Secondary poisoning of eagles following intentional poisoning of coyotes with anticholinesterase pesticides in western Canada.

Records of eagles, coyotes (Canis latrans), and red foxes (Vulpes vulpes) necropsied at the Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada, between 1967 and 2002 were reviewed for cases suggestive of anticholinesterase poisoning. From 1993 to 2002, 54 putative poisoning incidents involving 70 bald eagles (Haliaeetus leucocephalus) and 10 golden eagles (Aquila chrysaetus) were identified. Of these, 50 incidents occurred in Saskatchewan, two were in Manitoba, and one occurred in each of Alberta and the Northwest Territories. The diagnosis was confirmed in eight instances by demonstration of pesticide in ingesta from eagles or known use of pesticide at the site together with brain cholinesterase (AChE) reduction of >50% in at least one animal. A presnmptive diagnosis of poisoning was made in 33 incidents based on brain AChE reduction of >50% in at least one animal; 13 incidents were considered suspicious because of circumstantial evidence of the death of eagles in association with other species and limited AChE reduction. Other wild species were found dead in 85% of the incidents involving eagles. Coyotes, foxes, black-billed magpies (Pica pica), and striped skunks (Mephitis mephitis) were associated with 34, six, six, and three incidents, respectively. There were eight additional incidents that did not involve eagles in which poisoning was diagnosed in coyotes. Carbofuran was identified in nine incidents. Carbamate poisoning was indicated on the basis of reactivation of brain AChE activity in two additional incidents. Brain AChE activity was not reduced from normal in eagles in four of seven incidents in which carbofuran was identified. The organophosplorous insecticide terbufos was found together with carbofuran in one incident. Brain AChE activity was measured in wild canids and in eagles in 15 incidents; in all of these incidents, brain AChE was redulced by >50% in at least one mammal, whereas this level of reduction occrred in eagles in only four incidents. Use of anticholinesterase pesticides to poison coyotes is illegal, but the practice continues and secondary poisoning of eagles is a problem of unknown proportions in western North America.

Antibodies to selected pathogens in free-ranging terrestrial carnivores and marine mammals in Canada.

Antibody titres to selected pathogens (canine adenovirus [CAV-2], feline herpesvirus [FHV], phocine herpesvirus [PHV-1], canine distemper virus, dolphin morbillivirus [DMV], phocine distemper virus [PDV], parainfluenza virus type 3 [PI3], rabies virus, dolphin rhabdovirus [DRV], canine coronavirus, feline coronavirus, feline leukaemia virus, Borrelia burgdorferi and Toxoplasma gondii) were determined in whole blood or serum samples from selected free-ranging terrestrial carnivores and marine mammals, including cougars (Fellis concolor), lynxes (Fellis lynx), American badgers (Taxidea taxus), fishers (Martes pennanti), wolverines (Gulo gulo), wolves (Canis lupus), black bears (Ursus americanus), grizzly bears (Ursus arctos), polar bears (Ursus maritimus), walruses (Odobenus rosmarus) and belugas (Delphinapterus leucas), which had been collected at several locations in Canada between 1984 and 2001. Antibodies to a number of viruses were detected in species in which these infections have not been reported before, for example, antibodies to CAV-2 in walruses, to PDV in black bears, grizzly bears, polar bears, lynxes and wolves, to DMV in grizzly bears, polar bears, walruses and wolves, to PI3 in black bears and fishers, and to DRV in belugas and walruses.

Pathogenesis of porcine Actinobacillus pleuropneumonia, part II: roles of proinflammatory cytokines.

The in vitro production of proinflammatory cytokines after stimulation with Actinobacillus pleuropneumoniae and the relation of these cytokines in vivo with the disease caused by A. pleuropneumoniae were investigated. Within 24 h, in vitro stimulation by A. pleuropneumoniae (serotype 1) preparations, including killed bacteria, bacterial culture supernatant, lipopolysaccharide, and bacterial extracts, porcine pulmonary alveolar macrophages (PAM) produced significant (P < 0.05) amounts of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) as measured by bioassays. The supernatants containing interleukin-8 from PAM after stimulation by bacterial preparations showed significant neutrophil chemotaxis, while bacterial preparations alone did not. After in vivo infection with A. pleuropneumoniae, the mean levels of TNF-alpha and IL-1 in serum, as measured by bioassays, were elevated 37- to 27836-fold for TNF-alpha and 11- to 5941-fold higher for IL-1 within 4 d post-infection, depending on the treatments, and remained elevated up to Day 7. Both cytokines were also detected in porcine lungs by bioassays and immunocytochemistry. The results indicated that both secreted and surface components of A. pleuropneumoniae can stimulate PAM to produce proinflammatory mediators. Neutrophil chemoattractants rather than bacterial components are the major factor causing acute lung inflammation. The elevation of TNF-alpha and IL-1 in pigs occurred coincident with the onset of acute clinical disease.

Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions.

Effects of Prudhoe Bay crude oil on hatching success and associated changes in pipping muscles in embryos of domestic chickens (Gallus gallus).

Fertile white leghorn chicken eggs were exposed to 0, 1, 2, 4, 6, 8 or 16 microliters of Prudhoe Bay Crude oil (PBCO) on day 9 of incubation. The effects of oil on pipping and hatching success, body weight gain after hatching, serum creatine kinase levels, and pathological changes in organ systems were assessed in embryos that had survived acute toxic effects and were alive on day 18 of incubation. Exposure to oil greatly reduced pipping and hatching success. Severe edema and hemorrhage in the pipping muscle, multifocal subcapsular hepatic necrosis, marked depletion of lymphocytes in the bursa of Fabricius with infiltration by heterophils, and occasional dorso-caudal subcutaneous edema were observed in treated embryos. Pipping muscles were heavier in oil-exposed embryos. Embryos exposed to 4 microliters of PBCO had significantly reduced gain in body weight post-hatching. Serum creatine kinase levels were significantly elevated in the oil-exposed embryos only at the time of hatching. There was no evidence that exposure to oil caused degenerative changes in pipping muscle cells.

Further studies of brain cholinesterase: cholinergic receptor ratios in the diagnosis of acute lethal poisoning of birds by anticholinesterase pesticides.

Cholinesterase (ChE) and the muscarinic (mAChR) and nicotinic (nAChR) cholinergic receptors were measured in the brains of 86 birds of 20 different species collected in Saskatchewan, Canada during 1991 and 1992. There was a strong correlation between ChE and mAChR, and the ratio of ChE: mAChR was 38% less variable than was ChE alone. In a hypothetical test for minimal acute lethal poisoning of all birds based on a reduction by 50% of the normal ChE measured in each, the use of a single diagnostic threshold value of the ChE: mAChR ratio had a diagnostic sensitivity of 98% and a specificity of 99%, thus making theoretically possible the diagnosis of acute lethal poisoning by anticholinesterase chemicals without the use of reference control values for ChE. Both ChE and mAChR were highly stable during 12 days of postmortem decomposition under several different daily temperature regimes. Substantial changes in these molecules occurred only under conditions of constant high temperature (36 C). Acute lethal poisoning by the organophosphate chlorpyrifos did not affect the density of mAChR. Postmortem decomposition did not appear to be an important confounding factor in the diagnostic interpretation of either ChE or the ChE: mAChR ratio except under hot climatic conditions.

The morphology and pathology of Besnoitia sp. in reindeer (Rangifer tarandus tarandus).

Four of five reindeer (Rangifer tarandus tarandus) obtained from a Besnoitia sp.- infected herd at the Assiniboine Park Zoo in Winnipeg, Manitoba, Canada, in October 1989, had evidence of mild dermatitis over the articular surfaces of carpal and tarsal joints. Cysts of Besnoitia sp., either surrounded by inflammatory reactions or without evident host response, were present within the dermis, submucosa of the nasal turbinates, periosteum, tendons, testes and hooves. The light microscopic and histochemical features of Besnoitia sp. from reindeer were indistinguishable from those of other Besnoitia spp. described in cattle, rodents and horses. The Besnoitia sp. cysts and organisms from reindeer were unique in that bradyzoite membrane micropores and cytoplasmic enigmatic bodies were not observed. Two cats were fed cysts of Besnoitia sp. but no oocysts were detected in feces for 90 days post-infection.

Parallel measurement of brain acetylcholinesterase and the muscarinic cholinergic receptor in the diagnosis of acute, lethal poisoning by anti-cholinesterase pesticides.

The activity of acetylcholinesterase (AChE) and the density of muscarinic cholinergic binding receptors (mCBR) were measured in brains from normal Japanese quail (Coturnix coturnix japonica) and from quail after lethal intoxication with diazinon. These were measured in brains from whole heads held at 25 C for 0 to 8 days after death. The maximum relative loss of activity due to post mortem decomposition alone during 8 days was 13% and 10% for AChE and mCBR, respectively. During post mortem decomposition, the ratio of AChE: mCBR activities remained constant at approximately 1.3:1 in normal brains while it was always less than or equal to 0.5:1 after intoxication with diazinon. Normal AChE activity could be estimated from mCBR density. Parallel measurement of AChE and mCBR may assist in the post mortem diagnosis of death due to acute poisoning with anti-cholinesterase pesticides when control specimens are not available.

Multicentric intramuscular lipomatosis/fibromatosis in free-flying white-fronted and Canada geese.

Over a period of 9 yr, seven white-fronted geese (Anser albifrons) and one Canada goose (Branta canadensis) with multiple intramuscular mesenchymal tumors were encountered in Saskatchewan (Canada) and one similarly affected Canada goose was seen on Prince Edward Island (Canada). The tumors in these birds consisted either of adipose tissue, fibroblastic tissue, or a mixture of both types of tissues. The high prevalence of this condition in white-fronted geese suggested a genetic influence.

Sequential study of the pathology of Prudhoe Bay crude oil in chicken embryos.

Ten microliters of Prudhoe Bay crude oil was applied to the shell of fertile leghorn chicken eggs on Day 9 of incubation. Gross and microscopic pathological changes were examined in embryos surviving 1, 2, 4, and 9 days after treatment. Liver necrosis, renal lesions, and extensive edema appeared 2 days after treatment and reached maximal prevalence 4 days after treatment. There was minimal repair of the lesions from Day 4 to Day 9 after treatment. Pathological changes, including liver necrosis, mineralization in the kidney, infiltration by a large number of heterophils in the liver and spleen, subcutaneous edema with formation of large blisters, and reduction in body weight and length were still present on Day 18 of incubation, close to hatching time.

The toxicopathology of Prudhoe Bay crude oil in chicken embryos.

Microliter amounts of Prudhoe Bay crude oil were applied to the shell of fertile leghorn chicken eggs on Day 9 of incubation. Gross and microscopic pathological changes were examined in embryos surviving 4 days after exposure. Gross lesions seen consistently were extensive edema, superficial zones of pale hepatic tissue, distension of the heart, and enlargement of the spleen. Histologically, the pale liver tissue corresponded to areas of hepatocyte necrosis. Additional histological lesions were cellular casts and mineralization in the renal tubules, distension of the glomerular capillaries, and accumulation of hematopoietic tissue in the liver and spleen. Dose-related increases in the number of mitotic figures in hepatocytes and in weights of liver, spleen, and heart were observed.

Comparative pathology of Prudhoe Bay crude oil and inert shell sealants in chicken embryos.

Pathological changes and body weights were compared in 9-day-old chicken embryos exposed to Prudhoe Bay Crude Oil (PBCO), pharmaceutical mineral oil, or a sealing film on the eggshell. Embryos exposed to 10 or 20 microliters PBCO developed marked ascites or subcutaneous edema, extensive liver necrosis, dilation of the heart, and cellular casts and mineralization in renal tubules. Wet and dry body weights were normal or above normal in embryos exposed to PBCO. No changes were found in embryos exposed to 10 microliters mineral oil; embryos exposed to 20 microliters mineral oil had slight dilation of the heart. Very few embryos survived in eggs that were wrapped in sealing film. All embryos that did survive had greatly reduced body weight and marked dilation of the heart. Four of these embryos had vacuolation of hepatocytes at the surface of the liver, one had superficial liver necrosis, and one embryo had slight ascites. These results indicate that the mechanism of the toxic action of PBCO on the avian embryo involves more than simple physical sealing of the shell.

An adenovirus infection of the kidney of Franklin's ground squirrels (Spermophilus franklini) in Saskatchewan, Canada.

During routine pathological studies of Franklin's ground squirrels (Spermophilus franklini) collected during a predator control program, basophilic intranuclear inclusions were found in the collecting tubule epithelium of the renal papillae in seven of 13 squirrels. This was associated with marked karyomegaly in affected cells. An inflammatory response was not seen in the adjacent tissues. Electron microscopic examination of affected cells demonstrated that the enlarged nuclei contained numerous virus-like particles. Autoculture and serial passage of renal medullary cells resulted in the isolation of virus particles producing intranuclear inclusions and cytopathic effect. The virus possessed properties typical of adenoviruses, but showed no evidence of hemagglutinating activity with a range of species of erythrocytes tested under several temperature conditions. The isolates were relatively host-cell specific; they failed to grow in hamster and rabbit kidney cell lines and in ground squirrel kidney cortical cells.

Multiple cutaneous mast cell tumors in a dog: a case report and brief review.

A spayed female yellow Labrador Retriever was presented to the small animal clinic at the Western College of Veterinary Medicine with an ulcerated, draining mass on the caudal aspect of the right thigh. Multiple small, hairless masses were also present on the thorax and in the flank folds. Fine-needle aspiration and cytological examination revealed well-differentiated mast cells and numerous eosinophils. A diagnosis of multiple cutaneous mast cell tumors was made. The clinical and cytological findings of this case are discussed, as well as the treatment and prognosis for mast cell tumors in general.

Clinical, gross, and histological findings in herring gulls and Atlantic puffins that ingested Prudhoe Bay crude oil.

Oral doses of 0, 1, 4, 5, 10, or 20 ml of Prudhoe Bay crude oil/kg body weight/day were given to herring gull and Atlantic puffin nestlings for 5 to 7 consecutive days. Gulls defecated substantial amounts of oil within 10 to 15 minutes after receiving a dose. Clinical signs and lesions occurred only in birds given greater than or equal to 10 ml oil/kg body weight/day. Gulls consumed less food and lost weight. Two categories of lesions were observed: those considered secondary to a primary toxic hemolytic disease, and those considered nonspecific reactions to stress. The former included phagocytosis of degenerate erythrocytes in the liver and spleen, hemoglobin resorption droplets in renal proximal tubule cells, and erythroid hyperplasia in the bone marrow; the latter included lymphocyte depletion in primary lymphoid tissues, an increase in heterophil: lymphocyte ratio in peripheral blood, lipid depletion and necrosis in adrenal steroidogenic cells, and an increased prevalence and severity of lesions in the bursa of Fabricius. These findings indicated that the primary target of oil toxicity was the peripheral red blood cell, but that significant stress-related lesions were also associated with ingestion of oil.

Hemograms and microscopic lesions of herring gulls during captivity.

Herring gulls (Larus argentatus) kept in captivity for a maximum of 28 days developed marked heterophilia and nonregenerative anemia. These hematologic findings remained abnormal throughout captivity. Concurrently, severe amyloid deposition was seen in vessel walls of the spleen and in other tissues, and vacuolar degeneration of catecholamine-producing cells of the adrenal glands was found in gulls necropsied at various intervals. Behavioral signs of stress were seen in the early stages of captivity, but were not seen in most gulls after day 3. Clinical signs referable to the microscopic lesions were not observed in this study. Therefore, gulls brought into captivity appear to adapt behaviorally, but maintain severe microscopic lesions and an abnormal hemogram.

Biochemical and functional disturbances in red blood cells of herring gulls ingesting Prudhoe Bay crude oil.

Heinz body hemolytic anemia developed in Herring Gull (Larus argentatus) nestlings given oral doses of 10 ml of Prudhoe Bay crude oil per kilogram of body weight per day for 5 days. Associated disturbances in red blood cells were increased amounts of reduced glutathione (GSH), peroxidation of membrane lipids, an increase in membrane permeability, and a decrease in the oxygen-carrying capacity of cyanomethemoglobin-convertible hemoglobin. Among groups of gulls given different cumulative doses of oil over a 6-day period, significant covariance with dose and dependence on dose was demonstrated for packed cell volume, hemoglobin, and red cell GSH. Rapid defecation of oil by gulls indicated that the effective dose was substantially less than the administered dose. Pronounced damage to red cells occurred in some birds administered oil for only 2 days. These data imply that the toxic effects of ingested oil may contribute significantly to the morbidity and mortality of oil-contaminated birds.

Morphological lesions in red blood cells from herring gulls and Atlantic puffins ingesting Prudhoe Bay crude oil.

Red blood cells from nestling herring gulls and Atlantic puffins that had ingested 10 ml or more of a Prudhoe Bay crude oil/kg body weight/day for four to five days were examined by light and electron microscopy. In stained smears, red blood cells from oil-dosed birds were characterized by anisocytosis, poikilocytosis, reticulocytosis, and Heinz body formation. In transmission electron micrographs, affected cells had intracytoplasmic and intranuclear Heinz bodies, a variety of abnormal cytoplasmic vesicles, degenerate mitochondria, absence of circumferential microtubules, abnormal shape, and crenulation of the plasma membrane. The latter two cell surface anomalies were evident in scanning electron micrographs. Identical lesions were present in red cells from gulls injected with phenylhydrazine. Reticulocytosis was the only change evident in blood from gulls made anemic by hemorrhage. These observations support the hypothesis that the toxicity of ingested Prudhoe Bay oil to red cells was exerted by oxidant chemical compounds.

Heinz-body hemolytic anemia from the ingestion of crude oil: a primary toxic effect in marine birds.

Hemolytic anemia developed in young herring gulls and Atlantic puffins given daily oral doses of a Prudhoe Bay crude oil. Anemia developed 4 to 5 days after the initiation of oil ingestion and was accompanied by Heinz-body formation and a strong regenerative response. The data evince a toxic effect on circulating red blood cells involving an oxidative biochemical mechanism and the first clear evidence of a primary mechanism of toxicity from the ingestion of crude oil by birds.