Feasibility Study of NMR Based Serum Metabolomic Profiling to Animal Health Monitoring: A Case Study on Iron Storage Disease in Captive Sumatran Rhinoceros (Dicerorhinus sumatrensis)

Abstract

A variety of wildlife species maintained in captivity are susceptible to iron storage disease (ISD), or hemochromatosis, a disease resulting from the deposition of excess iron into insoluble iron clusters in soft tissue. Sumatran rhinoceros (Dicerorhinus sumatrensis) is one of the rhinoceros species that has evolutionarily adapted to a low-iron diet and is susceptible to iron overload. Hemosiderosis is reported at necropsy in many African black and Sumatran rhinoceroses but only a small number of animals reportedly die from hemochromatosis. The underlying cause and reasons for differences in susceptibility to hemochromatosis within the taxon remains unclear. Although serum ferritin concentrations have been useful in monitoring the progression of ISD in many species, there is some question regarding their value in diagnosing hemochromatosis in the Sumatran rhino. To investigate the metabolic changes during the development of hemochromatosis and possibly increase our understanding of its progression and individual susceptibility differences, the serum metabolome from a Sumatran rhinoceros was investigated by nuclear magnetic resonance (NMR)-based metabolomics. The study involved samples from female rhinoceros at the Cincinnati Zoo (n = 3), including two animals that died from liver failure caused by ISD, and the Sungai Dusun Rhinoceros Conservation Centre in Peninsular Malaysia (n = 4). Principal component analysis was performed to visually and statistically compare the metabolic profiles of the healthy animals. The results indicated that significant differences were present between the animals at the zoo and the animals in the conservation center. A comparison of the 43 serum metabolomes of three zoo rhinoceros showed two distinct groupings, healthy (n = 30) and unhealthy (n = 13). A total of eighteen altered metabolites were identified in healthy versus unhealthy samples. Results strongly suggest that NMR-based metabolomics is a valuable tool for animal health monitoring and may provide insight into the progression of this and other insidious diseases.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Multivariate PCA analysis of serum metabolome from Sumatran rhinoceroses.

(A) PCA scores plots indicating the relationship between three rhinoceroses: Rhino-1 (red), Rhino-2 (blue), Rhino-3 (green), and their health status (x-healthy, ●-unhealthy). (B) The average PC scores of each animal corresponding to the plot (A). The error bars are indicating the standard deviations. (C) PCA scores plot (A) based on the health status of animals (x-Healthy-black, ●-Unhealthy). (D) The average scores of each animal corresponding to the plot (C). (E) PCA scores plots indicating the Rhino-1 health status (x-healthy, ●-unhealthy), and the average PC scores (F).

Fig 2. Univariate significant difference spectra (SDS) analysis.

SDS spectra obtained by subtracting the mean buckets (n = 980) of the unhealthy from healthy samples. Only the buckets (n = 178) with significant alterations based on t-test with FDR correction are plotted. BCAA: branched chain amino acids, 2HB: 2-hydroxyisobutyrate, Lac: lactate, Ala: alanine, Gln: glutamine, Cit: citrate, Cre: creatine, Gly: glycine, Glc: glucose, Man: mannose, Phe: phenylalanine.

Fig 3. Metabolite changes in relation to health status of rhinoceroses.

Bucket intensities of each altered metabolite from three rhinoceroses: Rhino-1 (red), Rhino-2 (blue), Rhino-3 (green), and their health status (x-healthy, ●-unhealthy). The averages of all healthy or unhealthy samples are shown in black. (A) leucine, (B) isoleucine, (C) valine, (D) phenylalanine, (E) creatinine, (F) phosphocreatine.