Lead poisoning in whooper and tundra swans

Six weak whooper swans (Cygnus cygnus) and two weak tundra swans (Cygnus columbianus) were found at Swamp Miyajima (Hokkaido, Japan) in May 1998. Anorexia, depression, green watery feces, pale conjunctiva, and anemia were observed. Radiographs showed from six to 38 suspected lead pellets in the gizzard. Blood lead concentrations were 2.5-6.7 microg/g (mean+/-SD=4.6+/-1.14 microg/g) on day 1. After blood collection, the birds were treated with calcium disodium ethylenediaminetetraacetate (CaEDTA) given intravenously and force fed. Despite treatment, seven birds died the next day. Green, bile-stained livers and pale or green kidneys were observed on necropsy. Microscopically, bile pigment was widespread in the liver and acid-fast intranuclear inclusion bodies were observed in renal tubular epithelium. Lead concentrations in livers and kidneys were 14.0-30.4 microg/g and 30.2-122 microg/g wet weight, respectively. Only one bird survived and this whooper swan continued to be treated with CaEDTA and activated charcoal. No lead shot was observed in the proventriculus and gizzard by radiography on day 64 and the blood lead concentration decreased from 2.9 microg/g to 0.09 microg/g during that same period. After 4 mo of rehabilitation, the whooper swan was returned to the wild. Lead intoxication continues to be a problem at Swamp Miyajima.     

Causes of mortality in the Mute swan Cygnus olor on the River Thames

Over the last 20 years there has been an unprecedented decline of the Mute swan on the River Thames. Between August 1979 and October 1981 a systematic attempt was made to collect as many swan corpses as possible. Of 94 swan corpses examined at post-mortem 57% died from lead poisoning due to ingestion of anglers' weights. Birds dying from lead poisoning had significantly higher liver and kidney lead levels. Kidney proved to be the most useful indicator tissue, the median lead level in birds with lead weights in their gizzard was 908 μg/g DM compared to 8 μg/g DM in birds without weights. More immature birds die from lead poisoning than other factors and significantly more corpses were found between the months of July-October.
Ultra-structure examinations revealed electron dense intra-nuclear inclusions in the cells of proximal convulated tubules of the kidney and X-ray analysis confirmed that these structures consisted of lead.     

Epidemiologic investigation of lead poisoning in trumpeter and tundra swans in Washington State, USA, 2000-2002

An observational study was conducted to determine the proportionate mortality of wild trumpeter (Cygnus buccinator) and tundra (Cygnus columbianus columbianus) swans that died during the winters of 2000-02 in northwestern Washington State, USA. Among 400 swans necropsied, 81% were lead poisoned (302/365 trumpeter swans; 20/35 tundra swans). Mortality started in mid-November and peaked from late December through mid-February; swan mortality that was not associated with lead poisoning was uniformly lower throughout the winter months. Lead poisoning was 24 times more likely to be the cause of death in swans found in Whatcom County compared to swans found in other locations in northwestern Washington State (95% CI: 12.7, 47.0). Mortality attributable to lead poisoning was twice as likely in adults as in juveniles (95% CI: 1.0, 4.2). Aspergillosis was documented in 62 trumpeter and two tundra swans, including 37 swans in which mortality was caused by lead poisoning. Males were twice as likely as females to have aspergillosis (95% CI: 1.1, 3.8). Traumatic injuries were documented in 37 trumpeter and seven tundra swans, including seven trumpeter swans with concurrent lead poisoning. Dead swans found outside Whatcom County were four times more likely to have traumatic injuries compared to those found in Whatcom County (95% CI: 1.6, 10.0). Overall, lead-poisoned swans were significantly less likely to have concurrent aspergillosis or traumatic injuries. There was no apparent association between grit ingestion (total mass or mass categorized by size) and lead poisoning or number of lead shot. Not surprisingly, lead-poisoned swans were more likely to have one or more lead shot compared to swans that died from other causes (OR 294; 95% CI: 92, 1,005); lead-poisoned swans were also more likely to have one or more nontoxic shot compared to swans that were not poisoned (OR 63; 95% CI: 19, 318). The source(s) of shot are unknown but likely are in or near Whatcom County, Washington.     

The louse Trinoton anserinum (Amblycera: Phthiraptera), an intermediate host of Sarconema eurycerca (Filarioidea: Nematoda), a heartworm of swans

The role of the louse Trinoton anserinum (F) as an intermediate host of Sarconema eurycerca (Wehr) was investigated in swans. 8.3% of healthy swans carried one to twelve lice per bird, dispersed contagiously. Injured and lead-poisoned swans were more heavily infected. The mouthparts appear designed to penetrate the hosts' skin; the mandibles are robust and asymmetric, and the maxillae have a serrated intercutting surface. 22% fed exclusively on blood and 33% on both blood and feather. All life-cycle stages fed upon blood and the barbs and barbules from down feathers; hooklets from contour feathers were only found in adults. 9% of lice were infected with developing nematode larvae in the head, thorax or abdomen. Lice labelled with Technetium 99 m moved towards the scapulas and the wings. Lice were found to be highly active and were mobile.     

Observations on a hybrid between the whooper swan (<Emphasis Type="Italic">Cygnus cygnus</Emphasis>) and the mute swan (<Emphasis Type="Italic">Cygnus olor</Emphasis>)

The swan obtained by hybridization between Cygnus cygnus and C.olor was delivered to the Moscow Zoo from the Leningrad Zoo in 1989. Between 2002 and 2004, it permanently remained with a female whooper swan in a large pond. The two swans behaved as a breeding pair, although the sex of the hybrid bird remained unknown. We observed no courtship displays addressed by the hybrid swan either to the female whooper swan or to other swans living in the pond. The hybrid swan was exteriorly closer to C. cygnus rather than to C. olor and showed increased aggressiveness. Its threatening behavior was intermediate between parental species in terms of threat displays and motor activity, although behavioral patterns characteristic of C.olor prevailed. The most frequent sounds in the vocal repertoire of the hybrid swan were close to the vibrant throbbing call of C.olor in terms of duration and frequency range. In contrast to these sounds, the call of the hybrid swan was noiselike, with distinct harmonic structure (as in the whooper swan except for extremely narrow interharmonic intervals). These vocal signals of the hybrid swan were shown to be intermediate between those of the parental species. In addition, the acoustic repertoire of the hybrid swan included rarely emitted sounds closely resembling elements of vocalizations of both C. cygnus and C. olor.     

Zinc toxicosis in a free-flying trumpeter swan (Cygnus buccinator)

A trumpeter swan (Cygnus buccinator) was observed near a mill pond in Picher, Oklahoma, USA. It became weakened and emaciated after about 1 mo, was captured with little resistance, and taken into captivity for medical care. Serum chemistry results were consistent with hepatic, renal, and muscular damage. Serum zinc concentration was elevated at 11.2 parts per million (ppm). The swan was treated for suspected heavy-metal poisoning, but died overnight. Gross postmortem findings were emaciation and pectoral muscle atrophy. Histopathologic lesions in the pancreas included mild diffuse disruption of acinar architecture, severe diffuse depletion or absence of zymogen granules, occasional apoptotic bodies in acinar epithelial cells, and mild interstitial and capsular fibrosis. Zinc concentration in pancreas was 3,200 ppm wet weight, and was similar to that reported in the pancreases of waterfowl known to be killed by zinc toxicity. Zinc concentrations in liver (154 ppm) and kidneys (145 ppm) also were elevated. Acute tubular necrosis of the collecting tubules of the kidneys was also possibly due to zinc toxicity. To the authors' knowledge, this is the first confirmed case of zinc poisoning in a trumpeter swan associated with mining wastes.     

A Fine Structural Study and Energy-dispersive X-ray Spectrometrical Analysis of the Metals Present in Ovarian and Other Tissues of Normal and Lead-poisoned Quail (Coturnix coturnix japonica)

Abstract The fine structure was investigated of body tissues from Japanese quails which had received lead acetate added to their diet. A double fixation using glutaraldehyde and postfixation in osmic acid was compared with a fixation in glutaraldehyde alone. Because of the lack of contrast obtained in the specimens from the latter fixation, the presence of metal residues within the tissues were readily observable under the electron microscope. The various metal residues found in storage bodies in ovarian tissues from lead-poisoned quails were analyzed by means of energy-dispersive X-ray spectrometrical analysis. Out of a total of eight electron-dense, metal-containing inclusions which were analyzed, two contained lead. Other elements found in the inclusions were chlorine, phosphorus, iron and calcium, the latter element not being present in detectable amounts in those inclusions which also contained lead. Some correlation was found between the morphology and the elemental composition of the metal residues.     

Biosorption and bioaccumulation of lead by Penicillium sp. Psf-2 isolated from the deep sea sediment of the Pacific Ocean

A lead resistant fungus was isolated from the Pacific sediment. It was associated with Penicillium according to its partial sequences of 18S and ITS. The fungus could grow in the presence of 24 mM Pb(NO₃)₂ in a liquid medium, and no growth inhibition was observed at 4 mM and below. When growing in the presence of 4 mM Pb(NO₃)₂, the fungus accumulated a large amount of lead granules in the cell, as well as adsorbed on the outer layer of cell wall, as observed under a transmission electron microscope. The intracellular lead deposited either in the vicinity of the cytoplasm membrane or in the vacuoles, and also could aggregate into large particles in the cytoplasm. However, lead was not adsorbed on the thick inner wall of the fungus. Energy dispersive X-ray spectroscopy analysis showed that these granules or particles mainly consisted of lead, and other elements could hardly be detected. Selected area electron diffraction analysis showed that there were regular crystalline lattices in the lead precipitates, indicating that they were actually in the form of crystals to some extent. Therefore, both intracellular bioaccumulation and extracellular biosorption had contributed to the high resistance of this fungus to lead. These results suggest that this fungus can be used in biotreatment as a lead trapper.     

Scanning electron microscopy of digest cells in the midgut epithelium ofBoophilus microplus

Scanning electron microscopy showed the presence of microvilli on the digest cells, but more importantly, it showed that the gut could be everted to expose the lumen surface of the cells to molecular analysis. X-ray microanalysis demonstrated the presence of iron and sulphur in the haematin granules within spent digest cells, confirming that these granules are end products of blood digestion in adult ticks.Part of material already accepted for a Ph.D. degree, University of Queensland, Brisbane, Australia.     

Experimental refuges for migratory waterfowl in Danish wetlands. I. Baseline assessment of the disturbance effects of recreational activities

1. Potential human sources of disturbance, including fishing, sailing, windsurfing and different types of waterfowl hunting, are described and their effects on autumn-staging waterbirds, including mute swan, wigeon and coot, were examined at a coastal wetland in Denmark from 1985 to 1988.
2. Bird and human distributions were superimposed on the distribution of submerged vegetation and water depths to identify the extent of spatial overlap; likewise, seasonal and diurnal temporal overlap in bird abundance, and human activities were defined. Behavioural and distributional reactions of waterbirds to different human activities, in terms of escape distances, disruption of activity patterns and redistribution, were assessed to establish their relative effects.
3. Sailing and windsurfing showed little spatial overlap, and fishing partial spatial overlap with bird distributions; these activities almost ceased before the peak in autumn bird numbers. Hunting showed a high degree of spatial and temporal overlap with bird presence.
4. Birds responded to windsurfing at greatest distances, whereas hunting (especially from mobile punts) caused the longest disruptions to activities of waterfowl. In terms of behaviour and redistribution, wigeon was more affected by shooting than was mute swan or coot. One or two mobile shooting punts reduced wigeon numbers, whereas numbers were unaffected by the presence of up to 4–6 stationary punts; fishing boats had no effect on wigeon abundance.
5. Hunting, especially shooting from mobile punts, was identified as the most disturbing human activity in relation to staging waterfowl in this area. Similar results and conclusions were reached in another comparable study area. The results have implications for refuge designs and zoning of disturbing recreational activities.     

Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X-Ray Microanalysis

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster , was examined by cytochemical methods and X-ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron-dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron-dense organelles and yolk granules.     

Effects of lead on the renal response to extracellular volume expansion

Subacute lead exposure has been observed to inhibit the natriuretic response to isotonic saline expansion in adult female rats. Three-week exposure to 0.5% lead acetate in drinking water resulted in a moderately high blood lead concentration of 57 micrograms/100 ml and up to 60% inhibition of the natriuretic response to extracellular volume expansion. This ability of lead to inhibit natriuresis following volume expansion (an induced stress) may be a more sensitive index of lead poisoning than alterations of renal function in nonstressed animals. Lead exposure had no effect on GFR or plasma aldosterone concentrations, and in the presence of large doses of DOCA (a mineralocorticoid) this inhibitory effect of lead was still persistent. Amiloride completely blocked the antinatriuretic effect of lead in volume-expanded lead-poisoned animals, causing a twofold increase in water and electrolyte excretion while having minimal effects on volume-expanded controls. It is concluded that lead interferes with the action of a "third factor," controlling natriuresis.     

High-performance liquid chromatographic determination of protoporphyrin and zinc protoporphyrin in blood

Zinc protoporphyrin and protoporphyrin free acid in blood were determined by high-performance liquid chromatography on a C₁₈ column. Results for 63 human blood samples obtained through a lead poisoning detection program compared favorably with the widely-used ethyl acetate—acetic acid extraction determination. Blood from 16 rats which had been maintained on water heavily spiked with chloroform or bromodichloromethane and blood from a lead-poisoned cow were examined by this procedure.     

Lead levels in the blood of Mute swans Cygnus olor on the River thames

In this paper blood lead levels in three categories of Mute swan are examined, (i) flock birds (ii) breeding birds and (iii) cygnets. From these regional, seasonal and sex variation for both 1980 and 1981 was examined. In addition a portable haematofluorometer was assessed to determine its possible use as an alternative to atomic absorption and spectrophotometry for determining a measure of lead exposure.
Very few swans on the River Thames had blood lead levels below the maximum acceptable level of 40 mgg/100 ml. In general lead levels increased with proximity to London and the swans on the tributaries consistently had the lowest levels which were always below the maximum acceptable level. Blood lead levels in flock birds were shown to be highest during the coarse fishing season and it was only during the close season that levels dropped to around 40 g/1OO ml. Breeding females had significantly higher lead levels than males and females with lead levels in excess of 200 g/1OO ml seemed to have a poor chance of producing cygnets or surviving to the next breeding season. Cygnet mortality was significantly higher on the lower Thames where blood lead levels were also known to be at their highest.     

Silicon-containing granules of rat liver,kidney and spleen mitochondria

Summary Isolated rat liver mitochondria containing granule aggregates (25–75 nm in diameter) and small (5–10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000g fraction consisting predominantly of granule aggregates (25–75 nm) composed of smaller granules (5–10 nm in diameter), and (2) a 10,000–30,000 g fraction of non-aggregated small granules (5–10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with ³¹Si and ⁶⁸Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.Supported by Grant GM-08229-13-15 from the National Institutes of Health, USPHSWe are grateful to the Perkin-Elmer Corporation and to their Western Branch Manager, Mr. Michael E. Mullen and Microscopist, Mr. Minoru Saito, for use of and assistance with the Hitachi H 500 transmission electron microscope with the scanning attachment, and to the Kevex Corporation for the use of the Kevex X-ray Spectrometer. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and X-ray spectra photographs and Darlene Lum for technical assistance in the laboratory     

Isolation and studies of the granules of the amebocytes of Limulus polyphemus,the horseshoe crab

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.     

Effectiveness of Action to Reduce Exposure of Free-Ranging California Condors in Arizona and Utah to Lead from Spent Ammunition

California condors (Gymnogyps californianus) released into the wild in Arizona ranged widely in Arizona and Utah. Previous studies have shown that the blood lead concentrations of many of the birds rise because of ingestion of spent lead ammunition. Condors were routinely recaptured and treated to reduce their lead levels as necessary but, even so, several died from lead poisoning. We used tracking data from VHF and satellite tags, together with the results of routine testing of blood lead concentrations, to estimate daily changes in blood lead level in relation to the location of each bird. The mean daily increment in blood lead concentration depended upon both the location of the bird and the time of year. Birds that spent time during the deer hunting season in two areas in which deer were shot with lead ammunition (Kaibab Plateau (Arizona) and Zion (Utah)) were especially likely to have high blood lead levels. The influence upon blood lead level of presence in a particular area declined with time elapsed since the bird was last there. We estimated the daily blood lead level for each bird and its influence upon daily mortality rate from lead poisoning. Condors with high blood lead over a protracted period were much more likely to die than birds with low blood lead or short-term elevation. We simulated the effect of ending the existing lead exposure reduction measures at Kaibab Plateau, which encourage the voluntary use of non-lead ammunition and removal of gut piles of deer and elk killed using lead ammunition. The estimated mortality rate due to lead in the absence of this program was sufficiently high that the condor population would be expected to decline rapidly. The extension of the existing lead reduction program to cover Zion (Utah), as well as the Kaibab plateau, would be expected to reduce mortality caused by lead substantially and allow the condor population to increase.     

Effects of ingested lead on antibody production in mallards (Anas platyrhynchos)

We examined the effects of lead ingestion on in vitro and in vivo indices of immune function in mallards (Anas platyrhynchos). Twenty-four mallard drakes were randomly divided into three groups (I, II, III), then assigned to treatment or control subgroups (n = 4). On day 0, all treatment birds were dosed orally with one number 4 lead shot (mean = 0.2 g). We challenged all individuals in each group with washed sheep red blood cells (SRBC) injected intraperitoneally on days 0 (Group I), 7 (Group II) or 14 (Group III), and collected blood for analyses 7 and 8 days after SRBC challenge. We measured and compared blood lead concentrations, in vitro lymphocyte transformation responses to phytohemagglutinin A and lipopolysaccharide, and hemagglutination titers to SRBC. Mean blood lead concentrations were elevated (P less than or equal to 0.04) in treatment birds at each sampling period. Large individual variability in lymphocyte stimulation responses precluded further analysis of those data. Hemagglutination titers to SRBC were lower (P less than 0.0001) in lead-poisoned ducks than in controls, suggesting that ingested lead may have immunosuppressive effects on mallards.     

鄱阳湖小天鹅越冬种群数量与行为学特征

2012年10月-2013年3月,采用直接计数法对鄱阳湖小天鹅Cygnus columbianus种群数量分布进行了5次调查。结果显示,各调查点小天鹅的种群数量均不稳定,可能与食物丰富度、水位等相关。各次调查中以第2次调查观测到的小天鹅数量最多,合计10756 只;调查区域中以鄱阳县白沙洲自然保护区调查到的小天鹅数量最多。5次调查中鄱阳县的小天鹅数量均占每次调查总数的56%以上,平均2239.80 只。小天鹅在各保护区不同湖泊的数量分布存在时间差异,但分布仅局限在几个湖泊中。调查显示,小天鹅的幼体比例为29.04%(n=1150)。采用瞬时扫描法对小天鹅越冬期的行为进行了研究,其用于静止(43.12%)和取食(27.44%)行为的时间较多,用于运动(16.82%)和梳理(11.48%)行为的时间较少,用于社会行为(1.14%)的时间最少。在小天鹅的日间行为节律中,静止行为高峰出现在07:00-08:00和17:00-18:00时段,取食行为高峰出现在16:00-17:00。下午取食强度的增加与冬季夜间长和气温低有关,鸟类必须获得足够的能量以维持其夜间的能量消耗。对小天鹅成幼体行为分配进行检验,发现除社会行为差异显著(Z=-2.310,P=0.021)外,其余行为差异均不显著。但成幼体的行为节律有较大差异,主要体现在静止和取食行为上。     

Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.