자유생활 아메바의 병원성과 phytoagglutinin에 의한 응집반응

Abstract

[한글]

자연환경에서 분리 배양하여 실험동물 마우스에 감염시켜 병원성을 관찰한 자유생활아메바와 이미 병원성이 확인된 자유생활아메바를 사용하여 Phytoagglutinin인 Concanalvain A에 의한 응집반응을 봄으로서 그 반응과 아메바의 병원성과의 관계를 과찰하였다.

자유생활아메바는 CGV배지 또는 CGVS배지(Willaert 및 LeRay, 1973)를 사용하여 배양하였고 15∼20gm 내외의 백색마우스 비강에 자유생활아메바 영양형을 떨어뜨려 감염시킴으로 그 병원성을 관찰하였다.

자유생활아메바 영양형은 0.01M 인산완충액 (pH 7.0∼7.2)을 함유한 0.9%(w/v)식염수로 세척하고 그 부유액을 일정농도와 Concanavalin A(Sigma)와 잘 혼합하여 일정시간 방치하였다가 현미경으로 응집여부를 관찰하였고 또한 응집의 정도를 측정하기 위하여 Spectr

ophotometer(Bausch and Lomb)를 이용하여 660nm에서 transmittance의 증가율을 측정함으로 응집율의 변동을 관찰하였다.

그 결과를 요약하면 붕어 아가미에서 분리한 비병원성인 Acanthamoeba sp.인 YM-4 영양형은 Concanavalin A 25μ/ml이상 용액에서 응집반응이 잘 일어났다.

장기간 계대배양하여 병원성이 약화된 Acanthamoeba culbertsoni 영양형은 Concanavalin A 25μg/ml이상 용액에서 응집반응이 일어남을 알 수 있었다.

마우스에 감염시켜 병원성이 확인된 Acanthamoeba lenticulate는 Concanavalin A 25μg/ml이상 용액에서 응집반응이 일어남을 관찰하였고 비병원성인 Acanthamoeba royreba는 Concanavalin A 100μg/ml용액에서, Acanthamoeba hatchotti는 Concanavalin A 25μg/ml이상 용액에서, Acanthamoeba triangularis는 Concanavalin A 100μg/ml용액에서 응집반응이 일어남을 알 수 있었다.

장기간 계대배양하여 병원성이 약화된 Naegleria fowleri영양형은 Concanavalin A 100μg/ml-용액에서도 응집반응이 일어나지 않았으며 마우스에 감염시킨 다음 뇌조직에서 분리한 병원성이 증강된 Naegleria fowleri영양형을 사용하였을 때도 응집반응이 일어나지

않았다.

Acanthamoeba sp.인 YM-4 영양형을 사용하여 specrtophotometer로 응집 반응을 관찰한바 YM-4 부유액의 영양형수가 많을수록 응집반응이 잘 일어났으며 30℃에서 반응 45∼60분후 응집반응이 최고치에 달하였다. 또한 Concanavalin A농도 100μg/ml에서 1∼10μg/m

l에 비해 응집반응이 잘 일어났으며 200μg/ml에서는 더욱 현저하였다.

이상 자유생찰아메바의 Concanavalin A에 의한 응집반응을 관찰하였던 바 Naegleria sp.에서는 병원성과 관계가 있으나, Acanthamoeba sp.에서는 직접적인 관계가 없는 것으로사료된다.

[영문]

Virulence or pathogenicity of free-living amoebae was been judged mainly in-vivo. However, experiment in-vitro has also been tried in recent years to avoid the complexity of animal experiment and to save time and expenses.

Visvesvara and Balamuth (1975) reported that the tests of cytopathic effect in tissue culture might be useful way to determine the virulence of the amoebae and Josephson et al. (1977) found that pathogenic Naegleria strains were agglutinated

by phytoaglutinin.

The present study is to visualize the relationship between the pathogenicity of free-living amoebae in considering with the agglutinabilily of respective strain.

The free-living amoebae teat in the stuffy were: Naegleria fowleri, Acanthamoeba culbertsoni, A. hatchotti, A.lenticulate, A. royreba, A. triangularis and Abanthamoeba sp. (YM-4). For reference, Entamoeba histolyica strains isolated from amoebic hepatic abscess cases were also teated.

Free-living amoebae were cultured in the non-nutrient agar plate (Kasprzak and Mazur, 1972) and CGV or CGVS medium(Willaert and LeRay, 1973). For the pathogenicity test white mice weighing 15-20 gm were used. Free-living amoebae trophozoites were inoculated into the nasal cavity of mouse under the anesthesia by

intraperitoneal injection of secobarbital, and pathologic behabiour and brain tissue pathology were observed during the experimentation.

Free-living amoebae trophozoites were washed three times with 0.9% (w/v) NaCl containing 0.01 M phosphate buffer (PH 7.0-7.2) and then suspended carefully. One drop of the cell suspension wart mixed with came amount of concanavalin A of various concentrations on the Boerner slide and incubated at 36℃ for given period of time. Microscopic observation to determine the occurrence of agglutination was done. And agglutination was also measured using spectrophotometer by transmittance of free-living amoebae suspension in the presence of concanavalin A.

Cell suspension in 0.9% (w/v) NaCl containing 0.01 M phosphate buffer (pH 7.0-7.2) was added to the same volume of concanavalin A of various concentration establishing a final volume of 2ml, and then agitated well. Experimental reading was done with spectrephotometer (Bausch and Lomb) at 660 nm at 30℃. Any increase in transmittance observed in each experimental readings was substracted from the zero time reading. Percent agglutination was calculated from the expression (Y-X/X)×100, where X is the transmittance at zero time and Y is the transmittance at any given incubation time.

Acanthamoeba sp. YM-4 tropzoites, that is confirmed to be nonpathogenic to the mouse, agglutinated easily by more than 25 μg/ml of concanavalin A. Pathogenic Acanthamoeba culbertsoni, which was subcultures for a long time was agglutinated by 25μg/ml of concanavalin A.

Pathogenic Acanthamoeba tenticulata was agglutinated by 25 μg/ml of concanavalin A, nonpathogenic Acanthamoeba royreba by 100 μg/ml, nonpathogenic Acanthamoeba hatchotti 25μg/ml, Acunthamoeba triangularis 100 μg/ml. But no agglutination was

obtained with Naegleria fowleri and Entamoeba histolytica at any of the concanavalin A concentrations tested.

In agglutination, the beat result was achieved using a cell density of 157×10**4 /ml of Acanthamoeba sp., YM-4. Agglutination reached to the highest rate after 45-60 minutes at the concanavalin A concentration of 100μg/ml.

The overall results suggest that concanavalin A-induced agglutination is related with the pathogeicity in the Naegleria sp. but net in the Acanthamoeba sp.