RESUMEN
DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation.
Asunto(s)
Infecciones por Haemophilus/veterinaria , Haemophilus parasuis/aislamiento & purificación , Hibridación in Situ/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Enfermedades de los Porcinos/microbiología , Actinobacillus pleuropneumoniae/genética , Animales , Calostro , Cartilla de ADN/química , ADN Bacteriano/análisis , Formaldehído , Infecciones por Haemophilus/diagnóstico , Infecciones por Haemophilus/microbiología , Haemophilus parasuis/genética , Hibridación in Situ/métodos , Hígado/microbiología , Adhesión en Parafina/veterinaria , Pasteurella multocida/genética , Pericardio/microbiología , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Bazo/microbiología , Porcinos/microbiología , Enfermedades de los Porcinos/diagnóstico , Fijación del Tejido/veterinariaRESUMEN
In focal lesions of feline infectious peritonitis (FIP), the cells involved in the delayed-type hypersensitivity were identified in formalin-fixed paraffin-embedded and frozen samples taken from 35 affected cats. The clinical diagnosis of FIP was confirmed by necropsy, histology and direct immunofluorescence against the coronaviruses on cryostatic sections. The immune cells were detected immunohistochemically by the Avidin-Biotin-Complex (ABC) method using either polyclonal antibodies against lymphoid antigens (CD3) or monoclonal antibodies against lymphoid (PAN-T, CD4, CD8) and myeloid antigens (MAC387). Better identification of T cells and macrophages was found on formalin-fixed paraffin-embedded sections than on cryostatic ones, while T lymphocyte subpopulations could be differentiated only in cryostatic sections. Type IV hypersensitivity was detected in focal feline infectious peritonitis virus (FIPV)-induced lesions from progressive activation of T lymphocytes, mainly CD4+, and the presence of granulocytes and macrophages. The FIPV-induced lesions could be studied as examples of granulomas caused by unconventional antigens, such as viruses or immune complexes.