Metastatic mineralization in blacktip reef sharks, Carcharhinus melanopterus

David Perpiñán¹ and Taiana Costa^1,2*

* Corresponding author.

Short running title: Metastatic mineralization in blacktip reef sharks

Abstract

An episode of morbidity and mortality was observed in captive adult blacktip reef sharks, Carcharhinus melanopterus (Quoy & Gaimard 1824), affecting 5 out of 12 animals over a period of 11 months. When present, clinical signs included reduced appetite, uncoordinated swimming, and increasingly shorter swimming times. All 5 sharks died or were euthanized within 3 days of development of severe clinical signs. Gross necropsy findings included skin wounds, atrophy of the hepatic fat, and diffuse small white irregular areas throughout the renal parenchyma. Histological analysis revealed hepatic lipid depletion and multifocal metastatic mineralization of kidney (renal tubules and glomeruli), central nervous system (meningeal vessels), stomach (submucosal and serosal gastric blood vessels), heart (vessels of the epicardium), and gill arches. A chronic excess dietary intake of a multivitamin and multimineral supplement for elasmobranchs occurred for at least 15 months and it was thought to be the cause of the metastatic mineralization.

Mineralization disorders have been poorly described in elasmobranchs, despite being widely reported in teleost fish. One retrospective study reported three types of soft tissue mineralization in elasmobranchs: nephrocalcinosis, calcinosis circumscripta, and metastatic mineralization (Garner 2013). Nephrocalcinosis in fish is characterized by the deposition of calcium and magnesium salts within the ureters and renal collecting ducts, and it is caused or exacerbated by either a prolonged exposure to high levels of carbon dioxide in the water or by a diet with magnesium deficiency or with selenium excess (Bruno 1996). Calcinosis circumscripta is an uncommon syndrome of soft tissue calcification in fish; it can be idiopathic or associated with tissue damage (e.g trauma, foreign body reactions, neoplasms) (Tafti, Hanna & Bourque 2005).

Metastatic mineralization, also known as metastatic calcification, is a form of pathologic calcification that occurs in previously healthy tissues, and is generally associated with a disturbance in calcium and phosphorus metabolism, particularly calcium and phosphorus excess with extracellular deposition of amorphous calcium phosphate or hydroxyapatite crystal (Han & Garner, 2015). Causes of metastatic mineralization described in other animals include dietary calcium and phosphorus excess, dietary vitamin D excess, renal diseases (with elevation of parathyroid hormone) or paraneoplastic syndromes (with elevation of parathyroid hormone-related protein) (Morrow & Volmer 2002; Myers & McGavin 2007; Craig, Dittmer & Thompson 2015; Kumar, Abbas & Aster 2015). Mineral deposition targets tissues with natural acid secretion or with an internal alkaline environment (Kumar et al. 2015), and the most commonly affected tissues are renal cortex and pelvis, gastric mucosa, pulmonary interstitium and venules, and arteries throughout the body (Kumar et al. 2015; Craig et al. 2015).

This article describes an unusual mortality event associated with metastatic mineralization in a group of captive adult blacktip reef sharks (Carcharhinus melanopterus) from a public aquarium. Animals affected included 3 males and 2 females from a total collection of 12 individuals. While some animals died suddenly with no apparent clinical signs, other animals showed hyporexia to anorexia, uncoordinated swimming (consisting on hitting the wall of the pool and swimming upwards, with the head partially outside the water) and increasingly shorter swimming times (with increasingly longer resting periods). All five sharks died within three days of development of severe clinical signs, although one of them was euthanized (intravenous injection of pentobarbital in the ventral tail vein) and another one them exhibited milder and intermittent clinical signs for about a month before death. All deaths occurred in a period of 11 months.

Necropsy was performed in all animals within 12 hours of death. External gross lesions were consistent in all 5 sharks and included moderate to severe loss of body condition (body weight varied between 6.9 kg and 9.0 kg), multifocal skin wounds and bruises (such as abrasions and erosions on the tip of the snout), and diffuse erythema on the ventral aspect of the body, including claspers and caudal peduncle. Internally, the stomach was empty and the liver was atrophic and had a dark red to brown discoloration. Mineralization was grossly evident in two sharks, where fine white granules were observed throughout the renal parenchyma.

Representative samples of multiple tissues were preserved in 10% neutral buffered formalin and processed for paraffin sections. All tissue sections were stained using hematoxylin and eosin (HE). Histologically, widespread metastatic mineralization was identified in all sharks, affecting kidneys, brain, stomach, heart, arterial trunk, and gills. The mineralization was characterized by aggregates of granular (occasionally lamellar) deep blue-purple material on HE staining. Duplicate tissue sections were stained using von Kossa’s method, and the mineralization was visualized as dark metallic silver aggregates, demonstrating their calcium phosphate or calcium carbonate salt composition (Figure 1a).

Kidneys were the most severely affected organs, with moderate to severe multifocal mineralization observed in all animals. Renal tubules were often distended by a large amount of intraluminal mineral deposition, disrupting the normal architecture of surrounding tissue (Figure 1b); in these cases, the tubules were surrounded by fibrosis and moderate interstitial nephritis, characterized by infiltration of large amount of lymphocytes, fewer macrophages and neutrophils, and rare multinucleated giant cells. Two sharks had very severe renal lesions, where about 40% of the renal tubules evaluated were affected. Mineralization was also present on renal tubular basement membranes, arterial wall, Bowman’s capsule and glomerular basement membrane. There was marked dilatation of renal tubules and collecting ducts, occasionally associated with the presence of intraluminal granular cast material and cellular debris (Figures 1b and 1c).

The brain showed a widespread moderate mineralization of basement membrane of meningeal and choroidal vessels (Figure 1d), with occasional involvement of neuropil capillaries. One shark also showed multifocal gliosis of the optic tectum (mostly associated to the presence of mineral deposits in neuropil capillaries), while another animal had mild neuronal mineralization and chronic mononuclear vasculitis. Similarly, stomach sections revealed mineralization of the submucosal and serosal blood vessels (occasionally associated with mild vasculitis) and, less frequently, gastric glands.

At the heart, moderate to severe mineralization was present in intramural arteries and epicardial blood vessels, occasionally associated with mild and focal nonsuppurative epicarditis. Severe mineral deposition was also observed within the arterial trunk. Multifocal intramural mineralization was observed in filamentary blood vessels, supporting arch and walls of the cavernous bodies of the gills (Figure 1e). Moderate to severe depletion of lipid storage within hepatocytes (Figure 1f) was also a consistent finding. Moreover, mild to moderate, multifocal degeneration of myofibers was occasionally observed on skeletal muscle.

All the sharks were fed by target feeding and received a commercial multivitamin and multimineral supplement for elasmobranchs. After an extensive investigation on the elasmobranch husbandry practices and feeding records, a chronic excess dietary intake of the vitamin and mineral supplement was confirmed (5 to 10 times the dose recommended by the manufacturer), which was taking place for at least 15 months prior to the onset of mortality. Moreover, the investigation revealed the routine use of a human multivitamin and multimineral dietary supplement on sick sharks, although its frequency and amount was not recorded at the feeding records. Cases were lost to follow up after 12 months of initiation of mortality.

The metastatic mineralization in multiple organs observed on these sharks was believed to be directly related to the chronic excessive dietary intake of the multivitamin and multimineral supplement for elasmobranchs. Fishes do accumulate fat-soluble vitamins (A, D, E and K) under conditions where dietary intake exceeds metabolic demand and excess accumulation may produce hypervitaminosis (Tacon 1992).

In mammals, the classic effect of vitamin D is to facilitate the intestinal absorption of calcium by mediating active calcium transport across the intestinal mucosa, and many cases of metastatic mineralization associated with vitamin D toxicity have been described (Kirui, Weisbrode & Kindig 1981; Morita, Awakura, Shimada, Umemura, Nagai & Haruna 1995; Estepa, Aguilera-Tejero, Zafra, Mayer-Valor, Rodriguez & Perez 2006; Martínez, Manteca & Pastor 2013; Han & Garner 2015). In elasmobranchs, however, the role of vitamin D is not properly understood. Vitamin D is abundant in the liver of sharks and other fishes (Rao & Raghuramulu 1999b), although some authors suggest that it may not have a significant effect in calcium absorption (Hilton & Ferguson 1982; Rao & Raghuramulu 1999a) as calcium is an abundant element in the sea (Rao & Raghuramulu 1999a). It has also been reported that fish are resistant to vitamin D toxicosis (Hilton & Ferguson 1982). One study found no detectable levels of vitamin D in blood plasma of Atlantic sharpnose sharks (Rhizoprionodon terraenovae), bonnethead sharks (Sphyrna tiburo) and spiny dogfish sharks (Squalus acanthias) (Haman, Norton, Thomas, Dove & Tseng 2012).

A recent retrospective study of diseases of elasmobranchs described a peculiar histological characteristic and distribution of soft tissue mineralization in sharks and rays, where the mineral deposition was surrounded by a narrow zone of macrophages and fibroblasts, resembling calcinosis circumscripta, but it was not limited to the peripheral connective tissues, as usually observed in mammals and reptiles with calcinosis circumscripta (Garner 2013). Further case reports and retrospective studies of mineral disorders in elasmobranchs are encouraged in order to support such findings.

In conclusion, despite being an essential part of the diet of captive elasmobranchs, the use of multivitamin and multimineral supplements for elasmobranchs may contribute to the risk of excessive intakes and, therefore, must be given with caution. The use of human multivitamin and multimineral dietary supplement in fishes is imprudent and should not be used in any circumstance. More investigation is needed to further characterize the function of vitamin D in calcium metabolism and its toxic effects in elasmobranchs.

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