Effect of methanolic extract of Balanites aegyptiaca fruits on enteral and parenteral stages of Trichinella spiralis in rats.

There is a considerable interest in developing new anthelmintic drugs including those from medicinal plants due to increasing evidence of parasitic resistance against present anthelmintic drugs and decreasing activity against encapsulated larval stages of parasites. This study was carried out to assess, for the first time, the effectiveness of methanolic extract of Balanites aegyptiaca (BAE) fruits against different stages (pre-adult, migrating larvae, and encysted larvae) of Trichinella spiralis in rats compared with commonly used anthelmintic albendazole. Oral administration of BAE at a dose of 1,000 mg/kg b.wt. for five successive days throughout the parasite life cycle led to a marked reduction of migrating and encysted larval rate by 81.7% and 61.7%, respectively, in the muscular tissue. This treatment was less effective against adults in the gut (47.8%). Albendazole treatment at a dose of 10 mg/kg b.wt. for five successive days resulted in a marked eradication of T. spiralis adult worms (94.4%) and less reduction of migrating and encysted larval infections of skeletal muscles (62.2% and 26.4%, respectively). BAE-treated groups showed marked decreases in serum-glucose levels, triglyceride concentrations, aspartate aminotransferase (AST), creatinine phosphokinase (CPK) activities, and lipid peroxide products (malondialdehyde, MDA) as well as an increase in glutathione level in both serum and muscular tissue compared to albendazole-treated- and infected-untreated groups. This result was confirmed by few numbers of living- and dead-encysted larvae and less destruction of the diaphragm and skeletal muscle tissues in BAE-treated groups compared to other treated groups. It can be concluded that the methanolic extract of B. aegyptiaca fruits has high effectiveness against parenteral stages of T. spiralis than albendazole. Albendazole is more effective against enteral stage of T. spiralis than the extract.

In vitro effect of artemether and triclabendazole on adult Fasciola gigantica.

Triclabendazole "Fasinex" is the drug of choice against fasciolosis because of its high efficacy against both mature and immature flukes, however parasite resistance against this drug is increasing. Hence, there is pressing need for new fasciolicidal drugs. In the present study, the in vitro effect of artemether on adult flukes was evaluated using scanning electron microscopy. After 24 h incubation with 10 microg/ml artemether, the tegument of the apical cone appeared to be slightly more swollen than normal. This swelling became so severe and the spines appeared sunken, with their tips protruding from a swollen and blebbed base, on increasing the concentration to 20 microg/ml. With the higher concentration of 30 microg/ml, extensive and severe tegumental swelling occurred in the apical cone region of the flukes. There were many blebs around ventral sucker, a number of which appeared to have burst causing lesion. The tegumental changes occurred following incubation in artemether were comparable with those observed with triclabendazole in its active sulphoxide metabolite form (TCBZ-SX).

Anthelmintic efficacy of Moringa oleifera seed methanolic extract against Fasciola hepatica.

The current study was carried out to assess in vitro and in vivo effects of Moringa oleifera seed methanolic extract on Fasciola hepatica to develop an alternative source of treatment. The in vitro ovicidal effect of M. oleifera seed extract on immature F. hepatica eggs has provided evidence of inhibitory activity on the vitality and hatchability of F. hepatica eggs. This inhibitory activity was concentration-dependent and also correlated strongly with the exposure time. In the in vivo trial, the oral administration of F. hepatica experimentally infected rabbits with doses of 150 mg/kg BW prepared extract per day for 3 consecutive days on the 63rd day post infection confirmed potent fasciolicide activity of the extract. A gradual decrease in fecal egg count (FEC) was detected from the 1st day post treatment until reaching 100% FEC reduction by the 7th day post treatment. No flukes could be found at post mortem examinations. Significant increments of serum total protein, globulin, the activities of ALT and AST, total cholesterol, triglycerides and urea were recorded during the period of infection, which were improved by treatment. Remarkable histopathological alterations were observed in the infected liver and gallbladder tissues which decreased clearly in the treated rabbits. This study proposes that the used extract has promising and potent fasciolicide activity.

An in vitro effect of propolis on adult worms of Fasciola gigantica.

The effect of Siwa propolis on adult flukes was evaluated using scanning electron microcopy. It gave an overview of the surface architecture of the tegument of Fasciola gigantica apical cone. The base of the spines appeared to be "flaking off" and showed severe blebbing after 24h incubation with 10 micro/ml propolis. This swelling became so sever and the spines were barely visible, on increasing the concentration to 20 micro/ml. Besides, there were many large blebs on the apical cone, a number of which appeared to have burst, causing lesions and the tegument was marked by a number of pits caused by the loss of spines. With the higher concentration of 30 micro/ml, erosion of the surface had occurred to such extent that no tegument remained, only a mass of fibrous structures. The tegumental changes occurred following incubation in propolis were compared with those observed with triclabendazole (TCBZ) "Fasinex" because of its high efficacy against both mature and immature flukes.

Evaluation of crude larval protein and recombinant somatic protein 26/23 (rHcp26/23) immunization against Haemonchuscontortus in sheep.

AIM: The aim of this study was to evaluate the potential possibility of crude larval and recombinant (rHcp26/23) antigens of Haemonchus contortus for immunization to control sheep hemonchosis. MATERIALS AND METHODS: A total of 21 lambs were divided into five groups. Lambs were immunized with larval and recombinant (rHcp26/23) proteins at day 0 and day 14 and after that challenged with 5000 infective larvae of H. contortus on day 42. An unvaccinated positive control group was challenged with L3 in the meantime. An unvaccinated negative control group was not challenged. RESULTS: Fecal egg count reduction taking after challenge for rHcp26/23 and larval antigens was 92.2% and 38.2%, respectively, compared with the positive control group. Vaccine incited protection in rHcp26/23 and larval immunization was reflected in significant (p<0.05) decreases in worm burden; 59.9% and 40.1%, respectively. CONCLUSION: Recombinant rHcp26/23 vaccine induced a partial immune response and had immune-protective effect against sheep hemonchosis.

Immunological evaluation of some antigens of Lucilia sericata larvae.

The present study aimed to select an antigen of Lucilia sericata larvae showing both high antigenicity and cross-reactive binding abilities with other related antigens of L. sericata larvae for obtaining a promising candidate vaccine antigen. The ELISA results primary concluded that among the excretory secretory (ES) and midgut (MG) antigens of the different larval instars of L. sericata, MGL2 could be characterized as antigen which was able to reflect the highest level of antigenicity and cross-reactivity with the other tested L. sericata antigens. The results were extended to spot the light on the relation between different protein bands in MGL2 and rabbit hyper- immune sera (HIS) raised against the other tested antigens using SDS-PAGE and Western blot technique. Analysis by SDS-PAGE of ES and MG antigens of the different larval instars of L. sericata revealed common protein bands at molecular weights of about 10, 12, 16, 20, 28, 33 and 46 kDa. Western blotting of MGL2 antigen transferred to nitrocellulose sheet revealed reaction by MGL2 HIS to five polypeptide bands; 20, 28, 33, 46 and 63 kDa. Three bands of 28, 33 and 63 kDa were the most prominent bands detected whereas; there was a weak reaction with bands of 20 and 46 kDa. But what was apparent in Western blot was a strong reaction of all tested HIS with a polypeptide band of 63 kDa. This band might be considered to be the main cause of cross reactive binding ability of MGL2 antigen that had been recorded previously in ELISA technique.

In vitro tegumental alterations on adult Fasciola gigantica caused by mefloquine.

Emergence of drug-resistant Fasciola strains has drawn the attention of many authors to alternative drugs. The purpose of this study is to explore the in vitro effect of the antimalarial mefloquine against adult Fasciola gigantica. Light and scanning electron microscopic observations could be used to determine the target of the drug following 6 and 12 h of incubation in medium containing mefloquine at three different concentrations 10, 20 and 30 µg/mL, as morphological changes could be observed. These changes occurred in definite sequences in response to mefloquine, and were consisted of swelling, vacuolization that was later disrupted, leading to desquamation of the tegument, resulting in exposure and disruption of basal lamina and the dislodging of spines. It is concluded that mefloquine presented itself as a drug that might become important in trematode chemotherapy, with the tegument being an important drug target.

Larvicidal activity of camphor and lavender oils against sheep blowfly, Luciliasericata (Diptera: Calliphoridae).

In this study, the essential oils of camphor and lavender were tested in vitro against the third instar larvae of Lucilia sericata for the first time, following dipping toxicity technique. The toxicity results revealed that L. sericata larvae were susceptible to the applied essential oils. Lavender oil was more effective than camphor in killing of L. sericata larvae. With 32 % concentration, the mortality percentages of larvae were 100 and 93.3 %, respectively. Light and scanning electron microscopic examinations were done to determine the cuticular changes of L. sericata larvae following exposure to the applied essential oils. Larvae showed cuticular swelling and distortion after oil treatment, but its level was greater with lavender oil. The current study suggested that an alternative, effective and natural product can be developed as larvicides against L. sericata using camphor and lavender oils.

Anthelmintics Resistance; How to Overcome it?

Many parasitic helminthes of veterinary importance have genetic features that favor development of anthelmintic resistance, this becoming a major worldwide constrain in livestock production. The development of anthelmintic resistance poses a large threat to future production and welfare of grazing animals. Development of variable degrees of resistance among different species of gastrointestinal nematodes has been reported for all the major groups of anthelmintic drugs. It has been observed that frequent usage of the same group of anthelmintic; use of anthelmintics in sub-optimal doses, prophylactic mass treatment of domestic animals and frequent and continuous use of a single drug have contributed to the widespread development of anthelmintic resistance in helminthes. The degree and extent of this problem especially with respect to multidrug resistance in nematode populations is likely to increase. Maintaining parasites in refugia and not exposed to anthelmintics, seems to be a key point in controlling and delaying the development of resistance, because the susceptible genes are preserved. Targeted selective treatments attract the interest of scientists towards this direction. Additionally, adoption of strict quarantine measures and a combination drug strategy are two important methods of preventing of anthelmintic resistance. Experience from the development of anthelmintic resistance suggests that modern control schemes should not rely on sole use of anthelmintics, but employ other, more complex and sustainable recipes, including parasite resistant breeds, nutrition, pasture management, nematode-trapping fungi, antiparasitic vaccines and botanical dewormers. Most of them reduce reliance on the use of chemicals and are environmental friendly. Finally, if new anthelmintic products are released, an important question will be raised about how they should be used. It is suggested that slowing the development of resistance to a new class are likely to be gained by releasing it in combination with one or more of the older anthelmintic classes, especially where efficacy of the older active(s) remains high.

Efficacy of methanolic extract of Balanites aegyptiaca fruits on Toxocara vitulorum.

In this study, the effect of the methanolic extract of Balanites aegyptiaca fruits (BAE) on adult Toxocara vitulorum was evaluated after incubating the parasites in Ringer solution containing 10, 30, 60, 120 and 240 µg/ml of the methanolic extract, for 24h using light and scanning electron microscopic observations. Differences in response to BAE action were concentration dependent. These changes occurred in definite sequences in response to BAE concentration and were consisted of slightly swelling which became pronounced and so severe, with lips showed wrinkled cuticular surface and deformed sensory papillae on increasing the BAE concentration. The strongest effects were reached with the highest BAE concentration, where disorganization of the cuticle and body musculature was observed. Additionally, the ovicidal effect of BAE, at the previous concentrations, on the development of T. vitulorum eggs was examined after 12h exposure. The inhibitory activity of BAE on egg development was concentration dependent and the highest value reached to 100% with the concentration of 240 µg/ml. These results were compared with those observed in the worm cuticle and eggs following incubation in albendazole, as it was a broad-spectrum nematodicidal compound with well-known ovicidal activity.

Efficacy of volatile oils (curzerene, furanoeudesma-1, 3-diene and lindestrene) on avian coccidiosis under laboratory conditions.

The coccidicidal efficacy of volatile oils (curzerene, furanoeudesma-1, 3-diene and lindestrene) against unsporulated and sporulated chicken Eimeria species oocysts was tested in three concentrations: 1, 2 & 3 microg/ml. Marked reduction in the number of living oocysts was recorded in exposed groups. The concentration of 3 microg/ml volatile oils induced the highest destructive effect. 58.1% of viable unsporulated oocysts were destroyed. A mean number of 153,800 oocysts was the difference between the total number of the produced oocysts per gram faeces in the group infected with exposed oocysts and that of the group infected with non exposed oocysts being less in the exposed group with more reduction in the vitality of shedding oocysts in the former group. At the meantime, the postmortem and histopathological microscopical examination of the intestine and caecum of the tested group revealed a reduction in the intestinal lesions in the group infected with the exposed oocysts.

The diagnostic efficacy of Fasciola gigantica coproantigen in naturally infected cattle and buffaloes.

Monoclonal antibody sandwich ELISA was used detect F. gigantica coproantigen in 33 selected animals according to the faecal history to evaluate sensitivity and specificity by using three different rabbit hyper-immune sera; Copro HIS, Egg HIS and ES HIS. The results showed that Copro HIS & ES HIS detected F. gigantica coproantigen in faecal of naturally infected cattle and buffaloes, but egg HIS failed. The 26-28 KDa coproantigen proved sensitivity (81.8%) and specificity (90.9%) in diagnosis of fascioliasis. Also, there was a positive statistical significance between number of F. gigantica egg per gm faeces (EPG) and mean sandwich ELISA OD. values for copro-antigen. For diagnostic value of F. gigantica coproantigen in comparison with ES antigen, EITB was done on field sera of cattle and buffaloes of known faecal history. The F. gigantica coproantigen bands of 27.6 & 72.1 KDa were specific for diagnosis animal fascioliasis, but the 72.1 KDa was less sensitive than the 27.6 KDs. The immunoblotting reaction was more intensive the with fractionated ES antigen than with fractionated coproantigen.

Efficacy of some Fasciola gigantica antigens.

The present study tested the antigenic relationship between the three Egyptian strain of Fasciola gigantica antigens; coproantigen, excretory-secretory and egg antigens, versus their related hyper-immune sera and select the most specific one. By using SDS-PAGE, a structural homology was demonstrated in F. gigantica ES and egg antigens. This homology was resided in the components of the similar molecular weights between both antigens. When no cross-reaction was recorded with the coproantigen, the intense cross-reaction occurred between ES and egg antigens in ELISA technique. This was attributed to the presence of common bands at 18.0, 20.4 and 27.6 KDa in between them. Consequently, the F. gigantica coproantigen and Copro HIS reflected the lowest level of the cross-reaction with the other evaluated F. gigantica antigens. The cross-reaction elucidated in the present study between the F. gigantica ES and egg antigens was mainly at the low serum dilutions. The distinction between the specific and the cross-reactive binding activities was clear ly marked with the highly diluted sera.

Inhibitory effect of Egyptian propolis on Fasciola gigantica eggs with reference to its effect on Clostridium oedematiens and correlation to chemical composition.

The chemical composition of the propolis samples was investigated by GC/MS and HPLC. 91 compounds have been identified, 26 compounds are being new to propolis. Siwa oasis propolis was characterized by the presence of diprenyl-dihydrocoumaric acids (4.15%), coumarate esters (10.93%), benzofuran lignans (13.47%) and valeric acids derivatives (11.42%). Matrooh sample was characterized by the presence of new ferulate esters, hydroxy acetophenones and furanon derivatives, furofuran lignans and sterol from pregnane type. Dakahlia propolis was a typical poplar propolis. The present study proved evidence of inhibitory activity of propolis on the vitality and hatchability of immature F. gigantica eggs, where three different localities in Egypt were tested for this purpose. The Siwa propolis showed the highest inhibitory effect than the other sources. The complete failure of development and death of all immature eggs were recorded at concentration of 200 microg mL(-1) of Siwa, 400 microg mL(-1) of Matrooh and 800 microg mL(-1) of Dakahlia propolis. The exposed F. gigantica egg shell revealed the same features as the non exposed egg shell when scanning with the electron microscopy. The three propolis samples showed inhibition in the growth of associated bacterium Clostridium oedematiens (Clostridium novyi) type B to fascioliasis. The inhibition varied according to the propolis origin.