Laboratory Animal Medicine, 2nd Ed.
Chapter 17
Biology and Diseases of Amphibians
Parts I and II
Pages reviewed: 793-814
Questions:
1.
Amphibian is derived from the greek word “amphibios”. What does it mean?
2.
What 3 orders are found in the class Amphibia?
3.
What are the 3 groups of Caudata?
4.
What is neotony?
5.
Which salamander species demonstrate neotony?
6.
Frogs can be divided into 3 groups. What are they? Give examples.
7.
How can you tell males from females in Rana catesbiana (bull frog), R. grylio (pig frog),
and R. clamitans (bronze frog)?
8.
How has Xenopus laevis (African clawed frog) been used in research?
9.
List other amphibians used in research.
10.
What are the most important considerations when choosing primary enclosures for
amphibians?
11.
What environmental conditions are amphibians susceptible to?
12.
Many amphibians will die from exposure to chlorine in water. How can water be
dechlorinated?
13.
Cloramines, more toxic than chlorine, are sometimes added in municipal water systems.
How are these removed?
14.
Most amphibians have thermal zones lower than reptiles. What are the preferred zones
for tropical and temperate amphibians?
15.
If salamanders of the genus Plethodon are maintained at temperatures over 200 C (68o F),
what will happen?
16.
What type of lighting and light:dark cycles are recommended?
17.
What relative humidity is recommended for most amphibians?
18.
Animal room airflows my dry out wet environments. How can this be alleviated?
19.
How often should the primary enclosures of amphibians be cleaned?
20.
Can phenols be used to clean amphibian enclosures?
21.
Normally amphibians have a protective mucous layer on the skin and have the ability to
secrete toxins through the skin when threatened. What is the proper way to handle these
animals?
22.
What are 5 methods of identifying amphibians?
23.
How long should amphibians be quarantined for long term studies?
24.
What are some zoonotic diseases that may be carried in amphibians?
25.
What are the 2 primary types of skin glands present in amphibians? What do they
secrete and why?
26.
What are the antimicrobial peptides called that are secreted in the skin of Xenopus laevis?
27.
What other chemical compounds can be secreted by granular glands?
28.
Frogs have pronounced ribs. T/F?
29.
What is the primary respiration surface of most amphibians?
30.
What forces air in and out of lungs in amphibians which have lungs?
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How many chambers are found in larval and adult amphibians?
What anatomical feature may impact the pharmacokinetics of drugs with hepatic
excretion?
The lymphatic system of amphibians drains directly into the venous system. The lymph
is first collected into lymph sinuses. Where is the most accessible place to inject a
substance that would be transported to the venous circulation?
Adult amphibians are herbivorous and larval stages are carnivorous. T/F
How dos Xenopus prehend food?
Can amphibians vomit?
What type of kidney is found in salamanders and frogs?
Do amphibians secrete ammonia or urea?
How many cranial nerves are present in amphibians?
A lateral line system (similar to fish) is well developed in larval amphibians and is
retained by adults of many aquatic species. What is its function?
Can amphibians detect color?
How do amphibians detect odors?
Do amphibians live longer in northern or southern climates?
How many years have Xenopus laevis been documented living ?
What 2 types of blood cells are nucleated in amphibians?
What is the preferred choice of food of most adult amphibians?
If only raw meat and organs are fed to growing amphibians, what may be the result?
Most whole vertebrate prey constitute a balanced diet. Why is this?
Crickets (and other insects) lack a Ca-rich skeleton. How can the crickets be treated to
supplement Ca?
How often should amphibians be fed?
Should amphibians be group housed?
Sexual dimorphism exists in many amphibian species. What are some male
characteristics?
What is amplexus?
Do frogs and salamanders have internal or external egg fertilization?
What is unusual about care of the young in the Surinam toad (Pipa pipa)?
What is unusual about care of the young in the midwife toad (Alytes abstricans)?
What unusual reproductive behaviors are seen in the Darwin’s frog and Rheobatrachus?
What regulates reproductive behavior?
How should axolotls be bred?
How does Xenopus tropicalis differ from X. laevis?
What is the natural habitat of Xenopus sp.?
Distinguish between male and female Xenopus.
What are some components of Xenopus skin secretions?
How does Xenopus eat?
How does Xenopus detect smell?
How does Xenopus respire?
How does Xenopus handle waste products during periods of drought?
What is the life span of Xenopus?
Xenopus tanks can be made of what types of materials?
Do Xenopus tanks need covers?
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Clawed frogs have been shown to prefer opaque containers to clear containers because
they do not feel threatened. What are other things that can be added to the frog’s
environment that will allow the frog to hide?
If static water systems are used, how often should the water be changed?
What are 2 other types of water systems in addition to static?
What process should water go through before adding the frog tank?
Population density within the Xenopus tank is a critical factor in growth and productivity.
What are suggested stocking densities?
What is the optimum temperature range for water in the Xenopus tank?
What is the recommended light:dark cycle of breeding year-round?
How should Xenopus be handled?
What are 3 methods that have been used to identify individual animals?
Historically, Xenopus were fed beef liver, beef heart, or ground meat. These were not
nutritionally complete. What is used now?
How often should clawed frogs be fed?
Additional treats include earthworms, mealworms, chick embryos, tubifex worms, gold
fish, and crickets. What is a possible complication of feeding too many crickets or
mealworms?
At what age do Xenopus reach optimum egg production?
How often are Xenopus females bred in the laboratory?
How are Xenopus induced to ovulate and breed?
How can the jelly coat be removed from the frog embryo within the eggs?
What are the components of FETAX solution and what does FETAX stand for?
What do Xenopus tadpoles eat for the first 4 days after hatching?
What do Xenopus tadpoles eat in the wild?
What are Xenopus tadpoles fed in captivity?
What is the recommended stocking density for Xenopus tadpoles?
What type of environment should be given to tadpoles undergoing metamorphosis?
What anesthetic agent is usually used on female frogs to surgically harvest oocytes and in
what concentration?
How is frog skin prepped for surgery?
What analgesics have been used in leopard frogs and will probably work in Xenopus post
op?
How often can oocytes be harvested surgically?
Blood samples from amphibians should collected in lithium heparinized tubes. Identify
blood collection sites in frogs and salamanders. (Use 26-27g needles.)
What are other diagnostic techniques used in amphibians?
Name common routes of injection.
How are amphibians most commonly euthanatized?
Answers:
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“double life” describes the aquatic larval stage and postmetamorphic terrestrial lifestyle
of many amphibians
Gymnophioma (caecilians), Caudata (salamanders), and Anura (frogs)
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Sirens (eel-like amphibians), primitive salamanders (hell benders and related species),
advanced salamanders (mudpuppies, amphiumas, axolotls, newts, and many terrestrial
species)
Condition where salamanders retain larval characteristics while becoming fully
functional reproducing adults
Hellbenders (Cryptobranchus sp.), Ambystoma mexicanum (Axolotl), Ambystoma
tigrinum (tiger salamander)
Primitive frogs-family Discoglossidae (fire bellied toad-Bombina orientalis and midwife
toad-Alytes obstetricans), Transitional frog-family Pelobatidae (spade foot
frog-Scaphiopus), Advanced frogs-family Pipidae (Pipa and Xenopus) and 16 other
families (Bufonidae, Dendrobatidae, Hylidae, Leptodactylidae, and Ranidae)
Male-the typanum is larger than the eye and , in females, the tympanum is the same size
as the eye.
Originally used in pregnancy tests (assays). Injection of pregnant woman’s urine into
the dorsal lymph sac of a female Xenopus would induce the frog to lay eggs. Used in
cell and develomental biology. Can inject with commercially available hormones to
ensure that the frogs reproduce year-round. Also Xenopus have been the subject of
developmental toxicology investigations using FETAX assays (frog embryo teratogenesis
assay: Xenopus). Xenopus has also been used in molecular biology by injecting
oocysts with foreign DNA or messenger RNA to allow expression of genetic material.
Mudpuppies (Necturus)-comparative anatomy, axolotls (Ambystoma
mexicanum)-developmental research, axolotls and newt-limb regeneration, frogs (bull
frogs-Rana catesbiana and leopard frogs-R. pipiens)-physiology and also effects of
emetics because they regurgitate easily, and antinociceptive (pain relieving) effects of
analgesia
Should be constructed of impermeable, easily sanitized material; able to withstand
multiple cage washings; adequate height to accommodate behavioral needs of climbing
and jumping species; and fitted with nonabrasive lids
Dessication if environment is too dry and acidic pH (soil or substrate)
Age open containers of water for 24-48 hours. Add sodium thiosulfate to water. Pass
tap water through activated carbon filters. Aeroate the water.
Use an unused activated charcoal system. Alternatively, add sodium thiosulfate which
reacts with the chloramines to form ammonia. The ammonia can be removed with
zeolites.
Tropical species-21-29o C (70-85o F), temperate species-18-22o C (65-72o F)
Prevents animals from assimilating food rapidly enough to meet the need of the increased
metabolic rate.
Direct exposure to bright light should be avoided. Use full-spectrum bulbs. Provide
shelter to escape light. 12:12 light cycles are adequate unless breeding or trying to
mimic natural habitat.
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Decrease room airflows and house in primary enclosures with minimal openings
Singly housed terrestrial animals with ample floor space can be cleaned every 2 weeks
with spot cleaning as needed. Group housed should be cleaned more frequently as
needed.
No, phenols are toxic and can be absorbed through the skin.
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Glove should be worn that are free of powder and moistened with dechlorinated water.
Do not use abrasive paper towels. Nets of soft fine mesh can be used. Small terrestrial
can be restrained in 1 hand. Larger salamanders should be grasped behind the head and
around the pectoral girdle with 1 hand and around the pelvic girdle with the other hand.
Do not pick up by the tail as it will release! (predator avoidance)
Individual color patterns on the animals. Toe clipping-ineffective in long term studies
because the toes regenerate. It also requires anesthesia, sterilized instruments, and
hemostasis. Tattooing and freeze or chemical branding has been tried but it is not
permanent. Glass or plastic beads have been sewn with nonabsorbable sutures to
amphibians but they must be secure to a muscle mass or they will be shed. Lastly,
passive integrated transponders have been implanted intracoelomically.
2-4 weeks minimum and longer is wild caught
Atypical mycobacterosis (Mycobacterium fortuitum, M. marinum. M. xenopi),
Salmonella, and Chlamydia psittaci
Mucous glands secrete a slimy protective layer which prevents mechanical damage to
skin, facilitates retention of body fluids, and provides a barrier against pathogens.
Granular glands synthesize and secrete a variety of compounds that protect against
predators, as well as chemicals that have antibacterial and antifungal properties.
Defensive compounds can have neurotoxic, cardiotoxic, myotoxic, hallucinogenic,
hypotensive and vasoconstrictive activity. Toxic alkaloids are found in poison dart frogs
(Dendrobatidae), Bufo marinus, and other species can cause vomiting, respiratory
paralysis, and sometimes death.
Magainins which inhibit G+ and G- bacteria, several fungi, and some protozoal species.
Pheromones-courtship and mating, dermorphin-potent opioid and endogenous analgesic,
and bioadhesives-temporary entrapment of predators or adhere to female during mating
False, ribs are absent or greatly reduced.
Skin
Movement of buccopharyngeal floor
Larval-2 chambers, adult-3 chambers
Hepatic portal veins drain the rear half of the amphibian’s body.
Dorsal lymph sacs which lie SC over sacral area and lateral to midline
False, adults are carnivorous and larval may be herbivorous, omnivorous, or carnivorous.
They direct food in with their front legs but many other species use their well developed
tongue.
Yes and often use vomition as a defensive mechanism. Some frog species evert part of
the stomach during regurgitation.
Mesonephric-lacks the ability to concentrate urine in excess of plasma levels
Aquatic amphibians secrete ammonia and terrestrial amphibians secrete urea.
10, the hypoglossal nerve (cranial nerve XII) is formed by branches of the first 2 spinal
nerves.
The linear arrangement of neuromasts on the head and along the body detect changes in
water pressure and currents and function in locating prey.
Yes, 2 types of rods (red, green) are responsible for color sensitivity.
Vomeronasal (Jacobson’s) organ
Northern
15 years
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Red blood cell and thrombocytes
Carnivorous
Calcium deficiency
Bones-Ca, P, and magnesium, liver/kidneys-vitamins, pancreas-zinc, thyroids-iodine
Dust a vitamin-mineral mix on crickets before feeding to amphibians or feed insects with
a diet that is vitamin-mineral rich (gut loaded) before feeding to amphibians.
Daily to twice weekly, depending on species, age of animal, and ambient temperature
No, especially terrestrial species, because they are territorial. They mark territories with
fecal pellets and granular gland secretions and will fight to defend territories.
Male bullfrogs, pig frogs, and bronze frogs have a tympanum larger than the eye. Vocal
sacs of male frogs become larger and more pigmented during breeding season. Cloacal
glands in salamanders become swollen which results in enlarged cloacal lips. Male
plethodontid salamanders develop hedonistic glands on the chin which are rubbed on the
female during courtship. Most male frogs develop keratin pads on their thumbs to better
grip females during amplexus. Certain frog and salamander males develop teeth to
abrade the skin of females. It allows introduction of chin gland secretions into the
female’s bloodstream.
Prolonged breeding embrace
Frogs have external fertilization and salamanders have internal.
After a circuitous amplexus, the fertilized eggs land on the back of the female where the
skin covers them until metamorphosis when froglets emerge.
The male wraps egg strands around his legs and keeps them moist until hatching.
The male Darwin’s frog takes the newly hatched tadpoles in his mouth and holds them in
vocal sacs until metamorphosis. The Rheotrachus female swallows fertilized eggs.
Eggs and tadpoles live in her stomach until metamorphosis. During this time all gastric
activity ceases due to release of prostaglandin.
Arginine vasotocin
Hold males and females separately for 1 week at 72o F then place them together in a
shaded tank at 54o F. Breeding and egg deposition will occur within 2 days. After
oviposition, remove eggs and incubate in separate containers.
X. tropicalis is smaller, has half the haploid genome size per nucleus and has 20
chromosone pairs (X. laevis has 36), generation time is 4-6 months, females lay 3000
oocytes/ovulation, and the embryos are less variable in development
Central to South America. Found in rivers, lakes swamps, ditches, and wells. It
appears to prefer still, cloudy water.
Adult males have dark inner surfaces on front limbs (nuptial pads). Females have large
cloacal papillae and are larger than males.
Thyrotropin releasing hormone, caerulein, xenopsin, and magainins (antimicrobial
compounds)
They shred prey with rear claws and use their front feet to shove food into their mouth
(no tongue).
Two distinct olfactory cavities. One detects scent in water and the other detects airborne
odors.
They must come to the surface to gulp air because cutaneous respiration is not as well
developed as in other species. Even tadpoles develop and use lungs as well as gills for
breathing.
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Xenopus produces urea instead of ammonia. They revert back to ammonia as soon as
ample water is available to dissipate it.
Ten years in the wild and 15 years in the lab
Fiberglass, glass, plastic, or stainless steel
Yes, Xenopus will jump out if the water level is low or if startled.
Partially cover tanks or add retreats such as PVC pipe, stainless steel rabbit feeders,
styrofoam, sponge, round ceramic tiles, terra cotta pots, and sandstone.
After the animal has been fed to remove excess food and defecated material.
Flow through-nonrecirculating and flow through-recirculating
Dechlorination
One frog/3 liters water to 4 frogs/5-10 liters
20-22o C (68-72o F)
12/12
They should be handled with a soft net or gloves to prevent skin abrasion and disrupting
the protective mucous membrane.
The first is sewing a bead on a thread to the skin of the frog. The thread or suture must
be anchored in a muscle mass to prevent sloughing. The second method is to graft pale
skin from the ventral surface of the frog to the back of the frog in the desired pattern.
Thirdly, microchips can be injected into the dorsal lymph sac.
Commercially prepared foods-salmon chow, trout chow, and sinking frog food
2-5 times/week
Intestinal obstruction
2-3 years and continue for several years
Every 1-4 months (ideal induced breeding interval)
HCG (human chorionic gonadotropin) is injected into the dorsal lymph sacs of both the
male and female in 2 separate injection 1-5 hours apart. Males receive 400
units/injection and females get 150-250 on the first injection and 650-850 units in the
second injection. Both frogs are placed into FETAX solution and allowed to breed. If
frogs are bred in containers with false bottoms, the fertilized eggs can be removed safely.
Swirl in 2% cysteine solution at pH 8.1 for 1-3 minutes, decant, rinse embryos with
FETAX solution 5-8 times
NaCl-625 mg/liter, NaHCO3-96, KCl-30, CaCl2-15, CaSO4.H2O-60, MgSO4-75, mixed
with deionized, distilled water. FETAX stands for “frog embryo teratogenesis assay:
Xenopus”.
They do not feed while they are absorbing the yolk. They start eating by Day 5.
The bulk of the diet is plankton suspended in water.
They can be fed strained baby food green vegetables or algae powder; nettle powder,
baker’s yeast, and bonemeal in a 7:2:1 ratio; or pelleted food to larger tadpoles.
1-10 tadpoles/liter of water
Shallow water to prevent drowning
Tricaine methane sulfonate (MS 222) 0.5-2.0 g/liter, buffered with sodium bicarbonate
0.42-1.05 g/liter
Apply dilute povidone iodine or chlorhexidine solution. Do not use scrub that will
disrupt the mucous layer.
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Morphine (10 mg/kg intracoelomic q 12 hrs), flunixin (25 mg/kg intracoelomic q 4 hrs),
butorphanol (25 mg/kg intracoelomic q 12 hours), or xylazine (10 mg/kg intracoelomic q
12-24 hrs).
Rest frogs at least 1 month between surgeries. The IACUC at UT allows a maximum of
6 survival surgeries/frog.
Midventral abdomenal vein in frogs and salamanders, ventral caudal vein in salamanders,
lingual venous plexus beneath tongue in frogs (use heparinized capillary tubes), and
cardiocentesis in frogs and salamanders (should be anesthetized)
Fecal exam, bacterial swabs, impression smears, skin scrapings, abdomenocentesis,
biopsy, radiographs, fluoroscopy, endoscopy, transillumination
Subcutaneous, intramuscular, intracoelomic, and dorsal lymph sac
Overdose of MS 222 or other immersion anesthetic at concentration exceeding 3 g/liter of
buffered water
Part III
Pages reviewed: 814-826
Questions
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What is the most common bacteria associated with red leg?
What other bacteria can cause similar lesions to red leg?
What clinical signs can be seen with red leg?
What 2 factors predispose amphibians to red leg?
Which antibiotics have been used successfully to treat red leg?
Pseudomonas is a Gram + rod that is an opportunist in aquatic and terrestrial species.
What clinical signs and necropsy lesions are seen amphibians infected with
Pseudomonas?
What are the antibiotics of choice for Pseudomonas infection?
How is mycobacteriosis most likely transferred between animals?
What are necropsy findings in amphibians with mycobacteriosis?
Is mycobacteriosis treated in amphibians?
Flavobacterium is a Gram-, aerobic rod found in both soil and water. What species have
been found in frogs?
What clinical signs have been described with these 3 species of Flavobacterium?
What antibiotic has been used successfully to treat Flavobacteriosis?
How is Salmonella spread among amphibians?
What signs are seen in amphibians infected with Salmonella?
What virus causes renal adenocarcinoma in Rana pipiens (Northern leopard frog)?
At what time of year does the tumor grow?
Iridovirus can cause disease in frogs and tiger salamanders. What are the necropsy
findings?
What is a differential diagnosis for iridovirus?
The frog erythrocytic virus is found in the cytoplasm of erythrocytes of wild populations
of Rana. How is it transmitted? What is the common clinical sign?
Chlamydia psittici can infect amphibians. Is it zoonotic? How is it spread?
What clinical signs are associated with Chlamydia infections in amphibians?
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What can be used to treat Chlamydia?
What protozoan infect amphibians?
How do these protozoans infect amphibians?
What are the 3 most commonly described nematodes of amphibians?
Where are the nematodes found in the host?
What are 2 common trematodes of amphibians and where are they found in the host?
What is a commonly encountered cestode of amphibians?
What other types of parasites may infect amphibians?
What fungal agents may be found in amphibians?
What causes metabolic bone disease in both adult and larval amphibians?
What are the signs of metabolic bone disease in tadpoles and adult frogs?
What is lipid keratopathy?
Amphibians may be predisposed to dehydration if the environment is not kept moist.
What are signs of dehydration?
Answers
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Aeromonas hydrophila
Flavobacterium, Pseudomonas, and Proteus
Petechiation and ulceration of the skin (especially legs and abdomen), lethargy, anorexia,
ascites, ocular and periocular inflammation, and neurologic signs
Stress with subsequent immunosuppression and the presence of Aeromonas in the tank
water
Tetracycline, aminoglycosides, chloramphenicol. gentamicin
Axolotls and other amphibian species have skin sloughing and ulceration. Necturus
become septicemic and die. Necropsy lesions in mudpuppies include necrotic gray foci
on gills and cutaneous hyperemia.
Gentamicin and chloramphenicol
Through traumatized skin of an immunocompromised animal. Not highly infectious.
Yellowish white dermal and visceral granulomas and ulcerations. Visceral granulomas
are most often found in the liver. Acid-fast organisms are found in the granulomas.
Yes, valuable animals can be treated with amikacin or enrofloxacin.
F. meningosepticum and F. indologenes have caused disease in leopard frogs. F.
meningosepticum and F. oderans have been found in X. laevis.
F. indologenes causes weight loss, edema, ascites, petechia, dyspnea, uveitis, corneal
edema, and incoordination. F. meningosepticum causes head tilt, circling, ataxia,
anorexia, and ocular opacity.
Trimethoprim sulfa (3 mg/kg SC, IM, PO q 24 hrs)
Fecal-oral route
Anemia, lethargy, anorexia, and diarrhea (consistent with enteritis and septicemia)
Lucke’ tumor herpes virus (LTHV)
The virus replicates during cool winter temperatures but tumors grow during warm
temperatures (spring/summer).
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Petechial and ecchymotic hemorrhage of the skeletal muscle and viscera, dermal
ulceration and necrosis, digit necrosis, and erythema of the skin are seen in frogs.
Hypertrophy of epidermal, gill, and liver cells are found in salamanders.
Aeromonas. In fact, iridovirus may cause the primary lesions and Aeromonas may be a
secondary opportunistic invader.
Mechanically by mosquitoes and midges. The common clinical sign is weakness due to
anemia.
Yes, fecal-oral route
Lethargy, bloating, disequilibrium, erythema, and patchy depigmentation of skin
Oxytetracycline and doxycycline
Entamoeba ranarum (ameba), Trichodina (ciliate), Oodinium and Trypanosoma
(flagellates), and Plistophora (microsporidian)
Entamoeba cysts are swallowed and directly colonize the colon. Trophozoites spread to
the liver and kidney. Oodinium and Trichodina are external parasites that affect the gills
and skin of aquatic amphibians. Ingestion of infected fly larvae is the likely source of
Plistophora myotropica in toads. Trypanosoma infects the blood of amphibians and has
an indirect
Pseudocapillaroides xenopi (X. laevis), Rhabdias (frogs, salamanders), and Foleyella
(frogs)
Pseudocapillaroides lives in the epidermis of Xenopus. Rhabdias larvae penetrate the
frog’s skin and migrate to the lungs where eggs are coughed up and swallowed. Eggs
and larvae live in the gut. Larval Foleyella are found in blood and adults live in the body
cavity and lymph spaces.
Polystoma live in the bladder of frogs. Gyrodactylus lives on the skin and gills of
aquatic amphibians.
Nematotaenia is found in the gastrointestinal tract.
Acanthocephalans, copepods, leeches, trombiculid mites, and toad flies
Saprolegniasis is caused by several fungi, including saprolegnia. Various pigmented
fungi cause chromomycosis, and Basidiobolus is most frequently from cases of
phycomycosis.
Diets deficient in Ca or with an improper Ca:P ratio
Tadpoles have folding fractures, scoliosis, and “rubber jaw”(inordinate mandibular
flexibility due to Ca loss). Adult frogs exhibit abdomenal bloating and tetany following
exertion.
Only female frogs have corneal thickening and opacity with vascularization, superficial
pigmentation, and cholesterol clefts. Possible etiologies include lipid and cholesterol
mobilization associated with egg production, and high dietary fat.
Dull dark skin, sunken eyes, lethargy, and dry sticky mucus