Journal of Zoo and Wildlife Medicine 38(2): 222–230, 2007
Copyright 2007 by American Association of Zoo Veterinarians
QUANTITATIVE ULTRASOUND, MAGNETIC RESONANCE
IMAGING, AND HISTOLOGIC IMAGE ANALYSIS OF HEPATIC
IRON ACCUMULATION IN PIGEONS (COLUMBIA LIVIA)
Jodi S. Matheson, D.V.M., Joanne Paul-Murphy, D.V.M., Dipl. A.C.Z.M., Robert T. O’Brien
M.S., D.V.M., Dipl. A.C.V.R., and Howard Steinberg, D.V.M., Dipl. A.C.V.P.
Abstract: Iron overload was induced by iron dextran i.v. in clinically healthy adult pigeons, Columbia livia, (n ?
8). Hemosiderosis was induced in all treated birds. Two control pigeons received no iron injections. Pigeons did not
show clinical signs of iron overload during the 6-wk study. Ultrasound examination of the liver in the pigeons receiving
iron dextran was performed on days 0, 13, 28, and 42. No ultrasound images were collected on the control pigeons.
Magnetic resonance imaging was performed on days 0, 13, 28, and 42 on all study pigeons and imaging sequences
were collected in three different imaging formats: T1, T2, and gradient-recalled echo (GRE). Surgical liver biopsies
were performed on pigeons receiving iron dextran on days 2, 16, and 45 (at necropsy). A single liver sample was
collected at necropsy from the control birds. Histologic examination, quantitative image analysis, and tissue iron analysis
by thin-layer chromatography were performed on each liver sample and compared to the imaging studies. Although
hemosiderosis was confirmed histologically in each experimental pigeon, no significant change in pixel intensity of the
ultrasound images was seen at any point in the study. Signal intensity, in all magnetic resonance imaging formats,
significantly decreased in a linear fashion as the accumulation of iron increased.
Key words: Avian, hemochromatosis, hemosiderosis, iron, magnetic resonance imaging, ultrasound.
Circulating iron is deposited as hemosiderin, pre-
dominantly in the liver but also in the spleen, bone
marrow, and reticulocytes of birds. Hemosiderosis
refers to excessive stainable iron deposits (hemo-
siderin) within cells and occurs when whole body
iron concentrations are substantially higher than
physiologic iron requirements. Clinical signs might
not be present at this stage. However, elevated iron
concentrations in tissues can damage hepatocytes,
eventually leading to hepatic fibrosis. Hemochro-
matosis is the clinical condition caused by iron ac-
cumulation producing pathology within hepato-
cytes. Hemosiderosis occurs in many taxa of cap-
tive birds, with hemochromatosis a common dis-
order within species of the families Paradiseadae,
Rhamphastidae, and Sturnidae. Clinical signs as-
sociated with hemochromatosis are attributed to de-
clining hepatic function, including chronic wasting,
abdominal distention, hepatomegaly, coelomic ef-
fusion, and dyspnea from coelomic fill.27
The pathophysiology of iron storage disease has
not been well described in the avian patient. Pos-
sible causes include genetic predisposition (primary
hemochromatosis) and excessive dietary iron intake
From the University of Wisconsin–Madison, School of
Veterinary Medicine, 2015 Linden Drive, Madison, Wis-
consin 53706, USA. Present address (O’Brien): Kansas
State University, O221 Mosier Hall, Manhattan, Kansas
66506, USA. Correspondence should be directed to Dr. J.
(secondary hemochromatosis). Histopathologic
findings, and because the disease is seen only rarely
in wild populations, suggest the disease in avian
species most closely resembles secondary hemo-
chromatosis in humans.13,21,22
Antemortem identification of hemosiderosis or
hemochromatosis in the avian patient is a diagnos-
tic challenge. Hepatomegaly can be confirmed by
radiographic imaging, but it is not pathognomonic.
Specific tests to assess total body iron stores in hu-
mans and other mammals include serum iron, total
iron binding capacity, serum transferrin concentra-
tions, and transferrin saturation. These tests provide
an indirect estimate of total body iron stores but are
affected by common clinical conditions such as in-
flammation, infection, hepatic disease, and hemo-
lysis.33Interpretation of iron storage serum values
in avian species is complicated by the wide species
variation in serum iron concentrations and the lim-
ited availability of normal reference ranges.3,10,12,14,15
Hepatic biopsy with histologic examination of
the tissue, iron-specific stains, and colorometric as-
say of iron content are currently the most reliable
and direct methods to quantitatively measure body
iron status in the avian patient.18However, anes-
thetic and surgical risk of sequential evaluations by
hepatic biopsy limits its acceptability, especially in
the small avian species. Even with the histologic
information, complete agreement on what consti-
tutes inappropriate iron concentrations for avian
species is not available in the literature. Avian spe-
cies are documented to exhibit wide variations in
MATHESON ET AL.—IMAGING AVIAN HEPATIC IRON ACCUMULATION
iron storage at various stages in the life cycle, by
both age and seasonal events such as migration and
molting.18Quantitative means to assess whole body
stores of iron that is noninvasive, safe, accurate,
and readily available would be useful to avian vet-
Two methods investigated in human medicine for
diagnosis of iron accumulation include magnetic
resonance imaging (MRI) and ultrasound. Multiple
human studies show a correlation between hepatic
MRI tissue signal intensity and the quantity of iron
present.1,4,7,16,17,19,23,24,30,31,32No standard imaging pro-
tocol for human evaluation is currently published.
Conventional MRI scanning is reliably used to
screen for iron overload rather than quantification
of the iron stored within the tissues.2,6,26
In this study, we evaluate MRI and ultrasono-
graphic imaging for quantitative measurement of
avian hepatic iron accumulation. The pigeon was
selected as an experimental model because hemo-
siderosis is well documented in this species without
development of clinical signs.9
MATERIALS AND METHODS
The research protocol was reviewed and ap-
proved by the Animal Care and Use Committee of
the University of Wisconsin, School of Veterinary
Medicine (UW-SVM). Pigeons were maintained at
the UW-SVM Animal Research Facility and pro-
vided water and commercial pigeon feed ab libitum
(Pigeon Chow, Purina Mills LLC, Grays Summit,
Missouri 63039, USA). Ten clinically healthy adult
pigeons (Columbia livia), as determined by physi-
cal exam, packed cell volume, total protein, and
fecal flotations, were evaluated in this study.
Weights ranged from 269.9 to 379.3 g. Birds were
treated prophylactically (0.2 mg/kg p.o. Ivermectin,
Ivomec, Merck & Co., St. Louis, Missouri 63150,
USA; 60 mg/kg p.o. trimethoprim/sulfamethotrex-
azole Septra, Burroughs Wellcome, Research Tri-
angle Park, North Carolina 27709, USA) for para-
sites before the study. Iron dextran (25 mg/kg
INFeD, Watson Pharmaceuticals Morristown, New
Jersey 07962) was given i.v. to eight pigeons to the
left or right medial metatarsal vein, once every 7
days for three treatments. Although the treated pi-
geons served as their own controls during the ul-
trasound portion of the study, two pigeons were
used as controls for the MRI imaging and received
no iron injections.
Hepatic ultrasound imaging
Ultrasound examination of the liver of the ex-
perimental birds was performed on days 0, 13, 28,
and 42. Control birds were not examined by ultra-
Pigeons were manually restrained in dorsal re-
cumbency for the 5–10-min examination. The im-
aging window was located at the caudal margin of
the keel, and feathers in this area were plucked.
Water-soluble ultrasound gel (Aquasonic 100, Park-
er Laboratories, Fairfield, New Jersey 07004, USA)
was used as a coupling agent. Ultrasound images
were obtained with a General Electric Logic 400
ultrasound machine equipped with a 7.5-MHz lin-
ear transducer and computer-assisted direct digital
analysis (GE Healthcare, Global Headquarters,
Waukesha, Wisconsin 53189, USA). Eight images
of each bird’s liver were collected at two gain set-
tings (78 and 88 MHz), with all other machine set-
tings remaining constant throughout the imaging
process. Because of the lack of an internal standard
on the ultrasound images, a standardized ultrasound
phantom was used to collect control images at the
same machine settings.
Analysis of the digitized ultrasound images de-
termined the pixel intensity of the liver images and
phantom images by NIH Image (Research Services
Branch, National Institute of Mental Health, Na-
tional Institutes of Health public domain http://
rsb.info.nih.gov/nih-image/). A ratio of the liver
pixel intensity to phantom intensity was calculated
to account for machine variances.
Hepatic magnetic resonance imaging
MRI was performed on days 0, 13, 28, and 42
on both the iron-treated and untreated pigeons.
Each pigeon was sedated (0.5 mg/kg i.m. xylazine,
Rompun, Bayer, Pittsburgh, Pennsylvania 15205,
USA) then placed in a stockingnette for restraint.
T1-weighted (SE, TR:400, TE: 14, EC: 1/1 15.6
KHz), T2-weighted (TR:4000, TE: 84/Ef, EC: 1/1
16 KHz), and gradient-recalled echo (GRE) (GR/
10?, TR:17, TE: 5.9/Fr, EC: 1/1 15.6 KHz) imaging
sequences were collected. The slice thickness was
3 mm with a 1-mm gap. This imaging was per-
formed with a 1.5-tesla EchoSpeed Plus System
with a knee coil manufactured by General Electric
Medical Systems (GE Healthcare). Regions of in-
terest were drawn around the digital images of liver
and pectoral muscle in consecutive slices to deter-
mine their respective signal intensities (SI). Mean
SI was determined from consecutive image slices
containing liver. The internal standard for SI was
the birds’ pectoral muscles in the same slice (Fig.
1). The liver-to-muscle SI ratio was used to com-
pare changes in SI at the different time periods.
JOURNAL OF ZOO AND WILDLIFE MEDICINE
cavity of iron-treated pigeons. Regions of interest were drawn on axial slices demonstrating liver (red) and pectoral
muscles (yellow). Note the change in liver signal intensity, from hyper- to hypointense, on all imaging sequences as
iron accumulates in the liver.
T1, T2, and gradient-recalled echo (GRE) weighted magnetic resonance images of the midcoelomic
Hepatic biopsy collection
Birds were anesthetized with isoflurane in oxy-
gen by facemask (IsoFlo, Abbott Laboratories,
North Chicago, Illinois 60064, USA) for induction,
intubation, and maintenance during biopsy proce-
dures. Butorphanol (2 mg/kg i.m. Torbugesic, Fort
Dodge, Fort Dodge, Iowa 50501, USA,) was ad-
ministered during anesthetic induction for periop-
erative analgesia. Liver samples were obtained via
a midline coelotomy and wedge resection of a liver
lobe margin. Hepatic biopsies (0.5–0.75 g of wet
tissue) were collected twice from each iron-treated
bird. Biopsies were taken before any administration
of iron and 4 days after administration of the sec-
ond iron dextran treatment. All study birds (n ?
10) were humanely euthanized at 45 days (1 ml/
pigeon i.v. Beuthanasia-D Special Pentobarbitol so-
dium/Phenytoin sodium, Schering Plough Animal
Health, Summit, New Jersey 07901, USA). The
third liver samples were collected at necropsy. He-
patic biopsies were collected from control pigeons
on day 0 and at necropsy.
Hepatic iron analysis
Half of the hepatic tissue sample was immedi-
ately sent for biochemical analysis of total iron con-
centration (Wisconsin Veterinary Diagnostic Labo-
ratory, Madison, Wisconsin 53705, USA). Hepatic
iron was analyzed by flame atomic absorption after
microwave acid digestion. Wet and dry matter de-
termination was performed on each liver sample (n
? 26, control birds n ? 2). Dry weight iron was
used for comparison to all other quantitative mea-
surements included in this study.
Histology and image analysis
The remaining half of each hepatic sample was
placed in 10% buffered formalin, processed rou-
tinely for histopathology, and embedded in paraffin.
Samples were cut at 5 ?m then stained with he-
matoxylin and eosin and Perl’s Prussian blue for
histologic examination to note which cells con-
tained iron and the degree of cytoplasmic granular-
ity within the hepatocytes. A grading system for
iron deposition was used to assign a semiquantita-
tive score on the basis of a subjective interpretation
of the amount of hepatocytes with iron present and
amount of iron present within those hepatocytes,
ranging from 0 (rare iron granules observed to 5
(majority of hepatocytes had a dense accumulation
Hepatic sections stained with Perl’s Prussian blue
were microscopically analyzed with an AxioVert
S100 equipped with an AxioCam HRC camera
MATHESON ET AL.—IMAGING AVIAN HEPATIC IRON ACCUMULATION
ror bars) hepatic iron concentration (?g/g dry weight) in
pigeons (n ? 8) given iron dextran on days 7, 14, and 21.
Mean (?) with maximum and minimum (er-
(Zeiss, Thornwood, New York 10594, USA). Ten
fields from each hepatic sample were digitally cap-
tured at ?20 magnification in bright field phase I
with the use of only those sections with subjective-
ly uniform staining. Image analysis software KS
300 3.0 (DFI Technologies, Inc., Sacramento, Cal-
ifornia 95834, USA) was used to evaluate the in-
tensity and percent area of blue-stained regions.
The mean value of area percentage was determined
for each specimen.
To determine whether the increase in biochemi-
cal hepatic iron values over time was due to factors
other than group variability, a one-way analysis of
variance (ANOVA) with Bonferroni’s multiple
comparison tests was run. This same statistical
analysis was performed on the mean hepatic SI val-
ues for each MRI sequence and mean ultrasound
hepatic pixel intensity to determine whether the
changes occurring over time were due to factors
other than group variability. Values of P ? 0.05
were considered significant.
The hepatic iron concentrations were used as a
standard reference to which all other parameters
were compared, including the relationship of liver-
to-muscle SI ratio of each MRI sequence, histologic
image analysis, ultrasound pixel intensity–to–phan-
tom pixel intensity ratio, and histologic scores.
Each set of comparisons were analyzed by means
of a scatter plot.
These results were inspected for linearity and
goodness of fit. All numeric data collected were
continuous variables; therefore, the correlation be-
tween each variable to the biochemical hepatic iron
concentrations was assessed and tested without the
assumption of Gaussian distribution with the Spear-
man correlation coefficient.
All statistical analyses were determined with
GraphPad Prism version 4.00 for Mac OS X
(GraphPad Software, San Diego, California 92130,
None of the pigeons in this study showed any
clinical signs of hemochromatosis during the 6-wk
treatment with iron dextran. Mean hepatic iron con-
centrations were significantly increased between the
16- and 45-day samples (Fig. 2). The mean bio-
chemical hepatic iron concentration increased sig-
nificantly from pretreatment concentrations on days
16 and 45 in all birds receiving iron dextran The
control pigeons had no significant change in the
mean biochemical hepatic iron concentrations.
Quantitative ultrasound image analysis did not
provide a significant change in mean pixel intensity
between pretreatment images and images collected
at any time point after treatments.
MRI signal intensity of the liver decreased sig-
nificantly in all the imaging formats (T1, T2, GRE)
when mean pretreatment values (day 0) were com-
pared with mean values obtained from images col-
lected on days 13, 28, and 42. When comparing
each MRI imaging point in time (Bonferroni’s test),
no significant change in signal intensity in the T1
and GRE imaging formats were determined be-
tween days 28 and 42 of the study. The changes in
T2 signal intensities were not significant between
days 13 and 28, 13 and 42, or 28 and 42 (Fig. 3).
The mean signal intensity of the pectoral muscles
was constant throughout the study with no signifi-
cant differences at any point during the study (Fig.
3). MRI signal intensity changes corresponded in a
linear manner to changes in biochemical hepatic
iron concentration. MRI signal intensity, in all im-
aging formats—T1 (r2? 0.85), T2 (r2? 0.52), and
GRE (r2? 0.79)—decreased as the accumulation
of iron increased (Fig. 4).
Each biopsy used for histologic grading and
quantitative image analysis was taken from the
same hepatic lobe as was used for the biochemical
analysis of iron. Hemosiderosis was induced in all
treated pigeons as evidenced by increased iron
staining and histologic changes in the hepatic tis-
sue. The control pigeons did not develop hemosid-
erosis. The histologic scores of treated birds were
significantly different from day 2 to 16 and from
JOURNAL OF ZOO AND WILDLIFE MEDICINE
change in signal intensity of liver and muscle tissue in pigeons (n ? 8). Pigeons were given iron dextran for three
treatments at indicated times (↓). Liver signal intensity decreased in all imaging formats as iron accumulation increased
in the tissue, whereas muscle signal intensity remained constant.
Comparison of three magnetic resonance imaging (MRI) formats (T1, T2, gradient-recalled echo [GRE])
MATHESON ET AL.—IMAGING AVIAN HEPATIC IRON ACCUMULATION
signal intensity of liver-to-muscle ratio in three different imaging formats (T1, T2, and gradient-recalled echo [GRE]).
Signal intensity decreased as iron concentration increased. Linear regression best fit analysis was greatest for T1 (r2?
Linear regression plots comparing hepatic iron concentration (?g/g dry weight) in 10 pigeons to MRI
JOURNAL OF ZOO AND WILDLIFE MEDICINE
day 2 to 45 but were not significantly different from
day 16 to 45. The correlation coefficient of histo-
logic scoring to hepatic iron concentration (r ?
0.96) was significant.
The mean and standard deviation of area per-
centage of hepatic tissue occupied by iron granules,
as measured by the image analysis system, varied
from 5.5% ? 7.3 in the pretreatment biopsies to
41.9% ? 19 in the necropsy biopsies. The mean
area and standard deviation of area percentage for
the control pigeons after 6 wk without treatment
was 5.1% ? 7.3, within the same range as the orig-
inal pretreatment values for treated pigeons. The
correlation coefficient of area percentage deter-
mined by histologic image analysis (r ? 0.84) was
Hemochromatosis continues to be a problem in
avian populations of zoological institutions, even
with the practice of feeding low-iron diets and add-
ing natural iron chelators to the diet (e.g., tan-
nins).8,18,29However, the true incidence of this con-
dition in captive avian species is unknown. A ret-
rospective study of 180 necropsy cases, represent-
ing 40 species of birds, determined that hepatic
hemosiderosis is a common histologic finding in
most avian species, although not necessarily asso-
ciated with overt liver disease.9In a survey of 19
North American zoological institutions, hemosid-
erosis was identified as a cause of hepatic disease
in 11% of the studied birds.9Currently, hepatic bi-
opsy is the most accurate way to measure iron
stores in the avian species. In this study, biochem-
ical analysis of iron in liver specimens was used to
determine dry matter iron concentrations to which
all other diagnostic tests were compared. Hepatic
biopsy is an invasive procedure and multiple bi-
opsies are not an acceptable option for monitoring
the progression of clinical disease or the effect of
therapy. Normal reference ranges for biochemical
hepatic iron concentrations are not available for
most avian species; for example, according to the
Wisconsin Veterinary Diagnostic Laboratory’s
(WVDL) poultry reference ranges, all 8 experimen-
tal pigeons had concentrations of hepatic iron in the
WVDL toxic range for avian species after two in-
jections of iron dextran. These toxic tissue concen-
trations did not correlate with absence of clinical
signs and the moderate histologic changes found in
the pigeons. Some of the variation in hepatic iron
content before and after exogenous iron adminis-
tration could be attributed to the ignorance of the
sex of the subjects because iron mobilization has
been shown to occur in female chickens during dif-
ferent phases of the reproductive cycle.25
Histologic examination of the liver was the only
method used in this study that could determine the
amount of cellular damage present in the liver. Sig-
nificant correlation between histologic scores and
biochemical hepatic iron concentration was found.
Yet, eight of the 24 samples had scores of 5, and
iron concentrations within this score varied from
7,500 to 23,100 ?g/g. The narrow scoring margin
of 0–5 did not discriminate when iron concentra-
tions were mildly elevated verses severely elevated.
This insensitivity was reflected by the lack of sig-
nificant change in score between the second and
third biopsies. Additionally, scoring is a subjective
evaluation that can vary between pathologists.
The quantitative analysis of histologic sections
correlated well with biochemical analysis of hepatic
iron concentration. Quantitative analysis is more re-
liable than the histologic grading of iron content
when the same histologic sections were evaluated.
Quantitative image analysis can be used to diag-
nose hemosiderosis in birds and provide objective
evaluation of sequential biopsy specimens, when
available, to monitor the effectiveness of treat-
A quick, noninvasive, accurate means to measure
and monitor hepatic iron stores antemortem would
be beneficial. Ultrasound imaging, although quick
and noninvasive, lacks accuracy in predicting liver
iron stores. Inherent challenges of avian ultraso-
nography are also present. The window for ultra-
sound imaging is difficult to identify on small avian
patients, including the pigeon, because of size,
shape, and frequency of common clinically avail-
able transducers. However, ultrasonography can be
useful for imaging the liver for biopsy guidance or
looking for gross structural changes of the liver.
This study demonstrates that MRI meets most of
these criteria, although the technology is expensive
and not routinely available in zoological practice.
MRI is a quick, noninvasive process that required
only mild sedation of the study subjects. It is likely
that avian species commonly kept in a zoological
setting would require heavy sedation or complete
anesthesia to accomplish the same procedure. Al-
though MRI imaging is not definitively diagnostic
for iron accumulation, other hepatic diseases that
produce lower signal intensity than normal liver are
imaged in humans as nodular regions of decreased
signal intensity, rather than the diffuse decrease in
signal intensity that is seen with hemosiderosis.11
The most reliable application of hepatic MRI
would be after the definitive diagnosis of hemosid-
erosis or hemochromatosis obtained from biopsied
MATHESON ET AL.—IMAGING AVIAN HEPATIC IRON ACCUMULATION
tissue. Then, MRI could be used to monitor chang-
es in signal intensity associated with changes in he-
patic iron accumulation over time, such as during
a course of treatment. For reliability and repeat-
ability of serial examinations of the same patient,
the same MRI machine would need to be used. Un-
til standards of normal liver imaging have been es-
tablished, MRI analysis will be limited in baseline
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Received for publication 10 November 2005