Effects of heat stress on peripheral T and B lymphocyte profiles and IgG

and IgM serum levels in broiler chickens vaccinated for Newcastle

disease virus
Bruno Takashi Bueno Honda, Atilio Sersun Calefi, Carolina Costola-de-Souza, Wanderley
Moreno Quinteiro-Filho, Juliana Garcia da Silva Fonseca, Viviane Ferraz de Paula, and
Joao Palermo-Neto1
Neuroimmunomodulation Research Group, Department of Pathology, School of Veterinary Medicine and Animal
Science, University of S
ao Paulo, S
ao Paulo 05508-900, Brazil
and Vaccinated (HS/V). The NC vaccine was administered twice on experimental day (ED) 7 and ED14, and
the heat stress (38 1 C) was applied from ED2 to
ED6. The data showed that HS increased the corticosterone serum levels in the HS group compared with the
control groups (C and C/V groups). At ED7, increased
concentrations of IgM were observed in birds in the HS
and HS/V groups compared with C and C/V animals;
chickens from the HS/V group presented increased IgG
levels compared with those in the birds of the C group.
The heat stress shifted the immune cell profile from
B-lymphocyte to a T-cytotoxic and T-helper lymphocyte profile, and this immune cell pattern persisted
until the end of the study period. It was concluded
that heat stress immunomodulated the immune function response of the chickens to the NC disease vaccine
challenge.

Key words: heat stress, Newcastle disease, lymphocytes, neuroimmunomodulation, immunoglobulins

2015 Poultry Science 94:23752381
http://dx.doi.org/10.3382/ps/pev192

INTRODUCTION
Many studies in the field of neuroimmunomodulation have been conducted in an effort to better understand the bidirectional interactions between central nervous system functions and the immune processes. Such
works are based on the existence of a single organic and
integrated system in which each component performs
a specialized function to maintain homeostasis in the
presence of endogenous or exogenous challenges (Ader,
2000; del Rey et al., 2012).
Environmental changes in poultry production are becoming increasingly important, particularly in regard
to the effects induced by stressful stimuli such as heat
stress and overcrowding on animal welfare, immune
status, and performance (Quinteiro-Filho et al., 2010,

C 2015 Poultry Science Association Inc.
Received December 17, 2014.
Accepted May 15, 2015.
1
Corresponding author: jpalermo@usp.br

2012; Gomes et al., 2014; Calefi et al., 2014). Heat

stress in broiler chickens has been reported to selectively suppress the immune system, leading to failures
in the chickens response to vaccination and immune
organ involution (Shini et al., 2008, 2010; Shini and
Kaiser, 2009)
High levels of circulating corticosterone were reported to induce the redistribution of leukocytes between blood and lymphoid and nonlymphoid tissues
(Dhabhar et al., 1996). This mechanism is considered
to be an adaptive, evolutionary, and conservative response that contributes to an improvement in immune
surveillance (mainly antibacterial) in stressed animals,
i.e., chickens (Dhabhar and McEwen, 1997; Dhabhar,
2013; Aschbacher et al., 2013).
Modern broiler chicken production systems are
strongly susceptible to the effects of stressors; indeed, stressors are known to induce immunosuppression and to increase the animals susceptibility to infectious diseases (Quinteiro-Filho et al., 2010, 2012;
Gomes et al., 2014; Calefi et al., 2014). Vaccination

2375

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ABSTRACT Multiple factors, such as environment, nutritional status, and disease, induce stress in animals
during livestock production. It has been shown that
poultry exposed to stressors for prolonged periods had
decreases in their performance parameters, mortality
and decreased host resistance to pathogenic agents. It
seems that early age stress may have long-lasting impact and could possibly modify the expression of their
genetic potential on growth performance and immunity. This study aimed to discuss the effects of earlyage heat stress on the blood lymphocyte phenotypes
(B and T lymphocytes) and plasma immunoglobulin
levels (IgM and IgG) in chickens vaccinated against
paramixovirus of the Newcastle (NC) disease (LaSota
strain). For this purpose, 96 male chickens (Cobb) were
divided into 4 groups: 1) control (C), 2) heat-stressed
(HS), 3) control vaccinated (C/V), and 4) heat-stressed

2376

HONDA ET AL.

and ED6; for the remaining days of the study (ED7 to

ED19), these chickens were kept at the same environmental temperature as the control group (30 1 C at
ED1 and ED7 and 28 1 C from ED8 to ED19). The
vaccinated groups (C/V and HS/V) received a Newcastle Disease vaccine (LaSota strain) via topical ocular at one drop/dose/chicken on ED7 and a booster on
ED14, according the manufacturers instructions. On
ED7, ED14, and ED19, the animals peripheral blood
was collected in EDTA tubes for analysis. At the end of
the study period, the chickens were euthanized in CO2
chambers.

Plasma Corticosterone Determination

Corticosterone plasma levels were determined using
a corticosterone ELISA kit (DetectX, Arbor Assays,
Ann Arbor, MI). The corticosterone plasma concentration was determined by a standard curve and expressed in picograms per milliliter of corticosterone in
serum. The assay was performed according to the manufacturers instructions, and the limit of detection was
16.9 pgmL1 .

MATERIALS AND METHODS

Animals

Newcastle Disease Virus Antibody

Detection

A total of ninety-six 1-day-old broiler chicks (Cobb

500) were acquired from a commercial breeder hatchery (Amparo, SP, Brazil). The breeders were vaccinated
with Newcastle diseases vaccine (LaSota strain) and
they are Salmonella spp.- and Mycoplasma spp.-free
certificated. The broiler chickens were kept in the Department of Pathology, School of Veterinary Medicine
and Animal Science, University of S
ao Paulo, according
to environmental standards established and approved
by the Committee on Care and Use of Laboratory Animals Resources, School of Veterinary Medicine and Animal Science, University of Sao Paulo (Protocol No.
2360/2011). The chickens were housed in 4 isolator
chambers (Alesco, Sao Paulo, Brazil) in 12 h artificial
light during the day, 12L:12D (light on at 7 AM; light
off at 7 PM). Water and food (hanging feeders) were
provided ad libitum to the chickens, and they were constantly observed for health status and behavior; any
deaths were recorded.

The NDV antibody detection was performed using a

commercial ELISA kit (Shenzhen Lvshiyuan Biotechnology Co., Shenzhen, Guangdong, China), according
to the manufacturers instructions. The assay was performed to obtain a qualitative evaluation of the Newcastle disease antibody production.

Group Formation, Heat Stress, and

Newcastle Disease Virus (LaSota Strain)
Vaccination Protocol

Peripheral Blood B and T Lymphocyte

Immunophenotyping

The chickens were randomly and equally allocated

into 4 groups: control (C), heat-stressed (HS), control vaccinated (C/V), and heat-stressed vaccinated
(HS/V). Birds in the control groups were maintained
at the recommended environmental temperatures [30
1 C from experimental day (ED) 1 to ED7 and 28
1 C from ED8 to ED19]. The heat-stressed birds received heat stress (38 1 C) for 12 h/d between ED2

Serum IgM and IgG Determination

The blood collection was performed from the brachial
vein, and the samples were stored in microtubes and
then centrifuged under refrigeration (1,500 g, 5 min,
8 C) to obtain the serum, which was stored at 80 C
until analysis (approximately 30 d). The IgG and IgM
plasma concentrations were determined using an ELISA
kit from Bethyl Laboratories (Montgomery, AL, USA),
as described by Gao et al. (2008). The immunoglobulin
plasma levels were determined using a standard curve
and were expressed in nanograms per milliliter serum.

Leukocytes were separated by the Percoll method.

Chicken blood was diluted in Roswell Park Memorial
Institute (RPMI) cell culture medium [1:1 (vol/vol)],
after the solution was added in Percoll [GE Healthcore, 1:3 (vol/vol)] in a 15-mL Falcon tube. The tubes
were centrifuged at 1,280 g for 5 min at 18o C. The
agglomerate of leucocytes were removed and transferred in a solution of PBS and centrifuged at 300
g for 10 min at 18 C. The supernatant were removed

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is a common and important field practice to provide chickens with protection against bacterial and viral pathogens during the animal production process
(Kapczynski et al., 2013). Thus, a better understanding
of immunosuppression-induced risk factors is essential
for poultry production. Indeed, animal welfare maintenance would allow the broiler chickens to use all of the
genetic potential that they present and adequately respond to the vaccination and nutritional programs that
have been invested in their production (Hoerr, 2010).
However, few studies have analyzed the relationship
between heat stress and humoral immunity in earlyage broiler chickens under a vaccine challenge. Distress could affect the development of immune and endocrine system. Indeed, minor changes in the early age
phase of broiler chicks might have long-lasting impact
on their genetic potential and immune response against
pathogens (Gross, 1989). Thus, the present study was
designed to analyze the effects of early-age heat stress
on the corticosterone serum levels, lymphocyte profiles,
and immunoglobulin levels in broiler chickens that were
vaccinated against Newcastle disease virus (NDV).

2377

NEWCASTLE-VACCINATION AND HEAT STRESS

Figure 1. Effects of heat stress on corticosterone serum levels at

ED7 in broiler chickens vaccinated against Newcastle disease. Different letters above the columns indicate significant differences (ANOVA
followed by Tukeys multiple comparisons test). The data are reported
as the means SD.

Table 1. Effects of heat stress on IgM and IgG

serum levels in Newcastle-vaccinated chickens.
Immunoglobulin Serum Levels
ED
7

Statistical Analysis
The statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software Inc., San
Diego, CA). All of the data were first analyzed by
Barletts test to determine the homogeneity of the
variances. Parametric data were analyzed by both a
one- and 2-way ANOVA followed by the TukeyKramer
multiple comparisons test. Nonparametric data were
analyzed by KurskalWallis test and Dunns post hoc
test for comparisons among groups. The differences
were considered significant at P 0.05. No comparisons were performed among the different time points of
experiments (ED7, ED14, and ED19). Data from each
ED were analyzed independently.

RESULTS
Newcastle Disease Virus Antibody
Detection
All of the vaccinated chickens (C/V and S/V groups)
presented a positive result for antibody detection by a
commercial ELISA (data not shown; Biorbyt, San Francisco, CA, USA). This test was performed to confirm
that the NDV was effective.

Corticosterone Serum Levels

Heat stress increases corticosterone concentration. Differences were found among the groups in the
corticosterone serum levels Analyzing the interaction
between the factors heat stress and NDV, we observed
that factor stress is significant (P = 0.038). Heat stress

14

19

Groups

IgM (ng/mL)

IgG (ng/mL)

C
HS
C/V
HS/V

147.8
365.9
171.1
337.8

35.2
171.01
54.58
91.721

994.7
1,516
1,388
1,939

196.2
374.2
355.6
579.62,3

C
HS
C/V
HS/V

693.3
849.9
544.7
704.3

130.6
202.3
177.0
295.5

1,888
2,272
1,321
1,313

534.2
752.9
404.2
137.4

C
HS
C/V
HS/V

524
788.8
466
892.2

208.0
285.7
153.2
251.0

2,544
2,169
1,635
1,049

702.5
947.9
518.2
408.52

P < 0.05 in relation of C group of each ED.

P < 0.05 in relation of C group of each ED.
3
P = 0.015 for interaction between HS and NDV.
1
2

(HS and HS/V groups) increased corticosterone serum

levels relative to that of chickens kept in a thermoneutral environment (C and C/V groups, P = 0.027) on
ED7. Corticosterone levels at ED7 are represented in
Figure 1. However, no significant differences were observed in the corticosterone serum levels among the different groups on ED14 and ED19 (P > 0.05) (data not
shown).

Plasma IgM and IgG Determination

Heat stress increased IgM and IgG on ED07,
and decreased IgG on ED19. Table 1 shows the immunoglobulin data. An interaction between heat stress
and NDV was observed in IgG results (P = 0.015) in
ED7 and ED19; and no interactions were observed for
IgM plasma levels for all time points (P = 0.31). However, heat-stressed chickens (HS and HS/V groups) presented an increase in IgM serum levels relative to the
chickens kept in a thermoneutral environment (C and
C/V groups) (P = 0.012) at ED7. A higher concentration of IgG was observed in birds in the HS/V group

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and the leukocyte bottom was resuspended in 1 mL

PBS. Leukocytes of each animal were incubated for
30 min in the dark with specific antibodies (Southern
Biotechnology Associates, Birmingham, AL, USA) for
each cell type analyzed: anti-CD3-fluorescein isothiocyanate (FITC; total T cells), anti-CD4-PE (T helper
lymphocytes), anti-CD8-PE (cytotoxic/suppressor T
lymphocytes) and anti-Bu1-PE (B-lymphocytes). After
incubation, samples were washed twice using Hanks
balanced salt solution following by centrifugation
(500 g/5 min/8 C). For each sample analyzed in the
flow cytometer (FACSCalibur, Becton Dickinson Immunocytometry Systems, San Jose, CA), 30,000 events
were counted using the CellQuest software (Becton,
Dickinson and Company, Franklin Lakes, NJ, USA).
Supplementary material 1 and 2 show a typical side
scatter and specific antibody dotplot of peripheral
chicken blood leucocytes and a histogram of the fluorescence of the analyzed cell populations of antiCD3-FITC (A), anti-CD4-PE (B), anti-CD8-PE (C),
and anti-Bu1-PE (D). The data are expressed as the
percentage of cells in the different populations studied, which were analyzed using the software FlowJo
(Treestar, Inc., San Carlos, CA).

2378

HONDA ET AL.

compared with those in the control group (C) (P =

0.015) on the same day (ED7). No differences in IgM
or IgG were observed among the groups on ED14 (P >
0.05). Significant differences in the IgM concentrations
were not observed among the groups on ED19 (P =
0.77); however, the IgG serum levels were lower on
ED19 in chickens in the HS/V group compared with
those in the C group (P = 0.021).

B Lymphocytes (Anti-Bu1) in Peripheral

Blood

T Lymphocytes (Anti-CD3)
Heat stress increases and vaccination acutely reduces T lymphocytes (total) in peripheral blood.
As presented in Figure 2, heat stress (HS and HS/V
groups) increased the percentage of T-lymphocytes in
chickens relative to those in the non-stressed groups (C
and C/V groups) (P = 0.032) on ED7. However, no
significant difference was observed in the CD3+ phenotyping on ED14. On ED19, vaccinated chickens (C/V
and HS/V) presented a decreased percentage of Tlymphocytes compared with the non-vaccinated chickens (C and HS groups) (P = 0.038). No interactions
between heat stress and NCV were observed for CD3+
at ED7, ED14, and ED19 (P = 0.41, P = 0.15, and
P = 0.09, respectively).

T Helper Lymphocytes (Anti-CD4)

Heat stress increases and vaccination reduces
T helper lymphocytes in peripheral blood. No interactions between heat stress and NCV were observed
for CD4+ at ED7, ED14, and ED19 (P = 0.32, P =
0.92, and P = 0.45, respectively). However, the HS per
se increased the percentage of T helper lymphocytes
(CD4+) compared with the nonstressed groups (C and
C/V groups) (P = 0.045) on ED7. On ED14 and ED19,
vaccinated chickens (C/V and HS/V groups) presented
a decreased percentage of T helper lymphocytes rela-

T Cytotoxic/Suppressor Lymphocytes
(Anti-CD8)
Heat stress increases T cytotoxic/suppressor
lymphocytes in peripheral blood. As depicted in
Figure 2, heat-stressed chickens (HS) presented an increased percentage of T cytotoxic lymphocytes (CD8+)
compared with chickens in the C/V group on ED7
(P = 0.0152). The HS/V group showed an increase
of the percentage of T cytotoxic lymphocytes in
relation to animals in the C/V group on ED14
(P = 0.021). Significant differences were not found
among the groups on ED19. No interactions between the factors HS and NDC were observed at
ED7, ED14, and ED19 (P = 0.72, P = 0.88, and
P = 0.73, respectively).

DISCUSSION
Stress is a reality in poultry production. The hallmark of papers on stress and immune system usually
refers to the occurrence of immunosuppression with
subsequent failures in immune system activity development, particularly when the animals are challenged
with vaccines, pathogenic viruses, bacteria, and other
microorganisms. (Wilder, 1995; Johnson, 1998; Shini
et al., 2008; Shini and Kaiser, 2009; Costa-Pinto and
Palermo-Neto, 2010; Kaiser, 2010; Kokaia et al., 2012).
One of the most important systems that integrates
the body during stressful situations and/or homeostatic disturbances is the hypothalamuspituitary
adrenal axis (Besedovsky and del Rey, 1996; McEwen,
2000).
Corticosterone is the keystone hormone that is
released in stress situations by the hypothalamus
pituitaryadrenal axis; this glucocorticoid has been
shown to dysregulate immune system responses and is
potentially harmful to the health of an organism if the
high hormonal levels remain increased for a long period
of time (Post et al., 2003). Indeed, an inverse correlation
was reported to exist between serum corticosterone levels after heat stress and immune response to pathogenic
challenges in chickens (Elenkov et al., 2000; QuinteiroFilho et al., 2010; Shini et al., 2010; Calefi et al., 2014).
These data agree with and reinforce the findings reported in this study: heat stress (38 2 C from ED2 to
ED6) increased the costicosterone plasmatic levels and
dysregulated both the lymphocyte phenotype characteristics and total antibody production in broiler chickens. However, Newcastle disease vaccination per se was
unable to change the corticosterone serum levels; consequently, it may not be taken as a stressor for the
chickens in our study model.
According to Post et al. (2003), high plasma corticosterone concentrations in chickens enable a de-

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Heat stress reduces B lymphocytes in peripheral

blood. The interaction between heat stress and NDV
was observed in B lymphocytes cells at ED14 (P =
0.012); however, no interactions were observed at ED7
and ED19 (P = 0.76 and P = 0.10, respectively). As depicted in Figure 2, heat stress reduced the percentage
of B-lymphocytes in chickens in the HS/V group compared with those in the C/V group (P = 0.034) at ED7.
Interestingly, C/V chickens presented a higher percentage of B-lymphocytes than did those in the HS/V (P =
0.010) and C groups (P = 0.017) on ED14, and no differences were found on this day between the HS/V and
C groups. Similarly, birds in the C/V group presented
increased B-lymphocyte percentages relative to those of
the C group on ED19 (P = 0.041) and a tendency to
increase compared with HS/V (P = 0.069).

tive to the nonvaccinated broiler chickens (C and HS

groups) (P = 0.00165). Data are presented in Figure 2.

NEWCASTLE-VACCINATION AND HEAT STRESS

2379

crease in the formation of antibodies against SRBC.

These findings were interpreted as a consequence of
corticosterone-induced humoral immunity depression
(Edens and Siegel, 1975; Khansari et al., 1990; Mashaly
et al., 2004).
On the other hand, increased levels of IgM were observed in heat-stressed chickens at ED7. Similarly, a
higher IgG serum concentration was observed in heatstressed and vaccinated chickens at ED7. These findings
agree with the data reported by Zhao et al. (2013), who
noted that cold stress increased the IgG concentration
in broiler chickens. Moreover, it was shown that the IgA
and IgM plasma levels were increased in broiler chickens submitted to overcrowding stress (Gomes et al.,
2014). Together, they strongly suggest that stressors
are able to increase the immunoglobulin levels at least
24 h after the end of stress stimuli application. However,
the present results also showed that vaccinated birds
(groups C/V and HS/V) presented a decrease in IgG
serum levels at ED19. This fact may be explained by
the data reported by Nasrin et al. (2013), who showed
that heat stressed and vaccinated chickens (NDV) presented increased IgG concentrations in the trachea, caecal tonsils, and Harderian gland. In other words, the
concentration of IgG in stressed birds is increased in
lymphoid tissues/organs, where the immune system activation occurs, but not in the peripheral blood.
The present findings suggest that heat stress might
guide the immune profile of chickens to a higher per-

centage of T-lymphocytes (total, helper, and cytotoxic/suppressor), simultaneously reducing the percentage of B-lymphocytes in the peripheral blood. Khajavi
et al. (2003) also observed that heat-stressed chickens
(39 C, 7 h/d for 6 d) presented a reduction in the number of T helper and T cytotoxic/suppressor lymphocytes and a simultaneous decrease in antibody titers
against SRBC. Zulkifli and Siegel (1994) showed that
the application of stressors in young chickens increased
the lymphocyte in peripheral blood, a fact that agrees
with our presently reported findings.
The current data showed that vaccinated chickens (C/V) presented the highest percentage of Blymphocytes and a decreased percentage of T helper
and T cytotoxic/suppressor lymphocytes on ED14 and
ED19, which was a somewhat expected phenomenon
because NDV vaccinated chickens predominantly develop humoral immunity (Al-Zubeedy, 2009). These
findings might be taken as a consequence of the recognition of virus epitopes, virus neutralization, and/or
the development of cellular memory. However, our results importantly showed that chickens that received
the vaccine and were heat-stressed (HS/V group) presented a decreased percentage of B-lymphocytes in relation to those of nonstressed chickens (C/V group);
in other words, heat stress application may have
changed the type of immune response developed by
the chickens. This idea appears to be reinforced by the
observed decrease of IgG levels in chickens in the

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Figure 2. Effects of heat stress and vaccination on the percentage of B-lymphocytes (anti-Bu1-PE), T lymphocytes (anti-CD3-FITC), T
cytotoxic/suppressor lymphocytes (anti-CD8-PE), and T helper lymphocytes (anti-CD4-PE) in peripheral blood. The analyses were independently
performed 3 times at different time points (ED7, ED14, and ED19). No comparisons were performed between the different time points. Different
letters above the columns indicate significant differences at P < 0.05 (ANOVA and Tukey post-test).

2380

HONDA ET AL.

HS/V group at ED19, which also shows the persistence of heat stress effects on vaccinated chickens. Together, these results point towards a stress-induced reduction in humoral activity against Newcastle disease
vaccination, i.e., a decrease in immunological memory
development.
Indeed, stressed chickens are known to be more susceptible to infection by NDV because they present a
reduced immunological response to the vaccine and are
thus more susceptible to real viral challenges (Mohamed
and Hanson, 1980; Njagi et al., 2012). Taking together,
the present data showed that heat stress in the early
life of broiler chickens impacts immune system development and activity for a long period of time, thus
impairing vaccine responses and consequently the response to pathogenic environmental challenge.

This study was supported by S

ao Paulo Research Foundation (FAPESP) under Grant Numbers
2009/51886-3, 2013/17408-2 and 2013/13736-5 and by
National Counsel of Technological and Scientific Development (CNPq) under Grant Numbers 300764/2010-3
and 474690/2013-0.

SUPPLEMENTARY DATA
Supplementary data is available at PSA Journal
online.

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