The veterinary diagnostic investigation into the causes of death of Oriental White-Backed Vultures (Gyps bengalensis) in Pakistan was initiated in November, 2000. Field studies between the 2000/2001 and 2003/2004 breeding seasons have shown catastrophic declines, primarily due to high mortality rates in the adult breeding population. Once we began to examine these dead adults, it became quickly apparent that most of this mortality was associated with the clinical syndrome of visceral gout – of 259 birds that were in good enough post mortem condition for detailed necropsies, 85% had gout. In addition, virtually all of these affected birds were in good physical condition with abundant body fat, indicating that this disease was very acute. Some birds were even found dead on the nest incubating an egg. Because death was so rapid in these birds, we rarely had the opportunity to observe antemortem disease signs. When affected birds were found alive, the signs included weakness, lethargy/depression, diarrhea, nasal and oral discharges, and in a few cases ataxia and seizures. However, no consistent or specific clinical signs were observed that could be incorporated into a case definition. Although head-drooping has been observed in Pakistan, it has not been correlated to the gout syndrome and large scale gout-associated mortality occurs in the complete absence of head-drooping.
Visceral gout in birds is due to kidney failure, which leads to high blood levels of uric acid that precipitate onto the surface of organs such as the heart, pericardium, liver, and kidneys. The usual causes of renal failure in birds include infectious diseases, toxins, nutritional deficiencies or excesses, and metabolic diseases. Dehydration alone without kidney damage (due to lack of water, inability to get to water, or loss of water from disease such as diarrhea) is often cited as a cause of gout, but this is poorly substantiated by experimentation. We also noted that most of the immature vultures we found that were known to have died of starvation and dehydration during the fledgling period did not die of gout, indicating that dehydration alone was unlikely to cause renal failure and gout. Thus, our diagnostic focus was to find a disease that was selectively and directly affecting the kidneys of adult and subadult.
Samples were collected from a subset of 42 cases that were freshly dead and suitable for detailed analysis. Diagnostic testing included histopathology, virology, electron microscopy, bacteriology, and toxicology. Of the 14 vultures without gout, we determined the cause of death in 57% which included trauma, intestinal foreign bodies, lead poisoning, organophosphate poisoning, and gunshot. In contrast, other than a single vulture with avian tuberculosis (infection with Mycobacterium avium), no obvious underlying disease or condition was identified as the cause of renal failure. Histopathology on the gout cases showed lesions in the kidney tubules that would lead to renal failure, but no other lesions that indicated the underlying disease. Most notably, inflammatory lesions compatible with a primary infectious disease of the kidneys or other organs were not identified in any birds other than the one case with M. avium infection. Various other lesions were occasionally noted (e.g. enteritis, meningitis, arteriolitis, tracheitis), but these were generally mild and probably subclinical, and were not present consistently in all the cases. This suggests that none of these lesions were indicative of a common underlying cause of visceral gout. Oxalate crystals in the kidneys indicating ethylene glycol toxicity, a well known cause of renal failure, were not observed in kidney sections. Also, virus identification studies (including attempts at virus isolation and molecular biology methods to detect viral DNA or RNA) and bacteriology results were negative.
The results were most compatible with acute renal failure due to a toxin, and thus toxicology was pursued extensively. Organochlorines (lindane, dieldrin, DDD, DDE, and DDT) were detected in fat samples from some vultures indicating exposure to these pesticides. However, the levels in brain or liver were all well below the levels associated with acute organochlorine intoxication. No organophosphates, organophosphate metabolites, or carbamates were detected in tissues either by methods to directly detect the actual chemicals, or by methods that indirectly detect exposure by measuring brain acetylcholinesterase levels. Heavy metal concentrations in tissues were analyzed for toxic and deficient levels. Lead poisoning was identified in one non-gout vulture. Toxic levels of arsenic, cadmium, copper, iron, manganese, molybdenum, lead, mercury, and zinc were not detected in any other birds. The levels of copper and molybdenum may have been deficient in some individuals, but a clear pattern of deficiency did not distinguish the gout from the non-gout birds. Zinc levels are also potentially deficient if compared to many species of birds, but are comparable to the levels in normal adult eagles. A number of vultures were found to have non-food items in their stomachs (pottery, wood, glass, nails, metal); however this behavior is common in the African Gyps spp. and is unlikely to represent pica or otherwise suggest a nutritional deficiency. The possibility of the avicide 3-chloro-p-toluidine, a toxin that specifically targets the avian kidney and results in visceral gout, has been frequently mentioned. However, this poisoning is very unlikely to be associated with the vulture deaths for the following reasons: avicide is not known to be used in Pakistan, and raptors are not generally secondarily poisoned since by the time a target bird dies the active ingredient is completely metabolized. Thus, secondary poisoning of vultures would be extremely unlikely.
With the exclusion of the usual causes of avian kidney failure, we began investigating more unconventional causes. Our focus was on pharmaceuticals used to treat domestic livestock, based on the observations that the Pakistani vultures feed almost exclusively on domestic livestock and that veterinary care of livestock is routine in the Punjab. A survey was first conducted to identify drugs commonly used to treat livestock. Drugs listed in the survey that could account for the vulture mortalities were identified by looking for those that met the following criteria: known to be toxic to avian kidneys, commonly used, and relatively new to the market. Two candidate drugs were gentamicin, an antibiotic, and diclofenac, a non-steroidal anti-inflammatory drug. However, since gentamicin is not absorbed orally (and exposure of the vultures would need to be by ingestion), this left only diclofenac. Subsequent testing then identified a 100% correlation between gout and tissue residues of diclofenac (25/25 cases with gout were positive, while 0/13 non gout cases were positive). Follow up surveys of veterinarians and veterinary pharmaceutical retailers confirmed that diclofenac is very widely sold over the counter and is routinely used to treat livestock. More detailed analyses of the economics and use of veterinary diclofenac are the subject of ongoing studies in collaboration with WWF-Pakistan. Requests to ban or regulate the use of diclofenac are also being presented to the governments of Pakistan, India, and Nepal. These details of this study can be found in:
Oaks, JL, M Gilbert, MZ Virani, RT Watson, CU Meteyer, BA Rideout, HL Shivaprasad, S Ahmed, M J Chaudhry, M Arshad, S Mahmood, A Ali, AA Khan. Diclofenac residues as a cause of population decline of White-backed vultures in Pakistan. Nature, In Press
OTHER FINDINGS
There have been very limited veterinary studies on Gyps vultures, particularly in the area of microbiology. Thus, it is not surprising that in the course of the diagnostic investigation there would be new discoveries. Although these are peripheral to the primary problem, they provide valuable information about the general biology of Gyps bengalensis. Two of these discoveries are presented here.
Mycoplasma vulturii – a new bacteria
Cell culture experiments to attempt to detect infectious agents isolated a novel intracellular bacterium from the tissues of one of the vultures that in addition to gout had very mild respiratory lesions. Subsequent biochemical, genetic, and ultrastructural (electron microscopy) characterization showed this bacterium was a novel mycoplasma which we have named Mycoplasma vulturii. Since we discovered this organism prior to the discovery of diclofenac, and it was recovered from a gout case, we undertook additional studies to determine if it were a possible cause of the gout-associated mortalities. We developed a molecular assay (polymerase chain reaction) based on the unique genetics of the organism, and then used this test to determine if other vultures were also infected. We tested tissues from 24 gout cases and 13 non-gout cases: the gout cases had a prevalence of 21%, while the non-gout cases had a prevalence of 31%. Thus, although this agent is new and may cause mild respiratory lesions in some vultures (lesions were only found in the index case), there was no evidence that it was a cause of gout. The details of this study can be found in:
Oaks, JL, SL Donahoe, BA Rideout, M Gilbert, MZ Virani. Identification of a novel Mycoplasma Species from an Oriental White-Backed Vulture (Gyps bengalensis). Submitted
Raillietiella trachea – a new parasite
While performing necropsies, Dr. Gilbert noted an unusual species of parasite in the trachea of one of the vultures. This parasite was subsequently identified as a pentastomid by Dr. Mike Kinsella and John Riley (University of Dundee, Dundee, Scotland). There is no reason to suspect that this parasite has any connection with gout, or with any other recognized disease of vultures. However, the description of this parasite has been of considerable interest to parasitologists since this group of parasites are normally found in reptiles and a few marine birds – this is the first to be described from a full terrestrial bird. The details of this study can be found in:
Riley, J, JL Oaks, and M. Gilbert. 2003. Raillietiella trachea n. sp., a pentastomid from the trachea of an Oriental White Backed Vulture (Gyps bengalensis) taken in Pakistan, with speculation about its life cycle. Syst Parasitol 56(2):155-61.
Report Prepared by:
J. Lindsay Oaks, DVM, PhD, Dip. ACVM
Department of Veterinary Microbiology and Pathology
Washington State University
Pullman, WA 99164-7040
Phone (509) 335-6044, Fax (509) 335-8529
email: loaks@vetmed.wsu.edu
Veterinary Collaborators:
Martin Gilbert, MRCVS, BVMS, BSc.
The Peregrine Fund
5668 West Flying Hawk Lane
Boise, ID 83709, USA
Bruce A. Rideout, DVM, PhD
Center for Reproduction of Endangered Species
Zoological Society of San Diego
Collaborators: Pathology
H. L. Shivaprasad, BVSc, MVSc, MS, PhD, MS, DACPV
Associate Professor, Avian Pathology,
California Animal Health and Food Safety Laboratory System, Fresno Branch,
University of California, Davis.
Carol U. Meteyer, DVM, Dip. ACVP
Wildlife Pathologist
National Wildlife Health Center
Madison, WI
Patricia A. Talcott, DVM, PhD, Dip. ABVT
Associate Professor, Veterinary Toxicology
University of Idaho
Val Beasley, DVM, PhD, Dip. ABVT
Professor of Veterinary, Wildlife, and Ecological Toxicology Department of Veterinary Biosciences
University of Illinois at Urbana-Champaign
Dr. Kimberlee B. Beckman, DVM, PhD, Research Scientist Department of Veterinary Biosciences
University of Illinois at Urbana-Champaign
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