한국다람쥐(Eutamias sibiricus asiaticus)를 이용한 BCG의 독력측정에 관한 연구

Abstract

[한글]

BCG는 Mycobacterium bovis를 약독변이시킨 균주로서, 인체 및 동물에 접종하였을 경우 무독성이고 병원성이 없으며 항결핵 면역성을 지리게 된 것이다.

BCG는 1909년 불란서 Pasteur연구소의 Calmette와 Guerin에 의하여 개발된이래 세계 여러 연구소에 분양보급되었고 항결핵 항원성 이외에도 항나(antileprosy) 및 항암(antitumor)의 면역원(immunogen)을 비롯한 각종 질환에 있어서 비특이 면역요법에 대한 연구가 활발히 전개되어 오고 있다.

그러나 세계 여러 연구소에 분양되어 계대보존되어온 BCG균주들이 유지보존하는 방법에 따라 각 연구소마다 보존된 균주의 생물학적 성상 및 독성 (virulence)에 변이를 가져오게 되어 이들 변이성에 대한 보고가 있으며, 경우에 따라서는 유독성 BCG로 환원변이된 B

CG에 의한 감염증의 보고도 있다. 이와같이 BCG사용에 허다한 혼란과 위험이 부수됨은 독력검정을 할 수있는 시험관내 검정방법이나 적절하고 합리적인 실험동물모델이 (experimental animal model)결여됨에 있다고 할 수 있다.

따라서 본 실험에서는 야생 한국 다람쥐(Eutamias sibiricus asiaticus)가 BCG균주의 독력및 독력의 차이를 합리적으로 측정하기 위한 적절한 실험동물모델로서의 개발가능성을 추구하고저, 대표적인 수종 BCG균주를 다람쥐에 접종하여 각 BCG균주의 독력측정과 독력의 차이를 비교관찰하였던 바 다음과 같은 결론을 얻었다.

1. 실험에 사용된 BCG균주 7주(BCG-K, BCG-Y, BCG-P, BCG-J, BCG-F, BCG-T, BCG-Yo)중 2주(BCG-T, BCG-Yo)는 생물학적 성상에서 성장온도의 범위가 20℃에서 45℃로 광범위했으며 배양속도도 1주만에 발육되었고, 집락의 형태도 달리하였으며 착색 및 광발색성은 나

타내지 않았다. 한편 생화학적 특성에서 7주의 BCG균주가 niacin시험 및 urease시험에서 독력대조군인 인형 결핵균과는 달리 모두 음성반응을 나타내었고 BCG-K주만은 catalase시험에서 음성반응을 나타내었다. 따라서 생물학적 성상 및 생화학적 특성의 차이에 의한

각 BCG균주간의 독력의 비교는 할 수 없었다.

2. BCG-Y균주는 다람쥐 및 마우스 정맥내에 10**5 균수를 접종한 후 8주동안 관찰한 바 다람쥐에 있어서만 비장 및 간장에 육안적, 병리 조직학적 병변이 인정되었으며, 이들 장기로부터 항산균의 검출 및 환원배양에서 항산균이 발육되었다.

3. BCG-Y균주를 다람쥐와 마우스에는 복강, 정맥 및 뇌내에 접종하고 기니픽에는 복강내에 10**5 에서 10**8 균수를 접종한 바 마우스 및 기니픽에서는 관찰 56일동안에 한마리도 사망하지 않았으며 부검장기에서도 병변이 인정되지 않았으나, 다람쥐에서는 15일에

서 52일에 전부 사망하였으며 이들의 부검시에 심한 육안적, 병리학적인 병변이 관찰되었으며, 세균학적으로 항산균의 검출과 장기에서의 항산균의 환원배양이 인정되었다.

4. BCG-P, BCG-J, BCG-Y 및 Mycobacterium tuberulosis(H^^37 Rv)를 다람쥐와 마우스의 뇌내로 10**5 균수를 각자 5마리 에 접종한 바 한국 다람쥐에서만 Mycobacterium tuberulosis 접종군은 접종 후 13인에서 21일에 전부 사망하였고, BCG-Y접종군은 24에서 36일에

전부 사망하였으며, 이들은 부검시 심한 육안적, 병리학적 및 세균학적 변화를 관찰할 수 있었으나, BCG-P접중군에서는 접종후 36일에 한마리가 사망하여 부검시 육안적, 병리학적 병변과 세균학적으로 항산균을 관찰할 수 있었고 접종후 8주에 나머지 4마리를 희생시켜 관찰한 바 3마리에서 뇌내에서 경중의 육아종성 결절과 항산균의 환원배양이 되었으며, BCG-J접종군에서는 8주까지 1마리도 사망하지 않았으며 부검소견에서도 아무런 변화를 관찰할 수 없었다.

이상의 결과에서 한국 다람쥐는 BCG균주에 대한 감수성이 각 균주에 따라 높게 나타내었으며, 짧은 시일내에 발중이 가능하므로 BCG균주의 독력측정 및 독력의 차이를 검정할 수 있는 적절한 실험동물 모델이 될 수 있다고 사료된다.

The Virulence Assay, of Various BCG Strains with the Korean

Chipmunk(Eutamias sibiricus asiaticus)

Jung Hee Choi

Department of Medical Science, The Graduated School, Yonsei University

(Directed by Professor Joon Lew, M.D.)

BCG is an attenuated strain of Mycobacterium bovis which is not pathogenic to

animal and human but immunogenic to elicite immunity to tubercle bacillus

infection.

Since BCG has been established by Calmette and Guerin at Pasteur institute in

1909, it has been used as a vaccine for prevention of tuberculosis, besides applied

for antitumor treatment which can be elicited by non-specific stimulation of host

immune system.

However, long-term aubculture might cause some changes on the biological anti

biochemical characters and virulence. There was no report on adequate experimental

animal model or in vitro assay method to carry out the virulence test of long-term

subcultured BCG.

In an attempt to develope an adequate experimental animal model for testing the

virulence of BCG strains, Korean chipmunks were inoculated with various strains of

BCG(BCG-K, Y, P, J, F, T and Yo) and the pathological changes were compared.

The results are summarized as follows :

1. In two of seven BCG strains, the optimal temperature of growth showed

20℃∼45℃ and the colonies could be detected within a week, but pigmentation and

photochromogenesis were not exhibited.

2. Seven BCG strains and Mycobacterium bovis (Ravenel) exhibited negative

reaction in niacin and urease tests in contrarily with Mycobacterium tuberculosis

(H^^37 Rv). BCG-K strain, however, showed negative reacton in catalase test.

3. Virulences in various strains of BCG by the biological and biochemical

characteristics showed no difference.

4. By intravenous inoculation of BCG-K, gross and histopathological changes were

observed in liver and spleen only in Korean chipmunks but not in mice. Acid fast

organisms were also detected from liver and spleen of Korean chipmunks.

5. Korean chipmunks inoculated with BCG-Y died between 15 and 52 days showed

severe pathological changes. The acid fast organisms were isolated from various

organs, but none of mice and guinea pigs showed Pathological changes during 50 days

observation.

6. In Korean chipmunks after inoculation with BCG-Y, all died between 24 and 36

days and showed high grade of gross and histopathological changed with acid fast

organisms. The Korean chipmunks inoculated with Mycobacterium tuberculosis also

died between 13 and 21 days and showed severe pathological changes.

In BCG-P inoculation group, only one chipmunk died 36 days after inoculation and

showed severe grosp, and histopathological changes with acid fast organisms. In

four of this group sacrificed at 8th week, mild granulomatous changes was observed

in brain and reisolation of acid fast organisms was possible.

None of the BCG-J inoculated Korean chipmunks died up to 8th wee? and no

pathological changes were observed in sacrificed chipmunks.

Through the results obtained, it is suggested that Korean chipmunk is verified as

an adequate animal model for virulence assay of BCG strain in behalf of its

susceptibility and rapid manifestation of pathological changes after the

inoculation.

[영문]

BCG is an attenuated strain of Mycobacterium bovis which is not pathogenic to animal and human but immunogenic to elicite immunity to tubercle bacillus infection.

Since BCG has been established by Calmette and Guerin at Pasteur institute in 1909, it has been used as a vaccine for prevention of tuberculosis, besides applied for antitumor treatment which can be elicited by non-specific stimulation of host immune system.

However, long-term aubculture might cause some changes on the biological anti biochemical characters and virulence. There was no report on adequate experimental animal model or in vitro assay method to carry out the virulence test of long-term subcultured BCG.

In an attempt to develope an adequate experimental animal model for testing the virulence of BCG strains, Korean chipmunks were inoculated with various strains of BCG(BCG-K, Y, P, J, F, T and Yo) and the pathological changes were compared.

The results are summarized as follows :

1. In two of seven BCG strains, the optimal temperature of growth showed 20℃∼45℃ and the colonies could be detected within a week, but pigmentation and photochromogenesis were not exhibited.

2. Seven BCG strains and Mycobacterium bovis (Ravenel) exhibited negative reaction in niacin and urease tests in contrarily with Mycobacterium tuberculosis (H^^37 Rv). BCG-K strain, however, showed negative reacton in catalase test.

3. Virulences in various strains of BCG by the biological and biochemical characteristics showed no difference.

4. By intravenous inoculation of BCG-K, gross and histopathological changes were observed in liver and spleen only in Korean chipmunks but not in mice. Acid fast organisms were also detected from liver and spleen of Korean chipmunks.

5. Korean chipmunks inoculated with BCG-Y died between 15 and 52 days showed severe pathological changes. The acid fast organisms were isolated from various organs, but none of mice and guinea pigs showed Pathological changes during 50 days observation.

6. In Korean chipmunks after inoculation with BCG-Y, all died between 24 and 36 days and showed high grade of gross and histopathological changed with acid fast organisms. The Korean chipmunks inoculated with Mycobacterium tuberculosis also

died between 13 and 21 days and showed severe pathological changes.

In BCG-P inoculation group, only one chipmunk died 36 days after inoculation and showed severe grosp, and histopathological changes with acid fast organisms. In four of this group sacrificed at 8th week, mild granulomatous changes was observed

in brain and reisolation of acid fast organisms was possible.

None of the BCG-J inoculated Korean chipmunks died up to 8th wee? and no pathological changes were observed in sacrificed chipmunks.

Through the results obtained, it is suggested that Korean chipmunk is verified as an adequate animal model for virulence assay of BCG strain in behalf of its susceptibility and rapid manifestation of pathological changes after the inoculation.