Sub-lethal impacts of lead poisoning on blood biochemistry, immune function and delta-aminolevulinic acid dehydratase (δ-ALAD) activity in Cape (Gyps coprotheres) and white-backed (G. africanus) Vulture chicks.

Although the prevalence of lead poisoning in southern Africa's Gyps vultures is now well-established, its finer physiological effects on these endangered species remain poorly characterised. We evaluated the sub-lethal impact of acute lead exposure on Cape and White-backed Vulture chicks from two breeding colonies in South Africa, by analysing its possible effects on key blood biochemistry parameters, immune function, packed cell volume and δ-aminolevulinic acid dehydratase (δ-ALAD) activity. All 37 White-backed Vulture nestlings sampled displayed elevated lead levels (>10 µg/dL), and seven had blood [Pb] >100 µg/dL. Eight of 28 Cape Vulture nestlings sampled had blood [Pb] exceeding background exposure, with one showing blood [Pb] >100 µg/dL. Delta-aminolevulinic acid dehydratase (δ-ALAD) activity was significantly and negatively related to blood [Pb] in nestlings from both species, with 50% inhibition of the enzyme predicted to occur at blood [Pb] = 52.8 µg/dL (White-backed Vulture) and 18.8 µg/dL (Cape Vulture). Although no significant relationship was found between % packed cell volume (PCV) and blood [Pb], the relatively lower mean PCV of 32.9% in White-backed Vulture chicks, combined with normal serum protein values, is likely indicative of depression or haemolytic anaemia. The leukogram was consistent in both species, although the presence of immature heterophils suggested an inflammatory response in White-backed Vulture chicks with blood [Pb] >100 µg/dL. Values for cholesterol, triglycerides, total serum protein, albumin, globulin, albumin/globulin ratio, alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) were consistent with values previously reported. Calcium and phosphorus concentrations suggested no adverse effects on bone metabolism. A significant decrease in urea: uric acid (U:UA) ratio at blood [Pb] >100 µg/dL in White-backed Vulture chicks, brought about by a decrease in urea production, raises the possibility of hepatic abnormality. These results suggest that δ-ALAD activity may serve as a sensitive biomarker of lead toxicity in both species, while highlighting the need to better understand the significant variability in sensitivity that is observed, even between closely related members of the same genus.

Markers of inflammation in free-living African elephants (Loxodonta africana): Reference intervals and diagnostic performance of acute phase reactants.

INTRODUCTION: Acute phase reactants (APRs) have not been investigated in free-living African elephants (Loxodonta africana), and there is little information about negative APRs albumin and serum iron in elephants. OBJECTIVES: We aimed to generate reference intervals (RIs) for APRs for free-living African elephants, and to determine the diagnostic performance of APRs in apparently healthy elephants and elephants with inflammatory lesions. METHODS: Stored serum samples from 49 apparently healthy and 16 injured free-living elephants were used. The following APRs and methods were included: albumin, bromocresol green; haptoglobin, colorimetric assay; serum amyloid A (SAA), multispecies immunoturbidometric assay, and serum iron with ferrozine method. Reference intervals were generated using the nonparametric method. Indices of diagnostic accuracy were determined by receiver-operator characteristic (ROC) curve analysis. RESULTS: Reference intervals were: albumin 41-55 g/L, haptoglobin 0.16-3.51 g/L, SAA < 10 mg/L, and serum iron 8.60-16.99 µmol/L. Serum iron and albumin concentrations were lower and haptoglobin and SAA concentrations were higher in the injured group. Serum iron had the best ability to predict health or inflammation, followed by haptoglobin, SAA, and albumin, with the area under the ROC curve ranging from 0.88-0.93. CONCLUSIONS: SAA concentrations were lower in healthy African vs Asian elephants, and species-specific RIs should be used. Serum iron was determined to be a diagnostically useful negative APR which should be added to APR panels for elephants.

Reference Intervals for Hematology and Clinical Chemistry for the African Elephant (Loxodonta africana).

The African elephant (Loxodonta africana) is listed as vulnerable, with wild populations threatened by habitat loss and poaching. Clinical pathology is used to detect and monitor disease and injury, however existing reference interval (RI) studies for this species have been performed with outdated analytical methods, small sample sizes or using only managed animals. The aim of this study was to generate hematology and clinical chemistry RIs, using samples from the free-ranging elephant population in the Kruger National Park, South Africa. Hematology RIs were derived from EDTA whole blood samples automatically analyzed (n = 23); manual PCV measured from 48 samples; and differential cell count results (n = 51) were included. Clinical chemistry RIs were generated from the results of automated analyzers on stored serum samples (n = 50). Reference intervals were generated according to American Society for Veterinary Clinical Pathology guidelines with a strict exclusion of outliers. Hematology RIs were: PCV 34-49%, RBC 2.80-3.96 × 1012/L, HGB 116-163 g/L, MCV 112-134 fL, MCH 35.5-45.2 pg, MCHC 314-364 g/L, PLT 182-386 × 109/L, WBC 7.5-15.2 × 109/L, segmented heterophils 1.5-4.0 × 109/L, band heterophils 0.0-0.2 × 109/L, total monocytes 3.6-7.6 × 109/L (means for "regular" were 35.2%, bilobed 8.6%, round 3.9% of total leukocytes), lymphocytes 1.1-5.5 × 109/L, eosinophils 0.0-0.9 × 109/L, basophils 0.0-0.1 × 109/L. Clinical chemistry RIs were: albumin 41-55 g/L, ALP 30-122 U/L, AST 9-34 U/L, calcium 2.56-3.02 mmol/L, CK 85-322 U/L, GGT 7-16 U/L, globulin 30-59 g/L, magnesium 1.15-1.70 mmol/L, phosphorus 1.28-2.31 mmol/L, total protein 77-109 g/L, urea 1.2-4.6 mmol/L. Reference intervals were narrower than those reported in other studies. These RI will be helpful in the future management of injured or diseased elephants in national parks and zoological settings.

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.

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.

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.

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.