Amphibian Medicine

Greg Lewbart M.S., V.M.D., Dipl. ACZM
greg_lewbart@ncsu.edu

Taxonomy and Natural History

There are three orders in the class amphibia. Two of these contain members that are frequently seen in laboratory animal practice and are kept as pets.

I.  Caudata

Salamanders and sirens. These long tailed amphibians usually have two pairs of limbs of approximately equal size (sirens lack hindlimbs). Caudata (sometimes referred to as Urodela) have a holarctic distribution with 4 suborders, 9 living families, 62 living genera and 352 living species.

Fertilization is internal except in the cryptobranchoidea and sirens. Eggs are usually deposited singly or in clumps or strings in water and hatch into aquatic larvae. Plethodontid (lungless) salamanders have direct development of terrestrial eggs and some salamanders are ovoviviparous or viviparous. Several families of salamanders, (Cryptobranchidae, Sirenidae, Amphiumidae and Proteidae), undergo incomplete metamorphosis and remain aquatic for life.

Important Laboratory and Pet Genera:

Ambystoma spp. - The mole salamanders, 26 species. Most breed in ponds in spring but some breed in autumn and lay eggs on land near water which floods the nest. These are heavy bodied burrowing animals and include the popular tiger salamanders and axolotls.

Necturus spp. - Mudpuppies, 5 species. These are obligate aquatic animals and are frequently used in developmental biology studies.

II.  Anura

Frogs and toads. These tailless amphibians usually have long hindlimbs. Anurans are cosmopolitan in distribution except for the high latitudes in the Arctic and Antarctic and some oceanic islands and xeric deserts. There are 21 living families of anurans with 301 living genera and 3,438 recognized species.

Fertilization is external with a few exceptions and usually the eggs are laid in water where the aquatic larvae develop and metamorphose. Some anurans lay terrestrial eggs that produce aquatic tadpoles or even terrestrial eggs with direct development. Ovoviviparity and viviparity are known in some species. Many anurans guard or transport eggs or tadpoles.

Important Laboratory and Pet Genera:

Xenopus spp - The African clawed frog and 13 other species. These amphibians are completely aquatic and their aquatic eggs hatch into filter-feeding tadpoles. Adults are carnivorous. Young frogs are common as pets but can grow to large size (over 1 lb for large females). Tadpoles require still water and adults prefer still water. Used extensively in embryological and development studies.

Rana spp. - Leopard, bull, green frogs (258 species). Semi-aquatic adults lay eggs in water which produce aquatic pond-type tadpoles. Direct development of terrestrial eggs occurs in some Asian species. Adults require access to a terrestrial environment, water at a depth to allow them to sit in the water and deeper water that allows them to hang their legs. Used in basic physiology studies and embryology. These are common pets of young children in rural and suburban areas.

Dendrobates spp. - Poison arrow frogs (48 species). One of four genera of poison arrow frogs. Commonly kept in zoos, studied for their toxins and kept as pets because of their bright coloration. Require humid terrestrial and arboreal habitat. Parental care of tadpoles.

III.  Gymnophiona

The caecilians. These are elongate, limbless burrowing amphibians with small eyes and a short pointed tail when present. Distributed pantropically in 6 families, 34 genera and 162 species.

Most caecilians are oviparous and use internal fertilization, but some species are viviparous. Most species have aquatic young, but some have their eggs develop directly to terrestrial young.

Not commonly seen in laboratories or as pets.

Marketing and Identification

Several methods are used with these animals. For species with dramatic color patterns, individuals are often identified by photographs. Toe clipping is used for short term identification. Many of these species can regenerate toes. Tattooing is used in light colored species, but is limited to larger specimens. Implantation of passive responder chips has been used extensively in some colony situations where subcutaneous implantation or intra-abdominal implantation will not interfere with the research. The designated standard for placement of these chips is in the dorsal lymph sac between the shoulder blades.

Gender Determination

Some species are sexually dimorphic with regard to size or color pattern, but most are not. It can be difficult to determine gender in the absence of reproductive maturity and breeding condition. In many salamanders a swelling of the base of the tail is noticed in reproductively active males. Females ripe with eggs can be identified by candling the abdomen with a light to observe the presence of maturing eggs.

Handling and Housing

The natural history of the species of amphibian being kept is important to the design of appropriate housing. Species from ponds and small bodies of quiet water generally prefer low flow systems and tolerate lower quality water better than stream living species. Species which live in streams and rivers require excellent water quality and do best in flow-through or highly filtered systems.

Day/night cycles are important to reproduction of many species and can have an impact on metabolism. Time of day of feeding can even affect how food is metabolized. Temperature requirements are relatively narrow for tropical species, but temperate species hibernate over winter. Some species of subarctic frogs actually appear to freeze during the winter and revive when thawed.

Many species of amphibians, particularly the frogs, are very susceptible to topical contact with toxins. Smokers should avoid handling smaller species of frogs. The nicotine on their hands can be fatal to the frogs. The poison arrow frogs may cause irritation to the skin of people sensitive to their toxins. In captivity these frogs tend to lose the ability to produce their toxins. Wash hands after handling these frogs or use latex gloves. Salamanders tend to be more resistant to environmental and topical toxins.

Nutrition

Most amphibians are insectivores and require high protein diets. Wild amphibians must be habituated to dead food and may fail to convert, requiring live feed. Captive reared specimens often readily accept prepared diets. Prepared diets offer the advantage of a controlled intake for experimental purposes, and easy and steady accessibility to a food source. Dietary balance is important to amphibians despite the frequent reporting of mineral imbalanced diets used for Xenopus frogs. The main reason researchers get away with calcium poor diets is the ability of most amphibians to absorb calcium from the water. It is not a good idea to rely on this ability. Frogs are susceptible to Metabolic bone disease (MBD). Other nutritional diseases reported in adult amphibians include gout in African bullfrogs, lipid keratopathy in hylid tree frogs, and anemia and vitamin A toxicity in animals fed diets of only liver.

Larvae may have completely different dietary needs from adults. Most tadpoles are herbivorous and convert to carnivory or insectivory after metamorphosis. Immediately after metamorphosis, most young frogs cannot digest food and should not be fed. Tadpoles fed spinach can develop renal oxalate calculi. Iodine deficiency can cause failure to metamorphose.

Anesthesia

Anesthesia of amphibians is relatively simple. A commonly used method is to use tricaine methane sulfonate (MS 222) topically. A 10 gm/L solution of MS 222 is applied to the back of the subject or the subject is made to sit in the solution (about 1 cm deep). Induction is relatively slow and there is considerable individual and species variation in susceptibility to the drug. The margin of safety is good. This method works very well on small specimens.

An alternative method has been recently developed (Letcher, 1992) for use in larger amphibians. It involves the intracoelomic injection of an aqueous MS 222 solution (125 mg/ml) made with sterile water. Although this solution will have a pH of approximately 1.75, no evidence of peritonitis or inflammation attributable to the injection was seen in experimental animals. 100 to 200 mg/kg is suggested in leopard frogs and bullfrogs tolerate doses up to 400 mg/kg.

Blood Collection

In larger specimens it is possible to obtain blood from the saphenous vein or the femoral vein. Some investigators use the collection of lymph from the lymph sacs as a substitute for blood in examining electrolyte levels and protein levels. Cardiac puncture can be accomplished but has a high fatality rate in small specimens. A large vessel is frequently available in the roof of the mouth. The midventral abdominal vein has been used successfully in American bullfrogs.

Injections

Intramuscular (IM) injections are usually made in the posterior muscles of the forelimb or hindlimb in salamanders and the hindlimb of frogs. Most medications are readily absorbed rapidly when applied to the skin of amphibians and this is a common route for medications that otherwise are injected intracoelomic (ICe) or subcutaneously (SQ). ICe injections are made in the right side of the abdomen in the posterior quadrant.

Important Diseases

Red Leg Disease

The term red leg is used to describe a set of symptoms which include erythema and ecchymoses on the underside of the legs and abdomen of frogs. Affected frogs may also show skin ulceration, anemia and ascites. These signs are typical of a number of bacterial septicemias and are not specific for infections with Aeromonas hydrophila but this bacteria is commonly cultured from and incriminated in the pathogenesis of this syndrome.

Aeromonas hydrophila is considered an opportunistic pathogen of amphibians. It is essentially ubiquitous in aquatic environments. It establishes itself in stressed or immunosuppressed animals. Latent infections are common. Control of the disease centers on proper sanitation and environmental quality. Particular care should be taken to avoid the build up of high levels of organic matter in tanks. In the face of an outbreak, antibiotics should be administered based upon culture and sensitivity results.

Mycobacteriosis

Amphibians are susceptible to infections by several atypical Runyon IV mycobacteria including Mycobacterium fortuitum, M. marinum, M. xenopi, and M. thamnopheos. These organisms are ubiquitous saprophytes, found commonly in nature. Normally amphibians have a high degree of natural resistance to disease. Infection with mycobacteria appears to require immunosuppression of the amphibian host.

There is a high incidence of chronic hepatic carriers of M. intracellulare in ranid frogs. The disease is usually chronic, although acute nasopharyngeal mycobacteriosis does occur. The acute form of the disease is characterized by catarrhal or suppurative nasal discharge. The chronic form results in granuloma formation in various organs.

Chlamydiosis

Chlamydia psittaci has been diagnosed in Xenopus and other anurans. Clinical signs mimic an acute bacterial septicemia. At post mortem examination there is necrosis of liver, spleen, kidneys and the heart. Typical intracytoplasmic inclusions are usually visible. No successful therapy regimen has been documented.

Chromomycosis

Amphibians frequently contract fungal infections. Chromomycosis is caused by pigmented fungi that result in characteristic pigmented granulomas disseminated throughout the internal organs. Skin lesions can also bee seen. Therapeutic efforts have been unrewarding to date. Experienced clinicians look for underlying causes of debilitation when this disease is seen.

Saprolegniasis

Fungi of the genera Saprolegnia and Achyla will colonize the wounds of aquatic amphibians. These fungi are considered secondary invaders. Treatment with dilute benzalkonium chloride or povidone iodine topically on the affected wound can be successful. Again, underlying causes for the disease must be investigated and eliminated.

Lung Worm

Rhabidias lung worms can cause pneumonia in captive amphibians. Amphibians may be a source of infection for reptiles where the parasite is considered a serious pathogen. Adult worms are found in the lungs of amphibians. Larvae are found in large numbers in the gastrointestinal system. Worms can also be found free in the coelomic cavity and in the lymph sacs. Ivermectin has been used at 200 to 400 ug/kg orally or SQ to eliminate Rhabidias with 3 to 4 treatments 10 to 14 days apart. Reinfection may occur in a very short period, so tank disinfection with glutaraldehyde based disinfectants and replacement of substrates should accompany treatment.

Cutaneous Capillariasis

Xenopus are susceptible to a capillarid infection of the skin which causes irritation, blotchy skin, skin sloughing, and may predispose toward bacterial superinfections which can cause death. The presence of these small (2-4 mm) nematodes can be determined in skin scrapings or by examining the sloughed epidermis debris. Therapy with ivermectin at the same dose used for Rhabidia infection, or administration of Fenbendazole (30 mg/kg topically) seem to be promising.

Entamoebiasis

Marine toads (Bufo marinus) are reported to develop suppurative nephritis due to Entamoeba marinus infections. Other species of amphibians have shown diarrhea and poor condition from mixed infections of ciliates, flagellates and amoebae. Treatment: 50mg/kg metronidazole (5g/kg diet).

Lucke's Tumor

Lucke's Tumor is a renal adenocarcinoma affecting the northern leopard frog (Rana pipiens) caused by a herpesvirus. This was the first neoplasm demonstrated to be caused by a herpesvirus. Lucke published his belief that a filterable virus was associated with the tumor in 1934. Koch's postulates were fulfilled in the 1970's.

There is a seasonal change in tumor prevalence with tumors being most common in early spring as frogs emerge from hibernation. This has allowed studies of herpes virus replication using this model. Eosinophilic intranuclear inclusions are visible after frogs are held in cold temperatures.

Transmission of the disease is probably during the time that adults occupy breeding ponds. Infection cannot occur without mature virus present and mature virus is not present during warm months. Maturation of the virus occurs after tumor-bearing frogs enter hibernation and release of the virus depends on the warming temperatures of spring. Virus is shed in frog urine. Oocytes and young embryos are susceptible to infection with virus.

Additional Information:

1.  Crawshaw, Graham, J. (1993) Amphibian Medicine. In: Zoo and Wild Animal Medicine Current Therapy 3. M. Fowler Ed. W.B. Saunders, Philadelphia, PA. Pp 131-139.

2.  Duellman, W.E. and L. Trueb. (1986) Biology of Amphibians. McGraw-Hill Book Company. New York.

3.  Hoff, G.L., F.L. Frye, and E. R. Jacobson (editors). (1984) Diseases of Amphibians and Reptiles. Plenum Press, New York.

4.  Letcher, J. (1992) Intracoelomic use of tricaine methanesulfonate for anesthesia of bullfrogs (Rana catesbeiana) and leopard frogs (Rana pipiens). Zoo Biology. 11:243-251.

5.  Raphael, B.L. (1993) Amphibians. In: The Veterinary Clinics of North America, W.B. Saunders, eds: Quesenberry and Hillyer, 23:6, 1271-1286.

6.  Veterinary Clinics of North America: Exotic Animal Practice, WB Saunders, various issues.

7.  Wright K and B. Whitaker. 2001 (in press). Amphibian Medicine. Krieger Publishing, Malabar, FL.

**These notes are a modified version of Dr. Michael Stoskopf's NCSU lecture notes on amphibian medicine. I thank him for permission to use this information.