Serum interleukin-6 (IL-6) and IL-10 concentrations in normal and septic neonatal foals.

Abstract

Previously it was reported that compared to surviving septic foals, non-surviving foals had a 35-fold increase in interleukin-10 (IL-10) and 15-fold increase in IL-6 gene expression in their peripheral blood mononuclear cells (PBMC). As gene expression profiles can be time-consuming, we sought to determine if serum IL-6 and IL-10 in foals would aid in the diagnosis and prognosis of septicemia. A prospective study of septic neonatal foals admitted to the Cornell University Equine Hospital during 2007 and 2008 was performed. Septicemia was confirmed in 15 foals using blood culture results and sepsis scores. Blood samples for measurement of serum IL-6 and IL-10 concentrations were collected at the time of admission (T0) and again 24 (T24) and 48 (T48) hours later. Blood samples from age-matched control foals (n=15) born at the Cornell Equine Park were obtained from foals 12-72h after birth (T0) and again 24 (T24) and 48 (T48) hours later. IL-6 and IL-10 concentrations were determined in the serum from dams of septic foals and serum and colostrum from dams of control foals. Serum IL-6 was also measured in healthy foals prior to ingestion of colostrum. Interleukin-6 was detected using an ELISA and IL-10 was detected using a bead-based fluorescent immunoassay. Group differences were detected using a Wilcoxon rank sum test with a Bonferroni correction applied to the p value. There were no significant differences in serum IL-10 concentration between the two groups of foals. Relative to control foals, septic foals had significantly lower serum IL-6 concentrations at all 3 time points. Relative to septic foals, control foals had significantly higher serum IL-6:IL-10 ratios. Serum IL-6 was undetectable in foals prior to ingestion of colostrum. However, colostral IL-6 concentration measured in the control mares was high (> or =215ng/mL) in all samples suggesting passive transfer of maternal IL-6 to the equine neonate. Colostral IL-10 was undetectable in 11/12 samples. Failure of passive transfer may directly influence the serum IL-6 concentration in septic foals. Neither serum IL-6 nor IL-10 alone, were useful diagnostic indices of sepsis in equine neonates. Although the number of animals involved in this study was too small for the identification of a concrete value, the serum IL-6:IL-10 ratio is likely to provide a valuable prognosticator for neonatal septicemia.

Full text

Page 1

Serum interleukin-6 (IL-6) and IL-10 concentrations in normal
and septic neonatal foals
A.B. Burtona, B. Wagnerb,*, H.N. Erbb, D.M. Ainswortha
aDept of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
bDept of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
1. Introduction
In systemic bacterial infections, the host response
includes the production of both pro-inflammatory and
anti-inflammatory cytokines. Pro-inflammatory cytokines
(e.g. TNF-a, IL-1b, IL-2, IL-6 and IFN-g) promote macro-
Veterinary Immunology and Immunopathology 132 (2009) 122–128
A R T I C L EI N F O
Article history:
Received 16 January 2009
Received in revised form 31 March 2009
Accepted 11 May 2009
Keywords:
Cytokine
Equine
Neonatal
Sepsis
A B S T R A C T
Previously it was reported that compared to surviving septic foals, non-surviving foals had
a 35-fold increase in interleukin-10 (IL-10) and 15-fold increase in IL-6 gene expression in
their peripheral blood mononuclear cells (PBMC). As gene expression profiles can be time-
consuming, we sought to determine if serum IL-6 and IL-10 in foals would aid in the
diagnosis and prognosis of septicemia.
A prospective study of septic neonatal foals admitted to the Cornell University Equine
Hospitalduring2007and2008 wasperformed.Septicemiawasconfirmedin15foalsusing
blood culture results and sepsis scores. Blood samples for measurement of serum IL-6 and
IL-10 concentrations were collected at the time of admission (T0) and again 24 (T24) and
48 (T48) hours later. Blood samples from age-matched control foals (n = 15) born at the
Cornell Equine Park were obtained from foals 12–72 h after birth (T0) and again 24 (T24)
and48(T48)hourslater.IL-6andIL-10concentrations weredeterminedintheserumfrom
dams of septic foals and serum and colostrum from dams of control foals. Serum IL-6 was
also measured in healthy foals prior to ingestion of colostrum. Interleukin-6 was detected
using an ELISA and IL-10 was detected using a bead-based fluorescent immunoassay.
Group differences were detected using a Wilcoxon rank sum test with a Bonferroni
correction applied to the p value.
There were no significant differences in serum IL-10 concentration between the two
groups of foals. Relative to control foals, septic foals had significantly lower serum IL-6
concentrations at all 3 time points. Relative to septic foals, control foals had significantly
higher serum IL-6:IL-10 ratios. Serum IL-6 was undetectable in foals prior to ingestion of
colostrum. However, colostral IL-6 concentration measured in the control mares was high
(?215 ng/mL) in all samples suggesting passive transfer of maternal IL-6 to the equine
neonate. Colostral IL-10 was undetectable in 11/12 samples. Failure of passive transfer
may directly influence the serum IL-6 concentration in septic foals. Neither serum IL-6 nor
IL-10 alone, were useful diagnostic indices of sepsis in equine neonates. Although the
number of animals involved in this study was too small for the identification of a concrete
value, the serum IL-6:IL-10 ratio is likely to provide a valuable prognosticator for neonatal
septicemia.
? 2009 Elsevier B.V. All rights reserved.
* Corresponding author at: Room S1-082, Schurman Hall, Dept of
Population Medicine and Diagnostic Sciences, College of Veterinary
Medicine, Cornell University, Ithaca, NY 14853, USA.
Tel.: +1 607 253 3813.
E-mail address: bw73@cornell.edu (B. Wagner).
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm
0165-2427/$ – see front matter ? 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2009.05.006

Page 2

phage activation and phagocytosis, enhance cell-mediated
immunity and up-regulate acute phase protein synthesis.
Anti-inflammatorycytokines(e.g.IL-4,IL-5,IL-10,IL-13and
TGF-b) have immunoregulatory functions and support
development of a humoral immune response by promoting
B-cell differentiation and antibody production (Romagnani,
1996;Gogosetal.,2000).Inhumaninfants,cytokineprofiles
in the peripheral blood have been used for the early
identification of sepsis (Ng et al., 2006, 2007; Lam and Ng,
2008). Likewise, cytokine responses in PBMC of septic
equine neonates also have been examined with a view to
their diagnostic and prognostic significance. Sepsis is a
major cause of morbidity and mortality in the equine
neonate (Paradis, 1994). Early diagnosis and prognosis are
important, as treatment of the septic foal is often costly and
associated with an unfavorable outcome. Pusterla et al.
(2006) found that relative to healthy foals, non-surviving
sick foals (septic and non-septic) exhibited marked
increases in the gene expression of IL-10 in PBMC. Gene
expression of several other cytokines, including IL-6, was
not increased in the non-survivors relative to the survivors.
In a recent prospective study completed in our laboratory,
we found that compared to healthy foals, IL-4 gene
expression was initially down-regulated and toll-like
receptor 4 (TLR4) expression up-regulated in PBMC from
septic foals (Gold et al., 2007). Furthermore, the IL-6 gene
expression in PBMC isolated from septic foals that died was
15-fold greater compared with septic foals that survived
(Gold et al., 2007).
The data from Pusterla et al. (2006) and Gold et al.
(2007) suggest that IL-6 and IL-10 mRNA concentrations
may be valuable prognosticators of survival in septic foals.
However, because of the time required for processing of
samples (isolation of RNA, preparation of cDNA, perform-
ing real-time PCR), a simpler and faster test that measured
serum concentrations of these cytokines is required. Based
upon the gene expression data, we hypothesized that
serum IL-6 and IL-10 protein concentrations would be
increased in septic neonatal foals and would be predictive
of survival. The three objectives of this study were to: (1)
developarapidtestforquantificationofequineIL-6andIL-
10 serum concentrations; (2) measure serum IL-6 and IL-
10 concentrations in septic foals; and (3) determine if
maternal colostral IL-6 and IL-10 concentrations poten-
tially influence the foal serum concentrations of these two
cytokines.
2. Materials and methods
2.1. Case selection
Two groups of foals were studied and consisted of
neonatal septic foals admitted to the Cornell University
Hospital for Animals (CUHA) during the 2007 and 2008
foaling seasons and healthy (control) foals born and raised
at the Cornell University Equine Park during the spring of
2007.
All sick neonatal foals less than 14 days of age admitted
to the CUHA that were tentatively diagnosed as septic
(based upon historical findings and initial clinical exam-
ination by the attending veterinarian) were initially
enrolled. Foals were retained in the study if they had a
positive blood culture or a sepsis score ?11 (Brewer and
Koterba, 1988). Following admission to the hospital,
historical, physical examination, thoracic and abdominal
ultrasonographic findings were recorded. Blood samples
were obtained for aerobic and anaerobic blood cultures,
IgG quantification (Snap Foal IgG, Idexx Laboratories,
Westbrook, ME), a complete blood count (CBC), a range of
common blood serum biochemical measurements and
fibrinogen concentration. In addition, a 5 mL blood sample
for serum analysis was obtained from each foal at the time
of admission (T0), and then again 24 (T24) and 48 (T48)
hours later. The serum was collected and stored at ?20 8C
until cytokine measurement. After initial evaluation, each
hospitalized foal received at least 1 L of hyperimmune
equine plasma (Foalimmune, Lake Immunogenics, Ontario,
NY or HiGamm-Equi, Lake Immunogenics, Ontario, NY),
broad-spectrum antimicrobials, IV fluids and other sup-
portive therapy as needed. One foal received a plasma
transfusion prior to hospital admission. A 3 mL sample of
each batch of commercial plasma used to treat these septic
foals was obtained for interleukin analysis.
Fifteen septic foals were enrolled, 7 females and 8
males, with a median age of 24 (range, 12–288) hours.
Breeds represented included 6 Thoroughbreds, 2 Stan-
dardbreds and 1 (each) Morgan, mixed-breed, Oldenburg,
Paint, Quarterhorse, Arabian and Shire. The median
gestational age, as reported by owners, was 340 (range,
305–345) days. Parturition was judged by the owners to be
normal in 9/15 foals, required assistance with recognized
complications in 2/15 foals and was unobserved in 4/15
foals.Themediansepsis score ofthe septic foalswas 16(8–
33). Blood cultures were positive in 9/14 foals, negative in
5/14foalsandnot obtainedin1foal.Theselast6foalswere
classified as septic based upon their individual sepsis score
of 13, 18, 22, 24, 25 and 33. Microbial isolates from blood
culturesincludedEnterococcusfaecium(2foals),Escherichia
coli(2 foals), Actinobacillusequuli (2foals), Corynebacterium
spp. (1 foal), Staphylococcus aureus (1 foal) and methicillin-
resistant S. aureus (1 foal). Median duration of hospitaliza-
tion was 13 (1–19) days. Five of the foals had been treated
with antimicrobialspriorto admission and all(15/15) foals
were treated with antimicrobials during hospitalization.
Two foals received non-steroidal anti-inflammatories
(NSAIDs) prior to hospitalization and 4/15 foals were
treated with NSAIDs during hospitalization. None of the
foals received glucocorticoids either before or during
hospitalization. Foals in the septic group were considered
as survivors if they were discharged from the hospital
alive. Non-survivors were foals that died or were
euthanized during hospitalization. Foals that were eutha-
nized because of financial constraints were not included in
the study. Thirteen foals survived to discharge and 2/15
foals died after less than 1 day of hospitalization.
Fifteen foals, 5 females and 10 males, with a median age
of36(12–96)hourswereenrolledinthe controlgroup.The
median gestational age or the age of control foals when
enrolled into the study was not significantly different from
that of the septic group.
Breeds represented were Warmblood (10), Thor-
oughbred (2) and 1 (each) Thoroughbred cross, Irish Draft
A.B. Burton et al./Veterinary Immunology and Immunopathology 132 (2009) 122–128
123

Page 3

and Canadian Sport Horse. Each of these foals had an
uneventful parturition, had a gestational age ?334 days
and lacked detectable abnormalities on physical examina-
tion. Blood samples were obtained from each foal and used
for all tests described above for the septic group. A 5 mL
blood sample for serum analysis was obtained from each
foal at the time of enrolment into the study (T0) and then
again at 24 (T24) and 48 (T48) hours later. Serum samples
were also obtained from 5 control foals before they
ingested colostrum. Based upon initial physical examina-
tion and clinical pathology findings, the median sepsis
score of this group was 0 (range, 0–2). All of the control
foals were maintained at the Cornell University Equine
Park under the daily supervision of the farm manager and
remained healthy for at least 6 months following their
initial evaluations.
Serum samples were obtained within the T0–T48 time
frame for measurement of IL-10 and IL-6 in 5/15 of the
control foal mares and 11/15 of the septic foal mares.
Colostrum samples, collected shortly after foaling, were
obtained from 12/15 of the control foal mares. No
colostrum samples were available from the septic foal
mares.
All experimental procedures were approved by the
Animal Care Committee of Cornell University and were in
accordance with guidelines established by the National
Institutes of Health. For the foals admitted to the Cornell
University Hospital for Animals, owner consent authoriz-
ing the investigators to obtain blood samples for the study
was obtained.
2.2. Determination of IL-6 and IL-10 in serum and colostrum
Interleukin-6 was measured in an enzyme linked
immunosorbent assay (ELISA) as previously described
for other equine cytokines (Wagner et al., 2006, 2008).
Here, a polyclonal goat anti-horse IL-6 antibody (AF1886,
R&D Systems, Inc., Minneapolis, MN) was used for coating
of the ELISA plates (Immunoplate Maxisorp, Nalge Nunc
Int., Rochester, NY). The antibody was diluted to a final
concentration of 1 mg/mL in carbonate buffer (15 mmol
Na2CO3, 35 mmol NaHCO3, pH 9.6) and incubated over-
night at 4 8C. Afterwards, the coating solution was
discarded and empty spaces on the plates were blocked
for 30 min at room temperature by addition of phosphate
buffered saline (PBS, pH 7.2) containing 0.5% (w/v) bovine
serum albumin. Plates were washed five times with
phosphate buffer (2.5 mmol NaH2PO4, 7.5 mmol Na2HPO4,
145 mmol NaCl, 0.1% (v/v) Tween 20, pH 7.2). A
recombinant equine IL-6 (1886-EL, R&D Systems, Inc.,
Minneapolis, MN) diluted in 2-fold serial dilutions ranging
from 50 to 0.78 ng/mL was used as standard to determine
IL-6 concentrations in the samples. The serum and
colostrum samples were diluted from 1:10 to 1:100,000
in phosphate buffer. All samples were applied to the ELISA
platesandincubatedfor90 minatroomtemperature.After
fivewashes(seeabove)plateswere filledwithbiotinylated
goat anti-horse IL-6 (AF1886, R&D Systems, Inc., Minnea-
polis, MN) diluted 1:100 in phosphate buffer, incubated for
60 min and washed again. Then, a streptavidin–horse-
radish peroxidase solution (Jackson ImmunoResearch Lab.,
West Grove, PA) was added to the plates for another
30 min. After a final wash, the plates were filled with
substrate buffer (33.3 mmol
NaH2PO4, pH5.0),supplemented
3,30,5,50-tetramethylbenzidine (TMB, Sigma, St. Louis,
MO) and 0.01% (v/v) hydrogen peroxide. Substrate solution
was incubated for 20 min in the dark and the reaction was
stopped by adding one volume of 0.5 mol H2SO4. Plates
were evaluated in a Synergy HT ELISA reader (Bio-Tek,
Winooski, VT) at 450 and 630 nm absorbance. The IL-6
ELISA had an analytical sensitivity of 780 pg/mL.
Interleukin-10 concentrations in serum and colostrum
were determined using a fluorescent bead-based assay
(Luminex Corp., http://www.luminexcorp.com) and a pair
of monoclonal anti-equine IL-10 antibodies (Wagner et al.,
2008). The assay was performed and analyzed in a Luminex
IS 100 instrument (Luminex Corp., http://www.luminex-
corp.com) as described previously (Wagner and Freer,
2009). Serum samples were used undiluted. Colostrum
sampleswerediluted1:3and1:10.Theanalyticalsensitivity
of the equine IL-10 singleplex assay was 4 pg/mL.
citricacid,
with
66.7 mmol
130 mg/mL
2.3. Statistical analysis
Thedistributionof thedata wasnon-Gaussian.
Differences between the two groups of foals in selected
demographic variables, clinical pathology data and serum
IL-10 and IL-6 concentration at the 3 time points (T0, T24,
T48 hours) were analyzed using the Wilcoxon rank sum
test. IL-6 and IL-10 ratios were compared between foals at
T0 using the Wilcoxon rank sum test. Because multiple
comparisons were made during the analysis, a Bonferroni
correction was calculated and applied. For statistical
significance, a p value ? .017 (?.05/3) was used. All
computations were performed by use of a statistical
software program (Statistix 8, Analytical Software, Talla-
hassee, FL).
Table 1
Median (range) clinicopathological data of control and septic foals.
Control (n = 15)Septic (n = 15)
Seg. neutrophils (cells/mL)
Band neutrophils (cells/mL)
Lymphocytes (cells/mL)
Fibrinogen (mg/dL)
IgG (mg/dL)
Total protein (g/dL)
Iron (mg/dL)
6600 (3100–10,900)
0 (0–200)
1300 (600–2900)
200 (100–600)
>800
5.9 (5.1–6.8)
175 (42–371)
3950 (800–17,700)
300 (0–1000)
1200 (700–2600)
300 (100–700)
>400–800 (<200 to >800)
4.4 (3.6–6.2)
246 (32–393)
A.B. Burton et al./Veterinary Immunology and Immunopathology 132 (2009) 122–128
124

Page 4

3. Results
3.1. Demographic and clinicopathologic data
Median values for selected clinicopathologic data from
the two groups of foals are displayed in Table 1. There was
no significant difference between the two groups in
lymphocyte counts. Statistical analysis was not performed
on other physical and clinicopathologic data that were
included in the sepsis score calculation, as sepsis scores
were significantly different between the two groups
(p < .0001).
3.2. Cytokine concentrations in serum and colostrum
3.2.1. Interleukin-6
Shown in Fig. 1 is the serum IL-6 concentrations of the
control and septic foals measured at the 3 time points.
Relativetocontrolfoals,septicfoalshadsignificantlylower
median serum IL-6 concentrations at each time point:
T0 = 30.5 ng/mL (p = .0003); T24 = 36.1 ng/mL (p = .0007);
T48 = 35.0 ng/mL (p = .0016). In contrast, the median
serum IL-6 concentrations of the control foals at T0, T24
and T48 were 1883, 1955 and 1659 ng/mL, respectively.
Interleukin-6 was not detectable in serum samples
obtained from 5/5 control foals prior to ingestion of
colostrum. Interleukin-6 was also not detectable in any of
the samples from the two commercial equine plasma
products tested (n = 5). Median serum IL-6 concentrations,
measured in 5 dams of control foals and in 11 dams of
septic foals are shown in Table 2. Median colostral IL-6
concentrations, measured in 12 dams of control foals
exceeded 11,000 ng/mL. No colostral samples were avail-
able from dams of septic foals for comparison.
3.2.2. Interleukin-10
Shown in Fig. 2 is the serum IL-10 concentrations of the
control and septic foals at the 3 time points evaluated.
There were no significant differences in serum IL-10
concentrations between the two groups of foals at any of
the three sampling times (all p values > .19). Interleukin-
10 was not detectable in any of the samples of the
commercial equine plasma products tested (n = 5). Serum
IL-10 concentrations were undetectable (<4.0 pg/mL) in
most of the serum samples obtained from the dams of
control and of septic foals (12/16) (Table 2). Colostral IL-10
concentrations were also undetectable in 11/12 samples
from control mares (Table 2). No colostral samples were
available from mares of septic foals for IL-10 quantifica-
tion.
3.2.3. Interleukin-6 and interleukin-10 ratios
We then examined the combination of IL-6 and IL-10
levels together in the control and septic foals. Fig. 3
indicates that healthy control foals have significantly
higher IL-6:IL-10 ratios compared to septic foals. The
analysis of IL-6:IL-10 ratios was performed using cytokine
Fig. 1. Serum IL-6 concentrations of the control and septic foals measured
at the 3 time points evaluated. Relative to control foals, septic foals had
significantly lower median serum IL-6 concentrations at each time point:
T0 = 30.5 ng/mL (p = .0003); T24 = 36.1 ng/mL (p = .0007); T48 = 35.0 ng/
mL (p = .0016). In contrast, the median serum IL-6 concentrations of the
control foals at T0, T24 and T48 were 1883 ng/mL, 1955 ng/mL and
1659 ng/mL, respectively. Serum samples with undetectable IL-6
concentrations were set as 0.1 ng/mL for statistical evaluation.
Table 2
Median (range) IL-10 and IL-6 concentrations in serum and colostrum of dams of control and septic foals.
Mares of control foals Mares of septic foals
IL-6 (ng/mL) (serum, T = 0–48)
IL-6 (ng/mL) (colostrum)
IL-10 (pg/mL) (serum, T = 0–48)
IL-10 (pg/mL) (colostrum)
3242 (1016–28,183) (n = 5)
11,429 (215–100,973) (n = 12)
<4 (<4–172) (n = 5)
<4 (<4–5173) (n = 12)
491 (<0.78–189,000) (n = 11)
NA
<4 (<4–963) (n = 11)
NA
The lower limit of detection is 4 pg/mL for the IL-10 assay and 0.78 ng/mL for the IL-6 ELISA.
Fig. 2. Serum IL-10 concentrations of the control and septic foals
measured at the 3 time points evaluated. There were no significant
differences in serum IL-10 concentrations between the two groups of
foals at any of the three sampling times (all p values > .19). For the
statistical analysis and in this figure all samples with undetectable IL-10
concentrations were set as 0.1 pg/mL.
A.B. Burton et al./Veterinary Immunology and Immunopathology 132 (2009) 122–128
125

Page 5

concentrations as the unit of comparison (p = .0025) and
also by using ranks as the unit of comparison (p = .0011;
data not shown). Both comparisons suggested that the IL-
6:IL-10 ratio in serum is a valuable prognosticator for
neonatal sepsis in foals.
4. Discussion
Although the gene expression of IL-10 (Pusterla et al.,
2006) and IL-6 (Gold et al., 2007) in PBMC has been
examined in healthy and sick (septic and non-septic)
equine neonates, to the authors’ knowledge, this is the first
time that serum IL-6 and IL-10 protein concentrations in
neonatal foals have been measured directly via rapid ELISA
(IL-6) and fluorescent bead-based assays (IL-10).
As has been applied in the human medical field, we
investigated the usefulness of these cytokine assays in the
very early detection of sepsis in neonatal foals. To classify
foals as septic, we used a combination of the sepsis score,
which is routinely used in equine neonatal medicine
(Brewer and Koterba, 1988), as well as the results of blood
cultures, considered to be the ‘gold standard’. Although
sepsis score and positive blood cultures generally agree
well, some discrepancies may arise. For example, in this
study 5 foals had high sepsis scores but negative blood
culture results. The most likely reason for this divergence
was deemed prior antibiotic administration that inhibited
bacterial growth. Conversely, 1 foal had a sepsis score of 8
but a positive blood culture (Corynebacterium spp.), which
was judged not to be a contaminant. These two situations
illustrate the usefulness of using both methods to define
the septic foal. Nevertheless, the moderate sensitivity and
specificity of the sepsis score (Peek et al., 2006) and the
time delay for blood culture results (average 2–3 days)
highlights the need for a more sensitive and specific rapid
test that can identify early, the septic neonate.
Interleukin-6 is a pro-inflammatory cytokine predomi-
nantly produced by leukocytes and hepatocytes in
response to infection and trauma. In human adults and
infants, an increased serum IL-6 concentration has been
found to be a sensitive indicator of sepsis. Furthermore,
persistent increases in IL-6 are associated with increased
duration of hospitalization and increased risk of dying
(Buck et al., 1994; Reyes et al., 2003; Goepfert et al., 2004;
Latifi et al., 2004; Pavcnik-Arnol et al., 2004; Harris et al.,
2005). In contrast we found that median serum IL-6
concentrations were highest in the control foals and
exceeded median serum IL-6 values in the septic foals
by 62-fold at T0, 54-fold at T24 and 47-fold at T48. These
results appeared to conflict with the previously reported
gene expression that demonstrated no difference in IL-6
mRNA levels in PBMC from healthy foals and from
hospitalized septic (Gold et al., 2007) or sick (septic and
non-septic) foals (Pusterla et al., 2006) on admission. Can
these differences be reconciled? One explanation could be
thatmRNAexpression andsecretedproteinconcentrations
are not always linked. However, in human PBMC
stimulated with LPS and C3a, IL-6 protein secretion into
the culture media was associated with increases in mRNA
expression (Fischer et al., 1999). IL-6 protein and mRNA
expression levels were also both increased after stimula-
tion of human cord blood mononuclear cells with
Streptococcus agalactiae, the major pathogen of neonatal
sepsis in humans (Berner et al., 2001). Likewise, Jorgensen
et al. (2001) examined effects of sirolimus, a potent
immunosuppressive andantiproliferative
inhibited cytokine production in human blood and in
human PBMC stimulated with various bacterial products,
including LPS. A good correlation between IL-10 protein in
serum and mRNA expression in CD14+cells was found
(Jorgensen et al., 2001). In equine neonates, the situation
seemed to be more complex due to the sometimes massive
transfer of IL-6 with the colostrum. Our data demonstrate
(1) that prior to ingestion of colostrum, serum IL-6
concentrations are undetectable in healthy foals and (2)
that equine colostrum, like colostrum of other animal
species, containsIL-6 (Bo ¨ttcheretal.,2000;Hagiwaraetal.,
2000; Nguyen et al., 2007; Zanardo et al., 2007). Thus, the
healthy control foals—those that most likely ingested
adequate quantities of colostrum at birth (as reflected by
serum IgG concentrations exceeding 800 mg/dL)—also
ingested higher amounts of colostral IL-6. Strong evidence
forthetransferofcytokines,includingIL-6,fromcolostrum
to serum in the neonate has already been demonstrated in
calves (Yamanaka et al., 2003) and piglets (Nguyen et al.,
2007). Although maternally derived IL-6 is likely to
influence the immune response of the neonate, its exact
function on the foal’s immune system is still unknown.
Since the leading cause of equine neonatal septicemia is
failure of passive transfer (i.e. inadequate colostral intake)
this would explain in part why IL-6 concentrations would
be higher in healthy foals compared to the septic foals.
Thus, it is possible that the relatively low serum IL-6
concentrations detected in the septic foals (30–36 ng/mL)
represented at least partially endogenous cytokine pro-
duction from PBMC responding to the bacteremia. Even so,
the response of the two non-surviving septic foals (IL-
6 = 30 and 179 ng/mL) was unremarkable, given that IL-6
expression in PBMC of non-surviving septic foals is 15-fold
greater than that of surviving septic foals (Gold et al.,
2007). Furthermore, the reason why the neonatal foal fails
to mount a vigorous IL-6 protein response, as opposed to
the septic human infant (Lam and Ng, 2008), is unknown
butmakesthiscytokinelessusefulasasinglemarkerinthe
early diagnosis of sepsis.
agentthat
Fig. 3. Ratio of serum IL-6:IL-10 concentrations of the control and septic
foals measured at T0. The ratio of serum IL-6:IL-10 concentration is
significantly higher in the control foals versus the septic foals.
A.B. Burton et al./Veterinary Immunology and Immunopathology 132 (2009) 122–128
126