JPC SYSTEMIC PATHOLOGY
Signalment (JPC #4008764): A Panamanian golden frog
HISTOPATHOLOGIC DESCRIPTION: Skin: Multifocally, there is moderate epithelial hyperplasia, characterized by keratinocytes that are piling up to six layers thick with increased mitotic figures, acanthosis, and mild, multifocal orthokeratotic hyperkeratosis. Within the stratum corneum, including the hyperkeratotic debris, there are numerous round, 5-15 um diameter chytrid thalli, which have 1-2 um thick walls. All three forms of thalli are present and include the following: multifocal zoosporangium contain multiple discrete basophilic 2-3 um zoospores and have an injection papillae that is oriented away from the epidermis; fewer multinucleate forms with finely granular basophilic cytoplasm, multiple nuclei, and internal septation; and rare uninucleate forms with homogenous basophilic cytoplasm and a single nucleus. Within the hyperkeratotic debris and stratum corneum there are numerous empty thalli outlined by a 2 um thick eosinophilic walls. The hyperkaratotic debris also contains some necrotic debris admixed with numerous 1x3 um coccobacilli. Multifocally within the dermis there are low numbers of lymphocytes and plasma cells.
MORPHOLOGIC DIAGNOSIS: Skin: Epidermal hyperplasia, multifocal, moderate, with hyperkeratosis, minimal subacute dermatitis, and numerous intracorneal thalli, etiology consistent with Batrachochytrium dendrobatidis, ornate horned frog (Ceratophrys ornata), amphibian.
ETIOLOGIC DIAGNOSIS: Chytridiomycotic dermatitis
CAUSE: Batrachochytrium dendrobatidis
- Batrachochytrium dendrobatidis is a ubiquitous fungus in the phylum Chytridiomycota, class Chytridiomycetes (chytrids), which develops without characteristic hyphae, and are found in aquatic habitats and moist soils, where they degrade cellulose, chitin, and keratin
- Parasitic chytrids mainly infect plants, algae, protists, and invertebrates; the amphibian pathogen, Batrachochytrium dendrobatidis, is the only known chytrid to parasitize vertebrates
- Chytriomycosis is a highly virulent disease which causes dermal infection in both free-range and captive populations of frogs in the Americas, Australia, New Zealand, and Africa
- Batrachochytrium dendrobatidis infection is often restricted to the keratinized skin on the ventral surface of the host; in severe infections, chytrids can be found on the dorsal surface of the host
- Tadpole skin typically does not become infected as it is not keratinized; however, chytrids may infect the keratinized mouthparts of tadpoles
- Batrachochytrium salamandrivorans is causing a sharp decline in fire salamanders and newts in European pet trade
- Minimal hyperkeratosis when compared with anurans
- The exact pathogenesis is unknown
- In amphibians, the skin is important in maintaining hydration, osmoregulation, thermoregulation, and respiration; water absorption primarily occurs in the skin of the ventral pelvic region (area commonly affected by this condition)
- It is thought that chytrid-induced cutaneous changes interfere with these normal skin functions resulting in metabolic abnormalities and subsequent death
TYPICAL CLINICAL FINDINGS:
- Typically, clinical signs occur 14 days post exposure to fungus and include lethargy, abnormal posture, abducted hind limbs
- Excessive shedding and hyperemia; digits are the first areas affected
- Sudden death without clinical signs
TYPICAL GROSS FINDINGS:
- Mild gray-white discoloration of the skin over the hind legs, feet, and ventrum with the presence of partially shed skin
- Sloughed skin appears as thin wrinkled colorless material on the back and legs which gives the skin a roughened and dull appearance
- +/- erosions and ulcerations
TYPICAL LIGHT MICROSCOPIC FINDINGS:
- Stratum corneum affected with varying forms / stages of thalli
- Thalli - 7-15 um round to oval, thick-walled eukaryotic organisms with internal septa and/or zoospores; empty thalli common; thalli are in cytoplasm of epidermal keratinocytes
- 3 forms / stages of thalli
- Uninucleate form - rare; homogenous basophilic cytoplasm
- Multinucleate form - lightly stippled to vacuolated cytoplasm; +/- internal septation
- Cyst-like form (zoosporangium) - contain multiple 2-3 um round to oval basophilic spores
- Discharge papillae - short tubular flask-like extensions from thalli; oriented towards skin surface (may arise from the thalli in all forms of the organism)
- Mild to moderate epidermal hyperplasia and moderate to marked orthokeratotic hyperkeratosis
- Inflammation is rare; when present, mild superficial dermal infiltrates of neutrophils, lymphocytes, and macrophages
- Rare foci of epidermal degeneration and necrosis
- Superficial keratinocytes with intracytoplasmic thalli
- Uninucleate form - abundant cytoplasm with numerous clear vacuoles and mitochondria
- Multinucleate form - thalli cytoplasm subdivided by thin membranes
- Cyst-like form (zoosporangia) - multiple uninucleate, flagellated zoospores with thin cell membranes; no discernible cell walls
- Zoospores – flagella, kinetosome props, terminal plate in the axoneme core and mitochondria with plate-like cristae are highly diagnostic; perinuclear clusters of ribosomes; no visible connection between the nucleus and the kinetosome
- Rhizoid - long, thin, tubular extension from thallus; exits keratinocyte and enters deeper portions of epidermis
- Discharge papillae - wide tubular extensions from thallus; oriented toward skin surface
ADDITIONAL DIAGNOSTIC TESTS:
- Wet mounts of skin scrapings stained with Wright's or Diff-Quik stains
- Skin imprints (live frogs) - small, scattered clusters of keratinized epidermal cells with intracytoplasmic chytrid thalli; shed skin pieces - larger clusters of epidermal cells with many chytrids
- Thallus wall positive with periodic acid-Schiff (PAS) and Gomori's methenamine silver (GMS); spores in zoosporangia - gram-positive; stain weakly with Giemsa
- Culture; immunohistochemistry
- Gross lesions
- Pseudocapillaroides xenopi (Capillaria xenopodis) - 2-4 mm intraepidermal nematodes; causes shedding of skin, anorexia, color change and death; histology - intraepidermal tunnels containing nematodes and thin-shelled, bi-operculate and barrel-shaped eggs
- Aeromonas hydrophila - "Redleg"; acute septicemia, cutaneous hemorrhages and death
- Microscopic lesions
- Basidiobolus ranarum - Chytridiomycotic thalli are distinctive and easily differentiated from the broad, irregular, rarely septate Zygomycete, Basidiobolus ranarum
- Some chytrid species are obligate or facultative parasites of fungi, plants or invertebrates
- Bradley GA, Rosen PC, Sredl MJ, Jones TR, Longcore JE. Chytridiomycosis in native Arizona frogs. J Wild Dis. 2002;38(1):206-212.
- Churgin S, Raphael B, et al. Batrachochytrium dendobatidis in aquatic caecilians (Typhlonectes natans): a series of cases from two institutions. J Zoo Wildl Med. 2013;44:1002-1009.
- Daszak P, Berger L, Cunningham AA, Hyatt AD, Green ED, Speare R. Emerging infectious diseases and population declines. Emerg Infect Dis. 1999;5:735-746.
- Fites J, Ransey J, Holden W, et al. The invasive chytrid fungus of amphibians paralyzes lymphocyte responses. Science. 2013;342:366-369.
- Forzan MJ, Gunn H, Scott P. Chytridiomycosis in an aquarium collection of frogs: Diagnosis, treatment, and control. J Zoo and Wildl Med. 2008;39(3):406-411.
- Martel A, Blooi M, et al. Wildlife disease: Recent introduction of a chytrid fungus endagers western palearctic salamanders. Science. 2014;346:630-631.
- McMahon T, Sears B, et al. Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression. Nature. 2014;511:224-227.
- Mills G. Salamander plague on Britain’s doorstep. Vet Rec. 2015;176:88.
- Nichols DK, Lamirande EW, Pessier AP, Longcore JE. Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs. J Wild Dis. 2001:37:1-11.
- Pessier AP, Nichols DK, Longcore JE, Fuller MS. Cutaneous chytridiomycosis in poison dart frogs (Dendrobates ) and White's tree frogs (Litoria caerulea). J Vet Diagn Invest. 1999;11:194-199.
- Stice MJ, Briggs CJ. Immunization is ineffective at preventing infection and mortality due to the amphibian chytrid fungus Batrachochytrium dendrobatidis. J Wild Dis. 2010:46(1):70-77.
- Van Ells T, Stanton J, Strieby A, Daszak P, Hyatt AD, Brown C. Use of immunohistochemistry to diagnose chytridiomycosis in Dyeing poison dart frogs (Dendrobates tinctorius). J Wild Dis. 2003:39(3):742-745.
- Voyles J, Young S, Berger L, Campbell C, Voyles WF, Dinudom A, Cook D, Webb R, Alford R, Skerratt L, Speare R. Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian decline. Science. 2009: 326:582-585.