JPC SYSTEMIC PATHOLOGY
INTEGUMENTARY SYSTEM
August 2022
I-F10
Signalment (JPC# 4008764): A Panamanian golden frog
HISTORY: None
HISTOPATHOLOGIC DESCRIPTION: Skin: There is multifocal moderate epithelial hyperplasia characterized by keratinocytes that pile up to six layers thick, increased mitotic figures, acanthosis, and mild multifocal orthokeratotic hyperkeratosis. Within the hyperkeratotic stratum corneum there are numerous round, 5-15µm diameter, chytrid thalli with 1-2µm thick walls. All three forms of thalli are present, including: cyst-like zoosporangia that contain multiple discrete, basophilic, 2-3µm 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 2µm thick eosinophilic walls. The hyperkeratotic debris is admixed with some necrotic debris and numerous 1x3µm coccobacilli. Multifocally within the dermis there are low numbers of lymphocytes and plasma cells.
MORPHOLOGIC DIAGNOSIS: Skin: Epidermal hyperplasia, multifocal, moderate, with hyperkeratosis and numerous intracorneal thalli, etiology consistent with Batrachochytrium dendrobatidis, Panamanian golden frog (Atelopus zeteki), amphibian.
ETIOLOGIC DIAGNOSIS: Chytridiomycotic dermatitis
CAUSE: Batrachochytrium dendrobatidis
CONDITION: Chytridiomycosis
GENERAL DISCUSSION:
- 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
- Chytridiomycosis 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 the European pet trade
- Minimal hyperkeratosis when compared with anurans
- The effort needed to identify chytrids in histologic skin sections of some frog species (Physalaemus henselii and Pleurodema bibroni) required examination of 3.2 and 8.7 mm of skin sections for each frog species, respectively (Borteiro, 2019)
PATHOGENESIS:
- 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 the fungus and include lethargy, abnormal posture, and 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 partially shed skin
- Sloughed skin appears as thin wrinkled colorless material on the back and legs, giving 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 are common; thalli are in the 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 the skin surface (may arise from all forms of thalli)
- 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
ULTRASTRUCTURAL FINDINGS:
- Superficial keratinocytes with intracytoplasmic thalli; 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:
- Cytology:
- Skin scraping: Wet mounts stained with Wright's or Diff-Quik stains
- Skin imprints (live frogs): small, scattered clusters of keratinocytes with intracytoplasmic chytrid thalli
- Shed skin pieces: larger clusters of epidermal cells with many chytrid thalli
- Special/histochemical stains:
- 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
- PCR
DIFFERENTIAL DIAGNOSIS:
- 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; typically affects the dorsum
- "Redleg" (typically associated with Aeromonas hydrophila but can be due to septicemia caused by a wide variety of Gram-negative and a few Gram-positive bacteria): acute septicemia, cutaneous hemorrhages, and death
- Microscopic lesions, cutaneous fungal infection:
- Basidiobolus ranarum - broad, irregular, rarely septate Zygomycete (Chytridiomycotic thalli are distinctive and easily differentiated from these)
COMPARATIVE PATHOLOGY:
- Some chytrid species are obligate or facultative parasites of fungi, plants, or invertebrates
- Batrachochytrium salamandrivorans - Fatal disease in pet salamander populations (Pessier, Pathology of Wildlife and Zoo Animals 2018)
- Aquatic dimorphic water molds:
- Pythium insidiosum (I-F02)
- Horses (Cutaneous most frequently reported); Cattle/Sheep (Infrequent) - cutaneous - head, ventral abdomen, distal limbs; gastrointestinal; osteolytic and lymphatic lesions
- Dogs – GI form: enteritis thickened gastric and intestinal walls; Cutaneous reported less frequently (ulcerative dermatitis)
- Cat - rarely infected; cutaneous head and limb lesions, nasal and retrobulbar infection
- Lagenidium giganteum (Dogs)
- Multifocal cutaneous and/or subcutaneous lesions w/ regional lymphadenopathy
- Cutaneous – ulcerated, exudative, w/ necrosis and draining tracts; multiple, firm dermal or subcutaneous nodules
- Granulomatous lymphadenitis
REFERENCES:
- Borteiro C, Kolenc F, Verdes JM, Martínez Debat C, Ubilla M. Sensitivity of histology for the detection of the amphibian chytrid fungus Batrachochytrium dendrobatidis. J Vet Diagn Invest. 2019 Mar;31(2):246-249.
- Maxie, M, ed. Integument. In: Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals, 6th ed., St. Louis, MO: Elsevier, Inc.; 2016:657-659.
- Pessier AP. Amphibia. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals, Cambridge, MA: Elsevier Inc; 2018:930-932, 934, 936.
