|Year : 2014 | Volume
| Issue : 1 | Page : 93-102
In-vitro and in-vivo assessment of the effect of soybean extract on Fasciola gigantica infection in comparison with triclabendazole
Nashaat E. Nassef, Wafaa M. El-Kersh, Mona M El Sobky, Nancy M. Harba, Samar A El Refai Khalil
Department of Parasitology, Faculty of Medicine, Menoufia University, Shibin Al Kawm, Egypt
|Date of Submission||31-Mar-2013|
|Date of Acceptance||09-Sep-2013|
|Date of Web Publication||20-May-2014|
Samar A El Refai Khalil
Department of Parasitology, Faculty of Medicine, Menoufia University, Saad Zaghlool Street, Ashmoon, Shibin Al Kawm, Menoufia
Source of Support: None, Conflict of Interest: None
The aim of this study was to assess the effect of genistein (soybean extract) on fascioliasis.
Continued use of triclabendazole (TBZ) as the major antifascioliasis drug has resulted in resistance. Genistein, a major isoflavone in soybean extract, shows activity against different parasites.
Materials and methods
This was an in-vivo study that included 28 male New Zealand rabbits grouped as follows: GI, infected and nontreated; GII, infected and treated with TBZ; GIII, infected and treated with soybean extract; GIV, normal control group; GV1, not infected and treated with TBZ; and GV2, not infected and treated with soybean extract. Treatment started on the 80th day postinfection (d.p.i.) Stool examination by formol ether concentration and egg count per gram of feces were performed on day 0 of treatment, 3rd and 7th day post treatment (d.p.t.) Cure rate and fecal egg count reduction of TBZ and soybean extract were compared. Rabbits were killed on the 95th d.p.i. Liver sections were stained with hematoxylin & eosin (H&E) and with caspase-3 immunostain. The in-vitro study included 24 adult worms classified as follows: GI, control nondrug exposed; GII, TBZ exposed; GIII, soybean extract exposed; and GIV, incubated with DMSO (soybean solvent). Adult Fasciola DNA was extracted for electrophoresis.
Fecal egg count reduction of soybean extract was 41.6% on the 3rd d.p.t. and 95.8% on the 7th d.p.t. compared with 100% of TBZ on the 3rd and 7th d.p.t. The cure rate of this extract was 0% on the 3rd d.p.t. and 83.3% on the 7th d.p.t compared with 100% of TBZ on the 3rd and 7th d.p.t. Soybean extract improved most hepatic lesions shown by H&E staining and decreased the apoptotic changes detected by caspase-3 immunostaining. It also induced apoptotic damage of Fasciola gigantica DNA.
Soybean extract can be used in the treatment of fascioliasis to decrease the emergence of TBZ resistance.
Keywords: Caspase-3, electrophoresis, Fasciola gigantica, genistein, triclabendazole
|How to cite this article:|
Nassef NE, El-Kersh WM, El Sobky MM, Harba NM, El Refai Khalil SA. In-vitro and in-vivo assessment of the effect of soybean extract on Fasciola gigantica infection in comparison with triclabendazole. Menoufia Med J 2014;27:93-102
|How to cite this URL:|
Nassef NE, El-Kersh WM, El Sobky MM, Harba NM, El Refai Khalil SA. In-vitro and in-vivo assessment of the effect of soybean extract on Fasciola gigantica infection in comparison with triclabendazole. Menoufia Med J [serial online] 2014 [cited 2020 Mar 29];27:93-102. Available from: http://www.mmj.eg.net/text.asp?2014/27/1/93/132768
| Introduction|| |
Fasciola hepatica and Fasciola gigantica are two major parasitic species that are prevalent in temperate and tropical regions, respectively .
F. gigantica is restricted to tropical parts of Africa and Asia, where it is the most common Fasciola species infecting ruminants, whereas infections with F. hepatica are endemic in all continents .
Human cases of fascioliasis have been reported in South America, Europe, Australia, and the Far East. In Africa, human cases were reported in northern parts. The highest prevalence was recorded in Egypt, where the disease is distributed in communities resident in the Nile Delta .
The pathogenesis is remarkably complex, involving a liver migratory phase causing traumatic hepatitis and a phase of maturation and establishment of the adult parasite in the bile ducts causing hyperplastic obstructive cholangitis .
The major pathological changes in fascioliasis occur because of the immature migratory stage of the fluke. This stage of infection can cause extensive hemorrhage and fibrosis in the liver as the young flukes move through the liver .
Adults in bile ducts cause inflammation and edema, which in turn stimulate fibrosis in the walls of bile ducts (pipe stem fibrosis), resulting in atrophy of liver parenchyma, with concomitant cirrhosis and impaired liver function . In severe infections, the gall bladder is damaged and the walls of bile ducts are eroded completely; thus, the worms re-enter the liver parenchyma, again causing large abscesses .
At the molecular level, apoptosis, which is an organized DNA damage that causes nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation , has been reported to be triggered by many parasitic infections. The release of mitochondrial apoptotic proteins such as cytochrome c into the cytoplasm leads to activation of caspase-9 that activates caspase-3, which is a key event in apoptosis. Activated caspase-3 could be detected in situ by immunohistochemistry to detect apoptosis .
Triclabendazole (TBZ) is a very effective systemic antihelminthic drug used for the treatment of Fasciola infection . It can affect both juvenile stages of the fluke as early as 3 days old and the fully mature fluke, thus offering effective control for both acute and chronic fascioliasis .
Drug resistance has increased as a direct result of continued use of TBZ as a predominant means of Fasciola control . With respect to novel compounds for use as antihelminthics, there has been a growing interest in natural plant products that have been used as traditional medicines in developing countries .
Soybean extracts with genistein as a major component have been used previously in a variety of disorders including hypercholesterolemia, cardiovascular disease, renal disease, bone resorption, certain forms of cancer, obesity, and diabetes . Genistein and other isoflavones show activity against a range of nematode, cestode, and trematode parasites . Genistein had been used by Toner et al.  for the treatment of sheep liver fluke F. hepatica.
| Materials and methods|| |
This study was carried out on 28 male New Zealand rabbits; their weights ranged from 1.5-2.5 kg at the beginning of the experiment. Three successive stool samples were collected from each rabbit and examined using the formol ether concentration technique  to ensure that all rabbits were completely free from parasitic infection before the start of the study.
Encysted metacercariae of F. gigantica were obtained from the Schistosome Biological Supply Centre of Theodor Bilharz Research Institute, Giza Governorate. Each rabbit was infected orally by 25 encysted metacercariae of F. gigantica using a stomach tube .
The experimental rabbits were grouped into six groups as follows:
Group I (GI): Four infected rabbits served as the infected control group.
Group II (GII): Six infected rabbits treated orally with TBZ at a single dose of 10 mg/kg .
Group III (GIII): Six infected rabbits treated orally with soybean extract at a single dose of 100 mg/kg .
Group IV (GIV): Four noninfected nontreated rabbits.
Group V1 (GV1): Four noninfected rabbits treated with TBZ at a single dose of 10 mg/kg  (drug control 1).
Group V2 (GV2): Four noninfected rabbits treated with soybean extract at a single dose of 100 mg/kg  (drug control 2).
Treatment was administered at the 80th day postinfection (d.p.i.) and the following was done:
Formol ether concentration test
Stool examination using the formol ether concentration method was performed on day 0 treatment, 3rd, and 7th d.p.t. . Cure rates (CR) of TBZ and genistein were evaluated as follows: percentage of (rabbits with egg counts >0 before treatment that became negative after treatment/rabbits with egg counts >0 before treatment) .
Egg count per gram stool
Egg count per gram (EPG) of feces was performed using a McMaster slide . Fecal egg count reduction (FECR) was calculated as follows: percentage of (arithmetic mean of fecal egg count pretreatment-arithmetic mean of fecal egg count post-treatment/arithmetic mean of fecal egg count pretreatment) .
All rabbits were killed at the 95th d.p.i. and their livers were dissected and fixed in 10% formalin. Serial sections, 5 μm in thickness, were prepared and then embedded in paraffin and stained with hematoxylin & eosin (H&E)  in the Pathology Department, Menoufia University.
Liver sections were microwaved in citrate buffer for 9 min. Diluted primary polyclonal rabbit anti-active caspase-3 (Thermo Fisher Scientific, Fremont, California, USA) at a concentration of 5-10 μg/ml was then applied on the sections for 10-30 min. This was followed by cooling at room temperature for 20 min. The secondary antirabbit antibody (Thermo Fisher Scientific) was added. The sections were covered with horseradish peroxidase streptavidin complex. Diaminobenzidine chromogen substrate (Thermo Fisher Scientific) was added. It produces a brown precipitate in the presence of peroxidase .
Twenty-four adult mature F. gigantica flukes, of equal size, were selected from the livers of slaughtered animals at the Sentrees local abattoir, Menoufia Governorate. Worms were cleaned of blood and bile using PBS in small sieves. Worms were placed in sterile RPMI-1640 medium [pH 7.5, supplemented with penicillin (100 IU/ml), streptomycin (100 μl/ml), gentamycin (160 μl/ml), and 30% fetal calf serum] for 24 h at 37°C .
The worms were classified into four groups as follows:
Group (GI): Six nondrug-exposed F. gigantica worms served as a control group and were incubated in RPMI-1640 medium .
Group (GII): Six F. gigantica worms that were incubated in RPMI-1640 medium containing TBZ at a concentration of 20 μl/ml .
Group (GIII): Six F. gigantica worms that were incubated in RPMI-1640 medium containing soybean extract with genistein at a concentration of 0.27 mg/ml. Genistein was initially prepared as a stock solution in dimethylsulfoxide (DMSO) and added to culture medium to obtain a final solvent concentration of 0.1% .
Group (IV): Six F. gigantica worms that were incubated in RPMI-1640 medium containing 0.1% DMSO (solvent of soybean extract).
The four groups were left for 24 h and then subjected to the following:
DNA extraction and gel electrophoresis of DNA of adult Fasciola
Nucleic acid extraction was based on a modified salting-out extraction method in which DNA was precipitated by saturated NaCl . Fasciola worms were lysed with 200 μl lysing buffer (50 mmol/l NaCl, 1 mmol/l Na EDTA, 0.5% SDS). 200 ml of saturated NaCl was added. They were shaken gently and centrifuged at 12 000 rpm for 10 min. The DNA in the supernatant was precipitated in 700 μl cold isopropranolol by centrifugation for 10 min at 12 000 rpm. The pellets were washed with 500 μl 70% ethyl alcohol and centrifuged at 12 000 rpm for 5 min and to dry. Then, they were resuspended in 50 μl of buffer (10 mmol/l tris HCl, 1 mmol/l EDTA) supplemented with 5% glycerol for 30 min. The resuspended DNA was incubated for 30-60 min with 15 μl loading mix (5 μl of RNAase and 10 μl loading buffer). Finally, the samples were loaded into electrophoresis gel-wells. The intensity of apoptotic bands was measured using Software Gel Program (Biochemical solution company, University of California, San Francisco UCSF, USA).
| Results|| |
Results of the in-vivo study
Formol ether concentration test
Stool examination using the formol ether concentration method showed that all rabbits in GI, GII, and GIII were passing eggs at 80th d.p.i. (day 0 treatment). In the TBZ-treated group, at the 3rd d.p.t., all rabbits [6 of 6 rabbits (100%)] were negative for eggs in their stool. However, all rabbits [6 of 6 rabbits (100%)] treated by genistein continued to pass eggs on the 3rd day. There was a statistically significant difference between TBZ-treated rabbits in comparison with the infected nontreated group, (P1 < 0.01) and with the soybean extract-treated group (P3 < 0.01). At the 7th d.p.t., there was a significant decrease in the number of egg-passing rabbits in the soybean extract-treated group [5 of 6 rabbits (83.4%) were stool negative] in comparison with the infected nontreated group (P2 < 0.05), whereas there was no significant difference between the TBZ-treated and soybean extract-treated groups (P3 > 0.05) [Table 1]. The cure rate of TBZ at the 3rd d.p.t. was 100%, whereas that of soybean extract was found to be 0%, but at the 7th d.p.t., the cure rate of soybean extract was found to be 83.3%.
|Table 1: Comparison of number of egg-passing rabbits at different days among different groups|
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Egg count per gram stool
Egg count per gram of feces using a McMaster slide showed that there was no significant difference in EPG stool at day 0 treatment between the groups examined (P1 > 0.05, P2 > 0.05, and P3 > 0.05). At the 3rd d.p.t., there was a highly significant decrease in EPG stool in both the TBZ-treated and the soybean extract-treated group in comparison with the infected nontreated control group (P1 < 0.0001 and P2 < 0.0001). However, the TBZ-treated group showed a highly significant decrease in EPG stool when compared with the soybean extract-treated group (P3 < 0.0001). At the 7th d.p.t., there was a highly significant decrease in EPG stool in both the TBZ-treated and the soybean extract-treated groups in comparison with the infected nontreated group (P1 < 0.0001 and P2 < 0.0001), but no significant difference was detected between the TBZ-treated and soybean extract-treated groups in EPG stool (P3 > 0.05) [Table 2]. At the 3rd d.p.t., FECR for TBZ was 100%, whereas that of soybean extract was 41.6%. At the 7th d.p.t., FECR for soybean extract was 95.8%.
|Table 2: Comparison of egg count per gram stool among different studied groups at different days|
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Histopathological examination of liver sections of infected nontreated rabbits (GI) showed that F. gigantica infection produced degeneration, necrosis, and desquamation in the lining epithelium of the bile ducts with hyperplasia of their lining epithelium with polyp formation and the portal veins were dilated and congested. The central veins were dilated with dilatation and congestion of the sinusoides. The portal area showed severe edema, massive number of inflammatory cell infiltrates, and fibrosis. The hepatic parenchyma showed focal areas of necrosis. Some hepatocytes showed fatty changes and hydropic degeneration with pyknosis in their nuclei [Figure 1]. However, after treatment with TBZ, liver sections showed marked improvement in the pathological changes, with normalization of the lining epithelium of the bile ducts. There was only mild edema of the portal area remaining and little inflammatory infiltrates with normal capacity of the central vein and nondilated noncongested sinusoids with no degenerative changes in hepatocytes [Figure 2]. Also, liver sections of soybean extract treated rabbits showed improvement in the pathological changes including normalization of the lining epithelium of the bile ducts with remaining dilatation of central veins. The portal veins were also dilated and congested. There were also dilated congested sinusoids with mild edema in the portal area and few inflammatory cells surrounding the bile ducts at the portal area with no degenerative changes in hepatocytes [Figure 3].
Statistically, TBZ induced a significant improvement of all Fasciola-induced pathological changes when compared with the nontreated group (P1 < 0.05). Soybean extract also induced a significant improvement in these pathological changes (P2 < 0.05), except the portal vein changes, as four of six rabbits (66.6%) still showed dilated congested portal veins (P2 > 0.05). On comparing both TBZ and soybean extract, there was no significant difference in the improvement in the pathological changes (P3 > 0.05) [Table 3].
|Table 3: Comparison of histopathological changes among the different studied groups|
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In terms of the results of caspase-3 immunostain, it was found that infection of rabbits with F. gigantica caused marked apoptosis of liver cells as shown by marked expression of caspase-3 immunostain in the hepatocytes [Figure 4]a. Both TBZ-treated and soybean extract-treated groups showed a significant improvement in apoptosis in comparison with the nontreated group as shown by mild to moderate expression of caspase-3 (P1 < 0.05 and P2 < 0.05) [Figure 4]b and c and [Table 4].
|Table 4: Comparison of caspase -3 expression among the different studied groups|
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Results of the in-vitro study
In this study, TBZ induced damage of adult worm DNA, with a significant increase in MOD values of DNA apoptotic fragments at 800, 600, 400, and 200 bp when compared with the control nontreated group (P1 < 0.05, P1 < 0.01, P1 < 0.0001, and P1 < 0.01, respectively). Also, soybean extract induced damage of adult worm DNA, with a significant increase in the MOD values of DNA apoptotic fragments at 800, 600, 400, and 200 bp when compared with the control nontreated group (P2 < 0.01, P2 < 0.001, P2 < 0.0001, and P2 < 0.01, respectively), whereas, in comparison between TBZ and soybean extract, there was no significant difference at 800, 600, 400, and 200 bp (P3 > 0.05) [Figure 5] and [Table 5].
|Table 5: Mean OD values of DNA apoptotic bands at 800, 600, 400, and 200 bp in Fasciola worms of different groups|
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| Discussion|| |
TBZ is a very effective systemic antihelminthic used for the treatment of trematodes, such as Paragonimus spp., F. hepatica, and F. gigantica . However, resistance to the drug in fluke populations began to appear and spread . A number of strategies have been proposed in an attempt to maintain the efficacy of existing drugs and slow down the increased resistance. Genistein, which is the major component of soybean extract, was used by Toner et al.  to treat liver fluke F. hepatica.
In this study, stool samples were collected and examined using the formol ether concentration method and the eggs were counted per gram stool using the McMaster slide. In the TBZ-treated group, all rabbits (100%) were negative for eggs in their stool samples at the 3rd d.p.t. in comparison with the infected nontreated group (P1 < 0.01) and with the soybean-treated group (P3 < 0.01) [Table 1]. In terms of EPG stool at the 3rd d.p.t., there was a highly significant decrease in egg count in both TBZ-treated and soybean extract-treated groups in comparison with the infected nontreated group (P1 < 0.0001 and P2 < 0.0001). However, the decrease in the TBZ-treated group was highly significant compared with that in the soybean extract-treated group (P3 < 0.0001) [Table 2]. The efficacy of TBZ and genistein was evaluated qualitatively on the basis of the cure rate (20]. The cure rate of TBZ at the 3rd d.p.t. was estimated to be 100%, whereas that of soybean extract was found to be 0%. Quantitatively, treatment efficacy was based on the FECR . At 3rd d.p.t., FECR for TBZ was 100%, whereas that of soybean extract was 41.6%.
Complete disappearance of the eggs from the 3rd d.p.t. caused by TBZ in this work was in agreement with the results of Toner et al. , who studied the impact of treatment with TBZ on the female reproductive system of adult Fasciola and its impact on egg production. They found that all components of the female reproductive system including the ovary, vitelline glands, Mehlis gland, and uterus were affected by the drug and hence egg production was disrupted severely within 2 days only by TBZ treatment. This early onset of the effect of TBZ on egg appearance in stool was also reported by Hanna et al. , who found that egg production was severely disrupted within 24 h of TBZ and other benzimidazoles treatments and any eggs revealed in stool samples after this 24 hours duration could be explained by being stored in the uterus and released without being replaced. Besides, it was reported that the use of a single dose of TBZ in treated sheep led to complete clearance of the eggs from the stool  and can inhibit egg production by Fasciola worms even when it does not kill the worms as it affects the spermatogenic and vitelline cells of F. hepatica . Botros et al.  did not find Fasciola eggs in fecal samples of TBZ-treated cases after 1 week of treatment. This was in contrast to Mooney et al. , who found that TBZ induced only a 49% reduction in fecal egg count at the 7th d.p.t. and they reported that these results may be highly indicative of TBZ resistance.
At the 7th d.p.t., there was a significant decrease in the number of egg-passing rabbits in the soybean extract-treated group in comparison with the infected nontreated group (P2 < 0.05) as 83.4% of their rabbits (5 of 6) showed no eggs in their stool samples. However, in comparison with the TBZ-treated group, no significant difference was detected (P3 > 0.05) [Table 1]. Also, there was a highly significant decrease in EPG stool in the soybean extract-treated group in comparison with the infected nontreated group (P2 < 0.0001), but no significant difference was found in comparison with the TBZ-treated group (P3 > 0.05) [Table 2). The cure rate of soybean extract at the 7th d.p.t. was found to be 83.3% and its FECR was 100%.
The reduction in EPG stool in GIII treated with soybean could be attributed to the effect of genistein on Fasciola reproductive system as it induces severe disruption in the vitelline follicles, with an apparent block in the normal developmental sequence of the vitelline cells with affection of shell protein production by the vitelline cells, hence affecting the egg production by adult Fasciola . Also, this reduction may be related to the tegumental damage to the adult worms that leads to worm load reduction and worm elimination . In addition, Soliman  studied the effect of soybean oil extract on Schistosoma worms and found that it caused impairment in their egg production. Also, Kar et al.  reported that in-vitro treatment of Fasciolopsis buski with genistein increased nitric oxide synthase activity, leading to elevation in NO production. Overproduction of NO is known to cause oxidative stress, DNA damage, and disruption of energy metabolism, calcium homeostasis, and mitochondrial function that may affect the embryogenesis.
Histopathological examination of liver sections of infected nontreated rabbits showed that F. gigantica infection produced degeneration, necrosis, and desquamation in the lining epithelium of the bile ducts, with hyperplasia and polyp formation. These pathological changes in the wall of bile ducts were in agreement with Schmidt and Roberts , who attributed these to toxins produced by the larvae, mechanical irritation, toxic effects, and mechanical obstruction of the adult worms, which cause inflammation and edema in the walls of bile ducts.
In addition, the portal area showed edema, massive number of inflammatory cell infiltrates, and fibrosis. Also, the central veins and hepatic sinusoids were dilated. The hepatic parenchyma showed focal areas of necrosis with fatty changes and hydropic degeneration in hepatocytes. These changes were in agreement with Botros et al. , who attributed these to the effect of migrating juveniles in the liver tissue and the spiny body of the adult fluke. However, Schmidt and Roberts  reported that the changes in bile ducts lead to back pressure that causes atrophy and necrosis of liver parenchyma. In this study, some hepatocytes showed pyknosis of their nuclei as a sign of apoptosis. These results were supported by Botros et al. , who reported apoptosis in liver parenchyma of Fasciola-infected sheep.
However, TBZ liver sections showed a significant improvement in all Fasciola-induced pathological changes (P1 < 0.05) [Table 3]. These results were similar to those of Pérez et al. , who reported that administration of TBZ induced healing of the majority of hepatic lesions. These results were also supported by Botros et al. , who found that treatment of Fasciola-infected sheep with a single dose TBZ induced improvements in portal tracts with normal caliber and constituents with a decrease in the inflammatory cell infiltrates.
Liver sections of genistein-treated rabbits also showed a significant improvement in these pathological changes (P2 < 0.05), except portal vein dilatation and congestion, where only two of six rabbits (33.3%) showed normal portal veins (P2 > 0.05) [Table 3]. In the other trematode Schistosome, Soliman  reported that a combined soybean oil extract and an avocado extract treatment for murine schistosomiasis induced a significant reduction in the mean number of eggs per gram liver tissue and hence a reduction in all pathological lesions found in the liver.
Infection of rabbits with F. gigantica resulted in induction of marked apoptosis in liver cells as shown by marked expression of caspase-3 immunostain in hepatocytes. This result was supported by Sanchez-Campos et al. , who reported that infection with Fasciola increased the formation of reactive oxygen radicals including superoxide radicals, nitric oxide, and hydrogen peroxide, causing damage at the molecular level. Moreover, Wagih et al.  reported that F. gigantica infection decreased the level of the protective antioxidant superoxide dismutase, which is secreted by host cells as a defense mechanism aiming at suppression of oxygen radicals, resulting in a net effect of increased formation of reactive oxygen radicals.
In addition, El-Melegy et al.  found that F. gigantica infection induced apoptosis in hepatic cells of infected rabbits because of DNA damage as a result of oxidative stress. Also, Botros et al.  observed apoptosis in liver parenchyma of experimentally infected sheep with Fasciola.
In this study, both TBZ-treated and soybean extract-treated groups showed significant improvement in apoptosis in hepatocytes (P1 < 0.05 and P2 < 0.05) [Table 4], which was shown by mild to moderate expression of caspase-3. The antiapoptotic effect of TBZ could be attributed to the destructive effect of TBZ on adult worms that leads to a reduction in immunopathological reaction and improvement in the oxidative stress caused by Fasciola infection that causes DNA damage of hepatocytes. This was in accordance with El-Melegy et al. , who found that TBZ induced a significant improvement in Fasciola-induced apoptosis of hepatic cells. The improvement in hepatocyte apoptosis that was obtained by soybean extract was explained by Hsieh et al. , who found that the antioxidant activities of soybean isoflavones as genistein can reduce the oxidative DNA damage in different cell lines.
In terms of the molecular damage caused by both drugs on Fasciola DNA, there was a significant increase in MOD values of DNA apoptotic fragments at 800, 600, 400, and 200 bp [Table 5] in both drug-exposed groups when compared with the control nontreated group, without a significant difference between TBZ-exposed and soybean extract-exposed worms.
Apoptosis in TBZ-treated adult Fasciola has been described previously by Hanna et al. , who studied the effect of TBZ on the reproductive structures of adult Fasciola. They found that actively dividing cells tended to become rounded and condensed, with the detection of DNA strand breaks characteristic of apoptotic cell death.
In terms of DNA damage induced by soybean extract, it was found that in-vitro cultivation of Plasmodium falciparum with soybean emulsions resulted in interference with hypoxanthine incorporation into the parasite DNA, causing inhibition of the parasite protein synthesis . Another study assessed the production of nitric oxide in Fasciolopsis buski adult worms treated in vitro with genistein. A significant increase was found in the release of nitric oxide in the genistein-treated parasites. This caused oxidative stress and hence damage to the parasite DNA . Also, Hrckova and Velebny  reported that both natural and synthetic derivatives of genistein induced nuclear pyknosis (sign of apoptosis and DNA destruction) in cestodes.
| Conclusion|| |
From this study, it was concluded that F. gigantica infection caused severe histopathological changes in the liver of experimentally infected rabbits. Also, it induced apoptosis of hepatocytes as observed in caspase-3-stained liver sections of the infected nontreated group.
In terms of the in-vivo results, TBZ induced 100% disappearance of F. gigantica eggs from stool samples of all treated rabbits with a cure rate of 100% and FECR 100% as early as the 3rd d.p.t. However, with soybean extract at the 3rd d.p.t., 100% of rabbits were still passing eggs with a cure rate of 0%, but the mean EPG stool was decreased significantly in comparison with the nontreated group, with an FECR of 41.6%. At the 7th d.p.t., soybean extract caused a significant decrease in the number of egg-passing rabbits, with a cure rate of 83.3%. Also, it induced a significant decrease in the EPG in comparison with the nontreated group, with an FECR of 95.8%.
In terms of histopathological changes, both TBZ and soybean extract induced a significant improvement in the liver of experimentally infected rabbits with F. gigantica as shown by H&E staining, with the exception of portal vein dilatation and congestion in 66.6% of soybean extract-treated rabbits.
Both treatments caused a significant improvement in Fasciola-induced apoptosis of hepatocytes as shown by caspase-3 immunostain of liver sections.
This in-vitro study showed that both TBZ and soybean extract caused damage of adult F. gigantica DNA after 24 h of incubation with each treatment as shown by gel electrophoresis of the Fasciola DNA extracted. They induced a significant increase in the mean optical density values of DNA apoptotic fragments at 800, 600, 400, and 200 bp.
| Acknowledgements|| |
Conflicts of interest
| References|| |
|1.||Jayaraj R, Piedrafita B, Dynon K, Grams R, Spithill T, Smooker P. Liver fluke vaccines: vaccination against Fasciolosis by a multivalent vaccine of recombinant stage-specific antigens. Procedia Vaccinol 2009; 2 :82-85. |
|2.|| Anuracpreeda P, Songkoomkrong S, Sethadavit M, Chotwiwatthanakun C. Fasciola gigantica: production and characterization of a monoclonal antibody against recombinant cathepsin B3. Exp Parasitol 2011; 127 :340-345. |
|3.|| Mas-Coma S, Bargues M, Valero M. Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol 2005; 35 :255-1278. |
|4.|| Raina O, Nagar G, Varghese V, Prajitha G, Joshi P. Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins. Acta Trop 2011; 101 :22-42. |
|5.|| McConville M, Brennan G, Flanagan A. An evaluation of the efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica. Vet Parasitol 2009; 162 :75-88. |
|6.|| Keiser J, Xiao S, Tanner, M. Artesunate and artemether are effective fasciolicides in the rat model and in vitro. J Antimicrob Chemother 2009; 57 :1139-1145. |
|7.|| Schmidt G, Roberts L. Digeneans: foundations of parasitology. 8th ed. New York, USA: McGraw-Hill publishing Co.; 2009. 265-276. |
|8.|| Kerr J, Wyllie A, Currie A. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 2009; 26 :239-257. |
|9.|| Becker B, Mehlorn H, Andrewa P, Thomas H. Scanning and transmission electron microscope studies on the efficacy of praziquantel on Hymenolepis nana (Cestoda) in vitro. Z Parasitenkd 2010; 101 :121-133. |
|10.||1Hanna R, Edgar S, McConnell S, Toner E, McConville M, Brennan G. et al. Fasciola hepatica: histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha. Vet Parasitol 2010; 168 :240-254. |
|11.||1Halferty L, Brennan P, Hanna L, Edgar H, Meaney M, McConville M, et al. Tegumental surface changes in juvenile Fasciola hepatica in response to treatment in vivo with triclabendazole. Vet Parasitol 2008; 155 :49-58. |
|12.||1Flynn R, Mulcahy G, Elsheikha H. Coordinating innate and adaptive immunity in Fasciola hepatica infection: implications for control. Vet Parasitol 2010; 169 :235-240. |
|13.||1Toner E, Brennan G, Wells K, McGeon J, Fairweather I. Physiological and morphological effects of genistein against the liver fluke, Fasciola hepatica. Parasitology 2008; 135 :1189-1203. |
|14.||1Bhathena S, Velasquez M. Beneficial role of dietary phytoestrogens in obesity and diabetes. Am J Clin Nutr 2002; 76 :1191-1201. |
|15.||1Tandon V, Pal P, Roy B, Rao H, Reddy K. In vitro anthelminthic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India. Parasitol Res 1997; 83 :492-498. |
|16.||1Idris M, Al-Jabri A. Usefulness of kato-katz and trichrome staining as diagnostic methods for parasitic infections in clinical laboratories. J Sci Res Med Sci 2001; 3 :65-68. |
|17.||1Mahmoud IA Fascioliasis: infectivity, histopathology, histochemical immuneresponse in experimentally infected animals [MD thesis]. Menoufia, Egypt: Faculty of Medicine, Menoufia University; 2009. |
|18.||1El-Melegy M, Harba N, Abdou A, Abouelnour E, Hassab El-Nabi S. Oxidative stress on hepatic tissue of experimentally infected rabbits with Fasciola gigantica with evaluation of triclabendazole effects: parasitological, histopathological, biochemical and molecular changes. Egypt J Med Sci 2008; 29 :305-324. |
|19.||1McClain R, Wolz E, Davidovich A, Edward J, Bausch J. Reproductive safety studies with genistein in rats. Food Chem Toxicol 2007; 45 :1319-1332. |
|20.||2Albonico M, Bickle M, Ramsan A, Montresor L, Montresor L, Savioli M. Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bull World Health Organ 2003; 81 :343-352. |
|21.||2Jain P. General veterinary parasitology. 1st ed. Jaypee Brothers Medical Publishers (P) Ltd. London, United Kingdom: 2002; 62-63. |
|22.||2Vercruysse J, Behnke J, Albonico M, Ame S, Angebault C, Bethony J, et al. Assessment of the antihelminthic efficacy of albendazole in school children in seven countries where soil-transmitted helminths are endemic. PLoS Negl Trop Dis 2011; 5 :948. |
|23.||2Drury R, Wallington E. Histopathological techniques. 5th ed. Oxford: University Press; 1980. 129-130. |
|24.||2Vyas D, Robertson C, Stromberg P. Epithelial apoptosis in mechanistically distinct methods of injury in murine small intestine. Histol Histopathol 2007; 22 :623-630. |
|25.||2Hegazi A, Abd El Hady F, Shalaby H. An in vitro effect of propolis on adult worms of Fasciola gigantica. Vet Parasitol 2007; 144 :279-286. |
|26.||2Diab T, Mansour H, Mahmoud S. Fasciola gigantica: parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether. Exp Parasitol 2009; 124 :279-284. |
|27.||2Barduagni P, Hassanein Y, Mohamed M, Wakeel A, Sayed M, Hallaj Z, Curtale F, et al. Use of triclabendazole for treatment of patients co-infected by Fasciola spp. and S. mansoni in Behera Governorate, Egypt. Parasitol Res 2008; 102 :631-633. |
|28.||2Gajewska A, Smaga K, Wisniewski M. Pathological changes of liver in infection of Fasciola hepatica. Wiad Parazytol 2005; 51 :115-123. |
|29.||2Toner E, Brennan G, Hanna R, Edgar H, Fairweather I. Disruption of egg formation by Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host. Vet Parasitol 2011; 177 :79-89. |
|30.||3Hanna R, Forster F, Brennan G, Fairweather I. Fasciola hepatica: histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks. Vet Parasitol 2012; 14 :12. |
|31.||3Botros S, EL-Lakkany N, Badawy A, Mahmoud S. Mirazid shows insignificant activity against ovine fascioliasis. Ann Trop Med Parasitol 2009; 103 :605-616. |
|32.||3Duthaler U, Smith T, Keiser J. In vivo and in vitro sensitivity of Fasciola hepatica to triclabendazole combined with artesunate, artemether, or OZ78. Antimicrob Agents Chemother 2010; 54 :45-96. |
|33.||3Mooney L, Good B, Hanrahan J, Mulcahy G, Waal T. The comparative efficacy of four anthelmintics against a natural acquired Fasciola hepatica infection in hill sheep flock in the west of Ireland. Vet Parasitol 2009; 164 :201-205. |
|34.||3Lima C, Freitas F, de Morais L, Cavalcanti M, Silva L, Padilha R, et al. Ultrastructural study on the morphological changes to male worms of Schistosoma mansoni after in vitro exposure to allicin. Rev Soc Bras Med Trop 2011; 44 :327-330. |
|35.||3Soliman M. Evaluation of avocado/soybean unsaponifiable alone or concurrently with praziquantel in murine schistosomiasis. Acta Trop 2012; 122 :261-266. |
|36.||3Kar P, Tandon V, Saha N. Anthelmintic efficacy of Flemingia vestita: genistein-induced effect on the activity of nitric oxide synthase and nitric oxide in the trematode parasite, Fasciolopsis buski. Parasitol Int 2002; 51 :249-257. |
|37.||3Pérez J, Ortega J, Moreno T, Morrondo P, López-Sández C, Martínez-Moreno A, et al. Pathological and immunohistochemical study of the liver and hepatic lymph nodes of sheep chronically reinfected with Fasciola hepatica with or without triclabendazole treatment. J Comp Pathol 2002; 127 :30-36. |
|38.||3Sanchez-Campos S, Tunon M, Gonzalez P, Gonzalez-Gallego J. Oxidative stress and changes in liver antioxidant enzymes induced by experimental dicroceliosis in hamsters. Parasitol Res 1999; 85 :468-474. |
|39.||3Wagih A, Sheta E, Ismail H. Effect of fascioliasis and Schistosomiasis mansoni on the anti-oxidant defense mechanisms. Tanta Med Sci J 2007; 2 :56-66. |
|40.||4Hsieh H, Wu W, Hu M. Soy isoflavones attenuate oxidative stress and improve parameters related to aging and Alzheimer′s disease in C57BL/6J mice treated with d-galactose. Food Chem Toxicol 2009; 47 :625-632. |
|41.||4Deharo E, Krugliak M, Baccam D, Ginsburgp H. Antimalarial properties of soy-Bean fat emulsions. Int J Parasitol 1995; 25 :1457-1462. |
|42.||4Hrckova G, Velebny S. Pharmacological potential of selected natural compounds in control of parasitic diseases. New York Dordrecht London: Springer Wien Heidelberg; 2013. 31 :29-99. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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