Clinical Pathology of Amphibians: A Review

Veterinary Clinical Pathology ISSN 0275-6382
INVITED REVIEW
Clinical pathology of amphibians: a review

n1,2, Jill Heatley3, Karen E. Russell4, Barbara Horney2
Marıa J. Forza
1
Canadian Wildlife Health Cooperative; 2Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island,
Charlottetown, PE, Canada; 3Department of Clinical Sciences, Auburn University College of Veterinary Medicine, Auburn, AL, USA; and 4College of
Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA
Key Words Abstract: Amphibian declines and extinctions have worsened in the last
Amphibian, clinical chemistry, clinical 2 decades. Partly because one of the main causes of the declines is infec-
pathology, hematology, reference intervals, tious disease, veterinary professionals have increasingly become involved
review
in amphibian research, captive husbandry, and management. Health
Correspondence
evaluation of amphibians, free-living or captive, can benefit from
M.J. Forzan, Cornell Wildlife Health Laboratory, employing the tools of clinical pathology, something that is commonly
Cornell University School of Veterinary used in veterinary medicine of other vertebrates. The present review
Medicine, 240 Farrier Road, Ithaca, NY, 14853. compiles what is known of amphibian clinical pathology emphasizing
E-mail: mdf93@cornell.edu knowledge that may assist with the interpretation of laboratory results,
provides diagnostic recommendations for common amphibian diseases,
DOI:10.1111/vcp.12452 and includes RIs for a few amphibian species estimated based on peer-
reviewed studies. We hope to encourage the incorporation of clinical
pathology in amphibian practice and research, and to highlight the
importance of applying veterinary medicine principles in furthering our
knowledge of amphibian pathophysiology.
Contents Biochemical Panel

Glycerol and glucose in freeze-tolerant frogs
Introduction Cholesterol and corneal lipidosis in Tree frogs
Amphibians and veterinary medicine Nitrogenous waste-products, renal function, and hydration status
Amphibian physiology and anatomy of clinical relevance Electrolytes, osmolality, and blood gases
Osmoregulation, water, and electrolyte balance Proteins
Over-wintering adaptations Minerals, metals, and vitamins
Endocrine glands and metamorphosis Hormones
Hematopoiesis, blood, and lymph circulation Urinalysis
Life span Ancillary Diagnostic Tests for Common Amphibian Diseases
Sample Collection and Handling Acknowledgments
Cardiocentesis Disclosure
Maxillary (facial) vein References
Femoral and abdominal veins
Lingual venous plexus
Ventral caudal vein
Appendage amputation Introduction
Urine collection
Sample handling
Clinical pathology as a tool for health evaluation can
Hematology
Blood cell count
be as useful in amphibians as in higher vertebrates.
WBC differential blood cell count The stoic nature of amphibians and the great variety
Red blood cells of species in the class make it difficult to interpret
Thrombocytes external health criteria easily, thus increasing the
White blood cells value of laboratory tests that can be objectively inter-
Hemoparasites preted. Unfortunately, diagnostically relevant infor-
(continued) mation for hematology and clinical chemistry of
Vet Clin Pathol 0/0 (2017) 1–23 ©2017 American Society for Veterinary Clinical Pathology 1
Amphibian clinical pathology review Forzan et al
amphibians is scarce. Modern amphibians comprise that may assist with the interpretation of laboratory
over 7000 species and can be divided into 3 orders, results, provides diagnostic recommendations for com-
each with a distinct external morphology: Anura or mon amphibian diseases, and includes RIs for a few
Salientia (frogs and toads, the archetypical amphib- amphibian species estimated based on peer-reviewed
ians), Caudata or Urodela (salamanders and their studies.
aquatic counterparts, the newts, both of lacertilian
shape), and Apoda (caecilians, worm-like, and often
blind).1 Published information is mostly restricted to Amphibians and veterinary medicine
a few species in the first 2 orders, Anura and
Amphibians: This class of animals is distinguished
Caudata, henceforth referred to as “frogs” and “sala-
by a body cold and generally naked; a countenance
manders”, respectively, unless otherwise specified.
stern and expressive; voice harsh; colour mostly
Clinical pathology information is lacking for caecil-
lurid, and filthy odour: a few are furnished with a
ians. Most reports concerning native North American
horrid poison [. . .]; some undergo a metamorphosis
species are compiled with a biologic or ecologic per-
[. . .]; some appear to live promiscuously on land or
spective.2–6 Reports on laboratory frogs (Xenopus sp.)
in the water, and some are torpid during the winter.
are more clinically oriented but the number of
Carolus Linnaeus (1758) as translated by William
subjects studied is frequently small.7,8 The study of
Turton (1800)13
amphibian hematology and clinical chemistry is fur-
ther complicated by the numerous and varied species Although, when Linnaeus wrote his definition of
composing the class, the marked differences present the Amphibians he was also thinking of reptiles,
within a species depending on its life stage, and the which he placed in the same class (Linnaeus, 1800),
great influence that environmental factors have on his description conveys a slight distaste for cold and
amphibian physiology. Amphibian medicine would slimy creatures. Fortunately, from the early days
benefit from studies conducted under established of modern biology, other naturalists have found
guidelines, such as those by the American Society for amphibians both amenable experimental subjects
Veterinary Clinical Pathology (ASVCP)9, as exempli- and an intriguing group of vertebrates in their own
fied by recent reports on the hematologic profiles of right. Studies into the taxonomy and, later on, anat-
Australian Tree frogs10, the laboratory subject Xeno- omy and physiology of amphibians flourished in the
pus tropicalis11, and the North American Native Wood 18th and 19th centuries. In the 20th century, studies
frog.12 Although it is tempting to extrapolate findings on amphibian physiology, embryology, immunology,
in one species to others in the same genus, one and toxicology multiplied along with an increased
should use caution when relying on reference values interest in the natural history and ecology of wild
generated for a different species, even one closely populations.14 In the 1970s, naturalists and field
related to the amphibian of interest, as this may researchers working in the temperate forests of Aus-
result in misleading diagnostic information.10 The tralia and Central America began noticing marked
paucity of published or even laboratory-specific RIs decreases in some of the amphibian populations they
for most amphibian species often necessitates the use had studied for years. For 2 decades, while multiple
of conspecifics for comparative assessment of analytes theories were proposed but none substantiated, the
of interest in an individual animal. Conspecifics used declines continued and some turned into extirpations
as reference individuals must not only be of the same or extinctions. Finally, in an example of effective
species but also of a similar age (tadpole, juvenile, or trans-disciplinary collaboration and through the rig-
adult), fed and housed in the same way, and sampled orous diagnostic investigation of mortality events,
at the same time of day and under equal conditions the infectious disease chytridiomycosis was found to
of temperature and humidity as the individual in be responsible for the declines.15,16 Since then
question. Even when using values from individuals Batrachochytrium dendrobatidis, the fungus that causes
of the same species, life stage, and environmental chytridiomycosis, has been recognized as an emerg-
conditions, RIs should be considered for guidance ing pathogen worldwide and its impact on frogs as
only: interpretation of results must include consider- “the most spectacular loss of vertebrate biodiversity
ation for the amphibian’s clinical presentation, due to disease in recorded history”.17
results from conspecifics, and the variability of differ- The discovery that an infectious agent had played
ing analyzer and laboratory methodologies. a significant part in the amphibian population crisis of
The present review compiles what is known of the late 20th century propelled a growth in research on
amphibian clinical pathology emphasizing knowledge amphibian diseases. It also prompted the involvement
2 Vet Clin Pathol 0/0 (2017) 1–23 ©2017 American Society for Veterinary Clinical Pathology
Forz
an et al Amphibian clinical pathology review
of diagnosticians, microbiologists, and veterinarians in and reabsorbs water and electrolytes), the skin (cru-
amphibian research, a field that had been the almost cial in water uptake and electrolyte balance), and in
exclusive province of herpetologists and experimental frogs, the lymph hearts (paired structures adjacent
physiologists. The involvement of veterinarians in to the cloaca that pump the abundant lymph fluids
amphibian disease monitoring and research fostered back into general circulation through the renal por-
the progressive incorporation of standard veterinary tal system). Water and electrolyte balances are cru-
diagnostic techniques into amphibian studies, be it cial to the homeostasis of all amphibians, of course,
those on wild populations as indicators of environ- but the challenges are different depending on the
mental health, or on captive species used as experi- environment in which each species or life stage
mental models for infectious diseases, immunology, or inhabits: while terrestrial amphibians have devel-
ecotoxicology. oped mechanisms to avoid dehydration, aquatic
Amphibian medicine has also advanced signifi- ones must restrict water uptake to avoid plasma
cantly in the last few decades.1 This has improved con- dilution and decreased oncotic pressure.26 Both
ditions of captive amphibians in laboratories, served aquatic and terrestrial amphibians tolerate wide
frogs and salamanders kept as pets by a growing num- fluctuations in the osmolality and composition of
ber of amateur herpetologists, and improved captive their plasma. This adaptation is essential to conserve
rearing of endangered species in conservation and water as amphibian kidneys are incapable of con-
reintroduction programs. centrating urine above the osmolality of plasma. In
Caudata and Anura, the salamander and frog general, larvae and aquatic adults, such as those in
groups, be it native, exotic, or laboratory species, are the genus Xenopus, excrete ammonia through the
the most likely to be encountered by the clinician, kidneys (ammonotelia), skin, and gills, while terres-
diagnostician, or researcher (Table 1).18 Although trial species convert ammonia in the liver into urea
information contained in this review refers to various (ureotelia).1,14 Adults of some species of Tree frogs,
species, specific analyte values are reported only for such as the Waxy Monkey Tree frog (Phyllomedusa
the following: American Bullfrog (Rana [Lithobates] sauvagii), more concerned about water conservation,
catesbeiana)3, Northern Leopard frog (Rana [Litho- excrete uric acid (uricotelia) as the end product of
bates] pipiens)5, Wood frog (Rana sylvatica [Lithobates protein metabolism while their larvae, which are
sylvaticus])12, Cuban Tree frog (Osteopilus septentrion- developing in a more aquatic environment, are
alis)19, African Clawed frog (Xenopus laevis)7, African ureotelic.1,14 Some species of frogs are capable of
(Tropical) Clawed frog (X tropicalis)11, Sapito de switching from a water-demanding excretion pro-
Jardın (Bufo [Rhinella] fernandezae)20, Green (White’s) duct (ammonia) to a less toxic form (urea) in times
Tree frog (Litoria caerulea), White-Lipped Tree frog of environmental water shortage. Amphibians in
(Litoria infrafrenata)10, Japanese Newt (Cynops pyrrho- general are tolerant to high levels of blood urea and
gaste)21, and Eastern and Ozark Hellbenders (Crypto- only a few species are uricotelic. The latter may
branchus alleganiensis alleganiensis and C a bishop, explain why gout, so common in reptiles, is an
respectively).4 uncommon consequence of dehydration in amphib-
ians.26
Amphibian physiology and anatomy of clinical Over-wintering adaptations
relevance
Particularly important to frogs and salamanders
Amphibian veterinarians, biologists, and researchers inhabiting the northern hemisphere are adaptations
need to familiarize themselves with the various aspects for winter survival (over-wintering or hibernation).
in which each group varies from other vertebrates, and Over-wintering frogs and salamanders have
seek specific literature to enhance their understand- increased concentrations of fibrinogen, heat shock
ing.1,14,22–26 The present section is not meant to be a proteins, and glucose-transport proteins, and pro-
comprehensive review of amphibian physiology and duce ice-nucleating proteins in the blood to guide
anatomy, but a short summary highlighting the most ice crystal formation.22,23 They also accumulate low
distinct and clinically relevant characteristics of the molecular weight carbohydrates such as glycerol or
class. glucose in tissues and blood, and increase plasma
osmolality through dehydration. These changes
Osmoregulation, water, and electrolyte balance
lower the freezing point of tissues and ensure that
Osmoregulation in amphibians involves not only the ice crystals form in extracellular compartments, pro-
kidneys but also the urinary bladder (which stores tecting cells from shearing damage.22,23
Table 1. Amphibian species commonly kept as pets or laboratory subjects, captive-bred for conservation programs, or studied in the wild. Families
known to produce toxic secretions are given in bold. International Union for Conservation of Nature (IUCN) status18: least concern (LC), vulnerable (VU),
near threatened (NT), endangered (EN), critically endangered (CR), extinct in the wild (EW), data deficient (DD).14
Family Common Name Scientific Name IUCN Status
Ambystomatidae Eastern Tiger salamander Ambystoma tigrinum LC

Mexican Axolotl Ambystoma mexicanum* CR
Marbled salamander Ambystoma opacum LC
Spotted salamander Ambystoma maculatum LC
Bombinatoridae Oriental Fire-Bellied toad Bombina orientalis LC
Bufonidae African toad Amietophrynus regularis LC
Cane toad Rhinella marina LC
Cururu (Rococo) toad Rhinella schneideri LC
Gulf Coast toad Incilius nebulifer LC
Sapito de Jardın Rhinella fernandezae LC
Puerto Rican Crested toad Peltophryne lemur CR
Panamanian Golden frog Atelopus zeteki CR
Kihansi Spray toad Nectophrynoides asperginis EW
Calyptocephalellidae Helmeted Water toad Calyptocephalella gayi VU
Ceratophryidae Argentine Horned (Pac-man) frog Ceratophrys ornata NT
Brazilian Horned frog Ceratophrys aurita LC
Cranwell’s Horned frog Ceratophrys cranwelli LC
Pacific Horned frog Ceratophrys stolzmanni VU
Cryptobranchidae Eastern Hellbender Cryptobranchus alleganiensis NT
alleganiensis
Ozark Hellbender Cryptobranchus alleganiensis NT
bishop
Dendrobatidae Black-Legged Dart frog Phyllobates bicolor NT
Dyeing Poison frog Dendrobates azureus LC
Golden Poison frog Phyllobates terribilis EN
Golfo Dulce Poison Dart frog Phyllobates vittatus EN
Green & Black Poison Dart frog Dendrobates auratus LC
~ko
Ko e Poison Dart frog Phyllobates aurotaenia NT
Lovely Poison frog Phyllobates lugubris LC
Strawberry Poison Dart frog Oophaga pumilio LC
Eleutherodactylidae Puerto Rican Coquı Eleutherodactylus coqui LC
Hemiphractidae Andean Marsupial Tree frog Gastrotheca riobambae EN
Peru Marsupial frog Gastrotheca peruana LC
Hylidae Black-eyed (Morelet’s) Tree frog Agalychnis moreletii CR
Bleating Tree frog Phyllobates bicolor LC
Blue Mountains Tree frog Litoria citropa LC
Broad-Palmed frog Litoria latopalmata LC
Brown Tree frog Litoria ewingii LC
Cuban Tree frog Osteopilus septentrionalis LC
Dainty Green Tree frog Litoria gracilenta LC
Eastern Ddwarf Tree frog Litoria fallax LC
Green and Golden Bell frog Litoria aurea VU
Green (White’s) Tree frog Litoria caerulea LC
Growling (Southern bell) Grass frog Litoria raniformis EN
Leaf Green Tree frog Litoria phyllochroa LC
Littlejohn’s Tree frog Litoria littlejohni LC
Mimic Poison frog Ranitomeya [Dendrobates] imitator LC
Splendid Tree frog Litoria splendid LC
Mountain Stream Tree frog Litoria barringtonensis DD
Peron’s Tree frog Litoria peronii LC
Phantasmal Poison frog Epipedobates tricolor LC
Red-Eyed Tree frog (Australia) Litoria chloris LC
Red-Eyed Tree frog (Central America) Agalychnis callidryas LC
(continued)
Forz
Table 1. (continued)
Family Common Name Scientific Name IUCN Status
Striped Rocket frog Litoria nasuta LC

Tyler’s Tree frog Litoria tyleri LC
Waxy Monkey Leaf frog Phyllomedusa sauvagii LC
Whistling Tree frog Litoria verreauxii LC
White-Lipped Tree frog Litoria infrafrenata LC
Hyperoliidae Argus Reed frog Hyperolius argus LC
Mantellidae Black-Eared Mantilla Mantella milotympanum CE
Golden Mantilla Mantella aurantiaca CR
Microhylidae False Tomato frog Dyscophus guineti LC
Tomato frog Dyscophus antongilii NT
Myobatrachidae Corroboree frog Pseudophryne corroboree CR
Great Barred frog Mixophyes fasciolatus LC
Ornate Burrowing Frog Limnodynastes ornatus LC
Spotted Grass frog Limnodynastes tasmaniensis LC
Striped Marsh frog Limnodynastes peronii LC
Sudell’s frog Neobatrachus sudelli LC
Pipidae African Clawed frog Xenopus laevis LC
African (tropical) Clawed frog Xenopus tropicalis LC
African (Zaire) Dwarf frog Hymenochirus boettgeri LC
Pyxicephalidae African Bullfrog (pixie frog) Pyxicephalus adspersus LC
Ranidae American Bullfrog Rana [Lithobates] catesbeiana LC
Northern Leopard frog Rana [Lithobates] pipiens LC
Wood frog Rana sylvatica [Lithobates sylvaticus] LC
Rhacophoridae Mossy frog Theloderma corticale DD
Rhinodermatidae Darwin’s frog Rhinoderma darwinii VU
Salamandridae California Newt Taricha torosa* LC
Eastern Newt Notophthalmus viridescens LC
Lorestan Newt Neurergus kaiseri* CR
Oriental Fire-Bellied Newt Cynops orientalis LC
Japanese Newt Cynops pyrrhogaster LC
*Tail autotomy does not occur.
stage, is obligatory in several species of salamanders,

Endocrine glands and metamorphosis
the most famous of which is the Mexican Axolotl,
Endocrine organs in amphibians vary morphologically Ambystoma mexicanum. Neotenic species do not undergo
but have similar functions to those in other vertebrates. metamorphosis, and must remain permanently in an
Adrenal glands (also called interrenal glands, given their aquatic environment14, presumably retaining premeta-
location) produce 3 types of steroids in amphibians, morphic plasma characteristics into adulthood.
aldosterone, corticosterone, and cortisol.1,14
Hematopoiesis, blood, and lymph circulation
Metamorphosis occurs exclusively but not invari-
ably in amphibians and is a complicated process that The site of hematopoiesis varies depending on the spe-
transforms an aquatic limbless larva into a tetrapod cies, but there seem to be some characteristics shared
adult. During metamorphosis, glucocorticoid and thy- among members of the same order. Frogs produce lym-
roxine levels rise, and shifts in lymphocyte populations phoid, myeloid, and thrombocytic cells in their bone
are required to develop tolerance to a barrage of new marrow; erythrocytes are only produced in the spleen
adult antigens and a new definition of immunologic of adults while erythropoiesis occurs primarily in the
self.1,14 Changes in plasma biochemistry occur liver of froglets and in the kidneys of tadpoles. Sala-
in amphibians undergoing metamorphosis. Serum pro- manders produce lymphoid and myeloid cells in their
teins, particularly albumin, increase markedly as meta- bone marrow and liver, and erythroid cells in the
morphosis progresses in order to increase the osmotic spleen. In newts, hematopoiesis occurs exclusively in
pressure of blood and thus its water-retaining capac- the liver, kidney, thymus, and spleen.14
ity.14 Neoteny, or the acquisition of reproductive matu- Amphibian hearts have 2 atria but only one ven-
rity while retaining the external appearance of a larval tricle. The interatrial septum is fenestrated in most
salamanders, and complete in frogs. Blood from the During sample collection, handling must be mini-
caudal end of the body will pass through the kidney mized to avoid removing the protective mucous layer
before entering the postcaval vein, which may affect which maintains skin moisture, and to keep tempera-
the distribution of drugs administered to the caudal ture exchange between the handler and the amphib-
aspect of the body.22 Despite a lack of lymph nodes, ian to a minimum. Amphibians are poikilotherms
there is a well-developed lymphatic vasculature that (ectotherms) and rely on a combination of environ-
often runs parallel to large blood vessels. As in other mental temperature, heat- or cold-seeking behavior,
vertebrates, lymph drains into the blood stream.25 In peripheral vascular control, and color changes to
frogs, there is an extensive network of lymphatic ves- maintain their body temperature. An amphibian’s
sels, or lymph sacs, between the skin and underlying temperature is dependent on the immediate environ-
musculature. Large volumes of lymph flow through ment and has significant influence on hematologic and
the lymph sacs back into the renal portal system aided biochemical analyte values. Thus, increasing the
by the pumping mechanism of the lymph hearts, amphibian’s body temperature may alter test results in
located in the axillary region and alongside the the sample collected.1 Samples should be collected
cloaca.26 from animals kept at their optimum temperature
range, which depends on the species, season, and life
Life span
stage (Table 2).
Individuals in captivity have relatively long life spans. Clinical examination and observation of the frog
Native species like the American toad (Bufo [Anaxyrus] or salamander before sample collection should include
americanus) that lives for 5–10 years in the wild can assessing posture, blink reflex (by gently touching the
reach 35 years of age in captivity.24 Exotic species that eyes), righting reflex (place the amphibian on its back
are often kept as pets, such as the Bumblebee Poison and watch it right itself), and withdrawal reflex (care-
Dart frog (Dendrobates leucomelas) or Fire-Bellied toad fully extend the limbs and watch their retraction).1
(Bombina bombina), can live for 12 years or more. Loss of any of these reflexes or an abnormal posture
may indicate serious illness and influence the decision
to take a blood sample and the volume that should be
Sample Collection and Handling collected.32 One must always calculate the volume of
blood that can be safely drawn from a frog or salaman-
Plastic or nitrile gloves (without talcum powder), der to avoid causing metabolic disturbances, morbidity
rinsed with dechlorinated water are generally consid- or mortality. Minimal total blood volumes (TBV) are
ered best for handling frogs or salamanders, but care 7–10% of body weight in terrestrial amphibians and
should be taken to review the literature for species- 13–25% in aquatic ones (Figure 1). No more than
specific toxic reactions.22,27 Gloves protect the delicate 5–10% of the TBV should be drawn, ie only 5% from
skin of the frog or salamander, and reduce the han- sick individuals and up to 10% from healthy ones.
dler’s exposure to zoonotic pathogens or toxins. Choice of equipment and technique of blood col-
Amphibians can carry zoonotic pathogens such as lection from frogs, salamanders, or tadpoles depends
Salmonella spp.28 and Mycobacterium marinum29, and on the size of the animal and its anatomic characteris-
some species produce toxic secretions.30 Poisonous tics. Specifics for size and vein approach are given
frogs, found in Central and South America (some den- below in each section. Generally, 0.3- to 3-mL syringes
drobatidis and bufonids), Madagascar (mantellids), and 27- to 23-gauge needles are used, keeping in mind
and Australia (myobatrachids), have striking col- that a gauge of < 25 may result in hemolysis in species
orations that both mark them as undesirable prey and with large RBC. Superficial topical anesthetics such as
make them appealing pets. Several are endangered lidocaine or benzocaine creams or gels (EMLA cream,
species and the focus of intense conservation efforts AstraZeneca, Mississauga, ON, Canada or Oragel,
and captive breeding, like the Panamanian Golden frog Church & Dwight Co., Inc., Mississauga, ON, Canada)
(Table 1). Poisonous frogs have specialized skin glands may be used, albeit cautiously as overdosing can result
that secrete toxic alkaloids. As poisonous compounds in general anesthesia or, in extreme cases, death.
in frogs come from dietary sources, individuals main- Lithium or ammonium heparin is the preferred antico-
tained in captivity and fed nonpoisonous arthropods agulant for amphibian blood, unless the blood is
will not be able to synthesize the alkaloids.30 intended for bacterial culture. One may flush the
The exception is the Corroboree frog (Pseudophryne needle and syringe with the liquid solution or use hep-
corroboree) which synthesizes toxic alkaloids regardless arinized capillary tubes, such as Fisherbrand Microhe-
of the food source.31 matocrit (Fisher Scientific Cat. no. 22-362-566,
Forz
Table 2. Ambient and water temperature gradients (terrestrial and aquatic species/life stages), and water quality parameters (aquatic species/life
stages only*) recommended for amphibians (adapted from1).
Temperature (°C) Water Quality*
Type of Species Habitat Type Min Max Parameter Concentration
Terrestrial amphibians Tropical lowland 24 30 pH 6.5–8.5

Tropical montane 18 24 Salinity 0–5 ppt
Subtropical 21 27 Hardness 75–150 mg/L
Temperate 18 (10) 24 (16) Alkalinity 15–50 mg/L
(Winter hibernation) Dissolved oxygen > 80% saturation
Aquatic amphibians* Tropical lowland 24 30 Carbon dioxide < 5 mg/L
(and larvae of terrestrial species) Tropical montane 18 24 Unionized ammonia < 0.02 mg/L
Subtropical 21 27 Nitrite < 1 mg/L
Temperate, stream 16 21 Nitrate < 50 mg/L
Temperate, pond 18 (9) 24 (15) Chlorine Undetectable
(Winter hibernation)
Pittsburgh, PA, USA) 75-mm long capillary tubes (not Although the use of MS222 has been suggested to
to be confused with other shorter microhematocrit interfere with bacterial culture from samples taken
tubes), or green-cap microtainer collection tubes under anesthesia33, the suggestion originates from the
coated with dry lithium heparin (Becton, Dickinson misinterpretation of a study that examined the growth
and Company, Cat. no. 365965, Franklin Lakes, NJ, of Gram-negative bacteria in agar mixed with varying
USA).1 concentrations of the tricaine methane sulfonate.34
The study’s growth conditions did not mimic any stan-
dard protocol for bacterial culture as bacteria were
Cardiocentesis
invariably grown in MS222-free agar plates, never
Cardiac puncture can be used in any species, but this simply grown from samples collected in vivo from ani-
invasive procedure has potential for life-threatening mals under MS222-anesthesia. Its findings are thus
complications such as cardiac standstill, atrial fibrilla- irrelevant to routine bacterial cultures from animal
tion, and pericardial tamponade. General anesthesia is samples.
necessary and may be achieved by submerging the
amphibian in a buffered solution of 0.02–0.1% (0.2–
Maxillary (facial) vein
1 g/L) tricaine methanesulfonate (MS222; Sigma-
Aldrich Corporation, St. Louis, MO, USA) or a solution The maxillary (facial) and musculo-cutaneous veins
of benzocaine (Orajel, or similar gels, Orajel; Church can be sampled in ranid frogs (members of the
& Dwight Co., Inc.) at 0.005–0.01% for larvae and Ranidae family) and those with similar cranial anat-
0.02–0.03% for adults. Rinsing the amphibian with omy, such as Tree frogs and toads. The maxillary
clean dechlorinated water after the procedure is fi- vein runs parallel to the upper jaw toward the
nished will allow excretion of the MS222 and is neces- commissure of the mouth, becoming the musculo-
sary for recovery if benzocaine is used. First, the cutaneous vein as it passes the tympanum. Holding
anesthetized tadpole, frog, or salamander is placed on its the frog in one hand to expose only the head,
back on a moist paper towel; the cardiac pulse can then quickly insert and withdraw a small-caliber needle
be directly observed or detected using a Doppler probe through the skin between the upper jawline and
placed near the manubrium. A small 25- or 27-gauge the rostral side of the tympanum, in the slightly
needle is inserted into the ventricle, at a 45° to 60° sunken triangle formed by the back of the eye, the
degree angle to the skin, and blood is drawn either by front of the tympanum, and the maxillary bone
placing a capillary tube inside the hub of the needle or (Figure 2A–C, Video S1). Insertion follows a 30°
by gently aspirating with a 0.3-, 1-, or 3-ml syringe.1,32 angle to the skin, in a rostro-caudal direction to
When successfully employed, cardiocentesis is the col- access the maxillary vein, or in the opposite direc-
lection method most likely to yield the largest volume tion to access the musculo-cutaneous vein. This
of blood. The ease with which amphibian hearts can external collection method punctures the vein and
move within the coelomic cavity can make cardiocente- releases blood onto the skin surface for collection
sis challenging and potentially result in puncture of the with a capillary tube.35 Needles should be 30-gauge
bi-lobulated liver that wraps tightly around the heart. for frogs weighing under 25 g, and 25- to 27-gauge
Femoral and abdominal veins

Venipuncture of the femoral and ventral abdominal
veins can be performed on relatively large amphibians.
The veins should be visible through the skin unless the
individual is darkly pigmented, in which case transillu-
mination can facilitate visualization. Needles should be
27–30 gauge, depending on the size of the animal.
While syringes can range from 0.3 to 3 mL, aggressive
aspiration and thus vessel collapse should be avoided.
The technique cannot be used in small individuals. As
the ventral abdominal vein runs parallel to lymphatic
vessels and, in frogs, an extensive lymph sac network
is present in the subcutis, lymph contamination may
lead to a false interpretation of anemia, lymphocytosis,
or hypercalcemia.1,26
Lingual venous plexus

The lingual venous plexus may be accessed in most
frogs > 25 g, but cannot be used in species that lack a
proper tongue such as the pipid frogs, eg, African
Clawed frogs (Xenopus spp). The mouth should be care-
fully opened with a rigid but smooth instrument, such
as a rubber spatula. Drawing the tongue forward
exposes the lingual venous plexus (Figure 2E,F). After
puncturing the plexus with a 25- or 27-gauge needle,
the blood oozing out can be collected with a capillary
Figure 1. Normogram to calculate blood volume that can be drawn from tube.1 Releasing the tongue and allowing the frog to
an amphibian. Draw a line from the left column (body weight) through the close its mouth usually suffices to stop the bleeding.
type of species and health status (AH, aquatic healthy; AS, aquatic sick; TH, The contamination of the sample with saliva, a major
terrestrial healthy; TS, terrestrial sick): the right column indicates the vol-
disadvantage of this technique, can be reduced by
ume that can be drawn safely. Conservatively, this graph is based on total
blood volumes of 14% and 7% of body weight for aquatic and terrestrial
gently rubbing the lingual surface with a cotton swab
species, respectively; safe volumes to draw are 5% and 10% from sick and prior to sampling. This method, however, is cumber-
healthy animals, respectively. Normogram constructed by R. Vanderstichel. some for the practitioner and likely uncomfortable for
the frog.
for larger frogs. After collection, gentle pressure

Ventral caudal vein
over the area achieves hemostasis. The area may be
disinfected using the antiseptic spray Bactine (Bayer Venipuncture of the ventral caudal tail vein, which
Inc., Mississauga, ON, Canada) before or after blood runs immediately beneath the vertebrae, is practical
collection. Depending on sampling conditions, 0.02– for use in salamanders and newts. For salamanders
0.07 mL of blood (the latter approximating the weighing < 80 g, a 27-gauge needle is needed; in sala-
volume collected in one ammonium heparin capil- manders > 80 g, 25-gauge needles may be used.1
lary tube; Fisherbrand) can be collected from small Blood is slowly drawn into a 0.3- to 1-mL syringe. At
frogs; in large frogs, the puncture will yield larger least two-thirds of the Plethodontidae family, which
volumes. Tilting the capillary tube earthward uses includes most wild salamanders from northeastern
gravity to facilitate blood flow and increases the North America, is capable of autotomy, the ability to
speed of collection and the blood volume obtained. willfully detach the tail at predetermined zones of
Warmer environmental temperatures facilitate blood breakage (cleavage points). Shedding the tail allows a
flow. This technique does not require anesthesia salamander that feels threatened or injured to escape
and may be applied repeatedly, barring excessive and avoid predation.14 The tail may fully or incom-
sampling volumes. pletely regenerate, but coloration and internal
Forz
A B C
D E
Figure 2. Maxillary (facial) vein and lingual plexus venipuncture. (A–C) Maxillary vein venipuncture: A 27- to 30-gauge needle should be quickly inserted
and withdrawn in the area marked by the yellow-rimmed red triangle (A). Collect the blood that ebbs onto the skin surface with a heparinized capillary
tube; collection is aided by tilting the capillary toward the ground (B). Hemostasis is quickly achieved after a few seconds of gentle pressure in the area,
after the desired volume has been collected (C). (D and E) Lingual plexus venipuncture: Gently open the mouth of the frog with a soft instrument, such as
a rubber spatula (D). Bringing the tongue forward, the venus plexi behind and beneath the tongue are exposed and may be punctured with a 27-gauge
needle to collect blood with a capillary tube (E).
structure may differ from the original. Therefore, care amputation should occur at an interphalangeal joint,
is advised when collecting blood from wild-caught no more than one digit should be removed per limb,
North American salamanders via the ventral caudal and the digit chosen must not be involved in normal
vein as the animals may lose their tail during sampling behavior of the species (burrowing, climbing,
or any other stress-related intervention. The Mexican amplexus during pairing, nest excavation, or propul-
Axolotl (Ambystoma mexicanum), a common pet spe- sion) to avoid endangering the viability of the wild ani-
cies, does not practice tail autotomy.14 mal.1 Tail-and toe-clipping will result in short- or long-
term disfiguration. Furthermore, and perhaps more
importantly, toe-clipping provides either a small vol-
Appendage amputation
ume of blood or, often, none at all.
Tail-clipping is used in salamanders and tadpoles; toe-
clipping is used in frogs. These techniques are more
Urine collection
commonly used in field studies than in a clinical setting
and are the least advisable options for blood collec- Collection of urine from amphibians is dependent on
tion.36 The procedures are painful, so the use of local chance. Although most species have a thin-walled uri-
anesthetic, such as 2% lidocaine dripped gently onto nary bladder, catheterization or cystocentesis is not
the target digit, is recommended. Application of an usually performed.1 Some individuals will urinate
antiseptic, such as Bactine, may prevent secondary when they are captured or initially handled, but most
bacterial infections. If toe-clipping is performed, will not, particularly if they have spent time in a
transport container before being handled. Urinalysis and plasma enzymes can be determined. Diagnostic
tests (specific gravity/density, protein, etc.) can be run laboratories have equipment capable of processing
with amphibian samples to varying relevance. For minute amounts of plasma but need to be contacted
instance, specific gravity will not indicate renal func- prior to shipping to ensure optimal preanalytic proce-
tion but will reflect plasma density (see urinalysis sec- dures and a sufficient sample volume.
tion).1,14 Urinalysis should be performed within a few
hours of collection for best results.
Hematology
Sample handling Blood cell count

Given the small size of most amphibians, unless blood Hemocytometry remains the most accurate method
collection is terminal, only small sample volumes may available for the amphibian CBC (including WBC,
be obtained. However, small amounts will suffice to RBC, and thrombocyte determinations), as automated
obtain a CBC, make a blood smear for a differential analyzers cannot differentiate between amphibian
WBC count, evaluate RBC and WBC morphology and RBC, leukocytes, and thrombocytes. Because the man-
detect hemoparasites, determine the PCV, and mea- ual method is time-consuming (20–30 minutes for
sure total proteins and other select chemical analytes. complete counts of the 3 blood cell types) and requires
Prioritization of the sample depends on the analysis experience and skill, it is avoided by all but the most
desired but the best use of a small blood sample resolute clinicians and researchers. Hemocytometry
(0.07 mL or less) is to prepare a blood smear, and still provides a better estimate than indirect methods
determine the CBC, PCV, and total proteins. More from back-calculations on blood smears and is the
voluminous samples allow automated plasma bio- appropriate way to establish RIs for amphibian species.
chemistry analysis. Blood can be collected directly into Natt–Herrick’s solution, which can be stored in the
a capillary tube or into a syringe and then transferred refrigerator for up to 2 years, can be prepared follow-
to heparinized capillary tube(s) or microtainer(s), ing the original formulation37, purchased from a labo-
depending on the available volume. ratory or university, or from a commercial provider in
One should prioritize analyses to derive the most premeasured pipettes.
diagnostic information, particularly from small sam- To calculate the cell counts using a hemocytome-
ples (Figure 3). First, a small drop from the hep- ter, 20 lL of heparinized blood are transferred into a
arinized capillary tube (Fisherbrand) or syringe Unopette collection pipette (Becton Dickinson, Frank-
should be used immediately after collection to make a lin Lakes, NJ, USA), one of its analogs such as the
blood smear. The blood smear should be air-dried and Bryopette (Bioanalytic GmbH, Umkirch, Germany), or
fixed (70% methanol) or stained (Wright–Giemsa; any other pipette capable of measuring exactly 20 lL,
Siemens Healthcare, Erlangen, Germany or Diff-Quik; and are placed in a volume of a previously prepared
Siemens Healthcare Ltd., Oakville, ON, Canada) Natt–Herrick solution to achieve a 1:100 dilution (ie,
within 24–48 hours for best results. Then, the exact 20 lL of blood in 1.98 mL of Natt–Herrick solution).
volume of blood necessary for a CBC count (generally Both grids of a hemocytometer or Neubauer chamber
20 lL) is transferred into a purpose-specific micropip- are filled with the Natt–Herrick-diluted blood. The RBC
ette to be mixed with premeasured dye, such as Natt– are counted in the central and 4 corner small squares
Herrick solution (Figure 4A,B). Finally, the capillary within the central large square. Leukocytes are counted
tube is plugged and centrifuged to measure the PCV, in all 9 large squares, and thrombocytes in the center
and separated plasma is used to measure total solids large square.38 To ensure that no cell is counted twice,
(protein) with a refractometer. Care should be taken the cells touching the upper and left edges of a square
to avoid glass shards in the sample, as they will dam- are counted and the cells touching the right and lower
age the equipment. If more than one capillary tube is edges are ignored. If the discrepancy between WBC
collected, the plasma from one animal can be pooled counts from the 2 grids is > 15%, the full counting pro-
and measured and analyzed on an automated bench- cedure is repeated. The RBC 9 1012/L count equals
top analyzer for expanded biochemical analysis the number of erythrocytes counted in the 4 corner
instead of a refractometer reading. Alternatively, and one center small squares, multiplied by 0.005; the
plasma can be shipped to a diagnostic laboratory WBC 9 109/L count equals the number of WBC
where a more accurate total protein measurement counted in the entire chamber (ie, all 9 large squares)
can be obtained, and additional analytes, such as multiplied by 0.111; the thrombocytes 9 109/L count
urea, albumin, globulins, cholesterol, triglycerides, equals the number of thrombocytes counted in all 15
Forz
Figure 3. Stepwise algorithm to maximize diagnostic information

obtained from one anticoagulated amphibian blood sample.
small squares (multiplied by one). These formulae only B

apply at a 1:100 dilution.
Distinguishing between erythrocytes, leukocytes,
and thrombocytes of amphibians can be challenging.
Scanning the stained blood smear is helpful to gauge
the shape and size of the species’ blood cells before
beginning the hemocytometer count. Although cell
counting is usually performed soon after collection,
the cells in blood diluted in Natt–Herrick’s solution
right after collection and then refrigerated maintain
their integrity for up to 2 years11 due to the presence
of formalin, and, counts can be performed days or
weeks after collection without compromising the qual- C
ity of the results. Distinguishing cell types, however,
becomes difficult in older samples as differential stain-
ing properties are lost and leukocytes tend to aggre-
gate, and may thus be confused with thrombocytes.
Aggregation or clumping of leukocytes will also make
WBC counts less accurate.
Published CBC counts in amphibians are rare;
Figure 4. Amphibian blood processing for a CBC in the hemocytome-
most rely on indirect determinations from smears
ter using Natt–Herrick solution. Capillary action allows for transfer of
rather than on hemocytometer counts and follow dif- blood from syringe to heparinized capillary tube (A) and from capillary
ferent sampling and analyzing techniques, so that their tube to Unopette (B) or to any other pipette capable of measuring an
value to diagnostic investigation is minimal. Inten- exact volume for the CBC. The predetermined volume is mixed with
tional or unwitting inclusion of thrombocytes when Natt–Herrick solution, which stains cells allowing differentiation
reporting WBC counts further limits validity of some between erythrocytes, leukocytes, and thrombocytes in a hemocy-
reports. Clinicians, diagnosticians, and researchers tometer (Neubauer) chamber (C): Erythrocyte (oval unmarked cell),
leukocyte with dark granules (black arrow), and ovoid thrombocyte
should be wary of reports that do not mention throm-
(black arrowhead) of a Green frog, Rana (Lithobates) clamitans. Cells
bocytes specifically, as these cells could have been mis- that are difficult to identify (arrowheads) require fine adjustments of
takenly counted as lymphocytes. Lacking specific the microscope focus plane to visualize dark blue cytoplasm indicating
information regarding a particular amphibian species, a leukocyte (open arrowhead), and pale cytoplasm indicating a throm-
interpretation of abnormalities usually follows gene- bocyte (gray arrowhead). Natt–Herrick stain, Bar = 20 lm.
ralities established for related species or, more fre-
quently, other vertebrates. This extrapolation makes
assumptions that are unproved in amphibians. Only
WBC differential blood cell count
CBC and differential counts from a conspecific of simi-
lar age and maintained under equal environmental Leukocyte differential counts follow standard method-
conditions may be helpful in the interpretation of ology. Immediately after collection, one end of a
results from a particular individual. blood-filled capillary tube is gently tapped on a glass
slide to produce a small drop of blood that can then Red blood cell counts vary among amphibian spe-
easily be smeared following standard procedures, air- cies, mainly because of differences in erythrocyte size,
dried and stained with a Wright–Giemsa or Diff-Quick so values for one species are of little use as reference for
stain. At least 100 WBC should be counted, and another. Because of this difference in size, the PCV of 2
the absolute number of each type (ie, monocyte, lym- different species may be similar, while their RBC
phocyte, basophil, eosinophil and neutrophil) is then counts are different. For instance, RBC in Wood frogs
extrapolated from the hemocytometer WBC count.39 (Rana sylvatica or Lithobates sylvaticus) are roughly twice
Thrombocytes should not be included in the count. as large as those from African (tropical) Clawed frogs
Heparinized blood, which will likely be used to make (Xenopus tropicalis), so that even if their PCV is similar
the smear, may result in a diffuse blue hue to the cells (30% [19–41] vs 41% [27–54], respectively) their
and, occasionally, the background. actual RBC numbers are quite different (0.4 [0.3–0.6]
and 1.5 [1–2] 9 1012/L, respectively).11,12 A drop in
the PCV, probably along with a reduced RBC count
Red blood cells
and an increase in the percentage of immature ery-
Amphibian erythrocytes vary in size based on the spe- throcytes, has been found in intense infections with
cies but are ovoid and generally larger than those intraerythrocytic parasites, such as Hepatozoon spp., in
found in most other vertebrates.1,39 The erythrocytes Wild Green frogs, Rana [Lithobates] clamitans, and with
of the aquatic salamander Amphiuma sp., measuring unidentified hemogregarines in Australian Tree frogs,
close to 70 lm in length, are the largest of all verte- Litoria caerulea and L infrafrenata.10 In American
brates.40 Erythrocyte size (length and width) variation Bullfrogs (Rana [Lithobates] catesbeiana), PCV tends to
in vertebrates has been long known to be directly pro- increase as the environmental temperature decreases
portional to the size of the species genome: the longer in the winter.44
the genome, the larger the erythrocyte.41 Size differ-
ences between the salamander and frog groups of
Thrombocytes
amphibians are frequently inversely associated with
the intensity and extent of metamorphic change. Amphibian thrombocytes are often ovoid, occasionally
Thus, frogs, particularly those whose tadpoles inhabit round, and have a dark nucleus. Unlike lymphocytes,
vernal (temporary) pools and have only a short time however, chromatin in thrombocytes is not randomly
to complete metamorphosis, have shorter genomes clumped but condensed at the center of the nucleus,
and smaller erythrocytes.42 Salamanders, particularly and their cytoplasmic edges are seldom smooth (Fig-
neotenic species such as Amphiuma and Axolotl sps., ure 5B). With Natt and Herrick’s stain, thrombocytes
have much larger erythrocytes.43 Amphibian erythro- are ovoid or round, smaller than the erythrocytes, and
cytes are nucleated, both in larvae and adults, except with a small rim of pale blue cytoplasm that is lighter
in a few species of salamanders, such as the slender than that of the leukocytes (Figure 4C). Amphibian
salamanders of the genus Batrachoseps, where large thrombocytes are analogous to mammalian platelets
proportions are anucleate.1 Erythrocytes become and instrumental during coagulation. Thrombocyte
smaller and reduce their endoplasmic reticulum counts should be based on hemocytometer counting as
throughout metamorphosis. When using Natt–Herrick estimates from direct blood smear examination are
solution, erythrocytes are commonly the largest blood usually impeded by clumping and uneven distribution.
cells, ovoid, with a pale staining cytoplasm and
slightly darker nucleus; immature erythrocytes have a
White blood cells
slightly darker cytoplasm, are smaller and they may
be a bit rounder, but they usually retain their ovoid Leukocytes in amphibians include lymphocytes,
shape and are larger than leukocytes or thrombocytes monocytes, eosinophils, basophils, and neutrophils or
(Figure 4C). heterophils (Figure 6A–F).39 In vertebrate species, the
Red blood cells are counted via hemacytometry or term neutrophil is usually applied to polymorphonu-
estimated via PCV. Cell morphology and proportional clear cells whose granules contain myeloperoxidases
abnormalities in RBC, such as conspicuously immature and are colorless when stained with Wright–Giemsa,
erythrocytes (slightly smaller than other RBC and with whereas heterophils have prominent orange to pink
bluer cytoplasm), and circulating intra- or extracellular cytoplasmic granules that lack such enzymes.45 As nei-
pathogens, such as hemogregarines and trypanosomes, ther heterophils nor neutrophils contain visible cyto-
can be identified through microscopic examination of plasmic granules in images recorded for amphibian
the blood smear (Figures 5A and 6G–L). species and as the few that have been characterized do
Forz
information on WBC counts is scattered and frag-

A
mented.1,14
Attribution of specific significance to values
outside RIs is challenging. Metamorphosis alters
amphibian metabolism, and the elevated levels of
glucocorticoids required may result in neutrophilia
and lymphopenia.1,14 However, the precise effects of
metamorphosis on the numbers or proportions of cir-
culating leukocytes are poorly described and under-
stood, due to varying research methodologies among
researchers and small numbers of animals per study.
Similarly, an increase in the neutrophil:lymphocyte
(N:L) ratio can be associated with increased plasma
cortisol, and thus is considered an indicator of stress.47
B The N:L ratio is, however, nonspecific and needs to be
compared to an N:L ratio from conspecifics of the
same age, sex, and environmental conditions. A small
study in American Bullfrog tadpoles suffering from
chytridiomycosis suggests that severely affected ani-
mals may have a higher proportion of neutrophils
than those with mild lesions, although it was unclear
whether this was an inflammatory or a stress
response, or both.48 In reptiles, blue-gray intracyto-
plasmic viral inclusions have been reported in circu-
lating leukocytes infected with Ranavirus sp.49
Amphibians develop similar inclusions, albeit bright
pink-red when stained with Wright–Giemsa, as noted
in circulating leukocytes of Wood frogs experimen-
Figure 5. Amphibian blood smears. Wright–Giemsa, Bar = 20 lm. (A) tally infected with frog virus 3 (Ranavirus sp.).12 Both
Blood smear from a Green frog (Rana [Lithobates] clamitans). Among the
the inclusions found in Wood frogs and similar ones
3 immature erythrocytes (blue-staining cytoplasm), there is an atypical
cell with a large round nucleus and little deeply blue cytoplasm which
reported in circulating lymphocytes of European
may be a reactive lymphocyte or an immature erythrocyte. (B) Cuban Common frogs (Rana temporaria) that died of ranavi-
Tree frog (Osteopilus septentrionalis) blood cell with a cytoplasm full of rosis were immunohistochemically positive for rana-
fine black (melanin) granules (arrow). Thrombocytes are small and ovoid, virus antigen.12,50
with raggedy cytoplasmic edges and often clustering together (black Hematologic RIs for selected anuran and caudate
arrowhead); lymphocytes are round and slightly larger, with deeply blue species compiled from published literature are
cytoplasm and clumped nuclear chromatin (white arrowhead).
included in Tables 3 and 4. Data were used if they were
derived from at least 10 subjects, determined through
contain myeloperoxidase46, we will use the term known and acceptable methodologies, and included
neutrophil. diagnostically relevant analytes. The source informa-
Leukocytes are round, with moderate amounts of tion was standardized following the recommendations
pale cytoplasm that sometimes includes eosinophilic or of the ASVCP9 and is presented as mean (RI = mean
dark blue granules when stained with Natt and 2 SD, or 95% CI) or median (RI = 2.5th–97.5th
Herrick’s solution (Figure 4C). Absolute WBC counts quantile) for normally and nonnormally distributed
are based on hemocytometry. Morphology and pro- data, respectively. When distribution of the data was
portional properties including toxic change, the pres- not known, normality was assumed.
ence of atypical lymphocytes, and circulating intra- or
extracellular pathogens are identified through exami-
nation of the stained blood smear. Atypical cells, often Hemoparasites
difficult to categorize, are not uncommon in amphib-
ian smears (Figure 5). Leukocytes in amphibians are Hemoparasites are detected via direct examination of a
thought to have similar inflammatory and immune dried stained blood smear or via PCR. Hemoparasites
functions as those in other vertebrates but published found on examination of amphibian blood smears
A B C
D E F
G H I
J K L
Figure 6. Blood cells (A–L) and hemoparasites (G–L) of the Green frog (Rana [Lithobates] clamitans). Leukocytes resemble those in other vertebrates,
while erythrocytes and thrombocytes are nucleated. Wright–Giemsa stain, Bar = 10 lm. (A–L) Erythrocytes; (A) neutrophil; (B) eosinophil; (C) basophil; (D)
lymphocyte; (E) monocyte; (F) aggregate of thrombocytes. (G) Numerous gamonts of Hepatozoon sp in the cytoplasm of erythrocytes, accompanied by
numerous immature erythrocytes with blue cytoplasm (Polychromasia); (H) nuclear fragmentation associated with Hepatozoon clamatae infection; (I) con-
current infection with H clamatae and H catesbianae, the latter does not fragment the nucleus. (J–L) Trypomastigoes of Trypanosoma sp., spherical form
probably representing Trypanosoma chattoni (J), and elongated stages with a visible undulating membrane probably T rotatorium (K) and T pipiens (L).
include, but are not restricted to, intraerythrocytic intraerythrocytic rickettsial organisms such as those
gamonts of Hepatozoon sp., various species of Try- commonly found in some wild salamanders.51 These
panosoma spp. in the plasma (Figure 6G–L), and parasites can be found in wild-caught individuals or
Forz
captive-bred colonies with outdoor access. Frogs or, less frequently, glycerol) in tissues and blood23, and
acquire Hepatozoon spp. infections solely by feeding on thus have a physiologic glucosuria if sampled soon
infected Culex spp. mosquitoes, while the mosquitoes after thawing or during frozen hibernation. In Wood
must bite infected individuals to become vectors.52 frogs that have recently thawed (one hour) after hiber-
Interestingly, some frog trypanosomes remain in the nation, blood glucose levels can be > 400 mmol/L
renal vasculature during the night and circulate sys- (> 7000 mg/dL), while glucose in individuals that
temically only during the daytime. To detect infection, have not undergone freezing is only around
it is best to sample at mid-day.53 Infection with Try- 1.5 mmol/L (27 mg/dL). In another freeze-tolerant
panosoma spp. has not been linked to clinical disease. species, the Gray Tree frog (Hyla versicolor), response is
dependent on age: glucose levels of adults remain
unchanged during freezing, while glycerol concentra-
Biochemical Panel tions increase from 6.8 mmol/L to 423 mmol/L;
immature Gray Tree frogs have a mild increase in both
Most biochemical tests, including those for all ana- glycerol (0.1–16.3 mmol/L) and glucose (1.46–
lytes mentioned in this section, may be run on plasma 25.9 mmol/L).23
as well as serum, so blood collected for hematology
with a heparinized syringe or capillary tube may also
Cholesterol and corneal lipidosis in Tree frogs
be used for biochemistry panels. The normal color of
amphibian plasma ranges from clear to yellow or even Captive Cuban Tree frogs (Osteopilus septentrionalis),
blue, as in the case of the Japanese Giant salamander like some other tree frog species, are prone to obesity
(Andrias japonicus) and the White-Lipped Tree frog.1,10 and corneal lipidosis, which are associated with
Plasma should be separated immediately after cen- marked increases in serum cholesterol and triglyceride
trifugation and then kept refrigerated or frozen until concentrations. Cuban Tree frogs affected with corneal
processed or shipped to a diagnostic laboratory. Ide- lipidosis have cholesterol concentrations averaging
ally, RIs should be determined from conspecifics not 27.5 mmol/L or 1062 mg/dL, while cholesterol in
only of similar age and environment but also sampled wild-caught, nonaffected frogs averages 3.86 (0–8.26,
and tested by the same method, as lack of consistency 95% CI) mmol/L or 149 (0–319, 95% CI) mg/dL.19
in methodology diminishes the validity of RIs. Most Australian Green Tree frogs, also known as White’s
biochemical analyzers measure enzyme activity at a Tree frogs (Litoria caerulea) are also prone to obesity
mammalian body temperature (37°C), higher than and corneal lipidosis1, but no information regarding
the temperature at which amphibian enzymes func- the cholesterol levels of affected or nonaffected indi-
tion. Thus, measurements of enzymatic activity must viduals is available.
be interpreted with caution and may vary depending
on the instrument used, making it indispensable to
Nitrogenous waste-products, renal function, and
use the same instrument if comparisons are to be
hydration status
made between conspecifics or between samples from
an individual over time. Biochemical panels have Renal function and/or hydration status can be evalu-
been determined for few amphibian species. Bio- ated through measurement of nitrogen waste-products
chemical RIs for selected anuran and caudate species, in plasma, but it must be remembered that amphibians
compiled from published literature following the same in general have a high tolerance for urea in circulation
method used for the hematological RIs, are included as they need it to enhance water uptake through the
in Tables 5 and 6. Patchy information is available on skin.26 One must measure the appropriate form of
alterations in other species or groups. Almost nothing nitrogen, remembering that the type of waste product
is known of the diagnostic significance of hepatic, in amphibians depends on species-specific adaptations
pancreatic, muscular, or cardiac enzymes in amphib- to native environments: ammonia for aquatic adults
ian blood. and larvae (ammonotelic), urea for adult terrestrial
amphibians (ureotelic), and uric acid for some tree
frogs (uricotelic).1,14 Renal disease, be it infectious,
Glycerol and glucose in freeze-tolerant frogs
metabolic or neoplastic, is one of the main causes of
Freeze-tolerant over-wintering amphibians such as severe and generalized edema due to increased
Wood frogs, Spring Peepers (Pseudacris [Hyla] crucifer), glomerular filtration of protein (hypoproteinemia) or
and Western Chorus frogs (Pseudacris triseriata), accu- decreased tubular reabsorption of electrolytes (eg,
mulate low molecular weight carbohydrates (glucose hyponatremia).26
16
Table 3. Hematologic RI for select free-living (**) or captive (*) anuran species: American Bullfrog (Rana catesbeiana, Rc)3, Northern Leopard frog (Rana pipiens, Rp)5, Wood frog (Rana sylvatica, Rs)12,
African Clawed frog (Xenopus laevis, Xl)7, African (tropical) Clawed frog (X tropicalis, Xt)11, Sapito de Jardın (Bufo fernandezae, Bf)20, Green (White’s) Tree frog (Litoria caerulea, Lc), and White-Lipped Tree
frog (Litoria infrafrenata, Li).10
Northern African African Green

American Leopard Wood Clawed (Tropical) Sapito (White’s) White-Lipped
Analyte
Bullfrog* frog** Frog* Frog* Clawed Frog* de Jardın** Tree Frog** Tree Frog**
(Abbreviation) Units, SI
Data type Conventional Mean (RI)† Median (RI)‡
Sample size 302 12–56 26–40 10 33–41 17 80 66

Amphibian clinical pathology review
WBC 9109/L 20.5 (11.3–29.7) 5.5 (0.7–10.3) 7.65 (2.2–13.1) 4.59 (2.92–9.16) 20.7 (11.5–36.0)‡ 4.5 (1.18–7.82) 15.9 (6.7–34.9) 21 (6.5–47.9)
9103/lL
Absolute 9109/L 0.46 (0–0.9) 11.8 (3.7–26.7)‡ 0.79 (0.43–1.15) 3.3 (0.9–7.7) 4.2 (0.6–17.1)
neutrophils 9103/lL
Relative % 60.9 (36.1–85.7) 26.5 (3.7–49.3) 6.83 (0.4–13.3) 26.5 (7.5–45.5) 54.7 (26.7–82.7) 19 (0–45.8) 21.5 (7–42) 20 (5–50.3)
neutrophils
Absolute 9109/L 0.09 (0–0.25)‡ 0.2 (0–1.0)‡ 0.19 (0.11–0.27) 0.4 (0–3.1) 0 (0–2)
eosinophils 9103/lL
Relative % 5.8 (2.6–9) 7.3 (3.1–11.5) 1.55 (0–4.5)‡ 1.2 (0–5) 1.2 (0–4.0)‡ 5 (0–13.6) 2 (0–11) 0 (0–10.6)
eosinophils
Absolute 9109/L 0.8 (0.1–1.5) 0.4 (0–1.1)‡ 1.34 (0.78–1.9) 0 (0–1.1) 0 (0–4)
basophils 9103/lL
Relative % 3.5 (1.1–5.9) 4.4 (0–10.6) 10.65 (5.9–14.8)‡ 40.5 (16.5–64.5) 2.2 (0–7.0)‡ 32 (0–76.5) 0 (0–7) 0 (0–31)
basophils
Absolute 9109/L 5.76 (1.3–10.2) 7.2 (2.6–11.7) 1.71 (1.15–2.27) 10.7 (3.9–27.1) 12.2 (3.2–34.7)
lymphocytes 9103/lL
Relative % 26.8 (17–36.6) 53.4 (23.8–83) 76.85 (63.7–90.0) 30.1 (6.3–53.9) 37.2 (11.8–60.7) 41 (0–82.68 67.5 (40.2–88) 70 (33.4–85)
lymphocytes
Absolute 9109/L 0.13 (0–0.35)‡ 1.0 (0.2–2.5)‡ 0.01 (0–0.03) 1.3 (0.3–4.7) 1.1 (0.1–6.9)
monocytes 9103/lL
Relative % 2.9 (0.7–5.1) 11 (1.4–20.6) 1.64 (0–3.4) 1.6 (0–4) 4.7 (1.0–10.0)‡ 1 (0–2.52) 7 (2–18) 6 (1–21.3)
monocytes
RBC 91012/L 0.42 (0–1.82) 0.32 (0.16–0.48) 0.41 (0.25–0.57) 0.75 (0.53–1.09) 1.5 (1.0–2.0) 0.505 (0.24–0.76) 0.74 (0.42–1.02) 0.72 (0.4–1.12)
9106/lL
PCV % 30.1 (19.3–40.9) 29.5 (18.6–40.5) 40.8 (27.3–54.4) 38 (23–48) 30 (19.4–48.6)
HGB g/L 68 (38.4–97.6) 67.5 (27.5–107.5) 91.8 (35.8–147.8) 93 (41–126) 70 (33–117)
g/dL 6.8 (3.84–9.76) 6.75 (2.75–10.75) 9.18 (3.58–14.78) 9.3 (4.1–12.6) 7 (3.3–11.7)
HCT % 24.65 (5.51–43.79) 27.37 (12.01–42.73)
Thrombocytes 9109/L 7.3 (1.1–13.5) 8.3 (1.3–15.2) 18.52 (12–24.88) 14.6 (6.8–22.5) 4.81 (0–15.1) 27.3 (13.3–49.1) 31.9 (20–62.5)
9103/lL
Blood pH 7.36 (7.06–7.66)
† th th ‡
Reference interval = 95% CI or 2.5 –97.5 Quartile . Distribution of original data: normal (Rc, Xt), nonnormal (Rf, Lc, Li, Xt‡), unknown (Rp, Xl). Values below zero or incompatible with life are reported as 0.
Forzan et al
Vet Clin Pathol 0/0 (2017) 1–23 ©2017 American Society for Veterinary Clinical Pathology
Forz
Table 4. Hematologic RI for select short-term captive (*) or free-living (**) caudatan species: Japanese Newt (Cynops pyrrhogaster, Cp),21 Eastern and
Ozark Hellbenders (Cryptobranchus alleganiensis alleganiensis, Caa, and C a bishopi, Cab).4
Analyte Units, SI Conventional Japanese Newt* Eastern Hellbender** Ozark Hellbender**
Sample size 23 37–38 42

WBC 9109/L 3.9 (2.7–5.1) 4.6 (3.7–5.5)
9103/lL
Relative neutrophils** % 28 (3.06–52.94) 30.3 (21.3–39.2) 35.1 (29.2–41.1)
Relative eosinophils % 4 (0–10.72) 4.1 (1–7.3) 10.9 (8.8–13)
Relative basophils % 57 (26.3–87.7) 6.9 (4.3–9.4) 4.3 (2.7–6)
Relative lymphocytes % 3 (0–6.84) 54.6 (43.7–65.6) 49.3 (41.8–56.8)
Relative monocytes % 6 (0–15.58) 1.1 (0–2.3) 0.6 (0–1.3)
RBC 91012/L 2.28 (0–5.1)
9106/lL
PCV % 40 (21.78–58.22) 36.1 (31.7–40.6) 44.3 (41.3–47.4)
Thrombocytes 9109/L Not identified or counted Clumped, not counted Clumped, not counted
9103/lL
All data are reported as mean (RI = 95% CI) and calculated from a single population (Cp) or various populations weighted by sample size (Caa, Cab).
Distribution of original data: normal (Caa, Cab), unknown (Cp). Values below zero are reported as 0.
Electrolytes, osmolality, and blood gases Proteins

Osmolality, the concentration of active particles in an Blood proteins (albumin, globulins, total proteins,
aqueous solution, in this case serum or plasma, is usu- fibrinogen, and others) may be measured by refrac-
ally closely associated with the hydration state and the tometry, biochemical means (bromocresol green or
concentration of the most diagnostically important biuret method), or plasma protein electrophoresis
electrolytes: sodium and potassium. Various methods (EPH). Total protein in blood includes albumin and
of measuring osmolality and electrolytes exist, most globulins and is commonly estimated from the refrac-
require relatively large volumes of serum or plasma, so tive index measured with a refractometer or calculated
their determination may not be feasible except in the via spectrophotometric methods (biuret reaction) used
larger amphibians. in reference laboratories. Refractometry is a quick,
Amphibians are tolerant of wide fluctuations in dependable, and inexpensive way of measuring total
the osmolality and composition of their plasma. This proteins in serum or plasma. Total protein results
adaptation results in great variability in clinical chem- obtained with the refractometer may, however, be
istry values depending on environmental and physio- artificially inflated and unreliable when other solid
logic conditions and complicates interpretation of analytes occur in high concentrations in the plasma
results. Over-wintering frogs and salamanders inten- or serum, as the instrument measures total solids.
tionally undergo dehydration, thus healthy animals A refractometer reading above the RI, or significantly
close to or in hibernation should have increased higher than the reading from a conspecific, should be
plasma osmolality.14,22 In Green (White’s) Tree frogs, confirmed through chemical protein measurement as
reduced plasma osmolality, and sodium, potassium, high concentrations of bilirubin, cholesterol, and try-
magnesium, and chloride concentrations were glicerides increase the refractive index of plasma.39
found in 6 cases of severe B dendrobatidis infection The biuret reaction, used by most reference laborato-
(chytridiomycosis).54 Wild-caught Mountain Yellow- ries, is the most reliable method of measuring total pro-
Legged frogs (Rana muscosa) infected with B dendroba- teins, and it is recommended over refractometry if a
tidis developed hyponatremia and hypokalemia, while high concentration of other solid analytes such as
acid–base balance and blood gases remained unaf- cholesterol and triglycerides is suspected. Although
fected.55 changes in osmolarity may influence the total proteins
An acidic substrate in the habitat (pH ≤ 4–5) can measured by refractometry, the effect is seldom clini-
interfere with the sodium–potassium pump in the skin, cally significant.
and result in hyponatremia, often accompanied by The test used by most in-house automated analyz-
subcutaneous edema in frogs and toads. Mechanical or ers to measure albumin is based on the chemical bind-
infectious alterations to the lymph hearts also result in ing of albumin to bromcresol green dye; and, as
altered water and electrolyte imbalance and may result binding varies depending on the animal species, it may
in generalized subcutaneous edema.26 not be valid in all amphibians. It is also the least reliable
Table 5. Biochemical RIs for select free-living (**) or captive (*) anuran species: American Bullfrog (Rana catesbeiana, Rc)3, Cuban Tree frog (Osteopilus
septentrionalis, Os)19, African Clawed frog (Xenopus laevis, Xl)7, African (tropical) Clawed frog (X tropicalis, Xt)11, Green (White’s) Tree frog (Litoria caer-
ulea, Lc), and White-Lipped Tree frog (Litoria infrafrenata, Li).10
Cuban African African Green

Analyte Units, SI American Tree Clawed (Tropical) (White’s) White-lipped
(Abbreviation) Conventional Bullfrog* Frog** Frog* Clawed Frog* Tree Frog** Tree Frog**
Sample size 302 29 166 24 80 66

Data format Mean (RI)† Median (RI)‡
Albumin-to- 0.54 (0.3–0.78) 0.7 (0–3.28)
globulin ratio
Albumin g/L 15.8 (9.2–22.4) 10 (0–35.8)
g/dL 1.58 (0.92–2.24) 1 (0–3.58)
ALP U/L 157 (155.1–158.9) 148 (19–277)
ALT U/L 12.4 (10.52–14.28) 21 (0–46.8)
Amylase U/L 270 (0–682)
AST U/L 453 (0–1587) 91 (30–362) 67 (26–370)
Bilirubin, lmol/L 1.2 (0–6.35)
total mg/dL 0.07 (0–0.33)
Bilirubin, lmol/L 0.8(0–5.9)
indirect mg/dL 0.05 (0–0.3)
Bilirubin, lmol/L 0.3 (0–5.45)
direct mg/dL 0.02 (0–0.28)
Calcium mmol/L 2.08 (1.38–2.78) 2.2 (0.9–3.49) 2.94 (2–4.4) 2.45 (1.8–4.7)
mg/dL 8.31 (6.89–9.73) 8.9 (3.7–14.0)
Chloride mmol/L 108.6 (96–121.2) 82.5 (67–98)
mEq/L 108.6 (96–121.2) 82.5 (67–98)
Cholesterol mmol/L 1.6 (0.88–2.32) 3.86 (0–8.26) 6.01 (0–14)
mg/dL 61.8 (34.0–89.6) 149 (0–319) 232 (0–541.2)
CK U/L 432 (262–602) 1658 (0–6502) 470 (75–2555)* 399 (73–3420)
Creatinine lmol/L 42.7 (21.14–64.26) 35.4 (0–262.2)
mg/dL 0.48 (0.24–0.73) 0.4 (0–2.98)
Iron lmol/L 25.43 (14.8–36.0)
lg/dL 142.1 (82.7–201.1)
Fibrinogen g/L 7.9 (5.7–10.1)
mg/dL 268–.7 (193.1–343.54)
Globulins g/L 23 (0–48.77)
g/dL 2.3 (0–4.88)
Glucose mmol/L 2.77 (1.43–4.11) 2.9 (0.07–5.73) 3.6 (1.9–6) 3.3 (2–6.8)
mg/dL 49.9 (25.8–74.0) 53 (1.46–104.54) 64.9 (34.2–108.1) 59.4 (36–122.5)
GGT U/L 4 (0–29.8)
LDH U/L 117 (73–161) 1809 (0–4592)
Lipase U/L 98 (0–201)
Phosphorus mmol/L 2.85 (1.69–4.01) 2.39 (0.84–3.94) 1.33 (0.72–2.64) 1.3 (0.6–2.7)
mg/dL 8.82 (5.23–12.41) 7.4 (2.25–12.55)
Potassium mmol/L 3.62 (2.2–5.04) 4 (1.42–6.58) 5.9 (3.2–9.5) 3.7 (1.9–3.1)
mEq/L 3.62 (2.2–5.04)
Sodium mmol/L 118.6 (96.2–141) 123 (97.2–148–.8) 110 (101–123) 106 (99–114)
mEq/L 118.6 (96.2–141)
Total g/L 43.4 (30.2–56.6) 33 (7.23–58.77) 39.1 62 (39–85.9) 35 (18–56.3)
proteins g/dL 4.34 (3.02–5.66) 3.3 (.72–5.88) (24.2–54.0)
Triglycerides mmol/L 0.48 (0.26–0.7) 0.4 (0–1.12) 1.3 (0–3.36)
mg/dL 42.5 (23–61.9) 35.4 (0–99.1) 117 (0–297.38)
Urea mmol/L 3 (1.76–4.24) 1.8 (0–11.1)
Urea mg/dL 8.4 (4.93–11.9) 5 (0–30.8)
nitrogen
(BUN)
Uric acid lmol/L 79.7 (0–422.1) 11.89 (0–165.2) 25 (4–86) 12 (0–27)§
mg/dL 1.34 (0–7.1) 0.2 (0–2.78)
Reference interval = 95% CI† or 2.5th–97.5th Quartile‡. Data distribution: normal (Rc, Xt), nonnormal (Lc, Li), unknown (Os, Xl). Values below zero or
incompatible with life are reported as 0. §n = 65.
Forz
method in 2 species of Australian Tree frogs.10 If unavailable. If metabolic bone disease is suspected, a
plasma is deeply red-tinged, suggesting severe hemoly- whole-body radiographic examination is indicated to
sis, neither biuret nor bromcresol reactions will yield detect folding fractures, bone cortical thinning, or
valid protein or albumin measurements. decreased bone density.59 Metabolic bone disease in
Plasma EPH calculates the relative concentrations large amphibians may also be associated with a diet
of the various protein fractions and determines the that includes rats or mice, as the high levels of vitamin
concentrations of albumin and globulins when run A contained in rodents are thought to interfere with
alongside an accurate measurement of total protein the absorption and use of vitamin D.1
concentration. This is the most accurate, time-con- Heavy metals (Hg, Pb, Cd, Cr, and Co) in whole
suming, and expensive method for evaluating blood blood of free-ranging adult Eastern and Ozark Hellben-
proteins. It requires only very small volumes of plasma ders (Cryptobranchus alleganiensis alleganiensis and
or serum (~ 5 lL) and may be the most appropriate C a bishopi, respectively) have been reported.4 Because
when measuring albumin and globulins in at least none of the sites sampled included individuals from
some species of amphibians.10 If the test is run on both subspecies, differences in concentration between
plasma, heparin and fibrinogen bands or spikes may be the 2 species is difficult to distinguish from the effect of
present. The validity of changes such as increased location. However, Eastern Hellbenders may have
acute phase proteins or monoclonal gammopathies higher concentrations of Hg and Pb, while Co may be
determined by EPH in amphibians is unknown. higher in the Ozark subspecies. Ranges incorporating
Metamorphosis requires an increase in protein all animals sampled (lg/g of whole blood) are: Hg
concentrations from a relatively low level in tadpoles (0.08–0.65), Pb (0.013–0.180), Co (0.07–1.41), Cr
to a concentration equal to, or slightly higher than, the (0.13–6.87), and Cd (< 0.002–0.11). Mercury levels in
baseline of adults.14 Total proteins in plasma of Ameri- general increase proportionally to body mass and
can Bullfrog tadpoles increase incrementally during length.
metamorphosis from 14.6 g/L in early larval stages to Hypovitaminosis A is a commonly mentioned clin-
51.6 g/L in froglets56, values that are close to the upper ical concern in captive amphibians and may be associ-
end of the adult RI (43 [30–56] g/L).3 Albumin concen- ated with “short-tongue syndrome”, a condition in
trations also increase during metamorphosis of Bull- captive amphibians characterized by squamous meta-
frogs: from 0.6 g/L in early tadpole stages to 8.9 g/L in plasia of oral cavity and tongue epithelium.60 Little
froglets56, a concentration close to that of adult frogs information is available on adequate plasma or blood
(16 [9–22] g/L).3 Hibernating (over-wintering) levels of Vitamin A or D in most amphibian species,
amphibians have increased fibrinogen, heat shock pro- unfortunately. Recent reviews on vitamin A deficiency
teins, and glucose-transport protein concentrations.22 and supplementation of vitamin D and UV radiation in
Differential diagnoses for nonphysiologic increases in captive amphibians are available for those seeking
total proteins include active inflammation (globulin specific information.61,62
fraction) and dehydration (albumin portion).
Decreased total proteins may reflect a poor diet or
Hormones
suggest liver, gastrointestinal, or renal disease.1
Although amphibians have been the laboratory species
of choice when studying endocrinology, and reviews
Minerals, metals, and vitamins
on amphibian endocrinology exist63, most of the avail-
Calcium metabolism in some amphibian species able information has little or no clinical application.
changes with the season and life stage: in adults, cal- Free-ranging female Whistling frogs (Litoria ewingi)
cium level in plasma increases in spring and summer, have a marked increase in plasma corticosterone (from
decreases in winter, while in tadpoles, calcium < 1.8–13.8 ng/mL) after an episode of acute stress
increases as they approach metamorphosis.57 In free- (24 hours of captivity)47, suggesting that some
ranging Eastern Hellbenders (Cryptobranchus alleganien- endocrine responses may resemble those of other
sis)2 (Table 6) and wild-caught African Clawed frogs,8 vertebrates.
calcium levels in plasma and serum, respectively, are
slightly higher in females than males. A similar trend is
suggested for American Bullfrogs.58 Although meta- Urinalysis
bolic bone disease in frogs has been reported, informa-
tion on calcium concentration in amphibian plasma or Amphibian kidneys cannot concentrate urine above
on the calcium-to-phosphorus ratio are largely the osmolality of plasma. Therefore, in amphibians,
Table 6. Biochemical RIs for select free-living caudatan species: Eastern (Cryptobranchus alleganiensis alleganiensis, Caa)2,4 and Ozark Hellbenders
(C a bishop, Cab).4
Analyte (Abbreviation) Units, SI Conventional Eastern Hellbender Eastern Hellbender Ozark Hellbender
Sample size Males Females 37–38 42

Albumin g/L 12.2 (10.6–13.8) 11.9 (9.5–14.3) 9.8 (7.9–11.7) 11.3 (9.3–13.2)
g/dL 1.2 (1.1–1.4) 1.2 (0.9–1.4) 1 (0.8–1.2) 1.1 (0.9–1.3)
AST U/L 74.52 (0–150.72) 76.48 (0–170.72) 128.1 (84.8–171.5) 147.2 (117.1–177.4)
Calcium mmol/L 2.0 (1.72–2.28) 2.7 (1.66–3.74) 2.3 (1.9–2.7) 3 (2.7–3.3)
mg/dL 8.0 (6.7–9.0) 10.9 (6.7–15.1) 9.3 (7.7–10.8) 12.1 (11–13.3)
Chloride mmol/L 84.3 (81.7–86.9) 80.8 (79.0–82.7)
mEq/L
CK U/L 661.7 (0–2239.1) 488.52 (0–1088.7) 3703 (641–6765) 972 (0–3100)
Globulin g/L 26 (0–65.8) 20.3 (8.5–32.1) 21.7 (19.8–23.6) 22.3 (20.3–24.2)
g/dL 2.6 (0–6.6) 2.0 (0.8–3.2) 2.2 (2.0–2.4) 2.2 (2.0–2.4)
Glucose mmol/L 1.29 (0.59–1.99) 1.2 (0.6–1.8) 1.5 (0.9–2.1) 1.6 (1.1–2)
mg/dL 23.2 (10.6–35.8) 21.6 (10.8–32.4) 26.7 (31–5.6) 29.3 (21.6–37.1)
Phosphorus mmol/L 1.66 (0.8–2.52) 1.65 (0.77–2.53) 1.6 (1.2–2) 2.2 (2–2.4)
mg/dL 5.14 (2.48–7.8) 5.1 (2.38–7.83) 4.9 (0–9.9) 6.9 (6.3–7.5)
Potassium mmol/L 5.07 (2.23–7.91) 4.38 (2.02–6.74) 4.2 (3.4–5.0) 5.1 (4.5–5.6)
mEq/L 5.07 (2.23–7.91) 4.38 (2.02–6.74) 4.2 (3.4–5.0) 5.1 (4.5–5.6)
Sodium mmol/L 110.83 (109.33–112.33) 111 (108.6–113.4) 106.5 (104.3–108.8) 106.3 (104.7–107.9)
mEq/L 110.83 (109.33–112.33) 111 (108.6–113.4) 106.5 (104.3–108.8) 106.3 (104.7–107.9)
Total proteins g/L 31.5 (19.1–43.9) 31.9 (20.7–43.1) 31.8 (29–34.6) 32.5 (30.6–34.5)
g/dl 3.15 (1.91–4.39) 3.19 (2.07–4.31) 3.2 (2.9–3.5) 3.3 (3.1–3.4)
Urea mmol/L 0.8 (0.2–1.4) 1 (0.6–1.5)
mg/dL 2.5 (0.7–4.2) 3 (1.8–4.2)
Uric acid lmol/L 21.3 (13.5–29.2) 23.8 (17.3–30.3)
mg/dL 0.4 (0.2–0.5) 0.4 (0.3–0.5)
All data are reported as mean (RI = 95% CI); calculated from various populations weighted by sample size (Caa, Cab). Distribution of original data: normal
(Caa, Cab). Values below zero are reported as 0.
urine specific gravity/density is not an indicator of caused by a group of viruses in the Ranavirus genus
renal function but rather a reflection of plasma density. (Iridoviridae), are the most significant infectious dis-
A more relevant analyte to measure renal function eases currently affecting wild and captive amphib-
may be ammonia as its excretion increases during ians.66,67 Detection of infection with those pathogens
metabolic acidosis, particularly following episodes of can be achieved through PCR testing at various diag-
exhaustive exercise when ammonia concentration in nostic laboratories. A skin swab is sufficient to detect
urine may rise to over 200% of the upper reference Bd and Bsal by PCR. Unfortunately, reliable detection
limit.1 Crystals may be seen in urine from tadpoles of ranaviruses requires tissue samples, most often liver
fed oxalate-rich vegetables, such as spinach or kale. or kidney, which must be obtained postmortem. The
Development of oxalate crystals occurs in the mesone- exceptions are tail-clips, which can be diagnostic in
phron and usually results in death a few days after viremic salamanders and tadpoles, but are not advis-
metamorphosis.64,65 Urinalysis RIs are unknown for able in subclinical individuals68; toe-clips are equally
most species. Specific gravity (1.0075 [1.0007–1.0143]) nondiagnostic in juvenile or adult frogs.69 In general,
and pH (6.68 [5.26–8.1]) are only available for the samples for PCR testing should be placed in 70% etha-
American Bullfrog.3 Renal disease is one important dif- nol (prepared with PCR-quality distilled water) or kept
ferential diagnosis for the edematous amphibian, and dry and refrigerated prior to shipping, but collection,
its most common cause in some captive collections.62 fixation, and shipment instructions are best obtained
directly from the laboratory that will perform the tests.
The Amphibian Survival Alliance (ASA) and Amphib-
Ancillary Diagnostic Tests for Common ian Specialist Group (ASG) of the International Union
Amphibian Diseases for Conservation of Nature (IUCN) maintain an online
list that may serve as a source of contact details for
Chytridiomycosis, caused by the fungi B dendrobatidis diagnostic laboratories offering testing for amphibian
(Bd) and B salamandrivorans (Bsal), and ranavirosis, pathogens.70
Forz
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Acknowledgments Gilroy CV. Hematological reference intervals for Xeno-
The authors thank Dr Rapha€
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the normograph used in Figure 1, Drs Alfonso L opez and methods and reliability of long-term stored samples. Vet
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improved this manuscript. roy CV. Hematological reference intervals for Rana syl-
Disclosure: The authors have indicated that they vatica (Lithobates sylvaticus) and effect of infection with
have no affiliations or financial involvement with any Frog Virus 3 (Ranavirus sp, Iridoviridae). Vet Clin Pathol.
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vitamin D 3 in amphibian health, behaviour, diet and

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Veterinary Clinical Pathology ISSN 0275-6382

Clinical pathology of amphibians: a review

Contents Biochemical Panel

Family Common Name Scientific Name IUCN Status

Ambystomatidae Eastern Tiger salamander Ambystoma tigrinum LC

Family Common Name Scientific Name IUCN Status

Striped Rocket frog Litoria nasuta LC

*Tail autotomy does not occur.

stage, is obligatory in several species of salamanders,

Temperature (°C) Water Quality*

Type of Species Habitat Type Min Max Parameter Concentration

Terrestrial amphibians Tropical lowland 24 30 pH 6.5–8.5

Femoral and abdominal veins

Lingual venous plexus

for larger frogs. After collection, gentle pressure

Sample handling Blood cell count

Figure 3. Stepwise algorithm to maximize diagnostic information

small squares (multiplied by one). These formulae only B

information on WBC counts is scattered and frag-

Northern African African Green

Sample size 302 12–56 26–40 10 33–41 17 80 66

Analyte Units, SI Conventional Japanese Newt* Eastern Hellbender** Ozark Hellbender**

Sample size 23 37–38 42

Electrolytes, osmolality, and blood gases Proteins

Cuban African African Green

Sample size 302 29 166 24 80 66

Sample size Males Females 37–38 42

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Analyte Units, SI Conventional Japanese Newt* Eastern Hellbender Ozark Hellbender