Richard T. Watson

Diclofenac residues as the cause of vulture population decline in Pakistan.

The Oriental white-backed vulture (OWBV; Gyps bengalensis) was once one of the most common raptors in the Indian subcontinent. A population decline of >95%, starting in the 1990s, was first noted at Keoladeo National Park, India. Since then, catastrophic declines, also involving Gyps indicus and Gyps tenuirostris, have continued to be reported across the subcontinent. Consequently these vultures are now listed as critically endangered by BirdLife International. In 2000, the Peregrine Fund initiated its Asian Vulture Crisis Project with the Ornithological Society of Pakistan, establishing study sites at 16 OWBV colonies in the Kasur, Khanewal and Muzaffargarh–Layyah Districts of Pakistan to measure mortality at over 2,400 active nest sites. Between 2000 and 2003, high annual adult and subadult mortality (5–86%) and resulting population declines (34–95%) (ref. 5 and M.G., manuscript in preparation) were associated with renal failure and visceral gout. Here, we provide results that directly correlate residues of the anti-inflammatory drug diclofenac with renal failure. Diclofenac residues and renal disease were reproduced experimentally in OWBVs by direct oral exposure and through feeding vultures diclofenac-treated livestock. We propose that residues of veterinary diclofenac are responsible for the OWBV decline.

Lead bullet fragments in venison from rifle-killed deer: potential for human dietary exposure.

Human consumers of wildlife killed with lead ammunition may be exposed to health risks associated with lead ingestion. This hypothesis is based on published studies showing elevated blood lead concentrations in subsistence hunter populations, retention of ammunition residues in the tissues of hunter-killed animals, and systemic, cognitive, and behavioral disorders associated with human lead body burdens once considered safe. Our objective was to determine the incidence and bioavailability of lead bullet fragments in hunter-killed venison, a widely-eaten food among hunters and their families. We radiographed 30 eviscerated carcasses of White-tailed Deer (Odocoileus virginianus) shot by hunters with standard lead-core, copper-jacketed bullets under normal hunting conditions. All carcasses showed metal fragments (geometric mean = 136 fragments, range = 15–409) and widespread fragment dispersion. We took each carcass to a separate meat processor and fluoroscopically scanned the resulting meat packages; fluoroscopy revealed metal fragments in the ground meat packages of 24 (80%) of the 30 deer; 32% of 234 ground meat packages contained at least one fragment. Fragments were identified as lead by ICP in 93% of 27 samples. Isotope ratios of lead in meat matched the ratios of bullets, and differed from background lead in bone. We fed fragment-containing venison to four pigs to test bioavailability; four controls received venison without fragments from the same deer. Mean blood lead concentrations in pigs peaked at 2.29 µg/dL (maximum 3.8 µg/dL) 2 days following ingestion of fragment-containing venison, significantly higher than the 0.63 µg/dL averaged by controls. We conclude that people risk exposure to bioavailable lead from bullet fragments when they eat venison from deer killed with standard lead-based rifle bullets and processed under normal procedures. At risk in the U.S. are some ten million hunters, their families, and low-income beneficiaries of venison donations.

Vulture restaurants and their role in reducing diclofenac exposure in Asian vultures

Summary The provision of supplementary food at vulture restaurants is a well-established tool in the conservation of vulture species. Among their many applications, vulture restaurants are used to provide a safe food source in areas where carcasses are commonly baited with poisons. Rapid and extensive declines of vultures in the Indian subcontinent have been attributed to the toxic effects of diclofenac, a pharmaceutical used in the treatment of livestock, to which vultures are exposed while feeding on the carcasses of treated animals. A vulture restaurant was established at the Oriental White-backed Vulture Gyps bengalensis colony at Toawala, in Punjab province Pakistan, to test the effectiveness of the technique in modifying ranging behaviour and mortality at the colony. Six male vultures were fitted with satellite transmitters to describe variation in movement and home-range during periods when safe food was alternately available and withheld at the vulture restaurant. There was considerable variation in individual home-range size (minimum convex polygons, MCP, of 1,824 km 2 to 68,930 km 2 ), with birds occupying smaller home-ranges centred closer to the restaurant being more successful in locating the reliable source of food. Fixes showed that 3 of the tagged vultures fed at the vulture restaurant and the home-range of each bird declined following their initial visit, with a 23–59% reduction in MCP. Mean daily mortality during provisioning was 0.072 birds per day (8 birds in 111 days), compared with 0.387 birds per day (41 birds in 106 days) during non-provisioning control periods. Vultures tended to occupy greater home-ranges, cover greater distances each day and spend proportionately more time in the air during the late brooding and post-breeding seasons. Attendance at the vulture restaurant also declined during this period with fewer birds visiting less often and no tagged vultures visiting the vulture restaurant at all. These findings indicate that vulture restaurants can reduce, but not eliminate, vulture mortality through diclofenac exposure and represent a valuable interim measure in slowing vulture population decline locally until diclofenac can be withdrawn from veterinary use.

Suppression of Nuclear Factor-κB Activation and Inflammation in Microglia by Physically Modified Saline

Background: Microglial activation plays an important role in the pathogenesis of neurodegenerative disorders. Results: Taylor-Couette-Poiseuille flow-modified saline (RNS60) inhibits microglial inflammation via type 1A phosphatidylinositol 3-kinase-Akt-CREB-mediated up-regulation of IκBα and inhibition of NF-κB activation. Conclusion: These results delineate a novel biological function of a physically modified saline. Significance: RNS60 may be of therapeutic benefit in neurodegenerative disorders. Chronic inflammation involving activated microglia and astroglia is becoming a hallmark of many human diseases, including neurodegenerative disorders. Although NF-κB is a multifunctional transcription factor, it is an important target for controlling inflammation as the transcription of many proinflammatory molecules depends on the activation of NF-κB. Here, we have undertaken a novel approach to attenuate NF-κB activation and associated inflammation in activated glial cells. RNS60 is a 0.9% saline solution containing charge-stabilized nanostructures that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not normal saline, RNS10.3 (TCP-modified saline without excess oxygen), and PNS60 (saline containing excess oxygen without TCP modification) were found to inhibit the production of nitric oxide (NO) and the expression of inducible NO synthase in activated microglia. Similarly, RNS60 also inhibited the expression of inducible NO synthase in activated astroglia. Inhibition of NF-κB activation by RNS60 suggests that RNS60 exerts its anti-inflammatory effect through the inhibition of NF-κB. Interestingly, RNS60 induced the activation of type IA phosphatidylinositol (PI) 3-kinase and Akt and rapidly up-regulated IκBα, a specific endogenous inhibitor of NF-κB. Inhibition of PI 3-kinase and Akt by either chemical inhibitors or dominant-negative mutants abrogated the RNS60-mediated up-regulation of IκBα. Furthermore, we demonstrate that RNS60 induced the activation of cAMP-response element-binding protein (CREB) via the PI 3-kinase-Akt pathway and that RNS60 up-regulated IκBα via CREB. These results describe a novel anti-inflammatory property of RNS60 via type IA PI 3-kinase-Akt-CREB-mediated up-regulation of IκBα, which may be of therapeutic benefit in neurodegenerative disorders.

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.

Breeding and mortality of Oriental White-backed Vulture Gyps bengalensis in Punjab Province, Pakistan

Populations of Oriental White-backed Vulture Gyps bengalensis and Long-billed Vulture G. indicus declined in India between the mid 1980s and late 1990s. Regional reports from India described declines of 95–100% across a wide area. This study was conducted to investigate the breeding success and pattern of mortality in two vulture colonies (Dholewala and Changa Manga) within Punjab Province, Pakistan between December 2000 and June 2001. Breeding success was found to be 62% in Dholewala and 59% in Changa Manga. A total of 668 sick and dead vultures were collected of which 591 were less than one month post mortem . No significant variation was found in the weekly mortality rate of adult and subadult vultures during the study period spanning winter through summer. A peak in mortality rate was observed during late April and early May that corresponded to mortality of newly fledged juveniles. Minimum annual mortality rate in the adult breeding population was calculated to be 11.4% and 18.6% in Dholewala and Changa Manga respectively. In a subsample of dead vultures ( n = 185) visceral gout was found in 80% of adults, 63% of subadults, 19% of juveniles and 13% of nestlings. These mortality rates were consistent with a rapid population decline. Results imply that the mortality factor responsible for the decline in Gyps vultures described in India is also present in Pakistan and will potentially lead to a population decline of a comparable magnitude.

Rapid population declines and mortality clusters in three Oriental white-backed vulture Gyps bengalensis colonies in Pakistan due to diclofenac poisoning

The population declines affecting Asian Gyps vultures are among the most rapid and geographically widespread recorded for any species. This paper describes the rates and patterns of mortality and population change over 4 years at three Oriental white-backed vulture Gyps bengalensis colonies in Pakistan: Dholewala (initially 421 pairs), Toawala (initially 445 pairs) and Changa Manga (initially 758 pairs). Vulture mortality led to the extirpation of two of these colonies (Changa Manga and Dholewala) in 3 years, and a decline of 54.3% in the third. Visceral gout, indicative of diclofenac poisoning, was the largest single cause of death in vultures examined. Annual adult mortality from diclofenac poisoning was significantly positively correlated with annual population declines at each colony indicating a direct causal relationship. Visceral gout occurred in temporal and spatial clusters suggesting multiple point sources of diclofenac expo- sure. The spatial and temporal distribution of dead vultures and approximate time since death were used to estimate minimum rates at which colonies encountered carcasses with sufficient diclofenac to cause mortality of 1.26-1.88 carcasses per colony per month. By estimating total carcass consumption at each colony, the percentage of carcasses contaminated with diclofenac was calcu- lated as 1.41-3.02%, exceeding the minimum required to have caused the observed population decline. With populations declining by approximately 50% annually, the long term survival of Gyps vultures in South Asia will require the removal of diclofenac from vulture food and establishment of captive populations for future restoration once the environment is free from contam- ination.

Protection of Tregs, suppression of Th1 and Th17 cells, and amelioration of experimental allergic encephalomyelitis by a physically-modified saline.

In multiple sclerosis (MS) and other autoimmune diseases, the autoreactive T cells overcome the resistance provided by the regulatory T cells (Tregs) due to a decrease in the number of Foxp3-expressing Tregs. Therefore, upregulation and/or maintenance of Tregs during an autoimmune insult may have therapeutic efficacy in autoimmune diseases. Although several immunomodulatory drugs and molecules are available, most present significant side effects over long-term use. Here we have undertaken an innovative approach to upregulate Tregs and achieve immunomodulation. RNS60 is a 0.9% saline solution generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) and PNS60 (saline containing excess oxygen without TCP modification), was found to upregulate Foxp3 and enrich Tregs in MBP-primed T cells. Moreover, RNS60, but not NS, RNS10.3 and PNS60, inhibited the production of nitric oxide (NO) and the expression of iNOS in MBP-primed splenocytes. Incubation of the cells with an NO donor abrogated the RNS60-mediated upregulation of Foxp3. These results suggest that RNS60 boosts Tregs via suppression of NO production. Consistent to the suppressive activity of Tregs towards autoreactive T cells, RNS60, but not NS, RNS10.3, or PNS60, suppressed the differentiation of Th17 and Th1 cells and shifted the balance towards a Th2 response. Finally, RNS60 treatment exhibited immunomodulation and ameliorated adoptive transfer of experimental allergic encephalomyelitis, an animal model of MS, via Tregs. These results describe a novel immunomodulatory property of RNS60 and suggest its exploration for therapeutic intervention in MS and other autoimmune disorders.

Prioritizing species conservation: does the Cape Verde kite exist?

The Cape Verde kite (Milvus milvus fasciicauda) is considered to be one of the rarest birds of prey in the world and at significant risk of extinction. For this reason there is great interest in both the taxonomic and the population status of this group. To help resolve its taxonomic status, we provide phylogenetic analyses based on three mitochondrial genes for a sampling of kites in the genus Milvus, including a broad geographical sampling of black kites (Milvus migrans), red kites (Milvus milvus), Cape Verde kite museum specimens collected between 1897 and 1924, and five kites trapped on the Cape Verde Islands during August 2002. We found that the historical Cape Verde kites, including the type specimen, were non-monophyletic and scattered within a larger red kite clade. The recently trapped kites from the Cape Verde Islands were all phylogenetically diagnosed as black kites. Our findings suggest that the traditional Cape Verde kite is not a distinctive evolutionary unit, and the case for species status, as recently suggested by others, is not supported. We do find support for recognition of at least one clade of yellow-billed kites, traditionally considered as a black kite subspecies, as a distinctive phylogenetic species.

A physically-modified saline suppresses neuronal apoptosis, attenuates tau phosphorylation and protects memory in an animal model of Alzheimer's disease

Alzheimers disease (AD), the leading cause of dementia in the aging population, is characterized by the presence of neuritic plaques, neurofibrillary tangles and extensive neuronal apoptosis. Neuritic plaques are mainly composed of aggregates of amyloid-β (Aβ) protein while neurofibrillary tangles are composed of the hyperphosphorylated tau protein. Despite intense investigations, no effective therapy is currently available to halt the progression of this disease. Here, we have undertaken a novel approach to attenuate apoptosis and tau phosphorylation in cultured neuronal cells and in a transgenic animal model of AD. RNS60 is a 0.9% saline solution containing oxygenated nanobubbles that is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. In our experiments, fibrillar Aβ1-42, but not the reverse peptide Aβ42-1, induced apoptosis and cell death in human SHSY5Y neuronal cells. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), attenuated Aβ(1–42)-induced cell death. RNS60 inhibited neuronal cell death via activation of the type 1A phosphatidylinositol-3 (PI-3) kinase – Akt – BAD pathway. Furthermore, RNS60 also decreased Aβ(1–42)-induced tau phosphorylation via (PI-3 kinase – Akt)-mediated inhibition of GSK-3β. Similarly, RNS60 treatment suppressed neuronal apoptosis, attenuated Tau phosphorylation, inhibited glial activation, and reduced the burden of Aβ in the hippocampus and protected memory and learning in 5XFAD transgenic mouse model of AD. Therefore, RNS60 may be a promising pharmaceutical candidate in halting or delaying the progression of AD.