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.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

Structural insights into natural compounds as inhibitors of Fasciola gigantica thioredoxin glutathione reductase.

Fascioliasis is caused by the helminth parasites of genus Fasciola. Thioredoxin glutathione reductase (TGR) is an important enzyme in parasitic helminths and plays an indispensable role in its redox biology. In the present study, we conducted a structure-based virtual screening of natural compounds against the Fasciola gigantica TGR (FgTGR). The compounds were docked against FgTGR in four sequential docking modes. The screened ligands were further assessed for Lipinski and ADMET prediction so as to evaluate drug proficiency and likeness property. After refinement, three potential inhibitors were identified that were subjected to 50 ns molecular dynamics simulation and free energy binding analyses to evaluate the dynamics of protein-ligand interaction and the stability of the complexes. Key residues involved in the interaction of the selected ligands were also determined. The results suggested that three top hits had a negative binding energy greater than GSSG (-91.479 KJ · mol-1 ), having -152.657, -141.219, and -92.931 kJ · mol-1 for compounds with IDs ZINC85878789, ZINC85879991, and ZINC36369921, respectively. Further analysis showed that the compound ZINC85878789 and ZINC85879991 displayed substantial pharmacological and structural properties to be a drug candidate. Thus, the present study might prove useful for the future design of new derivatives with higher potency and specificity.

The in vitro anthelmintic activity of the ethanol leaf extracts of Terminalia catappa L. on Fasciola gigantica.

At present, there are no medicinal plant extracts currently available for treatment and control of fasciolosis. The present work could provide, for the first study, conclusions on the in vitro fasciolicidal properties of the ethanol extract of Terminalia catappa L. (TcCE) leaves against adult Fasciola gigantica after incubation with RPMI-1640 medium containing the TcCE at various concentrations and times when compared with triclabendazole (TCZ). The relative motility and survival index values of the TcCE-treated flukes decreased at a more rapid rate than the TCZ-treated flukes. The death of the parasites was observed after exposed to TcCE at 3 h incubation with 400, 800 and 1000 µg mL-1, and at 6 h incubation in 100 and 200 µg mL-1. Vacuolization, blebbings and partial disruption on the parasites' tegument were observed by light microscopy. When examined by scanning electron microscopy, TcCE caused similar tegumental alterations in the parasites as those observed in TCZ treatment but with larger damage at comparative incubation periods, consisting of swelling, blebbing, disrupted blebs, loss of spines, leading to the erosion, lesion and eventual disruption of the total tegument. Therefore, the TcCE may exert its fasciolicidal effect against F. gigantica by initially causing the tegumental alteration.

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.

In vitro screening of Cymbopogon jwarancusa and Conyza canadensis against liver flukes.

Aim of present study was to screen medicinal plants for flukicidal activity in vitro to develop alternative sources of treatment for trematodes infection. For this purpose, crude methanolic extracts (CME) of Cymbopogn jwarancusa and Conyza canadensis were prepared and live adult flukes viz; Fasciola gigantica, and Paramphistomum cervi isolated from liver and bile ducts of slaughtered buffalo were subjected to different drug concentrations as well as positive and negative control. Motility inhibition and paralysis leading to the death of parasites was considered as flukicidal activity of plants extracts. The results revealed that CME of C. jwarancusa and C. canadensis showed significant (P<0.05) flukicidal activity compared to positive control. Also there was a significant effect of different concentrations (P<0.05) and exposure of time on the flukes (P<0.05). Furthermore, ED50 for C. jwarancusa and C. canadensis against F. gigantica were 13.1 and 41.4 mg/ml, respectively. In the case of P. cervi, it was 10.8 and 29.0 mg/ml. It can be concluded that both tested plants showed greater flukicidal activity as compared to positive control with Albendazole till the 8(th) hr. These potent plants needs further studies invivo to elucidate their mode of action.

Evidence of Fasciola spp. resistance to albendazole, triclabendazole and bromofenofos in water buffaloes (Bubalus bubalis).

Fasciolosis caused by Fasciola spp. is considered the most important helminth infection of ruminants in tropical countries. Anthelmintic resistance has become a global concern. This study compared the efficacy of the commonly used anthelmintics, determined the toxicity level and any indication of resistance. Thirty two water buffaloes naturally-infected with Fasciola spp. were used to determine the efficacy of triclabendazole (TBZ), albendazole (ABZ), and bromofenofos (BRO) using Fecal Egg Count Reduction Test (FECRT). To test the toxicity of the drugs given, serum glutamic-pyruvic transaminase (SGPT) was evaluated before and within one week after treatment. One dose administration of ABZ registered an efficacy of 79.17%, 73.33% for TBZ and 70.83% for BRO. Efficacy in two dose- treatment group was 83.33% for both BRO and ABZ, and 90.00% for TBZ. Two dose-treatment was effective for TBZ (90%), ineffective for BRO and ABZ. SGPT levels were not significantly different between pre-treatment and post- treatment across all treatments. Giving one or two doses of anthelmintics, at one month interval, does not increase the efficacy of the three drugs tested. The study also implies that anthelmintic resistance may have developed in the animals.

Possible fasciocidal activity of methanol extract of Dregea volubilis leaves.

In eastern and southern part of India Dregea volubilis (Family Asclepediaceae) is widely used as anthelmintic in traditional system of medicine. The present study was conducted to evaluate the fasciocidal activity of the methanol extract of D. volubilis leaves (MEDV) and to observe the drug's effect on organisms through scanning electron microscopic (SEM) study. Live parasites (Trematode: Fasciola gigantica) were collected in 0.9% phosphate-buffered saline from the bile ducts of buffalo. Those were incubated in the said media at 37 ± 1 °C either as control, or with MEDV at 5, 10, 25, 50 and 100 mg/ml as test groups or with albendazole at 10mg/ml as standard group. The efficacy of the extract was determined on the basis of paralysis (temporary loss of spontaneous movement of the organisms) and/or death of the liver flukes. Death was confirmed when the organisms lost their motility permanently and their motility could not be revived even when vigorously shaken or dipped in warm water. MEDV at all concentration effectively paralyzed first and then killed the liver flukes (p < 0.001). Maximum fasciocidal activity was found with concentration of 100 mg/ml at 38.83 ± 3.41 min. Through SEM study, severe damages were observed in both the suckers as well as on the tegumental surfaces of the treated liver flukes. The study confirmed the fasciocidal activity of the MEDV.

Mirazid in treatment of three zoonotic trematodes in Beni-Sweif and Dakhalia Governorates.

A total of 60 patients with schistosomiasis (40), fascialiosis (15) and heterophyiasis (5) were selected Beni-Sweif and Mansoura Districts and subjected to history taking, clinical examination, Kato thick smear, sedimentation and hatching test (for schistosomiasis cases) at the beginning of the study, 2 & 3 months after treatment with Oleo-resin of Myrrh (Mirazid) in a dose of 10 mg/kg/day for 6 consecutive days an hour before breakfast. The results showed a significant improvement in symptoms with minimal negligible or no side effects. The cure rates, 2 & 3 months after treatment were 80.7%% & 11.8%% for schistosomiasis, 93.3% & 6.6% for fascioliasis, and 100% for heterophyiasis. The clinical picture of schistosomiasis before treatments were easy fatigability, anorexia, nausea, vomiting, epigastria pain, abdominal distention, right upper guardant pain, colicky abdominal pain, left upper and/or lower guardant pain, abdominal rumbling, dysentery, diarrhea, rectal bleeding, constipation, and alternating bowel habit. Those of fascioliasis were abdominal distention, dripping of saliva, right upper guardant, colicky abdominal pain, weight loss, easy fatigability, intermittent jaundice, anorexia, nausea, vomiting, epigastria, left upper and/or lower quadrant pain, right layer quadrant pain, loin pain, abdominal rumbling, diarrhea, constipation, and alternating bowel habit. The safety and efficacy of C. molmol extract in treating heterophyiasis (100%), fascioliasis (100%) and schistosomiasis (92.5%) were documented.

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica.

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.

Botanical study of Meryta denhamii Seem. and its antihelminthic activity against Fasciola gigantica.

The anthelmintic activity of the ethanolic extracts of stems and leaves of Meryta denhamii Seem. against adult liver flukes "Fasciola gigantica" was studied in vitro. Although leaves extract was inactive, stems extract exhibited anthelmintic activity and recorded LC50 and LC90 values, 16 and 26 gm/l respectively. The mode of action of the ethanolic extract of the stems on the adult flukes was evaluated by scanning electron microscopy (SEM). Tegumental sloughing, loss of spines and deformity of suckers were observed. These damages are responsible for the vermicidal effect of the ethanolic extract of the stems. The saponin contents of the stems (4.25%) and leaves (2.45%) were determined using haemolytic index. The plant was identified based on the macro and micromorphological features of the stem and leaf.

Myrrh and trematodoses in Egypt: an overview of safety, efficacy and effectiveness profiles.

Myrrh is an herbal product that has been used since ancient ages for traditional medication and other purposes. The revolution of myrrh as an antiparasitic agent in Egypt began in the 1990s through scientific evidence-based research. The human trematode infections in Egypt were the main focus of research with stories of success and disagreement, at times. The present paper reviewed the antiparasitary activity of myrrh with stress on its possible mode of action, its safety, efficacy and effectiveness on trematode infections in experimental studies and clinical trials in Egypt as well as its molluscicidal effects on the intermediate hosts of trematodes.

Fasciola gigantica: anthelmintic effect of the aqueous extract of Artocarpus lakoocha.

The effect of the crude extract of Artocarpus lakoocha (70% composition is 2,4,3',5'- tetrahydroxystilbene -THS) on adult Fasciola gigantica was evaluated after incubating the parasites in M-199 medium containing 250, 500, 750 and 1000 microg/ml of the crude extract, or triclabendazole (TCZ) at the concentrations of 80 and 175 microg/ml as the positive control, for 3, 6, 12 and 24h, using relative motility (RM) assay and observation by scanning electron microscope (SEM). Decreased contraction and motility were first observed after 3h incubation with TCZ at the concentration 80 and 175 microg/ml. TCZ markedly reduced the parasite's motility at the concentration of 175 microg/ml at 6h, and killed the worms after 12h exposure. The crude extract of A. lakoocha at all concentrations reduced the parasite's motility similar to TCZ at 3h incubation. In 250 and 500 microg/ml of the crude extract, the values were decreased from 3 to 12h, then they were stable between 12 and 24h and reduced to the level approximately 30-40% of the control. At 750 and 1000 microg/ml concentrations the crude extract rapidly reduced the RM values from the start to 12h and killed the parasites between 12 and 24h incubation. The crude extract also inhibited the larval migration by 75% and 100% at the concentrations of 250-500 and 750-1000 microg/ml, respectively. TCZ and the crude extract caused sequentially changes in the tegument including swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesion was formed which exposed the basal lamina. The damage appeared more severe on the dorsal than the ventral surface, and earlier on the anterior part and lateral margins when compared to the posterior part. The severity and rapidity of the damages were enhanced with increasing concentration of the crude extract. Hence, the crude extract of A. lakoocha, may exert its fasciolicidal effect against adult F. gigantica by initially causing the tegumental damage.

Laboratory assessment of the molluscicidal and cercaricidal activities of the Egyptian weed, Solanum nigrum L.

The molluscicidal properties of Solanum nigrum L. were tested against three Egyptian snail species (Biomphalaria alexandrina, Bulinus truncatus and Lymnaea natalensis), each an intermediate host of parasites causing human schistosomiasis or fascioliasis. The plant was collected in two regions within Egypt: Fayium and Giza. Snails were exposed for 24 and 48 h, to the dry powdered fruits and leaves or to crude water extracts of the powders, and mortality was recorded. The water extract of the leaves collected in Fayium (FLWE) had the highest molluscicidal activity, with median lethal concentrations (LC50) of 18.6 mg/litre for Bi. alexandrina, 14.5 mg/litre for Bu. truncatus and 17.7 mg/litre for L. natalensis. When Bi. alexandrina infected with Schistosoma mansoni were exposed to FLWE (20 or 25 mg/litre), they shed significantly fewer cercariae than unexposed snails (P < 0.02). The cercaricidal properties of FLWE were directly tested against S. haematobium, S. mansoni and Fasciola gigantica cercariae and a time-concentration relationship was observed; the concentrations needed to kill all cercariae (LC100) within 30 min of exposure were 30 mg/litre for both S. haematobium and S. mansoni and 40 mg/litre for F. gigantica.