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Veterinary Parasitology 176 (2011) 195–200

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsev ier .com/ locate /vetpar

RNA profile of Nellore calves after primary infection with
aemonchus placei

.M.G. Ibelli a, L.C. Nakataa, R. Andréob, L.L. Coutinhoc, M.C.S. Oliveirad,

.F.T. Amarantee, J. Furlongf, L.G. Zarosg, L.C.A. Regitanod,∗

Universidade Federal de São Carlos, 13565905, São Carlos, Estado de São Paulo, Brazil
Instituto de Química, Universidade Estadual de Paulista, Unesp, Araraquara, Estado de São Paulo 14801970, Brazil
Departamento de Zootecnia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, Estado de São Paulo 13418-900, Brazil
Embrapa Pecuária Sudeste, São Carlos, Estado de São Paulo 13560970, Brazil
Departamento de Parasitologia, Universidade Estadual Paulista, Unesp, Botucatu, Estado de São Paulo 18618000, Brazil
Embrapa Gado de Leite, Juiz de Fora, Estado de Minas Gerais 36038330, Brazil
Universidade Federal do Rio Grande do Norte, Natal, Estado do Rio Grande do Norte 59072970, Brazil

r t i c l e i n f o

rticle history:
eceived 26 August 2010
eceived in revised form
0 November 2010
ccepted 10 November 2010

a b s t r a c t

Haemonchus parasites are responsible for many losses in animal production. However, few
studies are available, especially of zebu cattle. In this study, we investigated mRNA differ-
ences of immune response genes in naïve Nellore calves infected with Haemonchus placei,
relating these differences to patterns of cellular infiltrate. Calves were infected with 15,000
H. placei L3 larvae and after 7 days lymph node and abomasum tissues were collected.
IL-2, IL-4, IL-8, IL-12, IL-13, IFN-�, MCP-1, lysozyme, pepsinogen and TNF-� genes were
eywords:
ytokines
eef cattle
aemonchus placei
os indicus
astrointestinal parasites

evaluated by qPCR. Mast cells, eosinophils and globular leukocytes were counted by abo-
masum histology. In the infected group, IL-4, IL-13 and TNF-� were up-regulated in the
abomasal lymph node. In the abomasum, IL-13 increased and TNF-� was down-regulated
(p < 0.05). No differences were detected for mast cells and eosinophil counts in abomasal
tissue (p > 0.05). We conclude that for this infection time, there was Th2 polarization but

ate in a
that cellular infiltr

. Introduction

One of the major constraints of the animal production
n the tropical regions is the presence of parasites. Losses
n Brazil due to gastrointestinal nematodes are estimated
o be about 68 million dollars a year (Honer and Bianchin,

987; Soutello et al., 2002). In Brazil, Haemonchus spp. and
ooperia spp. are the most prevalent nematodes (Bianchin
t al., 2007; Oliveira et al., 2009).

∗ Corresponding author at: Embrapa Pecuária Sudeste, Washington
uiz, Km 234, São Carlos, SP 1356097, Brazil. Tel.: +55 16 34115637; fax:
55 16 3411 5691.

E-mail address: luciana@cppse.embrapa.br (L.C.A. Regitano).

304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
oi:10.1016/j.vetpar.2010.11.013
bomasal tissue takes longer to develop.
© 2010 Elsevier B.V. All rights reserved.

Brazilian Nellore is a beef breed generally considered
resistant to many ectoparasites, but it is not clear if this
is true in the specific case of gastrointestinal parasites
(Holgado and Cruz, 1994; Oliveira et al., 2009). Although
the mechanisms involved in host defense are better under-
stood in Bos taurus (Sonstegard and Gasbarre, 2001), less
information is available for Bos indicus (Bricarello et al.,
2007, 2008; Zaros et al., 2010).

Genetic mechanisms underlying the variation of resis-
tance can be related to the development of different profiles
of Th1/Th2 cytokines (Meeusen et al., 2005; Huse et al.,

2006). The identification of the cytokine genes involved in
host response and the explanation of their function should
be pursued to establish the different degrees of host resis-
tance, allowing development of better methods of worm
control (Gasbarre et al., 2001; Glass et al., 2005).

dx.doi.org/10.1016/j.vetpar.2010.11.013
http://www.sciencedirect.com/science/journal/03044017
http://www.elsevier.com/locate/vetpar
mailto:luciana@cppse.embrapa.br
dx.doi.org/10.1016/j.vetpar.2010.11.013


ry Paras
196 A.M.G. Ibelli et al. / Veterina

It has been shown that Th2 cytokines confer protec-
tion to infections caused by endoparasites such as Trichuris
muris, H. polygirus, Haemonchus spp., Nippostrongylus
spp., Teladorsagia circumcincta (Else and Finkelman, 1998;
Claerebout et al., 2005; Craig et al., 2007; Zaros et al., 2010).
Worm resistance in sheep and cattle have been associated
with Th2 bias, where higher levels of interleukin 4 (IL-4),
interleukin 5 (IL-5), interleukin 13 (IL-13) are increased in
resistant animals (Gill et al., 2000; Zaros et al., 2010). Craig
et al. (2007) found high levels of IL-4 mRNA expression in
sheep infected with T. circumcincta. Claerebout et al. (2005)
found similar results when comparing infected and unin-
fected cattle exposed to Ostertagia ostertagi. IL-4 and IL-13
can increase muscle contractility in the region of infection
(Finkelman et al., 2006), and production of IL-5 causes the
expulsion of parasites (Bancroft et al., 1998; Garside et al.,
2000). Conversely, interferon gamma (IFN-�), interleukin
2 (IL-2) and interleukin 12 (IL-12) confer susceptibility to
that infection (Else and Finkelman, 1998; Zaros et al., 2010).

Histological changes have been described as a conse-
quence of gastrointestinal infections, such as mast cell
recruitments, eosinophilia in the mucosa, increase of IgG1,
IgG2, IgE, IgA and mucus secretion. Bricarello et al. (2007)
studied cattle resistant and susceptible to gastrointestinal
parasites, related high levels of IgE and eosinophils with
low fecal egg counts. These changes are also related to
cytokine polarization, which helps host response (Balic et
al., 2002; Meeusen et al., 2005).

In tropical areas such as Brazil, there is great interest in
understanding the mechanisms involved in host resistance
and the adaptation of breeding stock, such as Nellore cat-
tle, to learn what makes them more resistant to parasite
infections. However, the knowledge about this in cattle is
restricted to Bos taurus. Therefore, the aim of this study was
to evaluate mRNA levels of IL-2, IL-4, IL-8, IL-12, IL-13, IFN-
�, monocyte chemoatractant protein 1 (MCP-1), lysozyme,
pepsinogen and tumor necrosis factor alpha (TNF-�) genes
in the abomasum and abomasal lymph node, as well as
to determine the defense cells present in the abomasum
of naïve Nellore calves 7 days after primary infection by
Haemonchus placei.

2. Materials and methods

2.1. Animals

The experiment was carried out in Embrapa South-
east Cattle Station in São Paulo state, Brazil (22◦01′S and
47◦53′W). Ten Nellore calves descended from 10 cows and
three bulls born in November and December of 2005 were
used. To keep the animals free from parasite infections, the
calves were taken from their mothers immediately after
birth and received 2 L of frozen colostrum. They were then
kept in individual pens and received 4 L of milk twice a day,
along with hay and mineral salt ad libidum.
2.2. Calves infection

At 4–5 months of age, the calves were weaned and
assigned to two groups of five animals: (1) Infected Group,
which was orally infected with around 15,000 L3 H. placei
itology 176 (2011) 195–200

larvae provided by the Embrapa Beef Cattle Station, fol-
lowing the technique described by Roberts and O’Sullivan
(Ueno and Gonçalves, 1998) and (2) Control Group, which
was kept free from worms during the experimental period.
One calf sired by each bull was assigned to each group.

Twice a week samples of feces were collected to deter-
mine the EPG (eggs per gram) counts (Ueno and Gonçalves,
1998) to assure that animals had no contact with worms.
After 7 days of infection, the calves were sedated with
sodium pentobarbital (60 mg/kg of weight) and sacrificed
with 2% xylasin cloridrate (1 mL/100 kg of weight).

2.3. Tissue collection

Immediately after slaughter, samples of the abomasum
and abomasal lymph nodes were collected and split in two
samples: one stored in formalin solution (10%) for histo-
logical analysis and the other submerged in liquid nitrogen
and stored at −80 ◦C for gene expression analysis.

2.4. Histological analysis

Tissues were fixed in 10% formalin solution for 36 h,
washed and stored in 70% ethanol solution and then
dehydrated in a series of rising ethanol concentrations,
diaphanized with xilol and embedded in paraffin. Histo-
logical sections were stained with hematoxylin–eosin (HE)
for globule leukocyte and eosinophil counts. Toluidine blue
stained sections were used for mast cell counts under
an optical microscope. Leukocyte globules were counted
under an ultraviolet light microscope.

Cells were counted in 30 random fields of the abomasum
surface using a 10× eyepiece, with a 100-point grid and
100× objective. Cell counts were reported as arithmetic
means of cell number/mm2 of mucosa.

2.5. RNA extraction and cDNA synthesis

Frozen tissues (−80 ◦C) were macerated in liquid
nitrogen and total RNA was isolated using the Trizol©

(Invitrogen) reagent, following the manufacturer’s proto-
col. RNA concentration and purity were determined by light
absorption at 260 nm and OD260:OD280 ratio. Integrity
was verified in 1% agarose gel electrophoresis stained with
ethidium bromide (20 �g/mL).

Total RNA (5 �g) was used for first-strand cDNA synthe-
sis with the SuperscriptTM First-Strand Synthesis System
for RT-PCR (Invitrogen), using oligo dT priming, following
the manufacturer’s protocol.

2.6. Primers information

Primers for IL-2, IL-4, IL-8, IL-12p35, IL-13, MCP-1, TNF-
�, RPL-19, GAPDH, lysozyme and pesinogen genes were
described by Zaros et al. (2007), IFN-� by Coussens and

Nobis (2002) and HPRT-1 by Goossens et al. (2005). Ampli-
fication efficiencies were obtained by linear regression
(efficiency = 10 (−1/slope)), following Pfaffl (2001). Speci-
ficities were confirmed in 1% agarose gel and by melting
curve analysis (LightCycler, Roche Diagnostics, Mannheim,



y Parasitology 176 (2011) 195–200 197

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Fig. 1. . Mean of eosinophils and mast cells/mm2 in the abomasal mucus
of control and infected group of Nellore calves with Haemonchus placei.
A.M.G. Ibelli et al. / Veterinar

ermany) using the program from 70 ◦C to 95 ◦C at 0.1 ◦C/s
or all genes studied.

.7. Real time RT-PCR (qPCR)

Each real time RT-PCR reaction was carried out in 20 �L
f final volume containing 25 ng of cDNA, 0.2 mM of dNTP,
.1–0.2 �M of each primer, 2–3.75 mM of MgCl2, 1.5 units
f Taq Platinum DNA polymerase (Invitrogen), 0.04 �L of
ybr Green 100×, 2 �L of buffer 10× 50 mM of KCl, 10 mM
f Tris–HCl pH 9.0 (Invitrogen), and 0.5 �L of dimethyl sul-
oxide (DMSO; Sigma). For each sample, the cycle threshold
Ct) mean was obtained and normalized to a reference gene.

Three reference genes were evaluated: GAPDH
glyceraldehyde-3-phosphate dehydrogenase), HPRT-
(hypoxanthine phosphoribosyltransferase 1) and RPL-19

ribosomal protein L19).

.8. Statistical analysis

The relative quantification was evaluated by mathe-
atic modeling based on the PCR efficiencies (E) of the

arget and endogenous genes and on Ct variation of sam-
les from the experimental groups, according to Pfaffl et al.
2002). For this analysis, the Relative Expression Software
ool (REST©) was used, which applies a nonparametric
ignificance test called the Pair Wise Fixed Reallocation
andomisation Test©.

Histological data (eosinophils, mast cells and globule
eukocytes) were analyzed by the GLM procedure using the
AS program (SAS, 2002/2003).

. Results

.1. Histological analysis

The average eosinophil and mast cell counts were 30.96
±S.D. 5.55) and 10.31 (±S.D. 9) in the non-infected group
nd 28.41 (±S.D. 2.35) and 17.12 (±S.D. 1.95) in the infected
roup, respectively. No significant differences were found
etween groups for the eosinophil counts (p = 0.30) and
ast cell counts (p = 0.32) in the mucous membrane of the

bomasum (Fig. 1). No globular leukocyte was observed in
he slides in any group.

.2. Relative quantification

Among the three reference genes tested to be used in
he relative quantification, the RPL-19 gene was chosen
ecause it presented more constant Ct values (19 ± S.D. 2.3

n the abomasum and 20.7 ± S.D. 1.2 in lymph node) than
he other two genes analyzed.

Relative quantification of target genes showed that IL-
(14×; p = 0.002), IL-13 (26×; p = 0.003) and TNF-� (10×;
= 0.03) were up-regulated in the abomasal lymph nodes

f the infected group in comparison with control group
Fig. 2). In the abomasum tissue, IL-13 was up-regulated
4.8×; p = 0.03) in the infected group and TNF-� was down-
egulated (4.0×; p = 0.032) in the same group (Fig. 3). The
RNA levels of the other genes were not influenced by H.
Fig. 2. . Ratio of cytokine expression in abomasal lymph node between
infected and control group, normalized for reference gene (RPL-19);
*p < 0.05 and **p < 0.01.

placei larval exposure in the abomasal lymph node, as well
as in the abomasum tissue (p > 0.05).

4. Discussion

In this study, we compared cytokine gene expression of
Nellore calves in primary infection with H. placei infections
caused by helminths have been studied in many species
and have usually been associated with Th2 response in
infected animals. In cattle, these infections are not char-
acterized by a severe immune response and most become
chronic. Reduction in the number of adult parasites begins

after exposure to the parasite and at the same time there is
a significant increase in eosinophil, globular leukocyte and
mast cell counts in the sites of the infection (Grencis, 2001;
Bricarello et al., 2004). Cytokine polarization is indispens-
able for the correct immune response activation and TCD4+



198 A.M.G. Ibelli et al. / Veterinary Paras

During gastrointestinal infections, increases of mast
Fig. 3. . Ratio of cytokine expression in abomasum between infected and
control group, normalized for reference gene (RPL-19); *p < 0.05.

cells are effectors in Haemonchus contortus infections (Gill
et al., 1993).

IL-4 and IL-13 are frequently studied because they may
be the first genes to have increased levels in response to
extracellular parasites, leading to Th2 polarization (Else
and Finkelman, 1998) and resistance to animals (Zaros
et al., 2010). In this study, the IL-4 mRNA levels in the abo-
masal lymph node of the infected group were up-regulated
14-fold in comparison to the control group (Fig. 2). Similar
results were obtained by (Canals et al., 1997), who observed
a significant up-regulation of IL-4 abomasal lymph node of
Bos taurus cattle infected with O. ostertagi on the fourth
day post primary infection, increasing gradually until the
28th day of infection. Claerebout et al. (2005) observed
an increase in the expression of IL-4 and IL-10 in lymph
nodes of immunized calves also infected with O. ostertagi,
after 3 weeks of infection. In contrast, in the present study
we found no difference in this interleukin in the abomasal
mucosa, corroborating the results obtained by (Li et al.,
2007) and contrasting with those of Lacroux et al. (2006),
studying sheep (which are more sensitive to this nematode
infection).

IL-13 acts in parasitic infections to promote allergic
response, mast cell increase and IgE production, among
other reactions. In the present work, severe induction of
IL-13 mRNA in abomasal lymph node was observed, about
30 times higher in the infected than in the control group
(Fig. 2). In the abomasal mucosa, IL-13 expressed the same
pattern, with a fivefold increase in the infected group
compared with the control group (Fig. 3). Bancroft et al.
(1998), studying knockout mice for IL-13, observed sus-
ceptibility to T. muris infection as well as a decrease in the
response of other Th2 cytokines, inhibiting the expulsion
of the parasites. An increase of IL-13 was also observed in
sheep immunized and infected primarily with H. contortus

(Lacroux et al., 2006), showing this cytokine is essential in
the protection against gastrointestinal nematode infection.

In fact, some IL-4 and IL-13 functions are redundant,
conferring protective response, resistance and expulsion of
itology 176 (2011) 195–200

the parasites (Else and Finkelman, 1998). We showed that
IL-13 had a strong up-regulation, in both tissues, indicat-
ing this cytokine could be a precursor of IL-4, stimulating its
increase and probably the protective response in the early
infection stage in Nellore cattle. This is possible because
it has been found that IL-4 starts to increase in the fourth
day post-infection of calves with O. ostertagi (Canals et al.,
1997). There is little data about this polarization in zebu
cattle, but it is clear that both cytokines are expressed syn-
ergistically and are essential to control this infection. These
interleukins act to decrease the fecundity and numbers of
helminths by increasing muscular contraction, providing
protection by promoting the expulsion of gastrointesti-
nal worms (Else and Finkelman, 1998; Fallon et al., 2002;
Lawrence, 2003; Wynn, 2003). In similar conditions, pri-
marily infected Bos taurus cattle and sheep (Claerebout
et al., 2005; Lacroux et al., 2006), as well as resistant Nellore
cattle (Zaros et al., 2010), had high levels of these cytokines.

TNF-� is a pro-inflammatory factor that may have an
important role in gastrointestinal infections. Hayes et al.
(2007) observed that TNF-� acts to increase both cytokines
Th1 (IFN-�) and Th2 (IL-13). Although this pattern is not
well established yet, functions related to Th2 polarization
have been reported (Artis et al., 1999). Some works con-
cluded that this pro-inflammatory factor is associated to
resistance in sheep (Pernthaner et al., 2005) and cattle (Li et
al., 2007). In our work, TNF-� was about eight times higher
in the lymph node (Fig. 2) and fourfold less expressed in the
abomasal mucosa (Fig. 3) of the infected animals compared
to the uninfected ones. A contrasting pattern was observed
in both tissues studied. The presence of TNF-� was charac-
terized to potentialize the expulsion of parasites by IL-13,
conferring protection in the host (Artis et al., 1999), as well
as, it was correlated to induction of host resistance in early
larval stages (Babu and Nutman, 2004). So, differences of
TNF-� found in the two tissues studied could be a result
of tissue collection when the immune response started to
be established and when changes in larval stages were still
ongoing. Then, although, at this time, this cytokine could
be helping the Th2 polarization in the lymph nodes, the
presence of parasitic secretions in the abomasum could
exert some local immunomodulation. It is known that the
Haemonchus spp. L4 larvae stage is capable of inducing
changes in the host immune profile to evade host response
(Allen and MacDonald, 1998). As in early infection stages,
TNF-� has been reported to promote parasite expulsion,
this molecule could be a target for immunomodulation
by the parasite (Maizels and Yazdanbakhsh, 2003). TNF-
� down-regulation may be caused by mast cell inhibition
and may turn resistant animals in susceptible (Behnke et al.,
2003; Pernthaner et al., 2005). Therefore, maintaining low
TNF-� level in the host would be beneficial for completion
of parasitic life cycle. Artis et al. (1999) found that low TNF-
� level delays the expulsion of parasites from the host and
that IL-4 and IL-13 levels remain up-regulated, as observed
in this work.
cells and eosinophils are usually observed (Gasbarre, 1997;
Else, 2005). In sheep, these cells are involved in rejection
of H. contortus. Eosinophils are recruited to the abomasum
of sheep during primary infection (Balic et al., 2000, 2002)



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A.M.G. Ibelli et al. / Veterinar

nd are related to death of the parasite (Balic et al., 2006). In
attle, higher numbers of mast cells and eosinophils were
bserved in resistant compared to susceptible Bos indi-
us (Zaros et al., 2010). However, no differences between
nfected and control groups were observed for these cells
n the present trial (Fig. 1). This might be due to the short
eriod of infection, only 7 days. In cattle infected with
. ostertagi it is known that cells accumulate after adult
orms are present for 1 or 2 days in the abomasum (Scott

t al., 1998; Simpson, 2000). In the present work, infec-
ion was caused by larvae and abomasums were obtained

days after infection, a period when most of the larvae
hould be in L4 stage, which marks the beginning of the
ematophagous phase and is expected to stimulate host
rotective response. From our results, it was demonstrated
hat, in animals that had not been exposed to H. placei
eforehand, this period of time was not enough to defla-
rate local response in the mucosa.

For the other genes evaluated (IL-2, IL-8, IL-12, IFN-�
nd MCP-1), as well as for pepsinogen and lysozyme no dif-
erence in mRNA profile was observed in any of the tissues
tudied. As in this work, infections with H. placei, Cooperia
pp. and Ostertagia spp. were found not to be associated
ith changes in the levels of IL-8 (Li et al., 2007; Bricarello

t al., 2008; Zaros et al., 2010). Nevertheless, variations
ere reported for some of these genes, as in the work by

i et al. (2007), which concluded that IL-2, IFN-� and IL-12
ere responsible for conferring resistance to Angus heifers

nfected with O. ostertagi. Other evidence for the involve-
ent of these genes in response to parasite has been found

n Nellore cattle infected with H. placei, where a strong Th2
rofile was detected in resistant animals (Zaros et al., 2010).

In conclusion, we suggest that IL-4 and IL-13 initiate the
mmune response in the abomasal lymph nodes and abo-

asal mucosa in the first infection of naïve Nellore calves
ith H. placei, evidencing a possible initial Th2 polarization.

t may be possible that TNF-� helps in this polarization and
hat this cytokine is modulated by the parasite. In contrast,
o significant increase in eosinophils and mast cells was
etected, indicating that the local inflammatory response
o H. placei occurs later during the infection of Bos indicus
ellore calves.

cknowledgments

We thank Dr. Ivo Bianchin, of the Embrapa Beef Cattle
esearch Center, for providing the larvae and Dr. Rogério
aveira Barbosa and Dr. Rui Machado for taking care of
alves. This work was financially supported by Prodetab
nd Embrapa. We thank CAPES for providing scholarships
o AMGI and LCN and CNPq for fellowships to LLC and LCAR.

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	mRNA profile of Nellore calves after primary infection with Haemonchus placei
	Introduction
	Materials and methods
	Animals
	Calves infection
	Tissue collection
	Histological analysis
	RNA extraction and cDNA synthesis
	Primers information
	Real time RT-PCR (qPCR)
	Statistical analysis

	Results
	Histological analysis
	Relative quantification

	Discussion
	Acknowledgments
	References