ESTABLISHING MARGINAL LYMPH NODE

ULTRASONOGRAPHIC CHARACTERISTICS IN
HEALTHY BOTTLENOSE DOLPHINS (TURSIOPS
TRUNCATUS)
Author(s): Molly E. Martony, D.V.M., Marina Ivančić, D.V.M., Dipl. A.C.V.R.,
Forrest M. Gomez, D.V.M., Jennifer M. Meegan, D.V.M., Dipl. A.C.Z.M.,
Hendrik H. Nollens, D.V.M., Ph.D., Todd L. Schmitt, D.V.M., Claire D.
Erlacher-Reid, D.V.M., Dipl. A.C.Z.M., Kevin P. Carlin, M.P.H., and Cynthia R.
Smith, D.V.M.
Source: Journal of Zoo and Wildlife Medicine, 48(4):961-971.
Published By: American Association of Zoo Veterinarians
https://doi.org/10.1638/2016-0251.1
URL: http://www.bioone.org/doi/full/10.1638/2016-0251.1

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 Journal of Zoo and Wildlife Medicine 48(4): 961–971, 2017
Copyright 2017 by American Association of Zoo Veterinarians

ESTABLISHING MARGINAL LYMPH NODE

ULTRASONOGRAPHIC CHARACTERISTICS IN HEALTHY
BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS)

Molly E. Martony, D.V.M., Marina Ivančić, D.V.M., Dipl. A.C.V.R., Forrest M. Gomez, D.V.M.,
Jennifer M. Meegan, D.V.M., Dipl. A.C.Z.M., Hendrik H. Nollens, D.V.M., Ph.D., Todd L. Schmitt,
D.V.M., Claire D. Erlacher-Reid, D.V.M., Dipl. A.C.Z.M., Kevin P. Carlin, M.P.H., and Cynthia R.
Smith, D.V.M.

Abstract: Pulmonary disease has been well documented in wild and managed dolphin populations. The
marginal lymph nodes of the dolphin thorax provide lymphatic drainage to the lungs and can indicate pulmonary
disease. This study standardized a technique for rapid, efficient, and thorough ultrasonographic evaluation of the
marginal lymph nodes in bottlenose dolphins (Tursiops truncatus). Thoracic ultrasonography was performed on 29
clinically healthy adult bottlenose dolphins. Reference intervals for lymph node dimensions and ultrasonographic
characteristics of marginal lymph nodes were determined from four transducer orientations: longitudinal,
transverse, oblique, and an orientation optimized to the ultrasonographer’s eye. The relationship between lymph
node dimensions and dolphin age, sex, length, weight, origin, and management setting (pool versus ocean
enclosure) were also evaluated. The mean marginal lymph nodes measured 5.26 cm in length (SD ¼ 1.10 cm,
minimum ¼ 3.04 cm, maximum ¼ 7.61 cm, reference interval [10th to 90th percentiles per node dimension] 3.78–
6.55 cm) and 3.72 cm in depth (SD ¼ 0.59 cm, minimum ¼ 2.64, maximum ¼ 5.38 cm, reference interval 2.98–4.50
cm). Sex, dolphin length, weight, and management setting had no effect on lymph node dimensions. Dolphins .30
yr of age had longer node lengths than dolphins 5–10 yr old. Node dimensions did differ between dolphins from
various origins. Most commonly, the lymph node was found to be hyperechoic relative to surrounding soft tissues
(98%) and to have irregular caudal borders (84%), ill-defined deep borders (83%), flat superficial border (67%),
triangular or rounded triangle shape (59%), irregular cranial border (55%), and moderate heterogeneity (34%).
The data reported in this study serve as a baseline reference that may contribute to earlier detection of pleural and
pulmonary disease of managed and wild cetacean populations.
Key words: Bottlenose dolphin, lymph node, marginal lymph node, pulmonary, thoracic ultrasound, Tursiops
truncatus.

INTRODUCTION tance of investigating diagnostic modalities that

It has been well established that there is a high
prevalence of pulmonary disease in both man-
aged and wild cetacean populations world-
wide.1,3,6,9–11,13,14,19,20,26,28,29 Rapid detection of
respiratory disease in cetaceans is critical, given
that they are capable of masking even severe
disease processes.18 The significance of pulmo-
nary disease in cetaceans underscores the impor-
From the National Marine Mammal Foundation, 2240
Shelter Island Drive, San Diego, California 92106, USA
(Martony, Ivančić, Gomez, Meegan, Carlin, Smith); and
SeaWorld California, Veterinary Services, 500 SeaWorld
Drive, San Diego, California 92109, USA (Martony,
Nollens, Schmitt, Erlacher-Reid). Present addresses (Ivan-
čić): Brookfield Zoo/Chicago Zoological Society, 3300 Golf
Road, Brookfield, Illinois 60513, USA; (Martony): Uni-
versity of Florida College of Veterinary Medicine, 2015 SW
16th, Gainesville, Florida 32608, USA. Correspondence
should be directed to Dr. Martony (mollymartony@gmail.
com).
facilitate early detection of respiratory pathology.
Thoracic ultrasonography and radiography are
the most commonly used diagnostic modalities to
screen for pulmonary disease in cetaceans.5,26,27
Ultrasonography remains the only diagnostic
imaging tool that can be performed in water, and
it is both rapid and noninvasive. However, due to
the reverberation artifact at the surface of normal
air-filled lung, only the periphery of the lung can
be evaluated in the absence of disease. Deeper
structures can only be seen ultrasonographically
when peripheral lung pathology is present.12 The
thoracic lymph nodes are of particular clinical
interest because enlargement and changes in
echogenicity can indicate respiratory disease.5
Ultrasonography has proven to be a valuable tool
for evaluation of lymph nodes in humans and
domestic animals.2,15,17,21,22,24,32 Several descriptions
of normal lymph node characteristics are avail-
able for humans and domestic animals, and these
descriptions have led to advances in ultrasono-
graphic detection of disease in those species.21

961
962 JOURNAL OF ZOO AND WILDLIFE MEDICINE
Bottlenose dolphin (Tursiops truncatus) lym-
phoid organs follow mammalian distribution and
organization patterns with the following excep-
tions: the presence of marginal lymph nodes,
diaphragmatic lymph nodes, and anal tonsil
complex and the lack of an appendix.8 Based on
their structure and anatomic location, the lymph
nodes of bottlenose dolphins are divided into
somatic, visceral, aortic arch, and lung-associated
groups.8 Lung-associated nodes include the hilar
nodes, diaphragmatic node mass, and bilateral
marginal nodes.8 The marginal and diaphragmatic
nodes serve as the primary lymphatic drainage of
the lungs, as evidenced by the reactivity of the
nodes in bottlenose dolphins with confirmed lung
pathology.5,8,16 The marginal lymph nodes are of
particular interest to veterinarians because their
superficial location allows for ultrasonographic
evaluation.
Each marginal lymph node is a large, always
present, and solitary structure.8 The ultrasono-
graphic acoustic window to the node varies
between animals, but it generally lies caudoven-
tral to the insertion of the pectoral flipper.26
Internally, the node lies caudolateral to the heart
on the ventral free border of each lung where the
lung intersects with the diaphragmatic surface
(Fig. 1).8,26 Typically, the edge of the lung is
adhered to the node across midline or to the
parietal pleura of the sternum, although occa-
sionally they are unattached.8 Postmortem evalu-
ations of stranded bottlenose dolphins have found
the node to measure approximately 5 3 2 3 2 cm.8
However, no peer-reviewed data on antemortem
marginal lymph node dimensions in healthy
bottlenose dolphins have been reported.
This study aims to 1) standardize a technique
for the ultrasonographic evaluation of the mar-
ginal lymph node in dolphins; 2) establish refer-
ence intervals for the dimensions of the marginal
lymph node in clinically healthy bottlenose dol-
phins; 3) describe the ultrasonographic character-
istics of these lymph nodes in clinically healthy
adult bottlenose dolphins; and 4) evaluate the
relationship between lymph node dimensions and
dolphin age, sex, length, weight, origin (Atlantic
or Pacific Ocean), and management setting (pool
or ocean enclosure). A clear understanding of
normal lymph node dimensions and ultrasono-
graphic characteristics will aid in differentiating
normal and abnormal nodes and facilitate earlier
detection of pleural and pulmonary disease, more
precise disease monitoring, and more effective
care of managed and wild cetacean populations.
Figure 1. Gross thoracic anatomy in a 31-yr-old
male T. truncatus from the US Navy Marine Mammal
Program. (a) Heart. (b) Left marginal lymph node. (c)
Left lung. (d) Diaphragm.
MATERIALS AND METHODS
Study subjects
Thoracic ultrasonography was performed on
bottlenose dolphins at SeaWorld of California
(SWC) and the US Navy Marine Mammal Pro-
gram (MMP), both in San Diego, California,
USA. Only dolphins .5 yr of age that were
considered clinically healthy as evidenced by
blood work, thoracic ultrasonography, absence
of clinical signs in the prior month, and no history
of respiratory disease in the prior year were
included. To evaluate the dolphins’ health, blood
was collected from the periarterial venous rete of
the ventral tail fluke as part of the routine
preventive veterinary exams, within 7 days of the
lymph node and pulmonary ultrasound exams. A
complete blood count (CBC), chemistry panel,
fibrinogen, and erythrocyte sedimentation rate
(ESR) were evaluated. Dolphins were excluded if
blood work revealed inflammation as evidenced
by two or more of the following abnormal
parameters: increased fibrinogen, ESR, or total
white blood cell count; or decreased alkaline
phosphatase or iron, relative to the reference
ranges established for each of the two facili-
ties.4,18,30 Thoracic ultrasonographic evaluations
were conducted on each animal within one week
of marginal lymph node evaluations using previ-
ously described methods.26 Dolphins diagnosed
with pulmonary or pleural disease within 1 year of
data collection were not included. Those with a
history of neoplasia or systemic inflammatory
disease were also excluded. Dolphins were not
included if they had been administered systemic
medications other than preventative medications
in the 3 months prior to imaging.
 MARTONY ET AL—DOLPHIN MARGINAL LYMPH NODES
Figure 2. Voluntary ultrasonographic examination
of the left marginal lymph node in T. truncatus,
demonstrating the approximate location of transducer
placement.
Ultrasound evaluation
Dolphins cared for by SWC were evaluated
using a GE Logiq e portable ultrasound unit and a
2–5-MHz curvilinear transducer (GE Healthcare,
Noblesville, Indiana 46060, USA). Animals cared
for by the MMP were evaluated using a portable
SonoSite Edge ultrasound unit and a 2–5-MHz
curvilinear transducer (Sonosite, Bothell, Wash-
ington 98021, USA). Exams were performed with
the dolphin voluntarily floating at the surface of
the water in both right and left lateral recumben-
cy. The ultrasonographer and equipment were
positioned at the water’s edge, adjacent to the
animal’s ventral surface (Fig. 2). All ultrasound
evaluations were performed in B-mode (two
dimensional or gray scale) ultrasound. Specific
image settings associated with the model and
make of the ultrasound units were adjusted for
each dolphin to optimize image quality.
To locate the marginal lymph node, the trans-
ducer was placed caudal and ventral to the
insertion of the pectoral flipper, in a craniocaudal
orientation. The ventral diaphragm and cranial
surface of the liver were identified. The transduc-
er was then advanced cranially until the apex of
the heart, ventral border of the lung, diaphragm,
and cranial liver could all be seen in one acoustic
window. Identification of the lymph node re-
quired careful fanning dorsally and ventrally in
this location. An obliquely oriented vessel of
approximately 4–5 mm in diameter is frequently
seen traversing the superficial (lateral) surface of
the lymph node, serving as an additional anatomic
landmark.
963
Figure 3. Ultrasound transducer orientation for
three different image views used to evaluate the
marginal lymph node in T. truncatus: (a) longitudinal,
(b) transverse, and (c) oblique. Note that the fourth
‘‘optimized image view’’ is not pictured due to variabil-
ity in its orientation, unique to each lymph node.
Four image views were used to evaluate both
marginal lymph nodes in each animal (Fig. 3).
Cine loops (ultrasound videos) 3–6 seconds in
length were obtained for each node in each image
view. The image views used to acquire cine loops
were as follows: 1) longitudinal to the node (along
the craniocaudal axis) fanning dorsally and ven-
trally through the node, 2) transverse to the node
(along the dorsoventral axis and orthogonal to the
longitudinal view) fanning cranially to caudally
through the node, 3) oblique to the long axis of
the dolphin (cranioventral-caudodorsal intercos-
tal space, parallel to the sternal ribs) fanning
through the node cranially to caudally, and 4) a
view optimizing the node to the ultrasonogra-
pher’s eye. The longitudinal and transverse image
acquisition techniques were collected as de-
scribed above. To obtain the oblique image view,
the transducer was placed in a craniocaudal
orientation, and then the cranial aspect of the
transducer was rotated ventrally and the caudal
aspect of the transducer dorsally until the trans-
ducer was parallel to the sternal ribs and the
acoustic shadowing of the ribs eliminated. The
optimized image view was acquired by maximiz-
ing the dimensions of the lymph node to the eye
while not confining the transducer to one speci-
fied imaging view. All imaging was performed by
964 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Table 1. Maximal perceived marginal lymph node dimensions of T. truncatus using various ultrasonographic
image views

Node dimension (cm) n Range Mean SD Reference interval

Maximal
Length 58 3.04–7.61 5.26 1.1 3.78–6.55
Depth 58 2.64–5.38 3.72 0.59 2.98–4.50
Longitudinal view
Length 58 2.05–6.61 4.43 1.15 2.91–5.94
Depth 58 1.92–4.57 3.38 0.66 2.59–4.33
Transverse view
Height 58 1.24–4.3 2.26 0.59 1.67–2.89
Depth 58 1.93–5.07 3.18 0.66 2.44–4.05
Oblique view
Length 58 2.85–7.05 4.78 1.03 3.38–6.14
Right side depth 29 2.56–5.55 3.78 0.64 3.08–4.37
Left side depth 29 1.99–4.53 3.48 0.68 2.60–4.25
Optimized view
Length 58 3.04–7.61 5.11 1.11 3.35–6.51
Right side depth 29 2.96–5.38 3.86 0.62 3.20–4.81
Left side depth 29 2.52–4.54 3.58 0.58 2.72–4.26

either a board-certified veterinary radiologist
(MI) or a veterinarian (MEM) under direct
supervision by the board-certified veterinary
radiologist.
Case animal
In addition to the clinically healthy dolphins,
one case animal with confirmed lung disease
(pulmonary coccidiomycosis) was included.16
The marginal lymph node dimensions and char-
acteristics were established in this 32-yr-old
female bottlenose dolphin with Atlantic Ocean
origins.
Data collection and analysis
Ultrasound exams were reviewed using open
source Digital Imaging and Communications in
Medicine viewing software (OsiriX 5.8.2 Image
Software, Pixmeo, Geneva CH-1233, Switzer-
land). The maximum length, height, and depth
(in centimeters) were determined for each lymph
node using electronic calipers from still images
derived from the four cine loops (Table 1). The
following ultrasonographic characteristics were
visually evaluated: superficial border characteris-
tics (flat, concave, or convex), deep border mar-
gination (ill-defined or defined), cranial border
margination (smooth or irregular), caudal border
margination (smooth or irregular), dorsal border
margination (ill-defined due to the lung or rib
acoustic shadowing vs defined), ventral border
margination (ill-defined due to the rib acoustic
shadowing vs defined), node shape (rounded
[generally ovoid to rectangular with softly round-
ed borders], rounded triangle [generally triangular
with rounded borders], or triangular [generally
triangular lacking rounding of the borders]),
echotexture (mildly heterogenous, moderately
heterogenous, markedly heterogenous, or homog-
enous), general echogenicity (hyperechoic, hypo-
echoic, or isoechoic relative to the adjacent soft
tissues including; the external abdominal oblique
muscle, heart, and liver), and echogenicity detail
(focal hyperechoic regions, focal hypoechoic re-
gions, mixed diffuse hypoechoic and hyperechoic
areas, or homogeneous).7 Each dolphin’s weight
(in kilograms), length (in centimeters, defined as
the tip of the rostrum to the caudal margin of the
tail flukes), age (in years), sex, dolphin origin
(progeny of animals in the Atlantic or Pacific
Ocean), and management setting (currently
housed in an ocean enclosure or pool) were
recorded.
Analyses were conducted using SAS version 9.2
(SAS Incorporated, Cary, North Carolina 27513,
USA). The respective lengths, depths, and heights
measured on the image views were compared
between left and right lymph nodes by using a
paired t-test (PROC TTEST). P values , 0.05
were considered significant. No significant differ-
ence was identified between left and right lymph
node length measurements, or between depth
measurements of the left and right lymph nodes
in the longitudinal image view. Similarly, no
significant difference was identified between left
and right lymph node width measurements, or
between height measurements of the left and right
 MARTONY ET AL—DOLPHIN MARGINAL LYMPH NODES
lymph nodes in the transverse image view. There-
fore, these measurements were combined for
calculating descriptive statistics for these image
views (Table 1). However, in the oblique and
optimized image views, the mean depth of the left
lymph node was significantly smaller than the
mean depth of the right lymph node, so each were
reported separately (P ¼ 0.026 and 0.019, respec-
tively; Table 1). The reference interval was
generated for nodes in each image view by
calculating the 10th to 90th percentiles for each
node dimension.
The maximal length and depth of the marginal
lymph nodes were defined as the longest length
(craniocaudal dimension) and depth (lateromedial
dimension or distance between the superficial and
deep borders of the node) measurement obtained
from any one image view for each lymph node.
The maximal dimensions were extracted from the
still image that delivered the longest length and
depth measurement; therefore, the same still
image was not always used for both dimensions.
The maximal length and depth of the right and left
lymph nodes did not significantly differ using a
paired t-test, so length and depth of the right and
left lymph nodes were combined for calculating
descriptive statistics (Table 1). The relationship
between the maximal marginal lymph node length
and depth, body weight, and dolphin length was
explored using a linear regression model (PROC
REG). The maximal marginal lymph node length
and depth measurements were compared between
sex, dolphin origin, age group (subadult, 5–10 yr
old; adult, 11–30 yr old; and geriatric, .30 yr old),
and management setting by using an analysis of
variance via the general linear model procedure
(PROC GLM). Significant differences between
groups with small sample sizes were confirmed by
computing exact P values by using the Kruskal-
Wallis test (PROC NPAR1WAY).
RESULTS
Study subjects
In total, 38 dolphins were initially included in
the study population. Five animals were removed
from the study group due to abnormal thoracic
ultrasound results, as evidenced by mild-to-mod-
erate alveolar interstitial syndrome.26,31 Four ad-
ditional animals were removed due to poor image
quality and thus the inability to accurately
evaluate the lymph nodes. Increased blubber
thickness may have played a role in the poor
image quality of particular dolphins; however, for
others the reason is less clear. Ultimately, 29
965
bottlenose dolphins were enrolled in the study,
including 10 (34%) males and 19 (66%) females.
The dolphins’ ages ranged from 5 to 50 yr (mean
22.2 yr), body weight ranged from 120 to 240 kg
(mean 188 kg), and body length ranged from 215
to 282 cm (mean 252.6 cm). Dolphin origins
included 4 Atlantic-Pacific hybrid animals, 1
Pacific animal, and 24 Atlantic animals. Eighteen
dolphins were housed in ocean enclosures and 11
were housed in land-based pool systems. The
blood results for one dolphin were acquired 12
days after the lymph node evaluation, five dol-
phins did not have fibrinogen evaluated, and one
dolphin did not have an ESR evaluated. These
dolphins were included in the study due to their
normal thoracic ultrasounds and lack of abnormal
clinical signs or inflammatory hemograms.
Ultrasonographic evaluation
In total, 58 marginal lymph nodes from 29
dolphins were evaluated. For each of the four
ultrasound image views, a range, mean, SD, and
reference interval were calculated (Table 1). The
maximal marginal lymph node length and depth
were found most commonly in the optimized and
oblique image views (67.2%, 39/58 and 29.3%,
17/58, respectively) and least commonly in the
longitudinal image view (3.4%, 2/58). The mean
marginal lymph nodes measured 5.26 cm in length
(SD ¼ 1.10 cm, minimum ¼ 3.04 cm, maximum ¼
7.61 cm, reference interval 3.78–6.55 cm) and 3.72
cm in depth (SD ¼ 0.59 cm, minimum ¼ 2.64,
maximum ¼ 5.38 cm, reference interval 2.98–4.50
cm). The maximal lymph node dimensions did not
differ between the right and left sides.
The most prevalent ultrasonographic charac-
teristics identified included appearing generally
hyperechoic relative to surrounding soft tissues
(98%, 57/58), irregular caudal borders (84%, 49/
58), ill-defined deep borders (83%, 48/58), flat
superficial border (67%, 39/58), triangular or
rounded triangle shape (59%, 34/58), irregular
cranial border (55%, 32/58), and moderate het-
erogeneity (34%, 20/58) (Table 2; Figs. 4 and 5).
The maximal lymph node dimensions were
found to have no significant correlation with sex,
length, body weight, or management setting
(Table 3). However, the oldest age group (geriat-
ric, .30 yr old) had borderline significantly longer
lymph nodes than the youngest age group (sub-
adult, 5–10 years old; P ¼ 0.049). The animals with
origins in the Pacific Ocean (including the four
Atlantic-Pacific hybrids and a full Pacific dolphin)
had smaller lymph node lengths compared to the
full Atlantic animals (Table 3), although this
966 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Table 2. Gray-scale ultrasonography characteristics node of the case animal appeared with a convex
evaluated for the marginal lymph node. superficial border, defined deep border, smooth
cranial border, smooth caudal border, rounded
No. of lymph nodes
Parameter (n ¼ 58) (%)
node shape, generally homogeneous echotexture,
generally hypoechoic to the surrounding soft
Superficial border characteristics tissues, and homogenous in echogenicity detail
Concave 7 (12)
(Fig. 6).
Convex 12 (21)
Flat 39 (67)
DISCUSSION
Deep border margination
Ill defined 48 (83) An effective and efficient technique for the
Defined 10 (17) ultrasonographic examination of the marginal
Cranial border margination lymph nodes of dolphins is described in this
Irregular 32 (55)
manuscript. The technique uses internal anatomic
Smooth 26 (45)
landmarks, specifically the heart, ventral margin
Caudal border margination
Irregular 49 (84) of the lungs, diaphragm, and cranial border of the
Smooth 9 (16) liver, because the lymph nodes’ location varied
Dorsal border margination relative to external landmarks. Lymph node
Ill defined due to lung or rib 54 (93) exams could be completed in under 5 minutes
Defined 4 (7) using the methods described. The node is located
Ventral border margination caudoventral to the insertion of the pectoral
Ill defined due to rib 58 (100) flipper, and this general region can be used as a
Defined 0 (0) starting point in locating the node. The node is
Node shape
most commonly closely associated to the inser-
Rounded 24 (41)
tion of the pectoral flipper but in some cases is
Rounded triangle 15 (26)
Triangular 19 (33) located significantly caudomedially, approaching
Echotexture ventral midline. This variation underscores the
Mildly heterogeneous 16 (28) importance of relying on internal organ land-
Moderately heterogeneous 20 (34) marks to locate the lymph node.
Markedly heterogeneous 10 (17) The longitudinal and transverse image views
Homogeneous 12 (21) underrepresented the true dimensions of the
General echogenicity marginal lymph node. The acoustic shadowing
Hypoechoic 0 (0)
from the ribs commonly prohibits complete
Hyperechoic 57 (98)
visualization of the lymph node margins in these
Isoechoic to muscle 1 (2)
Echogenicity detail
image views. Therefore, the longitudinal and
Focal hypoechoic 4 (7) transverse image views are not recommended for
Focal hyperechoic 3 (5) evaluating the maximal dimensions of the node.
Mixed diffuse hyperechoic/ 40 (69) For this reason, the height measurement was not
hypoechoic used for further analysis in the study. Conversely,
Homogeneous 11 (19) the oblique and optimized image views reliably
delivered the longest and likely most accurate
depth and length measurements. Subjectively,
difference was only significant for the right node these two image views were similar to obtain
length (Fisher exact test, P , 0.01). The marginal technically, and they delivered visually similar
lymph nodes of the Atlantic animals measured images. The optimized image view was created by
5.43 cm in mean length (SD ¼ 1.07 cm, minimum ¼ the ultrasonographer maximizing the dimensions
3.04 cm, maximum ¼ 7.61 cm, reference interval of the node to the eye. In practice, this commonly
4.18–6.62 cm) and 3.71 cm in mean depth (SD ¼ translated to positioning the probe in the oblique
0.57 cm, minimum ¼ 2.64 cm, maximum ¼ 5.17 image view first, to remove the acoustic shadow-
cm, reference interval 3.04–4.50 cm). ing from the rib. Next, the probe was manipulated
in the appropriate direction to catch an improved
Case animal angle, as opposed to adhering to the strict criteria
of the oblique view and only fanning toward the
The maximal dimensions of the right marginal cranial and caudal sternal ribs. The oblique and
lymph node of the case animal measured 8.59 cm optimized image views were the most valuable
in length 3 7.40 cm in depth. The marginal lymph views due to the ultrasound beam being parallel
 MARTONY ET AL—DOLPHIN MARGINAL LYMPH NODES 967

Figure 4. Four ultrasonographic images of the right marginal lymph node in a clinically healthy 23-yr-old male
T. truncatus. (a) Longitudinal view: cranial is to the left. The acoustic shadow of a rib (star) is obscuring the node’s
cranial margin. (b) Transverse view: dorsal is to the left. The dorsal margin of the node is obscured by the
reverberation artifact of the adjacent lung (star); however, the smooth ventral margin can be seen (white arrows).
(c) Oblique view: cranioventral is to the left. The lymph node has a flat superficial margin (black arrows),
triangular in shape, generally hyperechoic to the surrounding tissues, and moderately heterogeneous with mixed
hyper- and hypoechoic areas. (d) Optimized view: varying degrees of cranioventral rotation are to the left. The
lymph node has an ill-defined deep margin (white arrows) and a smooth cranial margin adjacent to the heart (star).

to or between the sternal ribs, which eliminated
acoustic shadowing from the ribs. Overall, when
evaluating the marginal lymph node, the authors
recommend striving to acquire images from both
the optimized and oblique image views for full
evaluation.
The marginal lymph nodes’ ultrasonographic
characteristics varied greatly between dolphins.
The node most commonly appeared with a flat
well-defined superficial border, which is likely due
to the node lying adjacent to the smooth thoracic
body wall. The dorsal and ventral borders were
most commonly ill defined, likely because of an
artifact created by acoustic shadowing from the
rib and air-filled lung.
The marginal lymph nodes of healthy dolphins
differed from normal lymph nodes of domestic
animals with regard to border margination and
shape. Dolphins commonly have irregular cranial
and caudal marginal lymph node borders that
differs from domestic veterinary species, in which
irregular lymph node borders are associated with
an infiltrative process such as neoplasia.21 Simi-
larly, normal superficial lymph nodes in domestic
veterinary species have been characterized as oval
shaped. A short-to-long axis ratio is a tool used to
quantitatively assess the shape and size in round-
ed lymph nodes of domestic small mammals and
to distinguish normal and reactive nodes from
pathologic nodes.21,22 The marginal lymph node of
dolphins commonly was of a triangular shape
968 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Figure 5. Variable ultrasonographic appearance of marginal lymph nodes in three clinically healthy T.
truncatus. (a) Longitudinal view: This node has a flat superficial margin (black arrows), smooth cranial margin
(gray arrows), ill-defined deep margin (white arrow), irregular caudal margin adjacent to the liver (star); and is
triangular in shape, homogeneous, and hyperechoic to the surrounding tissues. (b) Optimized view: This lymph
node is moderately heterogeneous, hyperechoic to the surrounding tissues, and triangular in shape. Placement of
measurements for maximum length and depth are pictured as white lines. (c) Oblique view: This lymph node is
homogeneous, hyperechoic to the surrounding tissues, and triangular in shape.

(59%); thus, a short-to-long axis ratio could not
accurately describe the marginal node and was
therefore not used.
The healthy dolphins’ marginal lymph nodes
also differed from normal lymph nodes of domes-
tic animals with regard to echotexture and echo-
genicity. In the healthy dolphins studied, the
marginal lymph node was typically heterogeneous
(79%), ranging from mildly to markedly. Individ-
ual dolphins were also found to have different
echotextures with regard to the right and left
nodes. Conversely, in domestic veterinary species,
mixed echotextures or hyper- and hypoechoic
areas within the node have been associated with
pathology including metastatic lesions, coagula-
tion necrosis, hemorrhage or edema within the
lymph nodes.2,21 In addition in the present study,
98% of the normal marginal lymph nodes evalu-
ated were found to be generally hyperechoic to the
surrounding soft tissues (including the liver,
heart, and thoracic musculature), with only one
lymph node appearing isoechoic to the external
oblique muscle. This also varies considerably
from domestic veterinary species where normal
lymph node echogenicity is generally described as
uniform and isoechoic or slightly hypoechoic to
surrounding tissues.22
The case animal’s marginal lymph node dimen-
sions and ultrasonographic characteristics dif-
fered from the healthy dolphins evaluated. The
dolphin with confirmed pulmonary coccidiomy-
cosis had a marginal lymph node that was

Table 3. P values for the association between marginal lymph dimensions and independent variables.

n Right node length Right node depth Left node length Left node depth

Age group 0.049 0.422 0.478 0.451

Subadult 7
Adult 13
Geriatric 9
Sex 0.691 0.313 0.275 0.194
Male 10
Female 19
Management setting 0.847 0.462 0.07 0.287
Ocean enclosure 18
Pool 11
Body weight 29 0.251 0.057 0.09 0.393
Dolphin length 29 0.319 0.11 0.325 0.658
Dolphin origin 0.001 0.254 0.394 0.568
Atlantic 24
Pacific 5
 MARTONY ET AL—DOLPHIN MARGINAL LYMPH NODES
Figure 6. Ultrasonographic image of an abnormal
right marginal lymph node in a 32-yr-old female T.
truncatus with confirmed lung disease. The left side of
the image is oriented cranially. The maximum length
and depth of the node are 8.6 and 7.4 cm, respectively.
The node has a smooth and convex superficial surface
(white arrows), is homogeneous and hypoechoic to the
surrounding tissues, and has defined deep and caudal
margins. The reverberation artifact from the lung
(black arrow) is obscuring the cranial margin. The liver
is seen caudal to the node (gray arrow).
diffusely enlarged, with dimensions outside the
reference interval for the healthy dolphins in the
study. It has been previously established that in
reactive lymph nodes, stimuli such as inflamma-
tion or primary or metastatic neoplasia lead to
enlargement of the node.22 Moreover, the case
animal’s marginal lymph node appeared generally
homogeneous, with well-defined borders circum-
ferentially. Likewise, previous abnormal lymph
node studies in domestic veterinary species have
also found reactive lymph nodes to have well-
defined borders and decreased echogenicity.21,22
Interestingly, the dolphin’s suspected reactive
marginal lymph node evaluated in this study was
also found to be notably hypoechoic to the
surrounding tissues; conversely, no hypoechoic
marginal lymph nodes were identified in the
healthy dolphin population evaluated. Further
characterization of marginal lymph nodes in
bottlenose dolphins with known pulmonary pa-
thology will aid in establishing the commonality
and differences between normal and abnormal
nodes.
The marginal lymph node size was not found to
be associated with the dolphins’ length, sex,
weight, or management setting. Sex was found to
have no correlation to lymph node size in
dolphins, which is also consistent with human
cervical lymph node and canine superficial ingui-
nal lymph node studies.17,32 In contrast, it has been
previously reported that the medial retropharyn-
969
geal, medial iliac, and superficial inguinal lymph
nodes dimensions in dogs are positively correlat-
ed with the patient’s body weight.17 For the
dolphin population studied (.5 yr old) increased
age was found to be positively associated with
lymph node size. This finding is consistent with
human studies of cervical lymph nodes that
correlated larger dimensions with older age
groups.32
The dolphins with origins in the Pacific Ocean
were found to have significantly smaller right
marginal lymph node lengths. Although the
sample size was small for the Pacific origin
animals (five dolphins), additional statistical anal-
ysis still confirmed the significance. Interestingly,
the authors noted subjective differences when
imaging the Pacific animals. Specifically, when
imaging the lymph node it appeared to be more
intimately associated with the ventral free border
of the lung, which resulted in a greater challenge
when attempting to optimize the field of view and
maximize node dimensions. Tursiops truncatus is
considered to be a single bottlenose dolphin
species; however, phenotypic differences are well
established among animals from different geo-
graphic locations. Likewise, previous studies
found the Pacific dolphins to have a 50% larger
lung tidal volume (91 ml/kg) than the Atlantic
dolphins (49–71 ml/kg).23 Therefore, a greater
lung volume may be contributing to difficulty in
imaging the marginal lymph node in these dol-
phins, resulting in a larger field of air-associated
artifact, obscuring complete visualization of the
node. Thus, these lymph node lengths reported
for the Pacific dolphins potentially underrepre-
sent the length dimension. Additional studies
using cross-sectional imaging (such as computed
tomography), postmortem data, or a combination
could be helpful in determining the actual maxi-
mum lengths and widths for these animals.
In the present study, all dolphins were clinically
classified as free from pleural and pulmonary
diseases; however, a limitation of the study is the
potential for subclinical pulmonary disease as
well as the known limits of thoracic ultrasound to
detect deep lung pathology. In the absence of
peripheral thoracic pathology, only the superficial
pleura and lung can be examined with ultrasound
because of the reverberation artifact created by
air-filled lung. Thoracic radiography or computed
tomography would be needed to more definitively
rule out all pulmonary disease.12 However, as the
study animals were deemed otherwise clinically
healthy, these additional diagnostics were not
performed. The potential for intrasonographer
970 JOURNAL OF ZOO AND WILDLIFE MEDICINE
variation in lymph node ultrasonography, and
interobserver variation in cine loop evaluations
are additional limitations. Two veterinarians per-
formed the ultrasonographic evaluations, and one
veterinarian completed all of the cine loop lymph
node evaluations. However, multiple marine
mammal veterinarians and specialists first stan-
dardized the examination techniques and cine
loop lymph node evaluations, before the start of
the study to minimize variability. In addition, still
ultrasound images were reevaluated and the
lymph node ultrasound exam was repeated by a
second veterinarian if any uncertainty in the data
was identified. The inability to obtain replicate
measurements is a final limitation, and future
lymph node evaluations could be strengthened by
doing so.
Transthoracic ultrasound of the marginal
lymph node in bottlenose dolphins was rapid
and effective when following the technique devel-
oped and described in this study. As a result,
expected reference intervals and marginal lymph
node characteristics in clinically healthy dolphins
were established, and they may be relied upon to
detect and monitor disease. Application of this
technique to clinically ill animals will help to
establish characteristics of abnormal marginal
lymph nodes. Moreover, application to free-rang-
ing cetacean species could provide valuable data
regarding pulmonary health of individual animals.
Use of this technique and application of these
data to both wild and managed cetaceans will
facilitate prompt detection of abnormalities and
enable more precise disease monitoring of respi-
ratory diseases.
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Accepted for publication 9 September 2017