High-quality reference genome of Fasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions.

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.

A Comparison of the Risk for Chronic Fascioliasis between Children 3 to 5 Years and Children 6 to 12 Years of Age in the Cusco Region of Peru.

School-age children bear the highest burden of fascioliasis in endemic countries. Few studies have addressed Fasciola in preschool children. We performed a secondary data analysis using two Fasciola databases from Cusco, Peru, comparing preschoolers with elementary school children. We included 2,630 children, 50% were female, the median age was 8.4 years (interquartile range [IQR] 6.1-10.5), and 15% (396/2,630) were < 5 years of age. Children < 5 years were less likely to be infected with Fasciola hepatica (P = 0.008) and Hymenolepis nana (P < 0.001) and more likely to have anemia (P < 0.001) and a lower median height for age Z (HAZ) score (P = 0.002). Fascioliasis was less common in younger children, but this group may be at higher risk for chronic complications caused by fascioliasis.

Association between milk yield and milk anti-Fasciola hepatica antibody levels, and the utility of bulk tank milk samples for assessing within-herd prevalence on organic dairy farms.

Fasciola hepatica is an important disease of livestock that is responsible for substantial economic losses worldwide. Estimates of the impact of infection on milk yield vary, likely reflecting different geographical locations, farm-level management, and diagnostic methods. Measuring anti-Fasciola antibodies on bulk tank milk (BTM) by ELISA provides a convenient herd-level diagnosis, but the utility of this test remains unclear. Therefore, we evaluated the utility of BTM ELISA test results in Danish organic dairy farms, including estimating the association between 305 day energy corrected milk yield (305d ECM) and F. hepatica infection both at individual and herd level. BTM samples from 218 organic farms were analysed using IDEXX ELISA and subsequently the farmers were interviewed during spring 2016 with the aim of characterising their management practices. The corresponding farm-level production data covering the period 2014-2017 were collected from the Danish national cattle registry. In the following year, 284 individual milk samples (4-7 per herd) along with BTM samples were collected from a subset of the same herds (n = 55). Linear mixed models were used to estimate the association between milk production and ELISA value at both individual and farm levels, and a generalised additive model was used to assess the relationship between within-herd prevalence and BTM ELISA. A dichotomised BTM result with positive outcome was associated with a reduction of 580.5 kg in average 305d ECM, and a positive outcome on individual-level ELISA was associated with a 919.5 kg reduction in milk yield for cows in their third or later lactations. A strong relationship between quantitative BTM ELISA sample to positive percentage (S/P%) and apparent within-herd prevalence based on dichotomised individual-level milk ELISA was also observed, although this relationship was non-linear in nature. We conclude that a useful indication of the within-herd prevalence of infection can be obtained from BTM ELISA following categorisation as negative, low, medium or high according to S/P% cut-offs of approximately 30, 80, and 150. This approach represents a cheap and useful diagnostic tool for monitoring the long-term success of control strategies for F. hepatica infections on a dairy farm.

The impact of liver fluke infection on steers in Ireland: A meta-analytic approach.

Post-mortem liver inspection results together with production parameters are often used to estimate the impact of liver fluke infection on farm animal populations. However, post mortem liver inspection is an imperfect method of determining the liver fluke infection status of cattle. This work estimates the difference in mean lifetime weight gain at 819 days (ΔLWG819) between steers assigned liver fluke negative (LFN) and liver fluke positive (LFP) status at post-mortem meat inspection, quantifies the potential impact of imperfect sensitivity and specificity on these results and estimates the economic impact of these differences. The study population is 32,007 steers that never moved from their birth herd in the Republic of Ireland and were slaughtered in one of two Irish meat processors in 2014. Individual animal-level data are used to generate 46 county - processor level estimates of ΔLWG819. Standard errors and confidence intervals for these estimates are derived using bootstrapping. A meta-analytic approach is then used to obtain 3 overall estimates of the effect of liver fluke status on the ΔLWG819 in all the county - processor combinations, assuming post - mortem liver inspection Se = Sp = 1, 0.99 and 0.95. A random effects model is used and 95% prediction intervals (95% PI) are calculated. Assuming Se = Sp = 1 for post - mortem liver inspection, the random effects summary estimate of ΔLWG819 (ΔLWG819(RE)) is 36 kg (95% PI: -1, 73). There is a minor change in ΔLWG819(RE) (38 kg, 95% PI: -1, 77) when Se = Sp = 0.99 is assumed but this increases to 46 kg (95% PI: -2, 94) assuming Se = Sp = 0.95. The corresponding cost in euros of these differences between the LFN and LFP steers, assuming a price per kg of €3.90, are €77.01 (95% PI: -2.57, 156.37), €80.65 (95% PI: -3.43, 164.74) and €98.67 (95% PI: -5.15, 202.27) respectively. Our results demonstrate an association between liver fluke infection and reduced weight gain. We show that the effect of liver fluke infection on weight gain in cattle is underestimated due to misclassification resulting from imperfection in post mortem meat inspection. These findings will aid researchers, farmers and veterinary practitioners to make informed decisions on the control of liver fluke on farms.

Numerous Fasciola plasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis.

Human fascioliasis is a worldwide, pathogenic food-borne trematodiasis. Impressive clinical pictures comprising puzzling polymorphisms, manifestation multifocality, disease evolution changes, sequelae and mortality, have been reported in patients presenting with neurological, meningeal, neuropsychic and ocular disorders caused at distance by flukes infecting the liver. Proteomic and mass spectrometry analyses of the Fasciola hepatica excretome/secretome identified numerous, several new, plasminogen-binding proteins enhancing plasmin generation. This may underlie blood-brain barrier leakage whether by many simultaneously migrating, small-sized juvenile flukes in the acute phase, or by breakage of encapsulating formations triggered by single worm tracks in the chronic phase. Blood-brain barrier leakages may subsequently occur due to a fibrinolytic system-dependent mechanism involving plasmin-dependent generation of the proinflammatory peptide bradykinin and activation of bradykinin B2 receptors, after different plasminogen-binding protein agglomeration waves. Interactions between diverse parasitic situations and non-imbalancing fibrinolysis system alterations are for the first time proposed that explain the complexity, heterogeneity and timely variations of neurological disorders. Additionally, inflammation and dilation of blood vessels may be due to contact system-dependent generation bradykinin. This baseline allows for search of indicators to detect neurological risk in fascioliasis patients and experimental work on antifibrinolytic treatments or B2 receptor antagonists for preventing blood-brain barrier leakage.

"Liver fluke": conceptos actuales sobre fascioliasis / Liver fluke: update on fascioliasis

Fascioliasis is a parasitic disease produced mainly by the fluke Fasciola hepatica. The human infection is mainly due to the accidental intake of metacercariae present in watercress and/or contaminated water. The human disease is uncommon in Chile, despite the high prevalence of animal infection, which affects almost the entire national territory and determines high economic and productive impacts. Human fascioliasis can manifest like acute or chronic phase of the disease (in Chile, the majority in chronic phase) and its identification requires a high index of suspicion, in individuals with abdominal pain, hepatomegaly and eosinophilia, where the epidemiological background of watercress ingestion is usually present. Diagnosis usually requires the integration of egg visualization in stools, serology and imaging studies. The treatment of choice with triclabendazole is usually highly effective and safe.

La fascioliasis es una enfermedad parasitaria producida principalmente por el trematodo Fasciola hepática. La infección en el hombre, quien es un huésped accidental, se debe principalmente a la ingesta de metacercarias presentes en berros y/o aguas contaminadas. La enfermedad en humanos es infrecuente en Chile, a pesar de la alta prevalencia de infección animal, que afecta a casi todo el territorio nacional y determina un alto impacto económico y productivo. La fascioliasis humana puede manifestarse en fase aguda o crónica (en Chile, la mayoría en fase crónica) y su identificación requiere un alto índice de sospecha, en individuos con dolor abdominal, hepatomegalia y eosinofilia, donde el antecedente epidemiológico de ingesta de berros suele estar presente. El diagnóstico, habitualmente requiere la integración de la visualización directa de huevos en las deposiciones, estudios de serología e imágenes. El tratamiento de elección con triclabendazol, habitualmente es altamente efectivo y seguro.

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

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

Human fascioliasis in South Africa.

Human fascioliasis has the widest latitudinal, longitudinal and altitudinal distribution of any vector-borne disease, yet only 3 cases have been reported from South Africa, the last in 1964. We report 2 cases from the same geographic area associated with local consumption of watercress, suggesting an endemic focus. 

Integrating fasciolosis control in the dry cow management: the effect of closantel treatment on milk production.

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.

Evaluation of oxidative status in patients with Fasciola hepatica infection.

BACKGROUND: F. hepatica infection is rare and mostly subclinical. Migration of juvenile forms of F. hepatica into the host's liver is accompanied by an inflammatory reaction followed by fibrosis and cirrhosis. OBJECTIVE: The aim of this study was to evaluate oxidative status by using a novel automated method in patients with Fasciola hepatica. METHODS: Twenty two patients with a diagnosis of F. hepatica and 26 healthy volunteers were enrolled in the study. Their Total antioxidant capacity status (TAC), total oxidant status (TOS) and catalase were measured in them and oxidative stress index (OSI) was calculated. These measurements were also taken for the control group and the values were compared. RESULTS: Plasma levels of total TOS and OSI were significantly increased in patients as compared with healthy controls (P<0.001, P<0.001, P=0.001, P=0.008) respectively. In contrast, TAC level was significantly lower in patients as compared with controls (p<0.05). There was no significant difference between the catalase results of the two groups (p>0.05). CONCLUSION: Total oxidative status and OSI were increased and total antioxidative status capacity was decreased in patients with F. hepatica infection. A high oxidative stress occurs during F. hepatica infection, which may cause severe damage in both the liver.

Clinical presentation and management of Fasciola hepatica infection: single-center experience.

AIM: To identify the characteristic clinical, laboratory and radiological findings and response to treatment in patients with fascioliasis. METHODS: Patients who were diagnosed with Fasciola hepatica infection were included in this prospective study. Initial clinical, laboratory and radiological findings were recorded. All patients were followed until a complete response was achieved or for 6 mo after treatment discontinuation. RESULTS: Fasciola hepatica infection was diagnosed in 30 patients (24 females; mean age: 42.6 years) between January 2008 and February 2011. Twenty-two (73%) patients had hepatic phase fascioliasis, 5 patients had biliary phase, and 3 patients had biliary phase associated with acute pancreatitis. Of the 8 patients with biliary phase fascioliasis, 2 patients displayed features that overlapped with both hepatic and biliary phase. Abdominal pain and right upper abdominal tenderness were the most prominent signs and symptoms in all patients. Eosinophilia was the most prominent laboratory abnormality in both patients with hepatic and biliary phase (100% and 50%, respectively). Multiple nodular lesions like micro-abscesses on abdominal computerized tomography were the main radiological findings in patients with hepatic phase. Small linear filling defects in the distal choledochus were the main endoscopic retrograde cholangiopancreatography (ERCP) findings in patients with biliary phase. Patients with hepatic phase were treated with triclabendazole alone, and patients with biliary phase were treated with triclabendazole and had live Fasciola hepatica extracted from the bile ducts during ERCP. CONCLUSION: Fasciola hepatica infection should be considered in the differential diagnosis of patients with hepatic or biliary disease and/or acute pancreatitis associated with eosinophilia.

Investigation of the persistence of nitroxynil residues in milk from lactating dairy cows by ultra performance liquid chromatography tandem mass spectrometry.

Nitroxynil is an anthelmintic used in the treatment of liver fluke. In this study, six dairy cows were treated during lactation with Trodax, a 34% solution containing nitroxynil as its N-ethylglucamine salt, indicated for the treatment of fascioliasis in cattle and sheep. Samples were collected twice daily for 16 days and later at weekly intervals up to 58 days post-treatment. Nitroxynil residues were extracted from milk samples using acetonitrile; magnesium sulfate and sodium chloride were added to induce liquid-liquid partitioning and purified by dispersive solid phase extraction for clean-up. Nitroxynil was determined by ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in negative ionization mode. The limit of detection (CCα) of the method is 0.24 µg/kg. Maximum concentration of nitroxynil in the samples was in the range of 688-1358 µg/kg, with levels persisting for 58 days in four of the six lactating cows. Incurred nitroxynil samples were treated with sulfatase and ß-glucuronidase from Helix pomatia ; the results indicated the presence of glucuronide conjugates in samples at early withdrawal times. At later withdrawal times the concentration of free nitroxynil was lower than the concentration in the control samples, indicating potential degradation during enzymatic treatment.

Glucose, lipid, and lipoprotein levels in sheep naturally infected with Fasciola hepatica.

This study was designed to investigate serum glucose, lipid, and lipoprotein in sheep naturally infected with Fasciola hepatica. Ten healthy sheep and 15 infected with F. hepatica were used in study. Serum concentrations of total protein (TP), albumin, glucose, cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), very-low-density lipoproteins (VLDL), and serum activities of AST, ALT, GGT, and LDH were measured using a Roche-Cobas Integra 800 auto-analyzer. At day 0 (prior to treatment) and on the 28th day (after treatment) the serum concentrations of TP, albumin, glucose, cholesterol, triglyceride, HDL, LDL, and VLDL values in sheep with F. hepatica were significantly lower than those of the control group, while serum activities of AST, ALT, GGT, and LDH of lambs with F. hepatica were significantly higher than those of the control group. At day 56 (after treatment), none of the variables was significantly different between control sheep and those that received treatment for fascioliasis (P > 0.05). Nutritional management may be used to reduce the impact of fascioliasis.

Eosinophil-nerve interactions and neuronal plasticity in rat gut associated lymphoid tissue (GALT) in response to enteric parasitism.

Intestinal lymphoid tissues and Peyer's patches (PP) are innervated sites of immune surveillance in the gastrointestinal tract. Following infection with F. hepatica, neuronal hyperplasia and significantly increased eosinophil and mast cell trafficking to colonic PP sites were evident in rat tissues. Nerve-eosinophil associations were significantly elevated in infected colon and colonic PP, as were colonic tissue levels of the circulatory recruitment factors IL-5 and eotaxin. Increased immunoreactivity for neuronal plasticity markers GAP-43 and neural cell adhesion molecule (NCAM) was also found in infected tissues. Such neuronal alterations in the PP during enteric parasitism may have functional consequences on particular or pathogen uptake.

Natural history, clinicoradiologic correlates, and response to triclabendazole in acute massive fascioliasis.

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.

Epidemiological review of human and animal fascioliasis in Egypt.

One of the neglected food-borne-diseases in the international public health arena is fascioliasis. It is a serious infectious parasitic disease infecting humans and animals worldwide and tops all the zoonotic helminthes. Human cases are being increasingly reported from Europe, the Americas, Oceania, Africa and Asia. Hence, human fascioliasis is considered now as a zoonosis of major global and regional importance. In Egypt, animal and human fascioliasis is an endemic clinical and epidemiological health problem. Doubtless, understanding the epidemiology of the parasitic diseases and factors affecting their incidence provides the foundation upon which effective prevention and control programs should be established. This article reviews the history, life cycles, transmission, incidence, geographical distribution, and environmental and human determinants that contribute to the epidemiological picture of fascioliasis with special reference to Egypt.

Excretory-secretory products from Fasciola hepatica induce eosinophil apoptosis by a caspase-dependent mechanism.

Eosinophils (Eo) are known to be important effector cells in the host defense against helminth parasites. Excretory-secretory products (ESP) released by helminths have shown wide immunomodulatory properties, such as the induction of cellular apoptosis. We investigated the ability of ESP from Fasciola hepatica to induce Eo apoptosis. In this work, we observed that ESP induced an early apoptosis of rat peritoneal eosinophils and that this phenomenon was time- and concentration-dependent. Furthermore, we demonstrated that activation of protein tyrosine kinases (TyrK) and caspases were necessary to mediate the Eo apoptosis induced by the ESP, and that carbohydrate components present in these antigens were involved in this effect. We have described for the first time the ability of ESP from F. hepatica to modify the viability of Eo by apoptosis induction. Besides that, we have observed Eo apoptosis in the liver of rats 21 days after F. hepatica infection. The diminution in Eo survival in early infection could be a parasite strategy in order to prevent a host immune response.

Biochemical changes in the livers of bovines naturally infected with Fasciola gigantica.

The present study was carried out to determine the total carbohydrate, total protein and total lipid content in the non-infected (Control) and naturally infected with Fasciola gigantica liver samples of buffaloes and cows. The estimated total protein and lipid content in the infected livers of these animals were found significantly higher as compared to their control samples. Total carbohydrate content in the infected livers of buffaloes and cows did not show any significant change as compared to their control animals.

High risk of bacterobilia in advanced experimental chronic fasciolosis.

Fasciolosis is recognized as an important human disease. Wistar rats experimentally infected with Fasciola hepatica were examined using data obtained in the advanced chronic state of the disease (200, 300 and 400 days post-infection, dpi). Pigment stones (PS) and bile specimens were collected. The same procedure was applied in control rats. Liver tests were determined using stored serum samples. Bacteriological bile culture revealed viable bacteria (Escherichia coli, 45% of cases, Enterococcus faecalis, 45% and Klebsiella pneumoniae, 10%). The presence of bacterobilia was associated with liver serum enzymes, including aspartate aminotransferase (AST or SGOT), alanine aminotransferase (ALT or SGPT), alkaline phosphatase (AP) and total bilirubin levels. Multivariate analysis suggested an association between bacterobilia and the following factors: duration of parasitic infection and intensity of parasitic infection supported the impression that the obstruction caused by advanced chronic fasciolosis in the rat may be related to biliary sepsis. Extrapolation to human infection in fasciolosis hyperendemic areas is discussed. In conclusion, the results of the rodent model should lead to a reconsideration of treatment features in human disease, i.e. therapeutic strategies should not only include a parasitic treatment but also consider the possibility of bacterial co-infection.