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Research Paper
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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.
120
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
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
122
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).
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
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).
milk production was 38.2 8.8 kg/d during the rst month
of lactation.
4. Discussion
124
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.
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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