자유생활 아메바의 병원성에 관한 실험적 연구

Abstract

[한글]

원발성 아메바성 뇌수막염(primary amoebic meningoencephalitis)의 원인 원충인 자유생활 아메바에 관한 연구보고는 우리나라에서 아직 찾아 볼 수 없다.

서울시 하천에서 Naegleria sp.와 Acanthamoeba sp.를 검출하였으며, 이 검출된 아메바의 병원성 여부를 규명하기 위하여 실험 동물에 감염시켜 다음과 같은 소견을 얻게되었다.

실험에는 건강한 20gm 내외의 백색마우스를 사용하였다. agar배지에서 배양한 자유생활 아메바 2×10**3 개를 마취된 마우스 비강에 떨어뜨려 감염시키고, 시일이 경과하면서 행동장애나 병적상태가 야기되지 않나 관찰하고 20일 경과 후 비강 점막, 중추신경계통, 폐장 조직을 적출, 배양하여 자유생활 아메바를 검출하였고, 특히 뇌는 육안적으로 염증의 여부를 관찰하고 뇌조직을 고정하고 hematoxylin 과 eosin염색하여 조직에서 자유생활 아메바의 감염여부를 확인하였다.

서울시내 사근동 청계천에서 분리한 주 YM-1은 씨스트의 형태, 영양형의 유사분렬시핵의 형태학적 특징, 운동성 등으로 보아 Naegleria sp.로 동정되었다. 또한 천호동 논웅덩이와 구의동 저수지에서 분리한 YH-2와 YM-3는 모두 Acanthamoeba로 동정되었다.

Naegleria sp.인 YM-1과 Acanthamoeba sp.인 YM-2와 YM-3를 마우스의 비강점막에 감염시키고 20일후 뇌조직과 척수, 비강점막과 폐장조직 일부를 적출하여 agar배지에 배양하였더니 Naegleria sp.와 Acanthamoea sp.가 검출되었다. 즉 YM-1을 감염시킨 마우스에서 보면 뇌에서는 물론 천수, 폐장에서도 Naegieria sp.를 검출할 수 있었고,YM-2를 감염시킨 마우스의 척수에서는 Acanthamoeba sp.를 검출할 수 없었고, YM-3를 감염시킨 마우스에서는 비강 점막에서만 Acanthamoeba sp.를 검출하였다.

YH-1을 감염시킨 마우스 7마리중 1마리가, YM-2는 5마리중 2마리가 사망하였고, YM-3를 감염시킨 마우스 5마리중 한마리도 사망하지 않았다.

육안적 소견으로는 뇌전체에 부종이 있고 후엽과 전두엽 앞부분에 부분적으로 출혈, 괴사를 볼 수 있었고, 비강은 염증성 삼출물이 충만되어 있었으며 폐장은 울혈되어 있었다.

염증이 있는 뇌조직 일부를 절제하여 hematoxylin과 eosin염색하여 현미경 관찰을 하였다. 뇌조직중 특히 피질에 염증세포의 침윤이 심하였다. 이러한 급성염증 세포사이에서 핵인(nucleolus)이 진하게 염색되고 세포질내에 여러개의 식포(vacuole)가 보이는 많은

자유생황아메바가 존재함을 알 수 있었다.

자유생활 아메바에 감염된 마우스에서 감염기간이 경과함에 따른 임상증세를 관찰하였다. 감염 3일후 까지는 정상이었으나 4일후 에는 식욕부진, 머리를 비비고, 5일후에는 머리를 떨어뜨리고 음식을 먹지 않았고, 8일후에는 발작, 마비등이 오고, 9일후에는 사망하였다.

이상과 같이 원발성 아메바성 뇌수막염의 원인 원충인 자유생활 아메바 특히 Naegieria sp.와 Acanthamoeba sp.를 서울시내 하천, 논웅덩이 및 저수지의 물에서 검출할 수 있었다. 또한 분리된 자유생활 아메바는 실험동물 마우스에서 병원성이 있음을 증명하였다.

따라서 우리나라에서도 원발성 아메바성 뇌수막염 환자가 발생할 가능성이 있음을 제시했다고 사료된다.

Experimental Study on the Pathogenecity of Free-living Amoebae

Han-Ky Hwang, M.D.

Department of Medical Science The Graduate School Yonsei University

(Directed by Professor Chin-Thack Soh, M.D. and Duk Jin Yun, M.D.)

Recently, free-living amoebae (Naegleria sp. or Acanthamoeba sp.) have been

proved to be a causative agent of primary amoebic meningoencephalitis(PAM) in

human. Cotter(1973)reported there were abort 100 cases of fatal primary amoebic

meningoencephalitis in the world due to the limax amoeba by the end of 1972. The

laboratory isolation of pathogenic free-living amoebae may be made from nasal

discharge, spinal fluid, or sectioned brain tissue. Willaert(1973) pointed out that

cases of PAM occurred generally in summer season, and that infection source of PAM

may be swimming pool, lake, water or muddy puddle, sea water pool, warm springs and

ethers.

Jamieson and Anderson(1973) isolated the free-living amoebae from the soil and

water of swimming pool and pond. Culbertson et al.(1966) found that Hartmanellid

amoeba may produce acute meningoencephalitis and also chronic granulomatous

encephalitis in mice after intranasal instillation. Kasprzak and Mazur(1972)

experienced small free-living amoebae which isolated from water were virulent for

mice when given intranasally or intracerebrally. Martinez et al.(1973) succeeded

PAM by intranasal inoculation of pathogenic Naegleria experimentally.

On the assumption that the free-living amoebae isolated from the waters of

swimming pool, sewage and pond are supposed to be pathogenic, present study was

designed to find whether isolated free-living amoebae from Seoul area is

pathogenic.

Materials and methods

Isolation of free-living amoebae

In order to isolate free-living amoebae, non-nutrient agar plate described by

Kasprzak and Mazur(1972) was applied; 1.5% Agar-Agar(Fisher) in distilled water

with addition of 14 mg of Neomycin sulfate(Upjohn) and 3,750 Unit of

Nystatin(Sigma) and 2.0 ml. of heat-killed (at 65℃ for 30 minutes) Escherichia

coli(ATCC 11105) suspension per 100 ml. of the medium. The bacterial suspension was

incorporated into the medium cooled and a thin layer of the bacteria was then

spread over the surface of the plates. Transfers to fresh plates were performed

once in a week.

Three strains of free-living amoebae from waters of three different area of Seoul

were cultured at 36℃.

Identification of free-living amoebae

The identification of the isolated amoebas was done by the fellowing references;

Singh(1772), Page(1970), Kingston and Warhurst(1969), Singh and Das(1970b) and

Chang(1971).

Procedures for primary amoebic meningoencephalitis

The virulence of the isolated amoebas was examined by infecting them to white

mice, weighing abort 20gm, which kept on a ordinary diet and water. Each mouse was

infected intranasally with 2×10**3 amoebas of three different strains under ether

anesthesia.

Clinical manifestation of infected mice was observed. Autopsies were performed

immediately after death, and those mice which survived over 20 days were etherized

and dissected. The brain, spinal cord, lung and nasal mucosa were placed on the

non·nutrient agar plate with killed Escherichia coli for detection of free-living

amoebae. Pathological observation was carried out and brain tissue for

histopathologic examinations were fixed in 10% formaldehyde and stained with

hematoxylin and eosin.

Results

Naegleria sp., YM-1, isolated from sewage was identified on the base of

pseudopodial and cyst character and mitotic pattern. Two strains of Acanthamoeba

sp., YM-2 and YM-3, were isolated from water puddle and a storing reservoir.

Naegleria sp., YM-1, was detected in the tissues of brain, spinal cord, nasal

mucosa and lung of the mice infected intranasally at 20th days. Acanthamoeba sp.,

YM-2, was detected except spinal cord, but YM-3 only in the nasal mucosa.

One out of 7 mice infected with Naegleria sp.,7M-1, died of PAM and Acanthamoeba

sp.,YM-2, 2 out 5 mice. None of 5 mice infected with Acanthamoeba sp,, YM-3, were

followed by death.

Examination of necropsy revealed typical lesions of acute PAM with brain edema.

The most extensive lesions occurred in the olfactory lobe and prefrontal area with

haemorrhage and necrosis. Nasal cavity was filled with purulent and haemorrhagic

exudate and lung showed to be engorged with blood.

Brain section stained with hematoxylin and eosin demonstrated many invading

amoebas among many inflammatory cells infiltrated markedly.

The nuclei with deeply staining red nucleoli and vacuolated cytoplasm of the

amoebas were noted from the infected cerebral cortex.

Up to 3rd day after infection with free·living amoebae, the condition of mice

were normal. At 4th day, visible signs of distress and central nervous system

disturbance appeared. Anorexia, altered behavioral patterns (less mobile and

scratch head) occurred first, followed by one or more of the following symptoms:

hunched head, fasting, loss of coordination, paralysis. Mice that assumed a acute

course was usually followed by acute meningoencephalitis with death in 9th day.

Summary

Free-living amoebae (Naegleria sp. and Acanthamoeba sp.), causative agent of

primary amoebic meningoencephalitis, was isolated from the sewage, water puddle and

a storing reservoir.

One strain 7f 1?aegteri? sp. and two strains of Aranthamoeba sp. preyed to be

pathogenic for mice infected intranasally.

Therefore, it is considered that primary amoebic meningoencephalitis may occur

due to free-living amoebae in Korea.

[영문]

Recently, free-living amoebae (Naegleria sp. or Acanthamoeba sp.) have been proved to be a causative agent of primary amoebic meningoencephalitis(PAM) in human. Cotter(1973)reported there were abort 100 cases of fatal primary amoebic meningoencephalitis in the world due to the limax amoeba by the end of 1972. The

laboratory isolation of pathogenic free-living amoebae may be made from nasal discharge, spinal fluid, or sectioned brain tissue. Willaert(1973) pointed out that cases of PAM occurred generally in summer season, and that infection source of PAM

may be swimming pool, lake, water or muddy puddle, sea water pool, warm springs and ethers.

Jamieson and Anderson(1973) isolated the free-living amoebae from the soil and water of swimming pool and pond. Culbertson et al.(1966) found that Hartmanellid amoeba may produce acute meningoencephalitis and also chronic granulomatous encephalitis in mice after intranasal instillation. Kasprzak and Mazur(1972)

experienced small free-living amoebae which isolated from water were virulent for mice when given intranasally or intracerebrally. Martinez et al.(1973) succeeded PAM by intranasal inoculation of pathogenic Naegleria experimentally.

On the assumption that the free-living amoebae isolated from the waters of swimming pool, sewage and pond are supposed to be pathogenic, present study was designed to find whether isolated free-living amoebae from Seoul area is pathogenic.

Materials and methods

Isolation of free-living amoebae

In order to isolate free-living amoebae, non-nutrient agar plate described by Kasprzak and Mazur(1972) was applied; 1.5% Agar-Agar(Fisher) in distilled water with addition of 14 mg of Neomycin sulfate(Upjohn) and 3,750 Unit of Nystatin(Sigma) and 2.0 ml. of heat-killed (at 65℃ for 30 minutes) Escherichia

coli(ATCC 11105) suspension per 100 ml. of the medium. The bacterial suspension was incorporated into the medium cooled and a thin layer of the bacteria was then spread over the surface of the plates. Transfers to fresh plates were performed once in a week.

Three strains of free-living amoebae from waters of three different area of Seoul were cultured at 36℃.

Identification of free-living amoebae

The identification of the isolated amoebas was done by the fellowing references; Singh(1772), Page(1970), Kingston and Warhurst(1969), Singh and Das(1970b) and Chang(1971).

Procedures for primary amoebic meningoencephalitis

The virulence of the isolated amoebas was examined by infecting them to white mice, weighing abort 20gm, which kept on a ordinary diet and water. Each mouse was infected intranasally with 2×10**3 amoebas of three different strains under ether anesthesia.

Clinical manifestation of infected mice was observed. Autopsies were performed immediately after death, and those mice which survived over 20 days were etherized and dissected. The brain, spinal cord, lung and nasal mucosa were placed on the non·nutrient agar plate with killed Escherichia coli for detection of free-living amoebae. Pathological observation was carried out and brain tissue for histopathologic examinations were fixed in 10% formaldehyde and stained with hematoxylin and eosin.

Results

Naegleria sp., YM-1, isolated from sewage was identified on the base of pseudopodial and cyst character and mitotic pattern. Two strains of Acanthamoeba sp., YM-2 and YM-3, were isolated from water puddle and a storing reservoir.

Naegleria sp., YM-1, was detected in the tissues of brain, spinal cord, nasal mucosa and lung of the mice infected intranasally at 20th days. Acanthamoeba sp., YM-2, was detected except spinal cord, but YM-3 only in the nasal mucosa.

One out of 7 mice infected with Naegleria sp.,7M-1, died of PAM and Acanthamoeba sp.,YM-2, 2 out 5 mice. None of 5 mice infected with Acanthamoeba sp,, YM-3, were followed by death.

Examination of necropsy revealed typical lesions of acute PAM with brain edema. The most extensive lesions occurred in the olfactory lobe and prefrontal area with haemorrhage and necrosis. Nasal cavity was filled with purulent and haemorrhagic exudate and lung showed to be engorged with blood.

Brain section stained with hematoxylin and eosin demonstrated many invading amoebas among many inflammatory cells infiltrated markedly.

The nuclei with deeply staining red nucleoli and vacuolated cytoplasm of the amoebas were noted from the infected cerebral cortex.

Up to 3rd day after infection with free·living amoebae, the condition of mice were normal. At 4th day, visible signs of distress and central nervous system disturbance appeared. Anorexia, altered behavioral patterns (less mobile and

scratch head) occurred first, followed by one or more of the following symptoms: hunched head, fasting, loss of coordination, paralysis. Mice that assumed a acute course was usually followed by acute meningoencephalitis with death in 9th day.

Summary

Free-living amoebae (Naegleria sp. and Acanthamoeba sp.), causative agent of primary amoebic meningoencephalitis, was isolated from the sewage, water puddle and a storing reservoir.

One strain 7f 1?aegteri? sp. and two strains of Aranthamoeba sp. preyed to be pathogenic for mice infected intranasally.

Therefore, it is considered that primary amoebic meningoencephalitis may occur due to free-living amoebae in Korea.