Gyps bengalensis (Oriental white-backed vulture), Gyps indicus (Long-billed vultures) and Gyps tenuirostris (Slender-billed vultures) are three species of vultures present in South Asia. Oriental white-backed vulture (OWBV) specie was one of the most common raptors in subcontinent including Pakistan but now in grave danger of extinction due to decline of more than 95% in population size since early 1990’s. The Peregrine Fund started its Asian Vulture Crises Project in collaboration with the Ornithological Society of Pakistan, in 2000, and selected 16 colonies of OWBV having 2400 active nests, as study sites in Layyah, Muzafargarh, Kasur and Khanewal districts of Pakistan to measure mortality. During the period of 2000-2003 there was high adult and sub adult mortality (5-86%) and population decline (34-95%) associated with visceral gout and renal failure. Then evidences were presented that showed the direct relationship between the residues of Diclofenac Sodium (Anti-inflammatory drug) and renal failure in vultures. These evidences were supported by the experimental reproduction of renal failure due to residues of Diclofenac Sodium given through direct oral route and through feeding in live experimental population of OWBV, hence it was purposed that residues of Veterinary Diclofenac Sodium in the dead carcass of animals are the real cause of severe decline in OWBV population in Pakistan.
During that period of study, gross post-mortem was performed on total of 259 adult and sub adult Oriental white-backed vultures, from which 219 birds (85%) had clear post-mortem lesions of visceral gout, such as urate deposits on the surface of internal organs. Renal failure is the most common cause of visceral gout in birds, because renal failure leads to hyperuricaemia and deposition of uric acid within and on the surface of the internal organs of the bod. Renal failure can be caused by metabolic, degenerative, toxic or infectious disease. During that study, detailed diagnostic testing and necropsies were performed to verify the renal failure and reveal the cause behind it, on the subset of 42 OWBV (28 with visceral gout and 14 without visceral gout), within the 24h of their death. The remaining were decomposed as much that they were unsuitable for diagnostic testing, but upon gross post-mortem there were clear lesions that allowed the presence or absence of visceral gout to be determined. After the completion of diagnosis it was revealed that from the sub-subgroup of 14 birds that were without visceral gout, 8 were died of trauma, lead poisoning, intestinal foreign bodies and organophosphate poisoning. Among the other sub-subgroup of 28 birds with visceral gout, 27 birds were having clear significant lesions of severe renal tubular necrosis and crystals of uric acid in body tissues and kidneys, there were no fibrosis, no inflammatory lesion of infectious disease. Out of 28 only one bird had clear identifiable infection of Mycobacterium avium. Additional tests were performed in the vultures with visceral gout to eliminate the other possible diagnosis, like toxicity of Mercury, Arsenic, Cadmium and Lead as well as infections with West-Nile, Infectious Bronchitis and Avian Influenza Viruses; in result all the tests were negative for toxicities and deficiencies of metals and infections with viruses.
Vultures are the carnivorous birds and the main source of food for vultures is the dead livestock. Therefore it was hypothesized during the study that, there might be any pharmaceutical drug responsible for the renal failure in these raptor birds. A survey was conducted in that region in which Veterinarians and Veterinary Pharmaceutical retailers were accessed to identify the drugs that were known to be absorbed orally and toxic for the renal system of birds. As a result of survey, only one drug met that criterion was Diclofenac Sodium, NSAID drug used as anti-inflammatory, analgesic and antipyretic drug in domestic livestock. Then mass spectroscopy and liquid chromatography of kidneys of vultures was conducted to detect the residues of Diclofenac Sodium in kidneys. The detected concentration of drug residue in the kidneys of those died of renal toxicity was 0.051-0.643µg/g and not detected in those died from other causes.
For the verification of renal toxicity due to Diclofenac in OWBVs, four captive, non-releasable juvenile OWBVs were considered as experimental birds and out of these four, two were administered with high single oral doses (2.5mg/kg) of Veterinary Diclofenac and other two were administered with low single oral doses (0.25mg/kg) of Veterinary Diclofenac. Within 36-58h of the administration of Diclofenac, both the birds’ recipient of high dose and one of the low dose recipient died as a result of acute renal failure and visceral gout showing the same microscopic lesions as the field cases. Diclofenac residue concentration was 0.29, 1.1 and 0.16µg/g in the kidneys of two high dose-vultures and one low dose-vulture respectively.
Another experiment was performed to verify that carcass of Diclofenac treated livestock contain sufficient concentration of drug to cause renal failure and ultimately death in vultures. For this purpose, a buffalo was administered with 2.5mg/kg Intramuscular Veterinary Diclofenac once daily for 3 days and then slaughtered after the administration of last injection and 10 OWBVs fed meat from that buffalo containing 5.7, 1.5 and 0.7 µg/g Diclofenac residues in kidney, liver and muscle respectively. Ten more OWBVs were fed on buffalo meat containing 6.4 µg/g Diclofenac residues. On the basis of amount of food consumed and concentration of residues in meat, eight OWBVs ingested 0.005-0.3 mg/kg Diclofenac, two of these vultures died of renal failure. Other six remained clinically normal upto 6 months. Two vultures ingested 0.5-0.6 mg/kg Diclofenac and one of them died after 1 day of exposure due to renal failure and other remained clinically normal. Ten OWBVs ingested the dose of 0.8-1.0 mg/kg and all those ten vultures died of renal failure. All those OWBVs died due to renal failure after ingesting Diclofenac containing meat were having same post mortem renal lesions as of those birds which were directly administered with Diclofenac or the field cases.
Moreover to verify that renal toxicity or visceral gout was not associated with captivity, all the experimental vultures were held in captivity upto 75 days before the start of experiment and exposure to the Diclofenac Sodium, and all vultures remained clinically normal in that period, so it was clear indication that visceral gout or renal failure was not associated with captivity. Mortality of 65% exposed vultures showed that there is a significant relationship between renal failure and Diclofenac sodium. Water contaminated with Diclofenac may be a source of renal failure but it was already proved that “renal failure caused by Diclofenac is highly dose dependent”, and the concentration of this NSAID in water was very low, unlikely to cause toxicity.
Pharmaceutical drugs of Veterinary field can exert potentially toxic effects on the ecosystem. The decline in the population of vultures due to Diclofenac and its identification may serve as a landmark and provides an opportunity for conservation of wildlife and ecosystem. Another example of ecosystem damage by veterinary pharmaceutics is the shedding of anthelmintic drugs in the faeces of livestock that could be toxic and could cause extinction of important invertebrate species.
Ammar Ahmad, *Yasra Sayyed and **Amna Mahmood
DVM-7th Semester, University of Agriculture Faisalabad Pakistan
*Pharm-D-9th Semester, University of Agriculture Faisalabad Pakistan
**MPhil-Zoology, University of Agriculture Faisalabad Pakistan