가금티푸스 감염에 대한 키토산의 면역반응

조경오,고흥범,김계엽,Cho, Kyoung-Oh,Koh, Hong-Bum,Kim, Gye-Yeop 대한수의학회 2004 大韓獸醫學會誌 Vol.44 No.1

Chitosan is similar in structure to cellulose and are the second most abundant polysaccharides in nature, comprising the horny substance in the exoskeletons of crabs, shrimp and insects as well as fungi. This study was conducted to access the effect of immunomodulation responses of chitosan(N-acetyl-${\beta}$-D-glucosamine) chicken infected with in Fowl typhoid(Salmonella gallinarum). One-day-old broiler chicks were divided into eight groups: The 1st group was inoculated intra-peritoneally with chitosan and challenged intra-peritoneally with S. gallinarum. The 2nd group was inoculated intra-peritoneally with chitosan. The 3rd group was feeding with chitosan and intra-peritoneally inoculated with cyclophosphamide and challenged intra-peritoneally S. gallinarum. The 4th group was feeding with chitosan and intra-peritoneally with cyclophosphamide. The 5th group was feeding with chitosan and challenged intra-peritoneally with S. gallinarum. The 6th group was feeding with chitosan. The 7th group was challenged intra-peritoneally with S. gallinarum. The 8th group was nontreated-uninfected control group. The results shows that $CD4^+$, $CD8^+$ and B lymphocyte in lymphoid organs of chickens treated with chitosan increased in especially $CD4^+$, $CD8^+$ lymphocytes (p<0.05). The group of feeding chitosan showed the significantly increased $CD4^+$, $CD8^+$ and B lymphocyte than inoculated intra-peritoneally with chitosan. As the result suggests that the feeding of chitosan induced immunostimulatant effect than the inoculation intra-peritoeally of chitosan.

마렉병 바이러스 강독주의 실험 접종에 의해 유발된 닭 피부병변에 침윤한 림프구 표현형의 변화

조경오,Cho, Kyoung-Oh 대한수의학회 2001 大韓獸醫學會誌 Vol.41 No.3

Marek's disease virus (MDV) can cause skin lesions including inflammatory to tumorous. The phenotypical changes of lymphocytes infiltrating in the skin lesions induced by MDV were not clear. Therefore, the skin biopsies taken at weekly intervals for 8 weeks from the same specific-pathogen free chickens inoculated with Md/5 MDV were examined to analysis the phenotypical changes of lymphocytes. Histologically skin lesions progressed from initial inflammatory to late tumorous. Sequentially CD4+ T lymphocytes increased gradually in number from initial skin lesions and were major composition cells in the tumor lesions. Regardless of inflammatory or tumor lesions, CD8+ T cells and ${\gamma}{\delta}$ T cells infiltrated particularly in the dermis and subcutaneous on which MDV was actively replicated in the feather follicle epithelium(FFE). In addition, IgG bearing B lymphocytes in considerable number infiltrated in the dermis and subcutaneous tissues. From these results, the development of MDV-induced skin lesions was inflammatory following tumorous. In addition, each CD8+, ${\gamma}{\delta}$ and CD4+ T cells and B cell might act to protect MDV replication in the FFE or tumor cells which turned on lytic cycle.

선충류의 주사전자현미경적 관찰을 위한 마이크로웨이브 고정법

이산수,조경오,신길상,신성식,Lee, San-Soo,Cho, Kyoung-Oh,Shin, Kil-Sang,Shin, Sung-Shik 대한수의학회 2007 大韓獸醫學會誌 Vol.47 No.2

Conventional processing of biological materials including nematode parasites for scanning electron microscopy includes fixation with glutaraldehyde and osmium, followed by dehydration in an ascending grade of ethanol, and finally freeze drying. This procedure takes about 8 to 12 h depending on the characteristics of samples. Microwave irradiation of 2,450 MHz enhance the action of cross-linking fixatives and can greatly accelerate various stages of tissue processing. In this study, samples of nematode parasites, Setaria digitata, were fixed by a combination of conventional chemical fixation and the microwave irradiation during the process. The microwave irradiation was also incorporated in the serial dehydration process with ethanol. The complete procedure from the initial fixation to the completion of dehydration with ethanol was reduced to 1 h with good preservation of the ultrastructural details of the specimens.

돼지 유행성 설사(Porcine Epidemic Diarrhea)의 진단을 위한 면역조직 화학적 기법의 응용

박남용,조경오,Park, Nam-yong,Cho, Kyoung-oh 대한수의학회 1994 大韓獸醫學會誌 Vol.34 No.4

Immunohistochemical study on the intestinal tissues obtained from the 21 pigs of the 14 terms in Korea in which the clinical and epidemiological features had indicated the possible outbreaks of porcine epidemic diarrhea(PED) was performed using the indirect immunofluorescence test and/or the immunoperoxidase method in order to detect PED viral antigens in the infected cells of the intestines, and histopathological features were described as well. By immunohistochemical analysis, PED viral antigens were detected in the epithelial cells covering the small intestinal villi and recognized slightly in the cells lining the colonic surface epithelium as well. Occasional fluorescence was also seen in a few intestinal crypt epithelium. On light microscopy, the piglets with PED showed marked villous atrophy and fusion, and severe enterocyte degeneration and desquamation. On the other hand, the older pigs more than 4 week old age was mild villous atrophy and fusion, severe villous epithelial cell proliferation, and moderate mononuclear cell infiltration.

In situ Hybridization에 의한 토끼출혈증(rabbit haemorrhagic disease)의 신속.간편한 진단

박남용,조호성,조경오,김상집,박형선,Park, Nam-Yong,Cho, Ho-Seong,Cho, Kyoung-Oh,Kim, Sang-Jip,Park, Hyung-Seon 한국수의병리학회 2001 한국수의병리학회지 Vol.5 No.2

Recently various molecular diagnostic techniques have been used to identify rabbit hemorrhagic disease virus (RHDV), a causative agent responsible for acute hepatitis and disseminated intravascular coagulation in rabbit. But they were hard to perform and time consuming. To detect RHDV in a rapid and easy way, we developed biotinylated oligonucleotide probe within ORF 1 region encoding the polyprotein of RHDV in formalin-fixed and paraffin-embedded tissues from various tissues of 20 rabbits naturally infected with RHDV, Our in situ hybridization (ISH) was quickly carried out within two hours by MicroProbe capillary action system. The ISH produced a positive reaction in liver, kidney and lung. In conclusion, ISH with a biotintlated oligonucleotide probe provided a useful diagnostic method for detecting RHDV.

In situ hybridization에 의한 소 바이러스성 설사증 바이러스의 검출

박남용,홍기강,정치영,조경오,이봉주,박영석,박형선,권창희,Park, Nam-yong,Hong, Ki-kang,Chung, Ci-young,Cho, Kyoung-oh,Lee, Bong-joo,Park, Young-seok,Park, Hyung-seon,Kweon, Chang-hee 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.1

Detection and distribution of bovine viral diarrhea virus(BVDV) was studied in formalin-fixed, paraffin-embedded tissues from two naturally infected cattle by in situ hybridization with a non-radioactive biotinylated probe. A 600 base pair cDNA probe from BVDV B-25 strain was used for probe. The whole procedure of ISH to diagnose was carried out within 1~2 hours in $Microprobe^{TM}$ capillary action system. The biotin-labelled probe was demonstrated after hybridization under standard conditions by the application of streptoavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a fast red TR/naphthol phosphatase and the sections were counterstained with hematoxylin. We have obtained the result of positive reactions in digestive tract(sm1.all intestine and colon) and epidermis of tongue in the state of the intact tissues. The result suggested that in situ hybridization method can be considered as a useful diagnostic technique for detection of specific nucleic acid sequences of BVDV.

면역억압계군에서 Salmonella gallinarum 감염에 대한 면역반응

김계엽,임재향,고홍범,조경오,김태열,오명화,Kim, Gye-Yeup,Lim, Jae-Hyang,Koh, Hong-Bum,Cho, Kyoung-Oh,Kim, Tae-Youl,Oh, Myoung-Hwa 대한수의학회 2002 大韓獸醫學會誌 Vol.42 No.3

The immune response against Salmonella gallinarum infection was investigated in immunosuppresed chickens. Newly hatched chickens were treated with cyclophosphamide at the first and second day after birth and were challenged intraperitoneally with S gallinarum ($1{\times}10^7CFU/m{\ell}$) on day 6. Group 1, Immunosuppresed and Challenged group, was treated with cyclophaiphamide and challenged with S gallinarum; group 2, Immunosuppressed group, was only treated with cyclophsphamide; group 3, Challenged group, was only challenged with S gallinarum; group 4, Control group. In each group, the localization of lymphocytes of the lymphoid organs and intestine was immunohistochemically compared using a variety of monoclonal antiboies ($CD4^+$, $CD8^+$, and B lymphocyte). Also, S gallinarum were assessed by Maccallum-Goodpasture stain and immunohistochemical analysis in the paraffin-embedded intestinal tissues. In S gallinarum challenged chickens, $CD4^+$ and $CD8^+$ lymphocytes of the intestinal organs such as duodenum, jejunum, ileum and colon were increased. However, in cyclophophamide treated chickens, $CD4^+$ and $CD8^+$ lymphocytes and especially B lymphocytes of the lymphoid organs such as thymus, spleen, and bursa of Fabricius were dramatically decreased. These results suggest that cyclophsophamide is an immunosuppressive agent that especially causes depletion of B lymphocytes, suppress humoral immunity and eventually suppresses avian immune responses. Its protection against S gallinarum infection is mainly dependent on both cell-mediated mechanism and the humoral immune response.

In situ hybridization에 의한 개 디스템퍼의 진단

조현,박남용,김용환,조경오,박형선,박영석,이봉주,정치영,임형호,Cho, Hyeon,Park, Nam-yong,Kim, Yong-hwan,Cho, Kyoung-oh,Park, Hyung-seon,Park, Young-seok,Lee, Bong-joo,Chung, Chi-young,Im, Hyung-ho 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.3

We have developed the in situ hybridization(ISH) technique for rapid diagnosis of canine distemper(CD) which is the major infectious disease in dogs. In our experiment, we rapidly detected distribution of the specific canine distemper viral genome without disrupting morphology of tissues or cells. Two oligonucleotide probes for ISH were synthesized chemically and labelled 5' end with nonisotopic biotin by DNA synthesizer. The whole procedures of ISH was completed within 1~2 hours using the Microcapillary action system. On histological study, typical cytoplasmic or intranuclear inclusion bodies were observed in the trachea, bronchiole, brain, and urinary bladder with the presence of prominent red positive signals on ISH, indicating specific CDV genome from the paraffin-embedded tissues of infected 13 cases. The results showed ISH can be used as a rapid and effective diagnostic method for diagnosis of CD.

In situ RT-PCR 및 In situ hybridization 기법에 의한 닭 뉴캣슬병의 진단법 개발

박남용,최효임,조호성,강성귀,조경오,Park, Nam-Yong,Choi, Hyo-Im,Cho, Ho-Seong,Kang, Sung-Kwi,Cho, Kyoung-Oh,Brown, Corrie 대한수의학회 2002 大韓獸醫學會誌 Vol.42 No.3

Newcastle disease (ND) is a highly contagious infection of poultry, Two pathology-based techniques, in situ RT-PCR and in situ hybridization (ISH) were applied to formalin-fixed, paraffin-embedded tissues from chickens naturally infected with velogenic ND virus (VNDV). Two pairs of primers and a probe for ISH and in situ RT-PCR, respectively, were selected from highly conserved region of matrix gene of NDV. The ISH experiment was carried out using MicroProbe$^{TM}$ capillary action system within 2 hours. In situ RT-PCR was performed using MicroProbe$^{TM}$ capillary action system and GeneAmp In Situ PCR system. With ISH and in situ RT-PCR, viral nucleic acid was detected in the central nervous system of chickens from infected with neurotropic velogenic Newcastle disease virus (NVNDV), whereas viral nucleic acid was detected in various organs or tissues of chickens from infected with viscerotropic velogenic Newcastle disease virus (VVNDV). In the NVND group, positive signals were characteristically defined in the cytoplasm of neuron, vascular endothelial cells, and perivascular mononuclear macrophages in the central nervous system. One of NVND group, chicken from one farm exhibited positive signals in the bronchial epithelium. The VVND group chickens showed positive reaction in the macrophages, vascular endothelium, and bronchiolar epithelium. Markedly, viral nucleic acid was detected in the macrophages of morphologically normal tissues which were peripheral or located in distant areas from lesions. The central nervous system of chickens infected with VVND virus had positive signals in the vascular endothelial cell, perivascular mononuclear macrophages and some neuron. The number and intensity of the positive cells by in situ RT-PCR were more and stronger, respectively, in comparison with those by ISH. Particularly, positive reaction was detected in macrophages infiltrating in cardiac muscle by in situ RT-PCR, but not obtained by ISH. Therefore, these results demonstrated that ISH is a rapid diagnostic method for detection of NDV and in situ RT-PCR can be used as an efficient method for detection of low viral load infection or subclinical viral infection of NDV.

닭 크립토스포리디움의 형태계측을 위한 전자현미경적 연구

박남용,김영섭,정치영,조경오,박영석,이봉주,박형선,Park, Nam-yong,Kim, Young-seop,Chung, Chi-young,Cho, Kyoung-oh,Park, Young-seok,Lee, Bong-joo,Park, Hyung-seon 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.3

Morphometrical analysis of chicken Cryptosporidium baileyi in various stages of life cycle in the bursa of Fabricius were carried out by electron microscope to establish a differential point for identification of C baileyi. By avidin-biotin complex method, protozoans of the bursa of Fabricius were identified Cryptosporidium spp. The size and area on each developmental stages of C baileyi, as measured by Morphomat 10 attached to electron microscope were as follows. Trophozoites' size with range of $3.21{\pm}0.70{\times}2.49{\pm}0.59{\mu}m$, area with range of $118.82{\pm}41.92{\mu}m^2$; meronts' size $3.99{\pm}1.07{\times}2.96{\pm}0.52{\mu}m$, area $210.11{\pm}57.11{\mu}m^2$; merozoites' size $1.98{\pm}0.43{\times}0.60{\pm}0.18{\mu}m$, area $24.10{\pm}5.97{\mu}m^2$; microgametes' size $1.36{\pm}0.83{\times}0.50{\pm}0.23{\mu}m$, area $20.23{\pm}6.73{\mu}m^2$; macrogametes' size $4.57{\pm}0.65{\times}4.02{\pm}0.55{\mu}m$, area $258.37{\pm}51.83{\mu}m^2$; oocytes' size $4.39{\pm}0.56{\times}3.44{\pm}0.50{\mu}m$, area $187.21{\pm}62.68{\mu}m^2$. In conclusion, the size and area on each developmental stages of Cryptosporidium baileyi is different from that of other Gryptosporidia spp. It suggests, with considering tissue tropism and life cycle, morphometrical analysis can be quite a good way to identify C baileyi.