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Iron overload disorder (IOD) can lead to organ dysfunction and may exacerbate other diseases in the critically endangered black rhinoceros ( Diceros bicornis). It is important to develop methods for monitoring the progression of iron storage (hemosiderosis), diagnosing the disease, and evaluating treatments in this species. Traditionally, an equine enzyme immunoassay (EIA) was used to measure rhinoceros ferritin, a serum protein correlated to iron stores. The goal of this study was to validate a rhinoceros-specific assay and investigate factors potentially associated with ferritin concentrations in black rhinoceros. A ferritin EIA developed for Sumatran rhinoceros was validated for black rhinoceros via Western blot analysis of liver ferritin and confirmed parallelism of serum samples to the EIA standard curve and used to analyze serum samples ( n = 943) collected from 36 black rhinoceros (<1-33 yr) at 14 U.S. institutions. Mean (±SEM) serum ferritin concentration was 6,738 ± 518 ng/ml (range: 85-168,451 ng/ml). Concentrations differed among individuals with eastern black rhinoceros (7,444 ± 1,130 ng/ml) having a higher mean ferritin than southern black rhinoceros (6,317 ± 505 ng/ml; P < 0.05) and higher mean values in wild-born (11,110 ± 1,111 ng/ml) than captive-born individuals (3,487 ± 293 ng/ml; P < 0.05). Ferritin concentrations did not differ between young rhinoceros (<5 yr old; 2,163 ± 254 ng/ml) and adults (7,623 ± 610 ng/ml) and were not correlated with age ( r = 0.143) or time in captivity ( r = 0.146, wild born; r = 0.104, all animals). Ferritin concentration was not impacted by sex (female: 2,086 ± 190 ng/ml; male: 8,684 ± 717 ng/ml), date, month, or season of collection ( P > 0.05). Data indicate ferritin concentrations are variable and not necessarily associated with IOD; ferritin is not recommended for diagnosing or monitoring IOD in black rhinoceros.

The aim of this study was to determine if ferritin is a reliable biomarker of iron overload disorder (IOD) progression and hemochromatosis in the Sumatran rhinoceros (Dicerorhinus sumatrensis) by developing a species-specific ferritin assay and testing historically banked samples collected from rhinos that did and did not die of hemochromatosis. Ferritin extracted from Sumatran rhino liver tissue was used to generate antibodies for the Enzyme Immunoassay. Historically banked Sumatran rhino serum samples (n = 298) obtained from six rhinos in US zoos (n = 290); five rhinos at the Sumatran Rhino Conservation Centre in Sungai Dusun, Malaysia (n = 5); and two rhinos in Sabah, Malaysia (n = 3) were analyzed for ferritin concentrations. Across all US zoo samples, serum ferritin concentrations ranged from 348 to 7,071 ng/ml, with individual means ranging from 1,267 (n = 25) to 2,604 ng/ml (n = 36). The ferritin profiles were dynamic, and all rhinos exhibited spikes in ferritin above baseline during the sampling period. The rhino with the highest mean ferritin concentration did not die of hemochromatosis and exhibited only mild hemosiderosis postmortem. A reproductive female exhibited decreases and increases in serum ferritin concurrent with pregnant and nonpregnant states, respectively. Mean (±SD) serum ferritin concentration for Sumatran rhinos in Malaysia was high (4,904 ± 4,828 ng/ml) compared to that for US zoo rhinos (1,835 ± 495 ng/ml). However, those in Sabah had lower ferritin concentrations (1,025 ± 52.7 ng/ml) compared to those in Sungai Dusun (6,456 ± 4,941 ng/ml). In conclusion, Sumatran rhino serum ferritin concentrations are dynamic, and increases often are not associated with illness or hemochromatosis. Neither a specific pattern nor the individual's overall mean ferritin concentration can be used to accurately assess IOD progression or diagnose hemochromatosis in this rhino species.

Iron overload disorder (IOD) is a significant health issue for captive black rhinoceros ( Diceros bicornis ). Measurement of serum ferritin with a validated rhinoceros ferritin ELISA has been used extensively to detect animals in U.S. zoos that are at risk of developing IOD. However, there is limited information on serum ferritin levels in free-ranging black rhinoceros using this same assay. Serum ferritin, iron, and gamma-glutamyl transpeptidase (GGT) were determined in 194 black rhinoceros from southern Africa. Mean ferritin in free-ranging black rhinoceros (290.54 ±247.4 ng/ml) was significantly higher than in free-ranging white rhinoceros (64.0 ± 102.4 ng/ml) sampled in this study from Kruger National Park, South Africa. However, there were no significant differences between genders or age groups. Ferritin values varied with geographical location of the black rhinoceros, although this was not clinically significant. Serum iron values were also higher in black rhinoceros (40.4 ± 19.1 μmol/L) compared to white rhinoceros (29.7 ± 10.7 μmol/L). There was no association between ferritin and GGT. This study provides serum ferritin, iron, and GGT values from free-ranging black rhinoceros that can be used for as comparative target values for captive animals.

The African black rhinoceros (Diceros bicornis) has adapted to a low iron diet during evolution and is thus prone to iron overload in captivity, which is associated with a number of serious disorders. A S88T polymorphism in the HFE gene has been suggested as a potential genetic basis of increased iron uptake in the black rhinoceros, while the Indian rhinoceros is thought to be unaffected by iron overload in captivity. In the present study, the histopathology and distribution of iron accumulations in five black rhinoceroses with iron overload syndrome were characterized and compared with three Indian rhinoceroses (Rhinoceros unicornis) and one African white rhinoceros (Ceratotherium simum). At necropsy examination, iron storage in black rhinoceroses was not associated with gross lesions. Microscopically, the most consistent and highest degree of iron load was found in the spleen, liver, small intestine and lung. There was minimal fibrosis and single cell necrosis in the liver. Endocrine organs, lymph nodes, heart and kidney were less often and less markedly affected. Unexpectedly, Indian rhinoceroses also showed iron load in the spleen and smaller amounts in organs similar to the black rhinoceros except for in the heart, while the white rhinoceros had only minor detectable iron storage in intestine, liver and lung. Sequence analysis confirmed the HFE S88T polymorphism in black but not in Indian rhinoceroses. The results indicate that Indian rhinoceroses may also be affected by iron storage in captivity, although in a milder form than the black rhinoceros, and therefore challenge the relevance of the S88T polymorphism in the HFE gene of black rhinoceroses as the underlying cause for iron overload., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Iron overload disorder afflicts captive rhinoceros but has not been documented in the wild. The specific cause for the disorder has not been identified but is likely associated with diet and management. Compared with wild counterparts, captive rhinoceros eat diets containing more iron, have greater fat stores, and exercise less. It has been suggested that the problem may be linked to development of insulin resistance in the captive population. Given that controlled experiments with sufficient numbers of rhinoceros are logistically not possible, an equine model was used to look for a relationship between iron status and insulin resistance; the nutritional requirements of horses are used as a guide for rhinoceros, because they have similar gastrointestinal tracts. Sixteen horses were tested to determine blood insulin responses to an oral drench of dextrose (0.25 g/kg bodyweight) and a meal of pelleted corn (1.5 g/kg bodyweight). Fasting blood samples were taken 30 and 0 min before administration. Further blood samples were taken every 30 min for 4 hr after administration to determine peak insulin and total area under the insulin curve (AUC). Fasting samples were tested for serum ferritin concentrations. Correlations were determined between ferritin and peak insulin concentrations and insulin AUC after administration of oral dextrose and pelleted corn. The strongest correlation was between ferritin and insulin AUC after dextrose administration (r = 0.61; P = 0.01) followed by AUC after feeding a meal of pelleted corn (r = 0.60; P = 0.01), with the correlation for peak insulin being 0.53 (P = 0.03) after dextrose administration and 0.56 (P = 0.02) after pelleted corn. When evaluating responses by gender, a significant correlation existed only for females, influenced by one insulin resistant individual. These data suggest a potential link between insulin resistance and body stores of iron and also suggest that approaches to reduce the susceptibility to insulin resistance should be incorporated into management of captive browsing rhinoceros.

Objective: To investigate the possibility that excessive maternal iron (overload) may contribute to development of congenital leukoencephalomalacia in captive black rhinoceroses., Sample Population: Tissue specimens and serum samples from 18 rhinoceroses in 2 kindreds harboring 4 (possibly 5) affected female calves., Procedure: Fresh and archival sera and necropsy tissue specimens were evaluated to determine the nature and extent of iron overload in captive and wild black rhinoceroses as well as other rhinoceros species., Results: Quantitative serum and tissue assays of iron and iron analytes, corroborated by histopathologic findings, indicated that these kindreds carried the greatest body burdens of iron yet found among captive black rhinoceroses. Fourteen of 18 rhinoceroses had the highest serum ferritin concentrations measured among 64 black rhinoceroses in captivity in the United States. Dams of affected calves had serum ferritin concentrations 2 orders of magnitude higher than clinically normal humans, equids, or free-ranging rhinoceroses. A neonatal serum sample from 1 affected female calf had a high ferritin concentration (approx 100-fold increase), but a male sibling of another affected female did not, suggesting a possible sex disparity in fetal response to maternal iron overload. Morphologic hallmarks of hemochromatosis were prominent in dams and grandams of affected calves., Conclusions and Clinical Relevance: Excessive maternal iron may affect female fetuses more than males, possibly inducing leukoencephalomalacia by catalyzing production of highly toxic hydroxyl free radicals during crucial periods of in utero development. Reduction of maternal iron overload may decrease the probability of developing leukoencephalomalacia and some other disorders commonly affecting rhinoceroses in captivity.

When rhinoceros species that are browsers in the wild are fed in captivity they become iron overloaded. Presumably, their iron-absorptive mechanisms have evolved to become highly efficient. In humans, mutations of the HFE gene cause increased iron absorption. To determine whether the HFE gene of rhinoceroses has undergone mutation as an adaptive mechanism to improve iron absorption from iron-poor diets, we have sequenced the entire coding region of the HFE genes of four species of rhinoceros. Two of these were browsing species and two were grazing species. Although the HFE gene has been well preserved across species, numerous nucleotide differences were found between rhinoceros and human or mouse, some of which changed deduced amino acids. Of these mutations, only one found in the black rhinoceros appears to be a viable candidate mutation that might adversely affect HFE function. This mutation, S88T, is in a highly conserved region that is involved in the interaction between transferrin receptor and HFE., (Copyright 2001 Academic Press.)