Reduced capability of refrigerated white rhinoceros whole blood to produce interferon-gamma upon mitogen stimulation.

Ante-mortem surveillance for Mycobacterium bovis (M. bovis) infection in the Kruger National Park (KNP) rhinoceros population currently relies on results from the QuantiFERON-TB Gold (In-Tube) Plus (QFT)-interferon gamma (IFN-γ) release assay (IGRA). However, same-day processing of rhinoceros blood samples for this test is a logistical challenge. Therefore, a pilot study was performed to compare mitogen-stimulated and unstimulated IFN-γ concentrations in plasma from rhinoceros whole blood processed within 6 h of collection or stored at 4°C for 24 and 48 h prior to incubation in QFT tubes. Replicate samples of heparinized whole blood from seven subadult male white rhinoceros were used. Results showed no change in IFN-γ levels in unstimulated samples, however the relative concentrations of IFN-γ (based on optical density values) in mitogen plasma decreased significantly with increased time blood was stored post-collection and prior to QFT stimulation. These findings support a need for same-day processing of rhinoceros blood samples for QFT-IGRA testing as per the current practice. Further investigation using TB-antigen stimulated samples is warranted to properly assess the impact of blood storage on TB test results in rhinoceros.

Epidemiology of Mycobacterium bovis infection in free-ranging rhinoceros in Kruger National Park, South Africa.

Mycobacterium bovis infection, which is a prominent cause of bovine tuberculosis, has been confirmed by mycobacterial culture in African rhinoceros species in Kruger National Park (KNP), South Africa. In this population-based study of the epidemiology of M. bovis in 437 African rhinoceros (Diceros bicornis, Ceratotherium simum), we report an estimated prevalence of 15.4% (95% CI: 10.4 to 21.0%), based on results from mycobacterial culture and an antigen-specific interferon gamma release assay from animals sampled between 2016 and 2020. A significant spatial cluster of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo (Syncerus caffer) herds in the vicinity of the rhinoceros sampling location. Since African buffaloes are important maintenance hosts for M. bovis in KNP, spillover of infection from these hosts to white rhinoceros sharing the environment is suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016­2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. These study findings provide a foundation for further epidemiological investigation of M. bovis, a multihost pathogen, in a complex ecosystem that includes susceptible species that are threatened and endangered.

Mycobacterium bovis Infection in Free-Ranging African Elephants.

Mycobacterium bovis infection in wildlife species occurs worldwide. However, few cases of M. bovis infection in captive elephants have been reported. We describe 2 incidental cases of bovine tuberculosis in free-ranging African elephants (Loxodonta africana) from a tuberculosis-endemic national park in South Africa and the epidemiologic implications of these infections.

ANTIBODY PREVALENCE TO AFRICAN SWINE FEVER VIRUS, MYCOBACTERIUM BOVIS, FOOT-AND-MOUTH DISEASE VIRUS, RIFT VALLEY FEVER VIRUS, INFLUENZA A VIRUS, AND BRUCELLA AND LEPTOSPIRA SPP. IN FREE-RANGING WARTHOG (PHACOCHOERUS AFRICANUS) POPULATIONS IN SOUTH AFRICA.

The warthog (Phacochoerus africanus) can be used as a model for investigating disease transmission at the human, wildlife, and livestock interface. An omnivore and scavenger, a warthog moves freely between natural ecotypes, farmland, and human communities and is susceptible to diseases of zoonotic, agricultural, and conservation concern. A retrospective study using 100 individual serum samples collected from May 1999 to August 2016 was performed to determine antibody prevalence to seven pathogens in warthogs from five locations in northeastern South Africa. Higher prevalence of antibodies to African swine fever virus and Mycobacterium bovis were detected in warthogs from the Greater Kruger National Park ecosystem in comparison to lower prevalence of antibodies to M. bovis and no antibodies to African swine fever virus in warthogs from uMhkuze Game Reserve. Low prevalence of antibodies to foot-and-mouth disease virus, Rift Valley fever virus, and influenza A virus was detected in all locations, and no antibodies against Brucella and Leptospira spp. were detected. No statistically significant difference in antibody prevalence was found between sexes for any disease. At the univariate analysis, M. bovis seropositivity was significantly different among age categories, with 49% (35/71) of adults found positive versus 29% (4/14) of juveniles and 9% (1/11) of sub-adults (Fisher's exact test, P=0.020), and between the sampling locations (Fisher's exact test, P=0.001). The multivariate model results indicated that juvenile warthogs had lower odds of testing positive to M. bovis antibodies than adults (juveniles' odds ratio [OR]=0.17, 95% confidence interval [CI]: 0.02-1.0), although this result was not statistically significant at the 5% level (P=0.052). For warthogs sampled at Satara Buffalo Camp, the odds (OR=0.22, 95% CI: 0.035-0.96) of being M. bovis antibody positive were significantly lower (P=0.043) than for warthogs sampled at Skukuza. Of particular interest in this study was the detection of warthogs seropositive for influenza A virus.

Development of a cytokine gene expression assay for the relative quantification of the African elephant (Loxodonta africana) cell-mediated immune responses.

Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African elephant (Loxodonta africana), is limited however due to lack of information on immune responses. Since many immunoassays require both identified biomarkers of immune activation as well as species-specific reagents, it is crucial to have knowledge of basic immunological responses in the species of interest. Cytokine gene expression assays (GEAs) used to measure specific immune responses in wildlife have frequently shown that targeted biomarkers are often species-specific. Therefore, the aim of this study was to identify elephant-specific cytokine biomarkers to detect immune activation and to develop a GEA, using pokeweed mitogen stimulated whole blood from African elephants. This assay will provide the foundation for the development of future cytokine GEAs that can be used to detect antigen specific immune responses and potentially lead to various diagnostic tests for this species.

EVALUATION OF A PARTIALLY REVERSIBLE IMMOBILIZATION PROTOCOL USING MEDETOMIDINE, BUTORPHANOL, ZOLAZEPAM-TILETAMINE, AND KETAMINE IN FREE-RANGING WARTHOGS (PHACOCHOERUS AFRICANUS) IN KRUGER NATIONAL PARK, SOUTH AFRICA.

Twenty-one free-ranging warthogs (Phacochoerus africanus) in the Kruger National Park, South Africa, were immobilized with a combination of medetomidine (0.07 ± 0.01 mg/kg), butorphanol (0.26 ± 0.04 mg/kg), tiletamine-zolazepam (0.69 ± 0.15 mg/kg), and ketamine (1.43 ± 0.21 mg/kg) administered intramuscularly by dart. Induction, immobilization, and recovery characteristics were evaluated using a standardized scoring system. In the immobilized warthogs, physiological variables were measured every 5 min and arterial blood gases were analyzed at 15-min intervals. At 45 min after initial drug administration, atipamezole (0.34 ± 0.050 mg/kg) and naltrexone (0.53 ± 0.079 mg/kg) were administered intravenously. Overall, induction quality after darting was scored as excellent and the mean time to safe handling was 5.9 ± 2.0 min. Based on muscle relaxation, and loss of palpebral and pedal reflexes, most subjects (17 out of 21) reached a plane of surgical anesthesia by 10 and 15 min; 20 out of 21 warthogs were in this plane for the duration of the monitoring period. In the immobilized warthogs the overall mean heart rate was 65 ± 15.3 beats per minute, mean respiratory rate was 14.7 ± 5.6 breaths per minute, and the mean rectal temperature was 37.9 ± 1.4°C during the 40 min. Arterial blood gas results showed hypoxemia (mean PaO2 62.1 ± 16.2 mmHg), hypercapnia (mean PaCO2 47.1 ± 5.1 mmHg), and acidemia (mean pH = 7.36 ± 0.04). Values for PaO2 and pH improved over the immobilization period. After antagonist administration, overall recovery quality from immobilization was scored as good, with animals standing at a mean time of 7.3 ± 4.9 min. The drug combination proved to be effective in the immobilization of free-ranging warthogs with rapid induction, good anesthesia, and limited cardiorespiratory changes. This anesthetic protocol produces effective, safe, and partially reversible immobilization in warthogs.

Evaluation of Physiological Parameters and Effectiveness of an Immobilization Protocol Using Etorphine, Azaperone, and Butorphanol in Free-Ranging Warthogs (Phacochoerus africanus).

Twenty free-ranging warthogs (Phacochoerus africanus) in the Kruger National Park, South Africa, were immobilized with a combination of etorphine (0.039 ± 0.005 mg/kg) and azaperone (0.44 ± 0.06 mg/kg) administered intramuscularly by dart. Butorphanol (1 mg per mg etorphine) was administered intravenously at t = 5 min. A standardized scoring system was used to record induction, immobilization and recovery characteristics. Physiological parameters were recorded at 5 min intervals and an arterial sample collected for blood gas analyses every 15 min. At 45 min after butorphanol administration, immobilization was partially reversed by administering naltrexone (40x etorphine dose in mg) intravenously. Overall, induction quality was good, with the mean time to safe handling 5.9 ± 1.4 min. The majority of immobilization scores (54%) over the entire monitoring period (40 min) were at level 3, consistent with a light plane in which palpebral and laryngeal reflexes were still present but the animal could be safely handled. Overall mean heart rate was 94.7 ± 15.3 beats per min, mean respiratory rate was 14.7 ± 9.8 breaths per min, and the mean rectal temperature was 38.5 ± 1.0°C. Significant hypoxia (overall mean oxygen arterial partial pressure 38.8 ± 8.4 mmHg), hypercapnia (mean carbon dioxide arterial partial pressure 63.3 ± 7.8 mmHg), and acidosis (mean pH 7.28 ± 0.04) were observed in immobilized warthogs. Following antagonist administration, warthogs were standing within 1.0 ± 0.4 min, with the majority of recoveries scored as excellent. The drug combination proved to be effective in the immobilization of free-ranging warthogs with rapid induction and recovery, but with significant cardio-respiratory changes. Therefore, this drug combination may be useful when rapid immobilization and recovery are indicated, but should be used cautiously in compromised warthogs.

An interferon-gamma release assay for the diagnosis of the Mycobacterium bovis infection in white rhinoceros (Ceratotherium simum).

Mycobacterium bovis (M. bovis), the cause of bovine tuberculosis, is endemic in Kruger National Park (KNP), South Africa. The risk of spread of M. bovis infection currently prevents translocation of white rhinoceros (Ceratotherium simum) from this population. Therefore, accurate assays are necessary for screening this threatened species. Interferon gamma (IFN-γ) release assays (IGRA) are commonly used for tuberculosis diagnosis in humans and other wildlife species. Hence, the aim of this study was to develop an IGRA for M. bovis detection in white rhinoceros. Heparinized whole blood was collected from immobilized white rhinoceros in KNP (n = 131) and incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes, after which the plasma was harvested following centrifugation. Tissue samples for mycobacterial culture were available from a subset of 21 rhinoceros. The concentration of IFN-γ in plasma samples was measured using the Mabtech equine IFN-γ ELISAPRO kit. An IGRA result was calculated as the difference in IFN-γ concentrations in the QFT Nil and TB antigen tubes. Using test results for the white rhinoceros with known infection status, a diagnostic cut-off value was calculated as 21 pg/ml. Additionally, cut-off values for IFN-γ concentrations for plasma from QFT Nil and QFT Mitogen tubes were calculated to increase confidence in IGRA result interpretation. The combination of the QFT stimulation platform and Mabtech equine IFN-γ ELISA is a promising diagnostic test to distinguish between of M. bovis-infected and -uninfected white rhinoceros.

Mycobacterium bovis Infection in African Wild Dogs, Kruger National Park, South Africa.

We screened African wild dogs (Lycaon pictus) in Kruger National Park, South Africa, for Mycobacterium bovis infection using an interferon-gamma release assay. We detected M. bovis sensitization in 20 of 21 packs; overall apparent infection prevalence was 83%. These animals experience high infection pressure, which may affect long-term survival and conservation strategies.

MYCOBACTERIUM BOVIS IN FREE-RANGING LIONS (PANTHERA LEO) - EVALUATION OF SEROLOGICAL AND TUBERCULIN SKIN TESTS FOR DETECTION OF INFECTION AND DISEASE.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis infection, causes morbidity and mortality in free-ranging lions in bTB-endemic areas of South Africa. However, the only currently used diagnostic test is the tuberculin skin test (TST). This test is logistically challenging to perform because it requires immobilization of lions twice in a 72-hr period. Blood-based diagnostic tests, such as serological assays, have been previously reported for M. bovis detection in lion populations, and have the advantage of only requiring a single immobilization. In addition, serological assays can be used for retrospective testing. Therefore, the aim of this study was to test free-ranging lions with the STAT-PAKt (Chembio Diagnostics Systems, Medford, NY 11763, USA) and DPPt VetTB (Chembio Diagnostics Systems) serological assays and compare those results with the tuberculin skin test. The serological assays were also used to determine prevalence in bTB-endemic and uninfected lion populations. The results showed that the serological assays could distinguish between M. bovis culture-positive and -negative lions. In addition, antigen-specific humoral responses were present in lions that had clinical signs of bTB disease or were shedding M. bovis antemortem. Although the seroprevalence of M. bovis infection in Kruger National Park lions was similar to that obtained from antemortem mycobacterial culture (4.8 and 3.3%, respectively), it was less than that estimated by the TST (72%). These findings support the hypothesis that assays based on cell-mediated immune responses are more sensitive than serology is in detecting M. bovis infection in lions. However, serological assays can have a role in bTB disease detection in lions and are especially useful for retrospective studies.

Fatal Tuberculosis in a Free-Ranging African Elephant and One Health Implications of Human Pathogens in Wildlife.

Tuberculosis (TB) in humans is a global public health concern and the discovery of animal cases of Mycobacterium tuberculosis (Mtb) infection and disease, especially in multi-host settings, also has significant implications for public health, veterinary disease control, and conservation endeavors. This paper describes a fatal case of Mtb disease in a free-ranging African elephant (Loxodonta africana) in a high human TB burden region. Necropsy revealed extensive granulomatous pneumonia, from which Mtb was isolated and identified as a member of LAM3/F11 lineage; a common lineage found in humans in South Africa. These findings are contextualized within a framework of emerging Mtb disease in wildlife globally and highlights the importance of the One Health paradigm in addressing this anthroponotic threat to wildlife and the zoonotic implications.

AN INTERFERON GAMMA RELEASE ASSAY FOR THE DETECTION OF IMMUNE SENSITIZATION TO MYCOBACTERIUM BOVIS IN AFRICAN WILD DOGS (LYCAON PICTUS).

In South Africa, the largest proportion of the African wild dog (Lycaon pictus) population resides in regions where buffaloes have a high prevalence of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). Recent reports of deaths of wild dogs associated with bTB have raised concerns regarding the threat this disease might pose for this species. In order to understand the potential impact of the disease in wild dogs, diagnostic tools are required to identify infected individuals. The interferon gamma (IFN-γ) release assay (IGRA) is commonly used for tuberculosis (TB) screening of humans, cattle, and other species, and the aim of this study was to develop an IGRA for wild dogs to detect immune sensitization. Blood was collected from immobilized wild dogs from the Ann van Dyk Cheetah Centre (AvDCC; n=9) and Kruger National Park (KNP; n=31). Heparinized whole blood was incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes and with selected mitogens, after which the plasma fraction was harvested. Three canine IFN-γ enzymelinked immunosorbent assays (ELISAs) were compared for detection of wild dog IFN-γ in plasma and the R&D Quantikine canine IFN-γ ELISA was selected for measurement of M. bovis-specific IFN-γ release in plasma samples. An IGRA result was calculated as the concentration in plasma derived from the QFT TB Antigen tubes minus that in the QFT Nil tube. An IGRA cut-off value was calculated using the IGRA results of M. bovis-unexposed individuals from AvDCC. Using this cut-off value, 74% (23/31) of M. bovis-exposed KNP wild dogs were IGRA positive, indicating immune sensitization to TB antigens in these animals. Three M. bovis culture-positive wild dogs from KNP had IFN-γ concentrations between 758 and 1,445 pg/mL, supporting this interpretation. This warrants further investigation into the prevalence of M. bovis infection in the KNP population.

Measuring antigen-specific responses in Mycobacterium bovis-infected warthogs (Phacochoerus africanus) using the intradermal tuberculin test.

BACKGROUND: Bovine tuberculosis (bTB) caused by Mycobacterium bovis has previously been diagnosed in warthogs and infection can be highly prevalent (> 30%) in endemic areas. Thus, warthogs could potentially be an important species to consider as sentinels for disease surveillance. However, disease surveillance is dependent on availability of accurate diagnostic assays and only a few diagnostic tests have been investigated for warthogs. Furthermore, the tests that have been used in this species require laboratory equipment and trained personnel to obtain results. Therefore, this study investigated the use of the intradermal tuberculin test (ITT) to screen warthogs for bTB, which can be done with minimal equipment and under field conditions by most veterinarians and other qualified professionals. Changes in skin fold thickness measurements at the bovine purified protein derivative (PPD) administration site, between 0 and 72 h, were compared with differential changes between the bovine and avian PPD sites, for 34 warthogs, to evaluate the performance when different interpretation criteria for the ITT was used. RESULTS: Using an increase of 1.8 mm or more at the bovine PPD site as a cut-off for positive responders, 69% of 16 M. bovis culture-positive warthogs had a positive test result, with 100% of the 18 culture-negative warthogs considered as test negative. When a differential of 1.2 mm or more in skin fold thickness at the bovine PPD compared to the avian PPD site was used as a cut-off for the comparative ITT, 81% of culture-positive warthogs were considered as test positive, with 100% of culture-negative warthogs considered as test negative. CONCLUSION: The findings in this study suggest that the ITT is a promising tool to use when screening warthogs for M. bovis infection.

Conservation of White Rhinoceroses Threatened by Bovine Tuberculosis, South Africa, 2016-2017.

During 2016-2017, when Kruger National Park, South Africa, was under quarantine to limit bovine tuberculosis spread, we examined 35 white and 5 black rhinoceroses for infection. We found 6 infected white rhinoceroses during times of nutritional stress. Further research on Mycobacterium bovis pathogenesis in white rhinoceroses is needed.

Parasites of domestic and wild animals in South Africa. XLIX. Ticks (Acari: Ixodidae) infesting white and black rhinoceroses in southern Africa.

The objectives of the study were to determine the species composition of ticks infesting white and black rhinoceroses in southern Africa as well as the conservation status of those tick species that prefer rhinos as hosts. Ticks were collected opportunistically from rhinos that had been immobilised for management purposes, and 447 white rhinoceroses (Ceratotherium simum) and 164 black rhinoceroses (Diceros bicornis) were sampled in South Africa, 61 black rhinos in Namibia, 18 white and 12 black rhinos in Zimbabwe, and 24 black rhinos in Zambia. Nineteen tick species were recovered, of which two species, Amblyomma rhinocerotis and Dermacentor rhinocerinus, prefer rhinos as hosts. A. rhinocerotis was collected only in the northeastern KwaZulu-Natal reserves of South Africa and is endangered, while D. rhinocerinus is present in these reserves as well as in the Kruger National Park and surrounding conservancies. Eight of the tick species collected from the rhinos are ornate, and seven species are regularly collected from cattle. The species present on rhinos in the eastern, moister reserves of South Africa were amongst others Amblyomma hebraeum, A. rhinocerotis, D. rhinocerinus, Rhipicephalus maculatus, Rhipicephalus simus and Rhipicephalus zumpti, while those on rhinos in the Karoo and the drier western regions, including Namibia, were the drought-tolerant species, Hyalomma glabrum, Hyalomma rufipes, Hyalomma truncatum and Rhipicephalus gertrudae. The species composition of ticks on rhinoceroses in Zambia differed markedly from those of the other southern African countries in that Amblyomma sparsum, Amblyomma tholloni and Amblyomma variegatum accounted for the majority of infestations.

Prevalence and Risk Factors for Mycobacterium bovis Infection in African Lions ( Panthera leo ) in the Kruger National Park.

Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), is endemic in the Kruger National Park (KNP), South Africa. African lions ( Panthera leo ) are susceptible to BTB, but the impact of the disease on lion populations is unknown. In this study, we used a novel gene expression assay for chemokine (C-X-C motif) ligand 9 (CXCL9) to measure the prevalence of M. bovis infection in 70 free-ranging lions that were opportunistically sampled in the southern and central regions of the KNP. In the southern region of the KNP, the apparent prevalence of M. bovis infection was 54% (95% confidence interval [CI]=36.9-70.5%), compared with 33% (95% CI=18.0-51.8%) in the central region, an important difference (P=0.08). Prevalence of M. bovis infection in lions showed similar patterns to estimated BTB prevalence in African buffaloes ( Syncerus caffer ) in the same areas. Investigation of other risk factors showed a trend for older lions, males, or lions with concurrent feline immunodeficiency virus infection to have a higher M. bovis prevalence. Our findings demonstrate that the CXCL9 gene expression assay is a useful tool for the determination of M. bovis status in free-ranging lions and identifies important epidemiologic trends for future studies.

Test performance of three serological assays for the detection of Mycobacterium bovis infection in common warthogs (Phacochoerus africanus).

Sporadic cases of bovine tuberculosis (bTB) have been reported in warthogs in Southern Africa and confirmed through mycobacterial culture. However, there are no validated ante-mortem tests currently available for bTB in warthogs. In this study, we evaluated the use of three serological assays for the detection of Mycobacterium bovis infection in warthogs; an indirect enzyme-linked immunosorbent assay (ELISA) using bovine purified protein derivative (PPDb) as a capture antigen (indirect PPD ELISA), as well as two commercial assays, the TB ELISA-VK® and DPP® VetTB Assay. Test performance of these assays was compared using sera from 35 warthogs of known Mycobacterium bovis infection status. All three assays were able to distinguish M. bovis-infected from uninfected individuals with high sensitivity (Se) and specificity (Sp) (indirect PPD ELISA Se: 88%, Sp: 89%; TB ELISA-VK® 88%, 79%; DPP® VetTB Assay 75%, 89%, respectively). The assays performed very similarly and the ELISA assays showed the greatest agreement (κ=0.89). These results indicate that M. bovis-infected warthogs develop measurable pathogen-specific humoral responses which can be used to distinguish them from uninfected animals. Therefore, serological assays have value as ante-mortem bTB diagnostic tests in warthogs.

Application of Rapid Serologic Tests for Detection of Mycobacterium bovis Infection in Free-Ranging Warthogs (Phacochoerus africanus)--Implications for Antemortem Disease Screening.

Warthogs (Phacochoerus africanus) have been implicated as potential maintenance hosts of Mycobacterium bovis. Our preliminary investigation of bovine tuberculosis in three warthogs describes pathologic findings and associated positive serologic results in two infected animals. This demonstrates the potential use of serodiagnostic tests for M. bovis infection in this species.

Serum iron and selected biochemical values in free-ranging black rhinoceros (Diceros bicornis) from South Africa.

Serum samples collected from 20 black rhinoceros (Diceros bicornis) were analyzed for iron values from six different areas in South Africa. In addition, biochemical profiles were performed on individual samples. Comparisons of iron values from free-ranging black rhinoceros and from 28 free-ranging white rhinoceros (Ceratotherium simum) were conducted by location and age. Among the free-ranging black rhinoceros, samples were compared from different regions to a set of samples from black rhinoceros that had been captured and held in bomas. Serum iron levels were not significantly different (P = 0.55) among the three locations with more than one animal (medians 5.57, 5.70, 6.47 ppm), but the median value from the boma group was significantly lower (2.91 ppm; P = 0.042), contrary to previous studies. Similar to reports in captive black rhinos, serum iron levels appeared to show a trend toward increasing values between subadult and adult animals, although differences were not statistically significant among black rhinoceros. Comparison of serum iron levels between free-ranging black and white rhinoceros showed significantly higher median value in black rhinoceros (5.73 ppm) versus white rhinoceros (3.38 ppm, P= 0.001). Other significant differences (P < 0.05) in biochemical values between species included lower median aspartate aminotransferase (37 versus 76.5 U/L), higher copper (1.50 versus 1.34 ppm), higher zinc (1.36 versus 0.37 ppm), lower total protein (8.0 versus 10.35 g/dL), higher gamma glutamyltransferase (13.0 versus 12.5 U/L), and lower globulin (6.6 versus 7.6 g/dL) in black rhinoceros. Further investigations should be conducted to examine the role of age, location, and time in boma confinement on iron values in South African rhinoceros to understand iron metabolism in these species.