Pathology Case of the Month - Foothill Yellow-legged Frogs

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Case History: In October 2019, a mortality event of foothill yellow-legged frogs (Rana boylii) in a small creek in Santa Clara, California, USA was reported. Mortality peaked in autumn, when nighttime temperatures approached freezing. At the time of submission, the estimated number of dead was 35.

Gross Findings: Submitted were three post-metamorphic frogs, ranging from 1.9 to 4.2 cm snout-vent length. All animals were in fair to good body condition and good post-mortem state. On external examination one animal had pinpoint red foci on the ventral abdomen (Fig. 1A). Another animal had a mild splenomegaly and the kidneys were diffusely reddened (Fig. 1B). 

Undersides of two frogs one of which shows organs  inside abdomen.

Figure 1: Two foothill yellow-legged frogs (Rana boylii) found dead in Santa Clara, California, USA. (A) One animal had pinpoint red foci on the ventral abdomen. (B) Another animal had a diffusely reddened kidney (arrow). (Credit: Saskia Keller, USGS National Wildlife Health Center. Public domain.)

Histopathological Findings: Ranavirus affects endothelial cells, hematopoietic tissue, and various epithelial cells, causing widespread areas of necrosis in a variety of tissues characterized by accumulation of cellular debris with karyorrhectic, karyolitic, and karyopyknotic nuclei (Fig. 2). Endothelial necrosis is accompanied by fibrin thrombi and can be found in almost all organs (Fig. 2C, 2D). General hematolymphoid necrosis of the hematopoietic and lymphoid tissue can be observed in spleen, liver, kidney, bone marrow, and thymus (Fig. 2C, 2D). Examples of affected epithelial cells include the skin, liver, and kidney (Fig. 2A, 2C, 2D). In the liver, large areas of randomly distributed coagulative necrosis are present, and surrounding hepatocytes are often shrunken and hypereosinophilic. A few hepatocytes contain intracytoplasmic, 1 to 2-µm, basophilic inclusion bodies (Fig. 2B).

Photomicropgraphs with arrows showing location of lesions.

Figure 2. Photomicrographs from a foothill yellow-legged frog (Rana boylii) found dead in Santa Clara, California, USA. (A) Small areas of epidermal necrosis with apoptotic keratinocytes and nuclear debris are multifocally present (arrow). (B) The liver shows randomly distributed, variably sized areas of coagulative necrosis (*). A few adjacent hepatocytes have intracytoplasmic, round, basophilic, 2 to 4-µm inclusion bodies (inset, arrowhead). (C) Within the bone marrow are necrotic hematopoietic cells (arrow) and endothelial cells with consecutive fibrin thrombi (*). (D) Different processes are present in the kidney. Glomeruli are affected due to necrosis of the endothelial cells (arrow). The interstitium, normally containing hematopoietic cells, show widespread hematolymphoid necrosis (*). Additionally, proximal tubules show epithelial necrosis with hypereosinophilic sloughed epithelial cells (arrowhead). (Credit: Saskia Keller, USGS National Wildlife Health Center. Public domain.)

Morphologic Diagnosis/es:

Condition: Ranavirosis/ Ranavirus-Infection

Etiology: Ranavirus, Iridoviridae (double-stranded DNA-virus)

Distribution: Worldwide, where amphibians are present (North America, South America, Europe, Asia, Africa, and Australia).

Seasonality: Ranavirus mortality events occur during spring and summer. 

Host range: Ranavirus has a broad host range in ectothermic vertebrates, including reptiles, amphibians, and fish. While some strains are clade-specific (e.g. Frog Virus 3 in frogs; Ambystoma tigrinum virus in salamanders), others may be able to infect animals from more than one class (e.g. amphibians and reptiles, or amphibians and fish). In amphibians, larvae and metamorphs are especially susceptible. Adult morbidity and mortality occur less often.

Transmission: Transmission occurs through direct contact with an infected animal or via environmental contamination of water or substrates. Ingestion of infected carcasses has also been suggested as a possible route of transmission.

Clinical signs: Suspicion of a ranavirus outbreak should be raised if multiple species of amphibians, mainly including larvae or recent metamorphs, are affected. Skin lesions characterized by reddening and later skin ulceration are often the first signs observed. Acute deaths with no previous clinical signs are possible. In tiger salamanders and Chinese giant salamanders, epithelial proliferation presenting as raised plaques and polyps have been reported.

Pathology: The virus has a tropism for endothelial cells, hematopoietic tissue, and a variety of epithelial cells, leading to widespread hemorrhages and necrosis, especially in skin, liver, spleen, bone marrow, kidney, and the gastrointestinal tract. Nonclinical carriers have been identified that can further shed and transmit the virus.

Diagnosis: Gross and histological examination can be suggestive of a ranavirus infection, but further tests are necessary to confirm cases. Molecular testing (PCR), virus isolation, immunohistochemistry, and transmission electron microscopy can be used to verify the presence of the virus. The virus should be isolated from inner organs, since environmental contamination of the skin is possible.

Public health concerns: None known.

Wildlife population impacts: Ranavirus can cause significant mortalities in amphibian populations, further contributing to the global decline of this class. During an outbreak, entire juvenile amphibian populations can be decimated within 1 to 2 weeks. Due to this potentially devastating impact, ranaviral disease was listed by the World Organization for Animal Health (OIE) as an internationally notifiable disease in 2008.

Management: Virus particles are highly resistant to desiccation and can remain infectious for long periods of time in certain environments. Fomites such as nets, boats, and other equipment can carry the virus to new locations. Decontamination of equipment used during a visit to an infected site is advised to reduce the potential for human-assisted dispersal.

References:

Pessier AP. 2018. Amphibia. In: Pathology of Wildlife and Zoo Animals, Terio KA, McAloose D, St. Leger J, editors. Academic Press, pp. 921-942.

Densmore CL, Green DE. 2007. Diseases of Amphibians. ILAR Journal 48(3): 235–254.

Gray MJ, Chanchar VG. 2015 Ranaviruses: lethal pathogens of ectothermic vertebrates. Springer International Publishing AG, Cham, Switzerland

International Office of Epizootics (OIE), Aquatic Animal Health Standards Commission. 2019. Manual of Diagnostic Tests for Aquatic Animals, www.oie.int/en/standard-setting/aquatic-manual. Accessed February 2020

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Date published: July 29, 2019
Status: Active

Pathology Case of the Month

Notable cases at the National Wildlife Health Center are highlighted here in the Pathology Case of the Month Series.