Pathology in Practice

Jennie M. Jankovsky Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Andrew C. Cushing Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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Linden E. Craig Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996.

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History

A captive 13-year-old castrated male African lion (Panthera leo) housed at a large cat sanctuary for the previous 5 years was found dead within its enclosure, and the carcass was submitted to the University of Tennessee Veterinary Medical Center for autopsy. This animal seemed to be healthy in the days preceding its death and had no notable medical history.

Gross Findings

At autopsy, the lion weighed 208.5 kg (458.7 lb) and was obese (body condition score, 5/5) with abundant subcutaneous and visceral adipose tissue. The pericardial sac contained 350 mL of thin, clear, pale yellow fluid. Affecting 40% of the myocardium were irregularly shaped tan areas with brown soft centers (Figure 1). The left ventricular free wall and right auricle were most severely affected. Other relevant gross findings included moderate hepatic and pulmonary congestion.

Figure 1—
Figure 1—

Photograph of the left ventricular free wall of an apparently healthy adult African lion (Panthera leo) that died suddenly without premonitory signs. Notice the pale tan depressed areas in the myocardium.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.355

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

Histopathologic and Microbiological Findings

Aerobic and anaerobic cultures of samples of the myocardium yielded no bacterial growth in a 7-day period. Histologic examination of sections of the myocardium revealed multifocal to coalescing myocarditis; areas of inflammatory reaction were composed of plasma cells, macrophages, and eosinophils with fewer neutrophils and lymphocytes and scattered mast cells. Inflammation and edema extended into the endocardium and epicardium and separated, surrounded, and replaced degenerate and necrotic cardiomyocytes that contained intracytoplasmic clusters of 2 × 4-μm protozoal tachyzoites (Figure 2). Similar individual tachyzoites were present extra-cellularly. Immunohistochemical staining of sections of myocardium with rabbit-derived polyclonal antibodies against Neospora caninum revealed strong reaction of individual and clustered tachyzoites (Figure 3). Results of immunohistochemical testing of sections of myocardium with goat-derived polyclonal antibody against Toxoplasma gondii or with rabbit-derived polyclonal antibody against Sarcocystis neurona were negative.

Figure 2—
Figure 2—

Photomicrograph of a section of myocardium from the lion in Figure 1. An intracytoplasmic cluster of protozoal tachyzoites is visible within a cardiomyocyte (arrowhead). Cardiomyocytes are separated by edema (asterisks) and mixed inflammation including eosinophils (arrow). H&E stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.355

Figure 3—
Figure 3—

Photomicrograph of a section of myocardium from the lion in Figure 1. Intracytoplasmic protozoal tachyzoites are highlighted by immunohistochemical reactivity for Neospora caninum. Anti-N caninum-specific antibody and 3,3′diaminobenzidine chromogen with hematoxylin counterstain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 254, 3; 10.2460/javma.254.3.355

Other histologic findings included mild focal histiocytic and plasmacytic glossal myositis, lymph node lymphoid hyperplasia with sinus histiocytosis and eosinophilia, minimal multifocal lymphohistiocytic meningoencephalitis, pulmonary and cerebral arteriosclerosis, moderate pulmonary interstitial fibrosis, laryngopharyngeal and tonsillar lymphoid hyperplasia with intralesional foreign material, and mild focal lymphoplasmacytic interstitial nephritis. Protozoa were not found via microscopic examination of any tissue sections other than those from heart. Other tissues examined included the eyes, liver, stomach, prostate, and skeletal muscle from the tongue, diaphragm, and hind limbs.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe, subacute, lymphoplasmacytic, histiocytic, eosinophilic, and necrotizing myocarditis with intralesional protozoal tachyzoites.

Case summary: myocarditis attributable to infection with N caninum in an adult lion.

Comments

For the case described in the present report, the possible causes of the cardiac lesions initially included infection with protozoa, bacteria, or fungi. Because the heart alone was grossly affected, protozoal myocarditis (caused by organisms such as T gondii, N caninum, Sarcocystis spp, Trypanosoma cruzi, or Leishmania infantum) was considered most likely, whereas infection with bacteria (including Streptococcus spp, Escherichia coli, and Bartonella spp), fungi (eg, Blastomyces dermatitidis), or viruses (including feline parvovirus, FIV, and feline infectious peritonitis virus) was considered less likely.

Neospora caninum is an apicomplexan coccidian organism for which canids are both the definitive and intermediate hosts.1 Oocysts are passed into the environment in feces and after sporulation are ingested by the intermediate hosts, most commonly cattle.1 Zoites are released within the intestinal tract, penetrate the intestinal epithelium, become tachyzoites, and replicate by endodyogeny. Released tachyzoites are likely spread by leukocyte trafficking; they parasitize multiple cell types and undergo multiple cycles of proliferation.2 Bradyzoite-containing tissue cysts form primarily within the CNS and uncommonly within the retinas and skeletal muscles. Tissue cysts generally remain quiescent until the host becomes immunosuppressed; ingestion of infected intermediate hosts that have these cysts by canids restarts the cycle.3

In dogs, clinical neosporosis is most often the result of transplacental transmission of tachyzoites; affected puppies often develop polymyositis and polyradiculoneuritis characterized by ascending hind limb paresis and paralysis, muscle atrophy, and rarely dysphagia.4 In older dogs, encephalomyelitis, polymyositis, myocarditis, dermatitis, and pneumonia may be associated with either acute infection following ingestion of infected prey or with reactivation of latent infection as a result of immunosuppression.1,5–7 In cattle, infection is usually subclinical in adults; the most common sequela of neosporosis is abortion, usually midpregnancy, as a result of transplacental transmission.2

Histologically, N caninum and T gondii in tissue sections cannot be distinguished without additional diagnostic testing, such as immunohistochemical analysis as performed in the case described in the present report. Prior to 1988, when N caninum was identified as a separate species, infections were likely misdiagnosed as being caused by T gondii.8 Neospora caninum and T gondii may also be differentiated ultrastructurally, because N caninum zoites have more rhoptries and micronemes; furthermore, N caninum tissue cysts have thicker cell walls than those associated with T gondii.9

Naturally occurring clinical neosporosis in either domestic or nondomestic felids has not previously been reported to our knowledge. Circulating antibodies against N caninum have been identified in naturally infected domestic cats, and vertical transmission has occurred in experimentally infected queens, including 1 cat that had been infected via ingestion of tissue cysts.3,10 Serum antibodies against N caninum in both wild and captive lions have also been detected, although the organism has previously not been detected histologically.11–13 Clinical neosporosis in nondomestic carnivores, including a pine marten, blue foxes, and a red fox (all of which were juveniles), has been described.14–16

In the lion of the present report, N caninum organisms were microscopically detected only within the myocardium. None were found within the CNS, eyes, or skeletal muscles (including the tongue, diaphragm, and hind limbs), although there was mild glossal myositis and minimal meningoencephalitis. Immunohistochemical analyses were performed only on sections of the myocardium; thus, it is possible that zoites were present in other tissue sections but not detected with H&E staining. Unfortunately, the precise location of the hyperplastic lymph node was not recorded; however, it was either mandibular or mediastinal and may have been affected secondary to neosporosis or the laryngopharyngeal foreign material. All other findings were considered incidental.

The mode of infection in the case described in the present report was unknown. No other nondomestic felids at the large cat sanctuary appeared to be affected, although serologic screening was not performed to rule out subclinical infections. Ingestion of sporulated oocysts was possible, but the affected lion had no known exposure to domestic dogs or wild canids, such as coyotes, within the preceding 5 years. The lion was kept in an approximately 12 × 18-m pen enclosed with 3.7-m-high chain-link fence, which would prevent entry by coyotes; fence-line checks were performed daily by staff. Domestic dogs were present on the property, but not within the area of this lion's pen. Although the lion was fed previously frozen meat and N caninum organisms are killed by freezing,3 infection via carnivorism cannot be ruled out, as small prey animals may have entered the enclosure and been consumed. Anti-N caninum-specific antibodies and N caninum DNA have been identified in rodents, lagomorphs, and wild birds, and N caninum tissue cysts have been found in rodents and chickens.17–22

In the lion of the present report, chronic neosporosis with reactivation of N caninum bradyzoites could not be ruled out, although tissue cysts were not identified and no underlying cause of immunosuppression that may have predisposed the animal to recrudescence was identified. Unfortunately, minimal medical history was available for the lion. It was apparently healthy and had no medical issues during the years it lived at the sanctuary. Given the number of animals at the sanctuary and the requirement for sedation for physical handling, regular examination of healthy animals was impractical.

To our knowledge, this is the first report of clinical neosporosis in a felid; infection via ingestion of infected prey was considered most likely. For the lion of the present report, the cause of death was myocarditis resulting in cardiac arrhythmia and serous tamponade. On the basis of the histologic similarities, a postmortem diagnosis of toxoplasmosis or neosporosis in lions (and other species) should be confirmed by immunohistochemical or transmission electron microscopic examination of affected tissue sections. Neospora caninum infection should be considered as a potential cause of sudden death or myocarditis in lions.

References

  • 1. Donahoe SL, Lindsay SA, Krockenberger M, et al. A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. Int J Parasitol Parasites Wildl 2015;4:216238.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Dubey JP, Buxton D, Wouda W. Pathogenesis of bovine neosporosis. J Comp Pathol 2006;134:267289.

  • 3. Dubey JP, Schares G, Ortega-Mora LM. Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev 2007;20:323367.

  • 4. Dubey JP, Lappin MR. Toxoplasmosis and neosporosis. In: Greene CE, ed. Infectious diseases of the dog and cat. 4th ed. St Louis: Elsevier Saunders, 2012;806827.

    • Search Google Scholar
    • Export Citation
  • 5. Bjerkås I, Mohn SF, Presthus J. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd 1984;70:271274.

  • 6. Robinson WF, Robinson NA. Cardiovascular system. In: Maxie MG, ed. Jubb, Kennedy and Palmer's pathology of domestic animals. Vol. 3. 6th ed. St Louis: Elsevier, 2016;1101.

    • Search Google Scholar
    • Export Citation
  • 7. La Perle KM, Del Piero F, Carr RF, et al. Cutaneous neosporosis in two adult dogs on chronic immunosuppressive therapy. J Vet Diagn Invest 2001;13:252255.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Dubey JP, Carpenter JL, Speer CA, et al. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 1988;192:12691285.

  • 9. Dubey JP, Sreekumar C, Knickman E, et al. Biologic, morphologic, and molecular characterization of Neospora caninum isolates from littermate dogs. Int J Parasitol 2004;34:11571167.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Dubey JP, Lindsay DS. Transplacental Neospora caninum infection in cats. J Parasitol 1989;75:765771.

  • 11. Cheadle MA, Spencer JA, Blagburn BL. Seroprevalences of Neospora caninum and Toxoplasma gondii in nondomestic felids from southern Africa. J Zoo Wildl Med 1999;30:248251.

    • Search Google Scholar
    • Export Citation
  • 12. Ferroglio E, Wambwa E, Castiello M, et al. Antibodies to Neospora caninum in wild animals from Kenya, East Africa. Vet Parasitol 2003;118:4349.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Spencer JA, Higgenbotham MJ, Blagburn BL. Seroprevalence of Neospora caninum and Toxoplasma gondii in captive and free-ranging nondomestic felids in the United States. J Zoo Wildl Med 2003;34:246249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. van der Hage MH, Kik MJL, Dorrestein GM. Neospora caninum: myocarditis in a European pine marten (Martes martes), in Proceedings. Eur Assoc Zoo Wildl Vet 4th Sci Meet 2002;217220.

    • Search Google Scholar
    • Export Citation
  • 15. Yu X, Chen N, Hu D, et al. Detection of Neospora caninum from farm-bred young blue foxes (Alopex lagopus) in China. J Vet Med Sci 2009;71:113115.

  • 16. Duhey JP, Whitesell LE, Culp WE, et al. Diagnosis and treatment of Neospora caninum-associated dermatitis in a red fox (Vulpes vulpes) with concurrent Toxoplasma gondii infection. J Zoo Wildl Med 2014;45:454457.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Jenkins MC, Parker C, Hill D, et al. Neospora caninum detected in feral rodents. Vet Parasitol 2007;143:161165.

  • 18. Sobrino R, Dubey JP, Pabon M, et al. Neospora caninum antibodies in wild carnivores from Spain. Vet Parasitol 2008;155:190197.

  • 19. Ibrahim HM, Huang P, Salem TA, et al. Prevalence of Neospora caninum and Toxoplasma gondii antibodies in northern Egypt. Am J Trop Med Hyg 2009;80:263267.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Darwich L, Cabezon O, Echeverria I, et al. Presence of Toxoplasma gondii and Neospora caninum DNA in the brains of wild birds. Vet Parasitol 2012;183:377381.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Medina-Esparza L, Macias L, Ramos-Parra M, et al. Frequency of infection by Neospora caninum in wild rodents associated with dairy farms in Aguascalientes, Mexico. Vet Parasitol 2013;191:1114.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Costa KS, Santos SL, Uzeda RS, et al. Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum. Int J Parasitol 2012;38:157159.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photograph of the left ventricular free wall of an apparently healthy adult African lion (Panthera leo) that died suddenly without premonitory signs. Notice the pale tan depressed areas in the myocardium.

  • Figure 2—

    Photomicrograph of a section of myocardium from the lion in Figure 1. An intracytoplasmic cluster of protozoal tachyzoites is visible within a cardiomyocyte (arrowhead). Cardiomyocytes are separated by edema (asterisks) and mixed inflammation including eosinophils (arrow). H&E stain; bar = 20 μm.

  • Figure 3—

    Photomicrograph of a section of myocardium from the lion in Figure 1. Intracytoplasmic protozoal tachyzoites are highlighted by immunohistochemical reactivity for Neospora caninum. Anti-N caninum-specific antibody and 3,3′diaminobenzidine chromogen with hematoxylin counterstain; bar = 20 μm.

  • 1. Donahoe SL, Lindsay SA, Krockenberger M, et al. A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. Int J Parasitol Parasites Wildl 2015;4:216238.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Dubey JP, Buxton D, Wouda W. Pathogenesis of bovine neosporosis. J Comp Pathol 2006;134:267289.

  • 3. Dubey JP, Schares G, Ortega-Mora LM. Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev 2007;20:323367.

  • 4. Dubey JP, Lappin MR. Toxoplasmosis and neosporosis. In: Greene CE, ed. Infectious diseases of the dog and cat. 4th ed. St Louis: Elsevier Saunders, 2012;806827.

    • Search Google Scholar
    • Export Citation
  • 5. Bjerkås I, Mohn SF, Presthus J. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd 1984;70:271274.

  • 6. Robinson WF, Robinson NA. Cardiovascular system. In: Maxie MG, ed. Jubb, Kennedy and Palmer's pathology of domestic animals. Vol. 3. 6th ed. St Louis: Elsevier, 2016;1101.

    • Search Google Scholar
    • Export Citation
  • 7. La Perle KM, Del Piero F, Carr RF, et al. Cutaneous neosporosis in two adult dogs on chronic immunosuppressive therapy. J Vet Diagn Invest 2001;13:252255.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Dubey JP, Carpenter JL, Speer CA, et al. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 1988;192:12691285.

  • 9. Dubey JP, Sreekumar C, Knickman E, et al. Biologic, morphologic, and molecular characterization of Neospora caninum isolates from littermate dogs. Int J Parasitol 2004;34:11571167.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Dubey JP, Lindsay DS. Transplacental Neospora caninum infection in cats. J Parasitol 1989;75:765771.

  • 11. Cheadle MA, Spencer JA, Blagburn BL. Seroprevalences of Neospora caninum and Toxoplasma gondii in nondomestic felids from southern Africa. J Zoo Wildl Med 1999;30:248251.

    • Search Google Scholar
    • Export Citation
  • 12. Ferroglio E, Wambwa E, Castiello M, et al. Antibodies to Neospora caninum in wild animals from Kenya, East Africa. Vet Parasitol 2003;118:4349.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Spencer JA, Higgenbotham MJ, Blagburn BL. Seroprevalence of Neospora caninum and Toxoplasma gondii in captive and free-ranging nondomestic felids in the United States. J Zoo Wildl Med 2003;34:246249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. van der Hage MH, Kik MJL, Dorrestein GM. Neospora caninum: myocarditis in a European pine marten (Martes martes), in Proceedings. Eur Assoc Zoo Wildl Vet 4th Sci Meet 2002;217220.

    • Search Google Scholar
    • Export Citation
  • 15. Yu X, Chen N, Hu D, et al. Detection of Neospora caninum from farm-bred young blue foxes (Alopex lagopus) in China. J Vet Med Sci 2009;71:113115.

  • 16. Duhey JP, Whitesell LE, Culp WE, et al. Diagnosis and treatment of Neospora caninum-associated dermatitis in a red fox (Vulpes vulpes) with concurrent Toxoplasma gondii infection. J Zoo Wildl Med 2014;45:454457.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Jenkins MC, Parker C, Hill D, et al. Neospora caninum detected in feral rodents. Vet Parasitol 2007;143:161165.

  • 18. Sobrino R, Dubey JP, Pabon M, et al. Neospora caninum antibodies in wild carnivores from Spain. Vet Parasitol 2008;155:190197.

  • 19. Ibrahim HM, Huang P, Salem TA, et al. Prevalence of Neospora caninum and Toxoplasma gondii antibodies in northern Egypt. Am J Trop Med Hyg 2009;80:263267.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Darwich L, Cabezon O, Echeverria I, et al. Presence of Toxoplasma gondii and Neospora caninum DNA in the brains of wild birds. Vet Parasitol 2012;183:377381.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Medina-Esparza L, Macias L, Ramos-Parra M, et al. Frequency of infection by Neospora caninum in wild rodents associated with dairy farms in Aguascalientes, Mexico. Vet Parasitol 2013;191:1114.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Costa KS, Santos SL, Uzeda RS, et al. Chickens (Gallus domesticus) are natural intermediate hosts of Neospora caninum. Int J Parasitol 2012;38:157159.

    • Search Google Scholar
    • Export Citation

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