Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

A importância do cão como reservatório de L. infantum chagasi no meio urbano tem estimulado a realização de inúmeros trabalhos de avaliação de técnicas de diagnóstico, uma vez que este procedimento, quando realizado corretamente, torna-se um importante passo na prevenção da doença em humanos. Dentre os métodos de diagnóstico, as técnicas moleculares têm adquirido destaque. Objetivou-se neste trabalho verificar o desempenho da Reação em Cadeia da Polimerase (PCR) e da PCR em tempo real (qPCR) para diagnóstico da Leishmaniose Visceral Canina (LVC) utilizando diferentes amostras biológicas. Para tanto foram utilizados 35 cães provenientes de uma área endêmica para LVC, onde foram utilizados para o diagnóstico molecular, aspirado de medula óssea, fragmentos de linfonodo e baço. Neste estudo a qPCR foi capaz de detectar um maior número de animais positivos quando comparada com a PCR. Já entre as diferentes amostras biológicas utilizadas não foi observada diferença significativa na detecção de DNA de L. infantumchagasi por meio da PCR e qPCR. Mesmo assim, considerando a facilidade de obtenção, o linfonodo pode ser considerada como a melhor amostra para diagnóstico molecular da infecção por L. infantum chagasi.

Mitose como fator prognóstico para biópsia de linfonodo sentinela em melanoma fino

Paciente, sexo feminino, 23 anos, com melanoma extensivo superficial em dorso, Breslow 0,35 mm, Clark II, sem ulcerações e com 2 mitoses / mm². Foi submetida à ampliação de margem e biópsia de dois linfonodos sentinela (axila esquerda). O exame anatomopatológico mostrou micrometástases, no seio subcapsular de ambos. Seguindo a recomendação do American Joint Commitee on Cancer 2009, a paciente foi submetida à linfadenectomia axilar total, sem outros linfonodos metastáticos. A aplicação da dermatoscopia vem permitindo maior precisão diagnóstica de melanoma cutâneo, contribuindo para maior proporção de melanoma fino ao diagnóstico. A taxa mitótica foi incluída como um importante fator prognóstico para melanomas finos pelo American Joint Commitee on Cancer 2009, sugerindo biópsia para esses pacientes

Aplicação da técnica de imunofluorescência direta para o diagnóstico da leishmaniose visceral canina em aspirado de linfonodo

Recentemente, foco de leishmaniose visceral canina (CVL) foi descrito na região noroeste do Estado de São Paulo - Brasil. O Hospital Veterinário - UNESP - Araçatuba, no ano de 2.000, desenvolveu 60 testes citopatológicos de casos suspeitos de leishmaniose usando aspirado por agulha fina (FNA). Os esfregaços de linfonodo foram corados pelo método de Romanowsky (Diff-Quik®) e observados em microscopia de luz. Os casos positivos mostraram formas amastigotas típicas de Leishmania livres ou em vacúolos de macrófagos. Sinais citopatológicos de reatividade do sistema linfo-histiocitário com ausência de parasitos foram também observados. Com o objetivo de implementar o diagnóstico da CVL, detectando parasitos e material antigênico nos esfregaços, aplicou-se a reação de imunofluorescência direta (IFD) usando anticorpo policlonal anti-Leishmania produzido em camundongo. Comparamos o método de IFD com a pesquisa direta do parasito em esfregaços corados pelo método de Romanowsky. Dos 60 cães com sinais clínicos da doença, o exame direto foi positivo em 50% (n=30), duvidoso em 36,7% (n=22) e negativo com reatividade do linfonodo em 13,3% (n=8). Quando os linfonodos foram submetidos a reação de IFD observamos reação positiva em 93,3% (n=56) e reação negativa em 6,7% (n=4). Nossos resultados mostraram que a reação de IFD apresentou alta sensibilidade quando comparada a pesquisa direta do parasito pela coloração de Romanowsky. A reação de IFD pode ser um método útil para confirmar os casos duvidosos da doença, onde as formas amastigotas não são identificadas com facilidade.

Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo (Dasypus novemcinctus)

We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo (Dasypus novemcinctus ).Pulmonal inflammatory area was evaluated by intravenous inoculation of 10(6) yeast cells of each isolates in young, male, ddY mice. Moreover, the partial sequence of GP43kDa gene of P. brasiliensis was analyzed. The lung inflammatory area was greater in animals inoculated with isolate D3S1. The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1. This study suggested that the same armadillo might be susceptible to multiple P. brasiliensis isolates simultaneously.

Cellular immune responses in mice immunized with Anisakis simplex larval antigens

Cellular immune responses to Anisakis simplex L3 antigens were investigated in BALB/c mice injected subcutaneously with a homologous crude extract (CE). Popliteal lymph nodes (PLN) were found to be increased in size and weight after A. simplex CE footpad injection. The effects of A. simplex CE in vitro proliferation were assayed with non-fractionated PLN cells or nylon-wool purified T cells derived from pooled lymph node cells of mice subcutaneously injected with CE. Spleen cells from immunized animals (antigen alone, or larva alone, or antigen plus larva) were studied by flow cytometry. The immunization induced a high proportion of CD4 + and TCR alpha beta + T cells. The number of B cells (CD45 + and TCR alpha beta-) in pre-immunized and infected mice was lower than that observed in animals subjected to infection only. The number of CD4 + T cells increased in the infected and in the pre-immunized and infected mice. In the latter, a decrease of CD8a + T cells was noted. The greatest increase in CD8a+ and TCR alpha beta- T cells was found in mice that had been subjected to infection only. Histological analysis showed that the most prominent lesions were gastric and intestinal in animals infected orally with one larva.

IMMUNOCYTOCHEMICAL DIFFERENTIATION OF REACTIVE HYPERPLASIA FROM FOLLICULAR LYMPHOMA USING MONOCLONAL-ANTIBODIES TO CELL-SURFACE AND PROLIFERATION-RELATED MARKERS

We tested the hypothesis that a panel of antibodies to cell surface, cytoplasmic, and nuclear antigens could reliably distinguish the cells composing reactive germinal centers from those composing follicular lymphoma. Immunocytochemistry was performed on deparaffinized sections of methacarn-fixed lymph node and tonsil (15 cases of reactive hyperplasia and 14 cases of follicular lymphoma) using antibodies to the nerve growth factor receptor (NGFR5), bcl-2 protein (124), proliferating cell nuclear antigen (PCNA; 19A2), and CD45RA (MT2). In 100% of cases of reactive hyperplasia, both MT2 and 124 showed positive immunostaining of mantle zone and scattered interfollicular lymphocytes, but in all cases there was a sharply demarcated absence of immunostaining of germinal center cells. However, diffuse immunostaining of follicular centers with MT2 (64%) and 124 (93%) and scattered intervening cells were seen in follicular lymphoma. The combination of antibodies to CD45RA and bcl-2 yielded positive immunostaining of follicular center cells in 93% of follicular lymphomas. The germinal center cells of reactive hyperplasia showed >75% nuclear positivity with antibodies to PCNA, in contrast to the follicular lymphoma cells, which showed variable PCNA indices ranging from 25 to >75%. A minority of follicular lymphoma cases (29%) showed PCNA indices comparable with those seen in cases of reactive hyperplasia. Antibodies to NGFR were positive in all cases of reactive hyperplasia and in 79% of cases of follicular hyperplasia, although the immunostaining intensity was generally decreased in follicular hyperplasia. In summary, antibodies to bcl-2 appear to be superior to those to CD45RA in distinguishing reactive hyperplasia from follicular lymphoma. Reactive hyperplasia cannot be discriminated from follicular hyperplasia using antibodies to PCNA or to nerve growth factor receptor.

SINUS HISTIOCYTOSIS WITH MASSIVE LYMPHADENOPATHY (ROSAI-DORFMAN DISEASE) - CYTOMORPHOLOGIC ANALYSIS ON FINE-NEEDLE ASPIRATES

Cytomorphologic characteristics of smears from fine-needle aspirates (FNA) of three cases of sinus histiocytosis with massive lymphadenopathy (SHML) (Rosai-Dorfman disease) are described. All aspirates showed mature lymphocytes, plasma cells, and large pale histiocytes with well-preserved lymphocytes within the cytoplasm (lymphocytophagocytosis). The cases were confirmed by surgical biopsy, and the immunohistochemical findings suggested a macrophage origin for this lesion. The value of FNA analysis as an aid to diagnosis of SHML is discussed.

Evidence of Leishmania spp. antibodies and DNA in bush dogs (Speothos venaticus) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interleukin-8 as a prognostic serum marker in canine mammary gland neoplasias

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histopathological evaluation in experimental envenomation of dogs with Crotalus durissus terrificus Venom

The present work evaluated histopathological aspects in experimental envenomation of dogs with Crotalus durissus terrificus venom. Twenty-eight mixed breed adult dogs were divided into three groups of seven animals each: Group I only venom; Group II - venom + 50ml antiophidic serum + fluid therapy; Group III venom + 50ml antiophidic serum + fluid therapy + urine alkalization. Lyophilized venom of Crotalus durissus terrificus was reconstituted in saline solution and inoculated subcutaneously at the dose of 1mg/kg body weight. Three animals of each group were subjected to euthanasia, and their muscular tissue, brain, spleen, kidneys, heart, lungs, stomach, small and large intestines, and popliteal lymph node fragments were collected for histopathological evaluation. There was myonecrosis in the inoculated limb, renal tubular degeneration, lymphoid hyperplasia of spleen, and unspecific reactive hepatitis. These results show the antigenicity and action of the venom on the immune system.

Effect of Amblyomma cajennense Ticks on the Immune Response of BALB/c Mice and Horses

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Different inflammatory stimuli in the footpad of mice influence the kinetics of resident peritoneal cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental infection of bovines with oocysts of Toxoplasma gondii

Calves aged 3 mth were readily infected with oocysts and cysts of Toxoplasma gondii administered by the oral route. Fever, respiratory distress, nasal discharge, and hyperemia of the conjunctivas were the most significant clinical signs noted in the infected animals. Parasitemia was demonstrated in all infected calves. It occurred on different days and up to 62 days after the infection. Toxoplasma was demonstrated in tissues of all infected calves, and the organ most frequently parasitized was the lymph node. Parasitism of the retina was demonstrated in 2 calves. All infected animals had antibody against T. gondii in their serum. The Sabin-Feldman dye test and the indirect immunofluorescent test were both useful in detecting antitoxoplasma antibody.

The cervical lymphatic system in newborn rats

In the present work the authors studied the morphology of the cervical lymphatic system using 26 new-born white rats of both sexes. The studies were performed by injecting China-ink under the skin of the angle of mouth and dorsal region of the auricle in order to visualize and then to proceed the dissection of lymph vessels and nodes under stereoscopic microscope. Morphological descriptions of superficial and deep lymph vessels and lymph nodes were made, recording: a dense capillary network of the superficial system that joins to larger branches going to the submaxillary lymph nodes, generally the only ones on each side; those lymph nodes send efferent branches to deep cervical lymph nodes generally also the only ones on each side; those lymph nodes give efferent branches which form the jugular lymphatic sac that joins the jugulo-subclavian junction; the jugular lymphatic sac may receive branches from the plexus, which accompany the omocervical and external jugular veins and branches coming from the thoracic limb, as well as may join the branches coming from the thorax and the branches in most of the cases form plexus branches and seldom appear by themselves.