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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1193-1202

Parasitological and histopathological effects of some antischistosome drugs in Schistosoma mansoni- infected mice


1 Department of Medical Parasitology, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Department of Medical Parasitology, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission06-Dec-2016
Date of Acceptance05-Mar-2017
Date of Web Publication04-Apr-2018

Correspondence Address:
Asmaa F Ibrahim
Department of Medical Parasitology, National Liver Institute, Menoufia University, Shebin Elkom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_672_16

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  Abstract 


Objective
To assess the in-vivo effects of artemisinin (ART), arachidonic acid (ARA), and nifedipine (NIF) compared with praziquantel (PZQ) on experimental Schistosoma mansoni infection in terms of worm burden, liver histopathological, immunohistochemical alterations, cytokines changes, and scanning electron microscopy (SEM) on both juvenile and adult stages of S. mansoni in experimentally infected mice.
Background
Reliance on a single drug for schistosomiasis control may promote the spread of drug-resistant parasites.
Materials and methods
The study was carried out in Theodor Bilharz Research Institute (Giza, Egypt) in April 2015 and included 120 laboratory-bred female albino mice. These mice were divided into nine groups: group 1) uninfected and untreated), group 2 (infected untreated), group 3 (infected and ART treated), group 4 (infected and ARA treated), group 5 (infected and NIF treated), group 6 (infected and PZQ treated), group 7 (infected and ART plus PZQ treated), group 8 (infected and ARA plus PZQ treated), and group 9 (infected and NIF plus PZQ treated). Mice were subjected to worm burden, liver histopathology, and SEM.
Results
ART plus PZQ-treated mice showed the lowest total worm burden (6.10 ± 5.30), the lowest schistosomula stage worm burden (11.0 ± 1.58), the lowest adult stage worm burden (1.20 ± 0.83), the lowest granuloma number (1.60 ± 0.54), and the smallest granuloma diameter (100.20 ± 8.25 μm). NIF plus PZQ were the most effective on the schistosomula and the adult stages of S. mansoni; however, PZQ was the least effective on the schistosomula and ART was the least effective on the adult of S. mansoni by SEM.
Conclusion
A combination of ART plus PZQ was the most effective in reducing total, schistosomula stage, and adult stage worm burdens, and also the number and diameter of granuloma. NIF plus PZQ were the most effective against the schistosomula and the adult stages of S. mansoni by SEM, but PZQ was the least effective against schistosomula stage worm burden reduction and was the least effective against it by SEM.

Keywords: arachidonic acid, artemisinin, arachidonic acid, Schistosomiasis mansoni


How to cite this article:
Abdel-Ghaffar MM, Saad AGE, Moharm IM, Sharaf OF, Badr MT, Ibrahim AF. Parasitological and histopathological effects of some antischistosome drugs in Schistosoma mansoni- infected mice. Menoufia Med J 2017;30:1193-202

How to cite this URL:
Abdel-Ghaffar MM, Saad AGE, Moharm IM, Sharaf OF, Badr MT, Ibrahim AF. Parasitological and histopathological effects of some antischistosome drugs in Schistosoma mansoni- infected mice. Menoufia Med J [serial online] 2017 [cited 2020 Apr 5];30:1193-202. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1193/229236




  Introduction Top


WHO ranks schistosomiasis as the second parasitic disease among diseases worldwide in terms of socioeconomic importance and the third most important parasitic disease in terms of public health impact[1].

Schistosomiasis mansoni is endemic in 54 countries in Africa, Middle East, South America, and the Caribbean[2]. A WHO report showed that the total number of individuals who required preventive chemotherapy globally in 2010 was over 237 million; of these, more than 108 million were school-age children, of whom only 13% received treatment[3].

Most schistosomiasis-related pathology is induced by cellular immune responses. The granulomatous reactions around the eggs are orchestrated by clusters of differentiation-positive (CD4-positive) T cells and involve eosinophils, monocytes, and lymphocytes[4].

Currently, praziquantel (PZQ) is the drug of choice in the treatment of the three major species of schistosomes infecting humans, namely S. mansoni, Schistosoma haematobium, and Schistosoma japonicum. The success of PZQ is explained by its good safety and efficacy profile against all schistosome species parasitizing humans, as well as its very cheap cost. The major shortcoming of PZQ is its much lower efficacy against young developing stages of Schistosoma (schistosomula) and this may have impact on the cure rate of the patients treated with this drug. The second shortcoming is the possibility of reinfection of individuals after treatment in areas of heavy schistosomiasis transmission[5].

Reliance on a single drug for schistosomiasis control may promote the spread of drug-resistant parasites. Indeed, S. mansoni isolates with reduced susceptibilities to PZQ have already been identified. Obviously, there is a need to develop a new antischistosomal drug with a broad spectrum of activity against all stages of the parasite[6].

The areas of endemicity of schistosomiasis and some other infectious diseases, such as malaria, overlap geographically. Coendemicity, as well as known similarities of both parasites, such as hemoglobin digestion, were the key rationales for in-depth studies on the potential antischistosomal effects of antimalarials[7].

The use of arachidonic acid (ARA), omega 6, for schistosomiasis therapy was based on the fact that ARA is an essential constituent of membrane lipids and is the base material used by the body to synthesize a key series of hormones referred to collectively as dienolic prostaglandins[8].

Several studies have shown that voltage-operated calcium (Ca 2+) channels are prime candidate targets for chemotherapy as they play a critical role in regulating the levels of intracellular Ca 2+ and are essential for a variety of parasite cellular events, including contraction, gene expression, and neurotransmitter release. Nifedipine (NIF) is a dihydropyridine calcium channel blocker that primarily blocks l-type calcium channels[9].

In the present study, we investigated the efficacy of some antischistosomal drugs on S. mansoni infection in a murine model.


  Materials and Methods Top


This study was carried out in Theodor Bilharz Research Institute (Giza, Egypt) from April 2015 to June 2015 on 120 laboratory-bred female albino mice 6–8 weeks old and weighing 24 ± 2 g. These mice were divided into nine groups: group 1 (five mice): uninfected untreated mice (negative control group). Group 2 (10 mice): infected untreated mice (positive control group); the group was equally divided into two subgroups (2A and 2B). Group 3 (15 mice): infected mice treated with artemisinin (ART); the group was equally divided into three subgroups (3A, 3B, and 3C). Group 4 (15 mice): infected mice treated with ARA; the group was equally divided into three subgroups (4A, 4B, and 4C). Group 5 (15 mice): infected mice treated with NIF; the group was equally divided into three subgroups (5A, 5B, and 5C). Group 6 (15 mice): infected mice treated with PZQ; the group was equally divided into three subgroups (6A, 6B, and 6C). Group 7 (15 mice): infected mice treated with ART plus PZQ; the group was equally divided into three subgroups (7A, 7B, and 7C). Group 8 (15 mice): infected mice treated with ARA plus PZQ; the group was equally divided into three subgroups (8A, 8B, and 8C). Group 9 (15 mice): infected mice treated with NIF plus PZQ, which were equally divided into three subgroups (9A, 9B, and 9C).

Subgroups 3A, 4A, 5A, 6A, 7A, 8A, and 9A (infected treated mice) were examined for the juvenile stage of S. mansoni 23 days postinfection (d.p.i.). Subgroups 3B, 4B, 5B, 6B, 7B, 8B, and 9B (infected treated mice) were examined for the adult stage of S. mansoni 44 d.p.i. Subgroups 3C, 4C, 5C, 6C, 7C, 8C, and 9C (uninfected treated mice) were killed 2 days after treatment to be examined for the effect of drugs.

Fresh drug solutions were prepared on each day of experiment. The dosing protocols were administered as a single dose on day 21 (against juveniles in the liver) and on day 42 (against adults stages in the mesenteries) after infection and killed 2 days after treatment.

ART was purchased from Sigma-Aldrich (Chemie GmbH, Darmstadt, Germany). Mice were treated by the intraperitoneal route with a single dose of ART (100 mg/kg of body weight diluted in 100 μl of dimethyl sulfoxide)[10].

PZQ (Biltricide) was purchased from Alexandria Co. for Pharmaceuticals and Chemical Industries (Alexandria, Egypt). Biltricide tablets were ground and used as a freshly prepared suspension in 2% Cremophor EL (Pancharatna Char Rasta, G. I. D. C., Vapi, Gujarat, India) by vortexing and administered by an oral gavage with a single dose of 300 mg/kg corresponding to a dose volume of 0.5 ml[11].

NIF (Epilat) was purchased from Epico Pharmaceutical Industrial Co. (Cairo, Egypt). Epilat was suspended in a 1% aqueous solution of Tween 80 and administered intraperitoneally at a dose of 20 mg/kg of body weight at a volume of 10 ml/kg[12].

ARA was purchased from Efamol Ltd (Surrey, UK). A single oral dose of 300 mg/kg corresponded to a dose volume of 0.5 ml[13].

Experimental infection: cercariae of S. mansoni Egyptian strain were obtained from infected Biomphalaria alexandrina snails (purchased from the Schistosome Biological Supply Center at Theodor Bilharz Research Institute, Giza, Egypt) following exposure to artificial light to induce cercariae shedding in the laboratory. All mice were subjected to stool examination to ensure that they were free from infection. Each mouse was infected through the abdominal skin by a subcutaneous injection with about 80 ± 10 preharvested cercariae of S. mansoni (Egyptian strain) to induce liver fibrosis. The mice were housed in standard individual mouse cages (five mice per one cage) that were placed in acclimatized rooms under controlled 12 h light/12 h dark cycles. Animals had been reared under free access to standard pellet animal diet and tap water, constant room temperature and humidity, bedded on wood shavings, and checked upon twice daily for health status. All mice were deprived of food the night before treatment and they were allowed to eat 1 h after treatment[14]. All procedures related to animal experimentations fulfilled the International Guiding Principles for Biomedical Research Involving Animals as issued by the International Organizations of Medical Sciences (http://www.cioms.ch/).

Parasitological parameter (worm burden): the mice were euthanized by an intraperitoneal injection of sodium thiopental (100 mg/kg) using a CO2 chamber. Adult worms recovered by hepatic and mesenteric perfusion were counted and sexed under a stereomicroscope. The worm burden (infectivity) was determined as the percentage of maturation of cercariae into adult worms recovered from the portal system and mesenteric veins[14]. The percentage reduction of worm burden in all infected groups was calculated according to the following equation[15]:



Histopathology and granuloma measurement: when mice died, pieces from liver samples of each mouse were taken aseptically and immediately fixed in 4% paraformaldehyde. The liver tissues from all different groups were embedded in paraffin and sliced into 4 μm sections, dewaxed and rehydrated, and then stained with hematoxylin and eosin staining for histological examination[16].

Immunohistochemical staining of CD4 cells was detected using the avidin–biotin complex method on 4 μm liver sections of all groups. Liver sections of 4 μm thickness were dewaxed, rehydrated, and washed in distilled water for 5 min, rinsed in PBS with Tween-20 (PBST) for 10 min, and incubated with 10% normal goat serum for 15 min to reduce nonspecific background staining. Then, the sections were incubated with CD4 anti-mouse monoclonal antibody (1: 800) (Abcam, Cambridge, Massachusetts, USA) for 1–2 h at room temperature. The sections, after five baths in PBST, were incubated with biotinylated goat anti-rabbit immuoglobulin (Abcam). The sections, after five baths in PBST, were further incubated with avidin–biotin complex (Nichirei, Tokyo, Japan) for 1 h at room temperature. The reaction mixture was developed using 20 mg 3,3'-diaminobenzidine tetrahydrochloride (Wako Pure Chemical Industries Ltd., Chuo-ku, Osaka, Japan) in 40 ml PBST, pH 7.2, containing 10 ml of hydrogen peroxide for 7–9 min in a dark room, followed by distilled water and then dehydrated and mounted. The criterion for a positive reaction confirming the presence of CD4 is a dark, brownish, intracytoplasmic precipitate[17].

Scanning electron microscopic examination: worms were fixed in 2.5% glutaraldehyde–phosphate buffer (0.1 mol/l, pH 7.4) at − 4C for 24 h and postfixed in1% osmium tetroxide for 1 h. They were dehydrated through a graded series of ethanol and dried in a Hitachi HCP-2 (Hitachi Koki, Tokyo, Japan) critical-point dryer machine using liquid carbon dioxide as a transitional medium. After drying, they were mounted on aluminum stubs and coated with platinum and palladium in an ion-sputtering apparatus (Hitachi E-102) at 10–15 mA for 6 min[18].

Enzyme-linked immunosorbent assay was used for the detection of mouse interferon-γ and interleukin-4 in serum samples (sandwich enzyme-linked immunosorbent assay): For serological analysis, blood samples from each mouse were collected through a small puncture of the caudal vein using a sterile needle. Samples were transferred to a 1.5 ml tube, clotted, and then centrifuged for serum separation. Serum samples were kept at −20°C until used[19].

Statistical methods

The data collected were tabulated and analyzed using the statistical package for the social sciences software (version 20; SPSS Inc., Chicago, Illinois, USA). Two types of statistics were calculated: descriptive statistics; percentage, mean, and SD. Analytic statistics: one-way analysis of variance (F-test) and Kruskal–Wallis test.

P value 0.05 or less indicates significance[20].


  Results Top


Total worm burden

All infected groups showed a significant reduction in the total worm burden compared with the control group (P< 0.05). The lowest total worm burden (6.10 ± 5.30) was detected in mice treated with ART plus PZQ, followed by NIF plus PZQ-treated mice (8.0 ± 6.46). On using a single drug, ART was the most effective (10.30 ± 3.09), followed by NIF-treated mice (13.30 ± 3.26), but PZQ recorded 14.50 ± 11.32 [Table 1].
Table 1: Total worm burden in Schistosoma mansoni-infected mice of all groups

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Schistosomulum stage worm burden

All 'A' groups showed a significant reduction in schistosomula stage worm burden compared with the control group (group 2) (P< 0.05). The lowest schistosomula stage worm burden (11.0 ± 1.58) was detected in mice treated with ART plus PZQ, followed by ART-treated mice (12.80 ± 1.92). On using a single drug, ART was the most effective (12.80 ± 1.92), followed by NIF-treated mice (16.20 ± 1.30) [Table 2].
Table 2: Worm burden of Schistosoma mansoni schistosomula stage in different. A subgroups of studied mice (23 days postinfection)

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Adult stage worm burden

All 'B' groups showed a significant reduction in adult stage worm burden compared with the control group (group 2) (P< 0.05). The lowest adult stage worm burden (1.20 ± 0.83) was detected in mice treated with ART plus PZQ, followed by NIF plus PZQ-treated mice (2.0 ± 1.22). On using a single drug, PZQ was the most effective (3.80 ± 0.83), followed by ART-treated mice (7.80 ± 1.48) [Table 3].
Table 3: Worm burden of Schistosoma mansoni adult stage in different B subgroups of studied mice (44 days postinfection)

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Number of granuloma

All 'B' groups showed a significant reduction in the number of granuloma compared with the control group (group 2) (P< 0.05). The least granuloma number (1.60 ± 0.54) was detected in mice treated with ART plus PZQ, followed by NIF plus PZQ-treated mice (2.0 ± 0.70). On using a single drug, ART was the most effective (2.20 ± 0.83), followed by PZQ-treated mice (2.40 ± 0.54) [Table 4].
Table 4: Number of granuloma among Schistosoma mansoni-infected mice of all B subgroups (44 days postinfection)

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Diameter of granuloma

All 'B' groups showed a significant reduction in the diameter of granuloma compared with the control group (group 2) (P< 0.05). The least granuloma diameter (100.20 ± 8.25 μm) was detected in mice treated with ART plus PZQ, followed by NIF plus PZQ-treated mice (149.0 ± 14.93 μm). On using a single drug, ART was the most effective (150.40 ± 15.27 μm), followed by PZQ-treated mice (151.80 ± 9.25 μm) [Table 5].
Table 5: Diameter of granuloma among Schistosoma mansoni-infected mice of all B subgroups (44 days postinfection)

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Histopathological liver analysis (23 days postinfection for A groups)

Untreated mice showed congested central veins, diffuse hydropic degeneration of hepatocytes, moderate congestion of hepatocytes, and hepatic sinusoids. However, ART-treated mice showed normal central veins, focal hydropic degeneration of hepatocytes, mild congestion of hepatocytes, and hepatic sinusoids. ARA-treated and also NIF-treated mice showed diffuse hydropic degeneration of hepatocytes, mild congestion of hepatocytes, and hepatic sinusoids. PZQ-treated mice showed congested central veins, diffuse hydropic degeneration of hepatocytes, moderate congestion of hepatocytes, and hepatic sinusoids. ART plus PZQ-treated mice showed mild congestion of hepatocytes and hepatic sinusoids. ARA plus PZQ and also NIF plus PZQ-treated mice showed congested central veins, focal hydropic degeneration of hepatocytes, and mild congestion of hepatic sinusoids [Table 6] and [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d, [Figure 2]a, [Figure 2]b.
Table 6: Histopathological liver analysis among Schistosoma mansoni-infected mice of all A subgroups (23 days postinfection)

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Figure 1: (a) Histopathological liver section of uninfected untreated mice (group 1). (b) Infected untreated mice 23 days after infection (group 2A). (c) Infected and artemisinin-treated mice 21 days after infection (group 3A). (d) Infected and arachidonic acid-treated mice 21 days after infection (group 4A).

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Figure 2: (a) Histopathological liver section of infected and nifedipine-treated mice 21 days after infection (group 5A). (b) Infected and praziquantel-treated mice 21 days after infection (group 6A). (c) Infected untreated mice 44 days after infection (group 2B). (d) Infected and artemisinin-treated mice 42 days after infection (group 3B).

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Histopathological liver analysis (44 days postinfection for B groups)

Untreated mice showed congested central veins, diffuse hydropic degeneration of hepatocytes, moderate congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 9.40 ± 1.14 and diameter of 293.20 ± 12.09 μm. However, ART-treated mice showed congested central veins, focal hydropic degeneration of hepatocytes, mild congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 2.20 ± 0.83 and diameter of 150.40 ± 15.27 μm. Also, ARA-treated mice showed congested central veins, diffuse hydropic degeneration of hepatocytes, moderate congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 3.40 ± 0.54 and diameter of 200.20 ± 20.17 μm. NIF-treated mice showed congested central veins, focal hydropic degeneration of hepatocytes, mild congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 3.60 ± 0.54 and diameter of 202.40 ± 13.90 μm. PZQ-treated mice showed congested central veins, mild congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 2.40 ± 0.54 and diameter of 151.80 ± 9.25 μm. Also, ART plus PZQ-treated mice showed mild congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 1.60 ± 0.54 and diameter of 100.20 ± 8.25 μm. However, ARA plus PZQ-treated mice showed mild congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 2.40 ± 0.89 and diameter of 149.60 ± 6.34 μm. In contrast, NIF plus PZQ-treated mice showed mild congestion of hepatic sinusoids, and bilharzial granulomas with a mean number of 2.0 ± 0.70 and diameter of 149.0 ± 14.93 μm [Table 7] and [Figure 2]c, [Figure 2]d, [Figure 3]a, [Figure 3]b.
Table 7: Histopathological liver analysis among Schistosoma mansoni-infected mice of all B subgroups (44 days postinfection)

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Figure 3: (a) Histopathological liver section of infected and arachidonic acid-treated mice 42 days after infection (group 4B). (b) Infected and arachidonic acid plus praziquantel-treated mice 42 days after infection (group 8B). (c) Uninfected and artemisinin-treated mice (group 3C). (d) Uninfected and artemisinin plus praziquantel-treated mice (group 7C).

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Histopathological liver analysis (uninfected and treated groups for C groups)

ART-treated mice showed congested central veins and very mild congestion of hepatic sinusoids, but ARA-treated mice showed mild congestion of hepatic sinusoids. Also, NIF-treated mice showed congested central veins and mild congestion of hepatocytes, but PZQ-treated mice showed congested central veins and very mild congestion of hepatocytes and hepatic sinusoids. However, ART plus PZQ-treated mice showed congested central veins and very mild congestion of hepatocytes and hepatic sinusoids, whereas ARA plus PZQ-treated mice showed congested central veins, and mild congestion of hepatocytes and hepatic sinusoids. Also, NIF plus PZQ-treated mice showed congested central veins, focal hydropic degeneration of hepatocytes, and mild congestion of hepatocytes and hepatic sinusoids [Figure 3]c and [Figure 3]d.

Scanning electron microscopy of the schistosomula of Schistosoma mansoni (23 days postinfection for A groups)

ART-treated mice showed damaged oral sucker and collapse of the tegument, but ARA-treated mice showed moderate to severe swelling of the tegument and few blebs. NIF-treated mice showed peeling, erosion, and few blebs of the tegument and PZQ-treated mice showed few blebs of the tegument. ART plus PZQ-treated mice showed peeling and numerous blebs protruding from the surface of the damaged tegument, whereas ARA plus PZQ-treated mice showed numerous blebs protruding from the surface of the tegument with moderate to severe swelling. Also, NIF plus PZQ-treated mice showed numerous blebs protruding from the surface of the tegument with moderate to severe swelling, peeling, and erosion of the tegument [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d.
Figure 4: (a) Scanning electron microscopy of the schistosomula of Schistosoma mansoni from infected untreated mice (group 2A). (b) Artemisinin-treated mice (group 3A). (c) Praziquantel-treated mice (group 6A). (d) Artemisinin plus praziquantel-treated mice (group 7A).

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Scanning electron microscopy of the adult stage of Schistosoma mansoni (44 days postinfection for B groups)

ART-treated mice showed few blebs in the oral sucker, but ARA-treated mice showed loss of spines on the surface of the tuberculae, peeling, and erosion of the tegument. However, NIF-treated mice showed collapse of the tegument around the ventral sucker, peeling, erosion, and shortening or loss of spines on the surface of the tuberculae of the damaged tegument. Also, PZQ-treated mice showed few blebs of the tegument around the oral and ventral suckers, swelling, vacuolization, and disruption of the tuberculae with complete loss of the spines, whereas ART plus PZQ-treated mice showed edema with blebs of the tegument around the oral sucker, shortening, or loss of spines on the surface of the tuberculae, with peeling and erosion of the damaged tegument. However, ARA plus PZQ-treated mice showed blebs of the tegument around the oral and ventral suckers, but NIF plus PZQ-treated mice showed numerous blebs protruding from the surface of the damaged tegument, loss, or shortening of spines on the surface of the tuberculae with abnormal elevated lines around the tuberculae, peeling, erosion, collapse, and disruption of the tegument [Figure 5]a,[Figure 5]b,[Figure 5]c,[Figure 5]d.
Figure 5: (a) Scanning electron microscopy of the adult stage of Schistosoma mansoni from infected untreated mice (group 2B). (b) Praziquantel-treated mice (group 6B). (c) Artemisinin plus praziquantel-treated mice (group 7B). (d) Nifedipine plus praziquantel-treated mice (group 9B).

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  Discussion Top


S. mansoni is a significant parasite of humans. It is the most widespread of the human-infecting schistosomes, causing the disease intestinal schistosomiasis[21]. Most schistosomiasis-related pathology is induced by cellular immune responses. The granulomatous reactions around the eggs are orchestrated by CD4-positive T cells and involve eosinophils, monocytes, and lymphocytes[4]. PZQ is used widely in the control program; however, it showed low cure rates in several countries, necessitating repeated treatments, and thus augmenting the chances of emergence of resistance and the need to develop new schistosomicides[22].

In the present study, in terms of the result of total worm burden in all groups, the reported changes were in agreement with Li et al.[23], who found that a single oral dose of dihydroartemisinin (300 mg/kg) reduced total worm burdens by 13.8–82.1%. Also, Abdul-Ghani et al.[24] documented that PZQ and derivatives of artesunate were shown to be effective against different stages of worm development. In addition, Silva-Moraes et al.[9] found that NIF strongly reduced schistosomula viability alone or in combination with PZQ. In contrast, El-Beshbishi et al.[25] reported that administration of compound naphthoquine phosphate (CO-ArNp) at a dose of 600 mg/kg significantly reduced the total worm count by 100%; this difference was because of the use of a lower dose in the current study.

However, in terms of the result of S. mansoni schistosomula stage worm burden in all groups, the reported changes were in agreement with Li et al.[23], who found that the greatest reductions were observed when a single oral dose of dihydroartemisinin (300 mg/kg) treatment was administered on 21 d.p.i. by 88.5–90.1%. Also, El-Beshbishi et al.[25] detected that administration of CO-ArNp at a single dose of 400 or 500 mg/kg 3 weeks after infection significantly reduced the total number of worms by 73.11 and 92.64%, respectively, compared with the corresponding infected nontreated controls and the PZQ group that showed a lower reduction rate of 42.83%.

Similarly, these results were in agreement with Utzinger et al.[26], who found that artemether at concentrations of 150 or 300 mg/kg resulted in worm reductions of 88–97%. In addition, El Ridi et al.[13] reported that ARA treatment during patency led to a highly significant decrease of 75% in worm burden. In contrast, Vimieiro et al.[27] documented that in the animals treated with 300 or 600 mg/kg artesunate 14 or 21 d.p.i., a small percentage of changes could be observed; this difference was because of later treatment and the use of a different drug derivative.

However, in terms of the result of S. mansoni adult stage worm burden in all groups, the reported changes were in agreement with Li et al.[23], who found that a single oral dose of dihydroartemisinin (300 mg/kg) reduced total worm burdens by 13.8–82.1% and the least reductions were observed when treatment was administered on 49 or 56 d.p.i. In addition, El-Beshbishi et al.[25] found that when CO-ArNp was administered once 6 weeks after infection at a dose of 500 or 600 mg/kg, there was a significant reduction in mean worm burden by 74.71 and 93.36% respectively. Similarly, Utzinger et al.[26] documented that total concentrations of 600 or 800 mg/kg artemether administered to mice with adult S. mansoni reduced the worm burden by 46–51%. Also, El Ridi et al.[13], in a series of in-vivo experiments for each schistosome species, found that oral administration of ARA after patency led to a highly significant reduction in worm burden compared with the PZQ group.

In terms of the result of granuloma number and diameter among S. mansoni- infected mice of all B subgroups (44 d.p.i.), the reported changes were similar to El Ridi et al.[13], who found that up to 10 granulomas/section were observed in 9 weeks after infection. However, liver sections of ARA-treated hamsters showed few granulomas. Granulomas in the liver of control infected hamsters were large, whereas granulomas in ARA-treated hamsters showed a significantly smaller size[13]. Similarly, El-Beshbishi et al.[25] reported that hepatic tissues of untreated-infected mice (6 weeks after infection) showed granulomata encircling recently deposited intact or partially degenerated ova with a mean number of 14.50 ± 2.07 and diameter of 288.83 ± 46.44 μm. However, in the PZQ group, the parenchymal changes were similar to those in the infected untreated controls, with the least reduction in the granuloma count and diameter of 36.55 and 22.34%, respectively[25].

In terms of the result of histopathological liver analysis 23 d.p.i., the reported changes were in agreement with El-Beshbishi et al.[25], who reported that histopathological examination of the liver sections of infected untreated controls (4 weeks after infection) showed marked inflammation of liver parenchyma and portal tract. Yet, on administering drugs 3 weeks after infection, CO-ArNp at 600 mg/kg resulted in complete absence of ova deposition and granulomatous reactions in the parenchymal and portal areas with the least inflammatory reactions in comparison with the PZQ group; this was because of the use of a lower dose of a different drug derivative in the current study[25].

In terms of the result of histopathological liver analysis 44 d.p.i., the reported changes were in agreement with El-Beshbishi et al.[25], who found that hepatic tissues of untreated-infected mice (6 weeks after infection) showed moderate cloudy swelling of the liver parenchyma and cellular irregularly outlined granulomata encircling recently deposited intact or partially degenerated ova. Also, El Ridi et al.[13] noted that the schistosomicidal effects of ARA were associated with an improvement with respect to liver histopathology.

In terms of the result of scanning electron microscopy (SEM) of the schistosomula of S. mansoni, the reported changes were in accordance with Xiao et al.[28], who reported that 24 h after artemether treatment, all male and female S. mansoni schistosomules examined showed evident changes in shape. Most of the schistosomules showed extensive and severe tegumental damage, consisting of mild or moderate swelling of tegumental ridges that usually resulted in loss of spines in the tail and suckers and a disordered arrangement and fusion of the ridges. Also, Silva-Moraes et al.[9] observed tegumental damage following the NIF treatment.

In terms of the SEM of the adult stage of S. mansoni, the reported changes were in agreement with Jiraungkoorskul et al.[29], who observed that 1 day after a single dose of 300 mg/kg artesunate was administered to mice infected with 49-day-old Schistosoma mekongi, a few blebs were observed on the oral sucker along with moderate to severe focal damage of the tegument. Three d.p.i., severe swelling, vacuolization, and disruption were observed. Some areas of the swollen tegument coalesced and formed a mass, with some cracks or superficial peeling and numerous small blebs protruding from the surface of the trabeculae. However, 1 day after the administration of 300 mg/kg of PZQ to mice infected with 49-day-old S. mekongi, all worms showed slight to moderate focal damage of the dorsal surface of the tegument. The worms showed changes in the trabeculae, namely, swelling and shortening or even loss of the spines on the surface and there were numerous blebs around the trabeculae[29].

Similarly, Shaohong et al.[30] reported that artesunate induced damage on the worm tegument, normal tubercles with small sharp spines on the tegument, and alterations on the tubercles of adult worms by SEM. The suckers of adult worms were damaged and collapsed, and the tegument ridges were focally swollen and fused. Also, El Ridi et al.[13] found that SEM analysis of worms recovered after 5 weeks of infection in untreated and ARA-treated hamsters indicated that ARA treatment led to tegument alterations. Silva-Moraes et al.[9] observed several lesions on the adult worm tegument in in-vitro experiments using NIF alone or combined with PZQ. Tegumental injures were more apparent following NIF exposure compared with PZQ, which induced significant disruption of the tegument.

In addition, these results were in agreement with El-Beshbishi et al.[25], who found that worms recovered from mice treated with ART were smaller, thinner, and with an irregular contour, disrupted tegument, granularity, and slower motility compared with those from the untreated and the PZQ-treated group. Also, Xiao et al.[28] reported that 4 h after treatment with 300 mg/kg of PZQ, swelling, erosion, and focal peeling of the oral sucker were observed. The worms showed changes in the tubercles, shortening, or loss of the spines. In the severely damaged ones, there was disruption of the tubercles and vacuolization on the tegumental ridges or on the tubercles, whereas 24 h after treatment, damage was generally more severe.


  Conclusion Top


A combination of ART plus PZQ was the most effective in reducing total, schistosomula stage, and adult stage worm burdens, and also the number and the diameter of granuloma. However, ARA was the least effective in reducing total and adult stage worm burden.

In contrast, NIF plus PZQ showed the most extensive pathology of the liver; they were the most effective against the schistosomula and the adult stages of S. mansoni by SEM, but PZQ was the least effective against schistosomula stage worm burden reduction and was the least effective against it by SEM.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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