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Veterinary Immunology and Immunopathology 165 (2015) 119126

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Veterinary Immunology and Immunopathology


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

Research Paper

Assessment of immune response in periparturient dairy cows


using ex vivo whole blood stimulation assay with
lipopolysaccharides and carrageenan skin test
N. Jahan a,b , A. Minuti a,c , E. Trevisi a,c,
a
Istituto di Zootecnica, Facolt di scienze Agrarie, Alimentari e Ambientali Universit Cattolica del Sacro Cuore, via Emilia Parmense 84,
29122 Piacenza, Italy
b
IUBAT International University of Business Agriculture and Technology, 4-Embankment Drive Road (Off Dhaka-Ashulia Road),
Sector-10, Uttara Model-Town, Dhaka 1230, Bangladesh
c
Centro di Ricerca sulla Proteomica e Nutrigenomica PRONUTRIGEN, Universit Cattolica del Sacro Cuore, via Emilia Parmense 84,
29122 Piacenza, Italy

a r t i c l e

i n f o

Article history:
Received 13 October 2014
Received in revised form 2 March 2015
Accepted 1 April 2015
Keywords:
Dairy cows
Transition period
Whole blood stimulation assay
Carrageenan skin test
Pro-inammatory cytokines

a b s t r a c t
The transition period is known to be the most critical phase in the life of high yielding dairy
cow. Changes in the immune functions have been observed during the transition period
which may account for the onset of clinical and subclinical (e.g. inammatory response)
problems at calving or at the beginning of lactation however this relationship has not
yet been adequately investigated. Thus, to establish the potential of the periparturient
dairy cows immune system to respond to stimuli, two challenges [an ex vivo whole blood
stimulation assay (WBA) with lipopolysaccharides and a carrageenan skin test (CST)] were
performed in addition to characterizing the metabolic and inammatory prole. The WBA
was performed using 0, 0.01 and 5 g LPS/mL on whole blood and CST was administered by
subcutaneous injection of 0.7 mL solution containing 4.2 mg of carrageenan to the shoulder
region of the cows. These tests were performed on 10 Holstein-Friesian cows at 45 2,
20 2, 3, 3, 7, 28 2 days from parturition (DFP). Cows were also monitored for health
status, body condition score, milk yield. The results demonstrate a higher production of
IL-1 and IL-6 from leukocytes after LPS stimulation around calving (from 3 to 3 DFP)
compared to 45 DFP (P < 0.05). Moreover, IL-6 (but not IL-1) was able to reach close to
the maximum response at the lower stimulus intensity (0.01 g LPS/mL), maintaining a
higher response over a longer time in early lactation. The release of higher levels of IL-6
in the transition period, with low LPS dose, suggests its crucial role in the regulation of
inammatory response around calving. The response of cows to CST decreased a few days
before calving (3 DFP) compared with response at 45 and 28 DFP (P < 0.05), and remained
low in the rst week of lactation. This result suggests the reduction of the functionality of
some vascular factors, which decreases diapedesis. Overall, the WBA and CST tests conrm changes in immunocompetence around calving. These tests are able to better describe
the changes of the innate immune response at a local and systemic level, mainly when
combined with conventional metabolic and inammatory indices.
2015 Elsevier B.V. All rights reserved.

Abbreviations: WBA, whole blood stimulation assay; CST, carrageenan skin test; DFP, days from parturition; PIC, pro-inammatory cytokines.
Corresponding author. Present address: Istituto di Zootecnica, Facolt di scienze Agrarie, Alimentari e Ambientali, Universit Cattolica del Sacro Cuore,
via Emilia Parmense 84, 29122 Piacenza, Italy. Tel.: +39 0523 599279; fax: +39 0523 599276.
E-mail address: erminio.trevisi@unicatt.it (E. Trevisi).
http://dx.doi.org/10.1016/j.vetimm.2015.04.003
0165-2427/ 2015 Elsevier B.V. All rights reserved.

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N. Jahan et al. / Veterinary Immunology and Immunopathology 165 (2015) 119126

1. Introduction
The primary function of the immune system is
to provide the host with a defense against invading
pathogenic organisms and as such, any changes in its efciency can be responsible for the prevalence of disease.
In this respect the transition or peripartum period (23
weeks prior to and immediately following parturition) is
widely recognized as a critical time for the health, productivity and protability of dairy cows (Drackley, 1999;
Van Knegsel et al., 2014) because several metabolic and
infectious diseases such as milk fever, metritis, acidosis,
mastitis, and lameness are very common at this time. During peripartum, cows are characterized by neutrophilia,
eosinopenia, lymphopenia, and monocytosis (Meglia et al.,
2001; Guidry et al., 1976). Despite the increasing number of circulating leucocytes during the transition period,
alteration of the immune and innate host resistance mechanism has been observed (Mallard et al., 1998) with an
overall decline in the ability to mount an immune response
(Goff and Horst, 1997; Lacetera et al., 2005). The changes
that compromise the functionality of the immune system have been associated with a higher incidence of
infectious diseases (Goff and Horst, 1997; Meglia et al.,
2001).
The ex vivo whole blood stimulation assay with LPS
(WBA) has been proven to be a useful tool to evaluate the
capacity, of leucocytes from several species, to secrete proinammatory cytokines (PIC) (Finch-Arietta and Cochran,
1991; Foster et al., 1993; Carstensen et al., 2005). In dairy
cows Rntved et al. (2005) used the WBA and established
this test as a fast and reliable method for repeated measurements of TNF- responsiveness in high yielding dairy
cows. Thus, we believe that PIC production during WBA can
indicate the potential pro-inammatory effect of an inammatory challenge and the ability of a subject to mount an
effective immune response.
Carrageenan, a high-molecular-weight sulphated
polysaccharide, is commonly used to induce an aseptic
inammation and to test the efcacy of non-steroidal
anti-inammatory drugs in laboratory animals (Winter
et al., 1962; Amdekar et al., 2012). Using this approach,
King (1993) used intradermal injection of carrageenan in
calves and suggested CST as a simple, humane and useful
model for dose estimation of new NSAID in ruminants.
Other authors (Di Rosa, 1972; Thomson and Fowler, 1981;
Nicklin and Miller, 1984) reported that the carrageenan
skin test (CST) can stimulate the peripheral immune
system. Similarly, Lacetera et al. (1999) and Agazzi et al.
(2007) used phytohaemagglutinin injections to assess the
immune response. The increase of the skin thickness after
injections of phytohaemagglutinin and carrageenan has
been interpreted in animals as an index associated with a
greater cell mediated immune response at the peripheral
level.
According with the above information, we hypothesize
that WBA and CST tests can be valuable tools to investigate functionality of the immune system in periparturient
dairy cows. This study is aimed at assessing the functionality of the immune system during the transition period
of the dairy cow using CST, and WBA, to determine any

association between these responses with metabolic and


inammatory blood parameters.
2. Materials and methods
2.1. Animals and diets
This study complied with Italian laws on animal experimentation (DL n.116, 27/01/1992) and ethics.
The study was carried out at the experimental dairy
farm of Universit Cattolica del Sacro Cuore (Piacenza,
Italy), using 10 multiparous (from 2 to 6 parity) Holstein
Friesian cows in the transition period. Cows were housed in
an articially lighted and ventilated tie-stall. Climatic conditions were maintained under almost constant settings:
environmental temperature around 20 C, relative humidity between 60% and 70% and photoperiod with 14 h of light
(from 5:00 to 19:00 h) and 10 h of dark. Cows were individually fed and diet was offered ad libitum: forages every
12 h and concentrates by an auto-feeder every 12 or 3 h in
dry and lactating phases, respectively. Details of the diets
(ingredients and main chemical composition) are reported
in supplementary materials (Table S1). On average, cows
received 912 kg of grass hay, 810 kg of maize-silage and
12 kg of concentrate per day. After calving, the daily diet
included 2 kg of grass hay and 3 kg of alfalfa hay, while concentrate and maize-silage were gradually increased (on day
30 of lactation, the cows received on average 1113 kg of
concentrate and 1820 kg of maize-silage).
2.2. Milk yield, body condition score and health problems
During the experimental period, the animals health
condition was checked every day by individual inspections. Rectal temperature was also measured daily in the
morning. The milk yield of individual cows was routinely
weighted at each milking during the experimental period.
Body condition score (BCS) was individually evaluated fortnightly, by the same observer, according to a ve-point
scale (ADAS, 1986).
2.3. Experimental schedule
The experimental days for the study were 45 2,
20 2, 3 1, 3, 7, 28 days from parturition (DFP). The
experimental days after calving corresponded to the real
DFP, while experimental days before calving were selected
according to the expected day of calving and consequently
with little variability. WBA with LPS was performed on
all experimental days whereas, because this test requires
9 days to complete the measurements CST was not performed at 3 DFP, to ensure no overlapping with the
challenges undertaken at 3 and 7 DFP.
2.4. Blood sampling and analyses of metabolic and
inammatory status
To assess the metabolic and inammatory status blood
samples were collected before feeding in the morning on all experimental days. Blood was collected from
the jugular vein into evacuated 10 mL tubes containing

N. Jahan et al. / Veterinary Immunology and Immunopathology 165 (2015) 119126

lithium-heparin as an anticoagulant (Vacuette, Kremsmnster, Austria) and immediately cooled in ice water. A
small amount of blood was used for packed cell volume
determination (Centrifugette 4203, ALC International srl,
Cologno Monzese, Italy); the remainder was centrifuged
at 3500 g for 15 min at 4 C and the plasma was frozen
at 20 C until further analysis. Plasma samples were
analyzed for glucose, non-esteried fatty acids (NEFA),
-hydroxybutyrate (BHBA), urea, creatinine, haptoglobin,
ceruloplasmin, total proteins, globulins, albumin, paraoxonase (PON), total bilirubin, aspartate amino-transferase
(GOT), -glutamil transferase (GGT), alkaline phosphatase
(ALP), total reactive oxygen metabolites (ROMs), total nitric
oxide metabolites (NOx ), nitrites (NO2 ), nitrates (NO3 ), Ca,
P, Mg, Na, K, Cl, Zn. The analytical procedure and results
through-out the experimental period are reported as support material of the study in the supplementary material.
2.5. Whole blood stimulation assay with LPS (WBA)
During blood sampling an additional 10 mL tube containing lithium-heparin as an anticoagulant (Vacuette,
Kremsmnster, Austria) was collected to perform the WBA.
The tubes were stored in vertical position in a warm bath
at a temperature of 38 C and transported to the laboratory
within 20 min for the stimulation procedure.
The WBA was carry out according to Rntved et al.
(2005). Briey, two LPS solutions of concentration of
0.5 g LPS/mL and 250 g LPS/mL were prepared by
diluting lipopolysaccharides from Escherichia coli O111:B4
(SigmaAldrich Company Ltd., UK, Cat. No. L3012) in Dulbeccos modied Eagle medium (DMEM; SigmaAldrich
Company Ltd., UK, Cat. No. D6046) and stored at 20 C
until the time of stimulation. Then, under the laminar
airow cabinet, 3 aliquots of 980 l of blood from each
cow were transferred in 2 mL tight tubes and stimulated
with 20 l of: DMEM (considered as the negative control samples), 0.5 g LPS/mL and 250 g LPS/mL solutions
respectively. Consequently, the nal stimulation doses
were 0 (CTR), 0.01 (low dose) and 5 g LPS/mL blood (high
dose) respectively. All the samples were positioned on a
rotator within a heated incubator (Grant Boekel, HIR10 M)
set to a temperature of 38 C and a rotation speed of 3
times/minute for 3.5 h. Then the plasma was collected after
centrifugation at 8500 g for 10 min at 4 C and stored at
80 C in 2 fractions for the measurement of IL-1 and IL-6.

121

Cat. No. 98.610) immediately before carrageenan injection


(0 days), then 2 and 9 days after the injection. The overall response of each challenge was calculated as the area
under the curve (AUC) of the thickness measured at day 2
and day 9 subtracting the thickness measured at day 0.
2.7. Plasma cytokines assay
The concentrations of IL-1 and IL-6 were analyzed by
commercial bovine ELISA kit screening set (Pierce, Thermo
Scientic, Meridian Rd., Rockford, USA). Each cytokine concentration was measured in duplicate in each sample. The
intra and inter assay coefcients of variation were 4.5 and
17.0% respectively for IL-1 and the corresponding values
were 3.5 and 13.4% for IL-6.
2.8. Statistical analysis
The fold change of the cytokines produced after low and
high dose of LPS were calculated as the ratio of the concentration measured after the low and high dose of LPS
challenge and the concentration of cytokines measured in
CTR.
Statistical analyses were performed with SAS software
(SAS Inst. Inc., Cary, NC; release 8.0). Each parameter was
tested for normal distribution using UNIVARIATE procedure of SAS and normalized when necessary (AUC of CST,
IL-1, IL-6, fold change of IL-1 and fold change of IL-6),
using natural logarithmic conversion. The data were subjected to ANOVA using the REPEATED statement in the
MIXED procedure of SAS. The statistical model for BCS, rectal temperature and the AUC of CST used DFP (45, 20, 3,
3, 7, 28) as xed factor and the cow as the random effect
(in CST +3 DFP was omitted). The statistical model for the
data of the production of IL-1 and IL-6 in whole blood
after stimulation of LPS were used as xed factors while
DFP (45, 20, 3, 3, 7, 28), dose of LPS (CTR, low dose and
high dose) and their interaction and the cow was the random effect. Each parameter was subjected to 4 covariance
structures: rst order autoregressive, compound symmetry, spatial power and toeplitz, and the best covariance
structure was retained (Littell et al., 1998). The Bonferroni
method was used for multiple comparisons. To investigate
the relationship between the WBA and CST with metabolic
and inammatory parameters, Pearson correlations were
calculated at each time point using the CORR procedure of
SAS. Statistical signicance was established at P < 0.05.

2.6. Carrageenan skin test (CST)


3. Results
The CST was carried out according to King (1993) but
sterile carrageenan (-Carrageenan, Wako Pure Chemical Industries, Japan) was dissolved in saline (instead
phosphate-buffered saline) to obtain a 0.7% solution. The
CST was administered by injecting 0.6 mL of carrageenan
solution under the skin (equal to 4.2 mg of carrageenan
for each treatment) on the shoulder region of the cows.
The injection was made alternatively in the left and right
shoulder, the site of treatment was different for each injection and area of injection was marked by an indelible
marker. The skin thickness was measured using a plicometer (Holtain T/W skinfold caliper, Holtain Ltd., Crymych, UK,

3.1. Production of IL-1 and IL-6 after WBA


Data on the production of IL-1 and IL-6 in the whole
blood after stimulation of LPS are shown in Fig. 1(A) and
(B), respectively and the fold changes in the cytokines production are reported in Table 1. The effect of DFP, dose of
LPS and their interaction were always signicant (P < 0.01)
for both cytokines. However, important differences were
evident from comparing the behavior of the two cytokines
and particularly considering the response to low and high
dose of LPS. The IL-1 showed a dose response effect for

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N. Jahan et al. / Veterinary Immunology and Immunopathology 165 (2015) 119126

Fig. 1. (A) Production of IL-1 (A) and of IL-6 (B) in whole blood during the transition period of dairy cows when stimulated with 0 g LPS/mL blood
(CTR), 0.01 g LPS/mL blood (low dose) and 5 g LPS/mL blood (high dose). Statistical differences between the dose used for stimulation of each days from
parturition (DFP) are marked with letters, a, b, c; treatment dose with no common superscript are signicantly different (P < 0.05).

all the experimental period, with lower concentration for


CTR, medium concentration for low dose and the highest concentration for high dose of LPS (Fig. 1A; P < 0.05).
On the contrary, IL-6 (Fig. 1 B) showed a similar increase
and an analogous fold change for low and high dose of
LPS (P > 0.05), with difference between the LPS stimulated
samples and CTR for all the experimental period (Table 1;
P < 0.01).
Considering the effect of the transition period, the concentrations of IL-1 and IL-6 in CTR were higher for the
whole dry period compared to the 1st month of lactation
(Table 1; P < 0.05). After stimulation with the low dose of
LPS, the production of IL-1 reached the maximum concentrations around calving (Fig. 1A) and the highest values
were found from 3 to 7 DFP (Table 1). After the stimulation with the high dose of LPS, the fold changes for IL-1
were higher from 20 DFP until 28 DFP compared to 45
DFP (P < 0.01) and again the highest values were observed
around calving (from 3 to 7 DFP; Table 1). The response of
IL-6 was similar for low and high dose of LPS and the highest concentration after stimulation was observed at 3 DFP,
while the fold change of IL-6 showed higher values from 3
to 28 DFP compared to 45 DFP (P < 0.01).

No signicant correlations were observed between


the results of WBA and metabolic and inammatory
parameters at 45 and 20 DFP, correlations between the
fold change of IL-1 with high dose of LPS (Fig. 2) and some
metabolic and inammatory parameters were observed
while close to calving and during lactation. In particular,
negative correlations were found between the fold change
of IL-1 at high dose of LPS with plasma zinc and albumin at 3 and 3 DFP respectively, and positive correlations
with bilirubin and non-esteried fatty acid at 7 and 28 DFP
respectively (Fig. 2). These correlations were conrmed
considering the fold change of IL-1 at low dose of LPS
and CTR. No correlation between blood parameters and IL-6
were found.
3.2. Carrageenan skin test (CST)
The results of CST are shown in Fig. 3. A wide variation
in responses was found among cows (16.84 4.42 AUC of
carrageenan response). The response of cows to CST was
high at 20 DFP (P < 0.05 compared with 45 and 3 DFP);
subsequently, the response decreased around calving, particularly from 3 to 7 DFP. Thereafter, the response of CST

Table 1
Effect of the transition period on the concentration of IL-1, IL-6 and fold change calculated as the ratio between the concentration of cytokines after
stimulation with low (0.01 g LPS/mL blood) or high (5 g LPS/mL blood) dose of LPS and CTR (0 g LPS/mL blood), during the transition period of dairy
cows. Statistically signicant difference in each days from parturition (DFP) with 45 DFP are marked as *P < 0.05; **P < 0.01.
Days from parturition

SEM

45

20

IL-1
CTR (pg/mL)
Low (fold change)
High (fold change)

222
4.8
12.9

254
8.1
22.5

231
11.7*
45.3**

115**
25.0**
84.4**

96**
18.1**
54.2**

107**
9.8*
28.8**

438
16.2

IL-6
CTR (pg/mL)
Low (fold change)
High (fold change)

736
2.9
3.3

784
3.3
3.7

749
8.5**
9.3**

482*
11.9**
15.4**

465*
7.8**
10.8**

430**
9.4**
13.6**

345
2.9

28

N. Jahan et al. / Veterinary Immunology and Immunopathology 165 (2015) 119126

123

Fig. 2. Pearson correlation between IL-1 fold change (calculated between the production of IL-1 after high dose of LPS and CTR) and plasma zinc at 3
days from parturition (A); plasma albumin at 3 days from parturition (B), plasma bilirubin at 7 days from parturition (C), plasma non-esteried fatty acids
at 28 days from parturition (D).

increased again, and the average level returned close to the


highest one at 28 DFP (P < 0.05 compared to 3 DFP). No
correlations were observed between the results of CST and
any other measured variables.

milk production was 38.2 8.8 kg/d during the rst month
of lactation.
4. Discussion

3.3. Milk yield, BCS and health condition

4.1. Production of IL-1 and IL-6 after ex vivo challenge


with LPS

All the animals during the experimental period were in


good health condition and no disease symptoms related to
organ systems were observed during the clinical inspection. The body condition score was 2.51 at 45 DFP
and was quite stable in the dry period. Thereafter, BCS
showed a gradual post-calving drop through the end of
the study period and an average of 0.44 points reduction
was observed in the rst month of lactation. The average
rectal temperatures of all cows were within physiological
values and showed a signicant increase as the time of calving approached. The temperature at 3 to 7 DFP (P < 0.05)
was higher compared to 45 DFP. No fever episodes were
observed during the experiment. Milk yield increased progressively during the 1st month of lactation and the average

The production of IL-1 to both doses of LPS increased


close to parturition from 20 DFP until 3 DFP. The results
for IL-6 showed some differences compared to IL-1. In
fact, the highest response of IL-6 was observed from 3
to 3 DFP, and the subsequent decline was not rapid like
IL-1. To the best of our knowledge, this is the rst
experiment that describes the response of IL-1 and IL6 after the LPS ex vivo challenge in the transition period
of dairy cows. Two previous researches (Rntved et al.,
2005; OBoyle et al., 2006) using WBA investigated the
effect on TNF- release, but with different experimental conditions. Interestingly, the maximum response of
both PICs tested in our study occurred around calving and
agrees with previous ndings of Rntved et al. (2005), who

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N. Jahan et al. / Veterinary Immunology and Immunopathology 165 (2015) 119126

Fig. 3. Response to carrageenan skin test (CST) in terms of area under the
curve (AUC) in transition dairy cows. Statistically signicant differences
(P < 0.05) were found among 20 vs. 45, 3 vs. 20 and +28 vs. 3 DFP.

observed an increased TNF- response during the week


before parturition and in early lactation when whole blood
was stimulated with 5 g LPS/mL of blood. Sordillo et al.
(1995) in an in vitro study, observed that mononuclear cells
isolated from periparturient dairy cattle produced more
TNF- in comparison to cells obtained from mid-lactation
animals. Together these studies suggest a high reactivity of leukocytes around calving and the possibility that,
whatever the pro-inammatory stimulus in this period, the
consequences can be stronger than during other physiological periods.
In our study we observed correlations between the fold
change of IL-1 and some markers of inammation (Fig. 2).
In the days close to calving there were negative correlations
between production of IL-1 and blood concentration of
zinc and albumin. These correlations are partially driven
by outliers (for Il-1b fold change value) and obtained with
a low number of cows, therefore they require a validation
in further investigation. However, these parameters are
well known inammatory markers and, typically, they are
reduced during inammation (Bertoni et al., 2008; Trevisi
et al., 2012). Moreover bilirubin, a marker of inammation,
showed a positive correlation with the production of IL1. In fact, its concentration increased in the plasma as a
consequence of impaired production of the hepatic enzyme
necessary for bilirubin clearance (Assenat et al., 2004). During inammation the synthesis of liver proteins is modied
and results in an increase of the synthesis of positive acute
phase protein and a decrease of negative acute phase protein (Gruys et al., 2005; Ceciliani et al., 2012). The high
reactivity of leukocytes around calving and its relationship
with inammatory markers that we observed is coherent
with the inammatory like condition, often observed at the
beginning of lactation in dairy cows (Bionaz et al., 2007;
Bertoni et al., 2008). It is to be noted that the high potential inammatory response of leukocytes occurs in a period
with the maximum risk of inammatory stimuli (e.g. injury
and trauma at calving, psychological stress for new experiences, diet modications, Loor et al., 2013).

Another interesting aspect in our study is the difference


between the behavior of the two cytokines when compared to different doses of LPS. Both the PIC showed a
signicant increase in response to the LPS dose. Nevertheless, the increase of IL-1 response was markedly higher
compared to IL-6. Consequently, there were differences
between the dose responses for the IL-1, while no difference was observed for IL-6. Specically, the increased
response of IL-1 to the higher dose compared to the lower
dose was about 300% while the increase was only 13% for
IL-6. The results of our study revealed that the IL-6 response
reached a very high level in all the time points studied
in transition period using the lower intensity of stimuli
(0.01 g LPS/mL). The slower post-calving decrease of the
IL-6 response to the LPS stimulation compared to IL-1
can be due to the higher sensitivity of some population of
leukocytes to release IL-6. This effect could be mediated by
monocytes, which often show a marked increase after calving (OBoyle et al., 2012) and produce preferentially IL-6
(Anglin et al., 2010). Our results also suggest that differences exist in the sensitivity of immune cells in accordance
to the dose of LPS.
In our experiment, blood from the CTR group was
subjected to manipulation but did not receive any LPS
treatment, only the addition of DMEM buffer. Under these
conditions the concentration of PIC can provide information about their physiological pattern of change during
the transition period. The higher concentration of both
cytokines in the dry period compared to the rst month
of lactation agree with the studies of Ishikawa et al. (2004)
and Jahan et al. (2013), where the cytokines were measured in plasma samples without manipulations. Likewise,
Trevisi et al. (2012), observed higher concentration of IL6 during the last month of pregnancy compared to during
the rst month of lactation in cows indicating good liver
functionality and good health status in the postpartum. In
the same experiment, cows with the worse liver functionality in the postpartum showed an increase of IL-6 after
calving reaching the same concentrations of the dry period
at the end of the rst month of lactation likely due to the
presence of health problems. Also in a murine model, Orsi
et al. (2006), found a raise of PIC (e.g. TNF-, IL-1 and IL6) in the serum during mid and late gestation. The reason
for post-calving reduction of PIC seems to be due to the
removal of placenta, as previously suggested by Vitoratos
et al. (2008), and also to the strong increase of 17-estradiol
(Rogers and Eastell, 2001) and cortisol (Oppert et al., 2005),
that are both markedly increased at calving time (Goff and
Horst, 1997). To demonstrate that calving is the major factor for these changes, a comparison between transition
period and other physiological phases of the cow can be
useful. Despite the fact that basal concentrations of PIC are
progressively reduced with the approach of calving, the
response of leukocytes stimulated ex vivo with LPS, measured as production of IL-1 and IL-6, increased at 3 and
3 DFP.
4.2. Response to carrageenan skin test (CST)
Carrageenan-induced aseptic footpad edema is used as
a model for the evaluation of anti-inammatory drugs

N. Jahan et al. / Veterinary Immunology and Immunopathology 165 (2015) 119126

(Morris, 2003; Whitely and Dalrymple, 1998; Srinivasa


et al., 2013; Amdekar et al., 2012). Subcutaneous injection of carrageenan in the gastrocnemius muscle and the
hind paw of rats produced local inammation but did
not increase the concentrations of TNF-, IL-1 and IL-6
in plasma (Loram et al., 2007). This suggests that subcutaneous carrageenan injection has no effect on systemic
inammatory responses.
In a previous study, King (1993) used intradermal injection of the carrageenan measuring the volume of swelling
as well as sensitivity of calves by pressure on inamed
area, as a model to assess the activity of non-steroidal antiinammatory drugs. Other studies (Lacetera et al., 1999;
Agazzi et al., 2007) rated the changes in skin thickness
after intradermal injection of phytohaemagglutinin (plant
lectin used to stimulate peripheral lymphocyte and thus
to assess immune function) as an indirect index of the
cell mediated immune response in sheep and kid, respectively. Contrary to our CST model that measured the long
term local immune response, the phytohaemagglutinin
challenge used by Lacetera et al. (1999) and Agazzi et al.
(2007), measured the short time skin thickness. Under
these circumstances the higher skin thickness increase
was interpreted as an index associated with a greater cell
mediated immune response of animals. The species, the
experimental purpose, the physiological conditions, the
inammatory agent and some experimental details are
different making it difcult to directly compare the cited
researches with our results. Moreover, none of the above
mentioned experiments were conducted during the transition period. Therefore, we used CST for the rst time
to evaluate the individual variation of local immune and
inammatory responses of transition dairy cows. The technique we propose is very simple and causes only local and
short-lived swelling and discomfort. Our results suggest
that dairy cows became very sensitive to CST at 23 weeks
before calving and this sensitivity decreased in the few days
around calving.
During the skin swelling in the CST model polymorphonuclear leukocyte inltration was observed in the
inamed area (Vinegar et al., 1987). This observation was
also conrmed by Di Rosa (1972), who found a reduction
of swelling by the inhibition of immune cells migration
(mainly monocytes). According to Vinegar et al. (1987)
this is the mechanism whereby anti-inammatory drugs
reduce swelling. Based on this mechanism, we can speculate that the high concentrations of anti-inammatory
hormones, which increase close to calving (e.g. cortisol
and 17-estradiol), act by reducing cell migration at the
site of challenge (e.g. peripheral level) and, consequently,
the measured response after the carrageenan stimulation
in term of skin thickness was lower. Our CST result partially agrees with previous studies (Kehrli and Goff, 1989a;
Kehrli et al., 1989b) which observed a reduction of lymphocyte proliferation and neutrophil function in blood of dairy
cows in late pregnancy and attributed these phenomena to
the increase of intra-mammary infections by opportunistic bacteria. Edmondson and Bramley (2008) and Stevens
et al. (2012) observed an ineffective response to coliform
infections in mammary gland of high yielding dairy cows,
particularly in early lactation, attributed to the poor rate

125

of diapedesis. Our data also indicates a reduction of diapedesis with the approach of the parturition. Nevertheless,
it is not possible to know whether the lower CST response
around calving is mainly due to the changes in the leukocytes functions or the reduction of the migration of some
population from the bloodstream to the infected area, as
suggested by Wang et al. (2007) in sheep and by Meglia
et al. (2001) in periparturient dairy cows.
5. Conclusions
The present study conrms that the immune system,
and in particular the inammatory response, shows important modications in late pregnancy. The basal plasma
concentrations of pro-inammatory cytokines were higher
during the dry period compared to the 1st month of lactation in healthy cows. The response in blood to LPS
stimulation, in term of IL-1 and IL-6 release, increased
around calving, but the circulating leukocytes show a
different ability to produce PIC in accordance with the concentrations of LPS. The higher release of IL-6 compared
to IL-1 at the low LPS concentration suggests different
ability of leukocytes to produce cytokines. The peripheral
immune response to an aseptic stimulus (carrageen) shows
a reduction of the response around calving, likely due to
the impairment of diapedesis, which can justify a lower
peripheral reaction in the case of infection of injuries.
Acknowledgements
This study was partially funded by the Linea D1-2011,
Universit Cattolica del Sacro Cuore, Largo Gemelli 1, Milan,
Italy. The authors gratefully acknowledge Dott.ssa Rosanna
Lombardelli for her support during ELISA procedures and
Professor J. J. Loor (Department of Animal Sciences, University of Illinois at Urbana-Champaign) for careful and critical
revision of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.vetimm.2015.04.003.
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