Conference: 08 - 2010    Case: 04     Kidney - Tortoise


Signalment:  

Adult, female, red-footed tortoise (Geochelone carbonaria).


History:  

According to the provided history, an adult female red-footed tortoise (Geochelone carbonaria) from a privately-owned Midwestern zoological garden presented with progressive lethargy. As the animal was nonresponsive to treatment, necropsy was performed on site following euthanasia, and tissues were submitted to the Diagnostic Center for Population and Animal Health, Lansing, MI, for histopathologic examination.


Histopathologic Description:

Kidney: Diffusely in a section of kidney, the renal tubular epithelium is variably degenerate and necrotic. Within affected tubules, epithelial cells are often dissociated, variably swollen, with eosinophilic finely vacuolated to floccular cytoplasm, and pyknotic to karyolytic to lost nuclei (degeneration and necrosis). Few large, irregular, and mildly atypical cells with prominent vesicular nuclei are also present (regeneration). Multifocally, tubules are filled with small to moderate amounts of eosinophilic smooth to floccular material (proteinosis), sloughed-off epithelial cells, and few heterophils. Occasional tubular epithelial cells contain up to 10, 10-15 μm, intracytoplasmic, retractile, amphophilic ellipsoidal organisms obscuring the cytoplasm and nucleus. These organisms contain two apical polar capsules, approximately 2 μm in diameter, and basophilic nuclear material in between polar capsules (morphology suggestive of myxozoans). Similar organisms are also present in tubular lumina, within sloughed-off epithelial cells or individually amidst intraluminal debris. Low numbers of lymphocytes and heterophils expand the renal interstitium, together with rare pigment-laden macrophages (hemosiderin). Diffusely within the section, glomerular tufts are segmentally to globally, mildly to moderately thickened. Multifocally, there is rare segmental synechiation and mild expansion of the urinary space, which is mostly devoid of contents (clear space). Rare intratubular basophilic casts (mineral) are also identified. Degenerate sloughed-off epithelial cells, heterophils, and myxozoan organisms are present in the renal pelvic lumen. Multifocally within the subepithelial stroma of the pelvis are occasional foci of coarse deeply basophilic salt deposition (dystrophic mineralization). The above described organisms were not highlighted by PAS, acid-fast, or Giemsa histochemistries. Additional findings in other organs (not included): multifocal myocardial mineralization and severe heterophilic hepatitis. No organisms were observed within any other examined tissues, including but not limited to liver, biliary ducts, and urinary bladder


Morphologic Diagnosis:  


1. Kidney: Moderate diffuse tubular degeneration, and necrosis, with intralesional, intraepithelial and intratubular myxozoa-type parasites (Myxidium spp.).
2. Kidney: Moderate diffuse membranous glomerulonephritis, with mild tubular proteinosis.
3. Kidney: Minimal multifocal dystrophic mineralization.


Lab Results:  

After a diagnosis was made, water from the pond where this tortoise was kept was submitted for parasitological analysis but yielded no significant results.


Condition:  

Tubular degeneration, membranous glomerulonephritis


Contributor Comment:  

Microorganisms of the phylum Myxozoa are spore-forming, metazoan parasites that infect the biliary, urinary, and gastrointestinal tracts of cold-blooded aquatic vertebrates, especially fishes but rarely also reptiles, amphibians, and birds, with alternate life cycle stages in invertebrates. (2,4,5) Infection is believed to be of little clinical importance in most species, although in the recent years, life-threatening disease has been documented in a variety of species.(2) In the reported cases, the kidneys, biliary tract, and liver are the most commonly compromised organs. Affected kidneys are often grossly pale and swollen, while histologically the infection is characterized by degeneration and necrosis of the tubular epithelium infected with intralesional spores.(5) All myxozoans known to infect reptiles are in the genus Myxidium, and all reports involve aquatic turtles.(5)

In an attempt to elucidate the species involved in this case, additional ancillary tests such as ultrastructural analysis, phase contrast microscopy, and molecular analysis of the samples were performed.

Ultrastructural analysis on infected kidney tissue revealed 60x20 μm spores, with a mean polar capsule dimension of 15x10 μm. Phase contrast microscopy and transmission electron microscopy demonstrated only mature spores, with two asymmetrical valve cells and a binucleated sporoplasm between two opposing polar capsules. Valve cells were longitudinally striated, with two overlapping sigmoidal capsule sutures. Polar capsules contained a single polar filament, coiled 5 to 7 times and surrounded by a double-layered wall. Based on spore morphology, these myxozoa were classified in the genus Myxidium.

Macerated formalin-fixed tissue yielded plentiful spores from which DNA extraction was attempted. At least two distinct extraction methods were used (using Myxozoan and Myxydium generic primers) with no success, likely as a result of formalin-fixation nucleic acid damage to the samples (additional fresh samples were not readily available).

To the best of the contributors knowledge, renal myxozoanosis has not been documented in terrestrial chelonids.


AFIP Diagnosis:  


1. Kidney: Tubular degeneration and proteinosis, multifocal, mild with rare tubular epithelial necrosis and intraepithelial and intratubular myxozoan parasites.
2. Adipose tissue, perirenal: Fat atrophy, diffuse moderate.


Conference Comment:  

Several participants included some form of glomerulonephritis in the histomorphologic diagnosis. The moderator pointed out that early studies and descriptions of the histomorphology of the chelonian kidney mistakenly identified aging changes as glomerulonephritis, and he assessed the glomeruli in this tortoise as essentially normal. Tortoises and other reptiles possess smaller glomeruli with reduced vascularity as compared to amphibians, birds and mammals.(3) This morphologic feature acts to conserve water for these animals that often live in an arid, dehydrating environment. The reduced glomerular size and vascularity results in an ostensibly thicker mesangium with pronounced mesangial cells, and these normal histoanatomical features may be mistaken for glomerulonephritis by pathologists lacking extensive experience in evaluating reptile tissues. Additionally, the moderator believed there was little corroborative evidence to support a diagnosis of glomerulonephritis, e.g. lack of interstitial inflammatory infiltrates.

A subtle, but important, histologic finding in this case is the depletion of perirenal adipose tissue. The moderator indicated this was the most striking lesion, as it indicates negative energy balance. When conducting gross and histologic examination of reptiles and amphibians, assessment of adipose tissue provides valuable insight into the metabolic state of the animal.

Participants discussed the various animal species affected by myxozoan parasites. In addition to cold-blooded vertebrates, the moderator indicated that several ducks, both native wild and captive exotic species, have been infected with Myxidium anatidum n. sp.(1) Myxozoan parasites which infect ducts preferentially infect biliary epithelium.


References:

1. Bartholomew JL, Atkinson SD, Hallett SL, et al. Myxozoan parasitism in waterfowl. Int J Parasitol. 2008;38(10): 1199-1207.
2. Garner MM, Bartholomew JL, Whipps CM, Nordhausen RW, Raiti P. Renal myxozoanosis in crowned river turtles Hardella thurjii: description of the putative agent Myxidium hardella n. sp. by histopathology, electron microscopy, and DNA sequencing. Vet Pathol. 2005;42:589-595.
3. Jacobsen ER. Overview of reptile biology, anatomy, and histology. In: Infectious Diseases and Pathology of Reptiles. New York, NY: CRC; 2007:13-14.
4. Roberts JF, Whipps CM, Bartholomew JL, Schneider L, Jacobson ER. Myxidium scripta n. sp. identified in urinary and biliary tract of Louisiana-farmed red-eared slider turtles Trachemys scripta elegans. Dis Aquat Organ. 2008;80:199-209.
5. Zwart, P. Renal Pathology in Reptiles. Vet Clin North Am Exot Anim Pract. 2006;9:129-159.
Conference: 08 - 2010 (Cases 1 - 4) write up  


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4-1. Kidney


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