International Journal for Parasitology 43 (2013) 697–706

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International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Parasitological and immunological aspects of early Ascaris spp.

infection in mice
Pedro Henrique Gazzinelli-Guimarães a, Ana Clara Gazzinelli-Guimarães a, Flaviane Nunes Silva a,
Vitor Luís Tenório Mati a, Lucas de Carvalho Dhom-Lemos a, Fernando Sérgio Barbosa a,
Lívia Silva Araújo Passos a, Soraya Gaze a, Cláudia Martins Carneiro b, Daniella Castanheira Bartholomeu a,
Lilian Lacerda Bueno a, Ricardo Toshio Fujiwara a,⇑
a
Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil
b
Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas/NUPEB, Universidade Federal de Ouro Preto, Brasil

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

Article history: Studies related to the immunobiological aspects of an Ascaris spp. infection are still scarce, especially
Received 4 December 2012 those that aim to elucidate the early events of the immune response. In this study, we demonstrated a
Received in revised form 15 February 2013 novel standardized method for early experimental Ascaris infection, providing additional information
Accepted 19 February 2013
about the infectivity of eggs embryonated in vitro as well as the influence of host age on development
Available online 9 May 2013
of the infection. Finally, we characterised the immunopathology of early infection, focusing on the tissue
and systemic cytokine profiles and the histopathology of infection in the lungs of BALB/c mice. Our results
Keywords:
demonstrated that the highest egg infectivity occurred on the 100th and 200th days of in vitro embryo-
Ascaris infection
Immune response
nation and that 8 week-old BALB/c mice were more susceptible to infection than 16 week-old mice. Asca-
Murine model ris-infected mice showed an early, significant level of IL-5 production in the lungs 4 days p.i., followed by
Immunopathology an increase in the level of neutrophils in the inflammatory infiltrate at 8 days p.i, which was correlated
Inflammation with the peak of larval migration in the tissue and a significant level of IL-6 production. The inflammatory
Cytokine profile infiltrate in the lungs was gradually replaced by mononuclear cells and eosinophils on the 10th and 12th
days p.i., respectively, and an increase in TNF levels was observed. The downmodulation of systemic
TCD4+ cell numbers might suggest that T cell hyporesponsiveness was induced by the Ascaris spp. larvae,
contributing to safeguarding parasite survival during larval migration. Taken together, the novel aspects
of Ascaris infection presented here enabled a better understanding of the immunopathological events
during larval migration, providing insight for further studies focused on immunisation and immunopro-
phylatic assays.
Ó 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Human ascariasis, caused by the helminth nematode Ascaris
lumbricoides (Linnaeus, 1758) (Family Ascaridae), is the most pre-
valent neglected tropical disease in the world. Its distribution is
cosmopolitan and estimates suggest that approximately 1.2 billion
people are infected globally (de Silva et al., 2003), mainly in rural
and poor areas of developing countries of southern, eastern and
southeastern Asia, Latin America and Sub-Saharan Africa (Dold
and Holland, 2011a). Another member of the Ascaridae family,
Ascaris suum (Goeze 1782) is the etiological agent of porcine asca-
riasis. The pig roundworm shows a widespread global distribution
⇑ Corresponding author. Address: Laboratório de Imunologia e Genômica de
Parasitos, Bloco E4, sala 167, Instituto de Ciências Biológicas, Departamento de
Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
Tel.: +55 3134092859; fax: +55 3134092970.
E-mail address: fujiwara@icb.ufmg.br (R.T. Fujiwara).
and infection with this parasite interferes with the health and
performance of pigs, resulting in reduced feed-to-gain ratios and
liver condemnation, which can incur important economic losses
(Stewart and Hale, 1988; Roepstorff et al., 1999).
Ascaris lumbricoides and A. suum are morphologically and ‘‘bio-
logically’’ indistinguishable to many researchers. However, these
nematodes have been considered to be distinct valid species based
mainly on epidemiological observations and differences in experi-
mental infections, although anatomical distinctions between the
denticles of human and pig strains of Ascaris have also been sug-
gested (Sprent, 1952). Due to the anthropozoonotic character of
A. suum (Takata, 1951; Anderson, 1995; Nejsum et al., 2005;
Arizono et al., 2010; Nejsum et al., 2012) and the zooanthroponotic
potential of A. lumbricoides (Galvin, 1968), both species are patho-
genic to humans, although there is evidence that human infection
with A. suum is more likely to cause a larva migrans-like syndrome
(Pawlowski, 1978; Maruyama et al., 1996), and the larvae may only
rarely reach sexual maturity (Pawlowski, 1978; Cooper, 2002). Re-

0020-7519/$36.00 Ó 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijpara.2013.02.009
698 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706
cently, Liu et al. (2012) provided additional genetic evidence that
A. lumbricoides and A. suum might actually be the same species,
which would represent an important epidemiological concern with
direct implications for the development and implementation of
any ascariasis control program (Zhou et al., 2012).
In this context, studies related to the biological aspects of Asca-
ris spp. infection are still scarce and further studies are necessary,
mainly to elucidate the early events in the immune response and
focusing on new immunoprophylatic strategies. Although A. suum
has been used over the years in rodent models (Massara et al.,
1990; Slotved et al., 1998; Enobe et al., 2006; Lewis et al., 2006),
the immunopathological aspects of experimental infection with
this parasite are still poorly understood.
The use of a murine experimental model for Ascaris infection is
crucial at present. Such murine models provide detailed information
on the biology of early infection with Ascaris spp. and their migra-
tion, which are difficult to describe in natural infections. Moreover,
this experimental model mimics the biological events that occur in
the natural host (Murrell et al., 1997; Slotved et al., 1998). Thus,
the aim of the present study was to characterise the parasitological
and immunological aspects of experimental infection with Ascaris
spp. in a murine model, providing additional information about
the infectivity of eggs embryonated under culture conditions as well
as the influence of host age on development of the infection. Further-
more, we characterised some aspects of the early immunopathology
of this infection using this experimental model, focusing on cellular
and humoral immune responses and histopathology during the lung
phase of infection with the parasite.
2. Materials and methods
2.1. Parasites
Adult A. suum worms were harvested from pigs at a Brazilian
slaughterhouse (Belo Horizonte, Minas Gerais, Brazil) and were
maintained in saline until processed at the Laboratory of Immunol-
ogy and Genomics of Parasites at the Universidade Federal de Min-
as Gerais, Brazil.
Adult A. lumbricoides worms were collected from the naturally
expelled feces of an infected patient from an endemic area of Minas
Gerais State, Brazil, who agreed to donate the parasite to this study.
2.2. Egg embryonation assay
The embryonation of A. suum eggs was performed as described
by Boes et al. (1998), with some modifications. The eggs were iso-
lated from female uteri via gentle mechanical maceration, purified
by straining and cultured to embryonation in 0.2 M H2S04. The eggs
were stored at a concentration of 25 eggs/lL in 50 mL tissue cul-
ture flasks and kept in the dark at 26 °C for up to 400 days (Eriksen,
1990). During incubation, the egg suspensions were oxygenated
three times per week by stirring. Embryonation was evaluated
microscopically every 10 days during culture and from this analy-
sis the percentage of embryonated eggs was calculated by three
independent experiments using three aliquots of 10 lL for each
time point. The isolation and embryonation of A. lumbricoides eggs
were performed as described for A. suum.
2.3. Egg infectivity test
The infectivity of A. suum eggs as a function of embryonation
time was evaluated by experimental infection of BALB/c mice
(8 weeks old, male), as described by Enobe et al. (2006). Egg infec-
tivity was evaluated every 5 days from days 20 to 50 of culture in
H2SO4. Infectivity tests were subsequently performed at 100, 200,
300 and 400 days of egg embryonation. At each embryonation time
point, six mice were inoculated via the intragastric route (gavage)
with 0.2 mL of an egg suspension containing 2,500 fully embryo-
nated eggs, followed by 0.1 mL of H2O to wash the remaining eggs
out of the syringe and needle. Prior to inoculation, fully embryo-
nated eggs were incubated with 5% sodium hypochlorite in a
humidified atmosphere (37 °C, 5% CO2) for 2 h to disrupt the outer
egg layer and, thus, facilitate larval hatching, followed by five
washes with a saline solution. Eight days after inoculation, all of
the mice were euthanized with a lethal dose of anesthetic
(120 mg/kg Ketamine together with 45 mg/kg Xylazine). The lungs
were then collected, sliced finely with scissors and placed in a
Baermann apparatus for 4 h in PBS buffer at 37 °C. The larvae were
finally recovered, fixed with formalin and counted via optical
microscopy.
2.4. Experimental infection
2.4.1. Influence of host age on early A. suum infection
To evaluate the influence of host age on early Ascaris infection, 12
male BALB/c mice, six of which were 8 weeks old, while six were
16 weeks old, were inoculated via the intragastric route with 2,500
fully embryonated eggs that had been incubated for approximately
150 days in H2SO4. The parasite burden was evaluated based on
the total number of larvae recovered in the lungs 8 days p.i.
2.4.2. Characterization of A. suum larval migration in mice
BALB/c mice (8 weeks old, male) were inoculated via the intra-
gastric route with 2,500 fully embryonated eggs that had been
incubated for approximately 150 days in H2SO4. Six A. suum-in-
fected mice were euthanized daily, from the first until the 14th
day of infection. The number of larvae recovered from the large
intestine, liver, lung and small intestine was evaluated each time.
2.4.3. Comparison between A. suum and A. lumbricoides experimental
infections
To compare the recovery indices for A. suum and A. lumbricoides
lung-stage larvae, experimental infections were assessed following
the standardized method described in the present work for A.
suum. Six BALB/c mice (8 weeks old, male) were inoculated via
the intragastric route with 1,000 fully embryonated A. lumbricoides
eggs that had been incubated for approximately 150 days in H2SO4,
and six BALB/c mice (8 weeks old, male) were inoculated with
1,000 fully embryonated A. suum eggs under the same conditions.
The parasite burden was evaluated based on the total number of
larvae recovered in the lungs after 8 days of infection. The egg inoc-
ulum was reduced due the limited number of A. lumbricoides eggs
that were fully embryonated when cultured in H2SO4 compared
with A. suum.
2.5. Lung histology of A. suum-infected BALB/c mice during lung-stage
larval migration
Six mice were euthanized at each of the following time points:
8, 10 and 12 days p.i. The lungs were removed and fixed in a 10%
neutral-buffered formalin solution. The tissues were then gradu-
ally dehydrated in ethanol (70%, 90% and 100%), cleared with xylol
and embedded in paraffin. The paraffin blocks were cut into 4–
5 lm thick sections and adjacent sections were stained with H&E
for evaluation of general lung damage, and with Masson’s tri-
chrome for evaluation of collagen tissue deposition. All images of
the tissue sections were collected under a light microscope (Leica
DM5000). Six non-infected mice were used as a control group.
 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706
2.6. Tissue cytokine profile in the lung during A. suum larval migration
The Th1/Th2/Th17 cytokine profile was evaluated in the lung of
infected BALB/c mice during A. suum larval migration (4, 8 and
12 days p.i.), and in the lung of non-infected BALB/c mice (n = 6)
used as a control group (time 0). The cytokines IL-2, IL-4, IL-6,
IFN-c, TNF, IL-10 and IL-17A were measured via flow cytometry
using a Cytometric Bead Array kit (CBA – BD Biosciences, USA),
and IL-5 was measured via ELISA (R&D Systems, USA), evaluating
the different periods of lung-stage larval migration. Briefly, 24
BALB/c mice (8 weeks old, male) were infected with 2,500 fully
embryonated A. suum eggs that had been incubated for approxi-
mately 150 days in H2SO4. Six mice were euthanized at each of
the following time points: 4, 8, 10 and 12 days p.i., and the lungs
were removed and homogenised with a tissue homogenizer
(Power Gen 125 – Fisher Scientific Pennsylvania, USA) in 100 mL
of PBS supplemented with protease inhibitors (0.1 mM phen-
ylmethilsulfonyl fluoride (PMSF), 0.1 mM benzethonium chloride,
10 mM EDTA and 20 KI aprotinin A) and 0.05% Tween 20. Following
centrifugation at 8,000g for 10 min at 4 °C, the supernatant was
used to determine cytokine production.
2.7. Systemic cytokine profile during A. suum larval migration
The intracytoplasmatic cytokines IL-4, IL-10, IFN-c, TNF-A and
TGF-b, produced by CD4+ T lymphocytes from the spleen, were
evaluated via flow cytometry. Briefly, 10 A. suum-infected BALB/c
mice were euthanized 8 days p.i. and 10 non-infected BALB/c mice
were used as controls. The spleens were harvested and transferred
immediately to RPMI 1640 culture medium (Sigma, USA) supple-
mented with 10% heat-inactivated FCS, 1.6% L-glutamine and 3%
antibiotics/antimycotics (Invitrogen, USA). To isolate cells, the or-
gans were mechanically macerated and the maceration product
was purified using a 70 lm cell strainer (BD Falcon, USA). Then
the spleen cells were incubated with Brefeldin A (Sigma, USA) for
3 h followed by incubation with EDTA for 10 min, and erythrocyte
lysis was performed via treatment with 0.15 M ammonium chlo-
rite for 10 min at room temperature (RT). The cells (1  107) were
subsequently washed with PBS and stained with specific monoclo-
nal antibodies conjugated with FITC targeting mouse CD4 cell sur-
face markers for 30 min in the dark at RT. After incubation, the cells
were washed with PBS-W (0.5% BSA, 0.1% sodium azide) and then
permeabilized with PBS-P (0.5% BSA, 0.1% sodium azide, 0.5% sapo-
nin) for 10 min. Finally, the CD4+ cells from each mouse were dis-
tributed equally (1  106) in several 5 mL polystyrene tubes (BD
Biosciences, USA) containing IL-4, IL-10, IFN-c, TNF-A and TGF-b
anti-mouse cytokines, one per tube, conjugated with PE (phycoer-
ythrin) and incubated for 30 min in the dark at RT. Prior to flow
cytometric acquisition, the stained cells were washed with PBS
and fixed with FACS fixation solution (10 g/L paraformaldehyde,
10.2 g/L sodium cacodylate, 6.65 g/L sodium chloride) for at least
15 min at 4 °C. Phenotypic analyses were performed via flow
cytometry with a FACScan flow cytometer (BD Biosciences, USA).
Data on the lymphocyte populations were collected (gated by for-
ward and side scatter) and analysed using Cell Quest Pro™ soft-
ware (BD Biosciences).
2.8. Ethics statement
The maintenance and use of animals were carried out in strict
accordance with the recommendations of the guidelines of the Bra-
zilian College of Animal Experimentation (COBEA). The protocol
was approved by the Ethics Committee for Animal Experimenta-
tion (CETEA) of the Universidade Federal de Minas Gerais, Brazil
(Protocol# 45/2012). All efforts were made to minimize animal
suffering.
699
2.9. Statistical analysis
The one-sample Kolmogorov–Smirnov test was used to deter-
mine whether the observed variability followed a normal distribu-
tion pattern. For parametric data, a one-way ANOVA test followed
by Bonferroni’s Multiple Comparison Test was used to evaluate the
statistical differences in the results of the infectivity assay as a
function of egg embryonation time. For non-parametric data, the
Kruskal–Wallis test followed by Dunn’s Multiple Comparison Test
was used for the analysis of tissue cytokine profiles. An unpaired
t test was used to determine the differences between parametric
variables regarding the influence of host age as well as in the com-
parison between the A. suum and A. lumbricoides experimental
infections. Correlation analysis was performed using the Spearman
test. The maximum residual test (Grubb’s test) was used to detect
possible outliers. All statistics were calculated using Prism 5.0 for
Windows (GraphPad Software Inc., USA) and the differences were
considered statistically significant when P < 0.05.
3. Results
3.1. In vitro A. suum eggs embryonation over time
The A. suum egg embryonation time was assessed in vitro after
incubation with 0.2 M H2SO4 over 400 days at 26 °C (Fig. 1A). As
shown in Fig. 1B, the newly recovered eggs from the uteri of adult
females (decorticated eggs due the absence of the outer mucopoly-
saccharide membrane) were unembryonated, presenting a solid
mass of germinative cells surrounded by two layers, with the inner
layer being formed mainly from lipids and proteins and the outer
from quitine. On the 10th day of culture, the embryonation index
was determined and no embryonated eggs were observed,
although the first signs of embryonation were observed, character-
ised by cellular organisation into eggs. After 14 days of culture, the
A. suum larvae had completed egg formation. On the 20th day, the
results revealed that 55.3% of the total eggs in culture were embry-
onated. Interestingly, the percentage of embryonated eggs re-
mained close to 50%, with no significant reduction or increase in
the embryonation index being observed over 400 days of culture
in H2S04 (53.66% on day 50, 51.86% on day 100, 45.82% on day
200, 42.33% on day 300 and 40.26% on day 400).
3.2. Ascaris suum egg infectivity as a function of embryonation time in
culture
Ascaris suum egg infectivity was evaluated through the recovery
of lung-stage migrating larvae on the eighth day after infection of
BALB/c mice with fully embryonated eggs that had been incubated
for varying lengths of time in H2S04 in culture. As shown in Fig. 1C,
the A. suum eggs became infective on the 35th day of culture,
showed a statistically significant increase in infectivity on the
50th day and finally reached the peak of infectivity on the 100th
and 200th days of culture in H2SO4. The infectivity of the eggs sub-
sequently decreased considerably on the 300th day, returning to
almost zero after 400 days in H2SO4 (Fig. 1C).
3.3. Influence of host age on the parasitic burden
After standardizing the egg embryonation time for maximum
infectivity, the influence of host age on A. suum infection burden
was assessed in infected BALB/c mice. The results revealed that
the 8 week old BALB/c mice presented a significant increase
(P < 0.0001) in the number of recovered larvae in the lung at 8 days
p.i. compared with 16 week old BALB/c mice (median: 180.6 and
51.5 larvae, respectively) (Fig. 2).
700 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706

Fig. 1. Ascaris suum egg infectivity as a function of the embryonation time in culture. (A) The percentage of A. suum eggs embryonated over 400 days in 0.2 M H2SO4 culture.
(B) Representative photographs of A. suum egg embryonation after different culture times. (C) Egg infectivity measured based on the recovery of A. suum larvae from the lungs
of BALB/c mice at 8 days p.i. Mice were infected with fully embryonated eggs which had been cultured in H2SO4 for different lengths of time. The data are presented as the
mean ± S.E.M. from six mice for each embryonation time point. Statistical differences (P < 0.05) found after 0, 30, 35, 50, 100, 200, 300 and 400 days of culture are indicated by
a–h, respectively.

day, and parasite elimination was observed on the 14th day

(Fig. 3D).

3.5. Comparison of the recovery of A. suum and A. lumbricoides lung-

stage larvae

The knowledge obtained from A. suum infection of mice was ap-

plied to evaluate A. lumbricoides infection. No statistical differences
between the recovery of A. suum and A. lumbricoides larvae in the
lungs of 8 week old BALB/c mice on the eighth day p.i. (16.2 and
12.6 larvae, respectively (P = 1.000)) (Fig. 4) was detected. Also,
no differences were observed in other parameters in the larval
migration patterns (data not shown).

3.6. Lung histology of A. suum-infected BALB/c mice during lung stage

Fig. 2. Influence of host age on the Ascaris suum parasitic burden. Six 8 week old
BALB/c mice and six 16 week old BALB/c mice were infected with 2,500 fully
embryonated Ascaris eggs and the parasitic burden was assessed based on the
number of larvae recovered from the lungs on the eighth day p.i. Significant
differences between the groups (P < 0.05) are represented by the P value given in
the graph.
3.4. Ascaris suum larval migration
The migration of A. suum larvae was completed 14 days p.i. This
includes the ingestion of infective eggs, larval migration through
the host up to the small intestinal lumen and further elimination,
as mice are non-permissive hosts to chronic Ascaris spp. infection
and therefore adult worm maturation does not occur in the gut
as has been observed in humans and pigs. The time line of the
infection was as follows: in the first 24 h of the infection, the
A. suum larvae hatched in the large intestine and reached the liver,
and they showed gradually increasing migration into the liver until
the fifth day of infection (Fig. 3A and B). After this time, the para-
sitic burden in the liver decreased gradually to zero on the 10th
day p.i. The lung phase of migration was initiated on the third
day, reaching a peak in this organ on the seventh and eighth days
of infection (Fig. 3C). The parasitic burden in the lung subsequently
decreased gradually to zero on the 14th day of infection. Finally,
L4s were observed in the lumen of the small intestine at 9 days
p.i., with the peak of larval numbers being observed on the 10th
larval migration
Fig. 5 present the results of histological analyses of the lungs of
both control (Fig. 5A and B) and A. suum-infected mice (Fig. 5C–M).
The A. suum-infected mice showed an increase in inflammatory
infiltrate levels from 8 (Fig. 5C and D) to 12 days (Fig. 5J–L) p.i.
Neutrophils were initially observed on the eighth day of infection
and were gradually replaced by mononuclear cells. On day 12,
the inflammatory infiltrate was more evident and consisted mainly
of mononuclear cells (Fig. 5J), mostly macrophages (Fig. 5L). How-
ever, many eosinophils were also seen on day 12 (Fig. 5K), in great-
er numbers than that observed on day 10 (Fig. 5G). Ascaris suum
larvae were found in the alveolar spaces (Fig. 5H) and inside the
bronchioles until the 10th day of infection. An increase in collagen
deposition was noted from days 8 to 12 (Fig. 5E, I, M) compared
with the control group (Fig. 5B).
3.7. Early immune response characterization: tissue-specific and
systemic cytokine profiles during A. suum larval migration
The pattern of tissue cytokine expression was evaluated in the
lung at different time points during A. suum larval migration (4,
8, 10 and 12 days p.i.) and in non-infected mice. Overall, the results
showed a polarised pro-inflammatory, innate response, associated
with significant increases of IL-6, TNF and IL-5 levels (Fig. 6A, C and
E). The levels of IL-2, IL-4, IL-10, IFN-c and IL-17A were below the
 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706
Fig. 3. Characterization of Ascaris suum larval migration in mice. The complete A. suum larval migration in BALB/c mice was evaluated. Six A. suum-infected mice were
euthanized daily between the first and the 14th day p.i., and larval migration was assessed in the large intestine (A), liver (B), lung (C) and small intestine (D).
Fig. 4. Comparison of Ascaris suum and Ascaris lumbricoides experimental infection.
The lung parasitic burden of six A. suum-infected BALB/c mice was compared with
that of six A. lumbricoides-infected BALB/c mice. Significant differences between the
groups (P < 0.05) are represented by the P value given in the graph.
limit of detection, indicating very low or no production of these
cytokines in the lung during A. suum infection at these time points
(data not shown). Notably, the production of the IL-6 cytokine pre-
sented a different pattern during lung-stage larval migration. Basal
levels of IL-6 at 0 and 4 days p.i. (0.00 and 13.35 pg/100 mg of lung
tissue, respectively) were observed. Beginning at day 8 p.i., the IL-6
levels were significantly higher in the infected mice than in the
control group. On the 10th day p.i., the IL-6 levels were lower than
on the eighth day (111.92 pg/100 mg of lung tissue) but remained
significantly higher compared with non-infected BALB/c mice. Fi-
nally, on the 12th day p.i., the levels of IL-6 decreased considerably
(47.65 pg/100 mg of lung tissue) and were significantly different
from the control (Fig. 6A). Interestingly, correlation analysis
showed a strong positive (r = 1.000) and significant (P = 0.016) cor-
relation between the amount of IL-6 produced and the number of
larvae in the lung, suggesting that this pro-inflammatory cytokine
is associated with the A. suum lung-stage larval migration (Fig. 6B).
When TNF levels were evaluated in the lungs of infected mice, a
different pattern of cytokine production during larval migration
was also observed. The TNF levels were significantly higher
(P = 0.0004) on the 10th and 12th days p.i. (36.54 and 150.75 pg/
100 mg of lung tissue, respectively), compared with the control
group (Fig. 6C). However, correlation analysis demonstrated that
the peak of TNF production in the lung was not associated with
the peak of larval migration (Fig. 6D).
IL-5 levels were significantly higher in the lungs of Ascaris-in-
fected mice from the fourth day p.i. compared with the non-in-
fected group (P = 0.041), until the 10th day p.i. (P = 0.034)
(Fig. 6E). Otherwise, no correlation was observed between the IL-
5 levels and the peak of larval migration in the lung (Fig. 6F).
Interestingly, evaluation of the systemic cytokine profile in
spleen cells showed a significant decrease in the numbers of
T CD4+ lymphocytes in the Ascaris-infected mice compared with
701
the non-infected mice (P = 0.0018) (Fig. 7A). Additionally, mice in-
fected by A. suum showed a decrease in the absolute number of
TCD4+IL-4+ (P = 0.0033) (Fig. 7B) and TCD4+TNF-A+ lymphocytes
(P = 0.0132) (Fig. 7C) in the spleen compared with the non-infected
group. The numbers of TCD4+IFN-c+ (Fig. 7D), TCD4+IL-10+ (Fig. 7E)
and TCD4+TGF-b+ (Fig. 7F) cells were not altered after infection in
the spleen.
4. Discussion
The use of mice as an alternative model for studying early Asca-
ris spp. infection has enabled a better understanding of the host-
parasite relationship (Enobe et al., 2006; Arizono et al., 2010), as
studies of A. lumbricoides worms in human hosts are limited due
to ethical considerations (Dold and Holland, 2011b). BALB/c mice
were employed in the present study partly because this strain
has often been used in immunological studies related to Ascaris
infection, particularly in the study of immunological changes in-
duced in the host by parasitic antigens (Paterson et al., 2002) and
their effects on subsequent challenge infections with larvae of A.
suum (Tsuji et al., 2003; Islam et al., 2005). However, studies fo-
cused on the immunopathological differences between uninfected
and infected BALB/c mice have remained scarce. Only initial stud-
ies on humoral immunity in this ascariasis model had been per-
formed (Crandall and Crandall, 1971) until recently, when Enobe
et al. (2006) evaluated the induction of airway inflammation and
hyperreactivity during experimental infection with the nematode.
Additional information about the biology of Ascaris spp. is still
required. We showed that the kinetics of larval migration in the li-
ver and lungs of BALB/c mice were similar to that described for this
strain by Lewis et al. (2006). However, in contrast to what is re-
ported in the literature, from the ninth day p.i. up to 13th day,
migrating larvae were observed in the small intestine. This finding
enables further studies focused mainly on early mucosal immu-
nity, which remains obscure for Ascaris spp. infection. Moreover,
we detected a greater susceptibility of 8 week old BALB/c mice
compared with older mice, confirming that younger hosts are more
susceptible to Ascaris infection, which is also true for A. lumbrico-
ides infection in humans (Holland, 2009).
No significant differences were observed in the parasitic burden
or migration pattern (data not shown) between the A. suum and A.
lumbricoides experimental infections, suggesting that the standard-
ized method for A. suum experimental infection described here
might also be used for A. lumbricoides and could contribute to res-
olution of the intriguing taxonomic debate regarding the two par-
asites. Indeed, the percentages of larval recovery for both species
from 8 week old BALB/c mice generally corresponded to what
has previously been observed in outbred mice infected with a Bra-
zilian strain of A. lumbricoides (Massara et al., 1990). However, in a
recent study (Peng et al., 2012), C57BL/6 mice were infected with
several genotypes of Ascaris from China. This study showed differ-
ences in the larval migration and distribution patterns in host
702 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706

Fig. 5. The histology of lungs from Ascaris suum-infected BALB/c mice. The lung histopathology of A. suum-infected BALB/c mice was assessed after 8 (C–E), 10 (F–I) and 12 (J–
M) days p.i. and was compared with non-infected animals (A and B).  Highlights the Ascaris larvae in the pulmonary alveoli and the arrows indicate eosinophils in
inflammatory infiltrate of the lung.

tissues, including in the peak recovery, between two genotypes (a
predominately human-derived nematode versus a predominately
pig-derived nematode). Furthermore, depending on the genotype
of the parasites, peculiarities in the development of the eggs were
suggested.
Here, we found that the hatching and/or migration of the para-
sitic larvae appear to be dependent on the time the A. suum eggs
spend in culture. Although A. suum eggs became infective on the
35th day of culture, a higher infectivity rate was observed on the
100th and 200th days of culture in H2SO4. Varying culture times
have been used for eggs in different studies involving experimental
infection with Ascaris (Anderson, 1995; Boes et al., 1998; Lewis
et al., 2006), and the number of larvae recovered and inflammatory
response induced by the parasite may be affected by this parame-
ter. Thus, the results suggested that more attention must be paid to
the time the eggs spend in culture when designing experiments re-
lated to infection with A. suum or A. lumbricoides.
In this present study, we also carried out an evaluation of the
cellular immune response during early infection with Ascaris via
joint analysis of the kinetics of larval migration, histopathology
and cytokine humoral responses at different host sites.
The earliest cytokine that was detected in lung homogenates
was IL-5 and Ascaris-infected BALB/c mice showed higher levels
of IL-5 on the fourth day p.i. compared with control mice. Interest-
ingly, in a related Toxocara canis murine infection, significant early
production of IL-5 was observed in the brain as a result of migrat-
ing larvae on the third day p.i. (Hamilton et al., 2008). These
authors suggested that this high level of IL-5 induced by brain-
stage migrating larvae may result in the infiltration of eosinophils
and their subsequent degranulation and release of toxic mediators
(Bandeira-Melo and Weller, 2005; Hamilton et al., 2008).
In Ascaris murine infection, the early production of IL-5 might
also be associated with the immunopathology induced by the first
migrating larvae, which reach the lung by the third or fourth day
p.i. and are responsible for activating the protective eosinophil-
mediated immune response. The finding of high levels of IL-5 early
in infection, before the possibility of production by CD4+ T (Th2-
type) lymphocytes, is surprising and difficult to explain because
 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706 703

Fig. 6. The cytokine pattern in mouse lung during Ascaris suum larval migration. The levels of IL-6 (A), TNF (C) and IL-5 (E) were assessed in the lungs of A. suum-infected
BALB/c mice at 0, 4, 8, 10 and 12 days p.i. The levels of IL-6, TNF and IL-5 (B, D and F, respectively) were further correlated with the number of lung-stage larvae found in the
tissues at 0, 4, 8, 10 and 12 days p.i. Statistical differences between the groups are indicated in the graph with the significant P values: ⁄P < 0.05, ⁄⁄P < 0.01 and ⁄⁄⁄P < 0.001. The
P and r values represent the statistical correlation analyses.

the typical source of this cytokine in helminth infection is CD4+ T
lymphocytes as part of the adaptive immune response (Artis,
2006). However, during early Ascaris larval migration, IL-5 produc-
tion may originate from the innate immune response of resident
lung cells. Recently, some studies have highlighted the role of in-
nate cell populations in triggering type-2 immunity. Maizels
et al. (2012) discussed the role of innate lymphoid cells (ILCs, also
termed nuocytes) as a source of IL-5 in the lung during early hel-
minth infection. Lung-stage migrating larvae also induce ‘alarmin’
cytokine (IL-25, IL-33) production by epithelial cells, which stimu-
lates nuocytes to produce type 2 cytokines, particularly IL-5
and IL-13 (Moro et al., 2010; Neill et al., 2010; Saenz et al.,
2010). Furthermore, manifestations similar to those seen in
bronchospasm (asthma) are induced by the migrating larvae of
A. suum in the lungs (Richards et al., 1983) and it has been observed
in murine models of allergic asthma that pulmonary ILCs are also
major producers of IL-5 and IL-13 (Klein Wolterink et al., 2012).
Thus, without a doubt, the role of these innate cells during early
helminth infection warrants further studies, and there is evidence
that IL-5 may also be produced by other cells in the airways,
particularly mast cells (Bradding et al., 1994).
It has been shown that IL-5, which is found at high levels in
helminth-infected hosts during the Th2 cytokine-biased immune
response, is responsible for helminth-induced eosinophilia (Behm
and Ovington, 2000). In fact, many eosinophils were observed via
histopathological analysis in the lung on the 10th and 12th days
704 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706

Fig. 7. The systemic cytokine profile of Ascaris-infected mice during lung-stage larval migration. The percentages of TCD4+ lymphocytes in the spleens of Ascaris-infected mice
(A) as well as the production of IL-4 (B), TNF-A (C), IFN-c (D), IL-10 (E) and TGF-b (F) by those cells were evaluated via flow cytometry and compared with non-infected mouse
cells. Significant differences between the groups (P < 0.05) are represented by the P values given in the graphs.

of Ascaris infection in mice in the present study. This finding cor-
roborates previous results showing that greater numbers of eosin-
ophils in the blood and enhanced levels of eosinophil peroxidase
activity in the lung occurred during the end of the second week
of infection (Enobe et al., 2006).
A variety of helminth models have been tested regarding IL-5-
dependent eosinophilia and protection (Meeusen and Balic,
2000), including Strongyloides venezuelensis (Korenaga et al.,
1991; Korenaga and Tada, 1994), Strongyloides ratti (Ovington
et al., 1998), Angiostrongylus cantonensis (Sasaki et al., 1993; Sugaya
et al., 1997) and Onchocerca lienalis (Folkard et al., 1996). Interest-
ingly, it has been demonstrated in humans that IL-5 presents a
strong negative association with reinfection by gastrointestinal
nematodes (Turner et al., 2003; Jackson et al., 2004). This finding
is in agreement with studies suggesting the crucial role of IL-5 in
the early phase of the infection to eliminate migrating-larvae stage
(Maizels and Balic, 2004).
We also observed a significant correlation between the number
of recovered larvae and IL-6 cytokine levels in the lungs. Interest-
ingly, the greater number of neutrophils in the inflammatory infil-
trate observed on the eighth day p.i. coincided with the highest
migration rate of A. suum larvae in the lung. During the acute
inflammatory response, neutrophils may produce IL-6 (Riedemann
et al., 2004). IL-6 plays a major role in regulating neutrophil traf-
ficking (Fielding et al., 2008) and, as a result, can regulate the tran-
sition from neutrophil to mononuclear cell infiltrates during
inflammation through a shift of the chemokine profile from IL-8
to monocyte chemoattractant protein-1 (MCP-1) (Barnes et al.,
2011). Indeed, IL-6 induces neutrophils to undergo apoptosis and
therefore controls the accumulation of the neutrophilic inflamma-
tory infiltrate after promoting the resolution of acute inflammation
(Marin et al., 2002; McLoughlin et al., 2003). In addition, IL-6 pos-
sesses pro-fibrogenic proprieties and has been shown to increase
fibroblast collagen and glycosaminoglycan production (Duncan
and Berman, 1991; Mihara et al., 1995). Thus, the increase in colla-
gen deposition observed 8 and 12 days p.i. may also be correlated
with IL-6 production during the early phase of A. suum experimen-
tal infection.
The increase in the number of mononuclear cells observed via
histopathology during the progression of this parasitic infection
could explain the observed increase in TNF levels. In fact, we
showed an increase in the number of macrophages at inflamma-
tory sites in the lung, and these cells are a common source of
TNF (Tracey and Cerami, 1990), although other cell types, such as
eosinophils and epithelial cells, are also involved in its production
in the airway (Finotto et al., 1994; Khair et al., 1994).
In immunisation assays in which A. suum antigens (As24 kDa or
As16 kDa) were administered to BALB/c mice, a mixed immune re-
sponse involving high levels of IL-2, IFN-c and IL-10 was found to
be associated with partial protection against infection with A. suum
(Oksanen et al., 1990; Duncan and Berman, 1991). However, in the
present study, the immune response induced by the lung-stage
 P.H. Gazzinelli-Guimarães et al. / International Journal for Parasitology 43 (2013) 697–706
migrating larvae was polarised toward a pro-inflammatory cyto-
kine profile, characterised by high levels of IL-6 and TNF-A, fol-
lowed by systemic modulation of IL-4-expressing CD4+
lymphocytes in the spleen. Our results agree with those of other
researchers obtained using a murine model of asthma (B10.A and
C57BL/6 mice), which demonstrated that stimulation of lung bron-
coalveolar cells with a crude extract of A. suum induced suppres-
sion of IL-4 and IL-5 (Lima et al., 2002).
The downmodulation of the systemic TCD4+ immune response
observed on the eighth day p.i. might suggest a pattern of T cell
hyporesponsiveness induced by the Ascaris spp. migrating larvae.
This could contribute to safeguarding parasite survival in the host
during the larval migration stage. Immunomodulation of the host
immune response can also be seen in other intestinal nematode
models, such as in the initiation of apoptosis in TCD4+ cells induced
by the excretory/secretory (ES) products of hookworm (Chow et al.,
2000; Gazzinelli-Guimaraes et al., 2013); the downmodulated coli-
tis proinflammatory response induced by dextran sodium sulfate
(DSS) (Cançado et al., 2011); immunomodulated human inflamma-
tory diseases (Gaze et al., 2012); and, finally, the stimulated regu-
latory T cell (Treg) response (Ricci et al., 2011).
In humans infected with a large number of A. suum eggs, the
most substantial pulmonary infiltrates and highest levels of IgE
associated with asthmatic reactions are also seen during migration
of the larvae, mainly between 10 and 14 days p.i., associated with
delayed blood eosinophilia (Phills et al., 1972). Interestingly, Coo-
per et al. (2000) demonstrated that A. lumbricoides-infected pa-
tients from endemic areas present a notably polarised Th2
immune response. This finding may suggest that the switch from
a pro-inflammatory response, as observed in murine models and
human experimental infections, to a Th2 response might occur at
the end of the tissue migration phase or, perhaps, after adult worm
patency in the lumen of the host, characterising chronic infection.
Finally, the present study provides a detailed characterization of
the immunobiology of early Ascaris spp. infection, enabling a better
understanding of the immunopathological events that occur dur-
ing larval migration, contributing to further studies focused on
immunisation and immunoprophylatic assays.
Acknowledgments
This work was financially supported by the Fundação de
Amparo a Pesquisa do Estado de Mina Gerais/FAPEMIG, Brazil
(Grant# CBB APQ-01202-09 and Grant# CBB – PPM-00296-11),
the Brazilian National Research Council (CNPq) (Grant# 478729/
2011-1 and Grant# 470613/2012-5), Pró-Reitoria de Pesquisa of
Universidade Federal de Minas Gerais and CAPES (Programa Nac-
ional de Incentivo a Parasitologia Básica). PHGG is supported by a
doctoral degree fellowship from the CNPq. RTF, DB, CMC are sup-
ported by CNPq fellowships.
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