Experimental Parasitology 195 (2018) 44–53

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Could Araucaria heterophylla resin extract be used as a new treatment for T

toxoplasmosis?
Nora L. El-Tantawya,∗, Amal F. Solimanb, Aida Abdel-Magieda, Doaa Ghorabc, Ashraf T. Khalilb,
Zein M. Naeemb, Kuniyoshi Shimizud, Saleh H. El-Sharkawyb
a
Department of Medical Parasitology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
c
Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
d
Division of Systematic Forest and Forest Products Sciences, Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University,
Fukuoka, 812-8581, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Toxoplasmosis is a worldwide parasitic disease responsible for serious health problems to human. The currently
Toxoplasma available drugs used for toxoplasmosis treatment showed a limited efficacy and cause serious host toxicity. The
Treatment in vitro screening for toxoplasmicidal activity of Araucaria heterophylla resin (AHR) extract and its major com-
Mice ponent 13-epi-cupressic acid (CUP) showed that both AHR (EC50 = 3.90) and CUP (EC50 = 3.69) have high
Araucaria heterophylla
toxoplasmicidal activity in comparison with standard cotrimoxazole (EC50 = 4.28). The antiprotozoal effects of
13-Epi-cupressic acid
AHR and CUP were investigated against acute and chronic toxoplasmosis using mice models. Two groups of
Swiss albino mice were infected by RH Toxoplasma strain intraperitoneally and by Me49 strain orally. Both
groups were treated with AHR and CUP in different doses. Their effects were evaluated by survival rate, peri-
toneal, spleen and liver parasite burdens, brain cyst burden, NO serum level and histopathological lesions. The
ultrastructural changes of tachyzoites of acutely infected mice were studied using scanning electron microscopy
(SEM). There is an evidence of toxoplasmicidal activity of AHR and CUP in acute and chronic experimental
toxoplasmosis. In the acute model, mice treated with AHR and CUP showed prolonged survival rates, a sig-
nificant decrease in the parasite density in peritoneal lavage and pathological insult in both liver and spleen
compared with that of untreated ones. SEM results denote evident morphological alterations of treated tachy-
zoites. In chronic experimental toxoplasmosis, AHR and CUP treated groups could significantly reduce brain cyst
burden by 96.05% and 98.02% respectively. This study indicates that AHR and CUP showed potent tox-
oplasmicidal activities experimentally and could be used as a potential natural nontoxic agent for treatment of
toxoplasmosis.

1. Introduction efficiency against T. gondii encysted bradyzoites (De Oliveira et al.,

Toxoplasmosis is a protozoan disease widely spread worldwide. It is
caused by Toxoplasma gondii (T. gondii). Toxoplasmosis is successfully
controlled by the host immune response in the immunocompetent in-
dividuals but remains dormant lifetime in the host tissues (Dupont
et al., 2012). Although infection in immunocompetent hosts results in
mild or non-specific symptoms (Lang et al., 2007), it represents serious
life-threatening disease in immunocompromised patients (Kim and
Weiss, 2004).
Currently, the combination of sulfadiazine and pyrimethamine is
the reference treatment of toxoplasmosis (McLeod et al., 2014). These
drugs possess a synergistic action against tachyzoites but have limited
2009). Further, severe side effects and adverse drug reactions including
hematological reactions, hypersensitivity, intolerance, teratogenic ef-
fects, bone marrow suppression, and gastrointestinal disturbances have
been reported (Furtado et al., 2011). Historically, natural products have
been the most productive sources for treating a wide range of infectious
diseases (Kheirandish et al., 2016). Medicinal plants represent a pro-
mising source in the discovery of the new natural therapies (Emami
et al., 2012). Recently, the naturally derived plant extracts as alter-
native therapies are getting increasing attention. Several studies have
been conducted concerning the use of herbal medications for tox-
oplasmosis treatment, but the research on relatively effective and low
toxic substances still required (Arab-Mazar et al., 2017).

∗
Corresponding author. Department of Parasitology, Faculty of Medicine, Mansoura University, Egypt.
E-mail addresses: noratantawy@yahoo.com, noralabeeb@mans.edu.eg (N.L. El-Tantawy).

https://doi.org/10.1016/j.exppara.2018.10.003
Received 21 May 2018; Received in revised form 25 September 2018; Accepted 13 October 2018
Available online 16 October 2018
0014-4894/ © 2018 Elsevier Inc. All rights reserved.
N.L. El-Tantawy et al.
Araucaria heterophylla Salisb trees (Araucariaceae) are coniferous
trees grown in Egypt as ornamental plants and known as the Christmas
tree. The resin exudates of Araucaria species are rich in diterpenes
(Caputo et al., 1974; Caputo and Mangoni, 1974). The secreted resin,
when the trunk of the tree is injured, acts as a bandage protecting the
plant from invading insects or pathogens (Langenheim, 2003), there-
fore, it is expected that the resin possesses antimicrobial and anti-
protozoal activities. The major component isolated from Araucaria
heterophylla resin (AHR) is the labdane diterpene 13-epi-cupressic acid
(CUP). AHR was reported to have anti-ulcerogenic and cytotoxic ac-
tivities against breast and colon cancer cell lines (Abdel-Sattar et al.,
2009). Moreover, the Libyan propolis (bee glue), a resinous dark ad-
hesive substance collected by honey bees, showed in vitro antiprotozoal
activity against Trypanosoma brucei and Lieshmania donovani (Siheri
et al., 2014). Phytochemically, both AHR and Libyan propolis were
found to possess similar labdane diterpenes, 13-epi-cupressic acid and
acetyl-O-13-epi-cupressic acid (Abdel-Sattar et al., 2009; Siheri et al.,
2014).
The shortfalls of the current anti-Toxoplasma treatment as well, in-
creasing interest in the use of natural products to treat infectious dis-
eases triggered the investigation of AHR and its derivative CUP for their
antiprotozoal activity. This is the first report to indicate the use of AHR
extract and its CUP derivative as non-toxic, potent and bioactive agents
for the treatment of acute and chronic toxoplasmosis.
2. Materials and methods
2.1. Plant materials; extract preparation and 13-epi-cupressic acid isolation
The resin exudates from the stems of Araucaria heterophylla Salisb
were collected from Mansoura University Gardens, Mansoura, Egypt in
February 2015. The plant identity was kindly confirmed by staff
members at the department of Horticulture, Faculty of Agriculture,
Mansoura University, Egypt. A representative specimen was deposited
at the department of Pharmacognosy, Faculty of Pharmacy, Mansoura
University (AHR-2-2015). One Kg of the resin was extracted with
chloroform: n-hexane 1:1 v/v. The extract was evaporated under re-
duced pressure to give about 500 g of a sticky brown translucent re-
sidue. About 50 g of the residues was chromatographed on a silica gel G
60 column (4.5 × 100 cm) and eluted first with n-hexane, then EtOAc/
n-hexane mixtures with increasing polarity (1% EtOAc step). Fractions
of 250 ml each were collected and monitored by silica gel GF254 TLC
plates. 13-epi-cupressic acid was eluted by 6% EtOAc/n-hexane, showed
violet spot on silica gel GF254 plates at Rf 0.18 using the solvent system
DCM - EtOAc (95:5) when sprayed with p-anisaldehyde reagent and
heated at 105 °C for 1 min. 13-epi-cupressic acid (Fig. 1) was obtained
as a yellow viscous liquid (7.5 g) in 15% yield and of 100% purity.
2.2. Spectral analysis of 13-epi-cupressic acid
IR (KBr, ν max) spectrum was recorded on infrared spectro-
photometer, Mattson 5000 (England). 1H-NMR, 13C-NMR, DEPT-135,
HSQC and HMBC experiments were recorded on BRUKER Ascend™ 400
spectrometer using CDCl3 as solvent. FAB-MS experiment was de-
termined using LC-MS-IT-TOF (Shimadzu, Tokyo, Japan). Spectral data
were in full agreement with those published for 13-epi-cupressic acid
(Abdel-Sattar et al., 2009).
13-epi-cupressic acid spectral data, IR νmax (KBr): 3418, 3082,
2930, 2850, 1696, 1645, 1262, 921 cm−1; FAB-MS: m/z 319 [M-H]-
calc. for C20H32O3;1H-NMR: δ 5.95 (1H, dd, J = 16, 12, H-14), 5.24
(1H, d, J = 16, H-15), 5.08 (1H, d, J = 12, H-15′), 4.87 (1H, s, H-17),
4.56 (1H, s, H-17′), 1.30 (3H, s, H-16), 1.26 (3H, s, H-18), 0.63 (3H, s,
H-20); 13C-NMR and DEPT-135: δ 183.0 (C-19 C), 148.0 (C-8, C), 145.0
(C-14, CH), 111.5 (C-15, CH2), 106.6 (C-17, CH2), 73.6 (C-13, C), 56.7
(C-9, CH), 56.4 (C-5, CH), 44.2 (C-4, C), 41.5 (C-12, CH2) 40.7 (C-10,
C), 39.2 (C-1, CH2), 38.4 (C-7, CH2), 38.0 (C-3, CH2), 29.2 (C-16, CH3),
45
Experimental Parasitology 195 (2018) 44–53
12 OH
14
11 13
20 15
1 16
9 17
2
10 8
3 4 7
5
6
COOH
18 19
Fig. 1. Structure of 13-epi-cupressic acid.
27.6 (C-18, CH3), 26.0 (C-6, CH2), 19.9 (C-2, CH2), 17.9 (C-11, CH2),
12.7 (C-20, CH3).
2.3. AHR extract, CUP formulation and doses
As the tested AHR and CUP materials were sticky and resinous, they
were formulated in emulsion using 2% tween 80 to facilitate their ad-
ministration (Gad et al., 2006). About 300 mg of AHR was dissolved in
0.4 ml tween 80 and then mixed with 20 ml of normal saline. Three
different doses of 0.10 ml, 0.20 ml, and 0.40 ml, corresponding to 50,
100, and 200 mg/kg were given orally to individual mice. 150 mg of
CUP was dissolved in 0.4 ml tween 80 and then mixed with 20 ml of
normal saline. Three different doses of 0.10 ml, 0.20 ml, and 0.40 ml,
corresponding to 25, 50 and 100 mg/kg were given orally to individual
mice.
2.4. Toxicity assay of the studied plant extract
Five naïve Swiss albino female mice were randomly assigned to
study the toxic effect of the AHR extract and another 5 females were
used for CUP testing. The third group of 5 untreated mice was used as
the control. High doses only were given orally for the 2groups (200 mg/
kg/day for AHR extract and 100 mg/kg/day for CUP) for two weeks.
The mortality, clinical signs, body weights, food consumptions, and
histopathological examination of major organs (brain, liver, kidney,
and spleen) were assessed.
2.5. Mice and experimental design
This study was performed in accordance with the “Guide for the
care and use of laboratory animals” published by the US National
Institutes of Laboratory Animal Resources (National Research Council
(US) Committee for the Update of the Guide for the Care and Use of
Laboratory Anima (2011). The Institutional Review Board of the Fa-
culty of Medicine, Mansoura University, Egypt has reviewed and ap-
proved the study protocol (IRB code no: R/17.10.123). All surgeries
were performed under isoflurane anesthesia, and all efforts were made
to minimize animal suffering. The experiment was done on outbred
female Swiss albino mice, 6–8 week old, weighing 25–30 gm which
were obtained from Medical Experimental Research Center (MERC),
Faculty of Medicine, Mansoura University, Mansoura, Egypt. The mice
were housed five per cage and were kept in a controlled environment
with a temperature of 22–25 °C in a 12:12 h light/dark cycle, with food
N.L. El-Tantawy et al.
commercial and water available ad libitum. 295 mice were in-
corporated into this study. From which, 280 mice were used to study
the effects of the AHR extract and its active component CUP on mice
infected with RH and Me49 Toxoplasma strains. Besides, another 15
mice were used to test the toxicity of high doses of AHR and CUP on
mice (5 for each group and 5 for control).
2.6. Parasites and their maintenance
The two strains of T. gondii were used for the experiments, virulent
RH T. gondii strain which cause acute toxoplasmosis and avirulent Me49
T. gondii strain responsible for chronic toxoplasmosis. Both strains were
obtained from the Medical Parasitology Department, Faculty of
Medicine, Alexandria University, Egypt.
2.6.1. Virulent RH T. gondii strain
T. gondii virulent strain was maintained in the laboratory of the
Medical Parasitology department, Faculty of Medicine, Mansoura
University, Egypt, by continuous intraperitoneal passages into labora-
tory-bred albino mice every three days (Mcleod et al., 1988). For an-
imal infection, tachyzoites were harvested from peritoneal exudates of
the infected mice on the 4th day of infection. Debris and host cells were
removed by filtration throughout a sheet of glass wool fibers. The fil-
trate was purified by washing three times and diluted with phosphate
buffer saline (PBS), pH 7.4. The tachyzoites count was estimated by
using a hemocytometer. It was re-suspended at a density of 2 × 103/ml
in saline to be inoculated intraperitoneally into the female mice (Eissa
et al., 2012).
2.6.2. Avirulent Me49 T. gondii strain
Avirulent Me49 T. gondii strain was maintained at the medical
Parasitology department laboratory, Faculty of Medicine, Mansoura
University, by passage through Swiss-albino mice. As for animal in-
fection, mice were inoculated orally by lavage with a brain suspension
containing T. gondii cysts (10 cysts/mouse). Brain suspensions were
obtained by harvesting brains of infected mice 8 weeks after oral in-
fection and homogenized in 1 ml buffered saline pH 7.2 by tissue
homogenizer. For cyst enumeration, 0.10 ml of the brain suspension
was placed on a slide and microscopically counted under the high
power lens (x40). The suspension was then diluted to a concentration of
100 cysts/ml (Djakovic and Milenkovic, 2001).
2.7. Reference drug (cotrimoxazole)
Septrin® oral suspension (GlaxoSmithKline, Egypt) was used.
Cotrimoxazole is a combination of sulfamethoxazole and trimethoprim
(200/40 mg/5 ml). To translate the cotrimoxazole dose given to the
human to a dose for mice, an equation was used according to (Nair and
Jacob, 2016). It was given orally at the same day of infection in a dose
of 370 mg/kg divided in 2 doses/12 h for one week for acute tox-
oplasmosis model and at the same dose for 2 weeks started 8 weeks post
infection for chronic Toxoplasma model (Eissa et al., 2015).
2.8. In vitro screening for toxoplasmicidal activity of the plant's extract
Tachyzoites from the virulent strain RH of T. gondii were collected
from intraperitoneal fluid and suspended in PBS, pH 7.2, 48 h after
infection, in a density of 106 parasites/ml. Serial dilutions of the tested
extracts (AHR and CUP) were prepared to get final concentrations of
100, 50, 25, 12.5, 6.25, 3.125 and 1.56 μg/ml. About 45 μl of tachy-
zoites suspension (106 parasites/ml) was incubated with 0.10 ml of
different concentrations of each compound for 24 h at 37 °C in 10% CO2
incubator (Kavitha et al., 2012). Tachyzoites viability was evaluated by
dye (0.2% Trypan blue) exclusion test using a hemocytometer. The
results were expressed as % mortality. Cotrimoxazole drug was used as
the drug control. The tested extracts were assayed in triplicate at each
46
Experimental Parasitology 195 (2018) 44–53
concentration. The mean effective concentration causing 50% inhibi-
tion (EC50) in μg/ml for tested compounds was calculated and com-
pared with that of cotrimoxazole (Kavitha et al., 2012).
2.9. Challenge infection with T. gondii and treatment schedule
280 mice were randomly divided into 18 groups. Nine groups to
study the effect of the extract on mice infected with the RH Toxoplasma
strain and another nine for mice infected with Me49 Toxoplasma strain
as shown in the Table 1. Groups (I, II, V, VIII, and IX) have 20 mice for
each, (subgroup a = 10) to study the effect of extract and (subgroup
b = 10) for estimating survival rate. While groups (III, IV, 214 VI and
VII), have 10 mice in each group for estimating tachyzoites number in
peritoneal lavage in acute toxoplasmosis model and Toxoplasma cyst
count and size in the brain besides in chronic toxoplasmosis model.
Moreover, NO serum level and histopathological studies was evaluated
in acute and chronic model.
2.9.1. Acute toxoplasmosis model
All infected groups were challenged by intraperitoneal injection of
2500 viable tachyzoites/mouse to induce acute infection model (Eissa
et al., 2012). Treatment by both AHR and CUP was initiated on the
same day of infection and continued for 6 successive days in doses
mentioned before. While mice from the untreated groups (group I & II)
were administrated orally with 0.1 ml 2% Tween 80 normal saline in
the same schedule. Mice of groups (Ia, IIa, III, IV, Va, VI, VII, VIIIa, IXa)
were euthanized and sacrificed on the 7th day from the start of the
experiment for their assessment. While, the second subgroups (Ib, IIb,
Vb, VIIIb, IXb) were followed daily to estimate the survival rate till all
mice have died.
2.9.2. Chronic toxoplasmosis model
Infected groups were induced by administering 10 cysts per mouse
intragastrically using 22-gauge blunt feeding needle. Treatment started
8 weeks post infection and continued for 2 weeks. Mice from the un-
treated groups (group I & II) were administrated orally with 0.1 ml 2%
Tween 80 in normal saline in the same schedule. Mice of groups (I, IIa,
III, IV, Va, VI, VII, VIIIa and IXa) were euthanized and sacrificed, one
month after the end of therapy to assess. Even as, the second subgroups
(Ib, IIb, Vb, VIIIb and IXb) were followed for 6 weeks to estimate the
survival rate.
2.10. Evaluation of therapy efficacy in acute toxoplasmosis
2.10.1. The survival rate
The number of survived mice and survival period were recorded
daily till all mice died. The survival rate was calculated in groups (Ib,
IIb, Vb, VIIIb and IXb).
2.10.2. Estimation of T. gondii tachyzoites count in peritoneal lavage,
spleen, and liver
After euthanasia, all mice were submitted to peritoneal lavage as
mentioned before; then, tachyzoites were counted by the hemocyt-
ometer. For impression smears, obtained liver and spleen samples were
dissected, and excess blood was removed on absorbent paper. The re-
sultant material was imprinted on glass slides, fixed by methyl alcohol
and stained with Giemsa stain. Counting of T. gondii tachyzoites was
carried out using oil immersion objectives (x100) lens. The extracellular
tachyzoites in each mouse were counted in 10 high power field (HPF)
and then the mean number of tachyzoites/10HPF was estimated. The
mean number of tachyzoites in each group of mice was calculated ac-
cording to Gasparotto Junior et al. (2016).
2.10.3. Histopathological study of the liver and spleen
All mice were sacrificed to assess histopathology at 7th day post-
infection. Specimens from liver and spleen were removed from
N.L. El-Tantawy et al.
sacrificed mice and fixed in formaldehyde (10% in PBS) at pH 7.2 for
subsequent ascending dehydration and paraffin embedding. Serial
sections, slices of 5μ in thickness were prepared and stained with he-
matoxylin and eosin (Fuentes-Castro et al., 2017). Liver sections were
scored in accordance with Batts and Ludwig (1995).
2.10.4. Nitric oxide estimation
The production of NO was assessed in serum samples from infected
mice, as well as infected and treated ones. The serum samples were
stored at −70 °C until being analyzed for measurement of No using NO
assay kit (Biodiagnostic, Egypt) according to manufacturer's instruc-
tions. NO levels were quantified using the colorimetric method
(Montgomery and Dymock, 1961).
2.10.5. Ultra-structural study of T. gondii tachyzoites by SEM
For field emission scanning electron microscopy, the peritoneal
fluid from infected untreated and infected treated groups was collected;
the parasite-rich peritoneal fluid was centrifuged at 70 g for 3 min to
remove cells and debris. The supernatant was then centrifuged at 900 g
for 10min to deposit the free Toxoplasma tachyzoites, fixed in 2.5%
glutaraldehyde phosphate buffer (0.1 mol/L, pH 7.4) at 4 °C for 24 h
and post-fixed in 1% osmium tetroxide buffer for 1 h. They were de-
hydrated through a graded series of ethanol. The parasites were em-
bedded in epon resin, 20 nm gold particles were used to be sputter
coated before examination under a JOEL-JSM-6510 LV, Japan scanning
microscope (Hayat, 2000).
2.11. Evaluation of therapy efficacy in chronic toxoplasmosis
2.11.1. The survival rate
The survival rate of groups (Ib, IIb, Vb, VIIIb and IXb) was recorded
every week for 6 weeks from the start of the therapy.
2.11.2. Estimation of Toxoplasma cyst count and size in the brain
At the end of the study, all remaining mice were euthanized. For
counting brain cysts, halves of the brain were suspended in PBS and
centrifuged at 1500 g for 10 min. Cysts number was counted in 10/HPF
and the mean number was determined by calculating the mean num-
bers of cysts in each infected group. Moreover, the size of the cysts was
measured using an ocular micrometer (Araujo et al., 1996).
2.11.3. Histopathological study of the brain
Parts of the other half of the brain, including grey and white matter
of the cerebrum, were removed and fixed in formaldehyde (10% in PBS)
at pH 7.2 for subsequent histopathological study using (H&E) stain
(Abdel Wahab et al., 1989). The inflammatory score in the brain was
determined as described by (Silva et al., 2010).
2.11.4. Nitric oxide estimation
NO was assessed as described in acute toxoplasmosis section.
2.12. Statistical analysis
Data were entered and statistically analyzed using the Statistical
Package for Social Sciences (SPSS) version 20. Quantitative data were
described as mean and standard deviation after testing normality by
Kolmogorov-Smirnov test. Quantitative parametric variables were
compared using One Way ANOVA test with post Hoc LSD test (for
pairwise comparison). Kaplan Meier curve was used for calculation of
median survival time with the comparison between groups using Log-
rank Chi-Square test. Percent of reduction was calculated using the
following formula (Mean of the control group - mean of the group with
reduced mean/mean of control group). “p-value ≤0.05” was con-
sidered to be statistically significant and < 0.01 was considered high
statistically significant. All tests were 2-tailed.
47
Experimental Parasitology 195 (2018) 44–53
Fig. 2. The in vitro toxoplasmacidal activity of the plant extract and its in-
gredients and cotrimoxazole using trypan blue assay.
3. Results
3.1. Results of in vitro assessment of toxoplasmicidal activity
The in vitro screening of toxoplasmicidal activity against T. gondii
RH strain tachyzoites was found in all the samples tested. Both AHR
(EC50 = 3.90) and CUP (EC50 = 3.69) showed high activity relative to
the standard cotrimoxazole (EC50 = 4.28), however, 13-O-acetyl-epi-
cupressic acid (EC50 = 36.67) showed a very low toxoplasmicidal ac-
tivity. Therefore, AHR and CUP were selected for in vivo estimation of
their anti-T. gondii activities (Fig. 2). There is no significant difference
between AHR, CUP, and cotrimoxazole regarding EC50 values.
3.2. Results of toxicity test of the plant extract
No recorded mortality of mice groups till the 8th day of scarification
and no abnormal clinical observations were noticed during this period.
The mean body weight and food consumption of the 2 treated groups
(received AHR and CUP treatment) are comparable to the control group
and both fluctuated from day to day without any tendency of increasing
or decreasing. No statistically significant difference between the three
groups in regard to the mean body weight and food consumption.
Grossly examining the organs revealed no obvious changes between the
three groups. Histopathological examination showed no obvious mor-
phological changes in the liver, kidney or brain in comparison with the
control group. No statistical difference (P > 0.05) was detected re-
garding NO serum level between the 3 groups (Mean ± SD of
0.89 ± 0.03, 0.82 ± 0.07 and 0.75 ± 0.03 were recorded for No
serum level for control, AHR received and CUP received groups, re-
spectively).
4. Results of acute toxoplasmosis murine model
4.1. The survival rate of acutely infected toxoplasmosis model
From the 4th-day post-infection, the survival rate of mice in group
(II) started to decrease and its survival curve declined progressively till
all mice died at 7th day. There is an increase in the survival rate of
infected treated groups with the highly statistically significant differ-
ence in comparison to infected untreated control group (Fig. 3). How-
ever, mice treated with CUP (group VIII) showed a significant increase
in survival rate to about 70% at the 7th-day post infection in comparison
to cotrimoxazole and AHR treated groups which have 40% survival rate
at the 5th day and 6th day respectively post infection.
N.L. El-Tantawy et al. Experimental Parasitology 195 (2018) 44–53

Fig. 3. Survival rate of the acutely (a) and the chronically (b) infected murine
toxoplasmosis model.

4.2. Estimation of T. gondii tachyzoites count in peritoneal lavage, spleen,

and liver

The mean tachyzoites count recorded in the peritoneal lavage was

(Mean ± SD 17.56 ± 0.32, 17.56 ± 0.32, 6.24 ± 0.04,
2.08 ± 0.02, 15.04 ± 0.03, 5.12 ± 0.06, 1.43 ± 0.06 and
3.85 ± 0.12 for group II, III, IV, V, VI, VII, VIII, and IX respectively).
There was a highly significant statistical difference between infected
control group II and other groups. Mice group treated with CUP in a
dose of 100 mg/kg showed significant reduction (91.9%) in the total
number of tachyzoites followed by mice group that received AHR in a
dose of 200 mg/kg (88.2%) and then cotrimoxazole treated group
(78.1%) in comparison to infected control group (group II). Regarding,
the spleen impression smear, all mice that were given AHR, CUP and
cotrimoxazole showed a significant reduction in the total number of
tachyzoites in comparison to the infected control group (Fig. 4). The
reduction percent was 24.6%, 56.5%, 77.05%, 70.5%, 82.8%, 92.6%
and 81.97% for group III, IV, V, VI, VII, VIII, and IX respectively and
consecutively. It was observed that the highest reduction percent was in
the group of mice that received CUP in a dose of (100 mg/kg) (92.6%).
The results of liver impression smear are shown in (Fig. 4) with re-
duction percent of 23.7%, 54.8%, 81.5%, 72.6%, 82.96%, 94.07% and
82.2% for III, IV, V, VI, VII, VIII and IX groups, respectively in com-
parison to infected control group.
Fig. 5. The liver histopathological results, A: Liver section from normal control
mice (group I) showing preserved hepatic architecture (H&E, × 400) B,C, D:
Liver section from infected, untreated control group (II), showing disturbed
4.3. Histopathological study of the liver and spleen architecture with aggregates of inflammatory cells, mostly neutrophils and
macrophages (black arrows), and areas of necrosis were seen in the hepatic
In case of liver, the histopathological study of infected untreated parenchyma and around the congested central veins. Toxoplasma tachyzoites
mice showed disturbed hepatic architecture with degenerative changes were observed free in the liver parenchyma (arrowhead) (H&E, × 400), E: Liver
sections from mice groups treated with AHR (100 mg/kg) and CUP (50 mg/kg)
showing mild inflammatory cellular aggregates (black arrows) (x200) F: Liver
section from mice group treated with cotrimoxazole and AHR (200 mg/kg) and
CUP (100 mg/kg) showing moderate inflammatory cellular infiltrate (H&
E, × 400) G: Liver sections from mice groups treated with AHR (200 mg/kg)
and CUP (100 mg/kg) showing mild inflammatory cellular aggregates (x400)
and H: Liver histopathology scoring figure for studies groups (*denotes statis-
tical difference in comparison to infected untreated control group).

was noticed in the form of moderate swelling of hepatocytes and

Fig. 4. Photomicrographs of spleen impression smear from A: infected un-
treated control group and B: mice group received cotrimoxazole and C: mice
group received CUP in a dose of 100 mg/kg (Black arrows indicate T. gondii
tachyzoites). D, E: Figures showing the count of tachyzoites in the liver and
spleen impression smears respectively. * denotes the level of significance.
Kupffer cell hyperplasia. Aggregates of inflammatory cells, mostly
neutrophils, and macrophages were seen in the hepatic parenchyma
and around the congested central veins (Fig. 5B). Hepatic sinusoids
were studded with inflammatory cells. Toxoplasma tachyzoites were
observed free in the liver parenchyma (Fig. 5C). While in mice groups
treated with either AHR or CUP, a trend of recovery was observed in the
liver tissue, especially the group of infected mice treated with high
doses (group V and VIII). There was no apparent damage in hepato-
cytes, no inflammatory foci and without vascular congestion (Fig. 5E, F,
G).
Regarding the spleen, mice infected with T. gondii and untreated
showed enlarged lymphoid follicles of variable size and shape, some
without a germinal center and with a reduced marginal zone. The red
pulp demonstrated mild diffuse congestion and infiltration of

48
N.L. El-Tantawy et al.
Fig. 6. The spleen histopathological results, A&B: Spleen section from infected,
untreated control group (II), showing white pulp follicular hyperplasia (black
arrows) and marked red pulp congestion with infiltration of multinucleated
giant cells engulfing Toxoplasma tachyzoites (arrowheads) (H&E, × 400), C&D:
Spleen section from mice group treated with cotrimoxazole, AHR (100 mg/kg)
and CUP (50 mg/kg) showing moderate inflammatory infiltrate and decreased
number of giant cells (X200)& ( × 400), E&F: Spleen sections from mice groups
treated with AHR (200 mg/kg) and CUP (100 mg/kg) showing reduced red pulp
congestion and white pulp follicular hyperplasia (mild inflammation) (H&E,
x200&X400).
multinucleated giant cells (Fig. 6A and B). Spleen of infected and
treated mice by AHR and CUP showed tissue recovery notably at high
doses, with well-structured lymphoid follicles, germinal centers and
marginal zones with normal appearance and size (Fig. 6C–F).
4.4. Nitric oxide serum level estimation
Serum level of NO was significantly decreased in infected treated
mice groups. The percent of reduction was 83.7%, 82.4%, 80.9%,
74.9%, 70.5%, 67.3% and 52.7% for group VIII, V, IX, VII, IV, VI and
III, respectively and in consecutive manner) comparable to infected
control group (group II) (Table 2).
4.5. SEM study of T. gondii tachyzoites
By SEM, the T. gondii tachyzoites in peritoneal fluid of infected
control group showed a smooth crescent-shaped body with a smooth
surface. Conversely, those retrieved from AHC and CUP treated groups
showed deformities, distortion in their shape, irregularities, and surface
showed deep irregular ridges. Some tachyzoites were expanded as an
inflated balloon. Protrusions in the surfaces and distortion in the apical
region were also demonstrated in some tachyzoites (Fig. 7).
49
Experimental Parasitology 195 (2018) 44–53
5. Results of chronic murine toxoplasmosis model
5.1. Survival rate
At the end of the experiment (6 weeks from the start of therapy), the
survival rate for the infected control group (II) was 80%. While AHR
(200 mg/kg) and cotrimoxazole treated groups’ survival rates were 90%
and the survival rate of CUP (100 mg/kg) reached to 100% (Fig. 3B).
5.2. Determination of Toxoplasma cyst count and size in the brain
Using the ordinary light microscope, T. gondii cysts isolated from
infected untreated mice were rounded, circumscribed with intact wall
and full of bradyzoites. However, cysts of AHR and CUP treated groups
were smaller in size, rounded to oval in shape with irregular borders
and unclear content. The size of the cyst of the infected treated groups
is significantly reduced in comparison to infected control group.
Regarding cyst burden, there is a significant decrease in cyst count in
treated groups in comparison to infected control group (Table 3). Al-
though there was a significant reduction in cyst density especially in the
group (VIII) (98.02%) and even their absence in some mice, no group
achieved complete eradication of Toxoplasma cyst.
5.3. Histopathological study of the brain
Brain sections from infected untreated mice (group II) showed
perivascular mononuclear infiltration, a well-defined tissue cyst and
dilated congested blood vessels (Fig. 8B). The inflammatory infiltrate is
composed mainly of lymphocytes and histiocytes. In contrast, the
treated groups especially (V and VIII groups) showed a marked di-
minution of cyst number and even their absence in some instances. The
inflammatory process was milder, and tissue structure was preserved
(Fig. 8C and D).
5.4. Serum NO level estimation
Serum level of NO was estimated in all groups (Table 2) and a
significant reduction in its level was detected in all groups in compar-
ison to the infected control group. The percent of reduction was 69.5%,
59.3%, 58.6%, 54.6%, 46.4%, 41.7% and 33.1% for group VIII, V, IX,
VII, IV, VI and III, respectively and in consecutive descending manner
comparable to infected control group (II).
6. Discussion
T. gondii is an extreme zoonotic tissue cyst-forming protozoan
causing severe fatal disease, especially in pregnant females and im-
munocompromised individuals. So far, the currently available drugs
cannot eliminate the parasite completely, but only limit its replication
during the acute stage and once the parasite encysts in tissues, the drug
will lose its efficacy (Innes, 2010). Moreover, these drugs have severe
side effects and adverse drug reactions (Kaye, 2011). Therefore, novel
efficient therapies of low toxicities for toxoplasmosis are critically
needed that will be of great medical and veterinary importance (Tan
et al., 2011). This is the first study to determine the possible efficacy of
Araucaria heterophylla Salisb plant resin extract and its active compo-
nent (13-epi-cupressic acid) in the treatment of toxoplasmosis experi-
mental models using two T. gondii strains.
To develop AHR and CUP into a safe treatment therapy for tox-
oplasmosis, it should be non-toxic or less toxic to normal tissues. So
herein, the study experienced a preliminary study for possible toxicity.
Some parameters were tested in two groups given high doses of each
extract in comparison to control group. No significant difference was
observed including the survival rate, food consumption, mean body
weight, body weight gain and gross or microscopic histopathological
examination among the three groups. These toxicity studies
N.L. El-Tantawy et al. Experimental Parasitology 195 (2018) 44–53

Table 1
Study groups and number of mice in each.
Acute (n = 140) and chronic (n = 140) toxoplasmosis model

Groups Mice number Name Groups Mice number Name

Group I Uninfected control mice Group I Uninfected control mice

Group Ia 10 Group Ia 10
a a
Group Ib 10 Group Ib 10
Group II Infected untreated control Group II Infected untreated control
Group IIa 10 Group IIa 10
a a
Group IIb 10 Group IIb 10
Group III 10 Infected and treated with AHR (50 mg/kg/day) Group III 10 Infected and treated with AHR (50 mg/kg/day),
Group IV 10 Infected and treated with AHR (100 mg/kg/day) Group IV 10 Infected and treated with AHR (100 mg/kg/day)
Group V Infected and treated with AHR (200 mg/kg/day) Group V Infected and treated with AHR (200 mg/kg/day)
Group Va 10 Group Va 10
a a
Group Vb 10 Group Vb 10
Group VI 10 Infected and treated with CUP (25 mg/kg/day). Group VI 10 Infected and treated with CUP (25 mg/kg/day).
Group VII 10 Infected and treated with CUP (50 mg/kg/day). Group VII 10 Infected and treated with CUP (50 mg/kg/day).
Group VIII Infected and treated with CUP (100 mg/kg/day). Group VIII Infected and treated with CUP (100 mg/kg/day).
Group VIIIa 10 Group VIIIa 10
a a
Group VIIIb 10 Group VIIIb 10
Group IX Infected and treated by cotrimoxazole (Drug control) Group IX Infected and treated by cotrimoxazole (Drug control)
Group IXa 10 Group IXa 10
a a
Group IXb 10 Group IXb 10

a
b groups for survival rate estimation.

demonstrate that AHR and CUP are the quite safe treatment for tox-
oplasmosis in animal models.
Our results of in vivo studies of acute toxoplasmosis model showed
that treated mice with either AHR extract or its component (CUP) have
significant increase in the survival rates of mice, decrease in the mean
number of tachyzoites in peritoneal fluid and the liver impression
smears of treated mice, decrease in NO serum level and improvement of
the pathological changes caused by toxoplasmosis in the liver and
spleen in comparison to results of infected untreated and cotrimoxazole
treated mice. Accordingly, treatment with AHR extract and its com-
ponent (CUP) proved to be effective in controlling acute murine tox-
oplasmosis.
Herein, AHR (200 mg/kg) and its component CUP (100 mg/kg)
showed evident morphological alterations of the extracellular T. gondii
tachyzoites that were harvested from the peritoneal fluid of infected
treated mice, indicating their toxoplasmocidal effect on tachyzoites. By
SEM, these morphological changes include surface abnormalities as
irregularities, protrusions, and ridges comparable to infected untreated
controls. It was assumed that the changes in the shape of the parasite
could be secondary to changes resulting from interfering action of
treatment with DNA synthesis of the parasite or interference with folic
acid cycle (Gaafar et al., 2014). These morphological changes could
explain the loss of penetration power and reproductive capacity of the
treated tachyzoites which consequently led to the reduction in parasite
load (El Zawawy et al., 2015). Similarly, morphological changes were
described by (Eissa et al., 2015) who studied the effect of miltefosine
(an alkyl phospholipid) as a therapy for murine toxoplasmosis model
using SEM and (El Zawawy, 2008) who studied the therapeutic efficacy
of artesunate in the treatment of toxoplasmosis.
Efficacy of AHR and CUP component assessment against chronic
toxoplasmosis induced by Me49 Toxoplasma strain is based principally
on the comparative study of mean survival times of untreated and
treated mice, count and size of brain cysts, serum NO level and
histopathological changes in the brain. Treating mice with AHR and
CUP resulted in increased survival rate along the duration of the ex-
periment and showed a statistically significant reduction in the mean
number of brain cysts and decrease in its size in comparison to infected
untreated control and cotrimoxazole treated groups. Since Toxoplasma
bradyzoites are enclosed within a cyst wall protecting them from the
toxic action of the drugs, neither atovaquone, cotrimoxazole nor any
other drugs investigated as having toxoplasmicidal activity attained a
total elimination of cysts and killing of enclosed bradyzoites (Araujo
et al., 1996; Eissa et al., 2015). In addition, in the trial to discover a
novel alternative treatment for chronic toxoplasmosis, no one has
achieved complete clearance of Toxoplasma cysts (Barbosa et al., 2012;
Junior et al., 2016). But herein, this study achieved 98.02% reduction
in the brain cyst count, which is better than other studies. A 77.7%
reduction percent of the brain cyst in experimental mice was reported
by Eissa et al. (2012) when investigating miltefosine as a treatment of
toxoplasmosis. Fuentes-Castro et al. (2017) reported a reduction per-
centage of 31% in the parasite load of the mice group treated with
dialyzable leukocyte extracts. We suggest studying the effect of higher
doses of the extract could anticipate achieving a 100% reduction of
Toxoplasma cysts.
The cell-mediated host immune response is critical for controlling
acute toxoplasmosis infection and preventing reactivation of
Toxoplasma cysts. Interferon (IFN)-γ is the main cytokine in defense
against T. gondii. In mice, IFNe γ induced anti-Toxoplasma reaction is
mostly attributed to the production of NO by inducible nitric oxide
synthase (iNOS) expression (Pfaff et al., 2008). Macrophages which
mediate killing of T. gondii during acute infection are the main source of
NO production (Khan et al., 1997). Several studies have reported that
NO has direct anti-Toxoplasma effect and prompts the conversion be-
tween tachyzoite to bradyzoite stages (Ihara and Nishikawa, 2014;
Dincel and Atmaca, 2016). Moreover, a reduction in inducible iNOS
expression in chronic toxoplasmosis might be associated with

Table 2
Serum NO level in acute and chronic murine toxoplasmosis models in the studied groups.
No serum level Group II Group III Group IV Group V Group VI Group VII Group VIII Group IX F p

Acute model 7.91 ± 0.17 3.74 ± 0.3* 2.33 ± 0.11* 1.39 ± 0.13** 2.59 ± 0.09* 1.98 ± 0.11* 1.29 ± 0.09** 1.51 ± 0.1** 1983 < 0.001**
Chronic model 3.02 ± 0.11 2.02 ± 0.06* 1.76 ± 0.07* 1.23 ± 0.06* 1.62 ± 0.08* 1.37 ± 0.06* 0.92 ± 0.04** 1.25 ± 0.03* 852.8 < 0.001**

F: F test (one way ANOVA); * indicates the level of significance compared to the control positive group (p < 0.05).

50
N.L. El-Tantawy et al. Experimental Parasitology 195 (2018) 44–53

F: F test (one way ANOVA); * indicates the level of significance compared to the control positive group (p < 0.05); ●The percent of reduction was 98.02%, 96.05%, 94.07%, 93.4%, 91.22%, 85.08% and 9.21% for groups
Test of significance

P < 0.001*

P < 0.001*
F = 2984

F = 6734 22.41 ± 0.17*

300 ± 115.4
Group IX

17.92 ± 0.22*
90 ± 73.7
Group VIII

24.52 ± 0.16*
270 ± 94.8
Group VII

30.4 ± 0.36*
680 ± 78.8
Group VI
Toxoplasma cyst count and size in the brain of studied mice groups of chronic murine toxoplasmosis model.

20.65 ± 0.11*
180 ± 78.8
Group
V

27.15 ± 0.36*
400 ± 66.6
Group IV

VIII, V, VII, IX, IV, VI and III respectively in comparison to group II.
14140 ± 157.7

34.63 ± 0.18*

Fig. 7. Photomicrographs of SEM of Toxoplasma tachyzoites from infected un-

Group III

treated and infected treated groups A: Infected untreated tachyzoites showing

crescent shape smooth body BeF: AHR (200 mg/kg) and CUP (100 mg/kg)
infected and treated groups showing B: Protrusions, C: Ridges D: Ballooning, E:
Deformities in shape and F: Distortion of the apex.
Control positive II

reactivation in cerebral toxoplasmosis (Schluter et al., 1999). In this

4560 ± 171.2

36.51 ± 0.27

work, there is a significant decrease in NO serum level in both acute and

chronic toxoplasmosis in mice received AHR and CUP therapies which
denote that they have a toxoplasmocidal effect which diminishes the
systemic inflammatory responses and consequently decrease in NO
production. This finding has been previously repeated by Dincel and
Atmaca (2015, 2016) which showed an increase in NO production
Cyst size Mean ± SD

during T. gondii encephalitis in mice.

Mean ± SD
Cyst count●

7. Conclusion
Table 3

In conclusion, based on our experimental data, there is a promising

therapeutic effect of AHR and its component (CUP) in both acute and

51
N.L. El-Tantawy et al.
Fig. 8. The brain histopathological results, A: Brain section from uninfected
control mice group (I) showing normal anatomic structure (X400), B: brain
section from infected untreated control mice show inflammatory cells (black
arrows), gliosis and tissue T. gondii cysts with bradyzoites (arrowheads) (X400),
C: mice group treated with AHR (100 mg/kg) and CUP (50 mg/kg) showing
moderate inflammatory infiltrate and decreased number of cysts (arrowheads)
(X400), D: brain sections from mice groups treated with AHR (200 mg/kg) and
CUP (100 mg/kg) showing mild inflammatory infiltrates and no Toxoplasma
cysts (X400) and E: Figure showing the histopathological brain scoring of stu-
died groups with strikes denoting level of statistical significance.
chronic murine toxoplasmosis models that may improve the current
anti-Toxoplasma therapy. This work emphasized the potential of AHR
and CUP be used in the development of novel therapeutic drugs against
Toxoplasma infection. This will provide the basis for future research to
thoroughly elucidate the mechanism involved in the therapeutic re-
sponse of the parasite to this plant extract.
Declarations of interest
None.
References
Abdel-Sattar, E., Abdel Monemb, A.R., Ezzatb, S.M., El-Halawany, A.M., Mouneird, S.M.,
2009. Chemical and biological investigation of Araucaria heterophylla Salisb. Resin. Z.
Naturforsch. 64, 819–823.
Abdel Wahab, R.M., Morsy, T.A., Bahgat, A.B., Abdel Rahim, M.I., Eissa, M.H., Alfy, Y.E.,
1989. The histopathological picture of concomitant infection with Leishmania major
and Toxoplasma gondii in albino mice. J. Egypt. Soc. Parasitol. 1–13.
Arab-Mazar, Z., Kheirandish, F., Rajaeian, S., 2017. Anti-toxoplasmosis activity of herbal
medicines: narrative review. Herbal Medicines Journal 2 (1), 43–49 1.
Araujo, F., Suzuki, Y., Remington, J., 1996. Use of rifabutin in combination with atova-
quone, clindamycin, pyrimethamine, or sulfadiazine for treatment of toxoplasmic
encephalitis in mice. Eur. J. Clin. Microbiol. Infect. Dis. 15, 394–397.
Barbosa, B.F., Gomes, A.O., Ferro, E.A., Napolitano, D.R., Mineo, J.R., Silva, N.M., 2012.
Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo
experimental models. Vet. Parasitol. 8 (1–2), 44–52 187.
Batts, T., Ludwig, J., 1995. Chronic hepatitis. An update on terminology and reporting.
52
Experimental Parasitology 195 (2018) 44–53
Am. J. Surg. Pathol. 19, 1409–1419.
Caputo, R., Dovinola, V., Mangoni, L., 1974. New diterpenes from Araucaria cunninghami.
Phytochemistry 13, 475–478.
Caputo, R., Mangoni, L., 1974. Diterpenes from Araucaria bidwilli. Phytochemistry 13,
467–470.
De Oliveira, T.C., Silva, D.A., Rostkowska, C., Béla, S.R., Ferro, E.A., Magalhães, P.M.,
et al., 2009. Toxoplasma gondii: effects of Artemisia annua L. on susceptibility to
infection in experimental models in vitro and in vivo. Exp. Parasitol. 122 (3), 233–241.
Dincel, G.C., Atmaca, H.T., 2015. Nitric oxide production increases Toxoplasma gondii
encephalitis in mice. Exp. Parasitol. 156, 104–112.
Dincel, G.C., Atmaca, H.T., 2016. Role of oxidative stress in the pathophysiology of
Toxoplasma gondii infection. Int. J. Immunopathol. Pharmacol. 29, 226–240.
Djakovic, D., Milenkovic, T., 2001. Murine model of drug-induced reactivation of
Toxoplasma gondii. Acta Protozool. 40, 99–106.
Dupont, C.D., Christian, D.A., Hunter, C.A., 2012. Immune response and im-
munopathology during toxoplasmosis. Semin. Immunopathol. 34, 793–813.
Eissa, M.M., El-Azzouni, M.Z., Mady, R.F., Fathy, F.M., Baddour, N.M., 2012. Initial
characterization of an autoclaved Toxoplasma vaccine in mice. Exp. Parasitol. 131,
310–316.
Eissa, M.M., Barakat, A.M., Amer, E.I., Younis, L.K., 2015. Could miltefosine be used as a
therapy for toxoplasmosis? Exp. Parasitol. 157, 12–22.
El Zawawy, L.A., 2008. Effect of artesunate on Toxoplasma gondii: in vitro and in vivo
studies. J. Egypt. Soc. Parasitol. 38 (1), 185–201.
El-Zawawy, L., El-Said, D., Mossallam, S.F., Ramadan, H.S., Younis, S.S., 2015. Triclosan
and triclosan-loaded liposomal nanoparticles in the treatment of acute experimental
toxoplasmosis. Exp. Parasitol. 149, 54–64.
Emami, S.A., Asgary, S., Naderi, G.A., Ardekani, M.R., Aslani, S., Airin, A., et al., 2012.
Investigation of antioxidant and anti-glycation properties of essential oils from fruits
and branchlets of Juniperus oblonga. Rev. Bras. Farmacogn. 22 (5), 985–993.
Fuentes-Castro, B.E., Reyes-García, J.G., Valenzuela-Vargas, M.T., Martínez-Gómez, F.,
2017. Histopathology of murine toxoplasmosis under treatment with dialyzable
leukocyte extract. Mem. Inst. Oswaldo Cruz 112 (11), 741–747.
Furtado, J.M., Smith, J.R., Belfort, R., Gattey, D., Winthrop, K.L., 2011. Toxoplasmosis: a
global threat. J. Global Infect. Dis. 3 (3), 281–284.
Gaafar, M.R., Mady, R.F., Diab, R.G., Shalaby, T.I., 2014. Chitosan and silver nano-
particles: promising anti-Toxoplasma agents. Exp. Parasitol. 143, 30–38.
Gad, S.C., Cassidy, C.D., Aubert, N., Spainhour, B., Robbe, H., 2006. Nonclinical vehicle
use in studies by multiple routes in multipler. Int. J. Technoethics (IJT) 25, 499–521.
Hayat, M.A., 2000. Principles and Techniques of Electron Microscopy: Biological
Applications. VNR Company, New York, USA.
Ihara, F., Nishikawa, Y., 2014. Starvation of low-density lipoprotein-derived cholesterol
induces bradyzoite conversion in Toxoplasma gondii. Parasites Vectors 7, 248.
Innes, E.A., 2010. Vaccination against Toxoplasma gondii: an increasing priority for col-
laborative research? Expert Rev. Vaccines 9, 1117–1119.
Junior, G.A., Cosmo, M.L., Reis Mde, P., Dos Santos, P.S., Gonçalves, D.D., Gasparotto,
F.M., Navarro, I.T., Lourenço, E.L., 2016. Effects of extracts from Echinacea purpurea
(L) MOENCH on mice infected with different strains of Toxoplasma gondii. Parasitol.
Res. 115 (10), 3999–4005.
Kaye, A., 2011. Toxoplasmosis: diagnosis, treatment, and prevention in congenitally ex-
posed infants. J. Pediatr. Health Care 25, 355–364.
Kavitha, N., Noordin, R., Chan, K., Sasidharan, S., 2012. In vitro anti-Toxoplasma gondii
activity of root extract/fractions of Eurycoma longifolia jack. BMC Complement
Altern. Med. 12, 91.
Khan, I.A., Schwartzman, J.D., Matsuura, T., Kasper, L.H., 1997. A dichotomous role for
nitric oxide during acute Toxoplasma gondii infection in mice. Proc. Natl. Acad. Sci.
U.S.A. 94, 13955–13960.
Kheirandish, F., Chegeni, R., Delfan, B., Jabari, M., Ebrahimzadeh, F., Rashidipour, M.,
2016. The cytotoxic and antileishmanial effects of satureja khuzestanica essential oil.
Herbal Med. J. 1, 11–17.
Kim, K., Weiss, L.M., 2004. Toxoplasma gondii: the model apicomplexan. Int. J. Parasitol.
34, 423–432.
Lang, C., Grob, U., Luder, C.G.K., 2007. Subversion of innate and adaptative immune
responses by Toxoplasma gondii. Parasitol. Res. 100, 191–203.
Langenheim, J.H., 2003. Plant Resins: Chemistry, Evolution, Ecology, and Ethnobotany.
Published by Timber Press, Inc. The Haseltine Building 133 S.W. Second Avenue,
Suite 450 Portland, Oregon 97204, U.S.A.
Mcleod, R., Frenkel, J.K., Estes, R.G., Mack, D.G., Eisenhanuer, P.B., Gibori, G., 1988.
Subcutaneous and intestinal vaccination with tachyzoites of Toxoplasma gondii and
acquisition of immunity to per oral and congenital Toxoplasma challenge. J.
Immunol. 140, 1632–1637.
McLeod, R., Lykins, J., Gwendolyn Noble, A., Rabiah, P., Swisher, C., Heydemann, P.,
et al., 2014. Management of congenital toxoplasmosis. Curr. Pediatr. Rep. 2,
166–194.
Montgomery, H.A.C., Dymock, J., 1961. The determination of nitrite in water. Analyst 86,
414–416.
Nair, A.B., Jacob, S., 2016. A simple practice guide for dose conversion between animals
and human. J. Basic Clin. Pharm. 7 (2), 27–31.
National Research Council (US), 2011. Committee for the Update of the Guide for the
Care and Use of Laboratory Animals (2011) Guide for the Care and Use of Laboratory
Animals, eighth ed. National Academies Press, Washington (DC).
Pfaff, A.W., Mousli, M., Sénégas, A., et al., 2008. Impact of foetus and mother on IFN-γ-
induced indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression
in murine placenta following Toxoplasma gondii infection. Int. J. Parasitol. 38,
249–258.
Schluter, D., Deckert-Schluter, M., Lorenz, E., Meyer, T., Rollinghoff, M., Bogdan, C.,
1999. Inhibition of inducible nitric oxide synthase exacerbates chronic
N.L. El-Tantawy et al.
cerebraltoxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does no-
treactivate the latent disease in T. gondii-resistant BALB/c mice. J. Immunol. 162,
3512–3518.
Silva, N.M., Manzan, R.M., Carneiro, W.P., Milanezi, C.M., Silva, J.S., Ferro, E.A.V.,
Mineo, J.R., 2010. Toxoplasma gondii: the severity of toxoplasmic encephalitis in
C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the
central nervous system and higher blood–brain barrier permeability. Exp. Parasitol.
53
Experimental Parasitology 195 (2018) 44–53
126, 167–177.
Siheri, W., Igoli, J.O., Gray, A.I., Nasciemento, T.G., Zhang, T., Fearnley, J., Clements,
C.J., Carter, K.C., Carruthers, J., Edrada-Ebel, R., Watson, D.G., 2014. The isolation of
antiprotozoal compounds from Libyan propolis. J. Phytother. Res. 28, 1756–1760.
Tan, F., Hu, X., Luo, F.J., Pan, C.W., Chen, X., 2011. Induction of protective Th1 immune
responses in mice by vaccination with recombinant T. gondii nucleoside triphosphate
hydrolase-II. Vaccine 29, 2742–2748.