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.

Microscopic alterations in Fasciola hepatica treated with the essential oils of Pelargonium graveolens and Citrus aurantium.

Fasciolosis is one of the most important parasitic diseases in animal health, affecting mainly ruminants, causing economic and productivity losses. This study aimed to evaluate the in vitro ovicidal and adulticidal activity of essential oils (EOs) from Pelargonium graveolens (geranium) and Citrus aurantium (sour orange) on Fasciola hepatica. Performed Gas Chromatography of EOs P. graveolens and C. aurantium, with major compound, citronellol (31.37 %) and limonene (93.89 %), respectively. For the cytotoxicity assay, the sour orange EO showed to be promising when used in lower concentrations. For the ovicidal tests, the eggs were incubated with geranium EOs at concentrations from 4.5 to 0.03375 mg/mL and sour orange at concentrations from 4.25 to 0.031875 mg/mL, along with controls. The viable eggs were counted on the 14th day post-incubation. Adult forms of F. hepatica were incubated containing the EOs and observed for 24 h after treatment, as well as the control groups. Later the specimens were fixed for histological analysis. Geranium and sour orange EOs in trematode eggs at the concentrations tested were 100 % effective in inactivating hatching (p < 0.05) when compared to the untreated control. In the adulticidal test, the essential oil of P. graveolens at both concentrations tested (0.0675 and 0.03375) within 15 h, promoted the death of flukes. For C. aurantium, 18 h was enough to inactivate all specimens, up to a concentration of 0.06375. The histological analysis, observed the accumulation of liquid in the tegument in the specimens incubated in C. aurantium and P. graveolens, with vacuolization in the tegument and spines, preventing externalization. The results of the study present OEs with efficient ovicidal and adulticidal activity.

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.

Ovicidal in vitro activity of the fixed oil of Helianthus annus L. and the essential oil of Cuminum cyminum L. against Fasciola hepatica (Linnaeus, 1758).

The fascioliasis is a parasitic disease of importance in veterinary medicine and public health. For this parasitosis, the treatment by synthetic fasciolicides is used and due to their intense use although they have been shown less effective because of the establishment of resistant Fasciola hepatica population to these drugs, with a global concern. The use of derived products of plants with biological activity has been shown promising in the control of parasites. In this context, we evaluated the chemical composition and action of ovicidal in vitro fixed oil of Helianthus annuus L. (FOH) and essential oil of Cuminum cyminum L. (EOC), as well as their combination (FOH + EOC) of F. hepatica. In the assay in vitro of F. hepatica were submitted to different concentrations of oils, such as FOH (2.3 mg/mL + 0,017 mg/mL); EOC (2.07 mg/mL + 0,004 mg/mL) and the combination of (1.15 mg/mL + 1.03 mg/mL to 0,0085 mg/mL + 0,008 mg/mL) as well as a positive control of thiabendazole (0.025 mg/mL) and a negative control with distilled water and tween. The identification of the majority chemical compounds was performed by gas chromatography. The -cell viability of the oils was tested in MDBK cellular line by the MTT method. The majority compounds in the FOH were the linoleic (53.6%) and oleic (35.85%) unsaturated fatty acids, and the majority phytochemicals compounds in the EOC were the Cumaldehyde (26.8%) and the 2-Caren 10-al (22.17%). The EOC and the combination presented effectiveness of 99% (±1) and of 94% (±1) in the concentration of 0.03 mg/mL and 0.035 mg/mL+0.03 mg/mL, respectively, and the FOH was insufficiently active as ovicidal. The cell viability at this concentration of EOC was 93%. From the results above we could infer that the EOC is promising as a new alternative for the fascioliasis control.

Efficacy of purified fractions of Artemisia ludoviciana Nutt. mexicana and ultraestructural damage to newly excysted juveniles of Fasciola hepatica in vitro.

The study aimed to evaluate the fasciolicidal efficacy of extracts and fractions of Artemisia ludoviciana and identify the active substance. Extracts from A. ludoviciana were obtained by using hexane, ethyl acetate and methanol. To test the extracts, newly excysted juveniles of Fasciola hepatica were artificially obtained. The extracts were tested at concentrations of 125, 250, 375 and 500 mg/L. In each test run, an untreated control group and control wells containing triclabendazole sulfoxide were also included. The flukes were examined at 24, 48 and 72 h after treatment. Ethyl acetate extract (ALEAE) showed 100 % efficacy at 48 h of exposure (P < 0.05). Then, this extract was fractionated by column chromatography (CC), and the obtained fractions were evaluated in vitro as previously mentioned. The results indicated that fraction 3 yielded 100 % efficacy at 48 h (P < 0.05). Subsequently, the purification of fraction 3 was performed. New fractions were obtained (A-L), with sub-fraction "J" exhibiting 100 % efficacy at 24 h (P < 0.05). These sub-fractions were submitted to phytochemical analysis, demonstrated the presence of sesquiterpene lactones. Likewise, were analyzed by HPLC/MS/DAD, and the results showed that artemisinin was the main compound. In addition, flukes treated were examined by scanning electron microscopy (SEM) showing areas of inflammation throughout the tegument.

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.

Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives.

Infections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases.

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.

Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.

Effects of miconazole/clotrimazole and praziquantel combinations against the liver fluke Opisthorchis felineus in vivo and in vitro.

The liver fluke Opisthorchis felineus (Rivolta, 1884) is the causative agent of opisthorchiasis felinea in Eurasia. Opisthorchiasis is a serious human and fish-eating animal's disease affecting bile ducts and the gall bladder. Currently, the main drug for specific therapy of opisthorchiasis is praziquantel. We have previously shown that azole inhibitors of O. felineus cytochrome P450 significantly reduced survival of the worms in vitro. Here, we studied in vitro anthelmintic effects of drug combinations involving azole substances approved by the US Food and Drug Administration together with praziquantel against adult or juvenile O. felineus liver flukes. A synergistic interaction was shown for praziquantel-clotrimazole (CI = 0.68) combination and for praziquantel-miconazole (CI = 0.68) combination against adult helminths in vitro. Praziquantel-miconazole (CI = 0.30) had a strongly synergistic effect against newly excysted metacercariae. We also tested anthelmintic effects of azole substances and their combinations with praziquantel in vivo in an animal model of chemotherapy. The treatment of juvenile worms (1 day postinfection) with 100 mg/kg miconazole resulted in a worm burden reduction (WBR) of 37.5% (P = 0.049), with 100 mg/kg clotrimazole causing a WBR of 31.25% (P = 0.025). The treatment of adult worms (5-6 weeks postinfection) with 100 mg/kg or 200 mg/kg miconazole yielded a WBR of 23.8% (P = 0.01) and 21.4% (P = 0.006), respectively. When praziquantel was administered together with clotrimazole or with miconazole, a WBR slightly greater than the effect of ED50 praziquantel was observed (WBR of 59.5 and 54.7%, respectively).In conclusion, the synergistic effect of the praziquantel-clotrimazole and praziquantel-miconazole combinations observed in vitro was not confirmed in vivo. Thus, this combination chemotherapy revealed no benefits over praziquantel monotherapy in the treatment of opisthorchiasis felinea.

A new method for laboratory rearing of Galba truncatula, the intermediate host of Fasciola hepatica.

In this study a relatively large and open top aquarium was designed, constructed and introduced as a suitable habitat for nutrition, growth and development as well as for egg laying and breeding of Galba truncatula under laboratory conditions. The soil and water used in the aquarium were collected from the locality in which the snails were collected. The aquarium was placed in a laboratory with temperature of 18-32 ºC and relative humidity of 22-37% respectively, according to the season. The artificial light was controlled by a light timer, giving 12 h of light and 12 h of darkness. The snails were fed with dried lettuce leaves, Cyperus alternifolius (aquatic plant), Spirulina (algae), Orthotrichum rupestre (moss) and cuttlebone (a supplementary source of calcium). Approximately five weeks after the start of study, there was evidence of reproduction and success in rearing of G. truncatula by the appearance of eggs and small snails (0.1-0.5 mm) in the aquarium. In conclusion, large scale laboratory rearing of G. truncatula is a feasible task. The method may be improved by balancing the temperature and relative humidity as well as by optimizing the soil type, the water quality and the type of food.

Disruption of egg production by triclabendazole-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh (Mirazid).

An in vitro study has been carried out to monitor changes to the female reproductive system in adult triclabendazole (TCBZ)-resistant Fasciola hepatica following treatment with a commercial preparation of myrrh ("Mirazid"). Flukes were immersed for 6 h and 24 h in myrrh extract at a concentration of 200 µg/ml, then processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis' gland, ovary and vitellaria. Egg production had become abnormal at 6 h post-treatment (pt), with the uterine lumen being filled with free vitelline cells and masses of shell protein material; few eggs were present. At 24 h pt, no eggs were present. Distinct changes to the ovary and Mehlis' gland were only observed after 24 h incubation in Mirazid. The ovary contained numbers of apoptotic oogonia and oocytes. In the Mehlis' gland, the S1 cells were disorganised and the processes from them were vacuolated, although the disruption was not significant. More severe changes were observed in the vitelline cells and follicles. After 6 h incubation in Mirazid, although the gross organisation of the vitelline follicles appeared to be normal, nuclear changes indicative of the early stages of apoptosis were observed in the stem cells and shell protein production by the mature cells had decreased. At 24 h pt, a distinct shift in cell population was evident, with the follicles containing mainly mature cells and spaces were present between the cells. The shell globule clusters in the mature cells were disorganised. In more severely-affected follicles, cells were seen to be breaking down, with karyolytic nuclei and disintegrating cytoplasm. Overall, the results have shown that exposure to Mirazid treatment had a severe impact on egg production by TCBZ-resistant flukes, an effect that was mediated by disruption of the vitelline cells and of the mechanism co-ordinating egg formation in the ootype.

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.

A study of the molluscicidal and larvicidal activities of Citrullus colocynthis (L.) leaf extract and its main cucurbitacins against the mollusc Galba truncatula, intermediate host of Fasciola hepatica.

BACKGROUND: The molluscicidal and larvicidal activities of the medicinal plant Citrullus colocynthis leaf extracts and its main cucurbitacins were tested against the mollusc gastropod Galba truncatula, the intermediate host of Fasciola hepatica. RESULTS: Our findings proved for the first time that the molluscicidal activity was correlated with the presence of terpenoids. A significant molluscicidal value was found in the ethyl acetate extract (LC50 = 12.6 mg L-1 ). Further fractionation of this extract led to the isolation of two main compounds identified to cucurbitacin E 1 and 2-O-ß-d-glucocucurbitacin E 2. Their molluscicidal activities were also investigated and they possessed close activities with LC50 = 9.55 and 10.61 mg L-1 for compounds 2 and 1, respectively. CONCLUSION: The ethyl acetate extract and both pure compounds proved the highest larvicidal activities, with a deterioration rate exceeding 89.2% (89.2-100%) and with no toxic effects against associated fauna. © 2016 Society of Chemical Industry.

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.

Molluscicidal activity of Solanum elaeagnifolium seeds against Galba truncatula intermediate host of Fasciola hepatica: Identification of ß-solamarine.

CONTEXT: The persistence of fascioliasis in many developing countries urges the search for simple, cheap, and effective substances. In this view, plants provide interesting molluscicidal activities thanks to the secondary metabolites they produce. The genus Solanum is known for its potent effect on vector snails. OBJECTIVE: The molluscicidal activity of Solanum elaeagnifolium Cav. (Solanaceae) seeds against Galba truncatula Müll. (Lymnaeidae), intermediate host of Fasciola hepatica L. (Fasciolidae), was evaluated. MATERIALS AND METHODS: Solanum elaeagnifolium seeds were powdered and successively extracted using n-hexane, methylene chloride, acetone, and methanol, for 20 h each. After filtration, solvents were evaporated. An acid-base treatment was conducted on seed methanolic extract to isolate total alkaloids and ß-solamarine. Total saponins fraction was obtained after successive macerations and evaporations. The molluscicidal activity was evaluated by subjecting snails, in groups of 10, for 48 h to 500 mL of extracts, fractions, and pure product aqueous solutions, each containing amounts, ranging from 1 to 50 mg of plant material in 5 mg increments. RESULTS: The methanolic extract of seeds, ß-solamarine isolated for the first time from this plant and total saponins fraction showed very potent activities on snails, giving respective median lethal concentrations (LC50) of 1.18, 0.49, and 0.94 mg/L. Total alkaloids fraction obtained from the methanolic extract was less active giving an LC50 value of 14.67 mg/L. DISCUSSION AND CONCLUSION: This study emphasizes that glycoalkaloids and saponins of Solanum elaeagnifolium are potent molluscicidal agents. Seed methanolic extract, ß-solamarine, and total saponins fraction may be used as molluscicides.