Infectious Diseases of Amphibians
“Red-leg” syndrome commonly refers to the hyperemia of the ventral skin that accompanies systemic infection in amphibians. Saprophytic, gram-negative bacteria such as Aeromonas, Pseudomonas, Proteus, and Citrobacter spp typically cause red-leg. Viruses, fungi, and other pathogens may cause similar lesions. Ventral hyperemia is a nonspecific sign and may also be seen with toxicosis. Malnourished, newly acquired amphibians that are maintained in poor-quality water or other inappropriate environmental conditions are particularly susceptible. Clinical signs include lethargy; emaciation; ulcerations of the skin, nose, and toes; and characteristic cutaneous pinpoint hemorrhages of the legs and abdomen. Hemorrhages may also be seen in the skeletal muscles, tongue, and nictitating membrane. In acute cases, these signs may be absent. Histologic evidence of systemic infection may include inflammatory or necrotic foci in the liver, spleen, and other coelomic organs. Blood or, if present, coelomic fluid, should be taken for culture before beginning therapy. Individuals can be treated initially with enrofloxacin (5–10 mg/kg/day, PO or IM), oxytetracycline (50 mg/kg, PO, bid), or chloramphenicol (50 mg/kg, PO, bid) before receiving culture and sensitivity results. If fungal infection is suspected, a 0.01% itraconazole bath (3.5 L fresh water mixed with 35 mL itraconazole solution; 5 min/day for 8 days) may be effective.
Mycobacteriosis, caused by acid-fast bacilli, including Mycobacterium fortuitum, M marinum, M chelonea, M abscessus, M avium, M szulgai, and M xenopi, is found principally in debilitated amphibians. Although often an infection of the integument, ingestion of infectious organisms may also lead to GI disease and systemic infection. Affected amphibians may exhibit gray nodules in the skin, liver, kidneys, spleen, lungs, and other coelomic organs. Infected amphibians may eat well but still lose weight. Acid-fast bacilli may be detected in feces and oropharyngeal mucus. A premortem diagnosis can be made by finding acid-fast bacilli in animals with external lesions. Culture of mycobacteria requires special media such as Lowenstein-Jensen agar (cultured at 23°, 30°, and 37°C) but is frequently unsuccessful. In cases in which staining is inconclusive, confirmation using PCR is recommended. Treatment is not recommended for this potentially zoonotic disease, and euthanasia should be considered.
Chlamydiosis is a serious infection of amphibians. Based on histologic lesions and the presence of inclusion bodies, these infections were originally attributed to Chlamydia psittaci. Using molecular methods such as PCR, it has since been shown that other species of Chlamydia have been associated with these infections, including C pneumoniae, C abortus, and C suis. Chlamydia spp have also been found in apparently healthy frogs, which raises the question of whether these animals are a reservoir or vector for these infectious organisms. The disease was originally recognized in a mass mortality of African clawed frogs (Xenopus laevis) fed uncooked beef livers. Infected frogs may die peracutely or exhibit lethargy, dysequilibrium, cutaneous depigmentation, petechiae, and edema. Histologically, intracytoplasmic basophilic inclusion bodies can be identified in sinusoidal lining cells of the liver and spleen. Secondary bacterial infections are frequently present in affected amphibians and must be treated appropriately. Antibiotic treatment, including doxycycline (5–10 mg/kg/day, PO) or oxytetracycline (50 mg/kg/day, PO), may be effective against chlamydial infection.
Many of the fungi that infect amphibians are difficult to distinguish grossly because they produce similar clinical effects, including lethargy and skin ulcerations. Some fungi can be identified via the examination of a wet mount prepared from a skin scraping, whereas others require culture, histology, and special stains. Treatment includes proper hygiene and the use of topical or systemic antifungal agents such as itraconazole. Other antifungal drugs such as fluconazole may also be effective.
Chytridiomycosis is the most serious fungal infection in amphibians and has been implicated in the decline of wild and captive frog, toad, and salamander populations around the world. The causative agent, Batrachochytrium dendrobatidis (Bd), is a nonhyphal zoosporic fungus identified in specimens preserved as far back as 1938, yet it was not until the 1990s that the organism was associated with large losses of Australian frogs. Chytrids are typically found in moist environments, where they feed on decomposing organic matter or are parasitic to plants and invertebrates. Amphibians are the only known vertebrate host for chytrid fungi. Postmetamorphic anurans are affected far more than caudates or larval amphibians. Tadpoles are usually subclinical, because Bd infects only tissues that contain keratin. Anuran larvae have keratin only in their mouthparts, which may become depigmented and sometimes damaged. Most caudate larvae do not have keratinized mouthparts. As larvae undergo metamorphosis and their skin becomes keratinized, Bd can spread over the animal, infect the stratum corneum of the epidermis, and cause mortality. The “drink patch,” specialized skin located in the ventral pelvic region that absorbs water, and the digits are especially affected. Diagnostic samples are best taken from these regions for this reason. Because pathogenicity of Bd appears to decline above 25°–27°C, it is speculated that some amphibian populations living at higher elevations have disappeared because of the cooler temperatures associated with the winter months and possibly climate change. Although the exact mechanism of infection is unknown, Bd causes skin hyperkeratosis and subsequent sloughing, osmoregulatory disruption, electrolyte disruption, and cardiac arrest. Infection intensity (>10,000 zoospores) appears to be associated with mortality. Some amphibians, including the bullfrog (Rana catesbeiana) and the African clawed frog (Xenopus laevis) are less susceptible to Bd and may serve as reservoirs for the disease. Mobile zoospores contribute to the loss of whole populations of amphibians. Clinical signs include abnormal posture, anorexia, lethargy, dehydration, hyperemia, excessive shedding of skin, pupillary miosis, and muscle incoordination. These signs may be mild to absent in infected caudates, emphasizing the importance of performing diagnostic tests. Visualizing the spherical, single-celled organisms in skin scrapings stained with Wright-Giemsa or Gram stains using a light microscope is diagnostic, but the organisms are not always readily seen. Real-time PCR performed on swabs of the integument or pieces of skin is diagnostic and provides rapid assessment of presence and quantity of zoospores. This is useful in screening and management of amphibian populations at risk, such as those undergoing transport or quarantine. On histopathology, zoosporangia containing zoospores are associated with hyperkeratosis and underlying dermal infection. Treatment includes the topical administration of itraconazole (0.01% bath for 5 min/day for 10–11 days) and maintaining animals well within their normal thermal range. The use of terbinafine (0.01% bath buffered using bicarbonate to a pH of 7.2–7.4 for 5 min/day for 5 days) may also be effective. If appropriate for the species, raising environmental temperatures for captive populations to >23°C may help halt the infection while medicated baths are used to eliminate Bd. Systemic antifungal drugs appear to be ineffective in treating this infection of the epidermis. Bd is a World Organization for Animal Health (OIE) notifiable disease.
Saprolegniasis refers to disease caused by several genera of opportunistic fungi or “water molds” that infect the gills and/or skin of aquatic and larval amphibians. When in water, newly affected animals appear to have a whitish cotton-like growth on their skin. As the fungal mat ages, it may become greenish due to the presence of algae. Once removed from water, the fungal mat collapses and is difficult to see. Other signs include lethargy, respiratory distress, anorexia, and weight loss. Skin ulcerations may occur as the infection progresses. A diagnosis of saprolegniasis is made by finding hyphae and the thin-walled zoospores in a skin scrape. Treatment with a malachite green dip (67 mg/L for 15 sec, once daily for 2–3 days) or copper sulfate (500 mg/L for 2 min, once daily for 5 days, then once weekly until healed) may be effective. Treatment of eggs with methylene blue may be effective. Secondary bacterial and parasitic infections may be present in animals with dermal ulcers. Poor water quality conditions should be corrected.
Chromomycosis is caused by pigmented or black fungi from several genera (eg, Cladosporium, Fonsecaea, Phialophora, Ochroconis, Rhinocladosporium, and Wangiella). These fungi may be found in organic substrates such as topsoil and decaying plant matter. Disease is either cuteaneous or disseminated systemic; both have been seen in captive and wild populations of amphibians. Signs may include anorexia, weight loss, granulomatous skin lesions or ulcers, coelomic distention, and neurologic disease. Diagnosis is usually made postmortem by finding disseminated granulomas with pigmented fungal cells and hyphae. Culture is frequently unsuccessful; histopathology may be necessary to confirm the diagnosis. Treatment using itraconazole (10 mg/kg/day, PO, for 30 days) may be given, but the prognosis is poor once the infection is disseminated.
Zygomycosis, caused by fungi of the class Zygomycetes (Mucor spp, Basidiobolus sp, and Rhizopus spp), affects both wild and captive populations of anurans. Clinical signs include lethargy and multifocal hyperemic nodules with fungal growth on the ventrum. Disease progresses rapidly and results in mortality within 2 wk. Zygomycetes are found in the environment, especially soils and decaying matter, and are a normal component of the amphibian's GI tract. Successful treatment has not been reported, but advanced antifungal agents may be tried.
Mesomycetozoans are fungus-like microrganisms at the animal-fungal boundary. Those reported in the literature to infect amphibians include Amphibiothecum (previously Dermosporidium), Amphibiocystidium, and Ichthyophonus; however, continued molecular characterization of these organisms is ongoing, resulting in nomenclature changes and taxonomic reorganization. Amphibiothecum and Amphibiocystidium are spore-forming organisms that typically produce a nonlethal infection in anurans. Clinical signs include multifocal nodules and pustules, usually on the ventrum, that resolve in 4–8 wk. Microscopically encysted spores contain large cytoplasmic vacuoles. Ichthyophonus is pathogenic to salamanders and frogs living in the eastern half of the USA. Pre- and post-metamorphic life stages are infected. Clinical signs include muscle swelling in the thigh, rump, and tail and may appear nodular, especially in tadpoles. Debilitation may lead to mortality, especially in adults. Diagnosis is based on histopathology or finding characteristic spores through microscopic examination of material from the lesions. There is no treatment other than supportive care.
Many of the protozoa and metazoa found in and on amphibians are not associated with disease unless the host amphibian is stressed or immunocompromised. Recently caught or transported amphibians are particularly susceptible to parasitism, as are those kept in poor hygienic conditions and outside their POTZ. Parasites with indirect life cycles tend to die out when wild-caught amphibians are brought into captivity if the intermediate or final host is not present. Conversely, infections by parasites with a direct life cycle may be magnified in a closed environment. Excellent hygiene is essential for parasite control and includes the routine removal of sloughed skin, fecal material, uneaten food, and carcasses from animal enclosures.
External parasites may be found by close examination of amphibians using magnification and a bright, cool light. A skin scrape or biopsy may be required to identify parasites causing nodules or epidermal lesions. Internal parasites are often identified through examination of fresh fecal samples. Some small frogs are translucent enough to allow the visualization of nematodes using transillumination. In some cases, metazoan and protozoan parasites are found only at necropsy. Finding flagellates, ciliates, and opalinids in the feces is normal and does not require treatment in healthy amphibians. Although many larval nematodes found in the feces are nonpathogenic, treatment is recommended because pathogenic and nonpathogenic species cannot be readily distinguished.
Most protozoans found in the GI tract, including ciliates, opalinids, and flagellates, are commensals. The ciliate Tetrahymena, although normally nonpathogenic, has been associated with mortality of salamanders. Trichodinids may be found in the urinary bladder or on the skin of amphibians and require treatment. Hemoflagellates are occasionally found and generally nonpathogenic but can result in anemia. Greater-than-normal loads of GI flagellates and other protozoa may be found in debilitated amphibians and require treatment aimed at restoring balance and not eliminating the protozoa. External dinoflagellates (eg, Piscinoodinium) and flagellates (eg, Ichthyobodo) can cause significant mortality, especially in larval amphibians. Fecal samples collected by placing the amphibian on clean, moist paper towels helps to prevent contamination from free-ranging protozoa. Cloacal wash, gill clips, and skin scrapes are also diagnostic. Treatment using metronidazole is often effective for external and GI organisms. Sporozoans such as coccidia (Eimeria and Isospora) and microsporidians (Microsporidium, Pleistophora, and Alloglugea) may be incidental findings or parasitic. Clinical signs are nonspecific and include poor body condition and wasting. Myxozoa occasionally cause disease in amphibians and result in specific host/disease agent lesions. Treatment efforts are focused on providing supportive care.
Metazoa parasites include myxozoa, helminths, and arthropods. Myxozoa infections in amphibians generally do not cause mortality. Helminths that are pathogenic to amphibians include trematodes (Ribeirola, Clinostomum) and nematodes (Rhabdias, Strongyloides, Pseudocapillaroides). Arthropods, such as the common fish parasites Argulus and Lernaea, may infect amphibian aquatic life cycle stages, whereas ticks and mites affect postmetamorphic terrestrial animals. Larval dipterid flies may consume amphibian eggs and embryos and feed on the tissues of adults. Two of the most significant metazoan infections in captive amphibians are caused by Rhabdias sp and Pseudocapillaroides xenopi.
Rhabdiasis, caused by the lungworm Rhabdias sp, commonly causes pulmonary damage and secondary infections in captive amphibians. This nematode has a direct life cycle with free-living phases. Adult worms live in the lungs, where they deposit larvated eggs that are coughed up, swallowed, and then excreted into the environment. Infective L3 larvae then burrow through the skin of a new host, where they mature and migrate to the lungs. Affected animals may appear anorectic, thin, and generally debilitated. A premortem diagnosis may be made by finding ova or worms in oral and nasal secretions. Infection should be suspected when nematode larva and larvated eggs are found in fresh feces from an animal with clinical signs. When rhabdiasis is suspected, treatment using fenbendazole (100 mg/kg/day, PO, for 2 days then repeated 12–14 days later) or ivermectin (200–400 mcg/kg, PO, once, repeated 12–14 days later) is recommended. After the second of each 2-day fenbendazole treatment or each dose of ivermectin, the animals should be moved into a newly established environment to prevent reinfection from free-living life stages. Some reactions to fenbendazole have been reported, so animals should be monitored closely and treatment discontinued if necessary.
The capillarid nematode Pseudocapillaroides xenopi burrows into the skin and is known to affect colonies of the aquatic African clawed frog. Signs include discoloration, roughening, pitting, and ulceration of the skin. As the infection progresses, lethargy, anorexia, and skin sloughing occur. Diagnosis is made by finding small, white nematodes beneath the mucus on the skin; skin scrapings may show larvae and ova. Treatment by adding thiabendazole (0.1 g/L) to the water may be effective. Levamisole and other anthelmintics may also be effective. Frequent water changes with removal of shed skin containing the parasite are required to prevent the amplification and spread of infection to cage mates.
Renal adenocarcinomas (Lucké tumors), caused by ranid herpesvirus-1, are relatively common in leopard frogs (Rana pipiens) wild-caught in the northeastern and north central USA. Few frogs with tumors are seen in the summer, because viral replication is temperature-dependent. Virus particles and intranuclear inclusion bodies are seen when frogs are in hibernation, at 41°–50°F (5°–10°C). Metastasis of the tumor to liver, lungs, and other organs is common; both the primary and metastatic tumors can become very large. There is no treatment. The neoplasm is a model of herpesvirus-induced cancer.
Ranaviruses, which are DNA-based viruses in the genus Ranavirus, family Iridoviridae, have been identified as the cause of mass mortality in wild populations of anurans and caudates across the world. Environmental conditions, reservoir species, persistence in the environment, direct and indirect transmission, stress, and host immunity contribute to the impact of ranaviruses on amphibian populations. Species of Ranavirus that infect amphibians include frog virus 3 (FV3) and FV3-like viruses, Bohle iridovirus, and Ambystoma tigrinum virus. These viruses are highly virulent and may produce 90%–100% mortality in tadpoles and adults. Transmission occurs through exposure to contaminated water or soil, contact with infected individuals, and consumption of infected tissues. Fish and reptiles (especially turtles) are reservoirs for ranaviruses. Clinical signs are nonspecific, develop rapidly in a large number of cohorts, and include abnormal swimming behavior, swelling of the limbs or body, edema, hydrocoelom, erythema, ventral skin hemorrhage (especially in the hind region), and occasionally skin ulcerations. Lesions may appear very similar to those of bacterial dermatosepticemias. The original viral lesions may be overwhelmed by secondary invaders, and many outbreaks of “red-leg” may have had underlying and undiagnosed viral infection. Death typically results from multiple organ failure. Amphibian larvae undergoing metamorphosis or recently metamorphosed juveniles seem to be most susceptible to infection. For this reason, mortality events often occur in the spring/summer. Survivors appear to acquire some immunity to future infections. Diagnosis is made using PCR, primary cell culture, and/or microscopy. Ranaviruses can persist in the aquatic environment without a host for weeks, which contributes to their ability to infect naive amphibian populations and emphasizes the need for biosecurity measures when transporting amphibians or working in environments where the virus exists. Disinfection using bleach (1%) and chlorhexidine (0.75%) are effective. Ranavirus disease is notifiable to the World Organization for Animal Health (OIE).