Introduction
Three species of Gyps vultures endemic to South Asia have undergone catastrophic population declines since the mid-1990s (Gilbert et al. Reference Gilbert, Watson, Virani, Oaks, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2006, Prakash et al. Reference Prakash, Green, Pain, Ranade, Saravanan, Prakash, Venkitachalam, Cuthbert, Rahmani and Cunningham2007, Chaudhary et al. 2012) and are now listed as ‘Critically Endangered’ (IUCN 2011). A wide range of evidence has established that veterinary use of diclofenac, a non-steroidal anti-inflammatory drug (NSAID), is the main cause of these population declines (Green et al. Reference Green, Newton, Shultz, Cunningham, Gilbert, Pain and Prakash2004, Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Shultz et al. Reference Shultz, Baral, Charman, Cunningham, Das, Ghalsasi, Goudar, Green, Jones, Nighot, Pain and Prakash2004). Vultures are exposed to diclofenac when they feed upon carcasses of domestic ungulates treated with this drug shortly before death. Birds consuming a toxic dose die within 1-2 days of exposure from renal failure and have clinical signs of extensive visceral gout, characteristic of NSAID toxicity (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Swan et al. Reference Swan, Cuthbert, Quevedo, Green, Pain, Bartels, Cunningham, Duncan, Meharg, Oaks, Parry-Jones, Shultz, Taggart, Verdoorn and Wolter2006), along with diclofenac residues in their kidney and liver tissues (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Shultz et al. Reference Shultz, Baral, Charman, Cunningham, Das, Ghalsasi, Goudar, Green, Jones, Nighot, Pain and Prakash2004).To date the toxic effects of diclofenac has been determined in five species of Gyps vultures (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Swan et al. Reference Swan, Cuthbert, Quevedo, Green, Pain, Bartels, Cunningham, Duncan, Meharg, Oaks, Parry-Jones, Shultz, Taggart, Verdoorn and Wolter2006, Naidoo et al. Reference Naidoo, Wolter, Cuthbert and Duncan2009, Das et al. Reference Das, Cuthbert, Jakati and Prakash2011) and all eight species in this genus are likely to be susceptible (Johnson et al. Reference Johnson, Lerner, Rasmussen and Mindell2006). Concerns have been raised about the potential impact of diclofenac on other scavenging species in the region, including the Red-headed Vulture Sarcogyps calvus and Egyptian Vulture Neophron percnopterus that have undergone rapid declines in India over the same period as the Gyps vultures (Cuthbert et al. Reference Cuthbert, Green, Ranada, Saravanan, Pain, Prakash and Cunningham2006). However, testing of diclofenac on other species thus far has found no evidence for a high sensitivity to this drug to species outside the Gyps genus. This includes testing on Turkey Vultures Cathartes aura and Pied Crows Corvus alba which were unaffected by diclofenac even at very high doses (Rattner et al. 2008, Naidoo et al. Reference Naidoo, Mompati, Duncan and Taggart2011) and domestic chickens Gallus gallus domesticus which were only susceptible at doses 50–100 times larger than estimated for the White-rumped Vulture Gyps bengalensis (Naidoo et al. Reference Naidoo, Duncan, Bekker and Swan2007). Notably, besides Gyps species, no other species of raptor from the family Accipitridae has been tested. If other carrion-eater species were found to be sensitive to diclofenac then a much wider range of species could potentially be affected by veterinary misuse of diclofenac in South Asia.
Methods
On 13 February 2012, carcasses of dead raptors were noticed at the Jorbeer cattle carcass dump (latitude N27°57’59.4”, longitude E73°22’35.7”), situated 10 km from Bikaner city, Rajasthan, India (Sharma and Gopi Sunder Reference Sharma and Gopi Sunder2009). An investigation into the causes of death was carried out at the site on 17 February 2012 by expert veterinarians who undertook necropsy examinations of two Steppe Eagles Aquila nipalensis. All gross lesions were recorded and morbid materials were collected from kidneys, liver, heart, spleen and joints and fixed immediately in vials containing 10% neutral buffered formalin (NBF) for subsequent histopathological examination. Each tissue (0.5 cm thickness) was processed by conventional techniques to obtain 4 µm thick paraffin embedded sections (Luna Reference Luna1972). Staining was done with routine haematoxylin and eosin (HE) stain and/or with De Galantha stain for demonstration of urate crystals in the sections. Sections were then evaluated for pathological changes associated with nephrotoxicity. Images of representative and consistent changes were captured using a trinocular microscope with attached camera (Leica Microsystems, Wetzlar, Germany). Additional tissue samples from liver, kidney and muscle from one Steppe Eagle were frozen upon collection and brought to the laboratory in cold conditions and were analysed for diclofenac using an indirect competitive ELISA, following procedures detailed in Saini et al.(2012).
Results and Discussion
Necropsy examinations indicated the presence of extensive visceral gout in the carcasses of both Steppe Eagles (Figure S1A in the online Supplementary Materials). This finding was supported by histological examinations of kidney tissue sections, which demonstrated renal tubular nephrosis and the deposition of urate crystals (Figure S1B). The synovial membrane of the knee joints also showed uric acid crystals, which were formed into numerous large discs with needle-thin peripheral radiations, and occasional inflammatory mononuclear cell foci (Figure S1B). These results were suggestive of diclofenac toxicity, as they are the same clinical signs (extensive visceral gout) and same histopathology (lesions in the kidneys, liver and spleen with extensive uric acid crystal deposition) as reported for diclofenac toxicity in Gyps vultures (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Swan et al. Reference Swan, Cuthbert, Quevedo, Green, Pain, Bartels, Cunningham, Duncan, Meharg, Oaks, Parry-Jones, Shultz, Taggart, Verdoorn and Wolter2006, Daset al. 2011). Testing of kidney tissue samples of one Steppe Eagle with indirect competitive ELISA indicated this bird contained residues of diclofenac, with diclofenac levels estimated at 0.051 µg g-1 in this bird’s kidney tissues. This residue level of diclofenac in the Steppe Eagle is comparable with those found within kidney and liver tissues of wild Gyps vultures found dead with extensive visceral gout, where diclofenac residues ranged from 0.051-0.643 µg g-1in Pakistan (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004) and 0.004-0.160 µg g-1for vultures from India and Nepal (Shultz et al. Reference Shultz, Baral, Charman, Cunningham, Das, Ghalsasi, Goudar, Green, Jones, Nighot, Pain and Prakash2004).
Our findings of extensive visceral gout in the carcasses of two Steppe Eagles and the co-occurrence of gout with diclofenac residues in the one bird tested are the first case of diclofenac related mortality outside the Gyps genus. While this result is only correlative, there is a perfect positive association between gout and diclofenac in Gyps vultures: with all birds dying from gout having diclofenac residues; and birds dying without gout containing no residues (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Shultz et al. Reference Shultz, Baral, Charman, Cunningham, Das, Ghalsasi, Goudar, Green, Jones, Nighot, Pain and Prakash2004). Consequently, the finding of gout and diclofenac in Steppe Eagles is given added credibility by the 100% association in Gyps vultures. Further experimental testing conclusively established that oral dosing of Gyps vultures with diclofenac and feeding them carcasses of animals dosed with diclofenac will result in mortality and the same clinical signs of visceral gout and diclofenac residue levels (Oaks et al. Reference Oaks, Gilbert, Virani, Watson, Meteyer, Rideout, Shivaprasad, Ahmed, Chaudhry, Arshad, Mahmood, Ali and Khan2004, Swan et al. Reference Swan, Cuthbert, Quevedo, Green, Pain, Bartels, Cunningham, Duncan, Meharg, Oaks, Parry-Jones, Shultz, Taggart, Verdoorn and Wolter2006). A similar study is recommended for verifying the toxicity of diclofenac to Steppe Eagles. If such results are borne out, then the finding of diclofenac toxicity in this species has important implications for wintering populations of this species in South Asia and also considerably widens the potential diversity of raptor species, mainly facultative scavengers, that may be affected by diclofenac and other toxic NSAIDs used for veterinary purposes, such as ketoprofen (Naidoo et al. Reference Naidoo, Wolter, Cromarty, Diekmann, Duncan, Meharg, Taggart, Venter and Cuthbert2010). Steppe Eagles wintering in India are seen in large numbers at cattle carcass dumps (Sharma and Gopi Sunder Reference Sharma and Gopi Sunder2009) and frequently scavenge livestock carcasses (Naoroji 2006), thereby being exposed to diclofenac residues. Other species of Aquila eagles are also known to frequently scavenge at carcass dumps in the region including the Tawny Eagle A.rapax, Eastern Imperial Eagle A.heliaca and Indian Spotted Eagle A.hastata(Sharma and Gopi Sunder Reference Sharma and Gopi Sunder2009). For these species, scavenging is particularly important during the winter (Naoroji Reference Naoroji2006). If the finding of diclofenac sensitivity in the Steppe Eagle is confirmed then two phylogenetically distinct genera with the Accipitridae family (the Gyps vultures and Aquila eagles) will be affected by this compound, and potentially other species and genera within the Accipitridae that are known to scavenge on livestock could also be vulnerable. These include the Red-headed, Egyptian, and Bearded Vulture Gypaetus barbatus where there is also evidence for rapid population declines in the region (Cuthbert et al. Reference Cuthbert, Green, Ranada, Saravanan, Pain, Prakash and Cunningham2006, Acharya et al. 2010).
In summary, this study reports the first incidence of diclofenac-related mortality to species outside the Gyps genus of vultures and we recommend that further experimental testing is undertaken to establish the sensitivity of Steppe Eagles and other Aquila species, and to understand the wider threat of veterinary NSAIDs to scavenging raptors within South Asia.
Supplementary Material
The supplementary materials for this article can be found at journals.cambridge.org/bci
Acknowledgements
We thank the Director and Joint Director (Research) at IVRI and the Director of BNHS for providing the necessary facilities. Funding for this work came from the RSPB and the UK Government’s Darwin Initiative programme. We acknowledge laboratory assistance provided by Mohan Bhat at IVRI.
