痢疾아메바(entamoeba histolytica)의 溶血能에 關한 硏究

Abstract

The ingestion of red blood cells by Entamoeba histolytica was first observed by Losch in 1873, and it has been recognized as a part of nutrient of the protozoa. But recently Shaffer et al.(1961) reported that the red blood cells of rabbits produce toxic substances which inhibit the propagation of E. histolytica. Craig (1927) found that 5. histotytica had hemolytic ability, but Shaffer et al. (1963) reported that hemolytic ability of E. histolytica was different according to the strains.

The present study was designed to know whether the red blood cell had a toxic effect for the propagation of E. histolytica and, on the one hand, whether the protozoa had selective ability to lyse the red blood cell.

These studies were as follows.

1. Hemolytic ability of E. histolytica to human and various animal red blood cells.

2. Relationship between the strains of E. histdytica and the hemolytic bacteria.

3. Effect of the red blood cell for the propagation of E. histofytica.

4. Cytolytic ability of E. histolytica.

Materials and Methods:

1. Cultivation of E. histolytica

Modified diphasic medium containing 4 ml. of buffered saline was used for culture at 37℃., and subcultures at two days interval.

2. Strains of E. histolytica.

YS 1, 5, 9 and 10 which were sampled in the laboratory were used.

3. The hemolrtic ability of E. histolytica

a) R,B.C.........human, ok, pig., dog, rabbit and sheep.

b) R.B.C. suspension.

Each specimen of red blood cells was collected carefully in a double oxalate bottle. It was centrifuged for 15 minutes at 3,000 r.p.m. to sediment the cells. The cells were then washed 4 times with normal saline. Finally, the washed red blood cells ware resuspended in saline and centrifuged for10 minutes at 1,200

r.p.m. The sediment was defined as standard 100% erythrocyte concentration. A 5% suspension was made by adding I volume of the 100% erythrocytes to 19 volumes of saline. To minimize inclusion of white blood cells, sediment was withdrawal from near the bottom of the tube.

The media for the experiment were divided into 4 groups: bacteria group which was made by adding the 0.4 ml. supernatant fluid from the original culture media into new diphasic media; amoebae and bacteria group to which 0.5 ml. sediment from the

bottom of the slant of the same original culture medium was added: metabolites group to which Scitz filtrates of all the original culture media was added, and a control group. Two-tenth milliliter of the 5% suspension of R.B.C. was added to each tube. Each group was incubated at 37℃. for 14-20 hours and examined the hemoglobin content as well as the viability of the amoebae.

In order to define the hemoglobin measure, two-tenth ml. of 5% suspension of R.B.C. was added in 4 ml. distilled water to make the 100% solution of hemolysis as standard. The optical density of each soulution was measured on the spectrophotometer in 540 mμ wave length to obtain the standard line of the various species of R.B.C. Comparing with the standard line, hemolytic ranges were defined as follows:

0-10% hemolysis.................negative

10-25% hemolysis................moderate positive

25-100% hemolysis...............positive

Osmolarity of each group was determined with Osmometer (Fiske)

4. Relationship of E. histolytica and hemolytic bacteria

a) E. histoltica strains............YS 1, 9, 10.

b) Bacteria.........................Bacillus subtilis Staphylococcus aureus

c) Red blood cell...................Human R.B.C.

d) Method: Active amoebae were inoculated in fresh culture media and one loop of the bacterial colony was added to it (amoebae and bacteria group).

In another series, the hemolytic bacteria were added only to new culture media (bacteria group). 0.2% ml. of 5% human ref cells were added to above two groups and the hemolysis examined at 20 hours after the culture.

5. E. histolytica and the hemolytic substances which were produced from enteric bacteria and hemolytic bacteria.

Hemolytic substances ware obtained by Seitz filter Procedure. Hemolysis was observed dividing into two groups; human red cells and hemolytic substances, human red cells combined with amoebae and hemolytic substances.

6. Red cells for the propagation of E. histolytica.

YS 9 strain was used for this experiment. All the amoebae in 0.02 ml. of media ware counted under the microscope, and the number par 1 ml. was calculated. 0.2ml. of 5% rabbit and sheep red calls, and the known number of amebae were inoculated in new modified diphasic medium, and incubated at 37℃. for 2 days.

7. Gelatinolytic ability of E. histolytica.

YS 1, 9, 10 strains of E. histolytica were smeared on 15% gelatin media. As control the bacteria derived from the amoebae culture media were smeared on another gelatin media. After incubation for 24 hours at 37℃., the media ware plated in 4℃. refrigerator for 30 minutes in order to confirm the gelationlysis.

Results:

A. Hemolytic ability of E. histolytica.

1) The hemolytic ability was different affording to the strains of E. histotytica. YS 1 strain lysed the red cells of pig and sheep and slightly the ox, but not from rabbit, dog, and human. YS 5 strain lysed the red cells from ok, but net rabbit, dog, pig, sheep and human. YS 9 strain lysed the red cells of sheep,

but not from the others, and YS 10 strain lysed the red cells of dog and sheep slightly, but not from the others.

2) Metabolic products of each strain of 5. histolytica could not inhibit the hemolytic action of the substances from the supernatants of the culture.

B. Relationship of E. histolytica and hemolytic bacteria.

Bacillus subtilis hemolysed the red blood cells but showed slight hemolysis when combined with YS 1 and YS 9 strains, arid did not with YS 10,

Staphylococcus aureus showed no hemolysis when combined with YS 1, 9, 1O. Haemolytic substances of Bacillus subtilis, Staphylococcus aureus and Escherichia coli did not hemolyse the red blood cells when they were combined with E. histolytica.

C. The propagation of E. histolytica and red blood cells.

Red blood cells of sheep and rabbits were used for the experiment. Two sets of cultures, to which 0.2 ml. of 5% red blood cell suspension were added or not added. were used.

In the culture which rabbit red cells were added and in control, a standard number of 5800 amoebae per ml. were inoculated into each of them. Control tubes gave a yield of 19,600 per ml. at 48 hours from talc original number, but in the

red cell-treated tubes the prqpagation recovered on the 6th day and the average number was 24,750 per ml. and the R.B.C. were hemolysed on the 4th day.

In the culture which sheep red cells were added and in control, counts of the control tubes at 48 hours gave a yield of 19,300 per ml. from 5,100 originally seeded. A yield of 23,500 per ml. in the red cell-treated tubes was maximum at 4th day. Complete hemolysis of sheep R.B.C. occured on the 2nd day.

D. Gelatin liquifaction test.

In the amoebae (YS 1, 9, 10) arid bacteria groups, gelatin liquifaction was positive, but in the both bacteria and control groups were negative.

Conclusion:

Four strains of Entamoela a histolytica (YS 1, 5, 9, 10) were sampled from Korea, and the hemolytic and cytolytic action of the protozoa were studied.

1. Each strain of E. histolytica has selective ability to hemolyse the red blood cells of human, pig. dog, sheep, and rabbit.

2. E. histolytica inhibits the hemolytic action of enteric bacteria.

3. Metabolic products of E. histolytica does not interfere the hemolytic action of bacteria.

4. The normal red blood corpuscles inhibit the multiplication of E. histolytica but when the cells are hemolysed it accelerates the multiplication of the protozoa.

5. E. histolytica can liquify the gelatin.

From the above results it is concluded that the hemolytic ability of Entamoeba histolytica is different according to the species, and inhibits the hemolytic action of enteric bacteria, but multiplication is inhibited in medium containing normal red cells.