Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858

https://doi.org/10.1007/s12639-023-01632-4

ORIGINAL ARTICLE

Evaluation of structural changes of Echinococcus granulosus

protoscoleces following exposure to different protoscolicidal solutions
evaluated by differential interference contrast microscopy
Elaheh Karpisheh1 · Seyed Mahmoud Sadjjadi1 · Ali Akbar Nekooeian2 · Yosef Sharifi1

Received: 13 June 2023 / Accepted: 4 November 2023 / Published online: 13 November 2023
© Indian Society for Parasitology 2023

Abstract
The present study was aimed to assess the structural changes in protoscoleces of Echinococcus granulosus sensu stricto
following exposure to different natural and chemical protoscolicidal agents using differential interference contrast (DIC)/
Nomarski microscopy. Protoscoleces of sheep’s liver cysts were collected aseptically. Individually, about 1000 protoscoleces
were exposed to 0.5% silver nitrate, 20% hypertonic saline solution, 0.5% cetrimide solution and two different concentrations
of garlic chloroformic extraction as well as phosphate-buffered saline (PBS). The protoscoleces viability was assessed using
0.1% eosin solution, and structural modifications in the protoscoleces were examined by DIC/Nomarski microscopy. The
results revealed the degeneration of the tegument, disorganization of the hooks, and reduction of the size of the protoscoleces
exposed to cetrimide, hypertonic sodium chloride, and silver nitrate. Furthermore, calcareous corpuscles became blurred
and opaque and their numbers decreased in all the exposed samples except, those in PBS. The exposed protoscoleces to
cetrimide and hypertonic sodium chloride solution showed extensive degeneration of the tegument and disorganization of
the hooks. In the group exposed to 200 mg/ml chloroformic garlic extract, the protoscoleces’ width decreased. The length,
width, and number of calcareous corpuscles also decreased significantly in the silver nitrate-exposed protoscoleces. The
study concludes that protoscoleces exposed to different solutions; cetrimide 0.5% and hypertonic sodium chloride 20%
caused more pronounced structural changes in the exposed protoscoleces. These changes were well demonstrated by DIC
microscopy and can be used as a supplementary tool to evaluate the effects of protoscolicidal agents.

Keywords Echinococcosis · Garlic · Differential interference contrast microscopy · Nomarski microscopy · Morphological
structure · Protoscolicidal agents

Introduction (Eckert and Deplazes 2004). Despite the recent advances in

Cystic echinococcosis (CE) is one of the most important
zoonotic diseases. It is caused by the larval stage of Echi-
nococcus granulosus senu lato with worldwide distribution
(Deplasez et al. 2017). The definitive host of the disease is
Canidae, with livestock being intermediate. The disease is
transmitted to humans by ingestion of the eggs of parasites
* Seyed Mahmoud Sadjjadi
smsadjjadi@sums.ac.ir; sadjjadi316@gmail.com
1 are intolerant to surgery (Moro and Schantz 2006; Jaén-
Department of Parasitology and Mycology, School
of Medicine, Shiraz University of Medical Sciences, P.O.
Box 71345‑1735, Shiraz, Iran
2
Department of Pharmacology, School of Medicine, Shiraz
University of Medical Sciences, Shiraz, Iran
the control of CE, it continues to be a major public health
concern in many countries (Andrabi et al. 2020). The dis-
ease is associated with the formation of cysts in human and
domestic ungulates, mainly in the liver and lungs (Eckert
and Deplazes 2004). Diagnosis of the disease is based on
imaging and serological tests (Brunetti et al. 2010;). The dis-
ease is treated by radical and conservative surgery, applica-
tion of anthelmintics such as albendazole and mebendazole,
and also puncture, aspiration, injection of a protoscolicidal
solution, and re-aspiration (PAIR). The PAIR is less inva-
sive than surgery, and is usually used for the patients who
Torrejimeno et al. 2020). However, discharge of the cyst
fluid containing protoscoleces during surgery may result in
the recurrence of the disease at a rate of 3–30% (Goja et al.
2018). Various chemical and natural protoscolicidal agents

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Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858
such as silver nitrate, cetrimide, hypertonic sodium chloride
and Allium sativum have been used to deactivate the contents
of the cyst (Prasad et al. 1991; Napooni et al. 2019; Barzin
et al. 2019). Moreover, garlic chloroformic extract at a con-
centration of 200 mg/ml has a high protoscolicidal effect and
may be used as a protoscolicidal agent in surgery (Barzin
et al. 2019;). The viability assessment in cystic echinococ-
cosis is important for in vitro and in vivo studies and also for
drug experiments (Hemphill et al. 2010). Different methods
have been used for the assessment of viability of the proto-
scoleces for in vitro studies, including a series of vital stains
such as neutral red, methylene blue, eosin, Papanicolaou,
Giemsa, Ziehl–Neelsen, toluidine blue and trypan blue of
them methylene blue, eosin and trypan blue have shown the
same effect (Yildirim et al. 2007).
A combination of staining by 0.1% eosin solution, elec-
tron microscopy, colorimetric assay of caspase-3 like activ-
ity has also been used as viability tests (Shi et al. 2016).
Proscoleces motility and the presence and proliferation of
metacestode vesicles have also been documented as prelimi-
nary indicators of parasite viability (Laurimae et al. 2020).
Moreover, muscular movements of the protoscoleces, activ-
ity of the flame cell, whole body morphological perfectness,
and evagination have been recorded as protoscolices viabil-
ity assessment (Smyth and Barrett 1980; Casado et al. 1986;
Elissondo et al. 2004; Younes et al. 2011; Ismail and Saad
2017; Elowni et al. 2019). In case of metacestodes viability,
it has been shown that detection of parasitic antigens gives
no indication of viability. Presence of calcification which is
more frequent in CE4 and CE5 is not reliable as an indicator
of non-viability and may be observed at all stages (Hosch
et al. 2007). In this regard, antigen B (AgB) proteoforms
hold promise as markers of metacestodes viability (Pagnozi
et al. 2018). Moreover, proteomics investigations has been
shown to be used as biomarker for cure achievement after
surgical removal of human liver cysts (Sedaghat et al. 2021).
Besides, staining techniques using trypan blue, eosin and
methylene blue are more common for in vitro assessment of
protoscoleces viability (Yildirim et al. 2007; Li et al. 2013).
Many researchers use 0.1% eosin as a vital stain for the via-
bility assessment of the protoscoleces. However, the reli-
ability of staining assay as a reliable viability test in terms
of sensitivity and specificity is questionable (Robinson et al.
1985). Moreover, assessment of viability using eosin exclu-
sion might overestimate true viability and underestimate
drug efficacy (Morris et al. 1988). Due to these limitations, it
has been suggested that exposed protoscolices to protoscoli-
cidal agents have to be injected into the mouse peritoneum
for confirmation of its protoscolicidal effect (Robinson and
Arme 1985).
This method has also its limitations, as a number of viable
protoscoleces may be stained while they are alive ( Robin-
son and Arme 1985). Therefore, finding new simple and
851
applied methods for evaluation of viability of protoscoleces
is necessary. So, combination and simultaneous evaluation
of viability tests using staining by a: eosin dye, b:observing
flame cell movements c: enzyme digestion including pepsin
or trypsin digestion and d: in vitro culture to strobilar stage
is necessary (Smyth and Barret 1980). Many researchers
have used viewing the structural changes of protoscoleces
after exposure to protoscolicidal agents by Scanning Elec-
tron Microscopy (SEM) (Elissando et al. 2008; Verma et al.
2014; Shi et al. 2016). It is a hard work and the changes
of protoscoleces can be observed after killing the proto-
scoleces due to protocols of SEM. However, using a simple
way like application of DIC/Nomarski microscopy which
can show the trend of damage and killing of protoscoleces,
has not been evaluated, so far. The present study was aimed
to evaluate the morphological and morphometric changes
of protoscoleces after exposure to protoscolicidal agents,
including silver nitrate, cetrimide, hypertonic sodium chlo-
ride, and garlic chloroformic extract, using DIC/Nomarski
microscopy and observing the dynamic process of dam-
ages in the protoscoleces during exposure to protoscolicidal
agents.
Materials and methods
Compounds
Silver nitrate, cetrimide, Sodium chloride solutions and, two
concentrations of garlic chloroformic extract were used as
protoscolicidal agents. PBS was used as negative control.
Preparation of garlic extract
100 g of powdered dried garlic cloves were added to 400 ml
of chloroform and were placed in a soxhlet extractor while it
was being warmed for 24 h. To remove the extra chloroform
solvent from the extract, a rotary evaporator was used to
desiccate the extract. The remaining powder in the form of
crystal was used for the tests (Barzin et al. 2019).
Preparation of protoscolicidal solutions
Protoscolicidal solutions were prepared as 0.5% silver
nitrate, 0.5% cetrimide, 20% hypertonic sodium chlo-
ride( Merck, Germany) and, garlic chloroformic extract at
two different concentrations of 100 and 200 mg/ml. PBS
composed of sodium phosphate monobasic 2.4 g, sodium
phosphate dibasic 2.84 g and sodium chloride 8.5 g in 1 L
deionized water also in some formulas using potassium
phosphate monobasic 2.72 g, potassium phosphate dibasic
3.84 g, potassium chloride 8.5 g in 1 L with deionized water
(O’Bryan et al. 2009). 20% hypertonic sodium chloride was
13
852 Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858

Table 1  Comparison of morphometric structural changes of protoscoleces after exposure to 20% hypertonic saline solution, 0.5% silver nitrate,
0.05% cetrimide solution, 100 mg/ml garlic chloroformic extract and 200 mg/ml garlic chloroformic extract with PBS as negative control
Test reagents PL PW CL CW NC
Mean ± SD(N) Mean ± SD(N) Mean ± SD(N) Mean ± SD(N) Mean ± SD(N)

1 PBS (Negative control) 165.65 ± 19.3(23) 132.75 ± 12.3(23) 7.6 ± 1.1 (165) 6.2 ± 1.1 (165) 113.44 ± 17.3 (9)
2 20% hypertonic sodium chloride 102.2 ± 15.2(70) 80.96 ± 14.6 (70) 7.05 ± 1.12 (42) 5.8 ± 1.4 (42) 52.6 ± 6.9 (12)
3 0.5% Silver nitrate 119.49 ± 25.7(24) 98.99 ± 18.5 (24) 6.8 ± 1.6 (83) 5.4 ± 1.4 (83) 63.3 ± 29.4(9)
4 Garlic chloroformic extract, (100 mg/ml) 136.3 ± 31.11(13) 121.8 ± 21.15(13) 6.4 ± 0.8 (58) 5.2 ± 1.004 (58) 95.3 ± 16.55 (8)
5 Garlic chloroformic extract, (200 mg/ml) 113.91 ± 9.2(32) 96.69 ± 9.9(32) 6.6 ± 1.1 (112) 5.3 ± 1.07 (112) 75.4 ± 17.07 (22)
6 0.05% cetrimide 133.7 ± 8.4 (5) 111.5 ± 3.5 (5) 6.9 ± 0.7 (27) 5.9 ± 1.03 (27) 51.6 ± 8.8 (5)
p-value 1 vs 2: < 0.0001 1 vs 2: < 0.0001 1 vs 2: 0.0044 1 vs 2: 0.0485 1 vs 2: < 0.0001
1 vs 3: < 0.0001 1 vs 3: < 0.0001 1 vs 3: < 0.0001 1 vs 3: < 0.0001 1 vs 3: 0.0004
1 vs 4: 0.0013 1 vs 4: 0.0575 1 vs 4: < 0.0001 1 vs 4: < 0.0001 1 vs 4: 0.0437
1 vs 5: < 0.0001 1 vs 5: < 0.0001 1 vs 5: < 0.0001 1 vs 5: < 0.0001 1 vs 5: < 0.0001
1 vs 6: 0.0014 1 vs 6: 0.0008 1 vs 6: 0.0016 1 vs 6: 0.1868 1 vs 6: < 0.0001

PL: Protoscoleces Length; PW: Protoscoleces Width; CW: Calcareous corpuscles width; CL: Calcareous corpuscles Length; NC: Number of
calcareous corpuscles
prepared from 20 g of NaCl in 1 L of sterile DW (Chaúque
and Rott 2021). 0.5% Silver nitrate was prepared from 0.5 g
silver nitrate in 100 ml deionized water (Venkatesham et al.
2014). To prepare two concentrations of garlic chloroform
extract, we dissolved 100 and 200 mg of garlic chloroformic
extract in1 ml of DMSO. Cetrimide 0.5% was prepared by
diluting 0.5 ml of commercial cetrimide (Savlon) in 100 ml
of sterile DW.
Viability assay
The viability of protoscoleces was assessed by flame cell
movements under light microscopy, as well as imperme-
ability to 0.1% eosin solution as is usual. In addition, pro-
toscoleces mortality was calculated and reported as the
ratio of dead protoscoleces to total protoscoleces (Barzin
et al. 2019).

Assessment of the morphological structure

Protoscoleces collection and genotype
identification
Sheep’s liver cysts were obtained from a local slaughter-
house in Shiraz, Iran and, protoscoleces were collected
under sterile conditions for the preparation of protoscoleces
and viability tests (Barzin et al. 2019). To determine the
genotypes of the cysts used, DNA extraction from the proto-
scoleces and PCR amplification was performed using a Tis-
sue Genomic DNA Extraction Mini Kit (YTA, Yekta Tajhiz
Azma, Iran), according to the manufacturers’ protocol. The
PCR products were sequenced (Codon genetic group, Iran)
and compared with available reference sequences in Gen-
Bank (Siyadatpanah et al. 2020).
of protoscoleces using DIC/Nomarski microscopy
The changes of morphological structures of protoscoleces,
including changes in the structure of calcium corpuscles,
disorganization of the hooks, and destruction of the tegu-
ment, were observed in the control and test groups by dif-
ferential interference contrast (DIC) / Nomarski microscopy.
The structure of the protoscoleces was assessed qualitatively
(morphological changes, degeneration of the tegument, dis-
organization of the hooks, transparency or opacity) and
quantitatively (length and width of protoscoleces, calcare-
ous corpuscles) after exposure.The digimizer was also used
for morphometry and measurement of the length and width
of the protoscoleces and the calcareous corpuscles.

In vitro protoscolicidal activity
Protoscoleces were exposed to 0.5% silver nitrate, 20%
hypertonic saline solution, 0.5% cetrimide solution
and two different concentrations of garlic chloroformic
extract (100 mg/ml and 200 mg/ml). Experiments were
performed in triplicate.
Statistical analysis
Statistical analysis was performed using GraphPad Prism
software v.8.0.0 for Windows (GraphPad, USA). Statistical
differences between control groups were determined using
Student’s t-test with 95% confidence intervals (CI). Results
were expressed as mean ± S.D. and p < 0.05 were reported
as significant.

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Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858
Fig. 1  Protoscoleces in PBS observed by DIC/Nomarski microscopy:
Comparison of alive protoscolex tegument with dead protoscolex
tegument a, intact and stable tegument uniformed b, clear calcareous
Results
In PBS negative control group, the morphology of proto-
scoleces were observed as normal with intact, stable and
uniformed tegument (Fig. 1a, b and f). In addition, clear cal-
careous corpuscles were observed throughout the cell perim-
eter, and normal rostral hooks arrangement was observed
(Fig. 1c and d, Supplementary file 1). The size of the
853
corpuscles c, normal arrangement of hooks on the rostellum d, com-
parison of protoscoleces size with dead protoscoleces size e, no expo-
sure to protoscolicidl agents f. (scale bar is shown under each photo)
protoscoleces was larger than dead protoscoleces(Fig. 1e).
Figure 1 In the hypertonic sodium chloride group, no move-
ment was observed after exposure. The results also showed
the disorganization of the hooks (Fig. 2a) and the destruc-
tion of the tegument (Fig. 2b). Additionally, smaller pro-
toscoleces in sizes were observed in this group than other
protoscolicidal agents groups. The calcareous corpuscles
were blurred and opaque, and their number were decreased.
13
854
Fig. 2  Photomicrograph of protoscolex after exposure to hypertonic
sodium chloride: Disorganization of the hooks a and destruction
of tegument b Photo of protoscolex after exposure to silver nitrate:
Destruction of tegument c, disorganization of the hooks d Changes of
calcareous corpuscles to rosset shape e. Photo of protoscoleces after
exposure to 100 mg/ml garlic chloroformic extract: The calcareous
corpuscles decreased or disappeared f. Photomicrograph of proto-
In the silver nitrate group, the calcareous corpuscles were
blurred after exposure. The results also revealed a decrease
in the size of the protoscoleces as well as the destruction of
the tegument (Fig. 2c). Disorganization of the hooks was
13
Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858
scoleces after exposure to to garlic 200 mg/ml chloroformic extract:
The calcareous corpuscles blurred and their number decreased g.
Photomicrograph of protoscoleces after exposure to 0.05% cetrim-
ide The tegument is separated from the surface of the protoscolex h,
destruction of tegument i and, disorganization of the hooks j. (scale
bar is shown under each photo)
detected (Fig. 2d). Rose-like calcareous corpuscles were
also observed in a number of samples (Fig. 2e). In some
protoscoleces, the calcareous corpuscles decreased or dis-
appeared in the 100 mg/ml of garlic chloroformic extract
Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858
group after exposure (Fig. 2f). In the group exposed to
200 mg/ml garlic chloroformic extract: The calcareous cor-
puscles were blurred and their number decreased in some
protoscoleces(Fig. 2g). In the cetrimide exposed group:
Almost all the protoscoleces were stained with eosin and
reduced in size after exposure. In addition, the calcareous
corpuscles were blurred, the tegument was separated from
the surface of the parasite and destroyed (Fig. 2h and i),
and the hooks were disorganized (Fig. 2j). Figure 2 The
results of the morphometric changes after exposure to pro-
toscolisidal agents compared to PBS (negative control) are
shown in Table 1. A significant decrease in the length of the
protoscoleces exposed to hypertonic sodium chloride 20%,
Silver nitrate 0.5%, garlic chloroformic extract 200 mg/ml
with PBS group were observed (P < 0.0001), The exposed
protoscoleces to 100 mg/ml garlic chloroformic extract and
0.5% Cetrimide showed a significant difference with PBS
group (P = 0.0013 and p = 0.0014). A significant difference
was also observed in the width of the protoscoleces in 20%
hypertonic sodium chloride, 0.5% Silver nitrate, 200 mg/ml
garlic chloroformic extract and Cetrimide 0.5% with PBS
group. Nonetheless, there was no significant difference in
100 mg/ml garlic chloroformic extract with PBS group.
The results revealed a significant difference in the length
of the calcareous corpuscles in 0.5% Silver nitrate, 100 mg/
ml garlic chloroformic extract, 200 mg/ml garlic chloro-
formic extract, 20% hypertonic sodium chloride and 0.5%
Cetrimide with PBS group. Calcareous corpuscles’ width
were significantly different in 0.5% Silver nitrate, 100 mg/
ml garlic chloroformic extract, 200 mg/ml garlic chloro-
formic extract, 20% hypertonic sodium chloride and PBS
group. However; no significant difference was observed in
width of the calcareous corpuscles in 0.5% Cetrimide with
PBS group (P = 0.1868). The results indicated a significant
difference in the number of calcareous corpuscles in 20%
hypertonic sodium chloride, 200 mg/ml garlic chlorofor-
mic extract and, 0.5% Cetrimide, 0.5% Silver nitrate and
100 mg/ml garlic chloroformic extract with PBS group
(P < 0.0001, P = 0.0004 and P = 0.0437, respectively). One
of the limitations of staining with eosin was observing a
number of eosin stained protoscoleces while they were
alive and their movement can be seen (Suppplementary file
2). Based on PCR and sequencing, all used protoscoleces
samples were E. granulosus sensu stricto. The sequences
were deposited in the GenBank database with accession
numbers: MW363753.1, MW363754.1, MW363755.1, and
MW363756.1.
Discussion
Surgery, PAIR, chemotherapy, and the “watch and wait”
approach are treatment choices for human CE (Brunetti et al.
855
2010). Surgery is still the first choice treatment for compli-
cated cases of CE (Brunetti et al. 2010). However, discharge
of fluids rich in protoscoleces during surgery is the main
cause of the disease recurrence (Gholami et al. 2013). Thus,
protoscolicidal agents are injected into CE cysts to reduce
the risk of recurrence (Moazeni and Nazer 2010). The pro-
toscolicidal effects of cetrimide (Frayha et al. 1981), silver
nitrate (Caglar et al. 2008), garlic chloroformic extract (Bar-
zin et al. 2019), and hypertonic sodium chloride (Adas et al.
2009) have already been demonstrated in several in vitro
studies. Hypertonic sodium chloride solution is one of the
most commonly used protoscolicidal agents in the world
(Kayaalp et al. 2001). The in vitro protoscolicidal activity
of hypertonic sodium chloride 20% has been reported to be
100% effective in five minutes (Adas et al. 2009). In another
study, all protoscoleces were dead after 20 min of exposure
to silver nitrate 20% (Caglar et al. 2008). Moreover, lethality
rates of 99.58% and 96.06% were reported for garlic chloro-
formic extract at 200 and 100 mg/ml against protoscoleces,
respectively (Barzin et al. 2019). In vitro study on proto-
scolicidal effect of garlic chloroformic extract (200 mg/ml)
and 0.5% silver nitrate has been shown 100% and 93.8%
effects after 5 min of exposure time, respectively (Barzin
et al. 2019). The protoscolicidal effects of cetrimide 0.5%
for 1 min on protoscoleces showed 100% effectivity (Bar-
zin et al. 2019). However, all the above studies have used
vital stain for assessing the protoscolicidal activity, which
is not enough for assessing the viability of protoscoleces
(Robinson and Arme 1985). A previous report, using phase-
contrast and Nomarski microscopes to study the structure
of the adult E. granulosus, indicated that Nomarski micro-
scopes provided clearer details of parasites (Sadjjadi et al.
2019). Using these microscopes, create clearer images,
which might be due to the presence of a special polarizer
and a prism in the condenser. Moreover, produced images by
these microscopes results a distinctive and shaded appear-
ance that makes them look quasi-three-dimensionally real
(Allen and David 1969).
The present study also assessed the structural morphol-
ogy on the effects of protoscolicidal agent on exposed pro-
toscoleces. Structural studies using Nomarski microscopy
helped us to observe the effects of protoscolicidal agents on
the structure of protoscoleces both qualitatively and quanti-
tatively. The findings showed that garlic chloroformic extract
at both 100 mg/ml and 200 mg/ml concentrations reduced
the number of calcareous corpuscles and blurred them. In
cetrimide, hypertonic sodium chloride, and silver nitrate
groups, the size of the protoscoleces was reduced. Moreo-
ver, a reduction was observed in the size and number of the
calcareous corpuscles. The teguments were destroyed, and
the hooks were disorganized. After exposure to silver nitrate,
rose shape calcareous corpuscles were detected. Besides, the
number of involved protoscoleces was lower, and they had a
13
856
lower percentage of lethality compared to hypertonic sodium
chloride and cetrimide.
The antibiotic activity of garlic is mostly due to the pres-
ence of thiosulfate allicin (Münchberg et al. 2007). Recent
studies have pointed to the significant biological activity
of trisulfides and tetrasulfides in various Allium species.
Accordingly, a wide range of antibiotic properties and poly-
sulfides’ abilities can induce the death of specific cancer
cells. These effects involve a combination of several distinct
changes such as oxidation reactions, production of superox-
ide radicals, and production of peroxide. Moreover, inhibi-
tion of metal enzymes disrupts membrane integrity and cell
signaling pathways (Münchberg et al. 2007). These changes
were previously observed in protoscoleces following in vitro
exposure to rosmarinus oil (Albanese et al. 2009; Maggiore
et al. 2012), Thymus vulgaris and Origanum vulgare essen-
tial oils (Pensel et al. 2014), pestalotiopsis oil (Verma et al.
2013), gold nanoparticles (Napooni et al. 2019) and syn-
thetic drugs such as albendazole and albendazole sulfoxide
(Pérez-Serrano et al. 1994), praziquantel and albendazole
(Urrea-Paris et al. 2000), flubendazole and ivermectin (Elis-
sondo et al. 2009), thymol (Elissondo et al. 2008), retinoic
acid (Yones et al. 2014), chenodeoxycholic acid (Shi et al.
2016), sodium arsenite (Xing et al. 2016), and cyclosporin
A (Colebrook et al. 2004). The structural changes observed
by DIC/Nomarski microscopy such as contraction of the
soma region, formation of blebs on the tegument, rostellar
disorganization, loss of hooks and destruction of teguments
reported by Elissondo et al. are in some parts similar to our
observations (Elissondo et al. 2008). Changes in the tegu-
ments, loss and disorganization of the hooks may be due
to the “stress response” that occurs in protoscoleces under
harmful conditions (Pérez-Serrano et al. 1994). In the pre-
sent study, the size of the protoscoleces in hypertonic sodium
chloride was smaller compared to other cases due to osmotic
pressure. Some substances may have a selective effect on
protoscoleces’ teguments (Verma et al. 2013). On the other
hand, some substances may interfere with the activity of
proteins that have a protective role in teguments (Colebrook
et al. 2004). These agents seem to decompose and dissolve
the calcareous corpuscles, which are responsible for storing
materials and protecting parasites in acidic environments
(Vargas-Parada and Laclette 1999).
Conclusion
In vitro observations on the morphological changes of
protoscoleces of E. granulosus sensu stricto following
exposure to various concentrations of garlic chloroformic
extract, silver nitrate, cetrimide and hypertonic sodium chlo-
ride, using DIC/Nomarski microscopy showed the smaller
13
Journal of Parasitic Diseases (Oct–Dec 2023) 47(4):850–858
protoscoleces size, clear disorganization of the hooks, and
destruction of the tegument. The calcareous corpuscles were
blurred and opaque and their number decreased after expo-
sure. Moreover, cetrimide and hypertonic sodium chloride
had the greatest effects on the protoscoleces in the short
run. So, morphological observations and evaluation of pro-
toscoleces structural changes by DIC/Nomarski microscopy
could be a choice method for evaluation of in vitro proto-
scolicidal agents.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 12639-0​ 23-0​ 1632-4.
Acknowledgements This study was financially supported by the
office of Vice-Chancellor for Research Affairs of Shiraz University
of Medical Sciences. The authors would like to thank Dr Shams, Mrs
Kazemian and Dr. Mashhoudi for their kind helps. The helps of Ms. A.
Keivanshekouh at the Research Consultation Center (RCC) of Shiraz
University of Medical Sciences is acknowledged.
Author contributions SMS designed the study. EK, SMS and YS were
involved in the practical works of the study. SMS and EK photographed
the samples. SMS and EK analyzed and interpreted data. EK and SMS
prepared the original draft paper. SMS, EK, YS, and AAN reviewed
and edited the final version of the manuscript. All authors have read
and approved the final manuscript.
Funding This study was financed by the Office of the Vice-Chancellor
for Research of Shiraz University of Medical Sciences.(Grant number:
Research 17051).
Availability of data and material The datasets generated during and/or
analysed during the current study are available from the corresponding
author on reasonable request.
Declarations
Conflict of interest The authors have no competing interests to declare
that are relevant to the content of this article.
Ethical approval This study was approved by Shiraz University of
Medical Sciences with the ethical code: IR.SUMS.REC.1398.582.
Consent to participate Not applicable.
Consent for publication Not applicable.
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