Abstract

Two red-bellied short-necked turtles (Emydura albertisii) were presented with multiple periarticular nodules, 1–6 mm in diameter, on all limbs, located predominantly on digital joints. Clinical investigations and subsequent necropsy revealed that these nodules were periarticular deposits of calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). Similar lesions were described in the literature in two previous reptile cases as false gout or pseudogout. Because pseudogout in humans is always associated with articular deposits of calcium pyrophosphate dihydrate (CPPD), it is suggested to correspond to human nomenclature and use the term periarticular hydroxyapatite deposition disease (HADD) also in reptiles. The present report describes the clinical and pathologic features of HADD in two together-housed turtles. The etiology of the condition could not be determined but the simultaneous appearance of the lesions points to biochemical imbalances caused by nutritional or husbandry deficiencies.

Introduction

The crystalline arthropathies are a heterogeneous group of biochemical disorders that share the common feature of crystal accumulation in and around joints. In human medicine, they are classified as gout, which is the deposition of monosodium urate crystals in the synovial tissue following sustained hyperuricaemia, as pseudogout, which is the articular deposition of calcium pyrophosphate dihydrate crystals (CPPD), and as hydroxyapatite deposition disease (HADD).^1,12,13 The latter is usually periarticular, but intraarticular calcification can occur in a specific syndrome (Milwaukee shoulder-knee syndrome). HADD has been found in patients with chronic renal failure and secondary to connective tissue disorders.

In animals, CPPD cases have been reported in three dogs and one horse.^5,7-9 In reptiles, hydroxyapatite deposition has been described in an Egyptian spiny-tailed lizard (Uromastix sp.) and a red-eared slider turtle (Chrysemys scripta elegans).^4,6 In contrast to the human nomenclature and classification these reptile cases were designated as false gout or pseudogout, respectively. The purpose of this report is to describe two simultaneous cases of periarticular hydroxyapatite deposition in two red-bellied short-necked turtles, and to discuss possible etiologic factors of the disorder.

Case Report

Two juvenile, male, red-bellied short-necked turtles were presented for examination at the Division of Zoo Animals and Exotic Pets of the University of Zurich. This strictly carnivorous species lives in inshore waters in Papua New Guinea, and has a characteristic red-colored plastron, especially in juveniles and mature males.¹⁰ The turtles were brought to the clinic because both had multinodular periarticular swellings on all legs. The nodules had been first noticed less than 2 wk before and the alterations had since rapidly progressed. Both turtles were recently acquired from a pet store and housed together in an aqua-terrarium. Water temperature was held at around 26°C, recommended in the literature is 22°C.¹⁰ The turtles diet consisted of shrimps, commercial pellets, flies, and ground meat.

On physical examination, the animals were alert, eating and swimming normally. The larger one weighed 100 g, and the other 49 g. Abnormal findings included multiple, 1–6 mm in diameter, firm, white nodules, located predominantly around digital, but also around radiohumeral, radiocarpal, femorotibial, and tibiotarsal joints of all legs. Radiographic examination revealed amorphous, radiopaque densities at the sites of these periarticular nodules. Insertion of a hollow needle revealed a homogenous, strongly viscous white material. No microorganisms could be cultured from this sample. Cytologic examination showed non-cellular agglomerations of crystal-like clumps.

The owner declined any treatment, but permitted further investigations. The turtles were anesthetized with ketamine-hydrochloride (60 mg/kg body weight i.m.) and blood was collected by cardiocentesis for hematology and serum chemistry. Then the animals were euthanatized and submitted for postmortem examination. Heart, lung, liver, kidney and limb tissues were collected for routine histopathology, periarticular tissue was processed routinely for transmission electron microscopy, and two samples of the nodules were submitted for crystallographic analysis.

Because blood reference parameters for this species are not available, comparisons were made with general reference ranges of chelonians (Table 1).³ All values were within normal limits except for elevated glucose and urea values in the smaller turtle. The serum calcium-phosphorus ratio was 1.7 and 2.1, respectively. Hematology revealed a leucopenia in the smaller turtle. Except for the nodular swellings on the limbs and an additional similar nodule, 5 mm in diameter, subcutaneously on the ventral aspect of the neck of the larger animal, no gross pathologic lesions were found. The tophaceous periarticular swellings consisted of a solid yellowish mass which adhered to the surrounding subcutaneous and muscular tissues. Histologically, these nodules contained homogenous mineralized material which was delimited by a granulomatous inflammatory reaction with variable numbers of histiocytes and giant cells of the foreign-body type. In Ziehl-Neelsen and periodic acid-Schiff (PAS) stains, these granulomas were negative for acid-fast bacteria or fungal organisms, respectively. Electron microscopy of the deposits revealed tiny, needle-like, short, nanometer-sized crystals. On crystallographic examination the specimens examined consisted of 100% calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). On the basis of these findings a diagnosis of periarticular hydroxyapatite deposition disease was made.

Table 1. Hematology and serum chemistry of two red-bellied short-necked turtles (Emydura albertisii) with periarticular hydroxyapatite deposition disease

^aBeynon, et al. (1992)

Discussion

Differential diagnoses of periarticular swellings in reptiles include arthritis, viral papillomas, periarticular abscesses, mycobacterial infections, dermatophilosis, subcutaneous nematodes and filarid parasites, degenerative joint disease, articular forms of gout, metabolic bone disease, pseudogout, and neoplasms.^2,14 Radiographically, radiodense deposits are indicative of concrements with high calcium contents. In contrast to urate deposits, which usually can be easily removed at necropsy, the hydroxyapatite deposits adhere to the surrounding tissues.¹⁴ Furthermore, the apatite crystals can be differentiated by electron microscopy from the 2–20 µm long urate rods and the 2–10 µm long rhomboidal or rectangular CPPD crystals by their nanometric size and needle-like appearance. This is consistent with the radiographic, histopathologic, and electron microscopic findings in the two red-bellied short-necked turtles, and was confirmed by crystallographic detection of calcium-hydroxyapatite which is known as hydroxyapatite deposition disease (HADD) in humans. Because pseudogout in mammals including humans is always related to articular deposition of a different crystal, calcium pyrophosphate dihydrate (CPPD), it is suggested to correspond to this nomenclature and use periarticular HADD to describe this feature in reptiles.

In human cases, clinical diagnosis is confirmed by electron microscopic examination of synovial fluid samples because the crystals themselves are tiny—only 7–25 nm in diameter.¹² In addition to radiography, this would also be the method of choice for the clinical diagnosis of reptile samples because with routine cytologic light microscopy only non-birefringent clumps are detected and they could be mistaken for debris.

At variance with previously reported single cases in reptiles, two animals which were kept together showed HADD and were affected at the same time. This opens interesting epidemic aspects of the disease. Husbandry- and nutrition-related biochemical disorders, infectious and hereditary causes have to be considered. The low calcium-phosphorus ratio in these animals supports the hypothesis of a renal or nutritional origin of the disease. Ground meat is known to be low in calcium and to have a calcium-phosphorus ratio of 0.0411 and most captive diets have to be supplemented with calcium to avoid nutritional secondary osteodystrophy. No evidence of renal insufficiency could be determined—uric acid levels were within normal ranges in both turtles. Urea levels were moderately elevated in the smaller turtle but the kidneys were found to be histologically inconspicuous. The parathyroid glands were grossly not considered to be enlarged and therefore they were not investigated histologically. Markedly elevated glucose levels in the smaller turtle were considered to be stress-related. Leucopenia in the smaller animal could be a sign for a viral infection. However, no morphologic lesions indicative of an infectious disease were detected histologically. A relationship of the two turtles could not be determined. In humans with articular gout, apatite crystals were found together with urate crystals in samples of synovial fluid. A secondary apatite formation due to inflammatory processes primarily caused by articular gout is discussed for these patients (Prof. Asper, University Hospital, Zurich, Switzerland, personal communication). However, no urate crystals were detected in the two samples of the turtles by crystallography. Further investigations on the etiology of HADD in reptiles are needed.

Acknowledgments

Special thanks go to the laboratory staff of the Institute for Clinical Chemistry, University Hospital, Zurich, Switzerland (Prof. Asper) for performing the crystallographic work.

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