Pharmacokinetics of orally administered phenylbutazone in African and Asian elephants (Loxodonta africana and Elephas maximus)

The pharmacokinetic parameters of phenylbutazone were determined in 18 elephants (Loxodonta africana and Elephas maximus) after single-dose oral administration of 2, 3, and 4 mg/kg phenylbutazone, as well as multiple-dose administrations with a 4-wk washout period between trials. After administration of 2 mg/kg phenylbutazone, mean serum concentrations peaked in approximately 7.5 hr at 4.3 +/- 2.02 microg/ml and 9.7 hr at 7.1 +/- 2.36 microg/ml for African and Asian elephants, respectively, while 3 mg/kg dosages resulted in peak serum concentrations of 7.2 +/- 4.06 microg/ml in 8.4 hr and 12.1 +/- 3.13 microg/ml in 14 hr. The harmonic mean half-life was long, ranging between 13 and 15 hr and 39 and 45 hr for African and Asian elephants, respectively. There was evidence of enterohepatic cycling of phenylbutazone in Asian elephants. Significant differences (P < 0.0001) in pharmacokinetic values occurred between African and Asian elephants for clearance (27.9 and 7.6 ml/hr/kg, respectively), terminal half-life (15.0 and 38.7 hr, respectively), and mean residence time (22.5 and 55.5 hr, respectively) using 2-mg/kg dosages as an example. This suggests that different treatment regimens for Asian and African elephants should be used. There were no apparent gender differences in these parameters for either elephant species.     

Pharmacokinetics of orally administered ibuprofen in African and Asian elephants (Loxodonta africana and Elephas maximus).

The pharmacokinetic parameters of S(+) and R(-) ibuprofen were determined in 20 elephants after oral administration of preliminary 4-, 5-, and 6-mg/kg doses of racemic ibuprofen. Following administration of 4 mg/kg ibuprofen, serum concentrations of ibuprofen peaked at 5 hr at 3.9 +/- 2.07 microg/ml R(-) and 10.65 +/- 5.64 microg/ml S(+) (mean +/- SD) in African elephants (Loxodonta africana) and at 3 hr at 5.14 +/- 1.39 microg/ml R(-) and 13.77 +/- 3.75 microg/ml S(+) in Asian elephants (Elephas maximus), respectively. Six-milligram/kilogram dosages resulted in peak serum concentrations of 5.91 +/- 2.17 microg/ml R(-) and 14.82 +/- 9.71 microg/ml S(+) in African elephants, and 5.72 +/- 1.60 microg/ml R(-) and 18.32 +/- 10.35 microg/ml S(+) in Asian elephants. Ibuprofen was eliminated with first-order kinetics characteristic of a single-compartment model with a half-life of 2.2-2.4 hr R(-) and 4.5-5.1 hr S(+) in African elephants and 2.4-2.9 hr R(-) and 5.9-7.7 hr S(+) in Asian elephants. Serum concentrations of R(-) ibuprofen were undetectable at 24 hr, whereas S(+) ibuprofen decreased to below 5 microg/ml 24 hr postadministration in all elephants. The volume of distribution was estimated to be between 322 and 356 ml/kg R(-) and 133 and 173 ml/kg S(+) in Asian elephants and 360-431 ml/kg R(-) and 179-207 ml/kg S(+) in African elephants. Steady-state serum concentrations of ibuprofen ranged from 2.2 to 10.5 microg/ml R(-) and 5.5 to 32.0 microg/ml S(+) (mean: 5.17 +/- 0.7 R(-) and 13.95 +/- 0.9 S(+) microg/ml in African elephants and 5.0 +/- 1.09 microg/ml R(-) and 14.1 +/- 2.8 microg/ml S(+) in Asian elephants). Racemic ibuprofen administered at 6 mg/kg/12 hr for Asian elephants and at 7 mg/kg/12 hr for African elephants results in therapeutic serum concentrations of this antiinflammatory agent.     

Oxygen radical production by Asian elephant (Elephas maximus) heterophils and Holstein cattle (Bos taurus) neutrophils.

Oxidative metabolic burst activity by Asian elephant (Elephas maximus) heterophils and Holstein cattle (Bos taurus) neutrophils was indirectly evaluated by measuring the oxidation of nonfluorescent intracellular 2',7'-dichlorofluorescein (DCFH) to fluorescent 2',7'-dichlorofluorescein (DCF) with flow cytometry. The results were recorded as mean channel fluorescence intensity. Phorbol myristate acetate at 50 ng/ml was used to activate the cells. Elephant heterophils and bovine neutrophils exhibited similar abilities to oxidize intracellular DCFH to DCF, a fluorescent product. A wide range of mean channel fluorescence intensity (1,803-7,393) was observed among individual elephants, and the range of intensities was similar to that observed in bovine neutrophils, supporting the concept of functional heterogeneity of heterophils among elephants.     

Subclinical hypocalcaemia in captive Asian elephants (Elephas maximus)

The hypothesis that hypocalcaemia may play a role in dystocia in captive Asian elephants (Elephas maximus) was investigated. The objectives of the study were to measure the total calcium concentration in elephant plasma; assess the changes in parameters of calcium metabolism during a feeding trial; investigate a possible relationship between calcium metabolism and dystocia; and assess bone mineralisation in captive Asian elephants in vivo. The following parameters were measured: total and ionised calcium, inorganic phosphorous and magnesium, the fractional excretions of these minerals, intact parathyroid hormone, 25-OH-D(3) and 1,25-OH-D(3). Radiographs were taken from tail vertebrae for assessment of bone mineralisation. The mean (sd) heparinised plasma total calcium concentration was 2.7 (0.33) mmol/l (n=43) ranging from 0.84 to 3.08 mmol/l in 11 Asian elephants. There was no significant correlation between plasma total calcium concentration and age. Following feeding of a calcium rich ration to four captive Asian elephant cows, plasma total and ionised calcium peaked at 3.6 (0.24) mmol/l (range 3.4 to 3.9 mmol/l) and 1.25 (0.07) mmol/l (range 1.17 to 1.32 mmol/l), respectively. Plasma ionised calcium concentrations around parturition in four Asian elephant cows ranged from 0.37 to 1.1 mmol/l only. The present study indicates that captive Asian elephants might be hypocalcaemic, and that, in captive Asian elephants, the normal plasma concentration of total calcium should actually be around 3.6 mmol/l and normal plasma concentration of ionised calcium around 1.25 mmol/l. Given the fact that elephants absorb dietary calcium mainly from the intestine, it could be concluded that elephants should be fed calcium-rich diets at all times, and particularly around parturition. In addition, normal values for ionised calcium in captive Asian elephants should be reassessed.     

Oral bioavailability and pharmacokinetic characteristics of ketoprofen enantiomers after oral and intravenous administration in Asian elephants (Elephas maximus)

OBJECTIVE: To assess oral bioavailability (F) and pharmacokinetic characteristics of the R- and S-enantiomers of ketoprofen administered IV and orally to captive Asian elephants (Elephas maximus). ANIMALS: 5 adult Asian elephants. PROCEDURE: Elephants received single treatments of racemic ketoprofen at a dose of 2.2 mg/kg, administered IV and orally, in a complete crossover design. Blood samples were collected at intervals during the 24 hours following treatment. At least 4 weeks elapsed between drug administrations. Samples were analyzed for R- and S-ketoprofen with a validated liquid chromatography-mass spectroscopic assay. Pharmacokinetic parameters were determined by use of noncompartmental analysis. RESULTS: The enantiomers of ketoprofen were absorbed well after oral administration, with median F of 101% for R-ketoprofen and 85% for S-ketoprofen. Harmonic mean half-life ranged from 3.8 to 5.5 hours, depending on route of administration and enantiomer. The area under the concentration-time curve, mean residence time, apparent volume of distribution, plasma clearance, and maximum plasma concentration values were all significantly different between the 2 enantiomers for both routes of administration. CONCLUSIONS AND CLINICAL RELEVANCE: Ketoprofen has a long terminal half-life and complete absorption in this species. Based on the pharmacokinetic data, a dosage of ketoprofen of 1 mg/kg every 48 hours to 2 mg/kg every 24 hours, PO or IV, is recommended for use in Asian elephants, although the safety and efficacy of ketoprofen during long-term administration in elephants have not been determined.     

Long-term cannulation of the ovarian vein in mares.

A cannulation technique was developed to collect blood samples from the ovarian vein of mares over an extended period. Ovarian venous cannulae placed in 4 mares remained patent for a mean (+/- SEM) duration of 36.8 (+/- 6.2) days. During mid-diestrus, concentrations of progesterone in the ovarian vein ipsilateral to the corpus luteum (1,663.8 +/- 238.8 ng/ml) were significantly (P less than 0.001) higher than concentrations measured in paired samples from the jugular vein (6.1 +/- 0.3 ng/ml). Concentration of estradiol in the ovarian vein ranged from a mean of 1,053.2 +/- 303.1 pg/ml during diestrus to 3,353.8 +/- 1,052.8 pg/ml during estrus, whereas values for 74% of samples collected concurrently from the jugular vein were near or below the sensitivity of the assay (10 pg/ml). Results of the study indicate that patent long-term ovarian vein cannulation can be achieved in mares. The cannulation technique provides access to important fundamental information on equine reproductive endocrinology, which to our knowledge, has not been available.     

Pharmacokinetics of a single dose of enrofloxacin administered orally to captive Asian elephants (Elephas maximus)

OBJECTIVE: To determine the pharmacokinetics of enrofloxacin after oral administration to captive elephants. ANIMALS: 6 clinically normal adult Asian elephants (Elephas maximus). PROCEDURE: Each elephant received a single dose of enrofloxacin (2.5 mg/kg, PO). Three elephants received their complete diet (pellets and grain) within 2 hours after enrofloxacin administration, whereas the other 3 elephants received only hay within 6 hours after enrofloxacin administration. Serum concentrations of enrofloxacin and ciprofloxacin were measured by use of high-performance liquid chromatography. RESULTS: Harmonic mean half-life after oral administration was 18.4 hours for all elephants. Mean +/- SD peak serum concentration of enrofloxacin was 1.31 +/- 0.40 microg/mL at 5.0 +/- 4.2 hours after administration. Mean area under the curve was 20.72 +/- 4.25 (microg x h)/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of enrofloxacin to Asian elephants has a prolonged elimination half-life, compared with the elimination half-life for adult horses. In addition, potentially therapeutic concentrations in elephants were obtained when enrofloxacin was administered orally at a dosage of 2.5 mg/kg. Analysis of these results suggests that enrofloxacin administered with feed in the manner described in this study could be a potentially useful antimicrobial for use in treatment of captive Asian elephants with infections attributable to organisms, such as Bordetella spp, Escherichia coli, Mycoplasma spp, Pasteurella spp, Haemophilus spp, Salmonella spp, and Staphylococcus spp.     

Comparing Sex Steroid Levels During the Annual Cycles of Rainbow Trout (Oncorhynchus mykiss) Diploid Female (XX) and Triploid Female (XXX) Genotypic Sex

In this study, the annual cycle of the gonadal steroids testosterone (T), 11‐ketotestosterone (11‐KT), 17β‐estradiol (E2) and 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one (DHP) was determined using radioimmunoassay and then compared for two populations of rainbow trout, XX diploid females (n = 40) and XXX triploid females (n = 15). In females, E2 and DHP levels were found to be significantly related to body weight (r = 0.22513; p < 0.0001 and r = 0.15831; p > 0.001, respectively). In this group, E2 concentrations peaked in November (25.05 ng /ml), while maximum DHP levels, only measurable from October to April, were attained in February (64.14 ng /ml). No significant differences in hormone ranges related to egg output ability were observed. Finally, sex steroid concentrations were low in the triploid female XXX fish compared to the female XX population. Nevertheless, maximum T (33.85 ng /ml) and 11‐KT (32.35 ng /ml) levels were recorded in January, for XXX. The levels for these two hormones are relatively high and are also significantly associated (r = 0.8430; p < 0.0001). Diploid females showed significantly higher levels of E2 than triploids over the 12‐month study period. The female triploid fish produced the lowest steroid hormone levels, such that these would be the most suitable for human consumption.     

Control of musth in an Asian elephant bull (Elephas maximus) using leuprolide acetate.

The results of long-term administration of leuprolide acetate (LA) depot in a 52-yr-old Asian elephant bull (Elephas maximus) for control of musth are presented. Twelve injections were administered for 6 yr during our interpretation of early musth or "premusth." Intervals between musth periods during the study varied from 2 to 34 mo. Blood samples, drawn weekly, were assayed for serum testosterone concentrations; mean levels were 11.78 +/- 1.97 nmol/L throughout the first 26 mo of the study, 7.28 +/- 1.28 nmol/L during the following 21 mo, and 0.45 +/- 0.035 nmol/L in the last 34 mo of this study. Early musth signs ceased within 3 days of drug administration after 10 of 12 injections. The mean serum testosterone concentrations were significantly decreased by the last 34 mo of the study. The results suggest leuprolide is a suitable alternative for controlling or preventing (or both) musth in captive Asian elephants, although permanent reproductive effects may occur. Zoos and wildlife conservation institutions could benefit from the use of LA in Asian elephants to increase the male availability in captivity, consequently ensuring genetic diversity and the perpetuation of the species.     

Serum cortisol and progestin concentrations in pregnant and non-pregnant Asian elephants (Elephas maximus)

Blood samples were collected during the estrous cycle (n=3), throughout gestation (n=3), and during the periparturient period (n=11) to assess serum concentrations of cortisol in pregnant and non-pregnant Asian elephants whose reproductive status was being monitored by serum progestin determination. While serum cortisol concentrations remained constant throughout gestation, progestin concentrations decreased significantly (p<0.05) in the second half of pregnancy, declining to undetectable levels by 3 days before calving. During the non-luteal phase of the estrous cycle serum progestins varied from undetectable levels to 100pg/ml (53+/-10.7pg/ml) then increased steadily during the luteal phase (322+/-207.5pg/ml). There were no significant differences between serum cortisol concentrations during the luteal and those of the non-luteal phase (p>0.05). The mean cortisol concentration during the estrous cycle was about twice that during pregnancy (p>0.05). No substantial changes in maternal cortisol were found during the course of pregnancy or the periparturient period.     

A variant of the endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in European zoos.

Newly discovered, lethal elephant endotheliotropic herpesviruses (EEHV) have been identified in both Asian (Elephas maximus) and African (Loxodonta africana) elephants. Carried by otherwise healthy African elephants they can be fatal mainly for young Asian elephants. Since zoos often harbour both elephant species, we conducted a survey on the presence of EEHV in Asian elephants from 12 European zoos, 3 circuses and 1 Israeli zoo. Here, we demonstrate that all EEHV that have affected Asian elephants so far belong to the EEHV1 group. We also describe the detection and the partial sequencing of an endotheliotropic herpesvirus variant (named EEHV1b) in Asian elephants, being either an EEHV endogenous to Asian elephants or indicating different sources (African elephants) of infection.     

Serum concentrations of etorphine in juvenile African elephants

Eleven juvenile African elephants were given etorphine hydrochloride (2.19 +/- 0.11 micrograms/kg of body weight; mean +/- SD) as a single IM injection; 3 elephants were given additional etorphine (0.42 +/- 0.09 micrograms/kg) IV. After immobilization, each elephant was maintained in lateral recumbency by administration of a 0.5% halothane/oxygen mixture or by administration of multiple IV injections of etorphine. At postinjection hours 0.25 and 0.5 and at 30-minute intervals thereafter, blood samples were collected via an auricular artery, and serum concentrations of etorphine were determined by use of radioimmunoassay. The highest mean serum concentration of etorphine in 6 elephants given a single IM injection and subsequently maintained on halothane and oxygen was 1.62 +/- 0.97 ng/ml at postinjection hours 0.5; thereafter, the mean serum concentration decreased steadily. In 4 elephants maintained in lateral recumbency with multiple IV administrations of etorphine, a correlation was not found between the time to develop initial signs of arousal and serum concentrations of etorphine before arousal. After administration of the initial immobilizing dose of etorphine, the interval between successive IV administrations of etorphine decreased.     

Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in Asian elephants (Elephas maximus)

Endotheliotropic herpesvirus causes a fatal disease in young Asian elephants, but there are no methods for identifying latent carriers of the virus. During the postmortem study of one female African elephant and three male and two female Asian elephants, a lymph node located bilaterally caudoventral to the parotid gland, approximately 1.5 to 5 cm below the skin, was identified as suitable for transcutaneous ultrasound-guided biopsy. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. The calculated mean (se) volume of 10 active lymph nodes was 17.4 (6.9) cm(3), and that of three chronically active lymph nodes was 10.6 (1.0) cm(3), whereas the mean volume of 17 inactive lymph nodes was 3.1 (0.6) cm(3). The presence of lymph node tissue in samples obtained by ultrasound-guided biopsy from three animals that were maintained under conditions that allowed for additional sampling was confirmed histologically. The dna extracted from the lymphoid tissue and the whole blood of all the elephants was negative for endotheliotropic herpesvirus by PCR.     

Relationship between body temperature and ovarian cycle in Asian and African elephants

The aim of the present study was to investigate whether changes in body temperature are related to the ovarian cycle in elephants. Rectal, tongue or fecal temperature was measured for 2 Asian and 5 African elephants using an electric thermometer. Evaluation of ovarian cycles was based on the changes in serum or fecal progestin. The mean +/- SD values of the rectal, tongue, and fecal temperatures were 36.3 +/- 0.3 (2 Asian), 36.2 +/- 0.5 (1 African) and 36.5 +/- 0.3 C (4 African), respectively; the fecal temperature was the highest of the 3 temperatures (P<0.01). The longitudinal changes in body temperatures correlated with the ovarian cycle, with higher temperatures occurring during the luteal phase. The fecal temperatures of one acyclic African elephant did not change cyclically. These results suggest that measurement of body temperature can be used to easily evaluate the ovarian cyclicity of an individual animal, although it might not be able to determine the ovarian cycle length.     

Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine

OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species.     

The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.     

Plasma and erythrocyte fatty acids in captive Asian (Elephas maximus) and African (Loxodonta africana) elephants

The fatty acid components of the plasma triglycerides and the phospholipid fractions of the red blood cells of a captive group of two African (Loxodonta africana) and four Asian (Elephas maximus) elephants were investigated. All the animals received the same diet of hay, fruits and vegetables, and concentrates. A comparison with data from free-ranging African elephants or Asian work-camp elephants showed that the captive elephants had lower proportions of polyunsaturated fatty acids (PUFAs), and for several lipid fractions a higher n-6:n-3 ratio, than their counterparts in the wild or under the more natural, in terms of diet, work-camp conditions. The difference in PUFA content was smaller in the African than in the Asian elephants. The captive Asian elephants tended to have lower levels of n-3 and total unsaturated fatty acids in their red blood cells than the captive African elephants.     

Eradication of elephant ear mites (Loxoanoetus bassoni) in two African elephants (Loxodonta africana)

Elephant ear mites, not previously described in North America, were eradicated in two African elephants (Loxodonta africana) after six otic instillations of ivermectin at 2-wk intervals. The microscopic examination of a clear, mucoid discharge collected from the external ear canals of two wild-born African elephants housed in a New York State zoo for 25 yr revealed live mites (Loxoaneotus bassoni). The cytologic examination demonstrated no evidence of inflammation or infection. Both elephants were asymptomatic with normal hemograms and serum chemistry panels. A diagnosis of otoacariasis was made. Each elephant was treated six times with 5 ml of 1% ivermectin syrup instilled in each ear canal once every 2 wk. Microscopic examinations of clear mucus collected from each elephant's ear canals 9 days after the first instillation of ivermectin were negative for any life stages of ear mites. Microscopic examinations of mucus collected from both elephants' ear canals at 6, 11, and 16 wk, as well as annually post-treatment for 7 yr, confirmed eradication of the ear mites. The L. bassoni ear mite was first identified in the external ear canals of wild, asymptomatic, lesion-free, African elephants culled in Kruger National Park in South Africa. However, a new species in the same genus of mites (Loxoanoetus lenae) was identified at the necropsy of an 86-yr-old Asian elephant (Elephas maximus) living in a circus in Australia. The autopsy revealed a marked, ballooning distension of bone around the left external acoustic meatus, suggestive of mite-induced otitis externa, as seen in cattle infested with ear mites (Raillieta auris). Elephant health care providers should identify the prevalence of, and consider treatment of, elephants in their care infested with ear mites, given the possible risk for adverse health effects.