Cardiac troponin I immunoassay validation, reference interval determination and serum cardiac troponin I changes in translocated southern-central black rhinoceros (Diceros bicornis minor) and southern white rhinoceros (Ceratotherium simum simum)

Abstract

The abundance and range of occurrence of the two rhinoceros species in sub-Saharan Africa, the southern-central black (Diceros bicornis minor) and southern white (Ceratotherium simum simum) rhinoceros, have decreased dramatically. Common threats to rhinoceros conservation include poaching, habitat fragmentation and loss,international trade in illegal rhinoceros products, and reduced financial resources due to global financial recessions and pandemics. Translocation of wildlife species is a commonly used tool for the conservation of threatened and endangered animals, with a focus on restoring and enhancing populations. It plays an integral part in national and international conservation plans for African rhinoceros. Chemical immobilisation is often used during translocation, with etorphine forming the basis of most drug combinations used. Ensuring animal welfare during wildlife transport is critical and dehydration, electrolyte imbalances, a negative energy balance, muscle damage, protein catabolism, stress-induced immunomodulation, and oxidative stress are the main pathophysiological findings reported in rhinoceros translocated over long distances. Investigation into possible cardiomyopathy in transported rhinoceros has been hampered by the lack of validated immunoassays to measure serum biomarkers, specifically cardiac troponin I (cTnI), in rhinoceros. The broad objectives of this study were therefore to 1) sequence the cTnI gene in both rhinoceros species, to obtain the inferred amino acid sequences from the messenger ribonucleic acid (mRNA) transcript sequences, and assess the potential a^inity of several commercial cTnI immunoassays for detecting cTnI in African rhinoceros; 2) validate two point-of-care (POC) cTnI immunoassays for use in African rhinoceros; 3) to generate cTnI reference intervals (RIs) on the high-sensitivity (hs)-cTnI immunoassay in both rhinoceros species and apply subset partitioning in white rhinoceros that were chased by helicopter during capture versus those that were captured in a boma and therefore not chased; and 4) investigate the serum cTnI changes in long-distance translocated rhinoceros and in rhinoceros chemically immobilised with di^erent drug protocols during capture. Best practice guidelines for method validation, quality control (QC) and RI generation as published by the American Society for Veterinary Clinical Pathology were followed. Expert consensus recommendations on the clinical laboratory practice for hs-cTnI assays as advised by the International Federation of Clinical Chemistry Task Force on Clinical Applications of Biomarkers (IFCC TF-CB) were also complied with. The mRNA cTnI transcript sequences were obtained by RNA extraction from myocardium of deceased rhinoceros followed by primer design, complementary deoxyribonucleic acid (cDNA) synthesis using reverse transcription polymerase chain reaction, and Sanger sequencing. The percentage identity between black and white rhinoceros cDNA nucleotide sequences was 99%, while inferred amino acid sequences were identical. There were five amino acid di^erences between humans and rhinoceros in the epitope binding sites of immunoassay antibodies and five assays contained antibodies against epitopes that were not conserved. Nevertheless, only one assay was deemed unlikely to cross-react with rhinoceros cTnI and five assays were found to be suitable for further investigation into cTnI measurement in African rhinoceros.

The Siemens Stratus CS Acute Care troponin I cTnI and Siemens Atellica VTLi hs-cTnI were the two POC cTnI immunoassays selected for analytical method validation. Validation experiments included precision studies, reportable range, haemoglobin interference studies, recovery studies, and detection limit studies with results assessed against two total allowable error (TEa) performance goals, namely 30% and 70%. Imprecision was acceptable and met low cTnI concentration performance goals. Thereportable ranges were similar to the manufacturer’s specifications. For the Stratus CS, high haemoglobin concentrations in white rhinoceros resulted in bias. The QC validation results showed that a simple 13s QC rule using two levels of QC material and a TEa of 70% could be used in both analysers, except at very low cTnI concentrations in the Atellica VTLi. This study showed that both cTnI POC analysers are suitable for use in African rhinoceros and analytical performance goals for low cTnI concentrations in hs-cTnI assays were met.

To allow for the identification of cardiomyocyte injury in African rhinoceros, RIs were established for both species of rhinoceros using the validated hs-cTnI assay. Reference intervals were generated from 62 and 87 apparently healthy, free-living immobilised black and white rhinoceros, respectively. Additionally, the 99th percentile upper reference limits were also determined. Of interest is that subclass partitioning was valid for white rhinoceros based on whether they were immobilised in a boma or chased by a helicopter before being immobilised.

Although chemical capture and translocation (involving capture and long-distance transport) in African rhinoceros are essential components of conservation strategies aimed at improving the conservation status of the species, several adverse pathophysiological e^ects, specifically hypoxaemia, acidosis and muscle damage, are associated with these processes which negatively impact rhinoceros’ welfare. Serum cTnI concentration was measured using the Atellica VTLI hs-cTnI assay on stored serum samples collected during four long-distance translocation studies in black and white rhinoceros and in one chemical immobilisation study in white rhinoceros. Measurement of serum cTnI concentration in rhinoceros translocated over long distances showed significantly increased cTnI concentrations during transportation and at release when compared to concentrations at capture. The degree of cTnI increase was more significant in cohorts chased and darted from helicopters. Concurrent skeletal and cardiac muscle damage was demonstrated in transported black and white rhinoceros, indicative of capture myopathy (CM) in these animals. Furthermore, hypoxaemia, acidosis and a negative energy balance were correlated with elevated cTnI concentrations, highlighting specific areas in procedures involving chemical immobilisation, capture, and transport that need to be addressed to mitigate these adverse effects. The results of this study will allow wildlife veterinarians involved in African rhinoceros conservation procedures, and in the treatment of injured animals, to assess if cardiomyocyte damage is present. This assessment will allow for cardioprotective adjustments to be implemented in these procedures, resulting in improved animal welfare. Demonstration of concurrent elevated skeletal and cardiac muscle biomarkers in translocated rhinoceros will assist wildlife veterinarians in identifying animals at risk of developing CM that should be kept in confinement (boma) at the receiving end of the journey for monitoring and reduction in stress before release.