Experimental safety testing confirms that the NSAID nimesulide is toxic to Gyps vultures in India.

Population declines of Gyps vultures throughout South Asia were caused by unintentional poisoning by the NSAID diclofenac, which was subsequently banned. However, other vulture-toxic NSAIDs are available, including nimesulide, which, in experiments carried out in South Africa, was shown to be toxic to Gyps vultures. We report on safety-testing of nimesulide carried out on Himalayan Griffons G. himalayensis. We gave two vultures a dose of nimesulide by oral gavage at the maximum level of exposure, with two controls dosed with benzyl alcohol. In the two tested birds, plasma nimesulide concentrations peaked after six hours, while serum uric acid concentrations increased steadily up until 24 h post-treatment, after which both birds died, displaying severe visceral gout. The control birds showed no adverse clinical or biochemical signs. We confirm that nimesulide is toxic to Gyps vultures. Veterinary use of nimesulide should be banned in all Gyps vulture range countries in the region.

Metabolism of aceclofenac to diclofenac in the domestic water buffalo Bubalus bubalis confirms it as a threat to Critically Endangered Gyps vultures in South Asia.

Vulture declines in South Asia were caused by accidental poisoning by the veterinary non-steroidal anti-inflammatory drug (NSAID) diclofenac. Although veterinary use of diclofenac has been banned, other vulture-toxic NSAIDs are legally available, including aceclofenac, which has been shown to metabolise into diclofenac in domestic cattle. We gave nine domestic water buffalo the recommended dose of aceclofenac (2 mg kg-1 body weight), collected blood at intervals up to 48 h, and carried out a pharmacokinetic analysis of aceclofenac and its metabolite diclofenac in plasma. Aceclofenac was rapidly converted to diclofenac, and was barely detectable in plasma at any sampling time. Diclofenac was present within 20 min, and peaked 4-8 h after dosing. Aceclofenac is a prodrug of diclofenac, and behaves similarly in domestic water buffalo as it did in domestic cattle, posing the same risk to vultures. We recommend an immediate ban on the veterinary use of aceclofenac across vulture-range countries.

Experimental safety testing shows that the NSAID tolfenamic acid is not toxic to Gyps vultures in India at concentrations likely to be encountered in cattle carcasses.

Population declines of Gyps vultures across the Indian subcontinent were caused by unintentional poisoning by the non-steroidal anti-inflammatory drug (NSAID) diclofenac. Subsequently, a number of other NSAIDs have been identified as toxic to vultures, while one, meloxicam, is safe at concentrations likely to be encountered by vultures in the wild. Other vulture-safe drugs need to be identified to reduce the use of those toxic to vultures. We report on safety-testing experiments on the NSAID tolfenamic acid on captive vultures of three Gyps species, all of which are susceptible to diclofenac poisoning. Firstly, we estimated the maximum level of exposure (MLE) of wild vultures and gave this dose to 40 Near Threatened Himalayan Griffons G. himalayensis by oral gavage, with 15 control birds dosed with benzyl alcohol (the carrier solution for tolfenamic acid). Two birds given tolfenamic acid died with elevated uric acid levels and severe visceral gout, while the remainder showed no adverse clinical or biochemical signs. Secondly, four G. himalayensis were fed tissues from water buffaloes which had been treated with double the recommended veterinary dose of tolfenamic acid prior to death and compared to two birds fed uncontaminated tissue; none suffered any clinical effects. Finally, two captive Critically Endangered vultures, one G. bengalensis and one G. indicus, were given the MLE dose by gavage and compared to two control birds; again, none suffered any clinical effects. The death of two G. himalayensis may have been an anomaly due to i) the high dose level used and ii) the high ambient temperatures at the time of the experiment. Tolfenamic acid is likely to be safe to Gyps vultures at concentrations encountered by wild birds and could therefore be promoted as a safe alternative to toxic NSAIDs. It is manufactured in the region, and is increasingly being used to treat livestock.

Removing the threat of diclofenac to critically endangered Asian vultures.

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genus Gyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captive Gyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vulture Gyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered Asian Gyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40 G. africanus. Subsequently, six G. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species (Gyps bengalensis, Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity to Gyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.

Henipavirus infection in fruit bats (Pteropus giganteus), India.

We tested 41 bats for antibodies against Nipah and Hendra viruses to determine whether henipaviruses circulate in pteropid fruit bats (Pteropus giganteus) in northern India. Twenty bats were seropositive for Nipah virus, which suggests circulation in this species, thereby extending the known distribution of henipaviruses in Asia westward by >1,000 km.

Plasma biochemistry and hematologic values for wild-caught flying foxes (Pteropus giganteus) in India.

Although bats of the genus Pteropus are important ecologically as pollinators and natural hosts for zoonotic pathogens, little is known about their basic physiology. Hematology and plasma biochemistries were determined from wild-caught flying foxes (Pteropus giganteus) in northern India (n=41). Mean lymphocyte differential count was higher for juveniles than adults. Mean platelet count was lower than previously reported. No hemoparasites were observed. No differences were observed between plasma biochemistry values of male and female bats, juveniles and adults, or lactating and nonlactating females. Variation in aspartate aminotransferase (AST) was seen based on body condition score. Blood urea nitrogen and cholesterol concentrations were lower in P. giganteus than other mammalian groups, but were consistent with those reported from other Pteropus species. Alanine aminotransferase and AST concentrations were higher than those reported for Pteropus vampyrus, a closely related species. This study provides basic physiologic information that can be used in future health and disease studies of Indian flying foxes.

Diclofenac poisoning is widespread in declining vulture populations across the Indian subcontinent.

Recent declines in the populations of three species of vultures in the Indian subcontinent are among the most rapid ever recorded in any bird species. Evidence from a previous study of one of these species, Gyps bengalensis, in the Punjab province of Pakistan, strongly implicates mortality caused by ingestion of residues of the veterinary non-steroidal anti-inflammatory drug diclofenac as the major cause of the decline. We show that a high proportion of Gyps bengalensis and G. indicus found dead or dying in a much larger area of India and Nepal also have residues of diclofenac and visceral gout, a post-mortem finding that is strongly associated with diclofenac contamination in both species. Hence, veterinary use of diclofenac is likely to have been the major cause of the rapid vulture population declines across the subcontinent.

Avian scavengers and the threat from veterinary pharmaceuticals.

Veterinary use of the non-steroidal anti-inflammatory drug diclofenac on domesticated ungulates caused populations of resident Gyps vultures in the Indian sub-continent to collapse. The birds died when they fed on carrion from treated animals. Veterinary diclofenac was banned in 2006 and meloxicam was advocated as a 'vulture-safe' alternative. We examine the effectiveness of the 2006 ban, whether meloxicam has replaced diclofenac, and the impact of these changes on vultures. Drug residue data from liver samples collected from ungulate carcasses in India since 2004 demonstrate that the prevalence of diclofenac in carcasses in 2009 was half of that before the ban and meloxicam prevalence increased by 44%. The expected vulture death rate from diclofenac per meal in 2009 was one-third of that before the ban. Surveys at veterinary clinics show that diclofenac use in India began in 1994, coinciding with the onset of rapid Gyps declines ascertained from measured rates of declines. Our study shows that one pharmaceutical product has had a devastating impact on Asia's vultures. Large-scale research and survey were needed to detect, diagnose and quantify the problem and measure the response to remedial actions. Given these difficulties, other effects of pharmaceuticals in the environment may remain undetected.

Molecular sexing of threatened Gyps vultures: an important strategy for conservation breeding and ecological studies.

During the last two decades populations of three resident species of Gyps vulture have declined dramatically and are now threatened with extinction in South Asia. Sex identification of vultures is of key importance for the purpose of conservation breeding as it is desirable to have an equal sex ratio in these monogamous species which are housed together in large colony aviaries. Because vultures are monomorphic, with no differences in external morphology or plumage colour between the sexes, other methods are required for sex identification. Molecular methods for sex identification in birds rely on allelic length or nucleotide sequence discrimination of the chromohelicase-DNA binding (CHD) gene located on male and female chromosomes ZZ and ZW, respectively. We characterized the partial sequences of CHD alleles from Gyps indicus, Gyps bengalensis, Gyps himalayensis and Aegypius monachus and analysed the applicability of five molecular methods of sex identification of 46 individual vultures including 26 known-sex G. bengalensis and G. indicus. The results revealed that W-specific PCR in combination with ZW-common PCR is a quick, accurate and simple method, and is ideal for sex identification of vultures. The method is also suitable to augment ecological studies for identifying sex of these endangered birds during necropsy examinations especially when gonads are not apparent, possibly due to regression during non-breeding seasons.

Detecting diclofenac in livestock carcasses in India with an ELISA: a tool to prevent widespread vulture poisoning.

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), has caused catastrophic vulture declines across the Indian sub-continent. Here, an indirect ELISA is used to detect and quantify diclofenac in 1251 liver samples from livestock carcasses collected across India between August 2007 and June 2008, one to two years after a ban on diclofenac manufacture and distribution for veterinary use was implemented. The ELISAs applicability was authenticated with independent data obtained using LC-ESI/MS. Of 1251 samples, 1150 (91.9%) were negative for diclofenac using both methods, and 60 (4.8%) were positive at 10-4348 and 10-4441 µg kg(-1) when analysed by ELISA and LC-ESI/MS, respectively. The residue level relationship in the 60 positive samples was highly significant (p < 0.001, r(2) = 0.644). Data suggest that this immunological assay could be used not only for cost effective sample screening, but also for residue level semi-quantification.

The population decline of Gyps vultures in India and Nepal has slowed since veterinary use of diclofenac was banned.

Populations of oriental white-backed vulture (Gyps bengalensis), long-billed vulture (Gyps indicus) and slender-billed vulture (Gyps tenuirostris) crashed during the mid-1990s throughout the Indian subcontinent. Surveys in India, initially conducted in 1991-1993 and repeated in 2000, 2002, 2003 and 2007, revealed that the population of Gyps bengalensis had fallen by 2007 to 0.1% of its numbers in the early 1990s, with the population of Gyps indicus and G. tenuirostris combined having fallen to 3.2% of its earlier level. A survey of G. bengalensis in western Nepal indicated that the size of the population in 2009 was 25% of that in 2002. In this paper, repeat surveys conducted in 2011 were analysed to estimate recent population trends. Populations of all three species of vulture remained at a low level, but the decline had slowed and may even have reversed for G. bengalensis, both in India and Nepal. However, estimates of the most recent population trends are imprecise, so it is possible that declines may be continuing, though at a significantly slower rate. The degree to which the decline of G. bengalensis in India has slowed is consistent with the expected effects on population trend of a measured change in the level of contamination of ungulate carcasses with the drug diclofenac, which is toxic to vultures, following a ban on its veterinary use in 2006. The most recent available information indicates that the elimination of diclofenac from the vultures' food supply is incomplete, so further efforts are required to fully implement the ban.

Effectiveness of action in India to reduce exposure of Gyps vultures to the toxic veterinary drug diclofenac.

Contamination of their carrion food supply with the non-steroidal anti-inflammatory drug diclofenac has caused rapid population declines across the Indian subcontinent of three species of Gyps vultures endemic to South Asia. The governments of India, Pakistan and Nepal took action in 2006 to prevent the veterinary use of diclofenac on domesticated livestock, the route by which contamination occurs. We analyse data from three surveys of the prevalence and concentration of diclofenac residues in carcasses of domesticated ungulates in India, carried out before and after the implementation of a ban on veterinary use. There was little change in the prevalence and concentration of diclofenac between a survey before the ban and one conducted soon after its implementation, with the percentage of carcasses containing diclofenac in these surveys estimated at 10.8 and 10.7%, respectively. However, both the prevalence and concentration of diclofenac had fallen markedly 7-31 months after the implementation of the ban, with the true prevalence in this third survey estimated at 6.5%. Modelling of the impact of this reduction in diclofenac on the expected rate of decline of the oriental white-backed vulture (Gyps bengalensis) in India indicates that the decline rate has decreased to 40% of the rate before the ban, but is still likely to be rapid (about 18% year(-1)). Hence, further efforts to remove diclofenac from vulture food are still needed if the future recovery or successful reintroduction of vultures is to be feasible.

Phylogenetic analysis of the DNA polymerase gene of a novel alphaherpesvirus isolated from an Indian Gyps vulture.

The DNA polymerase gene of a novel herpesvirus, vulture herpesvirus (VHV), isolated from an Indian Gyps vulture was completely sequenced using primer walking and transposon insertion strategies. DNA sequencing analysis revealed a single open reading frame (ORF) of 3660 nucleotides (53% G-C content) able to encode 1219 amino acids. Identification was based on a nucleotide sequence identity of approximately 50% to other herpesvirus sequences found in Genbank. Nine motifs were identified that are conserved amongst all known herpesviruses and are found within the 3'-5' exonuclease and DNA binding functional domains of the DNA polymerase enzyme. Phylogenetic analysis using Clustal W with neighbour-joining revealed VHV to group within the subfamily Alphaherpesvirinae, more closely related to the avian herpesviruses than to those of other species. Partial sequence data also revealed VHV to contain other genes fundamental to the structure and replication of all herpesvirus genomes. A Real Time PCR Taqman assay specific for the VHV DNA polymerase gene was designed to detect the presence of VHV genomic material in post mortem tissue samples from diseased birds. Positive tissues included the spleen, rectum, thymus, kidney and brain. A herpesvirus specific to vultures may pose a threat to the management of captive breeding programs being established to assist the survival of wild populations of Gyps vultures.