Caroline Argenta Pescador

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.

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.

Intoxicação experimental por Trema micrantha (Ulmaceae) em caprinos

Green leaves of Trema micrantha were ground and mixed with water in a domestic blender and then administered by stomach tube to seven goats. One additional goat was fed ad libidum with the green leaves of T. micrantha. Clinical signs were observed in six goats that became ill 2 days after having been dosed with or fed the plant. There were five deaths, which occurred until 4 days after ingestion. Affected goats remained static during long periods and kept their heads low. Incoordination, rhythmical movements of the head, apathy, anorexia, and tenesmus were also noticed. Paddling movements and coma were seen in one goat. T. micrantha was toxic at dosages of 30 g/kg or higher. The most significant gross lesions were observed in the livers, which were yellowish, friable, and with pronounced lobular pattern. Their cut surfaces were reddened and depressed areas alternated with whitish ones. The liver of one goat was slightly but homogeneously reddened but did not show accentuated lobulation. Petechial haemorrhages in the region between the chest and scapula, in the epicardium, mediastinum and serosal membranes of the abdominal organs were also observed. The most important histologic finding was hepatic centrilobular coagulative necrosis, which was associated with congestion, haemorrhages and degenerative changes in the circumjacent hepatocytes. Additional microscopic lesions were found in the nervous system and included perineuronal and perivascular edema and swollen neurones, especially those of the frontal cortex.

Padronização da técnica de imuno-histoquímica para raiva em amostras de tecido do sistema nervoso central de bovinos fixadas em formol e emblocadas em parafina

For standardization of the rabies immunohistochemistry technique, five samples of central nervous system (CNS) of cattle naturally infected with rabies virus were examined. One polyclonal antibody and two monoclonal antibodies were used. The following reagents were evaluated for antigen retrieval: XIV protease, proteinase K and citrate buffer (pH 6.0) boiling at 100oC during 15 minutes in bain-marie. Detection of rabic antigen was possible with the three antibodies tested. The polyclonal antibody was superior to the monoclonal antibodies, demonstrating good results with the three antigen retrieval protocols. The highest intensity staining was obtained with the citrate buffer and heat. The immunohistochemistry technique demonstrated the presence of viral antigens in the cytoplasm of neurons, in form of aggregates or with round or oval shape. The antigens were found as single or multiples inclusion bodies in the neurons. Immunohistochemistry is a fast method that can be used in routine procedures in cases where rabies is suspected, especially when the brain is submitted to the laboratory as formalin-fixed fragments or when samples could not be immediately shipped. The technique is also useful for retrospective studies.

Perdas reprodutivas associadas com infecção por Toxoplasma gondii em caprinos no sul do Brasil

Toxoplasma gondii was implicated with reproductive losses in a goat herd in Rio Grande do Sul, Brazil. Gross changes were present in 2 out of 6 fetuses/offsprings submitted for diagnosis and included enlarged, pale mesenteric lymph nodes, and edematous, mottled red and tan lungs. Microscopic changes were observed in most fetuses and were especially characterized by lymphoplasmacytic infiltration in the brain and lungs. Other histological changes included lymphoplasmacytic interstitial nephritis, necrotizing lymphadenitis and periportal lymphoplasmacytic cell infiltrates. While bacteriological and direct immunofluorescent antibody tests for Leptospira sp. were negative in samples from all cases, immunohistochemical and PCR procedures for Toxoplasma gondii reacted positively in most of them. Antibody titles against T. gondii varying from 1:512 to 1:2048 were detected in serum samples from the mother goats of these aborted (1), stillborn (3) or dead newborn (2) kids. This paper describes the clinical, pathological, serological, molecular and immunohistochemical findings of a Toxoplasma gondii-infected goat flock.

Co-infection of Pneumocystis carinii f. sp. suis and porcine circovirus-2 (PCV2) in pig lungs obtained from slaughterhouses in southern and midwestern regions of Brazil.

EDNA M. CAVALLINI SANCHES, MAURO R. BORBA, ANDRÉIA SPANAMBERG, CAROLINE PESCADOR, LUÍS G. CORBELLINI, ANA P. RAVAZZOLO, DAVID DRIEMEIER and LAERTE FERREIRO Sections of Veterinary Mycology, Departamento de Patologia Clı́nica Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 9090, 90540-000, Porto Alegre, RS, Brazil, and Sections of Pathology, Departamento de Patologia Clı́nica Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 9090, 90540-000, Porto Alegre, RS, Brazil, and Sections of Immunology, Departamento de Patologia Clı́nica Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 9090, 90540-000, Porto Alegre, RS, Brazil

Co-infection by porcine circovirus type 2 and porcine parvovirus in aborted fetuses and stillborn piglets in southern Brazil

Porcine circovirus types 1 and 2 (PCV1, PCV2) and porcine parvovirus (PPV) are widespread in pig populations around the world. Nevertheless, only PCV2 has been associated with different clinical syndromes, thus representing a major problem to the pig industry. The association of cases of swine abortions and stillborns with PCV1 and PCV2 and PPV was studied retrospectively (2005-2007). Additional pathogens were also investigated in lesioned fetuses. The studied litters included stillborn piglets and several mummified fetuses of varied sizes. Ventricular dilatation, myocardial pale areas, and mesocolic edema were the gross lesions. Escherichia coli was detected as co-infecting with PCV2 the cases in which mesocolic edema was seen. Microscopic lesions included non-suppurative myocarditis, myocardial necrosis and fibrosis, mineralization foci and intranuclear inclusion bodies in cardiomyocytes, and interstitial mononuclear pneumonia. Samples from 7 (5.78 per cent) of 121 aborted fetuses and stillborn piglets had lesions consistent with a viral cause and showed both positive anti-PCV2 immunostaining as well as PCV2-PCR. In samples from 3 (2.47 per cent) of these 7 fetuses, co-infection with PPV was confirmed by Nested-PCR. Both viruses were detected in fetuses at different stages of gestation. Viral antigens of PCV2 were detected by immunohistochemistry mainly in macrophages and myocytes. PCV1 individually was not detected in any of these affected fetuses, but it was associated with PCV2 and/or PPV in some of them. These findings indicate that PCV2 alone or in association with PPV should be kept in mind when investigating causes of infectious abortion in pigs in Brazil.

Detection of Pneumocystis spp. in lung samples from pigs in Brazil

The genus Pneumocystis is composed of opportunistic fungi currently considered as specific pulmonary pathogens in humans and other mammals. In pigs, Pneumocystis pneumonia (PcP) could create significant economical losses due to its detrimental effects on growth, food conversion, and carcass/viscera condemnation. This study revealed that Pneumocystis organisms could be detected by Grocotts staining or immunohistochemistry in 36.9% of 564 slaughtered pigs from two geographic regions of Brazil. The prevalence of positive cases was 39.9% and 33.9% in pigs slaughtered in Rio Grande do Sul (RS) and Mato Grosso (MT) states, respectively. Among the positive cases in RS, Pneumocystis organisms were observed in 41.9% of 33 histologically normal lungs, and in 58.0% of lungs presenting with histological lesions. In contrast, the prevalence in MT in normal and abnormal lungs was 36.3% and 63.5%, respectively. Major histopathological findings in lungs of infected animals were bronchointerstitial pneumonia (47.6%), suggestive of enzootic pneumonia, and interstitial pneumonia (37.9%), compatible with PcP. The results of this survey strengthened the interest of detecting fungal pathogens, in addition to other infectious agents, and evaluating their financial impact on Brazilian pig industry. Preventive and/or therapeutic strategies should be developed in order to minimize the incidence of respiratory fungal infections in pigs and associated economic losses.

Spontaneous poisoning by larvae of Perreyia flavipes (Pergidae) in sheep

From a flock of 175 Texel sheep 25 animals died after consumption of a sawfly larvae subsequently identified as Perreyia flavipes. The disease occurred in June-July 2006 on a farm located in the county of Encruzilhada do Sul, Rio Grande do Sul, Brazil. Although there were 11 cattle in the same paddock, none of them was affected. High numbers of compact masses containing up to 150 larvae were scattered in the paddock where the animals were grazing. Most affected sheep showed severe apathy during 24-36 h before death, but weakness, muscular tremors and depression were also observed. Necropsy was performed on six sheep and the main macroscopic lesions were hemorrhages in the subcutaneous tissues, endocardium, gallbladder wall, and abomasal mucosa. In all animals was found hydrothorax, hydropericardium, ascites, and mild jaundice. Edema in the abomasal folds, mesentery, perirenal tissues, and gallbladder wall were also seen. The livers were yellowish with disseminated pinpoint hemorrhages in the parenchyma and had an enhanced lobular pattern. Perreyia flavipes larval body fragments and heads were found in the forestomach contents of the six sheep. Feces were scant, dry and formed balls coated by mucus and streaks of blood. Similar contents were also present at the end of the cecum. Prominent microscopic lesions included severe and diffuse periacinar or massive necrosis of hepatocytes associated with multifocal random hemorrhages. Diffuse necrosis of lymphoid follicles in lymph nodes and Peyers patches, lymphoid depletion and necrosis in germinative centers of the spleen, and diffuse vacuolization in the renal tubular epithelia were also seen.

Aspectos clínico-patológicos associados à circovirose suína no Rio Grande do Sul

No periodo de janeiro a julho de 2004, foram realizadas 97 necropsias de suinos que apresentaram subdesenvolvimento, aumento generalizado de linfonodos, palidez ou ictericia de mucosas e, ocasionalmente, problemas respiratorios. As principais lesoes macroscopicas encontradas incluiram aumento generalizado de linfonodos, pulmoes nao colapsados com bordos arredondados e areas de consolidacao, especialmente crânio-ventrais, alem de edema de septos interlobulares. Os rins estavam palidos, aumentados de volume e com pontos brancos que, difusamente distribuidos na superficie, infiltravam em forma de estrias ate a zona cortical. Alguns apresentavam pequenos pontos vermelhos, semelhantes a petequias, difusamente distribuidos no cortex renal. O achado histologico comum foi a presenca, em graus variaveis, de infiltrados linfo-histiocitarios em linfonodos, pulmoes e rins. O teste imuno-histoquimico utilizando anticorpo policlonal anti-circovirus suino tipo 2 foi positivo em amostras provenientes de 50 (89,2%) entre 56 suinos examinados.