Experiments were conducted on non-releasable Cape griffon vultures (G. coprotheres
) held captive within their normal aviaries in South Africa, and on wild-caught African white-backed vultures (G. africanus
) temporarily held captive in Namibia. Both G. coprotheres
and G. africanus
are as susceptible to diclofenac poisoning as G. bengalensis
(Swan et al. 2006a
; Naidoo et al. 2009
), and thus are suitable surrogates for safety testing. In order to assess the safety of ketoprofen at a range of doses, via different routes of exposure and to two species of Gyps
, the study was undertaken over four phases (). The range of doses (0.5–5 mg kg−1
) used included doses below and above the likely maximum level of exposure (MLE) that vultures could ingest in the wild. MLE was based upon residue levels of ketoprofen in kidney tissues of cattle (Bos taurus
) slaughtered 1 h after three daily injections of ketoprofen, each at 6 mg kg−1
cattle body weight and a vulture consuming 1.023 kg of kidney (see electronic supplementary material). Ketoprofen doses were within the recommended range for clinical treatment, based upon published guidelines for avian treatment (ranging from 1 to 10 mg kg−1
; see electronic supplementary material) and records indicating more than 22 birds (including six individuals from three Gyps
species) had been safely treated with ketoprofen at 1–9 mg kg−1
(Cuthbert et al. 2006
). Residue levels in kidney and liver tissues of cattle were analysed using LC-ESI/MS (Taggart et al. 2009
). Results showed that average tissue concentrations in cattle 1 h after the last of the three doses were 7.13 mg kg−1
in kidney and 0.16 mg kg−1
in liver, resulting in an MLE of 1.54 mg kg−1
. Ketoprofen used in the trials was sourced from India (Neoprofin, Ranbaxy) for phases 1 and 2, and from South Africa (Ketofen, Merial) for phases 2–4. Details of the route of dosing (oral gavage or cattle meat), phase of the experiment, dose volume and concentration, and sample sizes are reported in . Birds in phase 2 received tissues from five cattle that had received three daily injections of ketoprofen at 6 mg kg−1
and were slaughtered 1 h after the final dose. The kidneys of these cattle weighed 0.785–0.948 kg per animal. After removing 25 g of tissue for analysis, the remaining kidney was fed to the birds: the difference between the weight of kidney fed and 1 kg being made up with liver. Control birds in phase 2 were fed meat from a butcher and assumed free from NSAIDs. Following treatment, all birds in all phases had access to fresh water, food and shade.
Schedule, dose, route and sample sizes for testing of captive Gyps coprotheres and wild G. africanus vultures.
Blood samples were collected from the tarsal or wing vein prior to and at 24 and 48 h after dosing in phase 1, 3 and 4, and 16 h before and 48 h after dosing in phase 2 (to avoid birds regurgitating tissues). Plasma levels of uric acid, albumin, creatinine kinase (CK), alanine aminotransferase (ALT), sodium, potassium and calcium were analysed following standard procedures (see electronic supplementary material). The following observations were made: dose and dose volume of ketoprofen administered, feeding behaviour, time to first signs of unusual behaviour (if any), time to euthanasia (if required) or to mortality. A full necropsy, including gross pathology and histopathology (electronic supplementary material), was undertaken on all vultures that died or were euthanased. Statistical analysis of blood parameters was undertaken by two-way analysis of variance with log10-transformed values for the seven blood parameters as the dependent variables. Two models were fitted, the first considered three treatment–outcome categories as one factor (sham-treated and survived, dosed and survived, dosed and died) and two times of sampling (before and after dosing (at 24 h in phases 1, 3 and 4, and 48 h in phase 2)) as a second factor. Because one dosed bird was given emergency remedial treatment and survived, blood values from this individual were excluded from the analysis. The second model treated log10-transformed dose as a continuous independent variable and two times of sampling as a factor, as before, and the dose × time interaction.