article #5 ce diagnostic techniques and sample...

There is an increasing expectation forhigh-quality medicine for reptiles,whether they are pets or in a zoologic

institution. Unfortunately, there are very fewpathognomonic signs in reptiles; thus cliniciansmust rely on various diagnostic tests to obtain adefinitive diagnosis and instigate effective casemanagement. Inappropriately collecting sam-ples complicates matters by rendering resultsinvalid. Therapy based on poor laboratory datais often futile or transiently beneficial. Severalreviews have summarized diagnostic samplecollection from reptiles.1–4

Reptiles, like all other animal groups, cancause disease in humans. We do not believe thatthe risks of reptile-borne zoonoses are anygreater than those of other animal groups, andcareful attention to basic personal hygiene afterhandling patients or their biologic products

reduces these risks. Appropri-ate personal hygiene (e.g.,handwashing with soap andwater) and good laboratorypractice (e.g., no eating or

Article #5

ABSTRACT:

CE

Email comments/questions to

[email protected],

fax 800-556-3288, or log on to

www.VetLearn.com

Making a definitive diagnosis can be difficult when dealing with reptiles, which show fewpathognomonic signs and often have a chronic disease progression. Many clinicians havedifficulty making the transition from mammal to reptile diagnostics, although much mam-mal research applies to reptiles.This article is a basic guide to collecting biologic samplesthat can greatly assist in obtaining elusive diagnoses in reptiles.

drinking) are important in reducing all zoonoticrisks. There are also legal requirements, such asthose of the Occupational Safety and HealthAdministration, to protect employees. There-fore, all facilities that care for reptiles shouldhave written guidelines, appropriately trainedpersonnel, and adequately monitored proce-dures to ensure safety.

LABORATORY EQUIPMENT ANDSUPPLIES

The decision to develop an in-house labora-tory or solely use an external commercial labo-ratory should be based on the volume of workanticipated, cost analysis, quality control, andpersonal preference. The advantage of an in-house laboratory is essentially speed, whereascommercial laboratories can provide moreextensive service with greater accuracy andquality assurance. Basic quality-control mea-sures must also be practiced in every in-houselaboratory. The ability to conduct some basicinvestigations in a practice can be very reward-ing, and much can be accomplished with a few

COMPENDIUM 470 June 2004

Diagnostic Techniques andSample Collection in Reptiles

Stephen J. Hernandez-Divers, BSc(Hons), BVetMed,DRCVS ZooMed, CBiol, MIBiol, MRCVSUniversity of Georgia

John E. Cooper, DTVM, FRCPath, FIBiol, FRCVSStephen W. Cooke, BVSc, MRCVSGreendale Laboratories Ltd, Woking, Surrey, United Kingdom

Diagnostic Techniques and Sample Collection in Reptiles 471CE

essentials. The ability to examine wet preparations ofdiagnostic material is important. A compound, binocu-lar microscope with achromatic (economic) objectivesas well as appropriate cytology materials should beconsidered absolutely necessary. Phase contrast isexpensive, but a polarizer may help discern uric acidcrystals. Other investigations may be easily conductedwith this basic microscope, often leading to betterpatient assessment and greater clinical satisfaction (seebox on this page).

SAMPLE COLLECTION AND PROPERSUBMISSION

Successfully treating reptiles depends on making acorrect diagnosis. This requires samples that have beenappropriately collected at the proper time and submittedfor tests that have been correctly interpreted. Submit-ting abnormal and normal tissue (from the periphery ofthe lesion) is preferable. By far, the best samples arethose taken during the early stages of disease. The tim-ing of sample collection is of utmost importance. Some

June 2004 COMPENDIUM

Compound, Binocular Microscope• Achromat (economical) lenses

(×10, ×40, and ×100 oil immersion)• Phase contrast or a polarizer• Microscope slides• Coverslips• Mounting fluid

Cytologic Stains• Romanowsky

— Wright’s— Leishman’s— Giemsa— Combination stains (Wright-Leishman,

May-Grünwald Giemsa)• Rapid modifications of Romanowsky stains

— RapiDiff— Diff-Quik

• Gram’s stain reagents • New methylene blue stain

Microbiology Swabs• Standard tip (dry tubes)• Standard tip (bacterial and fungal transport media)• Microtip culturettes (bacterial and fungal

transport media) • Virology transport swabs and media

Blood Tubes • Heparin-separator gel tubes (0.6 ml) • Heparin tubes (0.6 ml) • Serum-gel separator tubes (0.6 ml) • Plain tubes of various sizes (0.6, 4, and 6 ml) • EDTA tubes (0.5 ml)• Pediatric blood culture tubes

Biochemical Equipment • Urinalysis strips

— SG10• Biochemistry analyzer capable of determining uric and

bile acids• Electrolyte analyzer

Equipment and Suppliesa

Centrifuge (capable of taking both microhematocritand sample vials)Hematology Equipment • Heparinized and plain hematocrit tubes • Hematocrit reader• Hematocytometer (preferably with improved

Neubauer ruling) • Diluents

— Rees-Ecker solution— Saline— Natt-Herrick solution

Diagnostic Endoscopy Equipment• Nova xenon light source (175 watts)• Light guide cable (3.5 mm × 230 cm)• Veterinary video camera II• Medical grade monitor• Insufflator (aquarium air pump or

carbon dioxide endoflator) and insufflation line• Hopkins telescope (2.7 mm × 18 cm, 30˚)• Operating sheath (14.5 Fr; 5-Fr instrument channel)• Examination and protection sheath (3.5-mm outside

diameter)• Biopsy forceps (5 Fr × 34 cm)• Grasping forceps (5 Fr × 34 cm)• Scissors (single action; 4 Fr × 34 cm)• Injection/aspiration needle (flexible with Teflon guide)Biopsy Devices• Tru-Cut or Biocut (Global Medical Instrumentation)

biopsy needles (14–20 gauge)• Spinal needles (14–25 gauge)• Bone biopsy and aspiration bone marrow needles

(14–18 gauge)• Michelle trephine or Jamshidi bone biopsy instrument

for large bone or shell biopsiesUniversal Containers, 10% Buffered Formal Saline, and

70% Ethanol

Dedicated Refrigerator and Freezer

Digital Camera (for documenting lesions and diseases)aReadily available from standard laboratory distributors.

COMPENDIUM June 2004

Diagnostic Techniques and Sample Collection in Reptiles472 CE

therapies can alter the pathologic picture (e.g., theeffects of exogenous steroids on tissue inflammation),whereas others (e.g., antimicrobials) may invalidatemicrobiologic results.

Some samples (e.g., blood, superficial skin samples)can be taken from conscious animals; however, mostanimals require some form of chemical restraint. It isimportant to appreciate that both physical and chemicalrestraint may induce artifacts, including changes inhematologic and biochemical values that may be mis-leading (e.g., increased creatinine kinase values, changesin blood glucose, stress leukograms, hemoconcentra-tion). In most cases, the detrimental effects of handlingconscious reptiles far outweigh the minor risks of anes-thesia.5 Inappropriately handling blood (e.g., using smallneedles and certain anticoagulants, shaking blood con-tainers) can also lead to hemolysis, causing inaccuratehematologic and biochemical results.

Inexperienced clinicians may lack confidence with unfa-miliar exotic species and may rely too much on commer-cial laboratories for the correct diagnosis. Certain labora-tories are known for their competence with exotic animalsand have an experienced veterinary staff to help clinicianswith cases. However, a laboratory interpretation is only asgood as the information provided with the sample; thusclinicians should consider providing their laboratory witha copy of the case records, including the following:

• A complete history, including details about hus-bandry and nutrition

• Details from the clinical examination

• An accurate description of lesions and, if possible, aprinted digital photograph

This information is immensely helpful and dramaticallyimproves the service that laboratories provide.

BLOODBlood volumes in reptiles vary from approximately 4%

to 8% of body weight. In general, 0.5 ml of blood can besafely collected from a healthy 100-g reptile. One to twodrops of blood can be obtained from a 30-g anole forhematocrit estimation and a blood smear. Collecting anonhemolyzed blood sample using an atraumatic tech-nique is essential for meaningful hematology and bio-chemical data. Unfortunately, venipuncture is generallya blind technique in reptiles, although skilled techni-cians can often raise the jugular vein in some tortoises(Figure 1). In all cases, anatomic knowledge of the posi-tion of veins is vital (Table 1). Heparin and heparin gelseparator tubes, microscope slides, povidone–iodine/alcohol gauze, and appropriately sized needles andsyringes should be prepared for use. It is essential toaseptically prepare the skin because postinjection infec-tion may result from skin contamination from fecal bac-teria.4 Aseptic preparation of the skin is especially vitalwhen conducting a blood culture.

Blood for hematology should generally be collectedinto heparin tubes because EDTA can lyze erythrocytes

Figure 1. Blood collection.

Blood collection from the caudal vein of a conscious green iguana(Iguana iguana) restrained using a towel.

Blood collection from the left jugular vein of an adult Africanspurred tortoise (Geochelone sulcata).This adult was consciousbut so debilitated that its head and neck were easily extendedwithout the usual need for sedation.

Tabl

e 1.

Blo

od

Co

llect

ion

fro

m R

epti

lesa

Sit

eL

oca

tio

nP

roce

dure

Snak

es

Ven

tral

coc

cyge

al (t

ail)

vein

Cau

dal t

o c l

oaca

,app

roxi

mat

ely

25%

–75%

dow

n th

e ta

il,av

oidi

ng th

e he

mip

enes

of m

ales

The

nee

dle

shou

ld b

e an

gled

at 4

5˚–9

0˚ (c

rani

odor

sal)

and

plac

ed a

t the

ven

tral

mid

line.

A 1

6- to

25-

mm

,22

- to

25-

gaug

e ne

edle

shou

ld b

e ad

vanc

ed w

hile

mai

ntai

ning

slig

ht n

egat

ive

pres

sure

.If t

he n

eedl

e hi

ts a

vert

ebra

l bod

y,w

ithdr

aw it

slig

htly

whi

le m

aint

aini

ng sl

ight

neg

ativ

e pr

essu

re a

nd re

dire

ct it

if n

eces

sary

.

Car

dioc

ente

sis

At a

poi

nt th

at is

20%

–33%

of

the

snou

t to

vent

leng

thT

he s

nake

sho

uld

be s

edat

ed o

r ane

sthe

tized

and

rest

rain

ed in

dor

sal r

ecum

benc

y.It

s he

art s

houl

d be

palp

ated

and

imm

obili

zed

betw

een

the

thum

b an

d fo

refin

ger.

A 1

6- to

36-

mm

,22-

to 2

5-ga

uge

need

lesh

ould

be

adva

nced

cra

niod

orsa

lly a

t 45˚

into

the

beat

ing

vent

ricl

e.B

lood

oft

en e

nter

s w

ith e

ach

hear

tbea

t.R

isks

incl

ude

card

iac

cont

usio

n,la

cera

tion,

and

hem

orrh

age.

Liz

ards

Ven

tral

coc

cyge

al (t

ail)

vein

Cau

dal t

o cl

oaca

,app

roxi

mat

ely

25%

–75%

do w

n th

e ta

il,av

oidi

ng th

e he

mip

enes

of m

ales

A 1

6- to

25-

mm

,22-

to 2

5-ga

uge

need

le sh

ould

be

angl

ed a

t 60˚

–90˚

and

pla

ced

at th

e ve

ntra

l mid

line

(i.e.

,25%

–75

% d

own

the

tail)

.The

nee

dle

shou

ld b

e ad

vanc

ed w

hile

mai

ntai

ning

slig

ht n

egat

ive

pres

sure

.If t

he n

eedl

e hi

ts a

vert

ebra

l bod

y,w

ithdr

aw it

slig

htly

whi

le m

aint

aini

ng sl

ight

neg

ativ

e pr

essu

re a

nd re

dire

ct it

if n

eces

sary

(Fig

ure

1).

Che

loni

ans

Jugu

lar v

ein

Lat

eral

asp

ect o

f the

nec

k,ru

nnin

g ca

udod

orsa

l fro

mbe

hind

the

tym

panu

m

A 1

6-m

m,2

3- to

25-

gaug

e ne

edle

shou

ld b

e po

sitio

ned

late

rally

,cau

dal t

o th

e ty

mpa

nic

scal

e,an

d di

rect

edca

udal

ly.T

his i

s the

blo

od c

olle

ctio

n si

te o

f cho

ice

beca

use

it av

oids

the

lym

phat

ic v

esse

ls.A

void

the

mor

eve

ntra

lly p

ositi

oned

car

otid

art

ery

beca

use

punc

turin

g it

ofte

n re

sults

in h

emat

oma

form

atio

n (F

igur

e 1)

.

Dor

sal c

occy

geal

vei

nD

orsa

l mid

line

of ta

ilA

16-

to 2

5-m

m,2

2- to

25-

gaug

e ne

edle

sho

uld

be a

ngle

d at

45˚

–90˚

and

pla

ced,

as c

rani

al a

s po

ssib

le,

at th

e do

rsal

mid

line

of th

e ta

il.T

he n

eedl

e sh

ould

be

adva

nced

whi

le m

aint

aini

ng s

light

neg

ativ

e pr

essu

re.

If th

e ne

edle

hits

a v

erte

bral

bod

y,w

ithdr

aw it

slig

htly

whi

le m

aint

aini

ng s

light

neg

ativ

e pr

essu

re a

ndre

dire

ct it

if n

eces

sary

.The

exa

ct p

ositi

on,s

ize,

and

pres

ence

of t

his

vess

el m

ay v

ary

amon

g sp

ecie

s.L

ymph

atic

con

tam

inat

ion

is n

ot u

ncom

mon

.

Subc

arap

acia

l sin

usM

idlin

e of

the

cran

iove

ntra

lca

rapa

ceT

he h

ead

can

be h

eld

exte

nded

or f

ully

retr

acte

d.A

16-

to 2

5-m

m,2

2- to

25-

gaug

e ne

edle

(ben

t to

70˚–

80˚)

shou

ld b

e in

sert

ed a

t the

mid

line

in th

e ve

ntra

l asp

ect o

f the

car

apac

e,ju

st c

auda

l to

whe

re th

e sk

in o

f the

nec

kat

tach

es to

the

cran

ial r

im o

f the

ven

tral

car

apac

e.A

dvan

ce th

e ne

edle

in a

dor

sal d

irect

ion

whi

le m

aint

aini

ng sl

ight

nega

tive

pres

sure

.The

lym

phat

ic v

esse

ls in

this

area

shou

ld b

e av

oide

d to

pre

vent

hem

odilu

tion.

Thi

s met

hod

isid

eal f

or ra

pidl

y co

llect

ing

larg

e vo

lum

es o

r col

lect

ing

sam

ples

from

neo

nate

s and

oth

er sm

all c

helo

nian

s.

a The

re a

re a

var

iety

of o

ther

blo

od c

olle

ctio

n si

tes,

incl

udin

g th

e br

achi

al a

nd fe

mor

al p

lexu

s of

che

loni

ans,

vent

ral a

bdom

inal

vei

n of

liza

rds,

and

pala

tine

vein

of s

nake

s.T

hese

site

s ha

ve b

een

omitt

ed b

ecau

se o

f inc

reas

ed r

isks

ass

ocia

ted

with

thei

r use

and

/or t

hey

prov

ide

sam

ples

that

are

infe

rior

in q

ualit

y an

d qu

antit

y.U

sing

nai

l clip

s to

col

lect

blo

od fr

om c

onsc

ious

rept

iles

is

inhu

man

e an

d pr

oduc

es p

oor-

qual

ity

sam

ples

.

Cro

codi

lians

Ven

tral

coc

cyge

al (t

ail)

vein

(sm

all s

peci

men

son

ly)

Cau

dal t

o c l

oaca

,app

roxi

mat

ely

25%

–75%

dow

n th

e ta

ilA

25-

to 7

5-m

m,1

8- to

22-

gaug

e ne

edle

shou

ld b

e an

gled

at 6

0˚–9

0˚ a

nd p

lace

d at

the

vent

ral m

idlin

e,ap

prox

imat

ely

25%

–75%

dow

n th

e ta

il.T

he n

eedl

e sh

ould

be

adva

nced

whi

le m

aint

aini

ng sl

ight

neg

ativ

epr

essu

re.I

f the

nee

dle

hits

a v

erte

bral

bod

y,w

ithdr

aw it

slig

htly

and

redi

rect

it.

Supr

aver

tebr

al v

ein

Dor

sal m

idlin

e,ju

st c

auda

l to

the

occi

put

A 2

5- to

50-

mm

,20-

to 2

2-ga

uge

need

le s

houl

d be

inse

rted

per

pend

icul

ar to

the

dors

al m

idlin

e,ju

st c

auda

lto

the

occi

put.

The

nee

dle

shou

ld b

e ad

vanc

ed to

the

supr

aver

tebr

al s

inus

(ove

rlyi

ng th

e sp

inal

can

al) w

hile

mai

ntai

ning

slig

ht n

egat

ive

pres

sure

.Lar

ger c

roco

dilia

ns s

houl

d be

sed

ated

or a

nest

hetiz

ed.B

ecau

se o

f the

loca

tion,

exce

ssiv

e ne

edle

pen

etra

tion

can

resu

lt in

spi

nal c

ord

dam

age.



in some reptile species, although heparin may cause cel-lular clumping.4 If sample size permits, submitting bothheparin and EDTA samples may be beneficial becausehematologists can make more informed decisions basedon both samples. Preparing one or more blood smearsusing fresh (nonanticoagulated) blood is essentialbecause cell morphology is often as informative as totalor differential cell counts. Smears should be made assoon as a sample has been obtained. Hematocrit tubescan be centrifuged to obtain a packed cell volume andtotal plasma solids count, whereas a blood smear can beused to obtain the following:

• An estimated total leukocyte count

• A leukocyte differential

• A leukocyte morphology assessment

• A hemoparasite evaluation

• An erythrocyte evaluation

A high level of skill and experience is required for accu-rate reptile hematology. It is prudent to check the com-

petence of technicians conducting such assessments, andsending duplicate samples to different laboratories canalso ensure confidence in the results.

For routine biochemistry, collection into heparin ispreferred because a given volume of blood yields moreheparinized plasma than serum. Separating heparinizedplasma from cellular components is essential and shouldbe done immediately; modern heparin-gel separatortubes are convenient and negate the need to decantplasma.

Blood can also be submitted for microbiologic culturewhen attempting to identify the agent responsible forvegetative endocarditis, osteomyelitis, or septicemia. Insuch situations, whole blood should be submitted indedicated media following the laboratory’s instructions.Pediatric blood culture vials are available to culture smallblood volumes. For nonroutine samples, it is advisable tocontact the laboratory for information on the samplesand volumes required for particular tests.

URINARY TRACTThe urinary system includes the kidneys, ureters,

bladder (present in many lizards and all chelonians [i.e.,tortoises, turtles, terrapins], but not in snakes), andurodeum of the cloaca. Urine passes from the kidneys,through the ureters, and into the urodeum of the cloaca,where it is redirected through the urethra and, in allchelonians and some lizards, into the bladder. In snakesand lizards that do not have bladders, urine and uratesmay be stored in distal flared ureters or within the distalcolon. Extrarenal tissues, including the bladder, distalcolon, and nasal salt glands, may also play pivotal rolesin osmoregulation. Postrenal modification (e.g., waterand electrolyte exchanges) of urine occurs in the bladderor colocloacal mucosa; therefore, unless urine is col-lected from the ureters, it is not necessarily representa-tive of renal function.

Urinalysis is less helpful in reptiles than in mammalsbecause the reptilian metanephric nephron lacks an ansanephroni (loop of Henle), making concentration of urineimpossible. In addition, most reptiles are uricotelic—they

produce more insoluble uric acid than urea. Therefore,specific gravity is generally not useful in evaluating renalfunction but may provide some composite information onboth renal and postrenal modification of urine. As urinepasses through the urodeum on its way to the bladder,even urine collected by cystocentesis may not be sterile,which limits the applicability of urine microbiology.4

In species that have bladders, urine can be collected bycystocentesis, catheterization, or simple digital stimulationof the cloaca, which often results in spontaneous urination.To avoid sample loss, clinicians should ensure that a suit-able collecting vessel is readily available. Lizards should berestrained vertically and aseptically prepared, and a 22- to25-gauge needle should be inserted in the lower coelom,avoiding the abdominal vein that lies in the ventral mid-line. Chelonians should also be restrained vertically and theneedle introduced via the prefemoral fossa. Needles areoften blocked by aspiration of semisolid uric acid, andthere is danger of postsampling leakage and coelomitis

Diagnosing disease in reptiles often requires demonstratinga host response (via cytology or histopathology) and,

if the disease is infectious, identifying the offending pathogen.

because the bladder is thinly walled and its contents areseldom sterile. Free-catch urine samples may be contami-nated by material from the intestinal and genital tracts.

Urine can be submitted for in-house reagent striptests. The flocculent and variably pigmented nature ofreptile urine, combined with a lack of reagent strip vali-dation, raises concerns about the accuracy of results.Microbiologic culture and, probably much more clini-cally important, sediment and cytologic evaluation arepreferred for reptile urine evaluation. Green to yellow(biliverdin- or bilirubin-stained) urates and urine havebeen associated with hepatopathy.4 A wet-mount sampleshould be examined for parasites immediately after col-

lection. Air-dried smears may be examined for the pres-ence of inflammatory cells, but bacterial contaminationis common and does not necessarily indicate primaryinfection.

Kidney disease is not uncommon in reptiles and,because many are uricotelic, end-stage renal failure canproduce gout. Assessing renal structure and function caninclude blood work and diagnostic imaging, but mostdiagnostic information can be obtained from renalbiopsy2,4 (Figure 2). Biopsies can be performed by thefollowing methods:

• Transcutaneous Tru-Cut biopsy using palpation orultrasound to guide the biopsy needle is most suitable



Craniolateral coccygeal approach to the caudal kidney of a greeniguana (Iguana iguana) for biopsy. The horizontal incision wasmade immediately behind the hindlimb between the dorsal andventral coccygeal muscles of the tail base.

Endoscopic examination and biopsy of an iguanid kidney.

Figure 2. Renal biopsy.

Exploratory celiotomy in a green iguana (Iguana iguana) revealed anenlarged kidney that was sampled using a Tru-Cut biopsy instrument.

Surgical biopsy of the kidney of a boa constrictor using vascularclips to isolate a renal lobule.



• Surgical biopsy following celiotomy ismore invasive but still practical insnakes and lizards (Figure 2). It is moredifficult in chelonians but still possiblevia a prefemoral, soft tissue approachto the lateral aspect of the kidney.

Biopsy samples are most valuable whenthey include both normal and abnormaltissue; therefore, visual techniques areoften more rewarding than blind ones,unless the renal changes are diffuse. Inaddition, iatrogenic damage to the kidneysand closely associated structures is greatlyincreased when blind techniques are used.Biopsy samples should be submitted in10% neutral buffered formalin for routinehistopathology (or ethanol to demonstrateuric acid and assess gout). Small biopsysamples should be placed into histologyfilters to avoid loss before evaluation, andtissue manipulation (especially the use ofrat-tooth forceps) should be relayed to thepathologist because crushing artifacts maybe apparent. Evaluation of renal functionin reptiles is still under development; how-ever, scintigraphy and iohexol excretionhave value in determining the glomerular

filtration rate, at least in green iguanas.

RESPIRATORY TRACTThe respiratory system of reptiles consists of the

nares, glottis, trachea, and lungs. Tracheal rings arecomplete in chelonians and crocodilians and incompletein snakes and lizards. Lungs tend to be sacculated withfaveolar, edicular, or trabecular gas-exchange areas. Tra-cheal swabs may be collected in conscious animals butare very prone to oral contamination.

Transtracheal lung lavage is much more useful andprovides material for both cytology and microbiology.Although transtracheal lung lavage is possible in con-scious animals, sedation or light anesthesia is preferred.To perform transtracheal lung lavage, a sterile catheterthat reaches the lungs should be inserted through theglottis, with care not to touch the oral mucosa (Figure3). In most reptiles, measuring from the snout to the endof the cranial third of the coelom is appropriate. Placinga sterile endotracheal tube can often help prevent oralcontamination of the lavage catheter. An empty syringe

Cloacocolonic lavage in a juvenile Uromastyx lizard (Uromastyx aegyptius) using a ball-tipped dosing needle. Blunt, lubricated catheters are less likely to cause trauma andperforate the thin colonic wall.

Figure 3. Lavage.

Transtracheal lung lavage in a sedated, blue tongue skink (Tiliqua scincoides). Note themetal gag to protect the clinician and catheter from bites.

for lizards and snakes with gross renomegaly butrelies on expertise and experience.

• The craniolateral coccygeal approach exposes thecaudal aspect of the kidney in lizards that haveintrapelvic kidneys (e.g., green iguanas [Iguanaiguana]; Figure 2). A small craniocaudal skin incisionshould be made in the tail base between the dorsaland ventral coccygeal muscles in the lateral midlinejust behind the pelvic limb. Blunt dissection revealsthe caudal pole of the intrapelvic kidney.

• Endoscopic biopsy using a rigid telescope forcelioscopy, visually guided biopsy, and kidney evalua-tion is suitable for most reptiles, especially lizards andchelonians (Figure 2). Paramedian or paralumbarentry in lizards and prefemoral entry in cheloniansprovide effective access to the coelom. Because che-lonian kidneys are retrocoelomic, a small endoscopicincision over the kidney is required before biopsy.Biopsies can be performed in any area, and the opticsof the telescope permit avoidance of the vas deferensand superficial renal vasculature.

should be attached and aspiration performed to removeexudates. If none can be removed, 0.5 ml of 0.9% sterilesaline per 100 g of body weight can be safely instilled.The reptile should be rotated and gentle aspiration reat-tempted. Excessive aspiration force may lead to lungentrapment and damage. In cases of unilateral lung dis-ease, the catheter can be carefully directed into one lung

using a curved stylet, and placement can be confirmedradiographically before aspiration and lavage.2 Alterna-tively, fine-needle aspiration may be attempted from thecraniodorsal aspect of the prefemoral fossa in cheloniansusing relatively long needles to penetrate the pulmonaryspace. However, because the risks associated with percu-taneous lung aspiration are greater, this technique shouldbe used with caution.

In snakes and lizards, fine-diameter (1 to 2.7 mm),rigid endoscopes can be used to examine the trachea andeven enter the lung(s) for examination and diagnosticsampling6 (Figure 4). In large snakes, ventrolateralceliotomy and pneumotomy (approximately 40% to 50%

of snout-to-vent length) permit introduction of anendoscope directly into the lungs. Using an endoscopeto examine chelonian lungs either via a prefemoral, soft-tissue approach through the septum horizontale (a thin,horizontal membrane that separates lungs from otherviscera in some chelonian species) or via a temporarycarapacial osteotomy permits both direct examinationand collection of tissue biopsies.7 In very large reptiles,flexible bronchoscopy techniques similar to those usedin mammals can be used.

Fresh samples should be immediately examinedmicroscopically as a wet preparation under both low(×40) and high (×400) magnification to identify proto-zoa and parasitic ova; adding a drop of Lugol’s iodineallows better visualization of protozoan parasites. Inaddition, a second sample should be preserved byadding 1 to 2 drops of ethanol per milliliter of sample.A microbiologic swab should be soaked with the samplestraight from the collection syringe and sent in transportmedia for bacterial and fungal culture. In addition, atleast one fresh, air-dried smear should be prepared forcytologic and microbiologic staining. This often yieldsenough information to aid diagnosis without resortingto cytoconcentration of the ethanol-preserved sample.

GASTROINTESTINAL TRACTReptiles often defecate during physical examination

or can be encouraged to do so by gentle cloacal manipu-

lation. Feces can be submitted for cytology, parasitology,electron microscopy, and microbiologic cultures. Inmany cases, it may be necessary to obtain feces usingcloacocolonic lavage: A relatively large, round-tippedcatheter should be lubricated and gently inserted andadvanced along the dorsal aspect of the cloaca, avoidingthe ventral urethra, if present2,4 (Figure 3). A similartechnique can be used to obtain samples from the stom-ach, with the catheter advanced to the midcoelom inmost reptiles (or as far as possible into the esophagus inlarge snakes). Lavage tubes should be measured fromthe snout to the midcoelom to prevent inadequate inser-tion (and regurgitation) or excessive insertion (and gas-



Figure 4. Endoscopic examination of an anesthetizedboa constrictor using a 2.5-mm flexible fiberscope.Endoscopic view of an acutely pneumonic boid lung (inset). Notethe edematous and inflamed lung parenchyma. Endoscopic biopsiesfor histology and microbiology are likely to be diagnostic.

Early definitive diagnosis of disease in reptiles directsappropriate case management and maximizes clinical success.



Figure 5. Sampling from musculoskeletal structures.

Synovial aspiration from a swollen stifle joint in a Greek tortoise(Testudo graeca). Note the aseptic precautions despite the highprobability that the joint is already infected.

Biopsy of a grossly swollen humerus in a green iguana (Iguanaiguana) using a cortical bone biopsy instrument.The diagnosis ofosteomyelitis was confirmed. Because of the proximity to thescapulohumeral joint, amputation was recommended andsuccessfully performed.

tric rupture). The lavage sample (centrifuged, if neces-sary) can then be used for wet-preparation directmicroscopy, microbiology, and cytology as previouslydescribed. A small amount of the sample should bemixed with warm physiologic saline and immediatelyexamined microscopically as a wet preparation for thepresence of protozoa and other parasites. Nematode ovamay also be visible if present in reasonable numbers.Mixing the sample with a small quantity of Lugol’siodine allows visualization of encysted Entamoeba sppbut kills live protozoa. Other aliquots may be subjectedto centrifugation and flotation techniques to concentrateorganisms (especially endoparasite ova) for quantifica-tion and identification. Acid-fast staining for Cryp-tosporidium spp should also be considered.

Stomatitis is a common owner complaint in tortoisesand snakes but less so in lizards. It is important that swabsnot be taken from superficial caseous debris because con-taminants are likely to be encountered. A better approachis to aseptically remove surface debris and obtain a swab orbiopsy sample from deeper tissues. Fungal, aerobic, andanaerobic bacterial cultures should be considered, althoughanaerobic infections are often missed because of poor sur-vival during the submission process.

Rigid and flexible endoscopes permit direct examina-tion of the intestinal tract from the mouth to smallintestine and cloaca to large intestine in many species.Endoscopic biopsies can be extremely helpful in makinga diagnosis and planning further treatment. Biopsies canbe performed on mucosal lesions of esophagus, stomach,and small intestine, but great care is required to avoidrupture or perforation. Biopsies of the thinly walledlarge intestine should be avoided.

MUSCULOSKELETAL SYSTEMThere are several indications for sampling the muscu-

loskeletal system, including gout, soft tissue (metastatic)mineralization, septic arthritis, osteomyelitis, neoplasia,and panthrombocytopenia or leukemia (bone marrowbiopsy). Distended joints are often associated with sep-tic arthritis, and, following aseptic preparation, jointfluid can be aspirated for cytology and cultures in muchthe same way as in mammals (Figure 5). A squashpreparation is appropriate for more viscous or granularsamples. Multiple air-dried smears are helpful if usingcytologic, microbiologic, and special staining. Cytologicstains could include Romanowsky stains (e.g., Wright’s,Giemsa, Leishman’s), their “quick” derivatives (e.g.,Diff-Quik, RapiDiff ), and new methylene blue stain. It

is impossible to conduct a productive cytologic exami-nation of a Gram’s- or Ziehl-Neelsen–stained smear: Aseparate smear for cytologic staining is required.

Muscles are best sampled using a spring-action Tru-Cut needle or sharp surgical biopsy. Depending on areptile’s size, spinal needles or pediatric or adult corebone biopsy instruments can be used to sampleosteomyelitis and other bony lesions from long bones(Figure 5). On rare occasion, it may be necessary tosample bone marrow to aid hematologic assessment. Inlizards, bone marrow is best aspirated via the tibial

tuberosity or greater trochanter of the femur, whereas insnakes it is necessary to remove a large rib and perform asplenopancreatic biopsy for histopathologic assessment.In some chelonians, bone marrow can be harvested fromthe long bones and plastrocarapacial bridge (i.e., the lat-eral connection between the plastron and carapace).

INTEGUMENTThe integument is probably the easiest structure to

sample, and, unless dangerous reptiles are involved,

many procedures can be performed in conscious animalsby judiciously using 1% lidocaine (maximum: 4 mg/kgby local infiltration) to effect a local, line, or perineural–local anesthetic block.5 The most common errors whensampling the integument are delayed sample collectionand contamination with superficial debris. Whenobtaining impression smears, aspirate, scrapes, or micro-biologic culturettes, the site should be gently but asepti-cally prepared before removing superficial scabs; samplesshould be taken from below these necrotic areas.Impression smears should be taken only after lesions

have been gently dabbed with sterile gauze swabs toremove excess blood and tissue fluids. Several impres-sions should be taken from each site so that differentstains may be used. Romanowsky (or “quick” deriva-tives), Gram’s, and Ziehl-Neelsen stains are most fre-quently used for skin smears.

A skin biopsy may be performed by subcutaneouslyinjecting local anesthetic and simply cutting off a singlescale. Scales contain both dermal and epidermal compo-nents and can be submitted for laboratory investigation.

Figure 6. Skin and shell biopsy.

Full-thickness skin biopsy in a Hermann’s tortoise (Testudohermannii) using a punch biopsy tool.

Full-thickness shell biopsy of the carapace in a red-eared slider(Trachemys scripta elegans) using a cortical bone biopsy instrument.



There are very few pathognomonic clinicalsigns of specific disease processes in reptiles.

However, more invasive, full-thickness skin biopsies arepreferred and generally require a combination of seda-tion and local anesthesia or general anesthesia. Sharpsurgical biopsies or mammalian punch biopsies can beperformed with the integument closed using an evert-ing, horizontal mattress pattern or tissue adhesive (Fig-ure 6). Shell lesions are sampled in a manner similar tothat of bones using a cortical biopsy instrument. Asepticpreparation and full-thickness shell biopsy should beperformed with care to avoid damaging the underlyingtissues. In many cases, pathology causes shells to soften,and collecting a full-thickness biopsy sample is not asdifficult as may be anticipated (Figure 6). After biopsy,the shell defect must be covered with a water-tightmaterial such as epoxy resin or veterinary acrylic.2

Biopsies can be performed on subcutaneous massesusing a Tru-Cut needle following aseptic preparation and astab incision through the skin (pushing a Tru-Cut needlethrough reptilian integument can damage the instrument).

COELOM AND VISCERAL ORGANSCoelomitis as well as cardiac, renal, and hepatic dis-

ease can cause accumulation of coelomic (ascitic) fluid,which can be aspirated at any convenient, gravity-dependent location, taking care to avoid the obviousvasculature (e.g., the ventral midline abdominal vein inlizards), intestinal tract, lungs, and bladder (if present).The dangers of iatrogenic trauma should be consideredand evaluated in terms of the risk:benefit ratio. Aspirateshould be handled as previously described.

Transcutaneous biopsy of the coelomic organs, partic-ularly the liver, can be performed using a fine Tru-Cutdevice through a small skin incision. This may be doneblindly by relying on anatomic knowledge and palpation,ultrasonography, or computed tomography. These tech-niques involve a greater risk of iatrogenic trauma andhemorrhage because of the lack of direct visual control.

Many practitioners may find surgical celiotomy to bemore diagnostic and reliable. Exploratory celiotomyoffers an excellent opportunity to examine the visceraand perform a biopsy on one or more organs using aTru-Cut or surgical (e.g., wedge) technique. Rigidendoscopy is less invasive but equally rewarding andpermits examination of most, if not all, coelomic struc-tures via a single coelioscopic entry point in mostlizards and chelonians.8,9 Coelomic insufflation isrequired with this technique. The magnified, color,real-time endoscopy images enable accurate biopsieswith minimal trauma.

CENTRAL NERVOUS SYSTEMA number of diseases and conditions may manifest as

primarily neurologic, including boid inclusion body dis-ease; ophidian paramyxovirus infection; bacterial, fungal,and protozoan meningitis or encephalitis; and spinaltrauma, abscessation, and osteomyelitis. Although stillin its infancy, collecting cerebrospinal fluid (CSF) fromreptiles may help diagnose neurologic disease. Unfortu-nately, the absence of a subarachnoid space and thepresence of a venous sinus lying dorsal to the spinal cordoften result in blood contamination of collected sam-ples. In such cases, a peripheral complete blood cellcount can help evaluate blood-contaminated CSF. CSFhas been successfully collected from snakes and lizardsfor cytologic and microbiologic evaluation using tech-niques similar to those in mammals. However, thesetechniques have not been validated in reptiles.2 A lum-bosacral approach has also been used in lizards andappears easier than a cisterna magna technique. In allcases, animals should be anesthetized and strict asepsisfollowed. Samples should be submitted as previouslydescribed, but individual laboratories may have particu-lar requirements for CSF submission. If only a very



1. Which venipuncture site is preferred for chelo-nian hematology?a. caudal tail veinb. dorsal coccygeal tail veinc. subcarapacial sinusd. jugular veine. brachial vein

2. Which sample is preferred for biochemistry pro-files in small reptiles?a. plasmab. serumc. whole bloodd. EDTA plasmae. blood (from a clipped toenail) drained into a

heparinized microhematocrit tube

3. What is the approximate safe volume of bloodthat can be collected from a 2.2-lb (1-kg) healthyreptile?a. 0.5 ml d. 5 mlb. 1 ml e. 10 mlc. 2.5 ml

4. Bacterial culture and sensitivity results fromurine collected by cystocentesis from a turtlemust be viewed cautiously becausea. bacterial cystitis is often secondary to other disease

processes in reptiles.b. bacterial sensitivity patterns in reptiles are not useful.c. normal bladder urine is frequently not sterile.d. enrofloxacin is preferred despite culture results.e. contamination of the sample by skin commensals is

unavoidable.

small volume of fluid is obtained, preparing a smear andsubmitting fluid for culture may be all that are possible.

EARTympanic disease, particularly abscessation, is com-

mon in chelonians, and the caseous material can be sur-gically removed and sent for a microbiologic culture iffuture treatment is required. There have also beenreported cases of aural cryptosporidiosis in lizards;therefore, material from fine-needle aspiration via thetympanum should also be submitted for smear examina-tion (i.e., Ziehl-Neelsen, acid-fast staining) and culture.

EYEThe inspissated nature of the reptilian inflammatory

response tends to result in caseous rather than liquid exu-dates. Therefore, chronic eye infections can cause whitemicroabscesses to develop on the surface of the cornea inlizards and chelonians. Under general anesthesia andusing magnification, it is possible to gently debride theselesions for laboratory submission. Care must be taken toavoid contaminating such small samples with skin com-mensals. In addition to submitting a microbiologic cul-turette, further material should be used to prepare at leasttwo smears for cytologic (i.e., Romanowsky) and Gram’sstaining. These smears should be made by the squash



method after mixing the sample with a small volume of0.9% sterile saline on the slide to break up the solid mate-rial. Snakes have spectacles (i.e., fused, transparent eye-lids), and infection can develop between the spectacle andcornea. Surgical removal of a wedge of the spectacle per-mits acquisition of samples, irrigation, and drainage.

REFERENCES1. Heard DJ: Diagnostic sampling and laboratory tests, in Raiti P (ed): Manual

of Reptiles. Cheltenham, England, British Small Animal Veterinary Associa-tion, 2004, in press.

2. Hernandez-Divers SJ: Diagnostic techniques, in Mader DR (ed): ReptileMedicine and Surgery. Philadelphia, WB Saunders, 2004, in press.

3. Frye FL: Reptile Clinician’s Handbook: A Compact Clinical and Surgical Refer-ence. Malabar, Florida, Krieger Publishing, 1995.

4. Fudge AM: Section two: Reptilian laboratory medicine, in Fudge AM (ed):Laboratory Medicine: Avian and Exotic Pets. Philadelphia, WB Saunders,2000, pp 185–264.

5. Heard DJ: Reptile anesthesia. Vet Clin North Am Exotic Anim Pract 4:83–117,2001.

6. Burrows CF, Heard DJ: Endoscopy in nondomestic species, in Tams TR (ed):Small Animal Endoscopy. St Louis, Mosby, 1999, pp 297–321.

7. Hernandez-Divers SJ: Pulmonary candidiasis due to Candida albicans in aGreek tortoise (Testudo graeca) and treatment using intrapneumonic ampho-tericin B. J Zoo Wild Med 32:352–359, 2002.

8. Hernandez-Divers SJ, Stahl S, Hernandez-Divers SM, et al: Coelomicendoscopy of the green iguana (Iguana iguana). J Herp Med Surg 14:10–18,2004.

9. Hernandez-Divers SJ: Endoscopic renal evaluation and biopsy in chelonia.Vet Rec 154:73–80, 2004.

ARTICLE #5 CE TEST This article qualifies for 1.5 contact hours of continuing education credit from the Auburn University College of Veterinary Medicine. Subscribers who wish to apply this credit to fulfill state relicensure requirements should consult their respective stateauthorities regarding the applicability of this program. To participate, fill out the test form inserted at the end of this issue. To take CE tests online and get real-time scores, log on to www.VetLearn.com.

CE

5. When investigating gout in reptiles, whichparameter should always be measured?a. uric acid d. creatineb. urea e. potassiumc. creatinine

6. Which technique provides the least informationon kidney structure?a. endoscopic biopsyb. cranial tail cut-down and biopsyc. surgical wedge biopsyd. ultrasound-guided, transcutaneous fine-needle

aspiratione. surgical Tru-Cut biopsy

7. What is the generally recommended safe vol-ume of 0.9% sterile saline to instill when per-forming lung lavage in a 300-g lizard?a. 0.5 ml d. 10 mlb. 1.5 ml e. 15 mlc. 5 ml

8. After collecting a lung lavage sample for labora-tory analysis,what samples should be submitted?a. fresh fluidb. culturettec. unstained smearsd. a and ce. a, b, and c

9. When performing a skin scrape and takingimpression smears from an exudative skin lesionon a nonaggressive corn snake, which method ofrestraint is preferred?a. sedation using ketamineb. anesthesia using isofluranec. local anesthesia using lidocained. physical restraint alonee. refrigeration (hypothermia)

10. Which statement regarding reptile diagnostics ismost correct?a. Reptiles are fragile and do not tolerate diagnostic

tests as well as mammals.b. Samples should be taken as early as possible in the

disease course.c. Diagnostics should be delayed until a course of

antibiotics has been administered.d. Because of a lack of knowledge, meaningful interpre-

tation of reptile histology is poor.e. A fecal culture for Salmonella spp should be con-

ducted on all sick reptiles.

483CE


article #5 ce diagnostic techniques and sample...

Documents