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Domoic acid (DA) is a neurotoxin reported to produce damage to the hippocampus, which plays an important role in memory. The authors inoculated rats intraperitoneally with an effective toxic dose of DA to study the distribution of the toxin in major internal organs by using immunohistochemistry, as well as to evaluate the induced pathology by means of histopathologic and immunohistochemical methods at different time points after toxin administration (6, 10, and 24 hours; 5 and 54 days). DA was detected by immunohistochemistry exclusively in pyramidal neurons of the hippocampus at 6 and 10 hours after dosing. Lesions induced by DA were prominent at 5 days following treatment in selected regions of the brain: hippocampus, amygdala, piriform and perirhinal cortices, olfactory tubercle, septal nuclei, and thalamus. The authors found 2 types of lesions: delayed death of selective neurons and large areas of necrosis, both accompanied by astrocytosis and microgliosis. At 54 days after DA exposure, the pathology was characterized by still-distinguishable dying neurons, calcified lesions in the thalamus, persistent astrocytosis, and pronounced microgliosis. The expression of nitric oxide synthases suggests a role for nitric oxide in the pathogenesis of neuronal degeneration and chronic inflammation induced by DA in the brain.

The effects of the neurotoxin domoic acid (DA) in the central nervous system of rodents (essentially rats and mice) after intraperitoneal administration have been profusely studied in the past. These observations have shown that the toxin induces similar symptoms and pathology in both species, but the lethality varies greatly. This article addresses the common and specific histopathological effects in rats and mice and the difference in sensitivity of these species to DA. Various sublethal and lethal doses were employed in mice (from 3 mg/kg to 8 mg/kg) to observe their neurotoxicity by using different histological techniques, and these results were compared with the pathological effects after the administration of LD50 in rats (2.5 mg/kg). Additionally we also detected the presence of this toxin in various tissues by means of immunohistochemistry. Our results showed that rats are more vulnerable than mice to the neurotoxic effects of DA after intraperitoneal inoculation: lethality was extremely high in rats and the toxin produced hippocampal damage in rats surviving the intoxication, while lesions were not observed in DA-inoculated mice. As for similarities between rats and mice, both displayed similar clinical signs and in both the toxin was detected in the hypophysis by immunohistochemistry, a brain region not reported to date as target of the toxin.

Pseudorabies virus (PRV) is an alphaherpesvirus that causes a neurological disease in many wild and domestic animals. The neuropathology elicited by PRV is quite consistent regardless of the host with the only exception of mink, in which it is characterized by a vasculopathy rather than by an encephalitis. In this study, we aimed to investigate the underlying pathogenic mechanism(s) of PRV infection in mink by using immunohistochemistry and laser capture microdissection (LCM) on material from naturally and experimentally infected animals. The inflammatory reaction induced by PRV was minimal or absent not only in the nervous system, where we identified a low number of macrophages and a few T lymphocytes, but also in the primary replication site, the oropharyngeal mucosa; however, the number of PRV-infected cells detected by immunohistochemistry was extremely high both in the peripheral mucosa and in the nervous tissue. On the other hand, the vascular pathology included parenchymal hemorrhages of various degrees and, in specific cortical areas of the brain, fibrinoid degeneration of the capillary walls. Detection of viral antigens by immunohistochemistry revealed infection of endothelial cells of capillaries situated both in the oropharyngeal mucosa and in the brain stem; the presence of PRV DNA in vessels was further demonstrated by PCR performed on LCM samples of brain capillaries. These results can be interpreted as supporting the idea that the different pathology of the disease in mink may be the consequence of an increased endotheliotropism of PRV in this species. Infection of the vessel wall may then lead to vascular pathology and impairment in endothelial cell function, resulting in a weak immune response to infection.

The general morphology of the vomeronasal vessels in adult cows was studied following a classic protocol, including optical, confocal and ultrastructural approaches. This anatomical work was completed immunohistochemically. The vomeronasal organ in cows is well developed, and its vessels are considerable in size. This fact allowed some functional properties of the vomeronasal arteries to be evaluated and, for the first time, their isometric tension to be recorded. Our functional studies were in agreement with the immunohistochemistry, and both corroborated the morphological data on the similarity between the vomeronasal vessels and those of the typical erectile tissue. In consequence, the vasoconstriction and vasodilation of the vomeronasal vessels would facilitate an influx and outflow of fluids in the vomeronasal organ, that is to say, this organ in cows would be able to work as a pump mechanism to send chemical signals to the vomeronasal receptor neurones.

Nitric oxide (NO) is a free radical gas with important roles in the host's immune response against viral infections. In this study, we examined the kinetics and distribution of nitric oxide synthase (NOS) expression during the early steps of infection of the porcine nervous system by the alphaherpesvirus pseudorabies virus (PRV). To this end, we examined changes in the expression of the three major NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), by immunohistochemistry in the trigeminal ganglia and brain of pigs inoculated intranasally with a virulent PRV strain. The results obtained show that infection of the porcine nervous system by PRV induced a rapid and progressive increment in NOS expression that coincided in timing, location, and magnitude with those of virus propagation in the nervous tissue. A major finding of this study was that PRV caused not only nNOS and iNOS induction in a variety of cell types, but also eNOS up-regulation in endothelial cells and neurons; therefore, all possible sources of NO are activated and probably contribute to the overproduction of NO during infection with the neurotropic alphaherpesvirus PRV in its natural host.

The enormous morphological diversity and heterogeneity of the vomeronasal system (VNS) in mammals--as well as its complete absence in some cases--complicates the extrapolation of data from one species to another, making any physiological and functional conclusions valid for the whole Mammalian Class difficult and risky to draw. Some highly-evolved macrosmatic mammals, like sheep, have been previously used in interesting behavioral studies concerning the main and accessory olfactory systems. However, in this species, certain crucial morphological peculiarities have not until now been considered. Following histological, histochemical and immunohistochemical procedures, we have studied the vomeronasal organ (VNO) and the accessory olfactory bulb (AOB) of adult sheep. We have determined: (1) that all structures which classically define the VNO in mammals are present and well developed, providing the morphological basis for functional activity. (2) that, conversely, there is only a scant population of scattered mitral/tufted cells. One morphological consequence of both details is that the strata of the AOB in adult sheep are not as sharply defined as in other species; moreover, the small number of the mitral/tufted cells in the AOB may imply that the VNS of adult sheep is not capable of functioning in the way a well-developed VNS does in other species. (3) the zone to zone projection from the apical and basal sensory epithelium of the VNO to the anterior and posterior part of the AOB, respectively, typical in rodents, lagomorphs and marsupials, is not present in adult sheep.

Summary An ultrastructural study was performed on the gills of juvenile Argentinian silverside, Odontesthes bonariensis. The gills are composed of two sets of four holobranchs and, in turn, each holobranch consists of a gill arch and two rows of caudolaterally projecting branchial filaments. From the dorsal and ventral surfaces of each filament, branchial lamellae radiate out as foldings of the epithelial layer. Gill rakers are present on each of the gill arches, on the anteromedial side of the arch opposite to the filaments. Gill rakers, gill arches and branchial filaments are covered by a stratified epithelium, whereas branchial lamellae essentially consist of a thin epithelial envelope containing capillaries. In the stratified epithelium, mucous cells, rodlet cells, granular cells, pavement epithelial cells and mitochondria-rich cells are identified. The thin epithelium that lines the lamellae comprises two cell types, outer and inner epithelial cells, and the capillary walls on the inside of the epithelial envelope are defined by pillar cells. The ultrastructure of all these cell types is described and our findings are discussed in light of the existing data on fish gill morphology. In the gills of juvenile Argentinian silverside is of particular interest the characteristics showed by mitochondria-rich cells, such as their arrangement in clusters of 2–3 cells and their small and depressed surface in contact with the aquatic milieu, features which strongly resemble those of euryhaline species.

In recent years, a new parasite that causes severe losses has been detected in farmed turbot ( Scophthalmus maximus ) in Northwestern Spain. Dead fish showed emaciation and cachexy caused by severe necrotizing enteritis that affected all portions of the digestive tract. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi . This study tested toltrazuril (symmetric triazinone) in order to determine whether this commercial compound can be used as an effective treatment for fish affected by E. scophthalmi . To this end, we used turbot experimentally infected with E. scophthalmi and treated with toltrazuril, and at various time points after exposure and treatment, their digestive tracts were assessed histologically. We found that the drug induced generalized vacuolization in the cytoplasm of parasitic cells, with destruction of mitochondria and enlargement of endoplasmic reticulum cisternae and of the perinuclear spaces. Despite the apparent pathological changes shown by the parasite, the particular dose, route and duration of toltrazuril administration used in this study did not ameliorate the clinical progress of the disease in turbot infected by E. scophthalmi.

Previous studies have shown that during acute infection of the porcine trigeminal ganglia (TG), Aujeszky's disease virus (ADV)-infected neurons are protected from apoptosis induced by the virus itself and by cells of the immune system. However, TG neurons productively infected by ADV finally die and are phagocytosed by adjacent cells, a fact that leads us to speculate that the inhibition of neuronal apoptosis by ADV may be temporary rather than absolute. To address this issue we used TG and brain stem from pigs during acute infection by ADV. Infected cells were detected by immunohistochemical staining of viral antigens, whereas apoptotic cells were identified with an anti-active caspase-3 antibody, the TUNEL assay and by transmission electron microscopy. The results obtained in this study support the contention that the inhibition of neuronal apoptosis by ADV is temporary, since activation of caspase-3 could be detected in infected neurons at late stages in infection and because foci of advanced neuronophagia contained neurons exhibiting typical ultrastructural features of apoptosis.

The morphological development of the vomeronasal organ (VNO) and accessory olfactory bulb (AOB) of the sheep from anlage to birth were studied by classical and histochemical methods using embryos and fetuses obtained from an abattoir with ages estimated from crown-to-rump length. Both VNO and AOB developed in a biologically logical sequence and completed their morphological development around day 98, at entry into the last third of the gestation period. A lectin with specificity for oligomeric N-acetylglucosamine labeled the sensory epithelium of the VNO, the vomeronasal nerves, and the nervous and glomerular layers of the AOB before birth. These results suggest that the vomeronasal system, which is well developed and functional in adult sheep, may be able to function at or even before birth in these animals (whereas in rodents, for example, this is precluded by the AOB not completing its development until after birth). Microsc. Res. Tech. 61:438–447, 2003. © 2003 Wiley-Liss, Inc.

Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.

The effects of the neurotoxin domoic acid (DA) in the central nervous system of rodents (essentially rats and mice) after intraperitoneal administration have been profusely studied in the past. These observations have shown that the toxin induces similar symptoms and pathology in both species, but the lethality varies greatly. This article addresses the common and specific histopathological effects in rats and mice and the difference in sensitivity of these species to DA. Various sublethal and lethal doses were employed in mice (from 3 mg/kg to 8 mg/kg) to observe their neurotoxicity by using different histological techniques, and these results were compared with the pathological effects after the administration of LD50 in rats (2.5 mg/kg). Additionally we also detected the presence of this toxin in various tissues by means of immunohistochemistry. Our results showed that rats are more vulnerable than mice to the neurotoxic effects of DA after intraperitoneal inoculation: lethality was extremely high in rats and the toxin produced hippocampal damage in rats surviving the intoxication, while lesions were not observed in DA-inoculated mice. As for similarities between rats and mice, both displayed similar clinical signs and in both the toxin was detected in the hypophysis by immunohistochemistry, a brain region not reported to date as target of the toxin.

This report describes the histological and immunohistochemical findings in an adult male genet ( Gennetta gennetta ) which died a few hours after being found lying in a forest in Lugo (north-western Spain). Subpleural, yellowish, firm foci were found in the lung. Histopathological examination confirmed a diagnosis of endogenous lipid pneumonia. Microscopical lesions that gave rise to a suspicion of canine distemper virus (CDV) infection included lymphoid depletion, non-suppurative encephalitis and demyelination in the central nervous system, and the presence of inclusion bodies in renal tubules. Immunohistochemical examination was performed with the streptavidin–biotin-complex method and a monoclonal antibody against the nucleocapsid protein (NP) of CDV. Antigen was detected in epithelial, nervous and lymphoid cells in several organs. This would appear to be the first report of distemper-like infection in a genet.

A 6-month-old female German Shepherd Dog died as a result of profuse oral bleeding. At postmortem examination, the oral cavity showed visible roots of the right mandibular fourth premolar and first molar teeth and, in addition, they were very mobile and compressible. Radiographs showed a generalized radiolucency in the body of the right mandible, with evidence of resorption of the affected alveolar bone. Histologically, the lesion of the right mandible was characterized by the lysis of bony structures and a non-malignant proliferation of blood-filled vascular spaces lined by a single layer of well-differentiated endothelial cells. The clinical, radiographic, and histologic presentation of this dog is consistent with that associated with Gorham-Stout disease, a rare bone disorder in humans.

A dog presented with cutaneous nodules, enlarged lymph nodes and oedema in limbs, face and abdomen. The diagnosis of visceral leishmaniasis was established by identification of Leishmania amastigotes within macrophages from skin and popliteal lymph node biopsies. At necropsy, lesions were found in different organs, but it was particularly striking to observe large areas of pallor in the myocardium. Histological examination revealed an intense chronic inflammatory reaction in many organs, and numerous macrophages were found to contain amastigote forms of Leishmania. The inflammatory reaction was especially severe in the heart, where large areas of the myocardium appeared infiltrated with huge numbers of mononuclear immune cells, causing cardiac muscle atrophy and degeneration. Despite the severe inflammation, the number of parasitized macrophages was low in the myocardium, as revealed by immunohistochemical staining of Leishmania amastigotes. Because cardiac involvement is not usually described in this condition, this dog represents a very rare case of canine visceral leishmaniasis with affection of the myocardium. SI

A young male Bernese mountain dog presented with neurologic abnormalities consisting of nonambulatory tetraparesis, generalized tremors, and depressed mental status. At necropsy only a mild enlargement of the lateral ventricles was seen. The histologic examination revealed the presence of eosinophilic deposits consistent with Rosenthal fibers (RFs) throughout the white matter of the central nervous system. There was also a marked proliferation of abnormally large astrocytes and limited myelin changes. RFs were most prominent in perivascular, subpial, and subependymal areas, where they were perpendicularly located, producing a pallisaded arrangement. immunohistochemically, RFs were strongly positive for glial fibrillary acidic protein (GFAP), and when they were examined ultrastructurally they appeared as electron-dense amorphous masses located within the processes of astrocytes, most particularly in the perivascular feet. The histologic and immunohistochemical findings of this canine case were consistent with the published neuropathologic descriptions of Alexander disease in humans and in a few dogs, a rare condition that in humans has been shown to be caused by dominant mutations in the GFAP gene.

The morphological development of the accessory olfactory bulb of the fetal pig was studied by classical and histo-chemical methods, and the vomeronasal organ and nasal septum were studied histochemically. Specimens were obtained from an abattoir and their ages estimated from their crown-to-rump length. The accessory olfactory bulb was structurally mature in fetuses of crown-to-rump length 21–23 cm, by which time the lectin Lycopersicum esculentum agglutinin stained the same structures as in adults (in particular, the entire sensory epithelium of the vomeronasal organ, the vomeronasal nerves, and the nervous and glomerular layers of the accessory olfactory bulb). These results suggest that the vomeronasal system of the pig may, like that of vertebrates such as snakes, be functional at birth.

The morphology of the soft tissue and supporting cartilage of the vomeronasal organ of the fetal pig was studied from early stages to term. Specimens obtained from an abattoir were aged by crown-to-rump distance. Series of transverse sections show that some time before birth all structures – cartilage, connective tissue, blood vessels, nerves, glands and epithelia – are well developed and very similar in appearance to those of the adult. Furthermore, in transmission electron microscopy photomicrographs obtained at this stage the vomeronasal glands exhibit secretory activity.

Squamous cell carcinoma of the oropharynx and esophagus was diagnosed in an adult Japanese bantam rooster. Grossly, a cauliflowerlike mass with irregular edges was found involving the ventrolateral surfaces of the caudal portion of the oropharynx and cervical portion of the esophagus. The large volume of the mass almost occluded the lumen of the alimentary passage. Histologically, the tumor consisted of irregular cords of pleomorphic epithelial cells that showed a disorganized pattern of growth and invaded the adjacent tissues. Keratinized epithelial cells and moderate numbers of keratin pearls were readily observed. The mitotic index was low, and, although the tumor was locally invasive, we found no evidence of vascular invasion or metastasis.

We examined the ability of pseudorabies virus (PRV) to induce and suppress apoptosis in the trigeminal ganglion during acute infection of its natural host. Eight pigs were intranasally inoculated with a virulent field strain of PRV, and at various early times after inoculation, the trigeminal ganglia were assessed histologically. PRV-infected cells were detected by use of immunohistochemistry and in situ hybridization, and apoptosis was identified by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Light and electron microscopy was also used for morphological studies. Apoptosis was readily detected among infiltrating immune cells that were located surrounding PRV-infected neurons. The majority of PRV-infected neurons did not show morphological or histochemical evidence of apoptosis, even including those neurons that were surrounded by numerous inflammatory cells and exhibited profound pathological changes. However, neuronal virus-induced apoptosis also occurred but at a sporadic low level. These findings suggest that PRV is able to block apoptosis of infected trigeminal ganglionic neurons during acute infection of swine. Furthermore, our results also suggest that apoptosis of infiltrating inflammatory cells may represent an important viral mechanism of immune evasion.

Objectives: To determine the relationship between skin collagen IV and basement membrane changes during aging, a total of 35 women who had been admitted for surgery, were studied. Methods: Subjects were arranged into six age-groups (from 35 to 60 years). Skin biopsies were performed in all patients and the samples were taken from a site 6 cm above the pubic symphysis. The collagen IV content and the epithelial basement membrane were analyzed by using immunohistochemical, transmission electron microscopy and computer-assisted image analysis methods. The skin collagen IV content was measured by an image analysis program and expressed in arbitrary units of integrate optical density, and, the basement membrane thickness was expressed in nanometers. Results: Type IV collagen content decreased with age after 35 years (r = −0.9561). The epithelial basement membrane thickness increased significantly with age (r = 0.98192; P < 0.01) and there is an inverse correlation between these two parameters (r = −0.990502). Conclusions: Although type IV collagen is a basement membrane component and declines with aging, the total thickness of this membrane increases, which suggests a reduction in tissue turnover.

Our paper describes pathologic and ultrastructural features of a canary (Serinus canarius) infected by Atoxoplasma sp. Histologically, numerous mononuclear cells were observed in intestinal mucosa, liver, and spleen. Most of these cells contained a variable number of protozoal-like organisms. These organisms were identified as Atoxoplasma sp. by ultrastructural examination of reprocessed paraffin-embedded tissues.