David Driemeier

Otimização da imunoistoquímica para detecção de herpesvírus bovino tipo 5 (BHV-5) em tecidos do sistema nervoso central fixados com formaldeído

In order to optimize immunohistochemical technique (IHC) for detection of Bovine herpesvirus type 5 (BHV-5) on formalin-fixed sections of central nervous system, different methods of enzymatic digestion, use of different antibodies and products for blocking of nonspecific reactivity were evaluated. The reactions showed the highest intensity of specific coloration and the minimum amounts of background when protease from Streptomyces griseus (0.1%) or proteinase K from Tritirachium album limber (0.05%) were used, incubating for 15 minutes at 37°C. Only two of the tested monoclonal antibodies specifically labelled BHV-5 antigen. The nonspecific reactions were blocked through incubation of tissues with casein (0.5%) for five minutes or powdered milk (2.5%) for 60 minutes or equine serum (2.5%) for 60 minutes. The optimized immunohistochemical method allowed the detection of BHV-5 antigen in histopathological archives.

Novel keratinase from Bacillus subtilis S14 exhibiting remarkable dehairing capabilities

ABSTRACT We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential.

Neosporosis as a cause of abortion in dairy cattle in Rio Grande do Sul, southern Brazil

Forty-six aborted bovine fetuses submitted to the Faculty of Veterinary, Department of Clinical Pathology, Universidade Federal do Rio Grande do Sul, were examined histopathologically. Non-suppurative inflammation was observed mainly in the brain and heart of 22 fetuses. Brain lesions consisted primarily of mononuclear inflammatory cell infiltrates with occasional foci of necrosis. Protozoa that reacted with Neospora caninum antisera were seen in 18 of the 22 (81.8%) brain specimens from fetuses with encephalitis. Blood samples collected from 223 Holstein cows on five dairy herds were tested for N. caninum antibodies by an immunofluorescent antibody technique. These samples were obtained from aborting cattle and normally calving cattle (control group). Overall, 11.2% of cows sampled had N. caninum antibodies at a dilution of 1:200. Seroprevalence was higher (P = 0.0053) in aborting (23.3%) than in non-aborting cows (8.3%). Association between seropositivity to N. caninum and abortion was found, with seropositive cows being 3.3 times more likely to abort than seronegative cows (OR = 3.33; 95% CI: 1.38, 8.062). Additionally, N. caninum antibodies were detected in sera from seven cows that had aborted fetuses with lesions suggestive of protozoal infection. These results suggest that N. caninum is an important cause of abortion in dairy cattle in Rio Grande do Sul State, Brazil.

Lysosomal Storage Disease Caused by Sida carpinifolia Poisoning in Goats

A neurologic disease characterized by ataxia, hypermetria, hyperesthesia, and muscle tremors of the head and neck was observed for 2 years in a flock of 28 Anglo-Nubian and Saanen goats on a farm with 5 ha of pasture. Six newborns died during the first week of life, and five abortions were recorded. The predominant plant in the pasture was Sida carpinifolia. The disease was reproduced experimentally in two goats by administration of this plant. Three goats with spontaneous disease and the two experimental animals were euthanatized and necropsied. No significant gross lesions were observed. Fragments of several organs, including the central nervous system, were processed for histopathology. Small fragments of the cerebellar cortex, liver, and pancreas of two spontaneously poisoned goats and two experimentally poisoned goats were processed for electron microscopy. Multiple cytoplasm vacuoles in hepatocytes, acinar pancreatic cells, and neurons, especially Purkinje cells, were the most striking microscopic lesions in the five animals. Ultrastructural changes included membrane-bound vacuoles in hepatocytes, Kupffer cells, acinar pancreatic cells, Purkinje cells, and the small neurons of the granular cell layer of the cerebellum. Paraffin-embedded sections of the cerebellum and pancreas were submitted for lectin histochemical analysis. The vacuoles in different cerebellar and acinar pancreatic cells reacted strongly to the following lectins: Concanavalia ensiformis, Triticum vulgaris, and succinylated Triticum vulgaris. The pattern of staining, analyzed in Purkinje cells and acinar pancreatic cells coincides with results reported for both swainsonine toxicosis and inherited mannosidosis.

Granulomatous encephalitis in a neurologically impaired goat kid associated with degeneration of Neospora caninum tissue cysts.

Congenital Neospora caninum infection was diagnosed in a Saanen goat from a farm in southern Brazil. The kid was unable to nurse and had difficulty rising, ataxia, and opistothotonos. The neurologic signs became more severe 3 days after birth, when it was euthanized. No gross lesions were observed at necropsy. Multifocal infiltrates primarily of mononuclear cells, nodular microgliosis, and perivascular cuffs of lymphocytes, plasma cells, and few neutrophils were observed in the brain, mostly in the cortex and adjacent to ventricles. Rare multinucleate giant cells were observed adjacent to inflammatory foci. Several tissue cysts with a thick wall that reacted strongly with polyclonal antiserum to N. caninum were in the cerebral cortex and medulla oblongata. Lesions were also present in heart, lungs, and liver, but N. caninum tachyzoites were not found. Distinguishing features in this goat kid included neurologic impairment resulting from congenital infection with N. caninum and the presence of granulomatous inflammation with rare giant cells associated with degeneration of tissue cysts.

Estudos histológico, histoquímico e ultra-estrutural de fígados e linfonodos de bovinos com presença de macrófagos espumosos ("foam cells")

Samples of liver, kidney, spleen, and hepatic, mesenteric, retromandibular, prescapular and mediastinic lymph nodes, collected from 12 groups of cattle of different ages from the State of Mato Grosso, were studied. A total of 120 bovines was examined. According to their history, the animals were kept in pastures where Brachiaria decumbens and Brachiaria brizantha were the predominant plants. Grossly there was a yellowish liver which was more evident after 24 hr. fixation with formalin. Hepatic and mesenteric lymph nodes had normal size, however, on the cut surface there were whitish grooves, extending from the cortex to the medular, and multiple small white nodular areas in the medular. Beside these areas there were red, sometimes hemorrhagic, foci. Microscopically all animals had cells with foamy citoplasm in the liver and in the hepatic and mesenteric lymph nodes, often forming multinucleated cells. In the lymph nodes these infiltrates were adjacent to necrotic, hemorrhagic areas. In the liver the foamy cells were irregularly disseminated throughout the parenchyma or formed nodules around the central vein. These cells did not stain with PAS but weakly with oil red. Ultrastructurally foamy cells and hepatocytes showed negative images of crystals involved partially or totally by membranes. Mediastinic, retromandibular and prescapular lymph nodes of the same animals showed no gross and microscopic alterations.

Study of experimentally induced lesions in sheep by grazing Brachiaria decumbens.

A histologic and ultrastructural study of the alterations found in the lymph nodes and livers of nine sheep with experimental cholangiohepatopathy by grazing on Brachiaria decumbens has been performed. Sheep were euthanized in three groups, on the 77th, 89th, and 150th days of the experimental feeding. The main gross lesions were whitish spots of multifocal distribution scattered throughout the hepatic parenchyma from all B. decumbens-grazed animals and whitish foci surrounded by reddened halos in the mesenteric and hepatic lymph nodes of sheep necropsied on the 150th. The principal histologic findings included hepatocellular cloudy swelling, marked multifocal cholangitis in the portal triads with bile duct proliferation and infiltration of macrophages and lymphocytes. Crystals were observed within bile ducts and surrounded by macrophages. Ultrastructurally, there were criytaloid structures within the macrophages and hepatocytes, which also presented hyperplasia of smooth endoplasmic reticulum. These findings suggest that hepatocytes were the initial target of the toxic effects, which depending on the degree of severity developed would cause both, subsequent cholangiopathy or occasional photosensitization. Additionally, the developmental stages of the hepatic lesions observed in this study have been presented.

Lysosomal storage disease in Sida carpinifolia toxicosis: an induced mannosidosis in horses

REASONS FOR PERFORMING STUDY This study reports a neurological disease unrecognised until now in ponies in southern Brazil. HYPOTHESIS Epidemiological data strongly suggests that the ingestion of Sida carpinifolia is involved in the aetiology. We tested the hypothesis that it is an acquired lyosomal storage disease. METHODS Following the death of 3 ponies, all ponies from the premises were closely monitored; epidemiological data and clinical findings carefully recorded. Fragments of several organs, including CNS, were fixed in neutral formalin and embedded in paraffin-wax. Sections were stained with haematoxylin and eosin. Representative sections of the cerebellum and trigeminal ganglia were submitted to lectin histochemical procedures. RESULTS The neurological disorder, characterised by stiff gait, muscle tremors, abdominal pain and death, was observed on a farm with 3 hectares of pasture. Three of 11 ponies died 15-20 days after they had been introduced into a new paddock heavily infested by the plant Sida carpinifolia. No significant gross lesions were observed. The main histological findings included multiple cytoplasmatic vacuoles in swollen neurones in the brain, cerebellum, spinal cord, autonomic ganglia (trigeminal and celiac ganglia), and submucosal and myenteric plexus of the intestines. In the kidneys, there was marked vacuolation of the proximal convoluted tubular cells. Sections of cerebellum and trigeminal ganglion were submitted to lectin histochemistry. The vacuoles in different cerebellar and ganglion cells reacted strongly to the following lectins: Concanavalia ensiformis, Triticum vulgaris and succinylated-Triticum vulgaris. CONCLUSIONS The pattern of staining coincides with that of both swainsonine toxicosis and inherited mannosidosis reports. The histopathological changes were similar to those described in S. carpinifolia spontaneous and experimental poisoning in goats. This disease seems to be similar to Swainsona, Oxytropis and Astragalus toxicosis. POTENTIAL RELEVANCE S. carpinifolia should be evaluated as a possible cause in the diagnosis of equine neuropathies.

Immunopathological investigations on bovine digital epidermitis.

Paraffin-embedded fragments of bovine digital skin lesions were sectioned and stained with Warthin-Starry, haematoxylin and eosin, Grocott’s methenamine silver and immunohistochemical techniques. Microorganisms observed in the silver-stained sections were classified into four major morphological groups. Spirochaetes were the most prevalent organisms, but bacillary and coccoid elements were also present in most sections. Immunohistochemical probing demonstrated that approximately 80 per cent, 46 per cent and 41 per cent of the digital and interdigital dermatitis sections stained positively with polyclonal antisera to Treponema pallidum, Campylobacter jejuni and Fusobacterium necrophorum, respectively. An unidentified branching filamentous organism (presumed to be an actinomycete) was consistently present in the sections of samples from mild interdigital lesions.

Aspectos clínicos e patológicos da intoxicação por Sida carpinifolia (Malvaceae) em caprinos no Rio Grande do Sul

This report includes the clinical and pathological studies of a lysosomal storage disease which spontaneously occurred in three flocs of goats e after consumption of Sida carpinifolia, the predominant plant in the paddocks where the animals were grazing. In the outbreaks a total of 25 out of 51 animals were affected. Post-mortem examination was performed on 11 goats. The disease was experimentally induced by dosing goats with Sida carpinifolia. The plant was administered in natura or dried to 3 animals. No clinical or pathological changes were observed in one goat dosed with Sida rhombifolia ad libidum during 40 days. Clinical signs of the poisoning were ataxia, hypermetria, muscle tremors in the head and neck and disorders of deglutition. The clinical signs were exacerbated by movement. After the surviving animals had been moved to other pastures and stopped eating the plant, clinical signs were still observed during 24 months. At necropsy, no significant gross lesions were observed. Microscopic lesions included various degrees of vacuolization in the cytoplasm of neurons and glial cells. Similar lesions were observed in the acinar pancreatic cells, hepatocytes, proximal convoluted tubular cells, follicular epithelial cells of the thyroid gland and macrophages of lymph nodes. In the surviving animals, mild neuronal cytoplasmic vacuolization was observed, and few cells were eosinophilic and shrunken. In these cases neurons, especially Purkinje cells, had disappeared. Through the histochemical study of the cerebellar sections, the lysosomal storage disease was characterized as an alpha-mannosidosis. The vacuoles within the Purkinje cells strongly reacted with lectins of Concanavalia ensiformis, Triticum vulgaris and succinylated Triticum vulgaris. The pattern observed in this investigation is similar to those seen in other poisonings by swainsonine-containing plants.