Parasitol Res (2015) 114:1563–1580 DOI 10.1007/s00436-015-4341-2
ORIGINAL PAPER
Transmission electron microscopic observations on ultrastructural alterations in Schistosoma mansoni adult worms recovered from C57BL/6 mice treated with radiationattenuated vaccine and/or praziquantel in addition to passive immunization with normal and vaccinated rabbit sera against infection Eman A. El-Shabasy & Enayat S. Reda & Sherif H. Abdeen & Ashraf E. Said & Allal Ouhtit
Received: 12 January 2015 / Accepted: 22 January 2015 / Published online: 18 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Although the current treatment of schistosomiasis relies largely on praziquantel (PZQ), it has not been successful in significantly reducing the overall rate of disease cases, one of the suggested reasons being the inevitable resistance to PZQ. Previous studies showed that radiation-attenuated vaccine provides protection against Schistosoma mansoni in a host of various species. In the present study, we evaluated the effect of various vaccination strategies in C57BL/6 mice, including single or multiple vaccination strategy, subcurative dose (20 mg/kg) of PZQ, and a combination of single vaccination with subcurative dose of PZQ. Treatment either with subcurative dose of PZQ or with a single vaccination of attenuated cercariae (500 per mouse), caused significant reduction in total worm burden, hepatic, and intestinal ova counts of 43.03, 73.2, and 59.5 and 37.97, 52.02, and 26.3 %, respectively. Furthermore, tegumental changes were observed. In multiple vaccinated group, there was an extensive lysis in
E. A. El-Shabasy (*) : E. S. Reda : S. H. Abdeen Department of Zoology, Faculty of Science, Mansoura University, Mansoura, Egypt e-mail:
[email protected]
tegumental layers. High deformations in gastrodermis, testis cells, vitelline cells, and oocytes were recorded. Also, this study is to explore the role of humoral immunity using highly resistant rabbits that had been exposed to three immunizations with ultraviolet (UV)-irradiated cercariae (8000 per rabbit in each immunization), and their sera were tested for their ability to transfer protection. The reduction in challenge worm burden had reached 32.76–43.64 % when compared with recipients of normal serum or no serum. The reduction in hepatic and intestinal ova counts reached to 74.4 and 71.08 % in group immunized with vaccinated rabbit sera. Swelling and extensive lysis of tegumental layers, gastrodermis lumen, spermatocytes, and deformation of oocytes were recorded with more severity than that recorded in normal rabbit sera group. Our findings recorded that multiple vaccination strategy is the most effective strategy then passive transfer of vaccinated rabbit. This gives guiding in the design the appropriate therapeutic strategy. Keywords Schistosoma mansoni . Vaccination . Attenuated cercariae . Ultrastructure . PZQ
A. E. Said Department of Zoology, Faculty of Science, Damietta University, Damietta, Egypt A. Ouhtit (*) Department of Genetics, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman e-mail:
[email protected]
Introduction Schistosomiasis is a disease caused by the blood fluke Schistosoma spp. (class Trematoda, family Schistosomatidae).
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Schistosoma haematobium, Schistosoma Japonicum, and Schistosoma mansoni are the three main species parasitizing humans (Utzinger and Keiser 2004; Gryseels et al. 2006). It has been estimated that more than 207 million people are infected worldwide, with 780 million at a risk of infection (Steinmann et al. 2006; Caffrey 2007). S. mansoni is the most prevalent, being endemic in 54 countries, mostly in Africa and parts of South America (Chitsulo et al. 2000). The annual mortality rate in Africa is estimated to be as high as 280,000 (van der Werf et al. 1980). Due to unavailability of schistosomiasis vaccine (Bergquist and Colley 1998; Katz 1999; Bergquist et al. 2002; Hagan and Sharaf 2003), the current strategy for the control of schistosomiasis depends only on a drug namely praziquantel, which is used in the treatment of three major species of schistosomes infecting humans (Fenwick et al. 2006; Doenhoff et al. 2009). However, drug resistance has been a challenging issue (Doenhoff et al. 2002; Sangster 2001) and therefore, development of a vaccine against S. mansoni could provide a powerful tool to prevent and cure the disease. Radiation-attenuated (RA) schistosome vaccine is highly effective under laboratory conditions but for ethical and practical reasons, it cannot be administrated to humans. However, it serves as a compelling model for the development of a recombinant vaccine (Coulson 1997; Wilson and Coulson 1999). The protective levels of RA vaccine were originally established in mice (Dean 1983) and subsequently extended to primates (Soisson et al. 1993; Yole et al. 1996a, b; Eberl et al. 2001). The tegument of an adult Schistosoma is a protective sheath that plays a role in defense as well as in the uptake of nutrients, osomoregulation, and excretion. Hence, the ultrastructural studies are necessary to potentially clarify aspects of drug- or vaccine-induced damage. Furthermore, protective immunity was not assessed by reduction of recovered worm burdens alone, but also via indirect estimates of reduction in hepatic and intestinal ova count as well as the level of schistosome antigens in the bloodstream. The data generated from Abdeen et al. (2012) has suggested that high levels of antibodies were developed in the vaccinated rabbit sera after multiple exposures to UV-attenuated cercariae of S. mansoni, explaining a passive transfer of protection from the infection and elimination of the worms. Reda et al. (2012) compared vaccine efficacy of a single or multiple vaccination strategy, subcurative dose (20 mg/kg) of praziquantel (PZQ), and a combination of single vaccination with subcurative dose of PZQ on the surface topography of adult S. mansoni. Therefore, as a continuation of our recent work, the present study aims to explore the effect of different vaccine strategies of adult S. mansoni worms using transmission electron microscopy.
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Material and methods Parasites and mice S. mansoni cercariae (Egyptian strain) were collected from infected Biomphalaria alexandrina snails. Female C57BL/6 mice weighing 35–40 g were obtained from the Biological Production Unit (BPU) of Theodore Bilharz Research Institute (Giza, Egypt). All the experiments described in this study were approved by the local ethical committee. Drug PZQ in the form of tablets was ground to white powder and suspended in 2 % Cremophore-EL, as it is insoluble in water. The drug was freshly prepared before oral administration using oral canola. The dose given was 20 mg/kg body weight at the fifth week post-infection. Immune serum preparation and transfer Four New Zealand White (NZW) rabbits, obtained from the BPU, Faculty of Dentistry, Alexandria University (Alexandria, Egypt), served as donors of normal rabbit serum (n=2) and immunized serum (n=2). Every rabbit was immunized thrice by receiving nearly 8000 UV-attenuated cercariae; immunizations were given 3 months apart as previously reported (Mangold and Dean 1992). Penetration of attenuated cercariae occurred through their alternate ears and shaved abdominal skin sides using ring method as previously reported (Smithers and Terry 1965). Prior to each immunization, rabbits were anesthetized by intramuscular injection with a mixture of ketamine (30 mg/kg) and xylazine (5 mg/kg). Blood samples (1 % of body weight) were collected after the last immunization (4 weeks) as previously reported by Mangold and Dean (1986). Blood samples were collected from rabbits (1 % of body weight) according to previously described protocols (Mangold and Dean 1986), and serum samples were prepared by standard procedures. Intraperitoneal injection of normal and vaccinated rabbit sera was applied. Experimental design Mice (n=50) were immunized with 500 attenuated cercariae per mouse or infected percutaneously for 1 h by tail immersion to 200 normal S. mansoni cercariae. Prior to mice infection or immunization, S. mansoni cercariae were shed from experimentally infected B. alexandrina after exposure to artificial light for 4 h, by looping method as previously reported by Sornmani et al. (1973). Fifty mice were used in the experiment, divided into three main groups as follows:
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1. The naive-infected group of ten mice (NC) which takes infection with normal cercariae (200 cercariae per mouse). 2. The UV-attenuated group which include two subgroups (ten mice each) (a) Vaccinated-challenged group (VC) which was immunized in the first week and challenged in the sixth week then sacrificed at 6 weeks postchallenge (b) The multiplied vaccinated-challenged group (V4C) which was immunized in the 1st, 5th, 11th, and 17th and challenged in 23 weeks and then sacrificed at the sixth week post-challenge 3. Chemotherapy group which include two subgroups (ten mice each) (a) Naive-infected chemotherapy group (NCC) which takes infection (200 cercariae per mouse) in the first week and chemotherapy at the fifth week after infection and sacrificed at week 6 post-infection (b) The vaccinated-challenged chemotherapy group (VCC) which takes UV-attenuated 500 cercariae per animal in the 1st week, challenged (200 normal cercariae per mouse) in the 6th week, followed by chemotherapy at the 11th week and sacrificed at week 6 post-challenge (infection) In passive transfer strategy, mice (n=21) were infected percutaneously for 1 h by tail immersion to 200 normal S. mansoni cercariae. Prior to mice infection, S. mansoni cercariae were shed from experimentally infected B. alexandrina after exposure to artificial light for 4 h, by looping method as
previously reported by Sornmani et al. (1973). The mice were divided into three groups as follows: 1. The first group of mice (n=7) were immunized by passive transfer of 400 μl of normal rabbit sera (NRS) on the fourth and the seventh day post-infection (200 μl each time/mouse) (NR group). 2. The second group of mice (n=7) were immunized by passive transfer of 400 μl of vaccinated rabbit sera (VRS) on the fourth and the seventh day post-infection (200 μl each time/mouse) (VR group). 3. The third group of mice (n=7) served as normal mice control group. All animals were sacrificed 6 weeks (42 days) postinfection (Table 1). Adult worms were collected from mice by perfusion method (Sornmani et al. 1973) using 0.1 M citrate in 0.15-M NaCl solution. The worms were washed several times with normal saline solution and fixed in 2.5 % glutaraldehyde–phosphate buffer (0.2 mol/l, pH 7.4) for transmission electron examination. Quantification of S. mansoni eggs in the liver and large intestine (ova count) The method of Cheever (1970) was followed with minor modifications. Hepatic and intestinal tissues were removed from mice after perfusion, washed with normal saline, dried, weighed, and placed in 5 ml of 5 % potassium hydroxide. Tissues were homogenized on ice for 20–30 s using a Polytron homogenizer (Brinkmann Instruments, Wesbury, NY, USA). The mixtures were allowed to incubate at 37 °C for 24 h. At the end of the incubation period, the mixture was shaken well, 100 μl samples were removed, and the total number of eggs in
Table 1 Experimental layout, indicating times of infection, vaccination with attenuated cercariae, PZQ administration, normal and vaccinated rabbit sera injection, and perfusion Groups
Subgroup n
Time (weeks) 0 Infection
Control (NC) – Chemotherapy (PZQ) NCC VCC Attenuated cercariae VC V4C
Passive transfer
vacc. Vaccination
NR VR
10 10 10 10 10
5
Infection PZQ First vacc. First vacc Second vacc. First vacc. Time (days) 0 day 10 Infection 10 Infection
6 11 Perfusion
12
Perfusion PZQ Perfusion Infection Infection Third vacc. Perfusion
4 days NRS VRS
14 17
Fourth vacc
7 days NRS VRS
18 20 23
29
Infection Perfusion
42 days Perfusion Perfusion
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the sample was counted under low power magnification. The results were reported as the mean total egg number of ten independently counted samples per gram tissue±standard deviation.
Table 2 Effect of single subcurative dose of PZQ, single vaccination, in combination with single vaccination and multiple vaccinations on the number of worm burden in S. mansoni-infected mice Group of mice
Normal mice, control group
NCC group
VCC group
VC group
V4C group
Transmission electron microscopic examination
Mean Std. deviation ±SE t Test P0.05
29.4 4.83 2.16 5.112 0.007a
13 3.39 1.5 9.207 0.001a
Worms were fixed in buffered 2.5 % glutaraldehyde phosphate (0.2 mol/l, pH 7.4) in 0.1 M sodium cacodylate. Postfixation was carried out in cold buffered 1 % osmium tetraoxide, followed by dehydration before embedding in Spur resin. Ultrathin sections were stained with 1 % uranyl acetate and lead citrate. Finally, the double-stained sections were examined with JEOL transmission electron microscope (Japan) operating at 60 kV. Statistical analysis
Significant difference to normal control group
respectively, in VC group while in V4C group, the reduction in the count was 90.7 and 65.79 %, respectively (Table 4). Effect of normal rabbit serum and vaccinated rabbit serum
Comparison between two groups was carried out using the Student’s t test. The data were considered significant when P≤0.05 and highly significant when P≤0.001. The percent reduction of the number of worms was determined for each group according to the formula described below (Fonseca et al. 2004): % Reduction ¼ 1−
a
Mean number of IM group 100 Mean number of IU group
where IU is the mean number of worms in the control group and IM is the mean number of worms in the immunized group.
Results Effects of single subcurative dose of PZQ alone and in combination with single vaccination The mean numbers of surviving worms were 47.43 ± 3, 27.857±2.77, and 82.4±1.6 in normal control (NC), single subcurative PZQ (NCC), and vaccinated chemotherapy (VCC) groups, respectively (Table 2). Moreover, the reduction of hepatic and intestinal ova counts were 73.2 and 59.5 %, respectively, in NCC group but 40.2 and 20.4 % in VCC group, respectively (Table 4). Effect of single and multiple vaccinations with attenuated cercariae The mean number of surviving worms was 29.4±2.16 (percent reduction=37.97 %) and 13±1.5 (percent reduction=72.57 %) in vaccinated one time (VC), and vaccinated four times (V4C) groups, respectively (Table 2). Moreover, the reduction of hepatic and intestinal ova count were 52.02 and 26.3 %,
The mean number of surviving worms in normal control group (NC) was 49.43±2.97, while in the NRS group, it was 41.43± 2.19, and in VRS group, it was 27.86±1.35 (Table 3). Moreover, while the reduction of hepatic and intestinal ova count was 36.4 and 34.1 %, respectively, in the NR group; in VR group, it was 74.4 and 71.08 %, respectively (Table 4). TEM of S. mansoni recovered from the normal mice NC Tegumental ultrastructure features of adult male S. mansoni recovered from infected, non-treated mice revealed no apparent damage. The spines appeared with triangular shape and were formed of electron-dense materials (Fig. 1a). High magnification of the tegumental surface showed normal sensory organelle (SO) surrounded with large number of short rod electron-dense secretary bodies (S3), small oval electronlucent secretary vesicles and large more or less round electron-lucent secretary bodies (Fig. 1b). The shape of the cavity was oval with a pigment cup and was located at the outer surface of the body (Fig. 1b). The gastrodermis appeared with normal nucleus and provided with numerous cytoplasmic extensions (Fig. 1c). In the testis, the testicular cells appeared normally with several primary spermatocytes with a normal nucleus (Fig. 1d). In female worms, the surface area of the tegument had no invagination (Fig. 1e). In tegumental matrix, small oval electronlucent secretary granules were observed. Furthermore, more or less round electron-lucent secretary bodies were recorded (Fig. 1e). The sensory organelle was recorded to have cilium and was connected to the body with cytoplasmic junction system which passes in between muscle bundles (Fig. 1e). The gut epithelia cells appeared normal and consisted of rough endoplasmic reticulum (RER) and numerous cytoplasmic extensions (Fig. 1f). The vitelline cells had a normal
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Table 3 Effect of normal rabbit serum or vaccinated rabbit serum on worm burden in S. mansoni-infected mice Group of mice
Normal mice, control group
Mice immunized with normal rabbit serum
Mice immunized with vaccinated rabbit serum
Mean Std. deviation ±SE t Test P0.05
27.857 3.579 1.35 6.728 0.001a
a
Significant difference to normal control group
appearance. The vitelline droplets were arranged in vitelline balls (Fig. 1g). In the oviduct, many oocytes were recorded. While some were recorded to be normal, the others have started to degenerate (Fig. 1h). TEM of S. mansoni recovered from mice treated with subcurative dose of PZQ (NCC group) The tegumental surface in male showed loss in their normal invagination and also lack of some tubercle spines in which were found not to be protruding in some areas (Fig. 2a). At the outer layer of the tegument, there was loss of architecture in the cytoplasm. Furthermore, the more or less round electronlucent and electron-dense secretary bodies were found to be prominent between muscle bundles (Fig. 2a). Also, the whole tegument underwent swelling in the outer and inner layers which was prominent in the muscular layers (Fig. 2a). Interestingly, extensive lysis was also seen in tegumental cells (i.e., there was necrosis in its cytoplasm) (Fig. 2b). The lumen of the gastrodermis appeared narrow (Fig. 2c). A part of the gastrodermis showed detachment of some cytoplasmic extensions, and the gut epithelial cells revealed loss in their definitions; the nucleus with infolded nuclear membrane was recorded (Fig. 2d).
Table 4
In testis cells, nucleus of early spermatides appeared pale and there was accumulation of mitochondria in the cytoplasm (Fig. 2e). The nucleus appeared very pale and small in size, while a few of them had degenerated completely (Fig. 2e). Degeneration and regression of late spermatids were also noted in testicular follicles of treated worms (Fig. 2f). Also, some of the spermatozoa showed abnormal morphology (Fig. 2f). In females, the tegumental surface showed short rod-like electron-dense and more or less round electron-lucent secretary bodies and the rod-like electron-lucent secretary bodies were rarely observed. The apparent damage in circular muscle bundles was apparently disrupted but the longitudinal muscles riddled with vacuoles as a result of lysis of some muscle fibers (Fig. 2g). There was accumulation of secretary bodies at the periphery of swollen and degenerated muscle bundles (Fig. 2g). Along with lysis in subtegumental matrix and tegumental cells appeared swollen with branched nucleus (Fig. 2g). The gastrodermis appeared with dark nucleus and consisted of very minute surface cytoplasmic extensions as compared to the control. Also, both, the blebbing of small components from the apical surface of the gastrodermis and degeneration of endoplasmic reticulum into the lumen were observed (Fig. 2h). In vitelline cells, the most significant alteration was in the form of extensive lysis in nucleus and appearance of residual bodies in the cytoplasm. Also, fusion of vitelline balls which resulted in formation of many vacuoles without vitelline droplets was recorded (Fig. 2i). Pyknosis of chromatin materials was also observed (Fig. 2i). In the ovary, the developing oocytes appeared with deformed nucleus and mature cortical granules at the periphery of the cell (Fig. 2j). TEM of male S. mansoni which recovered from the group that was vaccinated with irradiated cercariae before infection with normal cercariae (VC group) In the tegument, electron-dense secretary bodies were observed between muscle bundles (Fig. 3a). In some regions,
The effect of attenuated cercariae, PZQ, and passive transfer of NRS and VRS on hepatic and intestinal ova count in S. mansoni-infected mice
Animal groups
Hepatic ova count (mean±S.E.)
PR (%)
P value
Intestinal ova count (mean±S.E.)
PR (%)
P value
NC NCC VC VCC V4C NR VR
26,712.8±7614.02 7162.6±1118.04 12,815.6±1698.629 15,976.2±5854.62 2486±93.62a 16,997.4±3298.66 6845.2±495.3544
– 73.2 52.02 40.2 90.7 36.4 74.4
– 0.056 0.164 0.141 0.033* 0.359 0.062
14,859±5526.55 6011±386.689 10,954±1939.637 21,254.6±5218.19 5083±548.09 9791.2±1887.69 4296.600±280.726
– 59.5 26.3 20.4 65.79 34.1 71.08
– 0.173 0.485 0.285 0.137 0.425 0.125
a
Significant difference to NC group
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Fig. 1 TEM of S. mansoni recovered from the normal mice control group (NC). a Outer tegumental layers showing large triangular spines (S), circular muscles (CM), and longitudinal muscles (LM). Note the presence of more or less round electronlucent secretary bodies (S1) in tegumental matrix; ×10,000. b High magnification of the sensory organelle (SO). Note the presence of more or less round electronlucent secretary bodies (S1), short rod electron-dense secretary bodies (S3), and small oval electron-lucent secretary vesicles (thick arrow); ×13,000. c Gut epithelial cell with cytoplasmic extensions (stars), nucleus (N), and nucleolus (NU); ×10,000. d Fully developed spermatocytes (SC). Note the number of mitochondria (arrow); ×7500. e Tegument with sensory organelle (SO) and tegumental matrix with more or less round electron-lucent secretary bodies (S1). Note the presence of small oval electronlucent secretary granules (arrow), cilium (Ci); ×7500. f Gut epithelial cells with large amount of rough endoplasmic reticulum (RER). Note the cytoplasmic extensions (arrows); ×10,000. g Vitelline cell with normal nucleus (N). Note the vitelline balls (stars); ×7500. h Different oocytes in the oviduct. Some are normal (arrows) while others started to degenerate (thick arrows); ×5000
the tegument was disrupted and some longitudinal muscle bundles riddled with many vacuoles (Fig. 3a). There was large exovagination in the tegument with aggregation of more or less round electron-lucent secretary bodies and electron-dense secretary bodies in tegumental matrix (Fig. 3b). The tegumental cell appeared with vacuolated cytoplasm while the nucleus, on the other hand, appeared normal (Fig. 3c). The gut epithelial cells lost their normal definition with lysed nucleus, disappearance of gap between the nucleus and cytoplasm, deformed nucleolus, and detachment of cytoplasmic extensions were recorded (Fig. 3d).
In testis cells, the spermatocyte appeared with lysed cytoplasm and testicular tissue. Between spermatocytes, interstitial cell degeneration was appeared in the form of aviation of architecture (Fig. 3e). TEM of male S. mansoni which recovered from the group that was vaccinated with irradiated cercariae and then challenged with normal cercariae and treated with chemotherapy (VCC group) In this group, the invagination of tegument nearly disappeared (Fig. 4a). Further, there was swelling and focal
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Fig. 2 TEM of S. mansoni recovered from mice treated with subcurative dose of PZQ (NCC group). a Swollen tegument with some protruded spines (S). Note the swelling of muscle bundles and increasing of electron-lucent (thin arrows) and electron-dense secretary bodies (thick arrows); ×7500. b Tegumental cell with vacuolization in its cytoplasm (arrows). Note the lysis in surrounding matrix; ×7500. c Gastrodermis with very tight lumen; ×5000. d High magnification of a part of gastrodermis. Note separation of some cytoplasmic extensions and irregular shape of nucleus (thick arrow); ×10,000. e Testis cells with spermatocytes (SC) and abnormal spermatozoa (thick arrow). Note the degenerated early spermatid (ST1) and nucleus (N); ×5000. f Testicular tissue with many degenerated late spermatids (ST2); ×7500. g Tegument
with number of more or less round electron-lucent secretary bodies (S1) and short rod electron-dense secretary bodies (S3). Note the disruption of circular muscle bundles (arrowheads) longitudinal muscles riddled with vacuoles (small arrows), lysis around tegumental cell, and amoeboid nucleus of tegumental cell (large arrow); ×7500. h Gut epithelial cell with dark nucleus. Note degeneration of endoplasmic reticulum to lumen (small arrows) and blebs (large arrow); ×10,000. i Vitelline cell with degenerated nucleus (arrow). Note the fusion of empty vitelline balls (thick arrows); ×7500. j Ovary with developing oocytes with irregular shape of nucleus. Note mature cortical granules at periphery of the cell; ×5000
lysis of muscle bundles (Fig. 4a, b). More or less round electron-lucent secretary bodies appeared in large amounts in the tegumental matrix (Fig. 4a, b). Extensive
swelling and lysis of circular and longitudinal muscles were recorded in most regions (Fig. 4b) while in others, a number of mitochondria were seen between the
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Fig. 3 TEM of male S. mansoni which recovered from the group that vaccinated with irradiated cercariae before infection with normal cercariae (VC group). a Disrupted tegument showing lysis of its matrix and swelling of longitudinal muscles (LM). Note the lysis of muscle bundles (large arrow), some riddled with vacuoles (small arrows), and more or less electron-moderate (S5) and electron-dense secretary bodies (S4) between longitudinal muscle bundles; ×7500. b Exovagination of tegument with accumulation of more or less electron-lucent secretary bodies (S1) and electron-dense secretary bodies (S4); ×7500. c Tegumental cell showing vacuolated cytoplasm (arrows) with normal nucleus (N); ×7500. d Gut epithelial cell with detached cytoplasmic extensions and lysis nucleus; only deformed nucleolus founded (NU); ×10,000. e Section in the testis cells showing lysis in spermatocytes (SC) and testicular tissue (arrows). Note the swelling of spermatocytes (SC); ×7500
muscle bundles (Fig. 4a). Moreover, large number of more or less round electron-dense secretary bodies appeared in tegumental matrix and between muscle bundles (Fig. 4a). The cytoplasm of tegumental cell underwent degeneration in one cell, and the membrane of another cell was disrupted (Fig. 4c). The sensory organelle was damaged with lysis of its membrane and extensive lysis of tegumental matrix with spongy appearance and damaged mitochondria (Fig. 4d). The gut epithelial cell lost their definition, and there was a decrease in number of the surface cytoplasmic extensions when
compared to the control. There was blebbing of small components from the apical surface that appeared in gastrodermis lumen (Fig. 4e). Moreover, the nucleus was amoeboid in shape (Fig. 4e). In the testis cells, the spermatocytes appeared normal, possessed oval or round nucleus with a large spherical nucleolus and chromatin patches (Fig. 4f). Also, normal spermatids were observed in testis cells (Fig. 4f); however, in other cells, degenerated early spermatids and deformed spermatids were also recorded (Fig. 4g).
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Fig. 4 TEM of male S. mansoni which recovered from the group that vaccinated with irradiated cercariae then challenged with normal cercariae and treated with chemotherapy (VCC group). a Tegument with more or less round electron-lucent (S1) and electrondense secretary bodies (S4). Note the swelling and lysis of muscle bundles (arrow), mitochondria (arrowhead), and S4 between muscle bundles; ×7500. b Extensive lysis and swelling (arrows) of circular muscles (CM) and longitudinal muscles (LM); ×7500. c Tegumental cells appeared in degeneration of cytoplasm (arrows) or nuclear membrane (small arrow); ×7500. d High magnification of damaged sensory organelle (SO). Note the lysis of surrounding matrix (arrows) and damaged mitochondria (thick arrow). ×15,000. e Gut epithelial cell with detached cytoplasmic extensions. Note the amoeboid nucleus (arrows); ×10,000. f Section in testis showing the normal appearance of spermatocytes (SC) and normal spermatid (arrow); ×5000. g Section in another region of testis showing degenerated early spermatids (ST1) and abnormal spermatids (arrows); ×5000
TEM of S. mansoni which recovered from the group that vaccinated four times by attenuated cercariae before challenge with normal cercariae (V4C group) In male, there were extensive lysis of tegumental matrix resulting in the formation of some large vacuoles and disappearance of different types of secretary bodies (Fig. 5a). In tegumental cells, the major alterations included swelling of nucleus in the form of disappearance of gap between nucleus and perinuclear cytoplasm and extensive lysis of cytoplasm extending to the nucleus (karyolysis) (Fig. 5b). In addition, extensive swelling and lysis in surrounding tissue was also seen (Fig. 5b).
In the gut epithelial cell, severe damage was seen which revealed loss of cell definition with blurred nucleus, cytoplasm filled with vacuoles and disappearance of cytoplasmic extensions (Fig. 5c). The spermatocytes exhibited various degree of degeneration as they appeared to have swelling with large vacuoles (Fig. 5d). In females, the main alteration observed in the tegument was the separation between the outer tegument and muscle layers accompanied by appearance of large number of oval electron-dense secretary granules (Fig. 5e). There was swelling and focal lysis in the cytoplasm of tegumental cells which extended to surrounding matrix (Fig. 5e).
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Fig. 5 TEM of S. mansoni which recovered from the group that vaccinated four times by attenuated cercariae before challenge with normal cercariae (V4C group). a Tegument with separated spines (S) and large vacuoles (V). Note the swelling and lysis of muscles (arrows), more or less round electron-lucent secretary bodies (S1); ×7500. b Deformed tegumental cells with irregular shape of nucleus (N) and vacuolated cytoplasm (small arrows). Note the extensive lysis of the surrounding tissue; ×5000. c Gut epithelial cell lost its cytoplasmic extensions. Note the blurred nucleus (arrow) and vacuoles (V); NU nucleolus; ×10,000. d Degenerated spermatocytes (SC) with cytoplasmic vacuoles in cytoplasm (arrow). Note the swelling of nucleus (N); ×7,500. e Tegument showing detachment of tegumental matrix and muscle bundles (stars). Note the large number of oval electron-dense secretary granules (small arrows) and lysis of tegumental cells cytoplasm (matrix) (large arrow) with deformed nucleus (N); ×7500. f Gut epithelial cell showing emergence of vacuoles (arrows) in cytoplasm. Note the irregular shape of nucleus (N) and cytoplasmic extensions (star); ×10,000. g Vitelline cell showing disappearance of nucleolus and fusion of vitelline balls (arrow). Note the presence of endoplasmic reticulum (ER); N nucleus; ×7500. h Different stages of oocytes. Note the degenerated mitochondria (arrows); ×3000
The gut epithelial cell also showed a decrease in granular endoplasmic reticulum, emergence of vacuoles (more or less electron-lucent secretary bodies) in the cytoplasm, irregular shape of nucleus and numerous cytoplasmic directing into the lumen (Fig. 5f). In the vitelline cells, the most significant alteration was appearance of granular endoplasmic reticulum and some damaged mitochondria. The lysis of interstitial tissue between vitelline balls was recorded. Furthermore, fusions of vitelline balls which either consisted of or lacked vitelline droplets were also recorded (Fig. 5g).
In the ovary, different stages of developing oocytes were recorded and mature cortical granules in the cytoplasm at periphery of the cells were observed. The oocytes containing many deformed mitochondria were observed (Fig. 5h). TEM of S. mansoni which recovered from the group which was immunized by normal rabbit sera (NRS) after infection with normal cercariae (NR group) In males, the main alteration was disappearing of spines and permanent swelling of tegumental matrix with aggregation of large number of more or less round electron-dense and
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electron-lucent secretary bodies. In addition, there were atrophied sunken sensory organelles on the outer surface of the swollen tegument and they looked like an oval electrondense secretary bodies (Fig. 6a). The sensory organelle was damaged in another part as a result of separation of the bilayer membrane from each other (Fig. 6b). Rod-like electron-lucent secretary bodies, short rod-like electron-dense secretary bodies, and oval electron-lucent secretary granules were observed in the tegumental matrix (Fig. 6a). There were swelling, extensive lysis in longitudinal muscles (Fig. 6a, b) and appearance of more or less round electron-lucent secretary bodies between muscle bundles (Fig. 6b). In tegumental cells, there was a large alteration in shape; they appeared with vacuoles and irregular shape of nucleus (Fig. 6c). The vacuoles appeared as more or less electronlucent secretary bodies. There was also lysis in muscles surrounded the tegumental cells (Fig. 6c). In the gastrodermis, the gut epithelial cell lost its cytoplasmic extensions mostly and has irregular nucleus. Also, the muscle surrounded had a number of vacuoles and a decrease in rough endoplasmic reticulum (Fig. 6d). In testis cells, there was a large lysis in matrix of spermatocytes (Fig. 6e). In female, at the outer layer of the tegument, there was a loss of architecture in the cytoplasm. Although a number of rod-like and oval electron-lucent secretary bodies was observed (Fig. 6f). The sensory organelle appeared normal with sensory vesicles inside the cavity (Fig. 6f). Swelling in the tegumental muscles was observed (Fig. 6f). In the gastrodermis, the epithelial cell had a little number of cytoplasmic extensions and many blebs in the lumen were observed (Fig. 6g). In the vitelline cells, there was appearance of irregular shape of vitelline balls without vitelline droplets which resulted in presence of vitelline droplets in the cytoplasm (Fig. 6h). In addition, the nucleus appeared in an irregular shape (Fig. 6h). In ovary, many oocytes appeared mature, surrounded with immature cortical granules in the cytoplasm and mature cortical granules at the periphery of the cell (Fig. 6i).
(Fig. 7a). The tegumental cells appeared deformed like that in NR group (Fig. 7b). Also, large accumulation of large more or less round electron-moderate secretary bodies (lipid droplets) between tegumental cells was detected (Fig. 7c). In the gastrodermis, epithelial cells had a large number of cytoplasmic extensions, dark irregular shape of nucleus, and decrease in rough endoplasmic reticulum (Fig. 7d). The tegumental cell behind the gastrodermis showed vacuoles in its cytoplasm and disruption of nuclear membrane leads to movement of nuclear material to cytoplasm (Fig. 7d). In the testis cells, many spermatocytes appeared normal but the walls between cells started to disappear, resulting in combination of cytoplasm of two cells. Also, there were spermatocytes started to lysis and one appeared lysis (Fig. 7e). In female, the tegument showed extensive lysis in the tegumental matrix; just only oval electron-lucent secretary bodies could be seen (Fig. 7f). The sensory organelles appeared normal with secretary granules filled their cavity (Fig. 7f). Also, severe swelling in both circular and longitudinal muscle bundles was observed (Fig. 7f). Distortion of the tegumental cells was observed, including invasion of vacuoles in their cytoplasm or lysis of the cytoplasm completely (Fig. 7g). This lysis was extended also between tegumental cells, and the nucleus was irregular in shape (Fig. 7g). The lumen of gastrodermis appeared narrow with large number of cytoplasmic extensions and the nucleus was amoeboid in shape (Fig. 7h). In vitelline cells, some vitelline balls enlarged with vitelline droplets and others fused in irregular shape. In addition, the nucleolus was degenerated and some vitelline droplets were founded in the cytoplasm (Fig. 7i). In ovary, the oocytes were appeared in different stages of maturity and in irregular shape, smaller size than that of NR group were observed, and no mature cortical granules could be seen at periphery of cells (Fig. 7j).
TEM of S. mansoni which recovered from the group which immunized by vaccinated rabbit sera (VRS) after infection with normal cercariae (VR group)
The present study is the first to report the effects of vaccination with RA vaccine one or multiple times and subcurative dose of PZQ or/and in combination with one time vaccination against adult S. mansoni infection, by revealing unique ultrastructural alterations at level of transmission electron microscopy. Moreover, reporting the effect of vaccination with rabbit sera against adult S. mansoni infection. The present results supporting the hypothesis that RA vaccine is the most effective experimental vaccine available so far and the vaccinated rabbit sera might transfer protection against S. mansoni. Previous study showed that C57BL/6 strain of mice was more susceptible to the infection with Egyptian strain of
In male, little number of tegumental spines appeared and loss in architecture of the tegumental matrix with aggregation of large number of more or less round electron-lucent secretary bodies between spines; some parts appeared as a compact dark layer with blurred tegumental matrix (Fig. 7a). The sensory organelles were damaged as it appeared with disrupted membrane and some oval electron-lucent or electron-dense secretary granules appeared inside the cavity (Fig. 7a). There was extensive lysis and swelling in tegumental muscle bundles
Discussion
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Fig. 6 TEM of S. mansoni which recovered from the group which immunized by normal rabbit sera (NRS) after infection with normal cercariae (NR group). a Swollen tegument showing aggregation of more or less electron-lucent (S1), electron-dense secretary bodies (S4), and oval electron-lucent secretary granules (arrows). Note the presence of rod-like electron-lucent (S2), short rod-like electron-dense secretary bodies (S3), lysis in longitudinal muscles (LM), and sunken sensory organelles (thick arrows), circular muscles (CM); ×5000. b Damaged sensory organelle (SO). Note the swelling and lysis of muscle bundles (arrows) and electron-lucent secretary body (thick arrow); ×10,000. c Tegumental cells with vacuolated cytoplasm (small arrow), some vacuolated completely (large arrow), nucleus (N); ×7500. d Gut epithelial cell with few cytoplasmic extensions. Note the emergence of
vacuoles in epithelial cell and muscle surround (arrows), nucleus (N); ×10,000. e Section in testis showing large lysis of spermatocytes (SC); ×7500. f Tegument with normal sensory organelle (SO) and a number of more or less round (S1) and rod-like electron-lucent secretary bodies (S2). Note the swelling of longitudinal (LM) and circular (CM) muscle bundles; ×10,000. g Gut epithelial cell with irregular shape of nucleus and little cytoplasmic extensions. Note the presence of blebs in the lumen (arrow); ×10,000. h Vitelline cell showing irregular nucleus (white arrow) and presence of vitelline droplets in cytoplasm (thick arrow). Note the fusion of empty vitelline balls (thin arrow); ×7500. i Oocytes in ovary with mature cortical granules at periphery of cell. Note the pinocytosis in nucleolus (arrow); ×5000
S. mansoni than BALB/c mice strain (Bin Dajem et al. 2008) and is known to respond better to UV-irradiated cercariae
(Murrell et al. 1979; Richter et al. 1993; Mountford et al. 2001). We intentionally used C57BL/6 mice because they
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Fig. 7 TEM of S. mansoni which recovered from the group which immunized by vaccinated rabbit sera (VRS) after infection with normal cercariae (VR group). a High magnification of tegument showing damaged sensory organelle (SO), deformed tegumental matrix and large more or less round electron-lucent secretary bodies (S1). Note the emergence of secretary vesicles into sensory organelle (thin arrow) and swelling of muscle bundles (thick arrows), spine (S); ×10,000. b Tegumental cells (TC) appeared damaged with dark nucleus (N) and vacuolated cytoplasm (arrows); ×7500. c Subtegumental layer with large accumulation of large more or less round moderate electron secretary bodies (S5); ×7500. (d) Gut epithelial cells with large cytoplasmic extensions and dark nucleus (N). Note the deformed tegumental cell with dark nucleus (N) and vacuolated cytoplasm
(arrow); ×10,000. e Section in testis showing spermatocytes (SC) with separate walls (arrows). Note the one with degenerated nucleus (moderate arrow) and another nucleus degenerated completely (thick arrow), nucleus (N); ×5000. f Tegument with normal sensory organelles (SO), more or less round (S1), and rod-like electron-lucent secretary bodies (S2). Note the extensive swelling in circular (CM) and longitudinal muscle (LM) bundles; ×7500. g Distorted tegumental cells with vacuolated cytoplasm (thin arrow). Note the lysis of cytoplasm in some (thick arrows); ×7500. h Gastrodermis with narrow lumen. Note the amoeboid shape of the nucleus (N); ×10,000. i Vitelline cell with degenerated nucleus (N). Note the fusion of deformed empty vitelline balls (thick arrows); ×7500. j Ovary contains different stages of deformed oocytes; ×50,000
seem to be high responders than BALB/c mice who showed moderate response as reported by Mountford et al. (2001).
We used New Zealand White rabbits, known to develop primary antibody repertoire of IgGs (Lanning et al. 2000).
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Antibodies retrieved from vaccinated rabbit sera were passively transferred to induce immune humoral response in mice for worm elimination. For the assessment of anti-schistosomal agent or drug efficacy in S. mansoni-infected mice, it is important to study several criteria related to the parasitic intensity, stages, and distribution through the host tissues. Among these criteria, the worm burden, the percentage of ova pattern in parts of the intestine, and ova count in the liver and intestine were considered (Abdel-Ghaffar 2004; Abdel-Ghaffar et al. 2005). The reduction in challenged worm burdens had reached to 43.03 % in treated group with subcurative dose of PZQ and 72.5 % in group that vaccinated four times. Moreover, the reduction in challenged worm burdens had reached 32.76– 43.64 % when compared to recipients of normal serum or no serum. The number of ova per gram tissue is also an important criterion for the evaluation of the effect of anti-schistosomal agent on the infection (Mostafa 2005). Furthermore, the reduction of the eggs’ load in the hepatic tissue in treated mice may be attributed to the reduction in the worm burden as a result of treatment, the low productivity of the female already present, and the active destruction of the few eggs produced by the host’s tissue reaction (Mostafa 2001; Mostafa et al. 2011). There is significance reduction in both hepatic and intestinal ova by comparing V4C group with control group and by comparing VR group with NR group. The present study obviously showed that the subcurative dose of PZQ reduced both hepatic and intestinal ova by 73.2 and 59.5 %, respectively, and in group that vaccinated four times recorded reduction rates reached to 90.7 % in the liver and 65.79 % in the intestine. On other hand, in passive transfer of normal and vaccinated rabbit sera, the level of reduction in hepatic and intestinal ova count were 36.4 and 34.1 % in NR group and 74.4 and 71.08 % in VR group, respectively. Transmission electron microscope is an important tool in studying the different alterations due to different treatment modalities. The tegument of normal S. mansoni is an essential interface between the parasite and its intravascular environment in the host. Surface spines were a prominent feature of the tegument of the gynaecophoral canal of the male parasite and the entire length of the female parasite presented in this study mostly in posterior region. The possible function of the basal lamina and the invaginations of the tegument surface may be involved in the transport of molecules that acquired from the host blood stream, across the schistosome tegument (Skelly and Shoemaker 1996). Wilson and Barnes (1974a) examined the tegument of adult S. mansoni and noted that large bodies (rod or oval lucent secretary bodies) [referred by Inatomi et al. (1969) as secretary bodies] were similar in shape to those of other species (Inatomi et al. 1969; Leitch et al. 1984; Sobhon et al. 1984).
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Additionally, membranous (more or less round electron lucent bound vesicles) bodies were observed as recorded by Wilson and Barnes (1974b). This was suggested to be part of the maintenance of the apical membrane; however, the authors did not explain the process or the origin of the vesicles (Sobhon et al. 1984). These studies indicate that the apical membrane of schistosomes is a dynamic structure. The vesicles located at the base of tegument enfolding similar to that identified by Racoosin et al. (1999). The relative location of elongated bodies within the tegument matrix differed to that of the membranous bound vesicles. Most elongated bodies were observed in the medial and apical regions of the tegument matrix and frequently in very close proximity to the apical membrane. It has been suggested that elongated bodies may either contribute their contents to the tegument matrix or fuse directly with the apical membrane of the tegument (Wilson and Barnes 1974a; MacGregor et al. 1988). In this study of S. mansoni, there is a correlation between elongated bodies and the amount of direct exposure of the tegument surface to the host blood stream. This is in agreement with report of Gobert et al. (2003) on S. japonicum. Regions in close association with the host blood had higher levels of elongated bodies, while regions partially or totally protected (e.g., the gynaecophoral canal and the female tegument) had lower levels of the elongated bodies. Tegumental and subtegumental alterations induced by treatment or vaccination modalities used in the present investigation were characterized by swelling of tegument, tegumental muscles, disappearance of spines, lysis of tegumental cells and muscles, cytoplasm vacuolation, disappearance or accumulation of secretary bodies, degeneration in the cytoplasm and nuclear materials, and sunken as well as damage of some sensory papilla in the swelling tegument. The tegument of worms developed in mice treated with subcurative dose of PZQ (NCC group) showed moderate structural changes was extended to the gynaecophoral canal. These results were in agreement with Sobhon and Upatham (1990) who indicated the ultrastructural changes of the tegument induced by PZQ. Moreover, the present results compared with results obtained by Voge and Bueding (1980) who presented a detailed study on S. mansoni tegumental surface alterations induced by subcurative doses of the schistosomicide amoscanate. Mehlhorn et al. (1981) reported that the primary effect of praziquantel that eventually lead to the death of S. mansoni was the disruption of the tegument of those parasites that are chemotherapeutically susceptible to praziquantel. There is agreement with some tegumental alterations, as observed by Amin and Mikhail (1989). The tegument of S. mansoni was immediately destroyed after exposure in vitro to triclabendazole leading to the formation of defects in the surface of the worms (El-Sayed and Allam 1997). Also, Ro15-5458 and R-354 were recorded to
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cause tegumental damage of S. mansoni in the form of vacuolation (Fawzi 1999; Taha 2007). Furthermore, the worms developed in mice vaccinated one time with attenuated cercariae and treated with subcurative dose of PZQ (VCC group) showed similar changes to that of NCC group. While the worms developed in mice vaccinated one time with attenuated cercariae (VC group) showed greater changes; the tegument ruptured in many places, erosion of tegumental surface with exovagination, accumulation with secretary bodies in tegumental matrix, and damaged sensory organelles. The tegumental cells had highly vacuolated cytoplasm. On other hand, the group that vaccinated multiple times with attenuated cercariae showed highly severe swelling in distal cytoplasm process usually observed and resulted in formation of large vacuoles in the tegument, lyses of muscle bundles, and tegumental cells. Shaw and Erasmus (1983) suggested that these vacuoles rose from dilation of the basal membrane and that they enlarged because of water and ion imbalance. Moreover, it is believed that this imbalance may result in the impairment of transport function of the tegument leading to the surface swellings. This damage extended to sensory organelles and gynaecophoral canal. Sometimes, the detachment between tegument and muscle bundles was recorded in female worms. These results were basically similar to those induced by praziquantel and artemether (Becker et al. 1980; Mehlhorn et al. 1981; Xiao et al. 1981, 2002, 2006). For immunization with passive transfer, in the group that immunized by normal rabbit sera (NRS), the tegument of recovered worms showed severe structural changes. In the group that immunized by vaccinated rabbit sera, the tegument of recovered worms showed extensive lysis of tegumental matrix (blurred tegumental matrix), swelling of tegumental muscles, the tegumental cells with dark nucleus and highly vacuolated cytoplasm. This damage was also recorded in the gynaecophoral canal. Moreover, large accumulation of lipid granules between tegumental cells was recorded. These alterations were compared in accordance with results reported by Mostafa and Soliman (2010) and Xiao et al. (2010). The schistosomal gastrodermis has been a little attention in several ultrastructural investigations although it has been recognized, because of its role in the physiology and development of organism (Senft 1969; Erasmus 1977). The gastrodermis in male of NCC group which appeared with narrow lumen may be due to obvious or high multiplication and folding of the plasma membrane of the gut. In higher magnification, the gut epithelia appeared with darken or deformed nucleus and endoplasmic reticulum introduced to the lumen. While in the VC group showed severe detachment of cytoplasmic extensions and deformed nucleus. In the VCC group, the damage was less severe than VC group. These results could be compared with that recorded by
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Zhang et al. (2009) who recorded a pronounced dilatation of the gut adult worms, accompanied by focal or extensive peeling of the gut epithelial cells post-treatment with mefloquine. The group that vaccinated multiple times recorded severe damage to gastrodermis with development of vacuoles and focal lysis of cytoplasmic extensions. This result was in agreement with the result obtained by Leitch and Probert (1990) on S. haematobium treated with astiban and artemether administration (Xiao et al. 2002, 2006). The changes of gastrodermis in NR group were closely related to that occurred in V4C group. In addition to decrease in endoplasmic reticulum, large blebs in lumen were observed. Finally, the lumen of gastrodermis in the VR group appeared narrow with high intensity of cytoplasmic extensions and amoeboid shape of nucleus. The increase in number of gastrodermal vacuoles has been documented by Bogitsh (1975) and Clarkson and Erasmus (1984). It is important to mention that the treatment or vaccination modalities in the present study affected the testes in the schistosomes. Various alterations in testicular structure of S. mansoni have been recorded as a result of treatment with subcurative dose of PZQ or vaccination one time and in combination of treatment with PZQ post one time vaccination. These alterations included lysis of testicular tissue and degeneration of early and late spermatids with pale nucleus. ElShennawy et al. (2007) reported that the testicular cells were damaged also due to the use of enaminone 4hydroxyquinoline-2(1H)-one derivatives against S. mansoni. The present results could be compared with results reported on S. mansoni by Otubanjo (1981), Mohamed (1999), and Soliman (2008). Multiple vaccination with attenuated cercariae resulted in cellular swelling which resulted in vacuoles in early spermatids. The vacuolation of non-germinal cells was reported in testis of S. haematobium treated with Ro15-5458 (Fawzi et al. 2001). Moreover, Jiraungkoorskul et al. (2005) reported swelling of testicular tissue followed by degeneration, leaving several hollow spaces in Eurytrema pancreaticum after using PZQ and triclabendazole. In the present study, chromatin patches disappeared from most groups while mitochondria abnormally increased and appeared as dark bodies with indistinct cristae or with degenerated cristae. Leitch and Probert (1984) similarly reported abnormal increase of mitochondria as evident after treatment of S. haematobium with amoscanate. In addition, Basch and Clemens (1989) recorded damaging to the primary and secondary spermatocytes and spermatids, and disruption of mitotic process post-treatment with anticancer drug procarbazine; this in accordance to results in the present study. In the NR group, large lyses of early spermatids were recorded. While in the VR group, lysis or regression of spermatocytes partially or completely with degenerated nucleus was observed. The vacuolation within the testis and regression of spermatocytes observed in the present study
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were comparable to those recorded by Irie et al. (1989) in their work on S. mansoni treated with PZQ and dextro-PZQ and You et al. (1992) after artemether administration on S. japonicum. In addition to the ultrastructural changes in tegument, musculature, tegumental cells and significant changes in gut epithelial cells. The changes of vitelline cells of female worms were also seen, which may disturb the digestive function and block the oviposition of female worms. However, these lesions might not be the cause of worm death induced by treatment or vaccination, because in worms that survived after the treatment or vaccination, severe dilation of gut was universal and could recover to normal gradually post-treatment. Also, recovery of ova formation and oviposition were observed in female worms that survived post-treatment or vaccination (Xiao et al. 2010). The alterations in vitelline cells were observed in all groups and characterized by fusion of full or empty vitelline balls and deformed nucleus. This result is in accordance with Xiao et al. (2010). In addition, there was appearance of endoplasmic reticulum between vitelline balls in V4C group. Ovary cells showed deformed nucleus and pinocytosis of nucleolus (in NCC and NR groups) while damaged mitochondria of oocytes appeared in the V4C group. The oocytes became smaller in size in the VR group than others in all groups, and some oocytes were degenerated completely.
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