보고서 정보
| 주관연구기관 |
한경대학교 |
| 보고서유형 | 최종보고서 |
|---|
| 발행국가 | 대한민국 |
|---|
| 언어 |
한국어
|
| 발행년월 | 2008-04 |
|---|
| 과제시작연도 |
2007 |
| 주관부처 |
농림부
Ministry of Agriculture and Forestry |
| 과제관리전문기관 |
농림기술관리센터
Agricultural Research & development Promotion Center |
| 등록번호 |
TRKO201400022655 |
| 과제고유번호 |
1385006920 |
| 사업명 |
농림기술개발 |
| DB 구축일자 |
2014-11-14
|
초록
▼
○ 연구결과
1. 한국산 겨우살이에 의한 비특이적 면역증강 효과
① 겨우살이 추출물 (KM-110) 을 250 ㎍에서 1,000 ㎍/mouse의 양으로 투여한 결과 혈중 IgGAM이 증가하였다.
② 분중 IgGAM과 IgA의 양이 혈청에서와 유사한 패턴을 보이며 증가하는 것으로 나타났다.
③ KM-110의 경비적 투여에 의한 분변 중의 IgGAM 및 IgG 분비량 증가
④ 경구투여는 아무런 영향이 없었으나, 경비투여는 약간의 체중감소를 동반 경구투여나 경비투여 모두 간손상을 유발하지는 않는 것으로 확인
○ 연구결과
1. 한국산 겨우살이에 의한 비특이적 면역증강 효과
① 겨우살이 추출물 (KM-110) 을 250 ㎍에서 1,000 ㎍/mouse의 양으로 투여한 결과 혈중 IgGAM이 증가하였다.
② 분중 IgGAM과 IgA의 양이 혈청에서와 유사한 패턴을 보이며 증가하는 것으로 나타났다.
③ KM-110의 경비적 투여에 의한 분변 중의 IgGAM 및 IgG 분비량 증가
④ 경구투여는 아무런 영향이 없었으나, 경비투여는 약간의 체중감소를 동반 경구투여나 경비투여 모두 간손상을 유발하지는 않는 것으로 확인
⑤ 경구 혹은 경비적으로 투여할 경우 면역세포(비장세포)의 증식을 촉진
⑥ 시료의 점막투여는 마우스의 기초대사량을 높이는 것으로 나타났으며와는 매일 회씩 회 투여하면서 일째의 장내 량을 분변 중에서 측정 KM-PS KMF-WE 1 5 6 IgA
⑦ 한 결과 IgA분비를 증가시키는 것으로 나타났으며(Fig.11), 기초대사량도 유의하게 상승시키는 것으로 확인
2. 마우스에서의 항원 특이적 면역증강 시험
① 경비투여시 특이적 항원의 증가추세를 확인하였으며, 이것은 총 혈청과 질 세척액에서부터 각기 IgG와 IgA의 역가를 측정함으로써 확인하였다
② WHBP를 면역증강제로 사용하였을 때 10㎍의 농도와 LPS+10㎍의 농도에서 가장 높은 역가를 나타내었고, LPS를 대조군으로 사용했을 때보다 더 높은 수치를 나타내었다. 이것은 농도 의존적인 면역증강효과
③ WHBP의 경우 모든 농도에서 점막면역의 IgA를 증가시키는 효과는 없는 것으로 드러났다. 따라서 KM-110은 IgA와 IgG 모두를 증가시키나, HWBP는 IgG의 역가만을 증가시키는 것으로 나타났음
3. 닭을 이용한 파이로트 사양시험
(1) 실험실 수준의 육계 사양실험 결과.
① 사료섭취량 (Fig. 1), 증체량 (Fig. 2), 사료효율 (Fig. 3)에서는 차이를 나타내지 아니하였다. 이것은 겨우살이 추출물 급여를 통한 사료섭취량이나 육계의 생산성에는 1%까지 급여하여도 영양소 이용성에는 특별한 차이를 가져오지 아니하는 것으로 조사됨
② 겨우살이 0.5% 첨가는 ND/IBD 감염후 7일째에 LPS와 Con A로 자극한 PBMC의 증식도를 겨우살이가 첨가되지 않은 사료를 급여한 대조구에 비해 유의하게 증가시켰다(p<0.05)
③ 사료 내 겨우살이 0.5% 첨가로 ND/IBD 감염후 14일째에 LPS와 Con A로 자극한 비장세포의 증식도가 대조구에 비해 유의하게 증가
④사료중 겨우살이 0.5% 첨가는 ND와 IBD 감염시 육계에서 T 임파구의 증식을 증가시켜 세포성 면역(cell-mediated immunity)을 증강시키는 한편 B 임파구의 증식을 높여 항체에 의한 면역인 체액성 면역(humoral immunity)을 증강시킨다는 것을 나타낸다.
⑤ 사료 내 겨우살이를 0.5% 수준으로 첨가급여한 결과 ND의 항체역가가 대조군에 비해 증가한 반면 1% 수준으로 급여한 군에서는 오히려 낮아져 사료영양소의 이용성면이나 면역기능 조절을 위해서는 0.5% 이상 급여할 필요가 없는 것으로 조사되었다.
(2) 산란계를 이용한 사양실험 결과
① E.coli 88항원에 대한 항체 생산도 겨우살이 추출물 0.5% 첨가군에서 대조군에 비해 증가하였으며 1% 급여군에서도 2주, 4주 및 6주별 항체역가가 농도의존적으로 증가하는 것으로 나타나 겨우살이추출물이 산란계의 체액성면역기능을 강화시키는 것으로 판단됨
② 산란율이나 난각강도는 겨우살이 추출출 첨가급여군에서 대조군에 비해 차이는 없었으나 점토광물질과 혼합급여하였을 때 유의하게 개선되는 결과를 나타내어 겨우살이 추출물 첨가제를 개발할 경우 미량광물질이 함께 보완되면 매우 효능이 증가항 것으로 기대되었다.
③ 계란의 신선도, 난백고, 난각두께에서도 유사한 결과를 나타내어 산란생산성 및 난질에는 겨우살이 추출물 단독으로는 대조군과 차이를 보이지 않지만 미량광물질이 첨가되면 개선되는 것으로 나타나 산업화를 위한 주요한 기초자료를 얻었다고 할 수 있다.
가. KML-C 경비투여에 의한 항체반응 상승효과 해석
① 혈액 내의 HA특이적인 IgG titer에 있어서는 면역개시 1주 후의 혈청에서는 KML-C 및 CTB 모두 HA에 대한 항체가를 현저히 상승시키는 효과가 관찰되었다(Fig.1).
② 면역개시 3주 및 8주째의 혈청에 있어서는 항체가의 평균치는 KML-C+HA와 CTB+HA 모두 HA만을 면역한 마우스보다 높은 수치를 나타내었음
③ KML-C와 CTB 간의 활성비교에 있어서는 두 adjuvant는 항원 특이적인 IgG를 유도하는 작용은 거의 동등한 것으로 나타났다.
④ 혈청 중의 IgG와는 달리, 질액 중의 IgA에 있어서는 면역개시 3주째에 항체가의 상승이 관찰되었다
⑤ 혈청 IgG의 경우와 마찬가지로 질액 중의 IgA에 있어서도 KML-C와 CTB간의 항체유도 활성의 차이는 관찰되지 않았다. 하지만 CTB의 투여량이 1 ug/head이고, KML-C의 투여량이 0.1ug/head인 것을 감안하면 KML-C는 매우 낮은 투여량에서 활성을 나타내는 것으로 사료된다.
⑥ KML-C와 CTB 모두 항원 특이적인 IgG2b를 상승시키는 효과가 관찰되었으며(Fig.3), 이들 adjuvant는 거의 동등한 수준의 활성을 지니는 것으로 확인되었다.
나. KML-C 경비투여에 의한 세포성 면역 증강활성 해석
① HA항원으로 재자극하는 경우에 있어서 KML-C가 항원 특이적인 면역세포 증식반응을 현저히 상승시키는 것으로 나타났다(Fig.4).
② ConA나 LPS로 재자극한 경우에 있어서는 KML-C를 HA와 함께 투여한 그룹에서 가장 높은 활성이 관찰되었으나, 이들 mitogen에 의한 비특이적 활성화 유도에서는 항원 특이적인 활성화보다 증강효과가 크게 저하된 것으로 확인되었다. 즉 KML-C를 항원과 함께 경비투여 함으로서 항원 특이적인 세포성 면역의 증가를 유도할 수 있는 것으로 판단되었다.
③ FACS 이용하여 bromodeoxiuridine (BrdU) 에 의한 세포증식 반응을 측정한 결과에 있어서도 KML-C+HA 투여군에서 가장 높은 세포증식 반응이 관찰되었다(Fig.5).
④ 면역 마우스의 비장세포를 이용한 세포 증식반응 실험에서 관찰된 증식증가 효과가 T세포에서 기인하는 가를 알아보기 위하여 anti-CD4항체와 anti-BrdU항체로 동시에 염색하여 FACS분석을 행하였다. 그 결과 비장세포의 세포증식에서 나타난 세포증식 반응은 CD4+ T세포에 의한 것으로 확인되었다(Fig.5). ⑤ RT-PCR을 통해 IL-2, IFN-r, GM-CSF의 mRNA를 조사한 결과, KML-C를 HA와 함께 경비투여한 그룹에 있어서 이들 사이토카인에 대한 mRNA가 가장 강하게 유도되는 것으로 나타났다(Fig.6). 또한 IFN-r mRNA 발현에 대해 Real-time PCR을 통해 정량적 분석을 행한 결과, KML-C는 CTB보다도 약 3.5배 정도 이 사이토카인에 대한 mRNA의 발현유도 활성이 높은 것으로 확인되었다(Fig.7).
⑥ 이들 결과를 종합해 볼 때, KML-C는 HA와 함께 경비적 투여를 행할 경우 항체반응으로 대표되는 항원 특이적인 체액성 면역반응은 물론 T세포의 증식반응과 사이토카인의 분비 증가를 통해 세포성 면역반응을 높이는 것으로 확인되었다.
다. KML-C에 의한 T세포 활성화 작용
① KML-C는 항원(HA)과 함께 경비투여하였을 때, T세포를 활성화하여 세포성 면역을 높이는 것으로 확인되었다. 본 실험에서는 KML-C에 의한 T세포의 활성화를 세포수준에서 해석하기 위하여 FACS기법을 이용하여 활성화 T세포를 동정, 확인하였다. 먼저 면역개시 3주째의 비장세포내의 T세포/B세포의 비율을 조사한 결과, KML-C투여에 의해 이들 림프구의 population에는 영향이 없는 것으로 나타났다(Fig.8).
② CD8+ 세포에 대한 분석에서는 CTB와 KML-C는 모두 항원만을 면역한 마우스에 비해 CD8+ T세포의 분포가 거의 동등한 수준으로 증가하였다(Fig.9). 이 결과는 KML-C를 항원과 함께 경비투여할 경우, T세포와 B세포의 분포 상의 변화를 초래하지는 않지만, T세포에 있어서는 CD8+세포의 분포는 다소 높이는 경향이 있는 것으로 추정된다.
③ 활성화 T세포를 분석하기 위하여 T세포의 활성화 지표로 알려진 CD25와 CD69분자에 대한 monoclonal antibody를 이용하여 KML-C 투여에 의해 T세포 상에서 이들 활성화 분자의 발현이 상승하는 가를 FACS로 분석하였다. 그 결과 KML-C를 항원과 함께 경비투여한 마우스에서 CD25분자의 발현이 HA만을 투여한 마우스에 비해 현저하게 증가하는 것을 확인할 수 있었다(Fig.10).
④ CTB를 투여한 마우스에서는 HA만을 투여한 마우스와 거의 동등한 수준의 발현을 보였다. 또한 CD4 및 CD69분자에 대한 dual staining method를 통해 이들 분자를 공동발현하는 활성화 CD4+ T세포의 분포를 조사한 결과에 있어서도, CD25+ T세포 분석결과와 동일하게 KML-C를 투여한 마우스에서 HA 혹은 CTB+HA를 면역한 마우스보다 double positive T세포가 증가하는 것으로 확인되었다(Fig.11).
2. H9N2 인플루엔자 바이러스 공격접종 방어활성
가) 시험용 Influenza virus A/HS/K5/01 (H9N2)를 PBS로 10진 희석한 후 10일령 SPF 부화란의 요막강에 각각 접종하여 접종란의 폐사여부와 생존란의 요막액 에 대한 혈구응집 반응을 검사하여 바이러스 역가를 산출한 결과 108.0 EID50/ml로 확인되었다.
나) 시험물질 KM 투여 SPF 병아리에서 조류인플루엔자 바이러스 증식억제 시험을 실시한 결과 KM-110 10mg 투여군과 20 mg 투여군은 공격접종 대조군에 비하여 기관 및 맹장편도에서 바이러스 재분리율이 유의성 있게 저하되었으며, 시험물질 투여량에 비례하여 바이러스 증시 억제 효과가 관찰되었다.
다) 시험물질 HKM-110 투여 SPF 병아리에서 조류인플루엔자 바이러스 증식억제 시험을 실시한 결과 HKM-110 10 mg, 20 mg 투여군은 공격접종 대조군에 비하여 기관에서 바이러스 재분리율이 유의성 있게 저하되었고, HKM-110 5 mg, 10 mg, 20 mg 투여군은 공격접종 대조군에 비하여 맹장편도에서 바이러스 재분리율이 유의성 있게 저하되었으며, 시험물질 투여량에 비례하여 바이러스 증시 억제 효과가 관찰되었다.
라）시험물질 HKM-110 투여에 따른 SPF 병아리의 체중변화와 아울러 1일 사료소비량, 일일 음수소비량을 비교조사 한 결과 유의성 있는 변화가 관찰되지 않았다.
이상의 시험 결과 공시된 겨우살이에서 추출한 KM-110과 HKM-110은 SPF 병아리에 투여 시 병아리 생체 내에서 조류인플루엔자 바이러스 증식억제 효능이 관찰되어 항 조류인플루엔자 바이러스 효능이 있는 것으로 판단되었으며, 20 mg 투여 시 까지 병아리 체중과 사료 및 음수소비량에 변화가 없어 안전성이 확인되었으며, 향후 바이러스 증식이 억제 되는 기전연구와 아울러 항바이러스 효능이 있는 유효성분의 탐색, 치료 또는 예방 투여용량과 용법, 그리고 유효성분의 안전성 등에 대한 추가적인 시험을 실시 할 경우 향후 최근 전 세계적으로 문제시되고 있는 조류인플루엔자 바이러스에 유효한 기능성 식품이나 약품의 후보물질로 서의 가능성을 확인하였다.
Abstract
▼
AI(Avian Influenza) is a flu infection in birds. This disease is of concern to humans, who have no immunity against it. The virus that causes this infection in birds can mutate to easily infect humans. Such mutation can start a deadly worldwide epidemic. Historically, avian influenza viruses infecte
AI(Avian Influenza) is a flu infection in birds. This disease is of concern to humans, who have no immunity against it. The virus that causes this infection in birds can mutate to easily infect humans. Such mutation can start a deadly worldwide epidemic. Historically, avian influenza viruses infected the pigs and mixed with pig influenza viruses. The viruses changed their genetic information which led to formation of a new virus. This new virus could then infect humans and easily spread from person to person. Avian influenza virus was first detected to infect humans in Hong Kong in 1997, during an avian influenza on that island.
Since these initial outbreaks, HPAI(H5N1) continued to cause illness and death in variety of wild birds in Asia(United States Geological Survey, 2007). Asian lineage HPAI virus(H5N1) was first detected in Europe, where it was isolated from dead wild waterfowl in several European Union member states and neighboring countries(Sabirovic M, 2005).
This outbreak was linked to chickens and classified as avian influenza A (H5N1). This avian influenza A (H5N1) is highly pathogenic, hence this disease was classified as the first grade legal communicable disease or as a "A grade" by Office International des Epizooties. The avian influenza had crossed the species barriers in Asia to cause an increasing pandemic threat and resulted to serious damage to poultry industry.
The outbreak of avian influenza in Korea was first reported in 1996 in one of the layer farms in Choongcheungbukdo province, and reported again in the same place in 2003. It then spreaded out to the other cities and prefectures. It was reported that highly pathogenic avian influenza had occurred in Ansung last year and even this year. Roughly 2,400,000 poultry animals including 1,300,000 chickens were killed and buried.
This influenza is acquired or transmitted through inhalation of infectious droplets, and direct or indirect contact of either human foot, equipment, facilities, feces on egg surfaces onto upper respiratory tract and conjunctive mucosa.
Avian influenza viruses belong to the Influenza virus A genus of the Orthomyxoviridae family and are negative sense, single-stranded, segmented RNA viruses. Influenza viruses can be further categorized into subtypes according to the antigens of the haemagglutinin (H) and neuraminidase (N) projections on their surfaces. There are 14 haemagglutinin subtypes and 9 neuraminidase subtypes of influenza A viruses, and avian influenza viruses have representatives in all of these subtypes. However, to date all highly pathogenic AI viruses that cause generalized rather than respiratory disease belong to either the H5 or H7 subtypes. For example, the classical fowl plague virus is H7N7 and the virus responsible for the major epidemic in the eastern United States in 1983 and 1984 was H5N2 Avian Influenza.
The symptoms of AI are very variable according to pathogen of the infecting virus, but it generally shows respiratory problem, diarrhea, sudden decrease of egg production rate, blue color in cockscomb or head part, and some edema in front face.
The mortality of AI is pretty high (more than 50% at least), and symptoms are somewhat similar to newcastle disease, infectious laryngotracheitis(ILT) and mycoplasma. Therefore it is not easy to diagnose AI infection correctly. Mucosal membranes surrounding the epithelial tissues in mouth, respiratory tract, bronchi and genital organs are the first barrier in animal protection system. These mucosal membranes accomplish the role to protect the animal body from the invasion of contaminants, chemicals, various antigenic molecules including microorganisms.
In recent years, common mucosal immune system (CMIS) has been introduced. It is generally accepted that the immune stimulation through one of the mucosal membranes can activate the whole body mucosal immunity and further control the whole body immunity.
On this point of view, the objective of the present research was to develop the feed additive to suppress the respiratory infection by AI virus through fortification of mucosal immunity in chickens. Further, we aimed to accomplish the higher value-added agricultural managing techniques helpful for national economy and poultry industry through the scientific research data.
1. The first research subject
A. Feeding trial using broiler at the laboratory level
In order to investigate the effect of mistletoe extract on nutrient availability and immune function, Loss strain male chicks adapted to new environment for 3 days after hatching were fed dietary mistletoe at the level 0.5% or 1% for 4 weeks. After experimental feeding, body weight gain, feed efficiency, PBMC (Pheripheral Blood Mononuclear Cells) proliferation, blood antibody titer against ND (Newcastle Disease) and IBD (Infectious Bursal Disease) antigens were measured.
There were no significant differences in feed intake, body weight gain, and feed efficiency.
It was observed that addition of 1% mistletoe into the diet did not show any differences in feed intake and broiler productivity.
B. Investigation of immune modulation activity by dietary mistletoe
The effect of dietary mistletoe on immune response was evaluated through the proliferation of PBMC and splenocytes in chicks feed with experimental diets and challenged with ND and IBD virus infection.
In the present study, addition of 0.5% mistletoe showed significant increase in PBMC proliferation stimulated with both LPS and ConA after 7 days of ND and IBD challenge (P < 0.05).
Also, when compared to the control group, addition of 0.5% mistletoe in broiler feed for 14 days after ND/IBD infection increased the proliferation of the splenocytes stimulated with LPS and ConA.
In the present study, concanavalin A, one of mitogen used in measurement of PBMC and splenocytes proliferation, is the compound known to stimulate the proliferation of T lymphocytes, and LPS is known to stimulate the proliferation of B lymphocyte (Cochet et al., 1997).
Therefore, it was demonstrated that addition of 0.5% mistletoe in the diet reinforced cell-mediated immunity through increased proliferation of T lymphocytes and humoral immunity through the increased proliferation of B lymphocytes in broiler challenged with ND and IBD infection.
Also, it is assumed that the increase of B lymphocytes proliferation by 0.5% mistletoe addition might be related with the increase of antibody titer against ND and IBD infection.
C. ND and IBD antibody titer
The broilers serum was isolated and diluted 8 times. Then, antibody titer values were measured. As observed, the ND antibody titer value was increased by 0.5% dietary mistletoe when compared to the control group, but decreased in broilers fed the 1% dietary mistletoe. From these results, it is considered not to add more than 0.5% in the diet for nutrient availability and the modulation of immunity.
D. Feeding trial using laying hens
In order to investigate the effect of fermented clay mineral to obtain the synergic action of the mistletoe extract, laying hens were fed 0.5% fermented clay mineral (T1), mistletoe extract (T2) and fermented clay mineral+mistletoe extract (T3) diets, respectively for 6 weeks.
To investigate the immune modulation activity by the feeding of experimental diets, IgY titer was measured after 6 weeks of muscular injection of E. coli 88 microbe.
Though there were no significant differences in egg production rate and eggshell strength between the groups with or without mistletoe, it is strongly suggested that the addition of micro minerals into the diet may increase the synergic effect of mistletoe for the layer diet.
Haugh unit showing egg freshness, height of egg white, eggshell thickness were also similar among treatments.
From these results, it is demonstrated that very useful information for industrialization of the product was achieved through the improvement of egg product and egg quality by the blending with the micro minerals more than dietary feeding of mistletoe only.
2. The 2nd research subject
A. Non-specific immune stimulating effects of Korean mistletoe
1) The preparation of mistletoe extracts
In the present study, various extracts of Korean mistletoe were administered to the animal through the mucosal membrane (by oral or nose) to determine if the
mistletoe extracts will increase the non-specific immunity of host animal. Seven different mistletoe extracts were prepared. The immuno-functional study was undertaken for the firstly prepared KM-110 mistletoe extract.
B. Immunity modulation activity of KM-110 administered into the mucosa
KM-110 extract was firstly prepared from Korean mistletoe by cold water extraction, and then immune modulation activity and simple toxicity test were done after oral or intranasal administration of each extract into the mice.
After the administration of KM-110 once in a day during 7 consecutive days, the blood immunity-globulin IgG,A.M levels were measured by ELISA method. When mistletoe level was increased from 250ug to 1,000 ug/mouse, blood IgG,A,M levels were increased in all groups compared to the control group. The group administered with 250ug/mouse showed the highest blood IgG,A,M levels.
On the other hand, to investigate the synergistic effect of mistletoe extract by oral administration on the modulation of mucosal immunity, the immuno-globulin was measured from blood and feces. As a result, both blood and fecal IgG,A,M were increased in similar pattern. This result could be attributed to the stimulation of immunoglobulin secretion in feces by the modulation of immunity through oral administration of KM-110.
C. Investigation of immune modulation of by oral administration of KM-110
In order to investigate the immune modulation activity, mice were administered KM-110 through nose during 7 days and then fecal immunoglobulin was measured equally with oral administration to investigate immunity activity possibility by dose in hard ball of KM-110. As results, fecal IgGAM and IgG secretions were increased.
With the consideration that AI is a virus infectious disease through respiratory tract, the results showing increased immunity at the present study by the administration of extracts through nose are considered much meaningful for the control of AI virus.
As observed above, it has been clarified that consecutive administration of KM-110 by mouth or nose activated the non-specific immune response. At the next step, simple toxicity test was done to investigate if this administrative condition shows any toxicity to the host animal. The oral administration of KM-110 once in a day for seven days showed no side effects or negative effects, but there was slight decrease in body weight in mice administered with KM-110 by nose. However, it has been shown that there were no liver damage by both oral and nose administration of KM-110.
D. The comparison of immune modulation activities among KM-110, HKM-110 and KML-C
In order to investigate the activation of immune cells, mice were administered HKM-110 and KML-C including KM-110 five times through the mucosal membrane. It was found that both oral or nasal administration of these extracts stimulated the mouse splenocytes. Furthermore, the administration of these extracts increased the basal metabolism, and did not cause any weight loss or liver toxicity.
E. Immune modulation activities of KMF-WE and KM-PS
In order to investigate the immune modulation activity of crude polysaccharide extracts (KM-PS) or fruit extracts (KMF-WE) from Korean mistletoe, mice were administered with these extracts by mouth or nose 5 times, and then fecal IgA and basal metabolism were measured. In addition, simple toxicity test was done to check any toxicity through the change of their body weights.
As a result, it was observed that oral administration of KM-PS and KMF-WE once in a day for 5 consecutive days increased the intestinal IgA secretion, and basal metabolism was also clearly increased. By the observation that these extracts did not cause any weight loss, it has been considered that these extracts are comparatively safe samples.
F. Antigeen specific immune modulation ― activities in mice
On the view point that AI is influenza virus, mucosal immunity was investigated by intranasal administration of HA peptide antigen originated from influenza. From this study, it was found that antigen specific immunity was increased as measured by IgG and IgA titer values from samples of total serum and vaginal canal.
When KM-110 was used as immune modulator, highest titer activity was observed at 5㎍ administration and even higher immune modulation activity than that of control group stimulated with LPS. However, no dose-dependent results were observed among the titer activities at the level of 1㎍, 5㎍ and 10㎍. Therefore, it is supposed that more experiments are needed to clarify if the effects of extracts shows in dose-dependent manners.
When WHBP was used as a immune modulator, the highest titer values were observed in mice administered 10㎍ or LPS+10㎍ of extracts. These values were even higher than that of control group treated with LPS only.
G. Analysis of synergistic effect of antibody reaction by KML-C nasal administration.
The titer values of antigen specific IgG(immunoglobulin G) and IgA (immunoglobulin A) of the blood and vaginal fluid were measured using ELISA after 1week, 3weeks, 8weeks of immunization through the nasal administration of HA+KML-C or HA+CTB every other weeks. First, it was observed in blood HA specific IgG titer analysis that both KML-C and CTB increased antibody titer value against HA after a week of immunization.
Meanwhile, it appeared that blood antibody titer value was higher in mice administered KML-C+HA or CTB+HA than in mice administered HA only, but there was no significant statistical differences.
On the other hand, it was found that antigen specific IgG modulating activity was similar between KML-C and CTB. Unlike serum IgG, it was observed that IgA of vaginal fluid was increased after 3weeks of immunization. As in serum IgG, no difference was observed in antibody modulating activity by KML-C and CTB in IgA of vaginal fluid.
However, it was thought that, KML-C is acting under the very low dosage considering the administration concentration of CTB was 1ug/head and 0.1ug/head in KML-C.
In the present study, antigen specific antibody modulating activity was measured especially focusing on IgG2b antibody. As a result, it was observed that both KML-C and CTB increased antigen specific IgG2b. And these adjuvant have similar activity for antigen specific IgG2b antibody modulation.
H. Analysis of cell-mediated immunity modulation by intranasal administration of KML-C
In order to measure the antigen specific cell-mediated immune modulating activity, cell proliferation response were investigated by re-stimulation of splenocytes by antigen or mitogen (LPS or ConA) in vitro after 3 weeks of immunization. As a result, it appeared that, in case of re-stimulation with HA, KML-C remarkably increased antigen specific immune cell proliferation
In case of re-stimulation by ConA or LPS, it was observed that the highest activity was shown in group administered KML-C together with HA, but non-specific activation by mitogen was significantly decreased than antigen specific activation. In other words, it was thought that antigen specific cellular immunity was modulated by nose-administration of KML-C together with antigen.
In order to clarify antigen specific cellular immunity, cell proliferation was measured by bromodeoxiuridine (BrdU) using FACS. Cell proliferation was highest in group administered KML-C together with HA. In order to investigate if increased cell proliferation effect is due to T cell, FACS analysis was undertaken by using anti CD4 antibody and anti BrdU antibody. As a result, splenocytes proliferation was mediated through CD4+ T cell.
From the results of mRNA of IL-2, IFN-r, GM-CSF measured by RT-PCR, the highest mRNA expression was found in group administered KML-C together with HA by nose. Especially, IFN-r mRNA expression KML-C measured by quantitative real-time PCR was 3.5 times higher than CTB group. Altogether, it was clarified that KML-C administration by nose together with HA increased the cell mediated immune response by increasing of not only antigen specific humoral immunity but also T cell proliferation and cytokine secretion.
I. T cell activation by KML-C
When KML-C was administered by nose together with HA, it was clarified that cellular immunity was increased through T cell activation. In the present study, activated T cell was isolated and classified by FACS analysis to interpret the mechanism of T cell activation in the cellular level. First of all, it was clear that there was no change in lymphatic cell population by nose administration of KML-C measured by B cell/T cell ratio of splenocytes after 3 weeks of immune stimulation.
CTB or KML-C administration increased the CD8+ T cell population compared to those of groups immunized antigen only. This results shows that, when KML-C was administered together with antigen, there were no change in T cell and B cell population but CD8+ T cell population seems to be increased.
In order to analyze the activated T cell, FACS analysis was undertaken to know if protein expression of CD25 and CD69 proteins known as marker protein in T cell activation. As a result, CD25 protein expression was markedly increased in mice administered KML-C by nose compared to those of mice administered HA only by nose.
Meanwhile, mice administered CTB showed nearly similar level of CD25 and CD69 proteins expression to those of mice administered HA only. From the results of CD4 and CD69 molecules, it was clarified that, like CD25+ T cell analysis results, mice administered KML-C showed increased double positive T cell compared to those of mice administered HA or CTB+HA.
J. Protection activity against the H9N2 influenza virus challenging.
1) After the test Influenza virus A/HS/K5/01 (H9N2) was serially diluted with PBS, it was inoculated into the cavity of allantois of SPF eggs, which were previously incubated for 10 days. Mortality was determined and the EDI 50 virus titer value was measured by hemagglutinin assay. It was found that titer value was 108 EID50/ml.
2) The result of AI virus growth arrest test in KM-administered SPF chicks showed that 10mg and 20mg administration of KM-110 significantly decreased virus isolation rate in organ and caecum compared to control group challenged with test virus.
The arrest effect of test virus was shown in dose dependent manner of test sample concentration.
3) As a result of AI virus growth arrest test in SPF chicks, administration of 10mg and 20mg of HKM-110 decreased virus isolation rate in dose dependent manner compared to control group challenged with test virus.
4 There were no significant changes in body weight, daily) feed and water intake in SPF chicks administered with test sample of HKM-110.
Altogether, it was thought that test sample Korean mistletoe extract KM-110 and HKM-110 has anti AI virus activity from the observation of decreased AI virus growth in SPF chicks challenged with test virus.
With the results that there were no changes in body weight, feed and water intake of chicks administered 20mg, mistletoe sample is accepted safe sample.
In the present study, if further studies in mode of action for virus growth arrest, screening of functional compound showing anti virus activity, dosage and using guideline for therapy and protection, safety of functional compound are completed, mistletoe extract is considered to be one of the strong candidate compound to produce functional food and medicine useful against worldwide AI virus program.
3. The 3rd research subject
A. The results of field trials
The field test were undertaken to investigate if the mucosal immune modulation activity of KM-110 is also available in poultry farm.
After the experimental feeding of dietary mistletoe for 38 days, the production index, growing rates, final body weight increase were used as a availability of Korean mistletoe.
The results showed that treated groups had higher growing rates, body weight gain including production index than control group.
In the present study, though it was impossible to clarify the infection defensive effects, it has been suggested that administration of Korean mistletoe extract can increase the broiler growth by the effective suppression of respiratory disease, one of AI disease showing wasting syndrome, by fortifying the mucosal immunity. This speculation was supported by the results of increased final body weight in shipping.
B. The development of design of pilot product of feed additive and it's formulation
The original resources of pilot product consisted of Granitic Gneiss to fortify the absorptive function to reduce the ammonia, decomposition bacteria and mycotoxins. In addition to that, probiotics for stimulating the digestion and palatability, some other herbal extracts and mistletoe were added and well mixed. The design of trademark of pilot product was focused on symbolizing of mistletoe, intestinal mucosal membrane to emphasize the strengthening imucosal immunity; and virus adsorption.
목차 Contents
- 제출문 ... 1
- 요약문 ... 2
- SUMMARY ... 14
- 목차 ... 26
- 제 1 장. 연구개발과제의 개요 ... 27
- 1. 연구개발의 목표 및 내용 ... 27
- 제 2 장. 국내ㆍ외 기술개발현황 ... 28
- 제 3 장. 연구개발 수행 내용 및 결과 ... 29
- 제 1 절. 한국산 겨우살이에 의한 비특이적 면역증강 효과 ... 29
- 제 2 절. 마우스에서의 항원 특이적 면역증강 시험 ... 42
- 제 3 절. 닭을 이용한 파이로트 사양시험 ... 48
- 제 4 절. 한국산 겨우살이에 의한 점막면역강화 기전해석 ... 62
- 제 5 절. H9N2 인플루엔자 바이러스 공격접종 방어활성 ... 71
- 제 6 절. 현장 사양시험 결과 ... 77
- 제 7 절. 시제품 Formula 작성 및 제품디자인 개발 ... 80
- 제 4 장. 목표달성도 및 관련분야에의 기여도 ... 81
- 1. 연구목표 대비 달성도 ... 81
- 2. 핵심기술 ... 82
- 3. 연구결과별 기술적 수준 ... 82
- 제 5 장. 연구개발결과의 활용계획 ... 83
- 1. 각 핵심 연구 결과별 구체적 활용계획 ... 83
- 제 6 장 참고문헌 ... 84
- 끝페이지 ... 87
※ AI-Helper는 부적절한 답변을 할 수 있습니다.