CHRONIC UPPER RESPIRATORY TRACT DISEASE OF FREE-RANGING DESERT TORTOISES (XEROBATES AGASSIZII) Author(s): E. R. Jacobson, J. M. Gaskin, M. B. Brown, R. K. Harris, C. H. Gardiner, J. L. LaPointe, H. P. Adams, and C. Reggiardo Source: Journal of Wildlife Diseases, 27(2):296-316. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-27.2.296 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-27.2.296
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of Wildlife
Journal
Dsea.,es,
27(2),
C Wildlife
CHRONIC
UPPER
RESPIRATORY
FREE-RANGING
DESERT
E. R. Jacobson, C. H. Gardiner,2 ,
2
TRACT
TORTOISES
J. M. Gaskin,1 J. L. LaPointe,3
M. B. Brown,1 H. P. Adams,3
College of Veterinary Medicine, University of Rorida, Department of Veterinary Pathology, Armed Forces
Department New Mexico 4 Department
of Biology and Electron 88003, USA of Veterinary Science,
3
were from were
examined; a clinically significantly
significantly
in
1991
OF
(XEROBATES
AGASSIZI!)
R. K. Harris,2 and C. Reggiardo4 Gainesville, Ronda Institute of Pathology,
Laboratory, of Arizona,
New Tucson,
32610, USA Washington,
Mexico
State
Arizona
85721,
D.C.
University,
20306, Las
USA
Cruces,
USA
agassizii, with upper respiratory tract disease necropsy. Four normal control desert tortoises evaluated. Hemoglobin and phosphorus values sodium, urea, SGOT, and cholesterol values were controls. No significant differences in concentrations
desert
thirteen healthy (P
higher
University
Association
tortoises, Xerobates were euthanatized for population were similarly
Seventeen
ABSTRACT:
Microscopy
DISEASE
pp. 296-316
1991,
Disease
0.05)
tower
and
itt tortoises
serum
compared
to
of serum or liver vitamins A and E were found between the two groups. White no significant differences were found for concentrations of lead, copper, cadmium, and selenium, the tivers of illtortoises had higher concentrations of mercury and iron. Lesions were found consistently in the upper respiratory tract (URT) of ill tortoises. In all ill tortoises dense infiltrates of lymphocytes and
histiocytes
was
variably
revealed
small
association cultured sp. to
obscured
was the Key
organisms
The
mucosal
Electron
resembling
is often Viruses, at!
1978;
epithetium
microscopic
Mycoplasrna
few
toises,
rhinitis
and
1984;
et a!.,
diseases are
tortoises
cases.
of
studies
sp.,
in
close
common
snakes,
a necropsy
1978)
However,
re-
in captive
often
animal
a collection Since nasal with rhinitis
(Jackson and Needham, swabs taken of many often do not yield
organisms,
some
another
report,
tortoises
with
Pasteurella
sp.,
testudinis,
investigators agent and
tor-
Desert
although rhinitis,
have
not
isolated
Mycoplasrna
may tract
has 1988,
were
seen
Area
(DTNA),
(Snipes
the
Still,
exact
the
Desert Kern
and cause
not been elucidated. desert tortoises in
and
described testudinis, healthy and
be involved in disease (URTD)
tortoises
1982).
disease In
(Lawrence
A recently Pasteurella from both
with
an in
Biberof
this
URTD
Tortoise
Natural
County,
California
(USA) (K. H. Berry, pers. comm.). In 1989, a detailed survey of the DTNA and nearby areas in the Rand Mountains and Freemont Valley indicated that 43% of 468 live
fo!-
into
1983). tortoises bacterial
as the cause of Needham, 1983).
organism
1985). organism, isolated
desert
stein,
in
(Keymer,
and rhinitis in captive tortoises the introduction of a new
(Lawrence
a viral (Jackson
section.
as a suspect
captive
the
is a common occurrence Needham, 1985). Epidem-
pected disease
tissue
ill tortoises and upper respiratory
respiratory
Migaki
tract
fungi,
incriminated
reptilian
Although
lizards
vealed
in
Mycoplasrna
encountered bacteria,
been
of
1986).
144
mucosa
disease have
crocodi!ians,
the
disease,
Needham, bacterial has been
(Jacobson,
of
on
tract
listed
respiratory
low
pleomorphic
glands.
ulcerated.
INTRODUCTION
agents
Jacobson,
of
underlying
agassizii.
reptiles.
as causative
ics
nm),
organism present respiratory
parasites
survey
900
to
and
occasionally
and
Upper
Respiratory
in captive
tower
epithelium
with the surface epithetium of the URT of ill tortoises. Pasteurella testudinis was from the nasal cavity of all ill tortoises and one of four control tortoises. A Mycoplasrna cultured from the nasal passageways of four itt tortoises and was ultrastructuratly similar
Tortoise, Xerobates
disease
mucosal
hyperplastic,
(350
pleomorphic words:
and
the
dysplastic,
desert tortoises encountered surveyed showed Signs
sus-
(Knowles,
this In
627
tortoises
pled with
areas. signs
from
in
was 296
the
1989).
eastern
were of
of
on the this
Additionally,
carcasses
recovered
Free-ranging URTD Mojave
from
have
sections disease
desert been
Desert,
the
of sam-
tortoises seen also California
JACOBSON
(K. H. Berry, pers. comm.), Nevada (D. B. Hardenbrook, Beaver
Dam
Slope,
Utah
and
N. Coffeen, pers. comm.; comm.), and the Sonoran (C. Schwalbe,
pers.
undertaken
to
URTD in Clinicopathotogic are
Clark pers.
ET AL-UPPER
the
County, comm.),
Arizona
(M.
comm.).
This
study
the
free-ranging and
was
cause
desert microbiologic
of
tortoises. finds
Florida
(Gainesville,
Florida
clinicopathological mate
and
to
female
signs mean
306
117#{176}52’W),
32610,
evaluations.
one
with clinical 4,900 g and
were transported to Medicine, University
tortoises
of URTD, carapace
mm, Kern
desert
DTNA
California
for
1989, (Group
weighing lengths the
from County,
USA)
In May
11 I),
390
to of
(MCL)
(35#{176}12’N, (USA)
were
evaluated. In June 1989, four clinically healthy male desert tortoises (Group II), weighing 1,180 to 3,887
g and
MCL
of
199
to
280
mm,
from
Ivanpah Valley (35#{176}20’N,1 15#{176}22’W), San Bernardino County, California (USA) were evaluated. These tortoises were used as controls. In November 1989, one female desert tortoise (Group III), with URTD, weighing 2,090 g and a MCL of 229 mm, from the Red Hilt (37#{176}5’N, 1 13#{176}55’W) population north of St. George, Washington County, Utah (USA) was evaluated. This tortoise was collected in June 1989 and was healthy at the time of collection. It was subsequently housed with other captive desert tortoises, several of which manifested URTD. In September
1989,
this
tortoise
exhibited
signs
of
URTD. In December 1989, two male and two female desert tortoises (Group IV) with URTD, weighing 3,395 to 3,990 g and MCL of 224 to 292 mm, from private land (35#{176}12’N,1 17#{176}56’W), adjacent to the DTNA were evaluated. These tortoises were collected in July and August 1989 and were kept in an outdoor enclosure. All tortoises within Groups I, II, and III were euthanatized within 3 wk of arrival utilizing an intraperitoneal injection of a concentrated barbiturate solution. Once the tortoises showed complete muscle relaxation and were unresponsive to painful simulation, they were decapitated. Heads of seven tortoises in Group I, three tortoises in Group II, and the one tortoise in Group III were bisected longitudinally with an electric saw. Heads of one tortoise each in Groups I and II were transversely sectioned with an electric saw. The ptastron was removed from
viscera
TORTOISES
within
the
297
coelomic
exposed.
and serum
biochemical
evaluations
Hematologic
by
use
evaluations
red
counts, white blood cell (WBC) differential white blood cell counts, he(Hb) concentrations, and packed cell (PCV).
of an
21316, 33012,
The
RBC
electronic
Coulter USA).
The
count
counter
Diagnostics, The white
determined manually, niques for reptile and 1967).
were:
cell (RBC)
volumes
Four groups of tortoises the College of Veterinary
OF DESERT
At necropsy, blood samples were collected from tortoises in Groups I and II by intracardiac puncture. Portions of blood from each tortoise were placed in tubes containing lithium heparin for hematologic evaluations and silicone coated tubes for serum separation and biochemical de-
counts, moglobin
AND METHODS
Turtles
133
Hematologic
blood MATERIALS
of
and
were
terminations.
reported.
DISEASE
carapace,
cavity
C. Schwatbe, pers. Desert, Arizona
determine
RESPIRATORY
hemoglobin
was
determined
(Coutter
Counter
Hialeah,
Florida
blood cell count was using conventional techavian species (Schermer, concentration
was
de-
termined using an automated method (Coulter Hemoglobinometer, Coulter Diagnostics, Hialeah, Florida 33012, USA). The PCV concentration was determined using centrifugation. Serum biochemical evaluations included determinations of the amounts of glucose, sodium, potassium, chloride, C02, BUN (urea), creatinine, uric acid, calcium, phosphorus, alkaline phosphatase transaminase pyruvic
serum glutamic activity, serum
(ALP),
(SGOT) transaminase
(SGPT)
oxalacetic glutamic
activity,
total
cho-
lesterot, and total bitirubin; all evaluations were made utilizing an automated analyzer (Hitachi 737, BMD, Indianapolis, Indiana 46250, USA). Triglyceride concentrations were also determined utilizing an autoanalyzer (Encore Autoanatyzer, Baker Instruments, Allentown, Pennsylvania
18001,
USA).
Serum was collected from each sample and was electrophoresed on cellulose polyacetate strips at 200 V for 20 mm, stained with panceau-S in 7.5% trichtoracetic acid, and rinsed with 5% acetic acid. After clearing the strips in 40% aqueous
N-methyl
pyrrolidine,
the
strips
were
dried for 20 mm at 90 C. The separated proteins were densitometricalty quantitated using a scanning densitometer (GS300, Hoefer Instruments, San Francisco, California 94710, USA) at 540 nm. Concentrations of vitamin A and E in sera were determined for six tortoises in Group I and four tortoises in Group II using an HPLC method (Catignani and Bieri, 1983). Liver vitamin
and metal
determinations
Concentrations of the following mined on portions of liver from tortoises and four Group II tortoises:
were
deterGroup I vitamin A,
10
298
JOURNAL
OF WILDLIFE
DISEASES,
VOL. 27. NO. 2, APRIL
vitamin E, selenium (Se), iron (Fe), mercury (Hg), lead (Pb), and cadmium (Cd). Concentrations of vitamins A and E were determined according to the procedure described for sera above. Concentrations of selenium were determined by capillary gas chromatography with electron
capture
detection
(Poole
et
at.,
1977).
Amounts of copper and iron were determined by flame atomic absorption spectrometry (Instrumentation Laboratory Video 12 Atomic Absorption Spectrophotometer, Waltham, Massachusetts 02254, USA). Concentrations of lead and cadmium were determined utilizing graphite furnace atomic absorption (IL 655, Instrumentation Laboratory, Waltham, Massachusetts 02254, USA). Mercury values were determined using cold-vapor atomic absorption spectrometry (IL 400, Instrumentation Laboratory, Waltham, Massachusetts 02254, USA). Pathologic
investigations
A gross necropsy was conducted on alt euthanatized tortoises. An acetic acid digestion technique (Gotdstine et at., 1975) was used to locate the thymus of two tortoises in Group I. For histopathological studies, one-half of each longitudinally sectioned head of tortoises in Groups I, II, and III and transversely sectioned heads of one tortoise each in Groups I and II were fixed in neutral buffered 10% formalin (NBF), decalcified, embedded in paraffin, sectioned at 7 m, and stained with hematoxylin and eosin and by the Brown and Hopps method (Luna, 1968) for identification of gram-positive and gram-negative microorganisms. Tissues from the following visceral structures were collected, fixed in NBF, embedded in paraffin, sectioned at 7 iim, and stained with hematoxylmn and eosin: glottis, cranial trachea, tracheal bifurcation, right bronchus, left bronchus, right cranial-lung lobe, right mid-lung lobe, right caudat-lung lobe, left cranial-lung lobe, left mid-lung lobe, left caudal-tung lobe, thyroid, left and right thymus, spleen, heart, liver, cranial and caudal stomach, small intestine, pancreas, colon, right and left kidney, right and left reproductive tracts, bladder, and pectoral muscle. Liver sections of all tortoises in Groups I and II were also stained for iron by the Prussian Blue method. Sections of nasal mucosa from paraffin embedded tissues of one tortoise in Group I were deparaffinized, then fixed in Dalton’s osmium dichromate solution and embedded in polaron 812 (Potarbed, Micro Structure, PTY, Ltd., London, England). Thick sections were stained with toluidine blue and examined by light microscopy. Ultrathin sections were placed on copper grids, stained with uranyt acetate and lead citrate, and examined with a electron microscope.
1991
cavity mucosa of one tortoise in each I and Group III were separated from subadjacent tissues, cut into 1 mm cubes, placed in 2.5% glutaraldehyde, and post-fixed in osmium tetroxide. Some specimens were prepared for scanning electron microscopy (SEM) by critical point drying and sputter coating with gold. Other specimens were prepared for transmission electron microscopy (TEM) by embedding in epon-araldite and sectioning with an ultramicrotome. Formalin fixed nasal cavity mucosa of three tortoises in Group I and two tortoises in Group II was post-fixed in osmium tetroxide and similarly processed. Thick sections were stained with toluidine blue and examined by light microscopy. Ultrathin sections were placed on copper grids, stained with uranyt acetate and lead citrate, and examined with an electron microscope. Nasal
of Groups
Microbial
investigations
Swab specimens of choanae, nasal cavities, tracheal mucosa, and right lungs of alt tortoises in Groups I and II were collected and cultured on sheep blood agar and MacConkey’s agar, and incubated at 37 C for aerobic bacteria. A swab specimen of the nasal cavity of the Group III tortoise was similarly cultured. All aerobes were identified utilizing growth characteristics on various media and standard biochemicat tests. All isolates of organisms consistent with Pasteurella sp. were identified to species according to biochemical profiles listed for P. testudlnis (Snipes and Biberstein, 1982). The type specimen for P. testudlnis was purchased (American Type Culture Collection, Rockvitle, Maryland 20852, USA) and used for comparison. Swab specimens of choanae of five Group I tortoises and three Group II tortoises, liver samples of three Group I tortoises and three Group II tortoises, spleen samples of two Group I tortoises and three Group II tortoises, and colon contents of one Group I tortoise and three Group II tortoises were placed in minimal essential media (MEM; Remel, Regional Media Laboratories, Lenexa, Kansas 66215, USA) and submitted to the National Animal Disease Center, Ames, Iowa, USA, for chtamydial isolation attempts utilizing embryonated chicken eggs. Nasal
washings
utilizing
phosphate
buffered
saline (PBS) were collected from one Group I tortoise, the Group III tortoise, and from alt four tortoises in Group IV for isolation of mycoplasmas. Initial isolations were performed using modified Fabricant’s, arginine, Hayflick’s, 10 B and 5P4 broths and agars (Razin and Tully, 1983). Broth cultures were incubated at 22, 30 and 37 C. Agar plates were incubated in 5% CO2 at 22, 30 and 37 C. The initial isolates were
JACOBSON
obtained and this
were
with SP4 isolation medium at 30 medium and incubation temperature
used
weekly
for subsequent intervals,
growth
ET AL-UPPER
with
isolation
plates
C,
attempts.
were
DISEASE
etate and lead Hitachi H-7000
OF DESERT
citrate, STEM
TORTOISES
and examined with electron microscope.
for
microscope.
Statistical Student’s
t-test
was
used
to evaluate
ences between Group I and II tortoises following variables: hematologic values,
week
a single
tamins
agar.
When
plugs
were
broth,
passage
growth
was
removed
and
was
made
observed
treated
to 5P4
on agar,
aseptically,
placed
as described
for
a
analysis
Broth cultures were examined for a color change biweekly. If a color change was observed, the broth was subcultured to agar, fresh broth, and the remaining culture frozen at -70 C. At one
blind
299
At
examined
the aid of a dissection
RESPIRATORY
agar in
other
biochemical A
values, A
and
E,
of P
value
concentrations SE,
Pb,
0.05
serum
of serum
Cu,
was
differfor the
Fe,
Cd,
considered
vi-
and
Hg.
significant.
SP4
broth
RESULTS
cultures.
Aseptically from
collected
each
viral
tortoise
isolation.
was
Kidney
minced
and
solution
in
stirrer
at
tant
mm
to
was
a
pellet
the
decanted
in cell
fetal into
culture
and growth
cells. the
each on
a
Hemato!ogic
magnetic
C). The resulat 500 x g for 10 The supernatant fluid
celt
pellet
was
suspended
(MEM)
bovine serum. The suspension cell culture flasks and incubated
with
was
10%
placed at 30 C
with replenishment of media as necessary. When a confluent monolayer of cells was obtained, it was subdivided to form subcultures. This was repeated twice for each successfully initiated culture so that the primary, secondary, and ter-
tiary cultures could be velopment of cytopathic Swab
specimens
of the
monitored effects. nasal
for
passages
the
de-
for virus
isolation were suspended in MEM and were subsequently frozen at -85 C for virologic studies. When subcultures of primary kidney cell cultures were prepared, they were inoculated with 0.45 m membrane filtrates of the thawed, vortexed nasal specimens in MEM. The monolayers were observed every 48 hr for evidence of viral cytopathic effects (CPE). After 6 to 8 days, these inoculated cultures were subdivided to produce new monolayers which were observed in the same way. A second subculture was also made and
monitored.
microscopy a sample of agar concolonies of P. testudinis was fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and embedded in Spurrs low viscosity resin (Electron Microscopy Sciences, Ft. Washington, Pennsylvania 19034, USA). Equal porFor
electron
taining
tions
of
broth
containing
an organism
isolated
dl),
SGOT
stained
with
uranyl
for
ac-
(117
(305
phosphorus
(1.9
mg/d!)
lower
those
for
than
Liver vitamin
mg/dl)
tortoises
in
and metal
Values
for
tween vitamin
the
between
and
for
significantly
serum
vitamins
A
II are presented differences be-
in each
group.
recorded greater
value for a tortoise Liver values for
in that selenium,
cadmium, II are
and no
total
controls.
value times
ppm were
and values
determinations
liver
tortoises
I tortoises, ppm) and
IU/L) while
two groups were found. Serum E showed considerable variation
I, the highest was over 30
lead, I and
(153 mEq/ (0.2 mg/
were
and E for Groups I and in Table 3. No significant
selenium, tween the
ultrathin sections were
activity
cholesterol
For electron microscopy,
grids,
values
evaluations
cantly higher values for sodium L), BUN (100 mg/dl), creatinine
nificantly
on copper
biochemical
only hemoglobin concentration in the ill tortoises (7.6 g/dl) was significantly lower than that of controls (9.0 g/dl). Serum biochemical values for tortoises in Groups I and II are presented in Table 2. Il! tortoises were found to have signifi-
on SP4 agar and 5% glutaraldehyde were mixed, centrifuged, and the resulting pellet resuspended in 1% osmium tetroxide, then embedded in EM-BED-812 (Electron Microscopy Sciences). placed
and serum
Groups I (ill) and II (control) are presented in Table 1. Of the 14 values determined
(22
medium
Hematologic
tortoise
a trypsin-EDTA
flask,
centrifuged
were obtained Group II for
from with
temperature
was
I and
tissue
digested
trypsinizing
room
slurry
kidneys
Group
in
and
higher
group. copper,
mercury in Table
presented
the mean mercury
value (0.326
than
those
In Group
(6.32 sg/m!) than the lowest
for iron (1,526 ppm) were sigof controls
0.0287 ppm, respectively). differences for concentrations copper, lead two groups.
and
iron,
for Groups 4. In Group
cadmium
(361 There of be-
300
JOURNAL
TABLE
1.
OF WILDLIFE
Hematologic
DISEASES,
values
VOL. 27, NO. 2, APRIL
(mean,
1; standard
Group Determinant’
RBC (10’/l) WBC (4il) Hb (g/dl) PCV (%) MCV (fi)
9 9 9 9 9
(pg)
deviation,
SD)
for
Group
I and
Group
I
Group
f
b
1991
0.60 2,390 7.6 23.2 388
SD
n
0.09 1,032 1.1 4.3 62
4 4 4 4 4
II desert
tortoises.
11
2
SD
0.70
0.08 2,791
3,483
9.0 27.7 403
0.4 1.7 69
9
126
13
4
131
19
9
33
6
4
31
3
Heterophils
9
43
24
4
36
21
Lympohcytes
9
16
10
4
30
13
Monocytes
9
9
7
4
12
8
Eosinophils
9
1
1
4
2
2
Basophils
9
16
11
4
16
12
AGM’
9
5
12
4
0
0
AM’
9
10
10
4
5
5
MCH
(g/dl)
MCHC
Differential
. See
(%)
text
I.
Sample
.
Activated
d
Azurophilic
for
definition
of determinant
and
abbreviations.
size. granular
TABLE
monocytes.
monocytes.
2.
Serum
biochemical
profiles
(mean,
standard
t;
deviation,
SD)
for Group
I and
Group
II desert
tortoises.
Group Determinant
22 14 0.8 12 6
4 4 4 4 4
96 136 5.0 110 29
16 5 0.2 4 4
9
95 153 4.9 118 29 100
74
4
6
2
9
0.3
0.1
4
0.2
0
9
7.2
3.1
4
4.9
2.6
9
11.2
0.9
4
9.8
1.1
0.4 52 62 1
4 4 4 4
2.5 152 56 2
0.2 49 24 0.4
4
83
4
17
(mg/dI) (mEq/L) Potassium (mEq/L) Chloride (mEq/L) CO2 (mEq/L)
9 9 9 9 9
(mg/dl)
BUN
Creatinine Uric
(mg/dl)
acid
Calcium
(mg/dl) (mg/dl)
Phosphorus (mg/dl) ALP (U/L) SCOT (U/L) SGPT (U/L)
9 9 9 9
Cholesterol
9
114 117 2 305
9
18
(mg/dl)
Triglyceride
(mg/dl)
II
n
f
Sodium
Group SD
nb
Glucose
I
1.9
180 15
Z
SD
23 7
Bilirubin
(mg/dl)
9
0.1
0.1
4
0.05
0.05
Albumin
(g/dl)
9
0.8
0.2
4
0.9
0.2
a
9
0.6
0.2
4
0.5
0.1
a2
9
0.8
0.1
4
0.7
0.2
9 9 9
0.6 0.1
0.2
4
0.4
0.2
0.2
4
0
0
0.8
0.6
4
0.9
0.1
Globulins
(g/dl)
flu
fl2 ‘V . See b
text
Sample
for size.
definitions
of determinant
abbreviations.
JACOBSON
3. Liver tortoises.
TABLE
desert
and
serum
vitamins
A and
E
AL-UPPER
E (1,
mean;
Group n
SVA
(sg/ml)
LVA”
(sg/gram)
SVE
(sg/ml)
LVEd
(sg/gram)
‘Serum Liver
vitamin vitamin
RESPIRATORY
SD,
DISEASE
standard
OF DESERT
deviation)
for
I
Group
Group
I
SD
n
TORTOISES
I and
301
Group
II SD
I
6
0.155
0.086
4
0.153
0.106
10
40.72
38.70
4
14.58
8.02
6
3.1
2.8
4
1.14
1.08
10