Bull vet Inst Pulawy 56, 133-137, 2012
DOI: 10.2478/v10213-012-0024-2
DEVELOPMENT OF EARLY HUMORAL
AND CELL-MEDIATED IMMUNITY
IN PIGLETS WITH EXPERIMENTALLY INDUCED
SUBCLINICAL SWINE INFLUENZA
MAŁGORZATA POMORSKA-MÓL, IWONA MARKOWSKA-DANIEL,
1
AND JAROSŁAW RACHUBIK
Department of Swine Diseases, 1Department of Radiobiology,
National Veterinary Research Institute, 24-100 Pulawy, Poland
[email protected]
Received: February 22, 2012
Accepted: May 8, 2012
Abstract
Development of early immune response in piglets with subclinical swine influenza was investigated. Fourteen, seronegative
piglets were used. Ten of them were infected intranasally with swine influenza virus (SIV) H1N1 subtype. Temperature and clinical
signs were assessed daily. Leukocyte proportions and concentrations were analysed on a haematology analyser. Antibodies against
SIVs were measured by haemagglutination inhibition assay. To measure influenza-specific cell-mediated immunity (CMI), the
proliferation assay was performed. The real time reverse transcription PCR method was used for detection of SIV. No relevant
respiratory or systemic clinical signs were observed. The presence of SIV RNA in nasal swabs from all infected piglets was
confirmed between 2 and 5 dpi. The overall number of leukocytes did not differ during the study. The number of medium-sized cells
(MID) was significantly higher on 2 and 4 dpi, as compared to day 0 level. The percentage of lymphocytes decreased from 74% on
day 0 to 67.06% on 4 dpi, while the percentage of MID significantly increased at the same time. In control pigs no significant
changes were observed. All infected pigs exhibited specific antibodies between 7 and 10 dpi. Specific CMI was observed before
specific antibodies were present. Results of our research indicate that kinetics of the humoral and CMI response during subclinical
infection is similar to that observed in clinical form of the disease.
Key words: piglets, H1N1 swine influenza virus, subclinical infection, immune response.
Swine influenza (SI) is a highly contagious
respiratory disease caused by various subtypes of swine
influenza viruses (SIVs) (10, 15, 19). At present, three
main subtypes of SIVs are circulating in the swine
population throughout the world: H1N1, H1N2, and
H3N2, (1, 14, 15, 21). Typical SI outbreaks are
characterised by a rapid onset of high fever, loss of
appetite, laboured abdominal breathing, and coughing.
Mortality is low and recovery occurs within 7-10 d (10,
19). However, the infection is much more frequent than
the disease. Infection with swine influenza H1N1 virus
is frequently subclinical and typical signs are often
demonstrated only in 25% to 30% of a herd (1, 2).
While data regarding immune response during
acute course of SI in pigs are available, there is lack of
data concerning development of humoral and T-cell
response in pigs with subclinical infection. Therefore,
the aim of the present study was to investigate the
development of humoral and cell-mediated immunity in
piglets with experimentally induced subclinical swine
influenza (subSI), caused by H1N1 subtype of SIV.
Additionally, the changes in the white blood cell
populations during infection were assessed.
Material and Methods
Animals. Fourteen 7-week-old piglets of both
sexes were sourced from high health status herd and
prior to the start of the study were shown to be both
influenza A virus and antibody (subtypes H1N1, H1N2,
H3N2, pH1N12009) negative by Matrix (M) gene real
time reverse transcription PCR method (RRT-PCR) and
haemagglutination inhibition assay (HI), respectively.
Animal use and handling protocols were approved by
the Local Ethical Commission. Infected piglets were
housed separately from control ones.
Preparation of virus inoculum and challenge.
Swine influenza virus A/sw/Poland/KPR9/2004
(subtype H1N1) (hereafter referred to as SwH1N1),
which had been isolated from the lungs of fattening pig,
was used for experimental infection. The stock used for
inoculation represented the third passage in eggs. The
virus titre was evaluated in Madin-Darby canine kidney
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(MDCK) cells. Ten piglets were inoculated intranasally
with 3 ml of viral suspension containing 107.3 TCID50
SwH1N1/mL. Four piglets inoculated with PBS served
as controls.
Experimental design. On day 0, ten piglets
were inoculated with SwH1N1. In order to examine the
events taking place at the early stages of infection with
SwH1N1, two infected and one control piglets were
euthanised on days 2 and 4 post infection (dpi). The
remaining piglets were euthanised on 10 dpi. Necropsy
was performed immediately after the animals were
euthanised.
Clinical and postmortem examination. Rectal
temperatures were assessed daily and clinical signs of
disease were recorded. Fever was defined as body
temperature of >40°C.
Blood samples were collected on 0 (challenge),
1, 2, 3, 5, 7, and 10 dpi. Serum was harvested after
centrifugation and stored at -20ºC for further analyses.
Nasal swabs were taken on 2, 3, 4, 5, 7, and 10 dpi.
Complete necropsy was done on each animal, with
special emphasis on the respiratory tract. Gross lung
lesions were assessed for the presence or absence of
pulmonary cranioventral multifocal consolidation. Lung
samples and tracheas were collected aseptically and
frozen at -80°C until their use for viral RNA extraction.
PCR. The general swine influenza A RRT-PCR
method (the “perfect match” M gene RRT PCR) was
used for detection of SIV in nasal swabs, tracheas and
lung samples, as described previously (20). Samples
with Ct value <30 were considered to be M gene
positive, samples with Ct value 30-35 with
sigmoidal/logarithmic appearance were considered to be
weakly positive, samples with Ct value >35 were
considered to be negative.
Haematological examination. Whole blood
samples were analysed for different leukocyte
proportions and concentrations on a Abacus Junior Vet 5
haematology analyser (Diatron, Hungary). Proportions
of lymphocytes, medium-sized cells (MID) (represented
mainly by monocytes), and granulocytes were calculated
as a percentage of leukocyte concentration.
Humoral immune response. Antibodies
against SIVs were measured using a haemagglutination
inhibition (HI) assay. The HI assay was performed
according to the standard procedure, using 0.5% chicken
erythrocytes and 4HA units of strains H1N1 used for
inoculation (SwH1N1 virus). All sera were tested in
serial twofold dilutions, starting from 1:20. To estimate
the antibodies prevalence, titres equal or higher than 20
were considered positive.
Proliferation assay (PA). The proliferation
assay, used to measure influenza-specific T-cell
responses was performed on 0, 2, 4, 7, and 10 dpi, as
described for pseudorabies virus (9). Briefly, PBMC
were isolated from blood samples by centrifugation on
Histopaque 1.077 (Sigma, USA) and were washed twice
with PBS. The isolated PBMC were seeded in plastic
vials at a density of 1 x 106 viable cells per vial in 1 ml
medium (RPMI 1640 containing 10% foetal bovine
serum, 2 mM L-glutamine and 1% of antibiotic-
antimycotic solution). PBMC were restimulated in vitro
with 50 µl of medium containing live SwH1N1 virus
(titer 106 TCID50). In control vials, the cells were
incubated without the virus (mock-control) or with 5
µg/mL of concanavalin A (Con-A) (vitality control). All
samples were analysed in triplicate.
After 72 h of incubation at 37ºC in 5% CO2
atmosphere, the cultures were pulsed with 0.5 µCi [3H]thymidine (MP Biomedicals, USA). After 18 h of
incubation, the cells were harvested and the incorporated
radioactivity was measured in a liquid scintillation
counter (Tri-Carb 2500TR, Packard, USA). Proliferation
was expressed as a stimulation index (SIx). The SI was
calculated as the number of counts per minute (cpm) of
SwH1N1 restimulated PBMC divided by the number of
cpm of the mock-control-stimulated cells (in each cases
taking mean of triplicate vials).
Based on the SIx values observed before
infection (day 0) and in control animals, SIx values
pointing to influenza-specific proliferation were
established (as mean plus 3 x standard deviation). SIx
values ≥1.55 were considered to be positive.
Statistical analysis. The obtained data were
subjected to the W. Shapiro-Wilk’s test of normality and
the Levene’a test of equal variances. A nonparametric
Kruskal-Wallis test with post hoc multiple comparisons
for comparison of all pairs was used for comparison of
investigated parameters. Differences with α <0.05 were
considered as significant. All calculations were
performed with the Statistica 8.0 (Statsoft, Poland)
computer programme.
Results
Clinical signs. No relevant respiratory or
systemic clinical signs were observed in infected piglets.
However, five animals showed transient abnormal rectal
temperatures (>40°C) between days 1 and 3 post
infection. The general condition of the piglets and feed
intake were unchanged during the whole period of the
experiment.
Postmortem
examination.
Postmortem
examination revealed typical lesions, deriving from SIV
infection, in the lungs of eight out of ten infected piglets.
One pig euthanised on 2 dpi and one euthanised on 4 dpi
had no macroscopic changes in the lungs.
Presence of RNA of SIV in swabs and tissues
No SIV genetic material was found in the nasal swabs
taken before inoculation. M gene RRT-PCR assay, used
to confirm the presence of SIV in the nasal swabs
revealed positive results from all infected pigs between
2 and 5 dpi. No viral RNA was detected in swabs on 7
and 10 dpi. The presence of SIV RNA was also analysed
in the trachea and lungs. In all infected pigs, euthanised
on 2 and 4 dpi, the presence of SIV were confirmed for
both the trachea and lungs. No viral RNA was detected
in the lungs on 10 dpi as well as in the lungs of
uninfected pigs.
Haematological examination. The results of
the presented study demonstrated that the overall
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number of leukocytes did not differ significantly during
the study and ranged from 19.23 to 21.28 10 9/L
(Kruskal-Wallis, P>0.05). The absolute and relative
number of lymphocytes, MID, and granulocytes are
shown in Figs 1 and 2, respectively. The number of MID
was significantly higher (Kruskal-Wallis, P<0.05) on 2
and 4 dpi, as compared to day 0 level. The increase was
from 0.1 ±0.01 on day 0 to 0.46 ±0.16 and to 1.06 ± 0.1,
on 2 and 4 dpi, respectively. The percentage of
lymphocytes decreased from 74 ±1.84% on day 0 to
67.06 ±2.39% on 4 dpi (Kruskal-Wallis, P<0.05), while
the percentage of MID significantly increased at the
same time. The absolute and relative size of
granulocytes remained stable during the experiment
(Kruskal-Wallis,
P>0.05).
The
absolute
lymphocyte/MID ratio (Fig. 3.) decreased from 2 dpi
and remained significantly lower to 4 dpi (KruskalWallis, P<0.05). It was also true for relative
lymphocyte/MID ratio. In control piglets no significant
changes were observed.
Antibody response against SwH1N1. All
infected
piglets
exhibited
antibodies
against
haemagglutinin (anti-HA) in the serum, between 7 and
10 dpi. (Table 1). Sera from piglets in the control group
had no antibody titres (<20 HI titre). All uninfected
piglets remained seronegative to SwH1N1 till the end of
the experiment.
In vitro cellular response. The mean SIx
values in control piglets and piglets from experimental
group before inoculation (0 dpi) ranged from 0.71 to
1.16. Four days post infection in five out of eight
infected piglets an individual SIx was higher than 1.55.
Starting from 7 dpi all infected piglets developed an
antigen-specific proliferation. The mean SIx values
(±SD) in infected piglets are shown in Fig. 4.
Disscusion
In the presented study, the development of
antigen-specific humoral and cell–mediated immunity,
and changes in the white blood cell populations in
piglets with experimentally induced subSI were
investigated during the first 10 dpi.
***
**
Fig. 1. The absolute number of lymphocytes, MID, and granulocytes in infected piglets.
* P<0.05, ** P<0.01, *** P<0.001 - significance of differences as compared to day 0 level.
**
***
*
***
Fig. 2. The relative number of lymphocytes, MID, and granulocytes in infected piglets.
* P<0.05, ** P<0.01, *** P<0.001 - significance of differences as compared to day 0 level.
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136
**
***
Fig. 3. The absolute lymphocyte/MID ratio in infected
piglets.
* P<0.05, ** P<0.01, *** P<0.001 - significance of
differences as compared to day 0 level.
Stimulation index
10,85
10.85
9.30
9,30
7.74
7,75
6.20
6,20
4,65
4.65
3.10
3,10
1.55
1,55
0.00
0,00
0
2
4
7
10
dpi
Fig. 4. The mean (±SD) value of stimulation index in
infected piglets. The dashed line indicates value
considered as antigen-specific proliferation.
Table 1
Individual haemagglutination inhibition titres
in sera of infected piglets
No. of animal
dpi
1
0
-
5
-
7
20
10
80
2
-
-
-
40
3
-
-
-
20
4
-
-
40
160
5
-
-
20
80
6
-
-
-
20
Titres below 20 were assigned as (-).
Since clinical signs have only been reported
when intratracheal inoculation was performed (2), in our
study, the piglets were infected by intranasal rout.
Subclinical course of swine influenza is very common in
the field (1, 2), but data on the immune response during
subSI are currently lacking.
In the present study, a significant decrease in
relative number of lymphocytes and relative and
absolute number of MID, with no accompanying
leukopenia, were found between 2 and 4 dpi in all
infected piglets. There was almost a 10% drop in the
mean relative number of lymphocytes between 0 and 4
dpi, while total number of white blood cells remained
unchanged. Additionally, an over 300% increase in the
mean number of MID cells was observed between 0 and
2 dpi. On 4 dpi, the mean number of MID was up to 10
times higher than before infection. The mean number of
MID and percentage of lymphocytes returned to normal
values after 7 dpi. Previously, relative lymphopenia has
been found to be an early and reliable laboratory finding
of adult human patients with influenza A (3, 18).
In humans with positive test results for H1N1 a
relative lymphopenia is common, but leukopenia is not
present, which is in accordance to our findings.
Induction of lymphocyte apoptosis by death receptor
ligands including TNF-α and TNF-related apoptosisinducing ligand (TRAIL) may play an important role in
lymphopenia associated with influenza infection (22). It
was proposed in human medicine that relative
lymphopenia might be a marker for H1N1, and thus it
could also be used to prioritise H1N1 PCR testing, if the
emergency department’s ability is exceeded (4). The
peripheral lymphopenia was also reported in humans
infected with highly pathogenic avian influenza H5N1
(7). More studies are needed to evaluate if the same
diagnostic value of drop in lymphocyte relative number
may be proposed in pigs.
Increasing number of monocytes was
previously reported in human patients with H1N1 virus
infection (17). Monocytes and macrophages appear to
play a protective role during influenza virus infection, as
it was shown on a mice model (13). Blood monocytes,
or macrophages derived from blood monocytes,
recruited to the lungs, participate in the early host
response to influenza. An important beneficial activity
of monocytes/macrophages during influenza infection is
the phagocytosis of virus-infected apoptotic cells, which
correlates with clearance of the virus from the lungs (5).
Depletion of macrophages from pigs has been shown to
impair the response to influenza virus (8).
The adaptive immune response helps to restrict
the viral spread, eradicate virus, and finally to establish a
memory response resulting in a long-lived resistance to
re-infection with homologous virus; however, it requires
some days to be effective. Influenza infection induce
both systemic and local antibodies, as well as cytotoxic
T cell responses (11, 19). Serum HI antibodies are the
most commonly measured to determine the level of
protection against influenza. Usually, they can be
detected from 7-10 dpi. Peak titres are usually seen
between 14 and 21 dpi (6, 11).
In the present study, the HA-specific antibodies
in the serum of piglets with subclinical infection were
also detected from 7 dpi, and the HI titres increased over
time.
Regarding the cellular immune response, our
result showed that SIV – specific proliferation appeared
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137
very early, on 4 dpi (in five out of eight infected piglets).
Starting from 7 dpi an antigen-specific response was
noted in all infected animals and increased over time.
This evolution generally agrees with the result of Larsen
et al. (11). These authors found that antigen-specific Tcell response in pigs with acute influenza could be
detected from 7 dpi reaching peak levels on 14 dpi.
T lymphocyte influenza-specific responses in humans
also peak at about day 14 post infection (12). Cell
mediated immune response plays a role in recovery from
influenza infection and may prevent influenza-related
complications (19). Moreover, the level of SIV-specific
T lymphocytes correlates with the speed of viral
clearance from the respiratory tract (16).
In summary, this study defines the development
and kinetics of early immune responses to H1N1
influenza virus during subclinical infection of pigs.
Results of our research indicate that kinetics of the
humoral as well as T-cell immune response during
subclinical infection is similar to that observed in the
clinical form of the disease (11). The T-cell specific
immunity was observed before specific antibodies were
present. More studies are needed to evaluate the
significance of our findings on a decrease in the
percentage of lymphocytes with corresponding increase
in absolute and relative number of monocytes in the
diagnostics of influenza in pigs.
Acknowledgments:
This
work
was
supported by Project N N308 235938 founded by the
Ministry of Science and Higher Education.
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