Experimental Parasitology 121 (2009) 279–287

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

Toxoplasma gondii: Effect of maternal infection in the development

of lymphoid organs of BALB/c neonates
María Asunción Cabañas-Cortés a, Elba Reyes-Maldonado b, Laura Montiel-Cervantes b,c,
María Lilia Domínguez-López a, Luis Jiménez-Zamudio a, Ethel García-Latorre a,*
a
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, IPN. Prol. Carpio y Plan de Ayala s/n, Col. Plutarco Elías Calles Casco de Santo Tomás,
CP 11340 Mexico D.F., Mexico
b
Departamento de Morfología, Escuela Nacional de Ciencias Biológicas, IPN. Prol. Carpio y Plan de Ayala s/n, Col. Plutarco Elías Calles Casco de Santo Tomás,
CP 11340 Mexico D.F., Mexico
c
Hospital de Especialidades, Centro Médico Nacional La Raza, ‘‘UMAE Antonio Fraga Mouret”, IMSS, Seris y Zaachila s/n, Colonia La Raza, CP 02990 Mexico D.F., Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Toxoplasmosis is one of the worldwide parasitic zoonoses. Alterations in the lymphopoietic system are
Received 14 June 2008 still poorly studied. We analyzed lymphoid organs of BALB/c mice neonates from Toxoplasma gondii-intra-
Received in revised form 11 November 2008 peritoneally-infected mothers on 19th day of gestation, with 30 tachyzoites of strain RH. Normal non-
Accepted 28 November 2008
infected pregnant females were used as controls. At 7 days after birth, animals were classified as neonates
Available online 10 December 2008
from infected (NIM) and neonates from non-infected mothers (NNIM). Weight of the thymus and number
of thymic cells in NIM were decreased, percentage of apoptosis was significantly increased. Decrease in
Keywords:
lymphocytes and monocytes and an increase of plasma cells were observed in bone marrow of NIM.
Toxoplasma gondii
Maternal infection
Peripheral blood of NIM showed an increase of monocytes and neutrophils and a decrease in lympho-
Thymus cytes. Infection of the mother during the last day of gestation provokes in the neonates changes in the
Bone marrow lymphoid organs that could explain survival of 75% of them.
Neonates Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction About 10% of prenatal infection results in abortion or neonatal

Toxoplasma gondii (T. gondii) is an obligate protozoan parasite
responsible for both severe congenital birth defects and fatal toxo-
plasma encephalitis in immunocompromised hosts. Toxoplasmosis
is one of the more common parasitic zoonoses worldwide. Preg-
nant women infected with T. gondii for the first time can transmit
the infection to their fetuses across the placenta. The risk of con-
genital disease is lowest (10–25%) when acute maternal infection
occurs during the first trimester and highest (60–90%) when it oc-
curs during the third trimester (Jones et al., 2001). The mechanism
of transmission is not yet understood. A probable scenario is that
temporary parasitaemia in primarily infected pregnant woman
may result in invasion of the placenta by tachyzoites that then
multiply within placenta cells. Eventually, some of these tachyzo-
ites may cross the placenta and enter the fetal circulation or fetal
tissue (Barragan and Sibley, 2003).
Congenital toxoplasmosis may cause abortion, neonatal death,
or fetal abnormalities with detrimental consequences for the fetus
(Montoya and Liesenfeld, 2004). It may also significantly reduce
the quality of life of children who survive a prenatal infection.
* Corresponding author. Fax: +52 55 53423331.
E-mail address: ethelagarcia@hotmail.com (E. García-Latorre).
death. Another 10–23% of prenatally infected newborns show clin-
ical signs of toxoplasmosis at birth. Signs of the classic triad of
toxoplasmosis (retinochoroiditis, intracranial calcifications and
hydrocephalus) manifest in up to 10% of these newborns, while
the others show a variety of symptoms. The surviving infants suffer
from progressive mental retardation or other neurological defi-
ciencies that often require special education and residential care
(Tenter et al., 2000).
Mice have been found to be the most susceptible animal to
T. gondii infection and are a particularly interesting model to
study congenital infection (Freyre et al., 2006). In one model
BALB/c mice were infected with 5 cysts (Beverley strain) of
T. gondii eight weeks before mating, and on day 20 of pregnancy
they were re-infected with 20 cysts. Other mice received 20
cysts on day 12 of pregnancy only. Females infected eight weeks
before mating did not give birth to infected litters, even if they
were re-infected on day 12 of pregnancy. Mice that were in-
fected for the first time on day 12 of pregnancy approximately
50% of their offspring were infected. This study clearly demon-
strates that chronically infected BALB/c mice do not allow verti-
cal disease transmission and congenital infection in these mice
only occurs when the mother was infected for the first time dur-
ing pregnancy (Roberts and Alexander, 1992). In another similar

0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2008.11.015
280 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287
study, BALB/c mice were infected with the P strain of T. gondii
eight weeks prior to mating, after 60 days of infection, the fe-
males showed signs of uterine and ovarian atrophy, probably
resulting in reproductive failures, and only 2 of 49 female mice
gave birth (Fux et al., 2000). On the other hand, Roberts and Alex-
ander (1992) did not recorded reproductive difficulties with this
model. An important problem of many of these experiments is
that infection was performed with tissue cysts, variable amounts
of bradyzoites per cyst may be produced, so the initial parasite
load could significantly differ among and even within animal
groups. Other studies focus on defining the factors influencing
the outcome of infection with either a highly lethal RH strain
or a low lethal strain, ME49. Gavrilescu and Denkers (2001) com-
pared the immune response of animals undergoing acute infec-
tion with these high and low virulence parasite strains, they
suggested that virulence of a specific T. gondii strain relates to
the inherent ability of the tachyzoite stage to rapidly replicate
and disseminate, and to the immune response subsequently gen-
erated within the host. Infection of pregnant NYLAR, C57BL/6J
and BALB/c mice with T. gondii intraperitoneally, on day 7 of ges-
tation, resulted in fetal resorption, abortions and still-birth. Post-
natal pups were cachectic and growth-retarded, with some
developing hind limb weakness. A low percentage of pups sur-
vived. At necropsy, the histopathological study showed severe
damage in the organs (liver, spleen, kidney and lung). The path-
ogenesis of congenital toxoplasmosis has been characterized by
deregulation of homeostasis, perfusion failure, and multiple organ
dysfunctions (Stahl et al., 2004). A soluble tachyzoite antigen
(TSAg) entrapped within non-ionic surfactant vesicles (NISV)
can induce sufficient protection in female BALB/c mice, such that,
when they are infected with T. gondii during pregnancy, there is
no fetal death and congenital infection is significantly curtailed
(Roberts et al., 1994). These studies could lead to the design of
a vaccine using the appropriate antigen and adjuvant
preparations.
As shown above several aspects of toxoplasmosis have been
studied by different authors, but alterations in the lymphoid or-
gans and the contribution to disease are still poorly studied. It is
not known if in the neonatal stage the lymphoid organs are able
to control the disease or not. Lymphoid organs are organized tis-
sues containing large numbers of lymphocytes in a framework of
non-lymphoid cells. In these organs, interaction of lymphocytes
with non-lymphoid cells is important either for lymphocyte devel-
opment or for initiation of adaptive immune responses. Lymphoid
organs can be divided into central lymphoid organs, where lym-
phocytes are generated and differentiate into mature lymphocytes,
and peripheral lymphoid organs, where adaptive immune re-
sponses are initiated and where lymphocytes are maintained. Cen-
tral lymphoid organs are the bone marrow for B cells, and the
thymus for T cells. Peripheral organs comprise the spleen that col-
lects antigen from the bloodstream, the lymph nodes that receive
lymphocytes from lymph and the mucosal associated lymphocytes.
Once B and T lymphocytes have completed their maturation, both
enter the bloodstream, from which they migrate to the peripheral
lymphoid organs, returning to the bloodstream through the lym-
phatic vessels. Adaptive immune responses are initiated in periph-
eral lymphoid tissues: T cells that encounter antigen proliferate
and differentiate into antigen-specific effector cells, while B cells
proliferate and differentiate into antibody-secreting cells (Chaplin,
2003; Cyter, 2003; Crivellato et al., 2004).
In our laboratory we have designed an experimental model of
infection of pregnant BALB/c mice with 30 tachyzoites of the RH
strain on day 19 of gestation. This model allowed us to study the
lymphoid organs in the neonatal stage and correlate them with
qualitative changes in immune cells.
2. Material and methods
2.1. Mice and parasite
Four to six weeks old BALB/c mice were obtained from Harlan,
Teklan, USA and were allowed food and water ad libitum. All proce-
dures for care and use of experimental animals were performed
according to official Mexican norm NOM-062-ZOO-199 entitled
‘‘Technical specifications for the production, care and use of labora-
tory animals”.
BALB/c mice were inoculated with T. gondii RH strain. Tachyzo-
ites were recovered from the peritoneal cavity 72 h later by instill-
ing 4 ml of sterile 0.9% NaCl solution. The material was passed
three times through a 26 gauge needle, washed by centrifugation
twice at 200g for 10 min. Pellets were resuspended and washed
with sterile 0.9% NaCl solution. Tachyzoites were counted in a Neu-
bauer’s chamber and viability was tested with the Trypan blue
exclusion test (0.4% solution) and only samples with 95% or more
viable parasites were used and prepared at a concentration of
107 tachyzoites/mL, and diluted 10-fold in sterile 0.9% NaCl solu-
tion to obtain a concentration of 30 tachyzoites/mL.
2.2. 2. Experimental design
BALB/c mice weighing 22 g each were used for the model, one
male and two females were housed per cage, overnight, for mating.
The presence of vaginal plugs in the females was checked the fol-
lowing morning. The day of plug detection was considered as day
0 of gestation. Pregnant females thus identified were removed
from the mating cages. Each experiment consisted of a number
of pregnant females infected i.p. on day 19 of gestation with 30
tachyzoites of T. gondii and the same number injected with
0.5 mL sterile 0.9% NaCl solution as non-infected controls. Two
days after inoculation of parasites or saline solution offspring were
born. Newborns were enumerated at birth and visually examined
for size and appearance. At day 7 after birth, neonates were
weighted and classified as neonates from infected mothers and
neonates from non-infected mothers. All mothers were euthanized
at day 7 after birth and a visual examination of the uterus was
done. A total of four experiments were carried out to test for differ-
ent variables in 7 days old neonates. Table 1 show the experimen-
tal design used.
2.3. Isolation of thymocytes
Thymus from 30 neonates from infected mothers and 30 neo-
nates from non-infected mothers were aseptically isolated and
mechanically disaggregated. Freshly isolated cells were placed in
cold RPMI 1640 medium, supplemented with 10% FCS, 2 mM L-glu-
tamine, 0.5% non-essential amino acids, 0.5% sodium pyruvate,
200 U/mL of penicillin and 100 lg/mL of streptomycin obtained
from Gibco Invitrogen, USA. Cell suspension was washed, filtered,
and erythrocytes lysed by addition of an isotonic solution of ammo-
nium chloride warmed to room temperature. Cell numbers and via-
bility were determined by counting on a Neubauer’s chamber using
the Trypan blue dye-exclusion test. Final adjustment to a concentra-
tion of 1  107 cells/mL was done with supplemented RPMI 1640
medium. Aliquots of 1 mL of thymocytes (1  106 cells/mL) were
used for apoptosis analysis.
2.4. Analysis of apoptosis by flow cytometry
Thymic cells suspensions were analyzed by flow cytometry for
their DNA content as previously described by Nicoletti et al.
(1991), Telford et al. (1994). The 200g centrifuged cell pellet was
 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287
Table 1
Experimental design.
Experiment Variables studied
1 Numbers of neonates, body weight, thymic index, numbers of thymic cells and apoptosis
2 Bone marrow cell populations
3 Peripheral blood populations
4 Detection of Toxoplasma gondii by PCR
fixed in 1 mL cold 70% ethanol at 4 °C for 60 min. Cells were then
centrifuged, wash in 2 mL PBS and resuspended in 0.5 mL PBS.
For detection of cells containing sub diploid DNA, fixed permeabi-
lized cells were stained with propidium iodide (PI, Sigma, St. Louis,
MO, USA, 1 mg/mL) and RNAse (Type H Invitrogen, USA, 0.73 mg/
mL). Stained samples were analyzed in a FACScalibur cytofluorom-
eter (Becton Dickinson, San Jose, CA, USA) using Cell QuestTM Pro
program, version 5.2.1, and 10,000 events per sample were re-
corded. Analysis of apoptosis was performed by determination of
sub-G0 peak. Results were expressed as the percentage of apopto-
tic cells.
2.5. Bone marrow smears of neonates
Bone marrow analyses were carried out in 20 neonates from
infected mothers and 20 neonates from non-infected mothers. Fol-
lowing general anesthesia and euthanasia, femurs were immedi-
ately removed and excess muscle and fat trimmed. The bone
marrow was exposed and a small plug was gently extracted and
smeared onto glass slides. Slides were allowed to air dry and then
stained with Wright–Giemsa. Evaluation of cellularity and the
presence of bone marrow fat and megakaryocytes were assessed
using a 10 magnification objective. Evaluation of the proportion
and morphologic features of hematopoietic cellularity was as-
sessed with a 100 magnification immersion objective. Differen-
tial cell counts were done on 250–500 cells and morphological
differences were recorded (Travlos, 2006; Everds, 2004).
2.6. Differential peripheral blood cell counts in neonates
Blood smears for differential cell counts were made for each
mouse at the time of blood collection according to the method de-
scribed by Everds (2004). A single drop of blood formed at the end
of a 25 gauge needle attached to the syringe was used to make the
smear. Smears were allowed to air dry before staining. All slides
were stained with Wright–Giemsa. A total of 100 cells were
counted per smear and each type of cell was expressed as percent-
age of total cell count. Leucocytes were classified by morphology
into segmented and non-segmented neutrophils, lymphocytes,
monocytes, eosinophils and basophils.
2.7. Detection of T. gondii by PCR
The B1 gene of T. gondii is a 35-fold repetitive gene, with 2214
nucleotides in each repeat, and is highly conserved among Toxo-
plasma strains, thus making it a suitable target for detection by
PCR (Burg et al., 1989). Primers were designed and tested for spec-
ificity in the BLASTN data base and were predicted to be 100% spe-
cific for the B1 gene. Primers were: Tg1 (50 -AAAAATGTGGGAA
TGAAAGAG-30 ) and Tg2 (50 -ACGAATCAACGGAACTGTAAT-30 ). PCR
with these primers amplified a 469 bp DNA fragment of the B1
gene. These primers were used for detection of congenital
infection.
The detection of T. gondii DNA in spleen and peritoneal cavity
fluid was performed on 2 infected mothers and 2 non-infected
281
Pregnant mice n Neonates n
Non-infected Infected Non-Infected Infected
5 5 30 30
4 4 20 20
2 2 10 10
2 2 10 10
mothers. Parasite DNA was detected in liver and spleen of 10 neo-
nates from infected mothers and 10 neonates from non-infected
mothers. Fragments of liver and spleen and 1 mL of peritoneal cav-
ity fluid were stored at 20 °C before the extraction of DNA.
Approximately 25–30 mg of each organ was cut into small pieces
and homogenized in 200 lL of TE buffer (10 mM Tris, 1 mM EDTA,
and pH 8.0). The homogenized sample was transferred to a micro-
tube and DNA extraction was performed using Genomic DNA puri-
fication Kit, (Fermentas Life Sciences) in accordance with the
manufacturer’s instructions.
Following the protocol for PCR (Edvinsson et al., 2004) 1.5 mM
MgCl2, and 1 U of Taq DNA polymerase (Invitrogen, USA) per 25 ll
reactions were prepared; 2 lL de of template DNA was used in
each reaction. The reaction mixture was incubated for 10 min at
95 °C for initial denaturation followed by 40 cycles of 1 min at
94 °C, 30 s at 52 °C, and 1 min at 72 °C. A final extension step
was run at 72 °C for 7 min. PCR products (10 lL) and DNA base-pair
marker (100 bp ladder, Invitrogen, USA) were analyzed simulta-
neously by electrophoresis in 1.8% w/v agarose gel (Invitrogen,
USA) and amplified fragments of 469 bp were visualized under
UV illumination after staining with ethidium bromide for 10 min.
A positive control (DNA extracted from 14  106 tachyzoites)
and negative infection controls (DNA extracted from non-infected
mice) were tested in each experiment.
2.8. Statistical analysis
Differences between the groups were analyzed by the paramet-
ric t-Student test or the nonparametric Mann–Whitney U-test
using the Sigma Stat 2.03 program for Windows. The level of statis-
tical significance was p < 0.05.
3. Results
This experimental model was designed to study the effect of
maternal infection with T. gondii on the surviving neonates. In
experiment 1, neonates from 5 infected and 5 non-infected moth-
ers were studied. Litter size in infected mothers was about 8 pups
at birth but only 6 survived after one week. In contrast, in controls,
litter size was about 10 pups and all of them survived by day 7
(Fig. 1A). At birth offspring of infected mothers were very small
and congested compared to offspring of non-infected mothers
(data not shown). The uterus from infected mothers euthanized
at day 7 after birth showed uterine atrophy compared to the uterus
of non-infected mothers (data not shown). Live pups were weighed
when they were 7 days old (Fig. 1B). Data show that 30 neonates
from mothers infected intraperitoneally with T. gondii weighed less
than 30 neonates controls (2.9 g ± 0.52 SE, 6.0 g ± 0.15 SE, respec-
tively). Body weight of neonates from infected mothers was de-
creased by 52%. The average thymus weight in 30 neonates from
infected mothers was 0.0138 g ± 0.0008 SE compared to the weight
of the thymus in 30 neonates from non-infected mothers,
0.0417 g ± 0.0021 SE, (Fig. 2A). Thymus weights were also mark-
edly reduced in relation to body weights (thymic index) in 30 neo-
nates from infected mothers, 0.0048 g ± 0.0002 SE compared with
282 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287
Fig. 1. Numbers and body weight of neonates. (A) Numbers of neonates at birth per
mother. (B) Body weight of neonates recorded when they were 7 days old. Data in
(A and B) represent means ± SE from 30 neonates from 5 non-infected mothers and
30 from 5 infected mothers. Student’s t-test was used *p < 0.001.
30 neonates from non-infected mothers, 0.0068 g ± 0.0002 SE
(Fig. 2B). Reduction in the number of thymocytes was evident in
the thymus of neonates from infected mothers, 1.35  107 cells
± 0.22 SE compared with thymus of neonates from non-infected
mothers, 3.1  107 cells ± 0.18 SE (Fig. 2C). Frequency of spontane-
ous apoptotic cells in thymus of 30 neonates from infected mothers
showed a five times increase (2.9% ± 0.23 SD) compared to cells
from the thymus (0.57% ± 0.03 SD) of 30 neonates from non-in-
fected mothers (Fig. 2D).
In experiment 2, bone marrow of 20 neonates from infected and
20 from non-infected mothers were analyzed. Neonates from non-
infected mothers showed normal cellularity, cells were evenly dis-
persed together with adipocytes. Megakaryocytes and neutrophils
with their characteristic ring-form nucleus were observed (Fig. 3A
and B). Bone marrow of neonates from infected mothers showed a
high number of plasma cells, few adipocytes and normal numbers
of megakaryocytes and neutrophils (Fig. 3C). Plasma cells charac-
teristic appearance; with their basophil cytoplasm and eccentric
nucleus are seen (Fig. 3D). Differential counts of bone marrow cells
of neonates from both groups are shown in Fig. 4. A significant de-
crease in lymphocytes (3.16% ± 0.56 SD) of neonates from infected
mothers compared to neonates form non-infected mothers was ob-
served (9.6% ± 0.98 SD). On the other hand, the proportion of plas-
ma cells was significantly increased in neonates from infected
mothers (9.8% ± 0.80 SD) compared to neonates from non-infected
mothers (0.91% ± 0.21 SD). Values of the cellular composition of
the myeloid cells in bone marrow are shown (Table 2). Monocytes
of the neonates from infected mothers were significantly decreased
by half. There were no significant changes in the megakaryocytes,
neutrophils and erythroblast lineage.
In experiment 3, blood smears of 10 neonates from infected
mothers and 10 from non-infected mothers were studied.
Fig. 5A–D show monocytes with vacuoles and granules and neutro-
phils surrounded by small pieces of parasites in neonates from in-
fected mothers. Blood smears of neonates from non-infected
mothers did not show these changes, (Fig. 5E and F). Differential
cells counts also showed changes in lymphocytes, monocytes and
neutrophils (Fig. 6). Number of lymphocytes on differential smears
decreased in neonates from infected mothers, 31.5% ± 6.6 SD com-
pared to neonates from non-infected mothers, 62.5% ± 3.1 SD. Per-
centage of monocytes, 18.08% ± 5.2 SD and neutrophils, 49.58% ±
8.6 SD were significantly increased in neonates from infected
mothers compared to neonates from non-infected mothers,
10.18% ± 8.6 SD, 26.71% ± 7.8 SD, respectively. No basophils or
eosinophils were found on differential smears, (data not shown).
In experiment 4, parasites DNA in the liver and spleen of 10
neonates from infected mothers and 10 from non-infected mothers
were examined by PCR. Spleen and peritoneal cavity of 2 infected
and 2 non-infected mothers were analyzed for parasites DNA. In
a representative experiment shown in Fig. 7, parasite DNA was
not detected in non-infected mothers but it was found in the
spleen and peritoneal cavity of infected mothers (Fig. 7A), parasite
DNA was not detected in liver and spleen of neonates from infected
mothers (Fig. 7B and C).
4. Discussion
Little attention has been directed toward the alteration pro-
duced in the lymphoid organs associated with immune function
in neonates of mothers’ infected with the parasite. The experi-
mental model presented here was designed to study the effect
of maternal infection with T. gondii on the surviving neonates
at birth and at 7 days old, after inoculation of the mother on
day 19 of gestation with 30 tachyzoites. Infected mothers were
sick when they gave birth and continue to be sick on day 7
when they were euthanized. In our model 80% neonates from in-
fected mothers were born (8/10), but they were small and con-
gested. Besides, not all of these neonates survived; about 75%
(6/8) were alive by one week of age. In comparison with con-
trols, growth was significantly retarded in neonates from in-
fected mothers at day 7 after birth. They were small at birth
and continue to be smaller than controls at 7 days old. This
could be due to two possibilities placental atrophy and postnatal
malnutrition due to sickness of the mothers during the first
week of life. We observed that the uteri from infected mothers
at day 7 after giving birth were atrophied compared to non-in-
fected mothers. Similar to the human fetus, the neonates from
infected mothers manifested a marked low weight at birth;
probably caused by intrauterine growth retardation (IUGR). IUGR
is a major cause of morbidity and mortality in the perinatal per-
iod in humans. Placental damage is probably the primary cause
of fetal death. Although T. gondii causes pathological damage in
placenta, that is likely to impair placental function and probably
also fetal nutrition, the exact mechanism(s) affecting fetal
growth in association with placental toxoplasmosis are unknown
(Abbasi et al., 2003; Shiono et al., 2007).
Exposure of the fetus to T. gondii during this important develop-
mental period might alter and/or destroy the maturing lymphoid
organs, resulting in structural and functional anomalies of the in-
fant’s immune system (Huldt et al., 1973; Maródi, 2006; Petrova
and Mehta, 2007). Thymus is a primary lymphoid organ, able to
generate mature T cells that eventually colonize secondary lym-
phoid organs, and it is essential for peripheral T cell renewal (Cri-
vellato et al., 2004; Pearse, 2006). During the normal life span, this
organ undergoes atrophy in situations such as pregnancy, aging
and in the presence of a wide variety of infectious diseases (Elmore,
2006; Savino, 2006; Savino et al., 2007; Verinaud et al., 2004). In
our study, thymus from neonates from Toxoplasma gondii-infected
mothers showed clear acute atrophy expressed by loss of absolute
mass and reduction in the organ cellularity. The loss of thymocytes
appeared mediated, at least in part, by apoptosis or by a reduction
in the number of precursors migrating from the bone marrow to
the thymus. Percentage of apoptotic cells were low in the thymus
of neonates from non-infected mother’s maybe because they are
normally rapidly eliminated by phagocytes in vivo (Xu and Shi,
2007). Several mechanisms, including secretion of different medi-
ators could be responsible for apoptosis. One major pathway is re-
lated to the rise in glucocorticoid hormone levels in the blood. In
our experiments, the mother infection induces stress during the
last days of pregnancy, which could lead to elevated levels of glu-
 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287 283

Fig. 2. Effect of the mother infection on the thymus development of 7 days old neonates. (A) Thymic weight, (B) Thymic index, (C) Total number of thymic cells. Data
represent mean ± SE of 30 neonates from 5 non-infected and 30 neonates from 5 infected mothers. Student’s t-test was used *p < 0.001. (D) A typical histogram showing
percentages of cells falling within the apoptotic region is shown. Data represent median of 30 neonates from 5 non-infected and 5 infected mothers. The Mann–Whitney
U-test was used *p < 0.001.

cocorticoids (Knackstedt et al., 2005). Glucocorticoids within
maternal circulation will be inactivated at the placenta, which acts
as a feto-placental barrier to maternal glucocorticoids; however,
approximately 10–20% of the maternal glucocorticoids will pass
into the fetal system. Such steroids can trigger apoptosis in thymo-
cytes. Another mechanism could be the accidental consequence of
the inflammatory cytokine response to infection. Apoptosis result-
ing from inflammatory cytokine overproduction is associated with
high-virulence T. gondii strains (Gavrilescu and Denkers, 2001;
Shen et al., 2001).
Bone marrow is the major hematopoietic organ, and a primary
lymphoid tissue, responsible for the production of erythrocytes,
granulocytes, monocytes, lymphocytes and platelets. For hemato-
poiesis to occur it must be supported by a microenvironment that
is able to recognize and retain hematopoietic stem cells and pro-
vide the factors (e.g., cytokines) required to support proliferation,
differentiation and maturation of stem cells along committed lin-
eages (Travlos, 2006). Bone marrow is also sensitive to external
influences and can become suppressed in response to dietary
restriction, malnutrition, chronic inflammation, infections and sys-
284 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287

Fig. 3. Bone marrow smears. (A and B) Bone marrow smears from 7 days old neonates from non-infected mothers. An even dispersal of hematopoietic cells mixed with
adipocytes is observed. Typical mice big megakaryocytes (Meg) surrounded by neutrophils (N) with a ring-form nucleus is observed. (C and D) Bone marrow smears from 7
days old neonates from infected mothers. An increased numbers of plasma cells are observed. (D) Plasma cells have characteristic nuclei and abundant cytoplasm containing
dense rough endoplasmic reticulum (arrows). 100 immersion objective, Wright–Giemsa stain. Images shown are representative of 20 neonates from 4 non-infected mothers
and 20 neonates from 4 infected mothers.

mal numbers of megakaryocytes and neutrophils. Petakov et al.

Fig. 4. Lymphoid cells composition of bone marrow from 7 days old neonates. Data
represent means ± SD of 20 neonates from 4 non-infected mothers and 20 neonates
from 4 infected mothers. The Mann–Whitney U-test was used *p < 0.001.
temic diseases (Diebold et al., 2000). Bone marrow smears can be
evaluated for presence or absence of cell types, relative shifts in
cell populations, and alterations in maturation sequences and mor-
phologic changes in bone marrow cells (Everds, 2004; Diebold
et al., 2000). In our experiments, bone marrow of neonates from in-
fected mothers showed a high number of plasma cells, decreased
numbers of lymphocytes and monocytes, few adipocytes and nor-
(2002) showed that in acute infection of adult mice with T. gondii
(strain RH) bone marrow alterations were characterized by a de-
crease in the total number of nucleated cells. Acute T. gondii infec-
tion during gestation may disrupt the maternal-fetal
immunological balance for anti-parasitic pro-inflammatory cyto-
kines such as IFN-c, IL-12 and TFN-a, (Shiono et al., 2007). The vir-
ulent strain RH induces in BALB/c mice overproduction of IL-12 and
IFN-c (Mordue et al., 2001; Nguyen et al., 2003). The increase of
plasma cells observed by us in the bone marrow could be provoked
by the secondary action of IFN-c that the infected mothers secrete
that in turn, could activate the differentiation and proliferation of B
cells in bone marrow from their neonates. Macrophages, mast cells
and trophoblast are an important source of IL-6 at the feto-mater-
nal interface (Zenclussen et al., 2003). IFN-c and IL-6 are powerful
drives for clonal proliferation and expansion of the activated B cell
population. In neonates from non-infected mothers we found the
percentage of lymphocytes and plasma cells of bone marrow to
be approximately of 10% and 1%, respectively. Other authors have
made similar observations with respect to healthy mice and rats
(Bolliger, 2004; Sanderson and Philips, 1981). Results obtained in
primary lymphoid organs led us to look for changes in peripheral
blood.
Acute toxoplasmosis is characterized by dissemination and
intracellular growth of tachyzoites in a variety of host organs. In
the innate phase of the immune response, nonspecific inflamma-
tory cells, including neutrophils, monocytes and dendritic cells
are attracted to the site of infection. Defense against infection, espe-
cially in neonates with no immunological memory and low ability
to develop specific antibody to bacteria, virus, protozoa, and fungi
that they could be exposed to during intrauterine and post-partum
 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287 285

Table 2
Effect of the mother infection on the composition of the myeloid cells in bone marrow
of the 7 days old neonates.

Neonates from non-infected Neonates from infected p

mothers (%) mothers (%)
Neutrophils 31 ± 2.0 35 ± 2.1 0.229
Monocytes 0.50 ± 0.11 0.24 ± 0.07 0.022
Erythroblasts 55 ± 1.9 50 ± 1.72 0.056
Megakariocytes 0.78 ± 0.11 0.63 ± 0.09 0.353

Data represent medians ± SD values of four different experiments in which five

neonates from each group were included per experiment. The Mann–Whitney U-
test was used for statistical analysis. Percentage and p-values statistically different
are shown in bold.

life is predominantly dependent on innate immunity (Petrova and

Mehta, 2007). In peripheral blood of neonates from infected moth-
ers, percentage of monocytes and neutrophils were significantly in-
creased. The increase of monocytes in the peripheral blood could be
explained by migration of these cells from bone marrow were they
were diminished. In an acute natural infection, tachyzoites are ini-
tially disseminated from the gut to a variety of organs. The survival
of both the host and parasite is dependent on some of these tach-
yzoites becoming encysted or slowly dividing into bradyzoites,
while the remaining tachyzoites are eliminated (Tenter et al.,
2000). Monocytes play an important role in this phenomenon. They
lyse extracellular tachyzoites and, when infected they retard intra-
cellular tachyzoites division (Channon et al., 2000). In our experi-
ments monocytes from peripheral blood of neonates from
infected mothers showed an increase in granules, others were vac-
uolated and pieces of tachyzoites were observed inside vacuoles or
Fig. 6. Effect of the mother infection on the composition of the peripheral blood
cells of 7 days old neonates. Data represent means ± SD of 20 neonates from 4
non-infected mothers and 20 neonates from 4 infected mothers. The Mann–
Whitney U-test was used *p < 0.001, **p = 0.006 and ***p = 0.001
outside cells. Neutrophils showed a similar image. These results
indicate that these cells participate in the control of parasitemia.
Infected mothers died from the infection or were euthanized
after 7 days of giving birth. They were sick and presented signs
of illness (piloerection, huddling and tachypnea) in the moment
of childbirth but they continued feeding neonates until day 7
post-partum. They had large numbers of tachyzoites in peritoneal

Fig. 5. Peripheral blood smears of 7 days old neonates. (A) Monocyte with banded shaped or kidney shaped nucleus, with granules, is seen in a smear of a neonate from an
infected mother, (B and C) Monocytes of neonates from infected mothers with large vacuoles containing digested tachyzoites (arrows) and a neutrophil. (D) Neutrophil
surrounded by pieces of tachyzoites (arrows) in a smear of a neonate from an infected mother, (E) Typical segmented neutrophil on peripheral blood smear of neonates from
non-infected mothers. (F) Monocyte, the largest peripheral blood cell of mice, with its gray-blue cytoplasm and lobulated nucleus is observed in a smear of a neonate from a
non-infected mother. 100 immersion objective, Wright–Giemsa stain. Images shown are representative of 10 neonates from 2 infected mothers and 10 neonates from 2
non-infected mothers.
286 M.A. Cabañas-Cortés et al. / Experimental Parasitology 121 (2009) 279–287

Fig. 7. Detection of T. gondii DNA by PCR. (A) PCR of non-infected and infected mothers: lane 1: spleen, lane 2: liver, and lane 3: peritoneal cavity fluid of non-infected mother;
lane 4: spleen and lane 5: peritoneal cavity fluid of infected mother. (B) Five spleens of 7 days old neonates from non-infected mothers (lanes 1–5). (C) Five spleens of 7 days
old neonates from infected mothers (lanes 1–5). (D) Five livers of 7 days old neonates from non-infected mothers (lanes 1–5). (E) Five livers of 7 days old neonates from
infected mothers (lanes 1–5). Lanes N = distilled water (negative control); lanes P = T. gondii DNA (positive control). Lanes M = molecular size marker. The position of the
expected fragment (469 bp) is indicated on the left.

cavity (data not shown), but by PCR, parasite DNA was found in the
peritoneal cavity fluid and in the spleen. Parasite DNA in the liver
and spleen of the neonates was examined by PCR, also. It was not
detected in liver and spleen of neonates from infected mothers,
although the vacuolation observed in monocytes from peripheral
blood indicate that parasitemia could be controlled by phagocytes
in blood and they had not arrive to spleen or liver of neonates in
such a short time. Results suggest that the parasite was not present
in these organs at an early stage of infection. This fact may be re-
lated to inoculums’ size. Parasitemia could be transient and/or at
a very low level in the neonate.
In summary, after inoculation with tachyzoites in the peritoneal
cavity of pregnant mothers at day 19 of gestation, parasites reach
placental tissues via maternal leukocytes. In the placenta, maternal
cytokines, in concert with parasites, fever and hormonal and met-
abolic disequilibrium could disturb the uterine environment so
that neonatal immune tissues and cells are modified in such a
way that the neonate survive in the presence of parasites. This
work provides a murine model that could be used in the future
to understand the complex relations between mother and fetus
that could explain why the neonates survive by modulating their
immune system.
Acknowledgments
This work was supported in part by a grant from SIP-IPN 2007-
0509. E. Reyes-Maldonado, M.L. Domínguez-López, L. Jiménez-
Zamudio and E. García-Latorre are fellows of SNI, COFAA-IPN and
EDI-IPN. M.A. Cabañas-Cortes received during this study a scholar-
ship from Conacyt México for her Dsc studies (188857) and from
PIFI–IPN, 2005–2007. We thank E. Ramirez-San Juan for his helpful
assistance with the statistic analysis and L. Sánchez-Torres for her
expert help in apoptosis analyses.
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