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

Chronic upper respiratory tract disease of free-ranging desert tortoises (Xerobates agassizii).

Seventeen desert tortoises, Xerobates agassizii, with upper respiratory tract disease were examined; thirteen were euthanatized for necropsy. Four nor...
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