Fasciola Gigantica Research Articles

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.

15. KONSENTRASI PROTEIN ANTIGEN EKSKRETORI/SEKRETORI DAN SOMATIK PADA Fasciola gigantica DAN Eurytrema pancreaticum The Protein Concentration of Excretory/Secretory and Somatic Antigen from Fasciola gigantica and Eurytrema pancreaticum

The study was aimed at finding out the difference of protein content between excretory/secretory and somatic antigen of Fasciola gigantica as well as between excretory/secretory of Fasciola gigantica and Eurytrema pancreaticum. As many as 32 Fasciola gigantica and 30 Eurytrema pancreaticum were placed into 80 ml Roswell Park Memorial Institute (RPMI) 1640 and incubated for 6 hours. Excretory/secretory as well as somatic antigen was isolated and the concentration of protein was determined, using Lowry method. The result revealed the different of protein concentration. The concentrations of excretory/secretory antigen protein in Fasciola gigantica and Eurytrema pancreaticum were 3.386 mg/ml and 0.128, mg/ml respectively. The concentrations of somatic protein and Fasciola gigantica and Eurytrema pancreaticum were 8.028 mg/ml and 0.534 mg/ml, respectively. It can be concluded that the protein concentration from somatic tissue of Fasciola gigantica and Eurytrema pancreaticum is higher than the protein concentration from excretory/secretory of Fasciola gigantica and Eurytrema pancreaticum. The protein concentration from excerory/secretory antigen of Fasciola gigantica is higher than the protein concentration from excretory/secretory antigen of Eurytrema pancreaticum. ____________________________________________________________________________________________________________________ Key words: Fasciola gigantica, Eurytrema pancreaticum, protein concentation

Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling

Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes.

Patho-Surveillance and Pathology of Fascioliosis (Fasciola gigantica) in Black Bengal Goats

Patho-Surveillance and Pathology of Fascioliosis (Fasciola gigantica) in Black Bengal Goats

Phytotherapy of chlorophyllin exposed Lymnaea acuminata: A new biotechnological tool for fasciolosis control

Phytotherapy of chlorophyllin formulations against Fasciola gigantica infected Lymnaea acuminata under sunlight exposure was highly toxic against redia and cercaria larvae. Binary combinations (1:1 ratio) of chlorophyllin (CHL)+freeze dried cow urine (FCU) were more toxic against cercariae (8h LC50: 9.6mgL−1) than single treatment with chlorophyllin (8h LC50: 12.6mgL−1) in sunlight. The larvicidal activity of sunlight exposed CHL against rediae (8h LC50: 13.5mgL−1) and cercariae (8h LC50: 12.6mgL−1) was more pronounced than laboratory conditions CHL treatment (rediae- 8h LC50: 305.9mgL−1; cercariae- 8h LC50: 765.4mgL−1). Larvicidal activity of FCU was less than CHL and CHL+FCU against both redia and cercaria. Chlorophyllin and its formulations were more toxic against redia and cercaria larvae in sunlight than laboratory conditions. CHL and its different formulations may be used as potent larvicides against Fasciola gigantica larvae. Chlorophyllin formulations will be economical, ecologically sounder and their use in aquatic environment will be safe.

Chronic bovine fasciolosis associated cholangiolithiasis: a case report.

Cholangiolithiasis is a rare condition in animals wherein the choleliths are formed in the biliary tracts causing chronic inflammation. In the current study, choleliths which were firm and friable concretions of approximately 3-4cm in diameter were found in the bile ducts of the liver of an adult cattle infected with Fasciola gigantica. Two such irregularly shaped concretions were encountered in the two biliary tracts leading to common bile duct in the liver. There were localized irregular saccular dilatation and numerous liver flukes in the bile ducts. Further, several small hard concretions of varying sizes were also present in the ducts. The histological investigation revealed chronic cholangitis with bile duct hyperplasia and cirrhosis. The co-existence of F. gigantica and choleliths indicated the physical pre-disposition for the formation of bile concretions in the bile duct which is not a common site for the occurrence of gall stones.

Fasciola gigantica enolase is a major component of worm tegumental fraction protective against sheep fasciolosis

Infection of cattle and sheep with the parasite Fasciola gigantica is a cause of important economic losses throughout Asia and Africa. Many of the available anthelmintics have undesirable side effects, and the parasite may acquire drug resistance as a result of mass and repeated treatments of livestock. Accordingly, the need for developing a vaccine is evident. Triton-soluble surface membrane and tegumental proteins (TSMTP) of 60, 32, and 28kDa previously shown to elicit protective immunity in mice against challenge F. gigantica infection were found to be strongly immunogenic in sheep eliciting vigorous specific antibody responses to a titer>1:16,000 as assessed by enzyme-linked immunosorbent assay. Furthermore, the 60kDa fraction induced production of antibodies able to bind to the surface membrane of newly excysted juvenile flukes and mediate their attrition in antibody-dependent complement- and cell-mediated cytotoxicity assays, and significant (P<0.05) 40% protection of sheep against F. gigantica challenge infection. Amino acid micro sequencing of the 60 kDa-derived tryptic peptides revealed the fraction predominantly consists of F. gigantica enolase. The cDNA nucleotide and translated amino acid sequences of F. gigantica enolase showed homology of 92% and 95%, respectively to Fasciola hepatica enolase, suggesting that a fasciolosis vaccine might be effective against both tropical and temperate liver flukes.

A retrospective study of the prevalence of bovine fasciolosis at major abattoirs in Botswana.

A retrospective study covering a period of ten years (2001–2010) was conducted using postmortem meat inspection records of the Department of Veterinary Services in Gaborone to determine the prevalence of bovine fasciolosis in Botswana. Meat inspection records of monthly and annual returns from the two main export abattoirs in the country were examined, as well as the data collected on the total number of cattle slaughtered and the number of livers condemned due to Fasciola gigantica infection. Only 1250 of the approximately 1.4 million cattle slaughtered were infected with F. gigantica (0.09%, 95% confidence intervals [CI] 0.0% – 0.3%). No distinct seasonal pattern was observed in condemnation rates of livers. However, the pattern of distribution of fasciolosis was higher (but not significant) in cattle that originated from areas with high rainfall and more permanent water bodies than those from relatively low rainfall areas with a transitory water system. It is recommended that a longitudinal survey should be carried out at the abattoirs and farms to determine the prevalence of the disease in cattle of different ages, sex and breed as well as the place of origin in the country. The present study indicated that the prevalence of fasciolosis in cattle is low and the disease is therefore of less significance in Botswana than other African countries for which information is available.

Gambaran Histopatologi Selaput Lendir Kantung Empedu Sapi Bali yang Terinfeksi Cacing Fasciola gigantica (HISTOPATOLOGICAL ILLUSTRATION OF GALL BLADDER MUCOUS MEMBRANE INFASCIOLA GIGANTICA INFECTED BALI CATTLE)

This study aims were to describe lesions and their intensities of histopathological changes in gallbladder mucosa of bali cattle that infected with Fasciola gigantica. As many as 42 gall bladers of balicattle were used in this studi, i.e: 35 samples were infected and sefen were non-infected with F.gigantica.The samples were randomly gathered from Pesanggaran slaughter house in Denpasar. The gall bladderswere evaluated macroscopically and prepared for microscopically examination using haematoxylin andeosin staining.The result showed that there were histopathological changes observed in the mucosa of thegall bladder:necrosis cells of gall bladder with intencity 100%, hemorrhage 68.5%, hyperplasia serousgland and mucous hypertrophy gland 100%, collagen in filtration 100%, fibroblast proliferation 91.4%,and infiltration of inflammation cells, 74.2%. Histopathological changes in gallbladders mucous of balicattle infected F.gigantica experiencing severe lesons with frequency percentage above 50%.

Detection of Fasciola hepatica and Fasciola gigantica common and uncommon antigens, using rabbit hyper immune serum raised against their excretory-secretory and somatic antigens.

Fasciolosis is an important neglected helminth disease caused by two liver flukes, Fasciola hepatica and Fasciola gigantica. The two species of Fasciola are usually different in their morphological and molecular features. They have also common and uncommon antigens in both their somatic and excretory secretory metabolites. In this study, we compared somatic and excretory-secretory (ES) antigens of F. hepatica and F. gigantica, by using rabbit hyper immune serum raised against these antigens. Adult worms were collected from bile ducts of infected animals and species of the fluke was confirmed by RFLP-PCR. ES and somatic antigens of both species were prepared. Rabbits were subcutaneously immunized with either ES or somatic antigens to produce antibodies against these antigens. SDS-PAGE pattern of F. hepatica and F. gigantica somatic antigens was similar and both of them revealed 30 protein bands, ranging from 18 to 180kDa. In contrast, SDS-PAGE pattern of ES antigen of the two species was different. While protein bands with molecular weight of 18, 27, 29, 48, and 62kDa were common in both species, bands of 19, 45, 55 and 58kDa were only noticed in F. hepatica ES antigen. Rabbit polyclonal antibodies, raised against F. hepatica and F. gigantica ES antigen, reacted with main five protein bands, 25, 27, 29, 62 and 67kDa and polyclonal antibodies raised against somatic antigens of both species reacted with three protein bands, 25, 27 and 72kDa. Thus, the 25, 27 and 29kDa protein bands may serve as immunodominant antigens, which might be considered for serodiagnosis of fasciolosis. Moreover, bands of 62 and 67kDa in ES antigen and 72kDa in somatic antigens of both species were immunodominant and might be suitable candidate for development of serological assays for diagnosis of fasciolosis.

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

BackgroundTropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.MethodsIn this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.ResultsThe lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).ConclusionsThese two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Patho-Surveillance and Pathology of Fascioliosis (Fasciola gigantica) in Black Bengal Goats in Bangladesh

The aim of the study was conducted to investigate patho-surveillance and pathology of fascioliosis in Black Bengal goats in Sylhet region of Bangladesh. Patho-surveillance and Pathology on fascioliasis of Black Bengal goats were undertaken in such localities under different climatic conditions existing in Sylhet region of five different Upazilla. Goats were divided into young and adult groups. Affected liver samples from slaughtered goats were collected randomly and examined by grossly and histopathological study for hematoxylin and eosin staining. A total of 2000 Black Bengal goat livers were examined, of which 202(10.10%) livers were found to be affected with fascioliosis. Prevalence of fascioliosis was significantly higher in young goats (15.58 %) than in adult (9.59%) and female goats (13.10%) were more susceptible than male goats (7.10%). Seasonal prevalence of fascioliosis was also studied. Highest prevalence (16.51 %) was recorded during rainy season and lowest prevalence (4.70%) was recorded during summer season. Grossly, affected livers were enlarged with rounded edges and thickened capsule. In acute cases, numerous haemorrhagic spots were found on the parietal & visceral surfaces of the affected liver. In chronic form, liver was cirrhotic and reduced in size. The affected intra-hepatic bile ducts were protruded and engorged with flukes. Microscopically, migratory tracts were represented by the presence of haemorrhage, edema and infiltration of numerous eosinophils mixed with few lymphocytes. Fatty change, atrophy and necrosis of hepatocytes were recorded along with deposition of bile pigment in hepatic parenchyma and damage of portal tract area. The wall of bile ducts was thickened due to fibrosis and lining epithelia were hyperplastic. Cross sections of adult and immature flukes were found within the lumen of the thickened bile ducts and hepatic parenchyma respectively. The study to provides valuable in insight towards better understanding of epidemiology and pathogenesis of the disease in Black Bengal goats to adopt suitable control strategies against the disease and meat inspection at slaughter houses.

Toxicity of Chlorophyllin against Lymnaea acuminata at Different Wavelengths of Visible Light.

Fasciolosis is a water and food-borne disease caused by the liver fluke Fasciola hepatica and Fasciola gigantica. This disease is widespread in different parts of the world. Lymnaeidae and Planorbidae snails are the intermediate hosts of these flukes. Snail population management is a good tool to control fasciolosis because gastropods represent the weakest link in the life-cycle of trematodes. Chlorophyll can be extracted from any green plant. Chlorophyllin was prepared from spinach in 100% ethanol by using different types of chemicals. The chlorophyll obtained from spinach was transformed into water-soluble chlorophyllin. In the present paper, toxicity of chlorophyllin against the snail Lymnaea acuminata was time and concentration dependent. The toxicity of extracted and pure chlorophyllin at continuous 4 h exposure of sunlight was highest with lethal concentration (LC50) of 331.01 mg/L and 2.60 mg/L, respectively, than discontinuous exposure of sunlight up to 8 h with LC50 of 357.04 mg/L and 4.94 mg/L, respectively. Toxicity of extracted chlorophyllin was noted in the presence of different monochromatic visible lights. The highest toxicity was noted in yellow light (96 h, LC50 392.77 mg/L) and the lowest in green light (96 h, LC50 833.02 mg/L). Chlorophyllin in combination with solar radiation or different wavelength of monochromatic visible lights may become a latent remedy against the snail L. acuminata. It was demonstrated that chlorophyllin was more toxic in sunlight. Chlorophyllin is ecologically safe and more economical than synthetic molluscicides which have the potential to control the incidence of fasciolosis in developing countries.

Molecular characterization of Fasciola gigantica in Delhi, India and its phylogenetic relation to the species from South Asian countries.

The aim of this study was to phylogenetically analyze Fasciola gigantica (F. gigantica) from mainland India and to reveal the expansion history of F. gigantica in the Indian subcontinent. We analyzed 40 Fasciola flukes that were collected from Delhi, in the Indian mainland, and identified them as F. gigantica by using nucleotide analyses of the nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes. Based on the nucleotide sequence of mitochondrial NADH dehydrogenase subunit 1 (nad1) gene, the flukes had 18 haplotypes. The haplotypes were classified under haplogroup A, which is predominant in the F. gigantica of South Asia. The population genetics of haplogroup A revealed that Delhi population showed higher π value than eastern India population. These results suggest that F. gigantica of haplogroup A might have spread from the west to the east in India along with the artificial migration of the domestic Zebu cattle, Bos indicus.

Higher physiopathogenicity by Fasciola gigantica than by the genetically close F. hepatica: experimental long-term follow-up of biochemical markers.

Fascioliasis is caused by Fasciola hepatica and F. gigantica. The latter, always considered secondary in human infection, nowadays appears increasingly involved in Africa and Asia. Unfortunately, little is known about its pathogenicity, mainly due to difficulties in assessing the moment a patient first becomes infected and the differential diagnosis with F. hepatica. A long-term, 24-week, experimental study comparing F. hepatica and F. gigantica was made for the first time in the same animal model host, Guirra sheep. Serum biochemical parameters of liver damage, serum electrolytes, protein metabolism, plasma proteins, carbohydrate metabolism, hepatic lipid metabolism and inflammation were analysed on a biweekly basis as morbidity indicators. Serum anti-Fasciola IgG, coproantigen and egg shedding were simultaneously followed up. rDNA and mtDNA sequencing and the morphometric study by computer image analysis system (CIAS) showed that fasciolids used fitted standard species characteristics. Results demonstrated that F. gigantica is more pathogenic, given its bigger size and biomass but not due to genetic differences which are few. Fasciola gigantica shows a delayed development of 1-2 weeks regarding both the biliary phase and the beginning of egg shedding, with respective consequences for biochemical modifications in the acute and chronic periods. The higher F. gigantica pathogenicity contrasts with previous studies which only reflected the faster development of F. hepatica observed in short-term experiments.

Epidemiological Studies of Fascioliosis (Fasciola gigantica Infection) in Cattle

Epidemiological Studies of Fascioliosis (Fasciola gigantica Infection) in Cattle

An unusual recovery of Fasciola gigantica in the lungs of sheep

The present study was conducted in the Andhra Pradesh with the aim of determining prevalence and pathology of pulmonary lesions in sheep. A total of 988 slaughtered or spontaneously dead sheep were screened. Among 988 lungs examined, 187 lungs revealed definite lesions on gross and histopathological examination. The overall prevalence of pulmonary fasciolosis infection was found to be 3.21% (6/187). Grossly, lungs showed multiple dark red haemorrhagic areas. Cut section revealed dark, haemorrhagic tracts with leaf shaped fluke embedded inside the parenchyma. Microscopically, sections of fluke surrounded by haemorrhagic exudates were observed within the bronchus and in the lung parenchyma. Histopathological examination confirmed the disease in sheep as fasciolosis.

A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs in faeces.

Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method. In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs. This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99±0.08, 62.12±0.15 and 74.10±0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tm values. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites. Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections.

ARTIFICIAL INFECTION STUDY OF FASCIOLA GIGANTICA IN LOCAL SHEEP AND ITS IMPACT ON WEIGHT GAIN, HEMATOLOGY AND ENZYME GAMMA GLUTAMYL TRANSFERASE

This research was to investigate the effect of infection of Fasciola gigantica on weight gain, blood picture and the level of iiver damage of local sheep, and the correlation between the number of metacercariae infectH and the number of the fluke recovered. Twenty male local sheep, were allocated randomly into 4 different groups. One group used as uninfected control and a total of 200, 500 and 1.000 metacercariae was infected into animals of groups 2, 3 and 4, respectively. Variables evaluated include weight gain, blood picture, gamma glutamyl tons/erase (GGTase) activity, and the number of egg and the fluke recovered. All individual sheep were weighed every week, whole blood and sera were taken every 3 weeks, fecal samples were started to b« examined 8 weeks after infection and every week thereafter. Weight gain, hematology picture and enzyme GGTase activity were anafized by split-plot, the number of egg and fluke recovered were analized by correlation. All sheep were killed 18 weeks after infection. The results show that infection F. gigantica causes norntocytic normochromic anemia in local sheep. Increased GGTase acitivity is observed. The number of fluke recovered correlates with the number of metacercariae infected.

Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes

Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.