Immunization of ducks for type C botulism

A single subcutaneous immunization with a vaccine used for protecting ranch mink (Mustela vison) against type C botulism reduced morbidity and mortality in mallard (Anas platyrhynchos) and northern pintail (Anas acuta) ducks challenged with approximately 4.5 x 10(4) and 2.25 x 10(4) mouse lethal doses (MLD50), respectively, of botulinum toxin at 10 and 15 days post-immunization (pi). There was no significant protection at 5 days pi. Protection persisted in mallards for 90 days pi. To simulate use of vaccine as a part of treatment of sick birds in the field, mallards were exposed to toxin and, when clinical signs were evident, each bird was treated by intraperitoneal injection of type C botulinum antitoxin and one-half of the birds were immunized. Immunization had no significant effect on recovery from intoxication. At 10 days posttreatment, all birds were challenged with toxin. Clinical signs and mortality were significantly less frequent among immunized birds than among non-immunized birds after the second exposure. Immunization might be useful as part of the treatment regimen in botulism outbreaks.     

Avian botulism during late autumn and early spring in Saskatchewan

Two outbreaks of botulism in central Saskatchewan in which mortality of waterfowl continued into late autumn and then recurred in the same marshes the following spring are described. Small numbers of birds were involved in each instance. Dabbling ducks (predominantly mallards, Anas platyrhynchos and pintails, Anas acuta) and American coots, Fulica americana were affected most commonly in autumn, whereas only diving ducks (predominantly lesser scaup, Aythya affinis) were found to be involved in spring. Live maggots present in carcasses despite sub-freezing temperatures were the probable source of intoxication in the autumn; the source of toxin in the spring was not determined.     

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0xLD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.     

Health effects following chronic dosing with tungsten-iron and tungsten-polymer shot in adult game-farm mallards

Permanent approval of shot composed of tungsten-iron and tungsten-polymer for waterfowl hunting by the U.S. Fish and Wildlife Service was pending the results of the present study that examined the health and reproductive effects of the two shot types on mallards (Anas platyrhynchos) over a 150-day period. We collected data pertaining to the effects of tungsten-iron and tungsten-polymer shot on mortality, body weight, organ weight, tissue pathology, and shot erosion. Thirty-two bird groups (sexes equal) of adult mallards were dosed orally with eight #4 steel shot (control), eight #4 tungsten-iron shot, or eight #4 tungsten-polymer shot on days 0, 30, 60, 90, and 120 of a 150-day trial (26 January 1998 to 25 June 1998). An additional 12 mallards (sexes equal) were dosed orally with eight #4 lead shot (positive control) on day 0 of the study. All lead-dosed ducks died by day 25, whereas no ducks died in the other treatment groups. Significant liver hemosiderosis was present in all control and tungsten-iron-dosed males, in five of eight control and three of eight tungsten-iron-dosed females, and in one tungsten-polymer-dosed male examined. The rate of shot erosion was highest for tungsten-polymer shot (99%), followed by tungsten-iron (72%), and steel (55%) shot. Tungsten-iron or tungsten-polymer shot repeatedly administered to adult mallards did not have deleterious health effects during the 150-day trial based on mortality, body weights, organ weights, and histology of the liver and kidneys.     

Starvation, staphylococcosis, and vitamin A deficiency among mallards overwintering in Saskatchewan.

During January to March 1991, 38 mallards (Anas platyrhynchos) found dead from a group of approximately 600 overwintering on the South Saskatchewan River were examined. Thirty birds died from starvation, four had disseminated Staphylococcus aureus infection, and the cause of death of four birds was not determined. All six birds from which the esophagus was examined microscopically, including the four birds with staphylococcosis, had squamous metaplasia of the submucosal glands, a lesion pathognomonic for vitamin A deficiency. Vitamin A deficiency may occur in mallards and other waterfowl that overwinter north of traditional wintering areas and rely on grains deficient in carotenoids.     

Effects of lead shot ingestion on selected cells of the mallard immune system

The immunologic effects of lead were measured in game-farm mallards (Anas platyrhynchos) that ingested lead shot while foraging naturally, mallards intubated with lead shot, and unexposed controls. Circulating white blood cells (WBC) declined significantly in male mallards exposed to lead by either natural ingestion or intubation, but not females. Spleen plaque-forming cell (SPFC) counts were significantly lower in mallards intubated with lead pellets compared to controls. Declines in WBC and SPFC means with increasing tissue lead concentrations provide further evidence that lead exposure reduced immunologic cell numbers. Hormonal activity and diet may have influenced the immunologic effects of lead exposure in this study.     

Serum levels of luteinizing hormone, prolactin, estradiol and progesterone in laying and nonlaying mallards (Anas platyrhynchos)

Temporal changes of circulating serum hormones were measured to compare the reproductive endocrinology of laying and nonlaying mallards. In this study all sixteen control mallards left with their mates laid eggs, while only one of sixteen mallards stressed by daily movement into new pens, laid eggs. Serum levels of luteinizing hormone (LH), prolactin, estradiol, and progesterone were significantly lower (P less than 0.05) in stressed nonlaying mallards than in laying mallards over the 7-week period. Within 1 week of the rotation treatment, LH concentrations in stressed mallards averaged (means +/- SEM) 2.72 +/- 0.19 ng/ml and were significantly lower (P less than 0.05) than LH levels in the controls (3.62 +/- 0.18 ng/ml). After 7 weeks, injections of luteinizing hormone releasing hormone (LHRH) induced a greater change in circulating LH levels in stressed mallards (2.1 +/- 0.3 ng/ml) than in breeding control mallards (0.9 +/- 0.2 ng/ml). These data demonstrate that the lack of reproduction in stressed mallards was associated with LHRH-sensitive pituitary pools of LH, despite their low concentrations of serum LH. These data suggest that the block in reproduction is a failure of the hypothalamus to produce or release releasing hormones. The serum hormone levels of the control mallards varied temporally with stages in the nesting cycle. LH levels increased with the onset of nesting activity, and showed marked fluctuations during the laying period. LH levels fell at the onset of incubation but increased after loss of clutch. Estradiol levels were highest prior to the laying of the first egg and their peak coincided with the initial nest building behavior of the females. Progesterone levels increased sharply with the laying of the 2nd-4th eggs, decreased sharply with the laying of the 6th egg, and then increased slightly at the end of the nesting cycle. Prolactin levels were initially low but gradually increased with laying and incubation activity, declined with loss of clutch, and increased again with renesting activity. Prolactin levels in the stressed mallards also increased (P less than 0.01) over the 7-week period, but significantly less (P less than 0.05) than in layers.     

American wigeon mortality associated with turf application of diazinon AG500.

Nine fairways of a golf course located in Bellingham, Washington were treated with diazinon AG500 at a target application rate of 2.2 kg active ingredient (AI) per ha. The chemical application with a "boomless" sprayer resulted in a variable distribution of diazinon residues on the turf (associated with a deep thatch layer) that ranged from 1.0 to 6.2 kg AI/ha. The diazinon-treated turf was irrigated with 1.3 cm of water immediately following application. The post-irrigation diazinon residue levels ranged from 100 to 333 ppm (mean = 209; SD = 88; n = 8). These residue levels were higher than expected based on results of turf studies in other regions of the United States. Eighty-five American wigeon (Anas americana) died after grazing on one treated fairway on the day of application following irrigation. The brains of all 85 wigeon were analyzed for acetylcholinesterase (AChE) activity. Wigeon that died on the study area (n = 85) showed 44% to 87% depression of AChE (mean = 76%; SD = 7.1%) when compared to control wigeon (n = 3; AChE Activity = 1.86) AChE levels. Upper GI tract contents of 15 of the 85 dead wigeon contained 0.96 to 18.1 ppm diazinon. Extensive carcass searches revealed no other avian mortality attributable to diazinon toxicity on the treated study area. Although initial post-irrigation diazinon residues in grass samples were higher than expected, diazinon levels in grass samples on day seven post-application had declined to an average of 29 ppm. American wigeon appear to be vulnerable to exposure to diazinon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell surface expression of a GPI-anchored form of mouse acetylcholinesterase in Klpmr1Delta cells of Kluyveromyces lactis

The mouse acetylcholinesterase AChE(H) was expressed in the yeast Kluyveromyces lactis. The AChE(H) activity was detectable in intact cells whereas it was absent in the culture media. Glucanase treatment and immunoelectron microscopy data indicated that AChE(H) is anchored to plasma membrane and that the mouse GPI-signaling is compatible with the K. lactis targeting machinery. The AChE(H) was also expressed in a K. lactis strain carrying an inactivated allele of KlPMR1, the gene coding for a P-type Ca(2+)-ATPase of the Golgi apparatus. This mutant displays changes in protein glycosylation and cell wall structure. The AChE(H) activity detected in Klpmr1Delta cells was more than twofold higher than that observed in wild-type cells. The combination of AChE expression and anchoring with the characteristics of Klpmr1Delta strain of K. lactis resulted in yeast cells displaying high AChE activity. This could be regarded as a novel sensing unit to be employed for detecting AChE inhibitors as pesticides.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.     

Wild Mallards Have More “Goose-Like” Bills Than Their Ancestors: A Case of Anthropogenic Influence?

Wild populations of the world’s most common dabbling duck, the mallard (Anas platyrhynchos), run the risk of genetic introgression by farmed conspecifics released for hunting purposes. We tested whether bill morphology of free-living birds has changed since large-scale releases of farmed mallards started. Three groups of mallards from Sweden, Norway and Finland were compared: historical wild (before large-scale releases started), present-day wild, and present-day farmed. Higher density of bill lamellae was observed in historical wild mallards (only males). Farmed mallards had wider bills than present-day and historical wild ones. Present-day wild and farmed mallards also had higher and shorter bills than historical wild mallards. Present-day mallards thus tend to have more “goose-like” bills (wider, higher, and shorter) than their ancestors. Our study suggests that surviving released mallards affect morphological traits in wild population by introgression. We discuss how such anthropogenic impact may lead to a maladapted and genetically compromised wild mallard population. Our study system has bearing on other taxa where large-scale releases of conspecifics with ‘alien genes’ may cause a cryptic invasive process that nevertheless has fitness consequences for individual birds.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.     

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.     

二化螟体内乙酰胆碱酯酶的分布及纯化方法

研究了二化螟Chilo suppressalis乙酰胆碱酯酶（AChE）的体躯和亚细胞分布,并用凝胶过滤层析和2种亲和层析方法从二化螟幼虫体内分离、纯化乙酰胆碱酯酶。结果表明：二化螟幼虫乙酰胆碱酯酶的活性主要集中于头部和胸部,而成虫胸部乙酰胆碱酯酶的活性最低,显著低于头部和腹部。成虫体内AChE的活性明显高于幼虫。在亚细胞的分布上,乙酰胆碱酯酶主要位于膜上（86%）,近46%的活性存在微粒体中。在3种纯化乙酰胆碱酯酶的方法中,以3-羧基苯基-乙基二甲基铵作配体的亲和层析法纯化效果最佳,乙酰胆碱酯酶的最高纯化倍数为536.05倍,产率30.46%。     

Biochemical studies of the actions of ethanol on acetylcholinesterase activity: ethanol-enzyme-solvent interaction

1. Biochemical studies of the actions of ethanol on the activity of acetylcholinesterase (AChE), isolated from electric eel (Electrophorus electricus) and purified by affinity chromatography, were performed to elucidate ethanol-enzyme-solvent interactions. 2. Ethanol at a low concentration [( EtOH] = 2.7-200 mM) was found to enhance AChE activity slightly and systematically. 3. This observation was consistent with the result from enzyme-kinetic studies that ethanol might noncompetitively activate AChE activity at this lower concentration range. 4. If ethanol alters the hydrophobic site interaction on the enzyme and subsequently induces a favorable conformation for the active center of the enzyme, then a slight increase in the AChE activity in the presence of a low concentration of ethanol will be observed. 5. This speculation was supported by the finding of ethanol's ability to perturb the inhibition of AChE activity by tetrabutylammonium bromide and to affect hydrophobic interaction between this salt and AChE, as investigated by enzyme activity and microcalorimetric measurements. 6. The ethanol effect on the activity of this soluble AChE was found to be distinguishable from that on a membrane-bound AChE. 7. Furthermore, to elucidate the effect of ethanol-solvent interaction on AChE activity, enzyme activity in the presence of much higher concentrations of ethanol was also examined. 8. At [EtOH] greater than 800 mM, ethanol can perturb the structure of water around hydrophobic areas of AChE, causing an instability in the enzyme conformation and subsequently decreasing AChE activity.     

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.