Immunocytochemistry, In situ hybridization and electron microscopy for early diagnosis of Aujeszky's in living pigs |
Oun-kyong Moon1, Soon-bok Kim2, Jung-hyang Sur3, Geun-suk Song2, Whan-gook Nho4 |
1National Veterinary Research Institute 2College of Veterinary Medicine, Gyeongsang National University 3Detp. of Veterinary and Biomedical Sciences, University of Nebraska 4Pusan Veterinary Service Laboratory |
오제스키병의 생체 조기진단을 위한 면역세포화학, In situ hybridization 및 전자현미경적 연구 |
문운경1, 김순복2, 서정향3, 송근석2, 노환국4 |
1수의과학연구소 2경상대학교 수의과대학 3네브라스카대학교 수의학과 4부산가축위생시험소 |
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Abstract |
The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs. |
Key Words:
immunocytochemistry, digoxigenin-labeled in situ hybridization, electron microscopy, Aujeszky's disease virus, nasal cells, buffy coats |
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