Chronic upper respiratory tract disease of free-ranging desert tortoises

Full text

296
Journal of Wildlife Dsea.,es, 27(2), 1991, pp. 296-316
CWildlife Disease Association 1991
CHRONIC UPPER RESPIRATORY TRACT DISEASE OF
FREE-RANGING DESERT TORTOISES (XEROBATES AGASSIZI!)
E. R. Jacobson, J. M. Gaskin,1 M. B. Brown,1 R. K. Harris,2
C. H. Gardiner,2 J. L. LaPointe,3 H. P. Adams,3 and C. Reggiardo4
,College of Veterinary Medicine, University of Rorida, Gainesville, Ronda 32610, USA
2Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306, USA
3Department of Biology and Electron Microscopy Laboratory, New Mexico State University, Las Cruces,
New Mexico 88003, USA
4Department of Veterinary Science, University of Arizona, Tucson, Arizona 85721, USA
ABSTRACT: Seventeen desert tortoises, Xerobates agassizii, with upper respiratory tract disease
were examined; thirteen were euthanatized for necropsy. Four normal control desert tortoises
from a clinically healthy population were similarly evaluated. Hemoglobin and phosphorus values
were significantly (P 0.05) tower and serum sodium, urea, SGOT, and cholesterol values were
significantly higher in itt tortoises compared to controls. No significant differences in concentrations
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 obscured the mucosal epithelium and underlying glands. The mucosal epithetium
was variably dysplastic, hyperplastic, and occasionally ulcerated. Electron microscopic studies
revealed small (350 to 900 nm), pleomorphic organisms resembling Mycoplasrna sp., in close
association with the surface epithetium of the URT of ill tortoises. Pasteurella testudinis was
cultured from the nasal cavity of all ill tortoises and one of four control tortoises. AMycoplasrna
sp. was cultured from the nasal passageways of four itt tortoises and was ultrastructuratly similar
to the pleomorphic organism present on the mucosa in tissue section.
Key words: Upper respiratory tract disease, Mycoplasrna sp., Pasteurella testudinis, Desert
Tortoise, Xerobates agassizii.
INTRODUCTION
Respiratory disease is often encountered
in captive reptiles. Viruses, bacteria, fungi,
and parasites have at! been incriminated
as causative agents of reptilian respiratory
disease (Jacobson, 1978; Migaki et a!., 1984;
Jacobson, 1986). Although diseases of the
tower respiratory tract are common in
crocodi!ians, lizards and snakes, a necropsy
survey of 144 tortoises (Keymer, 1978) re-
vealed few cases. However, in captive tor-
toises, rhinitis is a common occurrence
(Lawrence and Needham, 1985). Epidem-
ics of rhinitis in captive tortoises often fo!-
low the introduction of a new animal into
a collection (Jackson and Needham, 1983).
Since nasal swabs taken of many tortoises
with rhinitis often do not yield bacterial
organisms, some investigators have sus-
pected a viral agent as the cause of this
disease (Jackson and Needham, 1983). In
another report, although not isolated from
tortoises with rhinitis, Mycoplasrna was
listed as a suspect organism (Lawrence and
Needham, 1985). A recently described
bacterial organism, Pasteurella testudinis,
has been isolated from both healthy and
ill tortoises and may be involved in an
upper respiratory tract disease (URTD) in
captive desert tortoises (Snipes and Biber-
stein, 1982). Still, the exact cause of this
disease has not been elucidated.
In 1988, desert tortoises with URTD
were seen in the Desert Tortoise Natural
Area (DTNA), Kern County, California
(USA) (K. H. Berry, pers. comm.). In 1989,
a detailed survey of the DTNA and nearby
areas in the Rand Mountains and Free-
mont Valley indicated that 43% of 468 live
desert tortoises encountered on the sections
surveyed showed Signs of this disease
(Knowles, 1989). Additionally, carcasses of
627 tortoises were recovered from the sam-
pled areas. Free-ranging desert tortoises
with signs of URTD have been seen also
in the eastern Mojave Desert, California

JACOBSON ET AL-UPPER RESPIRATORY DISEASE OF DESERT TORTOISES 297
(K. H. Berry, pers. comm.), Clark County,
Nevada (D. B. Hardenbrook, pers. comm.),
Beaver Dam Slope, Utah and Arizona (M.
N. Coffeen, pers. comm.; C. Schwatbe, pers.
comm.), and the Sonoran Desert, Arizona
(C. Schwalbe, pers. comm.). This study was
undertaken to determine the cause of
URTD in free-ranging desert tortoises.
Clinicopathotogic and microbiologic finds
are reported.
Turtles
MATERIALS AND METHODS
Four groups of tortoises were transported to
the College of Veterinary Medicine, University
of Florida (Gainesville, Florida 32610, USA) for
clinicopathological evaluations. In May 1989, 11
mate and one female desert tortoises (Group I),
with clinical signs of URTD, weighing 390 to
4,900 g and mean carapace lengths (MCL) of
133 to 306 mm, from the DTNA (35#{176}12’N,
117#{176}52’W), Kern County, California (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 Ber-
nardino County, California (USA) were evalu-
ated. These tortoises were used as controls. In
November 1989, one female desert tortoise
(Group III), with URTD, weighing 2,090 g and
aMCL 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 subse-
quently housed with other captive desert tor-
toises, 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 bar-
biturate solution. Once the tortoises showed
complete muscle relaxation and were unrespon-
sive to painful simulation, they were decapitat-
ed. 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
Iand II were transversely sectioned with an
electric saw. The ptastron was removed from
the carapace, and viscera within the coelomic
cavity were exposed.
Hematologic and serum biochemical evaluations
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-
terminations. Hematologic evaluations were: red
blood cell (RBC) counts, white blood cell (WBC)
counts, differential white blood cell counts, he-
moglobin (Hb) concentrations, and packed cell
volumes (PCV). The RBC count was determined
by use of an electronic counter (Coutter Counter
21316, Coulter Diagnostics, Hialeah, Florida
33012, USA). The white blood cell count was
determined manually, using conventional tech-
niques for reptile and avian species (Schermer,
1967). The hemoglobin concentration was de-
termined using an automated method (Coulter
Hemoglobinometer, Coulter Diagnostics, Hia-
leah, Florida 33012, USA). The PCV concen-
tration was determined using centrifugation.
Serum biochemical evaluations included de-
terminations of the amounts of glucose, sodium,
potassium, chloride, C02, BUN (urea), creati-
nine, uric acid, calcium, phosphorus, alkaline
phosphatase (ALP), serum glutamic oxalacetic
transaminase (SGOT) activity, serum glutamic
pyruvic transaminase (SGPT) 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 deter-
mined utilizing an autoanalyzer (Encore Au-
toanatyzer, 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 Instru-
ments, San Francisco, California 94710, USA)
at 540 nm.
Concentrations of vitamin Aand Ein sera
were determined for six tortoises in Group Iand
four tortoises in Group II using an HPLC meth-
od (Catignani and Bieri, 1983).
Liver vitamin and metal determinations
Concentrations of the following were deter-
mined on portions of liver from 10 Group I
tortoises and four Group II tortoises: vitamin A,

298 JOURNAL OF WILDLIFE DISEASES, VOL. 27. NO. 2, APRIL 1991
vitamin E, selenium (Se), iron (Fe), mercury
(Hg), lead (Pb), and cadmium (Cd). Concen-
trations of vitamins A and E were determined
according to the procedure described for sera
above. Concentrations of selenium were deter-
mined by capillary gas chromatography with
electron capture detection (Poole et at., 1977).
Amounts of copper and iron were determined
by flame atomic absorption spectrometry (In-
strumentation Laboratory Video 12 Atomic Ab-
sorption Spectrophotometer, Waltham, Massa-
chusetts 02254, USA). Concentrations of lead
and cadmium were determined utilizing graph-
ite furnace atomic absorption (IL 655, Instru-
mentation Laboratory, Waltham, Massachusetts
02254, USA). Mercury values were determined
using cold-vapor atomic absorption spectrom-
etry (IL 400, Instrumentation Laboratory, Wal-
tham, Massachusetts 02254, USA).
Pathologic investigations
Agross necropsy was conducted on alt eu-
thanatized 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, sec-
tioned 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 eo-
sin: 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, blad-
der, and pectoral muscle. Liver sections of all
tortoises in Groups Iand II were also stained
for iron by the Prussian Blue method.
Sections of nasal mucosa from paraffin em-
bedded tissues of one tortoise in Group Iwere
deparaffinized, then fixed in Dalton’s osmium
dichromate solution and embedded in polaron
812 (Potarbed, Micro Structure, PTY, Ltd., Lon-
don, England). Thick sections were stained with
toluidine blue and examined by light micros-
copy. Ultrathin sections were placed on copper
grids, stained with uranyt acetate and lead ci-
trate, and examined with a electron microscope.
Nasal cavity mucosa of one tortoise in each
of Groups Iand Group III were separated from
subadjacent tissues, cut into 1 mm cubes, placed
in 2.5% glutaraldehyde, and post-fixed in os-
mium tetroxide. Some specimens were prepared
for scanning electron microscopy (SEM) by crit-
ical point drying and sputter coating with gold.
Other specimens were prepared for transmis-
sion electron microscopy (TEM) by embedding
in epon-araldite and sectioning with an ultra-
microtome. 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 mi-
croscope.
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. Aswab
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 Pasteu-
rella sp. were identified to species according to
biochemical profiles listed for P. testudlnis
(Snipes and Biberstein, 1982). The type speci-
men 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 sam-
ples of three Group Itortoises and three Group
II tortoises, spleen samples of two Group Itor-
toises and three Group II tortoises, and colon
contents of one Group I tortoise and three Group
II tortoises were placed in minimal essential me-
dia (MEM; Remel, Regional Media Laborato-
ries, 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 mycoplas-
mas. 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 ET AL-UPPER RESPIRATORY DISEASE OF DESERT TORTOISES 299
obtained with SP4 isolation medium at 30 C,
and this medium and incubation temperature
were used for subsequent isolation attempts. At
weekly intervals, plates were examined for
growth with the aid of a dissection microscope.
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
week a single blind passage was made to 5P4
agar. When growth was observed on agar, agar
plugs were removed aseptically, placed in SP4
broth, and treated as described for other broth
cultures.
Aseptically collected kidneys were obtained
from each tortoise in Group Iand Group II for
viral isolation. Kidney tissue from each tortoise
was minced and digested with a trypsin-EDTA
solution in a trypsinizing flask, on a magnetic
stirrer at room temperature (22 C). The resul-
tant slurry was centrifuged at 500 x g for 10
mm to pellet the cells. The supernatant fluid
was decanted and the celt pellet was suspended
in cell culture growth medium (MEM) with 10%
fetal bovine serum. The suspension was placed
into cell culture flasks and incubated 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 monitored for the de-
velopment of cytopathic effects.
Swab specimens of the nasal passages for virus
isolation were suspended in MEM and were sub-
sequently frozen at -85 C for virologic studies.
When subcultures of primary kidney cell cul-
tures were prepared, they were inoculated with
0.45 m membrane filtrates of the thawed, vor-
texed 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.
For electron microscopy a sample of agar con-
taining colonies 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. Wash-
ington, Pennsylvania 19034, USA). Equal por-
tions of broth containing an organism isolated
on SP4 agar and 5% glutaraldehyde were mixed,
centrifuged, and the resulting pellet resuspend-
ed in 1% osmium tetroxide, then embedded in
EM-BED-812 (Electron Microscopy Sciences).
For electron microscopy, ultrathin sections were
placed on copper grids, stained with uranyl ac-
etate and lead citrate, and examined with a
Hitachi H-7000 STEM electron microscope.
Statistical analysis
Student’s t-test was used to evaluate differ-
ences between Group Iand II tortoises for the
following variables: hematologic values, serum
biochemical values, concentrations of serum vi-
tamins A and E, SE, Pb, Cu, Fe, Cd, and Hg.
Avalue of P 0.05 was considered significant.
RESULTS
Hematologic and serum biochemical evaluations
Hemato!ogic values for tortoises in
Groups I (ill) and II (control) are presented
in Table 1. Of the 14 values determined
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-
cantly higher values for sodium (153 mEq/
L), BUN (100 mg/dl), creatinine (0.2 mg/
dl), SGOT activity (117 IU/L) and total
cholesterol (305 mg/dl) while values for
phosphorus (1.9 mg/d!) were significantly
lower than those for controls.
Liver vitamin and metal determinations
Values for liver and serum vitamins A
and E for Groups I and II are presented
in Table 3. No significant differences be-
tween the two groups were found. Serum
vitamin E showed considerable variation
between tortoises in each group. In Group
I, the highest value recorded (6.32 sg/m!)
was over 30 times greater than the lowest
value for a tortoise in that group.
Liver values for selenium, copper, iron,
lead, cadmium, and mercury for Groups
Iand II are presented in Table 4. In Group
I tortoises, the mean value for iron (1,526
ppm) and mercury (0.326 ppm) were sig-
nificantly higher than those of controls (361
ppm and 0.0287 ppm, respectively). There
were no differences for concentrations of
selenium, copper, lead and cadmium be-
tween the two groups.

.See text for definitions of determinant abbreviations.
bSample size.
300 JOURNAL OF WILDLIFE DISEASES, VOL. 27, NO. 2, APRIL 1991
TABLE 1. Hematologic values (mean, 1; standard deviation, SD) for Group Iand Group II desert tortoises.
Determinant’
Group I Group 11
b fSD n2 SD
RBC (10’/l) 9 0.60 0.09 4 0.70 0.08
WBC (4il) 9 2,390 1,032 43,483 2,791
Hb (g/dl) 9 7.6 1.1 4 9.0 0.4
PCV (%) 9 23.2 4.3 4 27.7 1.7
MCV (fi) 9 388 62 4 403 69
MCH (pg) 9 126 13 4 131 19
MCHC (g/dl) 9 33 6 4 31 3
Differential (%)
Heterophils 9 43 24 4 36 21
Lympohcytes 9 16 10 4 30 13
Monocytes 9 9 74 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
.See text for definition of determinant and abbreviations.
I. Sample size.
.Activated granular monocytes.
dAzurophilic monocytes.
TABLE 2. Serum biochemical profiles (mean, t; standard deviation, SD) for Group I and Group II desert
tortoises.
Determinant
Group I Group II
nb f SD nZSD
Glucose (mg/dI) 9 95 22 4 96 16
Sodium (mEq/L) 9 153 14 4 136 5
Potassium (mEq/L) 9 4.9 0.8 4 5.0 0.2
Chloride (mEq/L) 9 118 12 4 110 4
CO2 (mEq/L) 9 29 6 4 29 4
BUN (mg/dl) 9 100 74 4 6 2
Creatinine (mg/dl) 9 0.3 0.1 4 0.2 0
Uric acid (mg/dl) 9 7.2 3.1 4 4.9 2.6
Calcium (mg/dl) 9 11.2 0.9 4 9.8 1.1
Phosphorus (mg/dl) 9 1.9 0.4 4 2.5 0.2
ALP (U/L) 9 114 52 4 152 49
SCOT (U/L) 9 117 62 4 56 24
SGPT (U/L) 9 2 1 4 2 0.4
Cholesterol (mg/dl) 9 305 180 4 83 23
Triglyceride (mg/dl) 9 18 15 4 17 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
Globulins (g/dl)
a 9 0.6 0.2 4 0.5 0.1
a2 9 0.8 0.1 4 0.7 0.2
flu 90.6 0.2 4 0.4 0.2
fl2 90.1 0.2 4 0 0
‘V 90.8 0.6 4 0.9 0.1

JACOBSON E AL-UPPER RESPIRATORY DISEASE OF DESERT TORTOISES 301
‘See text for definition of assay abbreviations.
TABLE 3. Liver and serum vitamins A and E (1, mean; SD, standard deviation) for Group I and Group II
desert tortoises.
Group I Group II
n I SD n I SD
SVA (sg/ml) 6 0.155 0.086 4 0.153 0.106
LVA” (sg/gram) 10 40.72 38.70 4 14.58 8.02
SVE (sg/ml) 6 3.1 2.8 4 1.14 1.08
LVEd (sg/gram) 10 <1.00 -4<1.00 -
‘Serum vitamin A.
Liver vitamin A.
Serum vitamin E.
Liver vitamin E.
Normal anatomy and histology
The external nares were continuous with
large nasa! cavities, which were separated
by an internasa! septum (Fig. 1A, B). Ven-
trally, the nasa! passageways were contin-
uous with the choanae (internal nares),
which opened into the pa!atine region of
the dorsal oral cavity.
Histologic examination revealed the in-
tegument continued through the external
nares into a short vestibule, which was mi-
tially lined by keratinized stratified squa-
mous epithe!ium. However, on the yen-
trolateral aspects of this vestibule, there
was an abrupt change to a mucous g!an-
du!ar epithe!ium. The vestibule subse-
quently opened into the nasal cavity. The
mucosa of the rostra! nasal cavity was com-
prised of areas of pseudostratified mucous
epithe!ial cells, with associated ciliated ep-
ithe!ia! cel!s (Fig. 2A) and areas of o!fac-
tory epithe!ium (Fig. 2B). Within the basal
cell layer of the mucosa, a cell type with
a large lightly basophi!ic nucleus was com-
mon!y seen. In the cauda! nasal cavity, the
olfactory epithe!ium was located on the
dorsal surface and the mucous epithe!ium
was ventral. Numerous serous and mucous
glands, vessels, nerve bundles, and clusters
of melanophores were present in the con-
nective tissue surrounding the URT. With
the exception of a small focal infi!trate of
lymphocytes in the propria-submucosa of
one tortoise, the URT of controls contained
few inflammatory cells.
The thymus was easy to find in controls
and was located on both sides of the tra-
chea, crania! to the base of the heart, ad-
jacent to the vagus nerve, and at the
branching of the subc!avian and carotid
arteries. The thymus was mu!ti!obulated
and measured from 1.2 to 2.0 by 0.7 to 1.5
cm. Histologically, there was a typically
dark staining cortex and a lighter staining
medul!a. The disparity in staining was due
to density of cells in each area. The cortex
contained densely packed thymocytes and
the medu!la contained significantly fewer
TABLE 4. Liver values (ppm; I, mean; SD, standard deviation) for minerals and heavy metals in Group I
and Group II desert tortoises.
Group I Group II
Assay’ n I SD n I SD
Se 10 0.383 0.141 4 0.212 0.059
Cu 10 8.67 7.90 4 9.16 2.800
Fe 10 1,526 674 4 361 64
Pb 10 0.035 0.022 4 0.012 0.017
Cd 10 0.51 0.26 4 0.41 0.15
Hg 10 0.326 0.086 4 0.0287 0.017

A
a
Cb
d
302 JOURNAL OF WILDLIFE DISEASES, VOL. 27, NO. 2, APRIL 1991
B
FIGURE 1. Diagrammatic representation of des-
ert tortoise heads, sectioned longitudinally (A) and
transversely (B). A large nasal cavity can be seen
cranial to the eyes (e). Lines in A correspond to cross-
sections in B. Sections a and b are opposite sides of
the same cut.
thymocytes, as well as thymic epithelial
cells, myoid cells and heterophils.
The spleen was located on the right side,
between the proximal duodenum and
transverse colon and was closely associated
with pancreatic tissue. Spleens of healthy
tortoises measured from 2.5 to 5 cm by 1.0
to 1.3 cm. Histologically, spleens were
composed of distinct areas of white and
red pulp. White pulp consisted of collec-
tions of lymphoid tissue surrounding blood
vessels. Red pulp, located between the
perivascular collections of the white pulp,
included red blood cells within sinusoids
and few tymphoid cells.
Pathologic findings
At necropsy, small amounts of fat were
found in the coelomic cavities of both
Group I and Group II tortoises. Controls
had more abundant subcutaneous fat in
the axillary and inguinal areas of both fore-
and hindlimbs.
Examination of longitudinally and
transversely sectioned heads of it! tortoises
revealed a moderate to large amount of
exudate within the nasa! cavity and nasal
passageways. Microscopic findings were
similar in the URT of all ill tortoises ex-
amined. In the nasal vestibule, there was
a diffuse loss of mucosal glands. Glandular
structures which remained were either re-
placed by and/or filled with proliferating
epithelia! cells (Fig. 3). In the more se-
verely affected areas, there were dense in-
filtrates of lymphocytes and histiocytes that
often formed a linear band which ob-
scured the mucosa! epithe!ium and ex-
tended into the deep !amina propria. It
was frequently perivascular, and effaced
mucosal glands. A moderate number of
plasma cells and occasional Mott cells (ac-
tivated plasma cells) were admixed with
the lymphocytes and histiocytes.
In the olfactory mucosa, the normal
multilayered arrangement of epithetial
cells could not be discerned (Fig. 4A). Mu!-
tifoca!ly, there were variable numbers of
heterophils in the tamina propria, trans-
migrating the mucosa! epithelium, filling
the lumina of mucosal glands, and accu-
mulating on the mucosal surface. Hetero-
phits were the predominant inflammatory
cell in a few areas. Within the mucosal
epithelium were small to moderate num-
bers of macrophages containing phago-
cytized cellular debris and, occasionally,
variable sized, up to 5 m in diameter,
pink, homogeneous, intracytoplasmic bod-

‘
.1, I.
JACOBSON ET AL-UPPER RESRATORY DISEASE OF DESERT TORTOISES 303
FIGURE 2. Photomicrographs of the nasal cavity of a healthy desert tortoise demonstrating an area of
mucous and ciliated epithelial cells (A) and an area consisting of a multilayered olfactory epithelium (B).
H&E. Bar =30 m.

,sI’*%’ 
‘‘
304 JOURNAL OF WILDLIFE DISEASES, VOL. 27, NO. 2, APRIL 1991
FIGURE 3. Photomicrograph of the nasal vestibule of a desert tortoise with URTD. There is a proliferation
of mucosal epithelial cells, reduction in glandular structure, and infiltrates of mononuclear inflammatory
cells. H&E. Bar =100 m.
ies (Fig. 4B). These structures were inter-
preted as either cell debris or phagocytized
heterophit granules.
In areas of inflammation, there was loss
of cilia, and atrophy and loss of gob!et cells
in the mucous segments of the mucosat
epithelium. In both mucous and olfactory
areas there was swelling and vacuolation
of the epithelium, loss of cell cohesiveness,
replacement of preexisting epithelium by
proliferating undifferentiated basa!oid
cells, and occasional squamous metap!asia.
The basaloid cell hyperplasia involved both
surface epithelium and under!ying g!ands,
with formation of irregular downgrowths
into underlying lamina propria and oc-
casional replacement of glands. There was
scattered individual epithe!ia! cell necrosis
and occasional erosion and ulceration of
the mucosal epithelium.
In several tortoises, an exudate com-
posed of necrotic cellular debris and het-
erophi!s partially filled the nasal passages.
Other changes observed in varying de-
grees were congestion and edema of the
lamina propria and deeper connective tis-
sue, and fibrosis of the lamina propria.
Scanning electron microscopical study
of the nasal cavity mucosa of one ill tortoise
revealed numerous pleomorphic organ-
isms on the cell surface (Fig. 5). Occasion-
ally, organisms were also seen to form
chains. Examination of nasal cavity mu-
cosa of four ill tortoises by transmission
electron microscopy revealed pleomorphic
organisms closely associated with cell
membranes of surface epithelial cells (Fig.
6A, B). These organisms often were situ-
ated in part between microvilti. The or-
ganisms lacked cell walls and measured
from 350 to 900 nm. Ultrastructurally, the
organisms were consistent with members
of the genus Mycoplasrna. No similar or-
ganisms were seen on the mucosal surface
of the nasal cavities of the two controls
examined.

JACOBSON Er AL-UPPER RESPiRATORY DISEASE OF DESERT TORTOISES 305
FIGURE 4. Photomicrograph of the olfactory epithelium of a desert tortoise with URTD. A. The normal
multilayered arrangement of epithelial cells is replaced by proliferating undifferentiated cells, with infiltrates
of lymphocytes, histiocytes, and heterophils. H&E. Bar =30 sm. B. At a higher magnification histiocytes
with a vacuolated cytoplasm (arrows) and heterophils (arrow heads) are seen within the mucosal epithelium.
H&E. Bar =60 Mm.

FIGURE 5. Scanning electron photomicrograph of the nasal cavity mucosa of a desert tortoise with URTD.
Pleomorphic organisms are diffusely seen on the mucosal surface. Bar =5 Mm.
FIGURE 6. Transmission photomicrographs of the nasal cavity mucosa of a desert tortoise with URTD.
Intimate associations between organisms and host cell membranes can be seen. A. Bar =1,000 nm; B. Bar
=400 nm.
306 JOURNAL OF WILDLIFE DISEASES. VOL. 27, NO. 2, APRIL 1991
Histological examination revealed two
Group I tortoises had focal proliferations
of the epithelium in the trachea and bron-
chi. Nodules observed within the right lung
lobe of one tortoise were composed of ma-
ture granulomas with caseous centers.
The thyroid of one Group I tortoise mea-
sured 2 by 1 cm, and was larger than thy-
roids of controls (0.7 to 1.2 cm by 0.7 to
0.8 cm). This thyroid consisted of prolif-
erating follicular epitheliat cells and welt-
developed follicles with abundant co!loid.
This was interpreted to be a cotloid goiter.
The thymus of only two of 12 Group I
tortoises could be located, and required
acetic acid digestion of surrounding tissues
for visualization. The thymus of these tor-
toises appeared to be smaller than those of
healthy tortoises. Since acetic acid diges-
tion was utilized for visualization, accurate
measurements could not be made. The
cortex and medullary areas of these thy-
muses appeared to be more densely packed
with thymocytes and the medullary area
contained fewer epithetial and myoid cells
compared to those of controls.
Gross examination revealed no remark-
able differences between spleens of Group
I and controls. However, microscopic ex-
amination revealed spleen of Group I tor-
toises contained less dense collections of
lymphocytes around the periarteriolar and
periellipsoidal vessels. In addition, the red
pulp of the ill tortoises had increased num-
bers of lymphocytes in the sinusoids.
With H&E staining, golden brown gran-

JACOBSON ET AL-UPPER RESPIRATORY DISEASE OF DESERT TORTOISES 307

308 JOURNAL OF WiLDLIFE DISEASES, VOL. 27, NO. 2, APRIL 1991
TABLE 5. Microbial isolates from respiratory tract of Group I and Group II desert tortoises.
Organism
Group I Grou p II
CH’ NC’ TR’ LU’ CH NC TR LU
Staphylococcus sp. 6/8b ND 2/8 1/8 3/4 0/4 1/4 0/4
Streptococcus sp. 3/8 ND 0/8 1/8 1/4 0/4 0/4 0/4
Corynebacterlum sp. 1/8 ND 0/8 0/8 0/4 0/4 0/4 0/4
Bacillus sp. 6/8 ND 2/8 0/8 0/4 0/4 0/4 0/4
Aeromonas hydrophlla 1/8 ND 1/8 1/8 1/4 0/4 1/4 0/4
KIebsiella oxytoca 1/8 ND 0/8 0/8 0/4 0/4 1/4 0/4
Enterobacter sp. 1/8 ND 0/8 0/8 0/4 0/4 1/4 0/4
Serratla sp. 0/8 ND 0/8 0/8 1/4 0/4 0/4 0/4
Pseudomonas sp. 0/8 ND 0/8 0/8 1/4 0/4 0/4 0/4
Pa,steurella testudlnis 1/8 6/6 0/8 0/8 1/4 1/4 0/4 0/4
Escherlchla coli 0/8 ND 1/8 2/8 0/4 0/4 0/4 0/4
CH, choanae; NC, nasal cavity; T, trachea; L, lung.
No. positive/No, cultured.
Not determined.
ules were seen within hepatocytes and
Kupffer cells throughout the livers of ill
tortoises. In livers of healthy tortoises, few-
er granules were seen. By the Prussian Blue
method, most of these granules stained
positive for iron.
Microbial investigations
Aerobic bacterial isolates from Groups
Iand II are presented in Table 5. A variety
of gram-positive and gram-negative bac-
teria were isolated from the nasal pas-
sageways of Group I tortoises. While Pas-
teurella testudinis was cultured from the
nasa! cavity of all tortoises sampled in this
group, it was only isolated from a choanal
swab of one tortoise. Compared to the URT,
fewer bacterial organisms were isolated
from the trachea and lungs of Group I
tortoises.
Few bacteria were isolated from the
choanae, nasa! cavities, tracheas and lungs
of controls. Pasteurella testudinis was iso-
!ated from the nasal cavity of one control.
Viruses and Chiamydia spp. were not
isolated from any of the tissues evaluated
from Group I and II tortoises.
Microorganisms resembling Mycoplas-
ma sp. were isolated from one tortoise in
Group I, the one Group III tortoise, and
from two tortoises in Group IV. All isola-
tions were made in SP4 medium at 30 C.
These isolates were relatively slow growers
(7 to 14 days) on primary isolation and
fermented glucose. Passage in antibiotic-
free medium did not cause the isolates to
revert to bacterial colonies. By electron mi-
croscopy, the microorganism was found to
measure 350 to 900 nm and lacked a cell
wall (Fig. 7A, B). The morphology and size
of the microorganism was consistent with
the microorganism seen by electron mi-
croscopy on the mucosal epithelium of the
nasal cavity of tortoises with URTD.
DISCUSSION
The URTD of free-ranging desert tor-
toises in the Mojave Desert was clinically
similar to that described for captive desert
tortoises (Snipes et at., 1980) and a variety
of captive tortoises imported into England
(Lawrence and Needham, 1985). We have
seen this disease also in the following cap-
tive tortoises submitted to the Veterinary
Medical Teaching Hospital, University of
Florida (Gainesville, Florida 32610 USA):
red-footed tortoises (Geochelone carbona-
na), leopard tortoises (Geochelone par-
dalis), Indian star tortoises (Geochelone
elegans), radiated tortoises (Geochelone
radiata) and gopher tortoises (Gopherus
polyphemus).
Anecdotal observations on captive des-
ert tortoises suggest that this disease is

I
.4,.
4, rr-
JACOBSON ET AL-UPPER RESPIRATORY DISEASE OF DESERT TORTOISES 309
s
 T
_____
FIGURE 7. A. Ultrastructural appearance of Mycoplasma isolated from a desert tortoise. Bar = 1,000 nm.
B. At a higher magnification, no cell wall can be seen. Bar = 500 nm.

310 JOURNAL OF WiLDLIFE DISEASES, VOL. 27, NO.2, APRIL 1991
highly infectious in nature (Rosskopf,
1988). In the DTNA and adjacent areas,
large numbers of desert tortoises became
ill and died in a relatively brief period of
time (Knowles, 1989). A similar decline in
desert tortoises has been seen on the Beaver
Dam slope of Arizona/Utah (USA), and
tortoises ill with URTD have been found
at this site (M. N. Coffeen, pers. comm.).
Because most tortoises affected and dying
of this disease are reproductive adults, the
consequences for severely affected popu-
lations may be disastrous. In addition, re-
cruitment of juveniles into the adult pop-
ulation of the western Mojave Desert, are
further compromised because of raven
predation (K. H. Berry, pers. comm.). Since
the desert tortoise is one of the longest lived
terrestrial vertebrates, requiring 12 to 20
yr to reach reproductive age in free-rang-
ing females (Woodbury and Hardy, 1948)
and producing small numbers of eggs in a
clutch (Turner et al., 1986), we cannot ex-
pect to see recovery of severely affected
populations in our life-time. As such on 2
April 1990, the Federal Government of the
United States listed desert tortoise popu-
lations north and west of the Colorado Riv-
er as threatened.
In desert tortoises and other tortoises,
the disease is seen as a rhinitis character-
ized by an intermittent serous discharge
flowing or bubbling from the nares. Tor-
toises will “wipe” the discharge using the
cranial surface of their forelimbs and the
surfaces may appear moist. On days where
a discharge is not observable, the nares will
appear dry. Captive tortoises in the early
stages of the disease can maintain a good
appetite.
As the disease progresses, the discharge
becomes more tenacious and contains large
numbers of inflammatory cells, desqua-
mated epithelial cells plus myriads of bac-
teria. In several free-ranging ill desert tor-
toises examined as part of this study, the
nasal passageways were occluded with ex-
udate, and the nares were dry. In the field,
when viewed at a distance, these tortoises
might be considered healthy since no dis-
charge would be seen draining from the
nares. Tortoises must be examined closely
to determine the patency of the flares.
Severely affected tortoises appear un-
healthy. The soft and hard integument may
appear dull. There may be palpebral ede-
ma and the globes may be recessed into
the orbits, indicating dehydration. Tor-
toises may appear listless and be anorectic.
The average duration of illness in free-
ranging desert tortoises in unknown. One
of the ill tortoises in Group I was first seen
with a nasal discharge in August 1988.
Clearly this disease is chronic in nature,
lasting up to 1 yr before tortoises eventu-
ally die. In captivity, ill tortoises may sur-
vive for several years before succumbing
to systemic disease. In our experience and
the experience of others (Rosskopf, 1988),
no antibiotic or combination of antibiotics
has been useful in successfully treating
captive tortoises with this disease.
The percentage of tortoises which re-
cover after showing clinical signs of illness
is unknown. During a survey conducted at
the DTNA and surrounding areas in the
spring of 1989, several dying tortoises were
found (Knowles, 1989). In all 627 carcasses
were found in 11 sampled areas. One per-
manent long-term trend plot was sampled
in 1989, and of 162 tortoises identified, 40
were dead (K. H. Berry, pers. comm.), sug-
gesting that about 25% of the tortoises
marked on the trend plot died within 1 yr.
Eighty-four percent of the carcasses were
in the sub-adult and adult age-size classes.
Obviously, tortoises, many of which are
>50 yr of age, were suddenly dying.
Hemoglobin concentrations for tortoises
ill with URTD were significantly lower
than those of controls. In domestic animals,
anemia is often a secondary response fol-
lowing or associated with disease (Coles,
1986). Infectious agents such as blood par-
asites, bacteria and viruses may all result
in excessive destructive or shortened
erythrocyte life span. The anemia in the
desert tortoises with URTD was probably
secondary to the chronic inflammation.
Liver iron values of ill tortoises were

JACOBSON ET AL-UPPER RESRRATORY DISEASE OF DESERT TORTOISES 311
significantly higher than those of controls.
Iron probably accumulated in the livers of
chronically ill tortoises as a consequence
of red blood cell breakdown and inability
to reutilize iron released from hemoglobin.
Hepatic hemosiderosis has been reported
in mammals with hemolytic anemia and
cachexia (Kelly, 1985). One of us (ERJ)
has seen many cases of hemosiderosis in
numerous species of recently imported ca-
chectic reptiles.
Serum sodium, BUN, SGOT activity,
and cholesterol were significantly higher
and phosphorus significantly lower for ill
tortoises compared to controls. Increases in
plasma BUN have been reported in cap-
tive hibernating Mediterranean tortoises
(Testudo graeca and T. herrnanni) (Law-
rence, 1987) and in Texas tortoises (Go-
pherus berlandieri) and desert tortoises
which were experimentally dehydrated
(Dantzler and Schmidt-Nielsen, 1966; Baze
and Home, 1970). In Texas tortoises, the
activity of the urea cycle enzyme, argi-
nosuccinate synthetase, also increased upon
dehydration and fasting; this probably ac-
counted for the increased urea production.
During dehydration and fasting, a switch
to greater protein catabolism may explain
the increase in activity of arginosuccinate
synthetase.
Ultimately, urea production may assist
in water conservation by elevating plasma
osmolality, thereby reducing water loss
through the soft integument and increas-
ing water uptake from the bladder. In the
desert tortoises with URTD, the increased
serum BUN and sodium may have been a
response to lack of water and inadequate
nutrient availability during the period be-
tween emergence and collection in late
May. From October to December, 1988,
there was only 25.6 mm of rain in Rands-
burg, approximately 6.3 km east of the
DTNA, and this was inadequate in spring
1989 for germination of annual plants nor-
mally eaten by desert tortoises (K. H. Ber-
ry, pers. comm.). Further, since ill animals
have higher caloric needs than healthy an-
imals (Selivanov and Sheldon, 1984), if nu-
trients are initially a compromising factor
in the health status of tortoises, then once
a tortoise becomes ill, it probably becomes
further compromised.
Serum cholesterol of ill tortoises was sig-
nificantly greater than that of controls.
Hypercholesterolemia has been seen in po-
nies and rabbits which were fasted exper-
imentally (Aladjem and Rubin, 1954;
Bauer, 1983). Although the exact mecha-
nism for elevated blood cholesterol con-
centrations in these animals is unknown,
mobilization of lipids from adipose tissue
is probably involved. The elevated serum
levels of cholesterol in the ill desert tor-
toises may similarly be a response to star-
vation and an attempt to meet energy needs
through lipid metabolism. Some investi-
gators have suggested that stored fats are
a source of both energy and water in tor-
toises (Woodbury and Hardy, 1948; Khalil
and Abdel-Messeih, 1962).
Glutamic oxalacetic transaminase is an
enzyme which is produced at multiple tis-
sue sites. Elevated serum levels of this en-
zyme reflect tissue damage (Coles, 1986).
Activity for captive desert tortoises ranges
from 10 to 100 IU/L (Rosskopf, 1982). In
the present study, SGOT activity for ill
tortoises was significantly elevated above
that of controls. Whether this is a response
to specific or diffuse tissue damage is un-
known.
Fowler (1980) believed that vitamin A
deficiency was a predisposing factor in
URTD of desert tortoises since squamous
metaplastic changes were seen in captive
desert tortoises with this disease. Squamous
metaplastia of mucosal epithelium was not
a major component of the disease in tor-
toises in the current study and it was con-
sidered a secondary response to the chronic
inflammation in the involved tissues. Fur-
thermore, serum and liver vitamin A val-
ues were not significantly different be-
tween ill and control tortoises. Hence
vitamin A does not appear to be a factor
in the pathogenesis of URTD.
Vitamin E, selenium, cadmium, copper
and lead values in liver of ill tortoises were

312 JOURNAL OF WILDLIFE DISEASES, VOL. 27, NO. 2, APRIL 1991
similar to those of controls. However, liver
mercury values (0.326 ppm) were signifi-
cantly greater than those of controls (0.0287
ppm). Although these values were below
those considered toxic for mammals (Kol-
ler et a!., 1977), several investigators have
reported altered host resistance to patho-
gens (Koller, 1975; Gainer, 1977), de-
pressed antibody responses to mitogen
stimulation (Koller, 1973; Koller et a!.,
1977), and thymic cortex and splenic fol-
licular atrophy with concomitant depres-
sion of 1gM and IgG antibody response to
mitogen stimulation (Blakley et al., 1980)
in rodents exposed to sublethal amounts of
mercury. The thymus could only be found
in two of 12 ill tortoises examined, where-
as, it was easily observed in all four con-
trols. Since malnutrition has also been re-
ported to result in thymic involution and
secondary infectious disease in turtles
(Borysenko and Lewis, 1979), it is not pos-
sible to determine if mercury or other fac-
tors were responsible to thymic atrophy in
the ill desert tortoises. Additional studies
are needed to identify the source and sig-
nificance of mercury in the desert tortoises
in the DTNA.
The illness in free-ranging desert tor-
toises appears to primarily involve the
URT, with minimal involvement of the
LRT. Since moribund tortoises were not
evaluated, it is unknown what ultimately
kills these animals. Possibly, systemic in-
vasion of opportunistic organisms may be
an end-stage event in severely debilitated
tortoises. In addition since ill tortoises ap-
pear to be in a negative nitrogen and en-
ergy balance, they may eventually die of
cachexia.
The size range and ultrastructural ap-
pearance of the pleomorphic organism on
the nasal mucosa suggested it was Myco-
plasma sp. (Razin, 1981). Mycoplasma sp.,
although suspected as a potential cause of
URTD of Mediterranean tortoises (Law-
rence, 1987), has not been isolated previ-
ously from tortoises with URTD. Recently,
Hill (1985) isolated Mycoplasma testudi-
nis from the cloaca of a Mediterranean
tortoise (T. graeca).
Organisms forming colonies typical of
Mycoplasma sp. were isolated from the
URT of four ill desert tortoises. Electron
microscopic evaluation of one of the iso-
lates confirmed it to be identical in size
and morphology to the Mycoplasma sp.
demonstrated in tissue section. Still, im-
munohistochemical staining will have to
be performed to clearly establish the iden-
tity of the organism seen in tissue section.
No attempts have been made to isolate
Mycoplasma sp. from clinically healthy
tortoises, and we do not know if a carrier
state exists in these animals.
Chlamydia or viruses were not isolated
from the URT of ill tortoises. If a virus is
involved in URTD of tortoises, it probably
would only be present in abundance in the
early stages of the disease. We have worked
on other diseases of reptiles which initially
commence as a viral infection and after a
relatively brief period of time become
complicated by secondary bacterial infec-
tion (Jacobson et al., 1986). Further viral
isolation attempts need to be conducted on
desert tortoises with URTD.
While a large number of gram-negative
and gram-positive bacteria were isolated
from nasal cavities of ill tortoises, relative-
ly few organisms were isolated from those
of healthy tortoises. The only bacterial or-
ganism consistently isolated from ill tor-
toises was Pasteurella testudinis; it was
also isolated from one healthy tortoise.
Pasteurella testudinis was originally iso-
lated from desert tortoises ill with URTD
and from healthy captive tortoises, and
from free-ranging tortoises from the Mo-
jave Desert in southern California (Snipes
et al., 1980; Snipes and Biberstein, 1982).
However, the disease could not be trans-
mitted experimentally using isolates of P.
testudinis in healthy desert tortoises.
Necrosis of the surface tissues and ab-
scess formation, which are commonly seen
in Pasteurella infections of mammals, were
not seen in URTD of desert tortoises. At-
rophy of nasal turbinates, with necrosis of

JACOBSON ET AL.-UPPER RESPIRATORY DISEASE OF DESERT TORTOISES 313
supporting bone, seen in rabbits with P.
multocida infection (DiGiacomo et al.,
1989) was not observed in tortoises. How-
ever, strains of P. testudinis with varying
pathogenicity may exist in desert tortoises
and maybe acting synergistically with an-
other organism.
Mycoplasmas are the etio!ogic agents of
respiratory disease in a number of hosts,
including humans (Mycoplasma pneu-
moniae), pigs (M. hyopneumoniae), cattle
(M. mycoides subsp. mycoides, M. bovis.
M. dispar, Urea plasma diversum), labo-
ratory rodents (M. pulmonis) and poultry
(M. gallisepticum, M. melagridis, M. io-
wae) (Casell et a!., 1985). With the excep-
tion of M. mycoides subsp. mycoides, the
mycoplasmas associated with respiratory
disease produce chronic, slowly progress-
ing infections which are frequently mu!-
tifactorial. In many cases, these pathogens
resemble opportunistic infections because
they can be isolated from seemingly nor-
mal carriers. However, studies with gno-
tobiotic animals have demonstrated their
pathogenic potential in the absence of oth-
er agents. In several cases, the interaction
between mycoplasmas and other infec-
tious agents is synergistic (Cassell et a!.,
1985; Gourlay and Houghton, 1985; Schoeb
et a!., 1985; Weinach et a!., 1985). In these
instances, the mycoplasma appears to be
the initial colonizer of the respiratory tract
and may predispose the host to secondary
agents.
Although the pathogenic mechanisms
are not fully defined, a concept for patho-
genesis has been proposed which is rele-
vant to most mycop!asmal infections (Cas-
sell et a!., 1985). Disease is dependent upon
attachment and association of the myco-
plasma with the host surface, elicitation of
an immune response which may be det-
rimental as well as beneficial to the host,
and interaction with the host immune sys-
tem which may enhance chronicity. At-
tachment to host tissue may require spe-
cific receptors on the host surface as well
as adhesins on the mycoplasma. To date,
only the P1 protein of M. pneumoniae has
been fully characterized (Su et a!., 1987).
Attachment may or may not involve a spe-
cialized attachment tip (Casse!! et a!.,
1985). Electron micrographs of the tortoise
isolate suggest that the mycoplasmas iso-
lated from diseased desert tortoises possess
a putative attachment structure. Once at-
tached to the host, mycop!asmas may cause
ce!l injury by a variety of methods, in-
cluding but not limited to ci!iostasis, hy-
drogen peroxide production, and toxin
production (Cassell et al., 1985; Gabridge
et al., 1985). It has been suggested that the
host defense system may influence lesion
development. Many mycoplasmas are
nonspecific mitogens, both for B and T
cells (Cole et a!., 1985). Recent evidence
suggests that M. pulmonis can act as both
a chemotactic and a chemokinetic agent
in addition to its role as a mitogen (Ross
et a!., 1989).
Predisposing factors are very likely in-
volved in the development and spread of
this disease in free-ranging populations.
Released, previously captive desert tor-
toises with URTD have been found on the
DTNA and elsewhere within the geo-
graphic range of the desert tortoise. Pos-
sibly, an extremely pathogenic organism
has been introduced into wild populations
at multiple sites. Since captive desert tor-
toises are often exposed to other pet tor-
toises, possibly a pathogenic organism has
been introduced by contact. We have re-
cently isolated Mycoplasma sp. from a
Greek tortoise (Testudo graeca) and leop-
ard tortoises (Geochelone pardalis) with
rhinitis. Furthermore, desert tortoises along
roads are collected commonly by local cit-
izens, tourists, and other travelers, and may
ultimately be released at other sites. The
ease with which tortoises can be collected
and transported probably have contrib-
uted to the spread of the disease. This
problem should be managed through mas-
sive education, within public schools, on
radio and television, and through litera-
ture given to travelers in airports, at road-
side rest areas, and border agricultural
check stations.

314 JOURNAL OF WiLDLIFE DISEASES, VOL. 27, NO.2, APRIL 1991
Since nutritional deficiency diseases are
known to result in immunosuppression
(Miller, 1985; Nockels, 1988; Ul!rey, 1986)
and important infections in human pop-
ulations are made more severe by the pres-
ence of malnutrition (Selivanov and She!-
don, 1984), habitat degradation and
reduction in quality of forage must be con-
sidered as another potential predisposing
factor in the severity and spread of this
disease. It may be that domestic animals,
by their grazing, are altering the phys-
ical environment and structure of plant
and animal communities of the Mojave
Desert (Webb and Stielstra, 1979). Since
desert tortoises are selective feeders, con-
suming annual forbs, annual and perennial
grasses, cacti, and on rare occasions some
other items (K. H. Berry, pers. comm.),
perhaps necessary nutrients are no longer
available in the DTNA. Additionally, it
seems unlikely that desert tortoises can
compete with domestic animals for the
same food resources. In one study, ap-
proximately 60% of the biomass of annuals
were consumed by sheep grazing on a study
plot in one day (Webb and Stielstra, 1979).
Superimposed are climatic factors which
influence plant production. Studies of rain-
fall patterns in the Southwestern U.S.
(Phillips et a!., 1984), indicated that au-
tumn rainfall in the Mojave Desert has
never been as great as it was during the
1940’s. Hastings and Turner’s (1965) anal-
yses of long-term changes of vegetation in
southeastern Arizona and Mexico showed
that profound changes in the vegetational
communities of this region have occurred
since the 1880’s. In considering causes, they
believed that a combination of climatic
and cultural stress was responsible. Long-
term studies will have to be done to cor-
relate changes in plant structure, i.e.,
changes in nutrient availability to desert
tortoises, with population dynamics of this
species. Since malnutrition is known to
cause thymic atrophy and immuno-
suppression in turtles (Borysenko and Lew-
is, 1979), structural and functional studies
of the immune system need to be done to
identify the interplay between forage
quality, immune status and URTD of des-
ert tortoises. In this way the pathogenesis
of URTD of desert tortoises may be better
understood.
ACKNOWLEDGMENTS
The authors of this report would like to thank
the fo!!owing individuals for information pro-
vided and scientific support for various aspects
of this study: Bobby Collins, College of Veter-
inary Medicine, University of Florida, Gaines-
ville, Florida, USA; Kristin Berry, Bureau of
Land Management, Riverside, California, USA;
Michael Weinstein, SAIC, Santa Barbara, Ca!-
ifornia, USA; Robert Benton, U.S. Fish and
Wildlife Service, Salt Lake City, Utah, USA;
Michael Coffeen, St. George, Utah, USA; James
Tappe, National Animal Disease Center, Ames,
Iowa, USA; and John Jenkins, Armed Forces
Institute of Pathology, Washington, D.C., USA.
This study was supported by the California De-
partment of Fish and Game and U.S. Bureau of
Land Management through contract No. CA
950-CT9-28 (BLM, California Desert District,
Riverside, California, USA). Published as Uni-
versity of Florida, Agricultural Experiment Sta-
tion Number R-00801.
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