Intraerythrocytic Inclusions in American Alligator (Alligator
mississippiensis) Hatchlings
Lauren J. Richey1, Kendal Harr2,
John W. Harvey2, Trenton R. Schoeb1,3; 1Department of Pathobiology, University of Florida
College of Veterinary Medicine, Gainesville, Florida 32611-0880; 2Department of Physiological Sciences, University of
Florida College of Veterinary Medicine, Gainesville, Florida, 32610; 3Current address: Department of Comparative Medicine,
University of Alabama at Birmingham, Birmingham, Alabama, 35294.
Abstract: Intracellular inclusions were found in
erythrocytes in the peripheral blood and bone marrow of two captive American alligator
hatchlings. The affected alligators had tail gangrene with intralesional gram negative and
positive bacteria. The inclusions were suspected to be associated with septicemia,
although the definitive cause was not determined.
Key Words: Alligator mississippiensis,
American alligator, erythrocytes, vacuoles, inclusions
Case Report
Two 5-months-old captive American alligator (Alligator
mississippiensis) hatchlings (body weights of 186g and 144g) presented with a 1-week
history of progressive dry gangrene of their distal tails (Figure 1). The animals became
moribund and were euthanized with an intravenous injection of sodium pentobarbital.
 |
| Figure 1. Dry gangrene of distal tail. |
These animals were part of a group of 76 animals housed indoors in
fiberglass tanks as part of a study on the effects of endocrine-disrupting contaminants on
the immune system of hatchling alligators. One animal was a dimethylsulfoxide (DMSO)
vehicle control and the other had been treated in ovo with 5 ppm p,p'-DDE (based on
total egg weight). Complete blood cell evaluations performed on these two animals two to
three weeks prior to their deaths were considered to be within the reference intervals for
this group of alligator hatchlings. No morphologic abnormalities had been noted at that
time in peripheral blood smears.
Just prior to euthanasia, blood was collected from the
supravertebral sinus in lithium heparin Vacutainer® tubes. Blood smears were
made within 1 minute of collection from the heparinized sample. No other hematologic
analyses were performed. Bone marrow for cytology was collected by bisecting the femur
longitudinally and scooping out the marrow, which was gently touched to a glass slide. Liver, brain, kidney, adrenal, heart, bone marrow, thymus, spleen, lung, thyroid, trachea,
stomach, intestines, and tail were collected and fixed in 10% neutral buffered formalin. Tissues were processed routinely, sectioned at 4 mm, and stained with hematoxylin and
eosin (H&E). Sections of tail and femoral bone marrow were stained with Brown &
Brenn (B&B) and periodic acid-Schiff (PAS).
Samples of peripheral blood from the supravertebral sinus and
femoral bone marrow were fixed in Trump's solution. Each sample was incubated with 1%
buffered osmium tetroxide, dehydrated in a graded ethanol series followed by acetone,
infiltrated in a graded epoxy resin series, and polymerized in epoxy resin. Ultrathin
sections (70nm) were collected on 200 mesh copper grids, stained with 2% uranyl acetate
followed by Reynold’s lead citrate, and examined on a transmission electron
microscope.
Cytology
Evaluation of peripheral blood smears revealed heterophils with
basophilic pleomorphic intracytoplasmic granules in addition to the usual rod-shaped
eosinophilic granules (Figure 2). This finding was interpreted to be a toxic change, and
similar lesions have been observed by the authors in the heterophils of adult alligators
and other reptiles with documented septicemia. Numerous round to oval intracytoplasmic
inclusions were found in mature and immature erythrocytes in peripheral blood smears and
in immature erythrocytes in bone marrow impression slides. The inclusions were clear to
lightly basophilic on Wright's stain, lightly basophilic on Wright Giemsa stain, periodic
acid-Schiff (PAS) positive, and Sudan black B negative (Figures 2 & 3).
 |
 |
| Figure 2. Inclusions in peripheral blood erythrocytes. Note toxic heterophil. Wright's
stain. |
Figure 3. Numerous intracytoplasmic inclusions in immature erythrocytes in femoral bone marrow. Wright's stain. |
Histology
Histologic lesions in tissue taken from the interface of the
viable and non-viable tail were characterized by severe diffuse necrosis and ulceration of
the epidermis with extension of necrosis into the dermis and underlying skeletal muscle. A
mixed population of numerous gram positive cocci and coccobacilli with fewer gram-negative
rods was present on the ulcerated surface. Occasional colonies of bacteria were noted deep
within the underlying tissues. Despite the extensive necrosis and numerous bacteria,
inflammatory infiltrates were absent. The spinal cord in these sections was diffusely
necrotic with associated hemorrhage.
Multiple clear intraerythrocytic vacuoles were noted in peripheral
erythrocytes in hepatic sinusoids and within areas of hemorrhage within the spinal cord. Immature erythrocytes lining sinusoids in the bone marrow of the femur and vertebrae of
the tail also contained numerous clear intracytoplasmic vacuoles that stained intensely
with PAS (Figure 4).
 |
| Figure 4. Numerous PAS positive inclusions in immature erythrocytes in femoral bone marrow. |
Electron Microscopy
On ultrastructural examination, erythroid cells in the peripheral
blood and bone marrow contained single to multiple vacuoles usually devoid of contents. Occasional vacuolar inclusions were bound by a distinct membrane, but a membrane was not
observed in the majority of cases. Some of the vacuoles contained amorphous electron dense
particles. Rare to occasional vacuoles contained degenerate organelles or ferritin
aggregates (Figures 5-7).
 |
| Figure 5. Intracytoplasmic vacuoles in peripheral blood erythrocyte with ferritin aggregates. |
 |
 |
| Figure 6. Ferritin aggregate within lighter staining erythrocyte inclusion. |
Figure 7. Vacuole containing degenerate organelles and ferritin aggregates. |
Discussion
Reported causes of intraerythrocytic vacuoles or inclusions in
other species include hematozoal parasites, viral infections (Telford and Jacobson, 1993;
Johnsrude et al. , 1997), bacterial infections and septicemia (Butler et al. , 1987;
Thompson, 1986), congenital erythrocyte dysplasias (Kenny et al. , 1978), erythremic
myelosis (Riddell and Davidson, 1968), erythroleukemia (Miura, 1976), febrile diseases
(Heino and Laitinen, 1987), and ethanol intoxication (Davidson and McPhie, 1980).
Intraerythrocytic inclusions have been reported in other reptilian
species. Noncrystalline hexagonal erythrocyte inclusions of an undetermined cause were
described in a rhinoceros iguana (Cyclura cornuta) with flaccid paralysis of four limbs
(Simpson et al. , 1980). Small acidophilic intraerythrocytic inclusions were reported in
flap-necked chameleons (Chamaeleo dilepsis) and Fischer's chameleons (Bradypodion
fischeri) (Telford and Jacobson, 1993) associated with iridovirus infection. Both
crystalline and noncrystalline intraerythrocytic inclusions were found in a Fer de Lance (Bothrops
moojeni) snake associated with iridovirus infection (Johnsrude et al. , 1997). Membrane-bound intracytoplasmic inclusions have been observed in erythrocytes of a boid
snake with an ulcerative dermatopathy by one of the authors (K. Harr, unpublished data). Inclusions were also present in the lymphocytes and thrombocytes of this snake, and Morganella
morganii and Clostridium sp. were cultured from the blood. Published examples
of vacuolated erythrocytes also exist for a royal python with chronic weight loss and in
erythroblasts of a Mediterranean spur-thighed tortoise with anemia and post-hibernation
anorexia (Hawkins and Dennett, 1989).
The experimental treatment was an unlikely cause of the condition
in the two alligators reported here, as one of the animals was a control. Although blood
cultures were not performed, the animals were probably septicemic, based on the presence
of numerous bacteria in the tail lesions and the presence of toxic changes in the
heterophils. The study alligators were frequently observed to bite each other during
feeding, which may have resulted in injury to the tails with subsequent gangrene. Although
gangrene in a distal extremity could also be consistent with an ischemic insult, the other
extremities (toes) were not affected in these alligators. The necrosis also appeared to
progress inward from the epidermis, rather than radiating outward from deeper tissues of
the tail, as one might expect from a vascular insult.
In the cases reported here, some of the vacuoles contained
degenerate organelles, leading to the possibility that the vacuoles may be autophagosomal,
formed during the normal maturation process of the erythrocyte. Autophagosomal vacuoles
have been shown to accumulate in acquired hemolytic anemia and other hematological
disorders associated with reticulocytosis (Kent et al. , 1966). This hypothesis may be
supported by the finding of more vacuoles in the immature erythrocytes than in the mature
peripheral erythrocytes.
Several other animals in the group were observed to have less
severe necrosis of their distal tails and were treated with chlorhexidine scrubs. Blood
smears were made on all 72 remaining animals at necropsy at 9 months of age. At this time,
none of the animals had active tail necrosis, and scarring was present at the tail tips of
the treated animals. Inclusions were not noted in any of the Wright's stained blood smears
at this time.
Acknowledgements
We thank Perry Bain,
Maureen Davidson, Jerry Davis, Carol Detrisac, and Rose Raskin for their comments. Electron microscopy services were provided by Karen Kelley of the University of Florida
Electron Microscopy Core Laboratory of the Interdisciplinary Center for Biotechnology
Research. Processing of tissues for histology was performed by Betty Hall of the
University of Florida College of Veterinary Medicine Veterinary Medical Teaching Hospital
Histology Laboratory. Funding for the histology and electron microscopy services was
provided by Environmental Protection Agency grant R826127.
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