Banca de QUALIFICAÇÃO: KELEN REGINA ASCOLI BALDI

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
DISCENTE : KELEN REGINA ASCOLI BALDI
DATA : 13/12/2019
HORA: 11:00
LOCAL: IFC Campus Concórdia
TÍTULO:

STANDARDIZATION OF DIRECT IMMUNOFLUORESCENCE  AND OPTIMIZATION OF IMMUNOHISTOCHEMISTRY  FOR   Listeria monocytogenes DIAGNOSIS IN PARAFFINIZED TISSUES.

PALAVRAS-CHAVES:

Listeriosis; immunological techniques; formalin-fixed tissue.

PÁGINAS: 45
GRANDE ÁREA: Ciências Agrárias
ÁREA: Medicina Veterinária
RESUMO:

Listeria monocytogenes is a bacterium that mayinfect humans and animals and cause listeriosis, a zoonotic disease characterized by encephalitis, septicemia or abortion. Sheep, goats and cattle aremainly affected by the disease.. Listeriosis also causes economic losses due to the animal death or sacrifice, drug spending and decreased production. Due its difficulty to isolate, it is necessary to implement immunological techniques for definitive diagnosis of listeriosis, such as immunofluorescence and immunohistochemistry. These techniques also allow the etiological diagnosis and retrospective studies in formalin-fixed and paraffin-embedded tissues. The aim of this work was to standardize the direct immunofluorescence and optimize immunohistochemistry as diagnostic tools for Listeria monocytogenes in fixed and paraffinized tissues. Tissue blocks from animals with a clinical history and/or lesions compatible with listeriosis were selected from the Veterinary Pathology laboratory archive at Instituto Federal Catarinense, Campus Concórdia, SC. For  technique standardization, , Positive and negative L. monocytogenes samples were selected by conventional PCR (polymerase chain reaction)  to be used as controls. For both techniques, tissue sections were cut by microtomy and transferred to positive charged slides. For immunohistochemistry, a diluted (1:200) polyclonal anti-L. monocytogenes primary antibody (BD, USA) was used, followed by a polymer-coupled secondary antibody (Polymer Kit,Dako, USA). Antibody-binding was detected with 3-Amino-9-ethylcarbazole (AEC, Dako, USA) and slide reading was assessed under an optical microscope. For immunofluorescence, same primary antibody was used, followed by fluorescein-labeled anti-rabbit IgG secondary antibody (Abcam, USA). Coverslips were  mounted with glycerol and slide reading was done using fluorescence microscope with excitation and emission filters of EX475/AF40 and  MS535/AF45, respectively. Each sample was classified for the presence or absence of immunostaining. Both immunohistochemistry and immunofluorescence showed  strong  labelingin tissue samples  from bovines experimentally infected with L. monocytogenes ATCC 7644, as well as nervous tissue samples from naturally infected ruminants. This study demonstrated that both techniques were efficient to detect the pathogen in  paraffin-embedded tissues, with immunolabeling at the same tissue sites. Immunofluorescence in paraffin-embedded tissues rather than fresh tissues is a new tool , as there are few reports using this technique in processed biological materials. However, immunofluorescence showed no difference in sensitivity when compared with immunohistochemistry, although the latter has a lower cost  and an easier  result interpretation. Definitive diagnosis of Listeria sp. is very relevant to public health. By controlling its occurrence in the herd, this pathogen is prevented from reaching  human being, as well as itreduces  economic losses due to animaldeath. The possibility of diagnosis using previously fixed tissues minimizes the biological risk of contamination during sample manipulation  in the laboratory. Tthus, this allows a sensitive and definitive diagnosis, even in the absence of fresh tissues for microbiological isolation.

MEMBROS DA BANCA:
Interna - 1989957 - ALESSANDRA FARIAS MILLEZI
Interno - 2017813 - RICARDO EVANDRO MENDES
Interna - 2408296 - SORAYA REGINA SACCO
Presidente - 1081425 - TEANE MILAGRES AUGUSTO GOMES