자유생활 아메바(Acanthamoeba species)의 숙주에 대한 면역학적 반응

Abstract

[한글]

숙주와 병원체의 관계에서 숙주에게 인위적 면역을 부여할 경우 병원체에 의한 발병을 예방 또는 약화시킬 수 있음은 잘 알려진 사실이며 숙주와 기생충간의 관계에서도 특정 기생충에 대한 숙주의 저항력이 강화되면 그 기생충에 대한 병해가 약화하거나 수명이 연

장된다. 이와 같은 숙주-기생충의관계를 토대로 근태에 와서 원발성 뇌수막염의 병원체로 알려진 자유생활아메바에 감염되면 숙주내에 면역체가 생성된다는 많은 보고가 있다. John등(1977)은 살아있는 자유생활아메바를 복강내에 접종면역시켰을때 숙주의 저항력이 증

가된다고 하였으며 Thong등(1978b)은 면역된 혈청을 실험동물에 주입하였을 때에도 병원성아메바에 대한 숙주의 저항력이 장기간 지속됨을 보고하였다. 본 실험에서는 한국에서 분리한 Acanthamoeba Species, YM-4를 마우스에 인위적으로 면역을 부여한 경우와 반대로

면역형성을 억제하는 경우에 본 원충에 대한 저항력 변화를 비교관찰함으로써 본 원충의외 저항력의 본태를 규명하며 아울러 치명적인 본 질환의 감염발병을 억제할 수 있는 가능성을 찾아 보고저 실험을 실시하였다.

실험동물로서는 8∼20g의 흰 웅성마우스를 사용하였으며 Acanthamoeba sp.,YM-4 strain은 CGV 배지에서 계대배양하였다. 실험군은 다음과 같이 4군으로 나누어 실험하였다.

1. 아무런 처치를 받지 않은 건강대조군

2. 면역 실험군

3. 프케드니소론 처치군

4. 감마선 조사군

면역실험군은 다시 3군으로 분류하였다.

1. 살아있는 아메바 5×10**5개를 1차 복강내 감염 면역된 군

2. 살아있는 아메바 5×10**5개를 2차 복강내 감염 면역된 군

3. 수용성 항원 0.2mg으로 피내접종 면역된 군

프레드니소론 처치는 격일로 10mg/kg씩 5회 근육주사하였으며 감마선은 총 300 rad.를 조사하였다. 1차 복강내 감염면역된 군은 6주 후에, 2차 복강내 감염면역된 군은 5주 후에, 수용성 항원으로 피내접종면역된 군은 5주후에, 프레드니소론처치 및 감마선 조사군은 2일 후에 각각 1x10**4개의 YM-4 strain을 뇌내감염시킨 후 30일간 관찰하였다. 실험 마우스가 사망하였을 때는 뇌조직을 분리하여 일부는 조직표본을 만들어 관찰하였고, 일부는 non-nutrient agar에 배양하여 아메바의 감염을 확인하였다. 실험마우스의 혈청내

항체가의 변동을 관찰하기 위해 간접 형광항체법을 이용하였다.

본 실험으로 다음과 같은 결과를 얻었다.

1. 각 실험군에 살아있는 Acanthamoeba sp., YM-4 1x10**4개를 뇌내감염시킨 후 30일간 관찰한바

1) 건강대조군은 7∼21일내에 64.0%,

2) 1차 복강내 감염면역된 군은 5∼21일내에 40.7%,

3) 2차 복강내 감염면역된 군은 3∼12일내에 8.3%,

4) 수용성 항원으로 피내접종면역된 군은 11∼23일내에 25.0%,

5) 프레드니소론 처치군은 2∼21일내에 75.0%,

6) 감마선 조사군은 5∼22일내에 64.0%가 각각 사망함으로써 면역이 부여된 군은 건강대조군에 비하여 사망율에 유의한 차이가 있었다. 면역억제된 군은 건강대조군에 비하여 사망율에는 별 차이가 없었으나 일찍 사망하는 경향을 관찰할 수 있었다.

2. 자유생활아메바에 대한 형광항체가는 면역접종에 의해 뚜렷이 증가하였으나 프레드니소론 처치군이나 감마선 조사군에서는 형광항체가의 상승이 둔화되었다.

이상의 결과를 볼 때 마우스에 있어서 자유생활아메바에 대한 인위적 면역을 부여할 때 본 원충에 대한 항체 생성이 증가됨으로써 치명적인 원발성아메바성뇌수막염의 감염발병을 억제할 수 있을 것으로 생각된다.

Immunologic Response of Mice Against Free-living Amoeba(Acanthamoebe sp.)

Young-Nam Whang

Department of Medical Science, Graduate School, Yonsei University

(directed by Professor Chin-Thack Soh, M.D.)

Several reports indicated that infection of free-living amebae produced immune

bodies in the host. John et al. (1977) reported that mice immunized by

intraperitoneal injection of live Naegteria fowleri acquired resistance to

challenge infection of the same amoebae. Thong et al. (1978b) reported that

protective immunity to Naegleria meningoencephalitis could be transferred by immune

serum. In the present study comarative observation of immunologic response of

immunized and immunosuppressed mice to Acanthamoeba sp. YM-4 was performed to

examine the possibility of acquiring protective imnunity. White male mice weighing

8 to 20g were used for experiments. Acanthamoeba sp., YM-4 strains were cultured in

CGV medium (Willaert and Le Ray, 1973). Mice were divided into four groups;

untreated control group, immnunized group, prednisolone treated group and gamma-ray

irradiated group. Immunized group was divided into 3 subgroups: intraperitoneal

injection of one dose of 5×10**5 Acanthamoeba sp., YM-4; intraperitoneal injection

of two doses of 5×10**5 Acanthamoeba sp., YM-4 one week apart; intradermal

injection of 0.2mg of YM-4 antigen. In the prednisolone treated group, the hormone

was injected intramuscularly in 5 doses of 10mg/kg every other day. In gamma-ray

group, mice were irradiated with total dose of 300 rad. 1×10**4 Acanthamoeba sp.,

YM-4 was injected into brain through occipital region under ether anesthesia

respectively to each mouse on: five weeks after immunization, 2 days after

prednisolone administration and 2 days after gamma-ray irradiation. On the designed

date, the animals were sacrificed and brain tissues were cultured in the

non-nutrient agar (Kasprzak & Mazur, 1979), or stained with hematoxylin-eosin to

examine the pathological changes of the tissue. Changes of antibody titer in the

sera of the experimental animals were checked by indirect flurolescent antibody

technique.

Immunized croup: Control animals died within 7 to 21 days in 64%-100%, one dose

of intraperitoneally immunized group died within 5 to 21 days in 40.7%, two doses

of intraperitoneally immunized group died within 3 to 12 days in 8.3% and

intradermal injection group died within 11 to 23 days in 25%. Thus, difference in

mortality was found between control group and immunized group, suggesting that mice

can acquire protective immunity to Acanthamoeba infection.

Immunosuppressed group: Mice of prednisolone administered group died within 2 to

21 days in 75% and mice of gamma-ray irradiation died within 5 to 22 days in 64%.

But, compared with untreated control group, mice immunosuppressed by prednisolone

administration or gamma-ray irradiation showed no significant difference in

mortality.

Indirect fluorescent antibody titer using Acanthamoeba sp. (YM-4 strain) as

antigen showed significant increases in mice by intraperitoneal or intradermal

immunization, but less increase in mice treated with prednisolone or irradiated

with gamma-ray: 1:32∼1:64 in immunized group but 1:16 or less in control group and

immunosuppression group. The above results may emphasize that artificial

immunization reduces the mortality of infected host with pathogenic free-living

amoeba, and give light to the immunoprophylaxis.

[영문]

Several reports indicated that infection of free-living amebae produced immune bodies in the host. John et al. (1977) reported that mice immunized by intraperitoneal injection of live Naegteria fowleri acquired resistance to challenge infection of the same amoebae. Thong et al. (1978b) reported that protective immunity to Naegleria meningoencephalitis could be transferred by immune serum. In the present study comarative observation of immunologic response of immunized and immunosuppressed mice to Acanthamoeba sp. YM-4 was performed to examine the possibility of acquiring protective imnunity. White male mice weighing 8 to 20g were used for experiments. Acanthamoeba sp., YM-4 strains were cultured in CGV medium (Willaert and Le Ray, 1973). Mice were divided into four groups; untreated control group, immnunized group, prednisolone treated group and gamma-ray irradiated group. Immunized group was divided into 3 subgroups: intraperitoneal

injection of one dose of 5×10**5 Acanthamoeba sp., YM-4; intraperitoneal injection of two doses of 5×10**5 Acanthamoeba sp., YM-4 one week apart; intradermal injection of 0.2mg of YM-4 antigen. In the prednisolone treated group, the hormone was injected intramuscularly in 5 doses of 10mg/kg every other day. In gamma-ray group, mice were irradiated with total dose of 300 rad. 1×10**4 Acanthamoeba sp., YM-4 was injected into brain through occipital region under ether anesthesia respectively to each mouse on: five weeks after immunization, 2 days after

prednisolone administration and 2 days after gamma-ray irradiation. On the designed date, the animals were sacrificed and brain tissues were cultured in the non-nutrient agar (Kasprzak & Mazur, 1979), or stained with hematoxylin-eosin to

examine the pathological changes of the tissue. Changes of antibody titer in the sera of the experimental animals were checked by indirect flurolescent antibody technique.

Immunized croup: Control animals died within 7 to 21 days in 64%-100%, one dose of intraperitoneally immunized group died within 5 to 21 days in 40.7%, two doses of intraperitoneally immunized group died within 3 to 12 days in 8.3% and

intradermal injection group died within 11 to 23 days in 25%. Thus, difference in mortality was found between control group and immunized group, suggesting that mice can acquire protective immunity to Acanthamoeba infection.

Immunosuppressed group: Mice of prednisolone administered group died within 2 to 21 days in 75% and mice of gamma-ray irradiation died within 5 to 22 days in 64%.

But, compared with untreated control group, mice immunosuppressed by prednisolone administration or gamma-ray irradiation showed no significant difference in mortality.

Indirect fluorescent antibody titer using Acanthamoeba sp. (YM-4 strain) as antigen showed significant increases in mice by intraperitoneal or intradermal immunization, but less increase in mice treated with prednisolone or irradiated with gamma-ray: 1:32∼1:64 in immunized group but 1:16 or less in control group and immunosuppression group. The above results may emphasize that artificial immunization reduces the mortality of infected host with pathogenic free-living amoeba, and give light to the immunoprophylaxis.