제주도지역의 아메바성 간농양에 관한 면역학적 연구

Abstract

[한글]

Immunological Studies on Amoebic Liver Abscess in Cheji Island

Soon-Ok Hong, M.D.

Department of parasitology Yonsei University College of Medicine, Seoul, Korea

(Directed by Drs. Chin-Thack Soh & Keun-Tae Lee)

Although a considerable number of reports related to liver abscess have appeared

in this country, serological investigations have done little to elucidate

immunological reactions in the infestation of Entamoeba histolytica.

During the years between 1966-1968, the author noted the frequent occurrence of

liver abscess and hepatitis in Cheju Islanders, and examined 64 patients who had

liver abscess, 106 cases of hepatitis, 14 cases of cyst carriers, and 55 cases of

normal healthy islanders.

The present paper deals with parasitological tests, microbiological cultures of

abscess of liver and immobilization tests of E. histolytica in each

group.Immobilization tests were also carried out with several strains of amebae

using immune rabbit sera. Furthermore, duration of immobilization antibodies was

determined several months after the initial treatment of amebiasis using the sera

of 54 immobilization test positive patients.

Two hundred and thirty eight islanders were divided into seven groups according

to their clinical symptoms; 1) Liver abscess(E. histolytica in liver), 2 Liver

abscess(E. histolytica in stool), 3) Liver abscess(E. histolytica not demonstrated

by examinations of abscess and stool), 4) Hepatomegaly(E. histolytica in stool), 5)

Hepatomegaly(E. histolytica not found in stool), 6) Cyst carrier, symptomless

healthy individuals, and 7) Control group. Stool examinations were performed 1-3

times by the formalin-ether concentration technique.

Collected material from the islanders was shipped via air freight to our

laboratory, in Seoul, as soon as possible. After arrival sera were stored in a deep

freezer (-30。C).

Sixty four liver abscess samples obtained by aspiration were cultured in the

media (EMG agar, SS agar, nutrient agar, Sabouraud's media) and the presence of

micro-organisms investigated. In the specimens containing Gram negative enteric

bacilli, the following investigations were carried out:IMVIC reactions,

fermentation of saccharides(lactose, sucrose, dextrose, mannitio, salicin),

motility test, urease test, TSI agar culture etc. in order to classify the bacilli.

Four strains of amebae were used in this study.

1) YS 9-strain. This strain was isolated in September 1966 from the feces of a 51

year-old man with liver abscess(Cheju islander) and subcultured every other day.

2) YS 14-strain. The ameba was isolated in January 1968 from 49-year-old healthy

cyst-passer's stool (man, Suwon inhabitant) and maintained by subculture.

3) YS 15-strain. The strain was isolated in January 1967 from a 51-year-old

healthy symptomless cyst carrier(man, Cheju islander) and maintained as above.

4) YS 15-strain. This was isolated in July 1968 from trophozoites in stool of an

acute amebic enteritis patient at Severance Hospital.

Both sexes of 16 white, weighing about 1.8kg and having a negative immobilization

reaction, were used in this study. Each animal received an intravenous injection of

200,000 trophozoites five times on alternate days. Control inocula were prepared

from the ameba inocula of each strain by heating for two hours in water bath(50。C)

in order to kill living parasites. The immunized animals were bled 12days following

the last injection and the serum separated under aseptic condition.

Immobilization test was performed as follow: Frozen sera sere liquefied in an

incubator(37。C). A drop of amebae, obtained from the bottom of a 48-hour-culture

was combined with a drop of serum on a clean slide. A cover slip was top of this

mixture and sealed with melted paraffin. The slide was then placed in an incubator

at 37。C. The slides were read by examining 100 amebae and counting the number of

immobile parasites. At the same time, another slide was prepared with amebae and

isotonic solution in place of serum as a control for spontaneous immobilization.

There were 189 males and 49 females out of a total of 238 examinees. In the liver

abscess group, the number of males was markedly greater than that of females, and

highest distribution was observed in the 30-39 age group. In cyst carriers group,

males numbered 8 and females 6, and half of total belonged to the age group from 10

to 19. In control group, out of 55 individuals, males numbered 31 and females 24,

and age distribution was 10-19, 20-29, 30-39 in decreasing order.

By the bacteriological examination of liver abscess, Gram negative enteric

bacilli were recovered in 27(42.1%) out of 64 cases, and fungi and cocci were not

detected. In Group 1, three cases were positive out of six cases. Two cases of

Cscherichia coli and one of paracolon group. In Group 2, five cases were positive

out of 13. Two cases showed Aerobacter aerohenes and the others were not

identified. In Group 3, nineteen cases(42.2%) were positive out of 45 cases. Eight

cases were Escherichia coli, six were alkaligenes fecalis, two were Aerobacter

aerogenes, one case was of the Paracolon group, and the other were not identified.

Rabbit immune sera were divided into an immune sera group and an inactivated

immune sera group(56。C, 30 minutes). The number of immobilized was counted four

times and the data were treated statististically.

1) Immune rabbit sera group. The reaction was observed every fifteen minutes for

4 hours. The highest immobilization occured from 45 minutes to 105 minutes after

the beginning of the test and remobilization of amebae took place gradually.

2) Inactivated immune rabbit sera group. The highest immobilization occured from

30 minutes to 135 minutes and then remobilization reaction took place.

At the immobilization reaction(I-R) on three strains of amebae(YS9,YS14, YS 16)

using rabbit sera which were immunized with YS 9-strain, YS 15-strain and their

control inocula, in general, I-R between homologous immuned sera and ameba occured

greater rate than heterologous sera and the parasite. However, little difference

and greater immobiization were observed in inactivated sera group.

At the hourly immobilization reaction check for the YS 9-Strain ameba using the

serum of amebiasis cases in Cheju Island, the highest peak was observed from 45

minutes to 90 minutes. Thereafter remobilization occured gradually.

In the immobilization test E. histoyltica(YS 9-strain) with the sera of amebiasis

cases in Cheju Island, ninety eight sera were sampled at random from the sera of

amebiasis cases and I-R was performed, and the rate over 51 was determined as I-R

positive. Positive cases were observed in 6 out of 6 in Group 1, 10 out of 10 in

Group 2, 15 out of 18 in Group 3,10 out of 12 in Group 4, 20 out of 22 in Group 5,5

out of 11 in cyst carrier group, and 6 out of 19 in control group.

Fifty six sera of amebiasis cases, which showed severe immobilization reaction,

were collected after the treatment for amebiasis. The sera were grouped according

to months after the beginning of treatment. Positive I-R were observed 2 out of 2

at one month, 1 our of 5 at two months, zero out of 1 at three months, 1 out 1 at

four months, and thereafter the results fluctuated. Total positives were 30 out of

56 cases. It was presumed that if the treatment were successful, reversal to

negative of the I-R occured from two to three months.

Conclusions

Parasitological, microbiological and immunologic studies were made in 238 Cheju

islanders, a highly endemic area of amebiasis in Korea. Ameba immobilization test

was carried out using immunized rabbit sera and several strains of E. histolytica.

1) The detection rate for micro-organisms in aspirated liver abscess was 42.1%.

Most of the recovered micro-organisms were Gram negative enteric bacilli;

Escherichia coli, Alkaligenes fecalis, aerobacter aerogenes, and Paracolon group.

Fungi and Cocci were not observed.

2) In the immobilization test using immune rabbit sera immunized with a 48 hour

culture of E. histolytica, the highest immobilization reaction occured 45-105

minutes after the beginning of the test and remobilization of the parasite took

place gradually. Immobilization of ameba continued for more hours and at higher

rate in the inactivated rabbit sera group and the differences among ameba strains

were not remarkable. In human amebiasis sera, the highest peak of immobilization

reaction occured at 45-90 minutes after testing with the parasite.

3) Positive rates for the immobilization test according to clinical feature were

83.2-100% in liver abscess cases, 83.3-90.7% in hepatomegaly cases, 45.4% in

asymptomatic cyst-passers and 31.5% in healthy controls.

4) For 56 cases who had showed a high rate of immobilization, the follow-up

positive rate after treatment for amebiasis was markedly reduced in 2-3 months.

[영문]

Although a considerable number of reports related to liver abscess have appeared in this country, serological investigations have done little to elucidate immunological reactions in the infestation of Entamoeba histolytica.

During the years between 1966-1968, the author noted the frequent occurrence of liver abscess and hepatitis in Cheju Islanders, and examined 64 patients who had liver abscess, 106 cases of hepatitis, 14 cases of cyst carriers, and 55 cases of

normal healthy islanders.

The present paper deals with parasitological tests, microbiological cultures of abscess of liver and immobilization tests of E. histolytica in each group.Immobilization tests were also carried out with several strains of amebae using immune rabbit sera. Furthermore, duration of immobilization antibodies was determined several months after the initial treatment of amebiasis using the sera of 54 immobilization test positive patients.

Two hundred and thirty eight islanders were divided into seven groups according to their clinical symptoms; 1) Liver abscess(E. histolytica in liver), 2 Liver abscess(E. histolytica in stool), 3) Liver abscess(E. histolytica not demonstrated by examinations of abscess and stool), 4) Hepatomegaly(E. histolytica in stool), 5) Hepatomegaly(E. histolytica not found in stool), 6) Cyst carrier, symptomless healthy individuals, and 7) Control group. Stool examinations were performed 1-3 times by the formalin-ether concentration technique.

Collected material from the islanders was shipped via air freight to our laboratory, in Seoul, as soon as possible. After arrival sera were stored in a deep freezer (-30。C).

Sixty four liver abscess samples obtained by aspiration were cultured in the media (EMG agar, SS agar, nutrient agar, Sabouraud's media) and the presence of micro-organisms investigated. In the specimens containing Gram negative enteric

bacilli, the following investigations were carried out:IMVIC reactions, fermentation of saccharides(lactose, sucrose, dextrose, mannitio, salicin), motility test, urease test, TSI agar culture etc. in order to classify the bacilli.

Four strains of amebae were used in this study.

1) YS 9-strain. This strain was isolated in September 1966 from the feces of a 51 year-old man with liver abscess(Cheju islander) and subcultured every other day.

2) YS 14-strain. The ameba was isolated in January 1968 from 49-year-old healthy cyst-passer's stool (man, Suwon inhabitant) and maintained by subculture.

3) YS 15-strain. The strain was isolated in January 1967 from a 51-year-old healthy symptomless cyst carrier(man, Cheju islander) and maintained as above.

4) YS 15-strain. This was isolated in July 1968 from trophozoites in stool of an acute amebic enteritis patient at Severance Hospital.

Both sexes of 16 white, weighing about 1.8kg and having a negative immobilization reaction, were used in this study. Each animal received an intravenous injection of 200,000 trophozoites five times on alternate days. Control inocula were prepared

from the ameba inocula of each strain by heating for two hours in water bath(50。C) in order to kill living parasites. The immunized animals were bled 12days following the last injection and the serum separated under aseptic condition.

Immobilization test was performed as follow: Frozen sera sere liquefied in an incubator(37。C). A drop of amebae, obtained from the bottom of a 48-hour-culture was combined with a drop of serum on a clean slide. A cover slip was top of this mixture and sealed with melted paraffin. The slide was then placed in an incubator

at 37。C. The slides were read by examining 100 amebae and counting the number of immobile parasites. At the same time, another slide was prepared with amebae and isotonic solution in place of serum as a control for spontaneous immobilization.

There were 189 males and 49 females out of a total of 238 examinees. In the liver abscess group, the number of males was markedly greater than that of females, and highest distribution was observed in the 30-39 age group. In cyst carriers group,

males numbered 8 and females 6, and half of total belonged to the age group from 10 to 19. In control group, out of 55 individuals, males numbered 31 and females 24, and age distribution was 10-19, 20-29, 30-39 in decreasing order.

By the bacteriological examination of liver abscess, Gram negative enteric bacilli were recovered in 27(42.1%) out of 64 cases, and fungi and cocci were not detected. In Group 1, three cases were positive out of six cases. Two cases of Cscherichia coli and one of paracolon group. In Group 2, five cases were positive out of 13. Two cases showed Aerobacter aerohenes and the others were not identified. In Group 3, nineteen cases(42.2%) were positive out of 45 cases. Eight cases were Escherichia coli, six were alkaligenes fecalis, two were Aerobacter aerogenes, one case was of the Paracolon group, and the other were not identified.

Rabbit immune sera were divided into an immune sera group and an inactivated immune sera group(56。C, 30 minutes). The number of immobilized was counted four times and the data were treated statististically.

1) Immune rabbit sera group. The reaction was observed every fifteen minutes for 4 hours. The highest immobilization occured from 45 minutes to 105 minutes after the beginning of the test and remobilization of amebae took place gradually.

2) Inactivated immune rabbit sera group. The highest immobilization occured from 30 minutes to 135 minutes and then remobilization reaction took place.

At the immobilization reaction(I-R) on three strains of amebae(YS9,YS14, YS 16) using rabbit sera which were immunized with YS 9-strain, YS 15-strain and their control inocula, in general, I-R between homologous immuned sera and ameba occured

greater rate than heterologous sera and the parasite. However, little difference and greater immobiization were observed in inactivated sera group.

At the hourly immobilization reaction check for the YS 9-Strain ameba using the serum of amebiasis cases in Cheju Island, the highest peak was observed from 45 minutes to 90 minutes. Thereafter remobilization occured gradually.

In the immobilization test E. histoyltica(YS 9-strain) with the sera of amebiasis cases in Cheju Island, ninety eight sera were sampled at random from the sera of amebiasis cases and I-R was performed, and the rate over 51 was determined as I-R positive. Positive cases were observed in 6 out of 6 in Group 1, 10 out of 10 in Group 2, 15 out of 18 in Group 3,10 out of 12 in Group 4, 20 out of 22 in Group 5,5 out of 11 in cyst carrier group, and 6 out of 19 in control group.

Fifty six sera of amebiasis cases, which showed severe immobilization reaction, were collected after the treatment for amebiasis. The sera were grouped according to months after the beginning of treatment. Positive I-R were observed 2 out of 2

at one month, 1 our of 5 at two months, zero out of 1 at three months, 1 out 1 at four months, and thereafter the results fluctuated. Total positives were 30 out of 56 cases. It was presumed that if the treatment were successful, reversal to

negative of the I-R occured from two to three months.

Conclusions

Parasitological, microbiological and immunologic studies were made in 238 Cheju islanders, a highly endemic area of amebiasis in Korea. Ameba immobilization test was carried out using immunized rabbit sera and several strains of E. histolytica.

1) The detection rate for micro-organisms in aspirated liver abscess was 42.1%. Most of the recovered micro-organisms were Gram negative enteric bacilli; Escherichia coli, Alkaligenes fecalis, aerobacter aerogenes, and Paracolon group. Fungi and Cocci were not observed.

2) In the immobilization test using immune rabbit sera immunized with a 48 hour culture of E. histolytica, the highest immobilization reaction occured 45-105 minutes after the beginning of the test and remobilization of the parasite took

place gradually. Immobilization of ameba continued for more hours and at higher rate in the inactivated rabbit sera group and the differences among ameba strains were not remarkable. In human amebiasis sera, the highest peak of immobilization reaction occured at 45-90 minutes after testing with the parasite.

3) Positive rates for the immobilization test according to clinical feature were 83.2-100% in liver abscess cases, 83.3-90.7% in hepatomegaly cases, 45.4% in asymptomatic cyst-passers and 31.5% in healthy controls.

4) For 56 cases who had showed a high rate of immobilization, the follow-up positive rate after treatment for amebiasis was markedly reduced in 2-3 months.