Trypanosomiasis in Introduced Southern White Rhinoceros (Ceratotherium simum simum) Gifts to Ex Situ Habitat in Aitong, Kenya.

During the opening of diplomatic relations in the 1990s, South Africa gifted 20 southern white rhinoceros (Ceratotherium simum simum) to Kenya. The species is not indigenous to Kenya, and management of the introduction was not clearly addressed in the legislation. Responsibility was left to the private sector and local authorities. Ten of the animals were introduced to land contiguous with the Maasai Mara National Reserve, an area with tsetse-trypanosomiasis challenges, and with rare cases of human sleeping sickness. Mortalities had been previously documented when indigenous naïve black rhinoceros were introduced to areas with tsetse; hence there was no consensus on the management of this introduction. Feasibility was only explored once before with the introduction of two animals in a monitored and managed translocation from Lewa Downs, Laikipia in 1992-1994. Ultimately, Kenyan experts were co-opted to address risk after trypanosomiasis occurred in many animals. Unfortunately, this finding was followed by gradual mortalities of most rhinoceros with only a few being saved by removal to highland private sanctuaries. This event was complicated by many factors. Samples were only sporadically collected, and mainly from sick animals. With no clear responsibility by government agencies, a collaboration between veterinarians and researchers resulted in characterization of the disease challenge, and when invited, assessment of health status. Laboratory diagnostics revealed common and sometimes severe infections with Trypanosoma brucei, a normally infrequent trypanosome. Infection was associated with disturbances in erythropoiesis, especially anemia. Symptoms varied from sudden death associated with intestinal atony, to a semiparalyzed animal that was partially responsive to treatment for trypanosomes. This event should be used as a caution to future movements of this species that are planned or ongoing in Africa, for conservation or other purposes.

Vesicular stomatitis virus in two species of rhinoceros at a California zoological park.

OBJECTIVE: To describe an outbreak of vesicular stomatitis virus (VSV) in southern white rhinoceros (SWR; Ceratotherium simum simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) at a safari park in San Diego, CA, from May to September 2023. ANIMALS: 21 SWR and 5 GOHR in professionally managed care. METHODS: Rhinoceros of both species presented with a range of clinical signs and severities. Lesion locations were categorized as cutaneous (coronary bands, heels and soles, limbs, ventrum, neck folds, and ears) and mucocutaneous (lips, nostrils, mucous membranes of the oral cavity, and vulva). Clinical signs included lethargy, lameness, difficulty with prehension, hyporexia to anorexia, and hypersalivation. Severely affected rhinoceros had clinical pathology findings consistent with systemic inflammation. RESULTS: Vesicular stomatitis New Jersey virus was confirmed via PCR from swabs of lesions in 10/26 (38%) rhinoceros. Of these 10 confirmed cases, 9 (90%) were SWR and 1 (10%) was a GOHR. A further 6/26 (24%) were considered probable cases, and 10/26 (38%) were considered suspect cases based on clinical signs, but the inability to appropriately sample due to the housing environment precluded confirmation. Histopathology samples from 3 rhinoceros were consistent with VSV, and viral RNA was localized in histologic lesions via RNA in situ hybridization for 1 case. All rhinoceros survived infection despite severe systemic illness in 2 animals. CLINICAL RELEVANCE: This case series describes the clinical appearance and progression of VSV in 2 rhinoceros species. To the authors' knowledge, this is the first report of VSV in a rhinoceros.

Dehorning Does Not Alter the Stress Response in Southern White Rhinoceroses (Ceratotherium simum simum) during Transport: A Preliminary Investigation.

Translocation and dehorning are common and important practices for rhinoceros management and conservation. It is not known if dehorning causes a stress response or negatively affects rhinoceroses during transport. Twenty-three subadult wild Southern white rhinoceros (Ceratotherium simum simum) bulls were immobilized and translocated >280 km for population management reasons. Ten animals were dehorned at capture, and 13 animals were transported without dehorning. For transport, five dehorned and six nondehorned rhinoceroses were sedated with azaperone (62.38±9.54 µg/kg) and five dehorned and seven nondehorned rhinoceroses with midazolam (64.61±9.28 µg/kg). Blood samples were collected at capture, start of transport, and after 6 h of transport. Measurements included 10 physiologic variables: hematocrit, total serum protein, creatine kinase (CK), aspartate aminotransferase, gamma-glutamyl transferase (GGT), creatinine, urea, cholesterol, ß-hydroxybutyrate, and glucose; and four stress response variables: cortisol, epinephrine, neutrophil-to-lymphocyte ratio, and leukocyte coping capacity. Using a linear mixed model, CK and GGT were higher in dehorned compared with nondehorned rhinoceroses. There were no significant differences in the other variables between the two groups. The likely cause of these differences is that dehorned animals spent more time in the crate before the start of transport than nondehorned rhinoceroses (3:11±0:54 h vs. 1:12±0:56 h, P<0.001). These results indicate that dehorning does not negatively alter the white rhinoceros' physiologic and stress responses during translocation, supporting its use for antipoaching measures.

Effects of Butorphanol on Respiration in White Rhinoceros (Ceratotherium simum) Immobilized with Etorphine-Azaperone.

This article reports on respiratory function in white rhinoceros (Ceratotherium simum) immobilized with etorphine-azaperone and the changes induced by butorphanol administration as part of a multifaceted crossover study that also investigated the effects of etorphine or etorphine-butorphanol treatments. Six male white rhinoceros underwent two immobilizations by using 1) etorphine-azaperone and 2) etorphine-azaperone-butorphanol. Starting 10 min after recumbency, arterial blood gases, limb muscle tremors, expired minute ventilation, and respiratory rate were evaluated at 5-min intervals for 25 min. Alveolar to arterial oxygen gradient, expected respiratory minute volume, oxygen consumption, and carbon dioxide production were calculated. Etorphine-azaperone administration resulted in hypoxemia and hypercapnia, with increases in alveolar to arterial oxygen gradient, oxygen consumption, and carbon dioxide production, and a decrease in expired minute ventilation. Muscle tremors were also observed. Intravenous butorphanol administration in etorphine-azaperone-immobilized white rhinoceros resulted in less hypoxemia and hypercapnia; a decrease in oxygen consumption, carbon dioxide production, and expired minute ventilation; and no change in the alveolar to arterial oxygen gradient and rate of breathing. We show that the immobilization of white rhinoceros with etorphine-azaperone results in hypoxemia and hypercapnia and that the subsequent intravenous administration of butorphanol improves both arterial blood oxygen and carbon dioxide partial pressures.

High-Throughput Sequencing Reveals a Dynamic Bacterial Linkage between the Captive White Rhinoceros and Its Environment.

Soil is an essential part of the animal habitat and has a large diversity of microbiota, while the animal body was colonized by a complex bacterial community; so far, the relationship between the animal host microbial community and the soil microbial ecosystem remains largely unknown. In this study, 15 white rhinoceros from three different captive grounds were selected and the bacterial community of the gut, skin, and environment of these rhinoceros were analyzed by 16S rRNA sequencing technology. Our results showed that Firmicutes and Bacteroidota were the predominant phyla in the gut microbiome, whereas skin and environment samples share similar microbiome profiles and are dominated by the phyla of Actinobacteriota, Chloroflexi, and Proteobacteria. Although the bacterial composition of the gut differs from that of the skin and environment, the Venn diagrams showed that there were 22 phyla and 186 genera shared by all the gut, skin, and environmental microbes in white rhinoceroses. Further cooccurrence network analysis indicated a bacterial linkage based on a complex interaction was established by the bacterial communities from the three different niches. In addition, beta diversity and bacterial composition analysis showed that both the captive ground and host ages induced shifts in the microbial composition of white rhinoceroses, which suggested that the bacterial linkage between the captive white rhinoceros and its environment is dynamic. Overall, our data contribute to a better understanding of the bacterial community of the captive white rhinoceros, especially for the relationship between the environment and animal bacterial communities. IMPORTANCE The white rhinoceros is one of the world's most endangered mammals. The microbial population plays a key role in animal health and welfare; however, studies regarding the microbial communities of the white rhinoceros are relatively limited. As the white rhinoceros has a common behavior of mud baths and thus is in direct contact with the environment, a relationship between the animal microbial community and the soil microbial ecosystem appears possible, but it remains unclear. Here, we described the characteristics and interaction of bacterial communities of the white rhinoceros in three different niches, including gut, skin, and environment. We also analyzed the effects of captive ground and age on the composition of the bacterial community. Our findings highlighted the relationship among the three niches and may have important implications for the conservation and management of this threatened species.

Effects of age, seasonality, and reproductive status on the gut microbiome of Southern White Rhinoceros (Ceratotherium simum simum) at the North Carolina zoo.

BACKGROUND: Managed southern white rhinoceros (Ceratotherium simum simum) serve as assurance populations for wild conspecifics threatened by poaching and other anthropocentric effects, though many managed populations experience subfertility and reproductive failure. Gut microbiome and host health are inextricably linked, and reproductive outcomes in managed southern white rhinoceros may be mediated in part by their diet and gut microbial diversity. Thus, understanding microbial dynamics within managed populations may help improve conservation efforts. We characterized the taxonomic composition of the gut microbiome in the managed population of female southern white rhinoceros (n = 8) at the North Carolina Zoo and investigated the effects of seasonality (summer vs. winter) and age classes (juveniles (n = 2; 0-2 years), subadults (n = 2; 3-7 years), and adults (n = 4; >7 years)) on microbial richness and community structure. Collection of a fecal sample was attempted for each individual once per month from July-September 2020 and January-March 2021 resulting in a total of 41 samples analyzed. Microbial DNA was extracted and sequenced using the V3-V4 region of the 16S rRNA bacterial gene. Total operational taxonomic units (OTUs), alpha diversity (species richness, Shannon diversity), and beta diversity (Bray-Curtis dissimilarity, linear discriminant analysis effect size) indices were examined, and differentially enriched taxa were identified. RESULTS: There were differences (p < 0.05) in alpha and beta diversity indices across individuals, age groups, and sampling months. Subadult females had higher levels of Shannon diversity (Wilcoxon, p < 0.05) compared to adult females and harbored a community cluster distinct from both juveniles and adults. Samples collected during winter months (January-March 2021) possessed higher species richness and statistically distinct communities compared to summer months (July-September 2020) (PERMANOVA, p < 0.05). Reproductively active (n = 2) and currently nonreproductive adult females (n = 2) harbored differentially enriched taxa, with the gut microbiome of nonreproductive females significantly enriched (p = 0.001) in unclassified members of Mobiluncus, a genus which possesses species associated with poor reproductive outcomes in other animal species when identified in the cervicovaginal microbiome. CONCLUSION: Together, our results increase the understanding of age and season related microbial variation in southern white rhinoceros at the North Carolina Zoo and have identified a potential microbial biomarker for reproductive concern within managed female southern white rhinoceros.

EFFECT OF BOMA CONFINEMENT ON HEMATOLOGIC AND BIOCHEMICAL VALUES IN FREE-RANGING WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN KRUGER NATIONAL PARK, SOUTH AFRICA.

Boma adaptation is an important component of rhinoceros translocations to allow transition to new diets, restricted space, and quarantine for disease screening. However, up to 20% of recently captured white rhinoceros (Ceratotherium simum) do not adjust to captivity, resulting in early release or even death. The causes and physiologic consequences of maladaptation to boma confinement are poorly understood. The aim of this investigation was to evaluate hematologic and serum biochemical changes in maladapted rhinoceros compared to animals that adapted under the same boma conditions. Ninety-six white rhinoceros were captured between 2009 and 2011 in Kruger National Park, South Africa and placed in bomas prior to translocation. Weight, complete blood count, and serum biochemical panel results were recorded when rhinoceros were placed in the boma and repeated on the day of release. In this study, the mean duration of boma confinement for maladapted white rhinoceros was 13 d (range 8-16 d) compared to 89.9 d (range 39-187 d) for adapted animals. Mean weight loss between capture and release was significantly greater in maladapted rhinoceros (224.0 versus 65.9 kgs; P<0.001). Although adapted rhinoceros had statistically significant changes in some hematologic and biochemical values, most were not considered clinically relevant. In contrast, the maladapted rhinoceros had significant changes at the time of early release from the boma, including evidence of leukocytosis with left shift, lymphopenia, eosinopenia, decreased red blood cell count and hematocrit, increased serum creatine kinase, and decreased serum calcium, phosphorus, and magnesium values. Along with loss of body condition, these findings were consistent with a stress-associated catabolic response. These changes occurred in the first 2 wk of confinement, and the results provide a foundation for evaluating adaptation in white rhinoceros. Future studies should focus on factors that improve adaptation and welfare of recently confined free-ranging white rhinoceros.

EFFECT OF CAPTURE AND IMMOBILIZATION ON BOMA ADAPTATION IN FREE-RANGING WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN KRUGER NATIONAL PARK, SOUTH AFRICA.

Ninety-six white rhinoceros (Ceratotherium simum) were captured between February and October 2009-2011 in Kruger National Park, South Africa and placed in boma confinement before translocation. Of these, 19 rhinoceros did not adapt to the bomas and required early release (n=18) or died (n=1). The available immobilization data and physiologic parameters, including blood gas analyses, were compared between adapted and maladapted rhinoceros to determine whether predisposing causes could be identified. There were no statistical differences in age category, sex, or body weight at capture between adaptation cohorts. The recorded immobilization data, physiologic values, blood gas analytes, hematologic, or serum chemistry values were not statistically different between adapted and maladapted rhinoceros at capture, except maladapted rhinoceros had lower median serum aspartate aminotransferase, blood urea nitrogen, and phosphorus values; however, these statistically different values were not clinically important. Therefore, observable demographic or capture-related factors did not appear to predispose white rhinoceros to maladaptation to boma confinement. Further investigations into factors affecting adaptation should be performed to minimize the effect on rhinoceros health and welfare.

Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa.

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal - CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC2-MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC2-MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC2-MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC2-MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study-ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)-providing a template for the assessment of ART procedures in projects involving other endangered species.

Arterial Blood Gases and Cardiorespiratory Parameters in Etorphine-Medetomidine-Midazolam Immobilized Free-Ranging and Game-Farmed Southern White Rhinoceroses (Ceratotherium simum simum) Undergoing Electro-Ejaculation.

With the rapid loss of individuals in the wild, semen cryopreservation has gained importance to safeguard the genetic diversity of white rhinoceroses (Ceratotherium simum). For semen collection via electro-ejaculation, immobilization of free-ranging individuals requires the potent opioid etorphine, which is routinely combined with azaperone, but causes hypoxemia, hypercarbia, acidemia, muscle rigidity, tachycardia, and systemic hypertension. In this study, the suitability of two alternative immobilization protocols including etorphine, medetomidine, and midazolam at different doses (high vs. low etorphine) was evaluated in adult white rhinoceros bulls in two different management systems (free-ranging vs. game-farmed) and undergoing electro-ejaculation. Fourteen free-ranging (Group 1) and 28 game-farmed rhinoceroses (Group 2) were immobilized with ≈2.5 µg/kg etorphine (high dose), ≈2.5 µg/kg medetomidine, ≈25 µg/kg midazolam and 1,500-1,700 IU hyaluronidase and received ≈2.5 µg/kg of butorphanol intravenously at first handling. Twenty game-farmed animals (Group 3) received ≈1 µg/kg etorphine (low dose), ≈5 µg/kg medetomidine, ≈25 µg/kg midazolam and 1,700 IU hyaluronidase. Respiratory rate, heart rate and peripheral hemoglobin oxygen saturation (SpO2) were measured at 5-min intervals; non-invasive oscillometric blood pressures and arterial blood gases at first handling and before reversal of the immobilization; serum clinical chemistry analytes and hematocrit at first handling. Generalized mixed models (fixed factors: group, time, recumbency; random factor: individual rhinoceros) were applied to compare longitudinal changes between free-ranging and game-farmed rhinoceroses immobilized with the higher etorphine dose (Groups 1 and 2), and between the two protocols tested in the game-farmed rhinoceroses (Groups 2 and 3). All animals were successfully immobilized, presented with normal lactate concentrations (<5 mmol/L), experienced no muscle tremors and recovered uneventfully. Hypoxemia and hypertension persisted throughout the immobilization in all groups. Acidemia and hypercarbia were absent in Group 1, but present in the game-farmed animals. The lower etorphine dose in Group 3 resulted in significantly longer induction times, however, tachycardia was not observed. SpO2 was higher for sternal vs. lateral recumbency. Semen-rich fractions were recovered following electro-stimulation in 46 out of the 62 animals. Our findings suggest that etorphine-medetomidine-midazolam provides effective immobilization with fewer side effects compared to previous reports in white rhinoceroses and is suitable for successful electro-ejaculation.

Preliminary Findings on How Different Management Systems and Social Interactions Influence Fecal Glucocorticoid Metabolites in White Rhinoceros (Ceratotherium simum).

White rhinoceroses (Ceratotherium simum) are the most social and gregarious species of all rhinoceroses known worldwide. One of the most critical effects of elevated glucocorticoid concentrations, especially in threatened species, is its relation to chronic stress, which could potentially lead to immunosuppression and reduced reproductive activity. Our aim is to determine how different social environments and management systems might be influencing the secretion of fecal glucocorticoids in white rhinoceroses. We have analyzed the concentration of fecal glucocorticoid metabolites in 658 fecal samples from 14 white rhinoceroses, seven free-ranging rhinos, and seven rhinos from two different managed captive populations. HPLC techniques were used to determine the main glucocorticoid metabolite found in this species, and a competitive EIA was used to establish fecal 5α-pregnan-3 3ß, 11ß, 21-triol-20-oneglucocorticoid metabolite (fGCM) levels. Our results reveal that management systems and social interactions had an influence on fGCM levels, suggesting that the more restrictive the management and social conditions are, the higher the glucocorticoid concentrations that are found. Additionally, sex was also found to influence fGCM levels, as in females, fGCM concentrations were higher than in males. We conclude that the analysis of glucocorticoids in relation to other factors is a powerful tool to assess adrenocortical response in white rhinoceros in order to broaden the knowledge of their reproductive biology and improve the management of the species.

Muscle tremors observed in white rhinoceroses immobilised with either etorphine-azaperone or etorphine-midazolam: An initial study.

Etorphine-azaperone is the most commonly used drug combination for chemical immobilisation of free-ranging white rhinoceroses, but causes several profound physiological disturbances, including muscle tremors. The addition of benzodiazepine sedatives, such as midazolam, has been proposed to reduce the muscular rigidity and tremors in immobilised rhinoceroses. Twenty-three free-ranging, sub-adult white rhinoceros bulls were darted and captured using a combination of etorphine plus either azaperone or midazolam. Skeletal muscle tremors were visually evaluated and scored by an experienced veterinarian, and tremor scores and distance run were compared between groups using the Wilcoxon rank sum test. No statistical differences were observed in tremor scores (p = 0.435) or distance run (p = 0.711) between the two groups, and no correlation between these variables was detected (r = -0.628; p = 0.807). Etorphine-midazolam was as effective as etorphine-azaperone at immobilising rhinoceroses, with animals running similar distances. Although the addition of midazolam to the etorphine did not reduce tremor scores compared to azaperone, it might have other beneficial immobilising effects in rhinoceroses, and further investigation is necessary to elucidate possible methods of reducing muscle tremoring during chemical immobilisation of rhinoceroses.

Historical population declines prompted significant genomic erosion in the northern and southern white rhinoceros (Ceratotherium simum).

Large vertebrates are extremely sensitive to anthropogenic pressure, and their populations are declining fast. The white rhinoceros (Ceratotherium simum) is a paradigmatic case: this African megaherbivore has suffered a remarkable decline in the last 150 years due to human activities. Its subspecies, the northern (NWR) and the southern white rhinoceros (SWR), however, underwent opposite fates: the NWR vanished quickly, while the SWR recovered after the severe decline. Such demographic events are predicted to have an erosive effect at the genomic level, linked to the extirpation of diversity, and increased genetic drift and inbreeding. However, there is little empirical data available to directly reconstruct the subtleties of such processes in light of distinct demographic histories. Therefore, we generated a whole-genome, temporal data set consisting of 52 resequenced white rhinoceros genomes, representing both subspecies at two time windows: before and during/after the bottleneck. Our data reveal previously unknown population structure within both subspecies, as well as quantifiable genomic erosion. Genome-wide heterozygosity decreased significantly by 10% in the NWR and 36% in the SWR, and inbreeding coefficients rose significantly by 11% and 39%, respectively. Despite the remarkable loss of genomic diversity and recent inbreeding it suffered, the only surviving subspecies, the SWR, does not show a significant accumulation of genetic load compared to its historical counterpart. Our data provide empirical support for predictions about the genomic consequences of shrinking populations, and our findings have the potential to inform the conservation efforts of the remaining white rhinoceroses.

A Model for Dental Age Verification Using Ultrastructural Imaging for Modern and Fossil Representatives of the Rhinocerotidae Family.

The analyses were performed on a right third premolar (P3) of a white rhinoceros female (Ceratotherium simum, Burchell 1817). The specimen was born in captivity at London Zoo (Zoological Society of London), then in the 1970s transferred to Kiev Zoo (Peremohy Avenue), Ukraine, and was kept there until it died at a documented chronological age of 48 years. The female died because of its age, which indicates it was kept in good conditions adequate to the requirements of this species. Photographs and micrographs with radiological documentation were taken on the said tooth. Its structural characteristics were determined, and on the occlusal surface areas and points of anatomical constitution of its crown were identified. The tooth was also histologically evaluated via sections taken horizontally in a mesial-distal plane through the crown, horizontally in a mesial-distal plane through the coronal portion of the root, and longitudinally in a lingual-buccal plane through the crown and the root. Preparations with ground sections were made and observed in white, polarized, and reflected light. In the subsequent stage X-ray and SEM imaging has also been used, for analysis of the distribution of annual growth layers of mineralized dental tissues of cement and dentine, counted from the root canal center to the buccal surface. An attempt was also made to confirm the annual season in which the animal died, based on cement growth lines. It was observed that the growth lines were visible in all the analyzed sections, in dentine and cement. In the cement, the lines were relatively few and did not represent the attested age of the animal. The analysis of the coloration of the cement lines indicated that the animal was regularly fed a diet that was not seasonally differentiated. From the X-ray examination comes a conclusion that the animal did not suffer from periodontal diseases. Visible growth lines were observed on the dentine. On the horizontal section through the crown growth lines in the dentine were few and unclear. On the longitudinal section, both on the caudal and rostral roots, these lines were clearly visible and much more numerous than expected considering the known age of the animal, as more than 50 were counted. On horizontal sections through the upper part of both roots, distinct growth lines were observed in the dentine, and their number-48 for both roots-corresponded precisely to the age of the animal. The results of our study indicate that this method has significant potential for application to verify the age at death for modern and fossil representatives of rhinoceros.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project's partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

Cytokine biomarker discovery in the white rhinoceros (Ceratotherium simum).

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, disrupts conservation programs of threatened species such as the white rhinoceros (Ceratotherium simum). Interferon gamma release assays have been developed for the diagnosis of M. bovis infection in rhinoceros, however, the discovery of additional diagnostic biomarkers might improve the accuracy of case detection. The aim of this pilot study was therefore to evaluate a novel unbiased approach to candidate biomarker discovery and preliminary validation. Whole blood samples from twelve white rhinoceros were incubated in Nil and TB antigen tubes of the QuantiFERON® TB Gold (In-Tube) system after which RNA was extracted and reverse transcribed. Using the equine RT2 profiler PCR array, relative gene expression analysis of samples from two immune sensitized rhinoceros identified CCL4, CCL8, IL23A, LTA, NODAL, TNF, CSF3, CXCL10 and GPI as upregulated in response to antigen stimulation. Novel gene expression assays (GEAs) were designed for selected candidates, i.e. CCL4, CXCL10 and IFNG, and analysis of QFT-processed samples showed the CXCL10 GEA could distinguish between five M. bovis-infected and five uninfected rhinoceros. These findings confirm the value of the equine RT2 profiler PCR array as a useful tool for screening biomarkers for the diagnosis of M. bovis infection in rhinoceros.

Micro-anatomy of the ear of the southern white rhinoceros (Ceratotherium simum simum).

The white rhinoceros is the largest of the five extant rhinoceros species. The population is declining rapidly because of intense poaching. However, normal anatomical descriptions in this species are lacking. The purpose of this study is to describe the osseous anatomy of the middle and inner ear of the southern white rhinoceros using micro-focus X-ray computed tomography imaging. Four temporal bones obtained from two 1-day old southern white rhinoceros preserved in 10% formalin were scanned. Tri-dimensional reconstructions were obtained and volumes of the middle ear ossicles and inner ear structures were calculated. Excellent high spatial resolution 3D images were obtained for all samples and virtual models of the auditory ossicles and bony labyrinth were generated. Visualization of the tympanic membrane, middle ear and inner ear structures was possible in all samples. Whereas the stapes and incus had a shape similar to their human or equine counterparts, the malleus showed a unique appearance with a long rostral branch projecting latero-distally to the manubrium. The cochlea described 2 turns rostro-laterally around its axis, with a medial direction of rotation. However, identification of the soft tissue structures of the middle ear was sometimes difficult and visualization of the small structures of the membranous labyrinth was not possible using this formalin fixation and alternative techniques should be investigated. Further investigations are needed in order to provide a complete virtual model including both soft and bone tissues of this difficultly accessible region.

Releasing Three Orphaned White Rhinoceroses (Ceratotherium simum) to the Game Reserve in South Africa. Rehabilitation, Translocation and Post-Release Observations.

White rhinoceros (Ceratotherium simum) is one of the most famous victims of poachers in Africa. One of the methods for dealing with decreasing rhino numbers is rehabilitating wounded and/or orphaned animals to successfully release them back into the wild. The status of rescued animal differs among individuals, but general procedures must be established and constantly improved. This study presents the history of successful release of three orphaned white rhino females; rehabilitated for 15 months in Wildlife Rehabilitation Centre in a private game reserve in South Africa. Female A was three years old, female B was one year old and the youngest female was three months old on arrival. The animals were rehabilitated together despite the differences in their age and size, with particular attention paid to keeping them as wild as possible. After being weaned and becoming old enough to go back to the wild, they were released at a distance from the rehabilitation centre, which required immobilization and translocation. Since the rhinos were released, they have been successfully living in the wild. All procedures used in this study proved to be sufficient for preparing the animals for life in the wild and can be recommended for other centres.

The VetMAX™ M. tuberculosis complex PCR kit detects MTBC DNA in antemortem and postmortem samples from white rhinoceros (Ceratotherium simum), African elephants (Loxodonta africana) and African buffaloes (Syncerus caffer).

BACKGROUND: Bovine tuberculosis and tuberculosis are chronic infectious diseases caused by the Mycobacterium tuberculosis complex members, Mycobacterium bovis and Mycobacterium tuberculosis, respectively. Infection with M. bovis and M. tuberculosis have significant implications for wildlife species management, public health, veterinary disease control, and conservation endeavours. RESULTS: Here we describe the first use of the VetMAX™ Mycobacterium tuberculosis complex (MTBC) DNA quantitative real-time polymerase chain reaction (qPCR) detection kit for African wildlife samples. DNA was extracted from tissues harvested from 48 African buffaloes and MTBC DNA was detected (test-positive) in all 26 M. bovis culture-confirmed animals with an additional 12 PCR-positive results in culture-negative buffaloes (originating from an exposed population). Of six MTBC-infected African rhinoceros tested, MTBC DNA was detected in antemortem and postmortem samples from five animals. The PCR was also able to detect MTBC DNA in samples from two African elephants confirmed to have M. bovis and M. tuberculosis infections (one each). Culture-confirmed uninfected rhinoceros and elephants' samples tested negative in the PCR assay. CONCLUSIONS: These results suggest this new detection kit is a sensitive screening test for the detection of MTBC-infected African buffaloes, African elephants and white rhinoceros.