Santarém, Vamilton Alvares; Tostes, Raimundo Alberto; Alberti, Haroldo; Sanches, Osimar de Carvalho
The purpose of this report was to register the presence of Fasciola hepatica in an adult female capybara (Hydrocherus hydrocheris). The macro and microscopical characteristics of hepatic lesions, together with the finding of F. hepatica in mature forms indicate the possible infection of capybara by the trematode.
Thioredoxin is a member of a group of small redox proteins that may protect parasites from the host immune response. Experiments were designed to localize thioredoxin anatomically within the adult and immature stages of Fasciola hepatica. SDS-PAGE and Western blotting were performed to determine t...
Antoniou, M; Lionis, C; Tselentis, Y
In Greece, Fasciola hepatica, the sheep liver fluke, is common in sheep but only three human cases of fascioliasis have been reported. An epidemiological study was conducted in central Crete which proved to be a focus of F. hepatica. Sheep and snails were found positive for this parasite, but none of the 205 persons tested serologically.
Ichikawa-Seki, Madoka; Ortiz, Pedro; Cabrera, Maria; Hobán, Cristian; Itagaki, Tadashi
The causative agent of fasciolosis in South America is thought to be Fasciola hepatica. In this study, Fasciola flukes from Peru were analyzed to investigate their genetic structure and phylogenetic relationships with those from other countries. Fasciola flukes were collected from the three definitive host species: cattle, sheep, and pigs. They were identified as F. hepatica because mature sperms were observed in their seminal vesicles, and also they displayed Fh type, which has an identical fragment pattern to F. hepatica in the nuclear internal transcribed spacer 1. Eight haplotypes were obtained from the mitochondrial NADH dehydrogenase subunit 1 (nad1) sequences of Peruvian F. hepatica; however, no special difference in genetic structure was observed between the three host species. Its extremely low genetic diversity suggests that the Peruvian population was introduced from other regions. Nad1 haplotypes identical to those of Peruvian F. hepatica were detected in China, Uruguay, Italy, Iran, and Australia. Our results indicate that F. hepatica rapidly expanded its range due to human migration. Future studies are required to elucidate dispersal route of F. hepatica from Europe, its probable origin, to other areas, including Peru.
Itagaki, T; Ichinomiya, M; Fukuda, K; Fusyuku, S; Carmona, C
Experiments on hybridization between Fasciola hepatica and Fasciola gigantica were carried out to clarify whether a reproductive isolating mechanism appears between the two Fasciola species. Molecular evidence for hybridization was based on the DNA sequence of the internal transcribed spacer 1 (ITS1) region in nuclear ribosomal DNA, which differs between the species. The results suggested that there were not pre-mating but post-mating isolating mechanisms between the two species. However, viable adults of the hybrids F1 and F2 were produced from both parental F. hepatica and F. gigantica. The hybrids inherited phenotypic characteristics such as ratio of body length and width and infectivity to rats from parental Fasciola hepatica and F. gigantica. These findings suggest that reproductive isolation is incomplete between Fasciola hepatica and F. gigantica. Adults of the hybrids F1 and F2 were completely different in mode of reproduction from aspermic Fasciola forms that occur in Asia and seem to be offspring originated from hybridization between F. hepatica and F. gigantica and to reproduce parthenogenetically.
Peng, Mao; Ichinomiya, Mie; Ohtori, Maiko; Ichikawa, Madoka; Shibahara, Toshiyuki; Itagaki, Tadashi
Parthenogenic Fasciola forms as well as bisexual Fasciola hepatica and Fasciola gigantica in mainland China have been identified on the basis of their spermatogenesis and genotypes in nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase I (NDI). The Chinese aspermic Fasciola would include forms originating in interspecific hybrids between F. hepatica and F. gigantica, since they showed the genotype of ITS1-Fh/Fg that had mixed sequences of the two Fasciola species or heterogeneous genotypes in ITS1 and NDI. Additionally, there were Chinese aspermic flukes in which the sequences of ITS1 and NDI genotypes completely coincided with those in aspermic forms from Japan, Korea, and Vietnam, suggesting that the aspermic forms from these four countries are offspring with a common provenance. The Fh-C4 haplotype in NDI was detected in both aspermic specimens and F. hepatica, indicating that aspermic forms showing the haplotype might come into existence in China. The ratio of body length and width in aspermic Fasciola specimens showed intermediate values between those of F. hepatica and F. gigantica.
Kaplan, Mustafa; Kuk, Salih; Kalkan, Ahmet; Demirdağ, Kutbettin; Ozdarendeli, Aykut
In this study, for the detection of Fasciola hepatica infection seroprevalence in Elaziğ region (Eastern Anatolia), the presence of F. hepatica IgG antibodies were investigated by using a home-made enzyme immunoassay, in the sera of 540 healthy volunteers (254 males, 286 females) from different age groups (age range: 0-81 yrs, mean age: 33 +/- 20 yrs). The total seropositivity rate in this region of our country was found 2.78%, with no differences between males and females (respectively, 2.76% and 2.8%). F. hepatica IgG positive subjects were then evaluated for the parameters of age, educational and socioeconomical status, and it was found that there were no statistically significant differences.
Haçarız, Orçun; Sayers, Gearóid
Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.
Waite, J H; Rice-Ficht, A C
Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.
Ichikawa-Seki, Madoka; Peng, Mao; Hayashi, Kei; Shoriki, Takuya; Mohanta, Uday Kumar; Shibahara, Toshiyuki; Itagaki, Tadashi
The well-known pathogens of fasciolosis, Fasciola hepatica (Fh) and Fasciola Gigantica (Fg), possess abundant mature sperms in their seminal vesicles, and thus, they reproduce bisexually. On the other hand, aspermic Fasciola flukes reported from Asian countries, which have no sperm in their seminal vesicles, probably reproduce parthenogenetically. The aim of this study was to reveal the origin of aspermic Fasciola flukes. The nuclear single copy markers, phosphoenolpyruvate carboxykinase and DNA polymerase delta, were employed for analysis of Fasciola species from China. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the Fh/Fg type, which includes DNA fragments of both F. hepatica and F. gigantica. China can be regarded as the cradle of the interspecific hybridization because F. hepatica and F. gigantica were detected in the northern and southern parts of China, respectively, and hybrids flukes were distributed between the habitats of the two species. The Chinese origin was supported by the fact that a larger number of mitochondrial NADH dehydrogenase subunit 1 (nad1) haplotypes was detected in Chinese aspermic Fasciola populations than in aspermic populations from the neighbouring countries. Hereafter, 'aspermic' Fasciola flukes should be termed as 'hybrid' Fasciola flukes.
Karadağ-Öncel, Eda; Ozsürekçi, Yasemin; Ozkaya-Parlakay, Aslınur; Celik, Melda; Cengiz, Ali Bülent; Haliloğlu, Mithat; Ceyhan, Mehmet; Kara, Ateş
Fascioliasis, an uncommon liver disease in children, is caused by the trematode Fasciola hepatica. Its clinical and laboratory findings may mimic several disorders of the liver, including malignancies. Diagnosis is usually made by demonstrating the presence of the parasite in liver tissue or the stool, or by serology, but many children are diagnosed incidentally. Described here are the clinical, laboratory and radiological features of five pediatric fascioliasis cases with different clinical pictures presenting over a period of five years, all of whom were successfully cured with oral triclabendazole.
Moazeni, Mohammad; Ahmadi, Amin
Fasciola hepatica is a well-known helminth parasite, with significant economic and public health importance all over the world. It has been known since more than 630 years ago and a considerable research work has been carried out on the life cycle of this important parasite. In the hepatic phase of the life cycle of F. hepatica, it is assumed that the young flukes, after about 6-7 weeks of migration in the liver parenchyma, enter into the bile ducts of the definitive hosts and become sexually mature. Even though the secretion of cysteine peptidases including cathepsin L and B proteases by F. hepatica may justify this opinion, because of several scientific reasons and based on the experimental studies conducted in different animals (reviewed in this article), the entry of parasites into the bile ducts, after their migration in the liver parenchyma seems to be doubtful. However, considering all the facts relating to the hepatic and biliary phases of the life cycle of F. hepatica, two alternative ideas are suggested: 1) some of the migrating juvenile flukes may enter into the bile ducts immediately after reaching the liver parenchyma while they are still very small, or 2) when newly excysted juvenile flukes are penetrating into the intestinal wall to reach the liver through the abdominal cavity, a number of these flukes may enter into the choleduct and reach the hepatic bile ducts, where they mature. According to the previously performed natural and experimental studies in different animals and human beings, the supporting and opposing evidences for the current opinion as well as the evidences that might justify the two new ideas are reviewed and discussed briefly. In conclusion, our present knowledge about the time and quality of the entry of F. hepaticas into the bile ducts, seems to be insufficient, therefore, there are still some dark corners and unknown aspects in this field that should be clarified.
Valencia-López, Natalia; Malone, John B; Carmona, Catalina Gómez; Velásquez, Luz E
A predictive Fasciola hepatica model, based on the growing degree day-water budget (GDD-WB) concept and the known biological requirements of the parasite, was developed within a geographical information system (GIS) in Colombia. Climate-based forecast index (CFI) values were calculated and represented in a national-scale, climate grid (18 x 18 km) using ArcGIS 9.3. A mask overlay was used to exclude unsuitable areas where mean annual temperature exceeded 25 °C, the upper threshold for development and propagation of the F. hepatica life cycle. The model was then validated and further developed by studies limited to one department in northwest Colombia. F. hepatica prevalence data was obtained from a 2008-2010 survey in 10 municipalities of 6,016 dairy cattle at 673 herd study sites, for which global positioning system coordinates were recorded. The CFI map results were compared to F. hepatica environmental risk models for the survey data points that had over 5% prevalence (231 of the 673 sites) at the 1 km2 scale using two independent approaches: (i) a GIS map query based on satellite data parameters including elevation, enhanced vegetation index and land surface temperature day-night difference; and (ii) an ecological niche model (MaxEnt), for which geographic point coordinates of F. hepatica survey farms were used with BioClim data as environmental variables to develop a probability map. The predicted risk pattern of both approaches was similar to that seen in the forecast index grid. The temporal risk, evaluated by the monthly CFIs and a daily GDD-WB forecast software for 2007 and 2008, revealed a major July-August to January transmission period with considerable inter-annual differences.
Rinaldi, Laura; Biggeri, Annibale; Musella, Vincenzo; De Waal, Theo; Hertzberg, Hubertus; Mavrot, Fabien; Torgerson, Paul R; Selemetas, Nikolaos; Coll, Tom; Bosco, Antonio; Grisotto, Laura; Cringoli, Giuseppe; Catelan, Dolores
Fasciola hepatica infection challenges health, welfare and productivity of small ruminants throughout the world. The distribution of F. hepatica in sheep in Europe is usually scattered and studies are generally concerned with a single area making it difficult to compare results from different environments, climates and management regimes. In order to elucidate the current scenario in terms of prevalence and intensity of F. hepatica infection in sheep farms across Europe, a standardized cross-sectional survey was conducted in three pilot areas in Ireland, Switzerland and Italy, all part of the EU funded GLOWORM project. Two consecutive field surveys (in 2012 and 2013) were conducted in the three countries in the same period (August-October) in 361 sheep farms in total. Harmonized procedures (from farm to laboratory) based on pooled samples and the highly sensitive and accurate, diagnostic FLOTAC technique were used. The georeferenced parasitological results were modelled (at the pilot area level) following a Bayesian geostatistical approach with correction for preferential sampling and accounting for climatic and environmental covariates. The observed F. hepatica prevalence rates did not differ between the two study years in any of the three pilot areas, but they did vary between the countries showing high values in Ireland (61.6%) compared to Italy (7.9%) and Switzerland (4.0%). Spatial patterns of F. hepatica distribution were detected by the Bayesian geostatistical approach in Ireland with a high risk of infection in the south-western part of the pilot area there. The latent factor analysis highlighted the importance of year-to-year variation of mean temperature, rainfall and seasonality within a country, while long-term trends of temperature and rainfall dominated between countries with respect to prevalence of infection.
Sampaio Silva, M L; Vindimian, M; Wattré, P; Capron, A
In patients infected by Fasciola hepatica, total IgE and specific IgE antibodies have been determined by radioimmunoassays, and IgG, IgA, IgM levels by radial immunodiffusion test (Mancini, 1965). Moreover, total and specific IgE levels have been related to parasite egg burden, age, clinical features and eosinophilia. Elevated total IgE and specific IgE antibodies levels have been found respectively in 76% and 48% of the patients whereas there was no significant variations in other immunoglobulins levels. However, though the amount of total and specific IgE was lower than in other helminthic diseases, it appears to be a significant data of the immune response to parasites as it has been reported and discussed previously. It has been shown a significant relationship between total and specific IgE levels, the number of lines by immunoelectrophoresis, and the results of the indirect haemagglutination and indirect fluorescent antibody techniques; each method appeared to be in equal value to perform the early diagnosis of human Fasciola hepatica. In addition, specific IgE antibodies levels were correlated with eosinophilia specially when it exceeds 15%. This results demonstrate the availability of their measurement in the diagnosis of fascioliasis versus other diseases with marked eosinophilia.
Beesley, N J; Cwiklinski, K; Williams, D J L; Hodgkinson, J
Diploid (2n = 2x = 20) and triploid (2n = 3x = 30) Fasciola hepatica have been reported in the UK, and in Asia diploid, triploid and mixoploid (2x/3x) Fasciola spp. exist but there is little information to indicate how common triploidy is, particularly in UK fluke. Here the ploidy of 565 adult F. hepatica from 66 naturally infected British sheep and 150 adult F. hepatica from 35 naturally infected British cattle was determined. All 715 of these parasites were diploid, based on observation of 10 bivalent chromosomes and sperm (n = 335) or, since triploids are aspermic, sperm alone (n = 380). This constitutes the first extensive analysis of the ploidy of F. hepatica field isolates from Great Britain and shows that most F. hepatica isolated from cattle and sheep are diploid and have the capacity to sexually reproduce. These data suggest that triploidy, and by extension parthenogenesis, is rare or non-existent in wild British F. hepatica populations. Given that F. hepatica is the only species of Fasciola present in Britain our results indicate that the parasite is predominantly diploid in areas where F. hepatica exists in isolation and suggests that triploidy may only originate in natural populations where co-infection of F. hepatica and its sister species Fasciola gigantica commonly occurs.
Novobilský, Adam; Engström, Annie; Sollenberg, Sofia; Gustafsson, Katarina; Morrison, David A; Höglund, Johan
Transmission patterns of Fasciola hepatica were investigated on beef cattle (n=3) and sheep (n=3) farms in Sweden between 2011 and 2012. The dynamics of fluke infection, particularly estimated time of infection, were screened each grazing season by ELISA detection of antibodies in lambs (n=94) and first grazing season calves (n=61). Colostral transfer of F. hepatica antibodies from seropositive ewes was detected in sheep up to 11 weeks of age. In sheep, the estimated time of infection differed significantly between herds and years. Typical 'winter infection' was observed on two sheep farms in 2012, but the most prevalent transmission pattern was found to be 'summer infection', characterised by infection of animals in late summer by F. hepatica originating from overwintered and/or spring-excreted eggs. In contrast, beef calves were infected mainly in September-October ('summer infection'). Furthermore, lymnaeid and succineid snails were collected on the pastures used by these animals both in spring and in the autumn each year. In total, 1726, 588, 138, 130, 93 and 42 specimens of Galba truncatula, Lymnaea palustris, Lymnaea glabra, Lymnaea fuscus, Radix peregra and Succinea putris, respectively, were collected and identified. These were subsequently examined for the presence of F. hepatica DNA by species-specific PCR and the findings compared against mean monthly rainfall and temperature data for each farm. The main intermediate host of the liver fluke was G. truncatula, with a prevalence range of F. hepatica infection from 0% to 82%. Only 1 out of 42 terrestrial S. putris tested positive for F. hepatica, casting doubt on the role of this species in transmission of F. hepatica in Sweden. In conclusion, two main peak periods of infection were observed: May-June (from overwintered infected snails='winter infection') and August-September (from metacercariae developed and produced by snails during summer='summer infection'). The occurrence and frequency of 'winter
Arafa, W M; Hassan, A I; Snousi, S A M; El-Dakhly, Kh M; Holman, P J; Craig, T M; Aboelhadid, S M
Infection by Fasciola species was investigated in seven districts of Dakhla Oasis, Egypt, through abattoir inspection of cattle livers for adult worms and sedimentation of faecal samples from local cattle to detect Fasciola eggs. In addition, lymnaeid snails collected from the study area were examined microscopically for developmental stages of Fasciola spp. Abattoir inspection revealed that 51 out of 458 cattle livers (11.1%) contained adult flukes, which were identified morphologically as Fasciola hepatica. Examination of the cattle faecal samples revealed that 142 out of 503 (28.2%) contained Fasciola eggs. The collected snails, identified as Galba truncatula and Radix natalensis, showed larval stages of Fasciola in 71 out of 731 (9.7%) G. truncatula, while R. natalensis showed no infection. Specific duplex polymerase chain reaction (PCR) targeting the mitochondrial cox1 gene of F. hepatica and Fasciola gigantica was carried out on DNA extracted from pooled infected snails and adult worms. The F. hepatica size amplicon (1031 bp) was obtained from both the infected G. truncatula and the adult worms isolated from cattle livers from different districts. The amplicon sequences were identical to the published sequences of F. hepatica mitochondrial cox1 gene. In conclusion, the zoonotic importance of Fasciola infection and appropriate hygienic measures must be taken into consideration in Dakhla Oasis, Egypt.
Fu, Yan; Chryssafidis, Andreas L.; Browne, John A.; O'Sullivan, Jack; McGettigan, Paul A.; Mulcahy, Grace
Background Fasciola hepatica is not only responsible for major economic losses in livestock farming, but is also a major food-borne zoonotic agent, with 180 million people being at risk of infection worldwide. This parasite is sophisticated in manipulating the hosts’ immune system to benefit its own survival. A better understanding of the mechanisms underpinning this immunomodulation is crucial for the development of control strategies such as vaccines. Methodology/principal findings This in vivo study investigated the global gene expression changes of ovine peripheral blood mononuclear cells (PBMC) response to both acute & chronic infection of F. hepatica, and revealed 6490 and 2364 differential expressed genes (DEGS), respectively. Several transcriptional regulators were predicted to be significantly inhibited (e.g. IL12 and IL18) or activated (e.g. miR155-5p) in PBMC during infection. Ingenuity Pathway Analysis highlighted a series of immune-associated pathways involved in the response to infection, including ‘Transforming Growth Factor Beta (TGFβ) signaling’, ‘Production of Nitric Oxide in Macrophages’, ‘Toll-like Receptor (TLRs) Signaling’, ‘Death Receptor Signaling’ and ‘IL17 Signaling’. We hypothesize that activation of pathways relevant to fibrosis in ovine chronic infection, may differ from those seen in cattle. Potential mechanisms behind immunomodulation in F. hepatica infection are a discussed. Significance In conclusion, the present study performed global transcriptomic analysis of ovine PBMC, the primary innate/adaptive immune cells, in response to infection with F. hepatica, using deep-sequencing (RNAseq). This dataset provides novel information pertinent to understanding of the pathological processes in fasciolosis, as well as a base from which to further refine development of vaccines. PMID:27661612
Le, Thanh Hoa; De, Nguyen Van; Agatsuma, Takeshi; Thi Nguyen, Thanh Giang; Nguyen, Quoc Doanh; McManus, Donald P; Blair, David
The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.
Foreyt, William J; Drew, M L
This experimental study was conducted to evaluate the susceptibility of American bison (Bison bison) to liver flukes, Fascioloides magna and Fasciola hepatica. Six bison were each experimentally inoculated with 600 metacercariae of Fascioloides magna, and three were later treated with triclabendazole suspension at 40 mg/kg of body weight. Four additional bison were each experimentally inoculated with 600 metacercariae of Fasciola hepatica. Five control bison were placebo controls. Two controls and all inoculated bison were euthanized 10 mo (Fascioloides magna) and 7 mo (Fasciola hepatica) after inoculation. None of the control bison or the bison inoculated with Fascioloides magna had flukes or lesions characteristic of fluke infection at necropsy. All four bison inoculated with Fasciola hepatica had characteristic liver fluke lesions at necropsy, and three of four bison contained four, 103, and 111 adult flukes, respectively. Fluke eggs were detected in feces of all Fasciola hepatica-inoculated bison during the experiment, but not from the Fascioloides magna-infected bison or control bison. Clinical signs of infection were not observed during the experiment, but hemoglobin and packed cell volumes were lower in the Fasciola hepatica bison when compared to controls, and eosinophil levels were increased. Triclabendazole at 40 mg/kg of body weight appeared to be safe in bison because no toxic reactions were observed. Results from this study indicated bison are susceptible to infection with Fasciola hepatica and are efficient definitive hosts. Because no Fascioloides magna were recovered, bison may have a decreased susceptibility or innate resistance to Fascioloides magna infection, which may account for a lack of reported infections in this host.
Vukman, Krisztina V; Adams, Paul N; Dowling, David; Metz, Martin; Maurer, Marcus; O'Neill, Sandra M
Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6(-/-) mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6(-/-) independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.
The investigations of the influence of Fasciola hepatica invasion on the assorted parameters of milk quality were performed in cows. Mean protein content in the milk from infected animals ammounted 3.60% and was higher than in the milk from uninfected cows (3.30%). Similar tendency was observed in the case of the fat (5.08% and 3.91%) and lactose (4.36% and 4.34%). The count of somatic cells in milk from infected cows was lower than in milk from uninfected animals (356.07 and 1776.92). It can indicate immunosupressive activity of Fasciola hepatica on the host.
Trueba, G; Guerrero, T; Fornasini, M; Casariego, I; Zapata, S; Ontaneda, S; Vasco, L
The presence of fascioliasis was assessed in four Andean communities using an enzyme-linked immunosorbent assay (ELISA) test to measure antibodies against Fasciola hepatica excretion-secretion antigens. Six percent (9 out of 150) of the individuals in one community were ELISA-positive for these antibodies. Fecal samples from two of the ELISA-positive individuals contained F. hepatica ova. All of the ELISA-positive cases, except for one, were children within the ages of 9 to 12 years.
Corba, J; Spaldonová, R
Results are presented on the effect of immunosuppressive substances such as chlorambucil, cyclophosphamide, azathioprine, amethopterine and a cortizone derivate of betamethasone, on the development of Fasciola hepatica in the rat. The suppression of the immune response of the host to immunosuppressants was reflected in an earlier start of migration of the flukes to the common bile duct, and in an earlier onset of egg production as compared with that in the controls. Of the substances employed, cyclophosphamide and betamethasone were the most effective ones within the period from week 2--6 p.i., which is the time during which the migration of the flukes in the liver parenchyma is highest. Pathological changes in the liver of the animals were less marked than those of the infected controls. Evidence was obtained on an increased pathogenicity of infective larval flukes causing a higher mortality of the hosts in comparison with that of the control animals. On the other hand, the administration of immunosuppressants did neither influence the total number of developed flukes nor the appearance of eosinophilia in the peripheral blood of the treated animals.
Niknam, Ramin; Kazemi, Mohammad Hassan; Mahmoudi, Laleh
Fasciola hepatica (F. hepatica) as a foodborne trematode can occasionally cause hepatobiliary diseases. We report a 67-year-old woman who was referred to our center because of the diagnosis of cholangitis. She was a resident of mountainous area with the history of unsafe water and contaminated vegetables. Endoscopic retrograde cholangiopancreatography (ERCP) was performed as a diagnostic and therapeutic modality for her. Three living F. hepatica was removed from biliary tract with a basket via ERCP. Clinical and laboratory condition of the patient improved after therapy of antibiotics and triclabendazole. PMID:26379355
Shu, Fan-Fan; Lv, Rui-Qing; Zhang, Yi-Fang; Duan, Gang; Wu, Ding-Yu; Li, Bi-Feng; Yang, Jian-Fa; Zou, Feng-Cai
On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.
Fox, Naomi J; White, Piran C L; McClean, Colin J; Marion, Glenn; Evans, Andy; Hutchings, Michael R
Fasciola hepatica (liver fluke) is a physically and economically devastating parasitic trematode whose rise in recent years has been attributed to climate change. Climate has an impact on the free-living stages of the parasite and its intermediate host Lymnaea truncatula, with the interactions between rainfall and temperature having the greatest influence on transmission efficacy. There have been a number of short term climate driven forecasts developed to predict the following season's infection risk, with the Ollerenshaw index being the most widely used. Through the synthesis of a modified Ollerenshaw index with the UKCP09 fine scale climate projection data we have developed long term seasonal risk forecasts up to 2070 at a 25 km square resolution. Additionally UKCIP gridded datasets at 5 km square resolution from 1970-2006 were used to highlight the climate-driven increase to date. The maps show unprecedented levels of future fasciolosis risk in parts of the UK, with risk of serious epidemics in Wales by 2050. The seasonal risk maps demonstrate the possible change in the timing of disease outbreaks due to increased risk from overwintering larvae. Despite an overall long term increase in all regions of the UK, spatio-temporal variation in risk levels is expected. Infection risk will reduce in some areas and fluctuate greatly in others with a predicted decrease in summer infection for parts of the UK due to restricted water availability. This forecast is the first approximation of the potential impacts of climate change on fasciolosis risk in the UK. It can be used as a basis for indicating where active disease surveillance should be targeted and where the development of improved mitigation or adaptation measures is likely to bring the greatest benefits.
El-Kouba, Maysa M A N; Marques, Sandra M T; Pilati, Célso; Hamann, Waldir
Sixteen nutria (Myocastor coypus) from a protected area in Curitiba, Brazil, were sampled to determine the prevalence of Fasciola hepatica eggs and intestinal parasites and the presence of snails in the habitat used by nutria. The overall prevalence rates were 56.25% (9) for F. hepatica eggs, 87.50% (14) for cestode eggs, 56.25% (9) for ascarid eggs, 50% (8) for coccidian (Eimeriidae) oocysts, and 56.25% (9) for Strongyloidea eggs. Fasciola hepatica eggs had an average size of 138 microm x 72 microm. The following mollusks were found: Physa cubensis, Physa marmorata, and Biomphalaria tenagophyla. These results suggest that the infected nutria could serve as a source of contamination for other animals, humans, and surface water. This is the first report about parasites in nutria in the state of Paraná, Brazil.
Lee, Jae-Hyung; Quan, Juan-Hua; Choi, In-Wook; Park, Gab-Man; Cha, Guang-Ho; Kim, Hyun-Ju; Yuk, Jae-Min; Lee, Young-Ha
Fasciola hepatica is a trematode that causes zoonosis, mainly in cattle and sheep, and occasionally in humans. Few recent studies have determined the infection status of this fluke in Korea. In August 2015, we collected 402 samples of freshwater snails at Hoenggye-ri (upper stream) and Suha-ri (lower stream) of Song-cheon (stream) in Daegwalnyeong-myeon, Pyeongchang-gun in Gangwon-do (Province) near many large cattle or sheep farms. F. hepatica infection was determined using PCR on the nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 402 samples, F. hepatica 1TS-2 marker was detected in 6 freshwater snails; thus, the overall prevalence in freshwater snails was 1.5%. The prevalence varied between collection areas, ranging from 0.0% at Hoenggye-ri to 2.9% at Suha-ri. However, F. gigantica ITS-2 was not detected in the 6 F. hepatica-positive samples by PCR. The nucleotide sequences of the 6 F. hepatica ITS-2 PCR-positive samples were 99.4% identical to the F. hepatica ITS-2 sequences in GenBank, whereas they were 98.4% similar to F. gigantica ITS-2 sequences. These results indicated that the prevalence of F. hepatica in snail intermediate hosts was 1.5% in Gangwon-do, Korea; however the prevalence varied between collection areas. These results may help us to understand F. hepatica infection status in natural environments. PMID:28285514
Lee, Jae-Hyung; Quan, Juan-Hua; Choi, In-Wook; Park, Gab-Man; Cha, Guang-Ho; Kim, Hyun-Ju; Yuk, Jae-Min; Lee, Young-Ha
Fasciola hepatica is a trematode that causes zoonosis, mainly in cattle and sheep, and occasionally in humans. Few recent studies have determined the infection status of this fluke in Korea. In August 2015, we collected 402 samples of freshwater snails at Hoenggye-ri (upper stream) and Suha-ri (lower stream) of Song-cheon (stream) in Daegwalnyeong-myeon, Pyeongchang-gun in Gangwon-do (Province) near many large cattle or sheep farms. F. hepatica infection was determined using PCR on the nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 402 samples, F. hepatica 1TS-2 marker was detected in 6 freshwater snails; thus, the overall prevalence in freshwater snails was 1.5%. The prevalence varied between collection areas, ranging from 0.0% at Hoenggye-ri to 2.9% at Suha-ri. However, F. gigantica ITS-2 was not detected in the 6 F. hepatica-positive samples by PCR. The nucleotide sequences of the 6 F. hepatica ITS-2 PCR-positive samples were 99.4% identical to the F. hepatica ITS-2 sequences in GenBank, whereas they were 98.4% similar to F. gigantica ITS-2 sequences. These results indicated that the prevalence of F. hepatica in snail intermediate hosts was 1.5% in Gangwon-do, Korea; however the prevalence varied between collection areas. These results may help us to understand F. hepatica infection status in natural environments.
Kim, Hwang-Yong; Choi, In-Wook; Kim, Yeon-Rok; Quan, Juan-Hua; Ismail, Hassan Ahmed Hassan Ahmed; Cha, Guang-Ho; Hong, Sung-Jong; Lee, Young-Ha
Fasciola hepatica is a trematode that causes zoonosis mainly in cattle and sheep and occasionally in humans. Fascioliasis has been reported in Korea; however, determining F. hepatica infection in snails has not been done recently. Thus, using PCR, we evaluated the prevalence of F. hepatica infection in snails at 4 large water-dropwort fields. Among 349 examined snails, F. hepatica-specific internal transcribed space 1 (ITS-1) and/or ITS-2 markers were detected in 12 snails and confirmed using sequence analysis. Morphologically, 213 of 349 collected snails were dextral shelled, which is the same aperture as the lymnaeid snail, the vectorial host for F. hepatica. Among the 12 F. hepatica-infected snails, 6 were known first intermediate hosts in Korea (Lymnaea viridis and L. ollula) and the remaining 6 (Lymnaea sp.) were potentially a new first intermediate host in Korea. It has been shown that the overall prevalence of the snails contaminated with F. hepatica in water-dropwort fields was 3.4%; however, the prevalence varied among the fields. This is the first study to estimate the prevalence of F. hepatica infection using the vectorial capacity of the snails in Korea.
Kim, Ah Jin; Choi, Chang Hwan; Choi, Sun Keun; Shin, Yong Woon; Park, Yun-Kyu; Kim, Lucia; Choi, Suk Jin; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae; Park, In Suh
We report here an ectopic case of Fasciola hepatica infection confirmed by recovery of an adult worm in the mesocolon. A 56-year-old female was admitted to our hospital with discomfort and pain in the left lower quadrant of the abdomen. Abdominal CT showed 3 abscesses in the left upper quadrant, mesentery, and pelvic cavity. On surgical exploration, abscess pockets were found in the mesocolon of the sigmoid colon and transverse colon. A leaf-like worm found in the abscess pocket of the mesocolon of the left colon was diagnosed as an adult fluke of F. hepatica. Histologically, numerous eggs of F. hepatica were noted with acute and chronic granulomatous inflammations in the subserosa and pericolic adipose tissues. Conclusively, a rare case of ectopic fascioliasis has been confirmed in this study by the adult worm recovery of F. hepatica in the mesocolon. PMID:26797440
Kim, Ah Jin; Choi, Chang Hwan; Choi, Sun Keun; Shin, Yong Woon; Park, Yun-Kyu; Kim, Lucia; Choi, Suk Jin; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae; Park, In Suh
We report here an ectopic case of Fasciola hepatica infection confirmed by recovery of an adult worm in the mesocolon. A 56-year-old female was admitted to our hospital with discomfort and pain in the left lower quadrant of the abdomen. Abdominal CT showed 3 abscesses in the left upper quadrant, mesentery, and pelvic cavity. On surgical exploration, abscess pockets were found in the mesocolon of the sigmoid colon and transverse colon. A leaf-like worm found in the abscess pocket of the mesocolon of the left colon was diagnosed as an adult fluke of F. hepatica. Histologically, numerous eggs of F. hepatica were noted with acute and chronic granulomatous inflammations in the subserosa and pericolic adipose tissues. Conclusively, a rare case of ectopic fascioliasis has been confirmed in this study by the adult worm recovery of F. hepatica in the mesocolon.
Ashrafi, K; Valero, M A; Panova, M; Periago, M V; Massoud, J; Mas-Coma, S
Fascioliasis is an important human and animal disease caused by Fasciola hepatica and Fasciola gigantica. In Iran, the distribution of these two species overlaps in most areas, including the northern human endemic province of Gilan where both fasciolids are simultaneously found in individual cattle and buffaloes. A phenotypic study of fasciolid adult flukes from naturally infected bovines from Gilan was carried out by means of an exhaustive morphometric analysis using traditional microscopic measurements and an allometric model. The Iranian fasciolids were compared to F. hepatica and F. gigantica standard populations, i.e. from geographical areas where both species do not co-exist (Bolivia and Burkina Faso, respectively). Although morphometric values somewhat overlapped, there were clear differences in allometric growth. The allometric function was adjusted to 25 pairs of variables. Results obtained revealed that Iranian F. hepatica-like specimens are larger than the F. hepatica standard and Iranian F. gigantica-like specimens are longer and narrower than the F. gigantica standard, but with smaller body area. Measurements which permit a specific differentiation in allopatric populations (distance between ventral sucker and posterior end of the body; ratio between body length and body width) overlap in the specimens from Gilan, thus proving the presence of intermediate forms. When compared to the standard populations, the different Iranian fasciolid morphs show greater differences in F. gigantica-like specimens than in F. hepatica-like specimens. This study shows that simple, traditional microscopic measurements may be sufficient for the morphometric characterisation of fasciolids, even in areas where intermediate forms are present.
Maggioli, Gabriela; Silveira, Fernando; Martín-Alonso, José M; Salinas, Gustavo; Carmona, Carlos; Parra, Francisco
Antioxidant systems are fundamental components of host-parasite interactions, and often play a key role in parasite survival. Here, we report the cloning, heterologous expression, and characterization of a thioredoxin glutathione reductase (TGR) from Fasciola hepatica. The deduced polypeptide sequence of the cloned open reading frame (ORF) confirmed the experimental N-terminus previously determined for a native F. hepatica TGR showing thioredoxin reductase (TR) activity. The sequence revealed the presence of a fusion between a glutaredoxin (Grx) and a TR domain, similar to that previously reported in Schistosoma mansoni and Echinococcus granulosus. The F. hepatica TGR sequence included an additional redox active center (ACUG; U being selenocysteine) located at the C-terminus. The addition of a recombinant selenocysteine insertion sequence (SECIS) element in the Escherichia coli expression vector, or the substitution of the native selenocysteine by a cysteine, indicated the relevance of this unusual amino acid residue for the activity of F. hepatica TGR. Rabbit vaccination with recombinant F. hepatica TGR reduced the worm burden by 96.7% following experimental infection, further supporting the relevance of TGR as a promising target for anti Fasciola treatments.
Howell, Alison; Baylis, Matthew; Smith, Rob; Pinchbeck, Gina; Williams, Diana
The liver fluke Fasciola hepatica is a trematode parasite with a worldwide distribution and is the cause of important production losses in the dairy industry. The aim of this observational study was to assess the prevalence of exposure to F. hepatica in a group of high yielding dairy herds, to determine the risk factors and investigate their associations with production and fertility parameters. Bulk milk tank samples from 606 herds that supply a single retailer with liquid milk were tested with an antibody ELISA for F. hepatica. Multivariable linear regression was used to investigate the effect of farm management and environmental risk factors on F. hepatica exposure. Higher rainfall, grazing boggy pasture, presence of beef cattle on farm, access to a stream or pond and smaller herd size were associated with an increased risk of exposure. Univariable regression was used to look for associations between fluke exposure and production-related variables including milk yield, composition, somatic cell count and calving index. Although causation cannot be assumed, a significant (p < 0.001) negative association was seen between F. hepatica exposure and estimated milk yield at the herd level, representing a 15% decrease in yield for an increase in F. hepatica exposure from the 25th to the 75th percentile. This remained significant when fertility, farm management and environmental factors were controlled for. No associations were found between F. hepatica exposure and any of the other production, disease or fertility variables. PMID:26093971
Howell, Alison; Baylis, Matthew; Smith, Rob; Pinchbeck, Gina; Williams, Diana
The liver fluke Fasciola hepatica is a trematode parasite with a worldwide distribution and is the cause of important production losses in the dairy industry. The aim of this observational study was to assess the prevalence of exposure to F. hepatica in a group of high yielding dairy herds, to determine the risk factors and investigate their associations with production and fertility parameters. Bulk milk tank samples from 606 herds that supply a single retailer with liquid milk were tested with an antibody ELISA for F. hepatica. Multivariable linear regression was used to investigate the effect of farm management and environmental risk factors on F. hepatica exposure. Higher rainfall, grazing boggy pasture, presence of beef cattle on farm, access to a stream or pond and smaller herd size were associated with an increased risk of exposure. Univariable regression was used to look for associations between fluke exposure and production-related variables including milk yield, composition, somatic cell count and calving index. Although causation cannot be assumed, a significant (p<0.001) negative association was seen between F. hepatica exposure and estimated milk yield at the herd level, representing a 15% decrease in yield for an increase in F. hepatica exposure from the 25th to the 75th percentile. This remained significant when fertility, farm management and environmental factors were controlled for. No associations were found between F. hepatica exposure and any of the other production, disease or fertility variables.
Rokni, Mohammad Bagher; Mirhendi, Hossein; Mizani, Azadeh; Mohebali, Mehdi; Sharbatkhori, Mitra; Kia, Eshrat Beigom; Abdoli, Hamid; Izadi, Shahrokh
Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes.
Villa-Mancera, A; Quiroz-Romero, H; Correa, D; Alonso, R A
The objective of this study was to assess the proteolytic activity of Fasciola hepatica cathepsins in liver sections from mice vaccinated with phage clones of cathepsin L mimotopes, using the film in situ zymography technique. Female BALB/c mice were immunized three times with 2.5 x 10¹¹ phage particles without adjuvant. Animals vaccinated with phage clones produced high titres of anti-mimotope antibodies and a significant reduction in fluke burden was observed following challenge with metacercariae of F. hepatica. The proteolytic activity in hepatic tissue was reduced after the immunization with phage clones.
Arifin, Maria Immaculata; Höglund, Johan; Novobilský, Adam
The liver fluke, Fasciola hepatica, is one of the major parasite threats to livestock industries world-wide. In sheep and cattle, F. hepatica infection is commonly diagnosed using a range of methods. Aside from conventional coprological and serological diagnostic methods, there are also several molecular methods available based on the detection of liver fluke DNA in faeces. In this study, the outcomes of faecal egg count (FEC), serology and coproantigen ELISA (cELISA) were compared with the performance of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) in diagnosis of F. hepatica from naturally infected cattle and sheep. A total of 64 individual faecal and serum samples were collected from four sheep and beef cattle herds with previous histories of F. hepatica infection. FEC and coproantigen levels were measured in faecal samples and anti-F.hepatica antibody levels were measured in serum samples. DNA samples isolated from faeces were examined both by PCR and LAMP, targeting the internal transcribed spacer 2 (ITS2) region of the F. hepatica genome. Results showed that F. hepatica eggs were present in 28 animals, while coproantigen and specific anti-F. hepatica antibodies were detected in 36 and 53 animals, respectively. Only 3 and 6 samples were positive by PCR and LAMP, respectively. To calculate method specificity and sensitivity, a combination of FEC and cELISA was selected as the composite reference standard (CRS). When compared to the CRS, PCR had a sensitivity of 10.7% and specificity of 100%, whereas LAMP had a sensitivity and specificity of 17.9% and 97.2%, respectively. PCR and LAMP in this field study were highly specific, but both had poor sensitivity compared with FEC and cELISA. Potential reasons for PCR and LAMP failure were inadequate amounts of amplifiable F. hepatica DNA, possibly due to the choice of DNA extraction procedure, amount of faecal material processed, as well as different faeces consistency and
Ahasan, Syed Ali; Valero, M Adela; Chowdhury, Emdadul Haque; Islam, Mohammad Taohidul; Islam, Mohammad Rafiqul; Hussain Mondal, Mohammad Motahar; Peixoto, Raquel V; Berinde, Lavinia; Panova, Miroslava; Mas-Coma, Santiago
Fascioliasis is an important food-borne parasitic zoonosis caused by two trematode species, Fasciola hepatica and Fasciola gigantica. The characterisation and differentiation of Fasciola populations is crucial to control the disease, given the different transmission, epidemiology and pathology characteristics of the two species. Lineal biometric features of adult liver flukes infecting livestock have been studied to characterise and discriminate fasciolids from Bangladesh. An accurate analysis was conducted to phenotypically discriminate between fasciolids from naturally infected bovines (cattle, buffaloes) throughout the country. Morphometric analyses were made with a computer image analysis system (CIAS) applied on the basis of standardised measurements and the logistic model of the body growth and development of fasciolids in the different host groups. Since it is the first ever comprehensive study of this kind undertaken in Bangladesh, the results are compared to pure fasciolid populations of F. hepatica from the European Mediterranean area and F. gigantica from Burkina Faso, geographical areas where both species do not co-exist. Principal component analysis showed that the biometric characteristics of fasciolids from Bangladesh are situated between F. hepatica and F. gigantica standard populations, indicating the presence of phenotypes of intermediate forms in Bangladesh. These results are analysed by considering the present emergence of animal fascioliasis, the local lymnaeid fauna, the impact of climate change, and the risk of human infection in the country.
Dreyfuss, G; Vignoles, P; Rondelaud, D
A total of 59 natural watercress beds in the Limousin region (central France) was surveyed over a 15-year period (1990-2004) to detect the contamination of watercress by the metacercariae of Fasciola hepatica and to determine the presence of larval forms in the two species of lymnaeids which live in these waterholes in June and July. The number of beds contaminated with F. hepatica metacercariae varied over the years, and the burden of the larvae on plants was low: a mean of 2.6-6.3 per bed. The same variability was also noted for natural infections of Galba truncatula with F. hepatica, as the annual prevalences ranged from 1.2% to 2.4%. Natural infections of Omphiscola glabra with F. hepatica were only detected from 1996 and the annual prevalences subsequently increased up to 1.4-1.8% between 2001 and 2004. However, for both lymnaeids, the variations in these prevalences with year were insignificant. The contamination of these beds with F. hepatica over the past 15 years was similar to that recorded in the same sites between 1970 and 1986. The main changes were the appearance of another digenea, Paramphistomum daubneyi, in the beds, and the possibility for O. glabra to naturally sustain the larval development of F. hepatica.
Rondelaud, D; Teukeng, F F Djuikwo; Vignoles, P; Dreyfuss, G
Experimental infections of Lymnaea glabra (two populations) with Fasciola hepatica were carried out during seven successive snail generations, to determine if prevalence and intensity of snail infection increased over time through descendants of snails already infected with F. hepatica. Controls were descendants coming from uninfected parents and infected according to the same protocol. No larval forms were found in the bodies of control snails coming from uninfected parents. In contrast, prevalence and intensity of F. hepatica infection in snails originating from infected parents progressively increased from the F2 or F3 to the F6 generation of L. glabra. In another experiment carried out with the F7 generations of L. glabra and a single generation of Galba truncatula (as controls), the prevalence of F. hepatica infection and the total number of cercariae were lower in L. glabra (without significant differences between both populations). If the number of cercariae shed by infected snails was compared to overall cercarial production noted in snails containing cercariae but dying without emission, the percentage was greater in G. truncatula (69% instead of 52-54% in L. glabra). Even if most characteristics of F. hepatica infection were lower in L. glabra, prevalence and intensity of parasite infection increased with snail generation when tested snails came from infected parents. This mode of snail infection with F. hepatica suggests an explanation for cases of fasciolosis occurring in cattle-breeding farms where paramphistomosis is lacking and G. truncatula is absent.
The reports deals with the results of testing seven new antihelminthics for Fasciola hepatica in the experimentally invaded Wistar rat. The greatest influence on juvenile flukes (2 and 4 weeks of age) was exerted by diamphenetid (Coriban) applied in a single dose of 100 mg kg-1. Hexachlorophene applied in the dose of 50 mg kg-1 showed the highest effect on sexually mature flukes. All the tested antihelminthics of the halogenated salicylanilide group were ineffective on juvenile stages and only slightly effective on mature F. hepatica flukes. It follows from the results that the effectiveness of some antifasciolics on laboratory animals need not always be in correlation with their effect in ruminants - hence it is necessary to verify the results obtained in laboratory animals and to check them on natural F. hepatica hosts.
Claridge, Jen; Diggle, Peter; McCann, Catherine M.; Mulcahy, Grace; Flynn, Rob; McNair, Jim; Strain, Sam; Welsh, Michael
Bovine tuberculosis (BTB) is a significant and intractable disease of cattle caused by Mycobacterium bovis. In the UK, despite an aggressive eradication programme, the prevalence of BTB is increasing with an unexplained, exponential rise in cases year on year. Here we show in a study involving 3026 dairy herds in England and Wales that there is a significant negative association between exposure to the common, ubiquitous helminth parasite, Fasciola hepatica and diagnosis of BTB. The magnitude of the single intradermal comparative cervical tuberculin test used to diagnose BTB is reduced in cattle experimentally co-infected with M. bovis and F. hepatica. We estimate an under-ascertainment rate of about one-third (95% Confidence Intervals 27-38%) among our study farms, in the hypothetical situation of no exposure to F. hepatica. This finding may in part explain the continuing spread of BTB and the failure of the current eradication programme in the UK. PMID:22617293
Figueroa-Santiago, Olgary; Delgado, Bonnibel; Espino, Ana M.
An indirect enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola hepatica saposin-like protein-2. The assay was compared with an indirect ELISA with excretory-secretory products (FhES) from adult F. hepatica. In an analysis of the sera of 37 patients infected with F. hepatica, 40 patients with other parasitic infections, and 50 healthy controls, the sensitivity of both ELISA assays was 100%. However, the FhSAP2-based ELISA was more specific (95.6%) than the FhES-ELISA (91.9%). These results demonstrated that FhSAP2 can be used in the serodiagnosis of chronic human fascioliasis with additional advantage that is relative cheap and easy to produce. Studies are in progress to evaluate this FhSAP2-ELISA assay in a large-scale prevalence surveys in endemic areas. PMID:21683266
Kang, Bong Kyun; Jung, Bong-Kwang; Lee, Yoon Suk; Hwang, In Kyeom; Lim, Hyemi; Cho, Jaeeun; Hwang, Jin-Hyeok; Chai, Jong-Yil
Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.
Afshan, K; Valero, M A; Qayyum, M; Peixoto, R V; Magraner, A; Mas-Coma, S
Fascioliasis is an important food-borne parasitic disease caused by the two trematode species, Fasciola hepatica and Fasciola gigantica. The phenotypic features of fasciolid adults and eggs infecting buffaloes inhabiting the Central Punjab area, Pakistan, have been studied to characterize fasciolid populations involved. Morphometric analyses were made with a computer image analysis system (CIAS) applied on the basis of standardized measurements. Since it is the first study of this kind undertaken in Pakistan, the results are compared to pure fasciolid populations: (a) F. hepatica from the European Mediterranean area; and (b) F. gigantica from Burkina Faso; i.e. geographical areas where both species do not co-exist. Only parasites obtained from bovines were used. The multivariate analysis showed that the characteristics, including egg morphometrics, of fasciolids from Central Punjab, Pakistan, are between F. hepatica and F. gigantica standard populations. Similarly, the morphometric measurements of fasciolid eggs from Central Punjab are also between F. hepatica and F. gigantica standard populations. These results demonstrate the existence of fasciolid intermediate forms in endemic areas in Pakistan.
Machicado, Claudia; Marcos, Luis A; Zimic, Mirko
Fasciola hepatica is considered an emergent human pathogen, causing liver fibrosis or cirrhosis, conditions that are known to be direct causes of cancer. Some parasites have been categorized by WHO as carcinogenic agents such as Opisthorchis viverrini, a relative of F. hepatica. Although these two parasites are from the same class (Trematoda), the role of F. hepatica in carcinogenesis is unclear. We hypothesized that F. hepatica might share some features with O. viverrini and to be responsible to induce proliferation of host cells. We analyzed the recently released genome of F. hepatica looking for a gene coding a granulin-like growth factor, a protein secreted by O. viverrini (Ov-GRN-1), which is a potent stimulator of proliferation of host cells. Using computational biology tools, we identified a granulin-like molecule in F. hepatica, here termed FhGLM, which has high sequence identity level to Ov-GRN-1 and human progranulin. We found evidence of an upstream promoter compatible with the expression of FhGLM. The FhGLM architecture showed to have five granulin domains, one of them, the domain 3, was homologue to Ov-GRN-1 and human GRNC. The structure of the FhGLM granulin domain 3 resulted to have the overall folding of its homologue the human GRNC. Our findings show the presence of a homologue of a potent modulator of cell growth in F. hepatica that might have, as other granulins, a proliferative action on host cells during fascioliasis. Future experimental assays to demonstrate the presence of FhGLM in F. hepatica are needed to confirm our hypothesis.
Shi, Yunliang; Toet, Hayley; Rathinasamy, Vignesh; Young, Neil D; Gasser, Robin B; Beddoe, Travis; Huang, Weiyi; Spithill, Terry W
The present study focussed on investigating CD59-like molecules of Fasciola hepatica. A cDNA encoding a CD59-like protein (termed FhCD59-1) identified previously in the membrane fraction of the F. hepatica tegument was isolated. This homologue was shown to encode a predicted open reading frame (ORF) of 122 amino acids (aa) orthologous to human CD59 with a 25 aa signal peptide, a mature protein containing 10 cysteines and a conserved CD59/Ly-6 family motif "CCXXXXCN". An analysis of cDNAs from two different adult specimens of F. hepatica revealed seven variable types of FhCD59-1 sequences, designated FhCD59-1.1 to FhCD59-1.7, which had 94.3-99.7% amino acid sequence identity upon pairwise comparison. Molecular modeling of FhCD59-1.1 with human CD59 confirmed the presence of the three-finger protein domain found in the CD59 family and predicted three disulphide bonds in the F. hepatica sequence. The interrogation of F. hepatica databases identified two additional sequences, designated FhCD59-2 and FhCD59-3, which had only 23.4-29.5% amino acid identity to FhCD59-1.1. Orthologues of the inferred CD59 protein sequences of F. hepatica were also identified in other flatworms, including Fasciola gigantica, Fascioloides magna, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Clonorchis sinensis, Opisthorchis viverrini, Taenia solium, Echinococcus granulosus and the free living Schmidtea mediterannea. The results revealed a considerable degree of sequence complexity in the CD59-like sequence families in F. hepatica and flatworms. Phylogenetic analysis of CD59-like aa sequences from F. hepatica and flatworms showed that FhCD59-2 clustered with the known surface-associated protein SmCD59-2 of S. mansoni. Relatively well-supported clades specific to schistosomes, fasciolids and opisthorchiids were identified. The qPCR analysis of gene transcription showed that the relative expression of these 3 FhCD59-like sequences varied by 11-47-fold during fluke
Pérez-Creo, Ana; Díaz, Pablo; López, Ceferino; Béjar, Juan Pablo; Martínez-Sernández, Victoria; Panadero, Rosario; Díez-Baños, Pablo; Ubeira, Florencio M; Morrondo, Patrocinio
In order to study the seroprevalence of Fasciola hepatica infection in goats from north-western Spain, a total of 603 serum samples from 47 herds were tested using a capture ELISA (MM3-SERO). The identification of risk factors was assessed by a mixed-effects logistic regression analysis. The results showed that F. hepatica is widespread in this area with 57.4% of the herds and 22.7% of the animals testing positive. Breed and age were identified as determining factors for caprine F. hepatica infection. Seroprevalence in cross-bred animals was significantly higher than in the autochthonous Cabra Galega breed. A significantly higher seroprevalence was observed in older animals. The use of locally adapted breeds and the implementation of suitable management practices could provide a substantial improvement over the current F. hepatica control measures carried out in goat herds and should be considered when designing new F. hepatica control programs.
Dreyfuss, G; Vignoles, P; Abrous, M; Rondelaud, D
Four freshwater pulmonate species (Lymnaea ovata, L. stagnalis, Physa acuta, Planorbis leucostoma) were living in several watercress beds known for their relationships with human cases of fasciolosis, whereas L. truncatula was never found. The aims of these studies were to determine the prevalence of natural infections with Fasciola hepatica in snails and to verify if these species might ensure the full larval development of this trematode (with cercarial shedding) when they were experimentally subjected to F. hepatica only, or to co-infections with an other trematode species. Investigations were so carried out in six snail populations living in watercress beds (including three for P. acuta) and in four others originating from three brooks or a pond (as controls). Snails naturally infected with F. hepatica were found in two watercress beds inhabited by L. ovata (prevalence of infection: 1.4%) and P. leucostoma (0.1%), respectively. The L. ovata from the watercress bed could be infected at a higher size than those from the control population and the prevalence of this infection was greater in the bed population. Similar findings were noted for L. stagnalis. Despite single or dual infections, the results obtained with the four populations of P. acuta were unsuccessful. In contrast, the co-infections of young P. leucostoma with Paramphistomum daubneyi and F. hepatica resulted in the shedding of some F. hepatica cercariae. According to the authors, the occurrence of fasciolosis in these watercress beds would be the consequence of frequent natural encounters between parasite and snails (L. ovata, L. stagnalis), or of co-infections with P. daubneyi and F. hepatica (P. leucostoma). In watercress beds only colonized by P. acuta, a lymnaeid species would have ensured the larval development of F. hepatica but it would have been eliminated by P. acuta, as this last species was known to be invasive and could colonize open drainage ditches on siliceous soil.
Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M.; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q.; Kilcoyne, Michelle; Hokke, Cornelis H.; Dalton, John P.; O'Neill, Sandra M.
Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. PMID:27466253
Moazeni, Mohammad; Sharifiyazdi, Hassan; Izadpanah, Afshin
The present study compared the genetic variation among 19 different isolates of Fasciola hepatica from cattle and sheep in different areas of Iran using sequence data for mitochondrial DNA gene, the subunit 1 of cytochrome C oxidase gene (CO1). Four different CO1 genotypes were detected among F. hepatica isolates that showed five variable nucleotide positions (accession nos.; GQ398051, GQ398052, GQ398053, GQ398054). Nucleotide sequence variation among 19 isolates for CO1 analyzed in this study ranged from 0% to 0.98% in Iran. Among the five polymorphism sites identified in this study, only one (T to G at position 51 in 5'end of GQ175362) resulted in putative amino acid alteration of phenylalanine (TTT) to leucine (TTG) in CO1. A phylogenetic analysis of the sequence data revealed that host associations and geographic location are likely not useful markers for Fasciola genotype classification. In addition, morphological analysis showed that the ratios of body length and body width of some (n = 5) of the 19 examined F. hepatica isolates were intermediate between F. hepatica and Fasciola gigantica, representing the substantial polymorphism of the F. hepatica species and the difficulty in the accurate recognition based on morphological features. In conclusion, Iranian F. hepatica exhibited the presence of considerable genetic diversity at CO1.
Rojas-Caraballo, Jose; López-Abán, Julio; Fernández-Soto, Pedro; Vicente, Belén; Collía, Francisco; Muro, Antonio
Background Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology. Methodology A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). Results We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. Conclusion The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F
Novobilský, Adam; Höglund, Johan
Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests. Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12–16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT). Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65–77%) and 97% (95% CI: 95–98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively. Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12–16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle. PMID:26448903
Novobilský, Adam; Höglund, Johan
Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests. Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12-16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT). Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65-77%) and 97% (95% CI: 95-98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively. Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12-16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle.
Brockwell, Yvette M.; Elliott, Timothy P.; Anderson, Glenn R.; Stanton, Rex; Spithill, Terry W.; Sangster, Nicholas C.
Triclabendazole (TCBZ) is the drug of choice for Fasciola hepatica control and reports of F. hepatica resistant to this drug from a wide range of geographic regions are very concerning. This study investigated the presence of TCBZ resistance in F. hepatica in naturally infected Australian beef and dairy cattle herds and evaluated methods of measuring the levels of resistance. Faecal egg count and coproantigen reduction tests (FECRT and CRT, respectively) were conducted on 6 South-eastern Australian beef properties and one dairy property where treatment failure by triclabendazole (TCBZ) was suspected. The CRT was conducted on an additional beef property. On each property 15 animals were treated with an oral preparation of TCBZ at the recommended dose and 15 animals remained as untreated controls. Fluke eggs in faeces were counted and coproantigen levels were measured before treatment and 21 days after treatment and in the untreated control animals. These data were evaluated using three different methods to calculate % reductions compared with controls. Resistance (<90% reduction) was detected on the dairy property using both FEC and CRT, and on 3/6 beef properties using FECRT and 4/7 beef properties using CRT. Using the FECRT, reductions of 6.1–14.1% were observed in dairy cattle and 25.9–65.5% in beef cattle. Using the CRT, reductions of 0.4–7.6% were observed in dairy cattle and 27.0–69.5% in beef cattle. Live flukes were recovered at slaughter following TCBZ treatment of 6 cattle from 3 of the beef properties, confirming the TCBZ resistance status of F. hepatica in these cattle. This is the first report of F. hepatica resistant to TCBZ in cattle in Australia and the results suggest that resistance is widespread in the South-eastern region. The CRT is shown to be a robust alternative to the FECRT for evaluation of TCBZ resistance in F. hepatica in cattle. PMID:24596668
Hillyer, G V; Sagramoso de Ateca, L
Antigens of Fasciola hepatica adult worms were chromatographed using concanavalin A-Sepharose 4B. Two unbound peaks appeared in the inclusion volume (DT-1 and DT-2), and one peak was eluted with alpha-methylglucoside (E1-1). At least seven peaks were obtained by isoelectric focusing of E1-1. The largest of these peaks, with an average pI of 4.0, contained the antigens reactive with antibodies to Schistosoma mansoni. Mice immunized with DT-2 or E1-1 and challenged with S. mansoni cercariae developed 39 to 82% fewer worms than controls. DT-1 had no protective effect. Combining DT-1 and DT-2 abolished this protection. These experiments demonstrate that F. hepatica glycoprotein antigens induce in mice significant protection to infection with S. mansoni and offer an interesting approach to the study of vaccines in experimental schistosomiasis. PMID:118932
Rondelaud, D; Vignoles, P; Abrous, M; Dreyfuss, G
Field investigations were carried out over a two-year period in 52 natural watercress beds located in the Limousin region of central France to list the mammal and bird species that frequented these sites. This enabled detection of the definitive hosts of Fasciola hepatica and determination of the prevalence of natural infection in snails. A total of 13 mammal and five bird species were listed in these watercress beds. Adult flukes were found in Lepus capensis (39.2%), Oryctolagus cuniculus (42.0%), and Sylvilagus floridanus (25.0%). No infection with F. hepatica was noted in the five species of rodents studied. Snails infected with F. hepatica were found in 14 watercress beds. The global prevalence of natural infection was 1.1% in Lymnaea truncatula and 0.3% in L. glabra. Among the other trematode larval forms detected, the most frequent was Haplometra cylindracea (0.5%). In the Limousin region, the presence of hares and rabbits in watercress beds ensured the continuation of the F. hepatica life cycle and permitted the subsequent infection of humans when this wild watercress was eaten.
Vignoles, Philippe; Dreyfuss, Gilles; Rondelaud, Daniel
As large numbers of metacercariae of Fasciola hepatica are necessary for research, experimental infections of Galba truncatula and Pseudosuccinea columella with this digenean were carried out to determine the better intermediate host for metacercarial production and, consequently, the most profitable snail for decreasing the cost price of these larvae. Pre-adult snails (4 mm in shell height) originating from two populations per lymnaeid species were individually exposed to two or five miracidia, raised at 23 °C and followed for cercarial shedding up to their death. Compared to values noted in G. truncatula, the survival of P. columella on day 30 post-exposure was significantly greater, while the prevalence of F. hepatica infection was significantly lower. In the four P. columella groups, metacercarial production was significantly greater than that noted in the four groups of G. truncatula (347–453 per cercariae-shedding snail versus 163–275, respectively). Apart from one population of G. truncatula, the use of five miracidia per snail at exposure significantly increased the prevalence of F. hepatica in P. columella and the other population of G. truncatula, whereas it did not have any clear effect on the mean number of metacercariae. The use of P. columella for experimental infections with F. hepatica resulted in significantly higher metacercarial production than that noted with G. truncatula, in spite of a lower prevalence for the former lymnaeid. This finding allows for a significant decrease in the cost price of these larvae for commercial production. PMID:25907356
Dracz, Ruth Massote; Ribeiro, Vinicius Marques Antunes; Pereira, Cintia Aparecida de Jesus; Lima, Walter Dos Santos
Fasciola hepatica is a parasite that affects the hepatic ducts of several species of domestic and wild vertebrates, causing huge economic losses to livestock rearing worldwide. Reports on occurrences of F. hepatica in capybaras are an important epidemiological aspect of this disease, since these rodents can be a source of contamination for other animals and humans. In the present study, conducted in a rural area of the municipality of Confins, Minas Gerais, fresh feces from capybaras were collected from the ground near a lagoon at the edge of the Ribeirão da Mata river. These were examined using the technique of four metal sieves. F. hepatica eggs were recovered. This trematode species was confirmed by observing morphological characteristics and measuring the eggs recovered from the capybara feces, and through experimental infection of Lymnaea columella (Say, 1817) by miracidia from these eggs and subsequent infection of C57/BL06 mice with metacercariae originating from these infected mollusks. The data suggest the occurrence of natural cycle of F. hepatica in this region and provide a warning that expansion of the geographical distribution of this parasite by means of this rodent is possible. It is therefore important to adopting measures for epidemiological control of this helminthiasis.
Georgieva, Katya; Georgieva, Liliya; Mizinska-Boevska, Yana; Stoitsova, Stoyanka R
The presence and distribution of surface carbohydrates in the tissues of Galba truncatula snails uninfected or after infection with Fasciola hepatica as well as on the surface of the snail-pathogenic larval stages of the parasite were studied by lectin labelling assay. This is an attempt to find similarities that indicate possible mimicry, utilised by the parasite as an evasion strategy in this snail-trematode system. Different binding patterns were identified on head-foot-mantle, hepatopancreas, genital glands, renopericardial complex of the host as well as of the snail-pathogenic larval stages of F. hepatica. The infection with F. hepatica leads to changes of labelling with Glycine max in the head-mantle cells and Arachis hypogaea in the tubular epithelium of the hepatopancreas. The lectin binding on the other snail tissues is not changed by the development of the larvae. Our data clearly demonstrated the similarity in labelling of G. truncatula tissues and the surface of the snail-pathogenic larval stages of F. hepatica. The role of glycosylation of the contact surfaces of both organisms in relation to the host-parasite interactions is also discussed. PMID:27384082
Cabán-Hernández, Kimberly; Espino, Ana M
FhSAP2 is a novel antigen isolated from the adult fluke of Fasciola hepatica. Based on sequence similarity with amoebapores and other related proteins, it belongs to the saposin-like protein (SAPLIP) family. FhSAP2 has been shown to be highly immunogenic and capable of inducing protective immune responses in mice and rabbits challenged with F. hepatica. Moreover, FhSAP2 is also reactive with sera from humans with chronic fascioliasis. In the present study, we investigated the expression of FhSAP2 in various developmental stages of F. hepatica by qPCR and demonstrated that FhSAP2-mRNA species are up-regulated in undeveloped eggs, newly excysted juveniles, and adults, but down-regulated in the miracidium stage. Monoclonal antibodies against FhSAP2 were produced, and two clones that are positive to F. hepatica whole-body extract, but not reactive with extracts from other trematodes, were selected, expanded and used for histolocalization studies. Confocal immunofluorescence revealed the presence of native FhSAP2 in epithelial cells surrounding the gut, toward the outermost part of the tegument, and toward the tegumental cells of both adults and newly excysted juveniles.
Vignoles, Philippe; Dreyfuss, Gilles; Rondelaud, Daniel
As large numbers of metacercariae of Fasciola hepatica are necessary for research, experimental infections of Galba truncatula and Pseudosuccinea columella with this digenean were carried out to determine the better intermediate host for metacercarial production and, consequently, the most profitable snail for decreasing the cost price of these larvae. Pre-adult snails (4 mm in shell height) originating from two populations per lymnaeid species were individually exposed to two or five miracidia, raised at 23 °C and followed for cercarial shedding up to their death. Compared to values noted in G. truncatula, the survival of P. columella on day 30 post-exposure was significantly greater, while the prevalence of F. hepatica infection was significantly lower. In the four P. columella groups, metacercarial production was significantly greater than that noted in the four groups of G. truncatula (347-453 per cercariae-shedding snail versus 163-275, respectively). Apart from one population of G. truncatula, the use of five miracidia per snail at exposure significantly increased the prevalence of F. hepatica in P. columella and the other population of G. truncatula, whereas it did not have any clear effect on the mean number of metacercariae. The use of P. columella for experimental infections with F. hepatica resulted in significantly higher metacercarial production than that noted with G. truncatula, in spite of a lower prevalence for the former lymnaeid. This finding allows for a significant decrease in the cost price of these larvae for commercial production.
Espinoza, Jose R; Maco, Vicente; Marcos, Luis; Saez, Sandra; Neyra, Victor; Terashima, Angelica; Samalvides, Frine; Gotuzzo, Eduardo; Chavarry, Elizabeth; Huaman, Maria Cecilia; Bargues, M Dolores; Valero, M Adela; Mas-Coma, Santiago
The performance of Fas2-ELISA for the diagnosis of Fasciola hepatica infection in children living in areas of high endemicity for fascioliasis in the Peruvian Andes is analyzed. Fas2-ELISA is based on the detection of circulating IgG antibodies elicited in infected individuals against a F. hepatica antigen termed Fas2. The study was conducted in three Andean localities, Huertas-Julcan in Junin, Asillo in Puno, and Cajamarca, with a total population of 634 children in an age range 1 to 16 years old. Child fascioliasis prevalence was 21.1% in Huertas-Julcan, 25.4% in Asillo, and 24% in Cajamarca, estimated by coprological inspection. The seroprevalence of F. hepatica infection, determined by Fas2-ELISA, was 27.8% in Huertas-Julcan, 44.6% in Asillo, and 29.1% in Cajamarca. The overall sensitivity of Fas2-ELISA was 92.4%, the specificity 83.6%, and the negative predictive value 97.2%. No association between OD(450) Fas2-ELISA and infection intensity measured by egg counting was observed. Results show that Fas2-ELISA is a highly sensitive immunodiagnostic test for the detection of F. hepatica infection in children living in human fascioliasis endemic areas.
Georgieva, Katya; Georgieva, Liliya; Mizinska-Boevska, Yana; Stoitsova, Stoyanka R
The presence and distribution of surface carbohydrates in the tissues of Galba truncatula snails uninfected or after infection with Fasciola hepatica as well as on the surface of the snail-pathogenic larval stages of the parasite were studied by lectin labelling assay. This is an attempt to find similarities that indicate possible mimicry, utilised by the parasite as an evasion strategy in this snail-trematode system. Different binding patterns were identified on head-foot-mantle, hepatopancreas, genital glands, renopericardial complex of the host as well as of the snail-pathogenic larval stages of F. hepatica. The infection with F. hepatica leads to changes of labelling with Glycine max in the head-mantle cells and Arachis hypogaea in the tubular epithelium of the hepatopancreas. The lectin binding on the other snail tissues is not changed by the development of the larvae. Our data clearly demonstrated the similarity in labelling of G. truncatula tissues and the surface of the snail-pathogenic larval stages of F. hepatica. The role of glycosylation of the contact surfaces of both organisms in relation to the host-parasite interactions is also discussed.
Corba, J; Spaldonová, R
We studied the specificity of the individual developmental stages of Fasciola hepatica for evoking immune response of the host to reinfection with this parasite, whereby the primary infection was eliminated by a dose of 150 mg/kg diamphenethide administered in various intervals. In rats we observed a state of hypersensitivity demonstrated by retarded migration and growth of the flukes and the reduction in the number of sexually developed parasites. The changes were most marked, if the elimination of the immunizing infection followed 8-10 weeks after primary infection.
Bouvry, M; Rau, M E
Seasonal changes in Fasciola hepatica egg passage were documented in 17 dairy herds in Portneuf County, Quebec (Canada). Egg passage was low or absent in spring and summer but rose in autumn to reach a peak in winter, then subsequently returned to low levels. This study suggests that there are two periods of transmission to the bovine host during the grazing season: a minor period in the spring, due to overwintered snails, and a major period in August and September due to snails that acquired the infection in May or June.
Fasciolosis, caused by the liver fluke (Fasciola hepatica) is an important issue for both human and animal health. The disease evokes economic losses which are a consequence of impaired animal productivity leading to higher costs of meat and milk production, as well as liver condemnation. The goals of this thesis were to: (1) elaborate a molecular method--PCR for the detection of F. hepatica DNA in intermediate and definite hosts; (2) estimate the usefulness of a recombinated cysteine proteinase produced in E. coli in the form of inclusive bodies in serological diagnosis of F. hepatica infection in definite hosts, using an enzyme-linked immunosorbent assay (ELISA); (3) conduct field research on the prevalence of infection among intermediate and definitive hosts (cattle) in chosen regions of Poland, utilizing the elaborated methods. Based on the results obtained in this study, it was established that it is possible to detect F. hepatica DNA in the feces of definite hosts with the elaborated PCR method. The amplification of a 124 base pair tandem repeat allows the detection of fluke larval stages in intermediate hosts within 12 hours of exposure and F. hepatica infection in definite hosts (by the 5th week in rats, 8th week in sheep and 10th week in cattle). Therefore, the PCR test is more sensitive than traditional microscopic methods. Furthermore, it was determined that, the recombinated cysteine proteinase in the form of inclusive bodies, after solubillization exhibits antigenic properties of the native protein and the ELISA method based on this antigen may be useful as a tool for diagnosing fasciolosis in sheep and cattle, in both serum and milk samples. The test achieves a greater sensitivity and specificity than an ELISA based on native excretory-secretory antigens. The results of field research indicate that Fasciola hepatica is a frequent parasite of cattle in central and eastern Poland. The mean prevalence was 34.86% (+/- 16.95) in all studied areas. The
Krump, L; Hamilton, C M; Sekiya, M; O'Neill, R; Mulcahy, G
Fasciola hepatica is a common parasite in cattle, and bovine fasciolosis causes significant production losses, as well as being a zoonotic disease of global importance. F. hepatica has been shown to have immunoregulatory effects and the aim of this research was to establish whether F. hepatica infection influences the response to vaccination against respiratory pathogens in calves. A total of 48 calves were randomly and equally allocated to two groups. The experimental group was infected with F. hepatica, while the other group was used as a control. At week 2 and 6 after infection calves from both groups were administered a vaccine containing inactivated PI-3, BRSV and Mannheimia haemolytica, pathogens commonly associated with bovine respiratory disease. Blood samples were taken weekly over 12 weeks to measure specific antibodies against all vaccine antigens and against F. hepatica, as well as IgG1 and IgG2 isotypes for PI-3 and BRSV specific antibodies. Faecal samples were examined for F. hepatica eggs and routine haematology and liver enzyme biochemistry were performed and cytokine production in vitro measured. Liver enzymes (GGT and GLDH) and eosinophils were significantly higher in the experimental group, whereas neutrophil numbers were higher in the control group. There was no significant difference between the groups in terms of vaccine-specific total responses to PI-3, BRSV and M. haemolytica. IgG1 levels were higher in comparison to IgG2 levels in both PI-3 and BRSV specific antibodies. IL-4 levels from stimulated and unstimulated PBMC were significantly higher in the control group. IFN-γ levels were significantly higher in PBMC from the control group when cultured in medium only. No significant differences were noted in the levels of other cytokines measured. In this work, no effect of early F. hepatica infection on the antibody responses to a range of respiratory vaccine antigens in calves was shown. However, differences in cytokine responsiveness of
Periago, M V; Valero, M A; El Sayed, M; Ashrafi, K; El Wakeel, A; Mohamed, M Y; Desquesnes, M; Curtale, F; Mas-Coma, S
Fasciola gigantica is the main fasciolid species in Africa; however, F. hepatica and F. gigantica overlap in some countries. Egypt deserves mentioning because of the emerging situation of human fascioliasis in the Nile Delta area. The morphometric characteristics of fasciolid adults infecting the main livestock species present in the Nile Delta human endemic area are analyzed through a computer image analysis system (CIAS) on the basis of standardized measurements known to be useful for the differentiation of both fasciolid species. This is the first time that such a study is performed in an African country and, therefore, the results are compared to (i) F. hepatica (European Mediterranean area) and F. gigantica (Burkina Faso) standard populations, i.e. geographical areas where both species do not co-exist, and (ii) F. hepatica and F. gigantica populations from geographical areas where both species do co-exist, including the presence of intermediate forms (Iran). Results indicate the presence of F. hepatica, F. gigantica and intermediate forms (Fasciola sp.) in Egypt for the first time, and demonstrate the usefulness of CIAS for the phenotypic characterization of liver fluke adults from a concrete fascioliasis endemic area. Body roundness, body length over body width, and distance between the ventral sucker and the posterior end of the body provide useful tools for studying inter- and intraspecific morphological diversity in Fasciola adults. The application of these markers to specimens from geographical areas where F. hepatica and F. gigantica co-exist, such as in Egypt and Iran, suggest a strong population-level variation in Fasciola adult morphology.
Le, Thanh Hoa; Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Le, Xuyen Thi Kim; Hoang, Chau Thi Minh; De, Nguyen Van
A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.
Ferraro, Florencia; Merlino, Alicia; dell´Oca, Nicolás; Gil, Jorge; Tort, José F.; Gonzalez, Mercedes; Cerecetto, Hugo; Cabrera, Mauricio
Background Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections. Methodology/Principle Findings We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1). Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 μM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells. Conclusions/Significance Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34) is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues
Mas-Coma, S; Funatsu, I R; Bargues, M D
Fascioliasis due to the digenean species Fasciola hepatica has recently proved to be an important public health problem, with human cases reported in countries of the five continents, including severe symptoms and pathology, with singular epidemiological characteristics, and presenting human endemic areas ranging from hypo- to hyperendemic. One of the singular epidemiological characteristics of human fascioliasis is the link of the hyperendemic areas to very high altitude regions, at least in South America. The Northern Bolivian Altiplano, located at very high altitude (3800-4100 m), presents the highest prevalences and intensities of human fascioliasis known. Sequences of the internal transcribed spacers ITS-1 and ITS-2 of the nuclear ribosomal DNA of Altiplanic Fasciola hepatica and the intermediate snail host Lymnaea truncatula suggest that both were recently introduced from Europe. Studies were undertaken to understand how the liver fluke and its lymnaeid snail host adapted to the extreme environmental conditions of the high altitude and succeeded in giving rise to high infection rates. In experimental infections of Altiplanic lymnaeids carried out with liver fluke isolates from Altiplanic sheep and cattle, the following aspects were studied: miracidium development inside the egg, infectivity of miracidia, prepatent period, shedding period, chronobiology of cercarial emergence, number of cercariae shed by individual snails, survival of molluscs at the beginning of the shedding process, survival of infected snails after the end of the shedding period and longevity of shedding and non-shedding snails. When comparing the development characteristics of European F. hepatica and L. truncatula, a longer cercarial shedding period and a higher cercarial production were observed, both aspects related to a greater survival capacity of the infected lymnaeid snails from the Altiplano. These differences would appear to favour transmission and may be interpreted as strategies
Alba, Annia; Vázquez, Antonio A; Hernández, Hilda; Sánchez, Jorge; Marcet, Ricardo; Figueredo, Mabel; Sarracent, Jorge; Fraga, Jorge
Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis - F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere.
Zafra, R; Pérez, J; Buffoni, L; Martínez-Moreno, F J; Acosta, I; Mozos, E; Martínez-Moreno, A
The proportions of CD4(+), CD8(+) and WC1+ T lymphocytes from peripheral blood using flow cytometry were investigated in goats infected with Fasciola hepatica and previously immunised with recombinant Cathepsin-L1 (rCL1) and Glutathione-S-transferase sigma class (GST). The immunisation trial did not induce protective responses, and no significant differences were recorded between immunised and non-immunised groups. However, there was a significant decrease in the proportion of CD4(+) T lymphocytes in the infected groups both at 5 weeks post-infection (wpi), coinciding with the migratory stage of the infection, and at 12 wpi in the biliary stage of the infection. The proportional decrease in this circulating population may be related to the recruitment of CD4(+) T cells in liver and hepatic lymph nodes and also to the immunomodulatory effect of the parasite through the interaction of F. hepatica excretory-secretory products (FhESP) with this cell population. To date, this is the first report about the effect of F. hepatica infection in peripheral lymphocyte subsets in goats.
Adams, P N; Aldridge, A; Vukman, K V; Donnelly, S; O'Neill, S M
The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory-secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune-modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2-like phenotype in vivo that modulates cytokine production from CD4(+) cells in response to anti-CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym-1/2 but not RELMα in FhTeg-stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL-13. FhTeg can induce IL-13-producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune-modulatory source during F. hepatica infection and sheds further light on helminth-macrophage interactions.
Haçarız, Orçun; Baykal, Ahmet Tarık; Akgün, Mete; Kavak, Pınar; Sağıroğlu, Mahmut Şamil; Sayers, Gearóid Patrick
Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed.
Presidente, P. J. A.; McCraw, B. M.; Lumsden, J. H.
Three white-tailed deer (Odocoileus virginianus) were inoculated with 1000 metacercariae of Fasciola hepatica and examined on days 7, 14, and 28 postinoculation to determine the early response of a resistant host to this infection. It was concluded that only small numbers of the metacercariae penetrated the intestinal wall into the peritoneal cavity. Flukes that migrated to the liver penetrated through Glisson's capsule, primarily on the parietal surface. Marked fibroplasia and cellular infiltration of the capsule were induced and flukes were killed and destroyed in granulomas immediately beneath the capsule. Migration in hepatic parenchyma was minimal and immature flukes or migratory tracks were not found. There were infiltrations of eosinophils and mononuclear cells, bile duct hyperplasia and fibroplasia in portal areas. A few flukes penetrated through the diaphragm within 14 days postinoculation and on day 28 granulomas were observed on the dorsal surface of the lung where F. hepatica had penetrated this organ. The early reaction of Glisson's capsule to F. hepatica infection in white-tailed deer has not been described in cattle, sheep or swine infected with this fluke. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8.Fig. 9.Fig. 10. PMID:4277445
Abdolahi Khabisi, S; Sarkari, B
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 180 kDa. 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 62 kDa were common in both species, bands of 19, 45, 55 and 58 kDa 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 67 kDa and polyclonal antibodies raised against somatic antigens of both species reacted with three protein bands, 25, 27 and 72 kDa. Thus, the 25, 27 and 29 kDa protein bands may serve as immunodominant antigens, which might be considered for serodiagnosis of fasciolosis. Moreover, bands of 62 and 67 kDa in ES antigen and 72 kDa in somatic antigens of both species were immunodominant and might be suitable candidate for development of serological assays for diagnosis of fasciolosis.
Marcos Raymundo, Luis A; Maco Flores, Vicente; Terashima Iwashita, Angélica; Samalvides Cuba, Frine; Gotuzzo Herencia, Eduardo
Recent studies show that human fascioliasis is an infectious disease with significance in Peru and in other Latin American countries. The purpose of this study is to describe the clinical characteristics with a criteria towards the diagnosis of chronic infection by hepatic Fasciola in children. Sixty five individuals with ages ranging between 4 and 15 years were included, all from the District of Asillo (fascioliasis endemic area) in the Province of Azángaro, Department of Puno, Peru. A clinical exam was performed, as well as egg count per gram of feces (EPG count) and eosinophiles count. The group of children with ages between 8 and 11 years was the most affected, accounting for 47.5% of the total. The most frequent result in the exams was abdominal pain (82%) with epigastric location (37.7%), the Murphy symptom showed in 25 children (41% and there was a jaundice record in 17 children (27.9%). The rest of the symptoms and signs were non-specific. In the leukocyte count, 43.5% presented eosinophilia. The average count of eosinophiles and the severity of the infection (EPG count) decreased as the child became older. We concluded that chronic fascioliasis in endemic zones presents abdominal pain localized in the epigastrium and the Murphy symptom as the most frequent clinical biliary characteristics, while the rest of the symptoms are non-specific and this is probably due to the high endemic degree of other intestinal parasitosis which are present in this population. Finally the eosinophilia of chronic fascioliasis is in inverse relation with age in individuals of endemic zones and the number of parasites probably decreases as the person grows older, probably due to a modulation in the immune response of the individual or by the natural death of the parasite.
Ibarra-Velarde, F; Vera-Montenegro, Y; Nájera-Fuentes, R; Sánchez-Albarran, A
A controlled trial of the efficacy of several anthelmintic compounds as a combined therapy in the treatment of gastrointestinal nematodes (GIN) and Fasciola hepatica (F. hepatica) in naturally infected cattle was carried out. Twenty crossbred calves, 8-18 months old, were selected for inclusion in the trial based on finding eggs of F. hepatica and GIN in the faeces. They were blocked in four groups of five animals each according to GIN fecal egg counts on day 0. Treatments were sequentially allocated to animals in each block as follows: Group 1 served as non-treated control; Group 2 was treated with netobimin orally at 20 mg/kg; Group 3 received triclabendazole orally at 12 mg/kg and levamisole was applied intramuscularly at 5.5 mg/kg; Group 4 received clorsulon administered subcutaneously (s.c.) at 2 mg/kg and ivermectin s.c. at 200 microg/kg. Six to eight days after treatment the animals were euthanatized in order to collect and identify the parasites. Results showed a reduction of GIN by 87.3, 95.8 and 99.5% in Groups 2, 3 and 4, respectively. The percentage reduction of immature flukes was 0.0, 72.5, and 67.5% and for adult flukes 91.0, 97.5 and 100% for Groups 2, 3 and 4, respectively. Compounds indicated against nematodes showed high efficacy and products directed against F. hepatica acceptably removed adult flukes. However, efficacy against immature stages was generally not satisfactory.
Rodríguez, Ernesto; Carasi, Paula; Frigerio, Sofía; da Costa, Valeria; van Vliet, Sandra; Noya, Verónica; Brossard, Natalie; van Kooyk, Yvette; García-Vallejo, Juan J.; Freire, Teresa
Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a trematode zoonosis of interest in public health and livestock production. Like other helminths, F. hepatica modulates the host immune response by inducing potent polarized Th2 and regulatory T cell immune responses and by downregulating the production of Th1 cytokines. In this work, we show that F. hepatica glycans increase Th2 immune responses by immunomodulating TLR-induced maturation and function of dendritic cells (DCs). This process was mediated by the macrophage Gal/GalNAc lectin (MGL) expressed on DCs, which recognizes the Tn antigen (GalNAc-Ser/Thr) on parasite components. More interestingly, we identified MGL-expressing CD11c+ cells in infected animals and showed that these cells are recruited both to the peritoneum and the liver upon F. hepatica infection. These cells express the regulatory cytokines IL-10, TNFα and TGFβ and a variety of regulatory markers. Furthermore, MGL+ CD11c+ cells expand parasite-specific Th2/regulatory cells and suppress Th1 polarization. The results presented here suggest a potential role of MGL in the immunomodulation of DCs induced by F. hepatica and contribute to a better understanding of the molecular and immunoregulatory mechanisms induced by this parasite. PMID:28360908
Rodríguez, Ernesto; Carasi, Paula; Frigerio, Sofía; da Costa, Valeria; van Vliet, Sandra; Noya, Verónica; Brossard, Natalie; van Kooyk, Yvette; García-Vallejo, Juan J; Freire, Teresa
Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a trematode zoonosis of interest in public health and livestock production. Like other helminths, F. hepatica modulates the host immune response by inducing potent polarized Th2 and regulatory T cell immune responses and by downregulating the production of Th1 cytokines. In this work, we show that F. hepatica glycans increase Th2 immune responses by immunomodulating TLR-induced maturation and function of dendritic cells (DCs). This process was mediated by the macrophage Gal/GalNAc lectin (MGL) expressed on DCs, which recognizes the Tn antigen (GalNAc-Ser/Thr) on parasite components. More interestingly, we identified MGL-expressing CD11c(+) cells in infected animals and showed that these cells are recruited both to the peritoneum and the liver upon F. hepatica infection. These cells express the regulatory cytokines IL-10, TNFα and TGFβ and a variety of regulatory markers. Furthermore, MGL(+) CD11c(+) cells expand parasite-specific Th2/regulatory cells and suppress Th1 polarization. The results presented here suggest a potential role of MGL in the immunomodulation of DCs induced by F. hepatica and contribute to a better understanding of the molecular and immunoregulatory mechanisms induced by this parasite.
Dalton, John P; Robinson, Mark W; Mulcahy, Grace; O'Neill, Sandra M; Donnelly, Sheila
The liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle), a global disease that is also an important infection of humans. As soon as the parasite invades the gut wall its interaction with various host immune cells (e.g. dendritic cells, macrophages and mast cells) is complex. The parasite secretes a myriad of molecules that direct the immune response towards a favourable non-protective Th2-mediate/regulatory environment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1), are under development as the first generation of fluke vaccines. However, this peptidase and other molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treat immune-related conditions in humans and animals.
Marcos, L A; Bussalleu, A; Terashima, A; Espinoza, J R
The prevalence of Fasciola hepatica infection, in endemic countries, in patients with established cirrhosis is unknown. We hypothesized that, in endemic countries, the presence of fascioliasis may be detected in a serum pool of cirrhotic patients. Forty-four previously stored serum samples of patients with established liver cirrhosis, in the Hospital Nacional Cayetano Heredia in Lima, Peru, were collected from 1998 to 2003 and assessed for hepatitis B, C and fascioliasis antibodies (Fas2 ELISA). Hepatitis B surface antigen (HBsAg) was positive in 8.8% (n = 34), hepatitis B core antibody (anti-HBc) in 32.5% (n = 34), hepatitis C antibodies (anti-HCV) in 9.1% (n = 33), and 9.1% (n = 44) were Fas2 ELISA positive. This disease is an example of an emerging tropical infection which can be present in chronic liver diseases, requiring greater clinician awareness especially in endemic rural areas. Further clinical studies are warranted.
Lopez, Martha; White, A Clinton; Cabada, Miguel M
There is a high prevalence of fascioliasis in the Peruvian highlands, but most cases remain undiagnosed. The burden of disease caused by chronic subclinical infection is largely unknown. We studied school-age children from a district in Paucartambo Province in Cusco, Peru to evaluate the burden of disease caused by subclinical fascioliasis. Parasite eggs and/or larvae were identified in 46.2% of subjects, including Fasciola hepatica in 10.3% of subjects. Fascioliasis was independently associated with anemia (adjusted odds ratio = 3.01 [1.10-8.23]). Subclinical fascioliasis was common among children and strongly associated with anemia. Anemia should be recognized as an important component of the burden of disease from fascioliasis.
Hammami, H; Hamed, N; Ayadi, A
Epidemiological investigations on Fasciola hepatica fasciolasis were carried out from July 2004 to June 2005 in the Gafsa oases (Tunisia) after the detection of a human case. Three habitats were studied: one in El Gsar and two in Ain Soltan. The prevalence of human infection was 6.6%. The presence of the parasite was detected through serology in 14.3% of cattle, 35% of sheep and 68.4% of goats. The plants Apium nodiflorum, Oxalis cernua and Sonchus maritimus were suspected to be at the origin of animal contamination and Apium nodiflorum was incriminated in human infection. The prevalence of the infection of the intermediate host Galba truncatula (G. truncatula) was 19.2% from July 2004 to June 2005. Gafsa oases constitute a new location for the development of fasciolasis in the southern west of Tunisia.
Sürücü, Erdem; Demir, Yusuf; Dülger, Ahmet C.; Batur, Abdüssamed; Ölmez, Şehmus; Kitapçı, Mehmet T.
A 48-year-old female with complaints of gastrointestinal symptoms such as abdominal pain, fatigue, vomiting, nausea, and weight loss was diagnosed with neuroendocrine tumor after removal of a 2 mm lesion from the stomach with endoscopic biopsy. Her magnetic resonance imaging that was performed due to on-going symptoms showed multiple linear hypointense lesions in the liver. Positron emission tomography/computed tomography (PET/CT) scan was performed for differential diagnosis, which showed high fluorodeoxyglucose (FDG) uptake in these lesions. Clinical and laboratory findings revealed the final diagnosis as Fasciola hepatica. The imaging features of this case is presented to aid in differentiating this infectious disease from malignancy and avoid misdiagnosis on FDG-PET/CT. PMID:27751978
Khan, Imran; Khan, Amir Muhammad; Ayaz, Khan, Sanaullah; Anees, Muhammad; Khan, Shaukat Ali
Fascioliasis is spread through contamination of water sources and cause morbidity throughout the world. In the current study 300 water samples were processed by PCR for detection of Fasciola hepatica. The overall prevalence in different water sources was 9.66 % (29/300). Highest prevalence was recorded in drain water16 % (16/100) followed by tube well water 10% (4/40), open well water 8 % (8/100) and the lowest was recorded in tap water 1.66 %(1/60). The significant difference P < 0.05 was recorded during data analysis. The highest prevalence was recorded in summer. It was concluded from the study that cleaning and filtration should be adopted to avoid the health hazards against water borne zoonotic parasites.
Vázquez-Prieto, Severo; Vilas, Román; Ubeira, Florencio M; Paniagua, Esperanza
We found low genetic differentiation between two temporal samples of Fasciola hepatica (2006 and 2008) collected from nine sheep of the same flock that shared the same pasture for at least 2 years. However, each sample, represented by four and five infrapopulations respectively, showed strong heterozygote deficits regarding Hardy-Weinberg expectations and a high degree of genetic structure at infrapopulation level. This is an unexpected result since genetic drift should increase temporal variation among years. Our findings are most likely explained by the fact that the parasite can survive many years in the definitive host. Temporal gene flow favored by high longevity probably increases levels of genetic variability of the population but could also contribute to the observed heterozygote deficits within temporal samples and infrapopulations if it favors the Wahlund effect. Despite the homogenizing effect of gene flow, the high genetic divergence observed between infrapopulations is most likely a consequence of strong genetic drift associated to the complexity of the life cycle.
Dunkel, A M; Rognlie, M C; Johnson, G R; Knapp, S E
A collection of lymnaeid snails in Montana was made over a 3 year period, in conjunction with a state-wide survey of the distribution of liver flukes in Montana. Collection areas were selected based on reports of infected cattle, sheep or wildlife, and with the intent of covering all geographic regions of the state. Snails were found at all 97 of the locations chosen for collections, with lymnaeids collected at 71 of the locations. The 97 sites were located in 28 of Montana's 56 counties. Nine lymnaeid species were collected, five of which have been reported either as natural or experimental intermediate hosts for Fasciola hepatica or Fascioloides magna. The two snail species most widely distributed over the areas enzootic for the flukes were Lymnaea modicella and Lymanaea caperata.
Espino, A M; Díaz, A; Pérez, A; Finlay, C M
In the present study the dynamics of antigenemia and coproantigens were studied in patients with Fasciola hepatica infection during an outbreak occurring in La Palma, Pinar del Río, in the West Province of Cuba. Stool and serum samples were collected from 67 patients and 40 healthy subjects. Stool samples were studied by a simple gravity sedimentation technique and an ES78 sandwich enzyme-linked immunosorbent assay (ELISA) for observation of eggs and detection of parasite coproantigens, respectively. Serum samples were also studied by the ES78 sandwich ELISA and an indirect ELISA to detect circulating antigens and antibodies, respectively. At the beginning of the study, 8 of 67 patients had patent infections and 59 had prepatent infections, which was determined by the recent consumption of lettuce contaminated with metacercariae of F. hepatica, the presence of clinical symptoms, and the absence of Fasciola eggs in their stools. Patients with prepatent infections were monitored by all techniques until patency. Circulating antigens were not detected in patients with patent infections. However, coproantigens were clearly detected in all patients with patent infections. On the other hand, 28.8% of patients with prepatent infections tested positive for circulating antigens and 81.4% tested positive for coproantigens in the first stool sample studied. Only two other coproantigen determinations were necessary to diagnose 93.2% of the patients. While circulating antigen levels diminished in all patients during the infection, coproantigen levels increased. The present study demonstrates that the ES78 sandwich ELISA is a better tool than parasitological examination for diagnosis of active early infection, since by the combination of the circulating-antigen detection assay and the coproantigen detection assay 91% of patients were able to be diagnosed at the beginning of the study. In contrast, a coprologic analysis repeated over several weeks was necessary to diagnose 100
Ali, H; Ai, L; Song, H Q; Ali, S; Lin, R Q; Seyni, B; Issa, G; Zhu, X Q
In the present study, 16 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from sheep and cattle from seven geographical locations in Niger were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361/362 bp, respectively, for all liver fluke samples sequenced. Comparison of the ITS sequences of the Niger Fasciola samples examined in the present study with that of Fasciola hepatica, Fasciola gigantica, and the "intermediate Fasciola" from elsewhere revealed that the Niger Fasciola samples examined represent two species, namely F. hepatica and F. gigantica. This is the first demonstration of the existence of both F. hepatica and F. gigantica in Niger by a genetic approach, which provides foundation for further studies on F. hepatica and F. gigantica in Niger and has implications for studying the population genetic structure of the Niger Fasciola and for the diagnosis and control of the disease they cause.
Bennema, Sita C.; Scholte, Ronaldo Guilherme Carvalho; Molento, Marcelo Beltrão; Medeiros, Camilla; Carvalho, Omar dos Santos
Fasciolosis is a disease of importance for both veterinary and public health. For the first time, georeferenced prevalence data of Fasciola hepatica in bovines were collected and mapped for the Brazilian territory and data availability was discussed. Bovine fasciolosis in Brazil is monitored on a Federal, State and Municipal level, and to improve monitoring it is essential to combine the data collected on these three levels into one dataset. Data were collected for 1032 municipalities where livers were condemned by the Federal Inspection Service (MAPA/SIF) because of the presence of F. hepatica. The information was distributed over 11 states: Espírito Santo, Goiás, Minas Gerais, Mato Grosso do Sul, Mato Grosso, Pará, Paraná, Rio de Janeiro, Rio Grande do Sul, Santa Catarina and São Paulo. The highest prevalence of fasciolosis was observed in the southern states, with disease clusters along the coast of Paraná and Santa Catarina and in Rio Grande do Sul. Also, temporal variation of the prevalence was observed. The observed prevalence and the kriged prevalence maps presented in this paper can assist both animal and human health workers in estimating the risk of infection in their state or municipality. PMID:24553606
Tegument protein extract from Fasciola hepatica adult flukes (FhTA) was obtained and assessed for its potential as a diagnostic agent for the serological detection of human fascioliasis using an indirect enzyme-linked immunosorbent assay (ELISA). In an analysis of sera from 45 patients infected with F. hepatica, sera from 41 patients with other parasitic infections, and sera from 33 healthy controls, the FhTA-ELISA showed sensitivity, specificity, and accuracy of 91.1%, 97.3%, and 95%, respectively. Specific IgG1 and IgG4 were the antibody isotypes mainly detected in sera from patients with fascioliasis. Polypeptides of 52, 38, 24 to 26, and 12 to 14 kDa were identified by Western blotting as the most immunoreactive components of the FhTA. A proteomic approach led us to identify enolase, aldolase, glutathione S-transferase, and fatty acid binding protein as the major immunoreactive components of the FhTA. PMID:23015645
Sampaio-Silva, M L; Da Costa, J M; Da Costa, A M; Pires, M A; Lopes, S A; Castro, A M; Monjour, L
The antigenic components of excretory-secretory products (ESP) of adult worms of Fasciola hepatica were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using sera from 20 patients infected with F. hepatica. Sera from 184 other parasitic infections and 20 healthy volunteers were also analyzed. It was found that the ESP were composed of more than 11 polypeptides; five components detected in fascioliasis sera had molecular weights of 12.4, 16.4, 19.4, 25, and 27 kilodaltons (kD). Only the 25- and 27-kD components were recognized by all 20 fascioliasis sera. Using the ESP as antigen, it was possible to perform an enzyme-linked immunosorbent assay with a sensitivity of 95% and a specificity of 97%. Sera from other parasitic infections had antibodies to antigenic components with apparent molecular weights of 37, 38.4, 52, 63, 73, 87, 109, and 116 kD that were also found in sera from fascioliasis patients. These findings suggested that the 25- and 27-kD antigenic components may be sensitive and specific for the diagnosis of human fascioliasis.
Marcos, Luis; Maco, Vicente; Samalvides, Frine; Terashima, Angélica; Espinoza, José R; Gotuzzo, Eduardo
We conducted a case-control study to clarify the risk factors for human fascioliasis in the Northern Peruvian Altiplano. Cases (n=61) were individuals who were diagnosed as having chronic fascioliasis by faecal and/or serologic (Fas2-ELISA) studies. Controls (n=61) had negative serologic and coprological results for Fasciola hepatica. We identified an association between fascioliasis and four variables (40 analysed): the habit of drinking alfalfa juice (OR=4.5; 95% CI 1.8-11.1; P<0.001); familiarity with aquatic plants (OR=4.3; 95% CI 1.8-10.6; P<0.001); dog ownership (OR=5; 95% CI 1.7-15.1; P=0.002); and raising more than five sheep (OR=0.3; 95% CI 0.1-0.8; P=0.01). According to clinical presentation and laboratory studies, dizzy spells (P=0.01), history of jaundice (P=0.01), peripheral eosinophilia (P=0.005) and Ascaris lumbricoides in stools (P=0.001) were associated with fascioliasis. The principal exposure factor for F. hepatica infection was drinking alfalfa juice. In conclusion, we suggest that human fascioliasis in Peru should be suspected in patients from livestock-rearing areas, who present with recurrent episodes of jaundice and who have a history of consumption of alfalfa juice or aquatic plants, or who have eosinophilia.
Bennema, Sita C; Scholte, Ronaldo Guilherme Carvalho; Molento, Marcelo Beltrão; Medeiros, Camilla; Carvalho, Omar Dos Santos
Fasciolosis is a disease of importance for both veterinary and public health. For the first time, georeferenced prevalence data of Fasciola hepatica in bovines were collected and mapped for the Brazilian territory and data availability was discussed. Bovine fasciolosis in Brazil is monitored on a Federal, State and Municipal level, and to improve monitoring it is essential to combine the data collected on these three levels into one dataset. Data were collected for 1032 municipalities where livers were condemned by the Federal Inspection Service (MAPA/SIF) because of the presence of F. hepatica. The information was distributed over 11 states: Espírito Santo, Goiás, Minas Gerais, Mato Grosso do Sul, Mato Grosso, Pará, Paraná, Rio de Janeiro, Rio Grande do Sul, Santa Catarina and São Paulo. The highest prevalence of fasciolosis was observed in the southern states, with disease clusters along the coast of Paraná and Santa Catarina and in Rio Grande do Sul. Also, temporal variation of the prevalence was observed. The observed prevalence and the kriged prevalence maps presented in this paper can assist both animal and human health workers in estimating the risk of infection in their state or municipality.
Incani, Renzo Nino; Vieira, Juan Manuel; Pacheco, Mercedes; Planchart, Sandra; Amarista, Manuel; Lazdins, Janis
A new case of human infection due to Fasciola hepatica is reported in the Venezuelan parasitological literature. The patient is an 81-year-old female asymptomatic, with an eosinophilia of 21% and critrosedimentation rate of 26 mm/h and was found during a routine check up. These values were normal at the time of treatment (4 months later), as were several tests of hepatic function, blood chemistry and peripheral blood haematological values. The number of eggs of the parasite were between 90 and 130/g of facees. The patient was successfully treated with the human formulation of trielabendazole at 2 single doses of 10 mg/kg, each separated by 24 h. The same laboratory tests mentioned above did not show modifications, except for a slight increase in the cosinophil counts at 2 and 11 days and erythrosedimentation rate at 11 days post-treatment. Coproparasitological observations carried out at 12, 18, 60 and 120 days post-treatment were negative. No clinical symptoms were registered up to 2 months after treatment. The patient most probably acquired the infection at home through the ingestion of commercially available lettuce from an endemic area of bovine fascioliasis, very distant from home, and between 4 and 7 months previous to treatment. We stress the need to investigate F. hepatica infections which he been neglected, probably because of limited knowledge by health workers, lack of specific symptoms and absence of more sensitive diagnostic procedures.
Issia, Laura; Pietrokovsky, Silvia; Sousa-Figueiredo, José; Stothard, J Russell; Wisnivesky-Colli, Cristina
Between autumn and spring 2006, a coprological survey was performed in two wildlife reserves located in the north of Argentine Patagonia to determine the prevalence of Fasciola hepatica and the number of parasite eggs per gram (epg) of feces in wild guanacos (Lama guanicoe), coypus (Myocastor coypus), and locally born and raised goats and sheep. Snails of the Family Lymnaeidae were collected in freshwater habitats, identified taxonomically and analyzed parasitologically. Prevalence of patent infection was 100% in sheep (n=69) and coypus (n=9), 84% in goats (n=20) and 0.5% in guanacos (n=224). No significant differences in epg were found among animals, but the median epg of coypus (160) and sheep (160) was higher than that of goats (80). For guanacos and goats, a negative binomial model estimating the population egg-count frequency could be fitted, while for coypus and sheep parasite egg-count frequencies trended toward a normal distribution, indicative of a more even, and much less aggregated distribution across sampled hosts. All snails (n=175) were Lymnaea truncatula and none of them was found infected. This is the first report of fascioliasis in free-ranging guanacos in Argentina. Coypu appears to be a major wildlife reservoir of F. hepatica, which was presumably introduced locally by livestock.
Di Maggio, Lucía Sánchez; Tirloni, Lucas; Pinto, Antonio F M; Diedrich, Jolene K; Yates Iii, John R; Benavides, Uruguaysito; Carmona, Carlos; da Silva Vaz, Itabajara; Berasain, Patricia
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite.
Falcón, Cristian; Carranza, Franco; Martínez, Fernando F; Knubel, Carolina P; Masih, Diana T; Motrán, Claudia C; Cervi, Laura
Fasciola hepatica is a helminth trematode that migrates through the host tissues until reaching bile ducts where it becomes an adult. During its migration the parasite releases different excretory-secretory products (ESP), which are in contact with the immune system. In this study, we focused on the effect of ESP on the maturation and function of murine bone marrow derived-dendritic cells (DC). We found that the treatment of DC with ESP failed to induce a classical maturation of these cells, since ESP alone did not activate DC to produce any cytokines, although they impaired the ability of DC to be activated by TLR ligands and also their capacity to stimulate an allospecific response. In addition, using an in vitro ovalbumin peptide-restricted priming assay, ESP-treated DC exhibited a capacity to drive Th2 and regulatory T cell (Treg) polarization of CD4(+) cells from DO11.10 transgenic mice. This was characterized by increased IL-4, IL-5, IL-10 and TGF-beta production and the expansion of CD4(+)CD25(+)Foxp3(+) cells. Our results support the hypothesis that ESP from F. hepatica modulate the maturation and function of DC as part of a generalized immunosuppressive mechanism that involves a bias towards a Th2 response and Treg development.
Fromm, B; Ovchinnikov, V; Høye, E; Bernal, D; Hackenberg, M; Marcilla, A
Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune system and live inside a hostile environment such as the mammalian liver and bile ducts. Recently, microRNAs, a class of short noncoding gene regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction, and recent data illustrate that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe) and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions that we review here.
Solana, María Victoria; Mera y Sierra, Roberto; Scarcella, Silvana; Neira, Gisela; Solana, Hugo Daniel
Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.
Vázquez, A A; Lounnas, M; Sánchez, J; Alba, A; Milesi, A; Hurtrez-Boussès, S
In this study we present the first approach to exploration of the genetic diversity of Cuban Fasciola hepatica populations using microsatellite markers, coupled with observed prevalence in slaughterhouses. Nine populations of flukes recovered from cows and buffalos were studied in the central-western region of Cuba. The observed infection rates of definitive hosts (bovines) were 70-100% in most cases. An important amount of polymorphism was found in the four loci explored. However, no apparent genetic differences were found between populations from different provinces or bovine species. The absence of deviations from Hardy-Weinberg equilibrium suggests a high rate of cross-fertilization between F. hepatica individuals. This result was confirmed when all multilocus genotypes were tested for clonal reproduction and only four individuals differed statistically (P sex< 0.05). High values of expected heterozygosity coupled with highly probable mixing among strains make the metapopulation genetically diversified but similar in terms of certain alleles (low F ST values). These results suggest a close relationship between parasite diversity and cattle management in Cuba. Our findings should be taken into consideration by veterinary authorities to help mitigate fasciolosis transmission.
Saric, Jasmina; Li, Jia V; Utzinger, Jürg; Wang, Yulan; Keiser, Jennifer; Dirnhofer, Stephan; Beckonert, Olaf; Sharabiani, Mansour T A; Fonville, Judith M; Nicholson, Jeremy K; Holmes, Elaine
We characterize the integrated response of a rat host to the liver fluke Fasciola hepatica using a combination of (1)H nuclear magnetic resonance spectroscopic profiles (liver, kidney, intestine, brain, spleen, plasma, urine, feces) and multiplex cytokine markers of systemic inflammation. Multivariate mathematical models were built to describe the main features of the infection at the systems level. In addition to the expected modulation of hepatic choline and energy metabolism, we found significant perturbations of the nucleotide balance in the brain, together with increased plasma IL-13, suggesting a shift toward modulation of immune reactions to minimize inflammatory damage, which may favor the co-existence of the parasite in the host. Subsequent analysis of brain extracts from other trematode infection models (i.e. Schistosoma mansoni, and Echinostoma caproni) did not elicit a change in neural nucleotide levels, indicating that the neural effects of F. hepatica infection are specific. We propose that the topographically extended response to invasion of the host as characterized by the modulated global metabolic phenotype is stratified across several bio-organizational levels and reflects the direct manipulation of host-nucleotide balance.
Di Maggio, Lucía Sánchez; Tirloni, Lucas; Pinto, Antonio F. M.; Diedrich, Jolene K.; Yates III, John R.; Benavides, Uruguaysito; Carmona, Carlos; da Silva Vaz Jr., Itabajara; Berasain, Patricia
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite. PMID:27600774
Keiser, Jennifer; Manneck, Theresia; Kirchhofer, Carla; Braissant, Olivier
Isothermal microcalorimetry (IMC) is an analytical tool that continuously measures the heat flow generated by chemical, physical or biological processes. We have demonstrated that IMC is a useful tool to analyze drug effects on helminths, including adult Fasciola hepatica. Here, we used IMC to examine the activity of triclabendazole and its metabolites triclabendazole sulphone and triclabendazole sulphoxide on juvenile and adult F. hepatica. Worms (one adult or 2-3 juveniles) were placed in 4 or 20 ml glass ampoules containing RPMI 1640 and the test compound (25-100 μg/ml) and the heat flow and motility of worms was examined with TAM48 and TAMIII isothermal microcalorimetry instruments. IMC was found to be precisely document drug effects on juvenile F. hepatica and confirmed the pronounced effect of the benzimidazole derivatives on the motor activity of F. hepatica. Juvenile F. hepatica incubated with 100 μg/ml triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements 8.3, 35 and 6h post-incubation (all p<0.001). The metabolic heat of triclabendazole sulphoxide treated worms (100 μg/ml) was reduced by 50% and 76% 24 and 120 h post-incubation, respectively. Limitations of calorimetric measurements were observed using adult F. hepatica as only three worms could be measured simultaneously and also control worms showed a considerable decrease in heat flow. Adult F. hepatica exposed to triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements after 31 (p=0.009), 49 (p>0.05) and 88 (p>0.05)h. In conclusion, IMC is useful to document drug effects on juvenile F. hepatica and since rapid technological developments in this field are occurring IMC might also hold promise to study adult F. hepatica in the near future.
Escamilla, A; Bautista, M J; Zafra, R; Pacheco, I L; Ruiz, M T; Martínez-Cruz, S; Méndez, A; Martínez-Moreno, A; Molina-Hernández, V; Pérez, J
The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n=5) of sheep were used; groups 1-3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3(+) eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3(+) apoptotic eosinophils in the periphery of necrotic foci was high (46.1-53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection.
Vukman, K V; Adams, P N; O'Neill, S M
Fasciola hepatica tegumental coat antigen (FhTeg) suppresses dendritic cell maturation and function by inhibiting IL-6, tumour necrosis factor (TNF)-α, IL-10 and IL-12 production and CD80, CD86 and CD40 cell surface marker expression in TLR4-stimulated dendritic cells. Fasciola hepatica also impairs dendritic cell function by inhibiting its phagocytic capacity and its ability to prime T cells. We have shown previously that activation of mast cells with bacterial ligands is also inhibited by FhTeg. Fasciola hepatica suppresses LPS-induced NF-κB and MAPK pathway (ERK) activation in these cells. Previously, we demonstrated that FhTeg induces expression of suppressor of cytokine signalling (SOCS)3, a negative regulator of the TLR pathway in mast cells. In this study, we show the same inhibitory effect of FhTeg on the activation of the other members of the MAPKs pathway (ERK, p38, JNK) in dendritic cells and demonstrate an enhanced expression of SOCS3, but not SOCS1, SOCS5 or PIAS3 in this process. These studies enhance our understanding of the immunomodulatory effect of helminth molecules on the TLR pathway.
Neyra, Victor; Chavarry, Elizabeth; Espinoza, Jose R
Circulating antibody against Fasciola hepatica antigens was determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis in alpacas naturally exposed to F. hepatica. Serological assay parameters were established by using sera from eight infected animals and seven controls with no record of this parasitic infection. Excretory--secretory (ES-) products, Fas1- and Fas2-ELISA were used to survey 307 alpacas from a F. hepatica endemic area in the Peruvian Andes. Seroprevalence of F. hepatica infection varied from 56.7, 64.8 and 66.8% measured by Fas1-, Fas2- and ES-ELISA, respectively. The sensitivity for ES-ELISA was 95%, corresponding Fas1- and Fas2-ELISA sensitivity values were 90 and 95%. In this population, 7% of animals were positive for F. hepatica eggs in faeces, other parasites detected were Trichuris sp. (40%), Nematodirus sp. (34.6%), Lamanema sp. (12.8%) and Eimeria sp. (11.8%). The results show that F. hepatica infected animals elicit circulating antibodies against ES, Fas1 and Fas2. Fas2-ELISA may be proposed as a sensitive assay for the immunodiagnosis of fasciolosis in alpacas.
Martínez-Valladares, M; Cordero-Pérez, C; Castañón-Ordóñez, L; Famularo, M R; Fernández-Pato, N; Rojo-Vázquez, F A
We have evaluated the efficacy in sheep of a combination drench formulation at the recommended dose rate of 0.2 mg moxidectin/kg bodyweight and 10 mg triclabendazole/kg bodyweight against an experimental infection with Fasciola hepatica and a natural infection with gastrointestinal nematodes. We confirmed that the efficacy of reducing fecal egg output was 98.3% for trichostrongyle eggs and 100% for F. hepatica eggs. Based on adult worm and fluke recovery, the efficacy varied according to the target species. A reduction was found in the number of Teladorsagia circumcincta, Trichostrongylus spp., Nematodirus spp., and Trichuris spp. greater than 95%, but the efficacy for Oesophagostomum spp. varied, with values below 90%. The reduction in F. hepatica was higher than 95% for all stages. The effectiveness of the formulation was also confirmed by an increase in total proteins and albumin following treatment.
Morphew, Russell M; Hamilton, Clare M; Wright, Hazel A; Dowling, David J; O'Neill, Sandra M; Brophy, Peter M
Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.
Scarcella, S; Solana, M V; Fernandez, V; Lamenza, P; Ceballos, L; Solana, H
Fasciolasis is a zoonotic parasitic disease caused by Fasciola hepatica and its control is mainly based on the use of triclabendazole (TCBZ). Parasite resistance to different anthelmintics is growing worldwide, including the resistance of F. hepatica to TCBZ. In the present work we evaluate "in vivo" the activity of xenobiotic metabolizing enzymes of phase I (carboxyl esterases) and phase II (glutathione S-transferases and carbonyl reductases) recovered of flukes from sheep treated with TCBZ. All three enzymes showed increased activity in TCBZ flukes returning 60h post-treatment at similar to baseline unexposed flukes. TCBZ action may induce secondary oxidative stress, which may explain the observed increment in activities of the analyzed enzymes as a defensive mechanism. The enzymes analyzed are candidates to participate actively in the development of resistance at TCBZ in F. hepatica.
Dar, Y; Lounnas, M; Djuikwo Teukeng, F F; Mouzet, R; Courtioux, B; Hurtrez-Boussès, S; Vignoles, P; Dreyfuss, G; Rondelaud, D
Two French populations of Galba truncatula were subjected to experimental infections with Egyptian and French isolates of Fasciola sp. miracidia, originating from cattle and sheep, to compare characteristics of snail infections in allopatric and sympatric groups. All sampled Egyptian isolates were identified as Fasciola hepatica using microsatellite markers. Compared to snails infected with French miracidia, snail survival at day 30 post-exposure was significantly greater in the Egyptian groups, while prevalence of infection was significantly lower (in an Egyptian group infected with cattle-derived miracidia) or did not show any significant differences in the other three cases. The total number of metacercariae was significantly higher in the four Egyptian groups. However, snail population and the mammalian origin of F. hepatica had also a significant effect on this parameter. The dissection of snail cadavers showed a significantly higher number of free rediae in the Egyptian groups, even if snail population also had a significant effect on the redial burden. Both Egyptian isolates of F. hepatica could easily develop in French snails, causing a low mortality in snails and inducing a metacercarial production higher than that noted in sympatric infections. However, the mammalian origin of F. hepatica eggs and the quality of snail populations as intermediate hosts had to be taken into account for studying local adaptation in reason of their effects on this process.
Dar, Y; Vignoles, P; Rondelaud, D; Dreyfuss, G
Experimental infections of three Egyptian Pseudosuccinea columella populations with sympatric miracidia of Fasciola sp., coming from cattle- or sheep-collected eggs, were carried out to determine the capacity of this lymnaeid to support larval development of the parasite. Using microsatellite markers, the isolates of Egyptian miracidia were identified as Fasciola hepatica. Apart from being independent of snail origin, prevalences ranging from 60.4 to 75.5% in snails infected with five miracidia of F. hepatica were significantly higher than values of 30.4 to 42.2% in snails with bi-miracidial infections. The number of metacercariae ranged from 243 to 472 per cercarial-shedding snail and was independent of snail origin, parasite origin and miracidial dose used for infection. If P. columella was subjected to two successive bi-miracidial infections with F. hepatica, prevalence of infection was 63.3%, with a mean of 311 metacercariae per snail. These values were clearly greater than those already reported for Radix natalensis infected with the same parasite and the same protocol. Successful experimental infection of P. columella with F. hepatica suggests that this lymnaeid snail is an important intermediate host for the transmission of fascioliasis in Egypt.
Esteban, J G; Flores, A; Aguirre, C; Strauss, W; Angles, R; Mas-Coma, S
Coprological studies of school children from four communities in the Northern Bolivian Altiplano were carried out in order to estimate the prevalences and intensities of Fasciola hepatica infection. Single stool specimens were collected at random from 558 school children (308 boys and 250 girls) aged 5-19 years old. Nineteen different parasite species (13 protozoan and six helminths) were detected. Of the children examined, 98.7% (96.5-100%) presented infection with at least one parasite species. The mean prevalence of 27.6% by Fasciola hepatica (range, 5.9-38.2%) was the highest not only with respect to the helminth species found in the Northern Bolivian Altiplano but also among the fasciolosis prevalences reported in children in other parts of the world to date. Prevalences were significantly different among the communities surveyed and was significantly higher in the 9-12 years age group. There were, however, no significant differences between sexes. Among the 154 children presenting F. hepatica eggs in stools, intensities ranged from 24-5064 eggs per gram of faeces (epg), with arithmetic and geometric means of 474 and 201 epg, respectively. Significant differences in mean egg output were detected between communities, sexes and age groups. Individual fasciolosis infections coexisting with other pathogenic parasite species (Entamoeba histolytica and/or E. dispar, Giardia intestinalis, Balantidium coli, Dientamoeba fragilis, Cryptosporidium sp., Hymenolepis nana, Taenia spp., Trichuris trichiura, Ascaris lumbricoides and Enterobius vermicularis) were detected. A significant positive association with F. hepatica was only found in the case of G. intestinalis. This coprological study not only verifies the existence of high prevalences of F. hepatica among humans in the Northern Bolivian Altiplano, but also demonstrates the need to expand the Southern boundaries of this high endemic zone to include the Southeastern region of Lake Titicaca.
Farjallah, Sarra; Ben Slimane, Badreddine; Piras, Cristina Maria; Amor, Nabil; Garippa, Giovanni; Merella, Paolo
The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n=66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus
Zawistowska-Deniziak, A; Wasyl, K; Norbury, L J; Wesołowska, A; Bień, J; Grodzik, M; Wiśniewski, M; Bąska, P; Wędrychowicz, H
Fasciola hepatica infections cause significant global problems in veterinary and human medicine, including causing huge losses in cattle and sheep production. F. hepatica host infection is a multistage process and flukes express papain-like cysteine proteases, termed cathepsins, which play pivotal roles in virulence through host entry, tissue migration and immune evasion. Expression of these proteases is developmentally regulated. Recent studies indicate that excystment of infective larvae is dependent on cysteine proteases and together FhCL3 and FhCB account for over 80% of total protease activity detectable in newly excysted juvenile (NEJ) fluke. This paper focuses on members of the cathepsin L gene family, specifically those belonging to the CL3 clade. The cDNA of two novel cathepsin L3 proteases--FhCL3-1 and FhCL3-2 were cloned. The mRNA transcript expression levels for these enzymes were significantly different at various time points in life development stages obtained in vitro, from dormant metacercariae to NEJ 24h after excystment. Maximum expression levels were observed in NEJ immediately after excystment. In all stages examined by Real Time PCR, FhCL3-2 was expressed at a higher level compared to FhCL3-1 which was expressed only at very low levels. Western blot and immunohistochemical analysis also indicated higher expression of the FhCL3-2 allele and its secretory nature. The ability of antibody responses from rats and sheep challenged with F. hepatica to recognize recombinant FhCL3-1 and FhCL3-2 was shown to differ. Differences were also confirmed through the use of anti-rFhCL3-1 and anti-rFhCL3-2 sera in Western blot analysis of juvenile excretory/secretory (ES) material separated by 2D electrophoresis. These results indicate analysis of relative expression of parasite virulence factors from different populations is required, as this will likely impact the effectiveness of vaccines based on these antigens.
Noya, Verónica; Brossard, Natalie; Berasaín, Patricia; Rodríguez, Ernesto; Chiale, Carolina; Mazal, Daniel; Carmona, Carlos; Freire, Teresa
Fasciolosis, caused by the liver fluke Fasciola hepatica, is a major parasitic disease of livestock that causes significant economic losses worldwide. Although drugs are effective against liver flukes, they do not prevent reinfection, and continuous treatment is costly. Moreover, resistant fluke strains are emerging. In this context, vaccination is a good alternative since it provides a cost-effective long-term prevention strategy to control fasciolosis. In this paper, we evaluate the Fhmuc peptide as a potential vaccine against fasciolosis. This peptide derives from a mucin-like protein highly expressed in the infective stage of Fasciola hepatica. Mucin-like molecules expressed by parasites can contribute to several infection processes by protecting the parasite from host proteases and recognition by the immune system. We show that the Fhmuc peptide induces Th1-like immune responses specific for F. hepatica excretion-secretion products (FhESP) with a high production of IFNγ. We also investigated whether this peptide could protect animals from infection, and present preliminary data indicating that animals treated with Fhmuc exhibited reduced liver damage compared to non-immunised animals and that this protection was associated with a recruitment of B and T lymphocytes in the peritoneum, as well as eosinophils and mature dendritic cells. These results suggest that the mucin-like peptide Fhmuc could constitute a potential vaccine candidate against fasciolosis and pave the way towards the development of vaccines against parasites.
Bąska, Piotr; Zawistowska-Deniziak, Anna; Zdziarska, Anna M; Wasyl, Katarzyna; Wiśniewski, Marcin; Cywińska, Anna; Klockiewicz, Maciej; Januszkiewicz, Kamil; Wędrychowicz, Halina
Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.
McNall, S J; Mansour, T E
Properties of forskolin activation of adenylate cyclase in the liver fluke Fasciola hepatica are described. Forskolin stimulated adenylate cyclase activity in cell-free fluke particles to levels more than 30-fold above the basal rate. This activation was not dependent on guanine nucleotides and, upon washing of the particles, was rapidly reversed. Forskolin potentiated the activation of adenylate cyclase by serotonin (5-HT) and lysergic acid diethylamide (LSD), resulting in both an increase in the maximal level of enzyme activity and a decrease in the apparent activation constant (KA). The 5-HT antagonist 2-bromo-LSD did not inhibit enzyme activation by forskolin. Furthermore, forskolin had no effect on specific [3H]LSD binding to fluke particles. Activation of adenylate cyclase by sodium fluoride or guanine nucleotides was modified in a complex manner by forskolin with both stimulatory and inhibitory effects present. The results suggest that forskolin does not interact directly with the 5-HT receptor coupled to adenylate cyclase. Instead, it appears that forskolin effects are, at least in part, due to its ability to alter the interaction between the regulatory and catalytic components of adenylate cyclase. Incubation of intact flukes with forskolin increased their cAMP levels 2- to 3-fold. The concentration dependence of this response was similar to that for forskolin activation of adenylate cyclase in fluke particles, with 300 microM forskolin giving the maximum response. Forskolin and other agents that increased fluke cAMP levels also stimulated fluke motility.
Falcón, Cristian R; Masih, Diana; Gatti, Gerardo; Sanchez, María Cecilia; Motrán, Claudia C; Cervi, Laura
The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.
Boukli, Nawal M; Delgado, Bonnibel; Ricaurte, Martha; Espino, Ana M
It is not unusual to find common molecules among parasites of different species, genera, or phyla. When those molecules are antigenic, they may be used for developing drugs or vaccines that simultaneously target different species or genera of parasite. In the present study, we used a proteomic-based approach to identify proteins that are common to adult Fasciola hepatica and Schistosoma mansoni. Whole-worm extracts from each parasite were separated by 2-dimensional electrophoresis (2-DE), and digital images of both proteomes were superimposed using imaging software to identify proteins with identical isoelectric points and molecular weights. Protein identities were determined by mass spectrometry. Imaging and immunoblot analyses identified 28 immunoreactive proteins that are common to both parasites. Among these molecules are antioxidant proteins (thioredoxin and glutathione-S-transferase), glycolytic enzymes (glyceraldehyde 6-phosphate dehydrogenase and enolase), proteolytic enzymes (cathepsin-L and -D), inhibitors (Kunitz-type, Stefin-1), proteins with chaperone activity (heat shock protein 70 and fatty acid-binding protein), and structural proteins (calcium-binding protein, actin, and myosin). Some of the identified proteins could be used to develop drugs and vaccines against fascioliasis and schistosomiasis.
Robles-Pérez, D; García-García, P; Martínez-Pérez, J M; Rojo-Vázquez, F A; Martínez-Valladares, M
Inter-simple sequence repeats markers were used to determinate the genetic variability of Fasciola hepatica populations recovered from sheep and cattle from Spain (Sp1, Sp2, Sp3 and Sp4), UK (Eng), Ireland (Ir) and Mexico (Mex). Twenty five primers were tested but only five produced 39 reproducible bands, being 71.79% polymorphic bands. This percentage ranged from 10.26% in Sp4 to 48.72% in Sp1, and per host between 28.21 and 48.72% in sheep and between 10.26 and 38.46% in cattle. This relatively low range of genetic diversity within populations, with a mean of 34.40%, implies that a large proportion of variation resided among populations. The population differentiation (Gst = 0.547) indicated that 54.7% of variation is due to differences between populations and 45.3% due to differences within population. The Nei's distance ranged between 0.091 and 0.230 in sheep and between 0.150 and 0.337 in cattle. The genetic relationships between populations and individuals were shown by a UPGMA dendrogram and a principal coordinate analysis; both grouped all populations separately from Sp4, a population of from the Midwest of Spain with the lowest level of diversity. Small genetic distances were observed between Eng and Ir, on the one hand, and Sp1, Sp2, Sp3, from the Northwest of Spain, together with Mex, on the other.
Villa-Mancera, A; Reynoso-Palomar, A; Olivares-Pérez, J; Ortega-Vargas, S; Cruz-Mendoza, I; Quiroz-Romero, H
The motility parameters of Fasciola hepatica miracidia were assessed at different temperatures and times post-hatching using computer-assisted sperm analysis. Eggs were incubated at 22 °C or 25 °C for 14 days. Five motion parameters were evaluated at different incubation temperatures up to 10 h post-hatching. No differences were observed in the percentage that hatched after incubation at the two different temperatures. However, the straight-line velocity of miracidia following incubation at 22 °C was significantly different from that observed at 25 °C (P< 0.01). All miracidium motion parameters at different post-hatching temperatures showed an overall decrease at the end of the experiment. Those miracidia hatching from eggs incubated at 25 °C had a higher velocity of 1673.3 μm/s compared with 1553.3 μm/s at 22 °C. Velocity parameters increased as the post-hatching temperature increased from 22 °C to 37 °C.
Guasconi, Lorena; Serradell, Marianela C; Garro, Ana P; Iacobelli, Luciana; Masih, Diana T
Fasciola hepatica releases excretory–secretory products (FhESP), and immunomodulatory properties have been described for the carbohydrates present in these parasite products. The interaction of FhESP with the innate immune cells, such as macrophages, is crucial in the early stage of infection. In this work we observed that peritoneal macrophages from naive BALB/c mice stimulated in vitro with FhESP presented: an increased arginase activity as well as Arginase I expression, and high levels of transforming growth factor-β and interleukin-10. A similar macrophage population was also observed in the peritoneum of infected mice. A partial inhibition of the immunomodulatory effects described above was observed when macrophages were pre-incubated with Mannan, anti-mannose receptor, Laminarin or anti-Dectin-1, and then stimulated with FhESP. In addition, we observed a partial inhibition of these effects in macrophages obtained from mice that were intraperitoneally injected with Mannan or Laminarin before being infected. Taken together, these results suggest the participation of at least two C-type lectin receptors, mannose receptor and Dectin-1, in the interaction of FhESP with macrophages, which allows this parasite to induce immunoregulatory effects on these important innate immune cells and may constitute a crucial event for extending its survival in the host. PMID:21595685
Falcón, Cristian R.; Masih, Diana; Gatti, Gerardo; Sanchez, María Cecilia; Motrán, Claudia C.; Cervi, Laura
The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite. PMID:25486609
Combining data from multiple analytical platforms is essential for comprehensive study of the molecular phenotype (metabotype) of a given biological sample. The metabolite profiles generated are intrinsically dependent on the analytical platforms, each requiring optimization of instrumental parameters, separation conditions, and sample extraction to deliver maximal biological information. An in-depth evaluation of extraction protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola hepatica, using ultra performance liquid chromatography and capillary electrophoresis coupled with mass spectroscopy is presented. The spectrometric methods were characterized by performance, and metrics of merit were established, including precision, mass accuracy, selectivity, sensitivity, and platform stability. Although a core group of molecules was common to all methods, each platform contributed a unique set, whereby 142 metabolites out of 14,724 features were identified. A mixture design revealed that the chloroform:methanol:water proportion of 15:59:26 was globally the best composition for metabolite extraction across UPLC-MS and CE-MS platforms accommodating different columns and ionization modes. Despite the general assumption of the necessity of platform-adapted protocols for achieving effective metabotype characterization, we show that an appropriately designed single extraction procedure is able to fit the requirements of all technologies. This may constitute a paradigm shift in developing efficient protocols for high-throughput metabolite profiling with more-general analytical applicability. PMID:22799605
Young, Neil D; Hall, Ross S; Jex, Aaron R; Cantacessi, Cinzia; Gasser, Robin B
Liver flukes of animals are parasitic flatworms (Platyhelminthes: Digenea) of major socioeconomic importance in many countries. Key representatives, such as Fasciola hepatica and F. gigantica, cause "liver fluke disease" (= fascioliasis), which is of major animal health significance worldwide. In particular, F. hepatica 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. This parasite is also a major food-borne pathogen of humans throughout parts of the Middle East, Asia and South America. Currently, there is a significant focus on the development of new approaches for the prevention and control of fascioliasis in livestock. Recent technological advances in genomics and bioinformatics provide unique opportunities for the identification and prevalidation of drug targets and vaccines through a better understanding of the biology of F. hepatica and related species as well as their relationship with their hosts at the molecular level. Surprisingly, despite the widespread socioeconomic impact of fascioliasis, genomic datasets for F. hepatica are scant, limiting the molecular biological research of this parasite. The present article explores specifically the transcriptome of the adult stage of F. hepatica using an integrated genomic-bioinformatic platform. The analysis of the current data reveals numerous molecules of biological relevance, some of which are inferred to be involved in key biological processes or pathways that could serve as targets for new trematocidal drugs or vaccines. Improved insights into the transcriptome of F. hepatica should pave the way for future, comparative analysis of the transcriptomes of other developmental stages of this and related parasites, such as F. gigantica, cancer-causing flatworms (Clonorchis sinensis and Opisthorchis viverrini) and blood flukes (Schistosoma mansoni and S. japonicum). Prediction of the
Rojas-Caraballo, Jose; López-Abán, Julio; Fernández-Soto, Pedro; Vicente, Belén; Collía, Francisco; Muro, Antonio
Fasciolosis is listed as one of the most important neglected tropical diseases according with the World Health Organization and is also considered as a reemerging disease in the human beings. Despite there are several studies describing the immune response induced by Fasciola hepatica in the mammalian host, investigations aimed at identifying the expression profile of genes involved in inducing hepatic injury are currently scarce. Data presented here belong to a time-course investigation of the gene expression profile in the liver of BALB/c mice infected with F. hepatica metacercariae at 7 and 21 days after experimental infection. The data published here have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE69588, previously published by Rojas-Caraballo et al. (2015) in PLoS One . PMID:26697343
Bąska, Piotr; Norbury, Luke J; Wiśniewski, Marcin; Januszkiewicz, Kamil; Wędrychowicz, Halina
The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts' liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein - FhPGK, had no impact on cell survival.
Arias, M S; Piñeiro, P; Sánchez-Andrade, R; Suárez, J L; Hillyer, G V; Díez-Baños, P; Paz-Silva, A; Morrondo, P
The aim of this work is to know the prevalence of Fasciola hepatica in 301 roe deer and in 676 beef cattle kept in an endemic area. Detection of antibodies was determined in roe deer using a homemade ELISA with excretory/secretory antigens (FhES) and a recombinant protein (FhrAPS). None of the deer passed eggs by faeces and none flukes in their livers were found. The seroprevalence of F. hepatica was 29% using FhES, with significantly higher values in the oldest ones (36%). Twenty-eight percent of the samples were positive to FhrAPS. Twenty-three percent of the cows eliminated eggs of F. hepatica and the seroprevalence was 67% using FhrAPS. No relationship between the seropositivity values of deer and cattle was demonstrated. The role of wild ruminants as reservoirs of F. hepatica is discussed. We encourage the use of ELISA to know the possibility of exposure to trematodes in wild ruminants.
Jang, Jung Hee; Park, Jin Bong; Kim, Sun Don; Lee, So Yeong; Hong, Sung-Jong; Ryu, Pan Dong
Fasciola hepatica causes biliary epithelial hyperplasia and obstructive jaundice in humans and animals. Using a planar lipid bilayer technique, we further characterized the single channel property of large conductance K(+)-permeable channels that were previously identified from F. hepatica. The single channel conductance was 254.7±17.9 pS under a symmetrical 200/200 mM (cis/trans) KCl gradient. Open state probability (P(o)) varied from channel to channel at a given membrane potential and Ca(2+) concentration, but increased with voltage (-60 to +40 mV) and cis Ca(2+) (1-200 μM). Under a near bi-ionic condition of 200 mM [K(+)](cis)/200 mM [Na(+)](trans), the permeability ratio of K(+) to Na(+) was 5.0. Charybdotoxin (1 μM) inhibited P(o), whereas tetraethylammonium reduced the conductance (K(D)=67.8mM). Taken together, the results show that the single channel properties of the large conductance K(+)-permeable channels in F. hepatica are similar to those of large conductance Ca(2+)-activated K(+) (BK) channels in general, but distinct from typical BK channels in the extent of voltage- and Ca(2+)-dependence, as well as permeability to Na(+). This study further reveals a variant BK channel in F. hepatica that could serve as a new drug target to treat fascioliasis.
Moazeni, Mohammad; Khademolhoseini, Ali Asghar
Fasciolosis is of considerable economic and public health importance worldwide. Little information is available on the ovicidal effects of anthelminthic drugs. The use of ovicidal anthelmintics can be effective in disease control. In this study, the effectiveness of the methanolic extract of ginger (Zingiber officinale) on the eggs of Fasciola hepatica is investigated. Fasciola hepatica eggs were obtained from the gall bladders of naturally infected sheep and kept at 4 °C until use. The eggs were exposed to varying concentrations of ginger extract (1, 5, 10, 25 and 50 mg/mL) for 24, 48 and 72 h. To investigate the effect of the ginger extracts on the miracidial formation, the treated eggs were incubated at 28 °C for 14 days. The results indicated that F. hepatica eggs are susceptible to the methanolic extract of Z. officinale. The ovicidal effect of ginger extract at a concentration of 1 mg/mL with 24, 48 and 72 h treatment time was 46.08, 51.53 and 69.09 % respectively (compared with 22.70 % for control group). The ovicidal effect of ginger extract at a concentration of 5 mg/mL after 24 h was 98.84 %. One hundred percent ovicidal efficacy was obtained through application of ginger extract at concentrations of 5 and 10 mg/mL with a 48 and 24 h treatment time respectively. The in vitro ovicidal effect of the methanolic extract of Z. officinale was satisfactory in this study, however, in vivo efficacy of this extract, remains for further investigation. To the best of our knowledge, this is the first report on the ovicidal effect of Z. officinale against F. hepatica eggs.
Valero, M Adela; Perez-Crespo, Ignacio; Periago, M Victoria; Khoubbane, Messaoud; Mas-Coma, Santiago
In trematodiases, shape and size of the fluke eggs shed with faeces are crucial diagnostic features because of their typically reduced intraspecific variability. In fascioliasis, the usual diagnosis during the biliary stage of infection is based on the classification of eggs found in stools, duodenal contents or bile. The aim of the present study is to validate the identification of Fasciola species based on the shape and size of eggs shed by humans, characterizing their morphometric traits using a computer image analysis system (CIAS). The influence of both the geographical location and of the host (human and livestock) has been analysed. Coprological studies were carried out in fascioliasis human endemic areas, where only F. hepatica is present (the northern Bolivian Altiplano and the Cajamarca valley in Peru), and where F. hepatica and F. gigantica coexist (the Kutaisi region of Georgia, the Nile Delta in Egypt, and the Quy Nhon province in Vietnam). Classically, it is considered that at the abopercular end of the shell of Fasciola eggs there is often a roughened or irregular area. Nevertheless, results show that the frequency of the presence of this feature in F. hepatica is population-dependent, and therefore is not a pathognomonic criterion in diagnosis. The study reveals that eggs shed by humans show morphological traits different from eggs shed by animals. In humans, F. hepatica eggs are bigger and F. gigantica eggs are smaller than reported to date from livestock, and their measurements overlap when compared. The material analysed in this study shows that the size of eggs shed by humans from Georgia and Egypt corresponds to the F. hepatica morph, while the size of eggs shed by humans from Vietnam corresponds to the F. gigantica morph. Measurements of F. hepatica and F. gigantica eggs originating from humans and animals from sympatric areas overlap, and, therefore, they do not allow differential diagnosis when within this overlapping range. In this sense
Background Fasciolosis affects different ruminant species and leads to great economic losses for cattle farmers worldwide. Thus, the current study aimed to evaluate bovine fasciolosis prevalence in the state of Espírito Santo, Brazil, using slaughter maps provided by slaughterhouses and verifying the origin of cattle. Methods A map was created based on analysis of epidemiological data. The ArcGIS/ArcINFO 10.1 software was employed in order to elaborate updated bioclimatic maps that displayed the fasciolosis prevalence within the state – per city– between 2009 and 2011. Results According to the bioclimatic map it was clear that 52.24% of the state’s total area comprise regions considered favorable for the development and survival of Fasciola hepatica. According to the data provided by slaughterhouses, the parasite was more frequent in the cities of Atílio Vivácqua, Itapemirim and Anchieta with respective prevalence of 28.41, 25.50 and 24.95%. Although the northern portion of the state is also favorable for the disease maintenance (reaching rates above 90%), several cities presented prevalence of only 0.99 and 1.94% respectively. These findings indicate that climatic and environmental factors only cannot be considered preponderant to fasciolosis occurrence. Regarding the slaughterhouse located in Anchieta city, the higher prevalence was registered in the cities of Jerônimo Monteiro, Alegre and Cachoeiro de Itapemirim, with mean prevalence of 1.21, 1.07 and 2.09% respectively. Conclusion Although the present findings suggest a pattern for the prevalence of fasciolosis, records of the cities for the occurrence of the disease usually do not reflect the true origin of animals. PMID:25101121
Maggioli, Gabriela; Bottini, Gualberto; Basika, Tatiana; Alonzo, Pablo; Salinas, Gustavo; Carmona, Carlos
The liver fluke Fasciola hepatica remains an important agent of food-borne trematode disease producing great economic losses due to its negative effect on productivity of livestock grazing in temperate areas. The prevailing control strategy based on anthelmintic drugs is unsustainable due to widespread resistance hence vaccination appears as an attractive option to pursue. In this study we evaluate the effect of vaccination in calves with a functional recombinant thioredoxin glutathione reductase (rFhTGR) from liver fluke, a critical antioxidant enzyme at the crossroads of the thioredoxin and glutathione metabolism in flatworms. The recombinant enzyme produced in Escherichia coli was tested in two vaccination experiments; in the first trial rFhTGR was administered in combination with Freund́s Incomplete Adjuvant (FIA) in a three-inoculation scheme on weeks 0, 4 and 8; in the second trial rFhTGR was given mixed with Adyuvac 50 or Alum as adjuvants on weeks 0 and 4. In both cases calves were challenged with metacercariae (400 in the first and 500 in the second trial) 2 weeks after the last inoculation. Our results demonstrate that two or three doses of the vaccine induced a non-significant reduction in worm counts of 8.2% (FIA), 3.8% (Adyuvac 50) and 23.0% (Alum) compared to adjuvant controls indicating that rFhTGR failed to induce a protective immunity in challenged calves. All vaccine formulations induced a mixed IgG1/IgG2 response but no booster was observed after challenge. No correlations between antibody titres and worm burdens were found.
Hilbe, M; Robert, N; Pospischil, A; Gerspach, C
In Switzerland, dicrocoeliasis is regarded as the most significant parasitic infection of llamas and alpacas. Fasciola hepatica infestation is also a problem but less common. The aim of the present retrospective study was to evaluate the lungs of New World camelids (NWCs) for evidence of arterial hypertension in association with liver changes due to liver fluke infestation. The lungs of 20 llamas and 20 alpacas with liver fluke infestation were histologically evaluated. The hematoxylin and eosin and van Gieson (VG)-elastica stains as well as immunohistology for the expression of α-smooth muscle actin (α-SMA) were used to visualize the structures of arterial walls. Parasitology of fecal matter (11 llamas and 17 alpacas) confirmed that most of these animals were infested with both Dicrocoelium dendriticum and other gastrointestinal parasites. In most cases (10/12 llamas, 4/6 alpacas), liver enzyme activity in serum was elevated. Histologically, arteries in the lungs of 9 of 20 llamas (45%) and 3 of 20 alpacas (15%) showed severe intimal and adventitial and slight to moderate medial thickening, which was confirmed with α-SMA and VG-elastica staining. All animals exhibited typical liver changes, such as fibrosis and biliary hyperplasia, in association with the presence of liver flukes. This study shows that liver flukes can induce proliferative changes in lung arteries in NWCs that resemble those seen with pulmonary arterial hypertension due to liver parasites in humans. However, the degree of liver fluke infestation was not correlated with the extent of liver damage, or with the amount of thoracic or abdominal effusion or pulmonary arterial changes.
Cwiklinski, Krystyna; de la Torre-Escudero, Eduardo; Trelis, Maria; Bernal, Dolores; Dufresne, Philippe J; Brennan, Gerard P; O'Neill, Sandra; Tort, Jose; Paterson, Steve; Marcilla, Antonio; Dalton, John P; Robinson, Mark W
Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular "machinery" required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.
Malone, J B; Williams, J C; Lutz, M; Fagan, N; Jacocks, M; Jones, E; Marbury, K; Willis, E
The efficacy, safety, and compatibility of fenbendazole (FBZ) and clorsulon (CLN) were tested after oral administration of label recommended and of higher (5x) dosage rates to calves naturally infected with gastrointestinal nematodes and Fasciola hepatica. Results for 42 calves allotted to 4 treatment groups indicated a similar efficacy against mature F hepatica by FBZ (5 mg/kg of body weight) and CLN (7 mg/kg) in a combined oral suspension, compared with CLN (7 mg/kg) alone (100 vs 99% reduction). A lesser efficacy was observed against immature flukes (88.6 and 84.9% reduction, respectively). Calves given 25 mg of FBZ/kg and 35 mg of CLN/kg had nearly complete reduction of both mature (99.6%) and immature flukes (99.1%). Fasciola egg counts were reduced by greater than 99.5% in all treated groups. Against Ostertagia ostertagi, the percentage of efficacy of the combined FBZ (5 mg/kg) and CLN (7 mg/kg) treatment was 94.3% against adults and 81.3% against inhibited larvae. Efficacy against all other nematodes was 100%, except against Cooperia spp adults (98.3%) and immature Oesaphagostomum radiatum (88.0%). At 5 x dosage rates for FBZ and CLN, percentage of removal of adults and inhibited larvae of O ostertagi was 99.3 and 99.0%, respectively, and 99 to 100% for other nematodes. Results indicate that FBZ and CLN are compatible when mixed together and administered as an oral suspension to cattle and that the efficacy is similar to that of the drugs individually. On the basis of further results, we suggest that summer treatment may be superior in preventive value for gastrointestinal nematodes and F hepatica, compared with spring treatment, because of seasonal infection dynamics of the major cattle parasites in Louisiana.
Alba, Annia; Hernández, Hilda M; Marcet, Ricardo; Vázquez, Antonio A; Figueredo, Mabel; Sánchez, Jorge; Otero, Oscar; Sarracent, Jorge
Fasciolosis is a globally distributed snail-borne disease which requires economic consideration due to its enormous impact on veterinary medicine. During recent decades, this parasitosis has also shown increasing prevalence in human populations worldwide. The dissemination and successful transmission of fasciolosis ultimately depends on the existence of susceptible snails that act as intermediate hosts. Therefore, to accomplish effective control of this disease, surveillance and detection of the infected intermediate host would be essential. The screening of trematodes within snails using classical parasitological examination of the larvae can be unreliable (sensitivity and specificity vary depending on the time of infection and the experience of the observer) and relatively costly when using molecular biological methods during large-scale monitoring. Here we propose a novel monoclonal antibody-based immunoenzymatic assay to detect ongoing Fasciola hepatica infection in lymnaeid snails. Anti-F. hepatica rediae mouse monoclonal antibodies were generated and used to develop a double monoclonal antibody-based ELISA for parasite detection. Fasciola hepatica-infected and uninfected laboratory-reared Galba cubensis and Pseudosuccinea columella were used for assessment of the developed ELISA. Experimentally infected snails were dissected and examined for parasite larvae as the "gold standard" method. Sensitivity results were 100% for both snail species, while specificity was 98% for G. cubensis and 100% for P. columella. No cross-reactivity was detected in lymnaeids infected with Trichobilharzia sp. or Cotylophoron sp. The ELISA enabled detection of the infection from day 8 p.i. in G. cubensis while in P. columella it was noted as early as day 4. To our knowledge no previous immunoassays have been reported to detect helminth-infected snails and the developed sandwich ELISA method is therefore suggested for infection status validation in natural populations of lymnaeid
Background The present study aims to assess the epidemiological role of different lymnaeid snails as intermediate hosts of the liver fluke Fasciola hepatica in Belgium and Luxembourg. Methods During summer 2008, 7103 lymnaeid snails were collected from 125 ponds distributed in 5 clusters each including 25 ponds. Each cluster was located in a different biogeographic area of Belgium and Luxembourg. In addition, snails were also collected in sixteen other biotopes considered as temporary wet areas. These snails were identified as Galba truncatula (n = 2474) (the main intermediate host of F. hepatica in Europe) and Radix sp. (n = 4629). Moreover, several biological and non-biological variables were also recorded from the different biotopes. DNA was extracted from each snail collected using Chelex® technique. DNA samples were screened through a multiplex PCR that amplifies lymnaeid internal transcribed spacer 2 gene sequences (500–600 bp) (acting as an internal control) and a 124 bp fragment of repetitive DNA from Fasciola sp. Results Lymnaeid snails were found in 75 biotopes (53.2%). Thirty individuals of G. truncatula (1.31%) and 7 of Radix sp. (0.16%) were found to be positive for Fasciola sp. The seven positive Radix sp. snails all belonged to the species R. balthica (Linnaeus, 1758). Classification and regression tree analysis were performed in order to better understand links and relative importance of the different recorded factors. One of the best explanatory variables for the presence/absence of the different snail species seems to be the geographic location, whereas for the infection status of the snails no obvious relationship was linked to the presence of cattle. Conclusions Epidemiological implications of these findings and particularly the role of R. balthica as an alternative intermediate host in Belgium and Luxembourg were discussed. PMID:24524623
Mezo, Mercedes; González-Warleta, Marta; Castro-Hermida, José Antonio; Manga-González, M Yolanda; Peixoto, Raquel; Mas-Coma, Santiago; Valero, M Adela
Fasciolosis is an emerging or reemerging human and animal disease in numerous parts of the world. In Galicia (NW, Spain), the wild boar (Sus scrofa) is the main wild ungulate in terms of abundance and distribution. Its population has continuously increased over the past decades and this population growth has been accompanied by a reduction of habitats, so that the wild boar populations encroach more and more frequently onto agricultural lands. The increase of the interface area between livestock and the wild boars frequently involves the sharing of pastures and water sources, so that the circulation of common pathogens is propitiated. This is the first report concerning the importance of the wild boar as a possible reservoir of Fasciola hepatica infection in Spain. Livers from 358 hunted wild boars were analyzed showing that 11.2% were parasitized by F. hepatica, with burdens ranging from 1 to 14 flukes (mean=2.3). Fecal analysis demonstrated that 40.0% of parasitized animals shed F. hepatica eggs with a mean excretion of 6.1 eggs per gram of feces (epg). The presence of coproantigens analyzed by MM3-COPRO ELISA was positive in 62.9% of infected wild boars. After incubation, the percentage of hatched eggs ranged between 41.0% and 90.0% suggesting that the wild boar is very likely to contribute to the environmental contamination with viable parasite eggs. Comparative morphometric data were obtained using a computer image analysis system (CIAS) on the basis of standardized measurements. F. hepatica from cattle, sheep and wild boars from the same geographical area presents a similar body development and gravidity. Our study shows for the first time that the F. hepatica uterus from the wild boar presents an intermediate size between that found in primary reservoir hosts such as cattle and sheep, i.e., the individual potential egg output capacity of the wild boar does not greatly differ from that detected in Galician livestock. These results show that F. hepatica in
Kołodziejczyk, Lidia; Podraza, Wojciech; Gonet, Bolesław; Dzika, Ewa; Kosik-Bogacka, Danuta I
The aim of this study was to determine the effect of an extremely low-frequency magnetic field (ELFMF) on the production of liver fluke larvae in a parasite-host system: Fasciola hepatica--Galba truncatula. Both F. hepatica eggs and F. hepatica-infected snails were exposed to an ELFMF (50 Hz, 2.0 mT) for 14 days and 36 days, respectively. F. hepatica-infected snails were divided into 4 groups, 10 specimens each. The snails of groups I and II were infected with F. hepatica larvae--miracidia obtained from control cultures, while the snails of groups III and IV were infected with miracidia reared from eggs that had been incubated in an ELFMF. After infection, the snails of groups II and IV were placed in an ELFMF, while those of groups I (control) and III were housed outside the ELFMF. At 36 days post-infection (dpi) there were no statistically significant differences between the number of F. hepatica larvae--cercariae and metacercariae, obtained from G. truncatula snails in the control group (group I) and the snail groups exposed to ELFMF (groups II, III and IV). However, a statistically significant difference between the average number of F. hepatica larvae in snail groups III and IV may indicate that the duration of exposure to ELFMF, i.e. embryogenesis period vs. the entire larval development, played a role in the production of F. hepatica larvae, and resulted in a reduction of their number.
Noya, Verónica; Brossard, Natalie; Rodríguez, Ernesto; Dergan-Dylon, L. Sebastián; Carmona, Carlos; Rabinovich, Gabriel A.; Freire, Teresa
Fasciolosis is a trematode zoonosis of interest in public health and cattle production. We report here the immunostimulatory effect of a 66 mer mucin-like peptide from Fasciola hepatica (Fhmuc), which synergizes with lipopolysaccharide (LPS) to promote dendritic cell (DC) maturation, endowing these cells with Th1-polarizing capacity. Exposure of DCs to Fhmuc in presence of LPS induced enhanced secretion of pro-inflammatory cytokines and expression of co-stimulatory molecules by DCs, promoting their T cell stimulatory capacity and selectively augmenting IFN-γ secretion by allogeneic T cells. Furthermore, exposure of DCs to Fhmuc augmented LPS-induced Toll-like receptor (TLR) 4 expression on the cell surface. Finally, Fhmuc-conditioned DCs induced parasite specific-adaptive immunity with increased levels of IFN-γ secreted by splenocytes from vaccinated animals, and higher parasite-specific IgG antibodies. However, Fhmuc-treated DC conferred modest protection against F. hepatica infection highlighting the potent immuno-regulatory capacity of the parasite. In summary, this work highlights the capacity of a mucin-derived peptide from F. hepatica to enhance LPS-maturation of DCs and induce parasite-specific immune responses with potential implications in vaccination and therapeutic strategies. PMID:28079156
Noya, Verónica; Brossard, Natalie; Rodríguez, Ernesto; Dergan-Dylon, L Sebastián; Carmona, Carlos; Rabinovich, Gabriel A; Freire, Teresa
Fasciolosis is a trematode zoonosis of interest in public health and cattle production. We report here the immunostimulatory effect of a 66 mer mucin-like peptide from Fasciola hepatica (Fhmuc), which synergizes with lipopolysaccharide (LPS) to promote dendritic cell (DC) maturation, endowing these cells with Th1-polarizing capacity. Exposure of DCs to Fhmuc in presence of LPS induced enhanced secretion of pro-inflammatory cytokines and expression of co-stimulatory molecules by DCs, promoting their T cell stimulatory capacity and selectively augmenting IFN-γ secretion by allogeneic T cells. Furthermore, exposure of DCs to Fhmuc augmented LPS-induced Toll-like receptor (TLR) 4 expression on the cell surface. Finally, Fhmuc-conditioned DCs induced parasite specific-adaptive immunity with increased levels of IFN-γ secreted by splenocytes from vaccinated animals, and higher parasite-specific IgG antibodies. However, Fhmuc-treated DC conferred modest protection against F. hepatica infection highlighting the potent immuno-regulatory capacity of the parasite. In summary, this work highlights the capacity of a mucin-derived peptide from F. hepatica to enhance LPS-maturation of DCs and induce parasite-specific immune responses with potential implications in vaccination and therapeutic strategies.
Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H
Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.
Mucheka, Vimbai T; Lamb, Jennifer M; Pfukenyi, Davies M; Mukaratirwa, Samson
The aim of this study was to identify and determine the genetic diversity of Fasciola species in cattle from Zimbabwe, the KwaZulu-Natal and Mpumalanga provinces of South Africa and selected wildlife hosts from Zimbabwe. This was based on analysis of DNA sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and 2) and mitochondrial cytochrome oxidase 1 (CO1) regions. The sample of 120 flukes was collected from livers of 57 cattle at 4 abattoirs in Zimbabwe and 47 cattle at 6 abattoirs in South Africa; it also included three alcohol-preserved duiker, antelope and eland samples from Zimbabwe. Aligned sequences (ITS 506 base pairs and CO1 381 base pairs) were analyzed by neighbour-joining, maximum parsimony and Bayesian inference methods. Phylogenetic trees revealed the presence of Fasciola gigantica in cattle from Zimbabwe and F. gigantica and Fasciola hepatica in the samples from South Africa. F. hepatica was more prevalent (64%) in South Africa than F. gigantica. In Zimbabwe, F. gigantica was present in 99% of the samples; F. hepatica was found in only one cattle sample, an antelope (Hippotragus niger) and a duiker (Sylvicapra grimmia). This is the first molecular confirmation of the identity Fasciola species in Zimbabwe and South Africa. Knowledge on the identity and distribution of these liver flukes at molecular level will allow disease surveillance and control in the studied areas.
Qin, Hongyu; Gao, Xiang; Wang, Hongbin; Xiao, Jianhua
Fasciola hepatica is an important trematode parasite of economic importance that infests sheep and cattle worldwide. We conducted a detailed investigation into the spatial distribution of F. hepatica infestation in farmed sheep in Qinghai (Wutumeiren) province, Mainland China. Mathematical modelling was used to assess the inter-relationships between meteorological and geographical factors and the risk of F. hepatica infestation across the province. A capture enzyme-linked immunosorbent assay (ELISA) test (MM3-SERO) was used to detect F. hepatica infestation. A niche model based on the maximum entropy method (MaxEnt) was used to estimate the influence of meteorological and geographical factors on the observed spatial distribution of F. hepatica infestation. Results of jackknife analysis indicated that temperature, precipitation, solar radiation, digital elevation and slope were associated with the occurrence of F. hepatica infestation, and that infestation rates were significantly higher among animals from districts with a high percentage of grassland habitat. The findings indicate that meteorological and geographical factors may be important variables affecting the distribution of F. hepatica infestation and should be taken into account in the development of future surveillance and control programmes for fascioliasis. PMID:28000591
Rickard, L G
A microenzyme-linked immunosorbent assay (dot-ELISA) was developed to detect serum antibodies against Fasciola hepatica antigens in llamas. Sera from five F. hepatica-infected and 11 non-infected llamas were used in initial test development. Nitrocellulose filter disks containing F. hepatica excretory-secretory product were placed in 96-well microtiter plates, washed, blocked with Tween-20, then incubated with four-fold serial dilutions of llama sera. After incubation with rabbit anti-llama IgG followed by peroxidase-conjugated goat anti-rabbit IgG, addition of precipitable substrate resulted in purple dots on white background (positives) easily read by eye. The technique was further evaluated at titers of 1:512 using an additional six known positive and eight known negative llamas. Test results showed 6/6 known positive as positive and 8/8 known negative as negative. Sera were collected, at approximately weekly intervals, from three llamas experimentally infected with F. hepatica. The dot-ELISA detected antibodies to F. hepatica as early as the second week post-infection in all llamas. In a serologic survey of 256 llamas from an F. hepatica endemic area, the dot-ELISA detected antigen-specific serum antibodies to F. hepatica in 42 (16%) of the llamas. Although no difference was noted in antibody prevalence between sexes, prevalence increased in llamas over 6 months of age.
Januszkiewicz, Kamil; Norbury, Luke J; Wilkowski, Przemysław; Zawistowska-Deniziak, Anna; Wesołowska, Agnieszka; Wedrychowicz, Halina
Fasciola hepatica infections cause large economic losses and are a serious veterinary medicine problem in many regions of the world. Recent studies examining fascioliasis in the bison population from Bialowieza National Park have shown that the prevalence of infection with this parasite is up to 100%. Liver flukes isolated from bison from Bialowieza National Park in Poland were compared with a fluke strain originally obtained from the Central Veterinary Laboratory, Weybridge, UK, to determine variations in cercarial production and establish the ability of their metacercariae to activate in vitro. Some small differences in cercarial production between the two isolates are shown, while significant differences in the ability of their metacercariae to activate in vitro were observed.
Zintl, Annetta; Talavera, Silvia; Sacchi-Nestor, Carlotta; Ryan, Marion; Chryssafidis, Andreas; Mulcahy, Grace
Fasciola hepatica is a common and economically important parasite of sheep and cattle. Although its marked genetic heterogeneity is well recognised, an association between haplotypes and specific phenotypic traits has yet to be identified. Using experimental infections in cattle this study investigated whether a fragment of mitochondrial DNA (coding for cytochrome c oxidase subunit III, transfer RNA histidine and cytochrome b) and 3 nuclear microsatellite loci (Fh15, Fh23 and Fh25) could be used as markers for the parasite's ability to complete its tissue migration and establish in the liver of the final host. While we did not detect any shift in the frequency of the various genotypes in the population of metacercariae used for the infection on the one hand and the flukes collected from the liver on the other, there was an indication that parasites with heterozygous microsatellite alleles may have a selective advantage over homozygote parasites during their migration in the final host.
Rathinasamy, Vignesh; Toet, Hayley; McCammick, Erin; O’Connor, Anna; Marks, Nikki J.; Mousley, Angela; Brennan, Gerard P.; Halton, David W.; Spithill, Terry W.; Maule, Aaron G.
Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving ‘molecular toolbox’ for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the “neoblast” stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in
Banford, Samantha; Drysdale, Orla; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J
A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.
Garcia-Campos, Andres; Cwiklinski, Krystyna; Dalton, John P.; Hokke, Cornelis H.; O’Neill, Sandra; Mulcahy, Grace
Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F
Garcia-Campos, Andres; Ravidà, Alessandra; Nguyen, D Linh; Cwiklinski, Krystyna; Dalton, John P; Hokke, Cornelis H; O'Neill, Sandra; Mulcahy, Grace
Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F
Medeiros, Camilla; Scholte, Ronaldo Guilherme Carvalho; D'ávila, Sthefane; Caldeira, Roberta Lima; Carvalho, Omar dos Santos
Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), “SpeciesLink” (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions. PMID:24879003
Kolodziejczyk, L; Laszczyńska, M; Masiuk, M; Grabowska, M; Skrzydlewska, E
We investigated the immunoexpression of the intermediate filament proteins, cytokeratin and desmin, and the morphological changes in the liver of rats during experimental fasciolosis at 4, 7 and 10 weeks post-infection. Rats were infected with 30 Fasciola hepatica metacercariae. Paraffin sections of the liver were stained using H & E, PAS and azan stains. Immunohistochemical reactions were performed using antibodies against cytokeratin and desmin. The experimental F. hepatica infection led to fibrosis and cirrhosis of the liver, and to inflammation of the common bile ducts. The expression of cytokeratin was increased in the epithelial cells of both the liver bile ductules at 4, 7 and 10 weeks post-infection and in the common bile ducts at 7 and 10 weeks post-infection compared to uninfected rats; expression in the common bile ducts was more intense. The myofibroblasts of the liver and smooth myocytes of the interlobular bile ducts and common bile ducts, showed a slight increase in desmin expression compared to the uninfected rats. The increased expression of cytokeratins in the hyperplastic rat common bile duct epithelium during the biliary phase of fasciolosis at 7 and 10 weeks post-infection may be explained by mechanical irritation by the parasite and an inflammatory reaction in the bile duct epithelium and in periductal fibrous tissue.
Japa, Ornampai; Hodgkinson, Jane E; Emes, Richard D; Flynn, Robin J
The helminth Fasciola hepatica causes fasciolosis throughout the world, a major disease of livestock and an emerging zoonotic disease in humans. Sustainable control mechanisms such as vaccination are urgently required. To discover potential vaccine targets we undertook a genome screen to identify members of the transforming growth factor (TGF) family of proteins. Herein we describe the discovery of three ligands belonging to this superfamily and the cloning and characterisation of an activin/TGF like molecule we term FhTLM. FhTLM has a limited expression pattern both temporally across the parasite stages but also spatially within the worm. Furthermore, a recombinant form of this protein is able to enhance the rate (or magnitude) of multiple developmental processes of the parasite indicating a conserved role for this protein superfamily in the developmental biology of a major trematode parasite. Our study demonstrates for the first time the existence of this protein superfamily within F. hepatica and assigns a function to one of the three identified ligands. Moreover further exploration of this superfamily may yield future targets for diagnostic or vaccination purposes due to its stage restricted expression and functional role.
Cuervo, Pablo F; Cataldo, Sophia Di; Fantozzi, M Cecilia; Deis, Erika; Isenrath, Gabriela Diaz; Viberti, Gabriela; Artigas, Patricio; Peixoto, Raquel; Valero, M Adela; Sierra, Roberto Mera Y; Mas-Coma, Santiago
Fascioliasis has recently been included in the WHO list of Neglected Zoonotic Diseases. Besides being a major veterinary health problem, fascioliasis has large underdeveloping effects on the human communities affected. Though scarcely considered in fascioliasis epidemiology, it is well recognized that both native and introduced wildlife species may play a significant role as reservoirs of the disease. The objectives are to study the morphological characteristics of Fasciola hepatica adults and eggs in a population of Lepus europaeus, to assess liver fluke prevalence, and to analyze the potential reservoir role of the European brown hare in northern Patagonia, Argentina, where fascioliasis is endemic. Measures of F. hepatica found in L. europaeus from northern Patagonia demonstrate that the liver fluke is able to fully develop in wild hares and to shed normal eggs through their faeces. Egg shedding to the environment is close to the lower limit obtained for pigs, a domestic animal whose epidemiological importance in endemic areas has already been highlighted. The former, combined with the high prevalence found (14.28%), suggest an even more important role in the transmission cycle than previously considered. The results obtained do not only remark the extraordinary plasticity and adaptability of this trematode species to different host species, but also highlight the role of the European brown hare, and other NIS, as reservoirs capable for parasite spillback to domestic and native cycle, representing a potentially important, but hitherto neglected, cause of disease emergence.
Scheerlinck, Jean-Pierre; Ansell, Brendan R. E.; Hall, Ross S.; Gasser, Robin B.; Jex, Aaron R.
Fasciola hepatica is a parasitic trematode that infects a wide range of mammalian hosts, including livestock and humans, in temperate and tropical regions globally. This trematode causes the disease fascioliasis, which consists of an acute phase (≤ 12 weeks) during which juvenile parasites migrate through the host liver tissues, and a chronic phase (> 12 weeks) following the establishment of adult parasites in the liver bile ducts. Few studies have explored the progression of the host response over the course of Fasciola infection in the same animals. In this study, we characterized transcriptomic changes in peripheral blood mononuclear cells (PBMCs) collected from sheep at three time points over the first eight weeks of infection relative to uninfected controls. In total, 183 and 76 genes were found to be differentially transcribed at two and eight weeks post-infection respectively. Functional and pathway analysis of differentially transcribed genes revealed changes related to T-cell activation that may underpin a Th2-biased immune response against this parasite. This first insight into the dynamics of host responses during the early stages of infection improves the understanding of the pathogenesis of acute fascioliasis, informs vaccine development and presents a set of PBMC markers with diagnostic potential. PMID:27438474
Scarcella, S; Miranda-Miranda, E; Solana, M V; Solana, H
The aim of the present study was to genetically characterize Fasciola hepatica strains from diverse ecogeographical regions (America and Europe), susceptible and resistant to Triclabendazole, using the random amplified polymorphic DNA fragments (RAPDs-PCR) technique to elucidate genetic variability between the different isolates. Ten different oligonucleotide primers of 10 bases with GC content varying from 50-70% were used. A polymerase chain reaction (PCR) was carried out in 25 μl of total volume. Duplicate PCR reactions on each individual template DNA were performed to test the reproducibility of the individual DNA bands. The size of the RAPD-PCR fragments was determined by the reciprocal plot between the delay factors (Rf) versus the logarithm of molecular weight ladder. The phenogram obtained showed three main clusters, the major of which contained European Strains (Cullompton and Sligo) showing a genetic distance of 27.2 between them. The American strains (Cedive and Cajamarca) on the other hand formed each their distinctive group but clearly maintaining a closer genetic relationship among them than that to their European counterparts, with which showed a distance of 33.8 and 37.8, respectively. This polymorphism would give this species enhanced adaptability against the host, as well as the environment. The existence of genetically different populations of F. hepatica could allow, against any selection pressure, natural or artificial (for use fasciolicides products and/or control measures), one or more populations of F. hepatica to be able to survive and create resistance or adaptability to such selective pressure.
Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa
Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis. PMID:26720149
Relf, V; Good, B; McCarthy, E; de Waal, T
Worldwide molluscs of the genera Lymnaea, Pseudosuccinea, Galba and Stagnicola act as intermediate hosts of the common liver fluke, Fasciola hepatica. Galba truncatula is the only recorded intermediate host of F. hepatica within Ireland and is the principle intermediate host within Europe. To investigate the transmission of liver fluke on the Teagasc hill sheep farm, Co. Mayo, Ireland, snail species other than G. truncatula, were collected from the lowland pasture (snail sp. 'A') in May (n=23) and from the hill area (snail sp. 'B') in March, April and May 2008 (n=78, 53 and 36 respectively). Morphological characteristics identified snail sp. 'A' as a Succinea sp. and snail sp. 'B' as Radix peregra. PCR and subsequent sequencing of the 18S gene and internal transcribed spacer 2 (ITS-2) region of the snail rDNA were used for molecular identification. The 18S ribosomal DNA (rDNA) sequences revealed a 99% similarity with Omalonyx matheroni (Succineidae) for snail sp. 'A', and for snail sp. 'B' a 99% and 98% similarity with Radix auricularia and R. peregra, respectively. The ITS-2 sequences of snail sp. 'B' revealed a 100% similarity with R. peregra. Using PCR F. hepatica DNA was identified in 73.9% (n=17) of snail sp. 'A' collected during May and 10.3% (n=8), 60.4% (n=32) and 61.1% (n=22) of snail sp. 'B' collected during March, April and May, respectively. A priori knowledge of habitat preference and environmental tolerances of G. truncatula has failed to explain the prevalence of infection recorded in the hill sheep flock on Teagasc hill sheep farm. These findings may explain the presence of infection in sheep populations grazing hill habitats considered too acidic to maintain G. truncatula populations.
Cancela, Martín; Santos, Guilherme B; Carmona, Carlos; Ferreira, Henrique B; Tort, José Francisco; Zaha, Arnaldo
Fasciola hepatica is the causative agent of fasciolosis, a zoonosis with significant impact both in human and animal health. Understanding the basic processes of parasite biology, especially those related to interactions with its host, will contribute to control F. hepatica infections and hence liver pathology. Mucins have been described as important mediators for parasite establishment within its host, due to their key roles in immune evasion. In F. hepatica, mucin expression is upregulated in the mammalian invasive newly excysted juvenile (NEJ) stage in comparison with the adult stage. Here, we performed sequencing of mucin cDNAs prepared from NEJ RNA, resulting in six different cDNAs clusters. The differences are due to the presence of a tandem repeated sequence of 66 bp encoded by different exons. Two groups of apomucins one with three and the other with four repeats, with 459 and 393 bp respectively, were identified. These cDNAs have open reading frames encoding Ser-Thr enriched proteins with an N-terminal signal peptide, characteristic of apomucin backbone. We cloned a 4470 bp gene comprising eight exons and seven introns that encodes all the cDNA variants identified in NEJs. By real time polymerase chain reaction and high-resolution melting approaches of individual flukes we infer that fhemuc-1 is a single-copy gene, with at least two different alleles. Our data suggest that both gene polymorphism and alternative splicing might account for apomucin variability in the fhemuc-1 gene that is upregulated in NEJ invasive stage. The relevance of this variation in host-parasite interplay is discussed.
Pereira, Cíntia A J; Oliveira, Laura L S; Coaglio, Aytube L; Santos, Fernanda S O; Cezar, Rodolfo S M; Mendes, Tiago; Oliveira, Fernando L P; Conzensa, Gustavo; Lima, Walter S
Fasciolosis, a parasitic disease caused by the trematode Fasciola hepatica underreported is expanding both in human and animal population, throughout the world. The constant use of synthetic drugs to treat this condition has led to the natural selection of resistant strains of the parasite. Hence, there is a growing focus on the potential anti-helminthic properties of medicinal plants and phytopharmaceuticals. The current study assessed the potential anti-fasciolicide action of Momordica charantia leaf extracts and fractions on the eggs of F. hepatica parasites. The lyophilized crude extract (CE) of M. charantia leaves and its sub-fractions, obtained from liquid-liquid partitioning with organic solvents, were analysed by High Performance Liquid Chromatography (HPLC), suspended in 1% DMSO and used in in vitro tests. Quadruplicates of 50F. hepatica eggs were incubated at 23°C with M. charantia leaf CE in different concentrations. After 12days no larvae were formed in eggs incubated with CE concentrations above 12.5mg/mL. Eggs incubated with CE sub-fractions at concentrations of 1000, 100, 10, 1, 0.1, 0.01μg/mL affected embryonic development, with n-butanol presenting the strongest inhibition of miracidia formation. In contrast, on the 12th day, 90% of the miracidia hatched in the control experiments using 0.03% DMSO whereas embryogenesis was completely abolished with any concentration of albendazole sulphoxide ABZ(SO). Chemical analysis of the CE and sub-fractions revealed a prominent presence of flavonoids. HPLC-MS confirmed Quercetin to be one of the main flavonoids present in the CE and the n-butanol subfraction. This is the first study to analyse the potential anti-fasciolicide action of M. charantia leaf CE and subfractions.
Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa
Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.
Beesley, Nicola J; Williams, Diana J L; Paterson, Steve; Hodgkinson, Jane
Fasciola hepatica, the liver fluke, is a trematode parasite of considerable economic importance to the livestock industry and is a re-emerging zoonosis that poses a risk to human health in F. hepatica-endemic areas worldwide. Drug resistance is a substantial threat to the current and future control of F. hepatica, yet little is known about how the biology of the parasite influences the development and spread of resistance. Given that F. hepatica can self-fertilise and therefore inbreed, there is the potential for greater population differentiation and an increased likelihood of recessive alleles, such as drug resistance genes, coming together. This could be compounded by clonal expansion within the snail intermediate host and aggregation of parasites of the same genotype on pasture. Alternatively, widespread movement of animals that typically occurs in the UK could promote high levels of gene flow and prevent population differentiation. We identified clonal parasites with identical multilocus genotypes in 61% of hosts. Despite this, 84% of 1579 adult parasites had unique multilocus genotypes, which supports high levels of genotypic diversity within F. hepatica populations. Our analyses indicate a selfing rate no greater than 2%, suggesting that this diversity is in part due to the propensity for F. hepatica to cross-fertilise. Finally, although we identified high genetic diversity within a given host, there was little evidence for differentiation between populations from different hosts, indicating a single panmictic population. This implies that, once those emerge, anthelmintic resistance genes have the potential to spread rapidly through liver fluke populations.
Xu, Min-Jun; Ai, Lin; Fu, Jing-Hua; Nisbet, Alasdair J; Liu, Qing-You; Chen, Mu-Xin; Zhou, Dong-Hui; Zhu, Xing-Quan
MicroRNAs (miRNAs) are key regulators of gene expression at the post-transcription level. The present study specifically explored and compared the miRNA expression profiles of F. gigantica and F. hepatica using an integrated sequencing and bioinformatics platform and quantitative real-time PCR. Nineteen and 16 miRNA candidates were identified from F. gigantica and F. hepatica, respectively. The two parasites shared 11 miRNAs, with 8 also showing similarity to miRNAs of Schistosoma japonicum. Another 8 miRNAs were identified as F. gigantica-specific and 5 as F. hepatica-specific, most of which were novel. Predicted target analysis with 11465 mRNA and EST sequences of F. hepatica and F. gigantica revealed that all of the miRNAs had more than one target, ranging from 2 to 398 with an average of 51 targets. Some functions of the predicted targets were only found in F. gigantica, such as "transcription regulator", while some others were only found in F. hepatica, such as "reproduction" and "response to stimulus", indicating the different metabolism and gene regulation patterns of the two parasites. The present study represents the first global comparative characterization of miRNA expression profiles of F. gigantica and F. hepatica, which has provided novel valuable resources for a better understanding of the two zoonotic trematodes.
Kantzoura, V; Kouam, M K; Demiris, N; Feidas, H; Theodoropoulos, G
Risk factors related to herd and farmer status, farm and pasture management, and environmental factors derived by satellite data were examined for their association with the prevalence of F. hepatica in sheep and goat farms in Thessaly, Greece. Twelve farms (16.2%) and 58 farms (78.4%) of 74 had evidence of infection using coproantigen and serology respectively. The average normalized difference vegetation index (NDVI) of farm location for 12 months before sampling was the most significant environmental risk factor for F. hepatica infection based on high seropositivity. The risk of infection increased by 1% when the value of NDVI increased by 0.01 degree. A geospatial map was constructed to show the relative risk (RR) of Fasciola infection in sheep and goat farms in Thessaly. In addition, geospatial maps of the model-based predicted RR for the presence of Fasciola infection in farms in Thessaly and the entire area of Greece were constructed from the developed model based on NDVI. In conclusion, this study demonstrated that Thessaly should be regarded as an endemic region for Fasciola infection and it represents the first prediction model of Fasciola infection in small ruminants in the Mediterranean basin.
McNulty, Samantha N.; Rosa, Bruce A.; Fontenla, Santiago; Choi, Young-Jun; Hallsworth-Pepin, Kymberlie; Kammili, Lakshmi; Latham, Patricia S.; Dell’Oca, Nicolas; Dominguez, Fernanda; Carmona, Carlos; Fischer, Peter U.; Mitreva, Makedonka
Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis’ gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans. PMID:28060841
Edwards, Jennifer; Brown, Martha; Peak, Emily; Bartholomew, Barbara; Nash, Robert J.; Hoffmann, Karl F.
Background Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control. Methodology/ Principle Findings Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and
Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh
Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately
Martínez-Ibeas, A M; González-Warleta, M; Martínez-Valladares, M; Castro-Hermida, J A; González-Lanza, C; Miñambres, B; Ferreras, C; Mezo, M; Manga-González, M Y
Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C. daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C. daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C. daubneyi and 0.001 ng of DNA from F. hepatica. This allowed
Vignoles, Philippe; Titi, Amal; Rondelaud, Daniel; Mekroud, Abdeslam; Dreyfuss, Gilles
As abrupt changes in water temperature (thermal shock) triggered a significantly greater cercarial emergence of Fasciola hepatica from experimentally infected Galba truncatula, laboratory investigations were carried out to study the influence of light on cercarial emergence in snails subjected to a thermal shock every week (a mean of 12 °C for 3 h) during the patent period. Thermal shock for these temperature-challenged (TC) snails was carried out outdoors under artificial or natural light, or indoors under constant artificial light. Compared with the infected control snails always reared indoors at 20 °C, the number of cercariae in TC snails subjected to a thermal shock and natural light outdoors was significantly greater. The repetition of this experiment by subjecting TC snails to the same thermal shock indoors under an artificial light level ranging from 600 to 3000 lux did not show any significant difference among the numbers of cercariae in the different subgroups. A detailed analysis of the results noted in the TC snails subjected to natural light during the thermal shock demonstrated that the number of cercariae-releasing snails was significantly higher between 601 and 1200 lux and for the highest nebulosity values (7–8 octas, which corresponds to a sufficiently or completely overcast sky). Contrary to the intensity of artificial light, which did not influence cercarial emergence, the natural light level had a significant effect on this process when F. hepatica-infected snails were subjected to a regular thermal shock during the patent period. PMID:24572174
Bottari, Nathieli B; Mendes, Ricardo E; Baldissera, Matheus D; Bochi, Guilherme V; Moresco, Rafael N; Leal, Marta L R; Morsch, Vera M; Schetinger, Maria R C; Christ, Ricardo; Gheller, Larissa; Marques, Éder J; Da Silva, Aleksandro S
The aim of this study was to evaluate the iron metabolism in serum, as well as antioxidant enzymes, in addition to the Delta-Aminolevulinic Acid Dehydratase (δ-ALA-D) activity in the liver of rats experimentally infected by Fasciola hepatica. Thirty male adult rats (Wistar) specific pathogen free were divided into four groups: two uninfected group (CTRL 1 and CTRL 2) with five animals each and two infected groups (INF 1 and INF 2) with 10 animals each. Infection was performed orally with 20 metacercariae at day 1. On day 15 (CTRL 1 and INF 1 groups) and 87 PI (CTRL 2 and INF 2 groups) blood and bone marrow were collected and the animals were subsequently euthanized for liver sampling. Blood was allocated in tubes without anticoagulant for serum acquisition to measure iron, transferrin and unsaturated iron binding capacity (UIBC). δ-ALA-D, superoxide dismutase (SOD), and catalase (CAT) activities were measured in the liver. A decrease in iron, transferrin and UIBC levels was observed in all infected animals compared to control groups (P < 0.05). Furthermore, iron accumulation was observed in bone marrow of infected mice. Infected animals showed an increase in δ-ALA-D activity at 87 post-infection (PI) (INF 2) as well as in SOD activity at days 15 (INF 1) and 87 PI (INF 2). On the other hand, CAT activity was reduced in rats infected by F. hepatica during acute and chronic phase of fasciolosis (INF 1 and INF 2 groups), when moderate (acute) and severe necrosis in the liver histopathology were observed. These results may suggest that oxidative damage to tissues along with antioxidant mechanisms might have taken part in fasciolosis pathogenesis and are also involved in iron deficiency associated to changes in δ-ALA-D activity during chronic phase of disease.
Machicado, Claudia; Machicado, Jorge D.; Maco, Vicente; Terashima, Angelica; Marcos, Luis A.
Background Fascioliasis has been sporadically associated with chronic liver disease on previous studies. In order to describe the current evidence, we carried out a systematic review to assess the association between fascioliasis with liver fibrosis, cirrhosis and cancer. Methodology and Principal Findings A systematic search of electronic databases (PubMed, LILACS, Scopus, Embase, Cochrane, and Scielo) was conducted from June to July 2015 and yielded 1,557 published studies. Among 21 studies that met inclusion and exclusion criteria, 12 studies explored the association of F. hepatica with liver fibrosis, 4 with liver cirrhosis, and 5 with cancer. Globally these studies suggested the ability of F. hepatica to promote liver fibrosis and cirrhosis. The role of F. hepatica in cancer is unknown. Given the heterogeneity of the studies, a meta-analysis could not be performed. Conclusions Future high-quality studies are needed to determine the role of F. hepatica on the development of liver fibrosis, liver cirrhosis, and cancer in humans. PMID:27681524
Stempin, Cinthia C.; Motrán, Claudia C.; Aoki, María P.; Falcón, Cristian R.
Macrophage plasticity is critical for controlling inflammation including those produced by helminth infections, where alternatively activated macrophages (AAM) are accumulated in tissues. AAM expressing the co-inhibitory molecule programmed death ligand 2 (PD-L2), which is capable of binding programmed death 1 (PD-1) expressed on activated T cells, have been demonstrated in different parasitic infections. However, the role of PD-L2 during F. hepatica infection has not yet been explored. We observed that F. hepatica infection or a F. hepatica total extract (TE) injection increased the expression of PD-L2 on peritoneal macrophages. In addition, the absence of PD-L2 expression correlated with an increase in susceptibility to F. hepatica infection, as evidenced by the shorter survival and increased liver damage observed in PD-L2 deficient (KO) mice. We assessed the contribution of the PD-L2 pathway to Th2 polarization during this infection, and found that the absence of PD-L2 caused a diminished Th2 type cytokine production by TE stimulated splenocytes from PD-L2 KO infected compared with WT mice. Besides, splenocytes and intrahepatic leukocytes from infected PD-L2 KO mice showed higher levels of IFN-γ than those from WT mice. Arginase expression and activity and IL-10 production were reduced in macrophages from PD-L2 KO mice compared to those from WT mice, revealing a strong correlation between PD-L2 expression and AAM polarization. Taken together, our data indicate that PD-L2 expression in macrophages is critical for AAM induction and the maintenance of an optimal balance between the Th1- and Th2-type immune responses to assure host survival during F. hepatica infection. PMID:27783986
Alvarez, L I; Mottier, M L; Lanusse, C E
The work reported here describes the comparative ability of albendazole (ABZ), fenbendazole (FBZ) and triclabendazole (TCBZ) to penetrate through the tegument of mature Fasciola hepatica, and the influence of the physicochemical composition of the incubation medium on the drug diffusion process. The data obtained from the trans-tegumental diffusion kinetic studies were complemented with the determination of lipid-to-water partition coefficients (octanol-water) for the benzimidazole (BZD) anthelmintic drugs assayed. Sixteen-week-old F. hepatica were obtained from untreated artificially infected sheep. The flukes were incubated (37 degrees C) over 60 and 90 min in incubation media (pH 7.4) prepared with different proportions of ovine bile and Krebs' Ringer Tris (KRT) buffer (100, 75, 50, 25 and 0% of bile) containing either ABZ, FBZ or TCBZ at a final concentration of 5 nmol/ml. After the incubation time expired, the liver fluke material was chemically processed and analysed by high performance liquid chromatography (HPLC) to measure drug concentrations within the parasite. Additionally, the octanol-water partition coefficients (PC) for each molecule were calculated (as an indicator of drug lipophilicity) using reversed phase HPLC. The 3 BZD molecules were recovered from F. hepatica at both incubation times in all incubation media assayed. The trans-tegumental diffusion of the most lipophilic molecules ABZ and FBZ (higher PC values) tended to be greater than that observed for TCBZ. Interestingly, the uptake of ABZ by the liver flukes was significantly greater than that measured for TCBZ, the most widely used flukicidal BZD compound. This differential uptake pattern may be a relevant issue to be considered to deal with TCBZ-resistant flukes. Drug concentrations measured within the parasite were lower in the incubations containing the highest bile proportions. The highest total availabilities of the 3 compounds were obtained in liver flukes incubated in the absence of
Rondelaud, D; Vignoles, P; Vareille-Morel, C; Abrous, M; Mage, C; Mouzet, R; Dreyfuss, G
Experimental investigations in eight open drainage ditches and furrows from central France were carried out to analyse the dispersal of floating metacercariae of two digenean species by running water and to determine the outcome of larvae which settled on Nasturtium officinale (watercress). The frequencies of larvae found after their transport by water ranged from 33% to 49.7%, thus indicating that more than half of the metacercariae used in this experiment had fallen to the bottom of the water during this transport. The nature of the site (furrow, or ditch supplied by a spring) had a significant effect on the distribution of floating larvae, while the digenean species had no effect. Low percentages of metacercariae on watercress were noted in furrows (3.5-4.3% of larvae) and ditches (0.8-1.3%). When the watercress grew, most larvae that had settled on leaves and stems died but there were always several living metacercariae on this plant (0.7-1.5% of larvae for Fasciola hepatica and 0.2-0.5% for Paramphistomum daubneyi). The weak buoyancy of these floating cysts on running water limited their dispersal and, consequently, led to a real diminution of risks incurred by definitive hosts towards these metacercariae.
Hamed, Néjia; Njeh, Fatma; Damak, Mohamed; Ayadi, Ali; Mezghani-Jarraya, Raoudha; Hammami, Hayet
Fasciolosis is a widespread parasitosis of farm live-stock in many developing countries. For this reason, it is necessary to search for new substances against parasitic diseases caused by flukes. Indeed, a wide variety of terrestrial plants have been subjected to chemical and pharmacological screening in order to discover their potential for human medicinal use. The molluscicidal and larvicidal activities of Atriplex inflata were tested on Galba truncatula and Fasciola hepatica larval stages infecting this snail in Tunisia. Phytochemical tests were conducted on extracts in order to establish a meaningful relationship with molluscicidal and larvicidal activities. The molluscicidal activity was evaluated by subjecting snails to sample aqueous solutions. Accordingly, hexane, ethyl acetate, methanol and methanol-water (8:2, v-v) were used as extraction solvents. As a result, hexane and ethyl acetate extracts showed potent activity, according to the World Health Organization, giving LC50 = 7.59 mg/L and 6.69 mg/L for hexane extracts of leaves and fruits, respectively. Ethyl acetate extracts gave LC50 = 5.90 mg/L and 7.32 mg/L for leaves and fruits, successively. Molluscicidal activities of powders were less potent on snails, but active according to the World Health Organization. Hexane and ethyl acetate extracts from leaves and fruits gave potent larvicidal activities with a delay rate exceeding 45.50% (45.50- 98.92%). Phytochemical tests showed that these activities may be attributed to the presence of triterpenoids and/or sterols.
Boulard, C; Carreras, F; Van Gool, F
The responses of cattle infected with Fasciola hepatica to treatment with nitroxynil or closantel were monitored by faecal egg counts and by ELISA assay of anti-fluke antibodies. A first trial with experimentally infected heifers showed an increase in anti-fluke antibody titre as early as 2 weeks post-infection. Eggs were first detected in the faeces 10 weeks after infection. Egg output increased steadily over the next 8 weeks and then rapidly decreased. Treatment of a 20-week infection with nitroxynil was followed by a slow decrease in antibody titre 4 weeks later. This decrease continued over the next 40 weeks, but returned to pre-infection levels in only 2 out of 4 animals. The faecal egg count fell to zero 2 weeks after treatment and remained so for the following 30 weeks, although 1 animal produced a few eggs 32 and 34 weeks post-treatment. Within this period, neither diagnostic technique discriminated between this persistently infected animal and the others. In a second trial, 45 cattle from a naturally infected herd were treated with nitroxynil or closantel. The faecal egg counts of the treated cattle were zero within the following 2 months, whereas there were eggs in the faeces of the control (untreated) group. Nevertheless, the treated cattle showed a small, non-significant drop in anti-fluke antibody titre. These results demonstrate the need for new tools to monitor and evaluate accurately the efficacy of anthelmintic treatment.
Valero, M Adela; Ubeira, Florencio M; Khoubbane, Messaoud; Artigas, Patricio; Muiño, Laura; Mezo, Mercedes; Pérez-Crespo, Ignacio; Periago, M Victoria; Mas-Coma, Santiago
During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4-7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3-6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33-66 adults), while it was for F. gigantica infection (burden of 17-69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.
Wesołowska, Agnieszka; Norbury, Luke J; Januszkiewicz, Kamil; Jedlina, Luiza; Jaros, Sławomir; Zawistowska-Deniziak, Anna; Zygner, Wojciech; Wędrychowicz, Halina
Not only do males and females of many species vary in their responses to certain parasitic infections, but also to treatments such as vaccines. However, there are very few studies investigating differences among sexes following vaccination and infection. Here we demonstrate that female Sprague-Dawley rats vaccinated with cDNA encoding a recently discovered cysteine proteinase of Fasciola hepatica (FhPcW1) develop considerably lower liver fluke burdens after F. hepatica infection than their male counterparts. This is accompanied by differences in the course of their immune responses which involve different eosinophil and monocyte responses throughout the study as well as humoral responses. It is evident that host gender influences the outcome of parasitic infections after vaccination and research on both sexes should be considered when developing new treatments against parasites.
Liu, Qing; Huang, Si-Yang; Yue, Dong-Mei; Wang, Jin-Lei; Wang, Yujian; Li, Xiangrui; Zhu, Xing-Quan
Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.
Fernández, V; Estein, S; Ortiz, P; Luchessi, P; Solana, V; Solana, H
The helminth parasite Fasciola hepatica causes fascioliasis in human and domestic ruminants. Economic losses due to this infection are estimated in U$S 2000-3000 million yearly. The most common method of control is the use of anthelmintic drugs. However, there is an increased concern about the growing appearance of F. hepatica resistance to Triclabendazole (TCBZ), an anthelmintic with activity over adult and young flukes. F. hepatica has eight Glutathione S-Transferase (GST) isozymes, which are enzymes involved in the detoxification of a wide range of substrates through chemical conjugation with glutathione. In the present work we identified and characterized the GST mu gene isolated from the TCBZ-susceptible and TCBZ-resistant F. hepatica strains. Total RNA was transcribed into cDNA by reverse transcription and a 657 bp amplicon corresponding to the GST mu gene was obtained. The comparative genetic analysis of the GST mu gene of the TCBZ susceptible strain (Cullompton) and TCBZ resistant strain (Sligo) showed three nucleotide changes and one amino acid change at position 143 in the GST mu isozyme of the TCBZ-resistant strain. These results have potential relevance as they contribute better understand the mechanisms that generate resistance to anthelmintics.
Cabada, Miguel M; Malaga, Jose L; Castellanos-Gonzalez, Alejandro; Bagwell, Kelli A; Naeger, Patrick A; Rogers, Hayley K; Maharsi, Safa; Mbaka, Maryann; White, A Clinton
Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)-based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization.
Valero, M Adela; Perez-Crespo, Ignácio; Khoubbane, Messaoud; Artigas, Patricio; Panova, Miroslava; Ortiz, Pedro; Maco, Vicente; Espinoza, José R; Mas-Coma, Santiago
Fascioliasis is a zoonotic parasitic disease caused by Fasciola hepatica and Fasciola gigantica. Of both species, F. hepatica is the only one described in the Americas, mainly transmitted by lymnaeid snail vectors of the Galba/Fossaria group. Human fascioliasis endemic areas are mainly located in high altitude areas of Andean countries. Given the necessity to characterize F. hepatica populations involved, the phenotypic features of fasciolid adults infecting sheep present in human fascioliasis endemic areas were analysed in the Cajamarca Valley and Mantaro Valley (valley transmission patterns) and the northern Bolivian Altiplano (altiplanic transmission pattern). A computer image analysis system (CIAS) was applied on the basis of standardized measurements. The aforementioned highland populations were compared to standard lowland natural and experimental populations of European origin. Liver fluke size was studied by multivariate analyses. Two phenotypic patterns could be distinguished in F. hepatica adult size: the valley pattern (Cajamarca and Mantaro, Peru) and the altiplanic pattern (northern Altiplano, Bolivia). Results showed that the Andean valley population and European standard populations presented a phenotypic homogeneity. The Altiplano population showed a large size range with a pronouncedly lower minimum size indicating that uterus gravidity is reached at a smaller size than in valley populations. The results of this study demonstrate that there is no apparent relationship between the shape of fasciolid adults with regard to altitudinal difference or geographical origin and that allometry-free shape appears as a more stable trait than size in fasciolid species. Results are analysed in terms of intensity/crowding effect aspects and permanent/seasonal transmission characteristics.
Acosta, Daniel; Cancela, Martín; Piacenza, Lucia; Roche, Leda; Carmona, Carlos; Tort, José F
Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host's homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn(2+), and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development.
O'Neill, S M; Parkinson, M; Strauss, W; Angles, R; Dalton, J P
Cathepsin L1 (CL1), an immunogenic cysteine proteinase secreted by juvenile and adult Fasciola hepatica, was assessed for its potential as a diagnostic agent for the serologic detection of human fascioliasis. Using ELISAs, we compared the ability of liver fluke homogenates (LFH), excretory/secretory (ES) products, and CL1 to discriminate between seropositive (infected) and seronegative (noninfected) individuals within a population of 95 patients from the Bolivian Altiplano. A high prevalence of human fascioliasis has been reported in this region. The division between the seropositive and seronegative individuals was poorly defined when LFH was used as the antigen. A greater discrimination between these populations was achieved with both ES and CL1. A K-means cluster analysis using the combined ES and CL1 ELISA data identified a cluster of seropositive individuals. Cathepsin L1 detected a subset (20) of these seropositive individuals while ES detected all 26; however, ES detected nine additional individuals that were in the seronegative cluster. The ratio of the mean absorbance readings between seropositive and seronegative individuals was markedly improved by using conjugated second antibodies to IgG4, the predominant isotype elicited by infection. In these IgG4-ELISAs, CL1 again identified fewer individuals as seropositive than did ES, but improved the discrimination between the seropositive and seronegative individuals and thus provided a more conclusive diagnosis. Sera obtained from patients infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas' disease were negative in these assays, which demonstrated the specificity of the IgG4-ELISA for detecting fascioliasis. Twenty of the 95 patients (21%) were seropositive for fascioliasis by the CL1 IgG4-ELISA, confirming the earlier reports of the high prevalence of disease in this region. A standardized diagnostic test for human fascioliasis, based on an ELISA that detects IgG4 responses to CL1
HAMED, Néjia; NJEH, Fatma; DAMAK, Mohamed; AYADI, Ali; MEZGHANI-JARRAYA, Raoudha; HAMMAMI, Hayet
Fasciolosis is a widespread parasitosis of farm live-stock in many developing countries. For this reason, it is necessary to search for new substances against parasitic diseases caused by flukes. Indeed, a wide variety of terrestrial plants have been subjected to chemical and pharmacological screening in order to discover their potential for human medicinal use. The molluscicidal and larvicidal activities of Atriplex inflata were tested on Galba truncatula and Fasciola hepatica larval stages infecting this snail in Tunisia. Phytochemical tests were conducted on extracts in order to establish a meaningful relationship with molluscicidal and larvicidal activities. The molluscicidal activity was evaluated by subjecting snails to sample aqueous solutions. Accordingly, hexane, ethyl acetate, methanol and methanol-water (8:2, v-v) were used as extraction solvents. As a result, hexane and ethyl acetate extracts showed potent activity, according to the World Health Organization, giving LC50 = 7.59 mg/L and 6.69 mg/L for hexane extracts of leaves and fruits, respectively. Ethyl acetate extracts gave LC50 = 5.90 mg/L and 7.32 mg/L for leaves and fruits, successively. Molluscicidal activities of powders were less potent on snails, but active according to the World Health Organization. Hexane and ethyl acetate extracts from leaves and fruits gave potent larvicidal activities with a delay rate exceeding 45.50% (45.50- 98.92%). Phytochemical tests showed that these activities may be attributed to the presence of triterpenoids and/or sterols. PMID:27049700
Zinsser, Veronika L; Lindert, Steffen; Banford, Samantha; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J
Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.
O'Neill, J F; Johnston, R C; Halferty, L; Brennan, G P; Fairweather, I
An in vivo study in the laboratory rat model has been carried out to monitor changes to the tegument and gut of adult Fasciola hepatica following treatment with artesunate. Rats infected with the triclabendazole-resistant Oberon isolate were dosed orally with artesunate at a concentration of 200 mg/kg and flukes recovered 24, 48, 72 and 96 h post-treatment (pt). The flukes were processed for scanning and transmission electron microscope examination. Changes to the external surface were limited to swelling and blebbing of the interspinal tegument. There was one exception, a specimen recovered 72 h pt, which had completely lost the syncytium over the posterior region of the fluke. Internal changes to the tegumental syncytium and cell bodies were more severe and were apparent from 48 h pt onwards. Increased numbers of secretory bodies were present in the apical region of the syncytium, the basal infolds were swollen and sloughing of the apical plasma membrane was seen at 96 h pt. In the cell bodies, there was swelling and vesiculation of the cisternae of the granular endoplasmic reticulum (ger), swelling of the mitochondria and a decrease in secretory body production. Changes to the gastrodermal cells were evident from 24 h onwards. They comprised swelling and vesiculation of the ger cisternae, swelling and lysis of the mitochondria and accumulation of autophagic vacuoles and lipid droplets. The nuclei of the cells were karyopyknotic by 96 h pt. The gut was consistently more severely affected than the tegument at all time points pt, pointing to an oral route of uptake for artesunate. This study has provided information on the primary subcellular targets for drug action in the fluke.
Hickey, Graeme L; Diggle, Peter J; McNeilly, Tom N; Tongue, Sue C; Chase-Topping, Margo E; Williams, Diana J L
The parasite Fasciola hepatica is a major cause of economic loss to the agricultural community worldwide as a result of morbidity and mortality in livestock, including cattle. Cattle are the principle reservoir of verocytotoxigenic Escherichia coli O157 (VTEC O157), an important cause of disease in humans. To date there has been little empirical research on the interaction between F. hepatica and VTEC O157. It is hypothesised that F. hepatica, which is known to suppress type 1 immune responses and induce an anti-inflammatory or regulatory immune environment in the host, may promote colonisation of the bovine intestine with VTEC O157. Here we assess whether it is statistically feasible to augment a prospective study to quantify the prevalence of VTEC O157 in cattle in Great Britain with a pilot study to test this hypothesis. We simulate data under the framework of a mixed-effects logistic regression model in order to calculate the power to detect an association effect size (odds ratio) of 2. In order to reduce the resources required for such a study, we exploit the fact that the test results for VTEC O157 will be known in advance of testing for F. hepatica by restricting analysis to farms with a VTEC O157 sample prevalence of >0% and <100%. From a total of 270 farms (mean 27 cows per farm) that will be tested for VTEC O157, power of 87% can be achieved, whereby testing of F. hepatica would only be necessary for an expected 50 farms, thus considerably reducing costs. Pre-study sample size calculations are an important part of any study design. The framework developed here is applicable to the study of other co-infections.
Hickey, Graeme L.; Diggle, Peter J.; McNeilly, Tom N.; Tongue, Sue C.; Chase-Topping, Margo E.; Williams, Diana J.L.
The parasite Fasciola hepatica is a major cause of economic loss to the agricultural community worldwide as a result of morbidity and mortality in livestock, including cattle. Cattle are the principle reservoir of verocytotoxigenic Escherichia coli O157 (VTEC O157), an important cause of disease in humans. To date there has been little empirical research on the interaction between F. hepatica and VTEC O157. It is hypothesised that F. hepatica, which is known to suppress type 1 immune responses and induce an anti-inflammatory or regulatory immune environment in the host, may promote colonisation of the bovine intestine with VTEC O157. Here we assess whether it is statistically feasible to augment a prospective study to quantify the prevalence of VTEC O157 in cattle in Great Britain with a pilot study to test this hypothesis. We simulate data under the framework of a mixed-effects logistic regression model in order to calculate the power to detect an association effect size (odds ratio) of 2. In order to reduce the resources required for such a study, we exploit the fact that the test results for VTEC O157 will be known in advance of testing for F. hepatica by restricting analysis to farms with a VTEC O157 sample prevalence of >0% and <100%. From a total of 270 farms (mean 27 cows per farm) that will be tested for VTEC O157, power of 87% can be achieved, whereby testing of F. hepatica would only be necessary for an expected 50 farms, thus considerably reducing costs. Pre-study sample size calculations are an important part of any study design. The framework developed here is applicable to the study of other co-infections. PMID:25779556
Cwiklinski, Krystyna; Allen, Katherine; LaCourse, James; Williams, Diana J; Paterson, Steve; Hodgkinson, Jane E
The liver fluke, Fasciola hepatica is an economically important pathogen of sheep and cattle and has been described by the WHO as a re-emerging zoonosis. Control is heavily reliant on the use of drugs, particularly triclabendazole and as a result resistance has now emerged. The population structure of F. hepatica is not well known, yet it can impact on host-parasite interactions and parasite control with drugs, particularly regarding the spread of triclabendazole resistance. We have identified 2448 potential microsatellites from 83 Mb of F. hepatica genome sequence using msatfinder. Thirty-five loci were developed and optimised for microsatellite PCR, resulting in a panel of 15 polymorphic loci, with a range of three to 15 alleles. This panel was validated on genomic DNA from 46 adult F. hepatica; 38 liver flukes sourced from a Northwest abattoir, UK and 8 liver flukes from an established isolate (Shrewsbury; Ridgeway Research). Evidence for null alleles was found at four loci (Fh_1, Fh_8, Fh_13 and Fh_14), which showed markedly higher levels of homozygosity than the remaining 11 loci. Of the 38 liver flukes isolated from cattle livers (n=10) at the abattoir, 37 genotypes were identified. Using a multiplex approach all 15 loci could be amplified from several life cycle stages that typically yield low amounts of DNA, including metacercariae, the infective life cycle stage present on pasture, highlighting the utility of this multiplex microsatellite panel. This study reports the largest panel of microsatellite markers available to date for population studies of F. hepatica and the first multiplex panel of microsatellite markers that can be used for several life cycle stages.
Dreyfuss, G; Vignoles, P; Rondelaud, D
Experimental co-infections of juvenile and pre-adult Pseudosuccinea columella with Calicophoron daubneyi and Fasciola hepatica (five miracidia of each digenean per snail) were carried out to determine the aptitude of this lymnaeid to ensure complete larval development of the former parasite, the latter or both. Snails infected with F. hepatica were found in the two groups of juveniles, i.e. 1 and 2 mm at exposure, and the four groups of pre-adults, i.e. 3-6 mm. The highest frequency of F. hepatica, i.e. 37.3%, was noted in the 4 mm group. Low frequencies were noted for C. daubneyi and co-infections of both digeneans in the 3, 4 and 5 mm groups. Two other groups of P. columella, measuring 3 and 4 mm at exposure, were also constituted to study the characteristics of these co-infections. Compared to controls infected only with F. hepatica, the frequency of this digenean infection and the mean number of metacercariae were significantly lower in co-infected snails, while the patent period was significantly shorter. In snails harbouring C. daubneyi only or both digeneans, lower values were noted for prevalence, the patent period and the number of metacercariae. Pre-adult P. columella (3-5 mm in shell height at exposure) were able to sustain larval development of C. daubneyi if they were co-infected with the sequence C. daubneyi +F. hepatica. Low values noted for the prevalence of C. daubneyi infection and the number of metacercariae would be in favour of a still incomplete adaptation between the snail population and the miracidial isolate.
Robinson, Mark W; Tort, Jose F; Lowther, Jonathan; Donnelly, Sheila M; Wong, Emily; Xu, Weibo; Stack, Colin M; Padula, Matthew; Herbert, Ben; Dalton, John P
Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser[downward arrow]His motif for
Alvarado, Raquel; To, Joyce; Lund, Maria E; Pinar, Anita; Mansell, Ashley; Robinson, Mark W; O'Brien, Bronwyn A; Dalton, John P; Donnelly, Sheila
The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL-1β, a cytokine that influences the development of both innate and adaptive immune responses. Helminth parasites secrete molecules that interact with innate immune cells, modulating their activity to ultimately determine the phenotype of differentiated T cells, thus creating an immune environment that is conducive to sustaining chronic infection. We show that one of these molecules, FhHDM-1, a cathelicidin-like peptide secreted by the helminth parasite, Fasciola hepatica, inhibits the activation of the NLRP3 inflammasome resulting in reduced secretion of IL-1β by macrophages. FhHDM-1 had no effect on the synthesis of pro-IL-1β. Rather, the inhibitory effect was associated with the capacity of the peptide to prevent acidification of the endolysosome. The activation of cathepsin B protease by lysosomal destabilization was prevented in FhHDM-1-treated macrophages. By contrast, peptide derivatives of FhHDM-1 that did not alter the lysosomal pH did not inhibit secretion of IL-1β. We propose a novel immune modulatory strategy used by F. hepatica, whereby secretion of the FhHDM-1 peptide impairs the activation of NLRP3 by lysosomal cathepsin B protease, which prevents the downstream production of IL-1β and the development of protective T helper 1 type immune responses that are detrimental to parasite survival.-Alvarado, R., To, J., Lund, M. E., Pinar, A., Mansell, A., Robinson, M. W., O'Brien, B. A., Dalton, J. P., Donnelly, S. The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.
Wesołowska, Agnieszka; Zawistowska-Deniziak, Anna; Norbury, Luke J; Wilkowski, Przemysław; Januszkiewicz, Kamil; Pyziel, Anna M; Zygner, Wojciech; Wędrychowicz, Halina
Immune responses of rats and sheep following vaccination with cDNA encoding phosphoglycerate kinase of Fasciola hepatica (cDNA-FhPGK/pCMV) and F. hepatica infection were investigated in the present study. cDNA-FhPGK/pCMV vaccinated female Sprague-Dawley rats were better protected by vaccination than their male counterparts - 48% reduction in fluke burden for females and no protection for males when compared with appropriate infection control groups. Moreover, male rats developed marked leukocytosis during the study with higher neutrophil, eosinophil and monocyte responses than females. Additionally, dynamics of eosinophil and monocyte responses varied between sexes. Increased titres of anti-FhPGK IgG1 and IgG2a correlated with the protective effect of vaccination that was observed among female rats. In the case of male sheep, no differences in worm burdens and in the course of the immune response were observed following vaccination. Titres of specific antibodies detected were low, and cellular responses were not significant. Apparently, sheep immune responses induced by cDNA-FhPGK/pCMV vaccination are not effective at controlling F. hepatica infection. Poor immunogenicity of DNA vaccines in large animals is still a major obstacle of this technology that has to be overcome.
Vázquez, Antonio A; Sánchez, Jorge; Alba, Annia; Pointier, Jean-Pierre; Hurtrez-Boussès, Sylvie
Natural infections of lymnaeid snails by Fasciola hepatica are of primary importance to study transmission. Also, infected snails in the field can be used to explore the existing compatibility in host-parasite interactions. This paper aimed to describe the infection rate of Galba cubensis populations in fasciolosis transmission areas. Eight sites were sampled in western Cuba and 24 infected snails at six sites were found. The mean prevalence was 2.94% and the maximum value was 11.4%. The intensity of parasite infection was assessed as the number of rediae inside a single snail. High variation within the sites examined was observed, but a maximum of 76 rediae was recovered from one individual. Although the presence of two other trematode families (Schistosomatidae and Paramphistomatidae) was discovered in dissected individuals, no co-infection with F. hepatica was observed. This is the first time a study of natural prevalence of F. hepatica infection is carried out in Cuba, considered a hyper endemic country for bovine fasciolosis. Our results suggest that fasciolosis transmission may occur even when the number of infected snails remains relatively low.
Gutierrez-Sanchez, Maria de Los Angeles; Luna-Herrera, Julieta; Trejo-Castro, Lauro; Montenegro-Cristino, Natividad; Almanza-Gonzalez, Alfredo; Escobar-Gutierrez, Alejandro; de la Rosa-Arana, Jorge Luis
We have studied the influence of both levamisole (AL) and Freund's adjuvant (AF) on the immunisation of mice with the secretory antigens of adults of the liver fluke Fasciola hepatica Linnaeus, 1758. Total IgG antibodies were detected in all groups where the F. hepatica antigen was administered, been levels of IgG1 increased respect to IgG2a antibodies. During immunisation, IL-4 and IFN-γ were only detected in AL and AF groups, but after infection, IL-4 boosted in all groups. IFN-γ increased two fold in AF and AL groups compared to the saline solution (AS) group. Worm recovering was of 32-35% in groups administered without antigen whereas in AS, AL and AF groups recovering was of 25%, 12% and 8%, respectively. Macroscopical lesions in the liver were scarce in AL and AF groups. Our data suggest that immunisation of mice with antigens of F. hepatica enhances the immune response avoiding both liver damage and worm establishment after challenge infection. The murine model of fasciolosis has appeared to be useful to elucidate the mechanism by which the parasite modulates immune responses toward a Th2 type but also the development of Th1 type-inducing vaccines.
Elliott, Timothy P; Spithill, Terry W
Triclabendazole (TCBZ) is widely used for control of Fasciola hepatica (liver fluke) in animals and humans and resistance to this drug is now widespread. However, the mechanism of resistance to TCBZ is not known. A T687G single nucleotide polymorphism (SNP) in a P-glycoprotein gene was proposed as a molecular marker for TCBZ resistance in F. hepatica (Wilkinson et al., 2012). We analyzed this Pgp gene from TCBZ-susceptible and TCBZ-resistant populations from Australia to determine if the SNP was a marker for TCBZ resistance. From the 21 parasites studied we observed 27 individual haplotypes in the Pgp sequences which comprised seven haplotypic groups (A-G), with haplotypes A and B representing 81% of the total observed. The T687G SNP was not observed in either of the resistant or susceptible populations. We conclude that the T687G SNP in this Pgp gene is not associated with TCBZ resistance in these Australian F. hepatica populations and therefore unlikely to be a universal molecular marker for TCBZ resistance.
Alba, Annia; Marcet, Ricardo; Otero, Oscar; Hernández, Hilda M; Figueredo, Mabel; Sarracent, Jorge
Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.
Marcos, Luis; Maco, Vicente; Terashima, Angélica; Samalvides, Frine; Espinoza, José R; Gotuzzo, Eduardo
High prevalence rates of human fascioliasis have been described in several regions of Peru. We surveyed 20 families in an endemic area of Peru in order to determine the proportion of infection with F. hepatica in relatives of diagnosed subjects and in order to identify associated risk factors. The study included feces and blood samples of 93 subjects. Ages ranged from one to 53 (mean = 18.6; SD = 14.2). The overall prevalence of fascioliasis by fecal examinations was 33.3% (n = 83) and by serology, 51.9% (n = 86). The prevalence in age group I (< or = 19 years old) by coprological and serological tests was 61.4% and 75.9%, respectively; in group II (> 19 years old) 15.4% and 37.5%. The main associated risk factor with fascioliasis was eating salads (OR = 3.29, CI = 1.2 - 9.0, p = 0.02). In conclusion, human fascioliasis is highly prevalent in the relatives of index cases and the most significant risk factor of acquiring fascioliasis in the family is eating salads in endemic areas.
Peritoneal lavage cells of Indonesian thin-tail sheep mediate antibody-dependent superoxide radical cytotoxicity in vitro against newly excysted juvenile Fasciola gigantica but not juvenile Fasciola hepatica.
Piedrafita, David; Estuningsih, Endah; Pleasance, Jill; Prowse, Rhoda; Raadsma, Herman W; Meeusen, Els N T; Spithill, Terry W
Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica.
Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran
Reaghi, Saber; Haghighi, Ali; Harandi, Majid Fasihi; Spotin, Adel; Arzamani, Kourosh; Rouhani, Soheila
Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. PMID:27733809
Rivera, Francheska; Espino, Ana M
Fasciola hepatica saposin-like protein-2 (FhSAP2) is a protein differentially expressed in various developmental stages of F. hepatica. Recombinant FhSAP2 has demonstrated the induction of partial protection in mice and rabbits when it is administered subcutaneously (SC) in Freund's adjuvant. Because FhSAP2 is overexpressed in bacteria in the form of inclusion bodies (IBs), we isolated IBs expressing FhSAP2 and tested their immunogenicity when administered SC in mice emulsified in two different adjuvants: QS-21 and Montanide TM ISA720. Animals received three injections containing 20 μg of protein two weeks apart and 4 weeks after the third injection, mice were infected with 10 F. hepatica metacercariae by oral route. The percentages of protection induced by FhSAP2-IBs were estimated to be between 60.0 and 62.5% when compared with adjuvant-vaccinated, infected controls. By determining the levels of IgG1 and IgG2a antibodies and IL-4 and IFNγ cytokines in the serum of experimental animals, it was found that both Th1 and Th2 immune responses were significantly increased in the FhSAP2-IBs vaccinated groups compared with the adjuvant-vaccinated, infected control groups. The adjuvant-vaccinated groups had significantly lower IgG1 to IgG2a ratios and lower IL-4 to IFNγ ratios than the FhSAP2-IBs vaccinated animals, which is indicative of higher levels of Th2 immune responses. Irrespective to the adjuvant used, animals vaccinated with FhSAP2-IBs exhibited significantly higher survival percentage and less liver damage than the adjuvant-control groups. This study suggests that FhSAP2 has potential as vaccine against F. hepatica and that the protection elicited by this molecule could be linked to a mechanism driven by the CD4-Th1 cells.
Ravidà, Alessandra; Aldridge, Allison M; Driessen, Nicole N; Heus, Ferry A H; Hokke, Cornelis H; O'Neill, Sandra M
Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica's tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg's binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg's ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host's immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.
Alba, Annia; Sánchez, Jorge; Hernández, Hilda; Mosqueda, Maryani; Rodríguez, Suanel Y; Capó, Virginia; Otero, Oscar; Alfonso, Carlos; Marcet, Ricardo; Sarracent, Jorge
Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites
These epidemiological studies concern 187 cases of human fasciolasis which occurred in the region of Limousin, France on a period of 24 years. Four species of plants have been eaten: Nasturtium officinale, Roripa silvestris, Taraxacum gr. officinale, Valerianella olitoria. These plants have been eaten during all months of the year, but with higher numbers in July-August and from November to February. The cases of human fasciolasis almost take place on all years, but their numbers are high in years showing a very important infestation of cattle by Fasciola hepatica. In 2/3 of cases, the plants come from stations situated out of cattle pastures. The water-cress almost comes from natural stations or from familial plantations. Five species of snails have been observed in these stations: Lymnaea glabra, L. palustris, L. peregra, L. stagnalis, L. truncatula. The young snails of these species experimentally have been observed. The significance of these results is discussed.
Sibille, P; Calléja, C; Carreras, F; Bigot, K; Galtier, P; Boulard, C
Two fasciolicide preparations have been compared in 130 rats experimentally infected with Fasciola hepatica. Parasitological, immunological, and biochemical parameters have been followed to monitor the efficacy of the treatments. While Fascinex (triclabendazole) efficiently cured both male and female rats when administered as soon as 4 weeks postinfection, treatment with Ivomec-D (clorsulon + ivermectin) displayed a low efficacy on either male or female rats at this time point (54 and 0%, respectively). Moreover, when administered 8 weeks postinfection, the Ivomec-D treatment proved highly efficient on male rats while it displayed little effect on the female population (100 and 53%, respectively). This unexpected result has been related to an overexpression of a P4503A isoform that is observed only in females that have been treated with Ivomec-D. The influence of this P4503A cytochrome on drug metabolism and the need for the incorporation of both genders in clinical trials are discussed.
Thomas, Charlotte M; Timson, David J
FhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.
Hernández Alvarez, Lilian; Naranjo Feliciano, Dany; Hernández González, Jorge Enrique; de Oliveira Soares, Rosemberg; Barreto Gomes, Diego Enry; Pascutti, Pedro Geraldo
Background Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica. Methodology/Principal Findings Starting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity. Conclusions/Significance The present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds
Ashrafi, Keyhan; Valero, M Adela; Peixoto, Raquel V; Artigas, Patricio; Panova, Miroslava; Mas-Coma, Santiago
Fascioliasis is a zoonotic disease emerging in numerous parts of the world. In any endemic area, the characterisation of scenarios and patterns of infection must always be considered the starting point before implementing any control measure. Fascioliasis is a parasitic disease of different epidemiological, pathological and control characteristics depending on the endemic area and the causal agent, Fasciola hepatica and Fasciolagigantica. Classically it has been accepted that F. hepatica is present worldwide, while the distribution of the two species overlaps in many areas of Africa and Asia. Fascioliasis caused by F. hepatica, F. gigantica and intermediate forms is present in Guilan province, a complicated epidemiological situation where the highest human infection rates have been described in Iran. Morphometric tools were used to analyse the possible relationship between liver-fluke metric traits and geographical and altitudinal distribution. This is the first study in which a detailed distribution of both Fasciola species is analysed in a human fascioliasis endemic area with a zonal overlap transmission pattern. An accurate analysis was conducted to phenotypically discriminate between fasciolids from naturally infected livestock (cattle, buffaloes, sheep and goats). The distribution of the % F. hepatica-like (F.h.) and F. gigantica-like (F.g.) flukes detected in each liver versus altitude (m) in each group was analysed. The presence of F.g. specimens mainly in locations below sea level (average: 11.23% F.h., 88.77% F.g.), the presence of both species with similar intensity at 1-99m (average: 56.95% F.h., 43.05% F.g.) and the presence of F.h. specimens mainly from 100 to 999m (average: 71.69% F.h., 28.31% F.g.) as well as in locations with an altitude above 1000m (average: 97.48% F.h., 2.52% F.g.) are noteworthy. A significant positive correlation was obtained between altitude and % F.h., and a significant negative correlation was obtained between altitude and
Haçarız, O; Sayers, G; Mulcahy, G
Fasciola hepatica, the liver fluke, is a highly evolved endo-parasite that uses various mechanisms to evade the host immune system. The immunosuppressive capabilities of the parasite's excretory/secretory products have been well demonstrated by previous independent studies. However, the role of the parasite's tegument in the immune responses remains to be investigated. In this study, the effect of the tegument and other fractions of adult F. hepatica (excretory/secretory, liver fluke homogenate and liver fluke homogenate without tegument) in the activation of naïve macrophages in vitro was investigated using an ovine model. In addition, an immunoproteomic approach was used to investigate the characteristics of humoral antibody responses developed in sheep against the tegument fraction. The results indicated significantly increased arginase expression in macrophages incubated with the tegument and excretory/secretory fractions. Two dimensional gel electrophoresis of the tegument demonstrated approximately 100 protein spots, with only four of these spots were highly reactive with the positive serum as determined by 2-DE immunoblotting. These results give a preliminary indication that the liver fluke tegument may play role in avoiding hosts' protective immune responses against itself.
Valero, M A; Mas-Coma, S
Fascioliasis due to Fasciola hepatica (Linnaeus, 1758) is an endemic disease on the Northern Bolivian Altiplano, where human prevalences and intensities are the highest known, sheep and cattle are the main reservoir hosts, and pigs and donkeys the secondary ones. Investigations were carried out to study the viability of metacercariae experimentally obtained from eggs shed by naturally infected Altiplanic sheep, cattle, pigs and donkeys. A total of 157 Wistar rats were infected with doses of 5, 10, 20 and 150 metacercariae. Metacercariae aged for different number of weeks were used to analyse the influence of age on their viability. The number of worms successfully developed in each rat was established by dissection. Results obtained show that metacercarial infectivity is dependent upon storage time, being lower when metacercariae are older. The maximum longevity is 31 weeks using doses of 20 metacercariae per rat and 48 weeks with 150 metacercariae per rat, although in the latter case only a very low percentage of worms is recovered. Age-related infectivity of metacercariae from Altiplanic F. hepatica does not significantly differ from that of the liver fluke in lowlands of other countries. Concerning the influence of the isolate according to host species, results indicate that metacercarial viabilities of pig and donkey isolates are similar to the viabilities of metacercariae of sheep and cattle isolates. Thus, pig and donkey have a high transmission potential capacity concerning this aspect. This fact is of great importance for the control of human and animal fascioliasis in this highly endemic zone.
Mendes, Ricardo E; Pérez-Ecija, Rafael A; Zafra, Rafael; Buffoni, Leandro; Martínez-Moreno, Alvaro; Dalton, John P; Mulcahy, Grace; Pérez, José
Protection against Fasciola hepatica in goats immunized with Peroxiredoxin (Prx) was assessed. The experimental trial consisted of three groups of seven animals; group 1 were unimmunized and uninfected, group 2 were immunized with adjuvant only and group 3 were immunized with recombinant Prx in adjuvant (immunized and infected). Immunization with Prx in Quil A adjuvant, group 3, induced a reduction in fluke burden of 33.04% when compared to adjuvant control, group 2, although this difference was not significant. The hepatic gross and microscopical morphometric study revealed lower damage in the Prx-immunized compared to group 2 (p<0.05). Furthermore, immunohistochemical studies revealed that the Prx-immunized group exhibited reduced infiltration of CD4(+), CD8(+), IFN-gamma(+) and TCR(+) (p<0.05); and CD2(+) and IL-4(+) (p<0.001) in hepatic lesions. Levels of anti-Prx serum IgG in group 3 showed a significant increase at the 4th week after challenge infection compared with group 2 (p<0.0001). This is the first report of ruminant immunization with recombinant Prx of F. hepatica. The study shows that this vaccine significantly reduces hepatic damage and encourages further studies to improve the vaccine efficacy.
Hanna, R E B; Moffett, D; Forster, F I; Trudgett, A G; Brennan, G P; Fairweather, I
The ultrastructure of the ovary of Fasciola hepatica collected from field-infected sheep, was compared with that of flukes from laboratory-infected rats harbouring the Oberon or the Cullompton fluke isolate. At the periphery of the ovarian tubules, in all flukes, interstitial tissue was identified that appears to provide physical support and facilitate the metabolism of the germinal-line cells. Oogonia undergo mitotic division to maintain the cell population and to produce oocytes. Early oocytes feature conspicuous synaptonemal complexes in the nucleoplasm, and these become less evident as the oocytes grow in size, move towards the core of the ovarian tubule, and synthesise osmiophilic bodies. The latter may represent cortical granules, and serve to block polyspermy. The identity of the synaptonemal complexes was confirmed by immunocytochemical labelling of synaptonemal proteins. The occurrence of synaptonemal complexes in the oocytes of all fluke types examined indicates that pairing of bivalent chromosomes, with the potential for genetic recombination and chiasmata formation, is a feature of the triploid aspermic parthenogenetic Cullompton flukes, as well as of the wild-type out-breeding field-derived and Oberon isolate flukes. In oocytes within shelled eggs in the proximal uterus of all flukes, condensed chromosomes align at meiotic metaphase plates. Following the reduction division, two equal pronuclei appear in each oocyte in the distal uterus. On the basis of these observations, a mechanism of facultative parthenogenesis for F. hepatica is proposed that accommodates the survival and clonal expansion of triploid aspermic isolates.
Chryssafidis, Andreas Lazaros; Fu, Yan; De Waal, Theo; Mulcahy, Grace
Fasciola hepatica and Calicophoron daubneyi, liver and rumen flukes respectively, infect ruminants throughout Europe. There is considerable interest in the development of vaccines and in testing new potential anthelmintic agents against these species. One potential target of new control measures is the parasite egg, as interference at this stage of the life cycle could aid in blocking the transmission of infection, and some experimental vaccines have been shown to affect egg viability. In this study, we describe the standardisation of protocols to evaluate the viability of eggs of these two parasites. Eggs were recovered from adult parasites collected in a commercial abattoir, from naturally infected cattle. A protocol for in vitro development of F. hepatica eggs was optimised based on previously published methods, with variations in duration and temperature of incubation. A new protocol for measurement of rumen fluke egg development in vitro was designed, based on testing different temperatures and periods of incubation, with or without light exposure. The protocols described here may be used in the future for comparing experimental groups when new technologies for parasite control are tested. In addition, the methods described for C. daubneyi present new information on the biology of this parasite.
Haydock, L A J; Pomroy, W E; Stevenson, M A; Lawrence, K E
Infections of ruminants with Fasciola hepatica are considered to be of regional importance within New Zealand but there is very little recent information on its prevalence or severity other than anecdotal reports. Generally they are considered to be of secondary importance compared to gastrointestinal nematode infections. Utilizing data from Virtual Climate Stations (n=11491) distributed on a 5km grid around New Zealand a growing degree-day model was used to describe the risk of infection with liver fluke from 1972 to 2012 and then to apply the predictions to estimate the risk of fluke infections within New Zealand for the years 2040 and 2090. The growing degree-day model was validated against the most recent survey of infection within New Zealand in 1984. A strong positive linear relationship for 1984 between F. hepatica prevalence in lambs and infection risk (p<0.001; R(2)=0.71) was found indicating the model was effective for New Zealand. A linear regression for risk values from 14 regions in New Zealand for 1972-2012 did not show any discernible change in risk of infection over this time period (p>0.05). Post-hoc comparisons indicate the risk in Westland was found to be substantially higher (p<0.05) than all other regions with Northland ranked second highest. Notable predicted changes in F. hepatica infection risk in 2040 and 2090 were detected although they did vary between different climate change scenarios. The highest average percentage changes in infection risk were found in regions with low initial risk values such as Canterbury and Otago; in these regions 2090 infection risk is expected to rise by an average of 186% and 184%, respectively. Despite the already high levels of infection risk in Westland, values are expected to rise by a further 76% by 2090. The model does show some areas with little change with Taranaki predicted to experience only very minor increases in infection risk with average 2040 and 2090 predicted changes of 0% and 29
French, Andrew S.; Zadoks, Ruth N.; Skuce, Philip J.; Mitchell, Gillian; Gordon-Gibbs, Danielle K.; Craine, Alexandra; Shaw, David; Gibb, Stuart W.; Taggart, Mark A.
Red deer (Cervus elaphus) are hosts of liver fluke (Fasciola hepatica); yet, prevalence is rarely quantified in wild populations. Testing fresh samples from remote regions by faecal examination (FE) can be logistically challenging; hence, we appraise frozen storage and the use of a coproantigen ELISA (cELISA) for F. hepatica surveillance. We also present cELISA surveillance data for red deer from the Highlands of Scotland. Diagnoses in faecal samples (207 frozen, 146 fresh) were compared using a cELISA and by FE. For each storage method (frozen or fresh), agreement between the two diagnostics was estimated at individual and population levels, where population prevalence was stratified into cohorts (e.g., by sampling location). To approximate sensitivity and specificity, 65 post-slaughter whole liver examinations were used as a reference. At the individual level, FE and cELISA diagnoses agreed moderately (κfrozen = 0.46; κfresh = 0.51), a likely reflection of their underlying principles. At the population level, FE and cELISA cohort prevalence correlated strongly (Pearson’s R = 0.89, p < 0.0001), reflecting good agreement on relative differences between cohort prevalence. In frozen samples, prevalence by cELISA exceeded FE overall (42.8% vs. 25.8%) and in 9/12 cohorts, alluding to differences in sensitivity; though, in fresh samples, no significant difference was found. In 959 deer tested by cELISA across the Scottish Highlands, infection prevalence ranged from 9.6% to 53% by sampling location. We highlight two key advantages of cELISA over FE: i) the ability to store samples long term (frozen) without apparent loss in diagnostic power; and ii) reduced labour and the ability to process large batches. Further evaluation of cELISA sensitivity in red deer, where a range of fluke burdens can be obtained, is desirable. In the interim, the cELISA is a practicable diagnostic for F. hepatica surveillance in red deer, and its application here has revealed considerable
Background In the present study we studied and updated the prevalence of the infections caused by gastrointestinal nematodes (GIN) and Fasciola hepatica in grazing sheep in the northwest (NW) of Spain for the last six years (2006–2011), and its relationship with the current climatic conditions. Methods We analyzed faecal samples from 110 flocks located in four different provinces of the Autonomous Community of Castilla y León: 76.4% of them were situated in León, 12.7% in Zamora, 9.1% in Palencia and 1.8% in Valladolid. Results The prevalence of GIN was 100% and the mean of eggs per gram (epg) in faeces was 237.2 (± 375.9) per flock. Regarding climatic conditions, we found a direct relationship between the GIN infection level and the maximum humidity (p<0.05) but inverse with the degree of solar radiation (p<0.05). The prevalence of fasciolosis was 59.3%, with a mean epg of 17.5 (± 33.9) per flock; these values were correlated with the minimum humidity and precipitations (p<0.05). Comparing our results in León with previous studies during the early 1990s, the mean epg of GIN was increased slightly (134.3 epg); regarding fasciolosis, the prevalence rose significantly, from 26.7% to 60.5%. Since the 1990s we observed that the maximum temperature is nowadays 0.45°C higher (17.0°C) and the minimum 0.5°C lower (5.2°C); the rainfall values were very similar in both decades but at the present time the humidity is higher (75.9%). Conclusions We found that the prevalence of GIN and F. hepatica infections was directly influenced by the humidity and also by precipitations in the case of F. hepatica. Comparing the current prevalence with studies carried out in the same area for the early 1990s, we observed that nowadays the mean epg of GIN is higher with a possible cause being the differences in climatic conditions depending on the sampling year. Regarding F. hepatica infection, its prevalence rose significantly probably favoured by an increase in irrigated areas in
Brockwell, Y M; Spithill, T W; Anderson, G R; Grillo, V; Sangster, N C
Three methods of diagnosing Fasciola hepatica (F. hepatica) infection (a coproantigen ELISA, Bio-X Diagnostics, Belgium, Faecal Egg Count (FEC), and a serum IgG ELISA,Bio-X Diagnostics, Belgium) were evaluated in artificially infected cattle, with and without drug treatment. Specifically, the potential value of the coproantigen ELISA in the quantitation of F. hepatica infection was sought. Twelve steers were each infected with 100, 200 or 500 metacercariae (n=4 cattle/group). On day 84, post infection (PI), 2 animals from each group were treated orally with triclabendazole (TCBZ). Faecal and blood samples were collected weekly after infection from all animals, as well as over 5 consecutive days (days 105-109 PI) for the six animals remaining infected to determine the repeatability of these assays. Cattle were killed 126 days PI and the coproantigen, FEC and IgG levels were compared with the number of fluke recovered. Animals first tested positive for infection with the serum ELISA, with 11/12 animals positive on day 28, and IgG responses increased to day 42 PI. The coproantigen ELISA was first positive on day 42 (3/12 animals), with all animals positive by day 56 PI. The first F. hepatica egg was detected on day 49 from an animal infected with 500 metacercariae; however only on one occasion (day 84) did all animals return positive FEC. Within one week of treatment with TCBZ, all six treated animals had returned to negative status by coproantigen ELISA and FEC whereas IgG levels persisted. Weekly variation in both coproantigen level and FEC was evident throughout the trial. Results from the consecutive daily collections varied greatly between days for both methods, with 2-6-fold differences in coproantigen levels and 2-4-fold variation in FEC. Strong correlations were observed between fluke burdens (day 126) and day 125 coproantigen levels (R(2)=0.8718) and FEC (R(2)=0.8368). The coproantigen ELISA was more sensitive than FEC (FEC displayed false negatives) and
Canevari, J; Ceballos, L; Sanabria, R; Romero, J; Olaechea, F; Ortiz, P; Cabrera, M; Gayo, V; Fairweather, I; Lanusse, C; Alvarez, L
The main goal of the current work was to develop and validate an in vitro fluke egg hatch test, as a method for the detection of albendazole (ABZ) resistance in the liver fluke, Fasciola hepatica. Fluke eggs (200/ml, n= 5) from six different isolates were used in the current experimental work. They were obtained from different geographical locations and named Cullompton (UK), CEDIVE (Chascomus, Argentina), INTA-Bariloche (Bariloche, Argentina), Rubino (Uruguay), Cajamarca (Perú) and Río Chico (Catamarca, Argentina). The fluke eggs were incubated (25 °C) for a 12-h period in the presence of either ABZ or its sulphoxide metabolite (ABZ.SO) (5, 0.5 or 0.05 nmol/ml). Untreated eggs were incubated as a control. Incubated eggs (with or without drug present) were kept in darkness at 25 °C for 15 days. Afterwards, the trematode eggs were exposed to daylight over a 2-h period. Hatched and unhatched eggs were evaluated using an optical microscope, and the ovicidal activity was assessed for each fluke isolate. A very low ovicidal activity ( ≤ 13.4%) was observed in the ABZ-resistant CEDIVE isolate for both ABZ and ABZ.SO. Conversely, in the INTA-Bariloche and Río Chico isolates, which are suspected to be susceptible to ABZ, ovicidal activities ≥ 70.3% were observed after incubation with ABZ at the lowest concentration tested (0.05 nmol/ml). This finding correlates with that previously described for the ABZ-susceptible Cullompton. Finally, the Cajamarca and Rubino isolates behaved as ABZ resistant, since no ovicidal activity was observed after eggs were incubated with ABZ at 0.5 nmol/ml. Considering the specific results obtained for each isolate under assessment, the egg hatch test described here may be a suitable method for detection of ABZ resistance in F. hepatica.
Scarcella, S; Hanna, R E B; Brennan, G P; Solana, H; Fairweather, I
Lambs infected with the Cullompton isolate of Fasciola hepatica were treated orally or subcutaneously with 10mg/kg of closantel at 16 weeks post-infection. Adult flukes were recovered from the liver of individual animals at 12h, 24h, or 36h post-treatment. The flukes were processed for histological analysis. In general, degenerative changes in the reproductive and somatic tissues were progressive, and were most marked in flukes exposed to closantel in vivo for 36h. However, flukes from a 12h subcutaneously-treated lamb showed marked deterioration of the testis, possibly because a portion of the dose has been delivered intravenously. Fewer intact eggs were seen in the uterus of flukes exposed to closantel for longer times (whether administered subcutaneously or orally to the host). The most conspicuous closantel-induced effect in flukes from treated hosts was progressive damage to the tegumental syncytium. While the flukes from 24h-treated hosts showed relatively minor damage to limited areas of the syncytium, towards the posterior end, the flukes from 36h-treated hosts (and flukes from the lamb that putatively received intravenous dosage) had lost large areas of the surface syncytium from the posterior end and dorsal surface, although the syncytium over the anterior end and the anterior ventral surface was largely spared. In areas where the syncytium had sloughed, the underlying structures such as the vitelline follicles, gut profiles and testis profiles, showed marked degeneration and breakdown. Other changes included cell depletion and early stage apoptosis in the testis, ovary and vitelline follicles. This study establishes a model for histological changes in closantel-sensitive F. hepatica exposed to closantel in vivo. Histopathological studies could be complementary to the efficacy controlled test for for closantel resistance in fluke populations.
Vignoles, P; Titi, A; Mekroud, A; Rondelaud, D; Dreyfuss, G
A retrospective study on different Lymnaea glabra samples collected from central France between 1993 and 2010 was carried out to determine the prevalence of natural co-infections with Calicophoron daubneyi and Fasciola hepatica, and to specify the composition of redial burdens. Experimental infections of L. glabra performed during the same period of time were also analysed to study metacercarial production of each digenean in co-infected snails. Controls were naturally or experimentally co-infected Galba truncatula. In natural co-infections, prevalence was 0.7% in L. glabra (186/25,128) and 0.4% in G. truncatula (137/31,345). Low redial burdens were found in these snails, with F. hepatica rediae significantly more numerous in L. glabra than in G. truncatula (7.5 per snail instead of 5.2). In contrast, the total numbers of C. daubneyi rediae in both lymnaeids were close to each other (4.3 and 3.0 rediae, respectively). In experimentally co-infected groups, prevalence was greater in G. truncatula than in the other lymnaeid (6.3% instead of 3.0%). Significantly shorter patent periods and lower metacercarial production for each digenean were noted in L. glabra than in G. truncatula. However, in both lymnaeids, the two types of cercariae were released during the same shedding waves and several peaks during the patent period were synchronous. In spite of a greater shell height for L. glabra, metacercarial production of both digeneans in co-infected snails was lower than that in G. truncatula, thus indicating a still incomplete adaptation between these French L. glabra and both parasites.
Fontenla, Santiago; Dell'Oca, Nicolás; Smircich, Pablo; Tort, José F; Siles-Lucas, Mar
The liver fluke Fasciola hepatica is a foodborne zoonotic parasite affecting livestock worldwide, with increasing relevance in human health. The first developmental stage that the host meets after ingestion of the parasite is the newly excysted juvenile, that actively transverses the gut wall and migrates to its final location in the liver. The regulation of the early developmental events in newly excysted juveniles is still poorly understood and a relevant target for control strategies. Here we investigated the putative involvement of small regulatory RNAs in the invasion process. The small RNA population of the newly excysted juvenile fall into two classes, one represented by micro (mi)RNAs and a secondary group of larger (32-33 nucleotides) tRNA-derived sequences. We identified 40 different miRNAs, most of those belonging to ancient miRNAs conserved in protostomes and metazoans, notably with a highly predominant miR-125b variant. Remarkably, several protostomian and metazoan conserved families were not detected in consonance with previous reports of drastic miRnome reduction in parasitic flatworms. Additionally, a set of five novel miRNAs was identified, probably associated with specific gene regulation expression needs in F. hepatica. While sequence conservation in mature miRNA is high across the metazoan tree, we observed that flatworm miRNAs are more divergent, suggesting that mutation rates in parasitic flatworms could be high. Finally, the distinctive presence of tRNA-derived sequences, mostly 5' tRNA halves of selected tRNAs in the small RNA population of newly excysted juveniles, raises the possibility that both miRNA and tRNA fragments participate in the regulation of gene expression in this parasite.
Rondelaud, D; Hourdin, P; Vignoles, P; Dreyfuss, G
As most natural watercress beds in central France are located upstream of the permanent habitats of two lymnaeid species, Galba truncatula and Omphiscola glabra, field investigations were made from 1999 to 2004 on 67 beds to determine why the contamination of watercress with Fasciola hepatica is irregular over time in these sites, while definitive hosts, especially lagomorphs, are regularly found infected around them. Snails are able to migrate upstream in winter and spring towards the beds, and a 4-year survey demonstrated the existence of annual variation in the colonization of these sites by snails. In the 45 beds irregularly contaminated with F. hepatica over time, 37.7-62.2%, according to the year, were not populated by lymnaeids, in spite of the presence of snail populations downstream. G. truncatula was found alone in 8.8-13.3% of sites and was the first colonizing snail in 24.3-33.3% when the two lymnaeid species successively settled in these waterholes. The colonizing ability of O. glabra was more limited, as it was observed alone in 2.2% of beds and was the first colonizing snail in only 2.2-20% of them. The distances covered by these snails significantly increased with increasing migration time. After migration, a few overwintering snails (3.8% of G. truncatula and 6.8% of O. glabra) are able to colonize the beds, but their numbers decreased when the distance of migration was greater. O. glabra migrated more quickly and reached more watercress beds than G. truncatula. However, this did not influence the natural infections of snails, which were more frequent in the F1 of G. truncatula. The variability observed in the colonization of beds by snails might explain the regular or irregular contamination of wild watercress by metacercariae of F. hepatica. One of the factors which may explain this variability is the occurrence of showers in spring, so that a few snails are able to reach watercress beds after their upstream migration.
Ravidà, Alessandra; Aldridge, Allison M.; Driessen, Nicole N.; Heus, Ferry A. H.; Hokke, Cornelis H.; O’Neill, Sandra M.
Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg. PMID:27104959
DNA multigene characterization of Fasciola hepatica and Lymnaea neotropica and its fascioliasis transmission capacity in Uruguay, with historical correlation, human report review and infection risk analysis
Gayo, Valeria; Sanchis, Jaime; Artigas, Patricio; Khoubbane, Messaoud; Birriel, Soledad; Mas-Coma, Santiago
Background Fascioliasis is a pathogenic disease transmitted by lymnaeid snails and recently emerging in humans, in part due to effects of climate changes, anthropogenic environment modifications, import/export and movements of livestock. South America is the continent presenting more human fascioliasis hyperendemic areas and the highest prevalences and intensities known. These scenarios appear mainly linked to altitude areas in Andean countries, whereas lowland areas of non-Andean countries, such as Uruguay, only show sporadic human cases or outbreaks. A study including DNA marker sequencing of fasciolids and lymnaeids, an experimental study of the life cycle in Uruguay, and a review of human fascioliasis in Uruguay, are performed. Methodology/Principal findings The characterization of Fasciola hepatica from cattle and horses of Uruguay included the complete sequences of the ribosomal DNA ITS-2 and ITS-1 and mitochondrial DNA cox1 and nad1. ITS-2, ITS-1, partial cox1 and rDNA 16S gene of mtDNA were used for lymnaeids. Results indicated that vectors belong to Lymnaea neotropica instead of to Lymnaea viator, as always reported from Uruguay. The life cycle and transmission features of F. hepatica by L. neotropica of Uruguay were studied under standardized experimental conditions to enable a comparison with the transmission capacity of F. hepatica by Galba truncatula at very high altitude in Bolivia. On this baseline, we reviewed the 95 human fascioliasis cases reported in Uruguay and analyzed the risk of human infection in front of future climate change estimations. Conclusions/Significance The correlation of fasciolid and lymnaeid haplotypes with historical data on the introduction and spread of livestock into Uruguay allowed to understand the molecular diversity detected. Although Uruguayan L. neotropica is a highly efficient vector, its transmission capacity is markedly lower than that of Bolivian G. truncatula. This allows to understand the transmission and
Gottstein, Bruno; Schneeberger, Marianne; Boubaker, Ghalia; Merkle, Bernadette; Huber, Cristina; Spiliotis, Markus; Müller, Norbert; Garate, Teresa; Doherr, Marcus G.
Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory–secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n = 20), patients with other parasitic infections (n = 87) and patients with malignancies (n = 121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls. PMID:24922050
Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings.
Baldissera, Matheus D; Bottari, Nathieli B; Mendes, Ricardo E; Schwertz, Claiton I; Lucca, Neuber J; Dalenogare, Diessica; Bochi, Guilherme V; Moresco, Rafael N; Morsch, Vera M; Schetinger, Maria R C; Rech, Virginia C; Jaques, Jeandre A; Da Silva, Aleksandro S
The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease.
D'Almeida, S C G; Freitas, D F; Carneiro, M B; Camargo, P F; Azevedo, J C; Martins, I V F
The aim of this study was to monitor the population density of Lymnaea columella, an intermediate host of Fasciola hepatica, in various aquatic habitats and in drinking water in the area of the Instituto Federal de Educação, Ciência e Tecnologia do Espírito Santo, on Caparaó Microregion, municipality of Alegre, state of Espírito Santo, Brazil. Monthly samplings were performed at certain points between drainage areas and drinking water in cattle and goat production systems during the years 2010 to 2013. The mean temperature, precipitation and the frequency of samples of L. columella were analysed graphically according the monthly average during the study period. A total of 2,038 molluscs were collected, 1558 of which were L. columella, that predominated in all sampled points. The highest average of specimens observed for L. columella was in the years 2010 and 2013 (51.0), and occurred decreased in 2011 (19.8). The temperature and precipitation averaged is 23.7 °C and 141 mm/year, respectively. Rainfall peak occurred in March (2011, 2013) and November (2012), during these periods the population of L. columella growth. There was no significant difference in the relationship between the specimens observed with seasons (dry-wet), thus the population of L. columella remained stable and can be found throughout the year.
Pérez, J; Martín de las Mulas, J; Carrasco, L; Gutierrez, P N; Martínez-Cruz, M S; Martínez-Moreno, A
Lesions produced by Fasciola hepatica in the liver, gall-bladder and hepatic lymph nodes (HLNs) of four groups of five goats are described; in addition, the distribution of CD3+ T lymphocytes and IgG-lambda light chain-bearing cells was analysed in the hepatic lesions and HLNs. One group of goats received a single oral dose of metacercariae, but the other four groups received four or five doses at different intervals over a period of 11 weeks. Animals that survived were killed 53-55 weeks after the first infective dose. Goats were more susceptible to multiple doses than to a single dose, even when the total number of metacercariae was the same. This greater susceptibility was manifested by the occurrence of deaths and the severity of hepatic lesions. CD3+ lymphocytes were sparse in the infiltrate surrounding the acute migratory tracts, suggesting inhibition of the local cell-mediated immune response. Goats with numerous hepatic calcareous granulomas showed the most severe hepatic damage, including marked cirrhosis, with a striking infiltrate of CD3+ T lymphocytes and lambda IgG- plasma cells, replacing extensive areas of hepatic parenchyma, in which hypertrophy of the smooth endoplasmic reticulum of hepatocytes was evident. These findings were observed mainly in the goats given more than one infective dose.
Escamilla, A; Zafra, R; Pérez, J; McNeilly, T N; Pacheco, I L; Buffoni, L; Martínez-Moreno, F J; Molina-Hernández, V; Martínez-Moreno, A
Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3(+) Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3(+) Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3(+) and CD3(+) T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3(+) and CD3(+) T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3(+) Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3(+) Tregs in acute and
Martínez-Sernández, Victoria; Mezo, Mercedes; González-Warleta, Marta; Perteguer, María J.; Muiño, Laura; Guitián, Esteban; Gárate, Teresa; Ubeira, Florencio M.
Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10−6 m−1. We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes. PMID:24280214
Bargues, M D; Artigas, P; Mera Y Sierra, R L; Pointier, J P; Mas-Coma, S
Although, in the endemic areas throughout the world, human fascioliasis presents varying patterns in its epidemiology, the species of lymnaeid snail that act as intermediate hosts and vectors are always crucial in the transmission of the causative parasites. Species in the Galba/Fossaria group of snails, such as Lymnaea cubensis, L. viatrix var. A ventricosa, L. viatrix var. B elongata and Galba truncatula, appear to be frequently involved in the transmission of Fasciola hepatica in Central and South America, although specific classification within this morphologically and anatomically confusing group is often very difficult. To explore the potential use of molecular analyses in the identification of vector snails, regions of the ribosomal DNA - the small subunit (18S) gene and internal transcribed spacers (ITS-2 and ITS-1) - and of the mitochondrial DNA - the cytochrome c oxidase subunit I (COI) - of wild-caught lymnaeid snails of L. cubensis, L. viatrix var. A ventricosa, L. viatrix var. B elongata and G. truncatula have been sequenced. The samples of the Latin American species included specimens from the respective type localities. The genetic distances observed and the results of phylogenetic analyses demonstrate that two different species exist within L. viatrix. Lymnaea neotropica n. sp. (=L. viatrix var. B elongata) is here proposed for specimens from Lima, Peru, and is differentiated from L. viatrix (=L. viatrix var. A ventricosa), L. cubensis and G. truncatula. The data collected on the 18S ribosomal-RNA gene indicate that the snails investigated may cover more than one supraspecific taxon. The ITS-2, ITS-1 and COI nucleotide sequences are clearly useful markers for the differentiation of these morpho-anatomically similar lymnaeid species. The numerous microsatellite repeats found within ITS-2 are potential tools for differentiation at population level.
Martin, Ivelisse; Cabán-Hernández, Kimberly; Figueroa-Santiago, Olgary; Espino, Ana M
TLR4, the innate immunity receptor for bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. There is a need to develop molecules that block either activation through TLR4 or the downstream signaling pathways to inhibit the storm of inflammation typically elicited by bacterial LPS, which is a major cause of the high mortality associated with bacterial sepsis. We report in this article that a single i.p. injection of 15 μg fatty acid binding protein from Fasciola hepatica (Fh12) 1 h before exposure to LPS suppressed significantly the expression of serum inflammatory cytokines in a model of septic shock using C57BL/6 mice. Because macrophages are a good source of IL-12p70 and TNF-α, and are critical in driving adaptive immunity, we investigated the effect of Fh12 on the function of mouse bone marrow-derived macrophages (bmMΦs). Although Fh12 alone did not induce cytokine expression, it significantly suppressed the expression of IL-12, TNF-α, IL-6, and IL-1β cytokines, as well as inducible NO synthase-2 in bmMΦs, and also impaired the phagocytic capacity of bmMΦs. Fh12 had a limited effect on the expression of inflammatory cytokines induced in response to other TLR ligands. One mechanism used by Fh12 to exert its anti-inflammatory effect is binding to the CD14 coreceptor. Moreover, it suppresses phosphorylation of ERK, p38, and JNK. The potent anti-inflammatory properties of Fh12 demonstrated in this study open doors to further studies directed at exploring the potential of this molecule as a new class of drug against septic shock or other inflammatory diseases.
Martin, Ivelisse; Cabán-Hernández, Kimberly; Figueroa-Santiago, Olgary; Espino, Ana M.
Toll-like receptor 4 (TLR4), the innate immunity receptor for bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. There is a need to develop molecules that block either activation through TLR4 or the downstream signaling pathways to inhibit the storm of inflammation typically elicited by bacterial lipopolysaccharide (LPS), which is a major cause of the high mortality associated with bacterial sepsis. We report here that a single intraperitoneal injection of 15μg Fasciola hepatica fatty acid binding protein (Fh12) 1 hour before exposure to LPS suppressed significantly the expression of serum inflammatory cytokines in a model of septic shock using C57BL/6 mice. Because macrophages are good source of IL12p70 and TNFα, and critical in driving adaptive immunity, we investigated the effect of Fh12 on the function of mouse bone marrow derived macrophages (bmMΦs). Whereas Fh12 alone did not induce cytokine expression, it significantly suppressed the expression of IL12, TNFα, IL6 and IL1β cytokines as well as iNOS2 in bmMΦs, and also impaired the phagocytic capacity of bmMΦs. Fh12 had a limited effect on the expression of inflammatory cytokines induced in response to other TLR-ligands. One mechanism used by Fh12 to exert its anti-inflammatory effect is binding to the CD14 co-receptor. Moreover, it suppresses phosphorylation of ERK, p38 and JNK. The potent anti-inflammatory properties of Fh12 demonstrated here open doors to further studies directed at exploring the potential of this molecule as a new class of drug against septic shock or other inflammatory diseases. PMID:25780044
Perez-Crespo, Ignacio; Chillón-Marinas, Carlos; Khoubbane, Messaoud; Quesada, Carla; Reguera-Gomez, Marta; Mas-Coma, Santiago; Fresno, Manuel; Gironès, Núria
Background Fascioliasis is a severe zoonotic disease of worldwide extension caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm and anemia is the main sign. Herein, the profile of the Th1/Th2/Th17/Treg expression levels is analyzed after reinfection, correlating them with their corresponding hematological biomarkers of morbidity. Methodology/Principal findings The experimental design reproduces the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 8 weeks (R8), and at 12 weeks (R12), and negative control rats. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. After 20 weeks of primary infection, PI did not present significant changes in the expression of those genes when compared to non-infected rats (NI), but an increase of Il4, Arg1 and Ifng mRNA in the spleen was observed in R12, suggesting the existence of an active mixed Th1/Th2 systemic immune response in reinfection. Foxp3, Il10, Tgfb and Ebi3 levels increased in the spleen in R12 when compared to NI and PI, indicating that the Treg gene expression levels are potentiated in chronic phase reinfection. Il17 gene expression levels in R12 in the spleen increased when compared to NI, PI and R8. Gene expression levels of Il10 in the thymus increased when compared to NI and PI in R12. Ifng expression levels in the thymus increased in all reinfected rats, but not in PI. The clinical phenotype was determined by the fluke burden, the rat body weight and the hemogram. Multivariate mathematical models were built to describe the Th1/Th2/Th17/Treg expression levels and the clinical phenotype. In reinfection, two phenotypic patterns were detected: i) one which includes only increased splenic Ifng
Rojas-Caraballo, Jose; López-Abán, Julio; Pérez del Villar, Luis; Vizcaíno, Carolina; Vicente, Belén; Fernández-Soto, Pedro; del Olmo, Esther; Patarroyo, Manuel Alfonso; Muro, Antonio
Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L+ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.
Evidence for high genetic diversity of NAD1 and COX1 mitochondrial haplotypes among triclabendazole resistant and susceptible populations and field isolates of Fasciola hepatica (liver fluke) in Australia.
Elliott, T; Muller, A; Brockwell, Y; Murphy, N; Grillo, V; Toet, H M; Anderson, G; Sangster, N; Spithill, T W
In recent years, the global incidence of Fasciola hepatica (liver fluke) infections exhibiting resistance to triclabendazole (TCBZ) has increased, resulting in increased economic losses for livestock producers and threatening future control. The development of TCBZ resistance and the worldwide discovery of F. hepatica population diversity has emphasized the need to further understand the genetic structure of drug susceptible and resistant Fasciola populations within Australia. In this study, the genetic diversity of liver flukes was estimated by sequencing mitochondrial DNA (mtDNA) encoding the NAD1 (530 bp) and COX1 (420 bp) genes of 208 liver flukes (F. hepatica) collected from three populations: field isolates obtained from abattoirs from New South Wales (NSW) and Victoria (Vic); three TCBZ-resistant fluke populations from NSW and Victoria; and the well-established TCBZ-susceptible Sunny Corner laboratory isolate. Overall nucleotide diversity for all flukes analysed of 0.00516 and 0.00336 was estimated for the NAD1 and COX1 genes respectively. Eighteen distinct haplotypes were established for the NAD1 gene and six haplotypes for the COX1 gene, resulting in haplotype diversity levels of 0.832 and 0.482, respectively. One field isolate showed a similar low level of haplotype diversity as seen in the Sunny Corner laboratory isolate. Analysis of TCBZ-resistant infrapopulations from 3 individual cattle grazing one property revealed considerable sequence parasite diversity between cattle. Analysis of parasite TCBZ-resistant infrapopulations from sheep and cattle revealed haplotypes unique to each host, but no significant difference between parasite populations. Fst analysis of fluke populations revealed little differentiation between the resistant and field populations. This study has revealed a high level of diversity in field and drug resistant flukes in South-Eastern Australia.
Rojas-Caraballo, Jose; López-Abán, Julio; Pérez del Villar, Luis; Vizcaíno, Carolina; Vicente, Belén; Fernández-Soto, Pedro; del Olmo, Esther; Patarroyo, Manuel Alfonso; Muro, Antonio
Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L+ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance. PMID:25122166
Fasciola hepatica: Specificity of a coproantigen ELISA test for diagnosis of fasciolosis in faecal samples from cattle and sheep concurrently infected with gastrointestinal nematodes, coccidians and/or rumen flukes (paramphistomes), under field conditions.
Kajugu, P-E; Hanna, R E B; Edgar, H W; McMahon, C; Cooper, M; Gordon, A; Barley, J P; Malone, F E; Brennan, G P; Fairweather, I
Chronic fasciolosis is often diagnosed by faecal egg counting (FEC), following concentration of the eggs in the sample by a zinc sulphate floatation method. However, concentration by a sedimentation technique gives improved sensitivity. Interpretation of FEC results for fasciolosis is complicated by factors such as the long pre-patent period and irregular egg shedding. Thus, FEC reduction tests (FECRT), when used alone, are not completely reliable for diagnosis of anthelmintic susceptibility or resistance in local fluke populations, especially when parasite burdens are small. A Fasciola hepatica coproantigen ELISA test has been introduced which more accurately reflects the presence of flukes in the host bile ducts in late pre-patent infections, and absence of flukes following successful chemotherapeutic intervention. The aim of the present study was to elucidate the specificity of the F. hepatica coproantigen ELISA technique, particularly regarding potential cross-reactivity with rumen fluke (paramphistome), gastrointestinal nematode and coccidian infections. The method involved parallel testing of a large battery of faecal samples from field-infected cattle and sheep using floatation and sedimentation FECs and coproantigen analysis. No evidence was found for significant false positivity in the F. hepatica coproantigen ELISA due to paramphistome, coccidian and/or gastrointestinal nematode co-infections. With sedimentation FECs less than 10 F. hepatica eggs per gram (epg), the likelihood of a positive coproantigen result for the sample progressively decreased. Diagnosis of fasciolosis should be based on consideration of both FEC and coproantigen ELISA findings, to ensure optimum sensitivity for pre-patent and low-level infections.
Fromm, B; Trelis, M; Hackenberg, M; Cantalapiedra, F; Bernal, D; Marcilla, A
MicroRNAs (miRNAs) are gene regulators that have recently been shown to down-regulate the immune response via extracellular vesicles in the mammalian host of helminthic parasites. Using the miRNA prediction pipeline miRCandRef, we expanded the current miRNA set of the liver fluke Fasciola hepatica (Platyhelminthes, Trematoda) from 16 to 54 miRNAs (42 conserved and 13 novel). Comparing the cellular expression levels with extracellular vesicles, we found all miRNAs expressed and enriched for miRNAs with immuno-regulatory function, tissue growth and cancer. Our findings support the hypothesis that miRNAs are the molecular mediators of the previously demonstrated immune modulatory function of extracellular vesicles.
Guasconi, Lorena; Chiapello, Laura S; Masih, Diana T
Fasciola hepatica excretory-secretory products (FhESP) induce immunomodulatory effects on macrophages. Previously, we demonstrated that these effects are dependent on Dectin-1. Therefore, the aim of this study was to determine how this affects the CD4 T-cells immune response. We observed that FhESP induce an increased expression of PD-L2 in macrophages via Dectin-1. Furthermore, in co-cultures with CD4 T-cell we observed a suppressive effect on proliferative response, down-modulation of IFN-γ and up-modulation of IL-10 via Dectin-1 on macrophages. These results suggest that FhESP induce T-cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way.
Ichikawa, Madoka; Itagaki, Tadashi
Molecular characterization is important for discriminating Fasciola specimens having the deoxyribonucleic acid (DNA) sequences of Fasciola hepatica, Fasciola gigantica, and both Fasciola species, since three Fasciola forms coexist in Asian countries. We have developed a restriction fragment length polymorphism of amplified DNA (PCR-RFLP) of the nuclear ribosomal internal transcribed spacer 1 (ITS1) region in Fasciola species. The band patterns of the fragments digested with a restriction enzyme, Rsa I, were accurately distinguished among the three forms of Fasciola. Amplicons with the sequences of F. hepatica and F. gigantica were divided into fragments of about 360, 100, and 60 bp, and 360, 170, and 60 bp, respectively, and amplicons with the sequences of both Fasciola species yielded fragments of 360, 170, 100, and 60 bp. The results of PCR-RFLP completely coincided with those of sequence analysis, and thus PCR-RFLP is a useful technique for determining the ITS1 type in Fasciola species.
Munita, M P; Rea, R; Bloemhoff, Y; Byrne, N; Martinez-Ibeas, A M; Sayers, R G
Completion of the F. hepatica lifecycle is dependent on suitable climatic conditions for development of immature stages of the parasite, and its snail intermediate host. Few investigations have been conducted regarding temporal variations in F. hepatica status in Irish dairy herds. The current study aimed to conduct a longitudinal study examining annual and seasonal trends in bulk milk seropositivity over six years, while also investigating associations with soil temperature, rainfall and flukicide treatment. Monthly bulk milk samples (BTM) were submitted by 28 herds between March 2009 and December 2014. In all, 1337 samples were analysed using a Cathepsin L1 ELISA. Soil temperature, rainfall and management data were obtained for general estimating equation and regression analyses. A general decrease in milk seropositivity was observed over the six year study period and was associated with an increased likelihood of treating for liver fluke (OR range=2.73-6.96). Annual and seasonal analyses of rainfall and F. hepatica BTM status yielded conflicting results. Higher annual rainfall (>1150mm) yielded a lower likelihood of being BTM positive than annual rainfall of <1000mm (OR=0.47; P=0.036). This was most likely due to farmers being more proactive in treating for F. hepatica in wetter years, although a 'wash effect' by high rainfall of the free living stages and snails cannot be ruled out. Higher seasonal rainfall (>120mm), however, was associated with increased ELISA S/P% values (Coefficient=9.63S/P%; P=0.001). Soil temperature was not found to influence F. hepatica to the same extent as rainfall and may reflect the lack of severe temperature fluctuations in Ireland. Flukicides active against both immature and mature F. hepatica were approximately half as likely to record a positive F. hepatica herd BTM status than a flukicide active against only the mature stage of the parasite (OR≅0.45; P<0.01). This study highlights the importance of examining both annual and
Fasciola hepatica: a comparative survey of adult fluke resistance to triclabendazole, nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and fluke histology.
Hanna, R E B; McMahon, C; Ellison, S; Edgar, H W; Kajugu, P-E; Gordon, A; Irwin, D; Barley, J P; Malone, F E; Brennan, G P; Fairweather, I
In order to investigate the incidence and distribution of adult fluke resistance to the fasciolicide tricalbendazole (TCBZ) amongst populations of Fasciola hepatica in sheep flocks in Northern Ireland (NI), individual rectal faeces samples were collected from 3 groups of 20 sheep, before (pre-dose), and 21 days after (post-dose) treatment of the animals with TCBZ, nitroxynil or closantel, on each of 13 well-managed sheep farms distributed across the province. The efficacy of each flukicide was determined for each farm, using faecal egg count reduction (FECRT) and F. hepatica coproantigen ELISA testing. In certain flocks, 2 sheep with high pre-dose faecal egg counts (FEC) were killed 3 days and 21 days respectively after TCBZ treatment, and the histology of the fluke reproductive organs was compared with that of flukes from untreated sheep, and from sheep treated with nitroxynil or closantel 2 days prior to death, using haematoxylin and eosin (H&E) staining and an in situ hybridisation method (TdT-mediated dUDP nick end labelling [TUNEL]) to demonstrate apoptosis. Results from FECRT revealed that in all flocks with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis, and this finding was generally supported by the results of the coproantigen reduction test (CRT). The histology of reproductive organs of flukes from TCBZ-treated sheep in these flocks was normal, when compared with untreated flukes, and this, together with the FECRT and CRT findings, indicated a likely diagnosis of TCBZ resistance in all the flocks with a high fluke burden. In contrast, nitroxynil and closantel were found to be fully effective against TCBZ-resistant flukes in each of the flocks bearing a high chronic fluke burden. All of the flocks with a high fluke burden and TCBZ resistance were managed on lowland in the South and East of NI. Upland flocks, in the North and West, had low fluke burdens, or were clear of infection; and FECs were too low to allow valid resistance
Fasciola hepatica: comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity.
Hanna, R E B; Gordon, A W; Moffett, D; Edgar, H W J; Oliver, L F; McConnell, S; Shaw, L; Brennan, G P; Fairweather, I
The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study, F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke
Shoriki, Takuya; Ichikawa-Seki, Madoka; Suganuma, Keisuke; Naito, Ikunori; Hayashi, Kei; Nakao, Minoru; Aita, Junya; Mohanta, Uday Kumar; Inoue, Noboru; Murakami, Kenji; Itagaki, Tadashi
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.
Hillyer, G V; Soler de Galanes, M; Buchón, P; Bjorland, J
A study was designed to determine by ELISA the seroprevalence of fasciolosis both in sheep (29 herds totaling 184 sheep), in samples collected in 1988, and in cattle (41 herds totaling 299 animals, samples collected in 1988; 34 herds totaling 147 animals, samples collected in 1989) in the same area of Corapata in which a seroprevalence survey had been done in humans. The results show high seropositivity in sheep (89%) and lower seropositivity in cattle (58% in 1988, and 57% in 1989). The seroprevalence in cattle in 1988 was essentially identical to that detected in 1989. Faecal examinations were also done in the 1988 sheep and 1989 cattle. Results of the study showed that of the 184 sheep examined, 22 were positive for F. hepatica eggs, while 163 were positive by serology. All of the 22 sheep which were positive parasitologically were also positive serologically for a sensitivity of 100%. On the other hand, of 147 cattle tested, 38 were positive parasitologically while 84 were positive serologically. Of the 38 positives for F. hepatica eggs, 31 were positive by serology (sensitivity 82%).
Sargison, Neil; Kelly, Robert F.; Bronsvoort, Barend M. deC.; Handel, Ian
The clinical and economic importance of fasciolosis has been recognised for centuries, yet diagnostic tests available for cattle are far from perfect. Test evaluation has mainly been carried out using gold standard approaches or under experimental settings, the limitations of which are well known. In this study, a Bayesian no gold standard approach was used to estimate the diagnostic sensitivity and specificity of five tests for fasciolosis in cattle. These included detailed liver necropsy including gall bladder egg count, faecal egg counting, a commercially available copro-antigen ELISA, an in-house serum excretory/secretory antibody ELISA and routine abattoir liver inspection. In total 619 cattle slaughtered at one of Scotland’s biggest abattoirs were sampled, during three sampling periods spanning summer 2013, winter 2014 and autumn 2014. Test sensitivities and specificities were estimated using an extension of the Hui Walter no gold standard model, where estimates were allowed to vary between seasons if tests were a priori believed to perform differently for any reason. The results of this analysis provide novel information on the performance of these tests in a naturally infected cattle population and at different times of the year where different levels of acute or chronic infection are expected. Accurate estimates of sensitivity and specificity will allow for routine abattoir liver inspection to be used as a tool for monitoring the epidemiology of F. hepatica as well as evaluating herd health planning. Furthermore, the results provide evidence to suggest that the copro-antigen ELISA does not cross-react with Calicophoron daubneyi rumen fluke parasites, while the serum antibody ELISA does. PMID:27564546
Choi, In-Wook; Kim, Hwang-Yong; Quan, Juan-Hua; Ryu, Jae-Gee; Sun, Rubing; Lee, Young-Ha
Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.
This review summarises the findings of a series of studies in which the histological changes, induced in the reproductive system of Fasciola hepatica following treatment of the ovine host with the anthelmintic triclabendazole (TCBZ), were examined. A detailed description of the normal macroscopic arrangement and histological features of the testes, ovary, vitelline tissue, Mehlis' gland and uterus is provided to aid recognition of the drug-induced lesions, and to provide a basic model to inform similar toxicological studies on F. hepatica in the future. The production of spermatozoa and egg components represents the main energy consuming activity of the adult fluke. Thus the reproductive organs, with their high turnover of cells and secretory products, are uniquely sensitive to metabolic inhibition and sub-cellular disorganisation induced by extraneous toxic compounds. The flukes chosen for study were derived from TCBZ-sensitive (TCBZ-S) and TCBZ-resistant (TCBZ-R) isolates, the status of which had previously been proven in controlled clinical trials. For comparison, flukes collected from flocks where TCBZ resistance had been diagnosed by coprological methods, and from a dairy farm with no history of TCBZ use, were also examined. The macroscopic arrangement of the reproductive system in flukes was studied using catechol/carmine stained whole mounts, and the histology of the main organs was examined using conventional haematoxylin-eosin stained sections. Validation of apoptosis in the fluke sections was carried out using an in situ hybridisation method designed to label endonuclease-induced DNA strand breaks. In TCBZ-S flukes exposed to TCBZ metabolites for 24-96 h in vivo, but not in TCBZ-R flukes, those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), were found to display progressive loss of cell content. This was due to apparent failure of cell division to keep pace with expulsion of the mature or effete products
This review summarises the findings of a series of studies in which the histological changes, induced in the reproductive system of Fasciola hepatica following treatment of the ovine host with the anthelmintic triclabendazole (TCBZ), were examined. A detailed description of the normal macroscopic arrangement and histological features of the testes, ovary, vitelline tissue, Mehlis’ gland and uterus is provided to aid recognition of the drug-induced lesions, and to provide a basic model to inform similar toxicological studies on F. hepatica in the future. The production of spermatozoa and egg components represents the main energy consuming activity of the adult fluke. Thus the reproductive organs, with their high turnover of cells and secretory products, are uniquely sensitive to metabolic inhibition and sub-cellular disorganisation induced by extraneous toxic compounds. The flukes chosen for study were derived from TCBZ-sensitive (TCBZ-S) and TCBZ-resistant (TCBZ-R) isolates, the status of which had previously been proven in controlled clinical trials. For comparison, flukes collected from flocks where TCBZ resistance had been diagnosed by coprological methods, and from a dairy farm with no history of TCBZ use, were also examined. The macroscopic arrangement of the reproductive system in flukes was studied using catechol/carmine stained whole mounts, and the histology of the main organs was examined using conventional haematoxylin-eosin stained sections. Validation of apoptosis in the fluke sections was carried out using an in situ hybridisation method designed to label endonuclease-induced DNA strand breaks. In TCBZ-S flukes exposed to TCBZ metabolites for 24–96 h in vivo, but not in TCBZ-R flukes, those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), were found to display progressive loss of cell content. This was due to apparent failure of cell division to keep pace with expulsion of the mature or effete products
YAKHCHALI, Mohammad; MALEKZADEH-VIAYEH, Reza; IMANI-BARAN, Abbas; MARDANI, Karim
Background: The trematodes of the genus Fasciola (the liver flukes) are among the well-known instances of food-borne parasites worldwide. Differentiation of Fasciola species is important because of their different transmission and epidemiological characteristics. The current study was undertaken to discriminate Fasciola species in the domestic ruminants of Urmia city, Iran. Methods: Adult flukes were isolated from the naturally infected livers of the slaughtered water buffaloes and sheep. The flukes were initially identified based on morphological and morphometric parameters. A 618-bp-long fragment of the 28SrRNA gene of Fasciola was amplified by polymerase chain reaction (PCR). The amplified fragment was digested by DraII or AvaII enzymes for a restriction fragment length polymorphism (RFLP) analysis and sequenced for the phylogenetic tree construction. Results: Based on the morphometric examination, the flukes belonged to F. hepatica, F. gigantica and an intermediate Fasciola form. The PCR-RFLP analysis was able to differentiate F. hepatica from F. gigantica. While the phylogenetic reconstruction justified, to some extent, the morphological diagnosis, it failed to segregate F. hepatica from F. gigantica identified in this and the previous studies. Conclusion: To resolve fully the problem of taxonomy and evolution in Fasciola species, employing a broad range of molecular and morphological approaches is necessary. This is crucial for epidemiological surveys and successful clinical management of their infection. PMID:25904945
Fasciola hepatica: histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks.
Hanna, R E B; Forster, F I; Brennan, G P; Fairweather, I
Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1-4days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the
Ai, L; Dong, S J; Zhang, W Y; Elsheikha, H M; Mahmmod, Y S; Lin, R Q; Yuan, Z G; Shi, Y L; Huang, W Y; Zhu, X Q
Among the helminths infecting ruminants in China are three taxa belonging to the genus Fasciola: F. hepatica, F. gigantica and the so-called 'intermediate form' that appears to lie between these two species. Based on the sequences of the second internal-transcribed spacers (ITS-2) within the parasites' nuclear ribosomal DNA (rDNA), a pair of primers (DSJf/DSJ3) specific for F. hepatica and a pair (DSJf/DSJ4) specific for F. gigantica were designed and used to develop PCR-based assays. These assays allowed the identification and differentiation of F. hepatica, F. gigantica and the 'intermediate' Fasciola, with no amplicons produced from heterologous DNA samples. The results of sequencing confirmed the species-specific identity of the amplified products. The assays showed good sensitivity, giving positive results with as little as 0.11 ng of F. hepatica DNA and 0.35 ng of F. gigantica DNA. This meant that the DNA from a single Fasciola egg or a single infected snail was sufficient for identification of the Fasciola taxon. The developed PCR assays could provide useful tools for the detection, identification and epidemiological investigation of Fasciola infection in humans, other mammals and snails.
Wannasan, Anchalee; Khositharattanakool, Pathamet; Chaiwong, Prasong; Piangjai, Somsak; Uparanukraw, Pichart; Morakote, Nimit
Molecular techniques were used to identify Fasciola species collected from Chiang Mai Thailand. Morphometrically, 65 stained and 45 fresh worms collected from cattle suggested the possible occurrence of both F. gigantica and F. hepatica. Twenty-two worms comprising 15 from cattle and 7 from human patients, were identified subsequently based on three genetic markers: mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2). All of them presented the F. gigantica type in maternally inherited mitochondrial sequences (nad1 and cox1), with six types in each sequence (FgNDI-CM1 to FgNDI-CM6 and FgCOI-CM1 to FgCOI-CM6, respectively). Remarkably, the predominant nad1 type, FgNDI-CM6, was identical to that of aspermic Fasciola sp. formerly reported from Thailand, Japan, Korea, China, Vietnam, and Myanmar. ITS2 sequences were analyzed successfully in 20 worms. Fifteen worms showed the F. gigantica type and five (including one worm from a patient) had mixed ITS2 sequences of both F. gigantica and F. hepatica in the same worms, with additional heterogeneity within both ITS2 types. This study revealed the intermediate form of Fasciola coexisting with F. gigantica for the first time in Thailand.
BOZORGOMID, Arezoo; NAZARI, Naser; RAHIMI, Hamzeh; BEIGOM KIA, Eshrat; HAJJARAN, Homa; MOHEBALI, Mehdi; ARYAEIPOUR, Mojgan; HEIDARIAN, Peyman; EZATI, Mohammad Saeid; ROKNI, Mohamad Bagher
Background: We evaluated the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Kermanshah Province, western Iran using PCR-RFLP method. Methods: We used PCR-RFLP analysis of ribosomal ITS1 fragment using RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (16 sheep, 28 cattle, 4 goats). The species of Fasciola were confirmed by sequencing the 700 bp region of ribosomal ITS1 gene. Results: In Kermanshah, F. hepatica was present in 96% of the samples, F. gigantica was found only in two cattle sample. No hybrid forms were detected in the present study. Conclusion: Our results contribute to clarify the dark spots of Fasciola genotyping in different parts of Iran. PMID:27957438
Meemon, Krai; Sobhon, Prasert
Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is one of the most neglected tropical zoonotic diseases. One sustainable control strategy against these infections is the employment of vaccines that target proteins essential for parasites' invasion and nutrition acquiring processes. Cathepsin proteases are the most abundantly expressed proteins in Fasciola spp. that have been tested successfully as vaccines against fasciolosis in experimental as well as large animals because of their important roles in digestion of nutrients, invasion, and migration. Specifically, juvenile-specific cathepsin proteases are the more effective vaccines because they could block the invasion and migration of juvenile parasites whose immune evasion mechanism has not yet been fully developed. Moreover, because of high sequence similarity and identity of cathepsins from juveniles with those of adults, the vaccines can attack both the juvenile and adult stages. In this article, the characteristics and vaccine potentials of juvenile-specific cathepsins, i.e., cathepsins L and B, of Fasciola spp. were reviewed.
Huang, W Y; He, B; Wang, C R; Zhu, X Q
Isolates of Fasciola (Platyhelminthes: Trematoda: Digenea) from different host species and geographical locations in Mainland China were characterised genetically. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified from individual trematodes by polymerase chain reaction (PCR), and the representative amplicons were cloned and sequenced. The length of the ITS-2 sequences was 361-362bp for all Chinese Fasciola specimens sequenced. While there was no variation in length or composition of the ITS-2 sequences among multiple specimens from France, Sichuan and Guangxi, sequence difference of 1.7% (6/362) was detected between specimens from France and Sichuan, and those from Guangxi. Based on ITS-2 sequence data, it was concluded that the Fasciola from Sichuan represented Fasciola hepatica, the one from Guangxi represented Fasciola gigantica and the one from sheep from Heilongjiang may represent an "intermediate genotype", as its ITS-2 sequences were unique in that two different ITS-2 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is almost identical to that of F. gigantica in that nucleotides at five of the six polymorphic positions represent F. gigantica. This microheterogeneity is possibly due to sequence polymorphism among copies of the ITS-2 array within the same worm. Based on the sequence differences, a PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay was established for the unequivocal delineation of the Fasciola spp. from Mainland China using restriction endonuclease Hsp92II or RcaI. This assay should provide a valuable tool for the molecular identification and for studying the ecology and population genetic structures of Fasciola spp. from Mainland China and elsewhere.
Chaudhry, U; van Paridon, B; Shabbir, M Z; Shafee, M; Ashraf, K; Yaqub, T; Gilleard, J
Fascioliasis is an important disease affecting livestock, with great costs to producers worldwide. It has also become a serious issue for human populations in some endemic areas as an emerging zoonotic infection. There are two Fasciola species of liver fluke responsible for this disease, which occur worldwide, Fasciola hepatica and Fasciola gigantica. Identifying these two species on the basis of adult or egg morphology requires specialist knowledge due to the similarity of characters, and may misidentify putative intermediate or hybrid forms. In this study we sequenced the internal transcribed spacer 2 (ITS-2) rDNA of liver flukes collected from multiple species of hosts from seven localities in the Punjab and Baluchistan provinces of Pakistan, to determine the distribution of these two species. All 46 flukes processed in this study, collected from seven sites, showed the rDNA ITS-2 genotype corresponding to F. gigantica, contradicting previous reports, based on adult and egg morphology, that both species are present in Pakistan, with F. hepatica being the more common.
El-Gozamy, Bothina R; Shoukry, Nahla M
Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.
Amor, Nabil; Halajian, Ali; Farjallah, Sarra; Merella, Paolo; Said, Khaled; Ben Slimane, Badreddine
Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n=25, 48.1%) and F. gigantica (n=20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n=7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic
El-Rahimy, Hoda H; Mahgoub, Abeer M A; El-Gebaly, Naglaa Saad M; Mousa, Wahid M A; Antably, Abeer S A E
Fascioliasis is an important disease caused by Fasciola hepatica and Fasciola gigantica. The distributions of both species overlap in many areas of Asia and Africa including Egypt. Fifty adult Fasciola worms were collected from livers of cattle and sheep slaughtered in abattoirs, Cairo, Egypt. They were subjected to morphological and metric assessment of external features of fresh adults, morphological and metric assessment of internal anatomy of stained mounted worms, determination of electrophorezed bands of crude adult homogenates using SDS-PAGE, and molecular characterization of species-specific DNA segments using RFLP-PCR. It was found that the correlation between conventional morphology and its morphotype was statistically significant (P value = 0.00). Using SDS-PAGE, 13 bands were detected among both genotypes of Fasciola (35.7, 33.6, 32.4, 29.3, 27.5, 26, 24.4, 23, 21.45, 19, 16.75, 12.5, and 9.1 kDa).The most prevalent bands were that with a molecular weight of 29.3, 26, and 19 kDa. Bands detected were common for both species, but protein bands could not distinguish between F. hepatica and F. gigantica. The result of PCR for the amplification of the selected 28S rDNA fragment with the designed primer set yielded 618 bp long PCR products for F. hepatica and F. gigantica. Different band patterns generated after digestion of the 618 bp segment by the enzyme AvaII obtained with F. hepatica showed segments of the length 529, 62, 27 bp, while with F. gigantica 322, 269, 27 bp bands were obtained. Genotyping revealed no equivocal results. The conventional morphological parameters for species determination of Fasciola spp. endemic in Egypt were evaluated versus protein bands characterization and genotyping. It was concluded that conventional morphological and metric assessments were not useful for differentiation between F. gigantica and F. hepatica due to extensive overlap in the relative ranges. Similar conclusion was reached concerning protein band
Itagaki, Tadashi; Kikawa, Masayuki; Terasaki, Kunio; Shibahara, Toshiyuki; Fukuda, Koichi
Nucleotide sequences of ribosomal internal transcribed spacer (ITS1) and mitochondrial NADH dehydrogenase I (NDI) gene were analyzed to genetically characterize aspermic Fasciola forms in Korea. From the difference in ITS1 sequences, Korean flukes were divided into 3 haplotypes represented by Kor1, Kor2 and Kor1/2, which had nucleotides identical to F. hepatica, F. gigantica and those overlapped between the two species, respectively. NDI sequences also showed that Korean flukes could be classified into 3 distinct haplotypes (Kor1: F. hepatica-type, Kor2a and Kor2b: F. gigantica-type). The sequences of Kor1 and Kor2a were 100% identical to those of the haplotypes Fsp1and Fsp2, respectively, which are major Fasciola forms in Japan. These findings strongly suggest that aspermic Fasciola forms in Korea and Japan originated from same ancestors and have recently spread throughout both countries.
Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan
The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.
GALAVANI, Hossein; GHOLIZADEH, Saber; HAZRATI TAPPEH, Khosrow
Background: Fascioliasis, caused by Fasciola hepatica and F. gigantica, has medical and economic importance in the world. Molecular approaches comparing traditional methods using for identification and characterization of Fasciola spp. are precise and reliable. The aims of current study were molecular characterization of Fasciola spp. in West Azerbaijan Province, Iran and then comparative analysis of them using GenBank sequences. Methods: A total number of 580 isolates were collected from different hosts in five cities of West Azerbaijan Province, in 2014 from 90 slaughtered cattle (n=50) and sheep (n=40). After morphological identification and DNA extraction, designing specific primer were used to amplification of ITS1, 5.8s and ITS2 regions, 50 samples were conducted to sequence, randomly. Result: Using morphometric characters 99.14% and 0.86% of isolates identified as F. hepatica and F. gigantica, respectively. PCR amplification of 1081 bp fragment and sequencing result showed 100% similarity with F. hepatica in ITS1 (428 bp), 5.8s (158 bp), and ITS2 (366 bp) regions. Sequence comparison among current study sequences and GenBank data showed 98% identity with 11 nucleotide mismatches. However, in phylogenetic tree F. hepatica sequences of West Azerbaijan Province, Iran, were in a close relationship with Iranian, Asian, and African isolates. Conclusions: Only F. hepatica species is distributed among sheep and cattle in West Azerbaijan Province Iran. However, 5 and 6 bp variation in ITS1 and ITS2 regions, respectively, is not enough to separate of Fasciola spp. Therefore, more studies are essential for designing new molecular markers to correct species identification. PMID:27095969
Ueno, H; Arandia, R; Morales, G; Medina, G
Fascioliasis caused by Fasciola hepatica was a serious problem for sheep and alpacas in the Altiplano Region of Bolivia. In some provinces close to Lake Titicaca, the raising of sheep was forced to discontinue, because infection with the fluke made it unprofitable and almost impossible. It was proved that in the Altiplano Region, two species of freshwater snails, Lymnaea viatrix and L. cubensis var., served as intermediate hosts for F. hepatica. In some subtropical areas of Bolivia, these snails could not be found, although other Lymnaea sp. was widely distributed there. As it is possible for Lymnaea sp. to be intermediate host for the fluke, further studies are required on the identification. Acute fascioliasis of sheep occurred in the Altiplano Region principally during a period from May to July, or the dry season. In some areas, the mortality rate of infected sheep was roughly estimated as 15 to 25% annually. Contamination with Fasciola metacercariae of herbage and semi-aquatic plants grown in a swamp in one of these areas was biologically assessed, using guinea pigs. Plants of Compositae and Eleocharis sp. were contaminated most intensely and those of Senicio sp. and Vallisneria sp. carried a fairly large number of cysts, while plants of Scirpus sp. and Ranunclaceae carried only a few cysts. No signs of Fasciola infection were observed in any animal given the plants of Liliaceae.
Ichikawa, Madoka; Itagaki, Tadashi
It has been speculated that populations of aspermic Fasciola flukes in Korea and Japan have a close phylogenetic relationship. To evaluate this, we analyzed 33 Korean aspermic Fasciola flukes on the basis of nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase 1 (cox1) sequences. Fh, Fg, and Fh/Fg types were detected in the ITS1 region and displayed the fragment patterns of F. hepatica, F. gigantica, and both species, respectively by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Additionally, three concatenated haplotypes of nad1 and cox1(nad1/cox1) were detected, and 2 of these, Kor1/Kor1 (Fsp1/Fsp1) haplotype and Kor2a/Kor2 (Fsp2/Fsp2) haplotype, were shared by Korean and Japanese aspermic flukes. The Fst value (0.019), calculated using the concatenated sequences, indicated that Korean and Japanese aspermic Fasciola populations were genetically undifferentiated. Interestingly, a combination of the Fh/Fg type and Kor1/Kor1 haplotype was found at the highest frequency in Korean aspermic flukes, whereas the Fg type and Fsp2/Fsp2 haplotype combination was found at a conspicuously high frequency in Japanese aspermic flukes. This indicates that a founder effect caused by the introduction of infected hosts may have played a key role in the introduction of aspermic Fasciola flukes from Korea into Japan.
Raina, O K; Nagar, Gaurav; Varghese, Anju; Prajitha, G; Alex, Asha; Maharana, B R; Joshi, P
Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300 μg of leucine aminopeptidase and a cocktail of 150 μg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.
Presidente, P J; McCraw, B M; Lumsden, J H
Six white-tailed deer (Odocoileus virginianus) and six sheep were inoculated with metacercariae of Fasciola hepatica. Two animals of each species were given 100, 500 or 2500 metacercariae. Clinicopathological features of these infections were determined by analyses of blood samples collected each week from inoculated deer and sheep as well as from two noninoculated animals of each species. One animal in each inoculated group was killed and examined at six weeks postinoculation and the remainder at 15 weeks postinoculation. Compared with the values obtained from noninoculated controls, eosinophilia, hyperproteinemia and hyperglobulinemia occured in inoculated deer. There were no other significant changes in hematological values or in serum aspartate aminotransferase levels. Marked leukocytosis and eosinophilia, with hyperproteinemia, hyperglobulinemia, hypoalbuminemia, elevated serum aspartate aminotransferase levels and mild macrocytic normochromic anemia characterized the infection in lambs. Although approximately 29% of the inoculum was recovered from the hepatic parenchyma of the sheep, F. hepatica was found in only one of six inoculated deer. A patent infection was established in this deer and constitutes the second report of mature F. hepatica in this host. Images Fig. 1. PMID:1125833
Amer, Said; Dar, Yasser; Ichikawa, Madoka; Fukuda, Yasuhiro; Tada, Chika; Itagaki, Tadashi; Nakai, Yutaka
Fascioliasis has a negative impact on the farming industry in both developed and developing countries, rather than a public health challenge. This study was performed to identify Fasciola sp. from different definitive hosts (buffalo, cattle, and sheep) based on the molecular parameters and spermatogenesis. Ninety-one adult flukes were collected from livers of slaughtered animals at abattoirs in different prefectures in Egypt. Microscopic examination of the analyzed flukes showed many normal spermatozoa in the seminal vesicles (spermic), suggesting that they have the ability of spermatogenesis. This study showed that no parthenogenic Fasciola species occurred in Egypt. Molecular analysis was performed utilizing genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Whereas 16 animals proved to have infection with a single Fasciola species, 2 were infected with both F. hepatica and F. gigantica. The results indicated that sheep were prone to F. hepatica (8 out of 10 animals) more than F. gigantica infection. Sequences of ITS1 and ITS2 ribosomal region indicated that the flukes were categorized into 3 groups F. hepatica-type (47), F. gigantica-type (42) and 2 flukes possessed sequences of both types indicating an existence of different alleles at the same loci. Unique overlapping of T/C bases were detected in both ITS1 (Position 96) and ITS2 (Position 416). Based on results of mitochondrial gene markers (NDI and COI), flukes were classified into F. hepatica-type and F. gigantica-type. Extensive intra-sequence polymorphism was detected at both markers. NDI and COI sequences of Egyptian strain of F. gigantica showed pronounced diversity compared with relevant sequences at database.
Shafiei, Reza; Sarkari, Bahador; Sadjjadi, Seyed Mahmuod; Mowlavi, Gholam Reza; Moshfe, Abdolali
The current study aimed to find out the morphometric and genotypic divergences of the flukes isolated from different hosts in a newly emerging focus of human fascioliasis in Iran. Adult Fasciola spp. were collected from 34 cattle, 13 sheep, and 11 goats from Kohgiluyeh and Boyer-Ahmad province, southwest of Iran. Genomic DNA was extracted from the flukes and PCR-RFLP was used to characterize the isolates. The ITS1, ITS2, and mitochondrial genes (mtDNA) of NDI and COI from individual liver flukes were amplified and the amplicons were sequenced. Genetic variation within and between the species was evaluated by comparing the sequences. Moreover, morphometric characteristics of flukes were measured through a computer image analysis system. Based on RFLP profile, from the total of 58 isolates, 41 isolates (from cattle, sheep, and goat) were identified as Fasciola hepatica, while 17 isolates from cattle were identified as Fasciola gigantica. Comparison of the ITS1 and ITS2 sequences showed six and seven single-base substitutions, resulting in segregation of the specimens into two different genotypes. The sequences of COI markers showed seven DNA polymorphic sites for F. hepatica and 35 DNA polymorphic sites for F. gigantica. Morphological diversity of the two species was observed in linear, ratios, and areas measurements. The findings have implications for studying the population genetics, epidemiology, and control of the disease.
Farahnak, Ali; Golmohamdi, Taghi; Eshraghian, Mohamadreza
Fascioliasis is an endemic disease in Iran and triclabendazole (TCBZ) is using for treatment of domestic animals and infected people. Excretory-secretory products (ESP) play an important role in the host biochemical defense by means of activities of detoxifying and antioxidant glutathione S-transferase (GST) and superoxide dismutase (SOD) enzymes respectively. Therefore, the aim of this comparative study was to evaluate fasciola protection against TCBZ drug by detection of enzymatic activities, GST and SOD, in TCBZ treated Fasciola hepatica / Fasciola gigantica and control ESP samples. F. gigantic and F. hepatica helminthes were collected and cultured within buffer media (TCBZ treated and untreated or control) for 4 h at 37 °C. Three TCBZ treated and 1 control ESP samples for each species were collected, centrifuged and supernatants were stored at -20°C. ESP samples protein concentrations were measured by Bradford method. SOD and GST enzymes activities of ESP samples were estimated photometrically. To determine the statistically significant difference between ESP of treated and control samples, t-test was conducted. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations in treated F. hepatica and F. gigantica ESP samples were estimated 204.88, 428, 130.4 and 288.2, 488.2, 308.2 µg/ml respectively. Protein concentrations in control samples were estimated 488.18 and 124.8 ug/ml respectively. SOD enzyme specific activities level in treated F.hepatica and F. gigantica ESP samples were determined 0.14, 0.31, 3.96 and 11.11, 13.54, 19.95 U/mg/protein respectively. SOD activities level in control samples were detected 70.69 and 10.92 U/mg/protein. GST specific activities level in treated F.hepatica and F. gigantica ESP samples were calculated 25.3, 85.5, 37.3 and 1823, 1314.3, 1320.8 U/mg respectively. GST activities levels in control samples were detected 98.6 and 1083.9 U/mg/protein respectively
Ullah, Rizwan; Rehman, Abdur; Zafeer, Mohd Faraz; Rehman, Lubna; Khan, Yasir A; Khan, M A Hannan; Khan, Shahper N; Khan, Asad U; Abidi, S M A
Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 μM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence
Ullah, Rizwan; Rehman, Abdur; Zafeer, Mohd Faraz; Rehman, Lubna; Khan, Yasir A.; Khan, M. A. Hannan; Khan, Shahper N.; Khan, Asad U.; Abidi, S. M. A.
Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 μM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence
Martínez-Pérez, J M; Robles-Pérez, D; Rojo-Vázquez, F A; Martínez-Valladares, M
The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-β at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.
Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; González-Warleta, Marta; Mezo, Mercedes; Ubeira, Florencio M.
ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1–10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis. PMID:27438470
Quadros, Rosiléia M; Pilati, Célso; Marques, Sandra M T; Mazzolli, Marcelo; Benedet, Rodrigo C
Capillaria hepatica was detected by histopathologic diagnosis in two cougars that were shot in April 2008 in Painel, Santa Catarina, Brazil. Macroscopic analysis of their livers revealed the presence of diffuse granulomas, and the histopathologic analysis indicated the presence of C. hepatica eggs, surrounded by mononuclear inflammatory cell infiltrate, small foci of necrosis, and mild-to-moderate fibrosis. This is the first report of C. hepatica in cougars (Puma concolor) in Brazil.
Hamir, A N; Rupprecht, C E
Tissues of 53 adult porcupines (Erethizon dorsatum) from Pennsylvania were obtained for histopathologic examination. Hepatic capillariasis was recorded in 9% of the porcupines. An additional 11% of the liver sections showed lesions that were compatible with migration by Capillaria hepatica. Because only 1 section of the liver per animal was examined microscopically, the documented prevalence of C. hepatica in Pennsylvania is considered conservatively low. However, this condition was subclinical, because none of the infected animals showed clinical signs, and none revealed severe pathologic changes in the affected livers. This seems to be the only report of C. hepatica in porcupines.
Hayashi, Kei; Ichikawa-Seki, Madoka; Mohanta, Uday Kumar; Singh, T Shantikumar; Shoriki, Takuya; Sugiyama, Hiromu; Itagaki, Tadashi
Fasciola flukes from eastern India were characterized on the basis of spermatogenesis status and nuclear ITS1. Both Fasciola gigantica and aspermic Fasciola flukes were detected in Imphal, Kohima, and Gantoku districts. The sequences of mitochondrial nad1 were analyzed to infer their phylogenetical relationship with neighboring countries. The haplotypes of aspermic Fasciola flukes were identical or showed a single nucleotide substitution compared to those from populations in the neighboring countries, corroborating the previous reports that categorized them in the same lineage. However, the prevalence of aspermic Fasciola flukes in eastern India was lower than those in the neighboring countries, suggesting that they have not dispersed throughout eastern India. In contrast, F. gigantica was predominant and well diversified, and the species was thought to be distributed in the area for a longer time than the aspermic Fasciola flukes. Fasciola gigantica populations from eastern India were categorized into two distinct haplogroups A and B. The level of their genetic diversity suggests that populations belonging to haplogroup A have dispersed from the west side of the Indian subcontinent to eastern India with the artificial movement of domestic cattle, Bos indicus, whereas populations belonging to haplogroup B might have spread from Myanmar to eastern India with domestic buffaloes, Bubalus bubalis.
Vichasri-Grams, Suksiri; Subpipattana, Pornpimol; Sobhon, Prasert; Viyanant, Vithoon; Grams, Rudi
A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.
Mowat, Vasanthi; Turton, John; Stewart, Jacqui; Lui, Kai Chiu; Pilling, Andrew M
Capillaria hepatica is a nematode parasite of wild rodents and other mammals. Adult worms inhabit the liver. Recently, during the necropsy examination of a group of 160 rabbits from a commercial supplier, firm pale or cystic areas (1-5 mm) were noted on the liver in thirteen animals. On further investigation, these animals were found to be infected with C. hepatica. The histopathological features of the infection in the rabbit are described for the first time and diagnostic features recorded. Lesions were identified predominantly in portal tracts consisting of dilated bile ducts with luminal debris, peribiliary inflammatory cell infiltrates, and fibrosis. Large granulomas (macrogranulomas) were evident in portal areas and involved the bile ducts. Macrogranulomas contained collections of characteristic C. hepatica eggs, macrophages, eosinophils, and lymphocytes. Small granulomas (microgranulomas), characterized by epithelioid macrophages surrounded by lymphocytes and eosinophils, were also identified. C. hepatica eggs were also observed in the lumina of the bile ducts and gall bladder. No adult C. hepatica worms were identified. Oocysts of Eimeria stiedae were also evident in the biliary epithelium in some animals. The unique characteristics of the C. hepatica life cycle are described, and the differential diagnosis of hepatic capillariasis is discussed.
Raymundo, Luis A Marcos; Flores, Vicente Maco; Terashima, Angélica; Samalvides, Frine; Miranda, Elba; Tantalean, Manuel; Espinoza, José R; Gotuzzo, Eduardo
A sample of 206 children (between 1 and 16 years-old) in Huertas (n = 110) and Julcán (n = 96) districts of the Mantaro Valley, Peru, were studied with a clinico-epidemiological interview, serology and fecal parasitologic tests to describe the prevalence and analyze risk factors of infection by Fasciola hepatica (Fh). Fh prevalence by fecal examinations in Huertas (H) was 28.3% and Julcán (J), 12.6%. Using serological tests, the prevalences were: for H, 36.3% and for J, 22.7%. Univariate analysis showed a significant association between Fh infection and 3 variables in H: living near small streams (OR= 4.8; P < 0.05) or farms (OR= 2.88; P < 0.05) and the habit of drinking hot herbal drinks "emolientes" (OR= 2.92; P < 0.05); and in other 3 variables in J: having a single room per house (OR= 21.0; P < 0.05), defecating in the fields (OR= 4.77; P < 0.05), history of taking antiparasitic medication in the last six months (OR= 4.61; P < 0.05). Multivariate analysis, applied to the whole set of data revealed as possible risk factors for infection with Fh: the habit of drinking "emolientes" (OR= 5.16; P < 0.05) and living near small streams (OR= 17.22; P < 0.05) or in farms (OR= 8.13; P < 0.05). According to these results one of the most important and less recognized risk factors is the habit of drinking "emolientes". Due to the clinical importance of human fascioliasis, we conclude that its presence in the Mantaro Valley is an important public health problem, effective early diagnosis would allow rapid treatment and avoidance of the serious impairment brought by chronic infection.
Shoriki, Takuya; Ichikawa-Seki, Madoka; Devkota, Bhuminand; Rana, Hari B; Devkota, Shiva P; Humagain, Sudeep K; Itagaki, Tadashi
Eighty-one Fasciola flukes collected from 8 districts in Nepal were analyzed for their species identification on the basis of their spermatogenic status and nuclear ribosomal internal transcribed spacer 1 (ITS1) and for their phylogenetic relation with Fasciola flukes from other Asian countries on the basis of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene. Sixty-one flukes (75.3%) were aspermic Fasciola sp., and 20 flukes (24.7%) were identified as Fasciola gigantica. All of the aspermic flukes displayed the Fh/Fg type in ITS1, which was predominant in aspermic Fasciola sp. from China, and most (60 flukes) displayed the Fsp-ND1-N1 haplotype in the nad1, which had an identical nucleotide sequence to the major haplotype (Fg-C2) of the aspermic flukes from China. These results suggest that aspermic Fasciola sp. was introduced into Nepal from China. Furthermore, the results of the diversity indices, neutrality indices, and median-joining network analysis with reference haplotypes from Asian countries suggest that aspermic Fasciola sp. rapidly expanded its distribution. In contrasts, F. gigantica displayed 10 nad1 haplotypes, which showed higher population diversity indices than the haplotypes of aspermic flukes, indicating that the F. gigantica population was clearly distributed in Nepal earlier than the aspermic Fasciola population. Although the F. gigantica haplotypes from Nepal formed a star-like phylogeny consisting of a main founder haplotype (Fg-ND1-N1), together with some F. gigantica haplotypes from Myanmar and Thailand, the Nepal population differed genetically from F. gigantica populations of neighboring countries as each country had distinct founder haplotype(s).
Molina-Hernández, Verónica; Mulcahy, Grace; Pérez, Jose; Martínez-Moreno, Álvaro; Donnelly, Sheila; O’Neill, Sandra M.; Dalton, John P.; Cwiklinski, Krystyna
Major advances have been made in identifying potential vaccine molecules for the control of fasciolosis in livestock but we have yet to reach the level of efficacy required for commercialisation. The pathogenesis of fasciolosis is associated with liver damage that is inflicted by migrating and feeding immature flukes as well as host inflammatory immune responses to parasite-secreted molecules and tissue damage alarm signals. Immune suppression/modulation by the parasites prevents the development of protective immune responses as evidenced by the lack of immunity observed in naturally and experimentally infected animals. In our opinion, future efforts need to focus on understanding how parasites invade and penetrate the tissues of their hosts and how they potentiate and control the ensuing immune responses, particularly in the first days of infection. Emerging ‘omics’ data employed in an unbiased approach are helping us understand liver fluke biology and, in parallel with new immunological data, to identify molecules that are essential to parasite development and accessible to vaccine-induced immune responses. PMID:25657086
Ichikawa-Seki, Madoka; Tokashiki, Minami; Opara, Maxwell Nwachukwu; Iroh, Gabriel; Hayashi, Kei; Kumar, Uday Mohanta; Itagaki, Tadashi
Fasciola gigantica is considered the major pathogen causing fasciolosis in Africa; however, molecular characterization of this fluke has not been adequately elucidated. It is important to scientifically elucidate the dispersal history of F. gigantica by analyzing its genetic diversity. Fasciola flukes from Nigeria were analyzed using nuclear and mitochondrial DNA markers. A total of 172 Fasciola flukes collected from cattle were identified as F. gigantica because they displayed the F. gigantica fragment pattern in multiplex PCR for the nuclear marker, phosphoenolpyruvate carboxykinase (pepck). In total, 70 haplotypes were detected from Nigerian F. gigantica on the basis of the concatenated sequence of mitochondrial NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase 1 (cox1). The index of neutrality (Fu's Fs) suggests rapid expansion of the Nigerian F. gigantica population. Although four haplogroups, Nigeria 1A, 1B, 2A, and 2B, were detected from Nigerian F. gigantica, a climate-specific genetic structure was not observed among F. gigantica populations from three agro-climatic regions (Sahel, Savannah, and Forest). This is probably because of the frequent transportation of livestock from one part of the country to the other. Nigeria 1A and 1B had close relationships with the Egyptian population of F. gigantica, whereas Nigeria 2A and 2B were comparatively related to the Zambian population. No haplotype was shared among the three countries, and it therefore is difficult to estimate the dispersal route of F. gigantica within the African continent.
Mas-Coma, Santiago; Valero, María Adela; Bargues, María Dolores
Fascioliasis, caused by liver fluke species of the genus Fasciola, has always been well recognized because of its high veterinary impact but it has been among the most neglected diseases for decades with regard to human infection. However, the increasing importance of human fascioliasis worldwide has re-launched interest in fascioliasis. From the 1990s, many new concepts have been developed regarding human fascioliasis and these have furnished a new baseline for the human disease that is very different to a simple extrapolation from fascioliasis in livestock. Studies have shown that human fascioliasis presents marked heterogeneity, including different epidemiological situations and transmission patterns in different endemic areas. This heterogeneity, added to the present emergence/re-emergence of the disease both in humans and animals in many regions, confirms a worrying global scenario. The huge negative impact of fascioliasis on human communities demands rapid action. When analyzing how better to define control measures for endemic areas differing at such a level, it would be useful to have genetic markers that could distinguish each type of transmission pattern and epidemiological situation. Accordingly, this chapter covers aspects of aetiology, geographical distribution, epidemiology, transmission and control in order to obtain a solid baseline for the interpretation of future results. The origins and geographical spread of F. hepatica and F. gigantica in both the ruminant pre-domestication times and the livestock post-domestication period are analyzed. Paleontological, archaeological and historical records, as well as genetic data on recent dispersal of livestock species, are taken into account to establish an evolutionary framework for the two fasciolids across all continents. Emphasis is given to the distributional overlap of both species and the roles of transportation, transhumance and trade in the different overlap situations. Areas with only one Fasciola
Hayashi, Kei; Ichikawa-Seki, Madoka; Allamanda, Puttik; Wibowo, Putut Eko; Mohanta, Uday Kumar; Sodirun; Guswanto, Azirwan; Nishikawa, Yoshifumi
Fasciola gigantica and aspermic (hybrid) Fasciola flukes are thought to be distributed in Southeast Asian countries. The objectives of this study were to investigate the distribution of these flukes from unidentified ruminants in western Java, Indonesia, and to determine their distribution history into the area. Sixty Fasciola flukes from western Java were identified as F. gigantica based on the nucleotide sequences of the nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold) genes. The flukes were then analyzed phylogenetically based on the nucleotide sequence of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene, together with Fasciola flukes from other Asian countries. All but one F. gigantica fluke were classified in F. gigantica haplogroup C, which mainly contains nad1 haplotypes detected in flukes from Thailand, Vietnam, and China. A population genetic analysis suggested that haplogroup C spread from Thailand to the neighboring countries including Indonesia together with domestic ruminants, such as the swamp buffalo, Bubalus bubalis. The swamp buffalo is one of the important definitive hosts of Fasciola flukes in Indonesia, and is considered to have been domesticated in the north of Thailand. The remaining one fluke displayed a novel nad1 haplotype that has never been detected in the reference countries. Therefore, the origin of the fluke could not be established. No hybrid Fasciola flukes were detected in this study, in contrast to neighboring Asian countries.
Rothenburger, Jamie L; Himsworth, Chelsea G; Chang, Victoria; LeJeune, Manigandan; Leighton, Frederick A
Capillaria hepatica is a parasitic nematode that infects the liver of rats (Rattus spp.), and occasionally other mammalian species, including humans. Despite its broad geographic distribution and host range, the ecology of this parasite remains poorly understood. We characterized the ecology of C. hepatica in urban Norway rats (Rattus norvegicus) in Vancouver, Canada. The overall prevalence of C. hepatica among Norway rats was 36% (241/671); however, there was significant variation in prevalence among city blocks. Using a generalized linear mixed model to control for clustering by block (where OR is odds ratio and CI is confidence interval), we found C. hepatica infection was negatively associated with season (spring [OR=0.14, 95% CI=0.05-0.39]; summer [OR=0.14, 95% CI=0.03-0.61]; winter [OR=0.34, 95% CI=0.13-0.84], compared to fall) and positively associated with sexual maturity (OR: 7.29, 95% CI=3.98-13.36) and presence of cutaneous bite wounds (OR=1.87, 95% CI=1.11-3.16). Our understanding of the ecology of C. hepatica in rats is hindered by a paucity of data regarding the main mechanisms of transmission (e.g., environmental exposure vs. active cannibalism). However, associations among infection, season, maturity, and bite wounds could suggest that social interactions, possibly including cannibalism, may be important in transmission.
HONG, Il-Hwa; KANG, Sun-Young; KIM, Jong-Hyun; SEOK, Seong-Hoon; LEE, Seong-Kyu; HONG, Seong-Jin; LEE, Seung-Yong; PARK, Se-Jin; KONG, Joo-Yeon; YEON, Seong-Chan
Capillaria hepatica is a zoonotic nematode that uses rodents and other mammals as hosts, especially rats and mice, and causes hepatic granuloma and eventually fibrosis/cirrhosis. However, C. hepatica infection in nutria, a large semiaquatic rodent, has rarely been reported, and histopathologic features of the infection have not been described in detail. We conducted necropsy on 36 wild nutrias. Some animals were found to have milky spots, parasitic eggs and worms within hepatic microgranuloma involving central calcification with cell debris, macrophages, eosinophils and multinucleated giant cells (MGCs). Interestingly, the eggs were closely surrounded by MGCs and appeared to be destroyed without inducing further chronic changes. Based on microscopical examination, C. hepatica infection was diagnosed, and we describe its histopathological characteristics in wild nutrias. PMID:27570138
Park, Jae-Hak; Novilla, Meliton N.; Song, Juha; Kim, Kyung-Sul; Chang, Seo-Na; Han, Ju-Hee; Lee, Byung Hee; Lee, Do-Hun; Kim, Hyun-Mac; Kim, Young-Ha; Youn, Hee-Jeong; Kil, Jihyon
This study reports the first case of Capillaria hepatica infection in a nutria in Korea. Ten nutrias, captured near the Nakdong River, were submitted to our laboratory for necropsy. White-yellowish nodules were found in the liver of 1 of the nutrias at necropsy. Histologically, the lesions were granulomatous, and infiltrations of lipid-laden macrophages, eosinophils, and several multinucleated giant cells were observed. The lesions consisted of numerous eggs and necrotic hepatocytes. The eggs were lemon-shaped and had polar plugs at the ends of both long sides. The eggs were morphologically identified as those of C. hepatica. Worldwide, C. hepatica infection in nutrias is very rare. Nutrias are a kind of livestock, as well as wildlife; therefore, an epidemiological study for parasitic infections needs to be conducted. PMID:25352702
Semyenova, Seraphima K; Morozova, Elena V; Chrisanfova, Galina G; Gorokhov, Vladimir V; Arkhipov, Ivan A; Moskvin, Alexander S; Movsessyan, Sergey O; Ryskov, Alexei P
Partial sequences of mitochondrial genes nad1 (316 bp) and cox1 (429 bp) were analyzed to estimate the variability of the liver fluke samples collected in 20 localities in Russia, Belarus, Ukraine, Bulgaria, Armenia, Azerbaijan, Georgia, Turkey, Turkmenistan, and China. The sequences had 4.1% (nad1) and 2.3% (cox1) of variable sites, and 13 and 10 haplotypes were identified among nad1 and cox1 genes, respectively. Spatial analysis of genetic and nucleotide diversity indicated little or no structuring of genetic variation between hosts or regions. The analysis of distribution of both separate and combined (nad1 + cox1) haplotypes revealed the existence of 2 well-defined lineages with 2 main haplotypes and a number of shared divergent haplotypes. Our study showed that the first lineage included the main N1-C1 haplotype, which was found in Australia, China, Georgia, Turkey, Armenia, Azerbaijan, and in all European populations (from Russia, Belarus, Ukraine, Bulgaria). The second lineage was found in all European populations and in populations from Armenia and Azerbaijan. It was suggested that one of the lineages (I) has an Asian origin. The possible source of mtDNA variability and associations between lineage divergence of parasite and its definitive hosts (cattle and sheep) are discussed.
Dreyfuss, Gilles; Vignoles, Philippe; Rondelaud, Daniel
Field investigations on the habitats colonized by Galba truncatula or Omphiscola glabra were carried out on 162 farms of the Limousin region, Central France, to determine whether there is currently a decline in the number and size of snail populations. Seven types of snail habitats were considered here. Compared to the numbers of snail populations recorded from 1976 to 1992, the values noted from 2013 to 2016 were significantly lower, with a decline rate of 34% for G. truncatula and 23% for O. glabra. Variations in this decline rate with the type of snail habitat were also noted. The greatest decreases in the numbers of snail populations were noted for spring heads located in meadows and for road ditches, while the lowest were noted for open drainage furrows present in meadows. The distribution of these habitats according to their area did not show any significant change over time. In contrast, overwintering snails were significantly less numerous in 2013–2016 in five types of habitats for G. truncatula and in three types only for O. glabra. Several causes underlie this population decline. Among them, the current development of mechanical cleaning in open drainage systems and road ditches, that of subsurface drainage in meadows, and regular gyro-crushing of vegetation around temporary spring heads were the most important. PMID:27774956
Duscher, Ruth; Duscher, Georg; Hofer, Johannes; Tichy, Alexander; Prosl, Heinrich; Joachim, Anja
In this study 595 lactating cows originating from 31 carinthian farms were investigated in accordance of liver fluke infection using individual and tank milk as well as individual blood and faecal samples. Two commercial ELISAs were used to test the milk and blood serum, and the results were compared with coproscopy and a commercial copro-antigen ELISA. Receiver operating characteristics (ROC) and two-graph operating characteristics (TG ROC) of tank milk results were conducted based on the individual milk to determine the minimum reliable in-herd antibody prevalence for the predominant condition in the investigation area. In 17.8% of the examined individuals located in 64.5% of the farms eggs were detected by coproscopy. The copro-antigen ELISA delivered 13.4% positive individuals from 54.8% of the farms. The milk ELISAs showed 42.7% (Euroclone) and 44.2% (Pourquier) positive cows on 90.3% of the farms. The blood samples were positive in 43% (Euroclone) and 45.2% (Pourquier) of the individuals from 90.3% to 96.8% of the herds, respectively. Based on the milk and the blood an average in-herd prevalence of 30-45% can be assumed. The serum and milk samples delivered correlating results with kappa values between 0.94 and 0.97, whereas the coproscopy and copro-antigen ELISA did not correlate well with the ELISA results. The two different ELISA tests highly correlated on individual and on herd level. Both showed a reliable minimum in-herd prevalence of ∼20%, meaning that one fifth of the individuals in a herd have to be positive to obtain a positive bulk tank milk result. In the investigated area a higher in-herd prevalence is expected, therefore the tank milk is useful as a monitoring tool and can be used as a basis for intervention strategies.
Miwa, Hidetsugu; Odrzykoski, Ireneusz J; Matsui, Atsushi; Hasegawa, Masami; Akiyama, Hiroyuki; Jia, Yu; Sabirov, Renat; Takahashi, Hideki; Boufford, David E; Murakami, Noriaki
An excess of nonsynonymous substitutions over synonymous ones has been regarded as an important indicator of adaptive evolution or positive selection at the molecular level. We now report such a case for rbcL sequences among cryptic species in Conocephalum (Hepaticae, Bryophytes). This finding can be regarded as evidence of adaptive evolution in several cryptic species (especially in F and JN types) within the genus. Bryophytes are small land plants with simple morphology. We can therefore expect the existence of several biologically distinct units or cryptic species within each morphological species. In our previous study, we found three rbcL types in Asian Conocephalum japonicum (Thunb.) Grolle and also found evidence strongly suggesting that the three types are reproductively isolated cryptic species. Additionally, we examined rbcL sequence variation in six cryptic species of C. conicum (L.) Dumort. previously recognized by allozyme analyses. As a result, we were able to discriminate the six cryptic species based only on their rbcL sequences. We were able to show that rbcL sequence variation is also useful in finding cryptic species of C. conicum.
Gupta, Ankita; Kesherwani, Manish; Velmurugan, Devadasan; Tripathi, Timir
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.
YAKHCHALI, Mohammad; BAHRAMNEJAD, Kia
Background: Fasciolosis, caused by the liver flukes of the genus Fasciola, is one of the most prevalent diseases of domestic livestock and human throughout the world, imposing considerable economic losses. The present study was aimed to assess the effects of different pH values on hatching rate of Fasciola miracidia. Methods: The flukes were isolated from the infected livers of the slaughtered ruminants at the abattoir of Urmia City, Iran, crushed thoroughly and sieved for isolation of the Fasciola eggs. The eggs were washed up several times by PBS (0.01N, pH 7.2). They were incubated at different pH values of 7±0.1 (control) and 3–9.5 (treatments) at 28°C for 16 days. Results: The maximum hatching rate was observed at pH 7 (14.93±0.65%), while no miracidia were hatched at pH 3 and/or pH 9–9.5. There were significant differences between the hatching rate of the treatments and that of the control group. Conclusion: Water pH is proven to be a crucial factor affecting the life cycle of Fasciola and its epidemiology. PMID:27095966
Camargo, A M; Teixeira, G G; Ortale, J R
In 30 normal adult livers the retrohepatic segment of inferior vena cava had a length of 6.7 cm and was totally encircled by liver substance in 30% of cases. Altogether 442 ostia venae hepaticae were found, averaging 14.7 per liver and classified as large, medium, small and minimum. The localisation of the openings was studied according to the division of the wall of the retrohepatic segment of the inferior vena cava into 16 areas. PMID:8655416
Sunita, Kumari; Singh, D. K.
Snail is one of the important components of an aquatic ecosystem, it acts as intermediate host of Fasciola species. Control of snail population below a certain threshold level is one of the important methods in the campaign to reduce the incidence of fascioliasis. Life cycle of the parasite can be interrupted by killing the snail or Fasciola larva redia and cercaria in the snail body. In vivo and in vitro toxicity of the plant products and their active component such as citral, ferulic acid, umbelliferone, azadirachtin, and allicin against larva of Fasciola in infected snail Lymnaea acuminata were tested. Mortality of larvae were observed at 2 h, 4 h, 6 h, and 8 h, of treatment. In in vivo treatment, azadirachtin caused highest mortality in redia and cercaria larva (8 h, LC50 0.11, and 0.05 mg/L) whereas in in vitro condition allicin was highly toxic against redia and cercaria (8 h, LC50 0.01, and 0.009 mg/L). Toxicity of citral was lowest against redia and cercaria larva. PMID:22132306
Floudas, Dimitrios; Held, Benjamin W; Riley, Robert; Nagy, Laszlo G; Koehler, Gage; Ransdell, Anthony S; Younus, Hina; Chow, Julianna; Chiniquy, Jennifer; Lipzen, Anna; Tritt, Andrew; Sun, Hui; Haridas, Sajeet; LaButti, Kurt; Ohm, Robin A; Kües, Ursula; Blanchette, Robert A; Grigoriev, Igor V; Minto, Robert E; Hibbett, David S
Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.
Floudas, Dimitrios; Held, Benjamin W.; Riley, Robert; ...
Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. In this paper, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot andmore » brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Finally, our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.« less
Floudas, Dimitrios; Held, Benjamin W.; Riley, Robert; Nagy, Laszlo G.; Koehler, Gage; Ransdell, Anthony S.; Younus, Hina; Chow, Julianna; Chiniquy, Jennifer; Lipzen, Anna; Tritt, Andrew; Sun, Hui; Haridas, Sajeet; LaButti, Kurt; Ohm, Robin A.; Kües, Ursula; Blanchette, Robert A.; Grigoriev, Igor V.; Minto, Robert E.; Hibbett, David S.
Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. In this paper, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Finally, our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.
Franco, F F; Silva, E C C; Barrios-Leal, D Y; Sene, F M; Manfrin, M H
The species of the Drosophila fasciola subgroup Wasserman represent the dominant section of the Drosophila repleta group Wasserman in the American rainforests and have a broad geographical distribution in the New World. However, despite of its wide range, the D. fasciola subgroup is one of the most overlooked D. repleta subgroups. Here, we report a molecular phylogenetic analysis focused on the D. fasciola subgroup using two mitochondrial [cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII)] and two nuclear [elongation factor-1alpha F1 (EF-alphaF1) and transformer (tra)] genes. Overall, we found that this subgroup is a monophyletic taxon, subdivided into two main internal branches: named Fas1 and Fas2 clades. The diversification of these clades is estimated to have begun in the middle Miocene, around 12 Ma [95% high posterior density (HPD) 9.0-15 Ma], and might be associated with the colonization of South America by Central America populations after the closure of Isthmus of Panama due to the temporal congruence between these events. The terminal branches had their origins estimated to be in the Pliocene or the Plio-Pleistocene transition. For the later estimates, both the geomorphological influences and the climatic oscillations of the Pleistocene may have played a role in shaping the diversification of the D. fasciola group.
Hassan, M M; Mostafa, N E; Ramadan, M; Nassar, A; Hassounah, O; Omar, O
Stool examination using modified Kato thick smear method was performed to detect Fasciola eggs and other parasites. Abdominal pain was the major presenting symptom (83.3%) followed by pallor (71.6%) and fever (16.7%). Anaemia and hepatomegaly were recorded in 70% of patients compared to 25% with splenomegaly. Abdominal ultrasonography revealed hepatomegaly and common bile duct dilatation in 70% of patients. Moreover, 6 cases showed Olympic game rings which is diagnostic. All of patients had positive IgG4 levels, 58 cases were found positive for specific total IgG and IgG1, whereas, only 36 cases had positive IgG2 levels (60%). All negative control group showed no cross reactions. On the other hand, ELISA detecting IgG4 showed the highest specificity (95%), followed by IgG2 (85%) and the least specific test was obtained with detection of IgG (70%) and IgG1 (65%). One week after treatment, 90% of patients were completely cured. One and 3 months after treatment, the cure rate was 83.3%. In completely cured patients, none of anti-Fasciola isotypes was significantly changed.
Singh, Divya Jyoti; Singh, Vinay Kumar; Singh, D K
Fasciolosis is a parasitic disease caused by Fasciola gigantica. The freshwater snail Lymnaea acuminata is the intermediate host of F. gigantica which cause endemic fasciolosis in the northern part of India. To investigate larvicidal activity of pure and laboratory extracted pheophorbide a (Pa) against cercaria larvae of F. gigantica, data were analyzed in different spectra of visible light, sunlight, and laboratory conditions. Photostimulation of chlorophyll derivative pheophorbide a (Pa) caused time and concentration dependent larvicidal activity against cercaria larvae of F. gigantica. Larvicidal activity of pure Pa under 650 nm and 400-650 nm (8 h LC50 0.006 mg/10 mL) was more pronounced than extracted Pa under same irradiations (650 nm LC50 0.12 mg/10 mL, 400-650 nm LC50 0.14 mg/10 mL). Lowest toxicity of pure (8 h LC50 0.14 mg/10 mL) and extracted Pa (8 h LC50 1.25 mg/10 mL) was noted under 400 nm. Pa was found to be toxic in laboratory conditions also. The results presented in this paper indicate that pheophorbide a possess potential larvicidal activity against Fasciola gigantica larvae in different wavelengths of visible light, sunlight, and laboratory conditions.
Singh, Divya Jyoti; Singh, Vinay Kumar
Fasciolosis is a parasitic disease caused by Fasciola gigantica. The freshwater snail Lymnaea acuminata is the intermediate host of F. gigantica which cause endemic fasciolosis in the northern part of India. To investigate larvicidal activity of pure and laboratory extracted pheophorbide a (Pa) against cercaria larvae of F. gigantica, data were analyzed in different spectra of visible light, sunlight, and laboratory conditions. Photostimulation of chlorophyll derivative pheophorbide a (Pa) caused time and concentration dependent larvicidal activity against cercaria larvae of F. gigantica. Larvicidal activity of pure Pa under 650 nm and 400–650 nm (8 h LC50 0.006 mg/10 mL) was more pronounced than extracted Pa under same irradiations (650 nm LC50 0.12 mg/10 mL, 400–650 nm LC50 0.14 mg/10 mL). Lowest toxicity of pure (8 h LC50 0.14 mg/10 mL) and extracted Pa (8 h LC50 1.25 mg/10 mL) was noted under 400 nm. Pa was found to be toxic in laboratory conditions also. The results presented in this paper indicate that pheophorbide a possess potential larvicidal activity against Fasciola gigantica larvae in different wavelengths of visible light, sunlight, and laboratory conditions. PMID:28127494
di Sisto, R. P.; Orellana, R.
El Observatorio de La Plata cuenta con un gran número de placas de asteroides y cometas obtenidas con el telescopio astrográfico, que cubren gran parte del cielo del hemisferio sur. En 1996 se recopilaron y clasificaron 2187 placas (Beca para estudiantes de la AAA 1996) de las cuales 2031 corresponden a asteroides. Los datos de cada placa se volcaron en una base de datos creada para facilitar su manejo y preservar la información. A partir de este trabajo se revisaron los MPC electrónicos y se identificaron aquellas placas de asteroides pertenecientes a nuestra base de datos cuyos resultados no fueron publicados en los mismos. De un total de 400 placas que no aparecían publicadas sobresalía un paquete constituído por 40 placas obtenidas en 1977. Estas últimas fueron reducidas utilizando las posiciones y movimientos propios de las estrellas de referencia obtenidas del catálogo SAO 2000 dadas para el sistema FK5. Las posiciones calculadas fueron enviadas y publicadas en los Minor Planet Circulars (MPC).
Background Human capillariasis caused by Capillaria hepatica (syn. Calodium hepaticum) is a rare disease with no more than 40 cases registered around the world. Classically, the disease has severe symptoms that mimic acute hepatitis. Natural reservoirs of C. hepatica are urban rodents (Mus musculus and Rattus novergicus) that harbor their eggs in the liver. After examining the feces of 6 riverine inhabitants (Rio Preto area, 8° 03'S and 62° 53' W to 8° 14'S and 62° 52'W) of the State of Rondonia, Brazil, and identifying C. hepatica eggs in their feces, the authors decided to investigate the real dimension of these findings by looking for two positive signals. Methods Between June 1st and 15th, 2008, 246 out of 304 individuals were clinically examined. Blood samples were collected, kept under -20°C, and test by the indirect immunofluorescence technique. Results The first positive signal was the presence of specific antibodies at 1:150 dilution, which indicates that the person is likely to have been exposed to eggs, most likely non-infective eggs, passing through the food chain or via contaminated food (total prevalence of 34.1%). A second more specific signal was the presence of antibodies at higher titers, thus indicating true infection. Conclusions The authors concluded that only two subjects were really infected (prevalence of 0.81%); the rest was false-positives that were sensitized after consuming non-embryonated eggs. The present study is the first one carried out in a native Amazonian population and indicates the presence of antibodies against C. hepatica in this population. The results further suggest that the transmission of the parasite occurs by the ingestion of embryonated eggs from human feces and/or carcasses of wild animals. The authors propose a novel mode of transmission, describing the disease as a low pathogenic one, and showing low infectivity. PMID:20187941
Aihara, Naoyuki; Chambers, James Kenn; Hosono, Kei; Kamiie, Junichi; Shirota, Kinji
Type II mixed cryoglobulinemia is characterized by systemic vasculitis with deposition of cryoprecipitatable-immunoglobulins containing rheumatoid factor. Pathogenesis of type II mixed cryoglobulinemia has not yet been completely clarified because of the lack of an experimental animal. Here, we report an animal model of type II mixed cryoglobulinemia that is induced by experimental infection with Capillaria hepatica in ICR mice. Capillaria hepatica is a nematode that causes necrotic hepatitis in several mammals. In this study, mice experimentally infected with C. hepatica eggs developed cryoglobulinemia at 20 and 30 days post injection. Using immunological analysis, cryoglobulinemia in infected mice was classified as type II mixed cryoglobulinemia by detection of monoclonal IgM rheumatoid factor and IgA in the cryoprecipitate of serum. Using immunofluorescence, we observed an increase in the number of double-positive cells for µ heavy and κ light chains of immunoglobulin in the spleens of infected mice. Histopathologically, this model was characterized by glomerulopathy associated with intense deposition of IgM and IgA filling in capillary lumina. Ultrastructural analysis showed that glomerular deposits consisted of stacks of twisted microtubular structures. These serological and histological features resembled those of type II mixed cryoglobulinemia in human. This is the first experimental animal model of type II mixed cryoglobulinemia that will enable detailed studies on the pathogenesis of cryoglobulinemia.
Massoud, Ahmad Mohamed; Shalaby, Hatem Abdel Mawgoud; El Khateeb, Rabab Mohamed; Mahmoud, Mona Said; Kutkat, Mohamed Abdel Aziz
Objective To evaluated the histological changes within the tegument of adult Fasciola gigantica (F. gigantica) that led to the gross changes that were visible externally. Methods The effects of oleoresin extract of myrrh (Mirazid®), myrrh volatile oil and triclabendazole sulphoxide (reference drug) on the tegumental structure of adult F. gigantica following treatment in vitro had been determined by light microscopy. Results The internal changes in the tegument observed in this study were compatible with surface changes seen in the previous scanning electron microscopy study, using the same drugs. The swelling of tegumental syncytium was a particular feature of their action, but its level was much greater with myrrh volatile oil, in which vacuolization of the tegument and loss of spines were observed. Conclusions The present study demonstrated the fasciocidal properties of Mirazid® oleoresin extract, and it might be possible to reinforce its fasciocidal activity by increasing its content of myrrh volatile oil. PMID:23730566
Huang, Wei-Yi; Zhao, Guang-Hui; Wei, Shu-Jun; Song, Hui-Qun; Xu, Min-Jun; Lin, Rui-Qing; Zhou, Dong-Hui; Zhu, Xing-Quan
Complete mitochondrial (mt) genomes and the gene rearrangements are increasingly used as molecular markers for investigating phylogenetic relationships. Contributing to the complete mt genomes of Gastropoda, especially Pulmonata, we determined the mt genome of the freshwater snail Galba pervia, which is an important intermediate host for Fasciola spp. in China. The complete mt genome of G. pervia is 13,768 bp in length. Its genome is circular, and consists of 37 genes, including 13 genes for proteins, 2 genes for rRNA, 22 genes for tRNA. The mt gene order of G. pervia showed novel arrangement (tRNA-His, tRNA-Gly and tRNA-Tyr change positions and directions) when compared with mt genomes of Pulmonata species sequenced to date, indicating divergence among different species within the Pulmonata. A total of 3655 amino acids were deduced to encode 13 protein genes. The most frequently used amino acid is Leu (15.05%), followed by Phe (11.24%), Ser (10.76%) and IIe (8.346%). Phylogenetic analyses using the concatenated amino acid sequences of the 13 protein-coding genes, with three different computational algorithms (maximum parsimony, maximum likelihood and Bayesian analysis), all revealed that the families Lymnaeidae and Planorbidae are closely related two snail families, consistent with previous classifications based on morphological and molecular studies. The complete mt genome sequence of G. pervia showed a novel gene arrangement and it represents the first sequenced high quality mt genome of the family Lymnaeidae. These novel mtDNA data provide additional genetic markers for studying the epidemiology, population genetics and phylogeographics of freshwater snails, as well as for understanding interplay between the intermediate snail hosts and the intra-mollusca stages of Fasciola spp.. PMID:22844544
Hayashi, Kei; Mohanta, Uday K; Neeraja, Tambireddy; Itagaki, Tadashi
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.
HAYASHI, Kei; MOHANTA, Uday K.; NEERAJA, Tambireddy; ITAGAKI, Tadashi
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. PMID:27301703
Kameoka, Shinichiro; Sakio, Hitoshi; Abe, Harue; Ikeda, Hajime; Setoguchi, Hiroaki
How phenotypic or genetic diversity is maintained in a natural habitat is a fundamental question in evolutionary biology. Flower color polymorphism in plants is a common polymorphism. Hepatica nobilis var. japonica on the Sea of Japan (SJ) side of the Japanese mainland exhibits within population flower color polymorphism (e.g., white, pink, and purple), while only white flowers are observed on the Pacific Ocean (PO) side. To determine the relationships between flower color polymorphism, within and among populations, and the genetic structure of H. nobilis var. japonica, we estimated the genetic variation using simple sequence repeat (SSR) markers. First, we examined whether cryptic lineages corresponding to distinct flower colors contribute to the flower color polymorphisms in H. nobilis var. japonica. In our field observations, no bias in color frequency was observed among populations on Sado Island, a region with high variation in flower color. Simple sequence repeat (SSR) analyses revealed that 18% of the genetic variance was explained by differences among populations, whereas no genetic variation was explained by flower color hue or intensity (0% for both components). These results indicate that the flower color polymorphism is likely not explained by cryptic lineages that have different flower colors. In contrast, populations in the SJ and PO regions were genetically distinguishable. As with the other plant species in these regions, refugial isolation and subsequent migration history may have caused the genetic structure as well as the spatially heterogeneous patterns of flower color polymorphisms in H. nobilis var. japonica.
Simões, Raquel O.; Luque, José L.; Faro, Marta Júlia; Motta, Ester; Maldonado, Arnaldo
The nematode Calodium hepaticum (syn. Capillaria hepatica) is a zoonotic helminth found mainly infecting rats. It was studied the prevalence of C. hepaticum infection in Rattus norvegicus in an urban area of Rio de Janeiro (Brazil), with low urban planning and sanitation. The presence of C. hepaticum was identified through visible yellowish-white lesions in liver tissue and histological analyses. The total prevalence of infection was 45%, with no significant differences between sex and age. The presence of infected rodents near the peridomestic area poses substantial risk to human health. PMID:25229230
Gupta, Ankita; Pandey, Tripti; Kumar, Bijay; Tripathi, Timir
The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites, and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx) system plays a key role in redox metabolism and defense against oxidative stress. In this study, biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage. Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by residues present in close proximity to the -CXXC- motif, through manipulation of the redox potential or the pKa of the active site Cys residues.
Changklungmoa, Narin; Kueakhai, Pornanan; Riengrojpitak, Suda; Chaithirayanon, Kulathida; Chaichanasak, Pannigan; Preyavichyapugdee, Narin; Chantree, Pathanin; Sansri, Veerawat; Itagaki, Tadashi; Sobhon, Prasert
Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 μg of rFgLAP combined with Freund's adjuvant (n = 10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3%, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat.
Chaichanasak, Pannigan; Ichikawa, Madoka; Sobhon, Prasert; Itagaki, Tadashi
We analyzed 147 Fasciola flukes obtained from cattle in Thailand based on their spermatogenetic ability, and nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial nicotiamide adenine dinucleotide dehydrogenase subunit 1 (ND1) genes as molecular markers. One hundred twenty-eight flukes, which had abundant sperm in their seminal vesicles (spermic) and showed the PCR-RFLP pattern of F. gigantica in the ITS1, were accurately identified as F. gigantica. The other 19 flukes that had no sperm in their seminal vesicles were aspermic Fasciola sp. with the RFLP patterns identical to that of F. gigantica. Twenty-nine ND1 haplotypes (Fg-ND1-Thai 2-30) were distinguished in the 128 F. gigantica flukes and were divided into haplotypes unique to Thailand and those common to other countries, suggesting the possibility that ancestral haplotypes were introduced into Thailand. Three haplotypes (Fg-ND1-Thai 7, 9 and 27) appeared to be the major haplotypes found in F. gigantica from Thailand. Only one haplotype (Fg-ND1-Thai 1) was found in the 19 aspermic Fasciola sp. flukes obtained from geographical regions, and the nucleotide sequence of Fg-ND1-Thai 1 was identical to that of the aspermic Fasciola sp. from Japan, Korea, China, Vietnam and Myanmar, suggesting that they were descendants with a common provenance and expanded to these countries in the relatively recent past.
SINGH, Divya Jyoti; SINGH, Dinesh Kumar
Fasciolosis is a food borne zoonosis, caused by the digenetic trematode Fasciola. Freshwater lymnaeid snails are the intermediate host of the trematodes. Chlorophyllin, a semi-synthetic derivative of chlorophyll and its formulations obtained from freeze dried cow urine (FCU) had their toxicity tested against redia and cercaria larvae of F. gigantica. The larvicidal activity of chlorophyllin and its formulations were found to depend on both, time and concentration used against the larvae. Toxicity of chlorophyllin + FCU (1:1 ratio) in sunlight against redia larva (8 h LC50: 0.03 mg/mL) was more pronounced than using just chlorophyllin (8 h LC50: 0.06 mg/mL). Toxicity of chlorophyllin + FCU in sunlight against redia (8 h LC50: 0.03 mg/mL) was higher than against cercaria (8 h LC50: 0.06 mg/mL). The larvicidal activity of chlorophyllin in sunlight (redia/cercaria larvae: 8 h LC50: 0.06 mg/mL) was more pronounced than under laboratory conditions (redia: 8 h LC50: 22.21 mg/mL/, cercaria 8 h LC50: 96.21 mg/mL). Toxicity of FCU against both larvae was lower than that of chlorophyllin and chlorophyllin + FCU. Chlorophyllin and its formulations + FCU were 357.4 to 1603.5 times more effective against redia/cercaria larvae in sunlight than under laboratory conditions. The present study has shown that chlorophyllin formulations may be used as potent larvicides against fasciolosis. PMID:27253741
Gupta, Ankita; Sripa, Banchob; Tripathi, Timir
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.
Singh, Divya Jyoti; Singh, Dinesh Kumar
Fasciolosis is a food borne zoonosis, caused by the digenetic trematode Fasciola. Freshwater lymnaeid snails are the intermediate host of the trematodes. Chlorophyllin, a semi-synthetic derivative of chlorophyll and its formulations obtained from freeze dried cow urine (FCU) had their toxicity tested against redia and cercaria larvae of F. gigantica. The larvicidal activity of chlorophyllin and its formulations were found to depend on both, time and concentration used against the larvae. Toxicity of chlorophyllin + FCU (1:1 ratio) in sunlight against redia larva (8 h LC50: 0.03 mg/mL) was more pronounced than using just chlorophyllin (8 h LC50: 0.06 mg/mL). Toxicity of chlorophyllin + FCU in sunlight against redia (8 h LC50: 0.03 mg/mL) was higher than against cercaria (8 h LC50: 0.06 mg/mL). The larvicidal activity of chlorophyllin in sunlight (redia/cercaria larvae: 8 h LC50: 0.06 mg/mL) was more pronounced than under laboratory conditions (redia: 8 h LC50: 22.21 mg/mL/, cercaria 8 h LC50: 96.21 mg/mL). Toxicity of FCU against both larvae was lower than that of chlorophyllin and chlorophyllin + FCU. Chlorophyllin and its formulations + FCU were 357.4 to 1603.5 times more effective against redia/cercaria larvae in sunlight than under laboratory conditions. The present study has shown that chlorophyllin formulations may be used as potent larvicides against fasciolosis.
Singh, Divya Jyoti; Singh, D K
Toxicity of chlorophyllin against redia and cercaria larvae of Fasciola gigantica was studied under irradiation of visible light. Highest and lowest toxicity of chlorophyllin against both larvae was noted under red (redia - 8 h LC₅₀ 7.88 mg/10 mL and cercaria - 11.99 mg/10 mL) and green (redia - 8 h LC₅₀ 32.12 mg/10 mL and cercaria - 8 h LC₅₀ 43.80 mg/10 mL) light irradiation respectively. The highest toxicity of chlorophyllin under red light irradiation against redia (8h LC₅₀ 7.88 mg/10 mL)/cercaria (8h LC₅₀ 11.99 mg/10 mL) was followed by white (8 h LC₅₀ redia - 20.48 mg/10 mL, 8 h LC₅₀ cercaria - 18.0 3mg/10 mL), blue (8 h LC₅₀ redia - 33.10 mg/10 mL/ 8 h LC₅₀ cercaria - 19.98 mg/10 mL) and yellow (8 h LC₅₀ redia - 23.87 mg/10 mL/ 8 h LC₅₀ cercaria - 23.48 mg/10 mL). Chlorophyllin treatment in darkness (control I) and without treatment of chlorophyllin, while all other conditions were same as treatment group (control II) caused no mortality of redia/cercaria larva. Chlorophyllin might be a promising new safe strategy to replace synthetic larvicide in fasciolosis control programme.
Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi
Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects.
Zhang, Xiao-Xuan; Cong, Wei; Elsheikha, Hany M; Liu, Guo-Hua; Ma, Jian-Gang; Huang, Wei-Yi; Zhao, Quan; Zhu, Xing-Quan
Fasciola gigantica is regarded as the major liver fluke causing fasciolosis in livestock in tropical countries. Despite the significant economic and public health impacts of F. gigantica there are few studies on the pathogenesis of this parasite and our understanding is further limited by the lack of genome and transcriptome information. In this study, de novo Illumina RNA sequencing (RNA-seq) was performed to obtain a comprehensive transcriptome profile of the juvenile (42days post infection) and adult stages of F. gigantica. A total of 49,720 unigenes were produced from juvenile and adult stages of F. gigantica, with an average length of 1286 nucleotides (nt) and N50 of 2076nt. A total of 27,862 (56.03%) unigenes were annotated by BLAST similarity searches against the NCBI non-redundant protein database. Because F. gigantica needs to feed and/or digest host tissues, some proteases (including cysteine proteases and aspartic proteases), which play a role in the degradation of host tissues (protein), have been paid more attention in the present study. A total of 6511 distinct genes were found differentially expressed between juveniles and adults, of which 3993 genes were up-regulated and 2518 genes were down-regulated in adults versus juveniles, respectively. Moreover, stage-specific differentially expressed genes were identified in juvenile (17,009) and adult (6517) F. gigantica. The significantly divergent pathways of differentially expressed genes included cAMP signaling pathway (226; 4.12%), proteoglycans in cancer (256; 4.67%) and focal adhesion (199; 3.63%). The transcription pattern also revealed two egg-laying-associated pathways: cGMP-PKG signaling pathway and TGF-β signaling pathway. This study provides the first comparative transcriptomic data concerning juvenile and adult stages of F. gigantica that will be of great value for future research efforts into understanding parasite pathogenesis and developing vaccines against this important parasite.
Dung, Bui Thi; Doanh, Pham Ngoc; The, Dang Tat; Loan, Ho Thi; Losson, Bertrand; Caron, Yannick
Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.
Klisiowicz, Débora do Rocio; Reifur, Larissa; Shimada, Márcia Kiyoe; Haidamak, Juciliane; Cognialli, Regielly Caroline Raimundo; Ferreira, Tatiane
Calodium hepaticum (syn. Capillaria hepatica) is a nematode of the Capillariidae family that infects rodents and other mammals. In Brazil, human spurious infections of C. hepaticum have been detected in indigenous or rural communities from the Amazon Basin, but not in the southern states of the country. Here, we report the highest occurrence (13.5% of 37 residents) of C. hepaticum human spurious infection detected in Brazil and the first record in a southern region, Guaraqueçaba. The finding is explained by the area being located in the Atlantic Forest of the state of Paraná, surrounded by preserved forests and because the inhabitants consume the meat of wild mammals. PMID:24676661
da Rocha, Elierson José Gomes; Basano, Sérgio de Almeida; de Souza, Márcia Maria; Honda, Eduardo Resende; de Castro, Márcio Botelho; Colodel, Edson Moleta; Silva, Jéssica Carolinne Damasceno e; Barros, Lauro Prado; Rodrigues, Elisa Sousa; Camargo, Luís Marcelo Aranha
Introduction: Hepatic capillariosis, caused by Capillaria hepatica (Calodium hepaticum) (Bancroft, 1893), Travassos, 1915 (Nematoda, Trichinelloidea, Capillariidae), is a common zoonosis in rodents but is rare in humans. Seventy-two cases in humans have been reported worldwide since the first case was described by MACARTHUR in 192417,27. This study aimed to determine the prevalence of Capillaria hepatica in humans and rodents in an urban area of Porto Velho, the capital of Rondônia, in Brazil. Methods: After conducting a census of the area, 490 residents were randomly selected, and, after signing a term of consent, provided blood samples that were screened for anti-Capillaria hepatica antibodies. Simultaneously, rats were captured to assess the prevalence of this parasite in rodents by histopathological examination in liver sections. Results: A prevalence of 1.8% was found among residents who had specific antibodies at a dilution of 1:150, indicating exposure to parasite eggs; 0.8% of the subjects also had positive titers at a dilution of 1:400, indicating true infection. The prevalence in rats was 2%. Conclusions: The prevalence of infection with this parasite among humans and rats was low. While the prevalence encountered among humans was within the limits reported in the literature, the prevalence among rodents was much lower. PMID:25651325
Hanlon, S.D.; Neves, R.J.
Recent efforts to restore remnant or extirpated populations of freshwater mussels have focused on artificial propagation as an effective and practical conservation strategy. Although artificially cultured juveniles have been produced and released to the wild at various times of the year, no study has investigated the best time of year to release these juveniles. Newly metamorphosed juveniles of the wavyrayed lampmussel (Lampsilis fasciola) were released into a stream-fed fish hatchery raceway during March, June, and September. Growth and survival rates were measured 32, 52, 72, and 92 days post-metamorphosis. Juveniles released in June experienced the greatest growth and survival rates. Juveniles released in September and March experienced high mortality within the first month of release and exhibited poor growth in the cool water conditions typical of those seasons. Overwinter survival exhibited a size-dependent relationship.
Sera from patients or experimental animals infected with Schistosoma, Fasciola hepatica, Trichinella spiralis, Taenia solium, Echinococcus...were used as antigens for immuno- precipitation with sera of patients infected with one of three species of Schistosoma or Trichinella spiralis, Taenia
Ayaz, Erol; Ertekin, Ali; Ozdal, Nalan; Taş, Zeynep
This study was performed in order to investigate the variations of some blood biochemical parameters as well as the levels of Vitamin. B(12) and some macro elements in sheep infected with endoparasites. The blood samples were taken from the sheep that were to be slaughtered in the Van Municipality Slaughterhouse while the stool samples were taken after the slaughtering of the same animals. The postmortem examinations were made to investigate for the presence of Fasciola spp., D. dendriticum and cyst hydatid infections. The stool samples were examined helminthologically using native, sedimentation, flotation and Baermann-Wetzel methods. The control group was composed of animals not showing any internal organ parasites or parasites in the stool examination. Following the macroscopic and the stool examination, the animals found to have the same type of parasites were considered to be the study group. According to the analyses performed on the animals, the levels of total protein (in Trichostrongylidae, hydatid cysts), globulin, amylase, chlorine, and Vit.B(12) were found to be increased significantly, while the levels of albumin, magnesium, and phosphorus were found to be decreased significantly. The other parameters analyzed were not significant statistically between the groups.
Molina, E C; Lozano, S P; Barraca, A P
The association between visual hepatic damage, burden of Fasciola gigantica, serum levels of gamma glutamyl transferase (GGT) and glutamate dehydrogenase (GLDH) is described from an abattoir study of 70 cattle in the Philippines. In another abattoir study of 60 cattle, the relationship between burden of F. gigantica and haematological indices was investigated. The degree of visual hepatic damage and burden of F. gigantica were significantly positively related to levels of GGT and GLDH. Red blood cell counts and packed cell volume were significantly inversely related to worm burden, but animals compensated for reduced numbers of red blood cells by increasing red cell haemoglobin content.
Arafa, Waleed M; Shokeir, Khalid M; Khateib, Abdelrahman M
This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A-E). On day zero; groups A-D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0 μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100 μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P < 0.0001), however, praziquantel did not record any reduction of Fasciola egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P < 0.01). In albendazole, the hatching varied according to the drug concentration. At the highest two concentrations; 0.2 and 2.0 μg/ml, the hatching percentages were 7.4 ± 1.6 and 5.6 ± 1.5 (P < 0.01) respectively. On the contrary, there were no significant differences in egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect.
Leonard, Jeremy A; Cope, W Gregory; Barnhart, M Christopher; Bringolf, Robert B
Androgen-induced masculinization of female aquatic biota poses concerns for natural population stability. This research evaluated the effects of a twelve day exposure of fadrozole hydrochloride on the metabolism and reproductive status of the unionid mussel Lampsilis fasciola. Although this compound is not considered to be widespread in the aquatic environment, it was selected as a model aromatase (enzyme that converts testosterone to estradiol) inhibitor. Adult mussels were exposed to a control and 3 concentrations of fadrozole (2μg/L, 20μg/L, and 50μg/L), and samples of gill tissue were taken on days 4 and 12 for metabolomics analysis. Gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Daily observed mussel behavior included female mantle display, foot protrusion, siphoning, and larval (glochidia) releases. Glochidia mortality was significantly higher in the 20μg/L treatment. Fewer conglutinate (packets of glochidia) releases were observed in the 50μg/L treatment, and mortality was highly correlated to release numbers. Foot protrusion was significantly higher in females in nearly all treatments, including the control, during the first 4days of observations. However, this sex difference was observed only in the 50μg/L treatment during the last 8days. Generally, metabolites were significantly altered in female gill tissue in the 2μg/L treatment whereas males were mostly affected only at the highest (50μg/L) treatment. Both sexes also revealed significant reductions in fadrozole-induced metabolic effects in gill tissue sampled after 12days compared to tissue sampled after 4days, indicating time-dependent mechanisms of disruptions in metabolic pathways and homeostatic processes to compensate for such disruptions.
Estuningsih, Endah; Spithill, Terry; Raadsma, Herman; Law, Ruby; Adiwinata, G; Meeusen, Els; Piedrafita, David
The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti-F. gigantica antibodies in serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood, feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than the anti-F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The coproantigen ELISA was able to detect 100% of the cattle with >15 flukes. A survey of 305 cattle in central Java over a 10-mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful diagnostic method to determine patent F. gigantica infections in cattle.
Imani-Baran, Abbas; Yakhchali, Mohammad; Malekzadeh Viayeh, Reza; Paktarmani, Razieh
Fasciolosis is an important disease in veterinary medicine worldwide, and is a cause of great economic loss in livestock husbandry in Iran. This study was aimed to determine prevalence of Fasciola gigantica infection in field-collected snails of Radix gedrosiana in northwestern Iran. The snails were collected from 28 perennial and seasonal freshwater habitats from May to December 2010 and identified. A fragment of 618 bp of 28s rRNA gene was amplified by polymerase chain reaction (PCR). The PCR products were subjected to restriction fragment length polymorphism (RFLP) using DraII and AvaII enzymes. PCR-RFLP patterns revealed that 3.12% of the snails were infected with F. gigantica. It was also found that the infected snails had a limited distribution over the water bodies located in the central part of the region. It was concluded that PCR-RFLP was a reliable approach to detect Fasciola infection in pond snails and may be useful to establish control measures for livestock and humans' fasciolosis in the region.
Lyngdoh, Damanbha; Sharma, Sunil; Roy, Bishnupada; Tandon, Veena
The parasitic flukes of the genus Fasciola (Platyhelminthes: Trematoda: Digenea) cause fascioliasis or liver-rot disease in ruminant livestock in tropical and sub-tropical regions of the world. Classically, two species of Fasciola- F. hepatica and F. gigantica, are universally recognized as taxonomically valid species. Our survey studies on ovid and bovid animals including yak and mithun from high altitudinal mountainous regions in Northeast India revealed the occurrence of Fasciola gigantica and also Fasciola sp.- an intermediate form, at altitudes between 5000 and 14,085 feet above sea level (asl). Two morphotypes- F. hepatica - like and F. gigantica - like, of Fasciola species were reported from the high altitudinal areas of Northeast India; most of these locales constitute new-locality and first records for the occurrence of these liver flukes.
Nogueira, Lygia S; Bianchini, Adalto; Wood, Chris M; Loro, Vania L; Higgins, Sarah; Gillis, Patricia L
The salinization of freshwater can have negative effects on ecosystem health, with heightened effects in salt-sensitive biota such as glochidia, the larvae of freshwater mussels. However, the toxicological mechanism underlying this sensitivity is unknown. Therefore, Lampsilis fasciola glochidia were exposed to NaCl (nominally 0.25 and 1.0 g/L) prepared in reconstituted moderately-hard water (control), as well as to a dilution of that water (1:4) with ultrapure reference water (diluted control). Unidirectional Na(+) influx (measured with (22)Na) was evaluated after 1, 3 and 48 h of exposure. In addition, unidirectional Cl(-) influx (measured with (36)Cl), whole-body ion (Cl(-) and Na(+)) concentrations, and glochidia viability (measured as the ability to close valves) were assessed after 48 h of exposure. Significantly reduced glochidia viability (56%) was observed after exposure to 1.0 g/L NaCl. Na(+) influx was significantly higher in glochidia exposed to both 0.25 and 1.0 g/L NaCl for 1h than in those kept under control conditions. After 3 and 48 h of exposure, differences in Na(+) influx rate between salt-exposed and control glochidia were generally reduced, indicating that larvae may be able to, at least temporarily, recover their ability to regulate Na(+) influx when exposed to elevated NaCl concentration. Compared to the moderately-hard water control, whole-body Na(+) and Cl(-) concentrations were relatively unchanged in glochidia exposed to 0.25 g/L NaCl, but were significantly elevated in glochidia exposed to 1.0 g/L NaCl and the diluted control. While Na(+) influx rate had recovered to the control level after 48 h of exposure to 1.0 g/L NaCl, Cl(-) influx rate remained elevated, being ~7-fold higher than the Na(+) influx rate. These findings suggest that the loss of viability observed when glochidia were exposed to a high NaCl concentration (1.0 g/L) could be caused by ionoregulatory disturbances mainly associated with an elevated Cl(-) influx.
Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert
Background Tropical 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. Methods In 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. Results The 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). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402
Zali, Mohammad Reza; Ghaziani, Tahereh; Shahraz, Saeed; Hekmatdoost, Azita; Radmehr, Ali
Background Fasciola hepatica primarily involves the liver, however in some exceptional situations other organs have been reported to be involved. The ectopic involvement is either a result of Parasite migration or perhaps eosinophilic reaction. Case presentation Here we report a known case of multiple myeloma who was under treatment with prednisolone and melphalan. He was infected by Fasciola hepatica, which involved many organs and the lesions were mistaken with metastatic ones. Discussion Presented here is a very unusual case of the disease, likely the first case involving the pancreas, spleen, and kidney, as well as the liver. PMID:15294025
Grabner, Daniel S.; Mohamed, Faten A. M. M.; Nachev, Milen; Méabed, Eman M. H.; Sabry, Abdel Hameed A.; Sures, Bernd
The liver fluke Fasciola gigantica is a trematode parasite of ruminants and humans that occurs naturally in Africa and Asia. Cases of human fascioliasis, attributable at least in part to F. gigantica, are significantly increasing in the last decades. The introduced snail species Galba truncatula was already identified to be an important intermediate host for this parasite and the efficient invader Pseudosuccinea columella is another suspect in this case. Therefore, we investigated snails collected in irrigation canals in Fayoum governorate in Egypt for prevalence of trematodes with focus on P. columella and its role for the transmission of F. gigantica. Species were identified morphologically and by partial sequencing of the cytochrome oxidase subunit I gene (COI). Among all 689 snails found at the 21 sampling sites, P. columella was the most abundant snail with 296 individuals (42.96%) and it was also the most dominant species at 10 sites. It was not found at 8 sites. Molecular detection by PCR and sequencing of the ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA) revealed infections with F. gigantica (3.38%), Echinostoma caproni (2.36%) and another echinostome (7.09%) that could not be identified further according to its sequence. No dependency of snail size and trematode infection was found. Both high abundance of P. columella in the Fayoum irrigation system and common infection with F. gigantica might be a case of parasite spill-back (increased prevalence in local final hosts due to highly susceptible introduced intermediate host species) from the introduced P. columella to the human population, explaining at least partly the observed increase of reported fascioliasis-cases in Egypt. Eichhornia crassipes, the invasive water hyacinth, which covers huge areas of the irrigation canals, offers safe refuges for the amphibious P. columella during molluscicide application. As a consequence, this snail dominates snail communities and efficiently transmits F. gigantica
Greter, Helena; Batil, Annour A; Alfaroukh, Idriss O; Grimm, Felix; Ngandolo, Bongo N; Keiser, Jennifer; Utzinger, Jürg; Zinsstag, Jakob; Hattendorf, Jan
At Lake Chad in central Africa, livestock fascioliasis caused by Fasciola gigantica represents a major veterinary health problem, particularly in cattle reared in mobile pastoralist husbandry systems. We assessed re-infection after a single dose of triclabendazole with fascioliasis in cattle in a mobile pastoralist setting towards the end of the dry season. Within the cattle herds of 14 groups of mobile pastoralists, 375 cattle were randomly selected. A faecal sample was obtained from each animal to determine the prevalence of F. gigantica. Animals were administered a single oral dose of triclabendazole (12mg/kg). A second faecal sample was obtained 6-month post-treatment after cattle had returned from the annual migration cycle. Faecal samples were fixed in sodium acetate-acetic acid-formalin (SAF), and examined for F. gigantica using the sedimentation technique. From the 375 cattle enrolled at baseline, 198 animals (53%) in 12 groups of mobile pastoralists were re-sampled at the 6-month follow-up. Baseline prevalence did not differ noteworthy between animals lost to follow-up and those re-examined. At baseline, bovine fascioliasis prevalence in cattle with follow-up data was 41.9% (95% confidence interval (CI) 35.2-48.9%). At the 6-month post-treatment follow-up, the prevalence was 46.0% (95% CI 39.2-52.9%), ranging between 0% and 75% at the herd level. The mean faecal egg counts at the unit of the herd were higher at follow-up compared to baseline. The observed persistent high prevalence of F. gigantica infection in cattle shows that a single pre-rainy season treatment does not prevent rapid re-infection despite the partial migration away from the high-risk areas at Lake Chad into drier areas. A locally adapted strategic control package for fascioliasis in cattle in the Lake Chad area ought to integrate targeted triclabendazole treatment and seasonal transhumance practices.
composites produced by the helminth Fasciola hepatica for biological microencapsulation . The natural microcapsule can withstand treatment with strong...can take advantage of large batch fermentation technology. 3. Methods for Microencapsulation Numerous methods exist for the production of microcapsules ...Biodegradable Bioadherent Microcapsules for Orally Administered Sustained Release Vaccines PRINCIPAL INVESTIGATOR: Cynthia L. Sheffield, Ph.D
... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Ivermectin and clorsulon. 522.1193 Section 522.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...) (Fasciola hepatica); grubs (parasitic stages) (Hypoderma bovis, H. lineatum); lice (Linognathus...
... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Ivermectin and clorsulon. 522.1193 Section 522.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...) (Fasciola hepatica); grubs (parasitic stages) (Hypoderma bovis, H. lineatum); lice (Linognathus...
... tapeworms (Moniezia benedeni and M. expansa); adult and 4th stage larvae of stomach worms (brown stomach... flukes (Fasciola hepatica and Fascioloides magna); heads and segments of common tapeworms (Moniezia expansa) and fringed tapeworm (Thysanosoma actinioides); adult and fourth stage larvae of stomach...
... tapeworms (Moniezia benedeni and M. expansa); adult and 4th stage larvae of stomach worms (brown stomach... flukes (Fasciola hepatica and Fascioloides magna); heads and segments of common tapeworms (Moniezia expansa) and fringed tapeworm (Thysanosoma actinioides); adult and fourth stage larvae of stomach...
... tapeworms (Moniezia benedeni and M. expansa); adult and 4th stage larvae of stomach worms (brown stomach... flukes (Fasciola hepatica and Fascioloides magna); heads and segments of common tapeworms (Moniezia expansa) and fringed tapeworm (Thysanosoma actinioides); adult and fourth stage larvae of stomach...
Nguyen, T G T; Le, T H; Dao, T H T; Tran, T L H; Praet, N; Speybroeck, N; Vercruysse, J; Dorny, P
A cross-sectional survey on bovine fasciolosis was conducted in the Binh Dinh province of Central Vietnam that was previously identified as hyperendemic for human fasciolosis. In Vietnam, both pure Fasciola gigantica and hybrid and/or introgressed populations of liverflukes bearing genetic material from both Fasciola hepatica and F. gigantica infect humans and animals. In this study, 825 cattle were randomly selected from 8 of the 11 provincial districts for faecal collection; blood samples were taken from 400 of these animals. Fasciola eggs and antibodies against Fasciola were detected by a quantitative sedimentation method and an Enzyme Linked Immunosorbent Assay, respectively. Overall, 54.9% of the animals were shedding Fasciola eggs while 72.2% were Fasciola seropositive. Animals under two years showed lower Fasciola infection rates than older animals. There were no differences in infection rates between districts. These results indicate a very high prevalence of Fasciola infections in cattle in Binh Dinh province. It is concluded that a fasciolosis control programme should be designed in this region aiming at reducing infection in both cattle and humans.
Espino, A M; Finlay, C M
A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive. PMID:8126178
Selemetas, N; Phelan, P; O'Kiely, P; Waal, T de
Fasciolosis caused by Fasciola hepatica is generally a subclinical infection of dairy cows and can result in marked economic losses on Irish dairy farms. This study investigated the exposure to F hepatica in 237 dairy cow herds, using an in-house antibody-detection ELISA applied to bulk tank milk (BTM) samples collected in the autumn of 2012. A total of 364 BTM samples were collected from 237 different herds, with 127 farmers submitting BTM samples in two consecutive months. Analysis of the BTM samples indicated that 67 per cent (n= 159) of the dairy herds had been exposed to F hepatica. Rainfall, temperature and soil types were significantly different between the exposed and non-exposed herds (P<0.05), highlighting the role of these variables to the exposure to F hepatica. Among the 127 herds that provided two monthly milk samples, 83 herds were exposed to F hepatica and 82 increased their F hepatica antibody levels at the later sampling time (P<0.01).The findings of this study confirm the high prevalence of F hepatica antibodies in Irish dairy herds and show the rise in antibody levels during autumn. This study is the first step towards assessing the spatiotemporal pattern of fasciolosis in dairy herds in Ireland.
Andrews, P.; Thomas, H.; Weber, H.
/sup 14/C-praziquantel was rapidly taken up by Schistosoma mansoni, Fasciola hepatica, Hymenolepis nana, and isolated strobilocerci of Taenia taeniaeformis. Schistosoma mansoni lost praziquantel rapidly to drug-free medium. Chromatography of extracts prepared after incubation of S. mansoni and H. nana yielded no indication that praziquantel was metabolized. Autoradiography revealed a uniform distribution of praziquantel throughout the tissues of S. mansoni and H. nana. Uptake was considerably slower in the nematode Heterakis spumosa and apparently via the oral route.
NO Arlington, VA 22217-5000 61153N RR4106 11 TITLE (Include Security Classification) (u) Synthesis of Stable Microcapsules from Trematode Eggshell...Continue on reverse if necessary and identify by block number) The trematode Fasciola hepatica produces a unique protein eggshell or microcapsule the...proteins to produce a hard quinone tanned microcapsule with unusual properties. The focus of this project is to i) characterize the protein components
Mokhtarian, Kobra; Akhlaghi, Lame; Meamar, Ahmad Reza; Razmjou, Elham; Manouchehri Naeini, Kourosh; Gholami, Samaneh; Najafi Samei, Masoomeh; Falak, Reza
In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.
Zhang, Xiao-Xuan; Feng, Sheng-Yong; Ma, Jian-Gang; Zheng, Wen-Bin; Yin, Ming-Yang; Qin, Si-Yuan; Zhou, Dong-Hui; Zhao, Quan; Zhu, Xing-Quan
The aim of this study was to determine the seroprevalence and risk factors of fascioliasis in yaks, Bos grunniens, from 3 counties of Gansu Province in China. A total of 1,584 serum samples, including 974 samples from white yaks from Tianzhu, 464 from black yaks from Maqu, and 146 from black yaks from Luqu County, were collected and analyzed using ELISA to detect IgG antibodies against Fasciola hepatica. The overall F. hepatica seroprevalence was 28.7% (454/1,584), with 29.2% in white yaks (284/974) and 27.9% in black yaks (170/610). The seroprevalence of F. hepatica in yaks from Tianzhu, Luqu, and Maqu was 29.2%, 22.6%, and 29.5%, respectively. Female yaks (30.9%) had higher F. hepatica seroprevalence than male yaks (23.4%). Also, F. hepatica seroprevalence varied by different age group from 24.1% to 33.8%. Further, the seroprevalence ranged from 21.8% to 39.1% over different seasons. Interestingly, the season and age of yaks were associated with F. hepatica infection in yaks in the investigated areas. These findings provided a basis for further studies on this disease in yaks from 3 counties of Gansu Province in northwestern China, which may ultimately support the development of effective control strategies of fascioliasis in these areas.
Zhang, Xiao-Xuan; Feng, Sheng-Yong; Ma, Jian-Gang; Zheng, Wen-Bin; Yin, Ming-Yang; Qin, Si-Yuan; Zhou, Dong-Hui; Zhao, Quan; Zhu, Xing-Quan
The aim of this study was to determine the seroprevalence and risk factors of fascioliasis in yaks, Bos grunniens, from 3 counties of Gansu Province in China. A total of 1,584 serum samples, including 974 samples from white yaks from Tianzhu, 464 from black yaks from Maqu, and 146 from black yaks from Luqu County, were collected and analyzed using ELISA to detect IgG antibodies against Fasciola hepatica. The overall F. hepatica seroprevalence was 28.7% (454/1,584), with 29.2% in white yaks (284/974) and 27.9% in black yaks (170/610). The seroprevalence of F. hepatica in yaks from Tianzhu, Luqu, and Maqu was 29.2%, 22.6%, and 29.5%, respectively. Female yaks (30.9%) had higher F. hepatica seroprevalence than male yaks (23.4%). Also, F. hepatica seroprevalence varied by different age group from 24.1% to 33.8%. Further, the seroprevalence ranged from 21.8% to 39.1% over different seasons. Interestingly, the season and age of yaks were associated with F. hepatica infection in yaks in the investigated areas. These findings provided a basis for further studies on this disease in yaks from 3 counties of Gansu Province in northwestern China, which may ultimately support the development of effective control strategies of fascioliasis in these areas. PMID:28285513
Zainalabidin, Fazly Ann; Azmi, Muhamad Syamsul Naim Noor; Bakri, Wan Normaziah Wan Omar; Sathaya, Geethamalar; Ismail, Mohd Iswadi
Fascioliasis, or trematode infestation, is an important disease caused by Fasciola hepatica and Fasciola gigantica. Both species are hepatic parasites that affect humans. We have examined the zoonotic aspects of fascioliasis. A total of 80 fresh liver samples were collected from 67 Kedah-Kelantan crossbred cattle and 13 Murrah buffalo at 4 local abattoirs in Perak, Malaysia. The samples were examined macroscopically to detect the presence of Fasciola spp. The results show 7.50% (6 of 80) of the animals were diagnosed with fascioliasis. Overall, 7.46% (5 of 67) and 7.69% (1 of 13) of cattle and buffalo samples were positive, respectively. There were only F. gigantica species identified in the samples. Our findings suggest that precautions should be taken because the disease has a zoonotic impact on public health. PMID:26868715
Carothers, Zane B.; Kreitner, Gerald L.
Spermatids of the liverwort Marchantia polymorpha were shown by analysis of thin-sections to contain a four-layered organelle, the Vierergruppe, lying between the flagellar bases above and a subjacent mitochondrial body. The uppermost layer consists of parallel tubules structurally similar to those of the axoneme. Some of the tubules extend beyond the four-layered region to form a long ribbon-like appendage. The second layer consists of low, vertical, membranous fins connected by numerous short links to the tubules above. The third layer, limited above and below by two horizontal membranes, consists of vertical partitions, apparently continuous with the fins. The fourth and lowermost layer consists of narrow parallel tubules aligned beneath alternate partitions. The long axes of the lower three layers are parallel to each other and lie at an angle of 48° to the tubule axis of the uppermost stratum. A graphic representation of a three-dimensional scale model interpreting organelle structure is given. The Vierergruppe supersedes Dreiergruppe interpretation in most if not all of the bryophytes studied to date. PMID:6033941
Carothers, Zane B.
An ultrastructural study of late-stage androgonial cells of Blasia pusilla, a thallose liverwort, showed the nearly spherical nuclei often lying close or appressed to the cell walls. In some cells the two membranes comprising the nuclear envelope separated, the inner membrane continuing intact as a limiting boundary of the nucleus and the membrane on the outer, cytoplasmic side recurving away from the nucleus to continue without evident interruption around the periphery of the cell as the plasma membrane. It is believed that Blasia offers the first completely convincing demonstration of the heretofore problematic continuity of cytoplasmic membranes. A possible sequence of events leading to this unusual relationship between nucleus and cytoplasm is suggested. The sequence includes blebbing of the outer membrane of the nuclear envelope and subsequent membrane proliferation, apparent isolation of cytoplasmic ground substance, fusion of internal membrane with the ectoplast, and migration that finally brings the nucleus into flat contact with the wall. While this manifestation of membrane continuity may be anomalous, it is not presently considered the result of cell injury. PMID:5061948
Bosco, Antonio; Rinaldi, Laura; Musella, Vincenzo; Amadesi, Alessandra; Cringoli, Giuseppe
The objective of the present study was to investigate whether climate change in recent years have influenced the onset of acute outbreaks of Fasciola hepatica in ovine farms in southern Italy. In May-June 2014, a severe outbreak of F. hepatica occurred in three sheep farms in the Campania region. Clinical, coprological and necroscopic examinations were performed. Morbidity and mortality due to F. hepatica were 3-67% and 3-50%, respectively. Coprological examinations showed high values of F. hepatica eggs per gram (EPG) of faeces (860-1,240). Similarly, high adult parasitic burdens were found in animals that had sucombed (124-426 flukes). The study area was georeferenced and climatic data (temperature, humidity, days of rain and total amount of rainfall) were recorded at four georeferenced meterological stations in the study area. Montly data were processed and analyzed for the period 2000-2013 to evaluate the change of the climatic parameters during these years. The results show that there was a significant increase (P<0.001) of temperature, increased rainfall and increase in the number of rainy days compared to previous years. In addition to the outbreak reported here, we discuss the potential effects of climate change on the epidemiology of F. hepatica and the implications for sheep farming in the Mediterranean area.
HOSSEINI, Ghasem; SARKARI, Bahador; MOSHFE, Abdolali; MOTAZEDIAN, Mohammad Hossein; ABDOLAHI KHABISI, Samaneh
Background: Fascioliasis, caused by Fasciola hepatica and F. gigantica, is one of the most important plant and water borne disease in Iran. This study aimed to determine the prevalence of fascioliasis and intestinal helminthes in inhabitants of rural areas of Boyer-Ahmad in Kohgiluyeh and Boyer-Ahmad province in Southwest of Iran. Methods: Stool samples (1025) were collected from inhabitant of 50 randomly selected villages in Boyer-Ahmad Township. Samples were evaluated with modified Telemann and formalin-ethyl acetate methods. Blood samples of Fasciola positive cases were assessed with ELISA and Western blotting. DNA was extracted from Fasciola eggs from stool of positive individuals and evaluated by molecular (PCR) method and the PCR products were sequenced and analyzed. Results: Of the 1025 participants, 473 (46.1%) were male and 552 (53.9%) were female. The mean age of the subjects was 20.25 (±15.86) years. Fasciola eggs were detected in stools of two cases (0.19%). Blood samples were obtained from the Fasciola positive cases and their infection was further confirmed by ELISA and Western blotting. Molecular analysis revealed that both cases are infected with F. hepatica. Furthermore, seven of participants (0.68%) were found to be infected with H. nana, 4 cases (0.39%) with E. vermicularis, and one case (0.09%) with Trichuris trichiura. Conclusion: Findings of this study showed that Boyer-Ahmad district is one of the endemic areas of human fascioliasis in Iran. The study also documented that the rate of helminthic infections in rural areas of the district has drastically declined over the past years. PMID:26744710
Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael
Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this
Carvalho-Costa, Filipe A; Silva, Adriano Gomes; de Souza, Alberto H; Moreira, Carlos J de C; de Souza, Daniela L; Valverde, Joanna G; Jaeger, Lauren H; Martins, Patrícia P; de Meneses, Viviane F; Araújo, Adauto; Bóia, Márcio N
We report the finding of eggs of Calodium spp. (syn. Capillaria spp.; Hepaticola spp.) in a fecal sample from an old woman living in a riverine community in the Negro River Basin and describe the associated epidemiological investigation. The case probably does not represent true parasitism; the eggs, which were compatible with the species Calodium hepaticum, were most likely ingested upon consumption of infected tapir (Tapirus terrestris) liver, subsequently passing through the gut and being eliminated. The evolution of these eggs to infective stages in the environment, given the poor sanitation background, could provide the risk of occurrence of hepatic disease in humans.
Marcos, Luis A; Tagle, Martin; Terashima, Angelica; Bussalleu, Alejandro; Ramirez, Cesar; Carrasco, Carlos; Valdez, Luis; Huerta-Mercado, Jorge; Freedman, David O; Vinetz, Joseph M; Gotuzzo, Eduardo
Fascioliasis is highly endemic in the Andean region of South America. Newer serological assays have improved our ability to diagnose acute fascioliasis. The diagnosis was established by Fasciola hepatica serology (Fas2-ELISA or Western blot) in 10 patients. Identifiable exposure included ingestion of watercress (N = 8), alfalfa juice (N = 5), and lettuce (N = 1). Computed tomography of the abdomen showed hepatomegaly (N = 9), track-like hypodense lesions with subcapsular location (N = 8), and subcapsular hematoma (N = 2). Radiologic sequelae included cyst calcifications detectable at least 3 years after treatment. Stool examinations were negative for F. hepatica eggs; serology was positive (Arc II [N = 2], Fas2-ELISA [N = 6], Western blot [N = 2]). The syndrome of eosinophilia, fever, and right upper quadrant pain, elevated transaminases without jaundice, hypodense liver lesions on CT, and an appropriate exposure history suggests acute fascioliasis. Fascioliasis is specifically treatable with a single dose of triclabendazole.
Carnevale, S; Rodríguez, M I; Guarnera, E A; Carmona, C; Tanos, T; Angel, S O
Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.
Hakyemez, Ismail Necati; Aktaş, Gülali; Savli, Haluk; Küçükbayrak, Abdülkadir; Gürel, Safiye; Taş, Tekin
Fascioliasis is a worlwide parasitic zoonosis, endemic in south-east mediterranean area, but uncommon in other areas. Clinical signs are usually non-specific. A 32 year old male patient was admitted to our hospital with complaints of abdominal pain, diarrhea, fatigue, nausea, lost of appetite, itching, cough, night sweats and weight loss. Complete blood count revealed hypereosinophilia. The abdominal ultrasound scan was normal. But computed tomography scan revealed irregular nodular lesions in periportal area of the liver. Based on these clinical and radiological signs and continuous hypereosinophilia, the patient was serologically investigated for Fasciola hepatica infection. F. hepatica indirect hemagglutination test in serum was positive at a titer of 1/1280. Single dose Triclabendasole 10mg/kg was administered and repeated two weeks later. Clinical and laboratory signs were completely resolved after treatment. Serological tests for fascioliasis should be included in all patients with hypereosinophilia and abnormal liver CT. PMID:22708044
Tiwari, Farindra; Singh, D. K.
Snail control is one of the most important tools in the campaign to reduce the incidence of fascioliasis. In order to attain this objective, the method of bait formulation in order to contain an attractant and a molluscicide is an expedient approach to lure the target snail population to the molluscicide. This study identifies certain carbohydrates, namely sucrose, maltose, glucose, fructose and starch, for preparing such baits. These were tested on Lymnaea acuminata, an intermediate host of the digenean trematodes Fasciola hepatica and Fasciola gigantica. The behavioural responses of snails to these carbohydrates were examined. Significant variations in behavioural responses were observed in the snail even when the five carbohydrates were used in low concentrations in snail-attractant pellets. Starch emerged as the strongest attractant for Lymnaea acuminata, followed by maltose.
Ai, Lin; Cai, Yu-Chun; Lu, Yan; Chen, Jia-Xu; Chen, Shao-Hong
Fascioliasis is a foodborne zoonotic parasitic disease. We report 4 cases occurring in the same family, in whom diagnosis of acute fascioliasis was established after series of tests. One case was hospitalized with fever, eosinophilia, and hepatic lesions. MRI showed hypodense changes in both liver lobes. The remaining 3 cases presented with the symptom of stomachache only. Stool analysis was positive for Fasciola eggs in 2 adult patients. The immunological test and molecular identification of eggs were confirmed at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China. The results of serological detection were positive in all the 4 patients. DNA sequencing of PCR products of the eggs demonstrated 100% homology with ITS and cox1 of Fasciola hepatica. The conditions of the patients were not improved by broad-spectrum anti-parasitic drugs until administration of triclabendazole. PMID:28285507
Bless, Philipp J; Schär, Fabian; Khieu, Virak; Kramme, Stefanie; Muth, Sinuon; Marti, Hanspeter; Odermatt, Peter
Large trematode eggs (LTE) resembling Fasciola spp. eggs were reportedly found in the stools of schoolchildren in Kandal province, Cambodia. This study aimed to assess the prevalence of LTE in the stools of children attending the affected school, identify potential risk factors for infection and ascertain the trematode species. We performed a cross-sectional study involving an in-depth questionnaire administered to schoolchildren at the affected school, and examined cattle droppings in the surrounding area and the livers of slaughtered cattle. Three stool samples were examined per child, using Kato-Katz and formalin-ether concentration techniques. In addition, blood serum enzyme-linked immunosorbent assay (ELISA) and coprological polymerase chain reaction (PCR) was conducted for species clarification. Cattle droppings were examined by cup sedimentation and coprological ELISA. LTE were observed in the stools of 106 schoolchildren (46.5%). Two blood serum samples from schoolchildren were positive for Fasciola hepatica in a first ELISA but were negative in a confirmation immunofluorescence antibody test. Out of 221 PCR samples, only one tested positive for Fasciola spp. and none for Fasciolopsis buski. The consumption of raw aquatic plants (odds ratio (OR)=3.3) and fermented fish sauce (OR=2.1) were significantly associated with LTE in the stool. Fasciola spp. flukes were observed in 18.3% of 191 cattle livers. The prevalence of fascioliasis in cattle droppings was 88.8%. The low prevalence of schoolchildren that tested positive for Fasciola spp. with specific molecular diagnostics and who had no diagnostic evidence of F. buski strongly indicates that the majority of microscopically observed LTE are from Echinostoma spp. Fasciola spp. transmission from cattle to human is possible and public health services need to be alerted accordingly.
Ayaz, Muhammad Mazhar; Raza, Muhammad Asif; Murtaza, Saeed; Akhtar, Saleem
The main purpose of this study was to investigate the prevalence of helminths of goats such as Fasciola hepatica, Haemonchus contortus, Paramphistomum cervi, Oesophagostomum columbian, Cotylophoron cotylophorum, Monezia expansa, Oestertagia oestertagi and Oestertagia circumcincta. The overall prevalence of all species of helminthes was 52% in goat. The study was designed to investigate the factors of helminths prevalence on the basis of sex and age of goat with the help of Chi-square. All the results obtained were non-significant due to some factors which directly affects the prevalence of helminths.
Noyer, Charles M; Coyle, Christina M; Werner, Craig; Dupouy-Camet, Jean; Tanowitz, Herbert B; Wiitner, Murray
Human infection with the sheep liver fluke Fasciola hepatica is a global zoonosis that usually parallels the prevalence of infection in sheep and other ruminants. The disease is endemic in South and Central America, Puerto Rico, the Caribbean region, many parts of Africa, Asia, the Middle East, Australia, and China. There have been a number of focal outbreaks reported from Europe, including southern France and the Mediterranean region. Since acute fascioliasis has rarely been reported in the United States, physicians in this country frequently overlook the diagnosis. Therefore, we report a case of acute human fascioliasis and review the pathogenesis, diagnosis, and treatment of this disease in a recently arrived immigrant.
Mas-Coma, S; Bargues, M D; Valero, M A
Fascioliasis and other food-borne trematodiases are included in the list of important helminthiases with a great impact on human development. Six plant-borne trematode species have been found to affect humans: Fasciola hepatica, Fasciola gigantica and Fasciolopsis buski (Fasciolidae), Gastrodiscoides hominis (Gastrodiscidae), Watsonius watsoni and Fischoederius elongatus (Paramphistomidae). Whereas F. hepatica and F. gigantica are hepatic, the other four species are intestinal parasites. The fasciolids and the gastrodiscid cause important zoonoses distributed throughout many countries, while W. watsoni and F. elongatus have been only accidentally detected in humans. Present climate and global changes appear to increasingly affect snail-borne helminthiases, which are strongly dependent on environmental factors. Fascioliasis is a good example of an emerging/re-emerging parasitic disease in many countries as a consequence of many phenomena related to environmental changes as well as man-made modifications. The ability of F. hepatica to spread is related to its capacity to colonise and adapt to new hosts and environments, even at the extreme inhospitality of very high altitude. Moreover, the spread of F. hepatica from its original European range to other continents is related to the geographic expansion of its original European lymnaeid intermediate host species Galba truncatula, the American species Pseudosuccinea columella, and its adaptation to other lymnaeid species authochthonous in the newly colonised areas. Although fasciolopsiasis and gastrodiscoidiasis can be controlled along with other food-borne parasitoses, fasciolopsiasis still remains a public health problem in many endemic areas despite sustained WHO control programmes. Fasciolopsiasis has become a re-emerging infection in recent years and gastrodiscoidiasis, initially supposed to be restricted to Asian countries, is now being reported in African countries.
Pintado, O. I.; Adelman, S. J.
Se determinan las abundancias químicas de estrellas de HgMn usando espectros obtenidos con EBASIM en CASLEO en un rango de longitud de onda comprendido entre los 400 y 890 nm. Los valores iniciales de temperatura efectiva y gravedad superficial se calculan con la fotometría uvbyβ. Las abundancias se calculan usando WIDTH9 y SYNTHE. Los resultados se comparan análisis realizados por los autores usando espectros obtenidos con el espectrógrado REOSC del CASLEO, el espectrógrafo echelle del Telescopio Anglo-Australiano y el espectrógrafo Coudé del Dominion Astrophysical Observatory.
Muñoz-Caro, Guillermo Manuel
Las nubes moleculares son regiones de formación de estrellas, con temperaturas cinéticas entre 10-50 K y densidades de 103-106 átomos cm-3. Su materia está formada por gas y polvo interestelar. Estas partículas de polvo están cubiertas por una fina capa de hielo, de unos 0.01 μm, que contiene H2O y a menudo CO, CO2, CH3OH y NH3. El hielo es presumiblemente irradiado por fotones ultravioleta y rayos cósmicos en las zonas poco profundas de las nubes moleculares y las regiones circunestelares. En un sistema de vacío, P ˜ 10-7 mbar, simulamos la deposición de hielo a partir de 10 K y la irradiación ultravioleta por medio de una lámpara de descarga de hidrógeno activada con microondas. La evolución del hielo se observa por medio de un espectrómetro infrarrojo. De este modo es posible determinar la composición del hielo observado en el medio interestelar y predecir la presencia de moléculas aún no detectadas en el espacio, que han sido producto del procesamiento del hielo en nuestros experimentos. También es posible calentar el sistema hasta temperatura ambiente para sublimar el hielo depositado. Cuando el hielo ha sido previamente irradiado, se observa un residuo compuesto por moléculas orgánicas complejas, algunas prebióticas, como varios ácidos carboxílicos, aminas, amidas, ésteres y en menor proporción moléculas heterocíclicas y aminoácidos. Algunas de estas moléculas podrían detectarse en estado gaseoso por medio de observaciones milimétricas y de radio. También podrían estar presentes en el polvo cometario, cuyo análisis químico está planeado por las misiones Stardust y Rosetta. Mientras tanto, nuestro grupo está llevando a cabo el análisis de partículas de polvo interplanetario (IDPs), algunas de las cuales pueden ser de origen cometario. Al igual que ocurre con los productos obtenidos por irradiación del hielo en nuestros experimentos, algunas IDPs son ricas en material orgánico que contiene oxígeno.
González, J. F.; Lapasset, E.
Complementando una línea de trabajo iniciada con anterioridad discutimos la estabilidad del espectrógrafo REOSC de CASLEO en DC para la medición de velocidades radiales en base al análisis de observaciones realizadas en enero y abril de 1997. En esas oportunidades obtuvimos 26 espectros de estrellas patrones y 27 espectros de 3 estrellas usadas como estrellas de referencia en nuestro programa de cúmulos abiertos. Además tomamos 26 espectros de crepúsculo con el telescopio en posiciones cubriendo el rango H=-4,+4 y δ =-90,+30. Mediante correlaciones cruzadas derivamos la velocidad de 19 órdenes en cada uno de estos espectros. En base a un análisis estadístico de los datos obtenidos discutimos la contribución de los distintos factores que afectan a la dispersión de lectura observada. En particular, la flexión del instrumento no introduciría errores significativos cuando se observa con masas de aire menores que 2.0. La dispersión de los valores de velocidad medidos para espectros de alta relación S/N de una misma estrella resultó del orden de 0.5 km/s. La comparación con los valores de velocidad publicados por distintos autores para las estrellas patrones no permite distinguir ninguna diferencia sistemática apreciable de las velocidades de CASLEO, siendo la media cuadrática de los residuos del orden de 1.0 km/s.
Charlier, Johannes; Hostens, Miel; Jacobs, Jos; Van Ranst, Bonny; Duchateau, Luc; Vercruysse, Jozef
The liver fluke Fasciola hepatica is a parasite of ruminants with a worldwide distribution and an apparent increasing incidence in EU member states. Effective control in dairy cattle is hampered by the lack of flukicides with a zero-withdrawal time for milk, leaving the dry period as the only time that preventive treatment can be applied. Here, we present the results of a blinded, randomized and placebo-controlled trial on 11 dairy herds (402 animals) exposed to F. hepatica to 1) assess the effect of closantel treatment at dry-off (or 80–42 days before calving in first-calving heifers) on milk production parameters and 2) evaluate if a number of easy-to-use animal parameters is related to the milk production response after treatment. Closantel treatment resulted in a noticeable decrease of anti-F. hepatica antibody levels from 3–6 months after treatment onwards, a higher peak production (1.06 kg) and a slightly higher persistence (9%) of the lactation, resulting in a 305-day milk production increase of 303 kg. No effects of anthelmintic treatment were found on the average protein and fat content of the milk. Milk production responses after treatment were poor in meagre animals and clinically relevant higher milk production responses were observed in first-lactation animals and in cows with a high (0.3–0.5 optical density ratio (ODR)), but not a very high (≥0.5 ODR) F. hepatica ELISA result on a milk sample from the previous lactation. We conclude that in dairy herds exposed to F. hepatica, flukicide treatment at dry-off is a useful strategy to reduce levels of exposure and increase milk production in the subsequent lactation. Moreover, the results suggest that treatment approaches that only target selected animals within a herd can be developed based on easy-to-use parameters. PMID:22916226
Doğan, Nihal; Koçman, Nazmiye Ulkü
It is known that infections caused by intestinal protozoa and helminths affect over 3.5 million people worldwide. In this case report, a patient with complaints of stomach ache for a long time who received thermal treatment is presented. During this thermal treatment, diarrhoea occurred and multiparasitism was diagnosed with two helminths; pseudoparasitism and multiprotozoa, simultaneously. Stool samples were collected from the patient on three consecutive days and one day after the treatment. All of the samples were prepared with formalin-ether sedimentation techniques after macroscopic and direct microscopic investigation. Cellophane-tape method for Enterobius vermicularis and Taenia spp. and Erlich-Ziehl-Neelsen staining method for coccidian parasites were used. At least four preparations were performed for each sample and serum physiologic, lugol' solution and trichrome stain were used for microscopic investigations.The motile segment she brought was investigated microscopically with Indian ink and identified as Taenia saginata. Under direct microscopy, Blastocystis hominis, Endolimax nana and Fasciola hepatica were seen. By formalin-ether sedimentation techniques, Ascaris lumbricoides, Fasciola hepatica, Blastocystis hominis, Endolimax nana and Entamoeba coli were identified. In recent years, intestinal parasitism is rarely seen in our city; therefore, multiparasitism in an adult and immunocompetent patient is interesting.
Caprino, Paola; Ferranti, Fabrizio; Passa, Giuseppe; Quintiliani, Alberto
The human infestation caused by Fasciola hepatica is a rare zoonosis, with an incidence of about 10 cases/year in Italy. We report a case of cholecystitis and obstructive jaundice in a patient affected by fascioliasis in which the diagnosis was secondary to the extraction of viable flukes from the bile duct during ERCP. The endoscopic examination permits, in addition to a rapid, correct diagnosis, direct clearance of the bile ducts. Oral drug therapy, when carried out following the endoscopic treatment, is aimed at killing any flukes potentially evading mechanical clearance. The healing achieved is confirmed by normalisation of antibody levels 6-12 months after therapy. Cholecystectomy is indicated and appropriate for the frequent occurrence of biliary colic related to acute and chronic cholecystitis and cholelithiasis, induced by the presence of the flukes. Infestation by Fasciola hepatica has to be considered among the differential diagnoses of obstructive jaundice. ERCP plays a major diagnostic and therapeutic role, and cholecystectomy, considering the pathogenetic effects of flukes on the organ, is mandatory.
MANOUCHEHRI NAEINI, Kouroush; MOHAMMAD NASIRI, Farnaz; ROKNI, Mohammad Bagher; KHEIRI, Soleiman
Background: Fascioliasis caused by Fasciola hepatica and F. gigantica is a foodborne and waterborne zoonotic disease with a worldwide distribution. The illness occurs in regions with intensive sheep or cattle production. In some parts of Iran the incidence of human infection has increased over the past decades. Since Chaharmahal and Bakhtiyari Province has been known as a main pole of traditional animal husbandry and there was no information about human fascioliasis in this region, the present study was carried out on human population to determine the seroprevalence of this infection. Methods: In this cross-sectional study 1475 serum samples collected from individuals attended to clinical laboratory of the province were subjected to ELISA to detect anti-F. hepatica IgG antibodies. The socio-demographic characteristics of the subjects were also collected through questionnaires. Results: In spite of expectation, the present study showed that out of the 1475 serum samples examined anti-F. hepatica IgG antibodies were found only in two of the subjects (0.135%). Therefore, because of the very few number of positive cases no statistical analysis was applicable. Conclusion: According to the finding of this investigation, human fascioliasis may not be an important health problem in this region of Iran. However, for a better understanding of situation of this parasitic infection in this area more comprehensive animal and human studies are needed. PMID:27648421
Rubel, Diana; Prepelitchi, Lucila; Kleiman, Florencia; Carnevale, Silvana; Wisnivesky-Colli, Cristina
An epidemiological focal study was performed in Loncopué, Neuquén, Argentina, in November 2002 to detect the origin of the infection in a human case of fascioliasis confirmed by an indirect-ELISA test, six months before the study. Thirty five individual fecal samples were taken from domestic livestock, and watercress plants and snails were collected from the irrigation ditches connected to a main canal in the surroundings of the patient's house. A new blood sample was taken from the already recovered patient. The patient was still seropositive to Fasciola hepatica antigens. No metacercariae were found in the 222 watercress leaves checked. All the snails collected (n=130) were identified as Lymnaea viatrix and two out of 101 (2%) were infected with F. hepatica larvae. Coprological analysis showed F. hepatica eggs in 100% of goats (10/10), 82% of sheep (9/11) and 86% of bovines (6/7). The number of eggs per gram shed by positive goats (median=20.7, Q1=6.2, Q3=34.5) and sheep (4, 1.8, 13) was significantly higher than in cows (0.3, 0.3, 1.7) (p < 0.01). Local veterinary control programs were apparently not effective in this case. Anthelmintics used and treatment schedule should be revised and small herds raised at households should also be included and treated.
Silva, Ana Elisa Pereira; Freitas, Corina da Costa; Dutra, Luciano Vieira; Molento, Marcelo Beltrão
Fasciola hepatica is the causative agent of fasciolosis, a disease that triggers a chronic inflammatory process in the liver affecting mainly ruminants and other animals including humans. In Brazil, F. hepatica occurs in larger numbers in the most Southern state of Rio Grande do Sul. The objective of this study was to estimate areas at risk using an eight-year (2002-2010) time series of climatic and environmental variables that best relate to the disease using a linear regression method to municipalities in the state of Rio Grande do Sul. The positivity index of the disease, which is the rate of infected animal per slaughtered animal, was divided into three risk classes: low, medium and high. The accuracy of the known sample classification on the confusion matrix for the low, medium and high rates produced by the estimated model presented values between 39 and 88% depending of the year. The regression analysis showed the importance of the time-based data for the construction of the model, considering the two variables of the previous year of the event (positivity index and maximum temperature). The generated data is important for epidemiological and parasite control studies mainly because F. hepatica is an infection that can last from months to years.
Getachew, M; Trawford, A; Feseha, G; Reid, S W J
The general prevalence and population composition of gastrointestinal and pulmonary helminths of working donkeys were studied. For the purpose 2935 working donkeys were coprologically examined for nematode and cestode, and 215 donkeys for trematode infections. Seven donkeys that died due to various health problems or were euthanased on a welfare ground were necropsied and the parasites were recovered and identified to the species level. The study was conducted during the periods 1996-1999.Coprological examination revealed 99% strongyle, 80% Fasciola, 51% Parascaris, 30% Gastrodiscus, 11% Strongyloides westeri, 8% cestodes and 2% Oxyuris equi infection prevalence. Over 55% of donkeys had more than 1000 eggs per gram of faeces (epg). Forty two different species of parasites consisting of 33 nematodes, 3 trematodes, 3 cestodes and 3 arthropod larvae were identified from postmortem examined donkeys. Among the nematodes 17 species of Cyathostominae and 7 species of Strongylinae were identified. Other parasites identified include, Habronema muscae, Draschia megastoma, Trichostrongylus axei, Strongyloides westeri, Anoplocephala perfoliata, Anoplocephala magna, Anoplocephaloides (Paranoplocephala) mamillana, Parascaris equorum, Fasciola hepatica, Fasciola gigantica, Gastrodiscus aegyptiacus, Dictyocaulus arnfieldi, Oxyuris equi, Probstmayria vivipara, Gasterophilus intestinalis, Gasterophilus nasalis, Rhinoestrus uzbekistanicus and Setaria equina. This study revealed that working donkeys in Ethiopia are infected with a range of helminths and arthropod larvae, which are representatives of the important pathogenic parasites found in equids worldwide.
Dutra, L H; Molento, M B; Naumann, C R C; Biondo, A W; Fortes, F S; Savio, D; Malone, J B
Fasciolosis, caused by Fasciola hepatica, is an endemic disease of ruminants that occurs in several countries of South America where it can lead to decreased production and fertility and, in severe cases, animal death. Although very prevalent, information on the epidemiology of the disease is incomplete in Brazil. The objective of the present study was to define the prevalence of F. hepatica in the livers of cattle from slaughterhouses and correlate the data with the animal's origin (climate and altitude) using a Geographic Information System (GIS). The data was used to create an epidemiological map of fasciolosis by state (Rio Grande do Sul, Santa Catarina, Paraná), by municipality (n=530) and by year (2003-2008). Information was analyzed using a databank from slaughterhouses with Federal Inspection Services of the Ministry of Agriculture. The highest cattle infection rate was found in the two most Southern states of Rio Grande do Sul (18.7%) and Santa Catarina (10.1%). Animals from the Campanha region of Rio Grande do Sul and from the central coast area of Santa Catarina had prevalences of greater than 40%. Cattle from low altitudes municipalities were significantly more likely to have the disease (p<0.05). No significant differences were found between high or low prevalence and ambient temperatures. Risk maps resulting from this study provide information on the epidemiology and transmission of F. hepatica in Southern Brazil needed for design of appropriate control measures to control economic impacts. F. hepatica may represent an important source of zoonotic infection of humans as well; therefore these findings may be complemented by future studies on human infections in high risk areas.
Use of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano.
Hillyer, G V; Soler de Galanes, M; Rodriguez-Perez, J; Bjorland, J; Silva de Lagrava, M; Ramirez Guzman, S; Bryan, R T
A collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotransfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Cobos, F. J.; Langarica, R.
A change in one dimension of the offset guider container is proposed. This modification based on image analysis is carried out in order to guarantee its universality of usage with minimal variations in the original design and minimal alterations in the actual configuration of the peripherical instruments. The performance of the offset guider with all periphericals is expected to be of better (or at least similar) quality than the one presently obtained.
Yilmaz, Hasan; Gödekmerdan, Ahmet
This investigation was carried out to determine the frequency of the fasciolosis in Erciş, a town in the Van province located in the Eastern Turkey. The study includes 500 asymptomatic subjects (322 males and 178 females) whose ages ranged from 5 to 75 years. In all subjects, stool samples were examined at initiation of the study and one week later to rule out pseudoparasitosis. The methods of flotation (in saturated saline solution) and sedimentation (in formalin-ether solution) were used in examination of the stool samples. Fasciola hepatica eggs were detected in nine (1.8%) out of 500 subjects. In conclusion, our data showed that the prevalence of human fasciolosis was high in our region because watercress consumption was common. We also think that human fasciolosis is more frequent than expected in Turkey. To determine the exact prevalence of the disease, extensive serological investigations should be performed in different regions of Turkey.
Moghaddam, A S; Massoud, J; Mahmoodi, M; Mahvi, A H; Periago, M V; Artigas, P; Fuentes, M V; Bargues, M D; Mas-Coma, S
The fascioliasis situation in humans and livestock of Iranian Mazandaran is analysed for the first time. Coprological studies showed 7.3% and 25.4% global prevalences in sheep and cattle, respectively. Studies in slaughterhouses indicate that sheep and cattle may be the main reservoir species, buffaloes may play local roles in the transmission, and goats and horses probably only participate sporadically. Morphometric studies by computer image analysis showed that forms intermediate between Fasciola hepatica and F. gigantica appear in addition to both species. A total of 107 infected humans were diagnosed during the 1999-2002 period, which suggests that fascioliasis may be widespread. The absence of differences in human fascioliasis among gender and age groups differs from other human endemic areas. Both human and animal infections show marked differences between western and eastern Mazandaran. Traditions in herbal condiments for human consumption, methods of animal husbandry and annual rainfall may explain the higher prevalences in western Mazandaran.
Rinaldi, Laura; Gonzalez, Sergio; Guerrero, Jorge; Aguilera, Luisa Carol; Musella, Vincenzo; Genchi, Claudio; Cringoli, Giuseppe
Fasciola hepatica infection is reported from many Latin American countries, with very high prevalence rates in both humans and livestock in the Andean countries. Due to its environmental characteristics, particularly suitable for liver fluke infection, the Cajamarca valley of Peru has often been chosen as a model to study the epidemiology of liver fluke infection in the Andes. In this paper we describe the profile of a project aimed at a multidisciplinary and integrated approach for the control of fascioliasis in animals and humans in this valley. The One-Health integrated approach applied here is based on accurate and sensitive diagnostics, namely the FLOTAC, and the use of geospatial tools for epidemiological scrutiny.
Parkinson, M; O'Neill, S M; Dalton, J P
Fasciolosis, caused by trematodes of the genus Fasciola, is an emerging disease of humans. One of the highest levels of human fasciolosis hepatica is found amongst the indigenous Aymaran people of the Northern Bolivian Altiplano. A meta-analysis of epidemiological surveys from 38 communities in the region demonstrates that fasciolosis has been endemic in the region since at least 1984 and is a zoonosis of rural communities. Human and bovine fasciolosis is associated with the communities lying in the plain from Lake Titicaca to La Paz, predominantly in the Los Andes province. In Los Andes incidences of up to 67% of population cohorts were found, and prevalence is age-related with the highest infection rate in children aged 8-11 years.
Mas-Coma, M S; Esteban, J G; Bargues, M D
The epidemiological picture of human fascioliasis has changed in recent years. The number of reports of humans infected with Fasciola hepatica has increased significantly since 1980 and several geographical areas have been described as endemic for the disease in humans, with prevalence and intensity ranging from low to very high. High prevalence of fascioliasis in humans does not necessarily occur in areas where fascioliasis is a major veterinary problem. Human fascioliasis can no longer be considered merely as a secondary zoonotic disease but must be considered to be an important human parasitic disease. Accordingly, we present in this article a proposed new classification for the epidemiology of human fascioliasis. The following situations are distinguished: imported cases; autochthonous, isolated, nonconstant cases; hypo-, meso-, hyper-, and holoendemics; epidemics in areas where fascioliasis is endemic in animals but not humans; and epidemics in human endemic areas.
Márquez-Navarro, Adrián; Nogueda-Torres, Benjamín; Hernández-Campos, Alicia; Soria-Arteche, Olivia; Castillo, Rafael; Rodríguez-Morales, Sergio; Yépez-Mulia, Lilián; Hernández-Luis, Francisco
The anthelmintic activity of 11 benzimidazole derivatives (A1-A11) and 2 thioureides N,N'-disubstituted (B1-B2) was determined. Each compound and albendazole was tested in vitro against Toxocara canis larvae and in vivo against Hymenolepis nana adult. Compounds A1-A6 and B1-B2 were designed as albendazole prodrugs. Compounds A8-A11 were designed as direct analogues of A7, which had previously proved to be an effective agent against Fasciola hepatica. Results of the in vitro screening showed that A6 was more active than albendazole at 0.18 microM (relative mobility 40% and 80%, respectively). Whereas that the in vivo evaluation against H. nana, compounds A7-A11 demonstrated significant activity in terms of removing cestode adults in the range of 88-97%, displaying better efficacy than albendazole (83%).
Koziol, Uriel; Marín, Monica; Castillo, Estela
Pumilio proteins are proposed to have a conserved primordial function in the maintenance of proliferation in stem cells through post-transcriptional regulation. In this work, a search for pumilio homology domain (PUM-HD) sequences of pumilio genes from several Platyhelminthes species was performed, including representatives form Cestoda, Trematoda and Tricladida. Only one PUM-HD sequence was found in each triclad species; however, two PUM-HD homologues were found in all the parasitic species. These sequences formed two clearly separated clades: PlatyPum1, with sequences from all species, and PlatyPum2, composed exclusively of neodermatan sequences. Therefore, at least one duplication of the pumilio gene must have occurred before the divergence of cestodes and trematodes. Further duplications of PUM-HD were found in Fasciola hepatica, but these consist of retropseudogenes. This is the first comparative analysis of PUM-HD sequences in the Platyhelminthes and, more generally, in any lophotrochozoan phylum.
Schubert, S; Granz, W
Taking into consideration the increasing intercontinental tourist traffic we are in an increasing degree confronted with diseases which above all or exclusively acquired in warm countries. Besides the diseases which occur in our country they must find differential diagnostic consideration in disturbances of the health. On account of their frequency gastrointestinal disturbances they are essential for these travellers. On the basis of the following instances of diseases problems of the differential diagnosis and the evaluation of findings are shortly demonstrated: colon amoeboma; colon carcinoma; colon polyposis; ascaridasis; lambliasis; trichuriasis, affection with fasciola hepatica; affection with fasciolopsis buski; schistosomiasis intestinalis. It is referred to peculiarities in inhabitants of the tropics who transitorily live in our climatic zone.
Mas-Coma, M. S.; Esteban, J. G.; Bargues, M. D.
The epidemiological picture of human fascioliasis has changed in recent years. The number of reports of humans infected with Fasciola hepatica has increased significantly since 1980 and several geographical areas have been described as endemic for the disease in humans, with prevalence and intensity ranging from low to very high. High prevalence of fascioliasis in humans does not necessarily occur in areas where fascioliasis is a major veterinary problem. Human fascioliasis can no longer be considered merely as a secondary zoonotic disease but must be considered to be an important human parasitic disease. Accordingly, we present in this article a proposed new classification for the epidemiology of human fascioliasis. The following situations are distinguished: imported cases; autochthonous, isolated, nonconstant cases; hypo-, meso-, hyper-, and holoendemics; epidemics in areas where fascioliasis is endemic in animals but not humans; and epidemics in human endemic areas. PMID:10327713
Mezghani-Jarraya, R; Hammami, H; Ayadi, A; Damak, M
The molluscicidal activity of Hammada scoparia leaf extracts and the principal alkaloids isolated from them (carnegine and N-methylisosalsoline) were tested against the mollusc gastropod, Galba truncatula, the intermediate host of Fasciola hepatica in Tunisia. The results indicated that the molluscicidal activity was correlated with the presence of alkaloids. A significant molluscicidal value, according to the World Health Organization, was found with the methanol extract (LC50 = 28.93 ppm). Further fractionation of the methanolic extract led to the isolation of two principal alkaloids: carnegine and N-methylisosalsoline. These alkaloids are isoquinolines that have not previously been characterised for their molluscicidal activity. The N-methylisosalsoline possesses the highest molluscicidal activity (LC50 = 0.47 microM against G. truncatula).
Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng
Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703
Walker, S M; Johnston, C; Hoey, E M; Fairweather, I; Borgsteede, F H M; Gaasenbeek, C P H; Prodohl, P A; Trudgett, A
Hares (Lepus europeanus) sharing pasture with cattle from six locations in the Netherlands were examined for the presence of liver fluke (Fasciola hepatica) and shown to have prevalences of infection ranging from 0 to 41%. The mitochondrial haplotypes of liver flukes present in the hare populations were determined and compared with those found in cattle from a farm where triclabendazole resistance has been reported. Phylogenetic analysis indicated that the flukes present in the hares belonged to the same clades as those present in the cattle. A consideration of the life cycle of the liver fluke and the seasonal breeding pattern and ecology of hares supports the suggestion that hares may act as a refugia for liver fluke and as a vector for the spread of drug-resistant genotypes.
Freites, Azael; Colmenares, Cecilia; Alarcón-Noya, Belkisyolé; García, María Eugenia; Díaz-Suárez, Odelis
In Venezuela, human Fasciolosis shows a low frequency. However, Mara Municipality is a highly endemic region for bovine fasciolosis and there are no reports of this parasite infection in humans. To determine the prevalence and associated factors to human fasciolosis in Mara municipality - Zulia state, a total of 51 blood and stool samples were collected. Serums were tested by ELISA and Western Blot (WB) assays, with excretion-secretion antigens of Fasciola hepatica (AFhES). The serum samples that resulted positive by these assays were tested by ELISA IgG anti Toxocara sp, Toxoplasma gondii and cysticerosis. Stool samples were concentrated by the Ritchie and rapid sedimentation techniques. Two serum samples were reactive to ELISA AFhES (3.9%) and these did not recognize the specific molecules of WB-AFhES detected by serum from patients with an absolutely demonstrated infection. Both participants were not positive to IgG anti Toxocara sp, Toxoplasma gondii, cysticerosis, and stool samples of these were negative to intestinal parasites. The general prevalence of intestinal parasites was 52.9% (27/51), being protozoa more frequent than helminthes. No Fasciola eggs were found. The two positives participants had in common that both had worked as fresh pasture cutters. These results suggest that the population had been in contact with F. hepatica, with no active infection because of the lack of specific molecules recognition and the absence of eggs in stool samples. Human fasciolosis has a low frequency in Venezuela and is underestimated and underrecognized by health workers and the general population.
Perez-C, Jorge Enrique; Giraldo-Pinzon, Etna Julieth; Aguilar-Marín, Sandra
Fascioliasis causes significant economic losses to the cattle industry and is considered a reemerging zoonosis. In Caldas-Colombia, an increase of bovine fascioliasis was detected at the Manizales Municipal Slaughterhouse, which is a potential risk to public health. The ecoepidemiology of human fascioliasis was analyzed in a region of bovine fascioliasis in Caldas-Colombia. The risk factors were studied. Samples were taken from 111 people who were directly related to the bovine milk production process. The immunoglobulin G frequency of Fasciola hepatica was determined in serum. A seriate stool test and a molecular analysis were conducted on those with positive results to look for parasite eggs and DNA, respectively. 6.3% of the samples were positive for the presence of antibodies; none was positive for the presence of eggs, while two samples showed a weak amplification band of the 124-bp DNA fragment of F. hepatica. Fifty-seven percent of the positive samples came from places located at 2026 meters above sea level (masl); 71% of people testing positive had been recently dewormed. Also, 86% had been in contact with cattle and handled grass and excrement. They eat salads and drink untreated water from the springs or ravines of the area. An outbreak of human fascioliasis was detected in Caldas, associated with risk factors for the disease. Clinical trials to confirm the presence of the parasite and implement public health control measures are required.
Ferreira, Jorge F S; Peaden, Paul; Keiser, Jennifer
Trematode infections negatively affect human and livestock health, and threaten global food safety. The only approved human anthelmintics for trematodiasis are triclabendazole and praziquantel with no alternative drugs in sight. We tested six crude plant extracts against adult Schistosoma mansoni, Fasciola hepatica, and Echinostoma caproni in vitro. Mortality was best achieved by ethanolic extracts of Artemisia annua (sweet Annie), Asimina triloba (paw-paw), and Artemisia absinthium (wormwood) which, at 2 mg/mL, killed S. mansoni and E. caproni in 20 h or less (except for wormwood), F. hepatica between 16 and 23 h (sweet Annie), or 40 h (paw-paw). Some extracts were active at 0.2 mg/mL and 20 μg/mL, although more time was required to kill trematodes. However, aqueous A. annua and methanol extracts of Fumaria officinalis had no activity. Chromatographic analysis of the three best extracts established that A. annua and A. triloba extracts contained bioactive artemisinin and acetogenins (asimicin and bullatacin), respectively. The anthelmintic activity of our extracts at such low doses indicates that their anthelmintic activity deserves further testing as natural alternative controls for parasites of both animals and humans. Our results also support recent evidence that synergistic effects of multiple bioactive compounds present in crude plant extracts is worth exploring.
Zoonotic parasites are animal parasites that can infect humans. The major zoonotic protozoa in the Republic of Korea are Babesia bovis, Chilomastix mesnili, Cryptosporidium parvum, Endolimax nana, Entamoeba coli, Entamoeba hitolytica, Giardia lamblia, Iodamoeba bütschlii, Pneumocystis carinii, Sarcocystis cruzi, and Toxoplasma gondii. The major zoonotic helminths in Korea include trematodes, cestodes, and nematodes. Trematodes are Clonorchis sinensis, Echinostoma hortense, Echinostoma spp., Fasciola hepatica, Heterophyes nocens, Metagonimus yokogawai, and Paragonimus westermani. Cestodes are Diphyllobothrium latum, Dipylidium caninum, Echinococcus granulosus, Hymenolepis nana, Raillietina tetragona, sparganum (Spirometra spp.), Taenia saginata, T. solium, and T. asiatica. Nematodes are Ancylostoma caninum, Brugia malayi, Capillaria hepatica, Dirofilaria immitis, Gnathostoma dololesi, Gnathostoma spinigerum, Loa loa, Onchocerca gibsoni, Strongyloides stercoralis, Thelazia callipaeda, Trichinella spiralis, Trichostrongylus orientalis, Trichuris trichiura, and Trichuris vulpis. The one arthropod is Sarcoptes scabiei. Many of these parasites have disappeared or were in decline after the 1990's. Since the late 1990's, the important zoonotic protozoa have been C. parvum, E. nana, E. coli, E. hitolytica, G. lamblia, I. buetschlii, P. carinii and T. gondii. The important zoonotic helminths have been C. sinensis, H. nocens, M. yokogawai, P. westermani, D. latum, T. asiatica, sparganum, B. malayi, T. orientalis, T. callipaeda and T. spiralis. However, outbreaks of these parasites are only in a few endemic areas. The outbreaks of Enterobius vermicularis and head lice, human parasites, have recently increased in the kindergartens and primary schools in the Republic of Korea.
Zoonotic parasites are animal parasites that can infect humans. The major zoonotic protozoa in the Republic of Korea are Babesia bovis, Chilomastix mesnili, Cryptosporidium parvum, Endolimax nana, Entamoeba coli, Entamoeba hitolytica, Giardia lamblia, Iodamoeba bütschlii, Pneumocystis carinii, Sarcocystis cruzi, and Toxoplasma gondii. The major zoonotic helminths in Korea include trematodes, cestodes, and nematodes. Trematodes are Clonorchis sinensis, Echinostoma hortense, Echinostoma spp., Fasciola hepatica, Heterophyes nocens, Metagonimus yokogawai, and Paragonimus westermani. Cestodes are Diphyllobothrium latum, Dipylidium caninum, Echinococcus granulosus, Hymenolepis nana, Raillietina tetragona, sparganum (Spirometra spp.), Taenia saginata, T. solium, and T. asiatica. Nematodes are Ancylostoma caninum, Brugia malayi, Capillaria hepatica, Dirofilaria immitis, Gnathostoma dololesi, Gnathostoma spinigerum, Loa loa, Onchocerca gibsoni, Strongyloides stercoralis, Thelazia callipaeda, Trichinella spiralis, Trichostrongylus orientalis, Trichuris trichiura, and Trichuris vulpis. The one arthropod is Sarcoptes scabiei. Many of these parasites have disappeared or were in decline after the 1990's. Since the late 1990's, the important zoonotic protozoa have been C. parvum, E. nana, E. coli, E. hitolytica, G. lamblia, I. buetschlii, P. carinii and T. gondii. The important zoonotic helminths have been C. sinensis, H. nocens, M. yokogawai, P. westermani, D. latum, T. asiatica, sparganum, B. malayi, T. orientalis, T. callipaeda and T. spiralis. However, outbreaks of these parasites are only in a few endemic areas. The outbreaks of Enterobius vermicularis and head lice, human parasites, have recently increased in the kindergartens and primary schools in the Republic of Korea. PMID:19885329
Turner, Joanne; Howell, Alison; McCann, Cathy; Caminade, Cyril; Bowers, Roger G.; Williams, Diana; Baylis, Matthew
Fasciola hepatica, common liver fluke, infects cattle and sheep causing disease and production losses costing approximately $3billion annually. Current control relies on drugs designed to kill the parasite. However, resistance is evident worldwide and widespread in some areas. Work towards a vaccine has identified several antigens of F. hepatica that show partial efficacy in terms of reducing worm burden and egg output. A critical question is what level of efficacy is required for such a vaccine to be useful? We have created the first mathematical model to assess the effectiveness of liver fluke vaccines under simulated field conditions. The model describes development of fluke within a group of animals and includes heterogeneity in host susceptibility, seasonal exposure to metacercariae and seasonal changes in temperature affecting metacercarial survival. Our analysis suggests that the potential vaccine candidates could reduce total fluke burden and egg output by up to 43% and 99%, respectively, on average under field conditions. It also suggests that for a vaccine to be effective, it must protect at least 90% of animals for the whole season. In conclusion, novel, partial, vaccines could contribute substantially towards fasciolosis control, reducing usage of anthelmintics and thus delaying the spread of anthelmintic resistance. PMID:27009747
Chen, Jia-Xu; Chen, Mu-Xin; Ai, Lin; Xu, Xue-Nian; Jiao, Jian-Ming; Zhu, Ting-Jun; Su, Hui-Yong; Zang, Wei; Luo, Jia-Jun; Guo, Yun-Hai; Lv, Shan; Zhou, Xiao-Nong
Fascioliasis is a common parasitic disease in livestock in China. However, human fascioliasis is rarely reported in the country. Here we describe an outbreak of human fascioliasis in Yunnan province. We reviewed the complete clinical records of 29 patients and performed an epidemiological investigation on the general human population and animals in the outbreak locality. Our findings support an outbreak due to Fasciola gigantica with a peak in late November, 2011. The most common symptoms were remittent fever, epigastric tenderness, and hepatalgia. Eosinophilia and tunnel-like lesions in ultrasound imaging in the liver were also commonly seen. Significant improvement of patients' condition was achieved by administration of triclabendazole®. Fasciola spp. were discovered in local cattle (28.6%) and goats (26.0%). Molecular evidence showed a coexistence of F. gigantica and F. hepatica. However, all eggs seen in humans were confirmed to be F. gigantica. Herb (Houttuynia cordata) was most likely the source of infections. Our findings indicate that human fascioliasis is a neglected disease in China. The distribution of triclabendazole®, the only efficacious drug against human fascioliasis, should be promoted.
Status and emerging issues in the use of praziquantel for treatment of human trematode and cestode infections are briefly reviewed. Since praziquantel was first introduced as a broadspectrum anthelmintic in 1975, innumerable articles describing its successful use in the treatment of the majority of human-infecting trematodes and cestodes have been published. The target trematode and cestode diseases include schistosomiasis, clonorchiasis and opisthorchiasis, paragonimiasis, heterophyidiasis, echinostomiasis, fasciolopsiasis, neodiplostomiasis, gymnophalloidiasis, taeniases, diphyllobothriasis, hymenolepiasis, and cysticercosis. However, Fasciola hepatica and Fasciola gigantica infections are refractory to praziquantel, for which triclabendazole, an alternative drug, is necessary. In addition, larval cestode infections, particularly hydatid disease and sparganosis, are not successfully treated by praziquantel. The precise mechanism of action of praziquantel is still poorly understood. There are also emerging problems with praziquantel treatment, which include the appearance of drug resistance in the treatment of Schistosoma mansoni and possibly Schistosoma japonicum, along with allergic or hypersensitivity reactions against praziquantel treatment. To cope with and overcome these problems, combined use of drugs, i.e., praziquantel and other newly introduced compounds such as triclabendazole, artemisinins, and tribendimidine, is being tried. PMID:24265948
Ai, Lin; Xu, Xue-Nian; Jiao, Jian-Ming; Zhu, Ting-Jun; Su, Hui-Yong; Zang, Wei; Luo, Jia-Jun; Guo, Yun-Hai; Lv, Shan; Zhou, Xiao-Nong
Fascioliasis is a common parasitic disease in livestock in China. However, human fascioliasis is rarely reported in the country. Here we describe an outbreak of human fascioliasis in Yunnan province. We reviewed the complete clinical records of 29 patients and performed an epidemiological investigation on the general human population and animals in the outbreak locality. Our findings support an outbreak due to Fasciola gigantica with a peak in late November, 2011. The most common symptoms were remittent fever, epigastric tenderness, and hepatalgia. Eosinophilia and tunnel-like lesions in ultrasound imaging in the liver were also commonly seen. Significant improvement of patients’ condition was achieved by administration of triclabendazole®. Fasciola spp. were discovered in local cattle (28.6%) and goats (26.0%). Molecular evidence showed a coexistence of F. gigantica and F. hepatica. However, all eggs seen in humans were confirmed to be F. gigantica. Herb (Houttuynia cordata) was most likely the source of infections. Our findings indicate that human fascioliasis is a neglected disease in China. The distribution of triclabendazole®, the only efficacious drug against human fascioliasis, should be promoted. PMID:23951181
van Schalkwyk, P C; Geyser, T L; Récio, M; Erasmus, F P
Anthelmintic trials were carried out to evaluate the efficacy of albendazole against helmi of 2,5 to 3,8 mg/kg administered orally, resulted in a 98,8 to 100% reduction of adult parasites of the genera Haemonchus, Ostertagia, Trichostrongylus, Nematodirus, Gaigeria, Oesophagostomum, Chabertia, Marshallagia and Cooperia. Against the immature stages of these genera, except for Marshallagia and Cooperia, which were not tested, a dose level of 2,5 to 3,8 mg/kg was 83,9-100% effective. Albendazole at 2,5 mg/kg was 99,0% effective against adult stages of Dictyocaulus; its activity at a dose of 3,8 mg/kg against the immature stages of D. filaria was 89,3%. In sheep naturally infested with Moniezia, 100% elimination was obtained at a dose level of 2,5 mg/kg. Dose levels of 3,8 mg/kg and higher were more than 76% effective against adult Fasciola hepatica, while a dose of 4,8 mg/kg was 63% effective against adult Fasciola gigantica.
Rodríguez, Diana Catalina; Pino, Nancy; Peñuela, Gustavo
When contaminated water is used to wash the udders of dairy cattle and milking utensils, raw milk may become contaminated with pathogens. Washing with high quality water is essential to reduce the microbial contamination of milk. Furthermore, the wastewater generated in dairy herds also contains high populations of pathogens, antibiotics and nutrients that more often are thrown into the water bodies without any treatment. In this work, both supply water and wastewater from 20 dairy farms from Antioquia, Colombia was monitored for 10months to determine the presence of pathogenic microorganisms. Both Cryptosporidium and Fasciola were determined by the Polymerase Chain Reaction (PCR) technique in real time. The results showed that the supply water used for drinking and activities involving the herd, has high populations of Fasciola hepatica and Cryptosporidium parvum, with percentages of about 53.7% and 64.75% respectively. Additionally high populations of Pseudomonas aeruginosa, Shigella, Salmonella, total coliforms and Escherichia coli were found in both types of water, with values around 9.4×10(7), 2.1×10(7), 1.8×10(7), 1.9×10(10) and 1.5×10(10) UFC/100 ml respectively for the wastewater and 3.1×10(4), 1.9×10(4), 7.3×10(3), 1.2×10(5) and 6.2×10(3) UFC/100 ml for the supply water.
Getachew, M; Innocent, G T; Trawford, A F; Reid, S W J; Love, S
A cross-sectional coprological survey in the tropical regions of Ada, Akaki, Bereh and Boset, and a retrospective post-mortem investigation were conducted to study the epidemiology of fasciolosis in working donkeys in Ethiopia. Faecal samples from 803 donkeys were collected, and the number of liver flukes recovered from 112 donkeys at post-mortem between 1995 and 2004 were analysed. There was a high prevalence of fasciolosis irrespective of the age of the donkeys. The overall prevalence of the infection was 44.4% in coprologically examined donkeys, and the prevalence in the donkeys examined post-mortem was 41.9%. The infection prevalence was significantly higher in Bereh and Ada regions than in Akaki and Boset regions. Bereh with 72.6% and Boset with 21.5% showed a significantly higher and lower infection prevalence, respectively, than the rest of the regions (P<0.001). There was no significant difference between different age groups of donkeys in the infection prevalence (P>0.05) but infection intensity was significantly higher in donkeys 8 years old and above (P<0.0001). Both Fasciola hepatica and Fasciola gigantica were identified.
Bui, T D; Doanh, P N; Saegerman, C; Losson, B
Vietnam is recognized to be endemic for fasciolosis. However, most of the available publications have not been published in international journals. This review is based on national and international Vietnamese publications and highlights the current status of fasciolosis in Vietnam. It also provides some information available for neighbouring countries. Updated data on responsible species, distribution, transmission and control aspects are summarized. The central region of Vietnam is reported as being highly endemic for fasciolosis, with a high number of human patients (more than 20,000 in 2011). Fasciola gigantica is reported to be the main species in Vietnam. However, hybrids between F. gigantica and F. hepatica were identified. Both humans and animals are infected by the ingestion of raw vegetables and possibly contaminated drinking water. Three lymnaeid snail species (Austropeplea viridis, Radix auricularia and Radix rubiginosa) may act as intermediate hosts of Fasciola spp. However, due to the likely misidentification of snail species and cercariae during the past decade the critical analysis of published data is difficult. A better understanding of transmission aspects of fasciolosis would allow the implementation of preventive measures of this important neglected zoonotic disease.
Bloemhoff, Yris; Danaher, Martin; Andrew Forbes; Morgan, Eric; Mulcahy, Grace; Power, Clare; Sayers, Ríona
Dictyocaulus viviparus, Ostertagia ostertagi (nematode parasites), and Fasciola hepatica (trematode parasite) result in productivity losses on dairy farms and impact on animal health through clinical and sub-clinical disease. Parasite control in livestock systems is largely based on the use of chemoprophylactic agents (anthelmintics), grazing management, or a combination of both. The objective of this study was to document current parasite control measures employed by Irish dairy farmers in a predominantly pasture-based livestock system. A questionnaire survey of 312 geographically representative farmers was completed in 2009 with a follow up survey completed in 2011. Statistical analysis highlighted significant differences in chemoprophylactic usage between 2009 and 2011. In particular, an increase in the use of albendazole for both trematode (19% in 2009 to 36% in 2011) and nematode (30% in 2009 to 58% in 2011) control was observed. This was most likely due to flukicide restrictions introduced in the Republic of Ireland in 2010 for dairy animals. Logistic regression highlighted regional differences in chemoprophylactic use. Farmers in southern parts of Ireland, an area with good quality soil, less rainfall, and a higher density of dairy farms than other regions, were approximately half as likely to dose for F. hepatica and were more likely (OR>2.0) to use albendazole for both nematode and fluke control. Approximately 30% of respondents who used a chemoprophylactic treatment for nematodes, used a product which was 'unsuitable for purpose' (e.g. ivermectin for the treatment of F. hepatica), highlighting the need for increased awareness, continuing research, and regionally targeted education tools regarding optimal parasite control.
Selemetas, Nikolaos; Phelan, Paul; O'Kiely, Padraig; de Waal, Theo
Fasciolosis caused by Fasciola hepatica is a widespread parasitic disease in cattle farms. The aim of this study was to detect clusters of fasciolosis in dairy cow herds in Munster Province, Ireland and to identify significant climatic and environmental predictors of the exposure risk. In total, 1,292 dairy herds across Munster was sampled in September 2012 providing a single bulk tank milk (BTM) sample. The analysis of samples by an in-house antibody-detection enzyme-linked immunosorbent assay (ELISA), showed that 65% of the dairy herds (n = 842) had been exposed to F. hepatica. Using the Getis-Ord Gi* statistic, 16 high-risk and 24 low-risk (P <0.01) clusters of fasciolosis were identified. The spatial distribution of high-risk clusters was more dispersed and mainly located in the northern and western regions of Munster compared to the low-risk clusters that were mostly concentrated in the southern and eastern regions. The most significant classes of variables that could reflect the difference between high-risk and low-risk clusters were the total number of wet-days and rain-days, rainfall, the normalized difference vegetation index (NDVI), temperature and soil type. There was a bigger proportion of well-drained soils among the low-risk clusters, whereas poorly drained soils were more common among the high-risk clusters. These results stress the role of precipitation, grazing, temperature and drainage on the life cycle of F. hepatica in the temperate Irish climate. The findings of this study highlight the importance of cluster analysis for identifying significant differences in climatic and environmental variables between high-risk and low-risk clusters of fasciolosis in Irish dairy herds.
Alasaad, S; Granados, J E; Cano-Manuel, F J; Meana, A; Zhu, X Q; Pérez, J M
Between 1995 and 2006, we surveyed the presence of Fasciola hepatica in Iberian ibex (Capra pyrenaica) from Andalucía (southern Spain) by both necropsy (n = 2,096) and coprological approaches (n = 380). Most of the samples came from the Sierra Nevada mountain range (n = 1,884 and 267, respectively), and all positive cases involved animals from this location. The prevalence reached 0.53% by necropsy and 1.87% by faecal examination. Taking into account both diagnostic methodologies and the total number of animals affected (n = 14), we obtained a yearly prevalence of 0.7 +/- 0.3%. The infection with F. hepatica was found not to be related to host sex, climatology or to co-infection with Sarcoptes scabiei (the most important parasite affecting Iberian ibex, with a prevalence of 49.27 +/- 7.90% in the examined animals). The prevalence of fasciolosis decreased significantly during the period under study and this would be explained by an increase of ibex resistance to this fluke as a result of a reduction of the parasite abundance in the area and/or a reduction of the host infection rate. There was no statistical difference between the two diagnostic methods for the examination of fasciolosis during the period in which both methods were used. Therefore, examination of faecal samples as a non-invasive procedure may provide a useful approach for monitoring fasciolosis in wild ungulate populations. The results of the present study provided foundation for the effective control of F. hepatica infection in Iberian ibex.
Zumaquero-Ríos, José Lino; Sarracent-Pérez, Jorge; Rojas-García, Raúl; Rojas-Rivero, Lázara; Martínez-Tovilla, Yaneth; Valero, María Adela; Mas-Coma, Santiago
Background The Atlixco municipality, Puebla State, at a mean altitude of 1840 m, was selected for a study of Fasciola hepatica infection in schoolchildren in Mexico. This area presents permanent water collections continuously receiving thaw water from Popocatepetl volcano (5426 m altitude) through the community supply channels, conforming an epidemiological scenario similar to those known in hyperendemic areas of Andean countries. Methodology and Findings A total of 865 6–14 year-old schoolchildren were analyzed with FasciDIG coproantigen test and Lumbreras rapid sedimentation technique, and quantitatively assessed with Kato-Katz. Fascioliasis prevalences ranged 2.94–13.33% according to localities (mean 5.78%). Intensities were however low (24–384 epg). The association between fascioliasis and the habit of eating raw vegetables was identified, including watercress and radish with pronouncedly higher relative risk than lettuce, corncob, spinach, alfalfa juice, and broccoli. Many F. hepatica-infected children were coinfected by other parasites. Entamoeba histolytica/dispar, Giardia intestinalis, Blastocystis hominis, Hymenolepis nana and Ascaris lumbricoides infection resulted in risk factors for F. hepatica infection. Nitazoxanide efficacy against fascioliasis was 94.0% and 100% after first and second treatment courses, respectively. The few children, for whom a second treatment course was needed, were concomitantly infected by moderate ascariasis burdens. Its efficacy was also very high in the treatment of E. histolytica/E. dispar, G. intestinalis, B. hominis, H. nana, A. lumbricoides, Trichuris trichiura, and Enterobius vermicularis. A second treatment course was needed for all children affected by ancylostomatids. Conclusions Fascioliasis prevalences indicate this area to be mesoendemic, with isolated hyperendemic foci. This is the first time that a human fascioliasis endemic area is described in North America. Nitazoxanide appears as an appropriate
Smith, David; Tikhonova, Irina G.; Jewhurst, Heather L.; Drysdale, Orla C.; Dvořák, Jan; Robinson, Mark W.; Cwiklinski, Krystyna; Dalton, John P.
Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host. PMID:27422822
Background Deep eosinophilic granulomatous abscesses, as distinguished from eosinophilic subcutaneous abscesses, are rare. Most reports are from the Far-East and India where the most commonly attributed cause is Toxocara. Sulaimaniyah in Northeastern Iraq has experienced an outbreak of eosinophilic granulomatous liver and gastrointestinal (GI) abscesses beginning in 2009. The purpose of this study was to determine the etiology and guide treatment. Methods The study was an ongoing investigation of patients having a histopathologic diagnosis of eosinophilic granulomatous abdominal abscesses in Sulaimaniyah hospitals from May 2009 to August 2012. Tissues were examined for organisms, and Enzyme Linked Immunoabsorbent Assays (ELISA) were performed for serum antibodies to Fasciola hepatica, Toxocara, and Echinococcus granulosus. Results Fourteen patients had granulomatous inflammation surrounding a central necrotizing eosinophilic exudate identified in surgical pathology specimens from abdominal surgeries. Two children and four adults had abscesses that formed GI masses. These patients included a 39 year old male with oropharyngeal and transverse colon disease, and a 48 year old male with liver and GI abscesses. All sites demonstrated a Zygomycete fungus surrounded by eosinophilic Splendori-Hoeppli material consistent with basidiobolomycosis. Five of the six patients with fungal disease were treated by surgery and 4 to 7 months of itraconozol. One child died of intestinal perforation while receiving IV amphotericin B; two adults required additional surgery for recurrent GI obstruction. Eight patients had isolated liver abscesses with no organisms identified by histopathology: ELISA results for F. hepatica were positive for five, borderline for one, and negative for two patients. These eight patients were treated for fascioliasis by surgical resection of localized abscesses and albendazol. One patient serologically positive for F. Hepatica was found to have a common duct
Law, David M.; Basile, Dominick V.; Basile, Margaret R.
Endogenous indole-3-acetic acid (IAA) was found in axenically cultured gametophytes of the leafy liverwort, Plagiochila arctica Bryhn and Kaal., by high-performance liquid chromatography with electrochemical detection. Identification of the methylated auxin was confirmed by gas chromatography-mass spectrometry. Addition of 57 micromolar IAA to cultures increased relative production of ethylene. This is the first definitive (gas chromatography-mass spectrometry) demonstration of the natural occurrence of IAA in a bryophyte. PMID:16664164
Redrobe, S P; Patterson-Kane, J C
Calodium hepaticum infection was diagnosed in the Bristol Zoo Gardens in 13 captive rodents of four species that died or were humanely killed over a 40-month period. Of these infected animals, nine were black-tailed prairie dogs (Cynomus ludovicianus), representing 45% of the members of this species examined during the study. A wild rat (Rattus norvegicus) found dead in an enclosure was also infected. To date few cases of C. hepaticum infection have been reported in the UK. The number of cases diagnosed in this urban zoo may be explained by the potentially high prevalence of infection in urban rat populations and increased risk of exposure of zoo animals kept in enclosures to which rats have access. As C. hepaticum is potentially zoonotic, members of staff in zoos should be careful to avoid soil-to-mouth contact, particularly in prairie dog enclosures.
Gordon, Catherine A.; Acosta, Luz P.; Gobert, Geoffrey N.; Jiz, Mario; Olveda, Remigio M.; Ross, Allen G.; Gray, Darren J.; Williams, Gail M.; Harn, Donald; Li, Yuesheng; McManus, Donald P.
The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis) have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD) technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle) from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively) and carabao (80.00% and 55.24%, respectively). The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively). The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines. PMID:25643317
Epe, C; Coati, N; Schnieder, T
The results of coproscopical examinations in horses, ruminants, pigs, dogs, cats, hedgehogs and rabbits between 1998 and 2002 are presented. In 4399 samples from horses 37.4% stages of strongylids, 1.4% anoplocephalids, 1.3% Strongyloides westeri, 0.9% Parascaris equorum, 0.04% Oxyuris equi, 0.04% Eimeria sp. and 0.04% Fasciola hepatica were found. In 998 samples of cattle 22.1% stages of strongylids, 11.2% of Eimeria spp., 3.5% of cryptosporidium, 2.9% of Moniezia spp., 1.3% of Trichuris spp., 0.7% of Dictyocaulus sp., 0.6% of Fasciola hepatica, 0.6% of Strongyloides sp., 0.5% of Nematodirus spp. and 0.4% of Capillaria sp. could be detected. In 524 samples of sheep 60.7% eggs of strongylids, 43.1% oozysts of Eimeria spp., 11.1% stages of Nematodirus spp., 9.5% of Moniezia spp., 7.8% of Trichuris spp., 6.7% of Strongyloides sp., 1.7% of Fasciola hepatica, 1% of Capillaria spp., 0.4% of protostrongylidae, 0.2% of Skrjabinema sp. and 0.2% of Dictyocaulus sp. were found. 33.9% of the 118 samples of goats that were examined were positive for oocysts of Eimeria spp., 30.5% for eggs of strongylids, 6.8% for Nematodirus spp., 4.2% for Trichuris spp., 3.4% for Moniezia spp., 0.8 for protostrongylids and 0.8% for Strongyloides sp. 5.7% of 1427 samples of pigs contained stages of strongylids, 1.5% of Ascaris suum, 0.4% of Isospora, 0.3% of Eimeria spp., 0.3% of Trichuris sp., 0.1% of Giardia sp., 0.1% of cryptosproidium as well as 0.1% of metastrongylids. In 1281 of the samples of dogs 2.3% Giardia sp., 2.3% Isospora sp., 2.2% Toxocara canis, 1.4% ancylostomids, 0.8% taeniids, 0.6% larvae of Crenosoma sp., 0.2% Capillaria sp, 0.2% Trichuris vulpis and 0.2% Hammondia-like oocysts were found. In 441 samples of cats 10.7% stages of Isospora sp., 3.9% eggs of Toxocara cati, 1.6% of ancylostomids, 1.4% of taeniids, 1.1% of Giardia sp., 0.7% of Toxoplasma-like oocysts, 0.7% of Aelurostrongylus abstrusus, 0.5% of Toxascaris leonina and 0.2% of Capillaria spp. were found
Olah, Sophie; van Wyk, Jan A; Wall, Richard; Morgan, Eric R
The liver fluke Fasciola hepatica causes considerable damage to the health, welfare and productivity of ruminants in temperate areas, and its control is challenged by anthelmintic resistance. Targeted selective treatment (TST) is an increasingly established strategy for preserving anthelmintic efficacy in grazing livestock, yet no practical indicators are available to target individuals for treatment against fluke infection. This paper evaluates the FAMACHA(©) system, a colour chart for the non-invasive detection of anaemia in small ruminants, for this purpose. FAMACHA(©) scores were collected from 288 sheep prior to slaughter during the winter period, when fluke infections were largely mature, and condemned livers were recovered and adult flukes extracted. Average FAMACHA(©) score was significantly higher (=paler conjunctivae) in animals whose livers were condemned (3.6, n=62) than in those whose livers were not condemned (2.1). The number of adult flukes recovered ranged from 2 to 485, and was positively correlated with FAMACHA(©) score (r(2)=0.54, p<0.001). Packed cell volume was correlated negatively with both FAMACHA(©) score (n=240, r=0.23, p<0.001) and fluke number (r=0.24, p<0.001). Nematode faecal egg count (FEC) did not correlate with FAMACHA(©) score, and selective treatment of individual sheep with FAMACHA(©) scores above 2 or 3 would have preserved between 27 and 100% of nematodes in refugia on the basis of FEC, depending on group and the threshold used for treatment. FAMACHA(©) holds promise as a tool for selective treatment of sheep against adult F. hepatica, in support of refugia-based control of fluke and nematode infections, and further field evaluation is warranted.
Background Fascioloides magna is a pathogenic fluke introduced to Europe ca 140 years ago. As it is spreading over the continent, new intermediate and definitive hosts might be involved in transmission of the parasite. In Europe, several studies reported potential new intermediate snail hosts (Radix spp.) for F. magna, and also several cases of fascioloidosis of wild and domestic animals were published. However, the data based on molecular and histological analyses confirming these findings remained unreported. This study aims to refer to unique findings of F. magna in European snails and domestic animals (the first observation in the Czech Republic in the last 30 years) and demonstrate the use of molecular techniques in determination of F. magna. Results Two snails of R. labiata naturally infected with F. magna were found; mature cercariae and daughter rediae were observed. Maturity of cercariae was checked by histological methods, however, their ability to encyst was not confirmed. Co-infection of F. magna and Fasciola hepatica in the liver of two highland cattle bulls was proved. Adult fasciolid flukes producing eggs were found in the liver pseudocysts (F. magna) and the bile ducts (F. hepatica). Identification of intermediate hosts, intramolluscan stages, adult flukes and eggs was performed by sequencing the ITS2 region. Connection of F. magna pseudocysts with the gut (via the bile ducts) was not confirmed by means of histological and coprological examinations. Conclusions For the first time, Radix labiata was confirmed as the snail host for F. magna under natural conditions and, together with the finding of F. magna infection in cattle, we can expect further transmission of F. magna from wildlife to livestock in localities shared by these hosts. PMID:24517409
Becker, Ann-Christin; Kraemer, Amelie; Epe, Christian; Strube, Christina
Coproscopical methods used in veterinary-parasitological diagnostics were validated according to their sensitivity (Se) and egg recovery rate [efficiency (Ef)]. Validation of the combined sedimentation-flotation method and the modified McMaster method was performed by using feces spiked with eggs of Ancylostoma caninum, Uncinaria stenocephala, Cooperia oncophora, cyathostomins, Ascaris suum, Toxascaris leonina, Toxocara canis, Trichuris vulpis, Moniezia expansa, and Anoplocephala perfoliata. For validation of the sedimentation method, Fasciola hepatica eggs were used. With the combined sedimentation-flotation method using ZnSO4 as flotation medium [specific gravity (SG) 1.30], 5 g fecal samples of all tested parasite species (concentration levels 1, 5, 10, 20, 40, 60, and 80 epg) were reproducibly detected "positive" (100 % Se) as of 80 epg. The Ef of the combined sedimentation-flotation method, defined as percentage of rediscovered eggs, revealed clear differences between parasites and showed the highest value for cyathostomins and the lowest for U. stenocephala and T. leonina eggs. The average Ef for all parasite species at 80 epg was 1.50 %. With the McMaster method (concentration levels 1, 30, 50, 80, 100, 500, and 1000 epg), all tested parasite species were detected reliably positive as of 500 epg with a mean Ef of 46.4 %. When evaluating the sedimentation method (concentration levels 1, 5, 10, 15, 20, 25, and 30 epg), F. hepatica eggs were reproducibly found in 5 g fecal samples as of 20 epg with 20.0 % Ef. The result that the combined zinc sulfate sedimentation-flotation method (SG 1.30) as flotation medium provides diagnostic certainty only as of 80 epg has to be considered at preventing zoonoses. If pet owners wish to prevent any zoonotic infection ("zero tolerance"), a monthly anthelminthic treatment should be advised instead of monthly fecal examinations.
Chaturvedi, Divya; Singh, Vinay Kumar
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. PMID:27688849
Cabada, Miguel M; Morales, Maria Luisa; Lopez, Martha; Reynolds, Spencer T; Vilchez, Elizabeth C; Lescano, Andres G; Gotuzzo, Eduardo; Garcia, Hector Hugo; White, Clinton A
Hymenolepis nana is the most common cestode infection in the world. However, limited information is available regarding its impact on affected populations. We studied the epidemiology and symptoms associated with hymenolepiasis among children 3-16 years old in 16 rural communities of the highlands of the Cusco region in Peru. Information on demographics, socioeconomic status, symptoms as reported by parents, and parasitological testing was obtained from the database of an ongoing Fasciola hepatica epidemiologic study. A total of 1,230 children were included in the study. Forty-five percent were infected with at least one pathogenic intestinal parasite. Giardia spp. (22.9%) was the most common, followed by Hymenolepis (17.4%), Fasciola (14.1%), Ascaris lumbricoides (6.1%), and Strongyloides stercoralis (2%). The prevalence of Hymenolepis infection varied by community, by other parasitic infections, and by socioeconomic status. However, only years of education of the mother, use of well water, and age less than 10 years were associated with Hymenolepis infection in the multivariate analysis. Hymenolepis nana infection was associated with diarrhea, jaundice, headaches, fever, and fatigue. Children with > 500 eggs/g of stool were more likely to have symptoms of weight loss, jaundice, diarrhea, and fever. Hymenolepis nana infection and age were the only factors retained in the multivariate analysis modeling diarrhea. Hymenolepiasis is a common gastrointestinal helminth in the Cusco region and is associated with significant morbidity in children in rural communities. The impact caused by the emergence of Hymenolepis as a prevalent intestinal parasite deserves closer scrutiny.
Background Freshwater lymnaeid snails can act as the intermediate hosts for trematode parasites such as the liver fluke Fasciola hepatica, that cause significant economic and biomedical burden worldwide, particularly through bovine fascioliasis. Transmission potential is tightly coupled to local compatibility with snail hosts, so accurate identification of lymnaeid species is crucial for understanding disease risk, especially when invasive species are encountered. Mendoza Province, in Argentina, is a center of livestock production and also an area of endemic fascioliasis transmission. However, the distribution of lymnaeid species in the region is not well known. Methods This study examined lymnaeid snails from seven localities in the Department of Malarguë, Mendoza Province, using morphological and molecular analyses and also describing ecological variables associated with snail presence. Results While morphological characters identified two species of lymnaeid, Galba truncatula and G. viatrix, molecular data revealed a third, cryptic species, G. neotropica, which was sympatric with G. viatrix. G. truncatula was exclusively found in high altitude (>1900 meters above sea level [masl]) sites, whereas mixed G. neotropica/G. viatrix localities were at middle elevations (1300–1900 masl), and G. viatrix was found alone at the lowest altitude sites (<1300 masl). Phylogenetic analysis using two mitochondrial markers revealed G. neotropica and G. viatrix to be closely related, and given their morphological similarities, their validities as separate taxonomic entities should be questioned. Conclusions This study highlights the need of a robust taxonomic framework for the identification of lymnaeid snails, incorporating molecular, morphological and ecological variables while avoiding nomenclature redundancy. As the three species observed here, including one alien invasive species, are considered hosts of varying susceptibility to Fasciola parasites, and given the economic
Chaturvedi, Divya; Singh, Vinay Kumar
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.
Zukowski, S H; Wilkerson, G W; Malone, J B
A geographic information system (GIS) model of habitat for Lymnaea bulimoides, the snail intermediate host of Fasciola hepatica and the rumen fluke, Calicophoron microbothrioides, on the Chenier Plain of southwest Louisiana was revised to incorporate broad (greater than 100 m) chenier (relict beaches) along with adjacent marsh and transitional soils associated with spatial distribution of L. bulimoides habitat. The proportion of farmland comprised of soils of the GIS model coincided with actual habitat from a previous survey with a sensitivity of 91.3% and a specificity of 80.1%, and regressed significantly against the proportion of farmland comprised of L. bulimoides habitat (n = 12, P = 0.01, r2 = 0.50, slope = 0.015). A soil model index was calculated to incorporate (1) the proportion of farmland comprised of soils of the GIS model and (2) stocking rate. Fluke egg shedding indices (mean egg count multiplied by prevalence) were calculated for each fluke species on nine farms at four sampling times over a 2-year period. The maximum egg shedding indices for each farm, taken to indicate potential fluke transmission intensity, were correlated for the two fluke species on herds not recently treated for fasciolosis (n = 9, P = 0.004, r2 = 0.72, slope = 0.2), although at no one sampling period were shedding indices of the two fluke species significantly correlated. Egg shedding of C. microbothrioides by cattle correlated with the proportion of farmland comprised of soils included in the GIS model (n = 9, Spearman's rank coefficient was 0.7, P = 0.05). We conclude that (1) the maximum of several observations of the C. microbothrioides egg shedding index may be useful as a surrogate for F. hepatica in estimating risk from snail habitat on a farm when regular flukicide treatment interferes with F. hepatica egg shedding, and (2) the GIS model may estimate site-specific differences in fasciolosis risk to cattle operations in the Chenier Plain based on the association of
Sulaiman, Azad A.; Zolnierczyk, Katarzyna; Japa, Ornampai; Owen, Jonathan P.; Maddison, Ben C.; Hodgkinson, Jane E.; Gough, Kevin C.
The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced
Biswal, Devendra Kumar; Ghatani, Sudeep; Shylla, Jollin A.; Sahu, Ranjana; Mullapudi, Nandita
Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest
Beltrame, Ludovica; Dunne, Toby; Rose, Hannah; Walker, Josephine; Morgan, Eric; Vickerman, Peter; Wagener, Thorsten
Liver Fluke (Fasciola hepatica) is a common parasite found in livestock and responsible for considerable economic losses throughout the world. Risk of infection is strongly influenced by climatic and hydrological conditions, which characterise the host environment for parasite development and transmission. Despite on-going control efforts, increases in fluke outbreaks have been reported in recent years in the UK, and have been often attributed to climate change. Currently used fluke risk models are based on empirical relationships derived between historical climate and incidence data. However, hydro-climate conditions are becoming increasingly non-stationary due to climate change and direct anthropogenic impacts such as land use change, making empirical models unsuitable for simulating future risk. In this study we introduce a mechanistic hydro-epidemiological model for Liver Fluke, which explicitly simulates habitat suitability for disease development in space and time, representing the parasite life cycle in connection with key environmental conditions. The model is used to assess patterns of Liver Fluke risk for two catchments in the UK under current and potential future climate conditions. Comparisons are made with a widely used empirical model employing different datasets, including data from regional veterinary laboratories. Results suggest that mechanistic models can achieve adequate predictive ability and support adaptive fluke control strategies under climate change scenarios.
In the 27 Member States of the European Union, zoonotic parasites transmitted by food are circulating with different prevalence according to the country, the environmental conditions, the human behaviour, and the socio-economic level. Foodborne parasites can be divided in two main groups according to the way of transmission to humans. These foodborne parasites reach the human beings through the consumption of raw infected food such as muscle tissues of different animal species (Toxoplasma gondii, Sarcocystis hominis, Sarcocystis suishominis, Diphyllobotrium latum, Taenia solium, Taenia saginata, Opisthorchis felineus, Anisakis spp., Pseudoterranova spp., Trichinella spp.), or vegetables (Fasciola hepatica), and contaminated food and water resources (Giardia duodenalis, Cryptosporidium spp., T. gondii, Echinococcus granulosus sensu latu, Echinococcus multilocularis, T. solium, Taenia multiceps). As a general role, the control strategies should be based on the education of the consumers, farmers and shepherds, the improvement of farming conditions, the improvement or the development of more sensitive methods to detect these parasites in slaughtered animals and in foodstuff, a control of sewage sludge on pastures and of drinking water resources, and the reduction of contacts between livestock and wild animals which frequently represent the most important reservoir of these pathogens.
González, L Carolina; Esteban, José Guillermo; Bargues, M Dolores; Valero, M Adela; Ortiz, Pedro; Náquira, Cesar; Mas-Coma, Santiago
A coprological survey including 476 2-18 year old school children from six rural localities between 2627 and 3061 m altitude was performed in Cajamarca province, Peru. Prevalences of fascioliasis ranging from 6.7 to 47.7% (mean 24.4%) proved to be the highest so far recorded in that human hyperendemic area. Higher prevalences in females and in the 2-5 year old group were not significant. Intensities ranged from 24 to 864 eggs per gram (arithmetic mean: 113; geometric mean: 68), the majority shedding less than 100, and without significant differences according to gender or age group. Fasciola hepatica was the most common helminth within a spectrum of 11-12 protozoan and 9-11 helminth species, 97.3% of the children showing infection with at least one parasite. The highest levels corresponded to coinfection with seven different species in females and subjects older than 5 years. Fascioliasis prevalence correlation with altitude appeared significant. An epidemiological characterisation of the valley transmission pattern of fascioliasis in Cajamarca is made by comparison with other better known hyperendemic areas. Results suggest that human fascioliasis may be widespread throughout different parts of Cajamarca province, even far away from the city, and that long-term fascioliasis chronicity and superimposed repetitive infections may be probably frequent.
Barber, Xavier; Conesa, David; Lladosa, Silvia; López-Quílez, Antonio
Modelling patterns of the spatial incidence of diseases using local environmental factors has been a growing problem in the last few years. Geostatistical models have become popular lately because they allow estimating and predicting the underlying disease risk and relating it with possible risk factors. Our approach to these models is based on the fact that the presence/absence of a disease can be expressed with a hierarchical Bayesian spatial model that incorporates the information provided by the geographical and environmental characteristics of the region of interest. Nevertheless, our main interest here is to tackle the misalignment problem arising when information about possible covariates are partially (or totally) different than those of the observed locations and those in which we want to predict. As a result, we present two different models depending on the fact that there is uncertainty on the covariates or not. In both cases, Bayesian inference on the parameters and prediction of presence/absence in new locations are made by considering the model as a latent Gaussian model, which allows the use of the integrated nested Laplace approximation. In particular, the spatial effect is implemented with the stochastic partial differential equation approach. The methodology is evaluated on the presence of the Fasciola hepatica in Galicia, a North-West region of Spain.
Considered a secondary zoonotic disease until the mid-1990s, human fascioliasis is at present emerging or re-emerging in many countries, including increases of prevalence and intensity and geographical expansion. Research in recent years has justified the inclusion of fascioliasis in the list of important human parasitic diseases. At present, fascioliasis is a vector-borne disease presenting the widest known latitudinal, longitudinal and altitudinal distribution. Fasciola hepatica has succeeded in expanding from its European original geographical area to colonize five continents, despite theoretical restrictions related to its biology and in turn dependent upon environmental and human activities. Among the different epidemiological situations, human hypo- to hyperendemic areas, including epidemics, are noteworthy. A global analysis of the distribution of human cases shows that the expected correlation between animal and human fascioliasis only appears at a basic level. Areas presenting very high human prevalences and intensities, especially in children and females, have been recently described. In hypo- to hyperendemic areas of Central and South America, Europe, Africa and Asia, human fascioliasis presents a r