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C.L. DAVIS SYMPOSIUM on:
DIAGNOSTIC PATHOLOGY OF DISEASES OF AERIAL, TERRESTRIAL & AQUATIC WILDLIFE
Amphibian Diseases, Declines & Malformations
By D. Earl Green
U.S. Geological Survey, National Wildlife Health Center, Madison, Wisconsin, U.S.A.
I. General. Information in this presentation in based on necropsies of >9,000 amphibians conducted at the USGS National
Wildlife Health Center since 1999. Over 95% of examined amphibians were free-living; very few captive amphibians
contributed to this data.
II. Special Anatomical Considerations in Amphibians.
Anura, Anuran: The order of amphibians consisting of frogs & toads
Caudata, Caudate: The order of amphibians consisting of salamanders (including sirens, amphiumas, newts, etc)
Bidder’s organs: Remnants of ovaries at the anterior pole of each testis in male toads (Family: Bufonidae) only.
Endolymphatic system: These diverticula of the ears partially encircle the brain and spinal cord and store calcium carbonate;
symmetrical lateral pairs of out-pouchings along the spinal column are called endolymphatic (“lime”) sacs.
Lymphatic sacs: The “subcutis” of post-metamorphic frogs and toads consists of an extensive network of large chambers (that
are separated by thin septae) that drain fluids from the skin to lymph hearts.
Lymph hearts: Frogs & toads have multiple pairs of lymph hearts located just ventral to the urostyle and in the dorsal
neck/pectoral girdle. Lymph hearts are very thin walled and contain leiomyocytes (not cardiomyocytes). The pelvic lymph
hearts pump fluids into the interstitium of the mesonephroi.
Fat bodies: Frogs, toads & tadpoles have a pair of grass tuft-like fat bodies at the anterior poles of the mesonephroi within the
body cavity; adipose tissue is sparse or absent from the “subcutis” and elsewhere in the body. Fat bodies of salamanders often
are a pair of ribbon-like structures in the mid dorsal body cavity.
Gills: Aquatic larval (and neotenic) salamanders have external gills while tadpoles have internal gills; within the internal gill
chambers of tadpoles, the forelimbs form & then emerge during metamorphosis.
Lungs: Lungs develop early in larvae but may not become aerated (inflated) for several weeks after hatching. Amphibian lungs
are sac-like with a large central lumen. Some groups of terrestrial salamanders (plethodontids) lack lungs.
“Kidneys”: Larval amphibians have 2 pairs of “kidneys” at the time of hatching: the aglomerular pronephroi and the
mesonephroi. Pronephroi disappear during metamorphosis. Mesonephroi contain intermixed “adrenal” cells, referred to as the
interrenals. The mesonephroi of salamanders usually are located in the pelvic canal; observation & removal often requires
splitting the pelvic symphysis from abdomen to the vent opening.
“Metamorph”: This term has at least two meanings: an amphibian (usually anuran) which is actively undergoing
metamorphosis, or, an amphibian that recently has completed metamorphosis (see also “recent metamorph”)
“Recent Metamorph”: A vague and imprecise term that usually refers to an amphibian that recently has completed
metamorphosis.
Mouth: The oral disc of most North American tadpoles consists of the pigmented (black) jaw sheaths, pigmented toothrows
and labia; these structures are lost during metamorphosis. Larval aquatic salamanders have an adult-like mouth.
Pigmented Macrophage Aggregates (PMA): These clusters of macrophages occur mostly in the liver and spleen;
macrophages are laden with phagocytosed debris, melanin, and multiple carotinoid pigments. Their number and size have no
significance.
Salamander histology: Cells & nuclei of nearly all caudate organs are very large; nuclei of salamanders contain 10 to 100
times more DNA than fish, frogs, toads, reptiles, birds & mammals. Constantly be aware of magnification when examining
tissue sections!
III. Microbial Cultures. As in fish, most aquatic amphibians autolyze rapidly. Consequently, bacterial and fungal cultures on
viscera of amphibians that have been dead >2-4 hrs usually contain post-mortem invaders, foremost among these are
Aeromonas spp., Pseudomonas spp., Citrobacter spp., Flavobacterium spp., enterococci, and numerous other gut flora and
bacteria common in surface waters. E. coli is infrequently isolated from the GIT of wild amphibians; when found, it generally
indicates the surface water (habitat) is contaminated human sewage, domestic animals or congregations of waterfowl. As
recommended by fish diagnosticians, submission of live sick amphibians is encouraged, especially if bacterial and fungal
cultures are to be attempted.
INFECTIOUS DISEASES:
IV. Viruses.
A. Ranaviruses (Family: Iridoviridae; Genus: Ranavirus)
1. Epizootiology. Ranaviruses are a major cause of massive mortality events in amphibian larvae and recent metamorphs.
Onset typically is explosive. Hundreds or 1,000’s of sick and dead larvae often are found. In North America, adult amphibians
rarely suffer mortality. Carrier state in a low percentage of adult amphibians is suspected, but affected (infected) organs are
unknown.
2. Gross Findings. Tadpoles & salamanders: erythema, petechia and paintbrush hemorrhages in skin at base of hindlimbs,
around vent tube, around oral disc and scattered elsewhere on the ventral body, head and tail. Older tadpoles may have mild to
severe clear or mildly fibrinous effusions in lymphatic sacs of thighs, ventral abdomen & gular area. Internally, miliary foci of
necrosis in liver & spleen; occasional necrotic foci are hemorrhagic. Mesonephroi may have hemorrhagic glomeruli. Petechiae
& ecchymoses may occur in muscles, GIT and many other organs. Hydrocoelom (clear or fibrinous) is common. Adult frogs
& toads: Rarely affected in North America. Treefrogs may have multiple small irregular pigmented ulcers in dorsal skin. Oral
(palatal) ulcers occasionally found in recently metamorphosed frogs.
3. Histopathology. Ranaviruses are pantropic, with predilection for small vessels, PMAs, spleen, liver & mesonephroi.
Miliary fibrinocellular vasculitis (especially in gills, dermis, GIT, heart & lungs) with necrotizing glomerulitis is common.
Spleen and renal interstitial (or subcapsular hepatic) myeloid cells frequently show miliary or diffuse necrosis. Liver first
shows necrosis of sinusoidal endothelium, then PMAs, and then hepatocellular necrosis; characteristic basophilic
intracytoplasmic inclusion bodies are single or multiple and usually 25-50% of the size of the nucleus. Fortuitous sections of
epidermis may show micro-vesicles, erosions and ulcers; occasional basophilic cytoplasmic inclusions are present in
degenerating epidermal cells at margins of vesicles and ulcers. Secondary and opportunistic skin infections are common.
B. Lucke tumor herpesvirus (ranid herpesvirus-1)
1. Epizootiology. This is the first neoplasm proven to be caused by a virus. It induces renal adenocarcinomata in northern
leopard frogs (Rana pipiens) only. Virus must be transmitted to eggs/embryos during breeding. Historically, prevalence varies
greatly, but prevalence of renal neoplasms increases with age. Rarely seen in frogs <2 yrs old.
2. Gross Findings. Renal neoplasms are multi-nodular, slightly pale, single or multiple, microscopic or enormous, and
occasionally metastatic.
3. Histopathology. Tumor morphology is consistent with a renal adenocarcinoma with one notable additional feature: in
those neoplasms removed from frogs recently emerged from hibernation, many neoplastic cells will have large prominent
intranuclear inclusions (typical of a herpesvirus). However, in those frogs maintained at room temperatures, no inclusions are
present and viral particles cannot be found ultrastructurally. Viral replication is very temperature sensitive and occurs only at
cold temperatures. Metastases are uncommon but reported (reviewed by Green & Harshbarger 2001)
C. Frog Erythrocytic virus (FEV, formerly Toddia spp. and Pirhemocyton spp.)
1. Epizootiology. Presently considered an incidental finding in erythrocytes of adult frogs. Virus has not been cultured but
may belong in the family, Iridoviridae. Probably closely related to erythrocytic viruses of fish and lizards. Clinical signs and
mortality have not been reported in infected frogs, but one report mentions that recapture of infected frogs was less than
recapture rate of non-infected frogs.
2. Gross Findings. None.
3. Histopathology. None reported. Intracytoplasmic viral inclusions are detected in stained blood smear slides. Three types
of cytoplasmic inclusions are described: 1) most common are red or sometimes blue (in Giemsa stain) dense, usually spherical
(but occasionally ovoid or pleomorphic) cytoplasmic inclusions are 1-3 microns diameter; these inclusions may be single or
multiple; 2) occasionally, a single larger pale or clear, albuminoid, and vacuole-like inclusion is found; and 3) uncommonly, a
single large dense elongate or crystalloid rectangular or trapezoidal membrane-bound inclusion is 10-20 microns long and
often displaces the erythrocyte’s nucleus and may cause cellular distortion. Up to 90% of erythrocytes may contain the former
smaller inclusions, but more often 20-50% of erythrocytes are affected.
D. Adenoviruses of intestine
1. Epizootiology. Enteric adenoviruses are an infrequent incidental histological finding in tadpoles, frogs and toads.
Morbidity and mortality are not reported.
2. Gross Findings. None.
3. Histopathology: typical basophilic intranuclear inclusions in intestinal epithelial cells.
E. West Nile virus has not yet been documented in free-living or captive amphibians. Experimental transmission in bullfrogs
has been done, but morbidity, mortality, gross and histological findings were not reported (Klenk & Komar, 2003).
V. Bacteria. Contrary to pre-1995 literature, bacterial infections of free-living amphibians are rare, seldom cause morbidity or
mortality, and when detected, usually are secondary or opportunistic invaders.
A. “Red Leg Syndrome”
1. Epizootiology. Primarily Gram-negative bacilli are implicated in this classic syndrome. Aeromonas spp, Flavobacterium
spp., Klebsiella spp., Acinetobacter sp. and others are mentioned in older publications. Clinical signs of classically described
“red leg syndrome” are suspiciously indistinguishable from ranaviral infection. With the exceptions of Wolf et al. (1968) and
Tomasec & Valpotic (1968), it is noteworthy that virus cultures were not attempted in the vast majority of published reports of
“red leg syndrome” prior to 1995.
2. Gross Findings. Same as for ranaviral infection
3. Histopathology. Same as for ranaviral infection. Mere presence of bacteria in blood vessels and sinuses of internal organs
is not evidence of bacterial septicemia (or red leg syndrome). Bacteria need to be associated with necrotic cells, fibrin and
inflammatory cells; bacteria also should be present intracellularly (phagocytosed).
B. Flavobacteriosis.
1. Epizootiology. Bacterial septicemias caused by multiple species of flavobacteria are a threat to captive amphibian colonies
and are rarely encountered in free-living amphibians. F. meningosepticum, F. indologenes and F. oderans are most often
implicated. Mortality rates as high as 70% may occur (Green et al. 1999; Hayes et al. 2006). It is noteworthy that infected
amphibians had received non-lethal doses of multiple pesticides (Hayes et al. 2006) and that virus cultures and appropriate
tests for chytrid fungi (Batrachochytrium dendrobatidis) were not attempted.
2. Gross Findings: In adult Xenopus laevis, signs and gross findings were hydrocoelom, anasarca, dyspnea, marked lethargy,
congestion of toe-webs and dermal petechiae (Green et al. 1999). In recently metamorphosed Rana pipiens, findings included
panophthalmitis, anisocoria, meningitis, otitis interna, head tilt, loss of righting reflex, abnormal posture (sternal recumbency),
anorexia and emaciation (Hayes et al. 2006). Olson et al. (1992) found weight loss, corneal edema, effusions in the lymphatic
sacs, marked hemorrhage in major muscle groups, serosanguinous hydrocoelom, purulent pericarditis, hepatosplenomegaly and
distended gastrointestinal tract with viscous hemorrhagic contents.
3. Histological Findings: Reports of histological changes are sketchy. Green et al. (1999) reported infiltrates of
macrophages, neutrophils and lymphocytes in the liver, spleen, mesonephroi, serosal surfaces and epicardium; macrophages
contain Gram-negative bacilli. Olson et al. (1992) described neutrophils in the anterior chamber with retinal detachment and
severe conjunctival and corneal edema. Pericardium had a thick layer of fibrin, neutrophils and bacteria. Liver showed
hepatocellular degeneration and coagulative necrosis.
C. Mycobacteriosis.
1. Epizootiology. This disease is rare in free-living amphibians, however, certain mycobacteria may be chronically
troublesome in captive amphibian colonies. Generally a disease of mature amphibians. Rarely seen in larvae. The more
commonly identified species from amphibians include M. chelonei, M. fortuitum, M. marinum, M. xenopi, M. szulgai, M.
avium (Chai et al. 2006) and M. liflandii (Suykerbuyk et al 2007). The latter sp is associated with ulcerative skin lesions in
captive clawed frogs.
2. Gross Findings. Granulomas may be irregular and poorly demarcated, or large and encapsulated. Granulomas may be
solitary, miliary or prominent and coalescing. Liver, spleen, intestine and dermis are most often affected, but granulomas may
be present in nearly any organ or tissue. M liflandii infection causes skin ulcers, hydrocoelom and inability to dive.
3. Histopathology. Mycobacteria elicit granulomatous inflammation which in amphibians consists of epithelioid
macrophages, small lymphocyte-like macrophages and true lymphocytes. Early granulomas are solidly cellular, poorly
demarcated, and have been confused repeatedly for “lymphosarcoma” (Asfari 1988). Multinucleated giant cells may be
present in chronic granulomas but otherwise are uncommon. Caseation is variable and an unreliable feature. Mineralization and
cavitation are not reported. M liflandii infection causes skin ulcers that contain acid fast bacilli but also widespread infection of
the mucosal epithelium of gall bladder, oviduct, renal tubules, and ova. Acid fast staining (Ziehl-Neelsen) is recommended for
all caseous and non-caseous granulomata of amphibians, especially those in long-term captive situations.
D. Salmonellosis.
1. Epizootiology. This is a somewhat controversial “infection” in amphibians. Salmonella spp. are most often isolated from
amphibians at low elevations in neotropical sites. Although not reported as a cause of morbidity or mortality in amphibians, it
may be a serious/significant contaminant of frog legs for human consumption. Contrary to published conjecture that a majority
of amphibians carry Salmonella spp. in their GIT (Taylor et al., 2001) only 9 (0.7%) isolates have been obtained from >1300
selective cultures for Salmonella spp. from free-living amphibians nationwide (DE Green, unpubl. data).
2. Gross Findings. None reported
3. Histopathology. None reported
VI. Sub-bacteria: Chlamydiosis, Rickettsiosis.
A. Chlamydiosis.
1. Epizootiology. Agent: Chlamydophila (formerly Chlamydia) pneumoniae. This infection was formerly a significant
hazard to colonies of African clawed frogs (Xenopus laevis) being fed raw beef liver. Mortality rates of about 50% may occur
in colonies of adult clawed frogs. Recent surveys of amphibians in Switzerland and Russia by PCR methods only, suggest
amphibians may be asymptomatic carriers of multiple spp of chlamydiae.
2. Gross Findings. Emaciation, hepatosplenomegaly, hydrocoelom & lymphatic sac effusion
3. Histopathology. Liver is organ of choice for tissue section. Disseminated to diffuse lymphohistiocytic hepatitis is found
with scattered necrotic hepatocytes, fibrin in sinusoids, and intracytoplasmic granular basophilic inclusions. Irregular foci of
purulent inflammation also may be present in liver and spleen. Granulomatous or pyogranulomatous inflammation in lungs
(interstitial pneumonitis), heart (lymphohistiocytic myocarditis & epicarditis), spleen and mesonephroi (fibrinopurulent
glomerulitis) tend to have fewer cytoplasmic inclusions (Howerth 1984; Reed et al 2000)
B. Rickettsia.
1. Epizootiology. Agent: Aegyptianella (formerly Cytamoeba) ranarum is an anaplasma-like agent infecting erythrocytes of
bullfrogs, green frogs and mink frogs in USA and Canada.
2. Gross Findings. None reported.
3. Cytology. In blood smear slides, organisms are cytoplasmic, and measure 3 to 11 microns; organisms occasionally are
densely packed and arranged in parallel (Desser 1987). Histology is not reported.
VII. Fungi.
A. Chytridiomycosis (infection by Batrachochytrium dendrobatidis [“BD”])
1. Epizootiology. BD is a panzoonosis. BD is the only chytrid pathogen of vertebrates. BD is lethal in many/most postmetamorphic frogs and toads; impact on salamanders is less clear. Organism is keratinophilic and infects only keratinized cells
of the adult skin and larval anuran oral disc. Damage to mucous layers & cellular integrity of stratum corneum allows serious
secondary bacterial infections. BD probably has caused most amphibian extinctions and population extirpations worldwide in
the last 2 decades and it continues to spread (Lips et al 2006). American bullfrogs (Rana catesbeiana) may be worldwide
carriers of BD (Garner et al. 2006).
2. Gross Findings. Larvae: keratinized portions of the oral disc are black (but not due to melanin); infection of oral disc is
characterized by multifocal to diffuse loss of black color on jaw sheaths (“beaks”) and toothrows. Post-metamorphs show
increased ecdysis, thickened translucent-to-opaque molts, incomplete ecdysis (flakes & sheets of retained molt), and
occasionally erythema and paint-brush hemorrhages in ventral skin. Skin topism: infections are most intense in ventral
abdominal & thigh skin (“pelvic patch”), toe-tips and toewebs. Dehydration tends to occur terminally.
3. Histopathology. BD produces thalli and zoospores, never hyphae. Acute infections show scattered single or clumps of
thalli in cytoplasm of keratinized cells. Thalli usually are 50-100% of size of host nucleus; multiple thalli per cell are common.
Subacute infections show mild to severe hyperkeratosis (or multiple layers of retained, unshed molt) and mild to moderate
acanthosis. Inflammatory reaction is minimal, but may be marked if secondary bacterial (and other fungal) infections develop.
Infective stage is a uniflagellate zoospore; initial intracellular infection consists of 1-4 micron diameter, basophilic, circular to
slightly ovoid thalli; these thalli grow, and infrequently develop thin septae forming 2 to 4 internal chambers; enlarging thalli
become less basophilic; thalli have a thin but rigid cell wall (PAS-positive) and then core matures into spherical-appearing
zoospores. Thallus forms a characteristic discharge pore or tube for release of zoospores; the pore or tube may be very short or
nearly as long as the diameter of the thallus. In subacute infections, vast majority of thalli will appear empty but some may
contain plugs of invasive basophilic bacteria.
B. Watermolds (Oomycetes)
1. Epizootiology. Watermolds (eg, Saprolegnia spp.) are ubiquitous in surface water. The term, saprolegniasis, may be used
for infections by multiple genera of Oomycetes (eg, Leptolegnia, Achlya, Aphanomyces). Taxonomic status for watermolds is
unsettled. Even entries in GenBank are unreliable. Watermolds are principally saprophytes that invade dead or infertile eggs
and larval skin wounds; but organisms from dead eggs may spread to and infect adjacent healthy embryonated eggs causing
extensive mortality in the eggmass. Secondary infections of aquatic amphibians often are associated with anchorworms
(Lernaea sp.), leech bite wounds, other wounds, gangrene of extremities subsequent to gas bubble disease, and ranaviral skin
ulcers. Recently, epizootic saprolegniasis of the oral disc and head of tadpoles has been described with no associated
underlying disease (Berger et al 2001)
2. Gross Findings. White, pale tan or pale grey fuzzy cottony growths from the skin or egg capsule (“albumin”). Infection
may be focal or generalized. Best observed in the immersed specimen, since filaments collapse out of water and become very
difficult to detect lying on the skin.
3. Histopathology. Filaments of most watermolds are aseptate, sparingly branching and occasionally stain poorly. Epidermal
erosions and ulcers often are present. Extent of inflammation varies from negligible to moderate infiltrates of mononuclear
cells in the dermis. Deeper invasion into dermis and muscles is variable.
VIII. Mesomycetozoa & Alveolates. Mesomycetozoa are a newly erected clade of protistan organisms, formerly known by
the acronym “DRIPs”; they are principally pathogens of shellfish, fish and amphibians, but includes the mammalian pathogen,
Rhinosporidium seeberi (Herra et al 1999). Alveolates include the oyster pathogen, Perkinsus marinus (“dermo” disease) and
the newly recognized but unnamed pathogen of larval amphibians called a Perkinsus-like organism.
A. Ichthyophonus sp.
1. Epizootiology. Usually an incidental finding in tadpoles, frogs and some aquatic salamanders but may cause morbidity and
mortality in red-spotted newts, and rarely in bullfrogs, green frogs and wood frogs. Relationship of the amphibian pathogen to
those found in marine fish is uncertain. Occasionally mis-identified or mis-named as Histocystidium, Ichthyophthirius and
adiaspiromycosis. Life cycle and infective stage are unknown. In amphibians, organism is known only from states and
provinces bordering the Atlantic Coast and Great Lakes (and West Virginia & Vermont).
2. Gross Findings. Five forms of infection are reported.
a. Newts have subtle to moderate swellings of the dorsal axial, rump, thigh and tail muscles; overlying skin usually is
intact.
b. Recently metamorphosed ranid frogs may present with mild to marked swelling of the rump (around urostyle).
c. Adult frogs with massive infections usually are lethargic and emaciated. On cut surface, skeletal muscle is tannishbrown in subacute and chronic infections.
d. Clinically silent infections are common and mild, probably incidental, infections are occasionally detected
histologically.
e. Massive acute lethal infections with numerous mortalities occur infrequently in ranid larvae (D. Green & M. Gahl,
unpubl. data)
3. Histopathology. Organisms are rhabdomyotropic and initially intracellular. Pre-myocytic stages have not been described
in amphibians. Organism is weakly basophilic with lacy cytoplasm and numerous uniformly dispersed minute eosinophilic
micro-nuclei. A prominent hyaline wall is present with an artefactual clear space separating wall from cytoplasm. Organisms
are circular in cross section but may be ovoid to elongate and up to 700 microns long. Initial intracellular infections lack
inflammatory cells but subacute and chronic infections elicit intense granulomatous inflammation. Both forms (no
inflammation and intense granulomatous myositis) often are present in an animal.
B. Amphibiothecum (formerly Dermosporidium) penneri; Amphibiocystidium spp. in European amphibians
1. Epizootiology. A sporadic dermal infection of adult toads. Infection develops in autumn and matures during hibernation;
toads emerge from hibernation with pustules and granulomas filled with spherical microscopic spores. Spores are discharged
during spring breeding and then pustules are mostly healed by mid-summer. Route of infection, infective stage and
intermediate hosts are unknown (Jay & Pohley 1981; Feldman et al 2005)
2. Gross Findings. Pustules occur principally in ventral skin and around vent, are 1 to 5 mm diameter and often are
coalescing. Overlying epidermis is ulcerated (umbilicated) and the caseous discharge is white, pale grey or light tannishyellow; discharge consists entirely of spores. Within the dermis, the nodules consist of serpentine, entwined white tubules that
occasionally are present in the lymphatic sacs.
3. Histopathology. Encapsulated organisms may elicit slight to extensive granulomatous inflammation. An indistinct or thin
wall is present and organisms at the periphery usually are loosely packed, polygonal and separated by thin indistinct septae into
small packets. Centrally, organisms are more mature, individualized, spherical and 8-15 microns diameter.
C. Perkinsus-like organism
1. Epizootiology. After ranaviruses and chytridiomycosis, this organism is considered the third major cause of mortality
events in amphibians. This infection occurs predominantly in ranid larvae (Rana spp.) and may cause mortality rates of 8099% in a pond over the course of 2-6 weeks. Weakly swimming, bloated and floating tadpoles are found. Like “dermo” in
oysters, the amphibian pathogen may be temperature dependent. Route of transmission and method of infection are unknown,
but cannibalism among tadpoles may be a factor. Very thick walls of the organism suggest the spores probably are resistant to
drying. Predominantly occurs in Atlantic and Gulf Coast States with pockets of infected populations in central MN, Willamette
Valley of OR and Kenai Peninsula of AK.
2. Gross Findings. Tadpoles dead/dying of this infection consistently have very distended abdomen. Abdominal distension is
due to massively enlarged (2 to 10x normal size) pale (white or light yellow) liver. Spleen, pronephroi and mesonephroi also
are pale and exceptionally enlarged. White paintbrush streaking of intestinal wall may be seen. In acute (early) infections, only
mild hepatosplenomegaly may be detected (Green et al, 2002; Davis et al, 2007).
3. Histopathology. In early infections, liver has clumps of 5-8 microns diameter spherical basophilic organisms, probably
associated with initial infection of macrophages in PMAs. In advanced lethal infections, liver is completely replaced by solid
masses of organisms; it is difficult to identify the organ or find normal hepatocytes. In pronephroi and mesonephroi, organisms
initially are multifocal clusters in interstitium and then become diffuse and replace most tubules. Tiny (0.5 to 2 microns
diameter) eosinophilic spherical pre-spores may be detected within hepatocellular cytoplasm in early stages of infection.
IX. Protozoa.
Most studies of protozoan parasites in amphibians have been conducted by biologists and protozoologists. There is a need for
veterinary pathologists to report the gross and histological changes, if any, associated with this group of organisms.
A. Trypanosomiasis
1. Epizootiology. Trypanosomes are an incidental finding, usually in blood smear slides, in many amphibians. Transmission
is by the bite of leeches.
2. Gross Findings. None.
3. Cytology. Typical trypanosomes on stained blood smear slides or tissue squash-smears. Live organisms are readily
detected in a fresh whole-mount unstained drop of blood. No tissue abnormalities have been reported in North American
amphibians.
B. Coccidiosis
1. Epizootiology. Fairly common but usually innocuous infection of larvae and adult amphibians (Upton & McAllister
1988). May become a problem in tadpoles. Reduced growth (runting) and thickened intestinal wall have been observed in one
free-living population of leopard frogs (Rana pipiens).
2. Gross Findings. In heavy infections only, runting and thickened intestinal wall observed in free-living tadpoles.
3. Histopathology. Slight to massive numbers of oocysts are present in enteric epithelium; inflammation is variable but most
debris-laden macrophages in the intestinal wall are reactive to metamorphic restructuring of intestine rather than the protozoa.
C. Amoebiasis.
1. Epizootiology. This is an infrequent entero-invasive infection of amphibians in zoos and low elevation neotropical
locations. Subclinical non-invasive infections may be more common. Valentine and Stoskopf (1984) reported amoebic
mesonephritis in one captive giant (cane) toad (Bufo marinus) but failed to find intestinal infection. Multiple free-living Cuban
treefrogs (Osteopilus septentrionalis) and two giant toads from 2 Caribbean Islands had mild entero-invasive amoebae in their
cloacas (D Green & P. Burrowes, unpubl. data).
D. Ectoparasites: Trichodina & Epistylis
1. Epizootiology. Occasional ponds or larval amphibian populations may have slight to massive numbers of these innocuous
protozoa. Trichodina-like protozoa are free-living and glide across the surface of skin, gills, gill chambers and occasionally
into the vent (cloaca) and urinary bladder. Epistylis-like protozoa are tulip-like in shape and attach to the surface epidermis by
a thin stalk; oral disc, gills, ventral skin, ventral fin around vent opening and toe-tips are frequent sites of attachment.
2. Gross Findings. Organisms are most easily detected in the anesthetized, submerged amphibian.
3. Histopathology. Trichodina spp. have characteristic pinwheel rotors and are distinctly hemispherical in shape. Epistylislike protozoa are basophilic and pleomorphic in tissue sections and commonly found in/on the oral disc, pharynx, gill chamber,
ventral skin and skin of tail; attachment stalks are seen rarely in fortuitous sections.
X. Myxozoa & Microsporidia.
A. Myxidium spp. (Myxozoan cholecystopathy)
1. Epizootiology. Fairly common, widespread and mostly innocuous myxozoan of the gall bladder (Eiras 2005). Infective
stage, route of infection and intermediate host(s) are unknown.
2. Gross Findings. Often inapparent at dissection; may present as white flocculent material within the gall bladder. As yet
undocumented reports of severe gall bladder distention and mortality may occur.
3. Histopathology. Early stages of plasmodia may be found in liver adjacent to or within bile ducts. Plasmodia are
intraluminal, elongate and thin; in some plasmodia, spores may be seen.
B. Sphaerospora (formerly Leptotheca) ohlmacheri (Myxozoan mesonephropathy)
1. Epizootiology. Fairly common but innocuous myxozoan infection of only mesonephroi in larval and post-metamorphic
frogs and toads; occasionally seen in aquatic salamanders (Eiras 2005). Infective stage, route of infection and intermediate
host(s) are unknown.
2. Gross Findings. None.
3. Histopathology. Poorly-stained cellular-like debris within renal tubules (H&E) usually is this organism. In heavy
infections, tubules are mildly to moderately dilated and partially filled with myxozoan spores. Plasmodia are very small.
Giemsa stain demonstrates most myxozoan organisms, especially spores, well.
C. Alloglugea sp. (Xenomas)
1. Epizootiology. Alloglugea is a xenoma-producing microsporidia; described from cane toad tadpoles in the neotropics
(Paperna & Lainson 1995). Similar infections have been seen in anuran larvae in California. Some infections may resolve
during metamorphosis.
2. Gross Findings. Small (~1 mm) roughly spherical nodules in the intestine and occasionally other organs.
3. Histopathology. Typical rod-shaped microsporidial spores within cytoplasm. Inflammation may be absent in early
infections or granulomatous in subacute and chronic infections.
D. Plistosphora spp. (Microsporidian myositis)
1. Epizootiology. Sporadic infection of tadpoles and adult anurans. Intense muscular infections may cause lethargy,
emaciation and death (Canning et al 1964)
2. Gross Findings. Inapparent or mild to intense white streaking of skeletal muscles
3. Histopathology. Intracytoplasmic organisms within indistinct parasitophorous vacuoles; inflammation may be absent or
mildly lymphohistiocytic.
E. Spraguea-like microsporidia (Microsporidian gangliomyelitis)
1. Epizootiology. Newly recognized infection of tadpoles (Hyla spp., Rana spp.) in southern USA.
2. Gross Findings. None.
3. Histopathology. Intraneuronal clusters of microsporidia are found in spinal cord neurons and cranial and spinal ganglia.
Inflammation may be absent or slight to moderate lymphocytic or lymphohistiocytic.
XI. Trematodes & Monogenes.
A. Gyrodactylus sp.
1. Epizootiology. This monogene is principally an ectoparasite on the skin of fish; infrequently encountered on tadpoles from
SE USA.
2. Gross Findings. Usually necessary to examine live or anesthetized tadpoles while submerged in water; most of these
monogenes are too small to be seen with naked eye
3. Histopathology. None reported.
B. Ribeiroia ondatrae (metacercariae)
1. Epizootiology. Infections by metacercariae of Ribeiroia cause 2 major problems: a variety of limb malformations (amelia,
ectromelia, polymelia, polydactyly, etc) and acute death in young tadpoles. As few as 4 cercariae burrowing into skin of a
tadpole may be lethal at young stages (Schotthoefer et al 2003); with age, tadpoles may acquire 100’s of encysted
metacercariae without ill affect. Experimental studies show tadpoles must be at specific stages of development (Gosner stages
26-28) for hindlimb malformations to occur. This parasite is distributed nationally (except possibly in Alaska & Hawai’i).
2. Gross Findings. As early as Gosner stage 30, extra hindlimb buds may be induced by encysted metacercaria. By
metamorphosis, malformations of hindlimbs may include amelia, polymelia, polydactyly, ectromelia, brachyphalangy, taumelia
(folded long bones with bony triangles), and skin folds (across stifle or ankle) (Johnson et al 2001). Encysted metacercariae in
larvae usually are found in dermis at 1) base of hindlimbs, 2) proximal ventral fin, 3) inguinal area and 4) around vent tube. In
post-metamorphic amphibians, encysted metacercariae (usually <0.2 mm diameter) occur in same pelvic area and in ventral
dermis from tip of chin to vent, and in “subcutis” around urostyle.
3. Histopathology. Other than metacercarial size and location in host, the metacercariae have no known characteristic
features to distinguish them from many other genera. Inflammatory reaction to encysted metacercariae is negligible.
C. Clinostomum spp. (metacercariae)
1. Epizootiology. An occasional cause of mild to severe skin lumps in tadpoles, frogs and salamanders. Mortality not
reported, even in heavy infections. As with all metacercarial & mesocercarial infections, the presence of the immature
trematodes indicates a final host defecated in the pond and aquatic snails (1 st intermediate hosts) also are present in pond.
2. Gross Findings. Encysted metacercariae are 1 per cyst, and are the largest North American metacercariae yet encountered.
Each cyst is 1 to 3 mm diameter and often slightly yellowish; acutely encysted metacercariae may have blood in the cyst fluid.
Excysted live metacercariae are 2 to 10 mm long.
3. Histopathology. Size of encysted metacercariae is best feature for identification; no characteristic histological features for
the genus. Inflammatory reaction to cyst walls is negligible but may develop months after initial infection.
D. Echinostoma spp. (metacercariae)
1. Epizootiology. Very common, minute, metacercariae of mesonephroi of larvae, frogs, toads and aquatic salamanders.
Intensity of infection varies from <10 to >500. Metacercariae are present in glomeruli, tubules and interstitium. Clinical
disease, even in heavy infections, is poorly described; usually considered innocuous & incidental.
2. Gross Findings. Not visible to most naked eyes (too small). Usually 0.05 to 0.2 mm diameter; requires dissecting
microscope to detect at necropsy.
3. Histopathology. Initial infections lack inflammatory response and may have indistinct cyst wall. Dead metacercariae elicit
caseo-granuomatous inflammation. Metacercariae have characteristic intensely eosinophilic spines on tegument.
E. Diplostomatidae (mesocercariae)
1. Epizootiology. Immature trematodes that do no form cysts but freely move through lymphatic sacs, body cavity and most
viscera usually are mesocercariae. Mild to intense infections are common in tadpoles in SE USA. Over 500 live mesocercariae
may be flushed from body cavity of a tadpole.
2. Gross Findings. None, usually requires dissecting microscope to detect mesocercariae, but larger individuals may be up to
2 mm long.
3. Histopathology. Organisms lack cyst wall, usually are present free in body cavity and lymphatic sacs, and lack any
distinguishing histological features.
H. Lung flukes (Haematoloechus spp.)
1. Epizootiology. Widely distributed adult parasite of post-metamorphic anurans. Amphibians are final hosts; aquatic snails
are first intermediate hosts, and aquatic insects (especially dragonfly naiads) are 2 nd intermediate hosts; requires ingestion of
insect by amphibian. Usually incidental intraluminal pulmonary infection, but occasionally associated with pleural cysts.
2. Gross Findings. Adult flukes usually are present in central lumen of lungs or between caviae. Flukes often have striking tricolor: orangish-tan, white and black.
3. Histopathology. In chronic infections, eosinophilic interstitial pneumonitis is disseminated or diffuse.
XII. Nematodes.
A. Lungworms (Rhabdias spp.)
1. Epizootiology. Rhabdiasis is a potentially serious infection of captive amphibians because the parasite is parthenogenic
and has a direct lifecycle. Occasionally it is encountered in free-living adult frogs, toads and salamanders. Hazard is from
recently hatched larvae that actively burrow into skin of amphibians; large numbers of larvae may be fatal (Williams, 1960).
Because of this parasite alone, prophylactic testing or anthelmintic treatments of newly arrived amphibians in captive colonies
is warranted.
2. Gross Findings. Adult nematodes are present in central lumen of lungs; individuals usually are black and white striped
longitudinally. Tiny white larvae may be detected under dissecting ‘scope in body cavity.
3. Histopathology. Adults may elicit an eosinophilic interstitial pneumonitis in septae of caviae and pleura. Larvated ovoid
eggs may be present in lumina of lung and GIT. Larvae may be detected as tiny, basophilic, 1-3 micron diameter, non-encysted
nematodes in nearly any organ but especially dermis, intestinal villi and free in body cavity. Small size of larvae may be
confused with fungal hyphae.
B. Skin worm (Pseudocapillaroides xenopi).
1. Epizootiology. This intra-epidermal capillarid parasite occurs in captive colonies of African clawed frogs (Xenopus
laevis). Parasite has a direct life-cycle, hence intensity of infections often steadily increase in colonies. May be associated with
morbidity and mortality in captive adult clawed frogs, especially in husbandry situations where aquariums are not changed and
disinfected regularly.
2. Gross Findings. Reddened roughened thickened frayed skin with irregular patchy molting occurs in heavy infections;
epidermal erythema and ulcers may occur; mild infections may be clinically silent. Skin topism: principally dorsal body skin.
3. Histopathology. The adult parasite occurs in epidermis and leaves burrow holes. Eggs are typically capillarid and
embryonated (ovoid, brown with bipolar plugs). Inflammatory response is minimal but secondary bacterial and fungal
infections may elicit intense dermal and epidermal mixed inflammatory cells.
C. Pinworms (Gyrinicola batrachiensis and Batracholandros spp.)
1. Epizootiology. G. batrachiensis is common in many genera of anuran larvae (tadpoles). During metamorphosis,
amphibians usually expel G. batrachiensis and acquire Batracholandros spp. Both genera are considered innocuous.
2. Gross Findings. Pinworms of amphibians have typical gross morphology of oxyurids.
3. Histopathology. Typical morphology of oxyurids.
XIII. Leeches, Fish Lice & Anchorworms
A. Leeches.
1. Epizootiology. The genera, Batrachobdella, Macrobdella and Oligobdella, are most often seen on amphibians. Two
Batrachobdella sp. on a 1-5 g tadpole may cause lethal blood loss (Boltz 1997)
2. Gross Findings. Many leeches leave a roughly circular hemorrhagic skin ulcer that is 1-6 mm diameter. Leeches may
attach anywhere on head, body and tail. Bite wounds may become secondarily infected by bacteria and watermolds.
3. Histopathology. Not described.
B. Anchorworms (Lernaea sp.).
1. Epizootiology. This copepod parasite is infrequently encountered in those species of free-living tadpoles that require
permanent bodies of water (eg, larval bullfrogs & green frogs).
2. Gross Findings. Adult burrowing female anchorworms typically have exposed tail and pair of egg sacs. Most common site
of infection is junction of body and tail (Tidd 1970), but also oral disc, gill chamber and body. Saprolegniasis occasionally
develops at burrow hole of anchorworms and may obscure presence of the copepod’s tail and egg sacs.
3. Histopathology. Inflammatory reaction to embedded portion of anchorworm is mild to intense. Composition of
inflammatory cells varies greatly depending on presence or absence of secondary bacterial and fungal infections.
C. Fish lice (Argulus spp.)
1. Epizootiology. Infrequently encountered on the skin of larval amphibians, especially from permanent ponds/lakes with
fish. Argulus have 2 suction cups and feed by inserting a sting organ which injects digestive enzymes into the body; liquidized
body fluids are then sucked out with the proboscis organ. Secondary skin infections occur in fish, but apparently not reported
in amphibians.
2. Gross Findings. Attachment to epidermis of amphibian and feeding may cause skin erosions or ulcers and subsequent
secondary watermold or bacterial infections.
3. Histopathology. Not reported.
XIV. Chiggers (Hannemania spp.)
1. Epizootiology. Amphibian chiggers occur nationwide in terrestrial stages of amphibians (frogs, toads and salamanders).
Unlike chiggers of other vertebrates, amphibian chiggers burrow into the skin, encyst and remain for 6-10 months. Mortality is
not reported. Other genera of chiggers occur in amphibians from other continents.
2. Gross Findings. Raised orange or reddish-orange 1 mm nodules are present in toewebs, and skin of limbs and ventral
body. Gentle lancing of cyst reveals one orange chigger per cyst.
3. Histopathology. Cyst wall usually elicits minimal inflammatory cell reaction. Chiggers have standard organ morphology
with chitin and skeletal muscles.
XV. Oligochaeta: Dero (Allodera) hylae et floridana
1. Epizootiology. Parasitic (or symbiotic) oligochaetes are darkly pigmented, elongate, segmented worm-like organisms
within the lumina of the Wolffian ducts (“ureters”) of treefrogs (Hyla spp. & Osteopilus sp.) and rarely toads (Bufo sp.) in SE
USA (Harman & Lawler 1975). Morbidity or mortality is not reported, even in heavy infections.
2. Gross Findings. Multiple para-renal oligochaetes may cause marked dilation of the Wolffian ducts; in heavy infections,
organisms may be present in urinary bladder.
XVI. Myiasis
1. Epizootiology. At least 3 genera of blow fly-like infections occur in terrestrial salamanders, frogs and toads. Bufolucilia
sp. in Europe causes massive infection and destruction of the nasal cavity often with invasion of orbit and brain by the
maggots. In New World and Australia, 3 fly genera (Bufolucilia, Batrachomyia and Notochaeta) may invade lymphatic sacs in
smaller numbers. Some maggots stray into body cavity (Bolek & Coggins 2002); death of host may occur when maggots
emerge from amphibian to pupate.
2. Gross Findings. Typical deep skin ulcer with maggot’s spiracle usually is present in dorsal body, rump or base of a limb
(Bolek & Coggins 2002). Typical white maggots are found the nasal cavity in infected European amphibians.
XVII. Amphibian Declines
1. Geographic. Declining amphibian populations (DAP) are reported on nearly every continent, except possibly Africa.
Declines may include marked reductions in local populations (usually >90% of historic pre-1970 levels) or extirpation of
populations in most watersheds. Amphibian extinctions are increasing alarmingly worldwide, but especially in Australia,
Central America and some islands.
2. USA. Declines were first documented in the mid 1970’s here in Wisconsin and Minnesota, when populations of northern
leopard frogs sharply declined and biological supply companies were not allowed to collect adult frogs for dissection classes.
The cause of these declines was never determined. Noteworthy DAP have occurred in SE USA (flatwoods salamander, gopher
frogs) and western USA (boreal toad, Wyoming toad, Yosemite toad, mountain yellow-legged frog, California red-legged frog,
California tiger salamander, et al.).
3. Etiologies.
a. Habitat loss is usually the first cause listed for DAP, and in areas with intense agriculture and urban development,
this probably is true. However, many DAP have occurred in relatively pristine national parks, wilderness areas and undisturbed
national forests, hence habitat loss is only one possible factor.
b. Agri-chemical contamination. A growing number of biologist favor industrial pollutants (eg, acid rain), pesticides,
fertilizers and other industrial contaminants (eg, PCBs) as a major factor in DAP. The lines of evidence are three-fold: 1) in the
Sierra Nevada Mountains, aerosol drift of pesticides from the Central Valley has been documented in surface water and animal
tissue; 2) amphibians may be extremely sensitive to trace levels of some pesticides (or the surfactants and “inert ingredients”)
at <10 ppb concentrations; furthermore, metabolized chemicals may be 1 to 2 magnitudes more toxic to amphibians than the
parent compounds; and 3) some pesticides (or the surfactants and “inert ingredients”) are endocrine disruptors at parts-perbillion concentrations and may cause feminization and increased prevalence of intersexes (ie, ovo-testes) in male amphibians.
However, it is not clear whether this feminization is sufficient to alter male behavior (calling, breeding, amplexus) or fertility
(sperm counts, motility). The impact of these and other chemicals on other endocrine organs (ie, thyroid, interrenals) is just
beginning to be addressed. Cortisone and thyroid hormones are essential for metamorphosis.
c. Invasive species. The introductions and spread of the American bullfrog (Rana catesbeiana) and giant (or cane)
toad (Bufo marinus) have been linked to the decline and extirpation of native amphibians in Europe, South America, Australia,
and USA. Bullfrogs are associated with many DAP in western USA where they are an invasive species and in Europe where
they are an introduced (exotic) species. Importation and sporadic release of African clawed frogs (especially Xenopus laevis) in
many countries appears to have preceded many DAP. Introduction of trout into watersheds of western USA, Central America
and Australia also have been linked to DAP. The assumption about invasive and introduced fish and amphibians is that the
newly arrived species reduce feed, out-compete the native species and consume native species. A frequently overlooked aspect
of these introductions is that the new species also tend to be carriers of new infectious diseases that may be devastating to naïve
endemic populations.
d. Infectious diseases. The study of infectious diseases of free-living amphibians is in its infancy. Very few viral
infections are known from amphibians, and the literature is littered with poorly investigated amphibian mortality events (ie,
usually no attempts at virus cultures or chytrid fungus tests). Three major lethal infectious diseases of amphibians that may
cause high mortality rates leading to seasonal or persistent population declines are ranaviruses, BD (chytrid fungus) and a soon
to be described Perkinsus-like organism. Of these 3 infectious diseases, chytridiomycosis is most frequently linked to DAP,
extirpations and extinctions in western USA, Australia, Latin America, South America, Europe and Puerto Rico. Adult and
larval bullfrogs and adult clawed frogs may be carriers of BD.
e. Fish hatcheries. The outdoor and permanent ponds of many fish hatcheries usually harbor enormous populations of
amphibian larvae. In selling and moving fish from hatcheries to other locations, it is common that tadpoles also are transported
to the new location. A recent study of 4 hatcheries in SE USA has documented the occurrence of chytridiomycosis in some
tadpoles from the hatcheries’ ponds (Green and Dodd, 2007).
4. Research. In 2001, the US Congress authorized and funded the national Amphibian Research and Monitoring Initiative
(ARMI). Ten senior amphibian herpetologists are intensively surveying, monitoring and documenting amphibian populations
and diseases nationwide (Muths et al. 2006). After 5 years of monitoring and surveying, statistically-based trends in amphibian
populations soon will be available.
XVIII. Amphibian Musculo-Skeletal Malformations
1. Background. In 1995, a group of high school students in Minnesota received national attention in the popular press when
they “discovered” a population of recently metamorphosed frogs with an exceptionally high prevalence of hindlimb
malformations. Frogs with missing, partially missing or extra hindlimbs were common at their site. Wild speculation followed
as to the possible cause. Other field studies quickly confirmed the occurrence of similar malformations in many other
amphibian species nationwide. But the cause(s) remained unknown.
2. Research. While it is highly likely that this phenomenon (limb malformations) had occurred in recently metamorphosed
amphibians for eons, it also became apparent that herpetologists rarely studied or published observations on cohorts of newly
metamorphosed amphibians. The controversy over the causes of these amphibian malformations became a significant factor in
the US Congress funding the Amphibian Research and Monitoring Initiative (ARMI). Grants to study the malformation
problem also were provided by NGOs, NSF, NIH and other agencies.
3. Etiologies. Initially, it was presumed by herpetologists, ecologists, environmentalists and others that these malformations
were the result of contaminants, especially agri-chemicals, and the repeated confirmation of similar malformations in other
amphibians nationwide was mistakenly interpreted to support the contaminant theory. Some documented etiologies of musculo-
skeletal malformations follow:
a. Ribeiroia spp. metacercarial infection. Infection of amphibian larvae at precise ages (stages) of development by
cercariae (and then encysted metacercariae) by Ribeiroia ondatrae has been shown to be the principal cause of malformations
of the skin and bones of amphibian limbs (Johnson et al. 2001). The parasite does not induce partial limb atrophy, taumelia or
polymelia/polydactyly in late stage tadpoles or post-metamorphic amphibians (Schotthoefer et al. 2003), but in order to
produce malformations, the cercarial/metacercarial infection must occur at a precise, rather young, tadpole age. There appear
to be no published reports on the teratogenicity of other genera of trematodes (and their cercariae/metacercariae), hence it is
unknown whether other trematodes are capable of inducing dermal and musculo-skeletal malformations. To date, the absence
of Ribeiroia spp. in wood frogs (R. sylvatica) in Alaska (Arctic & Kenai National Wildlife Refuges), where limb
malformations also are found suggests other genera of trematodes may be teratogenic, or other agents (perhaps including
contaminants) are involved.
b. Other metacercariae. Some genera of metacercariae (and mesocercariae) are generalists and may infect fish and
amphibians, and may be found nearly anywhere in the body and viscerca (eg, Clinostomum spp.). However, some genera of
trematodes have cercariae with organ tropism (eg, Echinostoma spp. in mesonephroi) or selective tissue topism (eg, Ribeiroia
spp in para-vent and proximal hindlimb buds). Unidentified non-encysted and encysted metacercariae occasionally are detected
histologically in the notochord and thyroid of larval amphibians; for the most part, their identities are unknown. The impact of
damage to the notochord during metamorphosis on the vertebrae and inter-vertebral discs is unknown, but scoliosis is a distinct
possibility. Since thyroid hormones are essential to initiate and control metamorphosis in amphibians, the impact of ablation or
partial destruction of the thyroids by metacercariae could be serious. Such parasites are worthy of additional study.
c. Hyperthermia. Studies in Japan with captive amphibians suggest bilateral hypoplasia of hindlimb digits and distal
extremities occur in larval toads that are raised in warm water at or near their maximum thermal tolerance (Muto 1969).
d. Ultraviolet light. Elevated levels of UV radiation in captive situations may lead to bilateral hypoplasia of hindlimb
digits and distal extremities very similar to those induced by hyperthermia (Ankley et al. 1998).
e. DDT. This organochlorine insecticide is associated with very specific cranio-facial malformations in late stage
tadpoles and the rare frogs that successfully complete metamorphosis (Hayes et al. 1997). Ulcers and perforations occur in the
dorsal midline of the tadpole’s snout and may involve loss of the upper jaw sheath. Perforations into the mouth may occur.
Other abnormalities, including micrencephaly (small head) and asynchronous metamorphosis (ie, tail fully resorbed before
adult mouth is fully formed) also may occur. Experimentally, indistinguishable malformations occur in tadpoles that are dosed
with corticosterones.
f. Heavy metals. Most studies of heavy metals in amphibians involve determination of acute or subacute LD50; seldom
are dosed tadpoles followed through metamorphosis for assessment of the musculo-skeletal system. However, interesting and
consistent malformations are induced by lead in tadpoles (yet to be published by D. Sparling et al.).
g. Contaminants. Most published studies of pesticides and other contaminants in larval amphibians report a few
malformed amphibians in different dosage groups, but rarely are there multiple affected animals in each group and there
generally is no consistent dose response (pertaining to development of limb malformations).
4. Diagnosis. Prior to metamorphosis, the “skeleton” of larval amphibians consists mostly of cartilage; hence, larvae are
unsuitable for standard radiographs. At the completion of metamorphosis, the skeleton is nearly fully ossified, although some
exceptionally small recent metamorphs (eg, toads) may still have cartilaginous digits. The 2 basic methods for the study of
musculo-skeletal abnormalities in amphibians are
a. Radiography. Radiography is applicable to fully metamorphosed amphibians. Live amphibians may be
radiographed as well as eviscerated fixed carcasses. It is vital that malformed amphibians intended for radiography be carefully
fixed with their limbs and digits extended.
b. Clearing-&-Staining. This teratological technique is often used to examine vertebrate embryos, but also is useful
for examining adult fish, amphibian larvae and adult amphibians. Fixed carcasses are cleared in potassium hydroxide and then
two stains are applied which render bones and cartilage visible. This method is preferred to radiography, since cartilaginous
structures become visible. Unfortunately, the clear-&-stain technique has been used mostly by biologists.
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