초록

○ 연구결과
-. 돼지증식성 회장염균 분리 및 동정
-. 돼지 증식성 회장염균의 약독화 성공
-. 돼지 증식성 회장며균의 대량 배양법 확립
-. 살모넬라 정항성 유산균 선정
-. 돼지 로타바이러스 VP8*의 유산균 발현 성공

Abstract ▼

Proliferative enteropathy (PE) is one of the most important enteric diseases, caused by Lawsonia intracellularis (L. intracellularis), all over the world including Korea. Significant economic losses were reported in the PE-suffered farms in Korea because of poor growth rates and high mortality. In s

Proliferative enteropathy (PE) is one of the most important enteric diseases, caused by Lawsonia intracellularis (L. intracellularis), all over the world including Korea. Significant economic losses were reported in the PE-suffered farms in Korea because of poor growth rates and high mortality. In spite of economic losses and concerns, little is known about the introduction mechanism of PE due to difficulty of isolation and cultivation of L. intracellularis. Successful isolation of L. intracellularis and experimental reproduction of PE were reported in Europe and the Unites States. However, there was no report of isolation of L. intracellularis in Korea. The objectives of this study were to isolate Korean strain of L. intracellularis and to reproduce the disease using a pure culture of L. intracellularis as well as intestinal mucosa homogenate obtained from a pig showing PE. In addition, several parameters were evaluated, such as clinical signs, growth performances, the severity of lesions and serology, For isolation of L. intracellularis, McCoy cells were inoculated with the extract of intestine demonstrating proliferative hemorrhagic enteropathy (PHE). In vitro cultivation and maintenance of the bacteria were established at five passages. Identification of the isolate was accomplished by staining the infected cells with mouse monoclonal antibodies, IG4 and 2001MAb, as well as by sequencing PCR (polymerase chain reaction) product using primers specific for genomic DNA of L. intracellularis. Furthermore, 4-week-old pigs were challenged with intestinal mucosa homogenate and pure culture of L. intracellularis. All pigs, challenged with intestinal mucosa homogenate and the bacteria, developed clinical signs of PE, such as a severe diarrhea. Necropsy was carried out at 3 weeks post infection and significant macroscopic lesions, such as the corrugation with hyperemic and thick folds, and microscopic lesions were observed in all the pigs challenged with intestinal homogenate and pure culture of bacteria. L. intracellularis antigen was also demonstrated in intestinal tissues of the infected pigs by immunoperoxidase staining. Serological responses developed in about 2 weeks post infection in all of the challenged pigs. When hamsters, known as highly susceptible animal to L. intracellularis, were challenged with same methods described above, the similar pathogenecity was observed in infected hamsters. In conclusion, this study demonstrates the successful isolation and in vitro maintenance of Korean isolate of L. intracellularis (PHE/KK421) for the first time in Korea and also proves that the pure culture strain of L. intracellularis could induce the PE in the infected commercial pigs and hamsters.
In order to develop probiotics with protective activity against diarrheal diseases as well as with expression of interest gene such as protective antigen gene of Lawsonia intracellularis and VP8 of rotavirus, two kinds of lactic acid bacteria (LAB), Lactobacillus casei E5 and Bifidobacterium longum BL730, were evaluated with a Salmonella infection model in mice. Each strain was administered orally to the mice for eight consecutive days, and one day after the last feeding, the mice were orally challenged with Salmonella enteritidis at a dose of LD100 and then observed for 21 days. While the survival rate of the control group and L. casei E5-treated group was 14%, 71% of the B. longum BL730-treated group survived, indicating that feeding BL730 has a protective effect against Salmonella infection. The possible immune mechanisms involved in this protective activity were investigated. The proliferative response of splenocytes with mitogens and the production of inflammatory cytokines from macrophages did not increase, whereas the concentration of secretory IgA (sIgA) in intestinal contents of the mice fed with BL730 was 2.5 times higher than that of the control group, suggesting that local intestinal immunity plays an important roles in protecting against the Salmonella infection.
Rotaviruses are one of the most important causes of diarrhea in piglets. Mucosal immunity is important to protect rotavirus infection in small intestine. It has been known that one of protective antigens of rotavirus is VP8* of VP4 cleavage product. To effectively induce mucosal immunity, the VP8* antigen may be injected by oral administration rather than by intramuscular vaccination.
Since Lactobacillus is resistant to acid environment in stomach and commensals of the guts, it may be used as delivery vehicle. To ideally deliver VP8* antigen of trypsin cleavage product of porcine rotavirus VP4, one third of VP4 gene coding for VP8* antigen was amplified and cloned in shuttle vector pAT:pgsA of Lactobacillus casei and in pBESAF2 of Bifidobacteria longum. To confirm expression of recombinant VP8* in both expression system, Western blot was carried out using antisera against bovine rotavirus. Expression of VP8* antigen was detected in Lactobacillus casei but not in Bifidobacteria longum. These data indicate that Lactobacillus may be used as delivery system to protect enteric disease as well as rotavirus infection.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• Summary ... 8
• Contents ... 11
• 목차 ... 14
• 제1장 연구개발과제의 개요 ... 17
• 1. 연구개발의 필요성 ... 18
• 제2장 국내외 기술개발 현황 ... 21
• 제3장 연구개발수행 내용 및 결과 ... 22
• 제1절 돼지증식성회장염균의 분리 및 동정 ... 22
• 1. 서론 ... 22
• 2. 재료 및 방법 ... 25
• 가. 돼지증식성회장염균의 분리 및 배양 ... 25
• 1) 분리재료 ... 25
• 2) 조직 배양 ... 25
• 3) 장조직 유제의 세포감염 ... 26
• 4) 감염 세포조직의 맹목적 계대배양 ... 26
• 5) 감염 세포조직의 감염 여부 관찰 ... 27
• 6) 면역조직화학염색 ... 28
• 나. 돼지 증식성회장염의 재현성 실험 ... 28
• 1) 실험동물 ... 28
• 2) 조직배양된 돼지증식성회장염균의 접종 ... 29
• 3) 장조직유제액의 접종 ... 29
• 4) 접종량의 결정 ... 30
• 5) 접종 ... 30
• 6) 임상증상 평가 기준설정 ... 31
• 7) 분변에서의 균체 검사 ... 31
• 8) 혈청학적 검사 ... 31
• 9) 간접형광항체법 ... 32
• 10) 부검 ... 32
• 11) 조직학적검사 ... 32
• 3. 결과 ... 33
• 1) 국내에서 분리된 Lawsonia균의 배양과 유지 ... 33
• (1) 균분리에 사용된 재료 ... 33
• (2) Lawsonia균의 분리 ... 35
• (3) Lawsonia균의 유지배양 ... 35
• (4) 분리균주의 동정 ... 37
• 2) 실험적 증식성 회장염의 유도 ... 38
• (1) 임상증상 ... 38
• (2) 분변내 Lawsonia균 검출 및 혈중 항체가 검사 ... 39
• (3) 부검 소견 ... 42
• (4) 면역조직화학 염색 결과 ... 42
• 3) 햄스터에서의 증식성 회장염 유도 ... 44
• 4) 국내 돼지증식성회장염의 감염 분포 상황 ... 46
• 4. 고찰 ... 49
• 제2절 유산균의 Salmonellar균 감염 방어능 비교 ... 51
• 1. 서론 ... 51
• 2. 재료 및 방법 ... 53
• 가. 실험동물 ... 53
• 나. 유산균 급여 ... 53
• 다. 살모넬라 감염에 대한 유산균 급여 마우스의 방어효과 ... 53
• 라. 비장세포 증식 시험 ... 53
• 마. 탐식세포 활성화 시험 ... 54
• 바. 장관내 분비형 IgA의 정량 분석 ... 54
• 사. 면역화학적 검사 ... 54
• 아. 통계학적 분석 ... 55
• 3. 결과 ... 56
• 가. 비피도박터 유산균의 살모넬라균 감염에 대한 방어효과 ... 56
• 나. 비피포박터 유산균의 비장 T와 B세포 활성화 효과 ... 57
• 다. 비피도박터 유산균의 탐식세포 활성화 효과 ... 58
• 라. 비피도박터 유산균의 장내 분비형 IgA 증강 효과 ... 60
• 4. 고찰 ... 62
• 제3절 유산균을 이용한 돼지로타바이러스 VP8 유전자 발현 ... 65
• 1. 서론 ... 65
• 2. 재료 및 방법 ... 68
• 가. 세균 종류 및 배양배지 ... 68
• 나. plasmid와 DNA 조작 ... 68
• 다. shuttle vector pNZ123:SPO2/beta-galactose의 제작 ... 69
• 라. Lactobacillus와 Bifidobacteyium에서 발현할 셔틀벡터에 VP8의 크로닝 ... 69
• 마. 전기영동과 Western blot ... 70
• 3. 결과 ... 72
• 가. Shuttle vector pNZ123:SPO2:beta-galactosidase 의 제작 ... 72
• 나. XL1-Blue 에서의 beta-galactosidase 의 발현 ... 72
• 다. Shuttle vector pBESAF2/VP8* 의 제작 ... 72
• 라. E. coli와 Bifidobacteria 에서 VP8* 의 발현 ... 72
• 마. Shuttle vector pAT:pgsA/VP8*의 제작 ... 73
• 바. E. coli와 Lactobacillus casei에서 VP8*의 발현 ... 73
• 4. 고찰 ... 79
• 제4장 목표달성도 및 관련분야에의 기여도 ... 81
• 제5장 연구개발결과의 활용계획 ... 82
• 1) 학문적인 측면 ... 82
• 2) 기술적인 측면 ... 82
• 3) 경제, 산업적인 측면 ... 82
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 84
• 제7장 참고문헌 ... 85
• 끝페이지 ... 100