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ENDANGERED SPECIES RESEARCH
Endang Species Res
Vol. 33: 291–303, 2017
https://doi.org/10.3354/esr00814 Published May 18
INTRODUCTION
The explosion of the Deepwater Horizon (DWH) oil
platform on April 20, 2010 resulted in an unprece-
dented release of oil in the Gulf of Mexico (GoM).
Millions of barrels of oil were discharged into the
Gulf from the DWH well before it was successfully
capped on July 15, 2010 (United States District Court
2014). Common bottlenose dolphins Tursiops trunca-
tus are long-lived apex predators likely to be ex -
© The authors and (outside the USA) the US Government 2017.
Open Access under Creative Commons by Attribution Licence.
Use, distribution and reproduction are un restricted. Authors and
original publication must be credited.
Publisher: Inter-Research · www.int-res.com
*Corresponding author: sylvain.deguise@uconn.edu
Changes in immune functions in bottlenose
dolphins in the northern Gulf of Mexico associated
with the Deepwater Horizon oil spill
Sylvain De Guise1,2,*, Milton Levin1, Erika Gebhard1, Lindsay Jasperse1,
Leslie Burdett Hart3, 4, Cynthia R. Smith5, Stephanie Venn-Watson5, Forrest Townsend6,
Randall Wells7, Brian Balmer4,7, Eric Zolman4, Teresa Rowles8, Lori Schwacke4
1University of Connecticut, Department of Pathobiology and Veterinary Science, Storrs, Connecticut 06269, USA
2Connecticut Sea Grant Program, Groton, Connecticut 06340, USA
3College of Charleston, Department of Health and Human Performance, Charleston, South Carolina 29424, USA
4National Centers for Coastal Ocean Science, National Oceanic and Atmospheric Administration, Charleston,
South Carolina 29412, USA
5National Marine Mammal Foundation, San Diego, California 92106, USA
6Bayside Hospital for Animals, Fort Walton Beach, Florida 32547, USA
7Chicago Zoological Society, c/o Mote Marine Laboratory, Sarasota, Florida 34236, USA
8Office of Protected Resources, National Marine Fisheries Service, National Oceanic and Atmospheric Administration,
Silver Spring, Maryland 20910, USA
ABSTRACT: Following the Deepwater Horizon (DWH) oil spill, the Natural Resource Damage
Assessment conducted comprehensive health assessments on common bottlenose dolphins Tur-
siops truncatus in Barataria Bay (BB), Louisiana, in 2011, 2013 and 2014, as well as in Mississippi
Sound (MS) in 2013, to assess potential health effects from exposure to oil compared to Sarasota
Bay (SB), Florida dolphins not exposed to DWH oil. Immune functions demonstrated a consistent
increase in T (BB 2011) and B (BB 2011 and 2013) lymphocyte proliferation compared to SB. Cyto-
kine concentrations varied considerably in wild dolphin populations, and no significant differ-
ences were found; however, interesting trends were observed. The Th1 cytokines IL-2, IL-12, and
IFNγ, and the Th2 cytokines IL-5, IL-10, and IL-13, were 2- to 50-fold lower, and IL-4 was 3-fold
higher, in BB 2011 compared to SB. Overall, the changes observed were compatible with those
documented in other species following exposure to oil or PAHs and were most pronounced in BB
2011, at the place and time most affected by oil. The changes in T cell functions, and the trend
towards a cytokine balance tilted towards a Th2, rather than a Th1 response, are compatible with
intra-cellular bacterial infections such as Brucella, which has been identified as one of the poten-
tial contributory factors to perinatal dolphin mortalities, and changes in B cell functions are com-
patible with an increase in extra-cellular bacterial infections and primary bacterial pneumonia.
KEY WORDS: Oil spill · Bottlenose dolphin · Lymphocyte proliferation · Cytokines · Immunology ·
Immunotoxicology · Health
O
PEN
PEN
A
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Contribution to the Theme Section ‘Effects of the Deepwater Horizon oil spill on protected marine species’
Endang Species Res 33: 291–303, 2017
posed to oil or its by-products through respiratory,
alimentary, and dermal routes (Schwacke et al.
2014). Dolphins, as well as other cetaceans, may be
able to detect the presence of oil but do not necessar-
ily avoid it (Gubbay & Earll 2000).
Specific health effects reported from live common
bottlenose dolphin health assessments from Bara -
taria Bay (BB), Louisiana in 2011, an area that re -
ceived heavy and prolonged oiling, were compared
to a reference site, Sarasota Bay (SB), Florida, where
oil was not observed. These health effects in dolphins
likely exposed to oil included hypoadrenocorticism,
consistent with adrenal toxicity as previously re -
ported for laboratory mammals exposed to oil, and a
5 times increased likelihood of moderate to severe
lung disease (Schwacke et al. 2014). Consistent find-
ings of adrenal and lung abnormalities were also
identified in dolphins that stranded and died within
the DWH oil spill footprint, including BB. Specifi-
cally, dolphins that stranded post-DWH oil spill had a
higher prevalence of thin adrenal gland cortices,
severe pneumonia, and primary bacterial pneumonia
compared to a reference population of dolphins that
stranded outside of the DWH oil spill footprint and
time (Venn-Watson et al. 2015). Combined, these
studies supported the conclusion that exposure to
petroleum products from the DWH oil spill led to
adrenal and lung disease in dolphins and contributed
to the observed increase in dolphin mortalities
(Venn-Watson et al. 2015). In addition, there were
increased numbers of perinatal dolphin strandings
during the years following the DHW oil spill (Cole-
grove et al. 2016). Perinatal studies support the
hypothesis that most of these late-term pregnancy
losses were likely due to poor maternal health follow-
ing the DHW oil spill, including increased in utero
infections, such as brucellosis (Colegrove et al. 2016).
The immune system normally ensures a balance
between maintaining an individual’s homeostasis
and preparing for a potential pathogen invasion. It
includes adaptive and innate branches, each with
specific cell types and functions that can be quanti-
fied. The in vitro mitogen-induced lymphocyte pro -
liferation assay measures the ability of lymphocytes
to proliferate in response to a broad, not antigen-
specific stimulation, as a representation of an initial
and important first step of the adaptive immune
response (De Guise et al. 1996). Cytokines are small
cell-signaling protein molecules secreted by numer-
ous cells of the immune system, which determine the
direction and magnitude of an immune response.
These include interferon, interleukin (IL), and growth
factors. Commonly, cytokines are divided into pro-
inflammatory and anti-inflammatory groups. Pro-
inflammatory cytokines, secreted in the beginning of
an immune response, include IL-1, IL-6, IL-8, and
tumor necrosis factor (TNF), and are produced pre-
dominantly by macrophages, monocytes, and T
helper 1 (Th1) lymphocytes. Anti-inflammatory cyto-
kines, secreted to dampen an inflammatory res -
ponse, include IL-4, IL-10, and IL-13 and are secreted
predominately by T helper 2 (Th2) lymphocytes.
Cytokines can define the direction of an immune
response orchestrated by T helper cells. Th1 cells
secrete interferon gamma (IFNγ), IL-2, and IL-12,
which stimulate cell-mediated immunity to help
combat intracellular pathogens (e.g. viruses and
intra-cellular bacteria). On the other hand, Th2 cells
produce IL-4, IL-10, IL-6, and IL-13, which inhibit
cell mediated (Th1) immunity and promote humoral
(i.e. antibody mediated) immune responses to help
combat extracellular pathogens (e.g. extracellular
bacteria, parasites) (Kuby 1997).
During and following the DWH oil spill, significantly
elevated polycyclic aromatic hydrocarbon (PAH) lev-
els attributed to DWH oil were detected in coastal
GoM waters, including Louisiana, Mississippi, and
Alabama (Allan et al. 2012). PAHs are an important
class of environmental pollutants associated with oil
spills and have known immunotoxic effects (Davila et
al. 1995, Reynaud & Deschaux 2006, Zaccaria & Mc-
Clure 2013). A recent review concluded that exposure
to benzo(a)pyrene (BaP), a model PAH mostly of pyro-
genic origin, affects primary and secondary immune
tissues as well as peripheral (circulating) immune
cells, resulting in altered cellular proliferation, differ-
entiation and survival through mechanisms that are
complex and include aryl hydrocarbon receptor
(AhR)-dependent pathways (Zaccaria & McClure
2013). While several studies have investigated the im-
munotoxicity of individual or groups of PAHs, rela-
tively few have asses sed the effects of direct exposure
to oil. In a hallmark study, mink Neovison vison with
chronic experimental exposure to bunker C fuel oil
had increased T cell proliferation, increased absolute
numbers of specific peripheral blood lymphocyte sub-
sets (CD3+T cells) and monocytes, and increased
level of expression of functionally significant cell sur-
face proteins including MHC II and CD18 (Schwartz
et al. 2004). Those results are compatible with previ-
ously described immune suppression upon exposure
to high concentrations of PAHs, and immune activa-
tion (adjuvant effect) at lower dose described by
others (Burchiel & Luster 2001). Given the likely
chronic exposure of dolphins to PAHs and other pe-
troleum compounds following the DWH oil spill, and
292
De Guise et al.: Immune response of dolphins exposed to DWH oil spill
the findings of dolphins with primary bacterial pneu-
monia, the present study aimed to evaluate potential
changes in im mune functions in bottlenose dolphins
inhabiting the DHW oil spill footprint over the first
3 yr following the oil spill.
MATERIALS AND METHODS
Animals
Bottlenose dolphins were captured, sampled, and
released as part of health assessment programs
(which included the immunological data presented
here), as previously described in detail elsewhere
(Wells et al. 2004, Schwacke et al. 2014). Sampling
was conducted at 3 GoM sites following the DWH oil
spill: BB, Louisiana, an area that received prolonged
and heavy oiling (Michel et al. 2013), sampled in
2011, 2013, and 2014; Mississippi Sound (MS), Mis-
sissippi, an area relatively less heavily oiled, sampled
in 2013; and SB, Florida, an area where no oil was
observed following the DWH spill, sampled in 2011,
2012, 2013, and 2014. The well-studied SB popu -
lation of resident bottlenose dolphins was used as
reference in this study (Wells et al. 2004). Mice were
used as quality control for functional assays. All
procedures were performed under appropriate per-
mits and IACUC approval as detailed in the Sup -
plement at www. int-res. com/ articles/ suppl/ n033 p291
_ supp. pdf.
Blood sampling and immune functions
Dolphin blood was collected into BD Vacutainer®
tubes as part of the physical examinations, kept cool
and shipped overnight for functional immunological
assays, as routinely performed in previous health as -
sessments (Schwacke et al. 2010, 2012). In addition,
serum was collected and immediately frozen prior to
shipping on dry ice for cytokine analysis. Cell isola-
tion and details of the immune function tests per-
formed, quality control measures and statistical
approaches are described in the Supplement.
RESULTS
Functional immune assays
Results for mitogen-induced lymphocyte prolifera-
tion are presented in Fig. 1. All 3 mitogens stimula ted
lymphocyte proliferation in a dose-dependent fash-
ion. When results were expressed as optical density
(OD), there was a consistent and significant in crease
in T lymphocyte proliferation, in reponse to both
concanavalin A (Con A) and phytohemagglutinin
(PHA), in BB 2011 dolphins compared to SB, at both
sub-optimal and optimal concentrations of mitogens.
However, the increase was consistently more marked
at sub-optimal concentrations of mitogens (55% for
sub-optimal vs. 44% for optimal concentrations of
Con A, and 164% for sub-optimal vs. 65% for optimal
concentrations of PHA). The T cell proliferation fol-
lowing stimulation with sub-optimal concentrations
of PHA was also significantly higher in the 2013 BB
dolphins compared to SB, but to a lesser extent (96%
increase) than in 2011 BB dolphins (164% increase).
The T cell proliferation following stimulation with
sub-optimal concentrations of PHA in 2014 BB and
2014 MS was not significantly different from SB.
A significant increase in B cell proliferation was
also observed in BB compared to SB. Sub-optimal
stimulation significantly increased B cell prolifera-
tion in 2011 (135% increase), 2013 (125%) and 2014
(61%), while optimal stimulation increased B cell
proliferation only in 2011 (83%) and 2013 (136%). As
with T cells, changes were generally (2011 and 2014,
but not 2013) slightly more marked upon sub-optimal
lipopolysaccharide (LPS) stimulation than with opti-
mal LPS stimulation. In the absence of mitogens, pro-
liferation was significantly higher in BB (2011, 2013
and 2014) than in SB. No significant effects were
observed in MS compared to the SB.
When results were expressed as stimulation index
(SI), patterns were generally similar but the vari -
ability was greater (as it takes into account both the
variability of the mitogen-stimulated cells and that of
the unstimulated control), and results were statisti-
cally significant only for sub-optimal stimulation with
PHA.
The results for T cell proliferation in SB were ro -
bust enough (n > 40) to allow for the generation of
reference intervals (RIs). Sample sizes used for eval-
uations of sub-optimal and optimal levels of ConA
and PHA (hereafter SubConA, OptConA, SubPHA,
and OptPHA) differed due to laboratory quality con-
trol measures, but ranged between 41 and 48 in -
dependent observations (Table 1). No outliers were
detected for any parameter (Table 1), values did not
significantly differ by year or between sexes, and
results were not significantly correlated with age.
Therefore, RIs calculated for each parameter were
not partitioned by any demographic variables
(Table 2).
293
Endang Species Res 33: 291–303, 2017
T cell proliferation (OD) values
from dolphins sampled in BB and MS
were compared to threshold values
for the 95th percentile RI to identify
cases for each parameter (Table 3).
For all parameters, the prevalence of
cases among dolphins sampled in BB
during 2011 was significantly greater
than expected, with most cases above
the RI threshold (Table 3). In contrast,
a significantly high prevalence of
294
Parameter N Male Female Mean SD Median Range Outliers
SubConA 47 24 23 0.97 0.60 0.96 0.09−2.21 0
OptConA 48 24 24 1.29 0.61 1.30 0.09−2.34 0
SubPHA 41 20 21 0.47 0.42 0.26 0.08−1.64 0
OptPHA 44 23 21 1.05 0.67 1.06 0.02−2.23 0
Table 1. Summary statistics for optical density data used to construct 95th per-
centile reference intervals for T cell proliferation in response to exposure to
sub-optimal (sub) and optimal (opt) levels of the mitogens ConA and PHA
(SubConA, OptConA, SubPHA, and OptPHA) in blood samples taken from
free-ranging bottlenose dolphins sampled in Sarasota Bay, FL, in 2011−2014
A
B
0.0
0.5
1.0
1.5
2.0
2.5
NM subConA optConA subPHA optPHA subLPS optLPS
Proliferation (OD)
SB
BB-11
BB-13
BB-14
MS-13
*
*
*
**
*
*
*
*
*
*
*
*
*
0
5
10
15
20
25
subConA optConA subPHA optPHA subLPS optLPS
Proliferation (SI)
SB
BB-11
BB-13
BB-14
MS-13
Fig. 1. Lymphocyte proliferation in blood of bottlenose dolphins exposed to varying levels of oiling following the DWH oil spill
upon stimulation with optimal (opt) and sub-optimal (sub) concentrations of mitogens, compared to no mitogen stimulation
(NM), expressed as (A) optical density (OD) and (B) stimulation index (SI). Samples were obtained from Sarasato Bay (SB), FL,
in 2011−2014 (n = 60 dolphins), Barataria Bay (BB), LA, in 2011 (n = 29), 2013 (n = 31), and 2014 (n = 32) and in Mississippi
Sound (MS), MS, in 2013 (n = 20). Results are means, and error bars represent standard deviation. *Results significantly
different from SB (p < 0.05)
De Guise et al.: Immune response of dolphins exposed to DWH oil spill
cases was observed only for SubPHA among 2013 BB
dolphins. Similarly, the prevalence of SubConA, Opt-
ConA, SubPHA, and OptPHA cases was either 0.00
or not significantly greater than expected for dol-
phins sampled in MS and BB in 2014, and where
cases were detected (e.g. BB 2014 and MS 2013 Sub-
ConA, BB 2014 SubPHA), values were below the RI
threshold (Table 3).
Cytokines
IL-4, IL-12, and IFNγwere measured using both
porcine and human reagents. Average values for IL-4
were 700-fold higher using the porcine kit, which
yielded detectable concentrations in 85% of the sam-
ples tested, while the human kit only detected meas-
urable concentrations in 59% of the samples tested.
On the other hand, the human kit yielded average
values 1.5- and 9-fold higher for IL-12 and IFNγ, re -
spectively; measurable concentrations were ob tained
in 71 and 48% of the samples for IL-12 and IFNγ,
respectively, using the human kit, and in 54 and 5%,
respectively, using the porcine kit. Thus, reported
values herein for IL-4 are based on the porcine kit,
whereas values for IL-12 and IFNγare based on the
human kit.
Serum cytokines varied considerably between in -
dividuals (Fig. 2), as expected for samples from wild
populations of outbred animals with different ages
and health status. There were no statistically signifi-
cant differences between locations and times when
compared to the SB reference. However, some inter-
esting trends were observed. For example, the Th1
cytokines IL-2, IL-12, and IFNγwere approximately
2-, 20-, and 4-fold, respectively, lower in BB 2011
compared to the SB reference, with no such differ-
ences observed for BB 2013, BB 2014, or MS. Simi-
larly, the Th2 cytokines IL-5, IL-10, and IL-13 were
approximately 2-, 50-, and 10-fold, respectively, low -
er in BB 2011 compared to the SB reference, with no
comparable differences observed for BB 2013, BB
2014, and MS. IL-4 was 3-fold higher in BB 2011
compared to SB, with no such differences in BB 2013,
BB 2014 and MS. The pro-inflammatory cytokines
IL-1β, TNFαand IL-8 trended differently among
locations and time, compared to SB. GM-CSF was
10-fold lower in BB 2011, but was higher in BB 2013,
BB 2014, and MS, respectively, compared to SB.
DISCUSSION
Oil exposure and health effects
Significant differences were observed in bottle-
nose dolphin immune functions measured in the
northern GoM following the DWH oil spill compared
to a reference population of dolphins not exposed to
295
Parameter N Lower 95th Upper 95th
(90% CI) (90% CI)
SubConA 47 0.22 (0.09−0.30) 2.17 (1.84−2.21)
OptConA 48 0.28 (0.09−0.51) 2.22 (2.11−2.34)
SubPHA 41 0.10 (0.08−0.15) 1.52 (1.12−1.64)
OptPHA 44 0.10 (0.02−0.22) 2.11 (2.05−2.23)
Table 2. 95th percentile reference intervals (and respective
90% confidence intervals) for optical density measures of
T cell function in response to exposure to mitogens (for
parameters, see Table 1 legend) in blood samples taken
from free-ranging bottlenose dolphins in Sarasota Bay, FL,
in 2011−2014
Parameter Year n Cases Preva- 95% CI
Site (low/high) lence
SubConA
BB 2011 29 7 (2/5) 0.24* 0.10−0.44
BB 2013 31 0 (0/0) 0.00 0.00−0.11
BB 2014 32 1 (1/0) 0.03 0.00−0.16
MS 2013 20 2 (2/0) 0.10 0.01−0.32
OptConA
BB 2011 28 7 (1/6) 0.25* 0.11−0.45
BB 2013 31 2 (0/2) 0.06 0.01−0.21
BB 2014 32 0 (0/0) 0.00 0.00−0.11
MS 2013 16 0 (0/0) 0.00 0.00−0.21
SubPHA
BB 2011 27 9 (0/9) 0.33* 0.17−0.54
BB 2013 31 5 (0/5) 0.16* 0.05−0.34
BB 2014 32 2 (2/0) 0.06 0.01−0.21
MS 2013 20 0 (0/0) 0.00 0.00−0.17
OptPHA
BB 2011 24 6 (0/6) 0.25* 0.10−0.47
BB 2013 28 2 (0/2) 0.07 0.01−0.24
BB 2014 31 0 (0/0) 0.00 0.00−0.11
MS 2013 18 0 (0/0) 0.00 0.00−0.19
Table 3. Counts and prevalence of bottlenose dolphins with
T cell function indices above or below 95th percentile refer-
ence intervals in response to exposure to mitogens (for para -
meters, see Table 1 legend). Data is for dolphins from
Barataria Bay (BB), LA, and Mississippi Bay (MS), MS, af -
fected by oiling following the DWH oil spill, compared to
reference values from unaffected dolphins in Sarasota Bay,
FL (Tables 1 & 2). ‘n’ is sample size and ‘cases’ are numbers
of dolphins with values either above (‘high’) or below (‘low’)
reference interval thresholds. ‘95% CI’ is the binomial con-
fidence interval for the case prevalence. *Prevalence
significantly different than expected (p < 0.05)
Endang Species Res 33: 291–303, 2017
oil, in SB, Florida. While there was no oil exposure
directly documented on the animals sampled as part
of this study, the DWH oil spill resulted in the con-
tamination of prime marine mammal habitat in the
estuarine, nearshore, and offshore waters of the nor -
thern GoM, and tens of thousands of marine mam-
mals were exposed to the DWH surface slick, where
they likely inhaled, aspirated, ingested, physically
contacted, and absorbed oil components (DWH
NRDA Trustees 2016). Further, the presence of in -
creased coastal levels of bioavailable PAHs associ-
ated with the DWH oil spill, especially near Grand
Isle, Louisiana in BB have been confirmed (Allan et
al. 2012). It is therefore highly likely that dolphins in
BB were exposed to oil as part of the DWH spill,
although the exact timing and magnitude of expo-
296
Fig. 2. Serum cytokines (pg ml−1) in blood of bottlenose dolphins following the DWH oil spill, in samples from SB (n = 49
dolphins), BB 2011 (n = 32), BB 2013 (n = 31), BB 2014 (n = 32) and MS 2013 (n = 20). Measurements were made using the
Bio-Plex technology and human (Hu) or porcine (Po) reagents, for Th1 (left panels), Th2 (center panels) and pro-inflammatory
(right panels) cytokines. See Fig. 1 legend for abbreviations of study sites and ‘Introduction’ for abbreviations of cytokines.
Results are means, and error bars represent standard deviation
De Guise et al.: Immune response of dolphins exposed to DWH oil spill
sure have not been documented, and it is uncertain
how long oil products may have remained in the food
chain or in sediments, resulting in potential ongoing
dolphin exposure. Further, other studies have docu-
mented health effects, including effects on reproduc-
tion, respiratory system and adrenal glands, in the
same dolphins sampled in 2011, a year following the
DWH oil spill (Schwacke et al. 2014, Lane et al.
2015). In addition, some of those health effects were
also observed in dead dolphins that stranded in in -
creased numbers following the DWH oil spill (Venn-
Watson et al. 2015). It is also noteworthy that some
long-term impacts on marine mammal health have
been documented following the Exxon Valdez oil
spill, including in sea otters (Monson et al. 2000, Bod-
kin et al. 2012) and killer whales (Matkin et al. 2008).
Few studies have assessed the immunomodulatory
effects of oil exposure, and to the authors’ knowl-
edge, none have specifically addressed the effects of
exposure to petrogenic PAHs. However, there are
studies documenting the effects of other PAHs in
diverse species, although they represent an imper-
fect model for the effects of oil exposure. We examine
below the nature of the immunological changes
observed in view of those described in other species
exposed to oil (few studies) or PAHs more broadly,
and the potential consequences of those changes.
Oil exposure and T cells
The increase in T cell proliferation was specific in
nature and different from those observed in recent
bottlenose dolphin health assessments performed us-
ing similar methods. Previous health assessments of
wild coastal populations of wild bottlenose dolphins
using capture-release methods found a negative cor-
relation between T cell proliferation and ex posure to
high levels of polychlorinated biphenyls (PCBs) near a
superfund site on the coast of Georgia (Schwacke et
al. 2012), as well as with exposure to biotoxins and re-
lated eosinophilia in St. Joseph Bay, Florida (Schwa -
cke et al. 2010). The effects on T cells, measured as
the population average and using the prevalence of
animals outside the reference range, also appeared to
be site specific (BB but not MS) and/or generally asso-
ciated with the timing of the spill (2011 > 2013 > 2014).
Unfortunately, samples from MS were not collected
until 2013; therefore, it is impossible to say whether
the lack of observed differences for this area was due
to the lack of an effect, or whether effects similar to
those observed in BB 2011 did occur, but had dimin-
ished by 2013. It has been long established that im-
munotoxicology studies deal with immune alterations,
both stimulatory and suppressive (Burleson & Dean
1995). Further, extensive studies in laboratory animals
suggest that any change, whether an increase or de-
crease in immune functions, is potentially deleterious
(Luster et al. 1992). While exposure to PAHs is gener-
ally associated with suppression of mitogen-induced
lymphocyte proliferation using in vitro and in vivo
models in mammals (Dean et al. 1985, Thurmond et
al. 1988, Davila et al. 1996, Karakaya et al. 2004, Zac-
caria & McClure 2013) and fish (Reynaud & Deschaux
2006), several instances of increased lymphocyte pro-
liferation have been reported in the presence or ab-
sence of mitogen stimulation (Tahir & Secombes 1995,
McMurry et al. 1999, Reynaud & Deschaux 2005,
Connelly & Means 2010) referred to as ‘adjuvant ef-
fect’ (Burchiel & Luster 2001). In fact, there have been
suggestions of immune suppression upon exposure to
high concentrations of PAHs, with immune activation
at lower doses (Burchiel & Luster 2001). While it is dif-
ficult to quantify the magnitude of PAH exposure in
dolphins during the DWH spill compared to controlled
experimental studies, the increase in T cell prolifera-
tion in BB 2011 is similar in nature and magnitude to
that observed in mink in response to chronic experi-
mental exposure to bunker C fuel oil (Schwartz et al.
2004). Stimulatory effects of PAHs have included an
increase in nasal antigen-specific IgE and tilting of
the cytokine balance towards a Th2 response in hu-
mans (Diaz-Sanchez et al. 1997, Tsien et al. 1997).
Also, mice exposed to cyclophosphamide experienced
an increase in T lymphocyte proliferation at lower
doses and a decrease in T cell proliferation at higher
doses, which was attributed to the higher sensitivity of
regulatory T cells at low dose (Luster et al. 1993). In-
terestingly, humans with hypo adrenocorticism, a con-
dition also described in BB dolphins in 2011 (Schwacke
et al. 2014), showed a downregulation of regulatory T
cells (Treg) (Coles et al. 2005). It is therefore possible
that exposure to oil could have modulated the prolif-
eration of T cells through direct and indirect (adrenal
insufficiency) effects on Treg, whose role is to gener-
ally dampen the immune response.
Oil exposure and B cells
The increase in B cell proliferation observed in BB
in 2011 and 2013 was site specific and closely associ-
ated with the timing of oil exposure (effects in 2011
and 2013; there were some effects in 2014 at sub-
optimal LPS concentrations, but not at optimal con-
centrations). It is also specific and different from the
297
Endang Species Res 33: 291–303, 2017
effects observed in other locations (Schwacke et al.
2010, 2012). For example, 6-mo exposure to diesel
exhaust increased the proliferation of mouse splenic
B lymphocytes at the lowest dose used (30 µg m−3),
but not at higher doses (Burchiel et al. 2004). While
the proliferation of B lymphocytes was not assessed
directly, experimental exposure of mink to bunker C
fuel oil resulted in the expression of an increased
density of MHC II on B cells, suggesting B cell acti -
vation (Schwartz et al. 2004). Activation of B and T
cells was also observed in workers occupationally ex -
posed to PAHs (Biró et al. 2002). Such B cell activa-
tion, in the absence of an antigen, could increase their
response to a mitogen, as seen in our study.
Oil exposure and Th1/Th2 cytokines
Previous studies have documented the modulation
of cytokines upon exposure to PAHs. Similar to the
trend in our study for BB 2011, PAH exposure mostly
resulted in down regulation of Th1 cytokines. In vitro
IL-2 production in mouse splenocytes was suppres -
sed by dimethylbenz(a)anthracene (DMBA) (Thur-
mond et al. 1988), and DMBA-induced suppression
of CTL was restored with the addition of IL-2, sug-
gesting that DMBA suppressed IL-2 in vivo as well
(Dean et al. 1985). Human dendritic cells matured in
vitro in the presence of BaP secreted less IL-12 than
un exposed control cells (Laupeze et al. 2002). Simi-
larly, in a mouse allergy model, the broncho-alveolar
lavage fluid had suppressed IL-12 upon exposure to
PAHs extracted from diesel exhaust particles (Ste -
vens et al. 2009). IL-12 was also reduced following
dermal exposure to PAHs in humans (Borska et al.
2008). IFNγwas down regulated upon in vitro BaP
exposure of human cell lines (Tang et al. 2012) and
olive flounder Paralichthys olivaceus head kidney
cells (Hur et al. 2013). The mediation of IFNγap -
peared to be mediated through a calcium-dependent
pathway (i.e. nuclear factor of activated T cells
[NFAT]) as it was blocked by ethyleneglycol-bis-
tetraacetic acid (EGTA) (Hur et al. 2013).
The effects of PAH exposure on Th2 cytokines
have also been described in other species. For exam-
ple, human dendritic cells matured in vitro in the
presence of BaP secreted less IL-10 than unexposed
control cells (Laupeze et al. 2002). BaP administration
in rats (Rattus norvegicus) resulted in a 2.6-fold in -
crease in IL-4 mRNA in the blood (Al-Daghri et al.
2014). BaP and phenanthrene significantly enhanced
the production of IL-4 in human basophils upon in
vitro exposure (Schober et al. 2007). In a spatial-
temporal regression model over multiple time periods,
ambient PAH exposure in children was associated
with decreased IL-10 and increased IL-4, along with
increased IL-13 and IFNγ(Hew et al. 2015). Overall,
while not statistically significant, the trend was towards
lower Th1 and Th2 cytokines except for higher levels
of IL-4 in BB 2011, likely resulting in a tilt of the bal-
ance towards Th2 given the dominant role of IL-4 in
the Th1/Th2 regulation, which would be compatible
with the outcome of several experimental studies.
Further, depletion studies in mice have demonstrated
the ability of Treg to influence the Th1/Th2 cytokine
balance (Xiong et al. 2015), offering the possibility of
an indirect pathway.
Oil exposure and pro-inflammatory cytokines
Pro-inflammatory cytokines did not all vary simi-
larly in our study, with trends towards higher Il-1β
and IL-8 in BB 2011, as was the case in human
macrophage exposed to BaP in vitro (Sparfel et al.
2010), and in olive flounders exposed to BaP in vivo
and in vitro (Hur et al. 2013). In another report,
human macrophage exposure to BaP enhanced the
production of IL-8 in a dose-dependent manner
through the AhR pathway (Podechard 2008). TNFα
was 7-fold lower in BB 2011 as compared to the SB
reference site. BaP exposure of human monocytes in
vitro reduced the production of TNFαapproximately
9 fold (van Grevenynghe et al. 2003). However, this is
different from most studies where TNFαis increased
upon exposure to BaP, including in human models
(Lecureur et al. 2005, Sparfel et al. 2010) and in olive
flounder upon in vivo and in vitro exposures (Hur
et al. 2013). It is possible that differences between
species are associated with differences in pathway/
receptors, since BaP-induced modulation of TNFα
was reported to occur through a AhR-independent,
extracellular signal−regulated kinases (ERK)-related
pathway in human macrophages (Lecureur et al.
2005), while it was reported to occur via the AHR
pathway in olive flounder, as it was blocked by ANF
(Hur et al. 2013).
Oil exposure, immune functions and reproduction
The bottlenose dolphin unusual mortality event in
the northern GoM was associated with significant
increases in perinatal mortality (Venn-Watson et al.
2015). Maintenance of pregnancy requires a complex
series of immunological events leading to local (intra -
298
De Guise et al.: Immune response of dolphins exposed to DWH oil spill
uterine) tolerance of the embryo/fetus by the
mother’s immune system, rather than immunological
rejection. In humans, the major mechanisms involved
in intrauterine tolerance include the expression of
the non-classical human major histocompatibility
(MHC) class Ib and its inhibitory effects on uterine
natural killer (NK) cells, as well as the IL-10 mediated
interaction between Treg and specialized tolerogenic
dendritic cells (DCs) (Lynge Nilsson et al. 2014). If
immune processes in the dolphin are similar to hu -
mans, it is plausible that immune tolerance towards
the fetus may have been compromised if the ability to
produce intrauterine IL-10 was downregulated as
appeared to be the case systematically, with serum
levels in BB 2011 that were 50-fold lower than in SB.
Further, it is possible that immune tolerance may
have been compromised if intrauterine Treg cells
were downregulated, as described systemically in
humans with hypoadrenocorticism (Coles et al. 2005),
as evidenced in BB 2011 (Schwacke et al. 2014). Sev-
eral functional studies have shown that unexplained
infertility, miscarriage and pre-clampsia are often
associated with deficits in Treg cell numbers and
function, while normal pregnancy selectively stimu-
lates the accumulation of maternal Treg cells with
fetal specificity (La Rocca et al. 2014). Unfortunately,
we do not have data to directly support such poten-
tial mechanisms in dolphins from our health assess-
ment data.
Oil exposure, immune functions
and perinatal mortalities
Increased perinatal mortalities in the northern
GoM unusual mortality event were associated with a
high prevalence of in utero deaths, fetal distress, and
non-lungworm associated pneumonia in the fetus
compared to reference stranded dolphin perinatal
populations. In Mississippi and Alabama, stranded
perinates also had a high prevalence of Brucella
infections involving multiple genetic subtypes across
the region (Colegrove et al. 2016). The importance of
the Th1/Th2 paradigm for the control of Brucella
infection was demonstrated almost 2 decades ago in
mice, where the predominance of the Th2 cytokines
IL-4 and IL-10 in the BALB/c mice was associated
with increased susceptibility to Brucella, whereas
the predominance of the Th1 cytokine IFNγin the
C57BL/10 mice was associated with increased resist-
ance to Brucella (Fernandes et al. 1996, Baldwin &
Parent 2002). Further, mouse studies with IFN regu-
lation confirmed the importance of IFNγand IL-12 for
resistance to Brucella infection (Ko et al. 2002), espe-
cially in the first weeks after infection when relative
resistance to brucellosis correlated with increased
production of IFNγby CD4 T cells (Baldwin & Parent
2002). Mouse macrophages activated with IFNγwere
shown to have enhanced brucellacidal and brucella -
static activities (Jiang et al. 1993). Additional knock-
out studies in mice demonstrated that IFNγappears
to be more important than IL-12 for controlling the
magnitude of Brucella infections (Brandao et al.
2012). Susceptibility of MyD88 KO mice to Brucella
abortus was due to impaired dendritic cell matura-
tion and lack of IL-12 synthesis (Macedo et al. 2008).
The administration of recombinant IL-12 to suscept -
ible BALB/c mice ameliorated the IFNγhiatus asso -
ciated with susceptibility, resulting in a 1000-fold
reduction in colony-forming units (CFU) during
primary Brucella infection and increased survival
following secondary challenge (Sathiyaseelan et al.
2006). B. abortus vaccine strain 19 replicated much
better in IL-6-/- than in IL-6+/+ mice (Pizarro-Cerdá
et al. 1999). Altogether, those studies confirm the
detrimental effects of the Th2 cytokines IL-4, IL-6
and IL-10, and the positive effects of the Th1 cyto-
kines IFNγand IL-12 in resistance to Brucella infec-
tion in mice. Similarly, monocyte-derived macro-
phages from resistant cows had the ability to mount a
Th1 immune response against B. abortus infection,
which was impaired in cells from susceptible animals
(Rossetti et al. 2011). It is plausible that a Th1 reduc-
tion and tilt towards a Th2 response in BB 2011, if
biologically significant despite not being statistically
significant, would increase the susceptibility of those
dolphins to Brucella infection or other intracellular
pathogens. Increased susceptibility to bacterial chal-
lenges, including intra-cellular Mycobacterium mari -
num, upon PAH exposure was specifically confirmed
in several studies in fish (Bravo et al. 2011, Prosser et
al. 2011).
Potential causes and consequences of changes
in immune functions
A broad and comprehensive hallmark study in lab-
oratory animals concluded that there was a good cor-
relation between changes in immune function tests
and altered host resistance (Luster et al. 1993). In that
study, there were no instances of altered host resist-
ance without altered immune test results. However,
there were instances of immune changes without
detectable changes in host resistance, mostly attrib-
utable to the specificity of the immune function
299
Endang Species Res 33: 291–303, 2017
changes (mechanistically relevant) in relation to the
challenge, i.e. challenges with a pathogen that was
not relevant to the immune functions affected. The
same authors suggested that, considering the pres-
ence of background levels of infectious diseases in
natural populations, it is possible that any change in
immune functions could translate to changes in host
resistance if the population exposed is large enough
(Luster et al. 1993). In other words, if immune func-
tions of a natural population are affected, it is highly
likely that there will be a pathogen somewhere in the
environment of the population that will be relevant to
the immune functions affected. These authors sug-
gested that the relationship between immune dis-
functions and infectious diseases is linear rather than
involving a threshold (Luster et al. 1993).
A broad diversity of chemicals can alter immune
functions, and the changes observed here are com-
patible with, but not necessarily specific to, exposure
to oil. The effects of PCBs and other persistent
organic pollutants (POPs) on immune functions have
been widely described, and our group has previously
demonstrated immune alterations associated with
PCB exposure in bottlenose dolphins near a super-
fund site on the coast of Georgia (Schwacke et al.
2012). However, POP concentrations in Barataria Bay
bottlenose dolphins were in the lower half of the
range compared to previously reported concentra-
tions from other southeastern US sites, suggesting
that POPs were like ly not a primary contributor to the
poor health conditions and increased
mortality observed in northern GoM
dolphins following the DWH oil spill
(Balmer et al. 2015). Several patho-
gens can be associated with modula-
tion of immune functions, including
morbilliviruses. A thorough investi-
gation of the northern GoM un usual
mortality event associated in time and
space with the DWH oil spill con-
cluded that brucellosis and morbil-
livirus infections were detected in 7
and 11% of unusual mortality event
dolphins, respectively, and biotoxin
levels were low or below the detec-
tion limit, indicating that these were
not primary causes of the current
unusual mortality event (Venn-Wat-
son et al. 2015). Further, it was un -
likely that other pathogens would
have been causing widespread disea -
ses, based on the results of histo patho -
logical examinations (Venn-Watson
et al. 2015). Stress exposure has also long been asso-
ciated with immune system modulation via the se -
cretion of corticosteroids. However, the dolphins
sampled in this study suffered from hypoadreno -
corticism, with low serum cortisol and aldosterone
(Schwacke et al. 2014). It is therefore unlikely that
exposure to POPs, the most common pathogens asso-
ciated with dolphin morbidity and mortality, biotox-
ins, or stress would have been responsible for the
changes in immune functions reported here. It is pos-
sible that other emerging or legacy contaminants or
pathogens not measured as part of this investigation
may be associated with the immune changes ob -
served. However, this is unlikely compared to the
weight of evidence for the DWH oil spill and its asso-
ciation with other health effects.
It is possible that changes in T cell functions, and
possibly the cytokine balance (if the changes ob -
served were to be biologically significant, despite the
fact that they are not statistically significant, given
the broad variability in a diverse population of wild
and outbred animals), would have resulted in in -
creased susceptibility to intra-cellular pathogens,
while changes in B cell functions would have re -
sulted in increased susceptibility to extracellular
pathogens such as bacteria that could have been
involved in the high incidence of primary bacterial
pneumonia observed in the northern GoM unusual
mortality event. A conceptual model is presented in
Fig. 3 to summarize the potential relationships be -
300
Fig. 3. Conceptual model summarizing the potential relationships between
the changes in immune function and the health effects observed in live and
dead dolphins following the DWH oil spill. The conceptual model is based on
observations, and links supported by relevant literature. PAH: polycyclic
aromatic hydrocarbon; Th2: T helper 2 lymphocytes
De Guise et al.: Immune response of dolphins exposed to DWH oil spill
tween the changes in immune function and the
health effects observed in live and dead dolphins
associated with the DWH oil spill.
While our results demonstrate associations be -
tween dolphins living in DWH-contaminated waters
and changes in T and B lymphocyte proliferation,
as well as trends towards a Th2-biased cytokine
ba lance, they do not confirm a cause and effect
relationship. However, the alterations in T and B
lymphocyte proliferation were comparable in nature
and magnitude to those observed in mink with
chronic experimental exposure to oil (Schwartz et
al. 2004), and compatible with those observed in
humans exposed to PAHs (Biró et al. 2002). The
altered T and B lympho cyte proliferation was con-
sistent in time and space with exposure to oil from
DWH as it occurred mostly in BB 2011, the area
and time when sampled dolphins would be most
affected by oil, and some of the changes observed
were less severe or subsiding in subsequent years.
The increase in T and B lympho cyte proliferation
was specific to BB, and different from the negative
correlation between T cell proliferation exposure to
PCBs (Schwacke et al. 2010) or biotoxins (Schwacke
et al. 2012) observed in wild dolphin populations.
Further, the altered T and B lymphocyte prolifera-
tion observed, and the trends towards a Th2-biased
cytokine balance, if biologically significant, would
be expected to increase susceptibility to infectious
disease, and are consistent with the bottlenose dol-
phin unusual mortality event investigation results
that found increased respiratory disease, often as -
sociated with bacteria, but did not identify a
specific single pathogen. The increase in perinatal
mortality and associated Brucella infection, an
intra-cellular bacterial infection for which T cells
and a Th1 predominant response are particularly
important, could be at least in part explained with
the T cell changes observed here. The increase in
frequency of primary bacterial pneumonia could be
at least in part explained by the B cell changes
observed here. The sum of the lines of evidence
above, along with the lack of evidence for alterna-
tive causes, suggests that the immune changes
reported here could be associated with exposure to
oil from the DWH spill.
Disclaimer. This work was part of the DWH Natural
Resource Damage Assessment (NRDA) being conducted co -
operatively among NOAA, other federal and state Trustees,
and BP plc. The findings and conclusions in this paper are
those of the authors and do not necessarily represent the
view of NOAA or of any other natural resource Trustee for
the BP/DWH NRDA.
Acknowledgements. The authors wish to thank the numer-
ous researchers, staff members and volunteers who pro-
vided support for the dolphin health assessment field work.
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Editorial responsibility: Karina Acevedo-Whitehouse,
Queretaro, Mexico
Submitted: June 6, 2016; Accepted: January 29, 2017
Proofs received from author(s): April 26, 2017
