Masumi Sawada

Acute Pulmonary Toxicity Caused by Exposure to Colloidal Silica: Particle Size Dependent Pathological Changes in Mice:

To compare the pulmonary toxicity between ultrafine colloidal silica particles (UFCSs) and fine colloidal silica particles (FCSs), mice were intratracheally instilled with 3 mg of 14 nm UFCSs and 230 nm FCSs and pathologically examined from 30 minutes to 24 hour postexposure. Histopathologically, lungs exposed to both sizes of particles showed bronchiolar degeneration and necrosis, neutrophilic inflammation in alveoli with alveolar type II cell swelling and particle-laden alveolar macrophage accumulation. UFCSs, however, induced extensive alveolar hemorrhage compared to FCSs from 30 minutes onwards. UFCSs also caused more severe bronchiolar epithelial cell necrosis and neutrophil influx in alveoli than FCSs at 12 and 24 hours postexposure. Laminin positive immunolabellings in basement membranes of bronchioles and alveoli of UFCSs treated animals was weaker than those of FCSs-treated animals in all observation times. Electron microscopy demonstrated UFCSs and FCSs on bronchiolar and alveolar wall surface as well as in the cytoplasm of alveolar epithelial cells, alveolar macrophages and neutrophils. Type I alveolar epithelial cell erosion with basement membrane damage in UFCSs treated animals was more severe than those in FCSs-treated animals. At 12 and 24 hours postexposure, bronchiolar epithelial cells in UFCSs-treated animals showed more intense vacuolation and necrosis compared to FCSs-treated animals. These findings suggest that UFCSs have greater ability to induce lung inflammation and tissue damages than FCSs.

An Improved Isolation Technique for Bovine Neospora Species

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Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow.

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.

Deleterious effects in mice of fish-associated methylmercury contained in a diet mimicking the Western populations' average fish consumption.

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. Only a few contradictory epidemiological studies are currently available examining the impact of fish consumption on human populations. In the present study, we wanted to address whether a diet mimicking the fish consumption of Western populations could result in observable adverse effects in mice, and whether beneficial nutriments from fish were able to counterbalance the deleterious effects of MeHg, if any. In Europe and the United States, fish consumption varies widely between countries, from 11 to 100 g fish/day. A mid-range value of 25 g fish/day corresponds to a fish contribution to the total diet of 1.25% on a dry weight basis. We decided to supplement a vegetarian-based mouse diet with 1.25% of lyophilized salmon flesh (SAL diet), or 1.25% of a blend of lyophilized cod, tuna, and swordfish (CTS diet). Total mercury contents were 1.15±0.15, 2.3±0.1 and 35.75±0.15 ng Hg/g of food pellets for the control, SAL and CTS diets, respectively. After two months feeding, the CTS diet resulted in significant observable effects as compared to the control and SAL diets, encompassing decreased body growth, altered behavioral performance and increased anxiety level, modification of mitochondrial respiratory protein subunit concentrations in kidney and brain structures, modified gene expression patterns in kidneys, liver and muscles, and a decrease of dopamine concentrations in the hypothalamus and striatum. Our findings have health implications, firstly because 1.25% of CTS flesh in the diet corresponds to an average exposure to MeHg below the WHO provisory tolerable weekly intake (PTWI) (1.6 μg MeHg/kg of body weight/week), and secondly because many people in Western populations, among them women of child-bearing age, are exceeding the PTWI value (for instance, 35% of the French population inhabiting the Atlantic and Mediterranean coasts).

Systemic rhabdomyonecrosis and acute tubular necrosis in a dog associated with wasp stings

THE number of beeand wasp-sting fatalities in human beings is more than 30 per year in Japan; mass attacks by the hornet Vespa simillima are responsible for most cases. Reported clinical symptoms include anaphylactic shock, haemolysis and acute renal failure associated with elevated levels of serum enzymes and metabolites such as lactate dehydrogenase, creatine kinase and creatinine (Shilkin and others 1972, Tumwine and Nkrumah 1990, Franca and others 1994, Riches and others 2002). Haemoglobinuria and myoglobinuria may also accompany these clinical signs (dos Reis and others 1998). Cardiac dysfunction related to the release of catecholamines and/or myocardial necrosis, due to venom phospholipase A2 and melittin release, may be responsible for death (Ferreira and others 1994, 1995, Franca and others 1994). Systemic skeletal muscle necrosis and acute tubular necrosis are the major histological lesions associated with bee and wasp stings in human beings (Shilkin and others 1972, Mejia and others 1986, Tumwine and Nkrumah 1990, Franca and others 1994). Hepatocellular necrosis, myocardial necrosis and respiratory damage may also occur (Franca and others 1994). The precise pathogenic mechanisms of these necrotic changes in human beings are not known; the changes may result from direct damage by the venom or from secondary insult due to hypoxia in the tissues (dos Reis and others 1998). Pathological changes related to bee venom toxicity have been experimentally induced in rats (Azevedo-Marques and others 1992, Ferreira and others 1994, 1995, dos Reis and others 1998) and mice (Ownby and others 1997). A damaging effect of Africanised bee venom on skeletal muscle (AzevedoMarques and others 1992) and myocardium (Ferreira and others 1995) was demonstrated in experimentally envenomated Wistar rats. Melittin and phospholipase A2 from bee venom have the ability to induce skeletal muscle necrosis (Ownby and others 1997). To the authors’ knowledge, there are no reports concerning the pathology of natural cases of bee or wasp sting fatalities in the veterinary literature. This short communication describes the clinical and pathological features of a dog that suffered a fatal mass attack by hornets. A six-year-old, male crossbred dog with a history of being attacked by a hive of hornets (V simillima) (Fig 1), which were nesting in a tree near the dog’s home, showed signs of shock including hypotension, indicated by a weak femoral pulse and prolonged capillary refill time, dyspnoea and depression. The animal was presented at the Haruna Veterinary Clinic with a severely swollen lip and tongue, and evidence of stings was observed (Fig 2). Clinicopathological findings obtained from laboratory examination included: red blood cells 576 x 104/μl, white blood cells 272 x 102/μl, aspartate aminotransferase over 10,000 U/l, alanine aminotransferase 4910 U/l, total bilirubin 133 mg/l, blood urea nitrogen 860 mg/l, creatinine 122 mg/l and intense haemolysis. The animal was treated with fluid. Three days after the onset of clinical signs, the animal died. A complete postmortem examination was performed. Severe lung oedema with haemorrhage, tracheal collapse, dark kidneys (haemoglobin kidney) and haemorrhagic diathesis were observed in addition to the macroscopic lesions on the lip and tongue described above. Samples of the brain, liver, spleen, kidney, heart, lung, tonsil, oesophagus, stomach, large and small intestines, bladder, adrenal glands, pancreas, skeletal muscle from the foreand hindlimbs, skin and lymph nodes were collected and fixed by immersion in 10 per cent neutral buffered formalin. They were then dehydrated, embedded in paraffin wax, sectioned at 4 μm and stained with haematoxylin and eosin. Selected sections from the kidney were stained with haemoglobin stain using diaminobenzidine as a reagent (Hara and Ogawa 1978) and Berlin blue stain. Myoglobin immunohistochemistry was carried out with the aid of polyclonal anti-human myoglobin antibody (1:400, A0324; DakoCytomation) as a primary antibody and a streptavidin-biotin kit (DakoCytomation) (Shimada and others 1997). Sections from skeletal muscle were stained with phosphotungstic haematoxylin. Histological examination demonstrated signs of intravascular haemolysis and necrotic changes in the kidney (Fig 3) and skeletal muscle (Fig 4). There were numerous urinary casts in the lumen of the distal and collecting urinary tubules, which were crystal-like in shape, reddish-orange in colour (Fig 3a) and strongly positive for the haemoglobin stain (Fig 3b). Berlin blue stain demonstrated positive pigments in the cytoplasm of the urinary epithelium and in the lumen of the tubules (Fig 3c). Myoglobin immunoreactivity was shown in the lumen of the tubules and the luminal surface of the urinary tubules (Fig 3d). Skeletal muscle from the thoracic limb, pelvic limb, tongue, diaphragm and skin showed coagulative necrosis of myofibres with sarcoplasmic fragmentation (Fig 4a), occasional calcification and loss of striations (Fig 4b). Foci of necrosis with a mild degree of vacuolar degeneration of hepatocytes were observed throughout the liver. The lung lesions consisted of severe alveolar oedema and haemorrhage Short Communications

Severe Involvement of Cerebral Neopallidum in a Dog with Hepatic Encephalopathy

This report describes a unique distribution of cerebral cortical necrotic lesion, which was diagnosed as hepatic encephalopathy in a 2-year-old Maltese dog. The dog showed splenocaval shunt and small liver with marked hepatocellular fatty degeneration. Histopathologic examination revealed that diffuse laminar cortical necrosis composed of neuronal necrosis, marked infiltration of gitter macrophages, and astrogliosis were found bilaterally in the dorsolateral area of the cerebrum. No necrotic lesions were observed in the cerebral paleopallium and archipallium, the central gray matter, cerebellum, and brain stem. Astrocytes with large and pale nuclei (Alzheimer type II astrocytes) were apparent throughout the brain. Immunohistochemically, a decrease of immunostains for glutamine synthetase and glutamate transporter antibodies was seen in Alzheimer type II astrocytes and neuropil. This is, to our knowledge, the first report of extensive involvement of cerebral neopallidum in canine hepatic encephalopathy.

Isolation of Streptococcus canis from a Japanese raccoon dog with fibrinous pleuropneumonia

T. Murase, DVM, PhD, Department of Veterinary Microbiology, T. Morita, DVM, PhD, Y. Sunagawa, DVM, M. Sawada, DVM, PhD, A. Shimada, DVM, PhD, Department of Veterinary Pathology, K. Sato, DVM, PhD, Y. Hikasa, DVM, PhD, Department of Veterinary Internal Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan Isolation of Streptococcus canis from a Japanese raccoon dog with fibrinous pleuropneumonia

Inositol 1,4,5-triphosphate receptor protein immunohistochemistry of cerebellar Purkinje cells in two dogs with hypoglycemia.

Immunohistochemical study was performed on cerebellar Purkinje cells of two dogs with hypoglycemia using an antibody against the inositol 1,4,5-triphosphate receptor that is identical to the cerebellar Purkinje cell glycoprotein P400 (P400/InsP3R). In the cerebellar neocortex of an acute case of hypoglycemia, the P400/InsP3R staining of hypoglycemic Purkinje cells was heterogeneous: some peripheral dendrites, including spiny branchlets, were negative and others were stained with various intensities, although Purkinje cells were morphologically intact by hematoxylin and eosin (HE) stain. In a chronic case of hypoglycemia, almost all the dendrites of Purkinje cells of both the neo- and archicortex of the cerebellum were not stained with the P400/InsP3R antibody. This is in contrast to the normal dog where Purkinje cell bodies, axons, and dendrites, including spiny branchlets, are intensely stained by the P400/InsP3R antibody. These results suggest that P400/InsP3R immunolabeling of Purkinje cells decreased, despite their morphology being preserved by HE stain, and that the function of P400/InsP3R, especially in spiny branchlets that receive inputs originating from axon terminals of parallel fibers, may be impaired in hypoglycemia.

Bilateral Oedema with Angiopathy in the Dorsointernal Area of the Cerebral Hemispheres of a Domestic Cat

This report describes a feline case of oedema and angiopathy associated with the sudden onset of pyrexia and opisthotonus, followed by death one day later. Histologically, oedema and vascular lesions were found bilaterally in the dorso-internal area of the cerebral hemispheres. The vascular lesions consisted of perivascular exudation of periodic acid-Schiff-positive eosinophilic material and hyaline degeneration of the vascular walls. Immunohistochemically, the perivascular material reacted with anti-fibrinogen antibody; weak labelling for laminin was occasionally seen in the basement membranes of affected capillaries, arterioles and venules, in contrast to the strong labelling seen in the normal brain. Ultrastructurally, serum protein was observed in the space between the basement membranes of capillary endothelial cells and astrocytes, and vascular basement membranes were occasionally thickened. This is, to our knowledge, the first report of feline vasogenic oedema with a bilateral distribution in the cerebrum.