Evaluation of the ovicidal activity and fasciolicidal activity of the extract of ethyl acetate from Artemisia ludoviciana Nutt. spp. mexicana and of artemisinin against adult parasites of Fasciola hepatica.

The objective of this work was to evaluate the effect of the ethyl acetate extract from A. ludoviciana (EALM) and artemisinin against adult parasites and eggs of F. hepatica. For the ovicidal assay, cell culture plates with 24 wells were used, and 90 to 110 F. hepatica eggs were placed in each well. The eggs were exposed to concentrations of 100, 200, 300, 400, and 500 mg/L EALM and incubated for 16 days. Additionally, triclabendazole (TCBZ) was used as a reference drug at concentrations of 10 and 50 mg, and the response of artemisinin at concentrations of 10 and 20 mg was simultaneously assessed. Adult flukes were exposed to concentrations of 125, 250, 375, and 500 mg/L EALM. The results of the ovicidal action of EALM on the eggs showed that concentrations greater than 300 mg/L were significant, with ovicidal percentages greater than 60% observed on day 16 of incubation (p < 0.05). The maximum efficiency of EALM on adult flukes was reached 72 h post-exposure at a concentration of 125 mg/L (p < 0.05).

A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

Fasciola gigantica vaccine construct: an in silico approach towards identification and design of a multi-epitope subunit vaccine using calcium binding EF-hand proteins.

Continuous attempts have been made to pinpoint candidate vaccine molecules and evaluate their effectiveness in order to commercialise such vaccines for the treatment of tropical fascioliasis in livestock. The pathophysiology of fascioliasis can be related to liver damage brought on by immature flukes that migrate and feed, as well as immunological reactions to chemicals produced by the parasites and alarm signals brought on by tissue damage. Future research should, in our opinion, concentrate on the biology of invasive parasites and the resulting immune responses, particularly in the early stages of infection. The goal of the current study was to use the calcium-binding proteins from F. gigantica to create a multi-epitope subunit vaccine. The adjuvant, B-cell epitopes, CTL epitopes, and HTL epitopes that make up the vaccine construct are all connected by certain linkers. The antigenicity, allergenicity, and physiochemical properties of the vaccine construct were examined. The vaccine construct was docked with toll-like receptor 2, and simulations of the molecular dynamics of the complex's stability, interaction, and dynamics were run. After performing in silico cloning and immunosimulation, it was discovered that the construct was suitable for further investigation. New vaccination technologies and adjuvant development are advancing our food safety procedures since vaccines are seen as safe and are accepted by the user community. This research is also applicable to the F. hepatica system.

Development of a safe pediatric liquisolid self-nanoemulsifying system of triclabendazole for the treatment of fascioliasis.

Fascioliasis, a common parasitic infection observed in the pediatric patient population, is a leading cause of concern in countries with poor/unhealthy water resources. To treat this condition first line agent such as triclabendazole (TBZ) has been the choice therapy. However, there is a major hurdle in exploiting TBZ. Characterized with poor aqueous solubility (0.1 mg/L), its solubility has been the rate limiting factor, rendering requirement of large doses of TBZ. To address the same, the focus of the current study was to develop a self-nano emulsifying drug delivery system (TBZ-SNEDDS) for TBZ and developing dose customizable pediatric dispersible color-coded tablets. TBZ-SNEDDS were successfully formulated by using Kolliphor®EL, as a surfactant, a lipid phase of medium chain triglyceride and α-tocopherol in the ratio of (1:1), with dimethylacetamide (DMA) as a solvent. It was observed during in vitro release studies that there was a significant effect of fed conditions on the rate of TBZ release from the formulation. greater than 85 % TBZ was observed to release in fed conditions in comparison to fasted conditions. As currently TBZ is prescribed on a weight-based dosage regimen, it is imperative to develop a dose-customizable fast dissolving pediatric formulation. Hence, TBZ-SNEDDS could prove to be pivotal in helping countless children around the world in desperate conditions to get cheap yet effective therapy.

In vitro anthelminthic activity of fringed spiderflower (Cleome rutidosperma) ethanolic leaf extract against Fasciola spp.

Fasciolosis is considered as one of the leading causes of morbidity and mortality among ruminants in the Philippines. Though anthelmintic drugs are widely used to treat and control the condition, it is still worthwhile to search for alternative treatments especially when resistance to commonly used anthelmintic drugs has been reported. In this study, the ethanolic leaf extract of fringed spiderflower (Cleome rutidosperma) was evaluated for its in vitro anthelmintic activity against Fasciola spp. Specifically, the study compared the different concentrations of ethanolic leaf extract and the commonly used anthelmintic drug (albendazole) on the gross motility and histology of Fasciola spp. The study consisted of five treatments: treatment 1, 2, and 3 which contain 10%, 20%, and 40% leaf extract, respectively, treatment 4 with 10% albendazole as the positive control, and treatment 5 with nutrient broth as the negative control. The motility of the Fasciola spp in all treatments was visually analyzed based on the established criteria. In addition Fasciola spp. in different treatments were subjected to tissue processing and histological examination. Results showed that increasing concentrations of leaf extract resulted in a decreasing time for Fasciola spp. to have a motility score of zero. Specifically, 10%, 20%, and 40% leaf extract resulted in a cumulative time period of 55.00 ± 5.00 min, 26.67 ± 2.89 min, and 15.00 ± 0.00 min, respectively, for the Fasciola spp. to have a motility score of zero. On the other hand, albendazole resulted in a 240.00 min cumulative time before it can cause a motility score of zero. Histologic examination showed that the different concentrations of leaf extract affected the tegument and parenchyma of the Fasciola spp.

Evaluation of the therapeutic effect of propolis on Fasciola gigantica and Clostridium novyi type B infections in sheep.

The use of natural products for disease control is a promising approach to solving the problem of drug resistance. The aim of the research reported here was to evaluate the fasciolicidal and anti-Clostridium novyi type B activities of propolis administered orally to sheep infected with Fasciola gigantica and C. novyi type B. Sheep infected with both pathogens were divided into two groups: an infected treated group and an infected non-treated group. The treatment was oral administration of 50 mg propolis extract/kg daily for 15 days. The body weight of the sheep, fecal egg counts of F. gigantica, serum levels of F. gigantica IgG, concentrations of cytokines (IL-2, IL-10, and IL-17), and bacterial counts of C. novyi were evaluated. Following treatment, the sheep had increased body weight and a significant decrease in the egg count, which was reduced by 54.54% at 15 days post treatment. The level of anti- Fasciola IgG increased, whereas levels of IL-2, IL-10, and IL-17 decreased in propolistreated sheep. Treatment of sheep with propolis produced a significant reduction in fecal count of C. novyi, from 8 × 109 to 3 × 103 colony units per gram at 15 days post treatment. This research highlights the therapeutic potential of Egyptian propolis extract as a treatment against F. gigantica and C. novyi type B infections, and investigated its mode of action through its effect on some cellular and humoral responses in sheep with both infections.

Current status of prevalence and risk factors associated with liver fluke Fasciola hepatica in cattle raised in different altitudinal regions of Colombia.

Fasciola hepatica is a trematode parasite with a worldwide distribution that affects mammals, including humans, and is considered a major food safety and public health problem. A research was conducted in three hundred eighty-five livestock farms located in different altitudinal regions of Colombia between February 2017 and March 2018 with the aim to determine the coprological prevalence, seroprevalence (SP) and risk factors associated with liver fluke F. hepatica. A total of 5620 fecal sample were collected from cattle and processed using sedimentation technique. Additionally, 597 blood samples were collected and processed by an ELISA test. The coprological prevalence of F. hepatica in animals and herds was 6.4% and 38.4%, respectively. Regarding the prevalence by municipalities, there was statistical association (P < .05), indicating that the prevalence was higher in those municipalities above 2000 masl (meters above sea level). Regarding risk factors, cattle raised in municipalities at 2000 to 3000 masl showed about 4 (OR = 3.96 [CI95% 3.1-5]; P < .05) times higher probability for infection than those raised at lower altitude. Cattle from 2 to 3 year of age, showed almost 4 times (OR = 3.7[CI95% 2.5-5.4]; P < .05), increased infection risk. Females and dairy breeds showed almost 2 (OR = 1.9 [CI95% 1.3-2.6]; P < .05) and 10 times (OR = 10 [CI95% 7.8-12.8]; P < .05), respectively, increased infection risk. The presence of antibodies anti- F. hepatica (SP = 37.8%; [CI95% 35.8-39.7]; P < .05) in cattle of the region under study is confirmed, which suggests an endemic behavior of this parasitosis. The present study provides current information on the prevalence of F. hepatica in different altitudinal regions of Colombia, and these findings could be useful for designing new holistic control measures for the liver fluke F. hepatica infection.

A holistic model to assess risk factors of fasciolosis in Ankole cattle.

In recent decades, remote sensing (RS) technology and geographical information systems (GIS) were increasingly used as tools for epidemiological studies and the control of zoonotic diseases. Fasciolosis, a zoonotic disease caused by a trematode parasite (Fasciola spp.), is a good candidate for the application of RS and GIS in epidemiology because it is strongly influenced by the environment, i.e. the habitat of the intermediate host. In this study, we examined variables which may increase the fasciolosis risk of Ankole cattle in the degraded and overgrazed Mutara rangelands of north-eastern Rwanda. The risk variables considered included three environmental variables (normalized difference vegetation index, NDVI; normalized difference moisture index, NDMI; normalized difference water index, NDWI), two landscape metric variables (rangeland proportion, building density), two geological variables (poorly-drained soil proportion, elevation) and three animal husbandry variables (herd size, adult proportion and the body condition score). Fasciola spp. prevalence was used as the dependent variable, sampling season as a fixed factor and four principal components (PCs, condensed from the ten risk variables) as covariates in a univariate General Linear Model. Fasciola spp. prevalence was positively correlated to rangeland proportion, cattle herd size in rural areas, adult proportion and individual body condition. Moreover, high Fasciola spp. prevalence was found in densely vegetated areas with high moisture (high values of NDVI and NDMI), in combination with large proportions of poorly-drained soil at low elevations. Future investigations should focus on increased sampling across the Mutara rangelands to prepare a predictive, spatial fasciolosis risk map that would help to further improve sustainable land-use management.

Liver fluke in Irish sheep: prevalence and associations with management practices and co-infection with rumen fluke.

BACKGROUND: The present study aimed to identify the national prevalence of Fasciola hepatica in Irish sheep and to conduct a risk analysis assessment based on management and treatment practices in participating flocks. Also, co-infection with rumen fluke was quantified and its association with liver fluke and management practices was assessed. METHODS: A total of 305 sheep flocks were selected ensuring even national representation of the sheep population. Participating farms were asked to complete a survey questionnaire on farm management practices and submit faecal samples during the winter of 2014-2015. Pooled faecal samples were analysed for the presence of F. hepatica and co-infection with rumen fluke. Apparent and true prevalence were calculated, additionally, the rate of co-infection with rumen fluke was also obtained. Correlation and regression analyses were used for assessing associations between management practices, liver fluke infection and co-infection with rumen fluke. RESULTS: The national true prevalence of F. hepatica was 50.4% (n = 305). Regional prevalence varied from 41% in the east to 52% in the south. Co-infection with rumen fluke was observed in 40% of the studied population and correlated with increased F. hepatica egg counts (OR = 2.9; P ≤ 0.001). Predominant breeds were Suffolk, Texel and Horned Mountain breeds. Beef cattle were the most frequent type of other livestock present on farms and mixed species grazing was frequently reported (73%). More than half of the flocks reported a mid-to-late lambing period (March-April). Use of mountain land for grazing was of 32%. Flukicides were most commonly used twice over the autumn-winter period. Regression analyses highlighted significant association of F. hepatica status, with the presence of other livestock on farm, frequency of flukicides used during the winter and clinical presentation of liver fluke. A significant increase in eggs per gram of faeces was observed in Charollais sheep in comparison with all other breeds. Co-infection with F. hepatica and Calicophoron daubneyi was also significantly associated with the presence of other livestock on the farm, type of flukicide used and clinical fasciolosis. CONCLUSIONS: The present study provides up-to-date information on the prevalence of F. hepatica in Irish sheep and adds insight to the epidemiology of the disease. These findings will be useful for designing new holistic control measures for F. hepatica infection.

Assessment of the molluscicidal impact of extracted chlorophyllin on some biochemical parameters in the nervous tissue and histological changes in Biomphalaria alexandrina and Lymnaea natalensis snails.

Biomphalaria alexandrina and Lymnaea natalensis snails are the intermediate hosts of schistosomiasis and fasciolosis. The aim of the present study is to evaluate the molluscicidal activity of chlorophyll extract as a photodynamic substance against these snails and how it affected its tissues and the biological system. Chlorophyllin was extracted from deep-frozen Moringa oleifera leaves, and then it was transformed into water-soluble chlorophyllin. The present results showed that it had a molluscicidal activity on B. alexandrina snails (LC50 17.6 mg/l; LC90 20.9 mg/l) and L. natalensis snails (LC50 4.3 mg/l; LC90 6.8 mg/l). Exposing B. alexandrina snails to the sublethal concentrations (LC0, LC10, and LC25) resulted in a significant reduction in their survival rates. Regarding its effect on biochemical parameters, chlorophyllin significantly reduced the acetylcholinesterase activity, protein content, and alkaline and acid phosphatase activity in B. alexandrina nervous tissue compared to the control group. Histopathological changes occurred in the digestive gland of treated B. alexandrina snails where cells lost their nuclei, vacuolated, degenerated, and ruptured, and the lumen increased. Photosynthesizing materials like chlorophyllin are new approaches to control schistosomiasis and fasciolosis in developing countries by affecting their intermediate host. These materials were cheap and environmentally safe to replace the synthetic molluscicides for snail control.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

Screening the Pathogen Box for Identification of New Chemical Agents with Anti-Fasciola hepatica Activity.

Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry. For decades, triclabendazole has been the unique anti-Fasciola drug that can effectively treat this disease. However, triclabendazole resistance in fascioliasis has more recently been reported around the world, and thus, the discovery of novel drugs is an urgent need. The aim of this study was to investigate the fasciocidal properties of 400 compounds contained in the Pathogen Box. The first stage of the screening was carried out by measuring the fasciocidal activity on metacercariae at a concentration of 33 µM each compound (the standard dose). Subsequently, the activities of the most active compounds (n = 33) at their 50% inhibitory concentration (IC50) values against metacercariae were assayed, and the results showed that 13 compounds had IC50s of ≤10 µM. The second stage queried the activities of these compounds at 33 µM against adult flukes, with seven of the compounds producing high mortality rates of >50%. Four hit compounds were selected on the basis of their predicted nontoxic properties, and the IC50 values obtained for adult worms were <10 µM; thus, these compounds represented the best fasciocidal compounds tested here. A cytotoxicity assay on four types of cell lines demonstrated that three compounds were nontoxic at their most active concentration. In conclusion, three hit compounds identified in this proof-of-concept study are potential candidates in the discovery of new fasciocidal drugs. Further studies are warranted.

The Battle of Worldviews: A Case Study of Liver Fluke Infection in Khon Kaen, Thailand.

Control efforts to reduce infection from the parasitic flatworm Opisthorchis viverrini have progressed through understanding the epidemiology of Opisthorchis viverrini, antiparasitic drug developments, technological innovations, health education promoting cooking of fish, and improved hygienic defecation. Yet the problem persists. The case study method was used to examine the fundamental cause of the liver fluke infection problem. Evidence shows that the liver fluke-infected population does not care about living a long life. For them, suffering and death are simply a part of life, and expected. Thus, the cause(s) leading to death is not important. They believe morally bad actions, and predetermined fate associated with kamma in Buddhism, play a big role whether or not one is infected with the liver fluke. Health interventions may be made more effective if they take into account the liver fluke-infected population's worldviews about ethics, morality, life, and death. We researchers should not feel concerned only about medically determined causes of death.

Effects of anthelmintic treatment and feed supplementation on parasite infections and morbidity parameters in Cambodian cattle.

Helminth infections are the cause of morbidity in Cambodian cattle but other factors such as nutritional deficiencies and concurrent diseases may enhance the effects of parasites. The present study aimed to investigate the impact of anthelmintic treatment, feed supplementation, or both on gastrointestinal strongyle (GIS) and trematode infections as well as on morbidity parameters in Cambodian village cattle. At the beginning of the dry season, cattle populations in six villages were randomly assigned to a group: (A) receiving anthelmintic treatment (ivermectin+clorsulon) at week 0; (P) feed pellet supplementation during week 0-13 or both (AP). On five visits (week 0-29), faecal and blood samples were obtained for parasitological examination and haematocrit determination, respectively. Body condition (BCS), hind quarter fouling (HQFS), diarrhoea (DS), and conjunctiva colour (FAMACHA©) were scored and heart girth circumference was determined. To investigate the impact of treatment over time (week 0-29), a mixed model was used with treatment, time, and their interaction as fixed effects, and animal and village as random factors. At baseline, the proportion of GIS positive animals was high (67.9%), whereas trematode infections were low (Paramphistomum: 8.8%; Fasciola: 2.6%). Very thin to emaciated cattle (BCS 1-2) were more prevalent (11.4%) and FAMACHA© scores of ≤3 or below (65.8%) less prevalent than in an earlier study in the region. A Time ⨯ Treatment interaction was present for faecal egg counts (FEC) of GIS, GIS prevalence (both p<0.0001), PCV (p=0.0034), DS (p=0.0086) and HQFS (p=0.0241). For GIS FEC, treatment groups differed at a specific time point, with levels of treatment group P being higher than in A at week 6 (p=0.0054). For Paramphistomum prevalence as well as FAMACHA© scoring, heart girth and BCS, the interaction between treatment and time was not significant, yet, time in itself had a significant impact on all (p<0.0001). The beneficial effects of protein supplementation were unclear from the current study.

Transcriptome profiling of gene expression during immunisation trial against Fasciola hepatica: identification of genes and pathways involved in conferring immunoprotection in a murine model.

BACKGROUND: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. METHODS: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. RESULTS: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. CONCLUSION: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates.

Study on the Relationship between Liver Parasitic Infections and Serum Vitamin A and ß-Carotene Status in Cattle.

OBJECTIVE: This study was conducted to evaluate the relationship between serum levels of vitamin A and ß-carotene in the liver of cattle. METHODS: A total number of 150 samples were selected according to the type of parasitic infections of the liver after postmortem examination and confirmation. Parasitic lesions in the liver were subdivided into three major parasites, including Fasciola hepatica, Dicrocoelium dendriticum, and hydatid cysts. Fifty samples were obtained from cattle without any parasitic infection as a control group. Serum levels of vitamin A, ß-carotene concentrations (µg/dL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity were assayed. RESULTS: Naturally infected cattle with D. dendriticum, F. hepatica and hydatid cyst showed lower vitamin A levels. Serum ß-carotene levels were significantly decreased in all groups (p<0.05). ALT and AST activities in animals with parasitic diseases were statistically higher than in control group (p<0.01). A significant negative correlation was determined between the ß-carotene, vitamin A levels and enzyme activities of the liver in all the three types of liver infections (p<0.001). CONCLUSIONS: This study indicated that serum levels of vitamin A and ß-carotene decline was present in cattle with liver parasite infection and vitamin supplements should be supplied.

Ultrastructural changes to the tegumental system and gastrodermal cells of adult Fasciola hepatica following treatment in vivo with a commercial preparation of myrrh (Mirazid).

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 myrrh ('Mirazid'). Rats infected with the triclabendazole-resistant Dutch isolate were dosed orally with Mirazid at a concentration of 250 mg/kg and flukes recovered 2, 3 and 7 days post-treatment (pt). The flukes were processed for examination by scanning and transmission electron microscopy. A variety of changes to the external surface were observed, culminating in the sloughing of the tegumental syncytium. Internal changes to the syncytium and tegumental cell bodies were more severe and were evident from 2 days pt onwards. Swelling of the basal infolds (leading to flooding of the surface layer) and a decline in secretory body production were the major changes seen. The gastrodermal cells were less severely affected than the tegument, pointing to a trans-tegumental route of uptake for Mirazid by the fluke. Some loss of muscle fibres in the main somatic muscle layers was observed, which may be correlated with the decline in movement of flukes seen at recovery.

Anti-helminthic activity of Momordica charantia L. against Fasciola hepatica eggs after twelve days of incubation in vitro.

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.

Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50µg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.