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.

EVALUATION OF SERUM FERRITIN AND SERUM IRON IN FREE-RANGING BLACK RHINOCEROS (DICEROS BICORNIS) AS A TOOL TO UNDERSTAND FACTORS AFFECTING IRON-OVERLOAD DISORDER.

Iron overload disorder (IOD) is a significant health issue for captive black rhinoceros ( Diceros bicornis ). Measurement of serum ferritin with a validated rhinoceros ferritin ELISA has been used extensively to detect animals in U.S. zoos that are at risk of developing IOD. However, there is limited information on serum ferritin levels in free-ranging black rhinoceros using this same assay. Serum ferritin, iron, and gamma-glutamyl transpeptidase (GGT) were determined in 194 black rhinoceros from southern Africa. Mean ferritin in free-ranging black rhinoceros (290.54 ±247.4 ng/ml) was significantly higher than in free-ranging white rhinoceros (64.0 ± 102.4 ng/ml) sampled in this study from Kruger National Park, South Africa. However, there were no significant differences between genders or age groups. Ferritin values varied with geographical location of the black rhinoceros, although this was not clinically significant. Serum iron values were also higher in black rhinoceros (40.4 ± 19.1 µmol/L) compared to white rhinoceros (29.7 ± 10.7 µmol/L). There was no association between ferritin and GGT. This study provides serum ferritin, iron, and GGT values from free-ranging black rhinoceros that can be used for as comparative target values for captive animals.

EVALUATION OF CARDIORESPIRATORY, BLOOD GAS, AND LACTATE VALUES DURING EXTENDED IMMOBILIZATION OF WHITE RHINOCEROS (CERATOTHERIUM SIMUM).

Ten white rhinoceros (Ceratotherium simum) were immobilized for a total of 13 procedures in holding facilities in Kruger National Park using etorphine, azaperone, and hyaluronidase to assess the effect of extended immobilization on serial cardiorespiratory, blood gas, and lactate values. Butorphanol was administered intravenously following initial blood collection and physiologic assessment (t=0). Respiratory and cardiovascular parameters, body temperature, and arterial blood gases were monitored at 10-min intervals for a total of 100 min. Initial parameters at the time of recumbency revealed severe hypoxemia, hypercapnia, tachycardia, an increased alveolar-arterial (A-a) gradient, and mildly elevated lactate levels. At 10 min and 20 min, there were significant (P<0.05) changes in the following physiologic parameters: heart rate decreased [96 and 80 beats/min, respectively, vs. 120 beats/min], arterial partial pressure of oxygen (PaO2) increased [48 and 45 mm Hg, respectively vs. 30 mm Hg], arterial hemoglobin oxygen saturation increased [79% and 74%, respectively, vs. 47%], A-a gradient decreased [29.13 and 30.00 mm Hg, respectively, vs. 49.19 mm Hg], and respiratory rate decreased [5 and 5 breaths/min vs. 7 breaths/min]. Blood lactate levels also decreased from 2.54 mM/L to 1.50 and 0.89 mM/L, respectively. Despite initial improvements in blood oxygen levels at t=10 and 20 min, the rhinoceros remained severely hypoxemic for the remainder of the procedure (median PaO2=50.5 mm Hg, 95% confidence interval, 43.8-58.1). Median values for respiratory rate (5 breaths/min) and arterial partial pressure of carbon dioxide (PaCO2; 68.5 mm Hg) did not change significantly for the remaining 80 min. Median lactate, base excess, bicarbonate, and pH values improved between 20 and 100 min despite the persistent hypercapnia, indicating that the animals adequately compensated for respiratory and lactic acidosis. White rhinoceros were immobilized for 100 min with no negative effects, a desirable outcome if procedures require extended chemical immobilization without oxygen supplementation.

Use of butorphanol during immobilization of free-ranging white rhinoceros (Ceratotherium simum).

Forty free-ranging white rhinoceros (Ceratotherium simum) were anesthetized with etorphine, azaperone, and hyaluronidase in Kruger National Park, South Africa, between February and August 2009. Eighteen rhinoceros received butorphanol in the dart combination, and 22 rhinoceros had butorphanol administered intravenously within 15 min of darting. Body position, blood gas values, heart rate, respiratory rate, and temperature were measured at two time points after darting, approximately 10 min apart (sample 1 mean collection time after darting, 9.4 +/- 2.7 min; sample 2 mean collection time, 18.6 +/- 2.8 min). A significant number of field-captured rhinoceros remained standing at the first sample period when butorphanol was administered in the dart. Higher median values for arterial partial pressure of oxygen (PaO2) in combination with lower arterial partial pressure of carbon dioxide (PaCO2) in standing versus recumbent rhinoceros suggested improved ventilation in this posture (P < 0.05). When the effect of time, body position, and age was controlled, median values for respiratory rate, lactate, and pH were better in rhinoceros that received butorphanol in the dart (P < 0.05). There was also a trend toward higher median values for SO2 and bicarbonate in rhinoceros receiving butorphanol in the dart. Intravenous administration of butorphanol resulted in significantly decreased median PaCO2 and heart rate in recumbent rhinoceros (P < 0.05) without changes in PaO2 between sample periods 1 and 2. However, rhinoceros remained hypoxemic during the short anesthetic procedure despite butorphanol administration. Preliminary results suggest that administration of butorphanol (either in the dart or intravenously) improves some metabolic parameters in free-ranging recumbent white rhinoceros without significantly affecting ventilation. It is hypothesized that this may be due to a lighter state of immobilization. Addition of butorphanol to the dart provides handling and physiologic advantages because the majority of rhinoceros remain standing.

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.

Biochemical values in free-ranging white rhinoceros (Ceratotherium simum) in Kruger National Park, South Africa.

Biochemical panels were analyzed on 181 individual free-ranging white rhinoceros (Ceratotherium simum) from Kruger National Park, South Africa. These animals were immobilized between July 2006 and May 2010 for management purposes. Serum and heparinized plasma samples were analyzed using an in-house chemistry analyser (ABAXIS VetScan2). The objectives of this study were to establish biochemical references ranges for Kruger National Park's population of white rhinoceros; to assess differences in values obtained using sera or plasma; and to assess differences in values between gender and different age categories. Significant differences between plasma and serum values were found in most measured parameters except minerals (calcium and magnesium). Because all animals appeared clinically healthy at the time of blood collection, it is hypothesized that choice of anticoagulant may affect certain parameters. Comparison between age categories and gender also resulted in significant differences in a few measured parameters. Identifying differences are important when establishing baseline reference ranges for wildlife populations to allow accurate monitoring of trends that may change over time. The paucity of data on normal biochemical ranges for free-ranging white rhinoceros demonstrates the value of this study and importance of evaluating potential confounding variables.

Detection of antibodies to tuberculosis antigens in free-ranging lions (Panthera leo) infected with Mycobacterium bovis in Kruger National Park, South Africa.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become established in Kruger National Park, South Africa, in the cape buffalo (Syncerus caffer) population and in other species. TB in prey species has resulted in infection and morbidity in the resident lion (Panthera leo) prides. The only validated live animal test currently available for lions is the intradermal tuberculin test. Because this test requires capture twice, 72 hr apart, of free-ranging lions to read results, it is logistically difficult to administer in a large ecosystem. Therefore, development of a rapid animal-side screening assay would be ideal in providing information for wildlife managers, veterinarians, and researchers working with free-living lion prides. This study reports preliminary descriptive results from an ongoing project evaluating two serologic tests for M. bovis (ElephantTB Stat-Pak and dual path platform VetTB). Disease status was determined by postmortem culture and presence of pathologic lesions in 14 free-ranging lions. Seropositivity was found to be associated with M. bovis infection. Extended field studies are underway to validate these rapid animal-side immunoassays for antemortem screening tests for TB in lions.