Pathology in Practice

Carola Kaulfers Tierklinik TS AG, Burgerstrasse 11, CH-3600 Thun, Switzerland.

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Simone de Brot Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland.

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Judith Howard Diagnostic Clinical Laboratory, Department of Veterinary Clinical Medicine, Vetsuisse Faculty, University of Bern, CH-3027 Bern, Switzerland.

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Morena B. Wernick ExoticVet GmbH, Wäldliweg 6, CH-8645 Rapperswil-Jona, Switzerland.

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History

A 4-month-old 0.036-kg (0.079-lb) male bearded dragon (Pogona vitticeps) was evaluated at the Tierklinik TS AG, Thun, Switzerland because of a 1-week history of anorexia, weight loss, and signs of severe depression. The owner reported that the animal mostly lay motionless on the ground in a flattened position and made no attempt to climb or move. The bearded dragon was purchased in a pet shop at the age of 2 months together with a partner animal of the same age; that other bearded dragon died suddenly and without prior signs of illness 3 weeks earlier. The evaluated animal was housed individually since the death of its partner animal in a terrarium with sand (commercial product) as substrate and was provided UV lighting as well as several heating elements. The temperature within the habitat ranged from 25° to 40°C, and the humidity was approximately 40%. The bearded dragon's diet consisted of crickets (offered 3 times/wk), a supplemental proprietary multivitamin-mineral powder (offered 3 times/wk), and a daily selection of fresh salads and vegetables as well as occasional fruit.

Clinical and Gross Findings

At the clinic, the bearded dragon was in a very poor general condition, cachectic, and severely dehydrated (Figure 1). Physical examination revealed a large mass in the caudal portion of the coelomic cavity. The rest of the physical examination findings were unremarkable. Given the animal's poor condition and the owner's financial constraints, the owner declined further examination and elected to have the animal euthanized (by IM injection of medetomidine and ketamine, followed by intracardiac injection of pentobarbital). The bearded dragon was grossly examined by the clinic veterinarian. The only gross lesion found was a mass in the colon; the colon was excised in its entirety and submitted for histologic examination. All other organs appeared grossly unremarkable and were not examined histologically.

Figure 1—
Figure 1—

Photographs of a 4-month-old male bearded dragon (Pogona vitticeps) that was evaluated because of a 1-week history of anorexia, weight loss, and signs of severe depression (A) and a large mass that it had in the caudal portion of its colelomic cavity (B), which was removed following euthanasia. Notice the colon wall is focally and massively thickened by a large tan multinodular mass. Bar = 3 cm.

Citation: Journal of the American Veterinary Medical Association 250, 7; 10.2460/javma.250.7.759

On gross examination, the colon wall was focally massively thickened by a large (2 × 2 × 3-cm) tan, firm, multinodular mass (Figure 1). The mass had a smooth surface and contained several 1- to 10-mm fluid-filled cystic cavities on the cut surface. The intestinal lumen was focally markedly narrowed and restricted to a diameter of approximately 1 mm. The intralesional mucosa was diffusely ulcerated and covered with whitish fibrinous exudates.

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

Histopathologic Findings

Samples of the affected colon were fixed in neutral-buffered 10% formalin, routinely processed, paraffin-embedded, sectioned at 3-μm intervals, and stained with H&E stain for light microscopic examination. Histologically, the colon wall was massively thickened (up to 3 mm) with evidence of severe, diffuse, transmural granulomatous inflammation (Figure 2). Abundant macrophages and fewer heterophils with multifocal granuloma formation and large multifocal areas of necrosis were present. The mucosa was diffusely ulcerated, and the colonic lumen contained abundant fibrinous and mucoid exudates. At higher magnification (40X), numerous macrophages were markedly distended and contained large numbers of intracytoplasmic small (1- to 2-μm diameter), round to oval, pale basophilic microorganisms. The microorganisms were Gram stain positive but Ziehl-Neelsen stain negative, and contained periodic acid–Schiff stain–positive polar granules. Electron microscopic examination of the organisms revealed microsporidial spores and early sporoblasts inside a parasitophorous vacuole (Figure 3). The spores were round to ellipsoid, measuring 1.0 to 1.5 × 2.0 to 2.5 μm in diameter, with a thick electron-lucent inner wall (endospore), a thin electron-dense outer wall (exospore), 1 nucleus, and a single row of 4 to 7 filament coils. Some of the spores contained a posterior vacuole.

Figure 2—
Figure 2—

Photomicrographs of a section of the colon from the bearded dragon in Figure 1. Notice the massive transmural granulomatous and necrotizing colitis (asterisk) leading to a severe thickening of the colon wall. The colonic lumen (dagger) contains abundant fibrinous and mucoid exudates. H&E stain; bar = 2,000 μm. Inset—Higher magnification image of the main section. Numerous macrophages with abundant intracytoplasmic microsporidial organisms are present. Periodic acid–Schiff stain; bar = 200 μm.

Citation: Journal of the American Veterinary Medical Association 250, 7; 10.2460/javma.250.7.759

Figure 3—
Figure 3—

Transmission electron micrograph of a section of the colon from the bearded dragon in Figure 1. Spores and early sporoblasts within a parasitophorous vacuole are visible. The spores have a single row of 4 to 7 filament coils, consistent with Encephalitozoon spp. Uranyl acetate-lead citrate stain; bar = 1 μm.

Citation: Journal of the American Veterinary Medical Association 250, 7; 10.2460/javma.250.7.759

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe chronic focally extensive transmural granulomatous and necrotizing colitis with abundant intralesional intrahistiocytic microsporidia.

Case summary: intestinal microsporidiosis in a bearded dragon.

Comments

Microsporidia are obligate intracellular parasites, recently reclassified from protozoa to fungi, with an unusual life cycle including both merogenic and sporogenic phases.a Although reptiles are only rarely infected, microsporidiosis may lead to severe illness or death.12 Little is known about the epidemiology of microsporidiosis in bearded dragons,a but infection generally seems to occur in younger animals (≤ 4 months) and leads to acute to subacute disease. Reports3,a of microsporidiosis in bearded dragons are rare and describe a systemic infection with granulomatous inflammation in various internal organs, including the liver, kidneys, adrenal glands, colon, and ovaries. In the bearded dragon of the present report, the colon was the only organ affected grossly; other organs appeared grossly unremarkable but were not examined histologically. The clinical signs reported for bearded dragons with microsporidiosis include anorexia, cachexia, polydipsia, and sudden death.a

Microsporidia are a diverse group of organisms with > 100 genera and > 1,000 species. These organisms infect both invertebrate and vertebrate hosts, including insects, fish, and mammals.4 Among the microsporidia is the common and important rabbit pathogen Encephalitozoon cuniculi. In human medicine, microsporidia are generally opportunistic pathogens affecting persons with HIV infection or other forms of immunocompromise and cause chronic diarrhea and weight loss, although symptoms vary depending on the immune status of the host.5 Reported microsporidial infections in reptiles include Pleistophora sp in a tuatara (Sphenodon punctatus),5 Encephalitozoon lacerate in a common wall lizard (Podarcis muralis),6 Pleistophora (Gluega) danilewskyi in a European grass snake (Natrix natrix),7 Pleistophora atretii in a split keelback snake (Atretium schistosum),8 Encephalitozoon sp in inland bearded dragons (Pogona vitticeps),3,9 and Heterosporis anguillarum in a garter snake (Thamnophis sirtalis).4 In addition, a microsporidium morphologically similar to E lacerate has been described in the African skink (Mabuyaperrotetii).10 Microsporidial infection in reptiles seems to occur mainly through ingestion of infectious spores (fecal-oral route), although vertical transmission has also been suggested.a Although microsporidia are common parasites of several invertebrates, infection in reptiles has thus far not been associated with ingestion of crickets or other live food.4

In the case described in the present report, the differential diagnoses at the initial evaluation included abscess, granuloma, or neoplasia of a coelomic organ and sand impaction of the gastrointestinal tract. The diagnosis of microsporidiosis is generally only made at necropsy and relies on histologic examination of affected tissues combined with special staining (modified trichrome, Gram, Giemsa, or Warthin-Starry staining as well as fluorescent brightener or immunofluorescent antibody staining). Other stains, such as periodic acid–Schiff and Ziehl-Neelsen stains, are used to distinguish microsporidia from other micro-organisms such as mycobacteria. Polar granules, which are characteristic for microsporidia, react with periodic acid–Schiff stain.3 Transmission electron microscopy has been considered the gold standard for both detection and species differentiation of microsporidia and was therefore performed in the case described in the present report. Species differentiation is based on morphologic characteristics, such as size of spores; number of coils of the polar tubules; arrangement of the polar tubules, nucleus, and vacuole; and certain other special features.11 For the bearded dragon of the present report, the ultrastructure of the spores was consistent with Encephalitozoon spp. In human medicine, PCR-based methods are applied to improve sensitivity for identification of the infecting species. With regard to serology, only diagnostic tests for the diagnosis of E cuniculi infection in rabbits are currently available in veterinary medicine, and no specific diagnostic tests are commercially available for diagnosis of microsporidia infections in reptiles, to the authors’ knowledge. As described in a previous report,9 PCR assays with consecutive nucleotide sequencing that target parts of the small subunit rRNA genes and the internal transcribed spacer region in frozen tissue samples can be used for further identification of Encephalitozoon spp by allowing comparison of the obtained sequences with published sequences.

In humans, albendazole is generally considered the drug of choice for treatment of microsporidiosis.2 Although the treatment of choice in reptiles has yet to be elucidated, daily oral administration of fenbendazole (20 mg/kg [91 mg/lb]) for a period of 4 weeks has been previously recommended, but may be associated with adverse effects.12 Microsporidiosis is considered an important zoonotic disease,2 most generally affecting children, the elderly, or immunocompromised people. No direct transmission from reptiles to humans has yet been described but microsporidiosis in humans, and probably also in other animals, is believed to be widely underdiagnosed.2

Footnotes

a.

Mitchell MA, Garner MM. Microsporidiosis in bearded dragons: a case for vertical transmission (abstr), in Proceedings. Annu ConfAssoc ReptileAmphib Vet 2011;123.

References

  • 1. Greiner EC, Mader DR. Parasitology. Reptile medicine and surgery. St Louis: Saunders Elsevier, 2006;343364.

  • 2. Didier ES, Stovall ME, Green LC, et al. Epidemiology of microsporidiosis: sources and modes of transmission. Vet Parasitol 2004; 126: 145166.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Jacobson ER, Green DE, Undeen AH, et al. Systemic microsporidiosis in inland bearded dragons (Pogona vitticeps). J Zoo Wildl Med 1998; 29: 315323.

    • Search Google Scholar
    • Export Citation
  • 4. Richter B, Graner I, Csokai J. Heterosporis anguillarum infection in a garter snake (Thamnophis sirtalis). J Comp Pathol 2014; 150: 332335.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Liu S-K, King FW. Microsporidiosis in the tuatara. J Am Vet Med Assoc 1971; 159: 15781582.

  • 6. Canning EU. Encephalitozoon lacerate n. sp., a microsporidian parasite of the lizard Podarcis muralis. In: Parasitological topics. Lincoln, England: Society of Protozoologists. 1981;5764.

    • Search Google Scholar
    • Export Citation
  • 7. Guyenot E, Naville A. Recherches sur le parasitisme et l'evolution d'une microsporidie Glugea danilewskyi Pfr parasite de la couleuvre. Rev Suisse Zool 1922; 30: 161.

    • Search Google Scholar
    • Export Citation
  • 8. Narasimhamurti CC, Kalavati C, Ahamed SN. A new microsporidian, Plistophora atretii sp.n. from the fresh water snake, Atretium schistosum Gunther. Acta Protozoologica 1982; 21: 127137.

    • Search Google Scholar
    • Export Citation
  • 9. Richter B, Csokai J, Graner I, et al. Encephalitozoonosis in two inland bearded dragons (Pogona vitticeps). J Comp Pathol 2013; 148: 278282.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Koudela B, Didier ES, Rogers LB, et al. Intestinal microsporidiosis in African skink Mabuya perrotetii. Folia Parasitol Prague 1998; 45: 149155.

    • Search Google Scholar
    • Export Citation
  • 11. Franzen C, Mueller A. Molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia. Clin Microbiol Rev 1999; 12: 243285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Pantchev N. Parasitosen bei Schildkröten. In: Hnizdo J, Pantchev N, eds. Tierarztpraxis Schildkröten. Frankfurt. Germany: Edition Chimaira, 2011;215216.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Dr. de Brot's present address is School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, England.

Address correspondence to Dr. Wernick (info@exoticvet.ch).
  • Figure 1—

    Photographs of a 4-month-old male bearded dragon (Pogona vitticeps) that was evaluated because of a 1-week history of anorexia, weight loss, and signs of severe depression (A) and a large mass that it had in the caudal portion of its colelomic cavity (B), which was removed following euthanasia. Notice the colon wall is focally and massively thickened by a large tan multinodular mass. Bar = 3 cm.

  • Figure 2—

    Photomicrographs of a section of the colon from the bearded dragon in Figure 1. Notice the massive transmural granulomatous and necrotizing colitis (asterisk) leading to a severe thickening of the colon wall. The colonic lumen (dagger) contains abundant fibrinous and mucoid exudates. H&E stain; bar = 2,000 μm. Inset—Higher magnification image of the main section. Numerous macrophages with abundant intracytoplasmic microsporidial organisms are present. Periodic acid–Schiff stain; bar = 200 μm.

  • Figure 3—

    Transmission electron micrograph of a section of the colon from the bearded dragon in Figure 1. Spores and early sporoblasts within a parasitophorous vacuole are visible. The spores have a single row of 4 to 7 filament coils, consistent with Encephalitozoon spp. Uranyl acetate-lead citrate stain; bar = 1 μm.

  • 1. Greiner EC, Mader DR. Parasitology. Reptile medicine and surgery. St Louis: Saunders Elsevier, 2006;343364.

  • 2. Didier ES, Stovall ME, Green LC, et al. Epidemiology of microsporidiosis: sources and modes of transmission. Vet Parasitol 2004; 126: 145166.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Jacobson ER, Green DE, Undeen AH, et al. Systemic microsporidiosis in inland bearded dragons (Pogona vitticeps). J Zoo Wildl Med 1998; 29: 315323.

    • Search Google Scholar
    • Export Citation
  • 4. Richter B, Graner I, Csokai J. Heterosporis anguillarum infection in a garter snake (Thamnophis sirtalis). J Comp Pathol 2014; 150: 332335.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Liu S-K, King FW. Microsporidiosis in the tuatara. J Am Vet Med Assoc 1971; 159: 15781582.

  • 6. Canning EU. Encephalitozoon lacerate n. sp., a microsporidian parasite of the lizard Podarcis muralis. In: Parasitological topics. Lincoln, England: Society of Protozoologists. 1981;5764.

    • Search Google Scholar
    • Export Citation
  • 7. Guyenot E, Naville A. Recherches sur le parasitisme et l'evolution d'une microsporidie Glugea danilewskyi Pfr parasite de la couleuvre. Rev Suisse Zool 1922; 30: 161.

    • Search Google Scholar
    • Export Citation
  • 8. Narasimhamurti CC, Kalavati C, Ahamed SN. A new microsporidian, Plistophora atretii sp.n. from the fresh water snake, Atretium schistosum Gunther. Acta Protozoologica 1982; 21: 127137.

    • Search Google Scholar
    • Export Citation
  • 9. Richter B, Csokai J, Graner I, et al. Encephalitozoonosis in two inland bearded dragons (Pogona vitticeps). J Comp Pathol 2013; 148: 278282.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Koudela B, Didier ES, Rogers LB, et al. Intestinal microsporidiosis in African skink Mabuya perrotetii. Folia Parasitol Prague 1998; 45: 149155.

    • Search Google Scholar
    • Export Citation
  • 11. Franzen C, Mueller A. Molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia. Clin Microbiol Rev 1999; 12: 243285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Pantchev N. Parasitosen bei Schildkröten. In: Hnizdo J, Pantchev N, eds. Tierarztpraxis Schildkröten. Frankfurt. Germany: Edition Chimaira, 2011;215216.

    • Search Google Scholar
    • Export Citation

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