly correlated with the macrophage polarization axis. In the present study, we used the murine model of F. gigantica to explore the interaction of host and F. gigantica. We found F. gigantica NEJs promoted pathology and fibrosis of mice liver, and the enlargement of mice spleen. We also showed that macrophages were recruited to mice peritoneal cavity at 5 days post infection. By evaluating the expression of genetic markers of M2 macrophages such as Arg-1, Ym1 and RELMɑ, and genetic marker of M1 macrophages iNOS, we showed that M2 macrophages were induced by F. gigantica. M2 macrophages are central to the immune response during helminth infection, and our findings in this study provided insight into the immune interaction between F. gigantica and host.

Purification and characterization of two-domain glutaredoxin in the parasitic helminth Fasciola gigantica.

Glutaredoxins (Grxs) are small thiol-dependent proteins and key elements of redox signaling as they regulate the redox state of important cellular proteins. In the present study, the complete sequence of a glutaredoxin protein, obtained from the liver fluke Fasciola gigantica, was PCR-amplified and cloned. The 690-bp open reading frame (ORF) encodes a 230-amino acid protein with two conserved domains (FgGrxD1 and FgGrxD2) and has similarities with two monothiol Grxs of Saccharomyces cerevisiae, i.e., ScGrx3 and ScGrx4. The full-length FgGrx along with its two constituent domains were overexpressed in Escherichia coli as hexahistidyl-tagged proteins. The affinity chromatography resulted in almost pure and soluble proteins. The full-length FgGrx and the FgGrxD2 showed reddish-brown color, indicating the presence of bound iron in the second domain. In the insulin based reduction assay, both FgGrx and FgGrxD2 containing the active site motif CGFS exhibited a weak reducing activity, whereas FgGrxD1 was inactive. Additionally, FgGrx did not show any GSH-disulfide transhydrogenase activity when 2-hydroxyethyl disulfide (HED) or de-hydroascorbate (DHA) were taken as substrates. These results indicated the probable role of FgGrx in cellular iron-sulfur homeostasis. FgGrx was found to be reversibly S-glutathionylated, suggesting a potential redox regulation that is likely to take place at the active site Cys158. Since there is only one Cys in FgGrxD2, the Cys158 might be involved in FeS binding. This study is the first report on the presence of Grx in platyhelminthic parasites and provides a starting point for further characterization of the redox network in liver flukes.

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

Role of oxidative stress in concomitant occurrence of Fasciola gigantica and leiomyoma in cattle.

Fasciola gigantica is a parasitic helminth that predominantly infects the liver and bile ducts of cattle and causes great losses of cattle production in the southwestern regions of Iran. The purpose of the present study was to find out the possible relationship between the extent of liver destruction and oxidative stress by estimating the level of MDA, and SOD and GPX enzymes in the liver of cattle infected with F. gigantica. Studies were carried out on 49 infected and 20 healthy livers. Based on the results, the SOD activity of the infected livers was substantially lower than those of the healthy ones. As in the livers with more damage, a significant decrease was observed in the SOD activity. Although the GPX activity of the control livers and the livers with low damage showed no statistically significant variation this value was significantly different between the livers with high injury and the control ones. The MDA level of the infected animals was significantly higher than the non-infected animals. In addition, the level of MDA production showed a parallel correlation with the severity of infection. By histological examination, the lesions in infected livers were varied from tortuous, organized fibrotic areas in or around the hepatic lobules, cirrhosis, and chronic catarrhal cholangitis to fresh migratory tunnels filled with RBC and eosinophils. Multifocal and relatively homogenous populations of densely packed spindle cells with blunt-ended nuclei, arranged in broad interlacing fascicles were noted in samples of 10 infected livers. Based on the histological features by routine and special staining and the results of the immune labeling, the tumors were diagnosed as hepatic leiomyoma. To the best of our knowledge, this is the first report on the concomitant occurrence of Fasciola gigantica infection and leiomyoma in cattle. Therefore, there is considerable evidence indicating that the severity of hepatic damage in fasciolosis is causally associated with the extent of intrahepatic oxidative stress. Future research is necessary in order to clarify the complex host-parasite interactions and to better define the oncogenic implications of F. gigantica infection.

Neurological and ocular fascioliasis in humans.

Fascioliasis is a food-borne parasitic disease caused by the trematode species Fasciola hepatica, distributed worldwide, and Fasciola gigantica, restricted to given regions of Africa and Asia. This disease in humans shows an increasing importance, which relies on its recent widespread emergence related to climate and global changes and also on its pathogenicity in the invasive, biliary, and advanced chronic phases in the human endemic areas, mainly of developing countries. In spite of the large neurological affection capacity of Fasciola, this important pathogenic aspect of the disease has been pronouncedly overlooked in the past decades and has not even appear within the numerous reviews on the parasitic diseases of the central nervous system. The aim of this wide retrospective review is an in-depth analysis of the characteristics of neurological and ocular fascioliasis caused by these two fasciolid species. The terms of neurofascioliasis and ophthalmofascioliasis are restricted to cases in which the direct affection of the central nervous system or the eye by a migrant ectopic fasciolid fluke is demonstrated by an aetiological diagnosis of recovered flukes after surgery or spontaneous moving-out of the fluke through the orbit. Cases in which the ectopic fluke is not recovered and the symptoms cannot be explained by an indirect affection at distance may also be included in these terms. Neurofascioliasis and ophthalmofascioliasis cases are reviewed and discussed. With regard to fascioliasis infection giving an indirect rise to neurological affection, the distribution and frequency of cases are analysed according to geography, sex, and age. Minor symptoms and major manifestations are discussed. Three main types of cases are distinguished depending on the characteristics of their manifestations: genuine neurological, meningeal, and psychiatric or neuropsychic. The impressive symptoms and signs appearing in each type of these cases are included. Brain examination techniques and neuroimaging useful for the diagnosis of neurological cases are exposed. Within fascioliasis infection indirectly causing ocular manifestations, case distribution and frequency are similarly analysed. A short analysis is devoted to clarify the first reports of a human eye infection. The affection of related and close organs is discussed by differentiating between cases of the dorsal spine, pulmonary manifestations, heart and vessel affection, findings in blood vessels, skin and dermatologic reactions, cases of ectopic mature flukes, and upper body locations. The clinical complexity of the puzzling polymorphisms, the disconcerting multifocality of the manifestations, and their changes along the evolution of the disease in the same patient, as well as the differences between the clinical pictures shown by different patients, are highlighted. The many syndromes involved are enumerated. The pathogenic and physiological mechanisms underlying neurofascioliasis and ophthalmofascioliasis caused by ectopic flukes and the physiopathogenic processes indirectly affecting the central nervous system and causing genuine neurological, meningeal, psychiatric, and ocular manifestations are discussed. The diagnosis of neurological and ophthalmologic fascioliasis is analysed in depth, including clinical and paraclinical diagnosis, eosinophilia in the blood and cerebrospinal fluid, differential diagnosis from other parasitic infections such as helminthiases and myiases, an update of human fascioliasis diagnosis, and fluke and/or fluke egg recovery by surgery. Diagnostic analyses with faecal and blood samples for fascioliasis patients are updated. Therapy for patients with major neurological manifestations includes both antiparasitic treatments and anti-inflammatory therapeutics. Prognosis in fascioliasis patients with neurological manifestations is discussed, with emphasis on sequelae and fatal cases, and the care of patients with ophthalmologic manifestations is added. Conclusions indicate that neurological cases are overlooked in human fascioliasis endemic areas and also in developing countries in general. In remote zones, rural health centres and small hospitals in or near the human endemic areas do not dispose of the appropriate equipments for neurological analyses. Moreover, physicians may not be aware about the potential relationship between liver fluke infection and neurological implications, and such cases may therefore remain misdiagnosed, even in developed countries. Priority should henceforth be given to the consideration of neurological and ocular affection in human endemic areas, and efforts should be implemented to assess their characteristics and frequency. Their impact should also be considered when estimating the global burden of fascioliasis.

Estrogen-related fecundity reduction of Lymnaea ollula following Fasciola gigantica infection.

Infection by trematode parasites generally affects life history traits of their intermediate hosts. Reduction in life expectancy and reproductive capacity have previously been documented in Fasciola gigantica-infected Lymnaea ollula, but the influence of the endocrine system on this specific host­parasite interaction has not been previously examined. In the present study, we observed survival, growth pattern, and reproductive output of L. ollula following exposure to F. gigantica. Both the survival and the growth pattern of infected snails were similar to those of the non-infected control group. However, a significant difference was apparent in fecundity, as infected snails consistently showed lower levels of egg and embryo production throughout a 7-wk observation period. Semi-quantitative RT-PCR also revealed down-regulation of estrogen receptor expression in infected snails during the first 4 wk of infection. Nonetheless, the inhibition of estrogen signaling was transient, as they regained expression in the later phase of infection. It is, therefore, suggested that other hormones of the complex endocrine system may be involved in the reduced fecundity of L. ollula following F. gigantica infection.

Fasciola gigantica: cercarial shedding pattern from Lymnaea natalensis after long-term exposure to cadmium at different temperatures.

Little is known about the response of Fasciola spp., inside its snail host to pollutant toxicity. The effect of long-term exposure of Lymnaea natalensis to 0.1 microg/l, 10 microg/l, or 100 microg/l cadmium (Cd) on the infection rates with F. gigantica, percentage of cercariae-shedding snails and cercariae shedding pattern was investigated. The snails were exposed to Cd for 7 days either pre-infection with single Fasciola miracidium or during the late pre-patent period of the infection. The possible interaction between metal exposure and acclimatization temperature was also studied in three ranges; 16-18 degrees C, 23-25 degrees C and 30-32 degrees C. Results clearly showed that host exposure to 100 microg/l Cd significantly affected the pattern of Fasciola development inside its snail host, and that the acclimatization temperature was a key factor affecting the role played by Cd. Pre-infection exposure to Cd caused a significant reduction in the infection rates where the effect was temperature-dependent. Post-infection exposure to Cd significantly increased the percentage of cercariae-shedding snails; the effect was temperature-independent. Interestingly, post-infection exposure significantly altered the differential cercarial output pattern, while no clear impact on the total was found. The alteration in the differential cercarial output was represented as a significantly higher proportion of floating metacercaria as compared to fixed ones and also higher proportion of dead cercariae which may directly reduce the transmission of Fasciola to the final host. The mechanisms of cadmium impact are briefly discussed.

Zoonotic helminth infections of humans: echinococcosis, cysticercosis and fascioliasis.

PURPOSE OF REVIEW: Tissue parasites of humans are still prevalent in most regions of the world, and are also seen more frequently in developed countries due to increasing travel patterns. In particular, Echinococcus infections still account for hepatic and pulmonary pathology, cysticercosis is a major cause of seizures and epilepsy, and fascioliasis also causes significant liver pathology. This review summarizes current knowledge on clinical and epidemiologic aspects of zoonotic disease caused by tissue helminths. RECENT FINDINGS: Tissue helminth infections remain as a public health concern. Recent research has provided new insights into clinical disease in humans and improved methods for diagnosis, treatment and control, arising mostly from the application of new techniques for immune and molecular diagnosis, availability of data from controlled trials, and development of new vaccines. Specific antiparasitic therapies are now better characterized, and new control tools are available. SUMMARY: Recent research has provided new diagnostic technologies applicable to diagnosis, treatment and control, but effective interventions to reduce transmission are rarely applied. Despite some progress in their control, these zoonoses continue to be a major public health problem in many regions both in developing countries and in some more developed ones.

[Proteases in helminthic parasites]. / Les protéases chez les helminthes.

Proteases catalyse the cleavage of internal peptide bonds within peptides and proteins. They are classified into four major classes and are involved in a broad range of eukaryotic processes. Proteases have also been found to play a number of critical roles in the virulence of pathogenic agents, particularly of nematode parasites. Parasitic proteases are involved in different aspects of host-parasite interactions. They facilitate the invasion of host tissues and allow nutrition as well as the survival of the parasite in its host. Proteases also participate in the parasite's evasion from the host's immune response. The functional diversity and complexity of these enzymes are described in this review, with a particular focus on the principally identified proteases of four helminths: Schistosoma sp., Fasciola sp., Taenia sp. and Haemonchus sp. Some of these proteases, especially the cysteine proteases secreted by the parasitic trematode Fasciola hepatica, have been successfully tested in experimental immunodiagnosis. Proteases identified in different parasites are currently under study for a use as recombinant vaccines. In this respect, proteases are proposed as major potential targets for immunotherapy and chemotherapy against parasitic diseases.

Morphological comparisons and hypotheses on the origin of polyploids in parthenogenetic Fasciola sp.

It is known that Fasciola sp. from Japan and the Republic of Korea consist of diploids (2n = 2x = 20), triploids (2n = 3x = 30), and mixoploids with diploid and triploid cells. Triploids are distributed over Asia and Hawaii. Abnormal spermatogenesis and parthenogenetic reproduction are the main characteristics of Fasciola sp. Here we measured 21 different morphological parameters of diploid and triploid flukes of Fasciola sp. obtained from Japan and the Republic of Korea. Statistical analysis showed that diploid and triploid flukes were morphologically different. No bivalents or trivalents could be detected in diploid and triploid flukes, respectively. Based on our findings, we speculate that parthenogenetic diploids, triploids, and mixoploids (2x/3x) of Fasciola sp. are genetically related to each other.

Abnormal spermatogenesis in the common liver fluke (Fasciola sp.) from Japan and Korea.

Diploid and triploid specimens of Japanese and Korean Fasciola sp. showed abnormality in their spermatogenesis. Live germ cells obtained from the testes were observed under a differential interference contrast microscope. In the stages from spermatogonium to spermatid, the cells combined together at the central cytoplasmic bridge during a series of divisions. One spermatogonium becomes a cell group of 8 primary spermatocytes through 3 mitoses. Until the primary spermatocyte stage, cells are divided in a uniform manner. In most of the diploid specimens, the primary spermatocytes are irregularly divided into non-uniform secondary spermatocytes, however, some specimens perform a regular division. In the majority of triploid flukes, the primary spermatocytes are divided in a regular pattern, but some of the specimens perform an irregular division. The non-uniform spermatids do not perform a spermiogenesis. In the diploid specimens, no spermatozoa were found that were produced by spermiogenesis. Whereas in the triploid specimens, some spermatids distributed uniformly on the surface went through a spermiogenesis. We observed some moving spermatozoa in one triploid specimen. The spermatozoa possibly retain their normal reproductive function.

Compartive studies on the effect of bithionol, praziqantel and triclabendazole in rabbit's fascioliasis. 1. Parasitological studies.

Rabbits were infected with 30 metacercariae each. They were then divided into four batches each of two groups. One batch served as controls and the others were treated each with a drug. The first group from each batch was treated when worms were still immature, the other group was treated after maturity. The effect of the drugs was monitored by: (1) Stool examination for eggs and studying the percent of egg reduction. (2) Worm recovery at autopsy and calculating the percent of the drug efficacy. (3) Culturing some eggs and following their embryonation and hatching. Results pointed out that Triclabendazole was the best fasciolicide followed by Bithionol. Praziquantel had a negligible effect.

Liver and intestinal flukes.

A number of trematodes besides schistosomes parasitize humans and domesticated animals. Although they do not have as great a public health impact as schistosomiasis, they are prevalent in Southeast Asia as well as among the greater than 1 million immigrants from this region to North America. The human biliary flukes include C. sinensis, O. viverrini, and O. felineus. These chronic infections are often asymptomatic but over time may cause biliary thickening, cholangitis, and a predisposition to cholangiocarcinoma. Zoonotic trematode infections include the sheep liver fluke F. hepatica and the intestinal flukes Fasciolopsis, Echinostoma, Heterophyes, and Metagonimus.

Separation of outer cysts from metacercariae of Fasciola gigantica in an aqueous two-phase system.

The outer cysts (Oc) of Fasciola gigantica metacercariae (Mc) were separated in an aqueous two-phase system composed of polyethylene glycol and dextran in distilled water. A large number of separated Mc without Oc were recovered in the bottom layer rich in dextran in the system. Ten separated Mc in the layer were used to inoculate the mouse with a syringe and juveniles were recovered from the animals on the 14th day. When the number of juveniles recovered was compared with that from the mice infected orally with the same number of Mc, there was a significant difference between them (P < 0.05). The former method is easier than the latter for inoculation.

Intraocular fascioliasis: a case report.

We report the case of a 28-year-old male who had been ill with headache and motor weakness for a month and developed sudden pain and blindness of the right eye. During ophthalmoscopy, a worm was recognized penetrating the iris, occupying the anterior chamber for a brief period, returning back behind the iris, and leaving corneal edema with hyphyema. Enucleation was performed to prevent the worm's escape from the eye. The enucleated eye revealed areas of a focal degeneration of sclera and intraocular hemorrhage. Microscopic findings of an abrupt tissue defect and few inflammatory reactions in the uvea suggested very recent migration of a moving worm. The flatworm detected in the anterior chamber was identified to be a juvenile Fasciola sp. This case is presumed to be the first case of intraocular fascioliasis reported in the literature.

Several observations concerning cercarial sheddings of Fasciola gigantica from Lymnaea natalensis.

The shedding of cercariae from Lymnaea natalensis and their transformation into metacercariae occurred mainly at night, and 70.7% of the cysts were counted between 7 p.m. and 1 a.m. There is no infradian rhythm in the numerical distribution of the daily mean values as related to experiment duration or patent period duration. One quarter of the snails shed their cercariae in a single wave and the others in 2 to 13 waves. Floating cysts represent 35% of the total number of metacercariae and their number decreased during the two first waves of shedding. Aberrant metacercarial localizations were noted on the inner surface of the shell when the snail died.

Factors contributing to the resistance of chickens to infection with Japanese Fasciola sp.

Attempts were made to clarify the factors contributing to the resistance of chickens to infection with Japanese Fasciola sp. Infection was not successfully established in chickens by oral inoculation of metacercariae, nor by inoculation of excysted juvenile flukes into the body cavity or to the liver surface. Many metacercarial cysts were detected within two days in the feces of orally inoculated chickens. In the in vitro excystation test with chicken bile at 42 degrees C, metacercariae emerged successfully. These results indicate that the major resistant factors may not act during the migration from the mouth to the liver. Histopathological examination of the liver of experimental chickens could not prove the effect of a resistant factor. Excysted flukes were cultivated at 37-42 degrees C in RPMI1640 supplemented with calf serum, with the result that the survival rate of flukes fell with higher temperatures. When chicken serum was used instead of calf serum, flukes survived for a long period of time at 37 degrees C, while all died within four days at 42 degrees C. The higher body temperature of chickens than that of other mammalian hosts is considered to be the major factor contributing to the resistance of chickens to infection with Fasciola sp.