Gerry E. Swan

Toxicity of diclofenac to Gyps vultures

Three endemic vulture species Gyps bengalensis, Gyps indicus and Gyps tenuirostris are critically endangered following dramatic declines in South Asia resulting from exposure to diclofenac, a veterinary drug present in the livestock carcasses that they scavenge. Diclofenac is widely used globally and could present a risk to Gyps species from other regions. In this study, we test the toxicity of diclofenac to a Eurasian (Gyps fulvus) and an African (Gyps africanus) species, neither of which is threatened. A dose of 0.8 mg kg−1 of diclofenac was highly toxic to both species, indicating that they are at least as sensitive to diclofenac as G. bengalensis, for which we estimate an LD50 of 0.1–0.2 mg kg−1. We suggest that diclofenac is likely to be toxic to all eight Gyps species, and that G. africanus, which is phylogenetically close to G. bengalensis, would be a suitable surrogate for the safety testing of alternative drugs to diclofenac.

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 genusGyps 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 captiveGyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vultureGyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered AsianGyps 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 40G. africanus. Subsequently, sixG. 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 toGyps 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.

The race to prevent the extinction of South Asian vultures

Summary Gyps vulture populations across the Indian subcontinent collapsed in the 1990s and continue to decline. Repeated population surveys showed that the rate of decline was so rapid that elevated mortality of adult birds must be a key demographic mechanism. Post mortem examination showed that the majority of dead vultures had visceral gout, due to kidney damage. The realisation that diclofenac, a non-steroidal anti-inflammatory drug potentially nephrotoxic to birds, had become a widely used veterinary medicine led to the identification of diclofenac poisoning as the cause of the decline. Surveys of diclofenac contamination of domestic ungulate carcasses, combined with vulture population modelling, show that the level of contamination is sufficient for it to be the sole cause of the decline. Testing on vultures of meloxicam, an alternative NSAID for livestock treatment, showed that it did not harm them at concentrations likely to be encountered by wild birds and would be a safe replacement for diclofenac. The manufacture of diclofenac for veterinary use has been banned, but its sale has not. Consequently, it may be some years before diclofenac is removed from the vultures’ food supply. In the meantime, captive populations of three vulture species have been established to provide sources of birds for future reintroduction programmes.

Diclofenac toxicity in Gyps vulture is associated with decreased uric acid excretion and not renal portal vasoconstriction.

Diclofenac (DF), a non-steroidal anti-inflammatory drug (NSAID), is largely regarded as one of the most devastating environmental toxicant in recent times, after accidental exposure via their food-chain lead to massive mortalities in three vulture species on the Asian subcontinent. Although the use of diclofenac was recently banned on the Indian subcontinent, following the favourable safety profile of meloxicam, its mechanism of toxicity remains unknown. In an attempt to establish this mechanism, we test three hypotheses using models established from either the domestic chicken (Gallus domesticus) or the African White-backed vulture (Gyps africanus). We demonstrate that both DF and meloxicam are toxic to renal tubular epithelial (RTE) cells following 12 h of exposure, due to an increase in production of reactive oxygen species (ROS), which could be temporarily ameliorated by pre-incubation with uric acid (UA). When cultures were incubated with either drug for only 2 h, meloxicam showed no toxicity in contrast to diclofenac. In both cases no increase in ROS production was evident. In addition, diclofenac decreased the transport of uric acid, by interfering with the p-amino-hippuric acid (PAH) channel. We conclude that vulture susceptibility to diclofenac results from a combination of an increased ROS, interference with UA transport and the duration of exposure.

The pharmacokinetics of meloxicam in vultures

Vulture populations across the Asian subcontinent have declined dramatically in the last 15 years and are now on the verge of extinction. Although the cause of the population decline was initially unknown, the decrease has recently been conclusively linked to the use of the nonsteroidal anti-inflammatory drug diclofenac in cattle that inadvertently ended up in the vulture food chain. With the vulture numbers continuing to decline by up to 48% a year, the Indian, Nepali and Pakistan governments have recently banned the manufacture and importation of veterinary diclofenac. They have also suggested meloxicam as an alternate anti-inflammatory for use in cattle. This recommendation was based on extensive acute safety studies in the African White-backed vulture (Gyps africanus), which evaluated worst case scenarios of maximum intake based on a once in three day feeding pattern. However, the possible cumulative pharmacokinetic and pharmacodynamic effects in vultures receiving multiple daily doses of meloxicam over time were not assessed. At present very little pharmacokinetic or pharmacodynamic information is available to add further support for the safety of meloxicam in this animal species. This article discusses the oral and intramuscular pharmacokinetics of meloxicam in Cape Griffon vultures (Gyps coprotheres). Therapeutic drug monitoring was also undertaken in White-backed, Egyptian (Neophron pernopterus) and one Lappet Faced vulture (Torgos tracheliotos). In all these species, meloxicam was characterized by a short half-life of elimination. The rapid metabolism of meloxicam in combination with a short duration of effect in the studied species Gyps vultures shown in this study makes it unlikely that the drug could accumulate. This confirms the safety of repeated exposure to meloxicam in vultures of this genus.

Validating the domestic fowl as a model to investigate the pathophysiology of diclofenac in Gyps vultures

Diclofenac has recently been identified as a cause of the widespread vulture decline on the Indian subcontinent. Although the clinical signs and pathology have been described, the pathophysiology of toxicity remains unexplained. In the following study we attempt to validate the domestic fowl as a model, to allow for the further characterisation of diclofenacs mechanism of toxicity. In a lethal dose study, diclofenac was shown to have an approximate intramuscular LD(50) of 9.8mg/kg in 18-week old layers. Signs of toxicity in the affected birds were severe depression that persisted from 24h post-dosing to death with corresponding increased plasma uric acid concentrations. Post-mortem examinations showed signs of gout with deposits of urates (tophi) in the kidneys, liver, heart and spleen. The pharmacokinetics after both the intramuscular and oral route showed that diclofenac had a short half-life of elimination of approximately 1h, a volume of distribution of 0.09-0.24l/kg and relative oral bioavailability of 50% compared to intramuscular administration. With the similarity in the clinical signs, necropsy findings, histopatological lesions and clinical pathological changes, the fowl may be used in further studies to characterise the mechanism of toxicity of diclofenac. However, due to the large difference in susceptibility of the fowl, it is not a suitable model to simulate the dose-response relationship of the vulture to the other non-steroidal anti-inflammatory drugs.

ESTABLISHMENT OF SELECTED BASELINE BLOOD CHEMISTRY AND HEMATOLOGIC PARAMETERS IN CAPTIVE AND WILD-CAUGHT AFRICAN WHITE-BACKED VULTURES (GYPS AFRICANUS )

Despite the devastating collapse of three vulture populations on the Asian subcontinent as a result of their exposure to diclofenac, there is little available information on the normal physiology of many vulture species, including the African White-backed Vulture (Gyps africanus). Such information is needed to fully understand mechanisms for toxicity and to identify and prevent future health problems. The aim of this study was to establish baseline parameters for hematologic and selected serum chemistry parameters for this model species for further studies into the toxicity of diclofenac. Captive nonreleasable and wild African White-backed Vultures were used to determine reference values. For hematology, erythrocyte counts, hemoglobin concentration, hematocrit, packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin concentration, and total and differential leukocyte counts were measured. Chemical analytes measured included sodium, potassium, calcium, albumin, and globulin concentrations, aspartate aminotransferase, creatine kinase, and alanine aminotransferase activities. Uric acid and urea concentrations and the urea:uric acid ratio also were evaluated. Values are presented as means, standard deviations, and reference intervals. The serum chemistry parameters selected may provide a starting point for the evaluation of changes in renal and hepatic function; these organ systems are most severely affected by diclofenac. Results were also compared with values reported for G. africanus nestlings, and from these results it is evident that the clinical pathologic parameters are age related. This indicates that the use of nestling values for the evaluation of clinical pathologic findings in adults may be unreliable and could lead to incorrect assumptions.

Part VI. Antibiotic management and resistance in livestock production

The antibiotic use and levels of antibiotic resistance found in animal populations in South Africa are reviewed: firstly, the framework for antibiotic management in livestock production; secondly, patterns of consumption by sector and application; and thirdly, what is known about bacterial resistance rates. The bacteria discussed are pathogenic to animals, zoonotic organisms and commensal bacteria.

Comparative toxicity studies of NSAIDs in birds: A criticism of Reddy et al.

Comparative toxicity studies of NSAIDs in birds: A criticism of Reddy et al. Richard Cuthbert a, Deborah J. Pain a, Rhys E. Green a,b,∗, Gerry Swan c, Devendra Swarup d a Royal Society for the Protection of Birds, The Lodge, Sandy, Bedfordshire, United Kingdom b Conservation Science Group, Department of Zoology, University of Cambridge, Cambridge, United Kingdom c Department of Paraclinical Sciences and Biomedical Research Centre, Faculty of Veterinary Science, University of Pretoria, South Africa d Indian Veterinary Research Institute, Uttar Pradesh, India

Pretreatment of Urginea sanguinea Bulbs Used in Ethnoveterinary Medicine Influences Chemical Composition and Biological Activity

We investigated the effect of freezing on the chemical composition and biological activity of bulbs of Urginea sanguinea Schinz, a known ethnoveterinary medicinal plant. Freshly harvested bulbs were extracted as either fresh material or after oven drying. Frozen bulbs were extracted after overnight thawing at room temperature or after thawing and oven drying. The leaves were extracted dried at room temperature. Plant material was finally ground and extracted with acetone. The yield from frozen material exceeded that from nonfrozen material by more than 10%. The chemical composition of frozen bulbs, as analyzed by thin-layer chromatography (TLC), differed significantly between fresh and dried material and marginally between fresh and thawed material. Because U. sanguinea is used for diseases that could be infection-related, we examined the antibacterial activity of the extracts. The minimal inhibitory concentration against Staphylococcus aureus was 1.25 mg/ml for fresh dried and thawed dried. Other fractions had no substantial antibacterial activity. No extract had significant free-radical scavenging (antioxidant) activity. The mechanism of action in ethnoveterinary use is probably not via antibacterial or antioxidant activity or immune stimulation. Although the dried bulbs were more chemically complex, the extractable mass had decreased from fresh to dried material by at least 13%, possibly because of desiccation. Bulbous material is difficult to extract, and freezing before extraction may be a viable option for use in the herbal industry.