Pathology in Practice

Rory C. Chien 1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078.

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 DVM, MSc
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Jerry W. Ritchey 1Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078.

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History

Seven adult captive-bred American bullfrogs (Lithobates catesbeianus) were purchased from a vendor and transported from the East Coast region to Oklahoma for teaching purposes. All bullfrogs were apparently healthy, except one had a fractured digit and palmar abrasions on the left forelimb that were noted at the time of arrival. Two days later, 3 of the 7 bullfrogs were found dead in their enclosure; another bullfrog died a day later. The remaining 3 bullfrogs appeared unaffected and were without evident external abnormalities up to the time of euthanasia (animals were anesthetized with tricaine methanesulfonate [MS222] and euthanized by double pithing) 1 week after arrival. No experimental manipulation or intervention was performed on live animals. Only 2 of the 4 bullfrogs found dead, a male and a female, were available for full postmortem examination.

Clinical and Gross Findings

The male and female bullfrogs weighed 0.467 kg (1.03 lb) and 0.387 kg (0.85 lb), respectively, at the time of necropsy. The animals were in adequate body condition with abundant visceral adipose tissue stores. The skin of the ventrum of both bullfrogs was markedly and variably reddened, and there were disseminated, petechial hemorrhages on the hind limbs. The female bullfrog had multiple cutaneous erosions and ulcerations ranging from 0.5 to 2 cm in diameter on the ventrum, thighs, medial aspect of the right tarsus, plantar surfaces of the hind limbs, and around the mouth (Figure 1). The female bullfrog also had multiple acicular, dark red foci on the epicardium and multiple pale tan, pinpoint foci throughout the surface and parenchyma of the spleen. The male bullfrog had a fractured and missing digit with granulation tissue and palmar abrasions on the left forelimb, which appeared to be a chronic, trauma-related lesion. Both animals had approximately 5 to 7 mL of yellow-tan, clear fluid in the subcutis of the ventrocaudal portion of the coelom and medial aspect of the thighs. Both animals had multiple, 1- to 5-mm-diameter, dark red, flat foci in the hepatic parenchyma.

Figure 1—
Figure 1—

Photograph of an adult female American bullfrog (Lithobates catesbeianus) that had been transported with 6 other bullfrogs from the East Coast region to a laboratory facility in Oklahoma for teaching purposes. The bullfrog was 1 of 3 animals found dead in the enclosure 2 days after arrival. No experimental procedures had been performed. Notice the marked redness, petechial hemorrhages, and multiple ulcers on the skin. Both thighs were swollen and edematous.

Citation: Journal of the American Veterinary Medical Association 256, 9; 10.2460/javma.256.9.995

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page

Histopathologic Findings

Representative samples of various tissues from each bullfrog were collected, preserved in neutral-buffered 10% formalin, and routinely processed and stained with H&E stain for histologic examination. In the heart of both animals, large numbers of lymphocytes and plasma cells were present in the epicardium, especially at the heart base, and there was evidence of fibrinoid vasculitis, interstitial edema, and fibrin deposition. In the female bullfrog, the spleen was nearly devoid of lymphocytes with marked lymphocytolysis. Approximately 30% to 50% of the splenic pulp was obscured by multifocal, randomly distributed necrosis (Figure 2). The necrotic foci were sharply demarcated and composed of scattered pyknotic and karyorrhectic debris with minimal inflammatory infiltrates. Necrosis of the blood vessels and intravascular fibrin thrombi were also observed. In both bullfrogs, the kidneys had multifocal to coalescing necrotic foci that affected approximately 50% of the parenchyma. Scattered glomerular tufts were necrotic and obscured by karyorrhectic debris. Renal tubular degeneration and necrosis with pronounced dilation and proteinosis were common, accompanied by infiltrates of lymphocytes and heterophils in the interstitium. A few basophilic inclusion bodies were found within the cytoplasm of degenerate hematopoietic cells. In both animals, the liver had multiple areas of hepatocellular swelling, which contained variably sized cytoplasmic vacuoles. The skin of both animals had multiple erosions and ulcerations, which were bordered by small numbers of heterophils and lymphocytes. The underlying dermis and skeletal muscles were edematous. The remaining epidermal cells were variably swollen with clear cytoplasmic vacuoles and large numbers of variably sized (2- to 6-μm-diameter), basophilic intracytoplasmic inclusion bodies (Figure 3).

Figure 2—
Figure 2—

Photomicrographs of sections of the spleen (A) and 1 of the 2 kidneys (B) from the bullfrog in Figure 1. A—Prominent lymphocytolysis is present in the lymphoid follicle next to an arteriole. H&E stain; bar = 100 μm. B—Several glomeruli are necrotic and obscured by karyorrhectic debris (arrows). Necrosis and degeneration of renal tubules with proteinosis (asterisk) are commonly observed. A small amount of inflammatory infiltrate is present in the interstitium. H&E stain; bar = 100 μm. Inset—Some of the hematopoietic cells are necrotic, containing condensed, pyknotic nuclei, and some are slightly swollen with variably sized, basophilic intracytoplasmic inclusion bodies (arrowheads). H&E stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 256, 9; 10.2460/javma.256.9.995

Figure 3—
Figure 3—

Photomicrograph of a section of the skin from the bullfrog in Figure 1. The epidermal cells are moderately swollen, and some contain clear vacuoles and variably sized, basophilic intracytoplasmic inclusion bodies (arrowheads). H&E stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 256, 9; 10.2460/javma.256.9.995

Overall, the microscopic lesions in the female bullfrog were subjectively more severe than those in the male bullfrog. No notable microscopic abnormality was identified within the sections of lung, stomach, intestines, or brain; however, moderate to marked postmortem tissue autolysis in the intestinal tract could have hampered the microscopic examination. The bone marrow of either bullfrog was not examined.

Laboratory Test Findings

Pooled liver and spleen samples were submitted to a veterinary diagnostic laboratorya for testing with a real-time PCR assay for ranavirus (frog virus 3 [FV3]) DNA. The assay result was positive.

A piece of ulcerated skin was collected from the female bullfrog at the time of necropsy and underwent aerobic bacterial culture. Culture yielded moderate growth of a mixed bacterial population that included Aeromonas hydrophila, Streptococcus iniae, Streptococcus dysgalactiae, Shewanella spp, and Elizabethkingia miricola.

Morphologic Diagnosis and Case Summary

Morphologic diagnoses: marked, acute, fibrinoid vasculitis with lymphoplasmacytic epicarditis; marked, lymphoid depletion and multifocal splenic necrosis; marked, acute, multifocal to coalescing glomerular and tubular necrosis with lymphocytic and heterophilic interstitial nephritis and tubular proteinosis; and moderate, subacute, multifocal cutaneous ulcerations with intracytoplasmic viral inclusions.

Case summary: spontaneous FV3 (genus Ranavirus; family Iridoviridae) infection and death in American bullfrogs.

Comments

Gross and histopathologic findings in the bullfrogs of the present report were strongly indicative of an acute systemic disease as the cause of death. The presence of intracytoplasmic inclusions supported a viral infection, and FV3 (an iridovirus) infection was confirmed with a real-time PCR assay.

Ranavirus infection is an emerging disease that can result in mass deaths among wild, captive, and cultured amphibian species.1–3 Ranavirus infection has been associated with decreases in amphibian populations worldwide.1–5 Studies2–4 have provided increasing evidence of ranavirus infection in various animal species, including reptiles and fish; these species can serve as reservoirs of infection and facilitate viral transmission and evolution. Given the species-specific implications of ranavirus-associated mass amphibian die-offs and the potential impact of such die-offs on other ectothermic vertebrate populations, awareness of ranavirus infections has increased during the last decade. It is also listed by the World Organisation for Animal Health (OIE) as a reportable disease of aquatic animals.6

Several transmission routes of ranaviruses have been documented, including exposure to contaminated water or fomites, direct contact with infected animals, and ingestion of infected tissue through predation, cannibalism, or necrophagy.2,4,5 Ranavirus-infected animals may appear ill or have no clinical signs, depending on the susceptibility and immune status of the affected individuals, and ranavirus infection is not always fatal.3 It is believed that amphibians may become more susceptible to infection after encountering natural and environmental stressors.2,4 The virulence of the virus strain may also have a role in the development of disease. Results of a recent study7 indicate that ranaviruses have undergone gene recombination resulting in increased pathogenicity. This phenomenon appears to be associated with human activity, specifically the international amphibian trade.8 With regard to the bullfrogs of the present report, shipping and environmental change could have caused major stress to the animals, predisposing them to infection and resultant death. However, the source of the viral infection was uncertain. No natural ranavirus infection has been reported in Oklahoma.3 We suspected that these bullfrogs were subclinically infected before shipping and that the virus was reactivated because of the immunosuppressed state of the hosts.

Animals that die as a result of FV3 infection may have no grossly identifiable or merely nonspecific lesions, including multiple skin erosions, ventrally located edema, and petechial hemorrhages on the skin or intestinal serosa.5,6,9 In the bullfrogs of the present report that underwent necropsy, ulcerations and hemorrhages on the ventrum and plantar surfaces were observed. These gross findings mimicked other amphibian diseases such as chytridiomycosis and cutaneous bacterial infections.5 Interestingly, A hydrophila was recovered from the cultured ulcerated skin sample. Aeromonas hydrophila is ubiquitous in aquatic environments and historically had been considered the cause of bacterial sepsis (so-called red-leg syndrome) associated with deaths of amphibians in certain instances.10 However, the presence of A hydrophila as well as other bacteria in the skin of the affected bullfrogs most likely represented opportunistic infection secondary to cutaneous ulcers and the compromised immune status of the animals.

Typical microscopic lesions of lethal FV3 infection are necrosis of multiple tissues, predominantly hematopoietic and lymphoid tissues, kidneys, intestinal mucosa, and skin along with variable numbers of basophilic intracytoplasmic inclusions.2,9,11 Necrosis was observed in the kidney, spleen, and blood vessels in the hearts of both bullfrogs of the present report, a finding that was consistent with those of a previous study.9 Basophilic intracytoplasmic viral inclusions were found in multiple affected tissues from the bullfrogs, especially in viable cells at the periphery of necrotic foci. It is worth noting that the viral inclusions can be inconspicuous because of the mixture of karyorrhectic debris. Occasionally, large numbers of viral inclusions can present in the epidermis, consistent with the finding in wood frogs that were experimentally infected with FV3.9 The vasculitis and fibrinoid vascular necrosis in the hearts of the bullfrogs of the present report were likely attributable to FV3-induced endothelial damage, as suggested in 1 report.9 The endothelial damage likely contributed to the widespread petechial hemorrhages and edema as a result of increased vascular permeability.

Research has demonstrated that FV3 as well as other ranaviruses can inhibit protein synthesis and trigger apoptosis of the host cells.12,13 This strategy facilitates FV3 replication and spreading without induction of inflammatory responses. In the bullfrogs examined in the present report, marked lymphocytolysis in the spleen was not accompanied by extensive inflammatory infiltrates, which indicated that some of these cells died through apoptosis rather than acute cell-lytic necrosis.

Diagnosis of FV3 infection is generally based on characteristic gross and microscopic findings. However, the gross lesions caused by FV3 infection can be nonspecific. Careful histologic evaluation of affected tissues and microscopic identification of viral inclusions are important. To confirm FV3 infection, testing with a PCR assay and immunohistochemical analysis specific for ranaviruses are also helpful.6 Data from a study9 of FV3-infected wood frogs have indicated that liver tissue is the preferred sample for the ranavirus-specific PCR assay.

Ranaviruses have been recognized as threats to amphibian populations, and infections with these viruses likely have an important role in global decreases in numbers of amphibians.2–4 For laboratory facilities that conduct work with amphibian species and commercial aquaculture facilities, adequate animal quarantine, biosecurity, and disease surveillance are important for maintaining animal health and preventing the spread of disease.

Acknowledgments

The authors thank Jill Murray, Dr. Todd Jackson, and Dr. Patti Coan for providing a comprehensive clinical history for this report.

Footnotes

a.

Veterinary Diagnostic Laboratory, University of Illinois, Urbana, Ill.

References

  • 1. Miller DL, Rajeev S, Gray MJ, et al. Frog virus 3 infection, cultured American bullfrogs. Emerg Infect Dis 2007;13:342343.

  • 2. Miller D, Gray M, Storfer A. Ecopathology of ranaviruses infecting amphibians. Viruses 2011;3:23512373.

  • 3. Duffus ALJ, Waltzek TB, Stöhr AC, et al. Distribution and host range of ranaviruses. In: Gray MJ, Chinchar VG, eds. Ranaviruses: lethal pathogens of ectothermic vertebrates. Cham, Switzerland: Springer International Publishing, 2015;957.

    • Search Google Scholar
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  • 4. Gray MJ, Miller DL, Hoverman JT. Ecology and pathology of amphibian ranaviruses. Dis Aquat Organ 2009;87:243266.

  • 5. Latney LV, Klaphake E. Selected emerging diseases of amphibia. Vet Clin North Am Exot Anim Pract 2013;16:283301.

  • 6. World Organisation for Animal Health (OIE). Chapter 2.1.2 Infection with ranavirus. Manual of diagnostic tests for aquatic animals 2017. Available at: www.oie.int/en/international-standard-setting/aquatic-manual/access-online/. Accessed Jan 8, 2018.

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  • 7. Claytor SC, Subramaniam K, Landrau-Giovannetti N, et al. Ranavirus phylogenomics: signatures of recombination and inversions among bullfrog ranaculture isolates. Virology 2017;511:330343.

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  • 8. Schloegel LM, Picco AM, Kilpatrick AM, et al. Magnitude of the US trade in amphibians and presence of Batrachochytrium dendrobatidis and ranavirus infection in imported North American bullfrogs (Rana catesbeiana). Biol Conserv 2009;142:14201426.

    • Crossref
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    • Export Citation
  • 9. Forzán MJ, Jones KM, Ariel E, et al. Pathogenesis of frog virus 3 (Ranavirus, Iridoviridae) infection in wood frogs (Rana sylvatica). Vet Pathol 2017;54:531548.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Mauel MJ, Miller DL, Frazier KS, et al. Bacterial pathogens isolated from cultured bullfrogs (Rana castesbeiana). J Vet Diagn Invest 2002;14:431433.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Cunningham AA, Tems CA, Russell PH. Immunohistochemical demonstration of ranavirus antigen in the tissues of infected frogs (Rana temporaria) with systemic haemorrhagic or cutaneous ulcerative disease. J Comp Pathol 2008;138:311.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Chinchar VG, Bryan L, Wang J, et al. Induction of apoptosis in frog virus 3 infected cells. Virology 2003;306:303312.

  • 13. Chinchar VG, Waltzek TB, Subramaniam K. Ranaviruses and other members of the family Iridoviridae: their place in the virosphere. Virology 2017;511:259271.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photograph of an adult female American bullfrog (Lithobates catesbeianus) that had been transported with 6 other bullfrogs from the East Coast region to a laboratory facility in Oklahoma for teaching purposes. The bullfrog was 1 of 3 animals found dead in the enclosure 2 days after arrival. No experimental procedures had been performed. Notice the marked redness, petechial hemorrhages, and multiple ulcers on the skin. Both thighs were swollen and edematous.

  • Figure 2—

    Photomicrographs of sections of the spleen (A) and 1 of the 2 kidneys (B) from the bullfrog in Figure 1. A—Prominent lymphocytolysis is present in the lymphoid follicle next to an arteriole. H&E stain; bar = 100 μm. B—Several glomeruli are necrotic and obscured by karyorrhectic debris (arrows). Necrosis and degeneration of renal tubules with proteinosis (asterisk) are commonly observed. A small amount of inflammatory infiltrate is present in the interstitium. H&E stain; bar = 100 μm. Inset—Some of the hematopoietic cells are necrotic, containing condensed, pyknotic nuclei, and some are slightly swollen with variably sized, basophilic intracytoplasmic inclusion bodies (arrowheads). H&E stain; bar = 10 μm.

  • Figure 3—

    Photomicrograph of a section of the skin from the bullfrog in Figure 1. The epidermal cells are moderately swollen, and some contain clear vacuoles and variably sized, basophilic intracytoplasmic inclusion bodies (arrowheads). H&E stain; bar = 20 μm.

  • 1. Miller DL, Rajeev S, Gray MJ, et al. Frog virus 3 infection, cultured American bullfrogs. Emerg Infect Dis 2007;13:342343.

  • 2. Miller D, Gray M, Storfer A. Ecopathology of ranaviruses infecting amphibians. Viruses 2011;3:23512373.

  • 3. Duffus ALJ, Waltzek TB, Stöhr AC, et al. Distribution and host range of ranaviruses. In: Gray MJ, Chinchar VG, eds. Ranaviruses: lethal pathogens of ectothermic vertebrates. Cham, Switzerland: Springer International Publishing, 2015;957.

    • Search Google Scholar
    • Export Citation
  • 4. Gray MJ, Miller DL, Hoverman JT. Ecology and pathology of amphibian ranaviruses. Dis Aquat Organ 2009;87:243266.

  • 5. Latney LV, Klaphake E. Selected emerging diseases of amphibia. Vet Clin North Am Exot Anim Pract 2013;16:283301.

  • 6. World Organisation for Animal Health (OIE). Chapter 2.1.2 Infection with ranavirus. Manual of diagnostic tests for aquatic animals 2017. Available at: www.oie.int/en/international-standard-setting/aquatic-manual/access-online/. Accessed Jan 8, 2018.

    • Search Google Scholar
    • Export Citation
  • 7. Claytor SC, Subramaniam K, Landrau-Giovannetti N, et al. Ranavirus phylogenomics: signatures of recombination and inversions among bullfrog ranaculture isolates. Virology 2017;511:330343.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Schloegel LM, Picco AM, Kilpatrick AM, et al. Magnitude of the US trade in amphibians and presence of Batrachochytrium dendrobatidis and ranavirus infection in imported North American bullfrogs (Rana catesbeiana). Biol Conserv 2009;142:14201426.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Forzán MJ, Jones KM, Ariel E, et al. Pathogenesis of frog virus 3 (Ranavirus, Iridoviridae) infection in wood frogs (Rana sylvatica). Vet Pathol 2017;54:531548.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Mauel MJ, Miller DL, Frazier KS, et al. Bacterial pathogens isolated from cultured bullfrogs (Rana castesbeiana). J Vet Diagn Invest 2002;14:431433.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Cunningham AA, Tems CA, Russell PH. Immunohistochemical demonstration of ranavirus antigen in the tissues of infected frogs (Rana temporaria) with systemic haemorrhagic or cutaneous ulcerative disease. J Comp Pathol 2008;138:311.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Chinchar VG, Bryan L, Wang J, et al. Induction of apoptosis in frog virus 3 infected cells. Virology 2003;306:303312.

  • 13. Chinchar VG, Waltzek TB, Subramaniam K. Ranaviruses and other members of the family Iridoviridae: their place in the virosphere. Virology 2017;511:259271.

    • Crossref
    • Search Google Scholar
    • Export Citation

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