Quantification of Enterobacteriaceae in faeces of captive black rhinoceros (Diceros bicornis) in relation to dietary tannin supplementation

Free-ranging browsing herbivores ingest a range of secondary plant compounds, such as tannins, with their natural diet. As many of these substances have been shown to have antibacterial properties, it could be speculated that a lack of such compounds in captive zoo diets could favour the growth of potentially pathogenic intestinal bacteria. The effect of a supplementation of a conventional diet (N, consisting mainly of grass hay and/or lucerne hay and pelleted compound feeds) fed to eight captive black rhinoceroses (Diceros bicornis) from three zoological institutions with either tannic acid (T), a source of hydrolysable tannins, or quebracho (Q), a source of condensed tannins, was investigated. The number of faecal colony forming units (CFU) of Enterobactericeae was determined by colony count of dilution series from fresh faeces applied to MacConkey agar plates. Tannins were added to the diets at approximately 5-15 g/kg dry matter, depending on the varying intake of roughage and compound feeds by the animals. There was no difference in the number of CFU between diets N (95.0 x 10(5) +/- 225.3 x 10(5)/g fresh faeces) and T (164.3 x 10(5) +/- 225.1 x 10(5)/g fresh faeces); in contrast, diet Q led to a significant reduction in CFU (4.3 x 10(5) +/- 6.5 x 10(5)/g fresh faeces) compared with the other diets. These findings suggest that condensed tannins could have the potential to reduce the number of potentially pathogenic intestinal bacteria, and that the deliberate inclusion of tannin sources in the diets of captive wild animals should be further investigated. The fact that tannic acid, shown to have antibacterial effects in various in vitro studies, did not have an effect in this study, emphasizes that the relevance of tannin supplementation for intestinal health must be verified in vivo.     

The influence of dietary tannin supplementation on digestive performance in captive black rhinoceros (Diceros bicornis)

Free-ranging browsers such as the black rhinoceros (Diceros bicornis) consume a diet that contains tannins, whereas the diets offered to them in captivity consist mostly of items known to contain hardly any such secondary plant compounds. Tannins could have potentially beneficial effects, including the chelation of dietary iron (iron storage disease is a common problem in black rhinos). Here, we tested the acceptance, and the consequences on digestion variables, of a low-dose tannin supplementation in captive animals. Eight black rhinoceroses from three zoological institutions were used. Faecal output was quantified by total faecal collection. Diets fed were regular zoo diets supplemented with either tannic acid (T, hydrolysable tannin) or quebracho (Q, condensed tannins); overall tannin source intake increased at 5-15 g/kg dry matter (DM) in relation to regular zoo diets. Adaptation periods to the new diets were >2 months. Additional data were taken from one hitherto unpublished study. Data were compared to measurements in the same animals on their regular zoo diets. All animals accepted the new diets without hesitation. There was no influence of tannin supplementation on digestion coefficients of DM and its constituents, or faecal concentrations of short-chain fatty acids or lactate. Water intake did not increase during tannin supplementation. Should the inclusion of dietary tannin sources be an objective in the development of diets for captive rhinoceroses, moderate doses such as used in this study are unlikely to cause relevant depressions of digestive efficiency and will not interfere with bacterial fermentation in a relevant way.     

Fatty acid status in captive and free-ranging black rhinoceroses (Diceros bicornis)

The fatty acid (FA) patterns of plasma/serum triglycerides (TG), phospholipids (PL) and cholesteryl esters (CE) of captive and free-ranging black rhinoceroses (Diceros bicornis) were investigated. Free-ranging animals (n = 28) stemmed from four different regions. Captive animals sampled included specimens from North American (n = 11) and three different European facilities (n = 6). The European animals were tested on 1-4 different diets, resulting in a total of 15 blood samples. Regardless of differences between the free-ranging animals from different regions, differences between captive and free-ranging animals were relatively uniform: captive animals had higher overall proportions of polyunsaturated fatty acid (PUFA), due to levels of linoleic acid (LA, 18:2n6) that were drastically increased as compared to free-ranging animals. In contrast, levels of alpha-linolenic acid (ALA, 18:3n3) were consistently lower on conventional zoo diets. n6/n3 ratios for TG, PL and CE were 1.6, 10 and 8 in samples from free-ranging animals, respectively, as compared to 4.1-16.3, 16-148 and 40-277 in samples from captive animals. There was a distinct correlation between the proportion of grain-based products (commercial concentrates, plain grains and bread) in the diets of the European animals and the measured levels of n6 PUFA. An animal from a facility with a very low proportion of grain products in the diet nevertheless had high LA readings, most probably due to the use of sunflower oil as 2% (dry matter basis) of its diet. One animal that received a high proportion of grass meal pellets due to an oral disease had increased ALA contents after the diet change. These results allow conclusions on the suitability of diets fed in captivity: the black rhinoceros is prone to several uncommon diseases that have been suspected to be linked to oxidative damage, possibly due to the disposition of this species to excessive iron storage. An unnatural dietary loading with PUFAs would exacerbate this problem. Additionally, n6 FAs are known as precursors of pro-inflammatory mediators, and their overrepresentation could therefore exacerbate any inflammatory processes. Therefore, the current practice of using grain-based feeds as major ingredients in captive rhinoceros diets is discouraged. Diet items containing ALA (a precursor of anti-inflammatory mediators) such as, fresh grass, fresh browse, the respective silages should be included at higher levels in diets for captive black rhinoceroses. Grass meal pellets, although a good source of ALA and linked with high levels of ALA in an animal of this study, must be chosen with care for black rhinoceroses due to their particular proneness for high iron contents.     

Overdose during chemical restraint in a black rhinoceros (Diceros bicornis)

A juvenile female black rhinoceros (Diceros bicornis) was successfully treated after overdose of drugs used for chemical restraint. Subsequent general anaesthesia for surgical reduction of a recurrent rectal prolapse was uneventful. Over a 25-minute period before transportation to the veterinary hospital, the animal received a total dose of 1.225 mg etorphine, 30 mg acepromazine and 30 mg detomidine. Based on an estimated mass of 200 kg, these corresponded to doses of 6.1 microg kg(-1) etorphine, 150 microg kg(-1) acepromazine, and 150 microg kg(-1) detomidine which constitutes considerable overdose for each drug given separately, notwithstanding the synergy that probably resulted when the three drugs were present concurrently. The estimated body mass may have substantially overestimated the actual body mass and exacerbated overdosage. The animal was recumbent and apnoeic on arrival at the hospital. Heart sounds were auscultated and a weak peripheral pulse was palpated; no pulse deficits were detected, although the heart rate was low. The trachea was intubated, inspired breath was enriched with oxygen and the lungs ventilated manually. Diprenorphine (1.5 mg) was given intravenously and spontaneous breathing resumed 11 minutes later. After induction of general anaesthesia using isoflurane, emergency surgery for correction of rectal prolapse was performed, from which the animal recovered uneventfully. The case highlights some of the practical problems that may be encountered in dealing with dangerous and unfamiliar species.     

Salmonella surveillance in a herd of asymptomatic captive black rhinoceros (Diceros bicornis) using fecal culture and PCR.

Feces were collected from captive black rhinoceros (Diceros bicornis minor) housed at Disney's Animal Kingdom to examine the frequency of Salmonella spp. shedding in asymptomatic animals using enrichment culture and broth culture- polymerase-chain-reaction (PCR) for detection. Three samples per animal were collected during the first week of each month between February 2001 and December 2003. During the study period, six different individual animals from one herd participated in the study, including two growing calves. A total of 550 cultures, using duplicate samples at two different laboratories, and 464 PCR tests were performed. When culture and PCR results were compared by the same laboratory, similar herd prevalence was found (2.4% positive cultures compared with 2.6% positive PCR tests). However, even though tests were performed on replicate samples, not every sample that was positive by culture was positive by PCR and vice versa. These results suggest that using multiple diagnostic methods and increasing the number of samples submitted may increase the likelihood of finding an asymptomatic Salmonella shedder. Although all of the rhinos shared the same environment throughout the study period, only four out of the six animals tested shed Salmonella spp. even though a minimum of 37 fecal samples were taken from each of the negative animals. Although this study followed a small number of rhinoceros, it suggests that asymptomatic shedding probably occurs more frequently in captive black rhinoceros than was previously believed. The prevalence appears to be similar to that reported for domestic ungulates.     

Morphology of the dorsal lingual papillae in the black rhinoceros (Diceros bicornis)

The dorsal lingual surface of a black rhinoceros (Diceros bicornis) was examined by scanning electron microscopy. The tongue was about 30 cm in length. There were about 60 vallate papillae on both sides. Filiform, fungiform and vallate papillae were found. The filiform papillae were distributed over the entire dorsal surface of the tongue. The papillae had a hair-like shape. The fungiform papillae were round in shape, and more densely distributed on the lingual apex. No foliate papillae were seen on the dorsal surface. The vallate papillae were located on both sides of the posterior end of the lingual body. Each papilla was surrounded by a groove.     

Globular periodontal cement dysplasia in a black rhinoceros (Diceros bicornis)]

A globular periodontal cementous dysplasia in a 18 years old black rhinoceros (Diceros bicornis) is diagnosed by gross pathology, X-ray, and by histological examinations. The findings are discussed.     

The importation of the black rhinoceros (Diceros bicornis) from Zimbabwe into Australia

SUMMARY The paper describes a program to import and breed black rhinoceros ex situ at Western Plains Zoo in Australia. Nine rhinoceros (7 females and 2 males) captured in 1992 in Chete National Park, Zimbabwe, were transported to Australia via Cocos Island. The veterinary treatment of the animals before and during quarantine in Zimbabwe and on Cocos (Keeling) Islands is described. Three animals died; an adult male on Cocos Islands and a juvenile male and an adult female at Western Plains Zoo, Dubbo, New South Wales. The juvenile male died as a result of trauma sustained shortly after arrival and the two adults after developing a severe hepatopathy. The group of 6 females and an additional 4 males imported from the USA in 1994 have adapted well to captivity and to the climate and environment of central west New South Wales.     

Urinalysis in three species of captive rhinoceros (Rhinoceros unicornis, Dicerorhinus sumatrensis, and Diceros bicornis)

This study reports urinalysis values for three species of captive rhinoceros (Rhinoceros unicornis, Dicerorhinus sumatrensis, and Diceros bicornis) and evaluates individual and species differences. Repeated urinalysis was conducted on 11 individuals to establish normal reference ranges. Although no individual or species differences existed in urinary values for pH, all species differed in specific gravity. Rhinoceros urine demonstrated many physical and chemical properties similar to that of the horse, but reliability of this comparison was limited. Urinary pH in the rhinoceros was within range of that established for the horse and other large herbivores. However, all rhinoceros species exhibited urinary specific gravities below the lower limit of the normal equine reference range. Comparative urinalysis using an outside laboratory source confirmed the results of this study and illustrated the value of conducting in-house analysis. These results are the first data available on reference ranges for urine parameters in the greater one-horned, Sumatran, and African black rhinoceros and provide a useful diagnostic tool for the veterinary care of individuals in captivity.     

Mineral absorption in the black rhinoceros (Diceros bicornis) as compared with the domestic horse

To test whether mineral recommendations for horses are likely to guarantee adequate mineral provision for black rhinoceroses (Diceros bicornis), we investigated the apparent absorption (aA) of macro- and microminerals in eight black rhinoceroses from three zoological institutions in a total of 32 feeding trials with total faecal collection, with additional data from three unpublished studies (18 feeding trials). Feeds and faeces were analysed for Ca, P, Mg, Na, K, Fe, Mn, Cu, Zn and Co. The resulting aA coefficients, and the linear relationships of apparently absorbable dietary mineral content to total dietary mineral content [per 100 g dry matter (DM)], were compared with data for domestic horses. Rhinoceroses had significantly higher aA coefficients for Ca and Mg (because of a higher calculated 'true' absorption), and lower ones for Na and K (because of calculated higher endogenous faecal losses). High absorption efficiency for divalent cations is hypothesized to be an adaptation to a natural diet of particularly high Ca:P ratio (approximately 14:1); an effective removal of Ca from the ingesta guarantees sufficient P availability at the fermentation site in the hindgut. Higher faecal losses of Na and K are hypothesized to be linked to a higher faecal bulk per DM intake in black rhinoceroses as compared with horses because of a generally lower digestive efficiency. There were no relevant differences in the absorption patterns of microminerals. In particular, there were no discernable differences in Fe absorption within the rhinoceroses for diets with and without tannin supplementation. Several of the zoo diets assessed in this study were deficient in Cu, Mn or Zn, and most contained excessive levels of Fe when compared with horse requirements. The findings of this study indicate that differences in mineral absorption between occur even between species of similar digestive anatomy; that in particular, Ca absorption might vary between hindgut fermenters with Ca:P ratio in their natural diet; that Na might be a particularly limiting factor in the ecology of free-ranging rhinoceroses; that moderate doses of tannins do not seem to markedly influence mineral absorption; and that diets for captive animals should contain adequate, but not excessive mineral levels.     

Acute lymphoblastic leukemia in a juvenile southern black rhinoceros (Diceros bicornis minor).

A 21-mo-old female southern black rhinoceros (Diceros bicornis minor) developed acute upper respiratory dyspnea in association with lymphadenopathy and marked immature lymphocytosis. A diagnosis of acute lymphoblastic leukemia was reached on the basis of the morphologic and cytochemical characteristics of peripheral lymphoblasts. Antineoplastic chemotherapy included administration of cytarabine, cyclophosphamide, vincristine, and doxorubicin, with clinical remission achieved 19 days after initiation of treatment. The rhinoceros died, however, of congestive heart failure, presumably secondary to doxorubicin cardiotoxicity and a particular sensitivity of rhinoceros myocardial tissue to free hydroxyl radicals. The pharmacologic effects of any therapeutic agent need to be carefully considered before use in the black rhinoceros, especially within the context of the unique physiology of this species.     

Idiopathic distal esophageal dilation in a southern black rhinoceros (Diceros bicornis minor).

An adult female southern black rhinoceros (Diceros bicornis minor) experienced intermittent periods of regurgitation while eating, suggesting an esophageal disorder. Endoscopy for evaluation of the trachea, esophagus, and stomach revealed a 30-mm nasopharyngeal orifice with associated recess located in the caudodorsal pharynx and a 10-cm dilated segment of the distal esophagus that was presumably the cause of regurgitation. Dietary management of esophageal dilation through short-term utilization of a "soft feed" program successfully eliminated the regurgitation. This is the first report of esophageal dysfunction in a rhinoceros.     

Effects of translocation on hematologic parameters of free-ranging black rhinoceros (Diceros bicornis michaeli) in Kenya.

Management of the endangered black rhinoceros (Diceros bicornis michaeli) in Africa frequently involves translocation. These procedures are not without risk, and protocols must be critically examined. Hematologic analyses can be used to evaluate the effects of translocation on animal health. Hematologic data obtained during routine translocation of free ranging black rhinoceros (n = 74) in Kenya between 1991 and 1995 were examined, and subsets of data from rhinoceros (n = 43) that were translocated to different regions of Kenya were compared. All animals showed an increase in total blood protein. Animals transported for longer periods and to lower altitude zones with higher ambient temperatures and trypanosomiasis developed anemia and showed neutrophilia, lymphopenia (males), and eosinopenia. The changes in packed cell volume (PCV), hemaglobin, and neutrophils were more marked in females, and the PCV drop was more significant in subadults. The red cell changes were most probably pathologic, involving the loss of red cells from circulation through sequestration or hemorrhage. The changes in white cell parameters are consistent with the effect of endogenous corticosteroids as a result of stress. Transport and confinement stress might lead to gastric ulceration with hemorrhage. In many animals, exposure to trypanosomes contributes to anemia.     

Serial temperature monitoring and comparison of rectal and muscle temperatures in immobilized free-ranging black rhinoceros (Diceros bicornis)

Control of body temperature is critical to a successful anesthetic outcome, particularly during field immobilization of wild animals. Hyperthermia associated with exertion can lead to serious and potentially life-threatening complications such as organ damage (including myopathy) and death. Methods for monitoring core body temperature must accurately reflect the physiologic status of the animal in order for interventions to be effective. The goal of this preliminary study was to compare serial rectal and muscle temperatures in field-immobilized black rhinoceros (Diceros bicornis) and evaluate a possible association. Twenty-four free-ranging black rhinoceros were immobilized between February and March of 2010 in Ethosha National Park, Namibia. Pairwise comparisons showed a correlation of 0.73 (95% CI; 0.70-0.75) between rectal and muscle temperature measurements. Results from a multivariable model indicate that muscle temperature readings were, on average, 0.46 degrees C (95% CI; 0.36-0.57 degrees C) higher than rectal temperatures while adjusting for repeated measurements on the same rhinoceros, effect of duration of immobilization, and effect of ambient temperature on rhinoceroses' temperature readings. As immobilization time increased, muscle and rectal temperature values within an individual rhinoceros tended to equilibrate. The overall temperatures decreased by an average of 0.00059 degrees C/min (95% CI; -0.0047 to -0.0035 degrees C/min; P = 0.779). As the ambient temperature at time of immobilization increased by 1 degree C, the average rhinoceros temperature increased by 0.09 degrees C (95% CI; 0.06-0.11 degrees C, P < 0.0001). Higher body temperature creates a potential for cellular damage leading to complications that include myopathy. Methods for monitoring rectal, muscle, and ambient temperatures should be incorporated into anesthetic monitoring protocols for large ungulates, particularly under field conditions.     

Digestion coefficients achieved by the black rhinoceros (Diceros bicornis), a large browsing hindgut fermenter

In contrast to the grazing white (C. simum) and Indian (R. unicornis) rhinoceros, the black rhinoceros (D. bicornis) is an exclusive browser. Due to the particular fermentation characteristics of browse, one would expect browsers to display both shorter ingesta retention times and lower digestion coefficients on comparable diets than grazers. In order to generate a database to test this hypothesis, we performed digestibility studies in eight black rhinoceroses (D. bicornis) from three zoological institutions, using total faecal collection for the quantification of faecal output. One to three regularly fed zoo rations of roughage, concentrates and varying proportions of browse material were used per animal. Additional data was taken from three hitherto unpublished studies as well as several published sources. When compared with horses on similar rations, black rhinoceroses achieved lower digestion coefficients for organic matter and CF. In general, an increase in dietary CF content led to a steeper decrease in organic matter and GE digestibility in black rhinoceroses than in horses. When comparing available data for rhinoceroses, browsing species showed a steeper decrease in organic matter digestibility than grazing species with increasing dietary cell wall content. Endogenous losses as determined by linear regression analysis were within the range reported for horses and Indian rhinoceroses. The results suggest that the horse is not a useful model animal for evaluating diets for black rhinoceroses energetically. In general, diets fed to captive black rhinoceroses seem to include higher proportions of concentrates than diets for other rhinoceros species, and an increase in browse or roughage would reduce digestion coefficients to levels observed in animals fed natural forage.     

Remote assessment of stress in white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) by measurement of adrenal steroids in feces.

This study monitored fecal cortisol and corticosterone levels in 14 black rhinoceroses (Diceros bicornis) and in seven white rhinoceroses (Certotherium simum) under various conditions of captivity, including translocation. Free cortisol and free corticosterone were measured in methylene chloride extracts of feces, using high-performance liquid chromatography (HPLC). The extraction-assay method was validated for quantitative measurement of these hormones by mass spectroscopy analysis, chemical derivitization, and radiolabel tracking and recovery. Both cortisol and corticosterone were extractable from feces and routinely detectable by HPLC. In three nonstressed, captivity-adapted white rhinoceroses monitored across 21 days of routine activity, fecal cortisol ranged from 2.0 to 7.3 ng/g dry feces and corticosterone from 4.0 to 10.8 ng/g dry feces, with no observable trend. Matched plasma, urine, and fecal samples in these rhinoceroses yielded corticosterone:cortisol ratios of 2.0:1.0, 2.7:1.0, and 2.2:1.0, respectively. Both black rhinoceroses (n = 5) and white rhinoceroses (n = 4) exhibited higher fecal cortisol (6.9- to 10.0-fold) and corticosterone (3.2- to 4.5-fold) levels in association with restraint-translocation than in limited free-roaming conditions. In five black rhinoceroses monitored across 6 wk after release from translocation, fecal levels of both cortisol and corticosterone decreased significantly between week 1 and weeks 4-6. In general, cortisol and corticosterone paralleled each other, with cortisol exhibiting a greater range of response. Measurement of either hormone in feces appears to be reliable for adrenal axis monitoring in the white and the black rhinoceroses.     

Effect of venipuncture site and anticoagulant on selected hematologic values in black rhinoceros (Diceros bicornis).

Paired blood samples were collected from the ear and radial vein of four captive healthy adult black rhinoceroses (Diceros bicornis). Samples were collected using heparin or ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Packed cell volume (PCV) and total protein (TP) values were compared between samples drawn from the two venipuncture sites and treated with the two anticoagulants to determine whether statistically significant variation occurred. No significant difference in the grouped values was observed when venipuncture sites (ear and radial vein) were compared using the same anticoagulant (heparin). However, when comparing different anticoagulants (EDTA and heparin) used to collect blood from the radial vein, the grouped-heparinized samples had higher mean PCV and TP values than did the EDTA-treated samples. These differences may be important when performing serial sampling in a sick rhinoceros and suggest that the choice of anticoagulant should be consistent, although selection of venipuncture site may be less important when monitoring selected hematologic values in black rhinoceroses.     

The correct name of the South-central black rhinoceros is Diceros bicornis keitloa (A.Smith, 1836)

In South Africa, the black rhinoceros (Diceros bicornis) is divided into two subspecies, the South-western in the west and the South-central in the east. The exact boundary between the ranges of these subspecies is uncertain, but has been defined to coincide with the administrative border between the Northern Cape and North West provinces. It is current practice to refer to the South-central black rhinoceros as Diceros bicornis minor, which has Zululand as the type-locality. This needs adjustment, because an earlier valid scientific name was given to a rhinoceros killed near Zeerust in the western part of North West province. In line with the rules of zoological nomenclature, the South-central black rhinoceros should be known as Diceros bicornis keitloa.     

Seminoma in a southern black rhinoceros (Diceros bicornis minor): diagnosis, surgical management and effect on fertility

A testicular mass was identified by ultrasonography performed during a routine reproductive evaluation of an adult male southern black rhinoceros ( Diceros bicornis minor ). Histological examination of a testicular biopsy supported a presumptive diagnosis of testicular neoplasia. Hemi-castration was performed to excise the affected testis and a pathological diagnosis of a seminoma was made. Assessment of semen suggested reduced fertility as a consequence of the neoplastic process, but hemi-castration prevented further growth and metastasis of the tumour and ensured the animal's breeding potential. This is the second documented case of a seminoma in a rhinoceros species and the first case in a black rhinoceros.