Pathology in Practice

Arno Wünschmann From the Minnesota Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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History

In June of 2019, 2 free-ranging adult male common loons (Gavia immer) were found dead in a lake in Northern Minnesota. The carcasses were frozen by the finder and submitted for postmortem examination.

Gross Findings

The birds were in a state of moderately (loon 1) and markedly (loon 2) advanced postmortem autolysis. Loons 1 and 2 weighed 4.3 kg and 4.5 kg, respectively. In both birds, there was a scant amount of internal adipose tissue, and the liver was diffusely moderately enlarged and diffusely tan (Figure 1). The spleen of each bird was moderately enlarged, measuring approximately 4 cm in length and approximately 2 cm in width (at its widest point in the midsegment). In loon 1, the small intestine was dilated along its entire length and its mucosa was tan.

Figure 1
Figure 1

Photographs of the body cavity of an adult male loon (loon 1) that was found dead along with another loon in a lake in northern Minnesota. A—The liver is diffusely enlarged and tan. The small intestine is dilated. Inset—Image of the liver of a control loon that was euthanized via IV injection because of tarsal joint inflammation. Notice the dark red color and size of the liver. B—The spleen is moderately enlarged. Inset—Image of the spleen of the control loon.

Citation: Journal of the American Veterinary Medical Association 259, 12; 10.2460/javma.19.07.0360

Formulate differential diagnoses, then continue reading.

Histopathologic Findings

Tissues from loon 1 only were deemed suitable for histologic examination. The liver had large, randomly distributed, confluent areas of necrosis. The hepatocytes in these areas were each hypereosinophilic and had a fragmented or pyknotic nucleus (Figure 2). Splenic ellipsoids were frequently replaced and surrounded by homogeneous, eosinophilic, acellular material, which was interpreted as fibrin. Numerous renal tubular epithelial cells were sloughed into the lumen of proximal tubules. The sloughed cells were each hypereosinophilic and had a fragmented or pyknotic nucleus. The intestinal mucosa appeared to be multifocally necrotic, but definitive interpretation was hampered by autolysis and freeze-thaw artifactual changes. Immunohistochemical analysis of sections of liver tissue with a peroxidase-based polymer system (Envision-HRP; DAKO) and an envelope protein–specific monoclonal antibody1 (clone 7H2; BioReliance) revealed that numerous hepatocytes and Kupffer cells were positive for West Nile virus (WNV) antigen (Figure 3). In all other examined organs, including the brain, heart, kidneys, intestines, liver, and lungs, endothelial cells were labeled for WNV antigen. In addition, numerous splenic arteries and ellipsoids were positive for WNV antigen, as were numerous tubular epithelial cells in the kidneys and crypt epithelial cells in the small intestines. The interrenal cells of the adrenal glands were strongly positive for WNV antigen.

Figure 2
Figure 2

Photomicrograph of a section of the liver of loon 1. The hepatocellular cords are in disarray. The cytoplasm of all hepatocytes is hypereosinophilic (arrows), and numerous nuclei are fragmented. H&E stain; bar = 60 µm.

Citation: Journal of the American Veterinary Medical Association 259, 12; 10.2460/javma.19.07.0360

Figure 3
Figure 3

Photomicrograph of a section of the liver of loon 1 after immunohistochemical staining for West Nile virus (WNV) antigen. Notice the strong immunoreactivity of the cytoplasm of numerous hepatocytes (arrows) and Kupffer cells to WNV-specific antibodies (arrowhead). WNV antigen–specific immunohistochemical reaction; bar = 60 µm.

Citation: Journal of the American Veterinary Medical Association 259, 12; 10.2460/javma.19.07.0360

Results of Ancillary Testing

Samples of splenic tissue from both loons were positive for WNV nucleic acid as determined by a PCR assay performed with a previously described protocol.1,2 Inductively coupled plasma atomic emission spectroscopy analysis of liver tissue from loon 1 revealed high mercury concentration (57.22 μg/g of tissue dry weight); hepatic lead concentration was below the detection limit of 0.9 μg/g. The hepatic selenium concentration was 20.91 μg/g. Aerobic bacterial culture of liver tissue from loon 1 yielded no growth.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: widespread hepatic necrosis, fibrinonecrotizing splenitis, and necrosis of renal tubular epithelial cells.

Case summary: systemic WNV infection in 2 common loons.

Comments

WNV, a flavivirus, was introduced into the US in 1999.3 The virus is primarily transmitted by mosquitoes. Consequently, occurrences of WNV-associated disease is seasonal in the Midwest of the US, with the highest virus activity between mid-June and the end of September.4 The virus has caused unprecedented morbidity and death among numerous avian species in North America.5,6 WNV not only has a remarkably diverse host spectrum—being capable of infecting a wide variety of birds, mammals (including humans), and reptiles—but also has a remarkably wide cell tropism, including epithelial cells, neuroepithelial cells, and mesenchymal cells. The lesions in a few avian species, such as crows and a variety of raptors, are well described.2,7,8,9,10 Interestingly, the clinical signs of WNV infection vary greatly, even within genera,9,11 and are reflected in the variety of gross and microscopic lesions as well as WNV antigen distribution in affected birds.12 As of May 2019, the deaths of only 5 loons as a result of WNV infection had been reported by ArboNET of the CDC, but the lesions were not described (N Lindsey, MS, CDC, email, July 22, 2019). Four of those loons were examined by the author in 2005 at the Minnesota Veterinary Diagnostic Laboratory. Those birds were a family of loons (2 parents and 2 chicks) that died within a 1-week period and had similar gross, histopathologic, and immunohistochemical findings to the loons of the present report. The major lesions in loon 1 of the present report were hepatic necrosis, splenic ellipsoid necrosis, and renal tubular necrosis; inflammatory lesions were not detected. These types of lesions, the high WNV antigen burden, and the widespread WNV antigen organ and cell distribution indicated that this bird died during a viremic phase. These observations suggested a high susceptibility of common loons to WNV infection, with rapid disease progression similar to that described for WNV-infected northern owls, such as snowy owls (Bubo scandiacus) and great gray owls (Strix nebulosa). In contrast, the clinicopathologic features of WNV infection in red-tailed hawks (Buteo jamaicensis), bald eagles (Haliaeetus leucocephalus), and golden eagles (Aquila chrysaetos) are characterized by a more protracted disease course and inflammation of the heart, brain, and eyes in the face of a variable abundance of detectable viral antigen in a limited number of tissues.9,12 In great horned owls, the disease course is even more protracted than that in hawks and eagles, as indicated by an invariably emaciated state of WNV-infected birds at necropsy, the otherwise usually subtle nature of the lesions, paucity of viral antigen concentration, and limitation of antigen distribution to the kidneys and possibly the cerebellum and heart.8 On the basis of this inference, the prevalence of West Nile disease in loons may be underreported. Loons spend their entire life on water except when nesting on offshore nest sites.13 This may explain why only few cases of WNV infection in loons have been recorded to date, despite close observation of these emblematic birds by many lakeside residents.

Besides hepatocytes, enterocytes, renal tubular epithelial cells, and adrenal interrenal cells, endothelial cells and cells of the monocyte-macrophage system (eg, Kupffer cells) appeared to be major targets for the virus in loon 1 of the present report. The targeting of endothelial cells and cells of the monocyte-macrophage system likely caused vascular damage and some of the resultant lesions. Although it was most likely that loon 1 was infected by mosquito transmission of WNV, the presence of viral antigen in intestinal epithelial cells suggested the animal may have shed virus via its fecal matter. This could have implications for nesting sites with respect to the potential of infecting hatchlings. Orofecal transmission of WNV among experimentally and naturally infected geese and crows has been documented.14,15

Major mortality factors in common loon populations include anthropogenic factors such as trauma, mercury and lead toxicoses, and oil spills.16 Even after anecdotal reports of WNV infection in common loons are taken into consideration, infectious diseases are uncommon causes of death in loons, with aspergillosis being most common and avian malaria being reported recently.16,17 Splenomegaly and hepatomegaly are features of avian malaria, but the livers of birds with avian malaria are usually dark brown to black as a result of malarial pigment deposition.18 Necrosis of the spleen and liver may be caused by bacterial sepsis, but there was no bacterial growth in the aerobic culture of liver tissue from loon 1 of the present report, and the presence of abundant WNV antigen in both its spleen and liver favored the virus as the cause of the observed lesions.19 Both chronic mercury and lead toxicoses may lead to immunosuppression.20,21 For loon 1, the hepatic mercury concentration was high but not considered to be at a toxic level, particularly given that the molar ratio of mercury to selenium22 was close to 1 (1.07). Clinically important comorbidity was not detected in this loon, although the relatively poor postmortem preservation of the bird limited the scope of the postmortem workup.

It is important to be aware that WNV is capable of causing disease and death in loons under natural conditions. In loons with necrotizing hepatitis and splenitis, WNV infection should be considered as a differential diagnosis.

Acknowledgments

The author thanks John Buchweitz from the toxicology section of the Michigan Veterinary Diagnostic Laboratory and Brian Nefzger from the Minnesota Department of Health for technical assistance.

References

  • 1.

    Wünschmann A, Shivers J, Bender J, et al. Pathological and immunohistochemical findings in red tailed hawks (Buteo jamaicensis) and Cooper's hawks (Accipiter cooperi) naturally infected with West Nile virus. Avian Dis. 2004;48(3):570580.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapid detection of West Nile virus from human clinical specimen, field collected mosquitoes, and avian samples by TaqMan reverse transcriptase-PCR assay. J Clin Microbiol. 2000;38(11):40664071.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Steele KE, Linn MJ, Schoepp RJ, et al. Pathology of West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet Pathol. 2000;37(3):208224.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Wimberly MC, Giacomo P, Kightlinger L, Hildreth MB. Spatio-temporal epidemiology of human West Nile virus disease in South Dakota. Int J Environ Res Public Health. 2013;10(11):55845602.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Campbell GL, Marfin RS, Lanciotti RS, Gubler DJ. West Nile virus. Lancet Infect Dis. 2002;2(9):519529.

  • 6.

    Marra PP, Griffing S, Caffrey C, et al. West Nile virus and wildlife. Bioscience. 2004;54(5):393402.

  • 7.

    Wünschmann A, Shivers J, Carroll L, Bender J. Pathological and immunohistochemical findings in American crows (Corvus brachyrhynchus) naturally infected with West Nile virus. J Vet Diagn Invest. 2004;16(4):329333.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Wünschmann A, Shivers J, Bender J, et al. Pathological and immunohistochemical findings in goshawks (Accipiter gentilis) and great horned owls (Bubo virginianus) naturally infected with West Nile virus. Avian Dis. 2005;49(2):252259.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Lopes H, Redig P, Glaser A, Armien A, Wünschmann A. Clinical findings, lesions, and viral antigen distribution in great gray owls (Strix nebulosa) and barred owls (Strix varia) with spontaneous West Nile virus infection. Avian Dis. 2007;51(1):140145.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Wünschmann A, Timurkaan N, Armien A, Bueno Padilla I, Glaser A, Redig PT. Clinical, pathological and immunohistochemical findings in bald eagles (Haliaaetus leucocephalus) and golden eagles (Aquila chryseatos) naturally infected with West Nile virus. J Vet Diagn Invest. 2014;26(5):599609.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Gancz AY, Smith DA, Barker IK, Lindsay R, Hunter B. Pathology and tissue distribution of West Nile virus in North American owls (Family Strigidae). Avian Pathol. 2006;35(1):1729.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Wünschmann A, Armien A, Höfle U, et al. Bird of prey. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. Academic Press; 2018:717740.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    McIntyre JW. The Common Loon: Spirit of Northern Lakes. University of Minnesota Press; 1988:228.

  • 14.

    Banet-Noach C, Simanov L, Malkinson M. Direct (non-vector) transmission of West Nile virus in geese. Avian Pathol. 2003;32(5):489494.

  • 15.

    Komar N, Langevin S, Hinten N, et al. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis. 2003;9(3):311322.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Sidor IF, Pokras MA, Major AR, et al. Mortality of common loons in New England, 1987–2000. J Wildl Dis. 2003;39(2):306315.

  • 17.

    Martinsen ES, Sidor IF, Flint S, Cooley J, Pokras MA. Documentation of malaria parasite (Plasmodium ssp.) infection and associated mortality in a common loon (Gavia immer). J Wildl Dis. 2017;53(4):859863.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Atkinson CT. Avian malaria. In: Atkinson CT, Thomas NJ, Hunter DB, eds. Parasitic Diseases of Wild Birds. Wiley-Blackwell; 2008:3553.

  • 19.

    Fisher ME, Trampel DW, Griffith RW. Postmortem detection of acute septicemia in broilers. Avian Dis. 1998;42(3):452461.

  • 20.

    Kenow KP, Grasman KA, Hines RK, et al. Effects of methylmercury exposure on the immune function of juvenile loons (Gavia immer). Environ Toxicol Chem. 2007;26(7):14601469.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Franson JC, Pain DJ. Lead in birds. In: Beyer NW, Meador JP, eds. Environmental Contaminants in Biota: Interpreting Tissue Concentrations. 2nd ed. CRC Press; 2011:563593.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Ohlendorf HM. Ecotoxicology of selenium. In: Hoffman DJ, Rattner BR, Burton GA, et al., eds. Handbook of Ecotoxicology. 2nd ed. CRC Press; 2003:465500.

    • Search Google Scholar
    • Export Citation
  • Figure 1

    Photographs of the body cavity of an adult male loon (loon 1) that was found dead along with another loon in a lake in northern Minnesota. A—The liver is diffusely enlarged and tan. The small intestine is dilated. Inset—Image of the liver of a control loon that was euthanized via IV injection because of tarsal joint inflammation. Notice the dark red color and size of the liver. B—The spleen is moderately enlarged. Inset—Image of the spleen of the control loon.

  • Figure 2

    Photomicrograph of a section of the liver of loon 1. The hepatocellular cords are in disarray. The cytoplasm of all hepatocytes is hypereosinophilic (arrows), and numerous nuclei are fragmented. H&E stain; bar = 60 µm.

  • Figure 3

    Photomicrograph of a section of the liver of loon 1 after immunohistochemical staining for West Nile virus (WNV) antigen. Notice the strong immunoreactivity of the cytoplasm of numerous hepatocytes (arrows) and Kupffer cells to WNV-specific antibodies (arrowhead). WNV antigen–specific immunohistochemical reaction; bar = 60 µm.

  • 1.

    Wünschmann A, Shivers J, Bender J, et al. Pathological and immunohistochemical findings in red tailed hawks (Buteo jamaicensis) and Cooper's hawks (Accipiter cooperi) naturally infected with West Nile virus. Avian Dis. 2004;48(3):570580.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapid detection of West Nile virus from human clinical specimen, field collected mosquitoes, and avian samples by TaqMan reverse transcriptase-PCR assay. J Clin Microbiol. 2000;38(11):40664071.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Steele KE, Linn MJ, Schoepp RJ, et al. Pathology of West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet Pathol. 2000;37(3):208224.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Wimberly MC, Giacomo P, Kightlinger L, Hildreth MB. Spatio-temporal epidemiology of human West Nile virus disease in South Dakota. Int J Environ Res Public Health. 2013;10(11):55845602.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Campbell GL, Marfin RS, Lanciotti RS, Gubler DJ. West Nile virus. Lancet Infect Dis. 2002;2(9):519529.

  • 6.

    Marra PP, Griffing S, Caffrey C, et al. West Nile virus and wildlife. Bioscience. 2004;54(5):393402.

  • 7.

    Wünschmann A, Shivers J, Carroll L, Bender J. Pathological and immunohistochemical findings in American crows (Corvus brachyrhynchus) naturally infected with West Nile virus. J Vet Diagn Invest. 2004;16(4):329333.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Wünschmann A, Shivers J, Bender J, et al. Pathological and immunohistochemical findings in goshawks (Accipiter gentilis) and great horned owls (Bubo virginianus) naturally infected with West Nile virus. Avian Dis. 2005;49(2):252259.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Lopes H, Redig P, Glaser A, Armien A, Wünschmann A. Clinical findings, lesions, and viral antigen distribution in great gray owls (Strix nebulosa) and barred owls (Strix varia) with spontaneous West Nile virus infection. Avian Dis. 2007;51(1):140145.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Wünschmann A, Timurkaan N, Armien A, Bueno Padilla I, Glaser A, Redig PT. Clinical, pathological and immunohistochemical findings in bald eagles (Haliaaetus leucocephalus) and golden eagles (Aquila chryseatos) naturally infected with West Nile virus. J Vet Diagn Invest. 2014;26(5):599609.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Gancz AY, Smith DA, Barker IK, Lindsay R, Hunter B. Pathology and tissue distribution of West Nile virus in North American owls (Family Strigidae). Avian Pathol. 2006;35(1):1729.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Wünschmann A, Armien A, Höfle U, et al. Bird of prey. In: Terio KA, McAloose D, St Leger J, eds. Pathology of Wildlife and Zoo Animals. Academic Press; 2018:717740.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    McIntyre JW. The Common Loon: Spirit of Northern Lakes. University of Minnesota Press; 1988:228.

  • 14.

    Banet-Noach C, Simanov L, Malkinson M. Direct (non-vector) transmission of West Nile virus in geese. Avian Pathol. 2003;32(5):489494.

  • 15.

    Komar N, Langevin S, Hinten N, et al. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis. 2003;9(3):311322.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Sidor IF, Pokras MA, Major AR, et al. Mortality of common loons in New England, 1987–2000. J Wildl Dis. 2003;39(2):306315.

  • 17.

    Martinsen ES, Sidor IF, Flint S, Cooley J, Pokras MA. Documentation of malaria parasite (Plasmodium ssp.) infection and associated mortality in a common loon (Gavia immer). J Wildl Dis. 2017;53(4):859863.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Atkinson CT. Avian malaria. In: Atkinson CT, Thomas NJ, Hunter DB, eds. Parasitic Diseases of Wild Birds. Wiley-Blackwell; 2008:3553.

  • 19.

    Fisher ME, Trampel DW, Griffith RW. Postmortem detection of acute septicemia in broilers. Avian Dis. 1998;42(3):452461.

  • 20.

    Kenow KP, Grasman KA, Hines RK, et al. Effects of methylmercury exposure on the immune function of juvenile loons (Gavia immer). Environ Toxicol Chem. 2007;26(7):14601469.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Franson JC, Pain DJ. Lead in birds. In: Beyer NW, Meador JP, eds. Environmental Contaminants in Biota: Interpreting Tissue Concentrations. 2nd ed. CRC Press; 2011:563593.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Ohlendorf HM. Ecotoxicology of selenium. In: Hoffman DJ, Rattner BR, Burton GA, et al., eds. Handbook of Ecotoxicology. 2nd ed. CRC Press; 2003:465500.

    • Search Google Scholar
    • Export Citation

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